UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA DOCTORAL SCHOOL

PhD THESIS

ABSTRACT

EPITHELIAL-MESENCHIMAL TRANSITION ROLE IN BLADDER UROTHELIAL CARCINOMA

PhD THESIS ADVISOR, CRĂIȚOIU ȘTEFANIA, PhD, PROFESSOR

PhD STUDENT, DROCAȘ IOAN ANDREI

CRAIOVA – 2016

2

CONTENTS

INTRODUCTION 3

LITERATURE REVIEW 3

CHAPTER 1

Histology and histophysiology of urinary system

3

CHAPTER 2

Bladder tumoral pathology

4

CHAPTER 3

Epithelial-mesenchimal transition role in bladder carcinogenesis

4

PERSONAL CONTRIBUTIONS

CHAPTER 4

Clinical-Epidemiology study of bladder urothelial carcinoma

4

CHAPTER 5

Morphological study of bladder urothelial carcinoma

6

CHAPTER 6

Imunohistochemical study of bladder urothelial carcinoma

8

GENERAL CONCLUSIONS 11

SELECTIVE BIBLIOGRAPHY 11

Key words: bladder cancer, epithelial-mesenchimal transition, urinary system,

imunohistochemical study, imunohistochemical markers

3

INTRODUCTION

Bladder cancer is most frequent cancer in urinary tract, after prostate cancer (Boring CC,

Squires TS, Tong T, 1995). It has important geographical variations, higher incidence rates is on

males living in developing contries. In Europe is the highest rate of bladder cancer around the

world. It is five times more frequent in males compared to females. Even is more frequent in

males, prognosis is improved compared to females and mortality rate is lower, bladder

carcinoma beeing diagnosed in initial stages (Shariat SF, 2010).

Urothelial carcinogenesis, recurrence, progression and metastasis can involve numerous

molecules that will be discovered. On this time there is no diagnosis, prognosis or therapeutics

molecular biomarkers for urothelial carcinoma (Liang Cheng, Darrell D. Davison, Julia Adams

et al, 2014).

Doctorate thesis is structurated in two major parts:

I Actual know, where in three chapters i have notice aspects regarding histology and

histofiziology of urinary sistem, bladder cancer pathology and role of epithelio-mezenchimal

transition in bladder cancer carcinogenesis.

II Personal contribution with main objective to found correlations between clinical

aspects, imagistic, histological and imunohistochemical and i realized one clinical-epidemiology

study, one for mophology and one imunohistochemical study, each with more objectives.

LITERATURE REVIEW

CHAPTER 1 HISTOLOGY AND HISTOFIZIOLOGY

OF URINARY TRACT

Urinary system have multiple functions: produce and eliminate urine, facilitating

cleaning the body from catabolic products, some highly toxic, maintaining acido-basic balance

and hidro-electrolitic, excretion of water and selective resorbtion for specific subtances and ions,

endocrine function including blood presure regulation by production of hormonal substance

(renin), stimulation of red blood cells by production of erytropoetin. All this functions performed

by urinary system contribute on body homeostasis.

Urinary system development is very close to genital system development, with

mesodermal origins. Intermediate mesoderm is segmented on cervical region, forming

nephrotoms (Sadler TW, 2010), from wich are developing three main structures: pronephros,

mesonephros and metanephros.

Urinary system is formed from kidney, pair located in retroperitoneum, in kidney lodge,

both parts of the spine, T11-L3, with secretion role, producing urine from intrarenal urinary

(small calics, large calics, urynari pelvis) and extrarenal (ureters, bladder, urethra), from wich

urine is transported, stocated and eliminated. (Chesbrough RM, Burkhard TK, Martinez AJ,

Burks DD, 1989; Crăiţoiu Ş, 2003; Sinescu I, Gluck G, 2008).

4

CHAPTER 2 BLADDER TUMOR PATHOLOGY

Bladder cancer is on second place between uro-genital cancers, beeing on the first place

on the urinary tract, after prostate cancer (Boring CC, Squires TS, Tong T, 1995). Incidence is

around 5% from new diagnosed cancer each year, on the 4th place on males after prostate cancer,

lung and colon cancer (Tomescu P, Pănuş A, 2006). Bladder cancer incidence seems to decrease,

probably because decreasing exposion to different risk factors like smoking or different chemical

agents (Sievert KD, Amend B, Nagele U et al, 2009). Bladder cancer is two times more frequent

on white males compared to black males, while on white females appeareance rates is with only

44% higher than for black females (Cutler SJ, Young JL, 1975).

This condition should not be regarded as a familial pathology (Kiemeney LA,

Schoenberg M, 1996; McCullough DL, 1975). Genetic study have discovered 7-10 cromosomial

modifications for patients with invasive bladder tumors (Richter J, Jiang F, Gorog JP et al, 1997;

Richter J, Beffa L, Wagner U et al, 1998).

Proffesional exposure is involved in approximately 20% patients with bladder cancer in

United States of America, with latence period between 30-50 years (Silverman DT, Levin LI,

Hoover RN, Hartge P, 1989; Silverman DT, Levin LI, Hoover RN, 1989; Maximilian B, James

WFC, Guido D et al, 2013). Bladder cancer incidence is proportional with period and number of

cigaretes smoked daily (Brennan P, Bogillot O, Cordier S et al, 2000; Joseph JC, Evanguelos X,

Luis AK et al, 2014). Diet, together with nutrition action as pro-factors or antitumor factors on

the tumors process in the urogenital tract (Cheng G, Xie L, 2011; Steineck G, Hagman

U, Gerhardsson M, Norell SE, 1990; Vena JE, Graham S, Freudenheim J et al, 1992; Xu C, Zeng

XT, Liu TZ et al, 2015).

CHAPTER 3 ROLE OF EPITHELIAL-MEZENCHIMAL TRANSITION IN BLADDER

CARCINOGENESIS

Epithelial-mesenchimal transition process (MET) was observed since embrional

development, beeing an important process for embrional development and forming different

tissues and organs (Savagner P, Boyer B, Valles AM et al, 1994). Interest for this process is

mainly because of his role in neoplasic progression (Thiery JP, Sleeman JP, 2006). In 1982 MET

theory was proposed, that reveals in epithelial cells cultures, they are morphological convert in

mesenchimal cells with gel mobility (Greenburg G, Hay ED 1982). MET represent dinamic

tranformation of epithelial cells with mesenchimal phenotype (van der Horst G, Bos L, van der

Pluijm G, 2012). MET is not specific to neoplasms, with important role in tissue differentiation,

organs development and wound healing (Peinado H, Olmeda D, Cano A, 2007). MET is reversible,

involved cells beeing able to regain epithelial phenotype (Chen J, Han Q, Pei D, 2012). MET

have three different subtypes, separated by production pathways and functions completed, one

associated with neoplasms. (Chaw SY, Majeed AA, Dalley AJ, 2012; Kalluri R, Weinberg RA,

2009).

5

PERSONAL CONTRIBUTIONS

CHAPTER 4 CLINICAL-EPIDEMIOLOGY STUDY OF BLADDER UROTHELIAL

CARCINOMA

Study was prospective and included 784 hospitalised patients for specific treatment

between 2013-2015 in Urology Department, Emergency County Hospital Craiova, diagnosed

with bladder tumor. For clinical analysis we followed patients distribution by years, age, gender,

patients origin, risk factors and associated symptoms, associated with imaging investigation (Fig.

4.1 - Fig. 4.5). Statistical processing used average values, standard deviations and comparative

tests (t Student, Chi pătrat, Pearson), completed with SPSS10 software (SPSS Inc., Chicago, IL,

USA). Table 4.1 presents investigated cases by years.

Tabelul 4.1 Cases repartition by years

Year 2013 2014 2015

Nr. Of Cases 205 262 317

% 26,1 33,4 40,5

592, 76%192, 24%

Males

Females

Fig. 4.1 Patients repartition by age Fig. 4.2 Patients repartition by gender

Fig. 4.3 Patients repartition by risk factors

Fig. 4.4 Patients repartition by presentation

symptoms

6

Parameter Variable Nr.cases % Tumor

number

unique

multiple

766

18

97,7

2,3

Size

<2cm

2-10cm

>10cm

686

82

16

87,5

10,5

2

Localisation

trigon

dome

anterior

posterior

lateral

160

102

152

137

215

20,4

13,1

19,4

17,5

27,4

Fig. 4.5 Patients repartition by number, size and tumor localisation

CHAPTER 5 MORPHOLOGICAL STUDY OF BLADDER UROTHELIAL CARCINOMA

Morphological study included 760 urothelial invasive bladder carcinoma, diagnosed in

Pathology Laboratory of Emergency County Hospital Craiova. Histopathological analyses

included tumoral differentiation grades (Fig. 5.1), of invasion (Fig. 5.2, Fig. 5.3, Fig. 5.4) and

pTNM stadialisation. We also followed tumor histology differences (Fig. 5.5, Fig. 5.6), vascular

invasion (Fig. 5.7) and perineural (Fig. 5.8), also histology aspects of tumor front, defined as

most profound invasion area of urothelial carcinoma on bladder wall or on resection pieces after

transurethral resection of bladder.

Histopatological parameters of urothelial carcinoma are important also for terapeuthics

strategies and for recurrence risk and survival period. Integrated analyses of clinical-imaging

parameters and histopathological can improve therapeutic management for better prognosis.

Fig. 5.1 High grade urothelial carcinoma col. HE, x200 Fig. 5.2 Peritumoral desmoplasic reaction, col. HE, x200

7

Fig. 5.3 Urothelial carcinoma invading lamina propria

(pT1), col. HE, x100

Fig. 5.4 Urothelial carcinoma invading entire wall

(pT3), col. HE, x100

Fig. 5.5 Urothelial carcinom, scuamose differentiation,

col. HE, x100

Fig. 5.6 Urothelial carcinoma, clear cell differentiation,

col. HE, x100

Fig. 5.7 Urothelial carcinoma, vascular invasion,

col. HE, x200

Fig. 5.8 Urothelial carcinoma, perineural invasion,

col. HE, x200

8

CHAPTER 6

IMUNOHISTOCHEMICAL STUDY OF BLADDER UROTHELIAL

CARCINOMA Imunohystochemical analysis was performed on a selected batch of 65 cases,which

included 35 cases in which it was practiced total cystectomy and 30 cases in which it was

practiced tumor resection transurethral.All the cases are from the samen batch that was clinical

and morphological analized withthesame processing in Urology department and Pathology

department from Emergency Hospital Craiova.

The principal markers investigated in this study were adressed to the main biomolecular

mechanisms : epitelio-mesenchymal tumor of bladder epithelial phenotype alteration,

respectively, the purchase mezenchimal phenotype, the cadherinic switch, transcription factors

(Table 6.1):

Tabel 6.1 Antibodies used for the investigation of the transition epithelial-mesenchymal at the

level of the bladder

Antibody Clona/ Producer Dillution Recuperarea

antigenică

Control extern

pozitiv

CK7 OV-TL 12/30

Dako

1:100 EDTA, pH 8 Urothelium

CK20 Ks 20.8/Dako 1:75 EDTA, pH 9 Colon

Vimentina SP20

Thermo Scientific

1:150

Citrat, pH 6 Colon

E-caderina NCH 38

Dako

1:100 Citrat, pH 6 glanad mamara

N-caderina 6G11/Dako 1:50

Citrat, pH 6 Amigdals

Twist Twist 1/LSbio 1:1000 Citrat, pH 6 Colon

Snail 1 Policlonal

Abcam

1:75 Citrat, pH 6 Placenta

The imunoexpression of the cytokeratin 7 was identified in all cases analyzed with

tumoral cells. The marking was variable intensity and located at the cytoplasmic level (fig. 6.1).

The imunoexpression of cytokeratin 20 was identified at the level of selected strain of tumor

cells in 62.9% of cases, intensity of the reaction was variable (fig. 6.2). The imunoexpression of

Vimentinei has been identified at the level of selected strain of tumor cells in 12 cases and

mesenchymal elements. The markings have been identified only in carcinomas of high-grade,

with his own invasion of muscle/whole wall or adjacent organs (pT2-T4) and in stages II to IV

(fig. 6.3, Figure 6.4). The imunoexpression of E-cadherin has been observed at the level of

tumor cells membrane. The intensity of reactions both at the front and intratumoral bleeds

invasion was variable and differences were observed in the number of cells marked (fig. 6.5 fig.

6.6). The imunoexpression was present at the level of the membrane and selected strain of tumor

cells, and stromal elements (fig. 4.2, fig. 4.2) . The immunoreaction Twist has been identified at

the kernel level of tumor cells and stromal elements such as fibroblasts, macrophages,

endothelial cells, lymphocytes (Fig. 4.4, fig. 6.10) . The imunoreactions Snail 1 were identified

at the level of selected strain and nucleus of tumor cells. The reaction was present at the level of

lymphocytes and stromali fibroblasts (fig. 6.11, 6.12.

9

Fig. 6.1 Low grade urothelial, invasion front, CK7, x

100 Fig. 6.2 High grade urothelial, front of invasion, CK20, x

100

Fig. 6.3 Urothelial carcinoma, invasion front, marked

Vimentina, x200

Fig. 6.4 Urothelial carcinoma, invasion,

double marked Vimentina (brun)/ CK7 (red), x200

Fig. 6.5 Low grade urothelial carcinoma, invasion front,

marked Ecadherin, x100

Fig. 6.6 High grade urothelial carcinoma, intratumor,

marked Ecadherin, x100

10

Fig. 6.7 Low grade urothelial carcinoma, invasion front N-

cadherin, x100

Fig. 6.8 High grade urothelial carcinoma, invasion front, N-

cadherin, x100

Fig. 6.9 Low grade urothelial carcinoma, intratumor,

marked Twist, x100

Fig. 6.10 High grade intratumor urothelial carcinoma,

marked Twist, x100

Fig. 6.11 Low grade urothelial carcinoma, invasion front,

marked Snail 1 x 100

Fig. 6.12 High grade urothelial carcinoma, intratumoral,

marked Snail 1, x100

11

CONCLUSIONS

Bladder urothelial carcinomas are lesions with increasing incidence, the patient profile

including over 60 years of age, male, smoker, with hematuria at presentation.

Hematuria was the most common symptom at presentation (56.2%) patients, and imaging

investigations have shown tumor predominența unice (97,7%), with sizes below 2 cm (87.5%)

and localization at the level of the lateral wall of the bladder and trigon. High grade urothelial carcinomas and advanced stage presents the invasion front level

ability to purchase of mezenchimal phenotype in epithelial phenotype conservation conditions. Cadherinic switch is present in MET urothelial tumour and offer informations regarding

lesions agresivity.

Markers study involved in urothelial carcionoma MET indicates specific

imunophenotipes reported with histopathological prognostic parameters, aspect that can select

them as therapeutic targets.

Cadherinic profile and transcription factors reported with histopathological parameters

ofurothelial carcinoma, can improve selection criteria of included patients in furder trials.

BIBLIOGRAPHY

1. Boring CC, Squires TS, Tong T. Cancer statistics 1995. Cancer J Clin, 1995;45:2.

2. Brennan P, Bogillot O, Cordier S, Greiser E, Schill W, Vineis P, Lopez-Abente G,

Tzonou A, Chang-Claude J, Bolm-Audorff U, Jockel KH, Donato F, Serra C,

Wahrendorf J, Hours M, T'Mannetje A, Kogevinas M, Boffetta P. Cigarette smoking and

bladder cancer in men: a pooled analysis of 11 case-control studies. Int J Cancer 2000,

86(2):289-94.

3. Chaw SY, Majeed AA, Dalley AJ, Chan A, Stein S, Farah CS. Epithelial to mesenchymal

transition (EMT) biomarkers--E-cadherin, beta-catenin, APC and Vimentin--in oral

squamous cell carcinogenesis and transformation. Oral Oncol. 2012;48(10):997-1006.

4. Chen J, Han Q, Pei D. EMT and MET as paradigms for cell fate switching. J Mol Cell

Biol 2012;4:66-9.

5. Cheng G, Xie L. Alcohol intake and risk of renal cell carcinoma: a meta-analysis of

published case-control studies. Arch Med Sci. 2011;7:648–57

6. Chesbrough RM, Burkhard TK, Martinez AJ, Burks DD. Gerota versus Zuckerkandl: The

renal fascia revisited. Radiology 1989;173:845-6).

7. Crăiţoiu Ş. Histologie specială. Ed. Medicală Universitară, Craiova, 2003.

8. Cutler SJ, Young JL Jr. Third National Cancer Survey: Incidence Data. Eds. Nat Cancer

Instit Monogr 1975; 41:20-25.

9. Greenburg G, Hay ED. Epithelia suspended in collagen gels can lose polarity and express

characteristics of migrating mesen¬chymal cells. J Cell Biol 1982; 95: 333 339.

10. Joseph JC, Evanguelos X, Luis AK, Malte R, Michael R, Shahrokh FS. Effect of

Smoking on Outcomes of Urothelial Carcinoma: A Systematic Review of the Literature,

EUROPEAN UROLOGY 2014;65:742 – 54

11. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest.

2009;119:1420–8.

12

12. Kiemeney LA, Schoenberg M, Familial transitional cell carcinoma. J Urol. 1996, 156:

867-872.

13. Liang Cheng, Darrell D. Davison, Julia Adams, Antonio Lopez-Beltran, Lisha Wang,

Rodolfo Montironi, Shaobo Zhang. Biomarkers in bladder cancer: Translational and

clinical implications, Critical Reviews in Oncology/Hematology 2014;89:73–111.

14. Maximilian B, James WFC, Guido D, Barton G, Harry H, Pierre K, Wassim K,

Lambertus AK, Carlo La V, Shahrokh S, Yair L. Epidemiology and Risk Factors of

Urothelial Bladder Cancer. European Urology, 2013; 63: 234 – 241.

15. McCullough DL, Lamma DL, McLaughlin AP3rd, Gittes RF, Familial transitional cell

carcinoma of the bladder. J Urol.1975, 113: 629-635.

16. Peinado H, Olmeda D, Cano A. Snail, Zeb, and bHLH factors in tumor progression: An

alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415–28.

17. Sadler T.W., Langman’s Medical Embriology, 11th edition, Lippincott, Williams and

Wilkins, Baltimore, 2010.

18. Savagner P, Boyer B, Valles AM, Jouanneau J, Thiery JP. Modulations of the epithelial

phenotype during embryogenesis and cancer progression. Cancer Treat Res 1994;71:229-

49.

19. Shariat SF, Pahlavan S, Baseman AG, Brown RM, Green AE, Wheeler TM, Lerner SP.

E-cadherin expression predicts clinical outcome in carcinoma in situ of the urinary

bladder. Urology 2001;57(1):60–5.

20. Sievert KD, Amend B, Nagele U, Schilling D, Bedke J, Horstmann M, Hennenlotter

J, Kruck S, Stenzl A. Economic aspects of bladder cancer: what are the benefits and

costs? World J Urol 2009;27(3): 295–300.

21. Silverman DT, Levin LI, Hoover RN, Hartge P. Occupational risks of bladder cancer in

the United States: I. White men. J Natl Cancer Inst 1989;81(19):1472–80.

22. Silverman DT, Levin LI, Hoover RN. Occupational risks of bladder cancer in the United

States: II Nonwhite men. J Natl Cancer Inst 1989;81(19):1480–3.

23. Sinescu I, Gluck G. Tratat de Urologie. Editura Medicală, 2008; 3:1947- 2142.

24. Steineck G, Hagman U, Gerhardsson M, Norell SE. Vitamin A supplements, fried foods,

fat and urothelial cancer. A case-referent study in Stockholm in 1985-87. Int J

Cancer. 1990 Jun 15;45(6):1006-11.

25. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal

transitions. Nat Rev Mol Cell Biol. 2006 Feb;7(2):131-42.

26. Van der Horst G, Bos L, van der Pluijm G. Epithelial plasticity, cancer stem cells, and the

tumor-supportive stroma in bladder carcinoma. Mol Cancer Res 2012;10:995-1009.

27. Vena JE, Graham S, Freudenheim J, Marshall J, Zielezny M, Swanson M, Sufrin G. Diet

in the epidemiology of bladder cancer in western New York. Nutr

Cancer. 1992;18(3):255-64.

28. Xu C, Zeng XT, Liu TZ, Zhang C, Yang ZH, Li S, Chen XY. Fruits and vegetables

intake and risk of bladder cancer: A PRISMA-compliant systemic review and dose-

response meta-analysis of prospective cohort studies. Med (Baltimore), 2015;

94(17):e759.