Title tbc

Anand Shah1,2, Jacqui Donovan3, Phil Marino4, Pallav L Shah1,2, Toby M Maher2,5, Robert

Wilson1,2, Michael Loebinger1,2.

1Host Defence Unit, Royal Brompton and Harefield NHS Foundation Trust, London, UK2Imperial College London, UK.3Department of Biochemistry, Royal Brompton and Harefield NHS Foundation Trust, London, UK4Department of Critical Care, Guy’s and St. Thomas’ NHS Foundation Trust, London, UK.5Interstitial Lung Disease Unit, Royal Brompton and Harefield NHS Foundation Trust, London,

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Corresponding Author: Dr Anand ShahManuscript Guarantor Host Defence Unit

Royal Brompton and Harefield NHS Foundation TrustSydney Street, London. SW3 6NPEmail: s.anand@imperial.ac.ukTel: +44 (0)20 7352 8121

Disclosures – Nothing to declare

There has been no financial support or other benefits from commercial sources received for

the work reported in the manuscript. The authors have no financial interests or conflicts of

interest to declare relating to this work.

Anand Shah (AS): A 74 year old male retired kitchen designer was transferred to the Royal Brompton hospital from a local district general hospital following increasing breathlessness, type 1 respiratory failure and expectoration of bronchial casts. The patient was raised in a children’s orphanage and recalls having recurrent chest infections alongside a cough with occasional haemoptysis as a young child. He had no prior history of asthma, atopy, tuberculosis or pertussis. At age 17 he was referred to a local chest clinic for recurrent expectoration of bronchial casts (see Figure 1A) and haemoptysis with right lower lobe opacification on his chest radiograph. There was partial resolution with antibiotic therapy but his symptoms intermittently continued. In the following years, a bronchogram revealed narrowed bronchi in the right lung which resolved on follow-up imaging and a normal pulmonary arteriogram. He was discharged from follow-up care with advice to use antibiotics as required. His symptoms of intermittent chronic recurrent bronchial cast expectoration continued alongside recurrent infections over the next twenty years. A fibreoptic bronchoscopy in 1979 at age 41 revealed erythemous bronchial mucosa in the right middle lobe with a ventilation/perfusion scan revealing decreased right middle lobe ventilation. Bronchial washings were unrevealing and due to concern that his symptoms were related to a pathology originating in the right middle lobe, he proceeded to have a right middle lobectomy. Histology showed non-specific inflammatory fibrosis around the lobar bronchus.

His symptoms of bronchial casts, however, did not resolve. Microbiological examination of his casts revealed no growth, and histological examination showed fibrin and mucus with some lymphocytes present. Over the forthcoming years, trials of corticosteroids, nebulised N-acetylcysteine, hypertonic saline, prophylactic azithromycin, systemic antifungals and oral cyclophosphamide did not result in any improvement. Further immunological and allergic tests (including total IgE, Aspergillus fumigatus RAST and IgG, immunoglobulins, serum electrophoresis and vasculitis screen) were normal.

His symptoms remained relatively stable over the years until in 2011 when his expectoration of casts and infections grew more frequent and severe. Prior to this admission, he had returned from a 2 week holiday in Spain where he required hospital admission for intravenous antibiotics and oral corticosteroids. Despite this, on return, his symptoms deteriorated requiring admission to his local district general hospital. He developed worsening type 1 respiratory failure and was transferred to our centre for further evaluation.

On arrival the patient was in respiratory distress. On a FiO2 of 60%, he had saturations of 90% with arterial blood gas analysis showing a pO2 of 6.72kPa, a pCO2 of 6.31kPa, a pH of 7.38 and a bicarbonate of 30.5mmol/L. A chest radiograph showed significant right sided consolidation (Figure 1B). Blood tests showed a normal white cell count with a C-reactive protein of 10mg/L. He was empirically started on intravenous Tazocin, Caspofungin, hydrocortisone, intravenous fluids, nebulised bronchodilators and transferred to a high-dependency ward for monitoring. Further history revealed that the patient had a background of ischaemic heart disease, hypertension and hypercholesterolaemia with previous stents to his left anterior descending and posterior descending coronary arteries. He was an ex-smoker with a cumulative 5 pack-year history. He lived with his wife and two children with no environmental exposures of note. Lung function two months prior to admission showed an forced expiratory volume in 1 second (FEV1) 1.05L/sec, a / forced vital capacity (FVC) of 1.05/2.37L and an FEV1:FVC 0.44. A high resolution CT scan was performed (Figure 1C).

AD or DH: …..

AS, ML and RW: This patient presented with a long history of what is termed ‘plastic bronchitis’. Plastic bronchitis is a rare but well described disease where there is formation of firm gelatinous mucous casts causing bronchial obstruction [1]. Historically, plastic bronchitis was classified histologically as either ‘inflammatory’ type I casts, which are cellular with abundant fibrin, eosinophils, and Charcot-Leyden crystals, or ‘non-inflammatory’ type II casts, which are acellular with mucin and little fibrin [2]. However, more recent attempts at classification are based on associated disease first and cast histology second [3]. The aetiology remains unknown, but plastic bronchitis is associated with a variety of cardiac and inflammatory conditions, including cyanotic congenital heart disease (particularly post Fontan procedure), respiratory infections, asthma, cystic fibrosis, malignancy, and sickle cell disease [4-8]. A number of cases of plastic bronchitis have additionally been associated with lymphatic abnormalities such as lymphangiectasis with expectoration of chylous casts [9-11].

In the above case, the underlying aetiology and classification of plastic bronchitis had not been established and to enable effective treatment, we focussed our initial attention on microbiological, biochemical and histopathological examination of the patient’s bronchial casts.

JD: Histological examination of the casts showed a relatively acellular cast with mucin and fibrin. Microbiological examination did not reveal any bacterial growth with fungal and mycobacterial stains negative. Centrifugation after liquefication of his casts, however, revealed a turbid fatty layer, with biochemical analysis confirming the chylous nature of his bronchial casts with a triglyceride level of 32.5mmol/L (Figure 1D).

AS, ML and RW: Following confirmation of chylous bronchial casts, we decided upon further management. As per the management of any chylous leak, a low fat, medium-chain triglyceride diet was instituted [12]. Given evidence in the management of chylothoraces, the patient was additionally started on Octreotide, a somatostatin analogue [13, 14]. The mechanism of action is uncertain, but it is proposed that octreotide causes mild vasoconstriction of splanchnic vessels, including hepatic venous flow. This leads to reduction in gastric, pancreatic and intestinal secretions as well as intestinal absorption collectively reducing the flow of chyle [15]. In addition to reducing chylous leak, the patient had significant bronchial obstruction from his bronchial casts and we required a means of either removal or liquefaction. A flexible bronchoscopy approach was considered.

PS: Although removal of bronchial casts by flexible bronchoscopy is possibly helpful, it is difficult [16, 17]. The casts are often too firm for removal by suction, yet too friable for removal by forceps. Patients are occasionally able to self-expectorate as in this case, however, severe exacerbation may limit this ability. Novel technology such as cryotherapy may be useful in these circumstances. Where casts are visible it is possible to adhere the cryotherapy probe to the proximal aspect of the cast by the freezing process and then to remove the bronchsocpe and cast en bloc , and hence bronchoscopy was attempted in a high-dependency environment [18]. Hence bronchoscopy was attempted in a high-dependency environment No bronchial casts were visible however with severely oedematous collapsed right lower and upper lobe bronchi. Washings and bronchial mucosal biopsies were taken but ano therapeutic procedures were able tocould not be performed. Washings did not reveal any microbiological growth and biopsies showed non-specific inflammation with the presence of some lymphocytes.

AS, ML and RW: As the removal of bronchial casts was not possible, we concentrated our efforts on liquefaction. The use of both nebulised dornase-alpha (DNase) and tissue plasminogen activator (t-PA) has been reported for the management of chylous bronchial casts [19-22]. As the patient was on dual antiplatelet therapy following implantation of drug-eluting coronary arterial stents, we were keen for confirmation of efficacy before commencement.

JD: A dried bronchial cast expectorated by the patient was incubated in 2ml of either 0.1mg/ml t-PA or 0.1mg/ml DNAse for 2hr or saline as control. The cast was removed from solution and air dried on nylon gauze and re-weighed, and the remaining drug solution was analysed for protein and fibrin break down products. 2 hour incubation with t-PA resulted in loss of structure, 62% loss of weight, 0.47g/L protein and >4x106ng/ml XDP release into supernatant. DNAase incubation resulted in no change in structure, 10% loss of weight, 0.07g/L protein and 40,000ng/ml XDP release in the supernatant. Saline control incubation resulted in no change in structure, 2% loss of weight, 0.0g/L protein and 1200ng/ml XDP release into the supernatant.

AS, ML and RW: Biochemical analysis clearly showed partial and high efficacy of DNAse and t-PA respectively in liquefaction of the patients bronchial casts. In a high-dependency environment, the patient was commenced on nebulised DNase 2.5mg BD and t-PA 5mg tds. There were no haemorrhagic complications from treatment and within 24 hours, there was good initial radiological improvement of the patient’s right upper and lower lobe obstruction. However, liquefaction led to subsequent difficulty in clearance, intermittent hypoxia and what appeared to be reformation of casts and further bronchial obstruction. As there was still clearly persistent chylous leak and bronchial cast formation, we subsequently focussed our efforts on understanding the pathophysiology of chylous bronchial cast formation in this patient.

PM: In patients with chylous bronchial casts post Fontan procedure with congenital heart disease, although the mechanism is unknown, it is thought high central and pulmonary venous pressures may play an aetiological role [23]. There have been documented case reports of the successful use of pulmonary vasodilator therapy such as Sildenafil and Epoprostenol [24, 25]. Given the above, an echocardiogram was performed. This showed a normal left ventricular wall thickness and cavity size with preserved systolic and diastolic function. There was mild hypokinesia of the basal to mid posterior inferior wall in keeping with previous ischaemic heart disease. His right heart was mildly dilated (RV inlet 42mm diastolic diameter) with normal longitudinal function (TAPSE – 2.8cm). There was no significant valvular disease, normal size right atrium and an estimated pulmonary arterial systolic pressure of 51mmHg (assuming RAP of 10mmHg). Given the evidence of right heart strain on echocardiogram and moderately elevated pulmonary pressures, the patient was started on pulmonary vasodilator therapy with Sildenafil increasing to 50mg tds.

AS, ML and RW: Patients with lymphangioleiomyomatosis (LAM) are known to have problems with recurrent chylothoraces [26, 27]. There have been recent reports regarding the use of the mammalian target of rapamycin (mTOR) inhibitor Sirolimus for management of recurrent chylothoraces [28, 29]. Given this, we consulted with our interstitial lung disease colleagues.

TM: LAM is a progressive cystic lung disease in women associated with inappropriate activation of the mTOR signalling pathway causing dysregulation of cellular

growth and lymphangiogenesis [30, 31]. This can result in recurrent chylothoraces and, on rare occasions, chyleoptysis [27]. There have been recent studies highlighting the successful use of the mTOR inhibitor Sirolimus in the management of both chylothoraces and also stabilisation of lung function in LAM [32-34]. There have also been isolated case reports showing successful use of Sirolomus in diffuse lymphangiomatosis [35, 36]. The mechanism of action in treatment of chylothoraces is unclear, but potentially relates to regression of the abnormal smooth muscle cellular proliferation causing obstruction and leak within the lymphatic system [37]. In the above gentlemanthe current case, the aetiology of his the chylous bronchial casts is not clear. Examination did not reveal any signs consistent with yellow nail syndrome. However, the presencethe reported symptoms since childhood may point to a diagnosis of diffuse pulmonary lymphangiomatosis (DPL) or primary thoracic lymphangiectasis [38, 39].

Lymphangiomas are congenital, benign, cystic, focal areas of lymphatic proliferation that can occur in any part of the body containing lymphatics. This pathological process can result in mass effect from infiltrative disease, restrictive and/or obstructive physiology, chylous effusions and respiratory failure. The disease is predominantly detected in children and young adults, and affects both sexes equally [40-42].

AS, ML and RW: Given the strong possibility of congenital or acquired lymphatic obstruction in the patient and published successful case reports of mTOR inhibitors, we started treatment with Sirolimus. This continued alongside regular DNAse, Octreotide, Sildenafil and an MCT diet. Gradually the patient started to improve and was eventually discharged from hospital. Subsequent lung function and HRCT imaging revealed almost complete resolution of his right sided lung changes and markedly improved lung function. To confirm the diagnosis of lymphatic obstruction a lymphangiogram was performed (Figure 1E). This showed x..

3 months post discharge, the patient was no longer productive of anyexpectorating bronchial casts. Gradually, over the forthcoming months his Octreotide and Sildenafil have beenwerestopped. He continues on Sirolimus and once daily DNase and tries to maintain a MCT diet where possible, but two years later has not had any significant further relapses or episodes of chylous bronchial cast formation.

Patient: …

AS: Sirolimus (Rapamycin) is emerging as a promising therapeutic option in the medical management of patients with lymphatic obstruction or chylous leak [43]. It is a macrolide produced by the bacterium Streptomyces hygroscopicus, is structurally related to the calcineurin inhibitor FK506 (Tacrolimus) and has potent immunosuppressive properties [44]. Sirolimus forms a complex with an intracellular binding protein (immunophilin) and binds with and inhibits mammalian target of rapamycin (mTOR), a serine/threonine kinase regulated by phosphoinositide-3-kinase (PI3K). By interfering with the function of mTOR, sirolimus inhibits the mTOR-mediated signal-transduction pathways, resulting in the arrest of cell cycle in G1 phase in various cell types [45]. This results in the inhibition of Il-2 induced proliferation of T cells making it a valuable immunosuppressant to prevent organ rejection in solid-organ transplant recipients [45]. Recently published genetic studies however have shown somatic mutations in the catalytic subunit of PI3K (PIK3CA) in patients with isolated and syndromic lymphatic malformations. This resulted in over-proliferation of lymphatic endothelial cells which improved on Sirolimus therapy [46]. This finding may help explain

the underlying mechanism behind the successful use of mTOR inhibitors, and importantly, could provide an avenue for future gene-therapy in congenital lymphatic obstruction cases.

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Additional reference for cryoextraction is Hetzel M, Hetzel J, Schumann C, Marx N, Babiak A. Cryorecanalization: a new approach for the immediate management of acute airway obstruction. J Thorac Cardiovasc Surg. 2004 May;127(5):1427-31