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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).
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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).
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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.
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