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Evaluation of E-Cadherin and Vimentin Expression As Prognostic Markers for Epithelial-Mesenchymal Transition and Tumor Aggressiveness in Breast Cancer

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Evaluation of E-Cadherin and Vimentin Expression As Prognostic Markers for Epithelial-Mesenchymal Transition and Tumor Aggressiveness in Breast Cancer Republic of Sudan Ministry of Higher Education and scientific Research Shendi University Faculty of Postgraduate Studies and Scientific Research Evaluation of E-Cadherin and Vimentin Expression as Prognostic Markers for Epithelial-Mesenchymal Transition and Tumor Aggressiveness in Breast Cancer A Thesis Submitted for Partial Fulfillment for Requirement of M.Sc. Degree in Medical Laboratory Sciences, (Histopathology & Cytology) By: Mohammed Awad Elkareem Abdelgadir Elzaki Abdelgadir B.Sc. In Histopathology, Cytology and Haematology Shendi University (2010) Supervisor: Dr. Ahmed Mohammed Ahmed 2018 آلاية بسم اهلل الرحمن الرحيم قال تعالى : )ولَ َق ْد آ تَيْنَا لُ ْقما َن الْ ِح ْْكَ َة َآ ِن ا ْش ُكر ِ ه ِّلِل ۚ وَم ْن يَ ْش ُكر فَانهما يَ ْش ُكر ِلنَ ْف ِس ِه ۖ وَم ْن َك َفر فَا هن ا ه َّلِل غَِِن َ َِحي د ( َ َ ْ َ ْ ِ َ ُ َ َ ِ ٌّ صدق اهلل العظيم سورة لقمان آلاية )21( Dedication To my mother, to her strength and patience in her stubborn fight with cancer Love and respect. To my father, brother, Sisters, I’m grateful for having you in my life. To my beloved friends, wherever they are. Acknowledgment First of all, I am thankful and grateful to my God (Allah) for making me able to do this research. I am grateful to my supervisor Dr. Ahmed Mohammed Ahmed for his help and advice. A lot of thanks to the department of histopathology and cytology, faculty of medical laboratory sciences (Shendi University); Dr. Ibraheem Bakhet, Dr. Mohammed abdulgadir, and Dr. Asmaa Al-Amir, for all help and support. Finally, I’m wishing all the luck to my colleagues in the master program. Abstract This is a descriptive retrospective laboratory based study, conducted at El-rahma medical centre, during the period from March to July 2018. The study aimed to evaluate the expression E-cadherin and Vimentin as prognostic markers for Epithelial-Mesenchymal Transition (EMT) and tumor aggressiveness in breast cancer using immunohistochemistry. Fifty six paraffin blocks were collected from archive for women patients previously diagnosed as breast cancer. Tissue Microarrays (TMAs) prepared from paraffin blocks, were cut by rotary microtome, and then stained by immunohistochemistry method (Dextran labeled polymer). The data obtained was analyzed using SPSS program version 22.0, Frequencies, mean, Chi-square test values and Pearson’s correlations were calculated. The age of patients ranged between 30 to 80 years with mean age of 51.1 years and standard deviation was 10.1. The study found that most patients were more than 40 years 45/56 (80%). The majority of the study cases were in age group of 50-59 representing 19/56 (33.9 %), followed by 40-49 representing 16/56 (28.6%), 60-69 representing 10/56 (17.9%), 30-39 representing 9/56 (16.1%) and 70-79 representing 2/56 (3.6%). The histopathological diagnosis of cases revealed that, the majority of diagnosed samples were invasive ductal carcinoma (IDC) in 40/56 (71.4%), medullary carcinoma (MC) in 14/56 (25%) and colloid (mucinous) carcinoma (CC) in 2/56 (3.6%). The histological grade of the study population showed, grade I in 6/56 (10.7%), grade II in 20/56 (35.7%) and grade III in 30/56 (53.6%). The study showed that, E-cadherin expression was positive in 49/56 (87.5%), negative in 7/56 (12.5%). Positive E-cadherin expression was weakly expressed in 18/49 (36.7%), moderately expressed in 27/49 (55.1%) and strongly expressed in 4/49 (8.2%), this result revealed a significant correlation between E- cadherin expression with age and histological grade (p-value = 0.028 – 0.027) respectively, but the correlation with the histological type is not significant (p-value = 0.126). Vimentin expression was positive in 55/56 (98.2%), negative in 1/56 (1.8%). Positive Vimentin expression was weakly expressed in 29/55 (52.7%), moderately expressed in 26/55 (47.3%) and strongly expressed in 0/55 (0%), this result showed a significant correlation between Vimentin expression with age and histological type (p-value = 0.016 – 0.004) respectively, but the correlation with the histological grade is not significant (p-value = 0.051). The study revealed an inverse correlation between E-cadherin and Vimentin (r = - 0.389) and strong significant correlation (p-value = 0.002). The study concluded that, decreased expression of E-cadherin and increased expression of Vimentin was associated with Epithelial-Mesenchymal Transition (EMT) and tumor aggressiveness. الخﻻصة أجريت هذه الدراسة المعملية الوصفية ، اﻹسترجاعية بمركز الرحمة الطبي خﻻل الفترة من ماار وحتى يوليو 8102. تهدف الدراسة لتقييم تعبير الكااررين الطﻻياي والفاايمنتين كمو تساماخ تيةيلاية للت ا تول الطﻻيي – الوسيطي واﻷورام العدوانيتة في سرطان الثدي باستةدام كيمياء اﻷنسجة المناعية. ُجمااس ساا مت وخمسااون مالاان ااامعي ماان اﻹرااايم لعيناااخ نساااء مراااى تاام تيةيلااهم مساابقا باا نهم ملابين بسرطان الثدي. ُح تضرخ ملفوفاخ اﻷنسجة اللغيرة من موالن اليمس ومُطعت باساتةدام الميارا الد توار و ُصبغت بواسطة طريقة كيمياء اﻷنسجة المناعية )بوليمر الديكستران المعلَّم(. واستُةدم برنامج ال ازم اﻹحلايية للعلوم اﻻجتماعية ، النسةة 88 لت ليل البيانااخ. ُحسابت التاررراخ ، المتوساق ، مايم رختباار مرباس كاي وارتباطاخ بيرسون. تراوحت أعماار المرااى باين 01 – 21 عاام بمتوساق عمار 11.0 سانة وكاان اﻹن اراف المعيااري 01.0. أظهرخ الدراسة أن معظام المرااى كانات أعماارهم أكثار مان 01 سانة. كانات لالبياة ال ااﻻخ ت ات الدراسااة فااي الفلااة العمريااة ماان 11-15 والتااي تمثاال 05\15 )00.5%( ، تليهااا 01-05 التااي تمثتاال 05\15 )82.5%( ، 51-55 التي تمثتل 01\15 )5..0%( ، 01-05 يمثتلاون 5\15 )05.0%( ، 1.-5. التاي تمثتال .)%0.5( 15\8 أظهر تيةيص الورم الةبيث لل ااﻻخ أن لالبياة العينااخ كانات سارطان اﻷمنياة الغا ياة فاي 01\15 )0.0.%( ، وسرطان النةاع التوسعي في 00\15 )81%( ، وسرطان ميوسيني في 8\15 )%0.5(. أظهر التمايز النسيجي لعينة الدراسة ، النوع اﻷول في 5\15 )..01%( ، والنوع الثاني فاي 81\15 )..01%( ، والنوع الثالث في 01\15 )%10.5(. أظهرخ الدراساة أن تعبيار الكااررين الطﻻياي كاان ريجابياا فاي 05\15 )1..2%( ، سالبيا فاي .\15 )08.1%(. التعبياار اﻹيجااابي للكاااررين الطﻻيااي كااان معبتاارا بيااكل اااعيم فااي 02\05 )0..0%( ، معبتاارا بيكل متوسق في .8\05 )11.0%( ، وتم التعبير عنهُ بواو في 0\05 )2.8%( ، كيفت هذه النتيجة عن ارتباط معنوي بين تعبير الكاررين الطﻻيي مس العمر والتمايز النسيجي )ميماة p = 1.182 – .1.18( علاى التوالي ، لكن لم يكن هناك ارتباط معنوي مس النوع النسيجي )ميمة p = 1.085(. كااان تعبياار الفااايمنتين ريجابيااا فااي 11\15 )52.8%( ، ساالبيا فااي 0\15 )0.2%(. التعبياار اﻹيجااابي للفايمنتين كان معبترا بياكل ااعيم فاي 85\11 )..18%( ، معبتارا بياكل متوساق فاي 85\11 )0..%0( ، ومعبترا عنه بواو في 1\11 )1%( ، كيفت هذه النتيجة عن ارتباط معنوي بين تعبير الفايمنتين مس العمر والنوع النسيجي ) ميمة p = 1.105 – 1.110( على التوالي ، لكن لم يكان هنااك ارتبااط معناوي ماس التماايز النسيجي ) ميمة p = 1.110(. كيفت الدراسة عن وجور عﻻمة عكسية باين الكااررين الطﻻياي والفاايمنتين ) r = -389( ، وارتبااط معنوي موي )ميمة p = 1.118(. خللت الدراسة رلاى أ تن انةفاات تعبيار الكااررين الطﻻياي و ياارة تعبيار الفاايمنتين يارتبق باﻻنتقاال الطﻻيي – الوسيطي وعدوانية الورم. List of Content Content Pages I اﻵية Dedication II Acknowledgment III Abstract (English) IV VI الةﻻصة List of content VIII List of tables X List of abbreviations XI Chapter One – Introduction 1.1 Introduction 1 1.2 Objectives 5 Chapter Two – Literature Review 2.1 Scientific background 6 2.2 Structure and function of the breast 6 2.3 Histology of the breast 7 2.4 Disorders of the breast 8 2.5 Epidemiology and risk factors of breast cancer 15 2.6 Diagnosis of breast cancer 20 2.7 Prognosis of breast cancer 22 2.8 Treatment of breast cancer 23 2.9 Epithelial-mesenchymal transition 24 2.10 EMT and breast cancer aggressiveness 25 Chapter Three – Materials and Methods 3.1 Materials 28 3.2 Methods 28 3.2.1 Study design 28 3.2.2 Study sampling and sample size 28 3.2.3 Study area 28 3.2.4 Sample processing 28 3.2.5 Immunohistochemical staining 28 3.2.6 Data analysis 29 3.2.7 Result interpretation 29 3.2.8 Ethical consideration 30 Chapter Four – Result 4.1 Results 31 Chapter Five – Discussion 5.1 Discussion 48 Chapter Six – Conclusion and Recommendations 6.1 Conclusion 11 6.2 Recommendations 12 References 13 Appendices 51 List of Tables Table No. Title Page Table (4.1) Mean and standard deviation of age distribution among 33 patients Table (4.2) Distribution of age groups among the Patients 34 Table (4.3) Distribution of histological type among the patient’s 35 sample Table (4.4) Distribution of the histological tumor grade among the 36 study cases Table (4.5) Distribution of E-cadherin expression among the study 37 cases Table (4.6) Distribution of E-cadherin positive expression 38 Table (4.7) Distribution of Vimentin expression among the study 39 cases Table (4.8) Distribution of Vimentin positive expression 40 Table (4.9) The correlation between age and expression of E- 41 cadherin Table (4.10) The correlation between age and expression of Vimentin 42 Table (4.11) The correlation between histological type and expression 43 of E-cadherin Table (4.12) The correlation between the histological type and 44 expression of Vimentin Table (4.13) The correlation between histological grade and 45 expression of E-cadherin Table (4.14) The correlation between histological grade and 46 expression of Vimentin Table (4.15) The correlation between E-cadherin and Vimentin 47 expression List of abbreviations ATM Ataxia Telangiectasia Mutated BRCA Breast Cancer Gene CC Colloid (mucinous) Carcinoma CEA Carcinoembryonic Antigen CECs Circulating Endothelial Cells CNB Core Needle Biopsy CT Chemotherapy DAB 3, 3 Diaminobenzidinetetrahydrochloride DCIS Ductal Carcinoma In-Situ DM Digital Mammography EC E-Cadherin
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