renal and pulmonary aspects of birt-hogg-dubé syndrome · general introduction and outline of the...

Renal and pulmonaRy aspects of BiRt-Hogg-duBé syndRome

Paul Christiaan Johannesma

Renal and Pulmonary Aspects of Birt-Hogg-Dubé syndrome.

Paul Christiaan Johannesma

Thesis, Faculty of Medicine, VU University medical center, VU University, The Netherlands

Proefschrift, faculteit der Geneeskunde, VU medisch centrum, Vrije Universiteit, Nederland

isBn: 978-94-6182-703-6

author: Paul Christiaan Johannesma

cover illustration: Arno Rozema

layout and printing: Off Page, Amsterdam

Copyright © P. C. Johannesma, Amsterdam, The Netherlands, 2016

All rights reserved. No parts of this publication may be reproduced, stored in a retrieval system, or transmitted in any

form or by any means, without written permission from the author or from the Publisher holding the comy right of the

published articles.

Publication of this thesis was financially supported by:

Vrije Universiteit, afdeling Heelkunde Jeroen Bosch Ziekenhuis, ABN AMRO N.V., Jeroen Bosch Academie, Rijnstate

Vriendenfonds, CARE10 HealthCare Products & Services, Myrovlytis Trust.

The sponsors who are gratefully acknowledged, had no involvement in any stage of the study design, data collection,

data-analysis, interpretation of the data or the decision to publish study results.

The research presented in this thesis is part of the research program of the VUmc Cancer Center Amsterdam (VUmc-

CCA). The studies were performed at the Department of Pulmonary Diseases and the Department of Urology of the VU

University medical center, Amsterdam, The Netherlands.

VRIJE UNIVERSITEIT

Renal and PulmonaRy asPects of BiRt-Hogg-duBé syndRome

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam,

op gezag van de rector magnificus

prof.dr. V. Subramaniam,

in het openbaar te verdedigen

ten overstaan van de promotiecommissie

van de Faculteit der Geneeskunde

op vrijdag 30 september 2016 om 9.45 uur

in de aula van de universiteit,

De Boelelaan 1105

door

paul christiaan Johannesma

geboren te Amstelveen

promotoren: prof.dr. P.E. Postmus

prof.dr. R.J.A. van Moorselaar

copromotoren: dr. F.H. Menko

dr. J.H.T.M. van Waesberghe

Voor mijn ouders

ta B l e o f c o n t e n t s

geneRal intRoduction

Chapter 1 Birt-Hogg-Dubé syndrome: a molecular and clinical overview. 15

Outline of the thesis 23

PaRt i PulmonaRy manifestations

Chapter 1.1 The pathogenesis of pneumothorax in Birt-Hogg-Dubé syndrome:

a hypothesis. 33

Johannesma PC, Houweling AC, van Waesberghe JH, van Moorselaar RJ,

Starink TM, Menko FH, Postmus PE.

Respirology. 2014 Nov;19(8):1248-50.

Chapter 1.2 Presence of pulmonary cysts in BHD patients with and without

a pneumothorax; a retrospective analysis of 61 patients. 41

Johannesma PC, van Waesberghe JHTM, Menko FH, van Moorselaar RJA,

Paul MA, Starink ThM, Reinhard R, Houweling AC, van de Beek I, Jonker MA,

Postmus PE.

(Submitted).

Chapter 1.3 Radiological features of primary spontaneous pneumothorax patients

with or without a mutation in FLCN. 51

Johannesma PC, van Waesberghe JHTM, Menko FH, van Moorselaar RJA,

Paul MA, Starink ThM, Reinhard R, Houweling AC, van de Beek I, Jonker MA,

Postmus PE.

(Submitted).

Chapter 1.4 How reliable are clinical criteria in distinguishing between Birt-Hogg-Dubé

syndrome and smoking as a cause for pneumothorax? 59

Johannesma PC, Thunnissen E, Postmus PE.

Histopathology. 2014 Jun;64(7):1045-6.

Chapter 1.5 Risk of spontaneous pneumothorax due to air travel and diving

in patients with Birt-Hogg-Dubé syndrome. 63

Johannesma PC, van der Wel JWT, Paul MA, Houweling AC, Jonker MA,

van Waesberghe JHTM, Reinhard R, Starink ThM, van Moorselaar RJA,

Menko FH, Postmus PE.

SpringerLink 2016 (Accepted for publication)

Chapter 1.6 Prevalence of Birt-Hogg-Dubé syndrome in patients with apparently

primary spontaneous pneumothorax. 75

Johannesma PC, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ,

Menko FH, Postmus PE; on behalf of the Amsterdam BHD working group.

Eur Respir J. 2015 Apr;45(4):1191-4.

Chapter 1.7 International guidelines for pneumothorax are not adequate for

treatment of spontaneous pneumothorax in patients with

Birt-Hogg-Dubé syndrome. 91

Johannesma PC, Paul MA, van Waesberghe JHTM, Jonker MA,

Houweling AC, van de Beek I, van Moorselaar RJA, Menko FH, Postmus PE.

(Submitted)

PaRt ii Renal manifestations

Chapter 2.1 Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome;

an analysis of 115 FLCN mutation carriers from 35 BHD families. 101

Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA,

van Spaendonck-Zwarts KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH,

de Jong MM, Bongers EM, Johannesma PC, Postmus PE, van Moorselaar RJ,

van Waesberghe JH, Starink TM, van Steensel MA, Gille JJ, Menko FH.

Br J Cancer. 2011 Dec 6;105(12):1912-9

Chapter 2.2 Are lung cysts in renal cell cancer (RCC) patients an indication for FLCN

mutation analysis? 119

Johannesma PC, Houweling AC, Menko FH, van de Beek I, Reinhard R,

Gille JJ, van Waesberghe JHTM, Thunnissen E, Starink TM, Postmus PE,

van Moorselaar RJ.

Fam Cancer. 2016 Apr;15(2):297-300.

Chapter 2.3 Renal imaging in 199 Dutch patients with Birt-Hogg-Dubé syndrome:

Screening, compliance and outcome. 127

Johannesma PC, van de Beek I, Reinhard R, Leter EM, Rozendaal L,

Starink ThM, Waesberghe JHTM, Horenblas S, Jonker MA, Menko FH,

Postmus PE, Houweling AC, van Moorselaar RJA.

(Submitted)

PaRt iii Relevant case RePoRts and case seRies

Chapter 3.1 In-flight pneumothorax: diagnosis may be missed because of

symptom delay. 141

Postmus PE, Johannesma PC, Menko FH, Paul MA.

Am J Respir Crit Care Med. 2014 Sep 15;190(6):704-5.

Chapter 3.2 Spontaneous pneumothorax as indicator for Birt-Hogg-Dubé syndrome

in paediatric patients. 149

Johannesma PC, van den Borne BE, Gille JJ, Nagelkerke AF,

van Waesberghe JT, Paul MA, van Moorselaar RJ, Menko FH, Postmus PE.

BMC Pediatr. 2014 Jul 3;14:171.

Chapter 3.3 Lung cysts as indicator for Birt-Hogg-Dubé syndrome. 157

Johannesma PC, Thunnissen E, Postmus PE.

Lung. 2014 Feb;192(1):215-6.

Chapter 3.4 Spontaneous pneumothorax as the first manifestation of a hereditary

condition with an increased renal cancer risk. 161

Johannesma PC, Lammers JW, van Moorselaar RJ, Starink TM,

Postmus PE, Menko FH.

Ned Tijdschr Geneeskd. 2009;153:A581.

Chapter 3.5 Facial fibrofolliculomas as indicator for renal cell cancer. 171

Johannesma PC, Starink TM, Van Moorselaar RJ, Postmus PE.

Jpn J Clin Oncol. 2014 Jun;44(6):609-10.

Chapter 3.6 Bilateral renal tumour as indicator for Birt-Hogg-Dubé syndrome. 175

Johannesma PC, van Moorselaar RJ, Horenblas S, van der Kolk LE,

Thunnissen E, van Waesberghe JH, Menko FH, Postmus PE.

Case Rep Med. 2014;2014:618675.

Chapter 3.7 A de novo FLCN mutation in a patient with spontaneous pneumothorax

and renal cancer; a clinical and molecular evaluation. 183

Menko FH, Johannesma PC, van Moorselaar RJ, Reinhard R,

van Waesberghe JH, Thunnissen E, Houweling AC, Leter EM, Waisfisz Q,

van Doorn MB, Starink TM, Postmus PE, Coull BJ, van Steensel MA, Gille JJ.

Fam Cancer. 2013 Sep;12(3):373-9.

PaRt iv summaRy, discussion and futuRe PeRsPectives

Chapter 4.1 Summary of the thesis 199

Chapter 4.2 Nederlandse samenvatting (voor leken) 209

Chapter 4.3 Conclusions and future directions 217

addendum

Review committee 228

List of co-authors and affiliations 229

List of abbreviations 232

List of publications 234

List of scientific meetings 239

Grants and Awards 239

Acknowledgements – Dankwoord 241

Curriculum Vitae Auctoris 248

"Een goed begin is het halve werk, maar een goed begin is maar de helft."

(Adapted from: De Jeugd van Tegenwoordig, Album De Lachende Derde,

Sterrenstof. Release: 5 november 2010)

g e n e R a l i n t R o d u c t i o n

c H a P t e R 0 . 1

general introduction and outline of the thesis

Paul C. Johannesma1

1 Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands

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History

In 1977, three Canadian physicians, Arthur Birt, Georgina Hogg, and James Dubé described a pedigree

in which multiple family members had characteristic skin lesions, consisting of fibrofolliculomas,

trichodiscomas and achrocordons (figure 1).1

At present, Birt-Hogg-Dubé syndrome (BHD) is defined as an autosomal dominant condition,

caused by germline mutations in the FLCN (folliculin) gene and clinically characterized by skin

fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal cancer (Online

Mendelian Inheritance in Man #135150). Clinical diagnostic criteria proposed by the European BHD

Consortium are outlined in figure 2.2

genetic and molecular aspects of Birt-Hogg-dubé syndrome

BHD is an autosomal dominant condition with high penetrance and variable expression. In 2001,

a BHD-associated locus was mapped to chromosome 17p11.2 by linkage analysis.3 4 Subsequently,

- in 2002 – in BHD probands truncating germline mutations were identified in a novel gene,

folliculin (FLCN).5 FLCN contains 14 exons and encodes folliculin, a protein of 579 amino acids

that has no major homology to any other human protein.6 Currently, in BHD kindreds, over

100 unique mutations in all coding regions of the FLCN gene have been reported in the LOVD

mutation database (www.lovd.nl).7 8 A hypermutable “hot spot” has been identified in a tract of

eight cytosines in exon 11.9 10 11 12 The majority of mutations are insertions/deletions, nonsense or

figure 1. Pedigree of the family described by Birt et al. (Adapted from Birt AR, et al. Hereditary multiple fibrofolliculomas

with trichodiscomas and acrochordons. Arch Dermatology 1977;113:1674-77).

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splice-site mutations that result in a shift in the reading frame and/or introduction of a premature

termination codon, indicating that loss of FLCN function is responsible for the clinical BHD.13

However, the precise role of folliculin requires further elucidation. The functions of folliculin remain

largely unknown, but are likely to include a tumor-suppressor function (figure 3).14 The mammalian

target of Rapamycine (mTOR) signaling pathway has been implicated in the pathogenesis of

several hereditary syndromes, including BHD. Several proteins, including FNIP1, FNIP 2, TSC1, TSC2

and AMPK, show an interaction with FLCN and abnormalities in the function of these are involved

in genetic disorders showing partial clinical overlap with BHD.15 Patients from a unique kindred

with a germline FNIP1 defect is show facial skin lesions resembling those found in BHD. Patients

with tuberous sclerosis complex due to TSC1 or TSC2 mutations have skin, lung and renal lesions,

a combination of features which also characterizes BHD.16

In ongoing studies the role of folliculin in multiple signaling pathways is investigated and these

include TGFβ/BMP signaling, PGC-1α-driven mitochondrial biogenesis, TFE3/TFEB transcriptional

regulation, cell polarity, Rho A signaling and regulation of the E-cadherin-LKB1-AMPK axis. 15

No clear genotype - phenotype correlations have been reported for BHD.17 It has been proposed that

in BHD patients with a cytosine deletion in the C8 tract in exon 11 of the FLCN gene, renal tumors are

less frequent than in those with a cytosine insertion. Differences in frequency of pulmonary cysts,

spontaneous pneumothorax, or fibrofolliculomas were not observed in this group. Another study

observed a trend towards more pneumothoraces in BHD patients with FLCN mutations in exons 9

and 12 and an association between FLCN mutations in exon 9 and an increased number of pulmonary

cysts have also been observed.18 19 20

Figure 2. Diagnostic criteria for BHD proposed by the European BHD Consortium (Adapted from Menko et al. Birt-

Hogg-Dubé syndrome: diagnosis and management. Lancet Oncology 2009;10:1199-206).

Panel: diagnostic criteria for Birt-Hogg-dubé syndrome (BHd; patients should fulfill one

major or two minor criteria for diagnosis)

major criteria

• At least five fibrofolliculomas or trichodiscomas, at least one histologically confirmed, of

adult onset *

• Pathogenic FLCN germline mutation

minor criteria

• Multiple lung cysts: bilateral basally located lung cysts with no other apparent cause, with

or without primary spontaneous pneumothorax

• Renal cancer: early onset (<50 years) or multifocal or bilateral renal cancer, or renal cancer

of mixed chromophobe and oncocytic histology

• A frist-degree relative with BHD

*Fibrofolliculoma and trichodiscoma are two possible presentations of the same lesion – for the differential diagnosis, angiofibroma in tuberous sclerosis should be considered. Childhood- onset familial fibrofolliculoma or trichodiscoma without other syndromic features might be a distinct entity.

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dermatological aspects of Birt-Hogg-dubé syndrome

The facial fibrofolliculomas typical for BHD are benign hair follicle tumors, consisting of epithelial

strands emanating from the outer root sheath of a deformed hair follicle (figure 4).21 These

lesions usually appear after the age of 20 years, as multiple, dome-shaped, whitish papules in

the face, neck and upper part of the back (figure 5).22 Occasionally they involve the lips, buccal

mucosa and gingiva. Fibrofolliculomas closely resemble angiofibromas and indeed, angiofibromas

have been reported in BHD.23 Other benign skin abnormalities possibly associated with BHD are

acrochordons (skin tags) and lipomas.24 Malignant skin lesions, including melanoma, have also

been observed in BHD.25 26 27

The diagnosis of BHD associated fibrofolliculomas is based on both clinical presentation and

histological examination. Biopsies and sectioning of on several levels show a unique histological

pattern, which confirms the clinical diagnosis.18 28 The penetrance of the skin lesions is age dependent

and an estimated 80% of FLCN mutation carriers will develop skin fibrofolliculomas. Expression is

quite variable: some patients have multiple facial papules whereas in other cases the lesions are

minimal and will only be recognized by expert dermatological examination.

figure 3. FLCN-FNIP axis and potential interactions with cellular pathways and processes. FLCN, FNIP and FNIP2

interact with AMPK and modulate mTOR signaling. Tumor suppressors are red. FLCN interactors are green. Potential

interactions of FLCN/FNIP1/FNIP2 with cellular pathways / processes are in yellow boxes. à indicates activation.

-| indicates inhibition. ? indicates evidence for both inhibition and activation (Adapted from Schmidt LS. Birt-Hogg-

Dubé syndrome: from gene discovery to molecularly targeted therapies. Fam Cancer 2013;12:357-64).

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For differential diagnosis tuberous sclerosis complex is important since the BHD associated

fibrofolliculomas may be mistakenly classified as angiofibromas due to TSC.2

Treatment of fibrofolliculomas is only needed for cosmetic reasons. Treatment options include

hyfrecation (electrocoagulation), followed if necessary by curettage. Alternative possibilities are

surgical removal, retinoic acid derivatives and ablative laser techniques (e.g. YAG, CO2). Targeted

local therapy by topical Rapamycin (mTOR inhibitor) has no obvious effect.29 30

Pulmonary aspects of Birt-Hogg-dubé syndrome

Pneumothorax is defined as the presence of air in the pleural cavity. It is a common condition with

a high incidence of between 1.2 and 18 cases per 100.000 persons per year. If not due to an obvious

external force (trauma, iatrogenic) the condition is described as spontaneous pneumothorax (SP).

SP is subdivided into secondary SP (SSP), due to various forms of lung pathology, and primary SP

(PSP) without an obvious underlying lung disease.31 The first episode of PSP usually occurs in the

third decade of life in males, who are often taller than age-matched controls, and the majority

has a history of smoking. Smoking increases the risk of PSP more than 100 times.32 PSP diagnosis is

usually based on history and confirmed by a standard erect chest X-ray during inspiration. A chest

CT is indicated in complicated cases, for example a recurrent or persistent air leak. In up to 90%

of uncomplicated cases cystic structures, usually described as (subpleural) blebs and bullae, are

found in the lung apices. In a subgroup of around 5-15% of PSP patients cystic abnormalities are

figure 4. Fibrofolliculomas; hair follicle

tumors, consisting of epithelial strands

emanating from the outer root sheath of

a deformed hair follicle. (Adapted from

Johannesma et al. Facial fibrofolliculomas

as indicator for renal cell cancer. Jpn J Clin

Oncol. 2014;44(6):609-10).

figure 5. Clinical facial fibrofolliculomas (Adapted from Menko

et al. Birt-Hogg-Dubé syndrome: diagnosis and management.

Lancet Oncology 2009;10:1199-206).

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0.1described in the apices and other areas of the lungs, also below the level of the main carina.33 34

Possibly in the latter group of patients the aetiology of pneumothorax differs from that in patients

with abnormalities restricted to the apices.

Overall 11.5% of patients with PSP report a positive family history of the disease.35 Familial occurrence

was described for the first time by Faber et al.36 In 1991, Albolnik et al. postulated two modes of

inheritance for familial primary spontaneous pneumothorax: autosomal dominant with reduced

penetrance and X-linked recessive.37

Hereditary predisposition for spontaneous pneumothorax can occur in specific syndromes

including Marfan syndrome, homocystinuria, Ehlers-Danlos syndrome, α1-antitrypsin deficiency,

tuberous sclerosis complex and Birt-Hogg-Dubé syndrome. The causes of hereditary predisposition

for spontaneous pneumothorax are summarized in table 1.35

table 1. Causes of hereditary predisposition for spontaneous pneumothorax (adapted from Chiu et al. Familial spontaneous pneumothorax. Current Opin Pulm Med. 2006;12:268-72).

disease gene(s) chromosomal location

Marfan syndrome Fibrillin 1 15q21.1

Homocystinuria Cystathionine β-synthase 21q22.3

Ehlers-Danlos syndrome Multiple Multiple

α1-Antitrypsin deficiency α

1-Antitrypsin 14q32.1

Birt-Hogg-Dubé syndrome Folliculin 17p11.2

SP may be the first and only apparent manifestation of BHD both in isolated and familial cases. In

most BHD patients without pneumothorax chest X ray shows normal lung parenchyma but multiple

lung cysts are commonly identified on CT, in about 90% of adult patients; approximately 50% of the

cysts are located in the subpleural area and 50% in the parenchyma (figure 6a and B).38 Zbar and

colleagues found a 50-times increase in the risk of pneumothorax for BHD-affected individuals.39

SP has been reported in BHD patients already at the age of 7 and 16 years, but the majority is over

18 years of age.40 41 Although about 90% of BHD syndrome patients have these multiple cysts, lung

function (measured by spirometry and diffusion capacity) is generally normal.42 Thus, patients

with pneumothorax due to BHD, usually have no preceding symptoms of pulmonary disease and

are therefore likely to be diagnosed as having PSP. The risk of pneumothorax is about 25% and

this risk is probably not due to specific gene defects since clear genotype phenotype correlations

have thus far not been demonstrated. While the recurrence rate in common primary SP has been

described up to 50% when treated in a conservative way, the recurrence rate of SP among BHD

patients has been reported to be much higher, up to 75%, despite even less conservative types of

treatment.43 44 45 46

The exact relationship between the presence of multiple lung cysts and the occurrence of PSP has

not been clarified. Most but not all BHD who develop pneumothorax have multiple basal lung cysts.

The presence of cysts does not per se result in pneumothorax.47 48

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Due to the variable expressivity of BHD, patients with only spontaneous pneumothorax, and without

other clinical manifestations, might in fact have BHD. It is important to diagnose the syndrome in

these apparently sporadic cases, since a diagnosis of BHD will lead to measures aimed at the early

detection and treatment of renal cancer, not only in the proven carrier but also in possibly affected

relatives. Thus far only one Chinese study described a prevalence of 9.8% of BHD in apparently

common PSP.46

Diagnosis of BHD in SP cases strongly depends on the demonstration of multiple lung cysts.

However, in uncomplicated SP cases imaging is restricted to X ray and does not include a CT scan.

Treatment of spontaneous pneumothorax is based on international guidelines. However, in BHD,

the naturally history of pneumothorax may differ from that in sporadic cases and possibly treatment

should be different for BHD associated pneumothorax, especially in complicated cases.49

Renal aspects of Birt-Hogg-dubé syndrome

Worldwide, renal cell carcinoma (RCC) accounts for about 150.000 new cancer cases and 78.000

cancer deaths annually, with a peak prevalence in the 6th and 7th decades, and a twofold excess

of males to females.50 51 The frequency of RCC has been increasing partly due to incidental

cases detected by abdominal imaging. Epidemiological risk factors associated with RCC include

cigarette smoking, obesity and hypertension.52 Approximately 3% of RCC is due to hereditary

predisposition and these hereditary cases include a dozen different syndromes.53 The most

common inherited syndromes associated with RCC are Von Hipple-Lindau disease, hereditary

papillary renal cancer, hereditary leiomyomatosis and renal cancer, succinate dehydrogenase

subunit mutations, chromosome 3 translocations, and Birt-Hogg-Dubé syndrome (BHD).54 An

overview of inherited disorders predisposing to RCC is given in table 1.50 Hereditary RCCs differ

from the far more common sporadic form in several aspects. Hereditary tumours often present at

an early age, are more often multifocal and / or bilateral and may have a characteristic histology.

figure 6. Thoracic CT scan: lung cysts in basal parts of the lung in BHD patient (Adapted from Johannesma et al.

The prevalence of Birt-Hogg-Dubé syndrome among patients with apparently primary spontaneous pneumothorax.

Eur Respir J 2015;45(4):1191-4).

a B

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0.1In addition, they may be associated with syndromic features besides renal cell cancer and clearly,

family history may be positive for RCC or features of the syndrome involved.55 In BHD the risk of

RCC is estimated to be about 15% with a mean age at diagnosis of 50 years (range 20-75 years).2 The

most common histological subtype in sporadic RCC is clear cell RCC (ccRCC).56 In BHD, the most

common subtypes are hybrid oncocytic, chromophobe renal cell carcinoma and oncocytoma

(figure 7).20 However, other subtypes including ccRCC may also occur. Given the high degree of

inter- and intra-familial variability of these features, it is likely that many cases of BHD associated

RCC currently remain unrecognized. The prevalence of BHD in (familial) renal cancer has been

investigated by Woodward et al. These authors found in selected patients (3/69) with RCC 4.3% of

cases with FLCN mutations.57

The main clinical consequence of a diagnosis of BHD is early diagnosis and treatment of renal cell

cancer and therefore periodic renal imaging is advised for all FLCN mutation carriers.20 55 Surveillance

at regular intervals by MRI, is advised from the age of 20 (figure 8). A disadvantage of MRI is its

availability and costs, which may lead to limited access in clinical practice. The role of ultrasound

(US) in the early detection of renal tumours is a matter of debate due to limited sensitivity for the

detection of small tumours.58 59 60 61

In general, BHD associated tumours are slowly growing tumours and the risk of distant metastasis is

strongly dependent on the size of the tumour. Therefore, surgical treatment is recommended when

the largest tumor reaches 3 cm in maximal diameter.59 This guideline has previously been adopted for

figure 7. Reported histological subtypes of BHD associated renal tumours are hybrid oncocytic, chromophobe renal

cell carcinoma and oncocytoma (Adapted from Houweling AC et al. Renal cancer and pneumothorax risk in Birt-Hogg-

Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. Br J Cancer. 2011 Dec 6;105(12):1912-9).

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Von Hippel-Lindau disease and is now also recommended for BHD.62 63 64 Local treatment should be

nephron sparing due to the high risk of metachronic new primary tumours. Partial nephrectomy, and

minimally invasive nephron sparing techniques such as cryoablation and radio frequency ablation

(RFA) are options for local treatment. Since BHD patients are at lifelong risk for the development

of new tumors, and cryoablation or RFA may complicate both long term evaluation and surgical

management, nephron-sparing surgery thus far is the safest and most effective treatment for BHD

associated RCC.2 55 59

other clinical findings in Birt-Hogg-dubé syndrome

In BHD, many other clinical features besides fibrofolliculomas, pneumothorax and renal cell cancer

have been observed, mainly in single case reports and small case series. Benign tumours with

BHD include multinodular goitre, parotid gland adenoma, colorectal polyp and adenoma, neural-

tissue tumour, trichoblastoma, connective–tissue nevus, focal-cutaneous mucinosis, lipoma,

angiolipoma and cutaneous leiomyoma. Malignant tumours include breast cancer, colorectal

cancer, sarcoma of the leg, tonsillar cancer, lung cancer, melanoma, basal and squamous-cell skin

cancer, dermatofibrosarcoma protuberans, cutaneous leiomyosarcoma and rhabdomyosarcoma.

Although in 1975 Hornstein and Knickenberg described a relationship between fibrofolliculomas

and colorectal polyps, this relationship remains uncertain. Also the suggested relationship between

parotid oncocytomas and BHD remains unclear.65 66 67 68 69 70 71 72 73 74 75 76

o u t l i n e o f t H e t H e s i s

The prevalence of BHD is unknown and the syndrome is likely unrecognized by doctors and

patients as the skin signs are often mild and the pulmonary and renal symptoms of BHD are hard to

distinguish from apparently common SP and sporadic RCC. Although over the last decade insight

into the clinical manifestations of BHD has expanded, most knowledge is based on case reports and

figure 8. Renal cell cancer on initial MRI (Johannesma et al. Prevalence of Birt-Hogg-Dubé syndrome in patients with

apparently primary spontaneous pneumothorax. Eur Respir J. 2015 Apr;45(4):1191-4).

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0.1small case series and not on large clinical datasets. Recognition of this syndrome is important to

optimize the effectiveness of surveillance and treatment. The VU University Medical Center (VUmc)

in Amsterdam, the Netherlands, is recognized as a NFU (Dutch Federation of University Medical

Centers) expert center for BHD providing the unique opportunity of evaluating a large cohort of

BHD families referred to the VUmc and its affiliated hospitals. We designed several clinical studies

aimed at the pulmonary and renal manifestations of BHD.

The thesis in subdivided into three parts. “Part one: pulmonary manifestations”, “Part two: renal

manifestations” and “Part three: relevant case reports”. In chapter 1.1 we evaluated the association

between BHD associated lung cysts and the development of spontaneous pneumothorax (SP). In

chapter 1.2 we studied the role of chest computed tomography (CT) in diagnosis and management

of BHD associated pneumothorax. We compared the radiological results of BHD patients with

and without a history of (recurrent) SP, to evaluate a possible relationship between lung cysts in

the development of SP. In chapter 1.3 we hypothesize that CT scanning in PSP patients could be

a sensitive tool in diagnosing BHD in PSP patients. This would be highly clinically relevant since

BHD is associated with an increased risk of renal cancer. Therefore we evaluated the findings of

chest CT in a group of PSP patients without a detectable FLCN mutation and compared these to a

group with a proven mutation in FLCN. In chapter 1.4 we discuss the reliability of clinical criteria in

distinguishing between BHD and smoking as a cause for pneumothorax. In literature an increased

prevalence of SP associated with air travel in patients with the interstitial cystic lung disease

lymphangioleiomyomatosis (LAM) has been described. Based on these results we hypothesize in

chapter 1.5 that BHD patients are also more prone to develop a SP during air travel or diving. This

might have possible implications for clinical management for pulmonologists and lung surgeons.

In chapter 1.6 we address the important clinical question whether all patients who present with

primary spontaneous pneumothorax (PSP) should be evaluated for BHD. We reviewed the available

thoracic CT scans in apparently PSP patients for the presence of basal cysts. In addition we perform

FLCN mutation analysis in a randomly selected group of PSP patients. The recurrence rate of SP in

BHD patients has been reported to be up to 75% despite different types of treatment. Therefore we

evaluated the recurrence rate and different treatment options of SP in BHD patients comparted to

SP patients without a pathogenic FLCN mutation in chapter 1.7.

“Part two: renal manifestations” focuses on the renal manifestations of BHD. In chapter 2.1 we

hypothesize that cysts under the main carina in patients diagnosed with “sporadic” RCC might be an

important diagnostic clue in unmasking BHD. Therefore, we test in a pilot study a cohort of patients

with formerly diagnosed RCC from the VU University Medical Center (VUmc) and the Netherlands

Cancer Center (NKI-AvL). In literature renal MRI is recommended for renal surveillance in BHD

patients from the age of 20. Unfortunately this suggestion is mainly based on expert opinion only

and other genetic renal diseases. In chapter 2.2 we retrospectively evaluate the compliance to, and

the outcomes of renal cancer surveillance in patients diagnosed with BHD in two Dutch centers

(VUmc and NKI-AvL).

“Part three: relevant case reports” contains several case reports and small case series. In chapter 3.1

we evaluate a case of missed diagnosis of pneumothorax in a BHD patient due to symptom delay.

In chapter 3.2 we highlight SP as indicator for BHD in two pediatric patients. In chapter 3.3 and

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0.1chapter 3.4 we evaluate SP as first manifestation in BHD. In chapter 3.5 we discuss the importance

of recognition of facial fibrofolliculomas as indicator for renal cell cancer. In chapter 3.6 we evaluate

a patient with bilateral renal cancer as indicator for BHD. In chapter 3.7 we describe a de novo FLCN

mutation in a patient with spontaneous pneumothorax and renal cancer. Finally, in part 4, the results

of the previous chapters are discussed, put into perspective, and summarized.

Table 2. Examples of inherited disorders predisposing to RCC (adapted from Maher ER. Genetics of familial renal cancers. Nephron Exp Nephrol 2011;118:e21-e26).

disorder type of Rccassociated tumours and other features gene and location

VHL disease Clear cell Retinal and CNS haemangioblastoma,

phaeochromocytoma, pancreatic

tumours, visceral cysts

VHL

3p25

Familial non-syndromic

clear cell RCC

Clear cell - ?

HPRC1 Papillary (type 1) - MET 7q31

Hereditary

leiomyomatosis and

renal cancer

Variable Cutaneous and uterine leiomyomas,

leiomyosarcoma

FH 1q25-32

Succinate dehydroge-

nase subunit mutations

Variable Extra-adrenal and adrenal

phaeochromocytoma, head and

neck paraganglioma

SDHB 1p36.1 – p36

SDHD 11q23

BHD syndrome Variable Fibrofolliculomas, lung cysts and

pneumothorax, colorectal polyps

BHD/FLCN 17p11.2

Hyperparathyreoidism-

jaw tumour

Papillary RCC, renal

hamartomas, Wilms

tumour

Parathyroid tumours, fibro-osseous

mandibular and maxillary tumours,

renal cysts

CD73/ HRPT2, 1q25

Chromosome 3

translocation

Clear cell - Various (FHIT, NORE1A,

etc.)

Lynch syndrome Transitional cell carci-

nomas of the renal

pelvis and ureter

Colorectal cancer, endometrial

cancer

MSH2 2p22-p21, MLH1

3p21.3, PMS2 7p22.2, PMS1

2q31.3, MSH6 2p16

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0.1R e f e R e n c e s 1. Birt AR, Hogg GR, Dubé WJ. Hereditary multiple

fibrofolliculomas with trichodiscomas and

acrochordons. Arch Dermatology 1977;113:1674-77.

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5. Nickerson ML, Warren MB, Toro JR, et al. Mutations

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40. Bessis D, Giraud, Richard S, et al. A novel familial

germline mutation in the initiator codon of the

BHD gene in a patient with Birt-Hogg-Dubé

syndrome. Br J Dermatol. 2006;155:1067-69.

41. Gunji Y, Akiyoshi T, Sato T, et al. Mutations in the

Birt-Hogg-Dubé gene in patients with multiple

lung cysts and recurrent pneumothorax. J Med

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42. Tobino K, Hirai T, Johkoh T, et al. Differentation

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lymphangioleimyomatosis: quantative analysis of

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chest in 66 females. Eur J Radiol. 2012:81:1340-6.

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44. Verhaert LL. A young man with bilateral spontaneous

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48. Gunji Y, Akiyoshi T, Sato T, Kurihara M, Tominaga S,

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55. Stamatakis L, Metwalli AR, Middelton LA, et al.

Diagnosis and management of BHD-associated

kidney cancer. Fam Cancer 2013;12(3):397-02.

56. Pavlovich CP, Walther MM, Eyler RA, et al. Renal

tumors in the Birt-Hogg-Dubé syndrome. Am J

Surg Pathol. 2002; 26:1542–52.

57. Woodward ER, Ricketts C, Killick P, et al. Familial

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2008 Sep 15;14(18):5925-30.

58. Choyke PL, Glenn GM, Walther MM, et al. Hereditary

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59. Jamis-Dow CA, Choyke PL, Jennings SB, et al. Small

(≤3cm) renal masses: detection with CT versus US and

pathologic correlation. Radiology 1996;198:785-88.

60. Hall EJ, Brenner DJ. Cancer risks from diagnostic

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parenchymal sparing surgery. J Urol 1999;161:1475-79.

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2001;52:1016-20.

p a R t 1

Pulmonary manifestations

c H a P t e R

the pathogenesis of pneumothorax in Birt-Hogg-dubé syndrome: a hypothesis

Paul C. Johannesma1, Arjan C. Houweling2, JanHein T.M. van Waesberghe3, R. Jeroen A. van Moorselaar4,

Theo M. Starink5, Fred H. Menko2 6, Pieter E. Postmus1 7

1 Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands2 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

3 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands4 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

5 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands6 Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands

7 Department of Thoracic Oncology, Clatterbridge Cancer Centre, Liverpool Heart & Chest Hospital, University of Liverpool, Liverpool, United Kingdom

Respirology 2014;19(8):1248-50

1 . 1

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a B s t R ac t

The development and natural course of lung cysts in patients with Birt-Hogg-Dubé syndrome is

still unclear and the relationship between the cysts and the development of pneumothorax has not

been fully clarified. Based on the follow up results of thoracic imaging in 6 patients with Birt-Hogg-

Dubé syndrome, we hypothesize that the pulmonary abnormalities of BHD patients are not due to

a progressive degenerative disease. The decreased potential for stretching of the cysts’ wall and

the extensive contact with the visceral pleura are likely to be responsible for rupture of the cyst wall

resulting in the increased risk for pneumothorax in BHD patients.

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i n t R o d u c t i o n

Pneumothorax can be caused by a blunt or penetrating chest injury, or may occur without an

identifiable cause and is then described as “spontaneous pneumothorax” (SP). If there is an obvious

underlying lung disease it is classified as secondary SP. All other cases are described as primary

SP (PSP), however, careful evaluation quite often reveals an underlying abnormality of the lungs,

or pleura, possibly related to the development of SP. The most common of these findings is the

presence of degenerative changes described as blebs, bullae or emphysema-like changes.1 These

lesions are especially found in areas with the highest degrees of pleural stress, the apices of both

lungs.2 In a subgroup of PSP patients somewhat comparable abnormalities are found in other

parts of the lung, especially below the level of the main carina in the parenchyma as well as in the

subpleural area.3 These abnormalities are typical for the Birt-Hogg-Dubé syndrome.4,5 Birt-Hogg-

Dubé syndrome (BHD) is a rare hereditary syndrome first described in 1977. It is characterized by

skin fibrofolliculomas, lung cysts, (recurrent) SP and renal cell cancer and is caused by germline

mutations in the folliculin (FLCN) gene on chromosome 17p11.2.6 Almost every adult BHD patient has

cysts in the lungs; however, the number detected on standard CT (slice thickness 3-5 mm) is much

smaller in those who have not been diagnosed with a pneumothorax.7 Around 50% of the cysts

are located in the subpleural area.8,9 It is unclear whether BHD-cysts are a sign of degeneration/

destruction of lung tissue like in lymphangioleiomyomatosis, Langerhans cell histiocytosis and

bullae in emphysema. If degeneration plays a significant role in the development of SP in BHD one

might expect an increase in number and/or size of the cysts over time. Information on the natural

course of cyst development in BHD patients is very limited. We evaluated the natural course of

pulmonary cysts in a patient previously treated for recurrent pneumothorax.

In this case study we provide the results of repeated pulmonary imaging at an interval of 3 years and

8 months in a 47 year old male BHD patient with recurrent pneumothorax and multiple basal lung

cysts and hypothesize on the causative mechanism of pneumothorax in BHD patients.

m e t H o d s

To evaluate the reproducibility of measurements of size and number of pulmonary cysts on CT we

evaluated 5 additional BHD cases, in whom for clinical reasons thoracic CTs were repeated, within 1

year after the baseline thoracic CT.

Thoracic CT scans were obtained using the 64-slice multi-detector CT system (Somatom

Volume Zoom, Siemens, Erlangen, Germany) in three patients and a 256-slice multi detector CT

system (Philips 256-slice Brilliance ICT, Best, The Netherlands) in two patients. Follow up of each

patient was performed using the same CT system. All CT scans were made at the end of inspiration

with the patient in the supine position; no intravenous contrast material was used. All images were

digitally reconstructed with a section thickness of 3-5 mm.

CT images were evaluated by one pulmonologist (observer). A pulmonary cyst was defined as an

air-filled space with a sharply demarcated thin wall (<2mm). The observer assessed the CT images for

the presence of pulmonary cysts. The size of each cyst was measured on the transversal maximum

diameter. The levels of the slides of the follow up scan were equalized to the level of the baseline

scan. All patients gave informed consent.

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R e s u lt s

Findings on the CT scans were comparable to those on subsequent scans as is shown in 5 sets of CTs

of 5 BHD control cases with CTs at a short interval (table 1). There was no increase in number of cysts

and the mean size (diameter) increased with a mean of 0.4 millimetres. Four of the patients had

suffered from recurrent pneumothorax. One was a former smoker, with a history of 23 pack-years.

The recurrent pneumothorax and the CT-abnormalities in our index patient were the reasons to

evaluate the possibility of a germline mutation in FLCN. A pathogenic splice site mutation was found

(c.1539-2A>G). The first pneumothorax at age 40 years was treated by VATS (in October 2006) the

subsequent ipsilateral recurrences were treated by total pleurectomy (in May 2007) and chemical

pleurodesis during VATS procedure (in September 2007) respectively.

table 1. Radiological characteristics of index patient and control group.

patientmean size cysts (in mm)

number cysts (in mm)

fu (months)

ptX (n=)

smoking (in py) FLCN mutation

initial fu initial fu

index 15.53 15.86 36 37 44 3 - c.1539-2A>G

control 1 11.07 10.80 44 44 12 2 23 FLCN.1539-2A>G

control 2 22.80 17.00 1 1 6 3 - FLCN.610_611DEL

control 3 11.59 11.75 76 76 9 6 - FLCN.1408_1418D

control 4 4.80 4.80 1 1 0.3 3 - FLCN.499C>T

control 5 - - 0 0 4 - - c.774_775delGTinsCAC

The thoracic CT in the index patient revealed multiple lung cysts mainly in the basal parts of both lungs

(figure 1). Despite the difference in slice thickness (5mm in the first CT versus 3mm in the second CT)

only one additional lung cyst (from 36 to 37) was found on the CT after 44 months follow up. The size

of the cysts was also not significantly different with a mean increase of 0.35 millimetres (range: minus

4.88 mm – plus 4.45 mm), which is comparable to the findings in the control group (table 1).

Figure 1. Thoracic computed tomography (CT) in the index patient revealed multiple lung cysts mainly in the basal

parts of both lungs.

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d i s c u s s i o n

The main finding of this case study was that within a period of 44 months there was no increase in size

or number of pulmonary cysts. If cyst formation and pneumothorax are signs of a degenerative disease

in BHD patients one might expect to see a higher prevalence of pneumothorax in older patients with

BHD, which has not been reported in literature.10 Little information is available on follow-up imaging

of lung cysts in individual BHD patients. Although 44 months follow up is not very long, we found

no indication of progression of the number and/or size of the cysts within this period. Ayo et al.

reported a “stable situation” of the cysts in all four patients during a follow up period between 3 and

66 months.11 Tobino et al. described no significant increase in size of the lung cysts in 3 patients who

had undergone follow up CT scans with an interval between 7 and 24 months. This differs from other

diseases with cystic changes such as pulmonary lymphangioleiomyomatosis (PLAM) and pulmonary

Langerhans cell histiocytosis (PLCH). Both are known to be progressive disorders.12

Despite the abnormalities in the lungs of BHD patients the lung function remains unaffected. This is

consistent with the normal lung parenchyma found in most patients with resected cysts.7

As most subpleural abnormalities in BHD patients are found in the lower parts of both lungs it is

not very likely that pleural stress, as found in the apices of PSP patients, is causally related to the

development of a pneumothorax.2 It is much more likely that the explanation comes from the

effect of the mutation in FLCN on the epithelial layer at the inside of the pleural cysts. Possibly

the down regulation of folliculin results in increased cell-cell adhesion, as proposed by Medvetz

and colleagues.13 If the increased cell-cell adhesion of cells in an epithelial surface results in less

potential to stretch, this might lead to rupture at the weakest spot of a continuous surface if the

stretching force is strong enough. This hypothesis is supported by the hypothesis that growth of

cysts is caused by fusion of smaller cysts by rupture of the wall between cysts resulting in larger

cysts in which often still parts of interlobular septa are found7. The areas in the lung where the

largest stretching force occurs are the lower parts of the lung, as is reflected by the mobility of lung

tumours.14 Direct connection between a cyst wall and the visceral pleura will result in considerable

stretching forces on the cyst wall adjacent to the pleura. If this results in rupture of this cyst wall one

might expect rupture of the visceral pleura as well and subsequently the possibility of development

of a pneumothorax. This visceral pleura – cyst wall connection and rupture may even be the case in

very small subpleural cysts not detectable by standard CT.15

In conclusion: based on our observations and that of others it is unlikely t the pulmonary

abnormalities of BHD patients are due to a progressive degenerative disease. It is much more likely

that the trend of development and recurrence of pneumothorax in BHD is related to the lack of

possibility of epithelial layers to stretch if forced to do so by connection to the visceral pleura.

Follow-up of more BHD patients is needed to expand knowledge on the natural course of these cysts.

This can be done by repeating thoracic CTs, for instance in cases with a recurrent or contralateral

pneumothorax. To prove that especially the cysts in the lower parts of the lungs are responsible for

development of a pneumothorax in BHD patients a therapeutic study is needed. A multicenter phase

II study aiming at complete obliteration of the pleural space, such as can be achieved by extensive

pleurectomy combined with chemical pleurodesis, should result in a much lower recurrence rate of

pneumothorax in these patients.

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We thank the BHD families for their cooperation.

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R e f e R e n c e1. Schramel FM, Postmus PE, Vanderschueren RG.

Current aspects of spontaneous pneumothorax.

Eur. Respir. J. 1997; 10(6):356-9.

2. Casha AR, Manché A, Gatt R, Wolak W, Dudek K,

Gauci M, Schembri-Wismayer P, Camilleri-Podesta

MT, Grima JN. Is there a biomechanical cause for

spontaneous pneumothorax? Eur. J. Cardiothorac.

Surg. 2014; 45(6):1011-6.

3. Smit HJM, Wienk MAThP, Schreurs AJM, Schramel

FM, Postmus PE. Do bullae indicate a predisposition

to recurrent pneumothorax? Br. J. Radiol. 2000;

73(868):356-9.

4. Ren HZ, Zhu CC, Yang C, Chen SL, Xie J, Hou YY,

Xu ZF, Wang DJ, Mu DK, Ma DH, Wang Y, Ye MH, Ye

ZR, Chen BF, Wang CG, Lin J, Qiao D, Yi L. Mutation

analysis of the FLCN gene in Chinese patients with


pneumothorax. Clin. Genet. 2008; 74(2):178-83.

5. Johannesma PC, Menko FH, Reinhard R, van

Waesberghe JHTM, van Moorselaar RJA,

Starink ThM, Postmus PE. Primary Spontaneous

Pneumothorax: a pilot study on the frequency of

FLCN mutation (Birt-Hogg-Dubé syndrome). Am.

J. Resp. Crit. Care Med. 2014;189:A6417.

6. Schmidt LS. Birt-Hogg-Dubé syndrome: from gene

discovery to moleculary targeted therapies. Fam.

Cancer. 2013;12(3):3578-64.


R, Gille JJP, van Moorselaar RJA, Houweling

AC, Starink ThM, Menko FH, Postmus PE. Birt-

Hogg-Dubé syndrome patients with and without

pneumothorax: findings on chest CT. Am. J. Resp.

Crit. Care Med. 2014;189:6416.

8. Kumasaka T, Hayashi T, Mitani K, Kataoka H,

Kikkawa M, Tobino K, Kobayashi E, Gunji Y, Kunogi

M, Kurihara M, Seyama K. Characterization of

pulmonary cysts in Birt-Hogg-Dubé syndrome:

histopathological and morphometric analysis of

229 pulmonary cysts from 50 unrelated patients.

Histopathology. 2014; 65(1):100-10.


R, Gille JJP, van Moorselaar RJA, Houweling AC,

Starink ThM, Menko FH, Postmus PE. Chest CT

for primary spontaneous pneumothorax (PSP):

findings: Birt-Hogg-Dubé versus non-Birt-Hogg-

Dubé patients. Am. J. Resp. Crit. Care Med.

2014;189:A6415.

10. Toro JR, Pautler SE, Stewart L, Glenn GM, Weinreich

M, Toure O, Wei MH, Schmidt LS, Davis L, Zbar

B, Choyke P, Steinberg SM, Nguyen DM, Linehan

WM. Lung cysts, spontaneous pneumothorax, and

genetic associations in 89 families with Birt-Hogg-

Dubé syndrome. Am. J. Resp. Crit. Care Med. 2007;

175(10):1044-53.

11. Ayo DS, Aughenbaugh GL, Yi ES, Hand JL, Ryu JH.

Cystic lung disease in Birt-Hogg-Dubé syndrome.

Chest 2007; 132(2):679-84.

12. Clarke BE. Cystic lung disease. J. Clin. Pathol.

2013;66(10):904-8.

13. Medvetz DA, Khabibullin D, Hariharan V,

Ongusaha PP, Goncharova EA, Schlechter T,

Darling TN, Hofmann I, Krymskaya VP, Liao JK,

Huang H, Henske EP. Folliculin, the product of

the Birt-Hogg-Dubé tumor suppressor gene,

interacts with the adherens junction protein

p0071 to regulate cell-cell adhesion. PLoS One.

2012;7(11): e47842.

14. van Sörnsen de Koste JR, Lagerwaard FJ, Nijssen-

Visser MR, Graveland WJ, Senan S. Tumor location

cannot predict the mobility of lung tumors: a 3D

analysis of data generated from multiple CT scans.

Int J Radiat Oncol Biol. Phys. 2003; 56(2):348-54.

15. Onuki T, Goto Y, Kuramochi M, Inagaki M, Bhunchet

E, Suzuki K, Tanaka R, Furuya M. Radiologically

indeterminate pulmonary cysts in Birt-Hogg-Dubé

syndrome. Ann. Thoracic. Surg. 2014; 97(2):682-5.

c H a P t e RPresence of pulmonary cysts in BHd patients

with and without a pneumothorax; a retrospective analysis of 61 patients

Paul C. Johannesma1, JanHein T.M. van Waesberghe2, Fred H. Menko3, R. Jeroen A. van Moorselaar4, Marinus A. Paul5, Theo M. Starink6,

Rinze Reinhard2 7, Arjan C. Houweling8, Marianne A. Jonker9, Pieter E. Postmus10


2 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands3 Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands4 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

5 Department of Thoracic Surgery, VU University Medical Center, Amsterdam, The Netherlands6 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands

7 Department of Radiology, Onze Lieve Vrouwen Gasthuis, Amsterdam, The Netherlands8 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

9 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands


Submitted

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a B s t R ac t

introduction

Multiple pulmonary cysts below the level of the carina are characteristic for Birt-Hogg-Dubé

syndrome (BHD), an autosomal dominant condition caused by germline mutations in the folliculin

(FLCN) gene. This autosomal dominant disorder clinically manifests in facial skin fibrofolliculomas,

renal cell cancer (RCC), lung cysts and (recurrent) spontaneous pneumothorax (SP). Although the

precise prevalence of BHD is unknown, two recent studies suggest that this syndrome is present in

5-10% of all PSP patients. The relationship between lung cyst characteristics and the development of

(recurrent) SP is unknown. Chest computed tomography (CT) in this patient group might therefore

be an useful tool for choice of treatment when developing a SP and might also play a role in advice

of lifestyle.

material and methods

We retrospectively collected the clinical data on all patients with a proven pathogenic FLCN mutation

and an available thoracic CT. We scored on demographics and medical history for (recurrent) SP.

The thoracic CT was reviewed for presence of abnormalities, and more specifically the presence of

cystic abnormalities and/or air filled cavities. If these were found the size, number, location below

and/or above level of main carina, in the parenchyma and/or subpleural area were noted.

Results

We included a total of 61 patients, 19 of them had a history of (recurrent) SP. We found a higher

number of lung cysts among BHD patients with a history of (recurrent) SP. No differences were

found in gender, size or location. We found no correlation between the number of cysts and age and

no genotype – phenotype correlation was found.

conclusion

CT scanning of BHD patients may lead to detection of abnormalities characteristic for BHD. There

seems to be a relationship between the number of cysts and development of (recurrent) SP. Chest

CT in this patient group might therefore be an useful tool for choice of treatment when developing

a SP and might also play a role in advice of lifestyle.

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Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition caused by germline mutations

in the folliculin (FLCN) gene. BHD is clinically characterized by skin fibrofolliculomas, pulmonary

cysts, (recurrent) spontaneous pneumothorax (SP) and renal cell cancer. Clinical manifestations

of BHD are variable and include patients and families with only skin, lung or renal abnormalities.1

Two recent studies suggest that this syndrome is presented in 5-10% of all primary spontaneous

pneumothorax (PSP) patients.2 3 SP may be the first and only manifestation of BHD in isolated

and familial cases. Most BHD patients have normal chest X-ray images but multiple lung cysts are

commonly identified on CT. Multiple pulmonary cysts below the level of the carina, detected by

thoracic CT, are characteristic for BHD as described in a few small thoracic CT studies and several

sporadic case reports or small retrospective studies with a small number of included patients.4 5 6 7

These studies show that most BHD patients (77-100%) have lung cysts, and it is assumed these to be

related to the development of SP, although this occurs in a minority (33-39%) of all BHD patients.8

Approximately 50% of these cysts are located in the subpleural area and 50% in the parenchyma.9 10

Although about 90% of BHD syndrome patients have these multiple cysts, lung function (measured

by spirometry and diffusion capacity) is generally normal.9 Thus, patients with pneumothorax due

to BHD often have no preceding symptoms of pulmonary disease and are therefore likely to be

diagnosed as having PSP. For BHD mutation carriers it is relevant to know if for an individual there

is a higher or lower risk of developing pulmonary complications, as this may affect their lifestyle,

such as flying or diving. What the relationship between the on CT found abnormalities and the

development of (recurrent) SP is, has not been described. In this study we compared the CT findings

in BHD patients with and without a history of SP to evaluate whether there are CT-characteristics

that differentiate BHD patients with SP from BHD patients without BHD.

m at e R i a l s a n d m e t H o d s

We retrospectively collected the clinical data on all patients with a proven pathogenic FLCN mutation

and an available thoracic CT known in the VU University Medical Center. Exclusion criteria were clinical

BHD without a proven FLCN mutation, secondary pneumothorax due to apparent underlying disease e.g.

emphysema, traumatic or iatrogenic pneumothorax. Furthermore, deceased patients were excluded.

One CT of the thorax of all the cases was scored, independently and blinded for final diagnosis, by an

experienced pulmonologist (PEP) and an experienced radiologist (JHvW). Blinding for diagnosis was

achieved by adding these cases to a much larger series of cases with a history of SP reviewed for other

purposes. In case of multiple CTs the one in time closest to a pneumothorax episode was used, when

available, after radiological resolution of the SP. For the BHD patients without a history of SP, the most

recent available CT was used. We assumed, based on our prior study, that all radiological findings remained

largely unchanged over time.10 Scoring consisted of presence of abnormalities, and more specifically the

presence of cystic abnormalities and/or air filled cavities. If these were found the size, number, location

below and/or above level of main carina, in the parenchyma and/or subpleural area were noted.

For FLCN mutation analysis genomic DNA was extracted from blood samples. Primers for the

amplification and sequencing of the 14 exons were designed as detailed by Nickerson et al. PCR

amplification was performed using a PE 9700 thermocycler (Applied Biosystems, Forster City,

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CA, USA). Sequencing reactions were performed using the Big Dye Terminator system (Applied

Biosystems) and run on an ABI 3100XL or ABI 3730 genetic analyzer (Applied Biosystems). For the

detection of deletions and duplications of one or more exons MLPA analysis was performed using

MLPA kit P256 (MRC Holland, www.mrc-holland.com).11

All statistical analyses were performed using R software (version 3.1.3.). To compare the results of

cysts on thoracic CT scans in BHD patients with and without history of (recurrent) SP, we used the

Wilcoxon rank sum test with continuity correction. To evaluate a possible correlation between age

and number of cyst and between number and size of cysts, we used the Spearman correlation test.

A p-value of less than 0.05 was considered significant. All patients gave written informed consent

for this study. The study was approved by the Ethics Committee of the VU University Medical Center.

R e s u lt s

We included a total of 61 BHD patients with a proven pathogenic FLCN mutation, 27 of them were

men and 34 of them were women. We included 19 BHD patients with a (recurrent) episode of SP and

42 BHD patients without a history of (recurrent) SP so far. We found in all included patients one or

more lung cysts on thoracic CT.

The mean number of cysts among BHD patients with a history of (recurrent) SP was 81.3 and the

mean number of cysts among BHD patients without a history of (recurrent) SP was 19.0 cysts, this

differed significantly between both groups (p<0.008). We found no significant difference in size,

predominant distribution subpleurally or in the parenchyma, shape, or presence of small pulmonary

artery and veins in the cysts. (table 1). As we found only a significant difference in number of cysts

between both groups, we performed a subgroup analysis. The number of cysts did not significantly

differ between men and women when both groups were taken together (figure 1; men=0 ; female =1).

The scatter of cysts in number vary much more widely in the group of BHD patients with (recurrent)

SP with a minimum of one cyst and a maximum of 250 cysts (mean number of pleural cysts 41.8 ± 37.0,

mean number of parenchymal cysts 53 ± 45.9), compared to the group of BHD patients without

a history of (recurrent) SP with a minimum of one cyst and a maximum of 80 cysts (mean number

of pleural cysts 8.4 ± 9.1, mean number of parenchymal cysts 10.5 ± 11.6) (figure 2). We found no

relationship (ρ=-0.027) between age and the number of cysts, which confirms the findings of our

former published study.14 Also when we perform this sub analysis in both groups separately, we

found no relationship between age and the number of cysts (figure 3). The scatter in size between

both groups are comparable, no predilection of size was found in both groups (figure 4). Also the

presence of cysts above and under the carina did not differ between both groups (figure 5). Finally

no genotype – phenotype correlation was found in both groups.

d i s c u s s i o n

In this study we evaluated a group of 61 patients, 19 of them had a history of (recurrent) SP. We

observed an important and significant difference between the two groups with respect to number

of cysts. In the BHD patients with a history of (recurrent) SP we found a significant higher number

of cysts in the lungs, both subpleurally and in the parenchyma. This might indicate that the visible

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table 1. Radiological characterisitics of cysts in BHD patients with a history of (recurrent) spontaneous pneumothorax and in BHD patients without a history of (recurrent) spontaneous pneumothorax.

BHd+ pneumothorax (sp)+ n=19

BHd+ pneumothorax (sp) – n=42 p-value

number of cysts (N=) 81.3 (9.5-114.5) 19.0 (2.0-33.0) 0,008

Distribution under main carina (%) 88.2 91.9 0.312

Distribution left lung (%) 49.6 54.8 0.419

Distribution right lung (%) 50.4 45.2 0.419

Diameter <1cm (%) 61.9 63.8 0.793

Diameter 1-2cm (%) 23.2 27.6 0.654

Diameter >2cm (%) 14.9 8.6 0.154

Distribution subpleural (%) 50.8 55.2 0.613

Distribution parenchyma (%) 49.2 44.8 0.613

Shape round (%) 32.2 29.9 0.813

Shape oval (%) 67.8 70.1 0.813

abutting pulmonal artery/vein (%) 15.2 9.9 0.548

figure 1. Number of cysts in male (0) and female (1) patients with BHD syndrome.

cysts on thoracic CT are related to the development of SP in BHD patients. Compared to SP patients

without a pathogenic FLCN mutation, lung cysts in BHD patients are smaller, are more equally

distributed both subpleural and in the parenchyma and predominantly located in the lower parts

of the lung.12 Compared to e.g. lymphangioleiomyomatosis (LAM), lung cysts in BHD are typically

described more uniform in size, multi-septated, round and diffuse in distribution.13

The pathogenesis of lung cysts and the relationship with SP in BHD has not been fully clarified yet.

A possible explanation can be the “stretch hypotheses” proposed by Kennedy et al. wherein they

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suggest that pulmonary cysts in BHD patients arise because of fundamental defects in cell-cell

adhesion, leading to repeated respiration-induced physical stretch-induced stress and, over time,

expansion of alveolar spaces particularly in regions of the lung with larger changes in alveolar

volume and at weaker “anchor points” to the pleura. Whether this happens trough a destructive/

inflammatory program or a proliferative program, remains unclear.14

Kumasaka et al analysed resected lung specimens of 50 BHD patients.15 Out of 229 cysts that were

found; 50% were located in the subpleural area and less than 5% abut on bronchioles. Based on this

result we hypothesized that one of the consequences of the mutation in FLCN is the difficulty of

respiratory epithelium to stretch, which likely will result in rupture of the wall of the cyst and leakage

of air from inside the cyst to the surrounding area. If this is the lung parenchyma there will probably

no problem occur, however if the cyst is in close connection with the overlying visceral pleura the

air may leak through the as well ruptured pleura into the pleural cavity. This might create a small

SP. If this ruptured cyst is in connection with the intrapulmonary airways, active leakage of more air

figure 2. Number of cysts in patients with BHD but without a history of (recurrent) spontaneous pneumothorax (left)

and number of cysts in patients with BHD and a history of (recurrent) spontaneous pneumothorax (right).

figure 3. Relationship between number of cysts and age.

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into the pleural cavity may follow, ultimately resulting in a symptomatic SP. Arguments to support

this mechanism come from observed delayed SP after considerable atmospheric pressure changes,

such as occur during flying.16 17 Our observation in this study, that SP patients with BHD have much

more cysts, supports the above described etiology as they have more possibilities to suffer from

cyst rupture than the non-SP patients with BHD. In this study, we found no difference between the

frequency of visible cysts in the subpleural area, in absolute numbers there certainly is. However, the

presence of subpleural cysts might be underestimated as small cysts, due to the way CTs were done,

will not be detected easily and these still could rupture.5 18 On the other hand rupture of a small cyst

will not that easily result in a symptomatic pneumothorax as the damage to the parenchyma, and

consequently leakage of air, will be much smaller and absorption of intrapleural gas might cope

with that. If we assume that the rupture of a subpleural cyst is the likely cause of the pneumothorax

in BHD patients, these patients might also need a different approach for treatment than is assumed

in current guidelines. As these cysts are found throughout the lungs with the majority in the lower

figure 4. Relationship between size of cysts and development of spontaneous pneumothorax.

figure 5. Presence of cysts above and under the carina.

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halves of the lungs, treatment to prevent recurrence should have as aim pleurodesis of the whole

pleura visceralis, and not only in the apical region. As we showed in a different study, this can be

done by extensive pleurectomy and/or talc pleurodesis.19

Unfortunately no genotype-phenotype association was found in this patient group. A history of

spontaneous SP was somewhat more common among c.1539-2A>G (4/19) and c.610_611delGCinsTA.

This is in line with another large study, wherein no genotype-phenotype correlation was found

either.20 Combining databases of BHD patients is needed to get more insight in possible prediposure

for fenotype patterns.

Based on 2 previous studies we hypothesized that BHD may not be as rare as assumed.6 7 In addition

to predisposing for a high risk of recurrent SP, BHD is also associated with a high risk of developing

renal cell cancer with an estimated life time risk of around 15%.21 If detected late, renal cancer is

likely to have a fatal outcome. Since BHD is an autosomal dominant condition, early diagnosis

enables screening for renal cancer in relatives of BHD patients who have a 50% chance of carrying

the mutation in FLCN. Identification of these at risk relatives, by screening initiated after diagnosing

BHD in an affected SP patient, was shown to result in earlier detection of renal cell cancer.22 Early

detection of a BHD in a patient presenting with SP may therefore be potentially life-saving not only

for the patient but also for affected relatives.

Although this is the first study in current literature that reviewed the relationship between lung cysts

and SP among BHD patients, this study has a few limitations. The main limitation is the small number

of patients we included. As this syndrome is relatively rare, it is difficult to gather a large cohort of

patients. Although there seems to be a clear distinction on thoracic CT between BHD patients with

and without a history of (recurrent) SP, the rarity of this syndrome may still lead to unawareness

among doctors who have to evaluate these thoracic CT’s. Although not all clinical information

regarding smoking history, familial inheritance on pneumothorax and prior (surgical) treatment of

pneumothorax was available, we suggest that thoracic imaging might play an important role in the

prediction for development of pneumothorax in BHD patients.

In conclusion: CT scanning of BHD patients may lead to detection of abnormalities characteristic for

BHD. There seems to be a relationship between the number of cysts and development of (recurrent)

SP. Chest CT in this patient group might therefore be an useful tool for choice of treatment when

developing a SP and might also play a role in advice of lifestyle. Current pneumothorax guidelines

might need to be discussed and revised.

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R e f e R e n c e s1. Menko FH, van Steensel MAM, Giraud S, et


management. Lancet Oncol 2009;10:1199-206.

2. Johannesma PC, Reinhard R, Kon Y, et al. Prevalence

of Birt-Hogg-Dubé syndrome in patients with

apparently primary spontaneous pneumothorax.

Eur Respir J 2015; 45: 1191–94.




pneumothorax Clin Genet 2008; 74: 178–83.

4. Bakan S, Kandemirli SG, Kilic F, et al. Birt-Hogg-

Dubé syndrome: A diagnosis to consider in

patients with renal cancer and pulmonary cysts.

Diagn Interv Imaging. 2016 Jan;97(1):117-8.

5. Onuki T, Goto Y, Kuramochi M, Radiologically


syndrome. Ann Thorac Surg. 2014 Feb;97(2):682-5.

6. Seaman DM, Meyer CA, Gilman MD, et al. Diffuse

cystic lung disease at high-resolution CT. AJR Am J

Roentgenol. 2011 Jun;196(6):1305-11.

7. Tobino K, Hirai T, Johkoh T, et al. Differentiation


lymphangioleiomyomatosis: quantitative analysis

of pulmonary cysts on computed tomography

of the chest in 66 females. Eur J Radiol. 2012

Jun;81(6):1340-6.


spontaneous pneumothorax, and genetic

associations in 89 families with Birt-Hogg-Dubé

syndrome. Am J Respir Crit Care Med 2007;175: 1044.

9. Gupta N, Seyama K, McCormack FX. Pulmonary

manifestations of Birt-Hogg-Dubé syndrome. Fam

Cancer. 2013;12:387-96.

10. Johannesma PC, Houweling AC, van Waesberghe

JH, et al. The pathogenesis of pneumothorax

in Birt-Hogg-Dubé syndrome: a hypothesis.

Respirology. 2014 Nov;19(8):1248-50.

11. Nickerson, M.L., Warren, M.B., Toro, J.R. et al.

Mutations in a novel gene lead to kidney tumors,

lung wall defects, and benign tumors of the

hair follicle in patients with the Birt-Hogg-Dubé

syndrome. Cancer Cell 2002: 2: 157-164.

12. Johannesma PC, van Waesberghe JHTM, Menko

FH, et al. Radiological features of primary

spontaneous pneumothorax patients with or

without a mutation in FLCN. (Submitted)

13. Agarwal PP, Gross BH, Holloway BJ, et al. Thoracic

CT findings in Birt-Hogg-Dubé syndrome. ARJ

2011;196;349-352.

14. Kennedy JC, Khabibullin D, Henske EP. Mechanisms

of pulmonary cyst pathogenesis in Birt-Hogg-

Dubé syndrome: The stretch hypothesis. Semin

Cell Dev Biol. 2016;52:47-52.

15. Kumasaka T, Hayashi T, Mitani K, et al.

Characterization of pulmonary cysts in Birt-

Hogg-Dubé syndrome: histopathological and

morphometric analysis of 229 pulmonary cysts

from 50 unrelated patients. Histopathology.

2014;65:100-10.

16. Johannesma PC, van de Beek I, van der Wel JWT,

et al. Risk of spontaneous pneumothorax due to air

travel and diving in patients with Birt-Hogg-Dubé

syndrome. (SpringerPlus, in revision).

17. Postmus PE, Johannesma PC, Menko FH, et al.

In-flight pneumothorax: diagnosis may be missed

because of symptom delay. Am J Respir Crit Care

Med. 2014 Sep 15;190(6):704-5.

18. Reference 18: Jawad H, Walker CM, Wu CC, Chung

JH. Cystic interstitial lung diseases: recognizing

the common and uncommon entities. Curr Probl

Diagn Radiol. 2014;43(3):115-27.

19. Johannesma PC, Paul MA, van Waesberghe JHTM,

et al. International guidelines for pneumothorax

are not adequate for treatment of spontaneous

pneumothorax in patients with Birt-Hogg-Dubé

syndrome. (Submitted)

20. Toro JR, Wei M-H, Glenn GM, et al. BHD mutations,




Genet 2008;45:321-31.




from 35 BHD families. Br J Cancer. 2011;105(12):1912-9.

22. Johannesma PC, Houweling AC, Menko FH, et al.

Are lung cysts in renal cell cancer (RCC) patients an

indication for FLCN mutation analysis? Fam Cancer.

6;15:297-300.

c H a P t e RRadiological features of primary spontaneous

pneumothorax patients with or without a mutation in FLCN

Paul C. Johannesma1, JanHein T.M. van Waesberghe2, Fred H. Menko3, R. Jeroen A. van Moorselaar4, Marinus A. Paul5, Theo M. Starink6,

Rinze Reinhard2,7, Arjan C. Houweling8, Irma van de Beek8, Hans J. Smit9, Jincey D. Sriram9, Marianne A. Jonker10, Pieter E. Postmus11


2 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands3 Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands 4 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

5 Department of Thoracic Surgery, VU University Medical Center, Amsterdam, The Netherlands6 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands

7 Department of Radiology, Onze Lieve Vrouwen Gasthuis, Amsterdam, The Netherlands8 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

9 Department of Pulmonary Diseases, Rijnstate Hospital, Arnhem, The Netherlands 10 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam,

The Netherlands 11 Department of Thoracic Oncology, Clatterbridge Cancer Centre, Liverpool Heart & Chest

Hospital, University of Liverpool, Liverpool, United Kingdom

Submitted

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introduction

In the past few years it has become apparent that several disorders with pulmonary involvement

are associated with an increased risk of primary spontaneous pneumothorax (PSP). One of these

disorders associated with a very high risk of recurring PSP, is the probably relatively common (5-10%

of all PSP patients) cystic lung disease Birt-Hogg-Dubé (BHD) syndrome. As over 90% of these

patients have cysts in basal parts of the lung, we hypothesized that the use of a low dose chest CT

might be an effective way to detect this syndrome in patients presenting with an apparently isolated

PSP. This is important as its inheritance is autosomal dominant, and is associated with a risk of renal

cell cancer of approximately16-30%.


We included 46 patients, 19 with a proven FLCN mutation and 27 without an identifiable FLCN

mutation, with a history of spontaneous pneumothorax. We retrospectively evaluated the thoracic

CT’s for cystic abnormalities and/or air filled cavities, size, number and site (below and/or above level

of main carina, presence in the parenchyma and/or subpleural area) were scored and compared to

findings in a both groups

Results

We found a higher prevalence of recurrent episodes of SP among patients with a proven pathogenic

FLCN mutation. Also the prevalence of (separate) episodes of SP in BHD patients was higher.

The number of cysts in BHD patients was significantly higher in patients with a FLCN mutation. Also

their distribution, location and size on thoracic CT differed significantly from patients without BHD

syndrome.

conclusion

CT scanning of PSP patients may by a useful tool in the detection of BHD in patients presenting with

an apparently isolated PSP.

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Idiopathic or primary spontaneous pneumothorax (PSP) is described as the presence of air in the

pleural cavity without a known pulmonary abnormality.1 Over time it has become clear that PSP might

be the result of a number of pulmonary abnormalities, ranging from smoking related subpleurally

located abnormalities 2 3, to inherited diseases like Marfan’s syndrome, alpha-1-antitrypsin deficiency,

Birt-Hogg-Dubé syndrome (BHD), lymphangioleiomyomatisis and homocysteinuria. 4 5 6 7 8

These conditions have in common that they may cause rupture of the visceral pleura, resulting in

leakage of air into the pleural cavity and, depending on the amount of air in the pleural cavity, the

development of a symptomatic pneumothorax.

In current British Thoracic Society guidelines and the guidelines of the American College of

Chest Physicians, recommendations on imaging of the lung parenchyma in PSP patients is very

restricted.9 10 In fact, for the first episode of pneumothorax the recommended diagnostic procedures

are limited to taking history, physical examination and an erect chest-X-ray.9 The rare conditions

associated with an increased risk of SP may remain unrecognized following the current guidelines.

A standardized more robust assessment of these patients might enable adequate diagnosis of

these conditions and lead to a more effective treatment and disease management. As almost half

of the PSP patients develop a recurrence, one of the main aims of guidelines on the diagnosis and

treatment of PSP is the prevention of recurrence in patients prone to recurring PSP.

One of the hereditary causes of pneumothorax, with a very high recurrence risk, is the relatively

common Birt-Hogg-Dubé (BHD) syndrome, estimated to be present in 5-10% of PSP patients.11 12

Furthermore, prevention of recurrence of pneumothorax with standard approaches is in these

patients very often ineffective.13

Current guidelines recommend the use of chest CT for recurrent pneumothorax. We evaluated the

findings of chest CT in a group of PSP patients without a detectable FLCN mutation and compared

these to a group with a proven mutation in FLCN to evaluate whether CT scanning in PSP patients is

a sensitive tool in diagnosing BHD in PSP patients. This would be highly clinically relevant since BHD

is associated with an increased risk of renal cancer.

m at e R i a l s a n d m e t H o d s

We evaluated the medical history of all FLCN mutation carriers known in the VU University

Medical Center and the Rijnstate Hospital. The medical records of patients with a history of SP

were examined (N=46). FLCN mutation negative patients with a history of PSP were recruited from

a prospective study we performed previously11, and retrospectively from the VUMC-database

of PSP patients tested negative for the FLCN mutation. The clinical details and number of all

pneumothorax episodes were collected of all patients in both groups (FLCN mutation positive and

FLCN mutation negative).

One CT-thorax of all the patients was scored independently by an experienced pulmonologist

(PEP) and an experienced radiologist (JHvW) blinded for the final diagnosis. In case of multiple

CTs the one made closest to the time of a pneumothorax episode was used, when available, after

radiological resolution of the pneumothorax. For the mutation negative patients to be included,

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the CT had to be performed within one year of the pneumothorax. We assumed, based on our prior

study, that for the BHD cases this time interval is less relevant as the findings on chest CT have were

found to remain largely unchanged over time.14 Scoring consisted of presence of abnormalities,

and more specifically the presence of cystic abnormalities and/or air filled cavities. If these were

found the size, number, location below and/or above level of main carina, in the parenchyma and/

or subpleural area were noted.


amplification and sequencing of the 14 exons were designed as detailed by Nickerson et al. PCR


CA, USA).15 Sequencing reactions were performed using the Big Dye Terminator system (Applied



MLPA kit P256 (MRC Holland, www.mrc-holland.com).

All statistical analyses were performed using R software (version 3.1.3.). To compare the results of

thoracic CT scans of SP patients with and without a FLCN mutation, we used the Wilcoxon test.

A p-value of less than 0.05 was considered significant. The study was approved by the Ethics

Committee of the VU University Medical Center.

R e s u lt s

The main characteristics of the 46 included patients are summarized in table 1. We included 46

patients with a history of (recurrent) SP. All patients were tested for FLCN mutations between

2005 and 2015. We included 19 patients with a pathogenic FLCN mutation and 27 patients without a

pathogenic FLCN mutation. In the included group of patients with a pathogenic FLCN mutation and

a history of (recurrent) SP, the mean age at the first episode of PSP was 31.2 years. The number of

patients with at least one recurrent episode was 68.4%. The mean number of episodes per patients

was 3.5 with a maximum of twelve episodes. As we scored both lungs separately, we found at least

one episode in the left lung in 72.2% of cases, 57.9% in the right lung and in 52.6% of cases at least

one episode (separate moment) in both lungs (table 1). In the group of patients with (recurrent) SP

(N=27) but without a detectable pathogenic FLCN mutation, the mean age at the first episode of

PSP was 27.8 years and 59.3% of cases underwent at least one recurrent episode in the same lung.

The mean number of episodes per patient was 1.5 episodes, with a maximum of four episodes.

In total 59.2% occurred in the left lung, 55.6% in the right lung and in 18.5% of cases (at separate

moments) in both lungs.

In table two the results of the scored thoracic CT’s are summarized. We found a large difference

between the two groups of patients in the number of cysts. The number of cysts in BHD patients

was significantly higher than in de group patients without BHD (p<0.001). We also found a significant

difference in the distribution of the cysts, as the majority of cysts in BHD patients were found under

the carina (p<0.001). Cysts in BHD patients are significant smaller (p<0.023), and are more equally

distributed both subpleural and in the parenchyma (p<0.001). Also the presence of small pulmonary

artery and veins in the cysts was only seen in the group of BHD patients (p<0.001).

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d i s c u s s i o n

Although the number of cases evaluated in this study is relatively small, we observed an important

and significant difference between the two groups in respect to the type of abnormalities found. In

the BHD patients the most commonly observed abnormality was the presence of one or more cysts

in the parenchyma or adjacent to the visceral pleura, predominantly in the lower lobes. In contrast,

in the FLCN mutation negative cases, we observed no abnormalities in the lower halves of the

lung, nor abnormalities in the parenchyma far from the visceral pleura. Most of the abnormalities

detected by CT were small (<2cm). This explains why these abnormalities could not be detected

using a standard erect chest X-ray.

The abnormalities found in the group of patients with a pathogenic FLCN mutation have in common

a probably important attribute, likely to be associated with an increased risk of pneumothorax:

spaces with trapped air. In the FLCN mutation negative cases the emphysema like changes in the

apical parts are caused by accumulation of trapped air behind a one-way valve caused by conditions

such as respiratory bronchiolitis.16 The vast majority of air-filled cysts in BHD patients are not

connected to the airways17, resulting in trapped-air. Rapid changes in atmospheric pressure are

likely to result in an increased chance of rupture of the wall of air-filled cavities or cysts potentially

resulting in a spontaneous pneumothorax. Epidemiological data on weather conditions support

this hypothesis18 19, but also individual cases of pneumothorax related to exposure to large pressure

changes have been reported.20 21

It is likely that in addition to air trapping other mechanisms play a role in the development of

a pneumothorax. In the apical parts of the lung the pleural stress is high and abnormalities in that

area are likely to increase the risk of rupture.22 In BHD-patients the majority of cysts are located in

the pleura in the lower lobes, which makes it likely that the wall of cysts connected to the visceral

pleura rupture easily.14 Important for this is the lack of stretching possibilities of the wall of lung cysts

of BHD patients.23

Based on 2 previous studies 11 12 we hypothesized that BHD may not be as rare as assumed. In addition

to predisposing for a high risk of recurrent pneumothorax, BHD is also associated with a high risk

of developing renal cell cancer with an estimated life time risk of around 15%24. If detected late,

renal cancer is likely to have a fatal outcome. Since BHD is an autosomal dominant condition, early

diagnosis enables screening for renal cancer in relatives of BHD patients who have a 50% chance of

carrying the mutation in FLCN. Identification of these at risk relatives, by screening initiated after

diagnosing BHD in an affected pneumothorax patient, was shown to result in earlier detection of

renal cell cancer. 11 25 Early detection of a BHD in a patient presenting with SP may therefore be

potentially life-saving not only for the patient but also for affected relatives.

There are a few limitations to our study. The main limitation is the small number of patients we

included. As this syndrome is relatively rare, it is difficult to gather a large cohort of patients.

Although there seems to be a clear distinction on thoracic CT between BHD patients and patients

without BHD, the rarity of this syndrome may still lead to unawareness among doctors who have

to evaluate these thoracic CT’s. Although not all clinical information regarding smoking history,

familial inheritance on pneumothorax and prior (surgical) treatment of pneumothorax was available,

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we suggest that the radiological distinction between BHD and patients without BHD can easily be

made on a low dose CT scan of the thorax. In conclusion: CT scanning of PSP patients may lead to

detection of abnormalities characteristic for BHD. Current pneumothorax guidelines might need to

be discussed and revised.

figure 1. Boxplot and histogram, number of cysts. Patients with pathogenic FLCN mutation (BHD syndrome) compared

to patients without a pathogenic FLCN mutation (no BHD syndrome).

table 1. Demographic results of all spontaneous pneumothorax patients, divided in patients with a pathogenic FLCN mutation and patients without a pathogenic FLCN mutation.

pathogenic FLCN mutation (n=19)

no pathogenic FLCN mutation (n=27)

Age in years (min-max) 31.2 (20-40) 27.8 (16-78)

Number of patients with at least one recurrent episode of SP (=%) 68.4 59.3

Mean number of episodes of SP (min-max) 3.5 (1-12) 1.5 (1-4)

Side (Left : Right : Both) 73.7 : 57.9 : 52.6 59.2 : 55.6 : 18.5

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R e f e R e n c e s1. Kjæergaard H. Spontaneous pneumothorax in the

apparently healthy. Acta Med Scand Suppl 1932; 43: 1–159.

2. Bense L, Lewander R, Eklund G, et al. Nonsmoking,

non-alpha 1-antitrypsin deficiency-induced

emphysema in nonsmokers with healed spontaneous

pneumothorax, identified by computed tomography

of the lungs. Chest 1993; 103: 433–38.

3. Mitlehner W, Friedrich M, Dissmann W. Value of

computer tomography in the detection of bullae

and blebs in patients with primary spontaneous

pneumothorax. Respiration 1992; 59: 221–27.

4. Dyhdalo K, Farver C. Pulmonary histologic changes

in Marfan syndrome: a case series and literature

review. Am J Clin Pathol 2011; 136: 857–63.

5. Daniel R, Teba L. Spontaneous pneumothorax and alpha

1-antitrypsin deficiency. Respir Care 2000; 45: 327–29.




syndrome. Am J Respir Crit Care Med 2007;175: 1044.

7. Bass HN, LaGrave D, Mardach R, et al. Spontaneous

pneumothorax in association with pyridoxine-responsive

homocystinuria. J Inherit Metab Dis 1999; 20: 831–32.

8. Taveira-DaSilva AM, Moss J. Clinical features,

epidemiology, and therapy of lymphangioleio-

myomatosis. Clin Epidemiol. 2015 Apr 7;7:249-57.

9. MacDuff A, Arnold A, Harvey J, and the BTS Pleural

Disease Guideline Group. Management of spontaneous

pneumothorax: British Thoracic Society Pleural Disease

Guideline 2010. Thorax 2010; 65 (suppl 2): ii18–31.

10. Baumann MH, Strange C, Heffner JE. Management

of spontaneous pneumothorax: an American

College of Chest Physicians Delphi consensus

statement. Chest. 2001;119:590-602.




Eur Respir J 2015; 45: 1191–94.




pneumothorax Clin Genet 2008; 74: 178–83.

13. Johannesma PC, Jonker MA, van der Wel MA, et

al. Management of spontaneous pneumothorax

in patients with or without Birt–Hogg–Dubé

syndrome. Eur Respir J 2014; 44: Suppl. 58, 752.


JHTM, van Moorselaar RJA, Starink ThM,

Menko FH, Postmus PE. The pathogenesis of

pneumothorax in Birt-Hogg-Dubé syndrome:

a hypothesis. Respirology 2014; 19: 1248-50.





Cancer Cell. 2002 Aug;2(2):157-64.

16. Cottin V, Streichenberger N, Gamondes J-P, et al.

Respiratory bronchiolitis in smokers with spontaneous

pneumothorax. Eur Respir J 1998; 12: 702-04.

17. Kumasaka T, Hayashi T, Mitani K, et al. Characterization

of pulmonary cysts in Birt-Hogg-Dubé syndrome:

histopathological and morphometric analysis of

229 pulmonarycysts from 50 unrelated patients.

Histopathology 2014; 65: 100–10.

18. Haga T, Kurihara M, Kataoka H, Ebana H. Influence

of wheather conditions on the onset of primary

spontaneous pneumothorax: positive association

with decreased atmospheric pressure. Ann Thorac

Cardiovasc Surg 2013; 19: 212-15.

19. Scott GC, Berger R, McKean HE. The role of atmospheric

pressure variation in the development of spontaneous

pneumothoraces. Am Rev Resp Dis 1989; 139: 659-62.

20. Araki K, Okada Y, Kono Y, To M, To Y. Pneumothorax

recurrence related to high-speed lift. Am J Med

2014; 127: e11-12.

21. Postmus PE, Johannesma PC, Menko FH, Paul MA.


due to delayed symptoms. Am J Resp Crit Care

Med 2014; 190: 704-5.

22. Casha AR, Manché A, Gatt R, et al. Is there a

biomechanical cause for spontaneous pneumothorax?

Eur J Cardiothorac Surg. 2014 Jun;45(6):1011-6.

23. Medvetz DA, Khabibullin D, Hariharan V, et al. Folliculin,

the product of the Birt-Hogg-Dubé tumor suppressor

gene, interacts with the adherens junction protein p0071

to regulate cell-cell adhesion. PLoS ONE 2012;7: e47842.


cancer and pneumothorax risk in Birt–Hogg–Dubé


from 35 BHD families. Br J Cancer 2011; 105: 1912–19.

25. Johannesma PC, Houweling AC, Menko FH, et al. Are

lung cysts in renal cell cancer (RCC) patients an indication

for FLCN mutation analysis? Fam Cancer 2016;16:297-300.

c H a P t e RHow reliable are clinical criteria to distinguish

between BHd and smoking as a cause for pneumothorax?

Paul C. Johannesma1, Erik Thunnissen2, Pieter E. Postmus1


2 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

Histopathology 2014;64(7)1045-6

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We congratulate dr. Fabre and colleagues with their interesting study1 as published reports of

histopathology of lung tissue of BHD patients are limited. In this study a retrospective analysis was

performed to compare histopathology and CT-imaging of 5 Birt-Hogg-Dubé (BHD) patients with

a matched cohort of five – assumed non-BHD - patients with a history of smoking and spontaneous

pneumothorax (SP).

Based on the study design and conclusion, we believe that some remarks should be made. It is

unclear why the author assume that the SP in their cases in the control group were caused by smoking

and not due to BHD. The final proof of non-BHD as causative factor is the lack of a pathogenic FLCN

mutation in a patient with SP. It is difficult to conclude on clinical and/or radiological characteristics

only, that SP is not caused by BHD. So far only in one study 102 unselected patients with SP were

tested for a pathogenic mutation, ten cases (9.8%) with a mutation were found.2 Although the

characteristics of these 10 cases, and of 4 relatives with a mutation and SP, were not reported in

a very detailed way, it is possible to conclude from this study that in at least 6 cases abnormalities

(cysts) were found in the upper lung or apical part, three of these were (former) smokers. In 5 of

these 6 cases the number of cysts was certainly < 10. In the study by Fabre et al. there were clear

differences between BHD cases and assumed non-BHD controls. However, the findings of Ren et

al. question the assumption of a lack of mutation as the characteristics of assumed non-BHD are

overlapping with at least a number of BHD cases and it is therefore far from certain that the 5

control cases were really free of a mutation in FLCN.

Furthermore the histopathological study by Furuya et al. confirms the presence of TTF-1 in BHD

cases, although controls are lacking.3 This suggests that TTF-1 staining patterns may be specific for

BHD, while this is not necessarily the case, as the results of TTF-1 in controls in this study are not

clearly mentioned. Furthermore it is far from certain that SP in a smoking BHD patient is always

related to BHD and not to the same pathological mechanism as in smokers with SP but without BHD,

at least in 1 of the 5 cases with BHD the authors report the same abnormalities as in the majority of

the “control” group.

The authors conclude that “….pathologists should remain vigilant when assessing ruptured

pulmonary bulla/bleb and bear in mind the possibility of BHD, especially in a non-smoking

woman….” Although the study design and results do not support this conclusion, we do not disagree

that pathologists (and clinicians) should be aware of the possibility of BHD in non-smokers with SP.

However, there is no reason to assume that gender is an important factor as the hereditary pattern is

autosomal dominant and it is therefore unlikely to expect a female predominance. A female gender

predilection is more related to a syndrome like pulmonary LAM.

What is clear from this small study is that those with a typical CT pattern (many cysts, punched-out)

would have been detected by CT at the time of SP, for those with a different pattern it remains

unclear. Whether this should be sufficient evidence to change the diagnostic approach of patients

with first time SP needs to be discussed.4

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R e f e R e n c e s1. Fabre A, Borie R, Debray MP, Crestaine B, Danel C.

Distinguishing histological and radiological features

between pulmonary cystic lung disease in Birt-Hogg-

Dubé syndrome and tobacco-related spontaneous

pneumothorax. Histopathology 2014;65:741-9.




pneumothorax. Clin Genet 2008:74 178-183.

3. Furuya M, Tanaka R, Koga S, et al. Pulmonary cysts

of Birt-Hogg-Dubé syndrome: a clinicopathologic

and immunohistochemical study of 9 families. Am

J Surg Pathol. 2012;36(4):589-600.

4. MacDuff A, Arnold A, Harvey J, et al. Management

of spontaneous pneumothorax: British Thoracic

Society Pleural Disease Guideline 2010. Thorax

2010;65 Suppl 2:ii18-31.

c H a P t e RRisk of spontaneous pneumothorax

due to air travel and diving in patients with Birt-Hogg-dubé syndrome

Johannesma PC1, van de Beek2, van der Wel JWT1, Paul MA3, Houweling AC2, Jonker MA4, van Waesberghe JHTM5, Reinhard R5,

Starink ThM6, van Moorselaar RJA7, Menko FH8, Postmus PE9


2Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 3Thoracic Surgery, VU University Medical Center, Amsterdam, The Netherlands

4Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands 5Radiology, VU University Medical Center, Amsterdam, The Netherlands

6Dermatology, VU University Medical Center, Amsterdam, The Netherlands 7Urology, VU University Medical Center, Amsterdam, The Netherlands

8 Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands 9 Clatterbridge Cancer Centre, Liverpool Heart & Chest Hospital, Department of Thoracic

Oncology, University of Liverpool, Liverpool, United Kingdom

Accepted for publication

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Background and objectives

Birt-Hogg-Dubé syndrome is an autosomal dominant disorder characterized by skin

fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal cell cancer due to germline

folliculin (FLCN) mutations. The aim of this study was to evaluate the incidence of spontaneous

pneumothorax in patients with BHD during or shortly after air travel and diving.

methods

A questionnaire was sent to a cohort of 190 BHD patients and the medical files of these patients

were evaluated. The diagnosis of BHD was confirmed by FLCN mutations analysis in all patients. We

assessed how many spontaneous pneumothoraces (SP) occurred within one month after air travel

or diving.

Results

In total 158 (83.2%) patients returned the completed questionnaire. A total of 145 patients had a

history of air travel. Sixty-one of them had a history of SP (42.1%), with a mean of 2.48 episodes

(range 1-10). Twenty-four (35.8%) patients had a history of pneumothorax on both sides. Thirteen

patients developed SP <1 month after air travel (9.0%) and 2 patients developed a SP <1 month after

diving (3.7%). We found in this population of BHD patients a pneumothorax risk of 0.63% per flight

and a risk of 0.33% per episode of diving. Symptoms possible related to SP were perceived in 30

patients (20.7%) after air travel, respectively in 10 patients (18.5%) after diving.

conclusion

Based on the results presented in this retrospective study, exposure of BHD patients to considerable

changes in atmospheric pressure associated with flying and diving may be related to an increased

risk for developing a symptomatic pneumothorax. Symptoms reported during or shortly after flying

and diving might be related to the early phase of pneumothorax. An individualized advice should be

given, taking also into account patients’ preferences and needs.

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Changes in atmospheric pressure are related to a higher incidence of spontaneous primary

pneumothorax. Atmospheric pressure changes from day to day are usually small. However, during

flying or diving significant changes in pressure will occur. Therefore it might be expected that

patients with a predisposition for air trapping, such as cystic lesions not connected to the airways,

have a considerable risk of developing a pneumothorax. Especially in diving, pulmonary barotrauma

may result in serious complications. The increased risk of pneumothorax associated with changes in

atmospheric pressure can be explained by a change in transpulmonary pressure in regions with air

trapping , and not in direct connection with the airways, resulting in a higher pressure compared to

the atmosphere.1 2 3 4 5 Air trapping may be caused by peripheral airway inflammation with a check-

valve mechanism resulting in obstruction of the airway.6 7 In addition, lung cysts, emphysematous

blebs or bullae may predispose to air trapping.8 Examples of diseases with cystic lesions in the

lung are lymphangioleiomyomatosis (LAM) and Birt-Hogg-Dubé syndrome (BHD). BHD is an

autosomal dominant condition caused by germline mutations in the folliculin (FLCN) gene, clinically

characterized by skin fibrofolliculomas, pulmonary cysts, (recurrent) spontaneous pneumothorax

and an increased risk of renal cell cancer. Pneumothorax may be the first and only manifestation of

BHD both in isolated and familial cases. Approximately 90% of BHD patients show multiple lung cysts

on standard CT. Lung function generally remains unaffected, demonstrated by normal spirometry

and diffusion capacity.9

Detailed analysis of resected lung specimens of 50 patients with BHD containing a total of 229

cysts was performed by Kumasaka and colleagues. Of the cysts, 50 % was located in the subpleural

area and less than 5% abutted bronchioles.10 These findings imply that almost all lung cysts in BHD

patients are spaces filled with air without a direct connection to airways and therefore pressure

changes outside the body will not rapidly affect the amount of air within the cyst. This situation of

trapped air is comparable to a “bag of chips” during air travel.11

According to Boyle’s law the inverse relationship between pressure and volume for gas in a closed

system at a constant temperature will result in an increased size of the cyst during flying and

subsequently risk of rupture of the cyst wall. During diving the pressure increases and the the cyst

will decrease in size reaching pressure equilibrium during the dive. However, after reaching pressure

equilibrium during the period under water, the cyst will re-expand to its original size during ascent

to the surface and the risk of rupture of the cyst wall is comparable to the situation during air travel.

Few data are available regarding the prevalence of pneumothorax associated with air travel in

patients with the interstitial cystic lung disease lymphangioleiomyomatosis (LAM). Two studies

retrospectively reviewed the occurrence of spontaneous pneumothorax related to air travel in

a group of patients with LAM and found an overall incidence of pneumothorax between 1.1 and 2.2

per 100 flights.12 13

These findings raise the important clinical question whether BHD patients are as well prone to

develop a spontaneous pneumothorax during air travel or after diving. As there is no clear definition

of “air travel related pneumothorax” we evaluated the prevalence of spontaneous pneumothorax

and adverse events in a period of one month both after air travel and after diving.

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study population

We retrospectively approached patients diagnosed in our center with BHD, confirmed by FLCN

mutation analysis. A survey including a letter summarising the study was sent to the last known

address to collect information on demographic data, time since BHD diagnosis, medical history,

use of cigarettes and inhaled soft drugs, history of spontaneous pneumothorax, number of SP

episodes, side of SP, treatment of SP, air travel and diving behaviour within one month before

the SP episode and adverse effects during/after flying and/or diving (dyspnoea, chest pain,

palpitations, anxiety, fatigue, nausea, dizziness, haemoptysis, headache, blue hands, shackles and/

or light headedness). To increase the response rate, a second mailing was sent to non-responders

after 6 weeks. The number of flights and the number of diving episodes was divided in none, 1-5

times, 6-10, 11-20 or more than 20. Air travel was divided in continental and intercontinental. Depth

during diving was categorized in 0-3 metres, 3-10 metres, more than 10 metres depth. Written

consent was obtained from all subjects.

Medical records of the responders were collected and reviewed for radiological evidence (chest

X-ray and/or thoracic CT) of spontaneous pneumothorax. In addition information regarding type

of treatment of the (recurrent) spontaneous pneumothorax was collected. Radiological studies for

the presence of pneumothorax and lung cysts were reviewed by a radiologist and pulmonologist.

The study was approved by the ethics committee of the VU University Medical Center.

R e s u lt s

demographics

In total 158 (83.2%) patients completed and returned the questionnaire. Of this group, 9.5% was

active smoker, 42.5% former smoker (mean: 19 pack years) and 7.6% had a history of inhaling drugs.

In total 145 BHD patients reported having traveled by airplane at least once. We defined a flight as a

single flight including one ascent and one descent. A total of 1582 single flights in Europe (mean 10.9

flights) and 946 single intercontinental flights (mean 6.5 flights) were reported. Fifty-four patients

(34.2%) had ever dived, all for recreational purposes. The majority of this group had dived between

1-5 times at a depth between 0-3 meters (56.3%). Depth was categorized in 0-3 meters (87%),

3-10 meters (48.1%), >10 meters (14.8%)

thoracic ct in BHd patients with a history of spontaneous pneumothorax after air travel or diving

A thoracic CT was available for 9 patients with a history of SP after air travel (N=8) or diving (N=1).

The number of cysts on standard thoracic CT varied between 1 and 140, with a mean of 57.2 cysts.

The majority of cysts was located in the basal parts of the lung equally divided subpleural and

parenchymal (Table 2). Whether the number of lung cysts is significantly higher in this patients

group, we scored the thoracic CT’s of 42 patients with BHD and a history of air travel and/or diving,

but without a history of (recurrent) pneumothorax. The number of cysts on thoracic CT varied

between 2 and 33, with a mean of 19 cysts. The number of cysts in patients BHD and a history of

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pneumothorax was significantly higher compared to the group of patients with BHD but without

a history of pneumothorax (p<0.008).

adverse events during air travel and diving

Complaints reported during air travel by BHD patients (N=145), included shortness of breath (4.1%),

chest pain (6.2%), palpitations (2.8%), anxiety (9.7%), abnormal fatigue (3.4%), nausea (4.1%),

dizziness (0.7%), abnormal headache (3.4%), abnormal chills (1.4%) and lightheadedness (4.8%).

These subjective symptoms were reported in 30 patients (20.7%) during flight.

In total 54 patients had “ever” dived, all recreational. Depth was categorized in 0-3 meters (87%),

3-10 meters (48.1%), >10 meters (14.8%). . Subjective symptoms were reported by 10 patients (18.5%)

during diving: shortness of breath (11.1%), anxiety (3.7%), dizziness (1.9%), abnormal fatigue (1.9%),

abnormal chills (1.9%), and hemoptysis (1.9%).

spontaneous pneumothorax after air travel

Sixty-one of 145 BHD patients (42.1%) had a history of both SP and air travel, with a mean of 2.48

episodes of SP (range 1-10). Twenty-four patients (39.3%) had a history of (separate) episodes

of spontaneous pneumothorax on both sides. Thirteen of 145 BHD patients (9.0%) developed

a SP <1 month after air traveling. 24.1% had travelled intercontinental. Two patients developed

a SP two times within one months after air travel. One patient developed a SP three times. For

5 patients this was the first episode of SP. The time interval between flight and radiographic

diagnoses of spontaneous pneumothorax are summarized in table 1. The diagnosis of

pneumothorax was confirmed in all patients with chest X-ray, additional thoracic CT was

performed in 5 patients. Retrospectively a thoracic CT was available in 8 patients (table 2).

Although these patients did not undergo radiographic imaging of the chest prior to their

flight, we assume that these patients developed a pneumothorax during air travel as these

patients had no subjective symptoms of pneumothorax before their flight. We calculated

a pneumothorax risk of 0.63% per flight.

spontaneous pneumothorax after diving

Two patients out of 54 that had ever dived (3,7%) developed a SP < 1 month after diving,

both after diving at depth between 3-10 meters. Although these patients did not undergo

radiographic imaging of the chest prior to their diving session, we assume that these patients

developed a pneumothorax during ascending to the surface, as both patients had no subjective

symptoms of pneumothorax before the diving session. The first patient dived between 6-10

times at a depth of 3-10 meters. After a diving session of 30 minutes, this patient developed

his first pneumothorax within 0-5 days. The second patient developed her third episode of

pneumothorax after her first diving session of maximum 30 minutes at a depth of 3-10 meters,

within 0-5 days (table 3). Thoracic CT was available in one patient. Both patients were treated

with a VATS procedure with surgical pleurodesis (table 4). We calculated a pneumothorax risk

of 0.33% per episode of session.

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table 1. Characteristics of 13 BHD patients with (recurrent) SP after air travel.

patient number

destination prior to sp

number of flights in europe

number of flights intercontinental

Recurrent episode of sp?

episode number of sp

type of imaging diagnosis sp

time interval (in days)

1 Unknown >20 >20 yes 2 X thorax 0-5

2 Europe >20 11-20 yes 3 X 0-5

3 Europe >20 1-5 yes 4 X thorax +CT 10-15

4 Europe >20 >20 no 1 X thorax 5-10

5 Intercontinental >20 >20 no 1 X thorax 0-5

5 Intercontinental >20 >20 yes 4 X thorax +CT 0-5

6 Intercontinental >20 >20 yes 2 X thorax 20-25

7 Europe 6-10 0 yes 10 X thorax 0-5

8 Europe 6-10 1-5 no 1 X thorax 0-5

9 Europe >20 0 yes 2 X thorax +CT 0-5

10 Europe >20 6-10 yes 2 X thorax 15-20

11 Europe >20 >20 yes 2 X thorax +CT 20-25

12 Intercontinental >20 >20 no 1 X thorax 15-20

13 Europe >20 >20 no 1 X thorax 5-10

13 Intercontinental >20 >20 yes 2 X thorax 0-5

13 Intercontinental >20 >20 yes 3 X thorax +CT 0-5

table 2. Imaging and treatment characteristics of 13 BHD patients with (recurrent) SP after air travel.

patient number

thoracic ct available

number of cysts

prior sp

prior pleurodesis

treatment sp after air travel FLCN mutation

1 yes 5 yes yes Pleurodesis c.610_611delinsTA

2 yes 1 yes no Pleurodesis c.499C>T

3 n/a# n/a# yes no Pleurectomy / pleurodesis c.250-?_1740+?del (del exon 5 – 14)

4 yes 99 no no Tube thoracostomy c.1285dupC

5 yes 140 no no Tube thoracostomy c.1408_1418del; p.Gly470fs

5 yes 140 yes yes Pleurodesis c.1408_1418del; p.Gly470fs

6 yes 4 yes no Pleurodesis c.619-1G>A

7 n/a# n/a# yes no Tube thoracostomy c.1539-2A>G

8 yes 74 no no Pleurodesis c.1539-2A>G

9 n/a# n/a# yes no Pleural rubbing c.610_611delinsTA

10 yes 19 yes yes Pleurodesis c.610_611delinsTA

11 n/a# n/a# yes no Pleural rubbing c.1065_1066delGCinsTA

12 n/a# n/a# no no Tube thoracostomy c.610_611delinsTA

13 yes 99 no no Tube thoracostomy c.1740dupC

13 yes 99 yes no Apical pleurectomy c.1740dupC

13 yes 99 yes no Total pleurectomy and

pleurodesis

c.1740dupC

#Not Available

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d i s c u s s i o n

To date, our study is the largest air travel and diving survey of patients diagnosed with BHD. In this

retrospective study we analyzed the self-reported history of air travel and diving of 158 patients

diagnosed with BHD. In total 13/145 (9.0%) and 2/54 (3.7%) patients developed a spontaneous

pneumothorax within one month after air travel or diving respectively. In total 30/145 (20.1%) and

10/54 (18.5%) of BHD patients experienced one or more adverse events during air travel and diving

respectively. We calculated a pneumothorax risk of 0.63% per flight and a pneumothorax risk of

0.33% per diving session.

The literature regarding the risk for pneumothorax related to diving in patients with lung cysts is

extremely limited. However, several case reports suggest a relationship between a bulla and an

increased risk for pneumothorax during ascent. So far, a standardised documentation of in-flight

medical and surgical emergencies (IMEs) has not been established.15 Recommendations by several

speciality society guidelines and literature reviews addressing time to travel from pneumothorax

diagnosis and or treatment vary widely and show some discrepancies, which is probably due to the

limited number of studies on this subject. The British Thoracic Society (BTS) guideline recommends

diving to be permanently avoided after an episode of spontaneous pneumothorax unless the

patient has undergone bilateral surgical pleurectomy and the lung function and postoperative

thoracic CT are normal.14 Recommended time to travel from diagnosis and/or treatment for

spontaneous pneumothorax varies between no time period noted up to 21 days after radiographic

resolution. Currently, there are no guidelines for patients with cystic lung diseases. We show here

that the incidence of pneumothorax in BHD associated with flying and diving might be higher than

in the general population and therefore recommendations with respect to air travel and diving for

patients with BHD have to be established. Pneumothorax occurring in-flight appears to be rare in

the general population. Coker and colleagues described the results on 500 patients with a variety of

lung diseases traveling by air. No in-flight emergencies, including pneumothoraces, were reported.15

table 4. Imaging and treatment characteristics of 2 BHD patients with (recurrent) SP after diving.

patient number

thoracic ct available

number of cysts

prior sp prior pleurodesis

treatment sp after air travel FLCN mutation

1 n/a# n/a# no no Pleurodesis IVS9+6 C>T and IVS8+36G>A

2 yes 74 yes Tube thoracostomy Pleurodesis c.1539-2A>G

#Not Available

table 3. Characteristics of 2 BHD patients with (recurrent) SP after diving.

patient number

total episodes of diving*

duration of diving prior to sp

type of imaging diagnosis sp

episode number of sp

depth of diving prior to sp

time interval (in days)

1 6-10 30 minutes X thorax 1 3-10 meters 0-5

2 1-5 30 minutes X thorax 3 3-10 meters 0-5

*All episodes at 3-10 meters depth

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Sand and colleagues identified 10.189 cases of in-flight medical emergencies in a retrospective study

of two European airlines; no cases of pneumothorax were documented.16 A recent evaluation among

11.920 patients by Peterson and colleagues, showed also no pneumothorax as in-flight emergency.

Between 1969 and 2012 a total of 38 episodes of pneumothorax during air travel are described in

literature.17 The majority of patients had the cystic lung disease LAM as underlying disease.18

Whether patients diagnosed with BHD are at an increased risk for developing spontaneous pneumothorax

associated to air travel or diving is unknown. So far only little data concerning BHD and air travel are

available. Hoshika and colleagues surveyed a small population with BHD syndrome (N=48). None of

them had experienced air travel related pneumothorax, although the length of the pneumothorax free

period after air travel was not specified.19 Hu and colleagues reported an overview of 12 reports of in-flight

pneumothoraces and associated outcomes but did not specify the disease characteristics of these

patients.17 In only one study the episode of pneumothorax during the flight was fatal.20

LAM, like BHD is a pulmonary disease associated with lung cysts. So far two studies including LAM

patients reported the incidence of pneumothorax during air travel. The first study found an in-flight

pneumothorax risk of 2.2% among 308 LAM patients and an estimated pneumothorax risk for LAM

patients of 4% per woman flying.12 The second study among 449 LAM patients reported an incidence

of 1.1 pneumothoraces per 100 flights and 2.9 pneumothoraces per 100 patients.11 This is a higher

incidence than we found in our study. An explanation could be that BHD patients have less cysts in

their lungs, as they only appear under the carina.

One may question whether a pneumothorax occurring days or weeks after the event with considerable

atmospheric pressure change should be considered as related to that event. The size of the connection

between airway and pleural cavity is likely to determine how fast a pneumothorax will increase in

size and it may therefore take days to weeks before causing symptoms.21 To determine whether a

pneumothorax indeed occurred during or directly after air travel, imaging before and directly after air

travel would be required. Our choice to make an inventory of SP within one month after air travel was

arbitrary and based on the assumption that these episodes may be related to flying.

Based on histological evaluation of the lung cysts in BHD patients this appears to be plausible. The

location of the cysts in the periphery of the lungs, at least 50% is located subpleurally and the lack

of a direct connection with the intrapulmonary airways supports this theory.21 22 23 Based on these

characteristics it is unlikely that rupture of a cyst will lead to a considerable flow of air into the

pleural cavity. On the contrary, if a subpleural cyst ruptures the overlying visceral pleura has to

rupture as well and resulting of the release of a very small amount of air into the pleural cavity. Larger

amounts of air can only get there through damage of surrounding parenchyma and/or airways in

the periphery adjacent to the ruptured cyst. Ultimately the size of the connection between airway

and pleural cavity determines how fast a pneumothorax will increase in size and it may therefore

take days to weeks before this will lead to symptoms.21

The more reported association between LAM and pneumothorax after air travel might be related to

the disease itself. Cysts in LAM are caused by air trapping behind an obstruction of the airways.24 25

Rupture of these cysts is therefore more likely to include the airway resulting in a larger connection

between airway and pleural cavity than in BHD and therefore leading to symptoms earlier.

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So far, a standardised documentation of in-flight medical and surgical emergencies (IMEs) has not

been established.15 Recommendations by several speciality society guidelines and literature reviews

addressing time to travel from pneumothorax diagnosis and or treatment vary widely and show some

disagreement, which is probably due to a lack of evidence-based support structure. Recommended

time to travel from diagnosis and/or treatment for spontaneous pneumothorax varies between no

time period noted up to 21 days after radiographic resolution, but is not suitable for patients with

cystic lung diseases. We show that the incidence of pneumothorax in BHD, and likely in other cystic

lung disease as well, is much higher than in the general population and therefore recommendations

with respect to air travel for patients with BHD have to be established.

Based on our current data, we suggest that patients with BHD are likely to be at an increased risk for

pneumothorax while flying. Possibly, it may take several days to weeks before a pneumothorax becomes

symptomatic. Patients with BHD should be advised that the presence of any clinical symptoms such

as shortness of breath or chest pain, during flying or shortly after air travel might indicate a (small)

pneumothorax. This should preclude flying and these persons should immediately undergo appropriate

radiologic testing, by chest X-ray or thoracic CT, before being approved for air travel or diving.

In summary, SP in BHD patients after air travel and diving might occur more often than in the general

population. We found a pneumothorax risk of 0.63% per flight and a pneumothorax risk of 0.33% per

diving episode. Although BHD is not mentioned in the BTS and ACCP guideline for pneumothorax

after air travel or diving, clinicians should be aware of the possible increased pneumothorax risk

in BHD patients. An individualized advice should be given, taking also into account patients’

preferences and needs. Further research is required to address the exact rate of pneumothorax

during and directly after air travel. Preferably, a healthy control group is used to address the

pneumothorax rate in the general population. Since pneumothorax during diving is reported to be

associated with serious complications, and screening for cysts has been suggested in professional

divers, we recommend that BHD patients are evaluated and counselled on the potentially associated

risk by a physician with experience in diving medicine.

Ac k n ow l e d g e m e n T s

We thank all the participants in this study for their extensive contribution.

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R e f e R e n c e s1. Scott GC, Berger R, McKean HE. The role of

atmospheric pressure variation in the development

of spontaneous pneumothoraces. Am Rev Respir

Dis. 1989; 139(3):659-62.

2. Suarez-Varel MM, Martinez-Selva MI, Llopis-

Gonzalez A, et al. Spontaneous pneumothorax

related with climatic characteristics in the Valencia

area (Spain). Eur J Epidemiol. 2000; 16(2):193-8.

3. Bense L. Spontaneous pneumothorax related to

falls in atmospheric pressure. Eur J Respir Dis. 1984;

65(7):544-6.

4. Bulajich B, Subotich D, Mandarich D, et al. Influence

of atmospheric pressure, outdoor temperature,

and weather phases on the onset of spontaneous

pneumothorax. Ann Epidemiol. 2005; 15(3):185-90.

5. Haga T, Kurihara M, Kataoka H, et al. Influence

of weather conditions on the onset of primary



Cardiovasc Surg. 2013; 19(3):212-5.

6. Smit HJ, Golding RP, Schramel FM, et al.

Lung density measurements in spontaneous

pneumothorax demonstrate airtrapping.

Chest 2004; 125(6):2083-90.

7. Cottin V, Streichenberger N, Gamondes JP, et

al. Respiratory bronchiolitis in smokers with

spontaneous pneumothorax. Eur Respir J. 1998;

12(3):702-4.

8. Edmonds C, Lowry C, Pennefather J, eds.

Pulmonary barotrauma. Diving and subaquatic

medicine, 3rd edn. Sydney, Australia: Butterworth-

Heinemann Medical, 1992:95–115.


R, et al. Chest CT for primary spontaneous

pneumothorax (PSP): findings: Birt-Hogg-Dubé

versus non-Birt-Hogg-Dubé patients. Am J Resp

Crit Care Med; 189:A6415.




morphometric analysis of 229 pulmonary cysts from

50 unrelated patients. Histopathology 2014;

65(1):100-10.

11. Baumann MH. Pneumothorax and air travel:

lessons learned from a bag of chips. Chest 2009;

136(3):655-6.

12. Taveira-DaSilva AM, Burstein D, Hathaway

OM, et al. Pneumothorax after air travel in

lymphangioleiomyomatosis, idiopathic pulmonary

fibrosis, and sarcoidosis. Chest 2009; 136(3):665-70.

13. Pollock-BarZiv S, Cohen MM, Downey GP, et al. Air

travel in women with lymphangioleiomyomatosis.

Thorax 2007; 62(2):1756-80.



pneumothorax: British Thoracic Society Pleural

Disease Guideline 2010. Thorax 2010; 65 Suppl 2:ii18-31.

15. Coker RK, Shiner RJ, Partridge MR. Is air travel

safe in thorse with lung disease? Eur Respir J 2007;

30:1057-63.

16. Sand M, Bechera F-G, Sand D, et al. Surgical and

medical emergencies on board European aircraft:

a retrospective study of 10189 cases. Critical Care

2009; 13(1):R3.

17. Peterson DC, Martin-Gill C, Guyette FX, et al.

Outcomes of medical emergencies on commercial

airline flights. N Engl J Med 2012; 368:2075-83.

18. Hu X, Cowl CT, Baqir M, et al. Air travel and

pneumothorax. Chest 2014; 145(4):688-694.

19. Hoshika Y, Kataoka H, Kurihara M, et al. Features of

pneumothorax and risk of air travel in Birt-Hogg-Dubé

syndrome. Am J Respir Crit Care Med 2012; 185:A4438.

20. Tiemensma M, Buys P, Wadee SA. Sudden death on

an aeroplane. S Afr Med J. 2010; 100(3):148-9.

c H a P t e Rthe prevalence of Birt-Hogg-dubé syndrome

among patients with apparently primary spontaneous pneumothorax

Paul C. Johannesma1, Rinze Reinhard2, Yael Kon3, Jincey D. Sriram4, JanHein T.M. van Waesberghe2, Marianne A. Jonker5, Theo M. Starink3,

Arjan C. Houweling6, Quinten Waisfisz6, Johannes J.P. Gille6, Erik Thunnissen7, Hans J. Smit4, R. Jeroen A. van Moorselaar8,

Fred H. Menko9, Pieter E. Postmus1


2 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands3 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands

4 Department of Pulmonary Diseases, Rijnstate Hospital, Arnhem, The Netherlands5 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam,

The Netherlands6 Department of Clinical Genetics, VU University Medical Center, Amsterdam,

The Netherlands 7 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

8 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 9 Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands

Eur Respir J 2015;45(4):1191-4

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a B s t R ac t

introduction

Primary spontaneous pneumothorax is a common condition often associated with apical lung blebs

or bullae. Birt-Hogg-Dubé syndrome (BHD) is a rare autosomal dominant predisposition due to

germline FLCN mutations and characterized by skin fibrofolliculomas, basally located lung cysts,

pneumothorax and renal cell cancer. At present, evaluation of PSP patients generally does not

include CT thorax. We hypothesized that a minority of PSP patients may in fact have underlying BHD.


We reviewed thoracic CT scans available for 69 apparently PSP patients for the presence of basal

cysts. In addition, in 40 apparently common PSP patients we performed FLCN mutation analysis,

thoracic CT scanning, renal MRI and skin examination.

Results

Among 69 PSP cases 7 (10.1%) had multiple basal cysts, indicating possible BHD. In two patients FLCN

mutation analysis was performed which revealed a pathogenic mutation in both cases. Among the

40 patients with apparently common PSP three had pathogenic germline FLCN mutations and one

of these had a positive family history for pneumothorax. All three patients had multiple basal lung

cysts. Asymptomatic renal cell cancer was detected in a first-degree family member of an identified

BHD patient.

conclusion

Among patients with apparently PSP up to 5-10% may have underlying BHD. Thoracic CT scanning

may reveal the multiple basal lung cysts typical for this syndrome. Periodic renal imaging is essential

for BHD patients since this may lead to early detection and treatment of BHD-associated renal cell

cancer.

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Pneumothorax is defined as the presence of air in the pleural cavity. If not due to an obvious

external force the condition is described as spontaneous pneumothorax (SP). This common clinical

condition is subdivided into secondary SP (SSP), due to various forms of lung pathology and primary

SP (PSP) without indication of an underlying lung disease, and diagnosis is usually based on history

and chest X-ray. The first episode of PSP most commonly occurs in the third decade of life in males

who are often taller than age-matched controls and the majority has a history of smoking. Smoking

increases the risk of PSP more than 100 times.1

For several reasons, such as recurrent or persistent air leak, a chest CT can be indicated for PSP

patients. In up to 90% of uncomplicated cases cystic structures, usually described as (subpleural)

blebs and bullae, are found in the lung apices.2 In a subgroup of around 5-15% of PSP patients cystic

abnormalities are described in other areas of the lungs, especially below the level of the main carina,

and not only in the apices.3 Possibly these patients have a different aetiology of pneumothorax than

the patients with abnormalities restricted to the apices. Multiple cysts below the level of the main

carina are characteristic for Birt-Hogg-Dubé syndrome (BHD, an autosomal dominant condition

caused by germline mutations in the folliculin (FLCN) gene. BHD is clinically characterized by

skin fibrofolliculomas, pulmonary cysts, recurrent pneumothorax and renal cell cancer. Clinical

manifestations of BHD are variable and include patients and families with only skin, lung or renal

abnormalities. SP may be the first and only manifestation of BHD in isolated and familial cases. Most

BHD patients have normal chest X-ray images but multiple lung cysts are commonly identified on

CT; approximately 50% of the cysts are located in the subpleural area and 50% in the parenchyma.4

Although about 90% of BHD syndrome patients have these multiple cysts, lung function (measured

by spirometry and diffusion capacity) is generally normal.4 Thus, patients with pneumothorax due

to BHD often have no preceding symptoms of pulmonary disease and are therefore likely to be

diagnosed as having PSP.5 6 7 8 9

In a prospective study in a Chinese population with apparently PSP testing for FLCN mutations was

performed by Ren and colleagues. A total of 10 cases of BHD were confirmed among 102 PSP cases.

Eight of these ten BHD cases had 10 or more cysts on CT, but unfortunately the exact location of the

cysts was not reported.10

These findings raise the important clinical question whether all patients who present with PSP should

be evaluated for BHD. To address this issue, we evaluated the presence of cysts on pulmonary CT

scans in PSP patients and performed FLCN mutation analysis in 2 patients with multiple cysts and

in addition, we evaluated a group of 40 patients with apparently common PSP for underlying PSP.

pat i e n t s a n d m e t H o d s

patients

Review of thoracic CTs

We retrospectively collected data on all patients, who had been treated for PSP at a medical

university and a general hospital (VU University Medical Center, Amsterdam and Rijnstate Hospital,

Arnhem, the Netherlands) in the years 2000-2012. Patients diagnosed with PSP according to the

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criteria proposed by the British Thoracic Society were included when they were over the age of 18

years at the time of diagnosis. Exclusion criteria were secondary pneumothorax due to apparent

underlying disease, traumatic or iatrogenic pneumothorax. Furthermore, deceased patients were

excluded (figure 1). All thoracic CTs, made within 1 year before or after the PSP, were collected and

scored by one pulmonologist (PEP) for the presence of abnormalities in the completely inflated lung

or lungs. Clinical information was not available at the time of scoring.

Evaluation for BHD characteristics

For the second part of the study a questionnaire was sent to the last known address of in total

316 patients registered in the VUMC database under the diagnosis of PSP. Additional information

on medical history (respiratory, dermatological, urological disease), any additional episodes of

pneumothorax and treatment, treatment complications, history of smoking, history of drug use and

family history of spontaneous pneumothorax were collected (n=93) (figure 1). All these participants

(n=42) gave written informed consent.

During an out-patient visit physical examination of the skin was performed by a dermatologist

familiar with BHD, a blood sample was taken for FLCN mutation analysis and thoracic CT scanning was

performed. In addition, all patients with a positive familial history for pneumothorax underwent an

figure 1. Flowchart study protocol BHD-P study.

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renal MRI. Renal MRI was also offered to those in whom a pathogenic FLCN mutation was identified.

Subsequently families of the patients diagnosed with BHD were invited for genetic counselling. This

study was approved by the ethics committee of the VU University Medical Center (NL31417.029.10).

All patients provided written informed consent.

mutation analysis


amplification and sequencing of the 14 exons were designed as detailed by Nickerson et al.11 PCR


CA, USA). Sequencing reactions were performed using the Big Dye Terminator system (Applied



MLPA kit P256 (MRC Holland, www.mrc-holland.com).

R e s u lt s

ct evaluation of psp patients

The main characteristics of the 69 radiological scored patients are summarized in table 1. Between

2000 and 2012 a total of 96 patients underwent one or more thoracic CT’s for spontaneous

pneumothorax. After exclusion of 6 non-evaluable cases (e.g. due to incomplete imaging of the

thoracic CT) and 21 cases reclassified as having secondary SP, a total of 69 patients were eligible

for scoring (figure 1). The mean age was 37.6 years, the recurrence rate was 28.2% and positive

family history for SP was noted in 16.2% of cases. A family history for renal cell cancer or skin

fibrofolliculomas was reported in none of the 316 medical records. Almost half of the patients

had at least one cyst on thoracic CT, of which the majority was located above the carina. Among

the 69 cases with PSP scored for lung cysts on thoracic CT, 14 patients (20.3%) showed one or

more cysts below the carina. In 7 patients >50% of the lung cysts were present below the carina

and therefore indicative for Birt-Hogg-Dubé syndrome (figure 2). In two of these patients the

diagnosis BHD syndrome had been confirmed previously by FLCN mutation analysis. For the other

5 patients FLCN mutation analysis was not available. The main characteristics of the 7 patients

are summarized in table 2. Two patients had a history of renal cell cancer. The first patient with

table 1. Characteristics of primary spontaneous pneumothorax patients with available thoracic CT.

main characteristics of the 69 psp patients with available thoracic ct n (%)

Male gender 52 (75.4)

≥ 1 cysts on thoracic CT 32 (46.4)

≥ 1 more cysts above carina 28 (40.6)

≥ 1 cysts under carina 14 (20.3)

Mean number of cysts above carina 5.37

Mean number of cysts under carina 1.97

≥ 50% of cysts under carina 7 (10.1)

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a proven pathogenic FLCN mutation had spontaneous pneumothorax at age 24 and developed

renal cell cancer at the age of 44. After needle biopsy, histopathology showed a chromophobe

renal cell carcinoma. The patient was treated with radiofrequency ablation (RFA). For the second

patient with renal cell carcinoma, FLCN mutation analysis was not available. This patient – with

an unremarkable medical history - was evaluated for haematuria at the age of 74. Computed

tomography (CT) of the abdomen showed an interpolar tumour in the right kidney (diameter of

10 cm) with lymph nodes metastasis and therefore classified as T3N2M1. The tumour was removed

during an uncomplicated total nephrectomy procedure. Histopathology showed a chromophobe

renal cell carcinoma, Fuhrman grade 4. Twelve months later the patient developed a spontaneous

pneumothorax.

table 2. Characteristics of the seven spontaneous pneumothorax patients with >50% of cysts under the carina.

patient

gender

(age at first psp)

Recu- rrence of psp

number of recurrences of psp

number of lung cysts under carina

(%) of cysts under carina

Renal cell cancer (age at diagnosis)

fibro-folli- culo- mas

positive family history of sp flcn mutation

1 M (24) yes 3 6 81% Yes (44) no no c.510C>G;

p.Tyr170X)

2 F (20) yes 8 13 84% no no no c.610_611delGCinsTA

p.Ala240X

3 M (62) no - 10 100% n/a n/a n/a Not tested

4 M(75) no - 3 100% Yes (74) n/a n/a Not tested

5 M (20) yes 2 3 67% n/a n/a n/a Not tested

6 M (29) no - 4 75% n/a n/a n/a Not tested

7 F (52) no - 35 52% n/a n/a No Not tested

figure 2. High resolution computed tomography of the chest of a non-tested PSP patient, clinically suspected for Birt-

Hogg-Dubé syndrome. The coronal and transversal coupe show multiple round and oval thin-walled pulmonary cysts

of varying sizes, localized mostly in the lower lobes of both lungs.

a B

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evaluation for presence of BHd

In total 93 patients returned a completed questionnaire and 42 of them gave written informed consent

to be contacted for further studies (outlined in the right arm of figure 1). The final study group consisted

of 40 cases after exclusion of two patients with extensive bullous emphysema on thoracic CT who were

reclassified as having SSP. The main characteristics of the 40 patients are summarized in table 3.

Three patients had a pathogenic FLCN mutation (7.5%, 95% confidence interval 1.5% – 20.3%).

Details of these patients are provided in table 4 and the pedigrees are shown in figure 3. The FLCN

mutations detected in these patients were a nonsense mutation, c.610_611delGCinsTA (p.Ala240X),

a frameshift mutation, c.1408_1418del (p.Gly470fs) and a splice site mutation, c.1539-2A>G.

The thoracic imaging performed in these three patients is shown in figure 4. For index patient B the

histological picture of a basal lung cyst for which bullectomy was performed is shown in figure 5.

Family members of the three probands with BHD were invited for genetic counselling and given

the option for pre-symptomatic DNA testing and further clinical evaluation. We identified four

additional FLCN mutation carriers (one in families A and B and two in family C). Renal MRI in a first-

table 3. Main characteristics of the 40 BHD-P study participants.

main characteristics of the 40 study participants n (%)

Male gender 25 (62.5)

History of smoking 29 (72.5)

Pack years (range) 7.68 (1-50)

History of drug abuse 11 (27.5)

Mean age at first pneumothorax (range) 33.8 (18-78)

Recurrence of pneumothorax 19 (47.5)

Mean number of recurrences (range) 1.6 (1-12)

Positive family history of pneumothorax 7 (17.5)

Pathogenic FLCN mutation 3 (7.5)

table 4. Characteristics of the three spontaneous pneumothorax patients who had pathogenic FLCN mutations.

patient (fam. no)

gender (age first psp)

delay between first symptom (psp) and final diagnosis BHd(in months)

Recu- rrence of psp

number of recurrences of psp

number of lung cysts

Renal tumor ff

smoking history flcn mutation

1 (84) F (20) 243 yes 8 13 no Minimal* no c.610_611delGCinsTA

(p.Ala240X)

2 (85) M (26) 153 yes 6 140 no Minimal* no c.1408_1418del

(p.Gly470fs)

3 (94) M (40) 81 yes 3 74 no no no c.1539-2A>G

* Very subtle minimal facial skin lesions, probably fibrofolliculomas. Clinical diagnoses, skin biopsy for histopathology not performed.

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a

B

c

figure 3. Pedigrees of families A, B and C. Symbols: in black: right upper quadrant: pneumothorax, right lower: multiple

lung cysts, left upper: skin fibrofolliculomas, left lower: renal cell cancer; Family A, patient II-7 and family B, patient III-6:

affected according to family history.

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figure 4. High resolution computed tomography of the

chest in the three patients with proven BHD revealed

multiple round and oval thin-walled pulmonary cysts

of varying sizes, localized mostly in the lower lobes,

which abutted to or enclosed the proximal portions of

lower pulmonary arteries and veins.

a B

c

figure 5. Histopathology of a lung cyst of the index patient of family B. The cyst walls are completely lined by

pneumocytes. The inner surfaces of the cysts stained positively for TTF-1 expression.

a B

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degree relative in family C who was a proven FLCN mutation carrier revealed a 15-mm solid mass

in the lower pole of the right kidney, compatible with a small renal cell cancer, shown in figure 6.

The patient underwent an uncomplicated nephron sparing therapy revealing a clear cell renal cell

carcinoma. The outcomes of all investigations are summarized in table 4.

Among the 37 cases with PSP who showed no pathogenic FLCN mutation, blebs and bullae in the

apical lung parts were seen in 21 (56.8%) patients, in none of these patients cysts were found in

the basal parts of the lungs. The six patients without pathogenic FLCN mutation but with a positive

family history for PSP showed no skin abnormalities or abnormalities on renal MRI.

d i s c u s s i o n

In this study we evaluated a group of 40 PSP patients with apparently common PSP for the presence

of BHD and found three (7.5) positive cases, which is in line with the findings previously reported for

a Chinese population group by Ren et al.10

All three cases had the typical pulmonary features of BHD, i.e. multiple lung cysts, localized in the

basal parts of the lungs. Apical blebs and bullae which are common in non-hereditary PSP were

absent in these three cases. In contrast, among the 37 non-hereditary PSP cases 21 patients (56.8%)

showed these apical lung abnormalities.

Our retrospective evaluation of CTs of PSP patients underscores these findings since among 69

patients seven had predominantly basal lung cysts and two of these had proven BHD. This also

indicates that in the common PSP study group indicating that the prevalence of BHD among PSP

patients may be up to 5-10%.

The three patients with a pathogenic FLCN mutation had undergone one or more thoracic CT’s prior

to our study. Although in retrospect lung cysts were visible on these scans, BHD was not suspected

previously. With current knowledge, the multiple lung cysts combined with the recurrent episodes of

pneumothorax would have been a reason to consider FLCN analysis at an earlier stage. A positive family

figure 6. Renal CT scan in patient III-IV, family C, showed a 15-mm solid mass (arrow) in the lower pole of the right

kidney, compatible with a small renal cell cancer.

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history for SP could also have been a reason for the treating pulmonologist to consider referring a patient

to a clinical genetics outpatient clinic, but this is not recommended in current BTS or ACCP guidelines.12 14

Among the group of 69 PSP patients scored for the presence of lung cysts, we found in 7 patients

(10.1%) a majority of cysts located under the carina. BHD was confirmed by FLCN mutation analysis

in two patients and was clinically suspected in five patients. Among these seven patients, renal cell

cancer was found in two patients, one of whom had a pathogenic FLCN mutation. The histopathology

in the second patient revealed a chromophobic renal cell tumour, which is suggestive for BHD.

Pneumothorax, the occurrence of air in the pleural cavity, is a common condition with a high

incidence of between 1.2 and 18 cases per 100.000 persons per year.13 The diagnosis is suggested by

patient’s history and findings on physical examination and is in most cases confirmed by a standard

erect chest X-ray during inspiration. Thoracic CT scanning is currently only recommended for

uncertain or complex cases.14 In PSP subpleural blebs/bullae are found among more than 76%

of patients during video-assisted thoracoscopic surgery.14 These cysts cannot be visualized by

standard chest X-ray and the relationship between these blebs/bullae and the development of

pneumothorax has not been fully clarified.15 Following the current guidelines, underlying lung

pathology as cause for SP might remain undiagnosed. Although cystic lesions on CT scanning in

PSP patients have been reported, the location of these cysts has not been studied extensively.16 In

a rather dated study a prevalence of cystic structures present only below the level of the carina

was reported in 8.4% (5 out 59 cases).3 Whether these abnormalities were related to a different

aetiology was not investigated at that time.

The expression of BHD is variable and patients may present with isolated pneumothorax. 5 9 17

The variation in clinical presentation is probably not due to specific gene defects since clear

genotype phenotype correlations have thus far not been demonstrated in this syndrome. Patients

with BHD who develop pneumothorax have multiple basal lung cysts in the majority of cases,

although the presence of cysts does not per se result in pneumothorax. Approximately 90% of

BHD patients have these lung cysts whereas only 24% of BHD cases develop a pneumothorax.16 The

relationship between these cysts and the pathogenesis of pneumothorax has not been clarified yet.

In our cohort of BHD families most probands were referred by their dermatologist after the

diagnosis of multiple skin fibrofolliculomas. On reviewing these families we found many cases with

pneumothorax that had been diagnosed initially as having common PSP.18 19 This is to be expected

given the lack of pulmonary symptoms and normal lung function in BHD patients.20 Therefore, we

hypothesized that common PSP may be diagnosed in cases that in fact have BHD. Furthermore there

might be an overlap in radiological abnormalities between BHD- and smoking related PSP.21 22 BHD

is reported to be increased in patients who report a positive family history for pneumothorax.16 17

There are several strengths and limitations of our study. This is the first study in a European population.

As the selection of cases for further testing in this study was done by voluntary participation, the

tested group (N=40) might be different from the “standard” PSP population although the mean age,

recurrence rate and percentage with a positive history for SP was comparable to the initial selected

PSP group of 316 patients and in none of the included patients a family history of renal cancer was

present (which would assumedly have resulted in a higher chance of participation to the study). Our

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retrospective evaluation of CTs of PSP patients more or less confirms our earlier findings and might

implicate that the prevalence of abnormalities indicating potential BHD on CTs of PSP patients is

around 5-10%. This percentage is comparable to the (limited) available literature.

In the study by Ren and colleagues, eight confirmed BHD patients had 10 or more lung cysts; two

had one and three cysts, respectively. This observation shows that multiple basal lung cysts may be

absent in BHD patients and we and others have made the same observation. 23 24 25 26

The association of BHD with multiple lung cysts was previously shown in a Japanese study in eight

patients with multiple lung cysts, seven of whom had had recurrent pneumothorax, five of them

had BHD.27 Other authors have selected patients with familial pneumothorax and demonstrated that

familial pneumothorax without other signs of BHD may indeed indicate BHD. 9 28 29 30

Patients with BHD have an increased risk of renal cell cancer. We calculated a 16% renal cancer risk

until the age of 70 years.17 Periodic renal imaging may result in early detection and treatment of BHD

associated renal cell cancer. Therefore diagnosing BHD as early as possible may have consequences

not only for the BHD patient presenting with pneumothorax, but also for their relatives. Indeed,

a first-degree relative of one of the three probands carrying the FLCN mutation identified in our

proband had an early stage solid small renal cell tumour and was treated successfully. Calculations

based on literature and our database indicate that of 100 BHD cases diagnosed through the presence

of SP around 60 cases of RCC will be detected during lifelong follow-up by MRI of the affected PSP

patients and detected family members.31

The three cases reported in our study lacked other signs of the syndrome. None had the typical

skin fibrofolliculomas and two had a negative family history for pneumothorax. Absence of skin

abnormalities and a negative family history for pneumothorax therefore do not exclude BHD and

DNA testing may therefore be needed to diagnose this syndrome. The suspicion of BHD would have

been much higher if at the time of diagnosis of PSP additional thoracic CT imaging would have been

analysed for the presence of basal cysts.

The main limitations of our study are the low response rate and a possible selection of cases.

The response rate was low, since only 40 out of a total group of 316 patients (9.9%) were fully

examined. Patients who were invited for the study may preferentially have opted for this possibility

due to certain characteristics for example young age at diagnosis, high recurrence rate or a positive

family history for the disease. In addition, after being informed on the characteristics of BHD

patients with skin lesions or with a personal or family history of pneumothorax of renal cancer may

have encouraged individuals to participate. As the results of the group of 40 patients is comparable

both with the scored thoracic CT group (N=69) and the much larger study by Ren and colleagues in

consecutive cases with PSP, selection bias may not have been a major factor in our study.

An important argument to diagnose BHD at an early stage might be the high recurrence rate of PSP

which might prompt a different and more aggressive therapeutic approach.32

Based on these results we advocate that prospective studies should lead to further insights in

the prevalence of BHD in PSP, any differences in response to therapy of pneumothorax and the

optimal diagnostic tools in diagnosing BHD among apparently non hereditary PSP cases. The most

attractive diagnostic routine for PSP patients may consist of thoracic CT as the first test, followed by

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dermatological examination, family history taking and FLCN mutation analysis in patients suspected

for BHD based on the findings on CT scan or their (family) history.

In summary, BHD among apparently PSP patients is probably not a rare event. In our PSP

group we identified a FLCN mutation in 3/40 (7.5%) patients. Although Birt-Hogg-Dubé is not

mentioned in the BTS and ACCP guideline for pneumothorax, clinicians should be aware of this

disease in the apparently common spontaneous pneumothorax population. Further research

is required to address the question whether current diagnostic procedures in spontaneous

pneumothorax should be changed, in order to allow detection of BHD - with its corresponding

increased risk on RCC – at an early stage.

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2. Donahue DM, Wright CD, Viale G, Mathisen DJ.

Resection of pulmonary blebs and pleurodesis

for spontaneous pneumothorax. Chest 1993;

104(6):1767-1769.

3. Smit HJ, Wienk MA, Schreurs AJ, Schramel FM,

Postmus PE. Do bullae indicate a predisposition

for recurrent pneumothorax? Br J Radiol

2000;73(868):356-9.


R, Gille JJP, van Moorselaar RJA, Houweling AC,

Menko FH, Postmus PE. Chest CT for primary

spontaneous pneumothorax (PSP): findings: Birt-

Hogg-Dubé versus non-Birt-Hogg-Dubé patients.

Am J Resp Crit Care Med;189:A6415.

5. Johannesma PC, Lammers JW, van Moorselaar RJ,

Starink ThM, Postmus PE, Menko FH. Spontaneous

pneumothorax as the first manifestation of a

hereditary condition with an increased renal cancer

risk. Ned. Tijdschr Geneeskd. 2009;153:A581.

6. Pierce CW, Hull PR, Lemire EG, Marciniuk

DD. Birt-Hogg-Dubé syndrome: an inherited

cause of spontaneous pneumothorax. CMAJ

2011;183(9):E601-3.

7. Verhaert LL. A young man with bilateral

spontaneous pneumothorax. Case Rep

Pulmonology. 2011;2011:414165.

8. Hopkins TG, Maher ER, Reid E, Marciniak SJ. Recurrent

pneumothorax. Lancet 2011;377(9777):1624.

9. Johannesma PC, Thunnissen E, Postmus E. Lung

cysts as indicator for Birt-Hogg-Dubé syndrome.

Lung 2014l1952(1):215-6.

10. Ren HZ, Zhu CC, Yang C, Chen SL, Xie J, HouYYm,

Xu ZF, Wang DJ, Mu DK, Ma DH, Wang Y, Ye ZR,

Chen BF, Wang CG, Lin J, Qiao D, Yi L. Mutation



pneumothorax. Clin Genet 2008; 74(2):178-183.

11. Nickerson ML, Warren MB, Toro JR, Matrosova V,

Glenn G, Turner ML, Duray P, Merino M, Choyke P,

Pavlovich CP, Sharma N, Walther M, Munroe D, Hill

R, Maher E, Greenberg C, Lerman MI, Linehan WM,

Zbar B, Schmidt LS. Mutations in a novel gene lead to

kidney tumors, lung wall defects, and benign tumors

of the hair follicle in patients with the Birt-Hogg-

Dubé syndrome. Cancer Cell 2002; 2(2):157-164.

12. Baumann MH, Strange C, Heffner JE, Light R,

Kirby TJ, Klein J, Luketich D, Panacek EA, Sahn SA,

for the ACCP Pneumothorax Consensus group.

Management of Spontaneous pneumothorax:

an American College of Chest Physicians Delphi

consensus statement. Chest 2001;119(2):590-602.

13. Sahn SA, Heffner JE. Spontaneous pneumothorax.

N Engl J Med 2000; 342(12):868-874.




Disease Guideline 2010. Thorax 2010; 65 Suppl

2:ii18-ii31.

15. Schramel FM, Postmus PE, Vanderschueren RG.


Eur Respir J 1997; 10(6):1372-1379.

16. Lesur O, Delorme N, Fromaget JM, Bernadac

P, Polu JM. Computed tomography in the

etiologic assessment of idiopathic spontaneous


17. Toro JR, Pautler SE, Stewart L, Glenn GM, Weinreich

M, Toure O, Wei MH, Schmidt LS, Davis L, Zbar

B, Choyke P, Steinberg SM, Nguyen DM, Linehan

WM. Lung cysts, spontaneous pneumothorax, and

genetic associations in 89 families with Birt-Hogg-

Dubé syndrome. Am J Respir Crit Care Med 2007;

175(10):1044-1053.

18. Houweling AC, Gijezen LM, Jonker MA, van Doorn

MB, Oldenburg RA, van Spaendonck-Zwarts KY,

Leter EM, van Os TA, van Grieken NC, Jaspars

EH, de Jong MM, Bongers EM, Johannesma PC,

Postmus PE, van Moorselaar RJ, van Waesberghe

JH, Starink TM, van Steensel MA, Gille JJ, Menko

FH. Renal cancer and pneumothorax risk in Birt-

Hogg-Dubé syndrome; an analysis of 115 FLCN

mutation carriers from 35 BHD families. Br J Cancer

2011; 105(12):1912-1919.

19. Leter EM, Koopmans AK, Gille JJ, van Os TA,

Vittoz GG, David EF, Jaspars EH, Postmus PE, van

Moorselaar RJ, Craanen ME, Starink TM, Menko

FH. Birt-Hogg-Dubé syndrome: clinical and

genetic studies of 20 families. J Invest Dermatol

2008; 128(1):45-49.

20. Sundaram S, Tasker AD, Morrell NW. Familial

spontaneous pneumothorax and lung cysts due

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to a Folliculin exon 10 mutation. Eur Respir J 2009;

33(6):1510-1512.

21. Fabre A, Borie R, Debray MP, Crestani B, Danel C.

Distinguishing the histological and radiological

features of cystic lung disease in Birt-Hogg-

Dubé syndrome from those of tobacco-related

spontaneous pneumothorax. Histopathology

201464(5):741-9.

22. Johannesma PC, Thunnissen E, Postmus PE. How

reliable are clinical criteria to distinguish between

BHD and smoking as a cause for pneumothorax?

Histopathology 2014;64(7):1045-6.

23. Fröhlich BA, Zeitz C, Mátyás G, Alkadhi H, Tuor

C, Berger W, Russi EW. Novel mutations in the

folliculine gene associated with spontaneous

pneumothorax. Eur Respir J 2008;32(5):1316-20.

24. Agarwal PP, Gross BH, Holloway BJ, Seely J, Stark

P, Kazerooni EA. Thoracic CT findings in Birt-

Hogg-Dubé syndrome. AJR Am J Roentgenol 2011;

196(2):349-352.

25. Menko FH, Johannesma PC, van Moorselaar RJ,

Reinhard R, van Waesberghe JH, Thunnissen E,

Houweling AC, Leter EM, Waisfisz Q, van Doorn

MB, Starink TM, Postmus PE, Coull BJ, van Steensel

MA, Gille JJ. A de novo FLCN mutation in a patient

with spontaneous pneumothorax and renal cancer;

a clinical and molecular evaluation. Fam Cancer

2013; 12(3):373-379.

26. Tomassetti S, Carloni A, Chilosi M, Maffe A, Ungari S,

Sverzellati N, Gurioli C, Casoni G, Romagnoli M, Gurioli

C, Ravaglia C, Poletti V. Pulmonary features of Birt-

Hogg-Dubé syndrome: cystic lesions and pulmonary

histiocytoma. Respir Med 2011; 105(5):768-774.


Takahashi K, Seyama K. Mutations of the Birt Hogg

Dubé gene in patients with multiple lung cysts

and recurrent pneumothorax. J Med Genet 2007;

44(9):588-593.

28. Graham RB, Nolasco M, Peterlin B, Garcia CK.

Nonsense mutations in folliculin presenting as

isolated familial spontaneous pneumothorax in

adults. Am J Respir Crit Care Med 2005; 172(1):39-44.

29. Painter JN, Tapanainen H, Somer M, Tukiainen P,

Aittomaki K. A 4-bp deletion in the Birt-Hogg-

Dubé gene (FLCN) causes dominantly inherited

spontaneous pneumothorax. Am J Hum Genet

2005; 76(3):522-527.

30. Yang CY, Wang HC, Chen JS, Yu CJ. Isolated

familial pneumothorax in a Taiwanese family with

Birt-Hogg-Dubé syndrome. J Postgrad Med 2013;

59(4):321-323.

31. Johannesma PC, Houweling AC, Reinhard R,

Thunnissen E, Menko FH, van Waesberghe JHTM,

Paul MA, Horenblas S, van Moorselaar RJA,

Postmus PE. Early detection of hereditary renal

cell cancer by improved evaluation of spontaneous

pneumothorax patients. Abstract ESMO congress

2014. (accepted)

32. Postmus PE, Johannesma PC, Menko FH, Paul MA.


due to symptom delay. Am J Respir Crit Care Med.

2014 Sep 15;190(6):704-5.

c H a P t e Rinternational guidelines for pneumothorax are not

adequate for treatment of spontaneous pneumothorax in patients with Birt-Hogg-dubé syndrome

Paul C. Johannesma1 , Marinus A. Paul2, JanHein T.M. van Waesberghe3, Marianne A. Jonker4, Arjan C. Houweling5, Irma van de Beek5,

Theo M. Starink6, R. Jeroen A. van Moorselaar7, Fred H. Menko8, Pieter E. Postmus9


2 Department of Thoracic Surgery, VU University Medical Center, Amsterdam, The Netherlands 3 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands


5 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 6 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands

7 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 8 Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands


Submitted

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An important genetic cause of spontaneous pneumothorax is Birt-Hogg-Dubé syndrome (BHD),

caused by mutations in the folliculin (FLCN) gene. It is suggested that approximately 7.5-10% of all

spontaneous pneumothorax (SP) patients may have this underlying disorder. As the recurrence

rate of SP in BHD has been described to be as high as 75%, we evaluated the effect of different

types of treatment. Current BTS and ACCP guidelines do not describe the treatment of SP in BHD

patients as a separate entity. In this study we compared the results of treatment in a comparable

group of BHD and non-BHD patients with (recurrent) SP. We found a recurrence rate of 64.5% after

conservative treatment and a recurrence rate of 11.1% after invasive treatment of SP in BHD patients.

This recurrence rate was significant higher when compared to patients without BHD. Therefore

invasive treatment seems to be the better option for BHD patients with (recurrent) SP. Our results

suggest that SP in BHD is an associated with a high recurrence rate after conservative treatment and

an invasive therapy would therefore be the best approach in this group.

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Pneumothorax is defined as the presence of air in the pleural cavity. If not due to an obvious external

force, the condition is described as spontaneous pneumothorax (SP). This common clinical condition

is subdivided into secondary SP (SSP) - due to various forms of lung pathology - and primary SP (PSP)

without indication of an underlying lung disease. This distinction is usually based on medical history

and chest X-ray at first presentation.1

In a minority of cases a positive family history is reported in individuals who present with a SP.

A possible genetic cause of familial SP in this situation is the Birt-Hogg-Dubé syndrome (BHD), due

to pathogenic mutations in FLCN. Two recent studies showed that approximately 7.5-10% of all SP

patients may have an underlying pathogenic FLCN mutation.2 3

While the recurrence rate in common primary SP has been described up to 50% when treated

conservative, the recurrence rate of SP among BHD patients has been reported to be much higher,

up to 75%, despite different types of treatment.1 4 Current international pneumothorax guidelines

e.g. by the British Thoracic Society (BTS) and the American College of Chest Physicians (ACCP), do

not distinguish common SP due to BHD as separate entity, and also in current published literature

the optimal treatment of SP in patients with BHD has not been evaluated so far.1,5 Therefore we

evaluated the recurrence rate and different treatment options of SP in BHD patients compared to SP

patients without a pathogenic FLCN mutation.


We collected data by sending a questionnaire to 54 patients with a proven pathogenic FLCN

mutation and a history of (recurrent) SP. Forty patients consented to the study and of 38 patients

the medical treatment details were available (table 1, table 2). As a control group, we included 47

randomly selected primary SP patients, who tested negative for a mutation in FLCN. To complete

the dataset, we collected all data from their medical charts, after written informed consent. All

included patients had one or more episodes of SP. We scored on general demographics, number of

episodes, side of SP, number of recurrences and type of treatment classified in conservative (one

time needle aspiration or tube thoracostomy) or invasive (chemical - or mechanical pleurodesis,

(partial) pleurectomy, lobectomy, bullectomy, or a combination of invasive treatment options).

Both lungs were scored separately. The Chi-square test was used for the qualitative variables. All

statistical analyses were performed using SPSS software (version 20, SPSS Inc., Chicago IL, USA).

A p-value of less than 0.05 was considered significant. The study was approved by the Ethics

Committee of the VU University Medical Center.

R e s u lt s

We found no significant difference in gender or mean age at first episode of SP between the FLCN

mutation positive- and negative groups. The mean number of episodes of SP was significantly

higher among patients with BHD, with a mean of 2.85 episodes versus 1.94 episodes in the non-BHD

group. Also the number of episodes of pneumothorax in both lungs (not at the same time) or same

lung was significantly higher in the group of 40 BHD patients (table 1). The mean age in the BHD and

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table 1. Spontaneous pneumothorax in BHD and non-BHD patients.

FLCN mutation + FLCN mutation - p-value

(n=) (%=) (n=) (%=)

Included patients with available medical

information and informed consent

40 47

Male : Female 19 : 21 47.5 : 52.5 29 : 18 61.7 : 38.3 NS#

Mean age (min-max) 32.2 (14-54) 31.6 (16-78) NS#

Mean number of spontaneous

pneumothorax (min-max)

2.85 (1-10) 1.94 (1-5) <0.05

Diagnosed first episode SP in:

Left lung

Right lung

Bilateral (same time)

Both lungs; separate episodes

11

9

2

18

27.5

22.5

5.0

45.0

19

21

1

6

40.4

44.7

2.1

12.8 <0.05

Total first episodes (left + right sided

pneumothorax)

60 54

Total episodes of pneumothorax (left

and right lung)

Right lung

Left lung

114

58

56

91

34

57

# Not significant β Calculated with X2 test

non-BHD group was almost the same; 32.2 years (14-54 years) versus 31.6 years of age (16-78 years).

The mean number of SP was 2.85 (1-10 episodes) among BHD patients and was significantly higher

compared to patients without BHD, with a mean of 1.94 episodes of SP (1-5).

We studied the number of SP episodes for each FLCN mutation. The number of included patients

carrying the same pathogenic FLCN mutation varied between 1 and 23 patients. The pathogenic

nonsense mutation in exon 6 (c.610_611delGCinsTA) was found most often and was identified in

seven different families. In one family, eight relatives with a history of (recurrent) SP carried mutation

c.1539-2A>G with a history of (recurrent) SP.

Recurrence of SP occurred in 64.5% BHD patients after conservative treatment, which was

significant higher when compared to the SP patients without BHD with a recurrence rate of 48.6%.

Immediate invasive treatment after a first episode of SP was performed in BHD patients more

frequently when compared to SP patients without BHD (46.6% versus 31.5%, p<0.05). Various types

of invasive treatment for pneumothorax showed good results in both BHD and non-BHD patient

groups. Especially the long-term results (>12 months) after invasive treatment gave good results in

the group of patients without BHD. Partial pleurectomy was not effective in all treated BHD patients

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table 2. Effect of type of treatment in BHD and non-BHD patients with a (recurrent) episode of SP.

FLCN mutation + FLCN mutation - p-valueβ

(n=) (%=) (n=) (%=)

Number of included patients* (N=) 38 47

Type of treatment not available (N=) 2# 0

Choice of treatment after first SP

(N= both lungs)

Conservative

Invasive

31/58#

27/58#

53.4

46.6

37/54&

17/54&

68.5

31.5

<0.05

Conservative treatment of first SP

No recurrence

Recurrence within ≤1 months

Recurrence within 1-12 months

Recurrence within >12 months

Total recurrence

11/31

7/31

4/31

9/31

20/31

35.5

22.6

12.9

29.0

64.5

19/37

4/37

6/37

8/37

18/37

51.4

10.8

16.2

21.6

48.6

<0.05

<0.05

Invasive treatment of first SP (via

thoracoscopy or VATS)

No recurrence

Recurrence within ≤1 months

Recurrence within 1-12 months

Recurrence within >12 months

Total recurrence

24/27

1/27

0/27

2/27

3/27

88.9

3.7

0.0

7.4

11.1

16/17

1/17

0/17

0/17

1/17

94.1

5.9

0.0

0.0

5.9

<0.05

<0.05

No recurrence after treatment:

Conservative (one time aspiration

/ tube thoracostomy)

(Talc) pleurodesis

Partial pleurectomy

Total pleurectomy

Bullectomy

Lobectomy

Combined invasive (bullectomy/

pleurectomy/pleurodesis)

12/53

20/28

0/3

4/4

0/2

NP$

22/24

22.6

71.4

0.0

100.0

0.0

NP$

91.7

18/46

24/26

2/2

2/2

4/6

NP$

4/7

39.1

92.3

100.0

100.0

66.7

NP$

57.1

NS#

NS#

<0.05

NS

<0.05

NA&

<0.05

*With completed information from questionnaire and patients charts and after informed consent. $ Not performed, & Not available, # Not significant β Calculated with X2 test # See table 1. In total 60 included separated lungs. In two patients with one sided pneumothorax no type of treatment known, therefore 60-2=58 included lungs with known type of treatment. & See table 1 for inclusion.

and also bullectomy resulted in recurrent episodes of SP in all BHD patients. Combined invasive

treatment or total pleurectomy resulted in excellent results in BHD patients. In non BHD patients all

type of invasive treatment resulted in good (long term) results.

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d i s c u s s i o n

In this study we compared a group of 54 pneumothorax patients with a pathogenic FLCN mutation with

47 pneumothorax patients without a pathogenic FLCN mutation. The main finding was the significant

higher number of recurrent episodes of spontaneous pneumothorax in BHD patients compared to

non-BHD patients, despite several types of treatment. We found a recurrence rate of 64.5% in our

BHD cohort, which is comparable with current literature.6 Invasive treatment as pleurodesis, total

pleurectomy or a combination of both gave good results in both groups (table 2). Recurrent episodes

of pneumothorax were found in all BHD patients after bullectomy and partial (apical) pleurectomy.

This seems explainable, as cysts in the basal parts of the lung are, very likely, often responsible for

pneumothorax, and bullectomy or partial pleurectomy is only performed in the lung apices.7 8 In current

literature very little information is available on treatment of pneumothorax among BHD patients. In

some papers surgical intervention with resection and pleurodesis is suggested to be an acceptable

option, even in BHD patients with a first episode of pneumothorax.4 9 10 11 These suggestions are only

based on the somewhat comparable (progressive) cystic lung disease lymphangioleiomyomatosis

(LAM).12 Pulmonary LAM is characterized by recurrent pneumothoraces (in 50-60% of mainly female

patients), chylothorax, progressive dyspnoea and pleural effusions.13 14 This progressive lung disorder

differs from BHD as pneumothorax occurs in approximately 30% of FLCN mutation carriers, and the lung

function remains unaffected despite multiple cysts in the basal parts of the lung. The similarity between

LAM and BHD is the high recurrence rate of up to 75% for pneumothorax, comparable to the 64.5%

recurrence rate we found in our BHD population. Current guidelines for the treatment of spontaneous

pneumothorax differ enormously and are based on older literature (BTS, ACCP).1 5 Recommendations

on the treatment of interstitial lung diseases as BHD or LAM are not discussed in these pneumothorax

guidelines. In a recently published Cochrane review on conservative versus interventional management

for primary spontaneous pneumothorax it was concluded that there is no strong evidence for

interventional management of PSP, despite widespread practice and recommendation.15

So far no gender predilection has been found in BHD with regards to development of pneumothorax.

Age and smoking status are not associated with pneumothorax in literature, in accordance with

the observations in our cohort. The two largest studies among BHD patients found no genotype/

phenotype correlation between pathogenic FLCN mutation and spontaneous pneumothorax.6 16 In

our cohort we observed no clear differences between the 15 different pathogenic FLCN mutations

and the number of episodes of pneumothorax, age and recurrence rates. We found no recurrent

episodes of pneumothorax in two different FLCN mutations, but both mutations were found in only

one patient respectively (data not shown).

In conclusion: based on our observations a conservative treatment is not accurate in BHD patients

who suffer from a high recurrence rate of spontaneous pneumothorax. All types of (combined)

invasive treatments –except for the more localized ones like bullectomy and partial pleurectomy-

appears to give better results for the treatment of spontaneous pneumothorax in BHD patients

compared to an initinal conservative approach. A large international multicentre phase II study is

needed to evaluate our results. Current pneumothorax guidelines might need to be discussed and

revised. Based on our results we suggest spontaneous pneumothorax in BHD needs a different and

more aggressive treatment for SP in BHD patients.

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R e f e R e n c e s1. MacDuff A, Arnold A, Harvey J, on behalf of the BTS

Pleural Disease Guideline Group. Management of

spontaneous pneumothorax: British Thoracic Society

pleural disease guideline 2010. Thorax 2010;65:ii18-ii31.




Eur Respir J. 2015;45:1191-4.




pneumothorax. Clin Genet 2008;74:178-83.

4. Gupta N, Seyama K, McCormack FX. Pulmonary

manifestations of Birt-Hogg-Dubé syndrome. Fam

Cancer 2013;13:387-96.

5. Baumann MH, Strange C, Heffner JE, et al.

Management of spontaneous pneumothorax:

An American College of Chest Physicians Delphi

Consensus Statement. Chest 2001;119:590-602.



associations in 89 families with Birt-Hogg-

Dubé syndrome. Am J REspir Crit Care Med.

2007;175:1044-53.

7. Agarwal PP, Gross BHD, Holloway BJ, et al. Thoracic

CT findings in Birt-Hogg-Dubé syndrome. AJR Am

J Roentgenol. 2011;196;349-52.


JHTM, et al. The pathogenesis of pneumothorax


Respirology. 2014 Nov;19(8):1248-50.

9. Rehman HU. Birt-Hogg-Dubé syndrome: report of

a new mutation. Can Respir J 2012;19:193-5.

10. Onuki T, Goto Y, Kuramochi M, et al. Radiologically


syndrome. Ann Thorac Surg 2014;97:682-5.

11. Dal Sasso AA, Belém LC, Zanetti G, et al. Birt-Hogg-

Dubé syndrome. State-of-the-art review with

emphasis on pulmonary involvement. Respir Med.

2015;109:289-96.

12. Taveirna-DaSilva AM, Moss J. Clinical features,

epidemiology, and therapy of lymphangioleio-

myomatosis. Clin Epidemiol 2015;7:249-75.

13. Ruy JH, Moss J, Beck GJ, et al. The NHLBI lymphan-

gioleiomyomatosis registry: characteristics of 230

patients at enrollment. Am J Respir Crit Care Med.

2006;173:105-11.

14. McCormack FX. Lymphangioleiomyomatosis:

a clinical update. Chest 2008;133:507-16.

15. Ashby M, Haug G, Mulcahy P, et al. Conservative

versus interventional management for primary

spontaneous pneumothorax in adults. Cochrane

Database Syst Rev. 2014;12:CD010565.





p a R t 2

Renal manifestations

c H a P t e RRenal cancer and pneumothorax risk

in Birt-Hogg-dubé syndrome (BHd); an analysis of 115 flcn mutation carriers from 35 BHd families

Arjan C. Houweling1, Lieke M. Gijezen2, Marianne A. Jonker3, Martijn B. A. van Doorn4 , Rogier A. Oldenburg5,

Karin Y. van Spaendonck-Zwarts6, Edward M. Leter1, Theo A. van Os7, Nicole C.T. van Grieken8, Elisabeth H. Jaspars8, Mirjam M. de Jong6,

Ernie M.H.F. Bongers9, Paul C. Johannesma10, Pieter E. Postmus10, R. Jeroen A. van Moorselaar11, JanHein T.M. van Waesberghe12, Theo M. Starink4, Maurice A.M. van Steensel2, Johan J. P. Gille1,

Fred H. Menko1

1Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 2Department of Dermatology, GROW School for Oncology and Developmental Biology,

Maastricht University Medical Center, Maastricht3Department of Mathematics, VU University, Amsterdam, The Netherlands

4 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands5 Department of Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam

6 Department of Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen

7Department of Clinical Genetics, Academic Medical Center, Amsterdam 8 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

9 Department of Department of Human Genetics, Radboud University Nijmegen Medical Center10 Department of Pulmonology, VU University Medical Center, Amsterdam, The Netherlands

11 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands12 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands

Br J Cancer 2011 dec 6;105(12):1912-9

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a B s t R ac t

Background

Birt-Hogg-Dubé syndrome is an autosomal dominant condition caused by germline FLCN mutations,

and characterized by fibrofolliculomas, pneumothorax and renal cancer. The renal cancer risk,

cancer phenotype and pneumothorax risk of BHD have not yet been fully clarified. The main focus

of this study was to assess the risk of renal cancer, the histological subtypes of renal tumours and

the pneumothorax risk in BHD.

methods

In this study we present the clinical data of 115 FLCN mutation carriers from 35 BHD families.

Results

Among 14 FLCN mutation carriers who developed renal cancer seven were <50 years at onset and/

or had multifocal / bilateral tumours. Five symptomatic patients developed metastatic disease. Two

early-stage cases were diagnosed by surveillance. The majority of tumours showed characteristics

of both eosinophilic variants of clear cell and chromophobe carcinoma. The estimated penetrance

for renal cancer and pneumothorax was 16% (95% minimal confidence interval: 6-25%) and 29% (95%

minimal confidence interval: 8-49%) at 70 years of age, respectively. The most frequent diagnosis in

families without identified FLCN mutations was familial multiple discoid fibromas.

conclusion

We confirmed a high yield of FLCN mutations in clinically defined BHD families, we found

a substantially increased lifetime risk of renal cancer of 16% for FLCN mutation carriers. The tumours

were metastatic in five out of 14 patients and tumour histology was not specific for BHD. We

found a pneumothorax risk of 29%. We discuss the implications of our findings for diagnosis and

management of BHD.

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Birt-Hogg-Dubé syndrome (BHD, OMIM #135150) is an autosomal dominant condition characterized

by fibrofolliculomas, pneumothorax, and renal tumours. BHD is caused by germline mutations

in the FLCN gene encoding folliculin.1 In the original kindred skin lesions were the only clinical

manifestation.2 Subsequently, renal cancer and pneumothorax were found to be part of the

syndrome.3 4 Furthermore, the risk for colorectal cancer might be slightly increased in FLCN

mutation carriers.5 The functions of folliculin have partly been clarified and might include a role

in the mammalian target of rapamycin (mTOR) pathway.6 Whereas many BHD kindreds exhibit all

three components of the syndrome, “pneumothorax-only” and “renal-cancer-only” families have

also been described.7 8 9 10 Among 69 patients with early-onset or familial clear cell renal cancer

without further characteristics of BHD, germline FLCN mutations were found in 4% of cases.9 In

recent reviews, the variable clinical manifestations, molecular pathogenesis and management

options for BHD were summarized.11 12 13

For optimal early detection and treatment of BHD-associated renal cancer, insight into the renal

cancer risk and the clinical picture of these tumours is essential. Among cohorts of BHD patients

a wide range of prevalence of kidney tumours has been observed, ranging from 6.5 to 34%.13 The

differences in prevalence are probably due to ascertainment in dermatological versus urological

clinics and age at examination. Notably, the lifetime renal cancer risk for FLCN mutation carriers

has not yet been established. In BHD, renal cancer is generally diagnosed at a relatively young age

and commonly presents as bilateral and / or multifocal disease. The renal neoplasms typically found

in BHD patients were described as hybrid tumours, containing elements of different histological

subtypes, in particular chromophobe tumours and oncocytoma. However, other subtypes including

clear cell renal carcinoma have also been reported.14 15

A 50 fold increased risk of spontaneous pneumothorax in BHD was reported.16 Among cohorts of

BHD patients the prevalence of pneumothorax ranged from 24-38%.17 Again, ascertainment has

varied for cohorts of patients and the lifetime risk of pneumothorax for FLCN mutation carriers

has not yet been established. On CT examination of the thorax, more than 80% of adult BHD

patients had multiple lung cysts, most often in the basal lung regions.17 The presence of lung

cysts is probably related to the increased risk for pneumothorax, which is often recurrent in BHD

patients. A positive family history for pneumothorax was associated with an increased risk of

pneumothorax and patients with a family history positive for renal cancer had an increased risk

of having renal tumours. However, a family history of renal cancer was not associated with an

increased pneumothorax risk.13

Previously, we described 25 FLCN germline mutation carriers from 11 BHD families.18 Here, we present

an update of this cohort and add evaluation of 24 new kindreds with pathogenic FLCN mutations. In

total, the clinical histories of 115 FLCN mutation carriers from 35 BHD families have been assessed.

The main focus of this study was to assess the risk of renal cancer, the histological subtypes of renal

tumours and the pneumothorax risk in BHD. Furthermore, we consider the yield of FLCN mutation

analysis and the clinical phenotype in kindreds without FLCN mutations.

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ascertainment of pedigrees

The BHD database at VU University Medical Center currently lists more than 65 Dutch families

referred for suspected BHD; 53 of these kindreds with completed family studies are considered in

this report. Forty of these 53 families were referred to our center, while other Dutch clinical genetics

centers contributed an additional 13 families with pathogenic FLCN mutations (table 1).

The index patient of 48 out of the 53 families was referred by a dermatologist after fibrofolliculomas

were diagnosed. In three BHD families the index patient had renal cancer (BHD families 33, 35 and

63), in one kindred the proband had recurrent pneumothorax (BHD 29). One patient without an

identifiable FLCN mutation was referred for multiple pulmonary cysts.

For ascertainment of pedigrees, the proband was requested to inform family members by means of

a written summary letter about BHD. After completion of the initial evaluation reminders were sent

to probands aimed at complete ascertainment of family members.

table 1. Ascertainment of 115 FLCN mutation carriers from 35 BHD families considered in this study.

subdivision of familiesclinical BHd & FLCN- mutation

clinical BHd without FLCN-mutation

other diagnoses

evaluation declined

40 families referred to our

center for suspected BHD1,#

22 probands &

67 mutation-positive

family members

5 92 4

13 FLCN- mutation-positive

families from other centers

13 probands &

13 mutation-positive

family members

1 Including families also described by in Leter et al., 2008 and Johannesma et al., 2009 2 Seven families (11, 17, 20, 24, 38, 45 and 50) had familial multiple discoid fibromas, described by Starink et al., 2011, in press; 2 index patients (26 and 39) were diagnosed with pulmonary emphysema and probable tuberous sclerosis complex and. # for calculation of renal cancer and pneumothorax penetrance the data of 86 FLCN mutation carriers from 21 kindreds for which complete family data were available were used

mutation analysis

After informed consent genomic DNA was extracted from blood samples. Primers for the amplification

and sequencing of the 14 exons were detailed previously by Nickerson et al. (2002).1 PCR amplification

was performed using a PE 9700 thermocycler (Applied Biosystems, Forster City, CA, USA). Sequencing

reactions were performed using Big Dye Terminator (Applied Biosystems) and run on an ABI 3100

genetic analyzer (Applied Biosystems). For the detection of deletions and duplications of one or more

exons the SALSA MLPA kit P256 obtained from MRC Holland was used (www.mrc-holland.com).

statistical analysis

Conditional on the mutation status for different individuals, we assumed the various expressions

of the BHD phenotype to be mutually independent and that for individual cases the risks for renal

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cancer and pneumothorax and the ages at which these traits were expressed are independent.

Finally, the penetrances of renal cancer and pneumothorax were assumed to be equal for male and

female mutation carriers.

For the estimates of the penetrance of pneumothorax and renal cancer we included 21 out of 22

FLCN-positive pedigrees investigated at our center. For these families medical records of all

mutation carriers and information on untested relatives were available. Out of these 21 pedigrees,

20 were ascertained via a proband referred by a dermatologist. One proband (BHD 29) was referred

by a pulmonologist for analysis of recurrent pneumothorax. Therefore, we included the proband

data in the estimation of renal cancer and pneumothorax risks, with the exception of the proband

referred for analysis of pneumothorax.

Major problems of penetrance estimates are missing data and possible preferential testing of

individuals affected with complications of BHD, i.e. renal cancer or pneumothorax. Indeed, if the

mutation status and phenotype had been known for all individuals in all pedigrees the penetrance

curves could have been assessed using the Kaplan-Meier estimator. Unfortunately, however, the

mutation status or phenotype was not known for all family members. Excluding the individuals for

whom the mutation status was unknown would likely lead to an overestimation of risk (see figure 1a),

assuming that non-affected individuals may be less willing to be genotyped. For our corrected risk

estimation we assumed that a negative family history for renal cancer or pneumothorax in a close

relative indeed reflects the absence of these complications in the untested relative. We imputed the

missing data as follows: for every individual in the data set who was not genotyped the probability

that he or she was a carrier was computed on the basis of the mutation status of his / her relatives.

Subsequently, using this carrier probability it was sampled whether he or she was a mutation carrier

or not. As a result, for every individual the mutation status was assigned and in combination with the

phenotype Kaplan-Meier curves and confidence intervals were computed. This strategy of sampling

the mutation status and estimating the penetrance functions by the Kaplan-Meier estimator was

repeated 10000 times. Next, the mean of the 10000 estimated curves and upper and lower bounds

of the confidence intervals were computed. These curves are plotted in figure 1a and B for renal

cancer and in figure 1c for pneumothorax. By using this strategy the estimator will be asymptotically

unbiased. The confidence interval found is slightly too narrow because there is a greater degree

of uncertainty than would have been the case if the missing mutation statuses had actually been

observed (as assumed after imputation). Therefore, the final confidence interval has a confidence

of slightly less than 95%. Although the exact confidences of the intervals are not exactly known we

included the calculated intervals in the figures in order to make those who would use the figures for

consultation aware of the uncertainty of the given risk estimates.

Results

The characteristics of 53 families referred for suspected BHD are presented in table 1. The main

features of the BHD kindreds with pathogenic FLCN germline mutations are listed in table 2. The

mutations detected are depicted in figure 2. Data on the renal tumours and pneumothorax are given

in tables 3 and 4, respectively. Among the 27 families with clinical BHD based on dermatological

evaluation at our center, 22 had pathogenic FLCN mutations (mutation detection rate 81%).

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a B

figure 1. (a) Estimate of the age-related penetrance function for renal cancer based on available data on both mutation

carriers and their untested relatives till the age of 70: 0.160 (continuous line n=86 mutation carriers and 84 untested

relatives), together with the Kaplan-Meier estimator based on the known mutation carriers only at age 70: 0.203 (n=86,

red dashed line). (B) Estimate of the age-related penetrance function for renal cancer based on available data on both

mutation carriers and their untested relatives together with a minimum 95% confidence interval. Estimated penetrance

at age 70: 0.160, 95% minimal confidence interval: [0.0626, 0.259] (c) Estimation of the penetrance function of age at

first pneumothorax and a minimum 95% confidence interval based on available data on both mutation carriers and

their untested relatives (n=85 mutation carriers and 84 untested relatives). Estimated penetrance at age 70: 0.288, 95%

minimal confidence interval: [ 0.0882, 0.487].

c

table 2. Main features of 115 FLCN mutation carriers from 35 BHD kindreds with pathogenic FLCN germline mutations.

fam. no. FLCN germline mutationno. of FLCN mutation carriers

no. of mutation carriers with pneumothorax

no. of mutation carriers with renal cancer

BHD1 c.610_611delinsTA 6 2 1

BHD3 c.420delC 2 0 0

BHD4 c.1285dupC 1 0 0

BHD6 c.1285dupC 4 0 2

BHD8 c.655dupG 2 0 0

BHD12 c.1285dupC 3 0 0


BHD15 c.619-1G>A 4 1 0


BHD18 c.[1301-7_1304del11; 1323delCinsGA] 2 1 0

BHD19 c.619-1G>A 1 0 0

BHD22 c.1285dupC 1 0 0

BHD23 c.319_320delGTinsCAC 11 1 1

BHD27 c.1408_1418delGGGAGCCCTGT 1 0 0


BHD30 c.1285dupC 1 0 0


BHD32 c.469_471delTTC 3 3 1

BHD33 c.619-1G>A 2 1 1

BHD35 c.1749_1753del 2 1 1



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figure 2. Overview of the FLCN mutations identified in this study. N: nonsense, SS: splice site, F: Frame shift, Del:

deletion, M: Missense. Exons are depicted as rectangles. The family numbers are shown after the mutations.

table 2. Main features of 115 FLCN mutation carriers from 35 BHD kindreds with pathogenic FLCN germline mutations.

fam. no. FLCN germline mutationno. of FLCN mutation carriers

no. of mutation carriers with pneumothorax

no. of mutation carriers with renal cancer

BHD40 c.1183_1198del 1 0 0



BHD46 c.1408_1418delGGGAGCCCTGT 6 0 1

BHD47 c.1177-2A>G 2 0 0

BHD48 c.1285dupC 1 0 0

BHD49 c.1300G>C 1 0 0

BHD51 c.1285dupC 1 0 0

BHD53 c.871+3_ 871+4delGAinsTCCAGAT 1 0 0

BHD57 c.250-?_1740+?del (del exon 5-14) 3 2 0

BHD 62 c.1301-?_1740+?del (del exon 12-14) 3 3 0

BHD 63 c.610_611delinsTA 2 1 1

BHD66 c.3G>A 1 0 0

table 2. (continued)

Characteristics of the five families without a detectable FLCN mutation are summarized in table 5;

they include two families with an unclassified exon 1 deletion. Analysis of these exon 1 deletions

is ongoing. In the cohort of our current study, no colorectal cancer was reported. Colorectal

adenomas were diagnosed by colonoscopy in four FLCN mutation carriers (age 40, 42, 83, these

patients were also included in our previous reports by Leter et al. 2008 and Nahorski et al. 2010 and

age 44, not reported previously).

Other malignancies than renal cancer were reported in 6 patients previously documented in Leter

et al. (2008).18 In addition to the patients reported by Leter et al. 2008, in five patients without renal

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cancer, tumours were reported including non-melanoma skin cancer in two patients and single

cases of melanoma, sarcoma, bladder and prostate cancer. The patient diagnosed with melanoma

was also diagnosed with a sarcoma. Three patients with renal cancer were diagnosed with additional

tumours. One patient had an oncocytic pituitary adenoma at age 9, a giant cell astrocytoma at age

12, pheochromocytoma at age 34 and renal cancer at age 34 and 35 (BHD 63, table 3). No evidence

for other tumour susceptibility was found. The other two patients had gastric carcinoma at age 55

and renal cancer at age 56 (BHD 43, M, table 3) and prostate carcinoma and renal cancer at age 50

(BHD 46, table 3), respectively.

Renal manifestations

Fourteen out of 115 (12%) FLCN mutation carriers from 12 families were diagnosed with renal cancer.

All available tumours were revised at our center by two pathologists. In addition, one mutation

table 3. Main features of 17 renal cancers in 14 FLCN germline mutation carriers from 12 BHD kindreds.

fam. no. m/f

age at diagnosis (yrs)

diagnosis #

clinical characteristics*

treatment§ outcome°localisation Histology tnm

BHD1 F 60 B 1 (L) 1 CC/Cph T1N0M0 T A (3)

BHD6 M 39 A 1 (R) 1 Pap/CC T3N2M1 T & Im D (40)

F 40 Aa 1 (L) 2 CC/Cph T2N0M0 T A (7)

BHD16 M 56 A 1 (R) 1 CC/Cph/Sa T3N0M1 T & Ch D (57)

BHD23 M 51 A 1 (L) 1 CC/Cph T1N0M1 T & Ch & R D (52)

BHD32 F 51 B 1 (R) 1 CC/Cph T1N0M0 P A (3)

BHD33 M 48

51

A

B

2 (L)

(R)

1

1

CC/Cph

unknown

T3N0M0

T1N0M0

T

P

A (6)

BHD35 M 38

40

A

B

2 (R)

(L)

2

2

CC

unknown

T1N0M0

T1N0M0

P

P

A (1)

BHD37 F 52 A 1 1 CC TxNxM1 Rth D (52)

BHD43 F 74 A 1 (L) 2 CC/Cph T1N0M0 T A (3)

M 56 A 1 (R) 2 CC/Cph T1N0M0 T A (2)

BHD44 F 43 A 1 (L) 1 CC/Cph TxNxM1 T & Me D (57)

BHD46 M 50 A 1 (L) 1 unclassifiedb T1N0M0 - D (50)

BHD63 M 34

35

A

B

2 (L)

(R)

2

1

CC/Cph

CC/Cph

T2N0M0

T1N0M0

T

P

A (3)

#: A: diagnosis after symptoms had developed; B: diagnosis after positive renal imaging of an asymptomatic individual * Localisation: Left column: 1/2: Unilateral/ Bilateral, in parentheses: L/ R: left-sided/ right-sided, Right column: 1: Unifocal, 2: multifocal. Histology: according to Lopez-Beltran et al., 2009; CC: clear cell, Pap: papillary, Cph chromophobe, Onc: oncocytoma; Sa: sarcomatoid component; CC/Cph: renal cell carcinoma with eosinophilic cytoplasm and characteristics of both CC en Cph, Ad: adenocarcinoma, classification not certain; TNM: classification according to tumour/ node/ metastasis status § Treatment: T: total nephrectomy, P: partial nephrectomy; Ch: chemotherapy, Im: immunotherapy; Rth: radiotherapy° A: alive, D: deceased; in parentheses: number of years of follow-up and age at death, respectivelya: coincidental finding at medical examination for gastrointestinal complaintsb: The tumour was found during autopsy and could not be reliably subclassified due to autolysis

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table 4. Main features of pneumothorax in 28 FLCN germline mutation carriers from 15 BHD kindreds.

fam. no. m/fage at diagnosis*

clinical characteristics

R/l* episodesage at diagnosis of renal cancer

BHD1 F 44 L 1

M 30 R 1

BHD14 M 36 L 1

BHD15 F U U 1

BHD16 M 67 L 1

F 47 U 1

BHD18 M 22 U 1

BHD23 M 22 L 1

BHD291 M 25# B 5

M 27# B 1

BHD32 F 39 U 1 51

F U U 1

M 23 U 1

BHD33 M U U 1

BHD35 M 38 R 2 38

BHD37 F 48 B 1 51

F 29 R 1

BHD 63 M 37 B 1

BHD 62 M 53 L 7

M 27 L 1

M 42 B 3

BHD43 F 38 L 1

M 37 R 4

F 74 L 1 74

M 32 L 3 55

F 18 R 2

BHD57 M 31 L 1

F 33 R 5

Age at first episode of pneumothorax; U: Available records did not state exact age/clinical characteristics. R: right lung, L: left lung, B: Bilateral# Multiple cysts on CT1: Described by Johannesma et al 2009. The relative diagnosed with a clear cell renal tumour was not tested for the FLCN mutation.Renal cancer: Age at diagnosis of renal cancer in patients with a history of pneumothoraxSkin: FF: confirmed fibrofolliculoma, U: unknown

carrier had renal oncocytoma (BHD 57). Five mutation carriers died of metastatic renal cancer

(BHD 6, BHD 16, BHD 23, BHD 37, BHD 44, table 3). All were diagnosed with renal cancer after

symptoms had developed. The histological classification of the renal tumours according to the

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WHO criteria is shown in table 3.19 Most of the tumours showed cells with granular / floccular

eosinophilic cytoplasm, as can be seen in both clear cell carcinoma (formerly called the eosinophilic

variant, table 3) and chromophobe carcinoma. This eosinophilic cell variation is also often seen

in sporadic clear cell carcinoma. 19 20 Since most tumours had mainly eosinophilic cytoplasm, with

moderately sharp cell borders, a vague perinuclear halo and moderately enlarged nuclei, we

classified them as intermediate between clear cell and chromophobe carcinoma (CC/Cph) (table 3

and figure 3). One of the tumours showed sarcomatoid changes, which can develop in both clear

cell and chromophobe carcinoma (table 3, BHD16). One other tumour had papillary structures in

combination with clear cell changes (table 3, BHD 6). Two additional patients from FLCN positive

families but with unknown mutation carrier status (BHD1, and BHD42) were diagnosed with renal

cancer. These two tumours also showed mixed chromophobe/clear cell histology. Two of the

14 renal tumours (BHD 1 and BHD 32, table 3) were detected by surveillance. For one of these

patients renal cancer was detected on the first ultrasound performed after the diagnosis BHD was

made (BHD 32, table 3). For the other patient renal cancer was detected by ultrasound four years

after the preceding normal ultrasound / MRI (BHD1, table 3); this latter patient did not undergo

standard yearly surveillance after the initial imaging.

table 5. Characteristics of BHD probands without an identified FLCN mutation.

BHd no. sex & age of proband clinical characteristics family data Remarks

BHD2 M 46 yrs Multiple fibrofolliculomas

on the face, neck and

trunk, starting at age

33 yrs

Colorectal cancer

in mother

Three unclassified variants

in FLCN:

exon 1 deletion

&polymorphisms

(IVS8+36G>A and

IVS9+6C>T)#


on thorax since childhood

Unclassified variant in FLCN:

IVS8+36G>A

BHD9 M 34 yrs More than 100 skin lesions

on the face, neck and

trunk, fibrofolliculomas.

Ulcerative colitis,

sacroiliitis

BHD25 F 70 yrs Multiple fibrofolliculomas

at age 66 yrs

Nephew had

pneumothorax

and died due to

renal cancer

Unclassified variant in FLCN:

exon 1 deletion


since age 40 yrs

# In BHD 2 an exon 1 deletion was detected in addition to two intronic FLCN variants, IVS9+6 C>T, and IVS8+36G>A. Variant IVS9+6 C>T was previously reported in a patient with suspected BHD who had multiple renal tumours and bilateral squamous papilloma of the eyelids 23. Both variants however, have since been reported to be rare polymorphisms (https://grenada.lumc.nl/LOVD2/shared1/home.php?select_db=FLCN).

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cutaneous manifestations

Most FLCN mutation carriers (91/115, 79%), underwent dermatological evaluation (68 of which were

evaluated by dermatologists at our center). Nineteen (21%) cases, aged 23-72 years, had no cutaneous

abnormalities; 14 (73%) of the patients without cutaneous manifestations were over age 40. Notably,

one patient who presented with metastatic renal cancer at age 51 (BHD 23) had no cutaneous lesions.

The youngest mutation carrier with histologically confirmed fibrofolliculomas was 25 years old (BHD

23). In five families without an identifiable FLCN mutation (BHD 2, 5, 9, 25 and 28) the clinical diagnosis

BHD was based on histologically confirmed multiple fibrofolliculomas; in one of these kindreds both

pneumothorax and renal cancer occurred (BHD 25, table 5). In seven families without FLCN mutations

the skin lesions consisted of multiple discoid fibromas of childhood onset (families 11, 17, 20, 24, 38, 45

and 50). In two of these families (24 and 45) a genealogical study showed common ancestry. No renal

or pulmonary signs were present in these seven families, except for one case with pneumothorax. For

two of these families the FLCN-locus was excluded by linkage analysis.21

Pulmonary manifestations

Among the 115 FLCN mutation carriers, 28 (24%)had a history of pneumothorax, recurrent in eight

patients (table 4). Four out of 28 patients with a previous pneumothorax were confirmed (former)

smokers. The medical records of the other patients did not state a history of smoking. The mean age

figure 3. Illustration of the histological pictures of renal cell carcinomas in our series. A: The most common pattern

found, classified as clear cell/chromophobe. The tumour cells show eosinophilic cytoplasm, moderate nuclear

pleiomorphism, vague perinuclear halos, no explicit cell borders and no vascular prominence. B: Classical picture of

clear cell carcinoma, as can be seen in many of the tumours in our series, but mostly only in part of the tumour cells C:

A clear cell/chromophobe renal cell carcinoma with partially clear cytoplasm, more prominent cell borders, no thick

walled vessels D: Renal cell carcinoma with sarcomatoid changes (in the right part of the picture).

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of the first pneumothorax was 36 years (range 18-74 years). We did not systematically subject mutation

carriers to CT-scanning of the lungs. For 12 FLCN mutation carriers the report of a CT-scan of the

thorax was available. Scans were performed either to confirm suspected BHD or because of pulmonary

complications of BHD. In five of these patients (aged 25-46 years), multiple cysts were reported in one or

both lungs. Of these, two had a history of recurrent or bilateral pneumothorax before the age of 30 years.

In seven FLCN mutation carriers no pulmonary cysts were detected (age 23-71, four were over age 40).

cumulative renal cancer and pneumothorax risk

The estimated renal cancer penetrance based on assessment of FLCN mutation carriers only was

0.20 at age 70 (red dashed line, figure 1a). In contrast, when considering both tested and untested

relatives, the estimated renal cancer penetrance at age 70 was 0.16 (continuous line, figure 1a and

figure 1B). The estimated penetrance for the first episode of spontaneous pneumothorax was 29%

(95% minimal confidence interval: 8-49%, figure 1c) at 70 years, again considering information on

both mutation carriers and their untested relatives.

d i s c u s s i o n

An important aim of this study was the estimation of renal cancer and pneumothorax penetrance

in Birt-Hogg-Dubé syndrome. By incorporating data on relatives who did not undergo DNA testing

we found an estimated penetrance for renal cancer of 16% and a penetrance for pneumothorax of

29% at the age of 70 years. The wide range of prevalence for renal cancer among cohorts of BHD

reported in literature is 6.5-34%. The renal cancer risk we found of around 16% is at age 70 years is

important for counselling of FLCN mutation carriers and their families ascertained in cancer family

clinics. Future studies with larger patient groups and comparison between cohorts investigated in

different populations may lead to further specification of the renal cancer risk.

The clinical presentation, histological pattern and biological behaviour of renal cancer in FLCN

mutation carriers are important for several reasons. First, a pathognomonic histological pattern

would be helpful for early diagnosis. Previously, The European BHD Consortium proposed clinical

diagnostic criteria for BHD which included early-onset, bilateral and multifocal renal tumours and

a mixed chromophobe and oncocytic histological pattern. In addition, FLCN mutation analysis

should be considered for patients who have familial cystic lung disease, familial pneumothorax,

familial renal cancer, or any combination of spontaneous pneumothorax and kidney cancer.

Notably, among 14 BHD patients in the current study who developed renal cancer, clinical signs of

hereditary disease (age at onset <50 years and / or multifocal / bilateral tumours) were present in

only seven cases. In addition, the histological picture of BHD- associated renal cancer in this cohort

was not typical for this syndrome. Ten out of 14 renal tumours revised in this series were difficult to

classify. None of them showed classical features of chromophobe renal cell carcinoma. Instead they

mainly exhibited characteristics of both eosinophilic variants of clear cell cancer and chromophobe

carcinoma. One of the tumours was a hybrid form of clear cell and papillary carcinoma and one

showed sarcomatoid changes. These histological patterns can also be found in sporadic RCC.

Therefore, late-onset unilateral, unifocal clear cell renal cancer also does not exclude BHD.

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Furthermore, although BHD-associated renal tumours were reported to metastasize rarely, five

of the 14 patients with renal cancer in our cohort developed metastatic disease underlining the

importance of early detection of these renal tumours by surveillance. In recently diagnosed families

not included in this report, we observed two BHD patients with renal cancer at the ages of 30 and in

the early twenties respectively, underlining that surveillance for renal cancer should be offered to

BHD patients from early age onward.

Of the 115 FLCN mutation carriers 28 had a history of pneumothorax, frequently recurrent and

bilateral. The mean ages at which pneumothorax and renal cancer occurred in our cohort were 36

years and 49 years, respectively. Among the 14 renal cancer patients described five had a history of

pneumothorax (table 3, 4), preceding renal cancer by several years in three patients (BHD 32, 37,

43, table 4). Although an increased risk for renal cancer in BHD families with a positive history for

pneumothorax has not been observed13, bilateral, recurrent or familial pneumothorax may serve as

an early indicator of BHD syndrome.22

Since, in 1975, Hornstein and Knickenberg23 described the combination of skin fibrofolliculomas

and colorectal polyps it is a matter of debate whether BHD is associated with an increased risk of

colorectal neoplasia. Whereas Zbar et al. found no significantly increased risk Khoo et al.32 proposed

that the risk might apply to specific subgroups only. Nahorski et al. (2010) found evidence that the

risk may be dependent on the FLCN genotype. Recently, in family BHD15, one of the FLCN mutation

carriers developed symptomatic colonic cancer at age 62 years. Late-onset colorectal cancer has

been diagnosed in several other FLCN mutation carriers from our BHD cohort not included in the

present study. Although colorectal cancer may well be coincidental in these cases the current data

call for further evaluation of the colorectal neoplasia risk in BHD.

Among 27 families with clinical BHD (multiple fibrofolliculomas) evaluated at our center 22 had

pathogenic FLCN mutations, i.e. a 81% yield for FLCN mutation analysis. Four patients referred for

suspected BHD declined genetic testing.

In two of the families (BHD 2 and 25 with clinical BHD but without a mutation in the coding region of

FLCN, a deletion of exon 1 was observed. Exon 1 is the first of three non coding exons. The exact size

and the effect of these deletions remains to be determined. Recently, after completion of our study,

intragenic duplications and deletions were reported in patients with BHD after completion.25 Using

a luciferase reporter assay, this study also showed that expression was strongly reduced when exon

1 was deleted. Analysis of the pathogenicity and co-segregation of the deletions detected in our

families are currently ongoing to prove pathogenicity. Therefore, these families were not included

in the calculation of the penetrance for renal cancer and pneumothorax although the deletions are

very likely to be pathogenic. In the current study, intragenic deletions were detected in two of the

BHD families (BHD 57, 62), underlining the importance of MLPA or CGH analysis in patients with a

clinical suspicion of BHD without an identifiable FLCN mutation.

Nine patients referred for possible BHD were diagnosed with other conditions. One patient had

probable tuberous sclerosis and one patient had pulmonary emphysema. Seven of the families were

diagnosed with familial multiple discoid fibromas (FMDF). In 1985, one of the authors described this

entity as a dominant condition distinct from BHD, showing childhood onset, preferential localisation

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of lesions on the ears, and distinct histology, which mimics the trichodiscomas in BHD.26 Thus far

no systemic complications have been noted for FMDF except for one patient with pneumothorax.

We have now excluded involvement of the FLCN locus in two FMDF kindreds using linkage analysis.

Currently, we use the renal cancer risk of around 16% by age 70 years for the counselling of patients

to emphasize the importance of surveillance for renal cancer. Since according to our histological

data, the renal cancers found in BHD were not evidently different from sporadic tumours, future

studies are aimed at the classification of BHD associated renal cancer in comparison with sporadic

disease are essential. Both the renal cancer risk and the pneumothorax risk (about 16% and 29% at

age 70 years, respectively) are based on a large set of data using a model incorporating available

data of family members not subjected to DNA testing.

Evaluation of larger patients groups and patients from other populations may yield other penetrance

figures in the future.

The European BHD Consortium proposed FLCN mutation testing in patients with early-onset renal

cancer (<50 years), in particular with multifocal or bilateral disease (or both) with chromophobe or

oncocytic histology and in familial renal cancer cases. Age at diagnosis of (the first) renal cancer in

our patient group was at or above 50 years of age in half of the patients and the tumour histology was

mixed in most patients but included clear cell elements in all cases. Therefore, it will be important to

study the yield of FLCN mutation testing using a wider set of criteria than proposed previously. The

histology and molecular pathology of renal tumours are associated with their biological behaviour

and reaction to systemic treatment of metastatic disease. Therefore, additional studies are needed

to monitor the success of surveillance for renal cancer, the results of surgical or other forms of local

treatment such as radiofrequency ablation and response to targeted therapies.

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c H a P t e R

are lung cysts in renal cell cancer (Rcc) patients an indication for flcn mutation analysis?

Paul C. Johannesma1, Arjan C. Houweling2, Fred H. Menko3, Irma van de Beek2, Rinze Reinhard4, Johan J.P. Gille2,

JanHein T.M. van Waesberghe4, Erik Thunnissen5, Theo M. Starink6, Pieter E. Postmus7, R Jeroen A van Moorselaar8


2 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 3 Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands

4 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands 5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

6 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands 7 Department of Thoracic Oncology, Clatterbridge Cancer Centre, Liverpool Heart & Chest

Hospital, University of Liverpool, Liverpool, United Kingdom 8 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

Fam Cancer 2016;15(2):297-300

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a B s t R ac t

Renal cell cancer (RCC) represents 2-3% of all cancers and is the most lethal of the urologic

malignancies, in a minority of cases caused by a genetic predisposition. Birt-Hogg-Dubé syndrome

(BHD) is one of the hereditary renal cancer syndromes. As the histological subtype and clinical

presentation in BHD are highly variable, this syndrome is easily missed. Lung cysts – mainly

under the carina - are reported to be present in over 90% of all BHD patients and might be an

important clue in differentiating between sporadic RCC and BHD associated RCC. We conducted

a retrospective study among patients diagnosed with sporadic RCC, wherein we retrospectively

scored for the presence of lung cysts on thoracic CT. We performed FLCN mutation analysis in 8 RCC

patients with at least one lung cysts under the carina. No mutations were identified. We compared

the radiological findings in the FLCN negative patients to those in 4 known BHD patients and found

multiple basal lung cysts were present significantly more frequent in FLCN mutation carriers and

may be an indication for BHD syndrome in apparent sporadic RCC patients.

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Renal cell carcinoma (RCC) represents 2-3% of all cancers and is the most lethal of the urologic

malignancies. There has been an annual increase about 2% in the incidence, with 88,400 new cases

of RCC worldwide in 2008.1 The incidence is approximately 5.8 per 100.000 and the mortality is

approximately 1.4 per 100.000.2 Currently over 50% of RCC are detected incidentally, as only a minority

(6-10%) of patients present with the classic triad; flank pain, gross haematuria and palpable abdominal

mass.3 These data underline the value of pre symptomatic identification and screening of patients

with an increased risk for RCC. A genetic predisposition for renal cancer is currently estimated to be

present in 3-5% of RCC patients, often showing recognisable features in addition to the increased

risk for renal cancer.4 A probably underdiagnosed autosomal dominant cancer disorder is the Birt-

Hogg-Dubé syndrome (BHD), clinically characterized by skin fibrofolliculomas, lung cysts in >90% of

cases, (recurrent) spontaneous pneumothorax (SP) and an increased lifetime risk for RCC between

16-35%. The gene associated with BHD encodes the protein folliculin (FLCN) which acts as a tumour

suppressor and interacts with mTOR and AMPK signalling pathways.5 The incidence is estimated about

1 in 200.000 people.6 So far, two large studies described the phenotypes of a large cohort of families

with BHD. The first study by Toro and colleagues showed 24 cases with a history of RCC. Of them 22/24

(91,7%) had multiple lung cysts on thoracic CT. In the remaining two patients, no thoracic CT scan

was available. The second study performed in our University centre, we found 17 cases of RCC among

a total of 115 FLCN mutation carriers. A thoracic CT was available in 13/17 (76.4%) which showed cysts in

one or both lungs in all cases. In the other four cases no thoracic CT was available.7 8

Therefore we hypothesized, that cysts under the main carina in patients with diagnosed with

“sporadic” RCC might be an important diagnostic clue in unmasking Birt-Hogg-Dubé syndrome.

We performed a pilot study to evaluate this hypothesis.


We retrospectively collected data on all patients (n=182), who had been diagnosed and treated for

RCC in the years 2003-2013. Patients diagnosed with RCC in our center were included when they

were over the age of 18 years at the time of diagnosis. Exclusion criteria were metastasis in the

kidney, no available thoracic CT or patients already known with a proven pathogenic FLCN mutation.

Furthermore, deceased patients were excluded (figure 1). All thoracic CT’s, made in the period

2003-2013 were collected and scored by one radiologist for the presence of one or more lung cysts,

below the level of the carina. Furthermore we collected the clinical data on familial occurrence on

SP, RCC and the history of SP. Clinical information was not available to the radiologist at the time of

scoring. We compared the radiological data to that of 4 FLCN mutation carriers diagnosed with RCC

and tested for potential significant differences in the number and size of lung cysts.

R e s u lt s

Patient data:

Proven FLCN mutation carriers (N=4)

In our BHD cohort of 250 FLCN mutation carriers, 4 patients were diagnosed with BHD after the

diagnosis of RCC and this enabled screening of their relatives. All 4 index patients had at diagnosis

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one or more symptoms of the classic triad; flank pain, gross haematuria and/or a palpable

abdominal mass at the time of diagnosis. All 4 patients had one or more RCC’s on the abdominal

CT. The mean age at time of diagnosis was 45.4 years of age (31-63 years). One patient had bilateral

RCC and one patient had two renal tumours in one kidney. Histopathology showed RCC with clear

cell and chromophobe elements in three patients and chromophobe elements only in one patient.

A thoracic CT was performed in all four patients that showed between 1 and 51 lung cysts in the basal

parts of the lung. Recurrent episodes of pneumothorax (3 episodes) occurred in one patient. Three

patients had multiple fibrofolliculomas in the face and upper neck. The family history for SP was

positive in three patients and was positive in two patients for RCC in two patients (table 1).

FLCN negative RCC patients (N=8)

For the evaluation for the potential presence of BHD in a “sporadic” RCC cohort, we included 182

patients with sporadic RCC in the medical history. Between 2003 and 2013 a total of 112 patients

underwent one or more thoracic CT’s, in the remaining 70 patients only a chest X-ray was performed.

Eleven patients met our inclusion criteria. Of these eight patients gave informed consent for a one

time visit at our outpatient clinic (figure 1). The mean age was 64.9 years (53-73 years) at time of

diagnosis. On thoracic CT all eight patients had at least one cyst in the basal parts of the lung. Four

patients had one cyst in the basal parts of the lung, three patients had two cysts in the basal parts of

the lung and one patient had 5 cysts in the basal parts of the lung respectively. None of the patients

had bilateral or multifocal RCC. The histopathology was clear cell in six patients, chromophobe in

one patient and sarcomatoid in one patient. The patient with 5 lung cysts had a history of three

figure 1. Study in- / exclusion flowchart.

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tab

le 1

. Cha

ract

eris

tics

of 4

RC

C p

atie

nts

wit

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atho

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CN

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atio

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nic

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episodes of SP; the other 7 patients had never experienced a SP. The familial history for SP and

RCC was negative in all eight patients. FLCN mutation analysis was performed; in none of the eight

patients a pathogenic FLCN mutation was found (table 1).

d i s c u s s i o n

To date, ten hereditary renal cancer syndromes have been defined, accounting for 3-5% of all RCCs.4

Based on the reported increased presence of multiple lung cysts under the carina in BHD patients

we performed FLCN mutation analysis in a pilot study setting among 8 patients with a history of

RCC and one or more lung cysts in the basal parts of the lung on thoracic CT. Although the history

of smoking was not available in all patients, it was possible to distinguish between smoking related

bullae in the apical parts of the lung and lung cysts on thoracic CT, as described by Fabre et al.9

We found no pathogenic FLCN mutations in this group. Although no pathogenic FLCN mutation

is found in this group, BHD is not excluded as only in 81-84% of clinical BHD cases a pathogenic

germline FLCN mutation is found.10 We compared the analysed the radiological findings to those

in 4 proven FLCN mutation carriers with renal cancer. We found that multiple basal lung cysts were

present significantly more frequent in FLCN mutation carriers and may be an indication for further

evaluation of BHD syndrome in apparently sporadic RCC patients. However, since solitary cysts were

found in both groups, the absence of multiple cysts does not appear to be a specific marker for the

absence of BHD, as one BHD patient had only one cyst on thoracic CT. We therefore advise that in

all RCC patients at least a concise family history is taken for the presence of RCC or SP and the skin

is evaluated for the presence of fibrofolliculomas. In the presence of a positive family history (SP or

RCC) or multiple basal lung cysts further investigation of BHD is indicated (e.g. by dermatological

evaluation or by DNA testing). The difficulty in unmasking BHD patients in apparently sporadic RCC

patients is illustrated by the negative family history for pneumothorax and RCC in the two FLCN

mutation carriers. Based on our results, we conclude that lung cysts in RCC patients can be an

indicator for underlying Birt-Hogg-Dubé syndrome, although the absence of presence of solitary

lung cysts doesn’t equate to the diagnosis of BHD. Further evaluation in larger RCC cohorts is

required to confirm our findings.

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R e f e R e n c e s1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C,

Parkin DM. Estimates of worldwide burden of

cancer in 2008L:GLOBOCAN 2008. Int J Cancer

2010;127:2893-917.

2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D.

Global cancer statistics. CA Cancer J Clin. 2011;61:69-90.

3. Lee CT, Katz J, Fearn PA, Russo P. Mode of

presentation of renal cell carcinoma provides

prognosticinformation. UrolOncol 2002;7:135-40.

4. Verine J, Pluvinage A, Bousquet G, Lehmann-Che

J, de Bazelaire C, Soufir N, et al. Hereditary renal

cancer syndromes: An update of a systematic

review. EurUrology 2010; 58: 701-10.

5. Baba M, Hong S-B, Sharma N, Warren MB, Nickerson

ML, Iwamatsu A, et al. Folliculin encoded by the

BHD gene interacts with a binding protein, FNIP1,

and AMPK, and is involved in AMPK and mTOR

signaling. Proc Nat AcadSci USA 2006;103:1552–57.

6. Maffé A, Toschi B, Genuardi M. Birt-Hogg-Dubé

syndrome (BHDS). Atlat Genet Cytogenet Oncol

Haematol. 2014; 18(7):521-5.

7. Houweling AC, Gijezen LM, Jonker MA, van Doorn

MB, Oldenburg RA, van Spaendonck-Zwarts KY,

et al. Renal cancer and pneumothorax risk in Birt-

Hogg-Dubé syndrome; an analysis of 115 FLCN

mutation carriers from 35 BHD families. Br J Cancer

2011;105:1912-19.

8. Toro JR, Schmidt LS, Nickerson ML, et al. Germline



syndrome. Am J Hum Genet. 2005;76:1023-33.

9. Fabre A, Borie R, Debray MP, Crestani B, Danel C.

Distinguishing the histological and radiological

features of cystic lung disease in Birt-Hogg-

Dubé syndrome from those of tobacco-related

spontaneous pneumothorax. Histopathology

2014;65(5):741-9.

10. Menko FH, van Steensel MAM, Giraud S, Friis-

Hansen L, Richard S, Ungari S, Nodenskjöld M,

Hansen TvO, Solly J, Maher ER. Birt-Hogg-Dubé

syndrome: diagnosis and management. Lancet

Oncol 2009;10:1199-206.

c H a P t e RRenal imaging in 199 dutch patients

with Birt-Hogg-dubé syndrome: screening, compliance and outcome

Paul C. Johannesma1, Irma van de Beek2, Rinze Reinhard3 4, Edward M. Leter5, Rence Rozendaal6, Theo M. Starink7,

JanHein T.M. van Waesberghe3, Simon Horenblas8, Marianne A. Jonker9, Pieter E. Postmus10, Arjan C. Houweling2, R. Jeroen A. van Moorselaar11

1 Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

3 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands

4 Department of Radiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands 5 Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands

6 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands7 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands

8 Urologic Oncology and Department of Urology, The Netherlands Cancer Institute, Amsterdam, The Netherlands



11 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

Submitted

2 . 3

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a B s t R ac t

Background information

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations

in the FLCN gene characterized by skin lesions, pneumothorax and an increased risk of renal

cell cancer (RCC). Renal surveillance in BHD is recommended, but the optimal imaging method

and screening interval remain to be defined. Local treatment is based on the ‘3 cm rule’, which

recommends surgical intervention when the (largest) lesion exceeds 3 cm in diameter. The aim

of our study was to retrospectively evaluate the compliance with and outcomes of renal cancer

surveillance in patients diagnosed with BHD in two centers.

methods

Surveillance data of 199 patients diagnosed with BHD in two hospitals were collected. Data were

collected form medical files and a questionnaire. In addition, we evaluated renal imaging follow up

data and the medical records of 23 BHD patients with renal cell carcinoma (RCC).

Results

Initial screening for RCC was performed in 171/199 patients (86%) and follow up data were available

from 117/171 patients (68%). A total follow-up period of 499 patient years was available. Of the

patients that performed follow-up surveillance, 85% was investigated at least yearly and 96% at least

every two years. A medical history of RCC was present in 23 patients, 38 tumours were diagnosed

with a mean age of the first tumour of 51 years. In 21 tumours ultrasound (US) was performed. Eleven

tumours sized 7-27 mm were visible on MR or CT and not detected using US.

conclusions

Our data indicate that compliance to renal screening is relatively high and that US might be

a sensitive, cheap and widely available imaging modality for detecting clinically relevant renal

tumours in BHD patients, since no tumours exceeding 3 cm were missed with US. Follow up studies

in BHD patient cohorts are required to further determine the optimal screening method and interval

in BHD patients.

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Birt-Hogg-Dubé syndrome (BHD, OMIM #315150) is an autosomal dominant condition characterized

by fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal tumours, due to germline

mutations in the FLCN gene encoding folliculin.1 In the initially reported family skin lesions were

the only clinical manifestations.2 Subsequently, renal cell carcinoma (RCC) and spontaneous

pneumothorax were found to be part of the syndrome.3 4 5 The functions of folliculin have partly

been clarified and are likely to include a role in the mammalian target of rapamycin (mTOR) pathway

and in ciliary function.6 7 8 Around 3-5% or of all RCCs are estimated to have a hereditary cause due to

genetic predisposition.9 Hereditary RCCs differ from the far more common sporadic form in several

aspects. Hereditary tumours often present at an early age, are more often multifocal and / or bilateral

and may have a characteristic histology. In addition, they may be associated with recognizable

syndromic features besides renal cancer. In addition, the family history may be positive for RCC or

syndromic clinical features. In BHD the prevalence of RCC is estimated to be 16-34% with a mean

age at diagnosis of 50 years (range 20-60 years).10 11 12 The most commonly reported histological

subtypes of BHD associated renal tumours are hybrid oncocytic, chromophobe renal cell carcinoma

and oncocytoma. However, other subtypes may also occur.10 12 13 14 15 16 Given the high degree of

inter- and intra familiail variability of these features, it is likely that many cases of hereditary RCC

currently remain unrecognized. Renal surveillance in BHD patients has been recommended from

age 20, preferably by annual MRI (Magnetic Resonance Imaging). This is based on the high sensitivity

and the lack of radiation exposure.17 However, its availability and cost may be limitations in clinical

practice. The aim of our study is to retrospectively evaluate the compliance to, and the outcomes of

renal cancer surveillance in patients diagnosed with BHD in two Dutch centers.


The suspected diagnosis of BHD in successively referred patients was based on patient and pedigree

data and confirmed by FLCN mutation testing. The collection of family data and the methods of

mutation analysis have been outlined in previous publications.10 18 For the current study we collected

data of 199 patients diagnosed with BHD at the VU University Medical Center and The Netherlands

Cancer Institute. Our cohort includes both symptomatic index patients and healthy family members

identified by pre-symptomatic DNA testing. We retrieved screening data from the medical files and we

sent a questionnaire on performed surveillance in other medical institutions. Patients were included

until June 2014. The study was approved by the ethical committee of the VU University Medical Center.

We collected all available renal imaging follow up data and the medical records of 23 BHD patients

with renal cell carcinoma (RCC). For evaluation of compliance with surveillance, we assessed

initial screening and follow up surveillance data. Initial screening was defined as the first renal

imaging after a maximum period of one year after the clinical or genetic diagnosis of BHD. Patients

diagnosed with (symptomatic) RCC before the diagnosis of BHD were excluded, since their tumours

were not detected by screening. Patients were included in the analysis of follow-up if de patient met

the following criteria; The last screening from which data was available, was performed in 2013 or

2014, the questionnaire was returned and no screening was performed in 2013 or 2014 and the last

screening was performed within one and a half years before death.

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R e s u lt s

screening compliance

Initial screening was performed in 171 patients (86%) and follow-up data were available of 117 patients

(68%). figure 1 shows a flowchart of the population. Of the 27 patients excluded from analysis of follow-

up, 10 were diagnosed with RCC before they were diagnosed with BHD, 2 decided not to undergo

screening because of older age and 2 died within 1,5 years after the diagnosis of BHD. It was unclear

whether or not initial screening was performed in 13 patients. No follow-up data was available for 54/174

patients, of which 16 had a recent diagnosis and initial screening was performed less than 1,5 year ago.

The mean age of initial screening was 50 (median 51, range 20-83). The data of initial screening and

follow-up screening are shown in table 1. Initial screening consisted predominantly of ultrasound

(US) and MRI. The follow-up screening was predominantly performed by US. The total follow-up

time was 499 patient years (mean 4,2; median 4; range 1-9). Most of the missed screening moments

occurred in the first year of follow-up (12%). From the second to ninth year approximately 3-7% of

patients per year missed a screening moment.

figure 1 . Flow diagram of screening data in the study population.

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The majority of patients (99/117, 85%) was screened at least once a year, 112/117 patients (96%) were

screened at least once every two years.

detected Rccs

A total of 23 patients (13 male) had a medical history of RCC. The mean age at diagnosis of the

first RCC was 51 (range 24-77). Ten patients developed RCC before they were diagnosed with BHD.

Nine and five patients were diagnosed at initial screening and during follow-up respectively (one

patient was diagnosed both at initial screening and follow-up). Eight patients were the first person

in the family to be diagnosed with BHD. Six of them were already known with RCC at the time of

diagnosis of BHD. The other 15 patients were diagnosed with BHD by (pre-)symtpomatic DNA-

testing. Even though there are many missing data, the available data suggest that the majority of

RCCs are asymptomatic.

table 2 shows tumour and patient characteristics. In total, 38 tumours were detected in 23

patients. Of one tumour no data was available since the treatment was performed 34 years ago.

The histology of available tumours (n=25) was either chromophobe (32%), clearcell (32%), mixed

chromophobe/clearcell (12%), papillary (12%), hybrid oncocytic/chromophobe (4%), sarcomatoid

(4%) or unclassified (4%).

There was a wide variability between the detection method(s) of these tumours. US was performed

in 21 tumours. Ten tumours, sized 20 to 120 mm, were detected by US. Eleven tumours, sized 7 to

27 mm, were seen on MRI or CT and missed by US. Five patients died, of which four had metastic

disease at the time of diagnosis of RCC. All four were diagnosed with RCC before they were

diagnosed with BHD. No patients were diagnosed with metastasized RCC during follow-up after

the BHD diagnosis.

Local treatment of RCCs consisted of either total nephrectomy, partial nephrectomy, cryo-ablation

or radio frequent ablation. All RCCs, except one, were treated independent of size.

table 1 . Overview of initial and follow-up screening per year.

initial screening (%) year 1 year 2 year 3 year 4 year 5 year 6 year 7 year 8 year 9

Total 171 117 101 88 69 46 30 25 15 8

MRI + US 119 (69,6) 18 17 14 12 11 7 5 2 0

US 9 (5,3) 60 56 45 41 28 15 10 8 8

MRI 31(18,1) 3 5 7 2 0 2 3 1 0

Other¥ 18 (10,5) 4 1 3 1 1 0 1 1 0

Unknown technique£ 3 (1,8) 18 19 14 9 4 4 5 2 0

No screening (%) NA 14 (12,0) 3 (3,0) 5 (5,7) 4 (5,8) 2 (4,3) 2 (6,7) 1 (4,0) 1 (6,7) 0

¥ CT, CT and US or CT and MRI£ Self-reported screening with questionnaire in which technique was not mentioned. NA: not applicable

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table 2. Detected RCCs: tumour and patient characteristics.

gender

age at diagnosis

tumour size (mm)

moment of diagnosis

sympto-matic

detected with

missed with

last screening outcome

F 74 40 (PA)

60 (PA)

20 (PA)

11 (PA)

<BHD

<BHD

<BHD

<BHD

AS CT

CT

CT

CT

7 years FU

M 51 69 <BHD S CT Metastasis

at diagnosis,

died

M 56 120 <BHD S CT + US Metastasis

at diagnosis,

died

M 31 18 Initial AS MRI (+2nd US) US 3 years FU

F 60 50 FU U US Unknown 5 years FU

F 64 16

7

5 (6 months later)

Initial AS MRI

MRI

MRI

US

US

4 years FU

F 49 17 Initial AS MRI + CT NA

M 43 10

14

Initial AS MRI + CT US (6

months

before)

NA

M 76 15

20

22

Initial

Initial

Initial

AS CT

CT + US

CT

US

US

4 years FU

F 55 27 Initial AS MRI US (2

months

before)

3 years

M 28 110 <BHD U MRI + US Metastasis

at diagnosis,

died

F 62 14 FU U MRI US 1 year ago:

MRI+US

NA

M 33

38

39

90

13

40

Initial

FU

FU

MRI +US

MRI

MRI+US

US

1 year ago:

MRI+US

2 years since

last tumour

M 69 3 Initial U MRI 3 years FU:

tumour now

9 mm

M 35 22 FU U MRI + US 1 year ago:

MRI + US

NA

M 24 70 <BHD U MRI+US Metastasis

at diagnosis,

died

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d i s c u s s i o n

To gain insight in optimal screening regimens in rare genetic cancer predisposition syndromes,

it is crucial to study data of patient cohorts collected from specialized centers. We were able to

collect data from 199 Dutch patients diagnosed with BHD in two Dutch centers. The recommended

screening program in the Netherlands has changed over the years. Initial screening in BHD patients

diagnosed at the VU University Medical Center, has consisted of both MRI and US followed by annual

US Currently, initial screening consists of MRI only. The goal of initial screening by both MRI and US

was to gain more insight in the optimal screening technique, since there are no evidence based

guidelines for screening in BHD patients. The change to MRI only was based on expert opinion. As

expected, the data show that a majority of initial screening consisted of both MRI and US (69,9%)

and that a majority (54,3%) of follow-up screening consisted of US only.

In some patients annual imaging is performed in the academic hospital. However, the majority

of patients is screened in local or regional non-academic hospitals. This makes it difficult to be

up to date about the screening compliance and outcomes. Using questionnaires, we tried to

collect these data of as many patients as possible. The presented data show that at least 87% of

the BHD patients performed initial screening. Excluding patients with a diagnosis of RCC before

the diagnosis of BHD, and patients that died within 1,5 year after the diagnosis of BHD, this number

increased to 91% (171/187). The true percentage might be slightly lower or higher, since no data was

available from 13 patients.

table 2. Detected RCCs: tumour and patient characteristics.

gender

age at diagnosis

tumour size (mm)

moment of diagnosis

sympto-matic

detected with

missed with

last screening outcome

F 40 30 <BHD U MRI + US 10 years FU

M 69 55 (multifocal)

15-20 (multifocal)

<BHD AS CT Unknown

F 77 11 FU AS CT US 6 months

before: US

1 year FU

F 25 110 (PA) <BHD S CT + US 21 years FU

M 42 14

8

<BHD AS CT

CT

2 years FU

M 56 51 (PA)

23 (PA)

17 (PA)

<BHD AS CT

CT

CT

Died due to

metastasis

of other

malignancy

F 63 19 Initial S CT 2 years FU

Gender: F ; female, M; maleTumour size: reported as size on imaging when available. In case of both US and CT/MRI is size on CT or MRI reported. In case of both CT and MRI, the largest described size is reported. PA; size measured at pathology, no imaging size available. Moment of diagnosis: < BHD; Diagnosis of RCC before the diagnosis of BHD, FU; tumour diagnosed at follow-up, Initial; tumour diagnosed at initial screening,Outcome: NA; not applicable

table 2. (continued)

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Our cohort of patients with BHD participating in the screening program, is highly compliant to the

recommended screening regimen (85%). The fact that the most missed screening moments were

in the first year after initial screening, might be discussed during genetic counseling. A possible

explanation is that patients are less aware of their own responsibility to organize the follow-up

screening via their general practitioner. The number of patients declining screening and their

reasons to do so remain unclear. The screening compliance is comparable to that described in

patients with Lynch syndrome (87%).19

It has been established that that most renal masses in the general population grow slowly. 20 21 22 23

In several studies the natural growth of presumed renal cancer in general (non-BHD) patients who

were not surgical candidates due to comorbidity was examined. The annual growth rate of the

renal masses was measured during active surveillance (AS) and was between 0.25 – 0.34 cm / year,

with a median follow-up of 34-36 months.20 22 24 One of these studies reported tumour size to be

a predictor of tumour growth rate, with lower growth rates of renal masses <2.45 cm compared

to masses >2.45 cm.20 However Chawla et al. concluded that renal tumour size at presentation

does not appear to be a reliable predictor for growth rate and to their opinion serial radiographic

data alone are insufficient to predict the natural history of renal masses.22 It is remarkable that

during active surveillance some benign lesions (e.g., AML and oncocytoma) increased in size,

while some biopsy-proven RCC’s did not grow and, in some cases even decreased in size.19 The

biological behaviour of renal cell cancer in several hereditary tumour syndromes is, in most

cases, reported to be indolent and metastases rarely occur at smaller tumour sizes. Taking into

account the relatively high frequency of multiple or bilateral tumours, parenchymal sparing

surgical treatment is important in patients with a hereditary predisposition for renal cancer.25 26

The aims of the treatment are local tumour control, preservation of renal function and prevention

of metastatic disease. Nephron sparing treatment is often possible, as tumours in patients who

undergo regular renal screening are usually discovered at a small size. Currently, the ‘3 cm rule’,

which recommends surgical intervention when the (largest) lesion exceeds 3 cm in diameter, is

often applied to patients with Von Hippel Lindau disease, hereditary papillary renal cell cancer

and BHD.9 25 27 28 In a study among patients adhering to this ‘3 cm rule’, no metastatic disease was

reported with more than 10 years follow-up in 49 patients with hereditary renal cell cancer. It must

be noted that in this study only 1 patient with BHD was included.26 In our group of 23 BHD patients

diagnosed with RCC, all but one patient underwent local treatment. In one patient, a tumour of

3 mm was detected. Three years later, this tumour was sized 9 mm and imaging was performed

frequently. The treatment of tumours smaller than 3 cm was probably at the request of the patient

due to anxiety for possible development of metastasis. Furthermore, the increasing availability

of less invasive treatment options as radiofrequent ablation or cryotherapy might play a role in

these decisions. When screening BHD patients, it is crucial to at least enable following the often

recommended 3 cm rule. Therefore, no tumours larger than 3 cm should be missed by the applied

screening technique and the screening interval must ensure that no tumours larger than 3 cm

develop during the screening interval.

The main techniques to be considered for imaging of renal tumours are CT, MRI and ultrasound.

These imaging modalities each have their strengths and weaknesses. CT is a fast and robust

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technique to display the kidneys three-dimensionally and with great anatomic detail. CT is the

gold standard for the diagnosis and staging of renal cell carcinoma29. Despite these qualities,

the use of CT for annual surveillance would lead to unacceptably high cumulative radiation dose

as patients will need many CT scans during their lifetime.30 MRI is a technique that provides

excellent soft tissue contrast without the use of radiation. It is powerful for the detection and

characterization of focal renal lesions, various RCC subtypes can be differentiated and patency of

blood vessels can be shown without the use of intravenous contrast. Drawbacks of MRI are long

examination times (30-45 minutes), variations in quality and scan protocol, limited availability

and high cost.28 In some patients MRI cannot be performed due to contra-indications like

claustrophobia, obesity, or the presence of metallic implants. US has the advantages that it is

widely available, fast, cheap and without use of ionizing radiation. However small renal lesions

can be missed due to limited spatial resolution of US depending on intrarenal location, isoechoic

aspect of a lesion, large patient size or obscuring bowel gas25 28 31. Furthermore US depends on

operator experience. Renal imaging with 1-3 year intervals has been proposed for BHD patients

without renal lesions on initial imaging.17 24 25.

In our cohort, 11/21 tumours were missed by US. All missed tumours were smaller than 3 cm (largest

27 mm). Five of our patients were imaged within 1 year before diagnosis of RCC. One tumour larger

than 3 cm (4 cm) was diagnosed within 1 year after a previous RCC in the same kidney, which was

treated with a partial nephrectomy. One year prior, this tumour was not seen on both MRI and US.

No metastatic disease occurred in this patient during a follow up period of 2 years.

Based on the findings in our BHD cohort, US screening may be an adequate strategy since no relevant

tumours were missed on US. However, a 40 mm tumour was found one year after imaging with MRI.

This suggests that the screening interval might be too long. However, given the often indolent

nature of BHD kidney cancer and the small number of patients, this remains to be further clarified.

In addition, the total number of RCCs was relatively small and therefore, further evaluation in larger

patient groups is required. Consistent with current literature, no metastatic disease occurred in

patients with RCC <3 cm.

c o n c l u s i o n s

Our main findings are that the vast majority of patients undergo initial screening and that 85% of

patients in the screening program are compliant with yearly renal imaging. In our patient cohort

no tumours larger than 3 cm visible on MRI or CT were missed with US and metastatic disease only

occurred in tumours larger than 3 cm diagnosed at initial screening. Our findings are in line with the

conclusions of Jamis-Dow et al that US is a sensitive method for detecting renal lesions larger than

3 cm in BHD patients.31 It might be important to give more attention to the recommended screening

program, especially the first follow-up moment after initial screening. Further studies are needed

to determine whether our results for BHD patients in this study can be reproduced, preferably in

a relatively large group of patients with screening by both MRI and ultrasound.

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R e f e R e n c e s1. Nickerson ML, Warren MB, Toro JR, et al. Mutations




Cancer Cell 2002;2:157-64.

2. Birt AR, Hogg GR, Dubé WJ. Hereditary multiple


acrochordons. Arch Dermatol. 1977;113:1674-7.

3. Chung JY, Ramos –Caro FA, Beers B, et al. Multiple

lipomas, angiolipomas, and parathyroid adenomas


Dermatol 1996;35:365-7.

4. Roth JS, Rabinowitz AD, Benson M, et al. Bilateral

renal cell carcinoma in the Birt-Hogg-Dubé

syndrome. J Am Acad Dermatol. 1993;29:1055-6.




Respir Crit Care Med 2007;175:1044-53.

6. Hasumi Y, Baba M, Ajima R, et al. Homozygous

loss of BHD causes early embryonic lethality and

kidney tumor development with activation of

mTORC1 and mTORC2. Proc Natl Acad Sci U.S.A.

2009;106:18722-7.

7. Hasumi Y, Baba M, Hasumi H, et al. Folliculin (Flcn)

inactivation leads to murine cardiac hypertrophy

through mTORC1 deregulation. Hum Mol Genet

2014;23:5706-14.

8. Luijten MN, Basten SG, Claessens T, et al. Birt-

Hogg-Dubé syndrome is a novel ciliopathy. Hum

Mol Genet 2013;22:4383-97.

9. Barrisford GW, Singer EA, Rosner IL, et al. Familial

renal cancer: molecular genetics and surgical

management. Int J Surg Oncol. 2011;2011:658767.





11. Khoo SK, Giraud S, Kahnoski K, et al. Clinical and


Med Genet 2002;39:906-12.





Genet 2008;45:321-31.

13. Benusiglio PR, Giraud S, Deveaux S, et al. Renal

cell tumour characteristics in patients with the

Birt-Hogg-Dubé cancer susceptibility syndrome:

a retrospective, multicentre study. Orphanet J

Rare Dis 2014;9:163.

14. Linehan WM, Pinto PA, Bratslavsky G, et al. Hereditary

kidney cancer: unique opportunity for disease-

based therapy. Cancer 2009;115(10Suppl):2252-61.

15. Murakami T, Sano F, Huang Y, et al. Identification

and characterization of Birt-Hogg-Dubé associated

renal carcinoma. J Pathol 2007;211:524-31.

16. Woodwared ER, Ricketts C, Killick P, et al. Familial



and mutation analysis of FLCN. Clin Cancer Res.

2008;14:5925-30.

17. Menko FH, van Steensel MA, Giraud S, et al. Birt-

Hogg-Dubé syndrome: diagnosis and treatment.

Lancet Oncol 2009;10:1099-206.

18. Leter EM, Koopmans AK, Gille JJ, et al. Birt-Hogg-



19. Newton K, Green K, Lalloo F, et al. Colonoscopy

screening compliance and outcomes in patients

with Lynch syndrome. Colorectal Dis 2015;17:38-46.

20. Jewett MAS, Mattar K, Basiuk J, et al. Active

surveillance of small renal masses: Progression

patterns of early stage kidney cancer. Eur Urol

2011;60:39-44.

21. Mason RJ, Abdolell M, Trottier G, et al. Growth

kinetics of renal masses: Analysis of a prospective

cohort of patients undergoing active surveillance.

European Urology 2011;59:863-7.

22. Abou Youssif T, Kassouf W, Steinberg J, et al.

Active surveillance for selected patients with renal

masses: updated results with long-term follow-up.

Cancer 2007;110:1010-4.

23. Chawla SN, Crispen PL, Hanlon AL, et al. The natural

history of observed enhancing renal masses: Meta-

analysis and review of the world literature. The

Journal of Urology 2006;175:425-31.

24. Rosales JC, Haramis G, Moreno J, et al. Active

surveillance for renal curtail neoplasms. J Urol

2010;183:1698-702.

25. Choyke PL, Glenn GM, McClellan WM, et al.

Hereditary renal cancers. Radiology 2003;226:33-46.

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26. Stamatakis L. Metwalli AR, Middelton LA, et al.


kidney cancer. Fam Cancer 2013;12:397-402.

27. Herring JC, Enquist EG, Chernoff A, et al.

Parenchymal sparing surgery in patients

with hereditary renal cell carcinoma: 10-year

experience. J Urol 2001;165:777-81.

28. Walther MM, Choyke PL, Weiss G, et al. Parenchymal

sparing surgery in patients with hereditary renal

cell carcinoma. J Urol 1995;153:913-6.

29. Sacco E, Pinto F, Totaro A, et al. Imaging of

renal cell carcinoma: state of the art and recent

advances. Urol Int 2011;86:125-39.

30. Sodickson A, Baeyens PF, Andriole KP, et al.

Recurrent CT, cumulative radiation exposure, and

associated radiation-induced cancer risks from CT

of adults. Radiology 2009;251:175-84.

31. Jamis-Dow CA, Choyke PL, Jennings SB, et al. Small

(< or = 3-cm) renal masses: detection with CT versus US

and pathologic correlation. Radiology 1996;198:785-8.

p a R t 3

Relevant case series and case Reports

c H a P t e R

in-flight pneumothorax: diagnosis will be missed due to symptom delay

Pieter E. Postmus1, Paul C. Johannesma2, Fred H. Menko3, Marinus A. Paul4



3 Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands 4 Department of Thoracic Surgery, VU University Medical Center, Amsterdam,

The Netherlands

Am J Respir Crit Care Med. 2014;190(6):704-5

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Boyle’s law describes the inverse relationship between pressure and volume for gas in a closed

system at constant temperature. Based on this mechanism, an air-filled lung cyst will increase in size

if brought in an environment with a lower atmospheric pressure. Patients with cystic lung disease

might therefore be at risk of developing pneumothorax during air travel.

Many persons have cystic changes in the lung, but the number of reported in-flight pneumothorax

is very low.1 This apparent discrepancy seems at first sight strange, but is in fact what one might

expect. Any cystic airspace in the lung will not expand in size if connected to the bronchial tree,

because there is no possibility to develop intracystic overpressure. However, if there is no connection

between an air-filled cyst and the bronchial tree, the cyst will increase in size with 25-30% which may

lead to rupture of the cyst.2 This is not sufficient for developing pneumothorax, the visceral pleura

needs to rupture as well. This may occur if a cyst is adjacent to the visceral pleura. But even then it

is very unlikely that this will result in symptomatic pneumothorax within a few hours since there will

be no or very little transport of air into the pleural cavity.

We hypothesize that a pneumothorax developed during air travel will become symptomatic hours,

or even more likely days, after air travel. We here report a patient with Birt-Hogg-Dubé syndrome

who developed pneumothorax following air travel and was manifested later.

c a s e R e p o Rt

A 38-year-old male was diagnosed to have BHD after his uncle had been diagnosed with facial

skin fibrofolliculomas. Three family members were found to carry the same pathogenic FLCN

germline mutation, his father and brother are asymptomatic, but the patient’s sister had a history

of recurrent bilateral pneumothorax. The patient works for a commercial international firm and

travels quite often, the mean number of flights per year during the last 6 years was 12. His first

pneumothorax, at the age of 34, was treated by drainage through a chest tube. Within 2 months he

developed a contralateral pneumothorax. Bilateral apical pleurectomy was performed. Four years

later he flew transatlantic. He had no complaints during or after this flight. He visited, while still

without complaints day after arrival one of the world’s highest towers and used the speed lift to 410

m in around 45 seconds. After descent to sea level he noticed acute serious shortness of breath.

Chest X-ray demonstrated a left-sided pneumothorax with considerable collapse in the basal part.

He flew back to Europe with a Heimlich valve connected to a chest tube and was referred for

additional treatment.

As the last event occurred during a rapid change in atmospheric pressure (speed lift) and shortly

after a transatlantic flight, he checked at home his old diaries for his flight pattern around the

earlier episodes of pneumothorax. The first pneumothorax was diagnosed on December 17, 2010,

complaints starting the day before diagnosis, he flew on the 8th and 12th that month within Europe.

At discharge from the hospital there was radiological proof of complete resolution. His contralateral

pneumothorax was diagnosed on January 31 after some shortness of breath starting the 30st, he

flew transatlantic on the 17th and 21st the same month.

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d i s c u s s i o n

BHD syndrome is autosomal dominant inherited, caused by germline mutations in the FLCN

(folliculin) gene and is clinically characterized by facial fibrofolliculomas, pulmonary cysts, recurrent

pneumothorax and an increased risk for renal cell cancer.3 All BHD patients with (recurrent)

pneumothorax have pulmonary cysts, especially in the lower lobes. The cysts are located both

in the parenchyma and subpleural area. We analysed the lungs by CT of 18 other BHD patients

with a history of at least one episode of pneumothorax, all patients had subpleural cysts and

50% of all cysts were located subpleural.4 Very small subpleural cysts might have been missed

on a standard CT.5 Several studies on the role of atmospheric pressure changes showed that the

interval after the trigger, assumed to be the change in atmospheric pressure, resulted with some

delay into clustering of admitted patients with a spontaneous pneumothorax.6 For instance Scott

and colleagues reported a delay up to 4 days after a significant change in atmospheric pressure.7

Considering the pressure changes during the subsequent flights as potential trigger for initiating

rupture of a subpleural cyst, this implies that the interval between air travel and the diagnosis

is respectively 5 and 9 days (1st episode), respectively 10 and 14 days (2nd episode) and 2 days or

immediately after use of a speed lift (3nd episode of pneumothorax). The latter gives a much faster

change (68 hPa/min) in outside pressure than during ascent (<13.3 hPa/min) in commercial air travel8

and might cause a rupture of a cyst by itself or, more likely based on start of patient’s complaints,

aggravate a small pneumothorax developed during the flight 2 days earlier. Hoshika and colleagues

reported no relationship between development of spontaneous pneumothorax and 2142 flights

among 48 BHD patients.9 Three patients reported tightness of the chest. Unfortunately there were

no details on the length of period after flying and the moment of becoming symptomatic of the

pneumothoraces, this makes it uncertain whether there might have been a possible delay after the

pressure change as in our patient. Striking is the reduction in number of flights in this series after

the patients became aware of the underlying cause. Although the reported patient flew more than

one week after complete resolution of his first pneumothorax, the character of the disease makes it

questionable whether current BTS guidelines on pneumothorax and flying are applicable for these

patients.10 One of the hallmarks of pneumothorax in BHD patients is the high recurrence rate, 59% in

53 cases.11 Rupture of a subpleural cyst is the likely cause of the pneumothorax in BHD patients. These

cysts are found throughout the lungs with the majority in the lower halves of the lungs, this implies

that treatment to prevent recurrence should have as aim pleurodesis of the whole pleura visceralis,

not only in the apical region. This can be done by extensive pleurectomy and/or talc pleurodesis.

From this perspective the initially performed bilateral apical pleurectomy after the pneumothorax

in the reported patient is inadequate as was also found in our retrospective series.11 Kumasaka et

al analyzed resected lung specimens of 50 BHD patients.12 Out of 229 cysts that were found; 50%

were located in the subpleural area and less than 5% abut on bronchioles. Subpleural cyst are by

definition far from the larger airways, so if a cyst would be connected to the bronchial tree the

size of connection to the airways is very small, resulting in small volumes of air transported into

the pleural cavity. Therefore it will probably take a long time before troublesome symptoms are

present. This concept of delayed symptoms after the actual rupture of a cyst is probably as well

relevant for other diseases with cystic changes in the lung, such as lymphangioleiomyomatosis or

Langerhans cell histiocytosis assuming there is no (large) connection between airways and cyst.

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Furthermore spontaneous resolution of a small pneumothorax after rupture of a subpleural cyst

may occur if there is no active transport of gas into the pleural cavity. Evidence that this occurs in

vivo comes from the presence of inflammation at the pleural side of the majority of the cysts13 and

the demonstration of pleural inflammation in patients with a pneumothorax.14

In a survey among 190 patients with BHD we found that of the group with a previous pneumothorax,

twelve other patients had a total of 13 episodes of pneumothorax within 31 days after a continental

or intercontinental flight.15 The interval between flying and diagnosis of pneumothorax was < 10

days in 6 cases, between 10 and 20 days in 4 and between 20 and 30 days in 3. This supports our

hypothesis that the delay of becoming symptomatic should be taken into account if a relation

between atmospheric pressure changes and pneumothorax is studied. Consequently BHD patients

who have minimal chest symptoms after the first flight should be checked for pneumothorax

before the return flight, because presence of pneumothorax, even if it is small, is a known risk

factor for flying .16

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R e f e R e n c e s1. Hu X, Cowl CT, Baqir M, Ryu JH. Air travel and

pneumothorax. Chest 2014; 145: 688-694.

2. BTS statement. Managing passengers with respiratory

disease planning air travel: British Thoracic Society

recommendations. Thorax 2002; 57: 289-304.

3. Menko FH, van Steensel MA, Giraud S, Friis-Hansen

L, Richard S, Ungari S, Nordenskjöld M, Hansen

TV, Solly J, Maher ER; European BHD Consortium.

Birt-Hogg-Dubé syndrome: diagnosis and

management. Lancet Oncol 2009; 10: 1199-1206.


R, Gille JJP, van Moorselaar RJA, Houweling

AC, Starink ThM, Menko FH, Postmus PE. Birt-

Hogg-Dubé syndrome patients with and without

pneumothorax: findings on chest CT. Am J Resp

Crit Care Med ajrccm-conference 2014.189.1_

Meeting Abstracts.A6416.

5. Onuki T, Goto Y, Kuramochi M, Inagaki M, Bhunchet

E, Suzuki K, Tanaka R, Furuya M. Radiologically


syndrome. Ann Thorac Surg 2014; 97: 682-685.

6. Haga T, Kurihara M, Kataoka H, Ebana H. Influence

of wheather conditions on the onset of primary



Cardiovasc Surg 2013; 19: 212-215.

7. Scott GC, Berger R, McKean HE. The role of

atmospheric pressure variation in the development

of spontaneous pneumothoraces. Am Rev Resp Dis

1989; 139: 659-662.

8. Araki K, Okada Y, Kono Y, To M, To Y. Pneumothorax

recurrence related to high-speed lift. Am J Med

2014. (in press)

9. Hoshika Y, Kataoka H, Kurihara M, Anod K, Sato T,

Seyama K, Takahashi K. Features of pneumothorax

and risk of air-travel in Birt-Hogg-Dubé syndrome

(abstract). Am J Resp Crit Care Med 2012; 185: A4438.

10. Josephs LK, Coker RK, Thomas M, on behalf of the

BTS air travel working group. Managing patients

with stable respiratory disease planning air travel: a

primary care summary of the British Thoracic Society

recommendations. Prim Care Respir J 2013; 22: 234-238.

11. Johannesma PC, Jonker MA, van der Wel JWT, van

Waesberghe JHTM, van Moorselaar RJA, Menko

FH, Postmus PE. Management of spontaneous

pneumothorax in patients with or without Birt-

Hogg-Dubé syndrome [abstract[. ERS 2014.

12. Kumasaka T, Hayashi T, Mitani K, Kataoka H, Kikkawa

M, Tobino K, Kobayasha E, Gunji Y, Kunogi M, Kurihara

M, Seyama K.Characterization of pulmonary cysts in

Birt-Hogg-Dubé syndrome: histopathological and

morphometric analysis of 229 pulmonary cysts from

50 unrelated patients. Histopathology 2014. (in press)

13. Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda

H, Takagi S, Hsu Y-H, Fujii T, Okada A, Kuroda N,

Moritani S, Mizino H, Nagashima Y,Nagaham K,

Hiroshima K, Yoshino I, Nomura F, Aoki I, Nakatani

Y. Pulmonary cysts of Birt-Hogg-Dubé syndrome: a

clinicopathological and immunohistochemical study

of 9 families. Am J Surg Pathol 2012; 36: 589-600.

14. Lichter I, Gwynne JF. Spontaneous pneumothorax

in young subjects. A clinical and pathological

study. Thorax 1971; 26: 409-417.

15. Johannesma PC, van der Wel JWT, Paul MA, Houweling

AC, Jonker MA, van Waesberghe JHTM, van

Moorselaar RJA, Menko FH, Postmus PE. Commercial

air travel and diving in patients with Birt-Hogg-Dubé

syndrome [abstract]. ACCP 2014. (submitted)

16. Ho BL. A case report of spontaneous pneumothorax

during flight. Aviat Space Environ Med 1975; 46:

840-841.

c H a P t e R

spontaneous pneumothorax as indicator for Birt-Hogg-dubé syndrome in pediatric patients

Paul C. Johannesma1, Ben E.E.M. van den Borne2, Ad F. Nagelkerke3, JanHein T.M. van Waesberghe4, Marinus A. Paul5,

R. Jeroen A. van Moorselaar6, Fred H. Menko7 8, Pieter E. Postmus1

1Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands

2 Department of Pulmonary Diseases, Catharina Hospital, Eindhoven, The Netherlands 3 Department of Pediatrics, VU University Medical Center, Amsterdam, The Netherlands

4 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands5 Department of Thoracic Surgery, VU University Medical Center, Amsterdam, The Netherlands

6 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 7 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

8 Department of Clinical Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands

BMC Pediatr. 2014 Jul 3;14:171

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a B s t R ac t

Background

Birt-Hogg-Dubé syndrome (BHD) is a rare autosomal dominant inherited disorder caused by

germline mutations in the folliculin (FLCN) gene. Clinical manifestations of the BHD syndrome

include fibrofolliculomas, renal tumours, lung cysts and (recurrent) spontaneous pneumothorax.

All clinical manifestations usually present in adults from the age 20 onward.


Two non-related patients with a history of (recurrent) pneumothorax from the age of 14 and multiple

basal lung cysts on thoracic CT were screened for a pathogenic FLCN mutation.

Results

A pathogenic FLCN mutation was found in both patients. No skin fibrofolliculomas or renal

abnormities were found.

conclusion

Although childhood occurrence of SP in BHD is rare, these two cases illustrate that BHD should be

considered as cause of SP during childhood.

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Birt-Hogg-Dubé (BHD) is a rare tumor syndrome first described in 1977. The syndrome is characterized

by skin fibrofolliculomas, lung cysts, (recurrent) spontaneous pneumothorax (SP) and renal cancer.

The underlying germline mutation is located in the folliculin (FLCN) gene on chromosome 17p11.2.

Although clinical manifestation usually appears after the age of 20, we here present two cases of

BHD wherein episodes of (recurrent) pneumothorax occurred from the age of 14.

pat i e n t p R e s e n tat i o n 1

A 14-year-old Caucasian apparently healthy boy, without any medical problems in the past, non-

smoker, was admitted to the Emergency Department for shortness of breath and right-sided chest

pain which increased when bending over. He had no fever or other signs of influenza. Breath sounds

over the right hemithorax were reduced. Routine laboratory tests showed no abnormalities. Chest

x-ray showed a right-sided pneumothorax with a complete collapse of the lung. After drainage

by catheter, thoracoscopic pleurodesis was performed. Approximately 8 months later he had

recurrence of right-sided pneumothorax which was treated with a partial right-sided pleurectomy.

In the subsequent 2 years he had 2 recurrences, both treated by drainage by catheter. Because

of the recurrent episodes of pneumothorax a thoracic computed tomography of his chest was

performed, which showed multiple cysts below the level of the carina in the parenchyma and

subpleural, especially right sided. Because of the recurrent pneumothorax and the CT-abnormalities

(CT-thorax, figure 1A, B) BHD was suspected. Genetic testing confirmed a pathogenic mutation of

the FLCN gene (c.1177-5_1177-3delCTC). Skin fibrofolliculomas and renal abnormalities were absent

in this patient. Subsequently other family members were counselled by the clinical geneticist. Three

family members were also affected. (pedigree, figure 2)

figure 1. (a) CT shows pneumothorax with intraparenchymal located cyst with small septa. (B) shows multiple cysts in

the lower lobes (under the carina), in the same patient after treatment of the pneumothorax.

a B

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pat i e n t p R e s e n tat i o n 2

A 19-year-old Caucasian male was presented at the Emergency Department for a recurrent right-

sided pneumothorax, the first at age 14, treated by video-assisted-thoracoscopy surgery (VATS).

A chest CT was made, which revealed a complete collapsed right lung with multiple bullae at the

ventero-cranial side of the right lower lobe. An uncomplicated VATS procedure was performed by

the thoracic surgeon. Although the thoracic CT showed multiple bullae, mainly in the basal parts

of the right lung, no bullae were seen at operation. A total pleurectomy was performed. Two years

later he was referred to our center for evaluation for BHD as it had been diagnosed in several family

members (pedigree, figure 3). A chest CT was performed again, which showed several cysts in both

lungs in the parenchyma and bullae mainly in the right lung (CT-thorax, figure 4A, B). Genetic

testing confirmed a pathogenic mutation (c.1301-7_1304del;1323delCinsGA) of the FLCN gene that

confirmed the diagnosis of BHD. Skin fibrofolliculomas and renal abnormalities were absent in

this patient. Other family members had skin fibrofolliculomas, but no history of renal tumours or

(recurrent) episodes of pneumothorax.

legend figure 2. Edited pedigree of “patient presentation 1”. Generation identifier numbers (I-III) are located to the

right of each generation. Each identifier numbers are listed below each family member (circle = female; square = male;

solid symbol = pathogenic FLCN mutation carrier; open symbol = no pathogenic FLCN mutation carrier; slash-through

symbol = deceased; arrow = proband).

legend figure 3. Edited pedigree of “patient presentation 2”. Generation identifier numbers (I-III) are located to the right of

each generation. Each identifier numbers are listed below each family member (circle = female; square = male; solid symbol =

pathogenic FLCN mutation carrier; open symbol = no pathogenic FLCN mutation carrier; slash-through symbol = deceased;

arrow = proband).

i

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d i s c u s s i o n

We report here two pediatric patients with recurrent spontaneous pneumothorax, as part of the

Birt-Hogg-Dubé (BHD) syndrome. Lung cysts were seen in both patients, predominantly located

below the level of the carina, located in both the parenchyma and subpleural. We assume that these

cysts are related to the (recurrent) SP.

The incidence of pediatric primary and secondary SP is approximately 4 per 100.000 males and 1.1.

per 100.000 females per year, suggesting that SP is probably less common than in adulthood.1 2 The

mean age at presentation in pediatric cases is between 13.8-15.9 years and occurs commonly in tall, thin

males.3 A specific racial/ethnic predominance has not been described in literature.2 The underlying

cause in primary SP in adulthood is often unknown but can be caused by an underlying disorder like an

inflammatory or connective tissue disease, infection, malignancy, foreign body aspiration or congenital

malformation.4 Subpleural bullae, mainly in the apex of the lung, are found in 76-100 percent of adult

patients during video-assisted thoracoscopic surgery (VATS) and thoracotomy.5 Among non-smokers

with a history of SP, 81 percent have bullae.6 The general underlying aetiology of SP in childhood seems

to be connective tissue changes, which predispose to spontaneous leaking of air from the airways

into the pleural space. Several case series suggest a relation between subpleural blebs/bullae and the

occurrence of SP.2 7 The incidence of blebs and bullae detected on CT in children with SP is between

45% and 100%.8 The clinical significance of these blebs and bullae remains unclear.4

SP in pediatrics is often diagnosed on clinical suspicion, history and physical examination, confirmed

by chest radiography. The additional value of chest CT is unclear. In the British Thoracic Society (BTS)

guidelines a CT of the chest is indicated when chest radiography is negative despite of clinical signs

for SP or for determining appropriate management strategies. Because blebs, bullae and cysts are

only visible on CT, which is performed in a minority of cases, the diagnosis BHD is likely to be missed.

Adult literature reports a recurrence rate in primary SP of approximately 30 percent, with a range of

16 to 52 percent.9 In pediatrics a recurrence rate up to 61 percent has been reported and seems to be

figure 4. (a) CT image of pneumothorax with cluster of lung cysts in the right lower lobe(4a). (B) Figure shows

lung cyst located subpleural in the right lower lobe, abutting the main fissure as shown after resolving of the

pneumothorax.

a B

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higher compared to the adult data.10 A smaller study by Ouanes-Berbes et al. reported a 19% overall

recurrence rate over 7 year follow-up in a young adult cohort, with a 72 percent incidence of bullae.11

Bullae and cysts have been associated in adults with the Birt-Hogg-Dubé syndrome, with a basal

rather than apical distribution of bullae and cysts. The literature on the incidence of BHD syndrome

in adults with SP is very limited. Ren and colleagues found a prevalence of 9.8% in 121 apparently

spontaneous pneumothorax adult patients.12 We found in our pilot study among 40 apparently

primary spontaneous pneumothorax patients in 3 patients (7.5%) a pathogenic FLCN mutation.13

SP has been reported twice in pediatric pathogenic FLCN-mutation carriers. The first patient had

one episode of SP at age of 7 years. Chest CT was not performed. The second patient at age of 16

years, with a positive family history for SP, had in total 6 episodes of SP on both sides, multiple basal

located cysts on chest CT were found.14 15

Birt-Hogg-Dubé syndrome [OMIM no. 135150] is a rare autosomal dominant inherited disorder

caused by germline mutations in the (FLCN) gene located on chromosome 17p11.2. The BHD gene

codes for the protein folliculin which is expressed in skin tissue, nephrons and type 1 pulmonary

alveolar epithelial cells. The function is not fully clarified yet, but it seems that folliculin acts as a

cancer-inhibitory gene.16 Clinically this syndrome consists of skin fibrofolliculomas, a 50-fold

increased risk for development of (recurrent) SP, multiple lung cysts predominantly in the basal

parts of the lung, renal cysts and renal cancer from the age of 20.17 18 Therefore patients have to be

lifelong screened annually by renal MRI or renal ultrasound. Because the clinical expression can vary,

BHD syndrome cannot be excluded when no renal abnormalities or skin lesions are found.18 The

differential diagnosis of patients with multiple lung cysts include emphysema, cystic bronchiectasis,

honeycomb change, cavitated infective nodules, pulmonary Langerhans cell histiocytosis (LCH),

lymphangioleiomyomatosis (LAM), lymphocytic interstitial pneumonia (LIP), follicular bronchiolitis,

amyloidosis, light chain deposition disease (LCDD) and the Birt-Hogg-Dubé (BHD) syndrome.19 The

cystic pattern in BHD differs from other lung diseases; multiple thin-walled pulmonary cysts of

various sizes, predominately distributed to the lower medial and subpleural regions of the lung with

cysts abutting or including the proximal portion of the lower pulmonary veins or arteries.20 21 22

Treatment of SP in pediatrics does not differ from adults and depends on size and underlying

cause. Both BTS and American College of Chest Physicians (ACCP) have published different

recommendations for treatment of primary SP in adults as well for pediatric patients. Multiple

studies in adult literature advocate aspiration as a first treatment option in PSP. For secondary

SP or recurrent SP the ACCP and BTS recommend tube thoracostomy and surgical recurrence

prevention.2 23 Whether treatment of pneumothorax in BHD patients need to be more aggressive

than in idiopathic SP is still discussed.18

In conclusion we report here two cases of Birt-Hogg-Dubé syndrome in children with recurrent

pneumothorax. As SP in the pediatric population is relatively rare, BHD should be considered as

underlying cause, especially when there is a positive family history for pneumothorax. BHD patients

have an increased risk for developing renal cancer, therefore we suggest that easy accessible low

dose chest CT and easy accessible genetic testing for BHD in pediatric patients with (recurrent)

spontaneous pneumothorax should be performed, even when skin manifestation are absent. We

suggest that more research on the prevalence of BHD in the pediatric population with a history of

(recurrent) spontaneous pneumothorax is needed.

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R e f e R e n c e s1. Healthcare Cost and Utilization Project (HCUP).

Kids’Inpatient Database (KID). 1997, 2000, 2003,

2006. Available at: http://www.hcup-us.ahrq.gov/

kidoverview.jsp

2. Dotson K et al. Pediatric spontaneous pneumothorax.

Pediatric Emergency Care 2012;28(7):715-21.

3. Zgangjer M, Cizmic A, Pajic A, et al. Primary

spontaneous pneumothorax in pediatric patients:

our 7-year expercience. J Laparoendosc Adv Surg

Tech A. 2010;20:195-98.

4. Robinson PD, Cooper P, Ranganathan SC.

Evidence-based management of pediatric primary

spontaneous pneumothorax. Paediatr Respir Rev.

2009;10(3):110-7.

5. Sahn SA, Heffner JE. Spontaneous pneumothorax.

N Engl J Med. 2000 Mar 23;342(12):868-74.

6. Bense L, Lewander R, Eklund G, et al. Non-

smoking, non-alpha 1-antitypsin deficiency-

induced emphysema in non-smokers with healed

spontaneous pneumothorax, identified by computed

tomography of the lungs. Chest 1993;103:433-8.

7. Smit HJ, Wienk MA, Schreurs AJ et al. Do

bullae indicate a predisposition to recurrent

pneumothorax? Br J Radiol 2000;73:356-9.

8. O’Lone E, Elphick HE, Robinson PJ.

Spontaneous pneumothorax in children:

when is invasive treatment indicated? Pediatr

Pulmonol. 2008 Jan;43(1):41-6.

9. Schramel FM, Postmus PE, Vanderschueren RG.


Eur Respir J 1997;10:1372-9.

10. Wilcox DT, Glick PL, Karamanoukian HL et al.

Spontaneous pneumothorax: a single-institution:,

12-year experience in patients under 16 years of

age. J Pediatr Surg 1995:30;1452-54.

11. Ouanes-Besbes L, Golli M, Knani J et al. Prediction

of recurrent spontaneous pneumothorax: CT scan

findings versus management features. Respir Med

2007;101:230-6.

12. Ren H-Z, Zhu CC, Yang C, et al. Mutation

Analysis of the FLCN gene in Chinese patients

with sporadic and familial associated isolated

primary spontaneous pneumothorax. Clin Genet

2008;74:178-83.

13. Johannesma PC, Reinhard R, Kon Y, et al. The

prevalence of Birt-Hogg-Dubé syndrome among

patients with apparently common primary

spontaneous pneumothorax. (submitted)

14. Bessis D, Giraud S, Richard S. A novel familial



syndrome. Br J Dermatol. 2006 Nov;155(5):1067-9.

15. Gunji Y, Akiyoshi T, Sato T et al. Mutations of the

Birt Hogg Dubé gene in patients with multiple


Genet. 2007 Sep;44(9):588-93.

16. Toro JR, Wei MH, Glenn GM et al. BHD mutations,




Genet 2008;45:321-331.

17. Houweling AC, Gijezen LM, Jonker MA, et al.

Renal cancer and pneumothorax risk in Birt-Hogg-

Dubé syndrome; an analysis of 115 FLCN mutation

carriers from 35 BHD families. Br J Cancer. 2011 Dec

6;105(12):1912-9.

18. Menko FH, van Steensel MA, Giraud S, et al. Birt-

Hogg-Dubé syndrome: diagnosis and management.

Lancet Oncol. 2009 Dec;10(12):1199-206.

19. Clarke BE. Cystic lung disease. J Clin Pathol

2013 Oct;66(10):904-8.

20. Johannesma PC, Thunnissen E, Postmus PE. Lung

cysts as indicator for Birt-Hogg-Dubé. Lung

2014;192(1):215-6.

21. Tobino K, Gunji Y, Kurihara M, et al. Characteristics


Thin-section CT findings of the chest in 12 patients.

European Journal of Radiology 2011;77:403-9.





from 50 unrelated patients. Histopathology 2014

Jan 7. (in press)

23. MacDuff A, Arnold A, Harvey J et al. Management

of spontaneous pneumothorax: British Thoracic

Society pleural disease guideline 2010. Thorax

2010;65:ii18-31.

c H a P t e R

lung cysts as indicator for Birt-Hogg-dubé syndrome

Paul C. Johannesma1, Erik Thunnissen2, Pieter E. Postmus1

1Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands

2 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

Lung 2014;192(1):215-6

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a B s t R ac t

In this case we describe a patients with a history of recurrent pneumothorax. Based on CT-thorax

and histopathology of the lung tissue, the Birt-Hogg-Dubé syndrome was suspected and confirmed

after genetic testing. Recognizing this syndrome by pulmonologists and radiologists is very

important as the risk on developing of renal cell cancer is high.

A 38-year-old former Olympic swimmer was referred to our hospital due to recurrent pneumothorax.

The patient was a non-smoker and had no positive family history for pneumothorax. From the age of

24 he developed 6 episodes of pneumothorax, three times on both sides. Treatment varied by VATS,

talkage, pleurodesis, bullectomy and pleurectomy. A lung function test showed a normal function.

High resolution computed tomography of the chest revealed multiple round and oval thin-walled

pulmonary cysts of varying sizes, localized mostly in the lower lobes between, which abutted to or

enclosed the proximal portions of lower pulmonary arteries and veins (Panel A,B,c).

Histopathology, the cyst wall were completely lined by pneumocytes. The inner surfaces of the

cysts stained positively for TTF-1 expression (Panel d,e), which might suggest that these cysts are

distinctly different from nonspecific blebs or bullae.1 A diagnosis of Birt-Hogg-Dubé syndrome

(BHDS) was made after molecular testing.

Birt-Hogg-Dubé syndrome BHDS [OMM no. 13515] is a rare autosomal dominant inherited disorder

caused by a mutation in the (FLCN) gene located on chromosome 17p11.2. BHD-patients have

a 50-fold increased risk for development of (recurrent) spontaneous pneumothorax. Lung cysts are

common in those with spontaneous pneumothorax.2 Recognizing this disease is important as the

risk on developing renal cell cancer is high and screening is indicated.

R e f e R e n c e s1. Furuya M, Tanaka R, Koga S et al. Pulmonary cysts

of Birt-Hogg-Dubé syndrome: A clinicopathologic

and Immunohistochemical study of 9 families. Am

J Surg Pathol. 2012;36:589-600.

2. Menko FH, van Steensel MAM, Giraud S. et al. Birt-

Hogg-Dubé syndrome: diagnosis and management.

The Lancet Oncology 2009;10:1199-1206.

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Figure 3. Cysts enclosing pulmonary vessel (arrow).

Figure 1. Frontal view of high-resolution CT

showing many round and oval thin-walled cysts

mainly in the lower lobes.

Figure 2. Cysts abutting pulmonary vessel (arrow).

Figure 4. Cysts walls lined by pneumocytes. Figure 5. Pneumocytes staining positive for TTF-1.

c H a P t e Rspontaneous pneumothorax

as the first manifestation of a hereditary condition with an increased renal cancer risk

Paul C. Johannesma1, Jan-Willem J. Lammers2, R. Jeroen A. van Moorselaar3, Theo M. Starink4, Pieter E. Postmus1, Fred H. Menko5,

on behalf of “Werkgroep Birt-Hogg-Dubé syndroom van het Centrum Familiaire Tumoren VUmc*”

*Hr. E.F.L. David, radioloog, hr. dr. J.J.P. Gille, klinisch moleculair geneticus, hr. P.C. Johannesma, student geneeskunde, hr. dr. E.M. Leter, klinisch geneticus,

hr. dr. F.H. Menko, klinisch geneticus, hr. dr. R.J.A. van Moorselaar, uroloog, hr. H.M. Ploeger, patiënten coördinator, hr. prof. dr. P.E. Postmus, longarts,

hr. prof. dr. Th.M. Starink, dermatoloog

1Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Pulmonary Diseases, Utrecht University Medical Center, Utrecht, The Netherlands

3 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands

5 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

Ned Tijdschr Geneeskd. 2009;153:a581

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a B s t R ac t

Spontaneous pneumothorax may be due to Birt-Hogg-Dubé syndrome (BHD), an autosomal

dominant predisposition for fibrofolliculomas, multiple lung cysts, pneumothorax and renal cancer.

The syndrome is due to germline mutations in the FLCN (folliculin) gene. Clinical presentation is

highly variable. Consequently, the syndrome is probably underdiagnosed. An illustrative kindred

is presented in which the index patient had recurrent episodes of pneumothorax without apparent

skin lesions or renal abnormalities. He had bilateral basally located lung cysts. Family members

had fibrofolliculomas, lung cysts, pneumothorax and clear cell renal cancer. Recognition of BHD is

important since mutation carriers can be offered surveillance for early detection and treatment of

renal cancer.

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dames en Heren,

Een pneumothorax (klaplong) ontstaat als er lucht komt tussen de pariëtale en viscerale pleura.

De long kan zich hierdoor niet meer volledig ontplooien. Pijn op de borst en kortademigheid zijn

de meest voorkomende symptomen. Naar oorzaak wordt pneumothorax ingedeeld in 1) spontane

pneumothorax, a) primair, zonder bekende oorzaak of b) secundair, als gevolg van een al bestaande

longaandoening, 2) traumatische en 3) iatrogene pneumothorax.1

Spontane pneumothorax kan een uiting zijn van het Birt-Hogg-Dubé syndroom (BHD). BHD

wordt gekenmerkt door fibrofolliculomen van de huid, multipele longcysten, pneumothorax en

nierkanker.2 3 4 5 Herkenning van het syndroom is belangrijk voor de patiënt en voor de familie. Door

jaarlijkse controle kan nierkanker in een vroeg stadium worden opgespoord en behandeld.

In deze klinische les willen wij aan de hand van vier ziektegeschiedenissen uw aandacht vragen voor

het Birt-Hogg-Dubé syndroom. Een positieve familie-anamnese voor pneumothorax leidde hier tot

herkenning van het syndroom.

Ziektegevallen

Patiënt A, een niet-rokende vitale man van 26 jaar met een blanco voorgeschiedenis kwam eind

juni 2007 op de spoedeisende hulp met kortademigheid en subfebriele temperatuursverhoging. Bij

lichamelijk onderzoek was er rechts op de borst hypersonore percussie en verminderd ademgeruis.

De thoraxfoto toonde een pneumothorax rechts. (Fig. 1A). Bij “Video Assisted Thoracoscopic

Surgery” (VATS) werden in de rechter bovenkwab multipele bulleuze afwijkingen waargenomen.

Behandeling vond plaats door talkage van de pleurabladen en zuigdrainage. In de weken daarop

trad driemaal opnieuw een pneumothorax aan dezelfde zijde op. Het eerste recidief werd

conservatief behandeld. Na het tweede recidief werd een restpleurectomie verricht gevolgd door

zuigdrainage. Het derde recidief werd behandeld met opruwen van de pleura van de onderste

thoraxhelft, talkage en zuigdrainage. Een week na ontslag traden opnieuw dyspnoeklachten op,

nu bleek er een pneumothorax aan de linker zijde. Een CT-scan van de longen (Fig. 1B) toonde

Figuur 1. (a) Patiënt A. De röntgenfoto van de thorax toont een pneumothorax rechts. (B) Patiënt A. De CT-scan van

de longen toont een pneumothorax rechts en bilaterale cysteuze longafwijkingen.

A B

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beiderzijds multipele cysteuze longafwijkingen, basaal meer uitgesproken dan apicaal. Behandeling

vond plaats door middel van thoracotomie, bullectomie, pleurectomie, opruwing van het diafragma

en zuigdrainage. Bij follow-up maakt patiënt het goed.

Patiënt B, een 60-jarige man, maakte op de leeftijd van 47 jaar een dubbelzijdige pneumothorax door.

Op een CT-scan van de longen waren multipele bullae in beide longen zichtbaar. Aan de gelaatshuid

heeft patiënt multipele huidkleurig tot wittig doorschijnende papels, passend bij fibrofolliculomen.

Histologisch onderzoek van één van deze laesies bevestigde deze diagnose (Fig. 2A, B).

Figuur 2. (a) Klinische aspect van multiple fibrofolliculomen in het gelaat bij patiënt B. (B) Histologische kenmerken

van een fibrofolliculoom: proliferatie van het perifolliculaire bindweefsel en netvormig vertakkende epitheelstrengen.

A B

Patiente c, een niet-rokende vrouw zonder bijzondere medische voorgeschiedenis, kreeg op

34-jarige leeftijd een pneumothorax rechts. Behandeling vond plaats via drainage. Na drie dagen

trad een recidief op. Als therapie werden drainage en pleurodese met talk toegepast. Een maand na

ontslag uit het ziekenhuis trad een tweede recidief op. Een CT-scan van de thorax toonde beiderzijds

in de longen dunwandige cysteuze afwijkingen. Bij mini-thoracotomie rechts werden twee grote en

meerdere kleine bullae gezien, verspreid over de long, maar niet in de longtop. Er wordt een totale

pleurectomie rechts verricht. Het postoperatieve beloop was voorspoedig.

dames en heren,

De patiënten A, B en C zijn familieleden van elkaar. De stamboom is weergegeven in figuur 4.

Bij een paternale oom van de indexpatiënt, patiënt d, werd op de leeftijd van 48 jaar nierkanker

geconstateerd. Er werd een thoraco-abdominale nefrectomie verricht, waarbij een groot

tumorproces werd verwijderd. Histologisch betrof het een heldercellig carcinoom (Fig. 3, Fig. 4).

Patiënt A werd wegens verdenking op Birt-Hogg-Dubé syndroom, op basis van het familiair

voorkomen van pneumothorax door de longarts verwezen naar de klinisch geneticus. Patiënt bleek

drager van een pathogene mutatie in exon 6 van het FLCN-gen (c.1065_1066delGCinsTA). Zijn vader,

patiënt B, bleek zoals verwacht ook drager van deze mutatie. Patiënt A had geen fibrofolliculomen;

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Figuur 4. Stamboom van de beschreven familie. De indexpatiënt A is met pijl aangegeven. De zwarte symbolen geven

de kenmerken van BHD aan: Pneumothorax , Fibrofolliculomen , en niercelkanker .

de kans is groot, dat deze typerende huidverschijnselen bij hem later alsnog zullen optreden. Bij

MRI en echoscopie van de nieren werden bij patiënt en bij zijn vader geen afwijkingen gevonden.

Tot op heden hebben overige verwanten geen DNA-diagnostiek laten verrichten. BHD bij de

patiënten C en D is een waarschijnlijkheidsdiagnose.

Birt-Hogg-dubé syndroom

In 1977 beschreven drie Canadese artsen een grote familie met “fibrofolliculomen, trichodiscomen

en acrochordons”. Vanaf de leeftijd van circa 25 jaar traden vooral in het gelaat multipele, kleine,

witte papels op.2 Nu staat het Birt-Hogg-Dubé syndroom bekend als een autosomaal dominant

erfelijke aandoening, gekenmerkt door fibrofolliculomen van de huid, multipele longcysten,

pneumothorax en nierkanker.

BHD wordt veroorzaakt door kiembaanmutaties in het FLCN (folliculine)- gen, dat gelokaliseerd is

op de korte arm van chromosoom 17 en codeert voor het eiwit folliculine. Het gen heeft kenmerken

van een tumorsuppressor-gen en komt tot expressie in onder andere huid, longen en nieren.6 7

Figuur 3. Histologisch beeld van het heldercellige niercarcinoom, opgetreden bij patiënt D.

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Bij patiënten met klinisch BHD kan in 70-80% van de gevallen een pathogene FLCN-mutatie worden

aangetoond. Dragers van een pathogene FLCN-mutatie hebben een sterk variabele expressie. Zo

zijn in families waarin alleen pneumothorax voorkwam - zonder fibrofolliculomen of niertumoren –

ook FLCN-mutaties gevonden.8 9

Huid. De huidverschijnselen bestaan uit multipele 1-5 mm grote witte of huidkleurige papels,

voornamelijk in het gelaat, maar ook in de hals en op de romp. Fibrofolliculomen zijn goedaardige

haarfollikeltumoren die uitgaan van het bovenste gedeelte van de haarfollikel. Zij bestaan uit een

proliferatie van het perifolliculaire bindweefsel en een epitheliale component met netvormig

vertakkende epitheelstrengen. Trichodiscomen zijn een histologische variant van fibrofolliculomen10

Indien om cosmetische redenen behandeling van de fibrofolliculomen wordt gewenst, kan met

lasertechnieken vaak een tijdelijke remissie worden bereikt.

Fibrofolliculomen zijn kenmerkend voor BHD: verwijzing van een patiënt met deze afwijkingen is

daarom steeds aangewezen.

Longen. Longcysten werden bij 80-90% van alle patiënten gevonden, vaker basaal dan apicaal

gelegen. De longcysten zijn de oorzaak van het optreden van pneumothorax.

Hoewel de meeste patiënten met BHD multipele longcysten hebben is de longfunctie als regel niet

gestoord. Ongeveer 25% van de patiënten met BHD krijgt een pneumothorax , meestal voor de

leeftijd van 50 jaar. Bij BHD is er een verhoogde kans op een recidief pneumothorax.5 11

Pneumothorax bij BHD wordt behandeld zoals bij andere patiënten met spontane pneumothorax,

ofwel conservatief ofwel invasief.

Er is geen reden voor standaard onderzoek naar longcysten bij een individu met aanleg voor BHD.

Dit onderzoek is wel nodig in omstandigheden met een verhoogde kans op een pneumothorax.

Zo wordt een CT-scan van de thorax geadviseerd voorafgaand aan een operatie onder algemene

narcose. Evaluatie door de longarts wordt ook geadviseerd in bijzondere situaties, zoals

beroepskeuze voor piloot of diepzeeduiken als hobby. Roken wordt aan individuen met BHD

sterk ontraden.

Nieren. In 1993 werd niercelkanker voor het eerst geassocieerd met BHD.12 FLCN-mutatiedragers

hebben een sterk verhoogde kans op nierkanker. De prevalentie van nierkanker bij patienten met

BHD loopt in verschillende studies echter sterk uiteen, van 6.5% tot 34%.5

Evenals bij andere erfelijke vormen van nierkanker ontstaan de niertumoren bij BHD op relatief

jonge leeftijd en treden zij vaak multifocaal en bilateraal op. Typerend voor BHD zijn chromofobe

carcinomen en oncocytomen. Er worden vaak mengvormen van deze histologische typen gezien.

Ook andere histologische vormen komen echter voor, waaronder het heldercellig niercarcinoom.13 14

Voor vroege detectie en behandeling van nierkanker wordt bij FLCN-mutatiedragers een jaarlijks

onderzoek verricht. Wij adviseren vanaf de leeftijd van 20 jaar eerst een MRI van de nieren en

nierechoscopie en vervolgens jaarlijks alleen echoscopie en MRI op indicatie (bij moeilijke

beoordeelbaarheid of bij mogelijke afwijkingen). Bij het vinden van nierkanker vindt na pre-

operatieve evaluatie zo mogelijk een niersparende operatie plaats.

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dames en Heren,

Primaire SP komt ongeveer even vaak voor als secundaire SP. In de huisartsenpraktijk wordt bij

24 mannen per 100.000 consulten per jaar en 10 per 100.000 vrouwen spontane pneumothorax

vastgesteld.15 Primaire spontane pneumothorax treedt voornamelijk op bij lange, dunne jongens

en mannen tussen de 10 en de 30 jaar. Roken is een belangrijke oorzakelijke factor. Bij de meeste

patiënten worden bij thoracoscopie subpleurale bullae gevonden, vooral apicaal gelegen. De kans

op een recidief pneumothorax is ongeveer 30%; de meeste recidieven treden binnen twee jaar na

de eerste episode op.16

Van secundaire spontane pneumothorax wordt gesproken, als de pneumothorax een complicatie is

van een longziekte, in het bijzonder chronische obstructieve longziekte, longinfectie, longkanker,

cystische fibrose, alfa-1-antitrypsine deficiëntie, sarcoïdose, Marfan syndroom, Ehlers Danlos syndroom,

lymfangioleiomyomatose, tubereuze sclerose complex, en het Birt-Hogg-Dubé syndroom.17

Tenslotte kan een pneumothorax optreden door een scherp of stomp thoraxtrauma of iatrogeen, in

het bijzonder als complicatie bij het inbrengen van een centrale centrale lijn in de vena subclavia of

bij het nemen van een pleurabiopt.18

Bij ongeveer 10% van alle patiënten met een pneumothorax komt de aandoening familiair voor

zonder enig ander verschijnsel. Verschillende vormen van overerving zijn hiervoor gesuggereerd

(autosomaal dominant, X-chromosomaal recessief). Daarnaast kan familiair optreden van

pneumothorax een uiting zijn van een erfelijk syndroom, waaronder het Birt-Hogg-Dubé

syndroom.19 Op basis van recente studies kan worden geschat, dat 15-25% van de familiaire gevallen

berust op een FLCN-mutatie.20 21

In Nederland zijn op dit moment meer dan 30 families bekend, waarin BHD op basis van

een pathogene FLCN-mutatie is aangetoond. De gegevens van een aantal van deze families

zijn onlangs gepubliceerd. [4, 22] Recent is het Europees BHD Consortium opgericht www.

europeanbhdconsortium.eu om het BHD syndroom beter in kaart te brengen en optimale

methoden van diagnostiek, surveillance en behandeling te ontwikkelen. Er is een internationale

patiëntenvereniging, met een website voor informatie: www.bhdsyndrome.org. Vermoedelijk

komt de aandoening vaker voor dan tot op heden wordt onderkend. Indien een patiënt een

spontane pneumothorax krijgt zonder al bekende longziekte, dan kan het gaan om BHD. [23] Bij

een dergelijke patiënt met pneumothorax zijn klinische aanwijzingen voor BHD multipele basaal

gelegen longcysten en het voorkomen van pneumothorax in de familie.19,20 In de besproken

familie werden de voor het syndroom kenmerkende fibrofolliculomen van de huid en nierkanker

gevonden bij het in kaart brengen van de familiegegevens.

Door het vinden van een pathogene FLCN-mutatie werd de diagnose Birt-Hogg-Dubé syndroom

bevestigd.

Dames en heren, de hier gepresenteerde casus tonen, dat de eerste aanwijzing voor een bijzondere

erfelijke aandoening kan worden verkregen door het nauwkeurig afnemen van een familie-

anamnese, door huisarts of behandelend specialist.

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R e f e R e n c e s1. Sahn SA, Heffner JE. Spontaneous pneumothorax.

N Engl J Med 2000; 342: 868-74.

2. Birt AR, Hogg GR, Dubé WJ. Hereditary multiple


acrochordons. Arch Dermatol 1977;113:1674-77.

3. Khoo, S.K., Giraud, S., Kahnoski, K., Chen, J.,

Motorna, O., Nickolov, R. et al. Clinical and genetic

studies of Birt-Hogg-Dubé syndrome. J. Med.

Genet. 2002; 39: 906-912.

4. Leter EM, Koopmans AK, Gille JJP, van Os TAM,

Vittoz GG, David EFL et al. Birt-Hogg-Dubé

syndrome: clinical and genetic studies of 20

families. J Invest Dermatol 2008; 128: 45-49.

5. Toro JR, Wei M-H, Glenn GM, Weinreich M, Toure

O, Vocke C, et al. BHD mutations, clinical and

molecular genetic investigations of Birt-Hogg-Dubé

syndrome: a new series of 50 families and a review of

published reports. J Med Genet 2008; 45: 321-31.

6. Nickerson, ML, Warren MB,.Toro JR, Matrosova V,

Glenn G, Turner ML et al. Mutations in a novel gene

lead to kidney tumors, lung wall defects, and benign

tumors of the hair follicle in patients with the Birt-

Hogg-Dubé syndrome. Cancer Cell 2002; 2:157–64.

7. Warren MB, Torres- Cabala CA, Turner ML, Merino

MJ, Matrosova VY, Nickerson ML. Expression of Birt-

Hogg-Dubé gene mRNA in normal and neoplastic

human tissues. Mod Pathol 2004;8: 998-1011.

8. Graham RB, Nolasco, M, Peterlin, B , Garcia CK.



adults. Am. J. Respir. Crit. Care Med. 2005; 172: 39-44.

9. Painter, J.N., Tapanainen, H., Somer, M. et al. A

4-bp deletion in the causes dominantly inherited

spontaneous pneumothorax. Am. J. Hum. Genet.

2005; 76: 522-527.

10. Vincent A, Farley M, Chan E, James WD. Birt-Hogg-

Dubé syndrome: a review of the literature and the

differential diagnosis of firm facial papules. J. Am.

Acad. Dermatol. 2003; 49: 698-705.

11. Toro JR, Pautler SE, Stewart, L, Glenn GM, Weinreich

M, Toure O, et al. Lung cysts, spontaneous

pneumothorax, and genetic associations in 89

families with Birt-Hogg-Dubé syndrome. Am J

Respir Crit Care Med 2007;175:1044-53.

12. Roth JS, Rabinowitz AD, Benson M., Grossman ME.

Bilateral renal carcinoma in the Birt-Hogg-Dubé

syndrome. J Am Acad Dermatol 1993; 29: 1055-1056.

13. Pavlovich CP, Grubb III RL, Hurley K, Glenn GM,

Toro J, Schmidt LS Evaluation and management of

renal tumors in the Birt-Hogg-Dubé syndrome. J

Urol 2005; 173: 1482-1486.

14. Woodward ER, Ricketts C, Killick P, Gad S, Morris MR,

Kavalier F. Familial non-VHL clear cell (conventional)

renal cell carcinoma: clinical features, segregation

analysis, and mutation analysis of FLCN. Clin. Cancer

Res. 2008; 14: 5925-5930.

15. Gupta, D., Hansell, A., Nichols, T. et al.

Epidemiology of pneumothorax in England. Thorax

2000; 55: 666-671.

16. Smit HJ, Devillé WL, Schramel FM, Schreurs JM,

Sutedja TG, Postmus PE. Atmospheric pressure

changes and outdoor temperature changes in

relation to spontaneous pneumothorax. Chest

1999;116:676-81.

17. Louis H, Los H, Lagendijk JH, de Graaff CS, Postmus

PE. Spontane pneumothorax bij jonge vrouwen:

mogelijk lymfangioleiomyomatose. Ned Tijdschr

Geneeskd. 1997;141:1924-8.

18. Tan ECTH, Vliet, van der JA. Late (spannings)

pneumothorax na het plaatsen van een

centraalveneuze lijn. Ned. Tijdschr. Geneeskd.

1999; 143: 1872-1874.

19. Chiu HT, Garcia CK. Familial spontaneous

pneumothorax. Curr Opin Pulm Med 2006;12:268-72.


Takahashi K et al Mutations of the Birt-Hogg-Dubé

gene in patients with multiple lung cysts and recurrent

pneumothorax. J Med Genet 2007; 44: 588-593.

21. Ren H-Z, Zhu C-C, Yang C, Chen S-L et al. Mutation



pneumothorax. Clin Genet 2008; 74: 178-184.

c H a P t e R

facial fibrofolliculomas as indicator for renal cell cancer

Jpn J Clin Oncol. 2014;44(6):609-10

Paul C. Johannesma1, Theo M. Starink2, R. Jeroen A. van Moorselaar3, Pieter E. Postmus1


2 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands 3 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands

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A 53-year old woman was referred to our hospital because of an increasing number of facial skin

papules on her cheeks and ears. A biopsy was taken from several of these papules. The location of

these lesions combined with the histology confirmed the diagnoses fibrofolliculomas (figure 1A+B).

Except for facial papules since the age of 26 years, her medical history was unremarkable. The

familial history was negative for renal cancer and spontaneous pneumothorax. She never smoked.

Her father died of pancreatic cancer at the age of 73 years and her mother of cardiac failure at

age 85. Based on the histology and location of the skin lesions, the patient was clinically diagnosed

as having Birt-Hogg-Dubé syndrome (BHD). Therefore she was referred for CT abdomen with

i.v. contrast, which showed a solid interpolar tumour in the lower pole of the right kidney, with

a diameter of 19 mm, classified as T1N0Mx (figure 2). The slices through the basal parts of the

lungs revealed multiple lung cysts (figure 3). Partial nephrectomy followed and a clear cell tumour,

Fuhrmann grade 2, was found. This combination of skin fibrofolliculomas, renal cell cancer and cysts

in the basal parts of the lungs is typical for the Birt-Hogg-Dubé syndrome (BHD), an autosomal

dominantly inherited cancer disorder, caused by pathogenic FLCN mutations.

figure 1. Skin fibrofolliculomas.

A B

figure 2. Renal tumour in the lower pole of the right kidney.

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Sequencing of the flcn gene showed a pathogenic splice site mutation in exon 12 (c.[1301-7_1304;

delCinsGA]), which confirmed the clinical diagnosis at DNA level.

Afterwards we identified four additional FLCN mutation carriers in her family. The family data are

summarized in figure 4. Magnetic Resonance Imaging (MRI) showed no local recurrence of the

removed renal tumour, respectively early detection of a new renal tumour during 18 months follow

up after surgery. Lifetime frequent renal MRI will be performed in this patient. As the prevalence of

renal cancer in BHD syndrome patients after initial renal imaging has been described in up to 27%

of cases, BHD should be considered when facial fibrofolliculomas are diagnosed and consequently

relatives should be stimulated to be screened for genetic testing.

figure 3. Thoracic CT: Multiple lung cysts in the basel parts of both lungs.

figure 4. Pedigree of the family.

c H a P t e R 3 . 6

Bilateral renal tumour as indicator for Birt-Hogg-dubé syndrome

Case Rep Med. 2014;2014:618675

Paul C. Johannesma1, R. Jeroen A. van Moorselaar2, Simon Horenblas3, Lisette E. van der Kolk4, Erik Thunnissen5, JanHein T.M. van Waesberghe6,

Fred H. Menko4 7, Pieter E. Postmus1


2 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 3 Urologic Oncology and Department of Urology, The Netherlands Cancer Institute,

Amsterdam, The Netherlands 4 Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands

5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands6 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands

7 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

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a B s t R ac t

Birt-Hogg-Dubé (BHD) syndrome is a cancer disorder caused by a pathogenic FLCN mutation

characterized by fibrofolliculomas, lung cysts, pneumothorax, benign renal cyst and renal cell

carcinoma (RCC).

In this case we describe a patient with bilateral renal tumour and a positive familial history for

pneumothorax and renal cancer. Based on this clinical presentation, the patient was suspected for

BHD syndrome, which was confirmed after molecular testing.

We discuss the importance of recognizing this autosomal dominant cancer disorder when a patient

is presented at the urologist with a positive family history of chromophobe renal cell cancer, or

a positive familial history for renal cell cancer and pneumothorax.

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Birt-Hogg-Dubé syndrome (BHDS), was originally described in 1977 and is nowadays known

as a rare autosomal dominant cancer disorder characterized by fibrofolliculomas, lung cysts,

pneumothorax, benign renal cyst and renal cell carcinoma (RCC). The mutated gene for BHD

encodes the protein folliculin (FLCN) which acts as a tumour suppressor and interacts with mTOR

and AMPK signalling pathways.1 Here we report a case of a patient with bilateral renal cancer and

a positive familial history for pneumothorax and renal cancer. Based on the bilateral renal tumour

and the positive family history for renal cancer and pneumothorax, Birt-Hogg-Dubé syndrome

was suspected.

c a s e R e p o Rt

In March 2011, a 44-year-old Caucasian male was evaluated for urolithiasis. He had no physical

complaints, no macroscopic haematuria or weight loss. His medical and social history were

unremarkable, he never smoked. His father had been treated for colorectal cancer, his mother

had three episodes of spontaneous pneumothorax and had been treated for a renal tumour.

Physical examination of the abdomen showed no abnormalities. Routine laboratory tests were

normal. Computed tomography (CT) of the abdomen showed an interpolar tumour in the left

kidney, diameter 14 mm (figure 1a, arrow), and a second tumour in the upper pole of the right

kidney, diameter 8 mm (figure 1B, arrow). After a needle biopsy of the largest tumour, revealing

a chromophobe renal cell carcinoma (figure 2a+2B), the tumour in the left kidney was treated

with radio frequency ablation (RFA). The CT findings in combination with the positive family history

for renal cancer and his mother’s episodes of pneumothorax suggested Birt-Hogg-Dubé (BHD)

syndrome. Sequencing of the FLCN gene showed a pathogenic heterozygous frameshift mutation

(c.155delc;p.Leu518Phefs*19) - which hasn’t been described before in literature - and confirmed

the diagnosis Birt-Hogg-Dubé syndrome. The index patient had neither siblings nor children. His

figure 1. Contrast enhanced CT shows in the arterial phase a hypervascular small lesion in both kidneys, representing

two small chromophobe renal cell carcinomas. The tumour in left kidney (a) was treated by radiofrequency ablation

(RFA), for the tumour in the right kidney (B) was follow up proposed.

A B

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parents died years ago, blood or tissue was not available for molecular testing. Frequent follow up

by Magnetic Resonance Imaging (MRI) will be performed for evaluation of the small tumour in the

right kidney and possible recurrence of the tumour in the left kidney.

d i s c u s s i o n a n d c o n c l u s i o n

Birt-Hogg-Dubé syndrome [OMIM #135150] is a rare autosomal dominant inherited disorder

caused by a mutation in the FLCN gene located on chromosome 17p11.2, which acts as a tumour

suppressor and probably interacts with mTOR and AMPK signalling pathways.1 BHD is clinically

characterized by skin fibrofolliculomas, lung cysts, (recurrent) spontaneous pneumothorax and

renal cancer.[2] In literature co-occurrence of BHD and a range of tumours, other than renal

cancer, has been described, but so far a causal relationship between BHD and these benign and

malignant tumours has not been proven.2 Skin fibrofolliculomas are multiple, dome shaped,

whitish papules located on the scalp, forehead, face and neck and are found in approximately

90% of families with confirmed BHD syndrome. Dermatologic consultation confirmed multiple

fibrofolliculomas on the forehead and face of the index patient. Although cosmetic therapeutic

options are limited, case reports suggest that laser ablation, using a YAG or fractional CO2 laser,

gives temporary improvement.3

BHD patients have a 50 fold higher risk to develop primary spontaneous pneumothorax (PSP)

compared to the normal population. PSP in BHD patients occur usually after the age of 20, although

it has been described already at the age of 7 years.4 Up to 90% of BHD patients have multiple lung

cysts, usually located in the basal regions.5 In our clinic we found an estimated penetrance for

pneumothorax of 29% (CI: 9-49%) at 70 years of age. BHD patients usually have a normal lung

function and no pulmonary symptoms. A CT of the chest performed in our index patient showed

no pulmonary cysts.

figure 2. Overview of needle biopsy of tumor in left kidney (a: Amplification X2.5) and detail (B: Amplification X40)

of a chromophobe renal carcinoma. Overview of needle biopsy (a: amplification X2.5) and detail (B: amplification

X40) of monotonous cellular pattern with mild nuclear pleomorphy and abundant partly eosinophilic cytoplasm with

perinuclear halo, compatible with chromophobe renal cell carcinoma.

A B

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Renal cell cancer (RCC) is the most lethal of the urologic malignancies, with an estimated 273.518

new cases diagnosed and 116.368 patient deaths in 2008, worldwide.6

RCC can be divided in sporadic and hereditary. The majority is of sporadic origin, presenting normally

after the age of 60 as one lesion, while the hereditary type mainly presents as multifocal and bilateral

at a far younger age. The most common hereditary renal cancer syndromes are associated with

hereditary leiomyomatosis and renal cell carcinoma (HLRCC), Von Hippel-Lindau syndrome (VHL)

and hereditary papillary renal carcinoma (HPRC). The prevalence of RCC in (familial) renal cancer

is unknown and might be underestimated, since the majority (>80%) of the BHD index patients are

referred by a dermatologist.

In a large published series by Pavlovich et al., 34 of 124 individuals (27.4%) with genetic confirmed

BHD had a median of 5 renal tumours at a mean age of 50.4 years (range 31-74 years).7 In a large

series published by Toro et al, 34% of individuals with BHD had renal tumours.5 The youngest patient

with BHD who developed renal cancer was 20 years old.8 Another study reported a history of renal

cancer and metastasis in the same year in a patient with BHD at age 27.9

In our Dutch study population published by Houweling et al., we found among 115 BHD patients,

14 patients with renal cancer, and calculated an estimated penetrance for renal cancer of 16% (CI:

6-26%) at 70 years of age.10 Most lesions are a mixture of solid and cystic components. The largest

published histological series of RCC among BHD patients demonstrates that these tumours contain

both oncocytoma and chromophobe elements.11 Houweling and colleagues confirmed these

findings, as they found in the majority of RCC tumours, cells with granular/floccular eosinophillic

cytoplasm, as can be seen in both clear cell carcinoma and chromophobe carcinoma.10

As the risk of developing RCC is high, imaging and follow up at regular intervals is advised by MRI

from the age of 20. The role of ultrasound (US) for detecting renal tumours is still extensively

discussed in literature. Surgical treatment, is recommended before the largest tumour reaches

3 cm in maximal diameter, which is based on the VHL guideline.9 Initially a nephron-sparing

surgery should be ideally pursued, which can help prevent chronic renal insufficiency in this

patient population. Minimally invasive nephron-sparing techniques such as cryoablation and

radio frequency ablation (RFA) are generally accepted as treatment of choice in patients with

a unifocal renal lesion. Since BHD patients are at lifelong risk for the development of new

tumours, and cryoablation or RFA can complicate both the long term evaluation and surgical

management, nephron-sparing surgery is so far the safest and most effective treatment for

hereditary renal tumours.12

In conclusion, in patients with a positive family history of chromophobe renal cell cancer or a positive

family history for renal cell cancer and pneumothorax, the diagnosis BHD should be considered.

Therefore we suggest easily accessible FLCN sequencing should be considered in patients and their

family because of the high incidence of renal cancer in BHD patients.

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R e f e R e n c e s1. Baba M, Hong S-B, Sharma N, et al. Folliculin encoded

by the BHD gene interacts with a binding protein,

FNIP1, and AMPK, and is involved in AMPK and mTOR

signaling. Proc Nat Acad Sci USA 2006; 103:1552–57.

2. Menko FH, van Steensel MAM,Giraud S, et


management. Lancet Oncol 2009; 10:1201-09.

3. Gambicher T, Wolter M, Altmeyer P, et al. Treatment

of Birt-Hogg-Dubé syndrome with erbium : YAG

laser. J Am Acad Dermatol 2000; 43:856-58.

4. Zbar B, Alvord WG, Glenn G, et al. Risk of renal and


in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol

Biomarkers Prev. 2002;11(4):393-400.



in 89 families with Birt-Hogg- Dubé syndrome. Am J

Respir Crit Care Med 2007; 175:1044-53.

6. Ferlay J, Shin HR, Bray F, et al. Estimates of

worldwide burden of cancer in 2008: GLOBOCAN

2008. Int J Cancer 2010;127:2893-2917.

7. Pavlovich CP, Grubb RL 3rd, Hurley K, et al.

Evaluation and management of renal tumors

in the Birt-Hogg-Dubé syndrome. J Urol 2005;

l173:1482-6.

8. Khoo SK, Giraud S, Kahnoski K, et al. Clinical and


Med Genet 2002; 39:906–12.

9. Kluijt I, de Jong D, Teerstra HJ, et al. Early onset

of renal cancer in a family with Birt-Hogg-Dubé

syndrome. Clin Genet 2009; 75:537-43.


cancer and pneumothorax risk in Birt-Hogg-


carriers from 35 BHD families. Br J Cancer

2011;105(12):1912-9.

11. Pavlovich CP, Walther MM, Eyler RA, et al. Renal

tumors in the Birt-Hogg-Dubé syndrome. Am J of

Surg Pathol 2002; 26:1542-55.

12. Stamatakis L, Metwalli AR, Middelton LA, et al.


kidney cancer. Fam. Cancer 2013;12(3):397-402.

c H a P t e R 3 . 7a de novo FLCN mutation in a patient with

spontaneous pneumothorax and renal cancer; a clinical and molecular evaluation

Fam. Cancer 2013;12(3):373-9

Fred H. Menko1, Paul C. Johannesma2, R. Jeroen A. van Moorselaar3, Rinze Reinhard4, Jan Hein van Waesberghe4, Erik Thunnissen5,

Arjan C. Houweling1, Edward M. Leter1, Quinten Waisfisz1, Martijn B. van Doorn6, Theo M. Starink6, Pieter E. Postmus2,

Barry J. Coull7, Maurice A.M. van Steensel7 8, Johan J.P. Gille1

1Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands


3 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands 5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

6 Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands 7 Department of Dermatology, GROW School for Oncology and Developmental Biology,

Maastricht University Medical Centre, Maastricht; The Netherlands8 Department of Clinical Genetics, GROW School for Oncology and Developmental

Biology, Maastricht University Medical Centre, Maastricht; The Netherlands

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a B s t R ac t

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition due to germline FLCN

(folliculin) mutations, characterized by skin fibrofolliculomas, lung cysts, pneumothorax and

renal cancer. We identified a de novo FLCN mutation, c.499C>T (p.Gln167X), in a patient who

presented with spontaneous pneumothorax. Subsequently, typical skin features and asymptomatic

renal cancer were diagnosed. Probably, de novo FLCN mutations are rare. However, they may be

under-diagnosed if BHD is not considered in sporadic patients who present with one or more of

the syndromic features. Genetic and immunohistochemical analysis of the renal tumour indicated

features compatible with a tumour suppressor role of FLCN. The finding that mutant FLCN was

expressed in the tumour might indicate residual functionality of mutant FLCN, a notion which will

be explored in future studies.

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In 1977 Birt, Hogg and Dubé described a three-generation pedigree affected with multiple

fibrofolliculomas.1 Subsequently, it was shown that patients with Birt-Hogg-Dubé syndrome (BHD)

can also develop renal cancer and pneumothorax.2 3 The causative gene was mapped to chromosome

17p11.2 and germline mutations in the FLCN (folliculin) gene were identified in BHD families.4 FLCN

mutations have now been detected not only in classical BHD families but also in pneumothorax

and renal cancer patients and families.5 6 7 The spectrum of FLCN mutations has been outlined in

detailed reports8 9 10 and summarized in two databases.11 12 The function of folliculin is complex and

involves several molecular pathways including mTOR and vesicular transport.13 14 15 The European

BHD Consortium recently summarized diagnosis and management of this syndrome.16

In autosomal dominant tumour syndromes a varying proportion of index patients have a de novo

mutation, which is an alteration in a gene that is present for the first time in a family member as

a result of a mutation in a germ cell of one of the parents, or a mutation that arises in the fertilized

egg itself during early embryogenesis. For example, in Lynch syndrome (hereditary nonpolyposis

colorectal cancer) about 1-5% of patients have de novo mutations in DNA mismatch repair genes17,

whereas in familial adenomatous polyposis de novo APC mutations occur in about 10-25% of

cases.18 The phenomenon of de novo mutations is important from a clinical point of view, since

it implies that a negative family history for syndromic features does not exclude an autosomal

dominant condition.

To our knowledge, in BHD de novo mutations have thus far not been reported. Here we describe

a patient who presented with spontaneous pneumothorax due to a de novo FLCN mutation.

Subsequently, skin fibrofolliculomas were diagnosed and Magnetic Resonance Imaging (MRI)

of the kidneys revealed a small left-sided renal cancer, treated by partial left nephrectomy.

Histologically, the tumour was a chromophobe renal cancer. Since the molecular pathogenesis of

renal cancer in BHD is incompletely understood, we analysed the tumour for loss of heterozygosity

and expression of FLCN.


Patient and family data

The proband, a 30-year-old man, had recently been admitted to our hospital due to pneumothorax.

He was referred to the department of clinical genetics by the dermatologist, who had identified

multiple skin-coloured centro-facial papules histologically compatible with fibrofolliculomas typical

for Birt-Hogg-Dubé syndrome (fig. 1). We collected patient and family data and performed FLCN

mutation analysis. In addition, renal imaging was performed by MRI and renal ultrasound.

mutation analysis

For FLCN mutation analysis genomic DNA was extracted from blood samples after the patient gave

informed consent. Primers for the amplification and sequencing of the 14 exons were designed

as detailed by Nickerson et al.4 PCR amplification was performed using a PE 9700 thermocycler

(Applied Biosystems, Forster City, CA, USA). Sequencing reactions were performed using the Big

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Dye Terminator system (Applied Biosystems) and run on an ABI 3100XL or ABI 3730 genetic analyzer

(Applied Biosystems). For the detection of deletions and duplications of one or more exons MLPA

analysis was performed using MLPA kit P256 (MRC Holland, www.mrc-holland.com).

Paternity confirmation

To confirm paternity and exclude sample mix-up DNA samples of the patient and both parents were

analysed using the Powerplex 16 system (Promega Madison, WI, USA).

molecular analysis of the renal tumour

(a) Mutation analysis

Genomic DNA was isolated from paraffin-embedded tumour material with the Macherey-Nagel kit

for FFPE material (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions.

Tumour tissue was isolated by needle scraping of macroscopically visible cancerous areas. Loss

of heterozygosity was assessed by amplifying exon 6 using a Corbett Rotorgene 6000 (Qiagen,

Venlo, the Netherlands) real-time system (primer sequences and conditions available on request).

Sequencing reactions were performed with the PCR primers using the ABI BigDye terminator (v 1.1)

kit according to the manufacturer’s instructions and analyzed on an ABI 3100 capillary system

(Applied Biosystems, Carlsbad, CA, USA). Sequence traces were assembled and examined using the

PhredPhrap-Consed software package. The amplified tumour DNA was cloned into the pCR2.1-TOPO

vector (Invitrogen, Groningen, The Netherlands) to determine whether the second hit had occurred

in cis or in trans to the germline mutation.

(b) Immunohistochemistry

Procedures used for immunohistochemistry have been described in detail elsewhere.19 Polyclonal FLCN

antibody (rabbit) was a kind gift of professor Arnim Pause (McGill University, Montreal, Canada). Four

µm formalin-fixed, paraffin-embedded (FFPE) sections of tumour sample, obtained during surgery,

were deparaffinized in xylene and dehydrated through graded ethanol concentrations. Endogenous

peroxidase activity was blocked by incubation in 3% (w/v) hydrogen peroxide (H2O

2) in methanol for

figure 1. Multiple smooth facial papules in the proband.

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30 minutes, followed by microwave treatment using 10 mmol/L citrate buffer (pH 6) for 10 minutes

(90W) to facilitate antigen retrieval. Non-specific protein binding was blocked using 3% bovine-serum-

albumin in tris-buffered saline tween-20. Primary antibody was diluted in Dako Antibody diluent and

incubated for 1 hour at room temperature. Secondary detection was done by use of the Envision

detection system (Dako Netherlands BV, Heverlee, Belgium) for 30 minutes and bound antibody was

visualized by using 3,3-diaminobenzidine (DAB) for 10 minutes. Tissue was counterstained with Gill II

haematoxylin, dehydrated and coverslipped. Dako Washbuffer was used throughout for washing.

R e s u lt s

Patient and family data

Both parents and the two siblings of the proband were healthy. The multiple fibrofolliculomas in the

proband which started to develop from the age of about 25 years are depicted in fig. 1. A few weeks

prior to referral the patient had been treated for left-sided spontaneous pneumothorax. He had

experienced two previous episodes of left-sided spontaneous pneumothorax, at the ages of 19 and

22 years, treated by drainage and drainage plus tetracycline pleurodesis, respectively.

A recent thoracic Computer Tomography (CT) showed a left-sided pneumothorax (fig. 2) but no

intrapulmonary cystic lesions apart from a small left-sided subpleural apical bleb.

Since pneumothorax had recurred despite former drainage and pleurodesis, video-assisted

thoracoscopic surgery was performed, which revealed a small rim of fibrosis in the left upper lobe

probably due to collapsed bullae. Excision of the apex of the left upper lobe and pleurectomy

were performed. Histologically, collapsed bullae associated with mild pleuritis, minor local

emphysematous changes and a small rim of fibrosis with focal excentric intima fibrosis of the

pulmonary artery were observed (fig. 3).

Abdominal MRI revealed a 18 mm left renal lesion which - notably - was not detected on renal

ultrasound. The differential diagnosis was renal cancer or oncocytoma. A follow-up MRI with

contrast made three months later showed that the lesion had not grown (fig. 4). After weighing

the various management options (follow-up, nephron sparing treatment) a left partial nephrectomy

was performed, which revealed a pT1aN0 chromophobe renal cancer (fig. 5).

figure 2. Left-sided pneumothorax; of note, pulmonary cysts were absent at all levels.

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figure 3. (a) 2,5x (H & E staining) overview of a lung bulla (asterisks) in the collapsed resection specimen. (B) and (c)

(elastin and H & E stain, respectively) show thick elastotic alveolar walls and fibrosis in subpleural alveolar spaces and

local marginal fibrotic thickening of the pleura.

B c

A

figure 4. The asymptomatic left renal tumour in the

proband as revealed by MRI.

figure 5. Histopathology of the chromophobe

kidney cancer (overview, 2.5x); the insert (40x)

shows the monotonous cytonuclear appearance.

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figure 6. Electropherogram of part of exon 6 of FLCN. (A) sequence of the DNA of the patient showing the FLCN

mutation c.499C>T; due to the mutation codon 167, which is underlined, is changed from CAG encoding Gln into TAG

encoding a stop; (B) wild type sequence.

figure 7. Sequence traces of cloned FLCN exon 6 fragments amplified from tumor DNA showing the second hit mutation

in the bottom panel, with a wild type sequence in the top for comparison. Since the sequence trace only shows one

mutation (the second hit) and not the germ-line change (not shown due to space constraints), it must have occurred in

trans to the germ line mutation. The reverse strand is shown as it had the best read quality. The 15bp deletion is shown

in the shaded area and is emphasized with a continuous line. The dashed lines indicate how the sequence has changed.

The intron-exon boundary is indicated by a vertical arrow.

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molecular data

In the proband a pathogenic FLCN mutation c.499C>T, p.Gln167X, located within exon 6 was

identified (fig. 6), which was absent in his parents and both siblings who had no signs of BHD. VNTR

markers were tested in the patient and his parents. For all markers tested the patient showed one

maternal and one paternal allele thus confirming paternity and excluding sample mix-up.

Mutation analysis of the tumour indicated the presence of a second somatic hit, c.397-7_404del15,

which deletes the exon 6 splice acceptor site and which is expected to result in exon skipping and

production of a truncated protein. There was no material available for RNA isolation and therefore

we were not able to analyze the consequences of the mutation. Subsequent cloning of amplified

tumour DNA showed that the somatic mutation had occurred in trans to the germline mutation

(fig. 7). Immunohistochemical staining of the tumour showed robust FLCN expression; normal

kidney tubules and skin also expressed FLCN (fig. 8).

d i s c u s s i o n

Among the more than 35 BHD families with pathogenic germline FLCN mutations identified in our

centre, most of which have been published 20 21, this is the only de novo FLCN mutation identified.

This type of FLCN mutation, c.499C>T, p.Gln167X, can be classified as pathogenic and causative of

BHD since it leads to a premature stop. The FLCN mutation identified in this case has not been listed

figure 8. Immunohistochemical staining of the renal tumor with a polyclonal FLCN antibody. (a) Unaffected kidney tissue

of the proband. Strong FLCN expression throughout the kidney tubules, but not in a glomerulus. (B) The kidney tumour

shows pronounced staining, consistent with expression of one or both the mutant alleles. (c) Positive control: normal skin

from an unrelated healthy individual. Uniform staining in the epidermis and hair follicle as previously reported. Note the

presence of FLCN in sebaceous glands. (d) Negative control, secondary antibody only on normal skin.

A

c

B

d

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in the FLCN mutation databases.11 12 Since the proband had all three major manifestations of BHD,

i.e. fibrofolliculomas, pneumothorax and renal cancer, a somatic mosaic of the FLCN mutation is

unlikely. The absence of the mutation in both parents is consistent with a de novo mutation, but

somatic mosaicism in one of the parents cannot be excluded. We cannot correlate the clinical

expression in this patient to characteristics of the specific mutation since thus far, in BHD, no clear

genotype-phenotype correlations have been found.

To our knowledge, de novo FLCN mutations have not been reported previously. The frequency of

de novo FLCN mutations is probably low, but may be under diagnosed if BHD is not considered in

sporadic patients who show one or more of the syndromic clinical features.

For clinical diagnosis the time scale for the various manifestations of BHD is also relevant. For example,

pneumothorax may be the first manifestation in a patient who will develop fibrofolliculomas and /

or renal cancer at a later age. If pneumothorax is the only manifestation the diagnosis BHD may

easily be missed. In de novo cases, the absence of a positive family history for syndromic features

increases the difficulty of recognizing the condition. Skin lesions may be absent in up to 20% of

FLCN mutation carriers and if typical skin lesions are present they may not be recognized as marker

lesions for the syndrome.

Apart from the unusual cause of BHD in this patient - a de novo FLCN mutation - his clinical features

are also remarkable and are therefore considered below in some detail.

The recurrent spontaneous pneumothorax in this patient was initially interpreted as primary, which

is the common form of this disease. The pathogenesis of primary spontaneous pneumothorax (PSP)

is incompletely understood. Probably, PSP is associated with a focal inflammatory process related

to the pleura and underlying lung. Small subpleural blebs or larger subpleural bullae, which are

found in the majority of these patients, are only one feature of a complex and not fully understood

pathogenetic mechanism. It remains unclear whether these emphysema-like changes are the cause

of the air leak or merely a coincidental phenomenon. Subpleural blebs or bullae are also observed in

about 15% of the normal population.22 23

In BHD, spontaneous pneumothorax has been linked directly to multiple bilateral and mostly basally

located lung cysts, which are found in the majority of patients. In the patient described here thoracic

CT did not show lung cysts but only a left apical subpleural bleb. The absence of lung cysts in a BHD

patient who exhibits pneumothorax is remarkable: all 48 BHD patients with pneumothorax described

by Toro et al.24 had multiple lung cysts. However, in BHD, the occurrence of pneumothorax without

lung cysts on thoracic CT has been described previously.25 26 Thus, the relationship between lung

cysts and pneumothorax in BHD has not yet been fully clarified.

Few case studies have reported lung histopathology in BHD and in general the findings were non-

specific.5 27 28 29 30 In the case described here the surgical specimen showed subpleural fibrosis in

pre-existing alveolar walls associated with focal excentric intima fibrosis in a small pulmonary artery.

Since these are morphologic changes of a small infarction, due to local hypoxia, a local infarction

may have led to a local bulla.

The molecular pathogenesis of renal cancer in BHD has been evaluated in a series of studies. Vocke

et al.31 demonstrated a second somatic FLCN mutation or loss of heterozygosity in the majority (70%)

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of 77 BHD-associated renal tumours. These findings were interpreted as supportive for a tumour

suppressor role for FLCN.

By immunohistochemical staining we detected strong FLCN expression. In theory, this could be

due to expression in cells in which a wild type allele is still present. However, when sequencing

cloned tumour DNA in order to determine whether the second hit we found was indeed in trans,

we detected no wild type sequences. We therefore suggest that at least one of the mutant alleles

is expressed in the tumour. The second hit mutation is predicted to result in skipping of exon 6

in which case the proper reading frame for FLCN is maintained. This mRNA encodes for a mutant

protein lacking the amino acids encoded by exon 6 and detected by the FLCN antibody, thus

explaining the IHC result. As FLCN truncations are considered to result in a non-functional protein,

our observations are consistent with a tumour suppressor role for FLCN.

An important goal of diagnosing BHD is prevention of disease burden and death due to renal

cancer. In the present case, early diagnosis of a chomophobe renal cancer allowed curative surgical

treatment. The renal tumour identified with MRI was not detected by renal ultrasound. Undoubtedly,

MRI is more sensitive than ultrasound for the detection of small renal lesions and therefore MRI is

generally advised for surveillance of individuals at high renal cancer risk.

In summary, the case described here with a de novo FLCN mutation shows that BHD should

be considered in patients with a negative family history who present with one or more of the

syndromic features. We also showed that mutant FLCN can be expressed in BHD-associated

renal cancer, raising the possibility that BHD-associated mutations do not result in complete

loss of functionality.

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R e f e R e n c e s1. Birt AR, Hogg GR, Dubé WJ Hereditary multiple


acrochordons. Arch Dermatol 1977;113:1674–1677.

2. Roth JS, Rabinowitz AD, Benson M Bilateral renal

cell carcinoma in the Birt-Hogg-Dubé syndrome. J

Am Acad Dermatol 1993; 29: 1055-1056.

3. Zbar B, Alvord WG, Glenn G et al. Risk of renal and


in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol

Biomarkers Prev. 2002; 11: 393-400.

4. Nickerson ML, Warren MB, Toro JR et al. Mutations

in a novel gene lead to kidney tumours, lung wall

defects, and benign tumours of the hair follicle


Cancer Cell 2002; 2:157-164.

5. Graham RB, Nolasco M, Peterlin B et al Nonsense

mutations in folliculin presenting as isolated

familial spontaneous pneumothorax in adults. Am

J Respir Crit Care Med 2005; 172: 39-44.

6. Painter JN, Tapanainen H, Somer M A 4-bp deletion

in the Birt-Hogg-Dubé gene (FLCN) causes

dominantly inherited spontaneous pneumothorax.

Am J Hum Genet 2005; 76: 522-527.

7. Woodward ER, Ricketts C, Killick P et al. Familial



and mutation analysis of FLCN. Clin Cancer Res

2008; 14: 5925-5930.

8. Schmidt LS, Nickerson ML, Warren MB et al. Germline



syndrome. Am J Hum Genet 2005; 76: 1023-1033.

9. Toro JR, Wei MH, Glenn GM et al BHD mutations,




Genet 2008; 45: 321-331.

10. Benhammou JN, Vocke CD, Santani A et al

Identification of intragenic deletions and duplication

in the FLCN gene in Birt-Hogg-Dubé syndrome.

Genes Chromosomes Cancer 2011; 50: 466-477.

11. Wei MH, Blake PW, Shevchenko J, Toro JR The

folliculin mutation database: an online database

of mutations associated with Birt-Hogg-Dubé

syndrome. Hum Mutat 2009; 30: E880-890.

12. Lim DH, Rehal PK, Nahorski MS et al. A new

locus-specific database (LSDB) for mutations in

the folliculin (FLCN) gene. Hum Mutat 2010; 1:

E1043-1051.

13. van Steensel MA, van Geel M, Badeloe S et al.

Molecular pathways involved in hair follicle

tumor formation: all about mammalian target of

rapamycin? Exp Dermatol 2009; 18: 185-191.

14. Hasumi Y, Baba M, Ajima R et al. Homozygous loss

of BHD causes early embryonic lethality and kidney

tumor development with activation of mTORC1

and mTORC2. Proc Natl Acad Sci USA 2009; 106:

18722-18727.

15. Nookala RK, Langemeyer L, Pacitto A et al. Crystal

structure of folliculin reveals a hidden function in

genetically inherited renal cancer. Open Biol 2012;

2: 120071.

16. Menko FH, van Steensel MA, Giraud S et al

European BHD Consortium. Birt-Hogg-Dubé

syndrome: diagnosis and management. Lancet

Oncol 2009; 10: 1199-1206.

17. Win AK, Jenkins MA, Buchanan DD et al.

Determining the frequency of de novo germline

mutations in DNA mismatch repair genes. J Med

Genet 2011; 48: 530-534.

18. Aretz S, Uhlhaas S, Caspari R, Frequency and

parental origin of de novo APC mutations in familial

adenomatous polyposis. Eur J Hum Genet 2004;

12: 52-58.

19. Preston RS, Phil A, Claessens T et al. Absence of the

Birt-Hogg-Dubé gene product is associated with

increased hypoxia-inducible factor transcriptional

activity and a loss of metabolic flexibility.

Oncogene 2011; 30: 1159-1173.

20. Leter EM, Koopmans AK, Gille JJ et al Birt-Hogg-


families. J Invest Dermatol 2008; 128: 45-49.

21. Houweling, AC, Gijezen, LM, Jonker MA et al. Renal

cancer and pneumothorax risk in Birt–Hogg–


carriers from 35 BHD families. Br J Cancer 2011; 105:

1912-1919.

22. Noppen, M. Spontaneous pneumothorax:

epidemiology, pathophysiology and cause. Eur

Resp Rev 2010; 19: 217-219.

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23. Grundy S, Bentley A, Tschopp J-M. Primary

spontaneous pneumothorax: A diffuse disease of

the pleura. Respiration 2012; 83: 185-189.

24. Toro JR, Pautler SE, Stewart L et al. Lung cysts,



Respir Crit Care Med 2007; 175: 1044-1053.

25. Fröhlich BA, Zeitz C, Mátyás G et al. Novel mutations

in the folliculin gene associated with spontaneous

pneumothorax. Eur Resp J 2008; 32: 1316-1320.

26. Kluger N, Giraud S, Coupier I et al. Birt-Hogg-Dubé

syndrome: clinical and genetic studies of 10 French

families. Br. J. Dermatol. 2010; 162: 527-537.

27. Butnor KJ & Guinee Jr DG Pleuropulmonary

pathology in Birt-Hogg-Dubé syndrome. Am J

Surg Pathol 2006; 30: 395-399.

28. Ayo DS, Aughenbaugh GL, Yi ES et al Cystic lung

disease in Birt-Hogg-Dubé syndrome. Chest 2007;

132: 679-684.

29. Koga S, Furuya M, Takahashi Y et al Lung cysts in

Birt-Hogg-Dubé syndrome: histopathological

characteristics and aberrant sequence repeats.

Pathol Internat 2009; 59: 720-728.

30. Furuya M, Tanaka R, Koga S et al. Pulmonary cysts

of Birt-Hogg-Dubé: A clinicopathological and

immunohistochemical study of 9 families. Am J of

Surg Pathol 2012; 36: 589-600.

31. Vocke CD, Yang Y, Pavlovich CP et al. High

frequency of somatic frameshift BHD gene

mutations in Birt-Hogg-Dubé-associated renal

tumors. J Natl Cancer Inst 2005; 97: 931-935.

p a R t 4

summary, discussion and future perspectives

c H a P t e R

summary

Paul C. Johannesma1


4 . 1

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Before the introduction of DNA-based diagnosis in the 1990’s, hereditary tumour syndromes

could only be recognized on the basis of clinical characteristics. These characteristics

included typical features in the index patient and a positive family history. Since the 1990’s the

genetic background of most hereditary tumour syndromes has been identified. This allowed

diagnosing these conditions based on germline mutation analysis and presymptomatic DNA

testing in healthy at-risk family members. Moreover, diagnostic testing for these syndromes

could now be performed in clinically equivocal cases, for example, in cases with only one or two

minor features of the condition. Indeed, DNA testing showed that the hereditary syndromes,

previously defined by the occurrence of multiple syndromic features had a much wider and

more variable clinical spectrum than originally recognized. Finally, insight into the molecular

pathogenesis of these syndromes has led to the development of targeted therapies aimed at

the deranged signaling pathway or pathways involved. These developments have even led to

the renaming of hereditary tumour syndromes from a clinically based definition to a DNA based

definition, for example PTEN hamartoma tumour syndromes has replaced the former name of

Cowden disease.

Birt-Hogg-Dubé syndrome is a good example of the developments outlined above. In 1977,

the dermatologist Birt described an extensive kindred, investigated in cooperation with his

colleagues Hogg, pathologist and Dubé, internist. The affected family members showed

typical benign skin pathology, described by the authors as fibrofolliculomas, trichodiscomas

and acrochordons. The pedigree showed an autosomal dominant inheritance pattern with

high penetrance, in which most affected individuals developed skin lesions from the age of 25

years onward. Subsequently, it was recognized that renal cell cancer (RCC) and spontaneous

pneumothorax (SP) were part of the clinical syndrome. In 2002 the associated folliculin (FLCN)

gene was identified. DNA testing now allowed confirmation of the diagnosis at the DNA level

and DNA testing could be diagnostic in cases in which BHD was suspected. For FLCN mutation

carriers surveillance for RCC was recommended aimed at the early detection and treatment of

BHD associated RCC.

Although the fibrofolliculomas seem to be specific for BHD, both SP and renal cell cancer

generally occur as sporadic disease. Indeed, the clinical features of SP in BHD cannot be easily

distinguished from those occurring in the common sporadic primary forms. Renal cell cancer

in BHD may show the typical characteristics of hereditary predisposition, i.e. early age at

diagnosis, bilateral and multifocal disease and mixed histology patterns, but common unilateral

and unifocal clear cell renal cell cancer in a middle-aged or elderly patient may be due to BHD.

DNA testing has been performed in apparently sporadic RCC cases enriched for early onset

of disease and familial occurrence and indeed FLCN mutations were found in apparently non-

syndromic cases. In several studies it has been demonstrated that familial occurrence of SP

without evidence of skin or renal manifestations can be due to germline FLCN defects. Thus,

the skin features that originally defined BHD may be absent in FLCN mutation carriers who

present with either renal or pulmonary manifestations or are healthy family members of an

index case. Here we summarize and discuss the most important renal and pulmonary features

of BHD described in this thesis.

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Part 1: Pulmonary manifestations

The pathogenesis and natural course of lung cysts which develop in patients with BHD is still unclear

and the relationship between these cysts and the development of SP has not been fully clarified. In

chapter 1.1 we evaluated several of these aspects. Based on follow-up results of thoracic imaging in

six patients with BHD we proposed that the pulmonary abnormalities of BHD patients are not due

to progressive degenerative disease. Instead, in BHD, a decreased potential for stretching of the

cysts’ wall and extensive contact with the visceral pleura are likely to be responsible for rupture of

the cyst wall resulting in SP. We evaluated the reproducibility of measurements of size and number

of pulmonary cysts on CT in six patients with BHD based on the findings in baseline thoracic CT and

in follow-up imaging. We found no increase in size or number of pulmonary cysts within a period

of 44 months there was. We interpreted these findings as follows. If in BHD cyst formation and SP

would be signs of degenerative disease in BHD patients one would expect to find a higher incidence

of SP in older patients, which, however, has not been reported in literature.1 This differs from the

findings in other diseases characterized by cystic pulmonary abnormalities including pulmonary

lymphangioleiomyomatosis (PLAM) and pulmonary Langerhans cell histiocytosis (PLCH). Both

conditions are progressive disorders.2 In summary, we propose that the pulmonary abnormalities

of BHD patients are probably not due to progressive degenerative disease. Development and

recurrence of SP in BHD may well be related to the lack of possibility of epithelial layers to stretch if

forced to do so by connection to the visceral pleura.

Only a few studies in current available literature describe the thoracic CT appearance in BHD

patients, and are usually limited to several sporadic case reports or small retrospective studies

with a small number of included patients. What the relationship between lung cyst characteristics

and the development of (recurrent) SP is, has not been described and therefore we evaluated

the possible relationship between cyst characteristics and SP in BHD patients. We hypothesized

that chest computed tomography (CT) in this patient group might therefore be an useful tool for

choice of treatment when developing a SP and might also play a role in advice of lifestyle. Therefore

we evaluated in chapter 1.2 the findings of chest CT in a group of BHD patients with a history of

(recurrent) SP and compared these patients with a group of BHD patients without a history of SP. We

evaluated the radiological results of in total 61 patients, the largest study so far.

We only found a significant difference (p<0.001) in number of cysts between the group of BHD

patients with a history of SP, compared to patients without a relationship of SP. We found no

relationship (ρ=-0.027) between age and the number of cysts. Therefore we suggested that BHD

patients with a history of SP lead to more possibilities to suffer from cyst rupture than the non-SP

patients. As thoracic CT does not always show detectable lesions, which are visible during VATS,

a comparable study between radiological findings and in vivo VATS might therefore be necessary.3

Based on the fact that over 90% of BHD patients have clinically detectable cysts in basal parts of the

lung, we hypothesized in chapter 1.3 that use of a low dose chest CT might be an effective way to

detect this syndrome in patients presenting with apparently isolated PSP. Early diagnosis of BHD is

important for the patient and his or her family members. inheritance is autosomal dominant, and

the condition is associated with a lifetime risk of renal cell cancer of approximately15%. In this study

we included 46 pneumothorax patients, 19 of which had proven BHD based on a FLCN germline

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mutation and 27 were negative in FLCN mutation analysis, thereby excluding BHD as good as

possible. We found a higher prevalence of recurrent SP among patients with a proven pathogenic

FLCN mutation, a higher incidence of episodes of pneumothorax and a higher number of cysts.

On thoracic CT the distribution, location and size of the cysts differed significantly from those in

patients without BHD syndrome. We found that in BHD cases the majority of cysts had a size < 2cm

and this probably explains why these abnormalities cannot be detected using standard erect chest

X-ray. It is likely that in addition to air trapping other mechanisms play a role in the development of

a pneumothorax. In the apical parts of the lung the pleural stress is high and abnormalities in that

area are likely to increase the risk of rupture.4 In BHD-patients the majority of cysts are located close

to the pleura in the lower lobes, which makes it likely that the wall of cysts connected to the visceral

pleura ruptures easily.5 Important for this is the lack of stretching possibilities of the wall of lung cysts

of BHD patients.6 As such this study is the largest study so far in literature, the main limitation remains

the small number of patients we included. As this syndrome is relatively rare, it is difficult to gather

a large cohort of patients. Although there seems to be a clear distinction on thoracic CT between

BHD patients and patients without BHD, the rarity of this syndrome may still lead to unawareness

among doctors who have to evaluate these thoracic CT’s. Despite lacking information in this study

on all clinical information regarding smoking history, familial inheritance on pneumothorax and

prior (surgical) treatment of pneumothorax , we suggested that the radiological distinction between

BHD and patients without BHD can easily be made on a low dose CT scan of the thorax.

The first episode of PSP usually occurs in the third decade in males, who are often taller than age-

matched controls, and the majority has a history of smoking. Smoking increases the risk of PSP more

than 100 times.7 PSP diagnosis is usually based on history and confirmed by a standard erect chest

X-ray during inspiration. Although we showed in chapter 1.2 and chapter 1.3 a significant difference

in thoracic imaging among BHD patients and patients without BHD who had a history of SP, it is

radiologically still difficult to distinguish between BHD and smoking as a cause for SP. This difficulty

is discussed in chapter 1.4. Therefore we describe a possible role for TTF-1 staining of the inner cyst

wall, which might be specific for BHD.

In chapter 1.5 we assessed the relationship between air travel or diving and the occurrence of

spontaneous pneumothorax in a large cohort of BHD patients with a proven pathogenic FLCN

mutation. A questionnaire was sent to a cohort of 190 patients and the medical files of these patients

were evaluated. In total 158 (83.2%) patients returned the completed questionnaire. Sixty-one of 145

patients who had ever travelled by airplane had a history of SP (42.1%), with a mean of 2.48 episodes

(range 1-10), 24 (35.8%) had a history of bilateral episodes. Thirteen patients developed SP <1 month

after air travel and 2 patients developed a SP <1 month after diving. Symptoms possibly related to

undiagnosed SP were perceived in 30 patients (20.7%) after air travel, respectively in 10 patients

(18.5%) after diving. Based on the results reported in this chapter, we suggest that exposure of BHD

patients to considerable changes in atmospheric pressure leads to an increased risk of developing a

symptomatic pneumothorax. Symptoms reported during or shortly after flying and diving might be

related to the early phase of pneumothorax.

The literature regarding the risk for SP after diving in patients with lung cysts is extremely limited.

The British Thoracic Society (BTS) guideline recommends that diving is permanently avoided after

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an episode of spontaneous pneumothorax unless the patient has undergone bilateral surgical

pleurectomy and lung function and postoperative thoracic CT are normal.8 9 10 Our results are

comparable to the study results of the interstitial lung disease lymphangioleiomyomatosis with an

incidence of 1.1 pneumothoraces per 100 patients.11 12

Based on the study of Ren et al, which showed in 9.8% of cases a pathogenic FLCN mutation, among

102 patients with apparently primary SP (PSP), we evaluated in chapter 1.6 the prevalence of BHD

among patients with apparently primary spontaneous pneumothorax (PSP).13 Among the 40 patients

with apparently common PSP, three had pathogenic germline FLCN mutations and one of these

had a positive family history for pneumothorax. All three patients had multiple basal lung cysts.

Asymptomatic renal cell cancer was detected in a first- degree family member of an identified BHD

patient. The main limitations of this study were the low response rate and a possible selection of cases.

The response rate was low, since only 40 out of a total group of 316 patients (9.9%) were fully examined.

Patients who were invited for the study may preferentially have opted for this possibility due to

certain characteristics for example young age at diagnosis, high recurrence rate or a positive family

history for the disease. In addition, the letter in which the study was explained contained information

on characteristics of BHD patients i.e. skin lesions or a personal or family history of pneumothorax

of renal cancer and this may have encouraged selected individuals to participate. However, as the

results of the group of 40 patients is in line with the much larger study by Ren13 and colleagues in

consecutive cases with PSP, selection bias may not have been a major factor in this study.

The recurrence rate of SP in BHD has been described in literature to be as high as 75%, therefore

we retrospectively evaluated the effect of different types of treatment in chapter 1.7. Current BTS

and ACCP guidelines do not describe the treatment of SP in BHD patients as a separate entity. In

this study we compared the results of treatment in a comparable group of BHD and non-BHD

patients with (recurrent) SP. We found a recurrence rate of 64.5% after conservative treatment and

a recurrence rate of 11.1% after invasive treatment of SP in BHD patients. This recurrence rate was

significantly higher when compared to recurrence risk in patients without BHD. Therefore invasive

treatment might be the better option for BHD patients with (recurrent) SP. Our results suggest that

SP in BHD is associated with a high recurrence rate after conservative treatment and an invasive

therapy would therefore be the best approach in this group.

Part 2: Renal manifestations

In chapter 2.1 the clinical data of 115 FLCN mutation carriers from 35 BHD families are evaluated.

Among 14 FLCN mutation carriers who developed renal cancer 7 were <50 years at onset and/or

had multifocal/bilateral tumours. Five symptomatic patients developed metastatic disease. Two

early-stage cases were diagnosed by surveillance. The majority of tumours showed characteristics

of both eosinophilic variants of clear cell and chromophobe carcinoma. The estimated penetrance

for renal cancer and pneumothorax was 16% (95% minimal confidence interval: 6-26%) and 29% (95%

minimal confidence interval: 9-49%) at 70 years of age, respectively. The most frequent diagnosis

in families without identified FLCN mutations was the rare syndrome of familial multiple discoid

fibromas identified in a large Dutch family. Based on these results we confirm the importance of

surveillance for renal cancer in BHD patients. As renal tumours in BHD patients do not evidently

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differ from sporadic tumours in growth or histological pattern, it remains difficult to clinically

distinguish between BHD-associated and sporadic renal cancer.

The prevalence of BHD among patients with apparently sporadic RCC is unknown. The histological

subtype and clinical presentation of RCC in BHD are highly variable. In chapter 2.2 we showed

the results of our retrospective study among patients diagnosed with sporadic RCC, wherein we

retrospectively scored for the presence of lung cysts on thoracic CT. We performed FLCN mutation

analysis in 8 RCC patients with at least one lung cysts under the carina. No FLCN mutations were

identified. We compared the radiological findings in the FLCN negative patients to those in four

BHD patients and found multiple basal lung cysts were present significantly more frequent in FLCN

mutation carriers. We therefore advise that in all RCC patients at least a concise family history is taken

for the presence of RCC or SP and that the skin is examined for the presence of fibrofolliculomas. In

the presence of a positive family history (SP or RCC) or multiple basal lung cysts further investigation

of BHD is indicated (e.g. by dermatological evaluation or by DNA testing). The difficulty in unmasking

BHD patients in apparently sporadic RCC patients is illustrated in this study by the negative family

history for pneumothorax and RCC in the two FLCN mutation carriers.

Notably, skin lesions can also be early signs of hereditary predisposition for RCC in other syndromes,

in particular hereditary leiomyomatosis and renal cell cancer.

In current literature renal surveillance in BHD is recommended, but the optimal imaging method and

screening interval remain to be defined. In chapter 2.3 we retrospectively evaluated the compliance

to, and the outcomes of renal cancer surveillance in patients diagnosed with BHD in two centers.

Screening data of 199 patients diagnosed with BHD in two hospitals were collected. All available

renal imaging follow up data and the medical records of 23 BHD patients with renal cell carcinoma

(RCC) were collected. Initial screening was performed in 171/199 patients (86%) and follow up data

were available from 117/171 patients (68%). The total follow-up period was 499 patient years. Of the

patients that performed follow-up screening, 85% was screened at least yearly and 96% at least

every two years. A medical history of RCC was present in 23 patients, 38 tumours were diagnosed

with a mean age of the diagnosis of the first tumour at 51 years. In 21 tumours ultrasound (US)

was performed. Eleven tumours, sized 7-27 mm, were visible on MR or CT and not detected using

US. This study indicated that compliance to renal screening is relatively high and that US might

be a sensitive, cheap and widely available imaging modality for detecting clinically relevant renal

tumours in BHD patients, since no tumours exceeding 3 cm were missed with US.

Part 3: Relevant case reports and case series

In chapter 3.1 we evaluated a patient with BHD that developed pneumothorax following flying and

was manifested later. We consider the pressure changes during the subsequent flights as potential

trigger for initiating rupture of a subpleural cyst, this implies that the interval between air travel

and the diagnosis is important. In other studies 50% of cysts were located in the subpleural area

and less than 5% abut on bronchioles. So if a cyst would be connected to the bronchial tree, the

size of connection to the airways is very small, resulting in small volumes of air transported into the

pleural cavity. Therefore it will probably take a long time before troublesome symptoms are present.

Also spontaneous resolution of a small pneumothorax after rupture of a subpleural cyst may occur

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if there is no active transport of gas into the pleural cavity. Based on this theory we concluded

that BHD patients who have minimal chest symptoms after the first flight should be checked for

pneumothorax before the return flight.

Although clinical manifestation usually appears after the age of 20, we discussed in chapter 3.2 two

cases of BHD wherein episodes of (recurrent) pneumothorax occurred from the age of 14. Lung cysts

were seen in both patients, mainly in the basal parts of the lung. SP has been reported only twice in

pediatric pathogenic FLCN-mutation carriers. 14 15 As SP in the pediatric population is relatively rare,

BHD should be considered as underlying cause, especially when there is a positive family history

for pneumothorax. Easy accessible genetic testing for BHD in pediatric patients with (recurrent)

spontaneous pneumothorax should be performed, even when skin manifestation is absent. Based

on this case series, more research on the prevalence of BHD in the pediatric population with

a history of (recurrent) spontaneous pneumothorax might be needed.

In chapter 3.3 we describe the case of a former Olympic swimmer who was referred to our hospital

due to recurrent episodes of pneumothorax. Although the patient never smoked and the patient

had multiple recurrences of SP, BHD was not suggested in this patient. This case shows the difficulty

of recognizing this syndrome and the difficulty to distinguish this syndrome from common PSP.

An illustrative kindred is presented in chapter 3.4 in which the index patient had recurrent episodes

of pneumothorax without apparent skin lesions or renal abnormalities. He had bilateral basally

located lung cysts. Family members had fibrofolliculomas, lung cysts, pneumothorax and clear cell

renal cancer. The described family highlights the importance for doctors to ask for family history

regarding pneumothorax and renal cancer.

In chapter 3.5 we evaluated a patient who presented with skin fibrofolliculomas. Based on the

diagnosis of multiple fibrofolliculomas BHD was suspected and an abdominal CT was performed. The

abdominal CT showed a solid intrapolar tumour in the lower pole of the right kidney. We concluded

in this chapter that BHD should be considered when facial fibrofolliculomas are diagnosed and

consequently relatives should be stimulated to undergo genetic testing.

A patient with apparently sporadic chromophobe renal cell cancer is discussed in chapter 3.6. As the

patient had a positive history for spontaneous pneumothorax and renal cancer, BHD was suspected,

which was confirmed after molecular testing. We evaluated in this chapter the importance of

recognizing this autosomal dominant cancer disorder when a patient is presented at the urologist

with a positive family history of chromophobe renal cell cancer, or a positive familial history for renal

cell cancer and pneumothorax.

Finally, in chapter 3.7 we describe a patient who was referred to the clinical geneticist due to

fibrofolliculomas. Although thoracic CT showed no lung cysts, the patient had a history of recurrent

episodes of pneumothorax. As BHD was suspected, an additional abdominal MRI was performed

which showed an asymptomatic tumour in the left kidney. A pathogenic FLCN germline mutation

was found in this patient, which was not found in both parents. This is the first publication that

describes a de novo FLCN mutation. De novo FLCN mutations are probably rare, but might be

under-diagnosed. We suggest that BHD should be considered in patients who present with one or

more of the syndromic features despite a negative family history.

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R e f e R e n c e s1. Toro JR, Pautler SE, Stewart L, et al. Lung cysts,



syndrome. Am. J. Resp. Crit. Care Med. 2007;

175:1044-1053.


2013;66: 904-908.



syndrome. Ann Thorac Surg. 2014;97:682-685.

4. Casha AR, Manché A, Gatt R, et al. Is there

a biomechanical cause for spontaneous

pneumothorax? Eur J Cardiothorac Surg.

2014;45:1011-1016.




Respirology 2014; 19: 1248-1250.

6. Medvetz DA, Khabibullin D, Hariharan V, et al.

Folliculin, the product of the Birt-Hogg-Dubé

tumor suppressor gene, interacts with the

adherens junction protein p0071 to regulate

cell-cell adhesion. PLoS ONE 2012;7: e47842.

7. Smit HJ, Chatrou M, Postmus PE. The impact of

spontaneous pneumothorax, and its treatment,

on the smoking behavior of young adult smokers.

Respir Med. 1998;92:1132-1136.





2:ii18-31.


pneumothorax and risk of air travel in Birt-Hogg-


185:A4438.



136:655-656.




fibrosis, and sarcoidosis. Chest 2009; 136:665-670.



Thorax 2007; 62:1756-1780.




pneumothorax. Clin Genet 2008; 74:178-183.

14. Bessis D, Giraud, Richard S, et al. A novel familial



syndrome. Br J Dermatol. 2006;155:1067-1069.

15. Gunji Y, Akiyoshi T, Sato T, et al. Mutations in the

Birt-Hogg-Dubé gene in patients with multiple


Genet 2007;44:588-593.

c H a P t e R

nederlandse samenvatting (voor leken)

Paul C. Johannesma1

1 Afdeling Longziekten, VU Medisch centrum, Amsterdam, Nederland

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Vóór de introductie van diagnoses middels DNA onderzoek - wat sinds 1990 mogelijk is - , werden

erfelijke tumor (kanker) syndromen alleen herkend middels een verzameling van uiterlijke

karakteristieken. Deze klinische (uiterlijke) karakteristieken bevatten kenmerken die bij de patiënt

gezien werden, waarbij er tevens andere familieleden waren met dezelfde klinische kenmerken.

Sinds 1990 zijn in toenemende mate erfelijke tumor syndromen op DNA niveau geïdentificeerd. Op

basis hiervan kunnen tegenwoordig “gezonde” patiënten – waarbij een familielid belast is met een

tumor syndroom - gescreend worden op DNA niveau om het erfelijke syndroom te bevestigen.

Een voordeel van het testen middels DNA, is dat bij patiënten met minimale uiterlijke kenmerken

het syndroom al bevestigd kan worden en hier in een vroeg stadium op geanticipeerd kan worden.

Doordat er tegenwoordig veel wetenschappelijk onderzoek gedaan wordt op DNA-niveau

naar tumorsyndromen, worden ook in toenemende mate meer verbanden tussen syndromen

ontdekt en worden er meer gerichte therapieën ontwikkeld voor het behandelen van de klinische

verschijnselen van deze syndromen.

Een goed voorbeeld van een syndroom dat in eerste instantie werd gediagnosticeerd middels

uiterlijke kenmerken, maar vele jaren later werd bevestigd op DNA niveau, is het zogenoemde

Birt-Hogg-Dubé syndroom. In 1977 beschreven de huidarts dr. Birt, de patholoog dr. Hogg en de

internist dr. Dubé een grote familie met dezelfde uiterlijke kenmerken, dat werd geduid als het Birt-

Hogg-Dubé syndroom. De familieleden hadden allen dezelfde karakteristieke huidafwijkingen, die

de drie dokters “fibrofolliculomen”, “trichodiscomen” en “acrochordons” noemden. De stamboom

liet een autosomale dominante overerving zien ( dus geen voorkeur voor geslacht, 50% kans op

overerving door volgende generatie) met uiterlijke kenmerken vanaf de leeftijd van 25 jaar. Later

ontdekte men dat het syndroom tevens gepaard ging met een verhoogde kans op het ontwikkelen

van nierkanker en (terugkerende) spontane klaplong (pneumothorax). In 2002 werd het syndroom

ontdekt op DNA niveau bij een afwijking op de korte arm van het 17e chromosoom. Doordat het

syndroom nu bevestigd kon worden op DNA niveau, is het tegenwoordig mogelijk nierkanker in

een vroeg stadium te ontdekken en behandelen.

Hoewel de huidafwijking “fibrofolliculoom” zeer specifiek is voor het Birt-Hogg-Dubé syndroom, zijn

de andere klinische kenmerken (klaplong en nierkanker) bij dit syndroom moeilijk te onderscheiden

van de niet-syndromale klaplong en nierkanker. Daarnaast komen de huidafwijkingen, nierkanker

en klaplong niet bij alle patiënten met dit syndroom tegelijkertijd voor, daarom is het soms lastig om

een patroon te vinden en het syndroom te herkennen.

De focus in dit proefschrift ligt op de afwijkingen in de longen en de nieren bij patiënten met het

BHD syndroom. Door de uiterlijke kenmerken, lichamelijke klachten en familiaire patronen van deze

patiëntengroep beter in kaart te brengen en verbanden aan te tonen, hopen we in de toekomst het

syndroom sneller te herkennen, wat zal leiden tot een snellere en meer optimale behandeling. De

belangrijkste bevindingen van dit proefschrift worden hieronder beschreven.

deel 1: Pulmonale (long) karakteristieken binnen het Birt-Hogg-dubé syndroom

Tot op heden is het nog steeds onduidelijk hoe cysten (holten) in de longen bij patiënten met het Birt-

Hogg-Dubé syndroom (BHD) zich ontwikkelen en waarom ze alleen onderin de longen voorkomen. In

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hoofdstuk 1.1 hebben we een follow-up (in tijd opvolgende) studie gedaan onder 6 patiënten met het

BHD syndroom. Hierin toonden we aan dat, hoewel cysteuze longaandoeningen normaal gesproken

progressief (verergerend) van aard zijn, dit niet het geval is bij patiënten met BHD. We veronderstelden

(hypothese) dat de holten in mindere mate kunnen oprekken, waardoor deze barsten. Doordat de

holten barsten kan lucht uit de long stromen en ontwikkelt de patiënt een klaplong. We evalueerden

de CT-scan van 6 patiënten, waarbij we de scan na 44 maanden bij dezelfde patiënten herhaalden en

vonden geen groei in grootte, aantal, locatie of vorm van de cysten (holten). We legden deze bevinding

naast de bevinding dat oudere patiënten met het BHD syndroom niet vaker een klaplong ontwikkelden,

en concludeerden op basis van deze gegeven dat pulmonale (long) BHD geen progressieve aandoening

is. Dat patiënten met het syndroom een klaplong ontwikkelen is dus mogelijk meer het gevolg van

drukverschil waardoor de holten knappen en dus niet leeftijd gerelateerd.1 2

In hoofdstuk 1.2 stelden we de hypothese dat een CT-scan van de longen een belangrijke rol

van betekenis kan spelen in het opstellen van de juiste behandeling van klaplong en het geven

van de juiste leefstijl adviezen. Daarom evalueerden we in dit hoofdstuk alle BHD patiënten met

een beschikbare CT-scan en deelden we deze patiënten op in twee groepen; met of zonder

doorgemaakte klaplong. We includeerden hiervoor 61 met bewezen BHD. De grootste studie tot

nu toe wereldwijd. Het enige verschil in de twee groepen bleek het aantal cysten in de longen te

zijn, waarbij er geen relatie met leeftijd was. Derhalve concludeerden we dat er mogelijk een relatie

tussen het aantal cysten en de kans op het ontwikkelen van een klaplong kan zijn.3

Meer dan 90% van alle patiënten met BHD hebben één of meer cysten in de onderste longvelden.

Derhalve kunnen cysten in de ondere longvelden een manier zijn om het BHD syndroom te herkennen

op een CT scan, wanneer een patient zich presenteert met (alleen) primaire spontane pneumothorax

Deze hypothese testten wij in hoofdstuk 1.3. We includeerden 46 patiënten, waarbij bij 19 patiënten het

BHD syndroom was bevestigd middels DNA-diagnostiek en bij 27 (controle) patiënten een pathogene

DNA mutatie was uitgesloten. In de groep patiënten met BHD vonden we een hogere incidentie

(voorkomen) van pneumothorax en een hoger aantal cysten in de longen. Daarnaast presenteerden

de cysten zich meer onderin de longen, en was er meer verschil in grootte van de cysten binnen de

BHD populatie ten opzichte van de controlegroep. De grote meerderheid van de cysten waren <2cm in

de BHD groep, derhalve zijn deze cysten waarschijnlijk niet te zien op een X-thorax.4 5 6

Hoewel het aantal geïncludeerde patiënten klein was, en het syndroom relatief zeldzaam is,

concludeerde we dat de CT scan van BHD patiënten toch echt duidelijk anders dan de CT scan van

niet-BHD patiënten. Dat veel dokters dit syndroom niet kennen, leidt mogelijk toch het veelvuldig

missen van deze aandoening in de praktijk.

In hoofdstuk 1.4 bespraken we opnieuw de meerwaarde van een CT-scan voor het diagnosticeren

van het BHD syndroom. We evalueerden welke factoreren invloed kunnen hebben op het missen van

het BHD syndroom bij het beoordelen van een CT scan, waarbij we met name zijn ingegaan op de

invloed van roken, waarbij een zelfde soort holten zich kunnen ontwikkelen in de longtop. Tenslotte

suggereerden we een mogelijke rol voor TTF-1 (long)kleuring als specifieke marker voor BHD.

In hoofdstuk 1.5 onderzochten we de relatie tussen vliegen of duiken en het ontwikkelen van een

klaplong onder 158 patiënten met het BHD syndroom. Hierbij waren we met name geïnteresseerd

of BHD patiënten (met longcysten) een verhoogd risico lopen op het ontwikkelen van een klaplong

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door drukverschil. We vonden een klein verhoogd risico voor het ontwikkelen van klaplong na

duiken of vliegen. Daarnaast hadden 1 op de 5 patiënten klachten als benauwdheid en pijn op

de borst tijdens duiken of vliegen. Het is niet met zekerheid te zeggen dat deze klachten ook

gerelateerd zijn aan het BHD syndroom. De resultaten in deze studie zijn vergelijkbaar met andere

cysteuze longaandoeningen zoals lymfangioleiomyomatosis. Huidige internationale richtlijnen

geven tot op heden geen specifiek advies betreft duiken en vliegen onder patiënten met een

cysteuze longaandoening.7 8 9 10 11

In hoofdstuk 1.6 onderzochten we de prevalentie van het BHD syndroom onder 40 patiënten met

een doorgemaakte klaplong, dat was geduid als primaire spontane pneumothorax. We vonden dat

3 van de 40 patiënten een pathogene mutatie had passend bij het BHD syndroom. 12 Alledrie de

patiënten hadden meerdere longcysten op de CT-scan en bij een eerstegraads familielid vonden we

nierkanker bij verder screenen van de familie.

Ongeveer drie op de vier BHD patiënten met een klaplong ontwikkelt opnieuw een klaplong na

reguliere behandeling. Dit is veel hoger percentage dan patiënten met een klaplong die geen BHD

syndroom hebben. Voor de behandeling van een klaplong wordt er op dit moment geen onderscheid

gemaakt tussen wel of geen BHD hebben. We vonden in hoofdstuk 1.7 een recidief percentage

van 65.5% na conservatieve behandeling (afwachten/zuigdrainage/ naald-decompressie) en 11.1%

na invasieve behandeling (plakken, opruwen longvlies, chirurgisch wegsnijden longblaasjes in de

longtop en/of longvlies). Dit recidief risico is veel hoger dan onder niet-BHD patiënten. Een initiële

invasieve behandeling lijkt hierdoor de voorkeur te genieten.

deel 2: Renale (nier) karakteristieken binnen het Birt-Hogg-dubé syndroom

In hoofdstuk 2.1 werden de patiëntkenmerken van 115 patiënten met BHD geëvalueerd. Binnen deze

groep hadden veertien patiënten nierkanker doorgemaakt, waarvan zeven patiënten jonger dan 20 jaar

waren en/of een niertumor hadden in beide nieren en/of op meerdere plaatsen in dezelfde nier. Vijf van

deze zeven patiënten ontwikkelden uitzaaingen. Van deze vijf patiënten waren er twee die bij initiële

beeldvorming al gediagnosticeerd werden met uitzaaingen elders in het lichaam. De meerderheid

van de patiënten hadden een niertumor met eosinofiele en/of heldercellige en/of chromophobe

histologische kenmerken. We berekenden een kans van 16% op het ontwikkelen van nierkanker (tot het

70e levensjaar) en een kans van 29% op het ontwikkelen van klaplong (tot het 70e levensjaar).

De prevalentie van het BHD syndroom in patiënten met sporadisch nierkanker is onbekend. Dit komt

mede doordat op basis van de histologie het onderscheid lastig te maken is tussen BHD en niet-BHD

patiënten. In hoofdstuk 2.2 onderzochten we hoeveel patienten met nierkanker, longcysten op

een CT-scan hadden. De patiënten met longcysten werden hierna getest op BHD, waarbij we geen

nieuwe patiënten vonden met het BHD syndroom. We vergeleken deze getestte patiënten met

vier patiënten met nierkanker en het BHD syndroom, en vonden in de laatste groep significant

meer longcysten. We benadrukten in dit hoofdstuk nogmaals het belang van het uitvragen van een

volledige (familie) anamnese om het BHD syndroom te diagnosticeren.

De optimale follow up van de nieren bij BHD patiënten is tot op heden onduidelijk. In hoofdstuk

2.3 hebben we middels een retrospectieve studie gekeken naar de resultaten hiervan onder 199

BHD patienten (VUmc en NKI-AvL). Initiële screening werd verricht onder 171 patiënten (86%),

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waarbij 85% zich jaarlijks liet screenen en 96% zich 1x per 2 jaar liet screenen. We vonden bij 23

patiënten een niertumor (totaal 38 tumoren) , bij een gemiddelde leeftijd van 51 jaar. Daarnaast

lijkt echo een betrouwbaar en goedkoop instrument te zijn voor deze screening, aangezien geen

tumoren boven de 3 cm gemist waren.

deel 3: Relevante casuistiek

In hoofdstuk 3.1 beschrijven we de casus van een BHD patient die een klaplong ontwikkelde vlak

na een vliegreis. We stelden in dit hoofdstuk, dat cysten tijdens de vlucht door drukverschil kunnen

knappen, maar het tijd kost daarna om een klaplong te ontwikkelen. Dit zou mogelijk kunnen

komen omdat maar 5% van de cysten verbonden is met de luchtwegen. Minimale klachten na een

vliegreis zou derhalve een alarmsymptoom moeten zijn voor een mogelijk aanwezige klaplong.

Hoewel in literatuur klachten bij het BHD syndroom worden beschreven vanaf de leeftijd van 20 jaar,

beschrijven we in hoofdstuk 3.2 twee patiënten met terugkerende episoden van klaplong vanaf

de leeftijd van 14 jaar. Hoewel er vaker klaplong in de familie voor kwam, werd er in eerste instantie

niet gedacht aan het BHD syndroom. Beide patiënten werden getest voor het BHD syndroom en het

syndroom werd bij beiden bevestigd. In dit hoofdstuk pleiten wij dan ook voor laagdrempelig inzetten

van genetisch onderzoek bij patiënten met een spontane klaplong, óók bij een leeftijd onder de 18 jaar.

In hoofdstuk 3.3 beschrijven we de casus van een voormalig Olympisch zwemmer die in het

verleden meermaals een episode van klaplong had doorgemaakt. Na genetisch onderzoek werd het

BHD syndroom bevestigd. We benadrukken in dit hoofdstuk nogmaals hoe moeilijk het is klaplong

door het BHD syndroom te onderscheiden van spontane klaplong zonder onderliggende ziekte.

In hoofdstuk 3.4 beschrijven we een familie waarbij de patiënt meermaals klaplong had doorgemaakt

zonder bijgaande nier- of huidafwijkingen. Deze nieren huidafwijkingen kwamen wel voor in de

familie. In dit hoofdstuk wordt benadrukt dat een adequate familie-anamnese onmisbaar is voor het

stellen van de diagnose BHD.

In hoofdstuk 3.5 beschrijven we een patiënt met het BHD syndroom waarbij – door het herkennen

van de huidafwijkingen passend bij BHD – een CT scan van de nieren verricht werd, en een niertumor

gevonden werd. Hierna kon de diagnose BHD makkelijk gesteld en – middels DNA onderzoek-

bevestigd worden.

In hoofdstuk 3.6 staat een patiënt centraal waarbij aan BHD werd gedacht doordat de patient zowel

nierkanker als meermaals klaplong had doorgemaakt. De boodschap in dit hoofdstuk is met name

gericht aan urologen, om aan BHD te denken wanneer een patiënt met nierkanker tevens klaplong

heeft doorgemaakt.

Tenslotte beschrijven we in hoofdstuk 3.7 een patiënt die doorgestuurd was door de dermatoloog

naar de klinisch geneticus in verband met verdenking op BHD. Hoewel de CT van de longen geen

cysten liet zien, werd er wel een niertumor gevonden op de MRI van de nieren. De diagnose BHD

werd middels DNA bevestigd. Hierop werden beide (biologische) ouders getest, die beiden geen

pathogene mutatie hadden. Derhalve stellen wij dat “de novo” mutaties mogelijk vaker voorkomen

dan gedacht. De familie-anamnese kan dus ook negatief zijn voor klaplong, huidafwijkingen of

nierkanker bij het BHD syndroom.

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R e f e R e n t i e s1. Toro JR, Pautler SE, Stewart L, et al. Lung cysts,



syndrome. Am. J. Resp. Crit. Care Med. 2007;

175:1044-1053.


2013;66: 904-908.



syndrome. Ann Thorac Surg. 2014;97:682-685.

4. Casha AR, Manché A, Gatt R, et al. Is there

a biomechanical cause for spontaneous

pneumothorax? Eur J Cardiothorac Surg.

2014;45:1011-1016.




Respirology 2014; 19: 1248-1250.

6. Medvetz DA, Khabibullin D, Hariharan V, et al.

Folliculin, the product of the Birt-Hogg-Dubé

tumor suppressor gene, interacts with the

adherens junction protein p0071 to regulate

cell-cell adhesion. PLoS ONE 2012;7: e47842.





2:ii18-31.


pneumothorax and risk of air travel in Birt-Hogg-


185:A4438.



136:655-656.




fibrosis, and sarcoidosis. Chest 2009; 136:665-670.



Thorax 2007; 62:1756-1780.





c H a P t e R

future perspectives

Paul C. Johannesma1


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In 1977, Birt-Hogg-Dubé syndrome (BHD) was described by three physicians (Arthur Birt, Georgina

Hogg and James Dubé)1. Since then, the molecular, genetic and clinical aspects have been

elucidated, and nowadays we have a much more detailed understanding of this rare, autosomal

dominant, hereditary disorder. The BHD-associated locus was mapped to chromosome 17p11.2 by

linkage analysis. The associated gene was identified in 2001, the starting point of exciting times for

researchers who conduct FLCN functional studies, and clinicians who manage BHD patients.

At the Department of Dermatology in VU Medical Center (VUmc), based on expertise in skin

adnexal tumours, a series of BHD patients had already been evaluated when - in 2001 - the FLCN

gene was identified. This led to the set-up of FLCN mutation analysis and evaluation of BHD

patients and families in collaboration with the Department of Clinical Genetics. Subsequently,

a multidisciplinary Birt-Hogg-Dubé working group. was established which resulted in publications

on BHD in 2008 and 2009 2 3

National cooperation was requested in order to collect clinical and molecular data on a large group

of Dutch BHD kindreds and colleagues from all clinical genetics centers were willing to contribute

to a central BHD database.

At present, BHD can be diagnosed by FLCN mutation analysis in clinically equivocal cases: BHD can

be diagnosed in patients with facial fibrofolliculomas, but also in patients with apparently primary

spontaneous pneumothorax (PSP) and sporadic renal cell cancer (RCC). At present, in 2016, we have

evaluated over 90 families with over 250 family members with a proven pathogenic FLCN germline

mutation. This cohort – which is one of the largest worldwide - has provided an unique opportunity

for research and can be the basis for collaborative research in the future.

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Pulmonary aspects of Birt-Hogg-dubé syndrome

Prevalence of BHD among pneumothorax patients

The incidence of primary spontaneous pneumothorax (PSP) is estimated to be approximately 1.2-18

per 100.000 per year.4 5 A positive family history for PSP is found in more than 10% of individuals

who present with spontaneous pneumothorax.6 PSP may occur in patients with several inherited

monogenic disorders.7 BHD seems to be an increasing topic of interest in current literature.

A prospective study of a Chinese patient cohort (n=102) with apparently PSP showed a prevalence

of 9.8% of cases with an underlying pathogenic FLCN germline mutation.8 Our study confirmed this

result since we found a prevalence of 7.5% FLCN mutation carriers among 40 randomly selected

apparently PSP patients.9 The main limitations of our study were the low response rate and the

selection of cases. Additional (international) prospective studies should lead to further insight into

the prevalence of BHD in apparently sporadic PSP. Based on these data any differences in response

to therapy of pneumothorax and the optimal diagnostic tools in diagnosing BHD among apparently

non hereditary PSP cases can be evaluated.

In this thesis we propose that an attractive diagnostic routine for PSP patients may consist of thoracic

CT as the first test, followed by FLCN mutation analysis in patients suspected for BHD based on the

findings on CT scan. Clearly, expert dermatological examination and detailed family history taking

may contribute to a suspected diagnosis of BHD. In children with pneumothorax, as a diagnostic

approach low dose CT might be considered to detect congenital or hereditary abnormalities, as

performed in the case series described in chapter 3.2 .

A collaboration between the departments of Pulmonary Diseases of the Rijnstate Hospital and

VUmc has been set up to investigate the prevalence of BHD among apparently PSP patients in a non-

academic patient population (n=200).

Development and natural course of lung cysts and pneumothorax in BHD

The development and natural course of lung cysts in patients with Birt-Hogg-Dubé syndrome is

still unclear and the relationship between the cysts and the development of pneumothorax has not

been clarified.To understand more of this it is necessary to follow BHD-patients with lung cysts for

several years and evaluate the found abnormalities after long intervals, preferably in those without

any interference by an episode of pneumothorax. Knowledge of this may have consequences for

the treatment in case of a pneumothorax. Questions such as, should larger cysts be resected if

adjacent to the pleura, or is a procedure to adhere both pleural layers sufficient, are difficult to

answer without this long-term evaluation. Furthermore, it is unclear whether in the long term there

will be a diminished pulmonary function due to destruction of lung tissue, repeated evaluation of

lung function by physiological tests is therefore needed.

Why do cysts rupture?

In this thesis we investigated the relationship between lung cysts and spontaneous pneumothorax

by testing several hypothesis in different studies.10 11 12 We concluded that decreased potential

for stretching of the cyst wall and the extensive contact with the visceral pleura are likely to be

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responsible for rupture of the cyst wall resulting in the increased risk for pneumothorax in BHD

patients. The VUmc BHD working group will extend their focus in the near future on the pathogenesis

of pulmonary cysts and the relationship between these cysts and the development of (recurrent)

spontaneous pneumothorax (SP). Currently several international laboratories perform research to

understand why pulmonary cysts in BHD patients rupture, and if there is a relationship between cyst

rupture and pneumothorax. A suggestion is that cysts in BHD arise because of fundamental defects

in cell-cell adhesion, leading to repeated respiration-induced physical stretch–inducted stress and,

over time, expansion of alveolar spaces, particularly in regions of the lung with larger changes in

alveolar volume and at weaker “anchor points” to the pleura.13 Another suggestion is that the BHDS

cysts possibly expand in size as the alveolar walls disappear at the alveolar-septal junction, and grow

even larger when several cysts fuse. As only 5% of the cysts connect to the bronchial three, cyst can

easily rupture, which might lead to the development of pneumothorax.14

Radiological thoracic imaging might also play an important role for a better understanding of

this question. Rare conditions – like BHD - associated with an increased risk of SP may remain

unrecognized following the current guidelines for diagnosis. In current literature results of

radiological thoracic imaging in BHD patients is very limited and reviewed in very small numbers of

patients.15 16 17 18 19 20 21. We concluded that cysts seem to be related to (recurrent) pneumothorax in

BHD patients, and several radiological parameters can suggest the presence of BHD. Therefore we

conclude that CT scanning can be a useful tool in the detection of BHD patients presenting with an

apparently isolated PSP. Secondly, periodic thoracic imaging in BHD patients might lead to a better

understanding of the mechanism of cyst cysts in BHD patients and possibly (partly) elucidate the

pathogenesis of PSP.

To evaluate our stretch hypothesis, a study in association with the University of Cincinnati (Ohio,

United States; dr. N. Gupta) is set up to evaluate the impact of air travel and diving on spontaneous

pneumothorax in a larger cohort of patient with pathogenic FLCN mutation, based on the material

and methods and results presented in this thesis.11

In addition, it might be worthwhile to evaluate in a prospective study of RCC and PSP patients on the

costs of screening for BHD in relation to detection of BHD related RCC. Examples of this model have

been suggested during the ESMO meeting in Madrid and the BHD meeting in Syracuse.22 23

How to treat PSP of BHD patients?

Current international pneumothorax guidelines e.g. by the British Thoracic Society (BTS) and the

American College of Chest Physicians (ACCP) do not classify SP due to BHD as separate entity, and

in literature the optimal treatment of SP in patients with BHD has not been evaluated so far. As

the recurrence rate of pneumothorax is up to 75% in BHD patients, in this thesis we evaluated the

recurrence rates after different treatment strategies. We found a recurrence rate of 64.5% after

conservative treatment and 11.1% after invasive treatment. These results suggest that reduction in

costs and morbidities of future hospitalizations and surgeries associated with future pneumothoraces

might be considerable when patients with BHD are treated in a more invasive manner compared to

treatment of patients with common PSP without an underlying disease. We therefore hypothesize

that invasive treatment for PSP in BHD patients can be considered, despite the potential for an

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increased complication rate and increased hospitalization time. We suggest a study in a larger cohort

of patients, for a cost-effectiveness evaluation of this hypothesis. Treatment might be optimal if

personalised based on individual characteristics. For example, an option might be to treat large

cysts might best be removed with the additional question if that would prevent the development of

new large cysts if pleural adhesive therapy would prevent stretching.

Renal aspects of Birt-Hogg-dubé syndrome

Pathogenesis

Inherited RCC is characterized by an early age at diagnosis in comparison with than in sporadic

cases and inherited RCC is often multifocal and/or bilateral. Renal tumours associated with BHDS are

diagnosed at an age ranging from 20-75 years and often occur before 50 years of age. In literature it

has been suggested that renal tumours occur in 25-35% of BHD patients. In chapter 2.1 of this thesis

we calculated a penetrance of renal cancer in FLCN gene mutation carriers in BHD patients of 16%

until the age of 70 years30 In literature a variety of histological RCC subtypes has been reported

including chromophobe, papillary clear cell RCC, oncocytoma and mixed histological patterns. This

role of FLCN in the pathogenesis of RCC is still not fully clarified. This is a hugh challenge, as different

groups suggest multiple possible cellular pathways in addition to the widely accepted role of FLCN

in the AKT-mTOR pathway.24

Signaling pathways of FLCN in BHD associated RCC’s will be evaluated at the VUmc. Furthermore,

a prospective study has been set up in collaboration with the Academic Medical Center, to evaluate

the prevalence of germline mutations in high-risk genes among patients diagnosed with RCC by using

a gene panel including over 50 genes. This study might provide additional insights in the development

of a second primary RCC and other malignancies in the tested patients. Secondly, the identification

of a pathogenic mutation in index-patients with RCC might lead to (pre)symptomatic examination

and screening of their relatives. This study will provide further insight in the prevalence of high-risk

mutations in seemingly sporadic RCC patients. Furthermore a collaboration between the VUmc and

the Institute of Medical Biology in Singapore (prof. dr. M.A.M. van Steensel) has been set up to

further elucidate the molecular aspects involved in renal cancer pathogenesis.

Renal imaging

In chapter 2.2 we conducted a retrospective study among patients diagnosed with sporadic RCC

aimed at the identification of BHD cases among RCC patients. We found that multiple basal lung

cysts were present significantly more frequent in FLCN mutation carriers than in sporadic cases

and therefore this finding on routine imaging may be an indication for BHD syndrome in apparent

sporadic RCC patients. Notably, the absence of presence of solitary lung cysts does not exclude the

diagnosis of BHD. As this is the first study in literature, we suggest an evaluation of these results

in a larger cohort. This might lead to early recognition of BHD associated RCC in patients with

apparently sporadic RCC.

In chapter 2.3 of this thesis25 we evaluated the advised regular renal imaging of 199 Dutch patients

with BHD. Our data indicated that compliance to renal screening is relatively high and that US might

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be a sensitive, cheap and widely available imaging modality for detecting clinically relevant renal

tumours in BHD patients, since no tumours exceeding 3 cm were missed with US. Nevertheless,

there is an ongoing discussion in our BHD working group how to optimize renal imaging in BHD

patients. American colleagues recommend that at-risk BHD family members must undergo baseline

surveillance by abdominal MRI with intravenous contrast or computed tomography and suggest

that ultrasound is not sensitive. They recommend follow up starting at 21 years of age at a frequency

of every 36 months in patients with no renal mass lesions at first imaging. This is a different

approach than we suggest in the Netherlands. In the future we will hope to conduct an international

collaborative study to determine prospectively? whether our results for BHD patients in our imaging

study25 can be reproduced, preferably in a relatively large group of patients with screening by both

MRI and ultrasound.

Treatment

In chapter 2.3 we evaluated the “3 cm rule” which recommends surgical intervention when the

(largest) lesion exceeds 3 cm in diameter. This rule is applied to patients with Von Hippel-Lindau

disease, hereditary papillary renal cell cancer and BHD. 26 27 The treatment of tumours < 3 cm in

the VUmc is often at the request of the patient due to anxiety for the possible development of

metastasis. Evaluation of ablative procedures (cryotherapy or radiofrequency ablation) in BHD

patients with RCC is not well evaluated so far, but is performed in the Netherlands. In The States

this type of therapy is generally not recommended as it is suggested that renal tumours are more

completely removed by surgical procedures. Evaluation of this type of therapy is needed.

Potential targeted therapies for BHD-associated (metastasized) renal tumours should also be

evaluated, as currently no approved therapeutic options other than surgical intervention is available.

The use of Everolimus as a second line systemic type of treatment is currently evaluated.28

genotype phenotype correlation in Birt-Hogg dubé syndrome

Up till now, over 100 unique germline mutations have been reported in the LOVD FLCN mutation

database.29 30 Insertion or deletion of a cytosine in a tract of 8 cytosines in exon 11 (c.1285dupC or

del) is a mutation “hot spot” which was identified in up to 50% of BHD patients. Until now no clear

genotype-phenotype correlations have been reported for BHD syndrome.31 32 33 Further research of

the clinical expression in large BHD cohorts is needed to evaluate and validate potential genotype-

phenotype correlations. International collaboration is essential to solve this ongoing quest.

other clinical aspects in Birt-Hogg-dubé syndrome

Finally, more research is necessary to evaluate the possible suggested risk of colonic neoplasms in

the Birt-Hogg-Dubé syndrome. One study reported colorectal polyps in 50% of BHD patients34, this

finding could not be confirmed by other studies. There might be a close link between colorectal

cancer and FLCN gene alterations in a subgroup of families with BHD. This will also be one of the

future topics of the VUmc working group in the coming years.

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R e f e R e n c e s1. Birt AR, Hogg GR, Dubé WJ. Hereditary multiple


acrochordons. Arch Dermatol. 1977;113:1674-1677.

2. Leter EM, Koopmans AK, Gille JJ, et al. Birt-Hogg-



3. Johannesma PC, Lammers JW, van Moorselaar

RJ, et al. Spontaneous pneumothorax as the

first manifestation of a hereditary codition with

an increased renal cancer risk. Ned Tijdschr

Geneeskd. 2009;153:A581.

4. Melton LJ III, Hepper NG, Offord KP, et al.

Incidence of spontaneous pneumothorax in

Olmsted country, Minnesota: 1950 to 1974. Am Rev

Respir Dis 1979;120:1379-1382.

5. Bense L. Wiman LG, Hedenstiema G, et al. Onset

of symptoms in spontaneous pneumothorax:

correlations to physical activity. Eur J Respir Dis

1987;71:181-186.

6. Albolnik IZ, Lossos IS, Zlotogora J, et al. On

the inheritance of primary spontaneous

pneumothorax. Am J Med Genet 1996;62:417-426.

7. Chiu TH, Garcia CK. Familial spontaneous

pneumothorax. Curr Opin Pulm Med. 2006;12:268-272.








Eur Respir J. 2015 Apr;45:1191-1194.


JH, et al. The pathogenesis of pneumothorax


Respirology. 2014 Nov;19(8):1248-1250.

11. Johannesma PC, van der Wel JWT, Paul MA, et

al. Risk of spontaneous pneumothorax due to air

travel and diving in patients with Birt-Hogg-Dubé

syndrome. (SpringerPlus, revised)

12. Postmus PE, Johannesma PC, Menko FH, et al.


because of symptom delay. Am J Respir Crit Care

Med. 2014;190:704-705.

13. Kennedy JC, Khabibullin D, Henske EP. Mechanisms

of pulmonary cyst pathogenesis in Birt-Hogg-

Dubé syndrome: The stretch hypothesis. Semin

Cell Dev Biol 2016;52:47-52.

14. Kumasake T, Hayashi T, Mitani K, et al.

Characterization of pulmonary cysts in Birt–

Hogg–Dubé syndrome: histopathological and


from 50 unrelated patients. Histopathology 2014;

65: 100–110.

15. Bakan S, Kandemirli SG, Kilic F, et al. Birt-Hogg-

Dubé syndrome: A diagnosis to consider in

patients with renal cancer and pulmonary cysts.

Diagn Interv Imaging. 2016 Jan;97:117-118.

16. Onuki T, Goto Y, Kuramochi M, Radiologically


syndrome. Ann Thorac Surg. 2014 Feb;97(2):682-5.

17. Seaman DM, Meyer CA, Gilman MD, et al. Diffuse

cystic lung disease at high-resolution CT. AJR Am J

Roentgenol. 2011 Jun;196(6):1305-11.

18. Tobino K, Hirai T, Johkoh T, et al. Differentiation


lymphangioleiomyomatosis: quantitative analysis

of pulmonary cysts on computed tomography

of the chest in 66 females. Eur J Radiol. 2012

Jun;81(6):1340-6.

19. Agarwal PP, Gross BH, Holloway BJ, et al. Thoracic

CT findings in Birt-Hogg-Dube syndrome. AJR Am

J Roentgenol. 2011 Feb;196(2):349-52.

20. Tobino K, Gunji Y, Kurihara M, et al. Characteristics


thin-section CT findings of the chest in 12 patients.

Eur J Radiol. 2011 Mar;77(3):403-9.

21. Ayo DS, Aughenbaugh GL, Yi ES, et al. Cystic lung

disease in Birt-Hogg-Dube syndrome. Chest. 2007

Aug;132(2):679-84.

22. Johannesma PC, Houweling AC, Reinhard R,

et al. Early detection of hereditary renal cell

cancer by improved evaluation of spontaneous

pneumothorax patients. Annals of Oncology

2014;25:(suppl_4): iv254-iv254.

23. Gupta, N., Langenderfer, D., McCormack, F.X.

HRCT screening for diffuse cystic lung diseases

in patients presenting with spontaneous

pneumothorax is cost-effective. Am J Respir Crit

Care Med. 2016;104:A6261.

24. Schmidt LS, Lineham WM. Clinical features,

genetics and potential therapeutic approaches for

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Birt-Hogg-Dubé syndrome. Expert Opin Orphan

drugs 2015;3:15-29.

25. Johannesma PC, van de Beek I, Reinhard R, et al.

Renal imaging in 199 Dutch patients with Birt-

Hogg-Dubé syndrome: Screening, compliance and

outcome. (Submitted)

26. Herring JC, Enquist EG, Chernoff A, et al.

Parenchymal sparing surgery in patients

with hereditary renal cell carcinoma: 10-year

experience. J Urol 2001;165:777-81.

27. Walther MM, Choyke PL, Weiss G, et al. Parenchymal

sparing surgery in patients with hereditary renal

cell carcinoma. J Urol 1995;153:913-6.

28. Clinical Trials. Gov Studies. https://www.

clinicaltrials.gov. “Everolimus therapy in people

with Birt-Hogg-Dubé syndrome (BHD)- associated

kidney cancer or sporadic chromophobe renal

cancer”.

29. Lim DH, Rehal PK, Nahorski MS, et al. A new locus-

specific database (LSDB) for mutations in the folliculin

(FLCN) gene. Hum. Mutat, 2010;31:E1043-1051.

30. European Birt-Hogg-Dubé Consortium. LOVD

Gene Homepage for FLCN. https://grenada.luc.nl/

LOVD2/shared1/home.php?select_db=FLCN; last

visisted 23-03-2016.





Genet. 2008 Jun;45(6):321-31.




syndrome. Am J Respir Crit Care Med. 2007 May

15;175(10):1044-53.

33. Houweling AC, Gijezen LM, Jonker MA, et al.

Renal cancer and pneumothorax risk in Birt-Hogg-


carriers from 35 BHD families. Br J Cancer. 2011 Dec

6;105(12):1912-9.

34. Kluger N, Giraud S, Coupier I, et al. Birt-Hogg-


French families. Br J Dermatol. 2010;162:527-37.

Paul C. Johannesma1


a d d e n d u mReview committee

co-authors and affiliationslist of abbreviations

list of publications list of scientific meetings

grants and awardsacknowledgements – dankwoord

curriculum vitae auctoris

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REVIEW

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R e v i e w c o m m i t t e e

Prof. dr. c.J.J. mulder (chair)

Professor of Gastroenterology,

Department of Gastroenterology and Hepatology, VU University Medical Center, Amsterdam,

The Netherlands

Prof. dr. ir. H.a. van swieten

Professor of Cardiothoracic Surgery,

Department of Cardiothoracic Surgery, Radboud University Medical Center, Nijmegen,

The Netherlands

Department of Cardiothoracic Surgery, HagaZiekenhuis, ‘s Gravenhage, The Netherlands

Prof. dr. m.P. laguna

Professor of Urological Oncology,

Department of Urological Oncology, Amsterdam Medical Center, Amsterdam, the Netherlands

dr. l.J. meijboom

Thoracic Radiologist,

Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands

dr. m. Kets

Clinical Geneticist,

Department of Clinical Genetics, Radboud University Medical Center, Nijmegen, The Netherlands

a d d i t i o n a l m e m B e R s o f t H e o p p o s i n g c o m m i t t e e

Prof. dr. th.m. starink

Professor of Dermatology,

Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands

Department of Dermatology, Radboud University Medical Center, Nijmegen, The Netherlands

dr. H.J.a.a. van geffen

Trauma Surgeon and Lung Surgeon,

Department of Surgery, Jeroen Bosch Hospital, ‘s Hertogenbosch, The Netherlands

&

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l i s t o f c o -a u t H o R s a n d a f f i l i at i o n s

irma van de Beek, md

VU University Medical Center, Department of Clinical Genetics, Amsterdam, The Netherlands

ernie m.H.f. Bongers, md Phd

Radboud University Medical Center, Departement of Human Genetics, Nijmegen, The Netherlands

Ben e.e.m. van den Borne, md Phd

Catharina Hospital, Department of Pulmonology, Eindhoven, The Netherlands

Barry J. coull, Phd

Maastricht University Medical Center, Department of Dermatology, GROW School for Oncology

and Developmental Biology, Maastricht, The Netherlands

martijn B.a. van doorn, md Phd

VU University Medical Center, Department of Dermatology, Amsterdam, The Netherlands

lieke m. gijezen, md



Johannes J.P. gille, Phd


nicole c.t. van grieken, md Phd

VU University Medical Center, Department of Pathology, Amsterdam, The Netherlands

Prof. simon Horenblas, md Phd

The Netherlands Cancer Institute, Urologic Oncology and Department of Urology, Amsterdam,

The Netherlands

arjan c. Houweling, md Phd


elisabeth H. Jaspars, md Phd


mirjam m. de Jong, md Phd

University Medical Center Groningen, Department of Clinical Genetics, Groningen, The Netherlands

marianne a. Jonker, Phd

VU University Medical Center, Department of Biostatistics and Epidemiology, Amsterdam, The

Netherlands

lisette e. van der Kolk, md Phd

The Netherlands Cancer Institute, Family Cancer Clinic, Amsterdam, The Netherlands

yael Kon, md


Prof. Jan-Willem J. lammers, md Phd

Utrecht University Medical Center, Department of Pulmonology, Utrecht, The Netherlands

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NS

edward m. leter, md Phd

Maastricht University Medical Center, Department of Clinical Genetics, Maastricht, The Netherlands


fred H. menko, md Phd

The Netherlands Cancer Institute, Family Cancer Clinic, Amsterdam, The Netherlands


Prof. R. Jeroen a. van moorselaar, md Phd

VU University Medical Center, Department of Urology, Amsterdam, The Netherlands

ad f. nagelkerke, md

VU University Medical Center, Department of Pediatrics, Amsterdam, The Netherlands

Rogier a. oldenburg, md Phd

Erasmus Medical Center, Department of Clinical Genetics, Rotterdam, The Netherlands

theo van os, md Phd

University of Amsterdam, Department of Clinical Genetics, Amsterdam, The Netherlands

marinus a. Paul, md Phd

VU University Medical Center, Department of Cardiothoracic Surgery, Amsterdam, The Netherlands

Prof. P.e. Postmus, md Phd

University of Liverpool, Clatterbridge Cancer Center, Department of Thoracic Oncology,

Liverpool, United Kingdom

VU University Medical Center, Department of Pulmonary Diseases, Amsterdam, The Netherlands

Rinze Reinhard, md

Onze Lieve Vrouwe Gasthuis, Department of Radiology, Amsterdam, The Netherlands

VU University Medical Center, Department of Radiology, Amsterdam, The Netherlands

Rence Rozendaal, md Phd


Hans J. smit, md Phd

Rijnstate Hospital, Department of Pulmonology, Arnhem, The Netherlands

Karin y. van spaendonck-Zwart, md Phd

University Medical Center Groningen, Department of Clinical Genetics, Groningen, The Netherlands

Prof. maurice a.m. van steensel, md Phd



The University of Dundee, Department of Dermatology, Dundee, United Kingdom

Nanyang Technological University, Lee Kong Chian School of Medicine, Singapore

Jincey d. sriram, md

Rijnstate Hospital, Department of Pulmonology, Arnhem, The Netherlands

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Prof. th. m. starink, md Phd

Leiden University Medical Center, Department of Dermatology, Leiden, The Netherlands


erik thunnissen, md Phd


JanHein t.m. van Waesberghe, md Phd

VU University Medical Center, Department of Radiology, Amsterdam, The Netherlands

Quinten Waisfisz, md Phd


J.W. tijmen van der Wel, Bsc

VU University Medical Center, Department of Pulmonary Diseases, Amsterdam, The Netherlands

&

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LIST OF A

BBREVIA

TION

S

l i s t o f a B B R e v i at i o n s

Ad Adenocarcinoma

AML Acute Myeloid Leukemia

AMPK AMP-activated Protein Kinase

APC Adenomatous Polyposis Coli

AS Active Surveillance

ACCP American College of Chest Physicians

BHD Birt-Hogg-Dubé syndrome

BTS British Thoracic Society

CC Clear Cell

CC/Cph Clear Cell / Chromophobe

Ch Chemotherapy

CI Confidence Interval

CO2 Carbon Dioxide

Cph Chromophobe

CT Computed Tomography

DNA Deoxyribonucleic Acid

ED Emergency Department

F Female

FB Follicular Bronchiolitis

FF Fibrofolliculomas

FFPE Formalin-Fixed, Paraffin-Embedded

FLCN Folliculin

FMDF Familial Multiple Discoid Fibromas

H2O2 Hydrogen Peroxide

HLRCC Hereditary Leiomyomatosis and Renal Cell Carcinoma

hPa Hectopascal (102 Pa)

HPRC Hereditary Papillary Renal Carcinoma

IME’s In-Flight Medical and Surgical Emergencies

LAM Lymphangioleiomyomatosis

LCDD Light Chain Deposition Disease

LCH Langerhans Cell Histiocytosis

LIP Lymphocytic Interstitial Pneumonia

M Male

Me Metastasectomy

MRI Magnetic Resonance Imaging

mRNA messenger RNA (Ribonucleic Acid)

&

233233

LIST OF A

BBREVIA

TION

S

mTOR Mammalian Target Of Rapamycin

OMIM Online Mendelian Inheritance in Man

Pap Papillary

PCR Polymerase Chain Reaction

PLAM Pulmonary Lymphangioleiomyomatosis

PLCH Pulmonary Langerhans Cell Histiocytosis

PSP Primary Spontaneous Pneumothorax

RFA Radiofrequency Ablation

RNA Ribonucleic Acid

Rth Radiotherapy

RCC Renal Cell Cancer

Sa Sarcomatoid component

SP Spontaneous Pneumothorax

SSP Secondary Spontaneous Pneumothorax

TNM Tumour/Node/Metastasis classification

TSC Tuberous Sclerosis Complex

TTF-1 Thyroid Transcription Factor 1

US Ultrasound

VATS Video Assisted Thoracoscopic Surgery

VHL Von Hippel Lindau syndrome

VNTR Variable Number Tandem Repeat

WHO World Health Organization

YAG Yttrium Aluminum Garnet (YAG, Y3Al

5O

12)

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l i s t o f p u B l i c at i o n s

Johannesma pc, van de Beek I, van der Wel T, Paul MA, Houweling AC, Jonker MA, van Waesberghe

JHTM, Reinhard R, Starink ThM, van Moorselaar RJA, Menko FH, Postmus PE. Risk of spontaneous

pneumothorax due to air travel and diving in patients with Birt-Hogg-Dubé syndrome. SpringerPlus

2016 July 11. [accepted for publication]

Balan TA, Jonker MA, Johannesma pc, Putter H. Ascertainment correction in frailty models for

recurrent events data. Stat Med. 2016 Apr 18. [Epub ahead of print]

Johannesma pc, Houweling AC, Menko FH, van de Beek I, Reinhard R, Gille JJ, van Waesberghe

JHWT, Thunnissen E, Starink ThM, Postmus PE, van Moorselaar RJ. Are lung cysts in renal cell cancer

(RCC) patients an indication for FLCN mutation analysis? Fam Cancer. 2016 Apr;15(2):297-300.

Merten H, Johannesma pc, Lubberding S, Zegers M, Langelaan M, Jukema GN, Heetveld MJ,

Wagner C. High risk of adverse events in hospitalised hip fracture patients of 65 years and older:

results of a retrospective record review study. BMJ Open. 2015 Sep 7;5(9):e006663.

Johannesma pc, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ, Menko FH, Postmus

PE; Amsterdam BHD working group. Prevalence of Birt-Hogg-Dubé syndrome in patients with

apparently primary spontaneous pneumothorax. Eur Respir J. 2015 Apr;45(4):1191-4.

Johannesma pc, Vonk Noordegraaf A. Pneumomediastinum and pneumopericardium due to high-

speed air turbine drill used during a dental procedure. Ann Thorac Surg. 2014 Dec;98(6):2232.

Johannesma pc, Houweling AC, van Waesberghe JH, van Moorselaar RJ, Starink TM, Menko FH,

Postmus PE. The pathogenesis of pneumothorax in Birt-Hogg-Dubé syndrome: a hypothesis.

Respirology. 2014 Nov;19(8):1248-50.

Postmus PE, Johannesma pc, Menko FH, Paul MA. In-flight pneumothorax: diagnosis may be missed

because of symptom delay. Am J Respir Crit Care Med. 2014 Sep 15;190(6):704-5.

Johannesma pc, van Bemmel AJ, Meijer J. An ill woman with persisting globus. Ned Tijdschr

Geneeskd. 2014;158:A7717.

Johannesma pc, van den Borne BE, Gille JJ, Nagelkerke AF, van Waesberghe JT, Paul MA, van

Moorselaar RJ, Menko FH, Postmus PE. Spontaneous pneumothorax as indicator for Birt-Hogg-

Dubé syndrome in paediatric patients. BMC Pediatr. 2014 Jul 3;14:171.

Johannesma pc, Starink TM, Van Moorselaar RJ, Postmus PE. Facial fibrofolliculomas as indicator

for renal cell cancer. Jpn J Clin Oncol. 2014 Jun;44(6):609-10.

Johannesma pc, van Moorselaar RJ, Horenblas S, van der Kolk LE, Thunnissen E, van Waesberghe

JH, Menko FH, Postmus PE. Bilateral renal tumour as indicator for Birt-Hogg-Dubé syndrome. Case

Rep Med. 2014;2014:618675.

Johannesma pc, Thunnissen E, Postmus PE. How reliable are clinical criteria in distinguishing

between Birt-Hogg-Dubé syndrome and smoking as a cause for pneumothorax? Histopathology.

2014 Jun;64(7):1045-6.

Johannesma pc, Thunnissen E, Postmus PE. Lung cysts as indicator for Birt-Hogg-Dubé syndrome.

Lung. 2014 Feb;192(1):215-6.

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Menko FH, Johannesma pc, van Moorselaar RJ, Reinhard R, van Waesberghe JH, Thunnissen E,

Houweling AC, Leter EM, Waisfisz Q, van Doorn MB, Starink TM, Postmus PE, Coull BJ, van Steensel

MA, Gille JJ. A de novo FLCN mutation in a patient with spontaneous pneumothorax and renal

cancer; a clinical and molecular evaluation. Fam Cancer. 2013 Sep;12(3):373-9.

Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts KY,

Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EM, Johannesma pc,

Postmus PE, van Moorselaar RJ, van Waesberghe JH, Starink TM, van Steensel MA, Gille JJ, Menko

FH. Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN

mutation carriers from 35 BHD families. Br J Cancer. 2011 Dec 6;105(12):1912-9.

Starink TM, Houweling AC, van Doorn MB, Leter EM, Jaspars EH, van Moorselaar RJ, Postmus PE,

Johannesma pc, van Waesberghe JH, Ploeger MH, Kramer MT, Gille JJ, Waisfisz Q, Menko FH.

Familial multiple discoid fibromas: a look-alike of Birt-Hogg-Dubé syndrome not linked to the FLCN

locus. J Am Acad Dermatol. 2012 Feb;66(2):259.e1-9.

Merten H, Lubberding S, van Wagtendonk I, Johannesma pc, Wagner C. Patient safety in elderly hip

fracture patients: design of a randomised controlled trial. BMC Health Serv Res. 2011 Mar 21;11:59.

Johannesma pc, van der Klift HM, van Grieken NC, Troost D, Te Riele H, Jacobs MA, Postma TJ,

Heideman DA, Tops CM, Wijnen JT, Menko FH. Childhood brain tumours due to germline bi-allelic

mismatch repair gene mutations. Clin Genet. 2011 Sep;80(3):243-55.

Johannesma pc, Lammers JW, van Moorselaar RJ, Starink TM, Postmus PE, Menko FH. Spontaneous

pneumothorax as the first manifestation of a hereditary condition with an increased renal cancer

risk. Ned Tijdschr Geneeskd. 2009;153:A581.

s u B m i t t e d m a n u s c R i p t s

Johannesma pc, Paul MA, van Waesberghe JHTM, Jonker MA, Houweling AC, van de Beek I, van

Moorselaar RJA, Menko FH, Postmus PE. International guidelines for pneumothorax are not adequate

for treatment of spontaneous pneumothorax in patients with Birt-Hogg-Dubé syndrome. (Submitted)

Johannesma pc, van Waesberghe JHTM, Menko FH, van Moorselaar RJA, Paul MA, Starink ThM,

Reinhard R, Houweling AC, van de Beek I, Jonker MA, Postmus PE. Presence of pulmonary cysts in

BHD patients with and without a pneumothorax; a retrospective analysis of 61 patients. (Submitted)

Johannesma pc, van Waesberghe JHTM, Menko FH, van Moorselaar RJA, Paul MA, Starink ThM,

Reinhard R, Houweling AC, van de Beek I, Jonker MA, Postmus PE. Radiological features of primary

spontaneous pneumothorax patients with or without a mutation in FLCN. (Submitted)

Johannesma pc, van de Beek I, Reinhard R, Leter EM, Rozendaal L, Starink ThM, Waesberghe JHTM,

Horenblas S, Jonker MA, Menko FH, Postmus PE, Houweling AC, van Moorselaar RJA. Renal imaging in

199 Dutch patients with Birt-Hogg-Dubé syndrome: Screening, compliance and outcome. (Submitted)

Luijten MNH, Starink ThM, Seifan S, Kenyon E, Land S, Easton JA, Houweling AC, van Doorn MB,

Leter EM, Jaspars EH, Johannesma pc, Hennekam RCM, Gille JJP, Müller F, Coull BJ, Menko FH,

Tee A, Steensel MAM, Waisfisz Q. Familial multiple discoid fibromas, a Birt-Hogg-Dubé lookalike, is

caused by a truncating FNIP1 mutation. (Under construction)

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l i s t o f p u B l i s H e d pa p e R s n ot i n c l u d e d i n t H i s t H e s i s

Balan TA, Jonker MA, Johannesma pc, Putter H. Ascertainment correction in frailty models for

recurrent events data. Statistics in Medicine 2016 Apr 18. [Epub ahead of print]

Merten H, Johannesma pc, Lubberding S, Zegers M, Langelaan M, Jukema GN, Heetveld MJ,

Wagner C. High risk of adverse events in hospitalised hip fracture patients of 65 years and older:

results of a retrospective record review study. BMJ Open. 2015 Sep 7;5(9):e006663.

Johannesma pc, Vonk Noordegraaf A. Pneumomediastinum and pneumopericardium due to high-

speed air turbine drill used during a dental procedure. Ann Thorac Surg. 2014 Dec;98(6):2232.

Johannesma pc, van Bemmel AJ, Meijer J. An ill woman with persisting globus. Ned Tijdschr

Geneeskd. 2014;158:A7717.

Starink TM, Houweling AC, van Doorn MB, Leter EM, Jaspars EH, van Moorselaar RJ, Postmus

PE, Johannesma pc, van Waesberghe JH, Ploeger MH, Kramer MT, Gille JJ, Waisfisz Q, Menko

FH.Familial multiple discoid fibromas: a look-alike of Birt-Hogg-Dubé syndrome not linked to the

FLCN locus. J Am Acad Dermatol. 2012 Feb;66(2):259.e1-9.

Merten H, Lubberding S, van Wagtendonk I, Johannesma pc, Wagner C. Patient safety in elderly hip

fracture patients: design of a randomised controlled trial. BMC Health Serv Res. 2011 Mar 21;11:59.

Johannesma pc, van der Klift HM, van Grieken NC, Troost D, Te Riele H, Jacobs MA, Postma TJ,

Heideman DA, Tops CM, Wijnen JT, Menko FH. Childhood brain tumours due to germline bi-allelic

mismatch repair gene mutations. Clin Genet. 2011 Sep;80(3):243-55.

p u B l i s H e d a B s t R ac t

Johannesma pc, van der Wel JWT, Paul MA, Houweling AC, Jonker MA, van Waesberghe JHTM,

van Moorselaar RJA, Menko FH, Postmus PE. Birt-Hogg-Dubé syndrome and the prevalence of

pneumothorax in relation to air travel and diving. Chest. 2014;146:448A.

Johannesma pc, Houweling AC, Paul MA, van der Wel JWT, Jonker MA, van Waesberghe JHTM, Van

Moorselaar RJA, Menko FH, Postmus PE. Clinical Cases: Recurrent Spontaneous Pneumothorax in 2

Patients With Birt-Hogg-Dubé Syndrome: A Causal Link With Air Travel? Chest. 2014;146:411A.

Johannesma pc, Jonker MA, van der Wel JWT, van Waesberghe JHTM, van Moorselaar RJA, Menko

FH, Postmus PE. Management of spontaneous pneumothorax in patients with or without Birt-Hogg-

Dubé syndrome. Eur Respir J 2014;44:P752.

Johannesma pc, van Waesberghe JHTM, Reinhard R, Gille JJP, van Moorselaar RJA, Houweling

AC, Starink ThM, Menko FH, Postmus PE. Birt-Hogg-Dubé syndrome patients with and without

pneumothorax: findings on chest CT. Am J Resp Crit Care Med 2014;189:A6416.

Johannesma pc, van Waesberghe JHTM, Reinhard R, Gille JJP, van Moorselaar RJA, Houweling

AC, Starink ThM, Menko FH, Postmus PE. Chest CT for primary spontaneous pneumothorax

(PSP): findings: Birt-Hogg-Dubé versus non-Birt-Hogg-Dubé patients. Am J Resp Crit Care

Med;189:A6415.

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Johannesma pc, van den Borne BEEM, Nagelkerke AF, van Waesberghe JHTM, Paul MA, Menko FH,

Postmus PE. Clinical cases; Spontaneous pneumothorax at the age of 14. Radiological evidence of

Birt-Hogg-Dubé syndrome. Am J Resp Crit Care Med 2014;189:A2591.

Johannesma pc, Menko FH, Reinhard R, van Waesberghe JHTM, van Moorselaar RJA, Starink ThM,

Postmus PE. Primary Spontaneous Pneumothorax: a pilot study on the frequency of FLCN mutation

(Birt-Hogg-Dubé syndrome). Am J Resp Crit Care Med 2014;189:A6417.

Johannesma pc, Binnendijk MJ, Reinhard R, Starink ThM, Kon Y, Terlou A, van Moorselaar RJA, van

Waesberghe JH, Houweling AC, Leter EM, Waisfisz, Q, Gille JJP, Menko FH, Postmus PE. Prevalence

of Birt-Hogg-Dubé Syndrome among patients with spontaneous pneumothorax: preliminary

results. Familial cancer 2013;12:442.

Binnendijk MJ, Johannesma pc, Gille JJP, Houweling AC, Reinhard R, Starink ThM, van Moorselaar

RJA, van Waesberghe JH, Leter EM, Waisfisz Q, Menko FH, Postmus PE. BHD versus non-BHD

patients with spontaneous pneumothorax: Chest CT findings. Familial cancer 2013;12:419.

Menko FH, Johannesma pc, Gille JJP, Houweling AC, Leter EM, Jonker MA, Kon Y, Terlou A, Starink

ThM, Jaspars EM, Reinhard R, van Waesberghe JH, van Moorselaar RJA, Postmus PE, Aalfs C, de Jong

MM , Bongers EMF, Oldenburg RA, van Os TA, van Spaendonck-Zwarts KY, van Steensel MA, Waisfisz

Q. An update of a Birt-Hogg-Dubé syndrome (BHD) database: evaluation of 89 families referred for

suspected BHD syndrome. Familial cancer 2013;12:422.

Binnendijk MJ, Johannesma pc, Gille JJP, Houweling AC, Reinhard R, Starink ThM, van Moorselaar

RJA, van Waesberghe JH, Leter EM, Waisfisz Q, Menko FH, Postmus PE. BHD patients with and

without pneumothorax: Chest CT findings. Familial cancer 2013;12:420.

Johannesma pc, Gille JJP, van Moorselaar RJA, Reinhard R, van Waesberghe JH, Houweling AC, Leter

EM, Waisfisz Q, van Doorn MBA, Starink ThM, Postmus PE, Menko FH. A de novo FLCN mutation in a

patient presenting with spontaneous pneumothorax. Familial Cancer 2011;10:113.

Reinhard R, van Moorselaar RJA, Houweling AC, Leter EM, van Doorn MBA, Starink ThM,

Johannesma pc, Postmus PE, Gille JJP, Waisfisz Q, Menko FH, van Waesberghe J-HTM. Renal

imaging in Birt-Hogg-Dubé syndrome: a comparison of initial renal MRI and ultrasound in 92 FLCN

mutation carriers. Familial Cancer 2011;10:109.

Houweling AC, Geijzen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts

KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EMHF, Johannesma

pc, Postmus PE, van Moorselaar RJA, van Waesberghe JH, Starink ThM, van Steensel AM, Gille JJP,

Menko FH. Renal cancer risk and cancer phenotype in Birt-Hogg-Dubé syndrome; analysis of 115

FLCN mutation carriers from 35 Birt-Hogg-Dubé families. Familial Cancer 2011;10:106.

Johannesma pc, FW Bloemers, FC Bakker, ESM de Lange-de Klerk, WP Zuidema. Survival and

outcome after hip fracture in individuals eighty years of age and older during hospital stay. Eur J

Trauma Emerg Surg; 2011;37 (Suppl 1):S115.

Johannesma pc, Houweling AC, Jonker MA, van Doorn MB, Oldenburg RA, van Spaerndonck-Zwarts

KY, de Jong MM, Postmus PE, van Moorselaar RJA, Leter EM, Ploeger HM, Kramer MT, van Waesberghe

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JH, Starink ThM, Gille JJP, Menko FH. Phenotypic expression of the c.774_775delGTinsCAC FLCN

germline mutation in 28 mutation carriers from an extended dutch kindred with Birt-Hogg-Dubé

syndrome. Familial Cancer 2010;9:8.

Starink ThM, Houweling AC, van Doorm MB, Leter EM, Jaspars E, van Moorselaar RJA, Postmus

PE, Johannesma pc, van Waesberghe JH, Ploeger HM, Kramer MT, Gille MT, Waisfisz Q, Menko

FH. Familial multiple trichodiscomas (discoid fibromas) is clinically distinct from Birt-Hogg-Dube

syndrome and not linked to the FLCN mutation. Familial Cancer 2010;9:10.

Houweling AC, Gijezen LM, Jonker MA, van Doorn MA, van Oldenburg RA, van Spaendonck- Zwarts

KY, Leter ME, van Os TE, de Jong MM, Gille JJ, Bongers EM, Ploeger HM, Kramer MT, Postmus PE,

Johannesma pc, van Moorselaar RJA, van Waesberghe JH, Starink ThM, van Steensel MA, Menko

FH. Evaluation and follow up of 54 families with suspected Birt-Hogg-Dubé syndrome; a multi center

study in the Netherlands.. Familial Cancer 2010;9:6.

Johannesma pc, Zuidema WP, Giannakopoulos GF, de Lange- de Klerk ESM, Bloemers FW, Bakker

FC. The impact of anaemia and blood transfusion on the outcome of elderly hip fracture patients.

Eur J Trauma Emerg Surg 2010;36:38.

Johannesma pc, Unger JM, de Lange-de Klerk ESM, Jukema GN. The influence of co-morbidity,

postoperative anaemia and complications on recovery and length of stay in the hospital after a hip

fracture in elderly in the Netherlands. Eur J Trauma Emerg Surg 2009;35:12.

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oral presentations

2016 Regionale Refereeravond Heelkunde, Utrecht, Nederland

2015 6th World Congress on Birt-Hogg-Dubé, Syracuse New York, Verenigde Staten

2014 7e Amsterdams Longchirurgie Symposium, Amsterdam, Nederland

2014 Longkring Longchirurgie NoordWest Nederland, Amsterdam, Nederland

2014 European Society Medical Oncology (ESMO), Madrid, Spanje

2014 American Thoracic Society (ATS), San Diego Californië, Verenigde Staten

2014 4th Joint Spring Conference of the UK /Dutch Clinical Genetics Societies &

Cancer Genetics groups, Leiden, Nederland

2014 VUmc Science Exchange Day, Amsterdam, Nederland

2014 Voorjaarsvergadering NVALT, Utrecht, Nederland

2013 5th World Congress on Birt-Hogg-Dubé, École du Louvre, Parijs, Frankrijk

2012 4th World Congress on Birt-Hogg-Dubé, Cincinnati Ohio, Verenigde Staten

2011 3th World Congress on Birt-Hogg-Dubé, Maastricht, Nederland

2010 2th World Congress on Genodermatology, Maastricht, Nederland

2010 2th World Congress on Birt-Hogg-Dubé, Washington DC, Verenigde Staten

2009 3th European Congress of Birt-Hogg-Dubé, Londen, Groot Britannië

Poster presentations

2014 Chest 2014 Annual Meeting, Austin Texas, Verenigde Staten

2014 European Respiratory Society (ERS), München, Duitsland

g R a n t s a n d awa R d s

2015 Myrovlytis Trust, Travel Grant

2015 Mr. Willem Bakhuys Roozeboom Foundation, Research Grant

2015 Prof. Dr. Jaap Swierenga Foundation, Research Grant

2014 European Society for Medical Oncology (ESMO), Research Award

2014 NRS Young Investigator Travel Grant




2008 Top 3 Excellent Student Award, VUmc Amsterdam

"Je gaat het pas zien als je het doorhebt”

Johan Cruijff

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ac k n ow l e d g e m e n t s – da n k wo o R d

Dit proefschrift is het resultaat van belangeloze en onuitputtelijke inzet en betrokkenheid van een

grote verscheidenheid aan mensen op velerlei manieren. Daarom wil ik hierbij al degenen bedanken

die hebben geholpen bij het tot stand komen ervan. Het is onmogelijk allen die aan dit proefschrift

hebben bijgedragen persoonlijk te bedanken, maar er zijn een aantal mensen die ik graag in het

bijzonder wil bedanken.

Dit proefschrift had niet tot stand kunnen komen zonder alle patiënten die bereid waren om deel

te nemen aan alle onderzoek die vaak veel tijd en inspanning vergden. Ontzettend veel dank voor

jullie inzet, medewerking, motivatie tijd en bereidheid.

Prof. dr. P.E. Postmus. geachte promotor, in mijn tweede studiejaar kwam er een vacature vrij

als student-assistent voor wetenschappelijk onderzoek onder uw supervisie naar spontane

pneumothorax. Ik had nooit verwacht dat dit de eerste stap zou zijn naar dit proefschrift. U

introduceerde mij binnen de Birt-Hogg-Dubé werkgroep als “lijm” tussen alle werkgroepleden van

de verscheidene afdelingen en gaf mij de mogelijkheid om mij wetenschappelijk te ontwikkelen,

waarbij niets teveel gevraagd was. U zag altijd mogelijkheden voor nieuwe ideeën en gaf het

proefschrift inhoudelijk vorm. U leerde mij dat ook een casereport of case-serie (deel III van dit

proefschrift) kan leiden tot nieuwe onderzoeksvragen en nieuwe inzichten. Daarnaast creëerde

u de mogelijkheid om als arts-onderzoeker na mijn studie fulltime onderzoek te doen naar het

Birt-Hogg-Dubé syndroom. Daarnaast zorgde u voor alle voorwaarden om de verscheidene

prospectieve studies, waardoor vele patiënten geïncludeerd konden worden voor aanvullende

diagnostiek, succesvol te laten verlopen. Alles was altijd bespreekbaar en alles was altijd mogelijk.

Tenslotte wil ik u enorm bedanken voor de tomeloze inzet en tijd die u voor mij heeft vrijgemaakt

om dit boekje te creëren. De vele uren waarbij u belangeloos thoracale CT-scans scoorde, waarbij u

mij daarbij klinische lessen gaf hoe een CT-scan of thoraxfoto te beoordelen waren geweldig en heb

ik nog steeds profijt van. Het is voor mij een grote eer dat ik de laatste promovendus ben die onder

u als promotor aan de VU mag promoveren.

Prof. dr. R.J.A. van Moorselaar. geachte promotor, naar mate het wetenschappelijk onderzoek

naar pulmonale afwijkingen bij het Birt-Hogg-Dubé syndroom (BHD) meer vorm begon te krijgen,

ontstond steeds meer de interesse naar de directe (klinische) relatie tussen de longen en nieren.

Uw visie en enthousiasme creëerde mogelijkheden om de klinische nierafwijkingen binnen het

syndroom nader te onderzoeken. U enthousiasmeerde mij voor de deze urologische afwijking, wat

leidde tot deel 2 van dit proefschrift. Tijdens de verscheidene BHD bijeenkomsten in binnen- en

buitenland heb ik u iets beter leren kennen, waarbij er mooie gesprekken volgden onder het genot

van een goed glas wijn. Uw enthousiasme, laagdrempeligheid en vriendelijkheid heb ik altijd enorm

gewaardeerd. Wanneer ik bij u spontaan langskwam op afdeling 4F nam u altijd uitgebreid de tijd

voor me en konden andere afspraken wel wachten. Dank voor alles en alle geboden mogelijkheden

dat heeft geleid tot dit proefschrift. Ik kijk uit naar de toekomst, naar de resultaten van de lopende

urologische follow up studies binnen het BHD syndroom en de papers die hieruit zullen gaan komen.

Dr. F.H. Menko, geachte co-promotor, beste Fred, waar moet ik beginnen? Ik leerde je kennen bij

de BHD werkgroep, en je bent de motor geweest achter het wetenschappelijk onderzoek van het

Birt-Hogg-Dubé syndroom binnen het VUmc. Véle, véle uren hebben we doorgebracht met het

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opzetten van studieprotocollen, uitvoeren van de studies en het opzetten van de papers. Jouw

kritische blik, waarbij jij vaak elk woord afwoog in een paper of presentatie heeft mij – ondanks

dat het me soms tot waanzin dreef - veel geleerd hoe wetenschap bedreven dient te worden.

Daarnaast wil ik je bedanken voor jouw vriendschap, openheid, vele gesprekken over wetenschap

maar ook over alle belangrijke zaken in het leven buiten werk. De deur stond en staat altijd bij je

open, in en zeker ook buiten het ziekenhuis. Vaak zei je “Paul, we gaan zo alles bespreken, maar

eerst een cup of tea”. Daarnaast dank ik je voor alle gezelligheid tijdens alle meetings in onder

andere Cincinnati, Madrid, Parijs, Maastricht en niet te vergeten in de mooiste stad van de wereld,

Amsterdam, in de Hortus Botanicus.

Dr. J.H.T.M. van Waesberghe, geachte co-promotor, beste dr. van Waesberghe, veel dank voor

de radiologische invulling van dit proefschrift. Dank voor de kritische beoordeling van alle

manuscripten, de tijd die u voor mij vrij maakte om alle CT-scans te beoordelen en de mooie

verhalen tijdens het scoren van alle radiologische kenmerken.

Geachte leden van de promotiecommissie, Prof. dr. C.J.J. Mulder, Prof. dr. ir. H.A. van Swieten, Prof.

dr. M.P. Laguna Pes, dr. L.J. Meijboom en dr. M. Kets, zeer veel dank voor de beoordeling van de

inhoud van mijn proefschrift en voor het plaatsnemen in de oppositie.

Prof. dr. Th.M. Starink, geachte professor, heel veel dank voor de samenwerking afgelopen jaren in

de Birt-Hogg-Dubé werkgroep. Ik heb uw aardige e-mails altijd enorm gewaardeerd waarbij u altijd

enorm meeleefde met de progressie en de vele struikelblokken van het promotietraject. Zoals u zelf

de beeldhouwer Brancusi citeerde in een van uw e-mails “It is not the doing of things that is difficult.

What is difficult is getting into the right mood to do them”. Dank voor de tijd die u voor mij heeft

vrijgemaakt om plaats te nemen in de oppositie.

Dr. H.J.A.A. van Geffen, beste Erwin, Ardennenweekend, Hap & Stap, Chirurgendagen, borrels bij

de KASerne, het is altijd feest met jou! Ik hoop komende jaren nog heel veel van je te kunnen leren

binnen de trauma- en longchirurgie in de JBZ kliniek. Dank voor de kritische beoordeling van het

proefschrift en het plaatsnemen in de oppositie.

Prof. dr. Anton Vonk Noordegraaf, geachte professor, veel dank voor de geboden mogelijkheid mijn

boekje af te kunnen maken na alle tumultueuze veranderingen op de afdeling.

Dr. Anco Boonstra, beste Anco, Veel dank voor de begeleiding en ondersteuning van het onderzoek

na alle onverwachte veranderingen bij de afdeling Longziekten.

Stafleden van de afdeling Longziekten van het VUmc, dank voor jullie interesse in mijn

promotieonderzoek.

Anny Kijk in de Vegte, Ella Wetser en Ellen Berkman van het Secretariaat Longziekten, dank voor

alle ondersteuning en hulp tijdens het lange BHD traject van student-assistent tot nu het boekje

eindelijk af is.

Secretariaat van de afdeling Klinische Genetica VUmc, altijd was er de mogelijkheid om gebruik te

maken van de faciliteiten van de afdeling. Dank voor alle ondersteuning tijdens de BHD-P studie.

Dr. Arjan Houweling, Beste Arjan, toen Fred vertrok van het VUmc naar het AvL-NKI nam jij het

stokje van hem over, en heb jij je enorm ingezet voor het BHD onderzoek binnen de VUmc. Jij hebt

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er (mede) voor gezorgd dat mijn promotieonderzoek nu vervolg gaat krijgen zowel in het lab als

klinisch. Syracuse was qua congres maar ook zeker qua sociaal programma onvergetelijk.

Drs. Irma van Beek, Beste Irma, ik vind het geweldig dat je mijn promotieonderzoek voorzet, waarbij

je de nadruk zal leggen op familiaire niertumoren. Dank voor je kritische blik op alle manuscripten,

ik kijk uit naar onze samenwerking komende tijd. Heel veel succes en plezier in Singapore in het lab

van Prof. van Steensel!

Drs. Jincey Sriram, Beste Jincey, dank voor de prettige samenwerking vanuit het Rijnstate Ziekenhuis.

Je onuitputtelijke inzet die je hebt geleverd naar onze BHD-P deel II studie lijkt z’n vruchten af te

gaan werpen, ik kijk enorm uit naar onze samenwerking naar meer pneumothorax onderzoek!

Beste leden van de BHD werkgroep die ik hierboven nog niet genoemd heb; dr. Hans Gille, prof.

Hanne Meijers – Heijboer, dr. Quinten Qaisfisz, prof. dr. Rick Hoekzema, dr. Erik Thunnissen,

dr. Rob Wolthuis, dr. Iris Glykofridis, dr. Annelinde Terlou, dr. Edward Leter, dr. Anco Boonstra,

dr. Martijn van Doorn, dr. Yael Kon en drs. Rinze Reinhard, veel dank voor alle werkgroepen en

samenwerking wat heeft geleid tot alle mooie papers over BHD. Onze samenwerking heeft ertoe

geleid dat we het grootste BHD centrum zijn van Nederland en op dit gebied een van de leidende

academische centra wereldwijd.

Alle co-auteurs van alle papers, heel veel dank voor de fijne samenwerking en kritische beoordeling

van de verscheidene manuscripten. Gelukkig hebben we er nog vijf gesubmit tijdens het drukken

van dit boekje, dus we hebben nog wat werk voor de boeg.

Stafleden, met in het bijzonder dr. Astrid Baan, en arts-assistenten Heelkunde van het Amstelland

Ziekenhuis, dank voor het geduld bij mijn eerst stappen in de kliniek. Dank voor jullie interesse en

flexibiliteit in het rooster, zodat ik ook nog fulltime aan mijn onderzoek kon zitten.

Stafleden en arts-assistenten, met in het bijzonder dr. Dylan de Lange, dr. Joost Meijer en dr. Leander

van den Ham, van de Spoedeisende Hulp in het UMC Utrecht. Dank voor de mooie leerzame tijd

en dank voor alles wat jullie voor mij gedaan hebben in de aanloop naar mijn sollicitatie voor mijn

opleidingsplek Heelkunde in Regio V.

Stafleden, met in het bijzonder opleider dr. Koop Bosscha, en (oud) arts-assistenten Heelkunde

van het Jeroen Bosch Ziekenhuis. Enorm veel dank voor het warme bad waar ik ruim anderhalf jaar

geleden in belandde met de aanvang van de opleiding Heelkunde. Het dagelijkse werkplezier in

het Bossche groeit nog elke dag. Dank voor jullie vertrouwen, de geboden mogelijkheden en jullie

(soms eeuwige) geduld om mij op te leiden. Elke dag sluit ik de dag weer af met meer kennis en

vaardigheden dan de dag ervoor.

Prof. dr. Menno R. Vriens, geachte professor, ik kijk er naar uit in uw academische kliniek - UMC

Utrecht- de opleiding Heelkunde voort te mogen zetten vanaf januari 2018.

Dear dr. Gupta, dear Nishant, our shared interest on the pathogenesis and clinical implications of

pulmonary BHD had lead to our current follow up study on pneumothorax. I’m looking foward to

our further transatlantic collaboration on BHD and rare lung diseases in the near future.

Claire van Hövell, collega en maatje, hoeveel gesprekken hebben wij niet gehad over mijn promotie

onderzoek, de wegen naar een opleidingsplek binnen Regio V en jouw ambitie en het wetenschappelijk

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onderzoek binnen de Plastische Chirurgie van het UMCU. Heel veel succes met het voortzetten van

je onderzoek in Los Angeles en de weg naar jouw opleidingsplek binnen de plastische.

Beste Tijmen, Brofessor, Broseidon, hier is dan eindelijk het boekje. Je ziet maar weer dat het meer

tijd heeft gekost dan eigenlijk gedacht, hoewel het in “Paul-minuten” eigenlijk nog best wel op tijd

is. Hoe mooi was het in Austin, Texas bij de CHEST meeting! Heel veel succes met het laatste deel

van je coschappen en je verdere carriere.

Kamergenoten van ZH-6D-120 en ZH-3F-013; Wouter Mellema, Romane Saouti-Schook, Pia Trip,

Cathelijne van der Bruggen, Mariëlle van de Veerdonk, Justine Kuiper en Onno Spruijt, wat was het

echt een supermooie tijd met elkaar.

Wouter, het was een mooie tijd die ik met je mee heb mogen maken, waarbij je in die periode

bent getrouwd, vader bent geworden, in opleiding bent gegaan tot longarts en vlak voor mij bent

gepromoveerd. Ik wens je al het goede toe in de toekomst.

Romane, heel veel succes met de laatste loodjes tot je opleiding tot huisarts. We hebben allebei

enorm uitgekeken naar onze promotie onder Prof Postmus, jij bent er al, ik hoop dat het net zo’n

mooie dag wordt.

Mariëlle, tijdens ons promotietraject waren we allebei druk bezig met onze optimale weg uit te

stippelen tot een opleidingsplek, jij tot cardioloog, ik tot chirurg. We hebben ondertussen allebei

onze droomplek bemachtigd. Ik was altijd onder de indruk van je doorzettingsvermogen en je

drukke agenda waarbij je 10 dingen tegelijk met elkaar combineerde. Dank voor de leuke tijd op 6D

en onze borrels met mooie gesprekken bij van Mechelen.

Jus, de grootste miemelaar ooit, uitvinder van het maximaal benutten van de mogelijkheid tot

“thuiswerkdag”, dank voor de mooie tijd op 6D. Gelukkig is je boekje nu ook bijna af. Jij weet als

geen ander hoe zwaar de laatste loodjes kunnen zijn tot het proefschrift écht klaar is. Succes met je

verdere opleiding tot Radiotherapeut.

Pia, gelukkig kon je de grote geluidsboxen op m’n bureau op 6D-120 enorm waarderen. Ook al

moesten we even aan elkaar wennen, het ijs was snel gesmolten en heb echt een supermooie tijd

met je gehad. Dank voor de mooie tijd met onze onderzoeksgroep in binnen- en buitenland tijdens

en buiten het werk. Succes met je verdere opleiding tot longarts in Nieuwegein en VUmc.

Cathelijne, mattie, “klein zusje”, en allergrootste Liefmans bier fan, wat heb ik enorm met je gelachen

afgelopen jaren op 6D en 3F. Altijd tijd voor een kletspauze tijdens en altijd in voor het plannen van

nieuwe party’s en weekendjes weg waar dan ook. Het geluid van je stem was ondoorgrondelijk, je

lach met hoge toon letterlijk onmisbaar. Het hoogtepunt was toch wel de dinsdag in San Diego met

de zoektocht om 5 uur ’s ochtends in de lokale supermarkt naar feest attributen.

Onno, waar moet ik beginnen? Toen ik bij 6D mijn werkplek kreeg, was er al direct een klik. Jouw

aanstekelijke vrolijkheid hielp iedereen altijd de dag door. Een allemansvriend, jouw sociale

vaardigheid is een gave, altijd lachen en vrolijk, maar vakinhoudelijk briljant, bijzonder scherp

en enorm hulpvaardig als ik er met statistiek niet uit kwam. Samen wielrennen; Dam tot Dam,

Gerrie Knetemann Classic, rondje Markermeer, rondje Hoep, en nog belangrijker de Radler

achteraf. Zoals een bekend groot wielrenner al ooit zei “de Tour win je in bed” Tenslotte jouw

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onnavolgbare Onno-move op de dansvloer, de soepelheid rechtevenredig met de stijging van

het alcoholpromillage, wat een feest altijd. Dank voor de mooie tijd. Ik waardeer onze ontstane

vriendschap enorm, en hoop deze nog lang voort te kunnen zetten.

Chris, bedenker van de meest uiteenlopende originele bijnamen, wat ben ik jou afgelopen jaren

gaan waarderen! Filosoferen op de trappen van het Edelwise terras tijdens de maandelijks borrel,

over kleuringen van longweefsel en de relatie tussen PH en BHD. We hadden altijd het idee dat

de Nobelprijs in zicht was met onze hypotheses. Daarnaast de vrijdagavonden bij Bar Bukowski

waren al legendarisch voor ze begonnen. Biomedische wetenschappen is ook maar biomedische

wetenschappen, muizen in het lab zijn mooi, maar het behandelen en toepassen van de geneeskunde

bij echte patiënten nog veel mooier. Wat een goede beslissing om nu nog de verkorte Geneeskunde

studie te gaan doen om dokter te worden. Ik ben onder de indruk van je doorzettingsvermogen en

ik kijk er naar uit jou later voor intercollegiaal consult te kunnen vragen. Ik wens je het allerbeste

met de laatste loodjes van je boekje en het begin van je 2e studie. Ik dank je voor onze vriendschap.

Bart Boerrigter en Gerrina “pizza” Ruiter, mijn voorlopers bij 6D, allebei bijna longarts, ik dank

jullie voor de mooie tijd in binnen en buitenland met elkaar tijdens de gezamenlijke weekendjes

weg en congressen.

Kirill Pavlov, MDL-aios pur sang, “een dag niet getoucheerd is een dag niet geleefd”. Liever een

pot vaseline in je doktersjas dan een stethoscoop. Elkaar leren kennen tijdens de introductie in het

Jeroen Bosch, sindsdien mijn favoriete niet-snijdende collega. Dank voor de mooie tijd afgelopen

anderhalf jaar.

Edgar Wong-Lun-Hing, mijn opleidingsmaatje in het JBZ. Zoals je vaak tegen me zegt “Much to

learn you still have little padawan”. Samen begonnen met de opleiding, samen voor hetzelfde

opleidingstraject ingedeeld, om uiteindelijk chirurg te worden. Ik zie jou elke dag zo enorm groeien

als AIOS, het vak is je op het lijf geschreven. Ik hoop nog vele cursussen, congressen, symposia en

najaarsvergaderingen met jou mee te maken.

Jacob de Bakker, elkaar jaren geleden leren kennen tijdens bedrijfshockey in het Heelkunde team

van het VUmc. Het hoogtepunt toch wel de ESTES in Milaan en het verblijf in het James Bond hotel

met diner bij Osteria del Binari. Dank voor onze regelmatig terugkerende diners waarbij de hele

Heelkunde, onze opleiding perikelen en alle andere zaken die belangrijk zijn in het leven uitgebreid

werden doorgesproken. Fijn dat je weer terug bent uit Malawi!

Boys, de Club van 12; Bart Keukenmeester, Bastiaan Cusell, Diederik Berendsen, Feiko Dols, Oscar

Everhard, Stephane Gaulard, Sebastiaan Heijman, Jasper Padding, Jeroen Stoffels, Maarten-

Paul Strasters, Mark Dijkstra en Sebastiaan Schippers, de rode draad sinds al zoveel jaren. Onze

vriendengroep is onovertroffen, allemaal verschillend, maar samen een enorme hechte groep.

Zoveel fijne momenten met jullie meegemaakt, hoe mooi is het om iedereen zo te zien ontwikkelen,

ieder op z’n eigen manier. Dank voor de mooie jaarlijkse kerstdiners met bijzonder slechte

gedichten, en onze jaarlijks terugkerende vakantie in het voorjaar georganiseerd door Dijkstra

Travels. Jullie interesse in mooie chirugische casuistiek en interesse in de ontwikkelingen van dit

boekje heb ik altijd enorm gewaardeerd. Ik hoop dat we onze vriendschap nog vele jaren voort

kunnen zetten met elkaar.

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Sebastiaan “Apo” Schippers, wat een mooie tijd hebben wij samen meegemaakt tijdens onze

studententijd toen we op Lange Leidse 41-II hoog woonden. Legendarisch en elke dag anders.

Hoewel dagen nachtritme soms ver te zoeken was, had ik die tijd niet willen missen.

Youri “ Poelie” Poelemeijer, sinds de introductie Geneeskunde 10 jaar geleden al onafscheidelijk.

Afgelopen 10 jaar van alles meegemaakt, van de legendarische “ hemiparese rechts” 10 jaar geleden

in Le Cube tot Alpe d’Huzes afgelopen jaar. Samen passie voor het snijdende vak, lopen onze

wegen zo goed als parallel. Als arts-onderzoeker nu aan de DICA /LUmc met speerpunt bariatrische

chirurgie om uiteindelijk binnen afzienbare tijd een opleidingsplek tot chirurg te bemachtigen. Je

vrolijkheid, enthousiasme, gedrevenheid en kunst om het altijd toch weer voor elkaar te krijgen

bewonder ik in je. Het paranimfschap leek nog bijna belangrijker voor jou dan de promotie voor mij,

mooi dat je naast me staat tijdens de verdediging, het wordt puur genieten.

Roderik “ Reaux” Francken, wie had dat gedacht toen we ruim 10 jaar geleden samen op de

reservebank zaten bij Athena. In gesprek geraakt over van alles en nog wat leidde tot een prachtige

vriendschap. Onafscheidelijk afgelopen 10 jaar, jut en jul. Je bent meester in het slap ouwehoeren,

de koning in het maken van selfies, bewonderaar van de unieke dingen in het leven. Al die

vrijdagavonden dat we hebben geborreld eindigden standaard op de kruising van de RAI met de

President Kennedylaan. Hier werd alles besproken wat ertoe deed en wat er eigenlijk ook niet toe

deed. Dank voor alles zover, prachtig dat je mijn paranimf bent. Op naar de toekomst!

Lieve familie, liebe Familie, chere famille, jullie interesse naar de vorderingen van het boekje en interesse

naar de lange weg om chirurg te worden heb ik altijd enorm gewaardeerd, heel veel dank hiervoor.

Bart, grote broer, misschien wel de meest bescheiden persoon die ik ken, ook al zijn we heel

verschillend, je interesse in mijn onderzoek was er niet minder om. Hoewel we allebei totaal iets

anders doen in het dagelijks leven toonde je altijd enorm veel interesse in alle ups en downs van

het wetenschappelijk onderzoek dat uiteindelijk heeft geleid tot dit boekje. De deur staat altijd

bij jou en Tam open, nooit is iets teveel, altijd sta je voor me klaar en altijd kan ik op je terugvallen.

Dat zijn precies de ingrediënten waarom ik minimaal net zo trots op jou ben als andersom. Tam

en Mees, fijn jullie in mijn leven te hebben. Dank voor alle interesse in werk maar ook zeker niet-

werkgerelateerde dingen. Dank voor jullie gastvrijheid op de Hackfort, ik waardeer het enorm.

Liefste Leonie. Samen is altijd leuker! Dat is het gevoel wat je mij altijd geeft. Ik leerde je pas kennen

tijdens het laatste deel van mijn promotie, waardoor je (gelukkig) weinig hebt gemerkt van alle

hobbels in de totstandkoming ervan. Desalniettemin was je enorm betrokken met alles wat nog aan

het boekje moest gebeuren, wat enorm fijn was. Met name als ik het overzicht weer even kwijt was

met alles wat nog moest gebeuren, schepte jij orde hierin. Dit boekje is nu af, maar ons hoofdstuk is

pas net begonnen, en is nu al het beste hoofdstuk ooit. Ik kijk enorm uit naar onze toekomst samen.

Lieve papa en mama, hoofdsponsor, dit boekje is voor jullie. Jullie zijn de basis en de fundering

geweest voor alles wat ik tot nu toe bereikt heb. Jullie onvoorwaardelijke vertrouwen in mij, de

financiële mogelijkheden die jullie mij altijd hebben geboden, nooit was iets teveel gevraagd en

alles was altijd bespreekbaar. Jullie onuitputtelijke inzet om mijn dromen en ambities te helpen

realiseren is meer dan ik jullie ooit voor kan bedanken.

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“It always seems impossible until it’s done.”

Nelson Mandela

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Paul Christiaan Johannesma was born on December 18th, 1983 in Amstelveen, The Netherlands. He

grew up in Amstelveen and attended pre-university secondary education at the Hermann Wesselink

College. After graduation he started the same year medical school at the VU University medical

center (Vumc) in Amsterdam, via the decentralized selection procedure. During medical study, he

set up a study on primairy spontaneous pneumothorax under supervision of dr. P.W.A. Kunst and

prof. dr. P.E. Postmus at the Department of Pulmonary Diseases of the VU University medical center,

which was the foundation of this thesis. In 2008 he joined the multidisciplinary Birt-Hogg-Dubé

Working Group headed by dr. F.H. Menko and prof. dr. P.E. Postmus. After obtaining his medical

degree in 2012, he continued working on his Ph.D. at the VUmc (prof. dr. P.E. Postmus, prof. dr.

R.J.A. van Moorselaar, dr. F.H. Menko and dr. J.H.T.M. van Waesberghe) and combined this with

clinical work as a resident at the Department of Surgery of the Amstelland Hospital, Amstelveen (dr.

S.C. Veltkamp) and as a resident at the Department of Emergency Medicine at the Utrecht University

Medical Center (UMCU), Utrecht (dr. D.W. de Lange). In January 2015 he commenced his six-year

training in general surgery at the Jeroen Bosch Hospital, ’s Hertogenbosch (dr. K. Bosscha) and will

continue his training in January 2018 at the Utrecht University Medical Center (prof. dr. M.R. Vriens).

renal and pulmonary aspects of birt-hogg-dubé syndrome · general introduction and outline of the...

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