UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA

DOCTORAL SCHOOL

PhD THESIS

ABSTRACT

THE ROLE OF IMMUNOHISTOCHEMICAL

EXAMINATION IN EARLY AND DIFFERENTIAL

DIAGNOSIS OF CERVICAL CANCER

PhD SUPERVISOR

Prof. Univ. Dr. ŞTEFANIA CRĂIŢOIU

PhD STUDENT

ILEANA BARBU

CRAIOVA - 2013

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CONTENTS

Introduction ................................................................................................... 3

STATE OF KNOWLEDGE ........................................................................... 3

CHAPTER I. Histology and uterus histophysiology....................................... 3

CHAPTER II. Epidemiology and pathogenesis of cervical cancer................ 4

CHAPTER III. Distinctive signs of Cancer.................................................... 4

PERSONAL CONTRIBUTIONS................................................................... 4

CHAPTER IV. Disability caused by cervical neoplasm in Olt county........... 4

CHAPTER V. Histopathological study of endocervical adenocarcinoma….. 5

CHAPTER VI. Immunohistochemical study of endocervical

adenocarcinomas .......................................................

7

CONCLUSIONS ……………………………...…………………………… 9

Selected Bibliography ……………………………………….……….……..

9

Keywords: cervical adenocarcinoma, invalidity, histopathological variants,

immunohistochemical markers.

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INTRODUCTION

Cervical carcinoma is the second most common cancer in women worldwide and

it remains a leading cause of cancer-related death for women in developing countries.

The diagnosis of cervical adenocarcinoma is clinically very important beacause of

its poorer prognosis and lower sensitivity to radiotherapy and chemotherapy in comparison

with squamous cell carcinoma [1, 2].

The purpose of this thesis is to supplement existing data in the specialized

literature on clinicomorphological aspects of cervical cancer and to highlight

immunohistochemical examination implications in early and differential diagnosis of

cervical adenocarcinomas, in order to apply corresponding therapeutic management.

In this respect, we performed a histopathological and immunohistochemical study

on 16 cases of endocervical adenocarcinoma selected from the casuistry of the

MorphoPathology Laboratory from the Slatina Clinical Hospital within 2005-2010.

We have also highlighted the adverse impact of cervical cancer at socioeconomic

level, particularly on disability in the county of Olt.

STATE OF KNOWLEDGE

CHAPTER I

HISTOLOGY AND UTERUS HISTOPHYSIOLOGY

The uterus develops along with the other female internal genital organs (fallopian

tubes and vagina) of Müller's channels (paramesonephrotic channels).

The Müller channels have three portions: one portion of skull, dilated (ostium), an

intermediate portion which opens through several orifices in the cloacal cavity and caudal

portion, in which the two Müller channels approach but remain separated by the septum

that will reabsorb in the 3rd

month of intrauterine life, when it will form the uterovaginal

canal, through the union of the two channels.

Thus, the skull portion and from the area from the beginning of the intermediate

portion will form the fallopian tube, the intermediate bottom portion, the body and the

cervix uteri, and lower portion of the uterovaginal canal will form the vagina.

Anatomically, the uterus has three parts: the upper section and the most dilated is

the body of the uterus, the isthmus, a narrow middle zone and the uterine cervix a lower

portion of cylindrical shape. The cervix is divided by insertion of the vagina in two par

areas: supra-vaginal and sub-vaginal (vaginal) that protrudes into the vagina.

At the level of these segments, there are three cavities: the cavity of the body of

the uterus, the isthmus canal and the cervical canal or uterine cervix canal [3].

Uterine wall has three tunics: tunica mucosa (endometrium), tunica musularis

(myometrium), tunica serosa (perimeter).

The cervix is the lower portion of the uterus and it has cylindrical shape. The wall

has three tunics: mucosa, muscular-elastic (smooth muscle fibers and connective tissue

rich in elastic fibers) and adventives.

The cervix has two areas: exocervix and endocervix. The exocervix is the portion

visible in the vaginal cavity, being covered by a nonkeratinized stratified squamous

epithelium and an aglandular chorion and the endocervix or endocervical canal, which

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continues the uterine isthmus, consisting of a simple cylindrical epithelium with numerous

mucous cells and glandular chorion.

CHAPTER II

EPIDEMIOLOGY AND PATHOGENESIS OF CERVICAL CANCER

Cervical carcinoma is the second most common cancer among women worldwide.

Global burden is enormous, with over 500,000 new cases of cervical cancer diagnosed

each year and 280,000 registered deaths [4].

While in developed countries screening programs have significantly reduced the

incidence of the disease, about 80% of cervical cancers still occur in developing countries.

Epidemiological studies conducted in the last 30 years have shown that the risk of

cervical cancer is strongly influenced by the following factors: sexual activity, smoking,

number of births, diet, infection with HPV, the use of birth control pills for a long time.

HPV strains present most frequently in precancerous lesions of the cervix and

cervical cancer are types 16 and 18.

CHAPTER III

HALLMARKS OF CANCER

Vastul catalog de genotipuri de celule canceroase este determinat de șase

modificări esențiale în fiziologia celulei, care determină creșterea malignă The broad

catalog of cancer cell genotypes is determined by six essential alterations in cell

physiology, determining the malignant growth [5]:

o self-sufficiency in growth signals;

o insensitivity to anti- growth signals;

o evading apoptosis;

o limitless replicative potential;

o sustained angiogenesis;

o tissue invasion and metastasis.

CHAPTER IV

DISABILITY CAUSED BY CERVICAL NEOPLASM

IN OLT COUNTY

Cervical cancer is the main cause of death among women aged between 15 and 44

years from Romania, when they are professionally active, this having a negative

socioeconomic impact.

I have conducted a clinicoepidemiological study and about the disability in Olt

County between 2005-2010.

The following materials were used: population cancer registries, dispensary

records of the patients with cervical cancer and statistics from the Department of Medical

Expertise and Work Capacity Recovery Olt,between 2005-2010.

The results obtained reveal that the majority of female patients are uninsured,

come from rural areas, have advanced disease stages and working capacity lost.

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Rural

Urban

0

20

40

60

80

2005 2006 2007 2008 2009 2010

T o t a l

S t a g e III/IV

Fig IV. 1 – Distribution of cervical cancer cases by area of

origin in Olt county in 2005-2010

Fig IV.2- Distribution of new cases of cervical cancer

by stage of disease during 2005-2010 in Olt county

0

10

20

30

40

50

60

70

80

2005 2006 2007 2008 2009 2010 total

uninsured

insured

Fig IV.3 –Distribution by insurance status in Olt county

2005-2010

0

10

20

30

40

50

60

70

C a s e s

Degree III

Degree II

Degree I

Fig IV.4 – The distribution of the disability degrees between

2005 -2010 in Olt county

CHAPTER V

HISTOPATHOLOGICAL STUDY OF ENDOCERVICAL

ADENOCARCINOMA In the histopathological study we investigated the main clinicomorphological

features of endocervical adenocarcinoma that might be involved in the prognosis of these

patients.

Histopathological material came from the casuistry of the MorphoPathology

Laboratory of the Emergency Hospital from Slatina County and was represented by 16

archived paraffin blocks.

In the morphological study we used the classic histological techniques including

paraffin. As staining methods we used:

� Hematoxylin eosin (HE) for diagnostic evaluation according to the criteria for

the classification of cervical tumors set by WHO (2003) [6];

� Masson trichrome of aniline blue to assess the degree of fibrosis tumor;

� Alcian Blue Periodic Acid Schiff (PAS) to assess the profile of mucins (neutral

versus acidic) secreted by tumor cells.

In the table below are presented the main clinicopathological features of

investigated casuistry (Table V.1).

40%

60%

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Tabelul V.1 The major clinicopathological features of the investigated cervical adenocarcinoma

Cer

vic

al

ad

eno

carc

inom

a

typ

e

Ag

e(y

ers)

Tu

mo

r si

ze(c

m0

Dif

fere

nti

ati

on

Dep

th o

f

stro

mal

In

va

sio

n

Dep

th o

f

mu

scu

lar

inv

asi

on

Pa

ram

etri

al

inv

olv

emen

t

Ly

mp

h n

od

e

met

ast

asi

s

Va

gin

al

marg

ins

TN

M

≤4 >4 W-M*

P* ≤1/2L

** >1/2L ≤1/2L >1/2L + - + - + -

Mucinous

endocervical 45 + + + + - - - IB1

Mucinous

endocervical 56 + + + + - - - IB2

Mucinous

endocervical 58 + + + + - - - IB2

Mucinous

endocervical 69 + + + + + - - IIB

Mucinous

endocervical 64 + + + + + - - IIB

Mucinous

endocervical 55 + + + + + + + IIIB

Mucinous

endocervical 61 + + + + + + + IIIB

Mucinous

intestinal 57 + + + + + - - IIB

Mucinos

villoglandular 37 + + + + - - - IB1

Mucinos

villoglandular 40 + + + + - - - IB2

Endometroid 59 + + + + + - - IIB

Endometroid 62 + + + + + - + IIIA

Endometroid 58 + + + + + + + IIIB

Endometrioid 63 + + + + + + + IIIB

Serous 47 + + + + - - - IB2

Serous 67 + + + + + - - IIB

- tumor degee of differentiation: W-M= well- moderate; P= poor; **

L= Layer

Fig. V.1 Cervical adenocarcinoma - mucinous

endocervical type..

HE stain, X100

Fig. V.2 Cervical adenocarcinoma - mucinous intestinal

type. – malignant cells with goblet cell morphology

that apically present a pool of acidic mucins.

AB – PAS stain, X200.

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Fig. V.3 Cervical adenocarcinoma. mucinous villoglandular type.

HE stain, X40.

Fig. V.4 Cervical adenocarcinoma endometrioid type.

HE stain, X40.

Fig. V.5 Cervical adenocarcinomm - serous type.

HE stain, X40.

In the investigated casuistry prevailed mucinous carcinoma cases (62.5%), with

the endocervical subtype as most common (43.75% from all cases investigated and 70%

from the all mucinous endocervical tumors). The second most common histopathological

type of endocervical carcinoma was the endometroid type which accounted for 25% of all

investigated cases.

Other variants diagnosed include: serous adenocarcinoma (12.5%), mucinous

villoglandular type (12.5%) and intestinal mucinous adenocarcinoma (6.25%).

The most aggressive form of endocervical adenocarcinoma investigated proved to

be the endometroid, the vast majority (75%) being diagnosed in advanced clinical stages

IIIA and IIIB.

A better prognosis had the serous, respectively the villoglandular varieties, which

were diagnosed in less advanced clinical stages (IB and IIB).

CHAPTER VI

IMMUNOHISTOCHEMICAL STUDY OF ENDOCERVICAL

CARCINOMAS

The aim of immunohistochemical study was to assess the expression of

immunohistochemical markers: CD105, VEGF, EGFR and c-erbB2 and their prognosis in

different types of endocervical adenocarcinoma.

In many situations, especially when a tumor involves both the lower uterine

segment and upper endocervix, the distiction between a primary endometrial and a

primary endocervical adenocarcinoma may be difficult. This differential diagnosis is

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imperative because the treatment plans and adjuvant therapies are totally different forthe

two forms of adenocarcinoma [7, 8].

This problem is partially solved by using appropriatepanels of antibodies. Several

studies have reported that typical immunoprofile of the primitive endocervical

adenocarcinomaseems to be ER-/PR-/Vim-/CEA+, wheares for primitive endometrial

carcinoma the characteristic profile is ER+/PR+/Vim+/CEA-[9,10,11,12].

Our investigations proved that this panel specific to the endocervical

adenocarcinoma was recorded in 62,5% of our cases [13].

In all investigated cases, we observed that the angiogenesis process identified by

the determination of MVD CD105 was more intensive in peritumoral area, regardless of

clinicomorphological features of thises cases (the degree of differentiation, depth of

muscular invasion or TNM stage).

For the first time, we revealed that the angiogenesis process seams to be

dependent on histopathological subtype, with the highest values of MVD CD105+

recorded in the mucinous variant of endocervical adenocarcinoma and the lower values in

serous type.

Investigation of VEGF expression showed that regardless of histophatological

variant, the highest values of VEGF reactivity corresponded to higher MVD CD105

values.

Reactivity for markers CD105, VEGF, EGFR-1 and C-erbB2 allow the

stratification of the patients with endocervical adenocarcinoma in order to determine the

most effective treatment options that will target these molecular markers.

Fig. VI.1 Cervical adenocarcinoma-

mucinous villoglandular type .ER

negative reaction in the nuclei of

neoplastic cells. DAB, X100.

Fig. VI.2 Cervical adenocarcinoma-

mucinous villoglandular type. PR

negative reaction in the nuclei of

neoplastic cells. DAB, X200.

Fig. VI.3 Cervical adenocarcinoma-

mucinous villoglandular type. VIM

negative reaction in the cytoplasm of

neoplastic cells but positive in blood

vessels endothelial. DAB, X200

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Fig. VI. Cervical adenocarcinoma-

mucinous serous type. CEA positive

reaction in the cytoplasm of neoplastic

cells.DAB, X100

Fig. VI.5 Cervical adenocarcinoma –

mucinous endocervical type. CD 105

positive reaction in blood vessels from

peritumoral DAB, X200

Fig. VI.6 Cervical adenocarcinoma

Intense cytoplasm EGFR reaction in the

mucinous adenocarcinoma DAB, X200

CONCLUSIONS

Cervical cancer remains a serious public health problem worldwide with

high mortality in developing countries, especially in Romania.

In our study, we highlighted that the clinical stage is the most important

prognostic factor and that to same extent, same histopathological features of these

tumors can condition their biological behavior.

We also noticed that in endocervical adenocarcinomas there is an intense

process of angiogenesis, which is controlled by the interrelation between VEGF,

EGFR and c-erbB2.

Further studies are needed to elucidate whether specific angiogenic

molecular profiles exist in different histopathological subtype of uterine

adenocarcinomas and which is their impact on prognosis and therapeutic outcomes

for these patients.

SELECTED BIBLIOGRAPHY

[1]. Hopkins M.P., Morley G.W. Acomparison of adenocarcinoma and squamous cell

carcinoma of the cervix. Obstet Gynecol 1991; 77:912–7;

[2]. Tinga D.J., Bouma J., Aalders J.G. Patients with squamous-cell vs. adeno(squamous)

carcinoma of the cervix; what factors determine the prognosis. Int J Gynecol Cancer

1992; 2:83–91;

[3]. Papilian V., Roșca Gh. Tratat elementar de histologie vol.1. Ed. Dacia Cluj Napoca,

1977;

[4]. Parkin D.M., Bray F., Ferlay J. Pisani P. Global cancer statistics, 2002. CA Cancer J

Clin 55: 74-108, 2005;

[5]. Hanahan D., Weinberg R. The Hallmarks of Cancer. Cell, Vol. 100, 57–70, January 7,

2000;

[6] Wells M., Ostor A.G, Crum C.P., Franceschi S., Tommasino M., Nesland T.M.,

Tumors of the cervix. In: World Health Organization, editor. Tumors of the breast and

female genital organs. Lyon: IARC Press, 2003. pp. 262-276;

immunohistochemical panel. Arch Pathol Lab Med. 2003 Dec;127(12):1586-90;

10

[7]. Lurain J.R., Bidus M.A., Elkas J.C. Uterine cancer, cervical and vaginal cancer. In:

Berek RS (ed) Novak’s gynecology, 14th edn. Lippincott Williams & Wilkins,

Philadelphia, 2007; pp 1343–1402;

[8]. Schorge J.O., Knowles L.M., Lea J.S. Adenocarcinoma of the cervix. Curr Treat

Options Oncol 2004; 5:119–127;

[9]. Alkushi A., Irving J., Hsu F., Dupuis B., Liu C.L,, Rijn M., Gilks C.B. ImmunoproWle

of cervical and endometrial adenocarcinomas using a tissue microarray. Virchows

Arch 2003; 442(3):271–277.

[10]. Castrillon D.H., Lee K.R., Nucci M.R. Distinction between endometrial,

endocervical adenocarcinoma: An immunohistochemical study. Int J Gynecol Pathol

2002; 21(1):4–10;

[11]. Dabbs D.J., Sturtz K., Zaino R.J. Distinguishing endometrial from endocervical

adenocarcinoma. Hum Pathol 1996; 27(2):172–177

[12]. McCluggage W.G., Sumathi V.P.,McBride H.A.,Patterson A. A panel of

immunohistochemical stains, including carcinoembryonic antigen, vimentin, and

estrogen receptor, aids the distinction between primary endometrial and

endocervical adenocarcinomas. Int J Gynecol Pathol 2002; 21: 11–15;

[13]. Barbu I., Crăiţoiu Ș., Mărgăritescu C. Cervical adenocarcinoma: a retrospective

clinicopathologic study of 16 cases. Rom J Morphol Embryol. 2012;53(3):615-24;

[14]. Castrillon D.H., Lee K.R., Nucci M.R. Distinction between endometrial and

endocervical adenocarcinoma: an immunohistochemical study. Int J Gynecol Pathol

2002, 21: 4–10;

[15]. Solomon D., Breen N., McNeel T. Cervical cancer screening rates in the United

States and the potential impact of implementation of screening guidelines. CA

Cancer J Clin. Mar-Apr 2007; 57(2):105-11;

[16]. Parazzini F., La Vecchia C. Epidemiology of adenocarcinoma of the cervix. Gynecol

Oncol. 1990 Oct; 39(1):40-6;

[17]. Eifel P.J., Burke T.W., Morris M., Smith T.L. Adenocarcinoma as an independent

risk factor for disease recurrence in patients with stage IB cervical carcinoma.

Gynecol Oncol 1995; 59:38–44;

[18]. Veikkola T., Alitalo K. VEGFs, receptors and angiogenesis. Semin. Cancer Biol. 9,

211–220, 1999;

[19]. Saigo P.E., Cain J.M., Kim W.S., Gaynor J.J., Johnson K., Lewis J.L. Jr. Prognostic

factors in adenocarcinoma of the uterine cervix. Cancer 1986;57:1584-93;

[20]. Grigore T., Cernea N. Uterine pathology. University Medical Publishing

Craiova, 1998;

[21]. Crăiţoiu Ş. Histology special. . University Medical Publishing, Craiova 2003;

[22]. Law 19/17 March 2000 on the public pension system and other social

insurance rights;

[23]. Șirjiță N., Ciuvică M.M, Gherman D., Statnic L. Elements of medical

examination and recovery of working capacity Tiparg 2004; 33;