The Value of Detection of Her2 Gene Amplification in Different Stages of Bladder Cancer

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1 The Value of Detection of Her2 Gene Amplification in Different Stages of Bladder Cancer Thesis for the fulfillment of M.D degree in Pathology By Dr. Heba Khalil Mohamed Under supervision of Prof. Dr. Ali Fouad El Hindawi Professor of Pathology and Head of Tumor Marker Unit - Pathology Department Faculty of medicine-cairo university Prof. Dr. Maha Mosaad Professor of Pathology Faculty of medicine-cairo university Prof. Dr. Olfat Ali Hammam Professor of Pathology Theodor Bilharz Research Institute Cairo University 2012

2 Acknowledgment I wish to express my deep gratitude to Prof. Dr. Ali Fouad El Hindawi, Prof. of pathology, and head of Tumor Marker unit, Faculty of Medicine, Cairo University for his constant guidance, Constructive supervision and following the performance and progress of this thesis. I would also like to express my profound gratitude and deep appreciation toward Prof. Dr. Maha Mosaad Prof. of pathology, Cairo University for her effective supervision and great help during the histopathological, immunohistochemical and FISH in this thesis and for her continuous encouragement throughout the course of the thesis. I am greatly indebted to Prof Dr. Olfat Ali Hammam, am, Prof. of pathology, Theodor Bilharz Research Institute for suggesting the topic of this research, setting up the plan and her valuable assistance in reading, revision, discussion and statistical studies of the results during the whole work in this thesis. I really enormously benefited from her creative thinking, valuable suggestions and constructive criticism. No words are sufficient to express my gratitude towards all the staff members of Pathology Department, Theodor Bilharz Research Institute, for their sincere encouragement and support throughout the study. Special thanks for technicians of Pathology Department, Theodor Bilharz Research Institute, for their help and cooperation. Finally, I appreciate the cooperation of our dear patients, I hope this work offers them a chance for their better health which they deserve. Heba Khalil M. Khalil

3 Abstract: The Her2/neu oncoprotein, also known as (NEU, EGFR2, or ERBB2) is one of the members of the Epidermal Growth Factor Receptor (EGFR) family, which includes EGFR or (ERBB1), EGFR3 or (HER3/ERBB3) and EGFR4 or (HER4/ERBB4), is known to contribute to physiological mechanisms of cell proliferation by intrinsic tyrosine-kinase-activity. Overexpression has been shown for several tumors and is known to influence malignant cell proliferation, metastasis and angiogenesis (Wülfing et al., 2005). Although the genes contain extracellular, transmembrane and intarcellular domains, the regions of greatest homology are the kinase domains contained within an intracellular domain. However, the genes have distinct properties: HER2 has strong kinase activity but has no identified ligand and ERBB3 lacks kinase activity due to substitutions in critical TK (Tyrosine Kinase) domain residues (Citri et al., 2003). All are capable of forming homodimers (with the possible exception of ERBB3) and heterodimers. EGFR and HER2 are dysregulated in many human cancers and play important roles in cancer development and progression (Slamon et al., 1978). Recent studies indicate the role of Her2 in the development of numerous types of human cancer. Her2 overexpression and/or amplification have been detected in 10%-34% of invasive breast cancers and correlates with clinical outcome, poor prognosis, and constitute a predictor factor of poor response to chemotherapy and endocrine therapy (Kaptain et al., 2001). Her2 Overexpression and/or amplification have been also detected in gastric carcinoma (Gravalos and Jimeno 2008), colonic carcinoma (Schuell et al., 2006) and bladder cancer (Eltze et al., 2005). Trastuzumab (Herceptin) is a monoclonal antibody which specifically targets Her2 protein by directly binding the

4 extracellular domain of the receptor. Trastuzumab enhances survival rates in both primary and metastatic Her2-positive breast cancer patients (Smith et al., 2007). The efficacy of Trastuzumab in breast cancer patients has led to investigate its antitumour activity in patients with Her2-positive cancers, including bladder cancers. Bladder carcinoma is one of the commonest malignancy in the Egypt. Approximately 93% of these tumors are derived from the epithelial lining and are termed urothelial or transitional cell carcinomas (Droller 1998). Similar to other epithelial malignancies, patients with carcinoma of the bladder have a high rate of survival (90%) if the disease is diagnosed at an early stage. However, the presence of even superficial invasion into the muscularis propria results in a dramatic fall in the 5-year survival rate to < 50%. Patients with metastatic bladder carcinoma are treated with systemic chemotherapy, but their disease remains a virulent disorder for which there is no cure. The disease is highly lethal, with a 5-year survival rate of < 5% (Thrasher and Crawford 1993). The standard treatment for patients with muscle-invasive bladder carcinoma is a radical cystectomy. Approximately 50% of patients who undergo this procedure ultimately will develop recurrences and die of their disease. Therefore, attempts have been made to reduce this recurrence rate by translating chemotherapy advances in metastatic disease into studies of neoadjuvant or adjuvant chemotherapy for patients with high-risk, organ-confined tumors. Unfortunately, after a decade of clinical trials exploring these issues, the role of chemotherapy in this patient remains largely undefined despite results of a recent U.S. Cooperative Group Trial (Southwest Oncology Group trial 8710)

5 reporting that neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin prior to cystectomy significantly improved survival compared with cystectomy alone (Lehmann et al., 2001). Fortunately, in the past decade, several new chemotherapeutic agents with activity and increased tolerability in bladder carcinoma have become available, including the nucleoside analog gemcitabine and the taxane paclitaxel. Several recent chemotherapy trials in patients with carcinoma of the bladder have incorporated paclitaxel in an attempt to improve outcomes and toxicity profiles. Those trials demonstrated that paclitaxelbased combinations are feasible, tolerable, and efficacious (Pectasides et al., 2000). Studies of bladder carcinoma that have analyzed gene amplification and/or increased protein expression of HER2 by a variety of methods have shown mixed results. Whereas several studies have reported its association with higher disease stage, increased tumor progression, and increased metastasis (Korkolopoulou et al., 1997). Other studies have shown no additional prognostic value over established criteria, such as disease stage and tumor grade (Mellon et al., 1996). Key words: Urinary bladder-her2- Immunohistochemistry-FISH.

6 LIST OF CONTENTS List of tables i List of figures ii,iii List of histograms iv List of abbreviations V Aim of the work 1 Review of literature.. 2 Material and methods 34 Results 42 Discussion. 78 Summary and Conclusion 85 Recommendations. 90 References.. 91 Arabic Summary 105

7 LIST TABLES Table (1): Sex distribution in different studied groups. Table (2): Age distribution in different studied groups. Table (3): HER2 protein immunoexpression in different bladder lesions. Table (4): HER2 gene expression by FISH in different bladder lesions. Table (5): HER2 protein immunoexpression in malignant lesions. Table (6): HER2 gene expression by FISH in malignant lesions. Table (7): HER2 protein immunoexpression by IHC in bilharzial associated and non bilharzial associated bladder lesions. Table (8): HER2 gene expression using FISH in Bilharzial and Non Bilharzial Bladder lesions. Table (9): HER2 protein immunoexpression in different stages of malignant lesions. Table (10): HER2 gene expression in different stages of malignant lesions. Table (11): HER2 protein immunoexpression in different stages of transitional cell carcinoma. Table (12): HER2 gene expression in different stages of transitional cell carcinoma. Table (13): HER2 protein immunoexpression in different stages of squamous cell carcinoma. Table (14): HER2 gene expression by FISH in different stages of Squamous Cell Carcinoma. Table (15): HER2 protein immunoexpression in different grades of transitional cell carcinoma. Table (16): HER2 gene expression in different grades of transitional cell. Table (17): HER2 protein immunoexpression in different grades of squamous cell carcinoma. Table (18): HER2 gene expression in different grades of Squamous Cell Carcinoma i

8 LIST OF FIGURES Fig. (1): Structure of the HER2 protein. Fig. (2): A diagram showing nucleus with DNA strands (Fluorescent in situ hybridization-wikipedia, the free encyclopedia.htm). Fig. (3): A diagram showing mechanism of FISH (Fluorescent in situ hybridization- Wikipedia, the free encyclopedia.htm). Fig. (4): A diagram showing steps of FISH (Fluorescent in situ hybridization- Wikipedia, the free encyclopedia.htm). Fig (5): Fluorescence microscope (Olympus x51) using 3 filters (green (WB), red (FITC) and DAPI). Fig. (6): A control case showing normal urothelial lining (H& E, x 100). Fig. (7): A control case showing negative HER2 protein immunoexpression, score 1+( IHC, Her2/neu, DAB, x200). Fig. (8): A case of Bilharzial cystitis showing living and dead Bilharzia ova in Lamina propria (H& E, x 10). Fig. (9): A case of polypoid cystitis showing negative HER2 protein immunoexpression, score 1+ in normal urothelial lining (IHC, HER2/neu, DAB, x100). Fig. (10): A case of non invasive low grade papillary urothelial carcinoma (H&E, X20). Fig. (11): A case of non invasive urothelial carcinoma, Grade II, showing strong HER2 protein immunoexpression in tumor cells (IHC, HER2/neu, DAB, x 200). Fig. (12): A case of infiltrating high grade urothelial carcinoma, grade III, infiltrating lamina propria and muscle (H&E, x100). Fig. (13): A case of moderately differentiated Bilharzial associated squamous cell carcinoma, strongly positive for HER2 protein ore 3+ (IHC, HER2/neu, DAB, x40). Fig. (14): A case of poorly differentiated urothelial (Transitional) cell carcinoma. Showing equivocal HER2 protein immunoexpression in tumor cells (IHC,HER2/neu DAB, x400). Fig. (15): A case of infiltrating poorly differentiated urothelial (Transitional) cell carcinoma strongly positive for HER2 protein immunoexpression, score 3+ (IHC, HER2/neu, DAB, x100). Fig. (16): A case of keratinizing moderately differentiated squamous cell carcinoma (H&E, X200). Fig. (17): A case of poorly differentiated urothelial (Transitional) cell carcinoma. Showing equivocal HER2 protein immunoexpression in tumor cells (IHC, HER/neu, DAB, x200). Fig. (18): A control case showing negative HER2 gene expression, two green signals and two red signals (FISH, HER2/neu gene, x1000). Fig. (19): A case of papillary urothelial (Transitional) cell carcinoma, positive for HER2 gene amplification, showing red clusters (FISH, HER2/neu ii

9 gene, x1000). Fig. (20): A case of squamous cell carcinoma, negative for HER2 gene amplification (FISH, HER2/neu gene, x1000). Fig. (21): A case of urothelial (Transitional) cell carcinoma, positive for HER2 gene amplification, showing red clusters (FISH,HER2 /neu gene, x1000). Fig. (22): A case of invasive urothelial (Transitional) cell carcinoma, negative for HER2 gene amplification ( FISH, HER2/neu gene, x1000). Fig. (23): A case of Bilharzial squamous cell carcinoma, positive for HER2 gene amplification, showing more than 6 red signals per cell ( FISH, HER2/neu gene x1000). Fig. (24): A case of squamous cell carcinoma, negative for HER2 gene amplification, FISH (FISH, HER2/neu, gene, x1000). Fig. (25): A case of high grade urothelial carcinoma showing polysomy of chromosome 17, more than 2 green signals in the cell (FISH, HER2/neu, gene, x1000) iii

10 LIST OF HISTOGRAMS Hist. (1): Sex distribution in different studied lesions. Hist. (2): Age distribution among different studied lesions. Hist. (3): HER2 Protein Immunoexpression in different Bladder lesions. Hist. (4):HER2 gene expression using FISH in different bladder lesions Hist. (5): HER2 protein immunoexpression in malignant lesions. Hist. (6): HER2 gene expression using FISH in malignant lesions. Hist. (7): HER2 protein immunoexpression in bilharzial and non bilharzial lesions. Hist. (8): HER2 gene expression in bilharzial and non bilharzial lesions. Hist. (9): HER2 protein immunoexpression in different stages of malignant lesions. Hist. (10): HER2 gene expression in different stages of malignant lesions. Hist. (11): HER2 protein immunoexpression in different stages of Transitional Cell Carcinoma. Hist. (12): HER2 gene expression in different stages of transitional cell carcinoma. Hist. (13): HER2 protein immunoexpression in different stages of squamous cell carcinoma Hist. (14): HER2 gene expression in different stages of squamous cell carcinoma. Hist. (15): HER2 protein immunoexpression in different grades of TCC. Hist. (16): HER2 gene expression in different grades of Transitional Cell Carcinoma. Hist. (17): HER2 protein immunoexpression in different grades of Squamous Cell Carcinoma. Hist. (18): HER2 gene expression in different grades of squamous cell carcinoma iv

11 LIST OF ABBREVIATIONS TCC: Transitional Cell Carcinoma SqCC: Squamous Cell Carcinoma CIS: Carcinoma In Situ EGFR: Epidermal Growth Factor Receptor (HER/erbB) FISH: Fluorescence In situ Hybridization. IHC: Immunohistochemistry TUR: Trans-Uretheral Resection Non. Bil.Cystitis:Non Bilharzial cystitis Bil.Cystitis: Bilharzial Cystitis. Non Bil.TCC: Non Bilharzial Transitional Cell Carcinoma. Bil.TCC: Bilharzial Transitional Cell Carcinoma. Non Bil.SqCC: Non Bilharzial Squamous Cell Carcinoma Bil.SqCC: Bilharzial Squamous Cell Carcinoma. v

12 Aim of work AIM OF THE WORK This work aims at: - To study the relationship between the expression of HER2 protein by using immunohistochemistry and gene amplification by using Fluorescence In situ Hybridization technique in different types and stages of cancer bladder. - To study the incidence of HER2 expression with the transitional cell neoplasia compared to squamous cell neoplasia in Bilharzial associated and Non Bilharzial associated cases. 1

13 Review of literatures Bladder Cancer Epidemiology of bladder cancer Urinary bladder cancer is a common disease worldwide. At any point in time 2.7 million people have a history of urinary bladder cancer (Ploeg et al., 2009). Urinary bladder cancer ranks ninth in worldwide cancer incidence (Ferlay et al., 2005). It is the seventh most common malignancy in men and seventeenth in women. Approximately 357,000 new bladder cancer cases (274,000 males and 83,000 females) occurred worldwide yearly (Parkin, 2008). Urinary bladder cancer is the second most common malignancy of all genitourinary tumors after prostate cancer and is nearly three times more common in men than in women (Jemal et al., 2005). In Egypt: Carcinoma of the bladder is the foremost oncologic problem, Egypt has the highest recorded incidence rate of bladder cancer in the world, and the incidence of 37.1 per 100,000 males is almost two times higher than in Western communities (Ferlay et al., 2005). At the National Cancer Institute (NCI), it constitutes 30.3% of all cancers, 40.6% of male cancers, and 14.3% of female cancers (El-Bolkainy et al., 2000). According to the International Agency for Research on Cancer (IARC) statistical study: Egyptian bladder cancer cases were 8842 male and 1911 female with an incidence crude rate reached 24.9 per for males and 5.5 per for females while deaths from bladder cancer were 7972 male and 1714 female with mortality crude rate reached 22.5 per for males and 5 per for females. In the same statistical study Egypt ranked first among Northern African and Arabian African countries in urinary bladder cancer incidence and mortality rates in both males and females (Ferlay et al., 2005). 2

14 Pathology of Bladder Cancer Review of literatures WHO Histological Classification of Tumors of the Urinary Tract (Eble et al., 2004) I. Transitional Cell Neoplasms Infiltrating urothelial carcinoma with squamous differentiation with glandular differentiation with trophoblastic differentiation Nested Microcystic Micropapillary Lymphoepithelioma-like Lymphoma-like Plasmacytoid Sarcomatoid Giant cell undifferentiated Non-invasive urothelial neoplasia Urothelial carcinoma in situ Non-invasive papillary urothelial carcinoma, high grade Non-invasive papillary urothelial carcinoma, low grade Non-invasive papillary urothelial neoplasia of low malignant potential Urothelial papilloma Inverted urothelial papilloma II. Squamous Cell Neoplasms A. Squamous cell carcinoma B. Verrucous carcinoma C. Squamous cell papilloma III. Glandular Neoplasms Adenocarcinoma Enteric Mucinous Signet-ring cell Clear cell A. Villous adenoma IV. Neuroendocrine Tumors A. Small cell carcinoma B. Carcinoid 3

15 Review of literatures C. Paraganglioma V. Melanocytic Tumors A. Malignant melanoma B. Nevus VI. Mesenchymal Tumors A. Rhabdomyosarcoma B. Leiomyosarcoma C. Angiosarcoma D. Asteosarcoma E. Malignant fibrous histiocytoma F. Leiomyoma G. Haemangioma H. Other VII. Haematopoietic and Lymphoid Tumors A. Lypmphoma B. Plasmacytoma VIII. Miscellaneous Tumors A. Carcinoma of Skene, Cowper and Littre glands B. Metastatic tumors and tumors extending from other organs The main two histological types of bladder cancer are identified: the transitional cell carcinomas (TCC), related to cigarette smoking and most prevalent in Western and industrialized countries, and the squamous cell carcinomas (SqCC), which are more frequently seen in some Middle Eastern and African countries, where urinary schistosomiasis is an endemic disease. I) Transitional Cell (Urothelial) Carcinoma (TCC): It comprises 90% of primary tumors of the bladder and 30-40% of carcinomas associated with shistosomiasis. In past decades, increased incidence of transitional cell carcinoma associated with shistosomiasis has been reported (Cardillo et al., 2003). Transitional cell carcinoma is defined as any malignant epithelial tumor of the bladder consisting entirely, partly or focally of anaplastic urothelium. These tumors differ from normal urothelium by having an increased number of epithelial cell layers with papillary folding of the mucosa, loss of cell polarity, 4

16 Review of literatures abnormal cell maturation from the basal to superficial layers, giant cells, nuclear crowding, increased nuclear-to-cytoplasmic ratio, prominent nucleoli, clumping of chromatin, and an increased number of mitoses (Koss et al., 1975). Transitional cell carcinomas manifest in a variety of patterns of tumor growth, including papillary, sessile, infiltrating, nodular, mixed, and flat intraepithelial growth (carcinoma in situ). Approximately 70% of bladder tumors are papillary, 10% are nodular, and 20% are mixed (Cardillo et al., 2003). A. Papillary transitional cell (Urothelial) carcinoma: It is defined as an urothelial tumor that does not invade beyond the basement membrane. According to Mostofi et al., 1999, it is graded on a scale of I to III in an ascending manner of severity as follows: 1. Grade-I TCC: The epithelium of grade I urothelial carcinomas has an orderly appearance, but with easily recognizable variations of architectural and cytological features. Mitoses are infrequent, but may occur at any level of the epithelium, usually the basal third. Nuclear grooving is prominent and a perpendicular orientation of basal cell is usually evident. Superficial cells may be present but flattened. 2. Grade-II TCC: They exhibit an intermediate degree of abnormality. They are designated from grade I by a predominantly disordered architectural pattern, but with retention of some elements of organization, e.g. polarity and nuclear uniformity. 3. Grade-III TCC: The papillae show complete disorder with loss of polarity and commonly loss of superficial cells, marked variation of all nuclear parameters and usually numerous irregularly distributed mitoses. With higher grade 5

17 Review of literatures tumors, there is variable epithelial thickness due to increasing loss of cellular cohesions. B. Infiltrating transitional cell (Urothelial) Carcinoma: It is defined as an urothelial tumor that invades beyond the basement membrane. As with the papillary tumors, these are graded on an I to III scale, depending on the degree of nuclear anaplasia. The most anaplastic areas determine the tumor grade (Fine et al., 2005). The WHO panel recognized that some centers prefer to use a two grade system for papillary and infiltrating transitional cell carcinoma that is designated as low grade and high grade lesions. The low grade lesions correspond to WHO grade I tumors, the high grade lesions correspond to WHO grade II and III tumors (Fine et al., 2005). C. Carcinoma in situ (CIS): It is a non-papillary, i.e. flat, lesion in which the surface epithelium contains cells that are cytologically malignant (Eble et al., 2004). De novo (primary) carcinoma in situ accounts for less than 1-3% of urothelial neoplasms, but is seen in 45-65% of invasive urothelial carcinoma (Wolf et al., 1987). Data suggest that de novo (primary) CIS is less likely to progress to invasive disease than secondary CIS (Orozco et al., 1994). II) Squamous Cell Carcinoma (SqCC): Squamous cell carcinoma of the urinary bladder, though uncommon in Europe and the United States, is the most common variety of bladder tumor in countries where urinary bilharziasis prevails (El- Sebaie et al., 2005). A. SqCC in the bilharzial bladder: The highest incidence of SqCC of the bilharzial bladder is in Egypt. In a report by Ghoneim et al., 1997, SqCC constituted 59% of 1026 cystectomy specimens at the Mansoura Urology and Nephrology Centre in the period between 1969 and Bilharzial eggs were seen in 85% of the 1026 specimens. 6

18 Review of literatures The tumor is diagnosed in the fifth decade, and five times more common in men than women. Bladder carcinogenesis is probably related to bacterial and viral infections, commonly associated with bilharzial infestation rather than the parasite itself. The presentation is often with irritative bladder symptoms and hematuria, and many patients present at an advanced stage, although most tumors are of low and moderate grades. At cystoscopy tumors are predominantly nodular and usually arise from the upper vesical hemisphere. Lymph-node metastasis occurs in 19% and significantly decreases survival; radical cystectomy remains the main treatment, giving a 5-year survival rate of 50% (Shokeir, 2004). B. SqCC in the non bilharzial bladder: The non-bilharzial type occurs in Western countries and represents <5% of all vesical tumours; it occurs most often in the seventh decade with a slight male predominance. The principal predisposing factor is prolonged indwelling urethral catheterization in patients with spinal cord injury and the main symptom is haematuria. Patients are usually diagnosed at an advanced stage and most of the tumours are of moderate and high grades. At cystoscopy tumours are predominantly ulcerative and commonly involve the trigone and lateral walls. Although distant metastasis is infrequent (8-10%) the prognosis is grave and most patients die after failure of loco-regional control; radical cystectomy provides the best therapy. To avoid non-bilharzial SqCC, patients with spinal cord injury should be free of catheterization if possible. The outcome can be improved by early detection with frequent cytology, cystoscopy and biopsy (Shokeir, 2004). Verrucous Carcinoma It is a special type of squamous carcinoma of border-line malignancy that is almost exclusively associated with S. hematobium infection representing 3% of total squamous bladder cancers. The majority of lesions are usually locally advanced (Khaled, 1993). 7

19 Review of literatures Tumor Staging Staging systems for cancer bladder have been based upon observation of the clinical course of different tumors in association with their depth of penetration through the bladder wall at presentation. Penetration is based primarily on tissues obtained at the time of transurethral resection TUR biopsy. However, physical examination and radiological imaging can provide complementary information. Bladder tumor treatment, prognosis, and the likelihood of metastatic disease are strongly depending on the depth of primary tumor invasion (Mundy, 1999). Currently, the most recent staging system is the one developed jointly by the World Health Organization (WHO) in 2004, also termed Tumor-Node- Metastasis (TNM). This staging system enables simultaneous description of primary tumor extent (T), status of lymph nodes (N), and extent of distant metastasis (M). The following table shows the 2004 TNM classification for staging of bladder cancer (Eble et al., 2004). 8

20 Review of literatures T T --- Primary Tumor Tx: Primary tumor cannot be assessed. T0: No evidence of tumor. Ta: Non-invasive papillary carcinoma. Tis: Carcinoma in situ: flat tumor. T1: Tumor invades subepithelial connective tisuue. T2: Tumor invades muscle T2a-Tumor invades superficial muscle (inner half). T2b-Tumor invades deep muscle (outer half). T3: Tumor invades perivesical tissue T3a-Microscopically T3b-Macroscopically (extravesical mass). T4: Tumor invades any of: prostate, uterus, vagina, pelvic wall, abdominal wall T4a-Invasion of the surrounding organs, (e.g.: prostate, uterus & vagina). T4b-Invasion of the pelvic wall, abdominal wall. N N --- Regional Lymph Nodes Nx: Regional lymph nodes cannot be assessed. N0: No regional lymph node metastasis. N1: Metastasis in a single lymph node 2 cm or less in greatest dimension. N2: Metastasis in a single lymph node more than 2 cm but not more than 5 cm in greatest dimension, or multiple lymph nodes none more than 5 cm in greatest dimension. N3: Metastasis in a lymph node more than 5 cm in greatest dimension. M M --- Distant Metastasis Mx: Distant metastasis cannot be assessed. M0: Absent distant metastasis. M1: Distant metastasis. 9

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