C erBB2 over expression in invasive breast carcinoma

C-ERBB2 OVER-EXPRESSION IN INVASIVE BREAST CARCINOMA SATHIYAMOORTHY SELVARAJAN (MBBS., DCP.) A THESIS SUBMITTED FOR THE DEGREE OF M.Sc (Clinical Science) DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE JULY 2003 ACKNOWLEDGMENTS It is a privilege to express my sincere and profound gratitude and appreciation to my supervisor, Associate Professor Bay Boon Huat, for his consistent and invaluable guidance, advice, as well as the encouragement, support, and patience throughout the course of this study. His exceptional supervision is embodied in the fresh ideas, sharp and critical comments, and many hours of thought provoking discussions, which are all essential for the completion of this study. What I have learned from him, not only with regard to science but also in daily life, will greatly benefit my career and life in future. I am indebted to my co-supervisor, Dr. Tan Puay Hoon, Consultant Pathologist, Department of Pathology, Singapore General Hospital (SGH), for her consistent interest, expert advice and especially for the many hours she has patiently spent, teaching me the fundamentals of basic pathology. I am very grateful to Professor Ling Eng Ang, Head, Department of Anatomy, National University of Singapore (NUS) for his understanding, kindness and support during my research. I also wish to extend my deep appreciation to Dr. Ivy Sng, Head, Department of Pathology, Singapore General Hospital (SGH) for her kindness and permission to do this study in SGH laboratories. I would also like to express my heartfelt thanks to: Dr. Tian Sim Leng, Head, Cytogenetics Section, Department of Pathology, Singapore General Hospital (SGH), for his help and kindness. Mrs. Christina Sivaswaren Rudduck, Scientific Officer, Cytogenetics Section, Department of Pathology, Singapore General Hospital (SGH) whose valuable teaching and guidance is very much appreciated. Mrs. Shalawati Mamat of Cytogenetics lab, Department of Pathology, Singapore General Hospital (SGH) for obliging me with technical assistance wherever I required help. Ms. Chng Mei Jiuan, Mr. Sivakumar and Ms. Maryam Hazly Hilmy of Immunohistochemistry Laboratory, Department of Pathology, Singapore General Hospital (SGH) for their kind assistance. All the staff of the Department of Anatomy for their assistance, co-operation and help during my stay. My friends and fellow graduate students for their friendship and support. All the staff of the Department of Pathology, SGH for the help, support and cooperation. Financial support from the National University of Singapore Postgraduate scholarship is gratefully acknowledged. This study was supported by a grant from SingHealth Cluster Research Fund No. BF006/2001 and Singapore Cancer Society. I gratefully acknowledged the support and encouragement of my family throughout the endeavor and their pivotal role in my progress. Last but not least, I am grateful to my wife, Mrs.Parameshwari, for her understanding and support during this important period of my academic career. I would like to dedicate this thesis to my wife and our daughter, Jothsna SP. CONTENTS ACKNOWLEDGEMENTS i CONTENTS iii ABBREVIATIONS ix LIST OF TABLES xiii LIST OF FIGURES xv LIST OF PUBLICATIONS xviii SUMMARY xx CHAPTER1. INTRODUCTION 1 1.1 Epidemiology of breast cancer 2 1.1.1 Breast cancer around the world 2 1.1.2 Breast cancer in Singapore 2 1.2 Classification of breast disorders 4 1.2.1 Benign breast disease 4 1.2.2 Malignant breast disease 6 1.2.2.1 Ductal carcinoma in situ 8 1.2.2.2 Lobular carcinoma in situ 8 1.2.2.3 Invasive ductal carcinoma 9 1.2.2.4 Invasive lobular carcinoma 11 1.2.2.5 Other forms of breast carcinoma 11 1.3 Clinicopathological parameters 12 1.3.1 Histologic grade 12 1.3.2 Pathological staging 14 1.3.3 Lymph node status 16 1.3.4 Hormone receptors 17 1.4 Biological markers 19 1.4.1 Ki-67 19 1.4.2 iNOS 20 1.4.3 C-myc 21 1.4.4 C-erbB2 and Epidermal growth factor receptor (EGFR) 22 1.4.4.1 C-erbB2 and breast cancer 25 1.4.4.2 Detection of c-erbB2 26 1.4.4.3 C-erbB2 and treatment 29 1.5 Scope of study 30 CHAPTER 2. MATERIALS AND METHODS 33 2.1 Patients and breast tissues 34 2.2 Histopathological diagnosis 34 2.3 Immunohistochemistry 35 2.3.1 Staining procedure for light microscopy 35 2.3.1.1 Sectioning, dewaxing and rehydration 35 2.3.1.2 Antigen retrieval 35 2.3.1.3 Blocking of endogenous peroxidase 35 2.3.1.4 Blockage of nonspecific binding sites 35 2.3.1.5 Specific binding of primary antibody 36 2.3.1.6 Detection of the specific primary antibody binding sites 36 2.3.1.7 Counterstain, dehydration and mounting 36 2.3.1.8 Controls of immunohistochemistry 2.3.2 Quantification of immunohistochemical staining 37 38 2.3.2.1 Assessment of c-erbB2 immunopositivity 38 2.3.2.2 Assessment of ER and PR immunostaining 39 2.3.2.3 Assessment of Ki-67 immunoreactivity 39 2.3.2.4 Assessment of iNOS immunostaining 39 2.3.2.5 Assessment of c-myc immunostaining 40 2.4 Fluorescence in situ (FISH) analysis of c-erbB2 oncogene 40 2.4.1 Breast cancer tissues 40 2.4.2 Fixation duration 40 2.4.3 Fixation protocols 41 2.4.4 Microwave oven fixation 41 2.4.5 Hybridization procedure 42 2.4.5.1 Sectioning, dewaxing and rehydration 42 2.4.5.2 Acid treatment and pretreatment with sodium thiocyanate 42 2.4.5.3 Protein digestion 42 2.4.5.4 Detergent treatment and dehydration 42 2.4.5.5 Probe protocol 43 2.4.5.6 Post-hybrid wash and mounting 43 2.4.6 Quantification of FISH signals 2.5 Nuclear morphometry 43 44 2.5.1 Breast cancer tissues 44 2.5.2 Image cytometry 44 2.6 Statistical analysis 45 CHAPTER 3. RESULTS 46 3.1 Histopathology 47 3.2 Hormone receptor status 50 3.3 C-erbB2 detection 52 3.3.1 C-erbB2 immunostaining at protein level 52 3.3.2 C-erbB2 amplification at gene level 56 3.3.2.1 C-erbB2 gene amplification and fixation duration 56 3.3.2.2 C-erbB2 gene amplification and fixation protocols 63 3.3.2.3 C-erbB2 gene amplification and microwave oven fixation 66 3.3.3 Correlation of c-erbB2 protein overexpression and gene amplification 67 3.4 Association of c-erbB2 overexpression with nuclear morphometry 68 3.5 Association of c-erbB2 overexpression with established clinicopathological and biological markers 71 3.5.1 Association of c-erbB2 overexpression and hormone receptor status 71 3.5.2 Relationship between c-erbB2 overexpression and clinicopathological parameters in invasive breast carcinoma 72 3.5.2.1 Association of c-erbB2 overexpression and histological grade 72 3.5.2.2 C-erbB2 overexpression and other clinicopathological factors 72 3.5.3 C-erbB2 overexpression with Ki-67 74 3.5.4 C-erbB2 overexpression with iNOS 76 3.5.5 C-erbB2 overexpression with c-myc 78 3.5.6 Association of biological markers with clinicopathological parameters in invasive breast carcinoma 80 3.5.6.1 Ki-67 80 3.5.6.2 iNOS 80 3.5.6.3 c-myc 80 3.6 Follow up and survival analysis 83 3.6.1 Kaplan-Meier survival analysis 83 3.6.2 Multivariate Cox regression analysis 89 CHAPER 4. DISCUSSION 90 4.1 C-erbB2 (HER2/neu) status in invasive breast carcinoma 91 4.1.1 Brief overview of c-erbB2 assessment 91 4.1.2 C-erbB2 protein overexpression by immunohistochemistry (IHC) 92 4.1.3 C-erbB2 gene amplification by Fluorescence in situ hybridization (FISH) 94 4.1.4 Concordance between c-erbB2 gene amplification and protein overexpression 96 4.2 C-erbB2 status and clinicopathological parameters 98 4.2.1 C-erbB2 overexpression and hormonal receptor status 98 4.2.2 C-erbB2 overexpression with histological grade and nuclear morphometry 100 4.2.3 C-erbB2 status and other clinicopathological parameters 103 4.3 C-erbB2 status and biological markers 106 4.3.1 C-erbB2 status and cell proliferation (Ki-67) 106 4.3.2 C-erbB2 status and iNOS 107 4.3.3 C-erbB2 status and c-myc 108 4.3.4 Clinicopathlogical parameters and biological markers: Ki-67, iNOS and c-myc 108 4.4 Follow up and survival analysis 110 4.5 Conclusion 111 4.6 Future study 114 REFERENCES 115 APPENDIX 147 ABBREVIATIONS µl Microliter A Amplified AJCC American Joint Committee on Cancer ANOVA One-way analysis of variance AR amphiregulin CEP 17 Centromere 17 C-erbB2 v-erb-B2 avian erythroblastic leukemia viral oncogene homolog 2 c-myc v-myc avian myelocytomatosis viral oncogene homolog CR-1 cripto-1 DAB 3, 3’ diaminobenzidine tetrachloride DAPI 4, 6 diamidino-2-phenylindoledihydrochloride hydrate DCIS Ductal carcinoma in situ DFS Disease free survival DNA Deoxyribonucleic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EIA Enzyme immunoassay ELISA Enzyme-linked immunosorption assay ER Estrogen receptor ERE Estrogen response elements FISH Fluorescence in situ hybridization GAP GTPase activating protein GFR Growth factor receptor H&E Hematoxylin and Eosin H2O2 Hydrogen peroxide HCl Hydrochloric acid HER2 Human epidermal growth receptor 2 hr/hrs Hour/hours IDC Invasive ductal carcinoma IGF-1 Insulin like growth factor IgG Immunoglobulin G IHC Immunohistochemistry ILC Invasive lobular carcinoma iNOS Inducible nitric oxide synthase kD Kilodalton KI Cell proliferation index LCIS Lobular carcinoma in situ LSI Locus specific M (Staging) Distant metastasis (staging) M Mole Min minute/minutes mm millimeter MMP Matrix metalloproteinase mRNA Messenger RNA N Axillary node metastasis NA Not amplified NaCl Sodium chloride Neg Negative NO Nitric oxide NP-40 Nonidet P - 40 NST No special type OH- hydroxyl ion OS Overall survival PLC-γ Phospholipase C-gamma Pos Positive PR Progesterone receptor pTNM Pathological staging RNA Ribonucleic acid RT Room temperature SD Standard deviation Silane 3-aminopropyltriethoxysilane SPSS Statistical package for social sciences SSC Sodium chloride sodium citrate T Tumor TBS Tris hydrochloride buffer saline TDLU Terminal duct-lobular units TGF-α Transforming growth factor-α TGF-β Transforming growth factor-β TNM Tumor, node and metastasis WHO World Health Organisation LIST OF TABLES Table 1 Ten most frequent cancers in Singapore women 3 Table 2 Categorization of benign breast lesions 5 Table 3 Classification of malignant breast disease 7 Table 4 Relative percentage of main histologic subtypes of invasive breast cancer in different studies 10 Table 5 Criteria for histological grading of invasive breast cancer 13 Table 6 AJCC - TNM staging of breast carcinoma 16 Table 7 Primary antibodies used for immunohistochemistry 37 Table 8 Scores for c-erbB2 overexpression using the DAKO Hercep protocol 39 Different protocols of protein digestion and pretreatments used for groups 41 Table 10 Clinico-pathological parameters of the patients in this study 48 Table 11 Hormone receptor status in invasive breast carcinoma 50 Table 12 C-erbB2 (HER2/neu) FISH and immunostaining of formalin fixed breast cancer tissues (12 hrs and 27 hrs) in group 1 57 C-erbB2 (HER2/neu) FISH and immunostaining of formalin fixed breast cancer tissues (2 hrs and 17.5 hrs) in group 2 59 C-erbB2 (HER2/neu) FISH and immunostaining of formalin Fixed breast cancer tissues (28.5 hrs and 541 hrs) in group 3 61 C-erbB2 (HER2/neu) FISH of formalin fixed paraffin-embedded archival breast cancer tissues of less than 12 months’ duration 64 C-erbB2 (HER2/neu) FISH of formalin fixed paraffin-embedded archival breast cancer tissues of more than 12 months’ duration 65 Table 9 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 C-erbB2 (HER2/neu) FISH of formalin fixed breast cancer tissues (microwave oven and routine fixation) Nuclear morphology of cancer cells in c-erbB2 positive and c-erbB2 negative tumors 66 68 Table 19 Nuclear morphology of malignant cells with respect to clinicopathologic markers of prognosis in c-erbB2 positive breast tumors 70 Table 20 Association of c-erbB2 overexpression and ER status 71 Table 21 Association of c-erbB2 overexpression and PR status 71 Table 22 Correlation between c-erbB2 overexpression and clinicopathological parameters 73 Table 23 Association of c-erbB2 overexpression with Ki-67 74 Table 24 Association of c-erbB2 overexpression with iNOS immunoreactivity 76 Table 25 Association of c-erbB2 overexpression with c-myc 78 Table 26 Distribution of clinico-pathological parameters and biological markers 81 Univariate analysis of biological markers and clinico-pathological parameters in breast carcinoma 82 Multivariate analysis of important prognostic factors for their relationship with overall survival 89 Comparison of methods used to detect c-erbB2 status 91 Table 27 Table 28 Table 29 LIST OF FIGURES Fig. 1 Mechanism of action of estrogen (E) in human breast cancer cells. 18 Fig. 2 Steps involved in growth factor (c-erbB2) signal transduction. 25 Fig. 3 Scope of study. 32 Fig. 4 A histologic grade 1 tumor showing predominant tubules with mild to moderately pleomorphic nuclei and hardly any mitoses. 49 A histological grade 2 tumor showing the presence of trabeculae with scanty tubules, moderately pleomorphic nuclei and few mitoses. 49 A histological grade 3 tumor displaying no tubules and nuclei which are irregular in size and shape and mitoses are obvious. 50 Invasive ductal breast carcinoma showing strong nuclear positivity for ER in cancer cells. 51 Invasive ductal breast carcinoma showing strong positive reactivity of the nuclei for PR. 51 Fig. 9 Intensity of c-erbB2 immunostaining in all patients. 52 Fig. 10 C-erbB2 immunostaining of invasive ductal breast cancer tissues. Negative control. 53 C-erbB2 immunostaining of invasive ductal breast carcinoma. 1+ staining (considered negative). 54 C-erbB2 immunostaining of invasive ductal breast carcinoma. 2+ positive staining. 54 C-erbB2 immunostaining of invasive ductal breast carcinoma. 3+ positive staining. 55 Ductal Carcinoma in situ component of invasive ductal breast carcinoma. 55 Fig. 15 FISH post 12 hr fixation. 58 Fig. 16 FISH post 27 hr fixation. 58 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 17 FISH post 2 hr fixation. 60 Fig. 18 FISH post 17.5 hr fixation. 60 Fig. 19 FISH post 28.5 hr fixation. 62 Fig. 20 FISH post extended duration of fixation. 62 Fig. 21 FISH of archival paraffin-embedded breast cancer tissues stored less than 12months’. 64 FISH of archival paraffin-embedded breast cancer tissues stored more than 12months’. 65 FISH post (A) 12 and (B) 27 hr fixation. (C)Immunostaining for c-erbB2 (HER2/neu) (3+), showing a complete, intense membranous pattern of positivity. 67 Nuclei of c-erbB2 positive invasive ductal carcinoma digitally outlined using a computer mouse. 69 Nuclei of c-erbB2 negative invasive ductal carcinoma digitally outlined using a computer mouse. 69 Positive Ki67 immunostaining showed nuclear positivity of the proliferating cancer cells of invasive breast carcinoma. 75 Negative control (omission of primary antibody) showed no Ki67 immunostaining ofthe cancer cells of invasive breast carcinoma. 75 iNOS immunostaining showed strong cytoplasmic positivity of the cancer cells of invasive breast carcinoma. 77 Negative control showed no iNOS immunostaining of the cancer cells of invasive breast carcinoma. 77 Positive c-myc immunostaining showed nuclear as well as cytoplasmic positivity of the cancer cells of invasive breast carcinoma. 79 Negative control showed no c-myc immunostaining of the cancer cells of invasive breast carcinoma. 79 Fig. 22 Fig. 23 Fig. 24 Fig. 25 Fig. 26 Fig. 27 Fig. 28 Fig. 29 Fig. 30 Fig. 31 Fig. 32 Kaplan-Meier curves for overall survival (OS) with regard Fig. 33 Fig. 34 Fig. 35 Fig. 36 Fig. 37 to c-erbB2 overexpression (n = 290). Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with node-positive tumors (n = 118). 84 85 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with histologic grade 1 and 2 tumors (n = 109). 86 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with ER positive tumors (n = 185). 87 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients in age group > 50 years (n = 159). 88 Flow chart of significance of c-erbB2 overexpression/ amplification in invasive breast cancer in Singapore women. 113 LIST OF PUBLICATIONS Parts of the present study have been published, are in press or have been submitted for publication in journals and conference proceedings. Journal Articles – International Refereed 1. Selvarajan S, Bay BH, Choo A, Chuah KL, Sivaswaren CR, Tien SL, Wong CY, Tan PH. Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma. J Histochem Cytochem 50:1693-1696, 2002 2. Selvarajan S, Bay BH, Mamat S, Choo A, Chuah KL, Sivaswaren CR, Tien SL, Wong CY, Tan PH. Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization. Histochem and Cell Biol 120:251-255,2003 3. Selvarajan S, Bay BH, Khoo KS, Tan PH. Overexpression of C-erbB2 Correlates with Nuclear Morphometry and Prognosis in Breast Carcinoma in Asian women. (Submitted for publication) Conference Papers (Abstracts): 1. Selvarajan S, Tan PH, Bay BH. Immunohistochemical expression of c-erbB2 in invasive breast carcinoma from Singapore Chinese women. 3rd ASEAN Micrcoscopy Conference and 19th Annual Conference of EMST, Chiang Mai, Thailand, 2002. (Oral presentation) 2. Selvarajan S, Lin VC, Jin R, Tan PH, Bay BH. Association of progesterone receptor with histologic grade in human invasive breast carcinoma. The 7th World Congress on Advances in Oncology and 5th International Symposium on Molecular Medicine, Hersonissos, Crete, Greece. 2002. (Abstract) 3. Selvarajan S, Bay BH, Tan PH. Nuclear morphometry in c-erbB2 positive invasive ductal breast carcinoma. The 7th World Congress on Advances in Oncology and 5th International Symposium on Molecular Medicine, Hersonissos, Crete, Greece. 2002. (Oral presentation) SUMMARY C-erbB2 belongs to the human epidermal growth factor receptor family that plays an important role in the regulation of fundamental processes such as cell growth, survival and differentiation. C-erbB2 or human epidermal growth factor receptor-2 (HER2) gene is a proto-oncogene mapped to chromosome 17q21 and encodes a 185-kD transmembrane glycoprotein, designated as p185HER2, which is often simply called the HER2 protein or receptor. C-erbB2 is known to be overexpressed, amplified or both in several human malignancies, including breast cancer. Amplification of c-erbB2 gene has been reported to occur in 10-34% of primary breast carcinomas. Furthermore, anti-HER2 monoclonal antibodies (Mabs) are known to inhibit the growth of tumors and human breast cancer cell lines overexpressing the c-erbB2 protein. The aim of this study is to explore the expression of c-erbB2 at the genetic and protein level and to correlate c-erbB2 overexpression with clinico-pathologic parameters, biological markers and prognosis. From 321 cases of breast cancer diagnosed at the Singapore General Hospital, c-erbB2 overexpression was detected by immunohistochemistry (IHC). Biological markers: Ki-67, iNOS, c-myc and ER and PR were also evaluated by IHC. Clinicopathological data was obtained from the Pathology Registry. Nuclear image cytometry was analysed. Follow up data were traced from patients’ case notes and Singapore Cancer Registry. Survival analysis of the patients for evaluating the prognostic significance of c-erbB2 overexpression was performed. In this current study, c-erbB2 overexpression was found to be expressed higher in grade 3 tumors compared to grade 1 and grade 2 tumors. This study also supported a positive correlation between c-erbB2 expression and histologic grade of breast cancer, with overexpression being less frequent in grade 1 and 2 than in grade 3 carcinomas (P = 0.017). C-erbB2 overexpressed cases showed an inverse association with ER and PR positivity (P = 0.001); ER positive tumors were more likely to be c-erbB2 negative than were ER negative tumors (74.1% versus 48.1%). When analyzing the association of nuclear morphometric data in relation to histopathologic parameters in the group of cerbB2 positive breast cancers, there was a significant difference in (a) nuclear area and perimeter between histological grade 3 and histologic grade 1 and 2 (P = 0.001 and P = 0.03 respectively) and (b) nuclear perimeter between tumor size ≤ 20 mm and tumor size > 20 mm (P = 0.046). There is significant difference in overall survival (P = 0.0166), between c-erbB2 positive and c-erbB2 negative cases, indicating an adverse prognosis for the c-erbB2 positive ones. Different combinations of tumor characteristics for possible additive prognostic capacities were also investigated. Node positivity, ER positivity, histologic grade 1 and 2 group and age at diagnosis >50 group showed diminished survival with regard to c-erbB2 overexpression. It is therefore concluded that c-erbB2 overexpression in invasive breast cancer in Singapore women has a similar pattern and trend as that reported in studies from other countries with mostly western populations. C-erbB2 overexpression is strongly associated with poorly differentiated breast carcinoma and inversely correlated with hormone receptor status. Overexpression of c-erbB2 in invasive breast cancer is associated with poor overall survival. Strong correlation is found in c-erbB2 expression at the genetic and protein expression level. CHAPTER 1 INTRODUCTION 1.1 Epidemiology of breast cancer 1.1.1 Breast cancer around the world Breast cancer is the second leading cause of cancer deaths in women today (after lung cancer) and is the most common cancer among women, excluding non-melanoma skin cancers. The incidence rates of breast cancer show considerable variations among different geographical locations. It ranges from high among women in North America, South America and Israel, to intermediate in much of Europe and Australia, and low in most of Asia (Deapen et al., 2002). In America, the breast cancer incidence rates in females increased from 82.6 per 100,000 person-years in 1973 to 118.1 per 100,000 person-years in 1998 (Howe et al., 2001). Within Asia, Hong Kong has the highest breast cancer incidence (Leung et al., 2002). In Japan, age-standardized rate of breast cancer which is the leading cancer in women is 41.8 per 100,000 in 1997 (Cancer research group., Japan, 2002). In Ho Chi Minh city, Vietnam, breast cancer has been reported to have the second highest incidence, after gastric carcinoma (Nguyen et al., 1998). Breast cancer is also the leading cause in women who die of cancers in Malaysia. A comparative study with the Singapore population showed that Singapore women presented with breast cancer at earlier stages with a smaller tumor size as compared to Malaysian women (Yip et al., 1996). 1.1.2 Breast cancer in Singapore Breast cancer is the commonest cancer among Singapore women for the last three decades. More than 90,000 persons in Singapore were diagnosed with invasive breast cancers from January 1968 to December 1992 (Chia et al., 2001). The ten most common cancers in Singapore women during the period 1993-1997 and 1998-1999 are listed in Table 1 (Chia et al., 2002). Table 1 Ten most frequent cancers in Singapore women (adapted from Chia KS et al., Singapore cancer registry report no.5; 2002) Ranking Site 1993-1997 1998-1999 Breast 1 1 Colorectum 2 2 Lung 3 3 Cervix 4 4 Stomach 5 5 Ovary 6 6 Skin (including Melanoma) 7 7 Corpus uteri 8 8 Thyroid 9 9 Lymphoma - 10 10 - Nasopharynx There were 20.2 breast cancer cases per 100,000 women in the period 1968-1972, which has increased to 38.8 per 100,000 in 1988-1992, leading to an average annual increase of 3.6% over the past 25-year period for all women (Seow et al., 1996). The incidence rate of breast cancer has increased 2.3 times in 1993-1997 compared to that in 1968-1972 with the peak incidence age group being 45-49 years. Breast cancer comprises 22.8% of all cancers in Singapore women and is associated with an annual mortality rate of 13.7 per 100,000 per year (Chia et al., 2000). The age-standardized incidence rate is 53.1 cases per 100,000 women per year over the period of 1998-1999 as compared with 46.1 cases per 100,000 women over the period of 1993-1997 (Chia et al., 2002). However, the incidence is less than 50% compared to that of American women i.e., 114.5 per 100,000 women per year (Wingo et al., 1998). The pattern of breast cancer incidence is also becoming more similar to the western population as evidenced by the shift of the peak age-specific incidence for breast cancer from premenopausal to postmenopausal years over the period of 1998-1999 (Chia et al., 2002). Among ethnic groups, there is controversy in different studies. Malay women have been reported to be at an increased risk (4.4%) of developing breast cancer as compared to Chinese and Indian women (1.4%) (Seow et al., 1996). In another study, Chinese women appear to be at an increased risk i.e., 10% to 20% higher, as compared to Malay and Indian women (Chia et al., 2000). 1.2 Classification of breast disorders 1.2.1 Benign breast disease Breast lesions are broadly classified as inflammatory, benign and malignant lesions. Benign breast disorders are a heterogeneous group of lesions that clinically and radiographically span the entire spectrum of breast abnormalities. Some benign breast lesions may mimic breast cancer on physical examination and imaging studies. Inflammatory and benign lesions are shown combined in Table 2. Categorization of benign breast lesions is in accordance with the criteria of Schnitt et al (2000). Table 2 Categorization of benign breast lesions (modified from Schnitt et al., 2000) Reactive and Inflammatory Mammary duct ectasia Fat Necrosis Foreign body reaction Mondor’s disease Sarcoidosis Diabetic mastopathy Infections Hamartoma Non-proliferative Cysts Papillary apocrine change Mild hyperplasia Proliferative Moderate or florid ductal hyperplasia Intraductal papilloma Sclerosing adenosis Atypical hyperplasia Atypical ductal hyperplasia (ADH) Atypical lobular hyperplasia (ALH) Benign tumors Fibroadenoma Tubular adenoma Lactating adenoma Juvenile papillomatosis Microglandular adenosis Radial scars Granular cell tumors Fibromatosis Miscellaneous, like apocrine changes, calcifications. 1.2.2 Malignant breast disease (Histologic subtypes) Breast carcinoma presents in a great variety of histological patterns, including specific types which have useful clinical correlates and prognostic implications. Morphological classification of invasive breast carcinoma has existed for several decades. The classification system currently followed is based on a descriptive terminology for patterns of tumor growth (histological typing) which has been outlined by the World Health Organization (WHO) (Azzopardi et al., 1981). Table 3 shows the classification of malignant breast disease modified from that described by Page and Anderson (1987), which recognizes the WHO classification system for histological typing. Table 3 Classification of malignant breast disease (modified from Page and Anderson, 1987) Epithelial origin Non-invasive Ductal carcinoma in situ Microinvasive carcinoma Lobular carcinoma in situ Invasive Ductal no special type (NST) Lobular Medullary Tubular Invasive cribriform Mucinous Metaplastic Mixed types Uncommon types Secretory Adenoid cystic Mucoepidermoid Invasive papillary Tubulolobular Inflammatory Rare types Signet ring Lipid rich Clear cell Myoepithelioma Carcinoid Mesenchymal origin Sarcomas Miscellaneous origin Hematopoietic Metastatic carcinoma 1.2.2.1 Ductal carcinoma in situ (DCIS) Ductal carcinoma in situ (DCIS) originates from the terminal duct-lobular unit (TDLU), and implies malignant transformation of lining epithelial cells restricted within the basement membrane. Myoepithelial cells are seen in DCIS, which is a distinct feature that differentiates it from invasive carcinoma. The recognition of DCIS as a separate entity distinct from hyperplasia and invasive carcinoma was gradual through the first half of the twentieth century. It is now a firmly accepted entity, and has been categorized into several architectural patterns (Page and Anderson, 1987). DCIS is an early, noninvasive phase of breast cancer, and also the purported forerunner of the majority of invasive breast cancers (Frykberg and Bland, 1994). DCIS is classified into subtypes based on architectural patterns viz., comedo, cribriform, micropapillary, papillary, solid types. Currently this morphologic classification system is replaced by schemes that attempt to predict the biologic potential of DCIS, particularly the risk of recurrence and likelihood of progression to invasive carcinoma (Shoker and Sloane, 1999; Ellis et al., 1998). After the introduction of mammography, which has enabled early diagnosis of breast cancer, DCIS detection has increased from 0.8% - 5% (Tan et al., 1999; Schnitt et al., 1988) to 15% - 20% (Lagios, 1990). In the Singapore breast screening project, it accounted for 25% of all screen detected breast cancers (Tan et al., 1999). 1.2.2.2 Lobular carcinoma in situ (LCIS) Lobular carcinoma in situ (LCIS) is a distinct entity from DCIS. It is predominantly a disease of premenopausal women between the ages of 40 to 50 years (Lishman and Lakhani, 1999). It is not clinically palpable and there are usually no mammographic changes. The pathological diagnosis of LCIS is made when there is a monomorphic population of small round cells with thin cytoplasmic rims and high nuclear-cytoplasmic ratios, sometimes with intracytoplasmic lumina, affecting the lobular units (Lishman and Lakhani, 1999). It is now regarded as risk indicator, rather than as a true forerunner of invasive breast cancer. Low nuclear grade solid DCIS may mimic LCIS, and pose diagnostic difficulty. The relationship between DCIS, LCIS and invasive breast cancer needs further elucidation. 1.2.2.3 Invasive ductal carcinoma (IDC) Invasive ductal carcinoma (IDC) is the most common type of invasive carcinoma of the breast, although exact figures derived from different publications vary. Table 4 shows the relative percentage of main pathological types of invasive breast cancer in different studies. The distribution of IDC ranges from 47% to 79.2% of all subtypes of invasive breast cancers (Li et al., 2003; Chia et al., 2000; Pereira et al., 1995; Ellis et al., 1992; Soomro et al., 1991) (Table 4). Table 4 Relative percentage of main histologic subtypes of invasive breast cancer in different studies Type Chia et al. 2000 Singapore (%) Pereira et al. 1995 UK (%) Ellis et al. 1992 UK (%) Soomro et al. 1991 UK (%) Dixon et al. 1985 UK (%) Ductal 79.2 50 47 42.3 66.8 Lobular 4.4 15.4 15 22.1 9.8 Medullary 1.2 2.8 7.3 8.7 4.6 Tubular 0.6 2.4 2.3 4 7.9 Mucinous 2.6 0.9 0.9 15.4 1.7 Cribriform 0.7 0.8 0.8 4 3.3 Papillary 1 0.3 - 2.7 1 Mixed 0.8 19.8 20.8 0.8 - Others 9.5 8.4 5.9 - 4.9 Histologically, an extreme variety of patterns without the usual regularity and uniformity is seen in IDC. Features of single cell infiltration, poorly cohesive islands of infiltrating cells, foci of poorly formed glands, or even well defined glandular patterns are seen throughout a single lesion. Because of this, the term “Carcinoma-No special type” (NST) has been used as a substitute (Dixon et al., 1985). IDC usually presents as a hard, palpable mass with an average size of 2 to 3 cm, but the size may vary from a tiny lesion of few millimeters to as large as to replace the entire breast. Macroscopically, IDC is usually whitish gray in color and of varying shapes like stellate, oval or sometimes irregular. The typical appearance of spicules radiating out from the central mass lesion is characteristic of cancer which literally means ‘the crab’. Microscopically, there are not many distinctive features, as IDC may be solid and highly cellular or paucicellular (Sharkey et al., 1996). 1.2.2.4 Invasive lobular carcinoma (ILC) It is the second most frequent form of invasive breast carcinoma and accounts for 4.5% to 15% of all invasive breast cancers (Li et al., 2003; Chia et al., 2000; Pereira et al., 1995; Ellis et al., 1992; Dixon et al., 1985). The incidence rate of ILC has continued to rise in the past 15 years (Li et al., 2003). ILC presents as a diffuse lesion that is not detectable by routine physical examination or by mammography. Histologically, ILC comprises uniform, small, round, poorly cohesive cells with rounded or oval nuclei and eccentrically placed cytoplasm, frequently containing intracytoplasmic lumina (du Toit et al., 1989). Tumor cells are arranged like narrow cords (“Indian file” pattern) in a desmoplastic stroma. Several subtypes or variants of invasive lobular carcinoma have been described: (a) classical variant (b) solid variant (c) alveolar variant (d) tubulolobular variant (e) pleomorphic variant and (f) mixed variant (Sloane et al., 1995; du Toit et al., 1989; Dixon et al., 1982). 1.2.2.5 Other forms of breast carcinoma These tumors are much less common than IDC or ILC and are called special forms due to the following reasons: (i) Specific architectural patterns, for example mucinous, medullary, tubular, adenoid cystic, apocrine and cribriform (ii) Distinct clinical behavior, for example inflammatory, metaplastic, Paget’s disease of nipple, the former two entities having aggressive behavior. Other than these types, a few rare forms of invasive breast cancers can also occur viz., pseudosarcomatous type, signet-ring cell type, invasive papillary, secretory carcinoma, etc. Primary sarcomas of the breast are rare. Yet the parenchyma and connective tissue elements are capable of giving rise to many soft tissue sarcomas. Malignant cystosarcoma phyllodes, a special type of sarcoma, is the most common of all. Other sarcomas that do arise from breast tissue are angiosarcoma, fibrosarcoma, liposarcoma, malignant fibrohistiocytoma etc. Studies have shown that recognition of the histological type can provide highly significant prognostic prediction. The special types (tubular, invasive cribriform, mucinous) and tubulo-lobular carcinoma carry an excellent prognosis, mixed types have good prognosis, classical lobular and medullary (all types) have an average prognosis; solid lobular and ductal (NST) types are of relatively poor prognosis (Ellis et al., 1992). 1.3 Clinicopathological parameters 1.3.1 Histologic grade Histologic grading is one of the most important pathologic parameters and an essential determinant of prognosis that also allows risk stratification of invasive breast carcinoma (Fitzgibbons et al., 2000; Brown et al., 1993). Sloane et al (1995) demonstrated the latest revised system of histologic grading of breast cancers. The histologic grading system gained its importance from the early quarter of the 20th century and has been well recognised by Scarff-Bloom-Richardson (1957), further modified by Elston and Ellis (1991) in the Nottingham study. The histological grading of breast cancer always has a potentially subjective element, but reproducibility can be achieved when specific guidelines are followed (Dalton et al., 1994; Elston and Ellis, 1991). Table 5 shows the grading criteria as outlined by the Nottingham group, which represent a semi-quantitative modification of the Bloom and Richardson criteria, therefore providing more specific guidelines for grading. Table 5 Criteria for histological grading of invasive breast cancer* Feature Score Degree of tubule formation Majority of tumor (>75%) Moderate (10-75%) Little or none ( 10% of tumor cells were regarded as 2+ and 3+ respectively. A score of 0 and 1+ was considered negative and 2+ and 3+ as positive (Table 8). One section from each case has been scored. Staining was considered ‘indeterminate’ when c-erbB2 immunostaining was also observed in adjacent stroma or inflammatory cells, or if benign epithelium also showed membranous reactivity (Fitzgibbons et al., 2000). Table 8 Scores for c-erbB2 overexpression using the DAKO Hercep protocol (DAKO HercepTest-Package insert, 1999). Score C-erbB2 overexpression assessment 0 Negative 1+ Negative 2+ Weak positive 3+ Strong positive Staining pattern No staining is observed or membrane staining is observed in 10% of tumor cells. The cells are only stained in part of their membrane A weak to moderate complete membrane staining is observed in >10% of tumor cells A strong complete membrane staining is observed in >10% of tumor cells 2.3.2.2 Assessment of ER and PR immunostaining Positive ER and PR staining was defined as when 10% or more of tumor cell nuclei were immunoreactive. 2.3.2.3 Assessment of Ki-67 immunoreactivity Immunoreactivity of Ki-67 was expressed as a percentage based on semiquantitative assessment of the proportion of immunopositive cells. The immunoreactivity of the Ki-67 antibody was distributed in 4 categories: negative proliferative index (no detected positive cells), low (≤ 5% positive cells), moderate (610% positive cells) and high (>10% positive cells). 2.3.2.4 Assessment of iNOS immunostaining Positivity of iNOS was confirmed when more than 10% of tumor cells showed cytoplasmic staining. 2.3.2.5 Assessment of c-myc immunostaining Cases in which more than 10% of tumor cells showed cytoplasmic staining were considered positive, irrespective of the nuclear positivity. Some tumors included cells with nuclear positivity. 2.4 Fluorescence in situ hybridization (FISH) analysis of c-erbB2 oncogene 2.4.1 Breast cancer tissues A total of 98 cases comprising both archival and more recent ones, were subjected to FISH evaluation. To study the retrievel of FISH signals from archival paraffin blocks of breast cancer, sections were cut from the blocks stored between the years 1995-2001. 2.4.2. Fixation duration Tumor tissues were divided into three groups based on duration of fixation in 10% buffered formalin. Two sets of tumor tissues from each case were allotted into three groups. They were fixed in 10% buffered formaldehyde for 12 ± 2.5 hrs (range 9-18 hrs, median 12 hrs) and 27 ± 3.0 hrs (range 20–32 hrs, median 27 hrs) in the first group; 2 hrs and 17.5 ± 1.5 hrs (range 16-20 hrs, median 17.5 hrs) in the second group; and 28.5 ± 2 hrs (range 26-30 hrs, median 28.5 hrs) and 541 hrs ± 285 hrs (range 193-1010 hrs, median 541 hrs) in the third group. All cases in the first group represented the usual range of fixation duration for breast surgical specimens routinely handled in the laboratory; while the second and third groups were used to test the effect of a shortened fixation duration (2 hrs) and an extended fixation duration (median 541 hrs) on FISH results, as compared with routinely fixed cases. 2.4.3 Fixation protocols The tumor tissues were divided into two groups: archived samples less than 12 months’ duration and archived samples more than 12 months’ duration. Paraffin sections cut from the stored paraffin blocks were subjected to routine and modified FISH protocols (Table 9). Table 9 Different protocols of protein digestion and pretreatments used for groups Modified Protocols Protocol I Protease 5mg/ml, for 1 hr; NaSCN at 80˚c for 30mins Protocol II Protease 1.5mg/ml, for 2hrs; NaSCN at 80˚c for 30mins Protocol III Protease 1.5mg/ml, for 2.5hrs; NaSCN at 80˚c for 30mins Protocol IV Protease 1.25mg/ml, for 1hr; NaSCN at 80˚c for 30mins Protocol V Protease 0.5mg/ml, for 40mins; NaSCN at 80˚c for 1hr Protocol VI Protease 0.5mg/ml, for 40mins; NaSCN at 80˚c for 30 mins 2.4.4 Microwave oven fixation In this arm, tumor tissue from 7 cases was divided into two groups. One group was fixed in 10% buffered formaldehyde hastened by microwave oven heating at 55˚C for 2.5 minutes and the other group was fixed in 10% buffered formalin for durations ranging from 12 to 27 hrs before paraffin-embedding. 2.4.5 Hybridization procedure 2.4.5.1 Sectioning, dewaxing and rehydration Three µm thick sections were cut for FISH analysis and mounted on glass slides coated with silane (3-aminopropyltriethoxysilane, Sigma). Briefly, slides were heated overnight (56oC). The sections were deparaffinized and pretreated to facilitate probe permeability using a Pretreatment Kit (Vysis, Downers Grove, IL) according to manufacturer’s instructions with minor modifications. This was followed by de-waxing in xylene for 10 min (3 changes), then rehydrated in absolute alcohol for 5 min (2 changes) and then air dried. 2.4.5.2 Acid treatment and pretreatment with sodium thiocyanate Air dried slides were treated with 0.2N HCl for 20 min at room temperature (RT). The slides were then immersed in deionised water for 1 min and then washed in buffer for 3 min. This was followed by incubation in 1M sodium thiocyanate in 2XSSC (0.5M NaCl, 0.015M sodium citrate) at 80oC for 30 min, followed by immersion in deionised water for 1 min, and in wash buffer for 5 min (2 changes). 2.4.5.3 Protein digestion The slides were digested with protease (0.5 mg/ml) for 30 min at 37oC then, immersed in wash buffer for 5 min (2 changes) and then air dried. 2.4.5.4 Detergent treatment and dehydration Slides are soaked in detergent solution (0.5% Tween-20 in 2X SSC, pH 7) at 37˚C for 40 min then dehydrated via a series of graded alcohols (70%, 85%, 95%, 100%, and 100%) for 2 min each. Then the slides are air dried and subjected to probe protocols. The pretreatment protocols were modified according to the categories and groups studied. 2.4.5.5 Probe protocol In situ hybridization was performed using the PathVysion HER2 DNA Probe Kit (Vysis, Downers Grove, IL) (Selvarajan et al. 2002). 3µl of the hybridization solution containing the probe (LSI/CEP17) was applied to the treated slides and the areas were covered with 12mm diameter coverslips and sealed with rubber solution. The slides were then placed in a slide thermocycler (Hybaid Omnislide) programmed to co-denature the probe and target the DNA. Slides were incubated with hybridization solution at 75oC for 5 min, and subsequently at 38oC for 16 hr overnight. 2.4.5.6 Post-hybrid wash and mounting After overnight incubation the slides were taken out of the thermocycler and the cover slips were peeled off the seal. The slides were washed 2 times in 0.1% NP-40 in 0.5X SSC at 70˚C for 3 min, then washed in wash buffer at RT for 2 min, counterstained in DAPI (0.1 g/ml in 2 x SSC), then mounted with antifade (Vectashield) and bordered with nail polish. 2.4.6 Quantification of FISH signals Once the slides were hybridized and post-hybridization washes done, they were mounted with antifade (Vectashield) and viewed under an epifluorescence microscope (Olympus), with the images captured (CytoVision). Sixty nuclei were counted. Signal enumeration was performed with the following conditions: overlapping nuclei were excluded and split signals were counted as 1 chromosome component (Hoang et al., 2000). Stromal and inflammatory cells were excluded from analysis on the basis of the morphologic features of their nuclei. The criteria for gene amplification were (a) >4 signals per cell and (b) the HER2/neu to CEP 17 ratio > 2 (Ellis et al., 2000; Ridolfi et al., 2000). 2.5 Nuclear morphometry 2.5.1 Breast cancer tissues Ninety-six cases were randomly selected for assessing nuclear morphometry. CerbB2 immunostained breast cancer tissues were used for analysis. Only the invasive breast carcinoma component was included for the image cytometry and not the DCIS component. 2.5.2 Image cytometry Nuclear image analysis was performed on a light microscope (Axioplan, Zeiss), equipped with a Carl Zeiss camera linked to a computer, using the Imagepro software. Cell nuclei from digitized images were outlined using a mouse. For each case, a minimum of 100 nuclei of cancer cells was randomly selected for morphometric analysis. Morphometric parameters measured were nuclear area, nuclear perimeter, and roundness. For comparison of nuclear size and shape between the two populations of stained and unstained cells in each c-erbB2 positive case, 50 cells from each group were analyzed. 2.6 Statistical analysis The results were analysed using the statistical software SPSS for Windows, version 11 and the GraphPad Prism statistical Package. Association of c-erbB2 membrane staining status with clinico-pathologic parameters like age group, tumor size, histologic subtype, histologic grade stage lymphovascular invasion, nodal status, ER and PR were analyzed using the Chi-square χ2 test. The Student’s paired t-test was performed to compare means and Chi-square χ2 test for comparing proportions in FISH analysis. For comparison of c-erB2 expression and nuclear morphometry, one-way ANOVA was performed to compare means. Association of c-erbB2 membrane staining status with biologic markers like Ki-67, iNOS and c-myc were analyzed using the Chi-square χ2 test. Association of biological markers like Ki-67 and iNOS with clinico-pathologic markers like ethnicity, tumor size, histologic subtype, histologic grade, lymphovascular invasion, nodal status, ER, PR and p53 were analyzed using the Chi-square χ2 test. Overall survival curves were calculated using the method of Kaplan and Meier and differences between the curves were analyzed by the log-rank test. P 50 Ethinicity Chinese Malay Indian Tumor size ≤ 20mm > 20mm Histologic subtype IDC Others Histologic Grade Grade 1 Grade 2 Grade 3 Staging Stage 1 Stage 2 Stage 3 Stage 4 Lymphovascular Invasion Present Absent Nodal status Negative Positive 321* 134 181 271 23 14 116 181 266 55 46 132 127 68 192 28 7 92 227 162 133 ER Negative Positive 107 211 PR Negative Positive * Missing values range from 2 to 26 169 149 Fig. 4 A histologic grade 1 tumor showing predominant tubules with mild to moderately pleomorphic nuclei and hardly any mitoses ( H & E stain, Original magnification x 310). Fig. 5 A histological grade 2 tumor showing the presence of trabeculae with scanty tubules, moderately pleomorphic nuclei and few mitoses. (H & E stain, Original magnification x 310). Fig. 6 A histological grade 3 tumor displaying no tubules and nuclei which are irregular in size and shape and mitoses are obvious. (H & E stain, Original magnification x 310). 3.2 Hormone receptor status As evaluated by immunohistochemistry in histopathologically diagnosed cases of invasive ductal breast carcinoma, 66.4% of the tumors were ER positive and 46.9% were PR positive (Table 11). Examples of ER and PR positive immunostaining are shown in Fig 7 and Fig 8 respectively. Table 11 Hormone receptor status in invasive breast carcinoma PR+ PR- Total ER+ 137 74 211 ER- 12 95 107 Total 149 169 318 Fig. 7 Invasive ductal breast carcinoma showing strong nuclear positivity for ER in cancer cells. (Hematoxylin counterstain; Original magnification x 310). Fig. 8 Invasive ductal breast carcinoma showing strong positive reactivity of the nuclei for PR. (Hematoxylin counterstain; Original magnification x 310). 3.3 C-erbB2 detection 3.3.1 C-erbB2 immunostaining at protein level-Immunohistochemistry (IHC) C-erbB2 immunopositivity was detected in 110 (34.3%) cases, with 208 (64.8%) cases defined as negative. The distribution of the different immunostaining intensities of c-erbB2 in this study is shown in Fig 9. In 3 cases (1%), the staining was indeterminate as the adjacent benign breast tissue was also stained. Frequency 200 100 0 0 1+ 2+ Intensity Fig. 9 Intensity of c-erbB2 immunostaining in all patients. 3+ The different patterns of immunostaining are shown in Figures 10-13. A component of ductal carcinoma in situ was found in 30% to 40% of cases (Fig 14). CerbB2 expression was also present in the in situ cancer cells, with similar staining distribution and intensity as the surrounding invasive elements. Fig. 10 C-erbB2 immunostaining of invasive ductal breast cancer tissues. Negative control. (Haematoxylin counterstain; Original magnification x 400). Fig. 11 C-erbB2 immunostaining of invasive ductal breast carcinoma. 1+ staining (considered negative). (Haematoxylin counterstain; Original magnification x 310). Fig. 12 C-erbB2 immunostaining of invasive ductal breast carcinoma. 2+ positive staining. (Haematoxylin counterstain; Original magnification x 310). Fig. 13 C-erbB2 immunostaining of invasive ductal breast carcinoma. 3+ positive staining. (Haematoxylin counterstain; Original magnification x 310). Fig. 14 Ductal Carcinoma in situ component (shown in arrows) of invasive ductal breast carcinoma . (H & E stain; Original magnification x 80). 3.3.2 C-erbB2 amplification at gene level-Fluorescence in situ hybridization (FISH) A total of 98 cases were subjected to FISH for detection of c-erbB2 gene amplification. All the cases were grouped into three arms based on specific criteria as elaborated in the methods. 3.3.2.1 C-erbB2 gene amplification and fixation duration The purpose of this arm was to ascertain if variations in fixation duration in buffered formalin had any impact on the reliability of HER2/neu amplification results obtained via FISH. Out of 35 cases, two sets of tumor tissues from each case were allotted into three groups. There was no significant difference in detection of FISH signals with respect to the 12 hour or 27 hour fixation protocols in the first group (P = 0.476, Table 12, Fig 15 and 16.) indicating that the range of fixation times used routinely in the surgical pathology laboratory did not affect FISH results. Out of 4 amplified cases in this group, 2 cases revealed very high levels of gene amplification as shown by the HER2/neu-CEP17 ratio of 13.24, 10.6 and 14.65, 9.5 for the 12 hr and 27 hr fixation protocols, respectively. In the second group, there was also no significant difference in FISH results between the 2 hr or 17.5 hr fixation protocols (P = 0.151, Table 13, Fig 17 and 18). In the third group, signals were detected in all 6 cases in the 28.5 hr fixation protocol but only 2 cases showed signals in the extended duration fixation protocol (cases 1 and 2, Table 14, Fig19 and 20). Table 12 C-erbB2 (HER2/neu) FISH and immunostaining of formalin fixed breast cancer tissues (12 hrs and 27 hrs) in group 1 12 HRS 27 HRS RATIO RATIO NA 1.054 1.29 POS 2+ 2 NA 1.008 1.008 NEG 0 3 NA 1.134 1.102 POS 2+ 4 NA 1.155 1.095 NEG 0 6 A 2.366 2.445 POS 3+ 7 NA 1.016 1.032 POS 2+ 8 NA 1.00 1.057 POS 2+ 9 NA 1.008 1.039 NEG 0 10 NA 1.015 1.064 POS 2+ 11 NA 1.2 1.2 NEG 0 12 A 13.238 14.651 POS 3+ 13 NA 1.058 1.074 NEG 1+ 14 NA 1 1.043 NEG 0 15 A 10.6 9.5 POS 3+ 16 NA 1.049 1.107 POS 2+ 17 A 2.451 2.58 POS 3+ 18 NA 1.04 1.056 NEG 1+ 19 NA 1.069 1.102 NEG 0 20 NA 1.339 1.627 NEG 0 21 NA 1.378 1.482 NEG 0 S.NO FISH 1 IHC IHC SCORES A- amplified; NA- not amplified; POS-positive; NEGnegative. RATIO-ratio of HER2/neu to CEP17 signals. Fig. 15 FISH post 12 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Fig. 16 FISH post 27 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Table 13 C-erbB2 (HER2/neu) FISH and immunostaining of formalin fixed breast cancer tissues (2 hrs and 17.5 hrs) in group 2 2 HRS 17.5 HRS RATIO RATIO NA 1.146 1.133 NEG 0 2 A 2.031 2.019 POS 2+ 3 NA 1.065 1.094 NEG 0 4 NA 1.057 1.073 NEG 0 5 NA 1.041 1.041 NEG 0 6 A 2.295 2.16 POS 2+ 7 NA 1.124 1.08 NEG 0 8 A 5.206 5.058 POS 2+ S.NO FISH 1 IHC IHC SCORES* A- amplified; NA- not amplified; POS-positive; NEG-negative.RATIO-ratio of HER2/neu to CEP17 signals. * 0 – No staining is observed in tumor cells; 2+ - A weak to moderate complete membrane staining is observed in >10% of tumor cells. Fig. 17 FISH post 2 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Fig. 18 FISH post 17.5 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Table 14 C-erbB2 (HER2/neu) FISH and immunostaining of formalin fixed breast cancer tissues (28.5 hrs and 541 hrs) in group 3 28.5 HRS 541 HRS RATIO RATIO A 6.06 4.27 POS 2+ 2 NA 1.025 1.025 NEG 0 3 NA 1.176 *** NEG 0 4 NA 1.363 *** NEG 0 5 NA 1.192 *** NEG 0 6 A 4.085 *** POS 2+ S.NO FISH 1 IHC IHC SCORES A- amplified; NA- not amplified; ***-no signals; POS-positive; NEG-negative. RATIO-ratio of HER2/neu to CEP17 signals. * 0 – No staining is observed in tumor cells; 2+ - A weak to moderate complete membrane staining is observed in >10% of tumor cells. Fig. 19 FISH post 28.5 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Fig. 20 FISH post extended duration of fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. 3.3.2.2 C-erbB2 gene amplification and fixation protocols The purpose of this arm was to evaluate the effect of different protocols in the retrieval of fluorescent signals from FISH analysis of the c-erbB2 gene in archival paraffin-embedded breast cancer tissue blocks. 63 archival paraffin-embedded breast cancer tissue blocks were grouped into two: archived samples less than 12 months’ duration and archived samples more than 12 months’ duration. All the archived specimens less than 12 months’ duration (mean 5.8 months; median 3.5 months) exhibited hybridization signals with the routine protocol (Table 15; Fig 21). In the group where the specimens were archived for more than 12 months’ duration (mean 47 months, median 49.5 months), we obtained signals in 10 specimens after manipulating the treatment procedure (Table 16; Fig 22). No fluorescent signals were detected with the rest of the 40 specimens even after attempting two protocols. Table 15 C-erbB2 (HER2/neu) FISH of formalin fixed paraffin-embedded archival breast cancer tissues of less than 12 months’ durationa CASE NO DURATION IN MONTHS PROTOCOL FISH RATIO 1 11.5 Routine A 2.58 2 5.0 Routine A 4.69 3 7.5 Routine A 8.8 4 10.2 Routine A 7.88 5 10.2 Routine NA 1.10 6 8.4 Routine A 8.50 7 3.4 Routine NA 1.16 8 2.6 Routine NA 1.24 9 2.9 Routine NA 1.08 10 3.5 Routine NA 1.10 11 3.5 Routine A 3.16 12 3.5 Routine A 3.84 13 3.2 Routine A 3.19 a A, amplified; NA, not amplified; RATIO, ratio of HER2/neu to CEP17 signals. Fig. 21 FISH of archival paraffin-embedded breast cancer tissues stored less than 12months’. Green signals show presence of centromere 17 copies and red signals represent multiple copies of c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. Table 16 C-erbB2 (HER2/neu) FISH of formalin fixed paraffin-embedded archival breast cancer tissues of more than 12 months’ durationa CASE NO DURATION IN MONTHS 1 83.7 PROTOCOL I FISH RATIO NA 1.26 2 64.9 V A 5.3 3 56.9 VI NA 1.7 4 57.9 III A 5.9 5 54.8 II A 4.2 6 24.5 VI A 9.0 7 12.6 IV A 3.37 8 53.7 VI A 5.8 9 37.5 I A 10.5 10 30.9 VI A 8.8 11 - 30 23.6 - 87.2 Routine and I *** - 31 - 40 38.1 - 56.6 Routine and VI *** - 41 - 50 27.8 - 45.9 Routine and V *** - a A, amplified; NA, not amplified; ***, no signals; RATIO, ratio of total HER2/neu to total CEP17 signals. Fig. 22 FISH of archival paraffin-embedded breast cancer tissues stored more than 12 months’duration (B). Green signals show presence of centromere 17 copies and red signals represent multiple copies of c-erbB2 (HER2/neu) gene. Original magnification x 100 objective. 3.3.2.3 C-erbB2 gene amplification and microwave oven fixation In this arm, there was no significant difference in the detection of FISH signals with respect to the microwave oven or routine fixation protocols in the 7 breast cancer cases (P = 0.637, Table 17). Table 17 C-erbB2 (HER2/neu) FISH of formalin fixed breast cancer tissues (microwave oven and routine fixation)a a CASE NO FISH MICROWAVE OVEN FIXATION RATIO ROUTINE FIXATION RATIO 1 NA 1.15 1.16 2 NA 1.20 1.24 3 NA 1.11 1.08 4 NA 1.10 1.11 5 A 3.28 3.16 6 A 3.47 3.84 7 A 3.26 3.19 A, amplified; NA, not amplified; Pos, positive; Neg, negative; RATIO, ratio of total HER2/neu signals to total CEP17 signals. 3.3.3 Correlation of c-erbB2 protein overexpression and gene amplification 35 cases in the first arm were used to compare the gene amplification and protein overexpression of c-erbB2. In cases where FISH signals were detected, there was a positive correlation between c-erbB2 gene amplification by FISH and protein expression by immunohistochemistry (P = 0.0002) in all the groups. All tissues with 3+ IHC staining disclosed c-erbB2 amplification, (Fig 23) whereas those with negative immunostaining (0 and 1+ IHC staining) showed no c-erbB2 gene amplification (Table 12, 13, 14). There was no gene amplification detected in the 6 immunopositive cases with 2+ IHC staining in group 1 of the first arm (Table 12), while in the other groups (Table 13, 14), it showed gene amplification in 2+ IHC cases. Fig. 23 FISH post (A) 12 and (B) 27 hr fixation. The green signal shows presence of centromere 17 copies while the red signal represents multiple copies of the c-erbB2 (HER2/neu) gene. (C)Immunostaining for c-erbB2 (HER2/neu) (3+), showing a complete, intense membranous pattern of positivity. Original magnification X 400. 3.4 Association of c-erbB2 overexpression with nuclear morphometry For nuclear morphometry, c-erbB2 positive and negative tumors differed significantly only in nuclear roundness, with malignant cells in c-erbB2 positive cases being less round (P = 0.0322, Table 18) (Fig. 24 & 25). There was no statistically significant difference in nuclear morphometry findings between immunopositive versus immunonegative malignant cells in the c-erbB2 positive cancers. When analyzing the association of nuclear morphometric data in relation to histopathologic parameters in the group of c-erbB2 positive breast cancers, there was a significant difference in (a) nuclear area and perimeter between histological grade 3 and histologic grade 1 and 2 (P = 0.001 and P = 0.03 respectively) and (b) nuclear perimeter between tumor size ≤ 20 mm and tumor size > 20 mm (P = 0.046) (Table 19). Table 18 Nuclear morphology of cancer cells in c-erbB2 positive and c-erbB2 negative tumors C-erbB2 positive tumors C-erbB2 negative P-value tumors Area*(µm) 119.2 ± 5.6 116.2 ± 5.1 0.7039 Perimeter* (µm) 41.91 ± 1.0 41 ± 0.9 0.5192 Roundness* 1.23 ± 0.009 1.2 ± 0.009 0.0322 * values are mean ± SD Fig. 24 Nuclei of c-erbB2 positive invasive ductal carcinoma digitally outlined using a computer mouse. Note that most of the nuclei are not so round or oval as in c-erbB2 negative patients. (IHC staining; Original magnification x 400). Fig. 25 Nuclei of c-erbB2 negative invasive ductal carcinoma digitally outlined using a computer mouse. Note that most of the nuclei are round or oval in configuration. (IHC staining; Original magnification x 400). Table 19 Nuclear morphology of malignant cells with respect to clinico-pathologic markers of prognosis in c-erbB2 positive breast tumors Histologic grade Tumor size LV Invasion Node status ER PR Area1 (µm2) Perimeter1 (µm) Roundness1 (µm) Feret Ratio2 Grade 1 or 2 107.56* ± 35.68 39.62* ± 7.87 1.2 ± 0.17 1.44 Grade 3 142.36*± 47.55 44.6* ± 9.62 1.2 ± 0.18 1.47 ≤ 20mm 110.76 ± 36.23 39.31* ± 7.29 1.2 ± 0.16 1.47 > 20mm 133.99 ± 45.43 44.05* ± 9.7 1.19 ± 0.17 1.42 Yes 122.22 ± 44.54 41.97 ± 9.13 1.28 ± 0.2 1.45 No 115.33 ± 37.2 40.88 ± 8.16 1.2 ± 0.16 1.45 Neg 119.06 ± 35.7 41.12 ± 8.06 1.2 ± 0.16 1.47 Pos 126.62 ± 45.48 42.93 ± 9.82 1.19 ± 0.18 1.41 Neg 125.8 ± 47.26 42.36 ± 9.39 1.2 ± 0.17 1.47 Pos 113.97 ± 34.49 40.57 ± 8.04 1.2 ± 0.18 1.43 Neg 117.32 ± 40.83 40.89 ± 8.43 1.2 ± 0.17 1.47 Pos 118.13 ± 64.04 41.58 ± 8.49 1.21 ± 0.18 1.43 1 – Values are Mean ± SD; 2 – Ratio; *-- P < 0.05 3.5 Association of c-erbB2 overexpression with established clinicopathological and biological markers 3.5.1 Association of c-erbB2 overexpression and hormone receptor status A significant negative association between c-erbB2 expression and ER and PR status was noted (P = 0.0001; Table 20 and 21). Table 20 Association of c-erbB2 overexpression and ER status ER Total Negative Positive C-erbB2 - 53 152 205 C-erbB2 + 53 57 110 Total 106 209 315 P-value 0.0001 Table 21 Association of c-erbB2 overexpression and PR status PR Total Negative Positive C-erbB2 - 94 111 205 C-erbB2 + 74 36 110 Total 168 147 315 P-value 0.0001 3.5.2 Relationship between c-erbB2 parameters in invasive breast carcinoma overexpression and clinicalpathological Immunohistochemical expression of c-erbB2 protein and other clinicopathological parameters in a series of Singapore women with invasive breast carcinoma are documented in Table 22. The findings were correlated with pathologic parameters such as age at diagnosis, tumor size, histologic subtype, histologic grade, pathological stage, lymphovascular invasion and lymph node status (Table 22). 3.5.2.1 Association of c-erbB2 overexpression and histological grade There was a significantly higher c-erbB2 overexpression in histological grade 3 tumors than in histological grade 1 and 2 tumors. 3.5.2.2 C-erbB2 overexpression and other clinicopathological factors There was no statistically significant difference in c-erbB2 overexpression with regard to patient age, histologic subtype, lymphovascular invasion, pathologic stage, axillary lymph node status and tumor size. Table 22 Correlation between c-erbB2 overexpression and clinicopathological parameters C-erbB2 immunoexpression (%) Parameter No. of Patients Total Age group ≤ 50 > 50 Ethinicity Chinese Malay Indian Tumor size ≤20mm >20mm Histologic subtype IDC Others Histologic Grade Grade 1 Grade 2 Grade 3 Staging Stage 1 Stage 2 Stage 3 Stage 4 Lymphovascular Invasion Present Absent Nodal status Negative Positive * Missing values range from 2 to 26 Positive P-value 134 181 50(37.3) 60(33.2) 0.443 271 23 14 86(31.7) 13(56.5) 8(57.1) 116 181 24(20.7) 44(24.3) 0.087 266 55 96(36.1) 14(25.5) 0.142 46 132 127 12(26.1) 37(28) 55(43.3) 68 192 28 7 15(22.1) 70(36.5) 11(39.3) 3(42.9) 92 227 35(38) 75(33) 0.386 162 133 50(30.9) 46(34.6) 0.569 321* - 0.017 0.142 3.5.3 C-erbB2 overexpression with Ki-67(cell proliferation index) The percentage of Ki-67 immunostaining (KI) detected in the 72 cases ranged from nil to 80%. An example of Ki-67 positive stained breast cancer section along with negative control is shown in Fig 26 and 27. There was no siginificant association between c-erbB2 overexpression and Ki-67 (Table 23). Table 23 Association of c-erbB2 overexpression with Ki-67 Ki-67 Negative Positive Positive Positive ≤ 5% 6 -10% > 10% Total C-erbB2 - 8 15 4 27 54 C-erbB2 + 3 2 2 10 17 Total 11 17 6 37 71 P-value 0.586 Fig. 26 Positive Ki67 immunostaining showed nuclear positivity of the proliferating cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). Fig. 27 Negative control (by omission of primary antibody) showed no Ki67 immunostaining of the cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). 3.5.4 C-erbB2 overexpression with iNOS Cells with more than 10% cytoplasmic staining for iNOS were considered positive. Out of 72 cases, 34 (47.2%) were considered positive and the rest were negative. An example of a strong iNOS positive section is shown in Fig 28, along with a negative control (Fig 29). There was no significant correlation between c-erbB2 overexpression and iNOS expression (Table 24). Table 24 Association of c-erbB2 overexpression with iNOS immunoreactivity iNOS Total Negative Positive C-erbB2 - 31 23 54 C-erbB2 + 7 10 17 Total 38 33 71 P-value 0.242 One case has no c-erbB2 value so is not included in the analysis Fig. 28 iNOS immunostaining showed strong cytoplasmic positivity of the cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). Fig. 29 Negative control showed no iNOS immunostaining of the cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). 3.5.5 C-erbB2 overexpression with c-myc The expression of c-myc was studied in 34 cases of invasive breast carcinoma. Cells with more than 10% cytoplasmic staining were considered positive. All carcinomas showed positive cytoplasmic staining except two cases. An example of an immunopositive section is shown, along with negative control in the Fig 30 and 31. C-myc gene expression did not show any association with c-erbB2 overexpression, (Table 25). Table 25 Association of c-erbB2 overexpression with c-myc c-myc Total Negative Positive C-erbB2 - 2 17 19 C-erbB2 + - 15 15 Total 2 32 34 P-value 0.195 Fig. 30 Positive c-myc immunostaining showed nuclear as well as cytoplasmic positivity of the cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). Fig. 31 Negative control showed no c-myc immunostaining of the cancer cells of invasive breast carcinoma. (Hematoxylin counterstain; Original magnification x 310). 3.5.6 Association of biological markers with clinicopathological parameters in invasive breast carcinoma Clinicopathological data in 72 cases which were subjected to immunohistohemical evaluation of Ki-67, iNOS and c-myc are shown in Table 26. Cases with missing values are eliminated from the statistical analysis. Univariate analysis of the association between clinicopathological parameters and these markers is displayed in Table 27. 3.5.6.1 Ki-67 Ki-67 showed a positive association with histologic grade and histologic subtypes. Remaining parameters did not have any correlation with cell proliferation. Histologic grade 3 tumors and invasive ductal carcinoma subtypes had higher proliferation indices than lower grade tumors and other histologic subtypes. 3.5.6.2 iNOS iNOS expression showed a positive correlation with lymph node status. 84.5% had axillary lymph node metastasis in those cases which showed iNOS expression (22/26). None of the other parameters had any association with iNOS expression. 3.5.6.3 C-myc C-myc protein expression did not show any association with any of the clinicopathological markers. Table 26 Distribution of clinicopathological parameters and biological markers Parameter Classification Total Patients Age Median Mean Range Ethnicity Tumor size Chinese Malay Indian & Others ≤25mm >25mm Histologic subtype Ductal Others Histological grade I II III Lymphovascular invasion Present Not present Lymph node status Positive Negative Stage C-erbB2 I II III IV Positive Negative Ki-67 0- 5% 5-10% > 10% iNOS Positive Negative No. of patients (%) 72* 54 55.35 36-85 60 (3.3%) 8 (11.1%) 4 (5.6%) 24 (35.8%) 43 (64.2%) 62 (86.1%) 10 (13.9%) 10 (14.3%) 34 (48.6%) 26 (37.1%) 40 (57.1%) 30 (42.9%) 19 (33.3%) 38 (66.7%) 7 (13.3%) 35 (66%) 6 (11.3%) 5 (9.4%) 17 (23.9%) 54 (76.1%) 28 (38.9%) 6 (8.3%) 38 (52.8%) 34(47.2%) 38 (52.8%) *-Not all 72 cases are reflected in all parameters. Cases with missing values are removed from statistical analysis. Table 27 Univariate analysis of biological markers and clinicopathological parameters in breast carcinoma Ki-67 (P-value) iNOS (P-value) 0.5 0.343 0.078 0.475 Histologic subtype 0.038* 0.24 Histological grade 0.007* 0.423 Lymphovascular invasion 0.675 0.782 Lymph node status 0.882 0.008* 0.714 0.797 0.13 0.928 0.214 0.42 0.119 0.424 Parameters Ethnicity Tumor size Stage ER PR p53 *-statistically significant values (P < 0.05). Invasive ductal subtype and histological grade 3 breast cancers expressed higher Ki-67 levels. 3.6 Follow up and survival analysis 3.6.1 Kaplan-Meier survival analysis In the 290 women with mean and median follow-up of 18 months and 17 months respectively, 15 deaths from breast carcinoma (5.2%) were documented. The OS for cerbB2 positive and c-erbB2 negative patients is illustrated in Fig 32, with mean survival of 43 months for c-erbB2 positive patients and 45 months for c-erbB2 negative ones. This difference in OS is statistically significant (P = 0.0166), indicating an adverse prognosis for the c-erbB2 positive cases. A similar trend was noted for DFS with mean survival of 10 months before detection of recurrence for c-erbB2 positive patients and 11 months for c-erbB2 negative ones, although the difference did not reach statistical significance (P = 0.7013) (figure not shown). Different combinations of tumor characteristics for possible additive prognostic capacities were also investigated. In the node positive group, statistically significant difference was observed in OS for c-erbB2 negative patients compared to c-erbB2 positive patients, with mean survival period of 40 months and 48 months respectively (P=0.0047; Fig 33). Patients with histologic grade 1 and 2 tumors experienced improved OS for c-erbB2 negative tumors compared to c-erbB2 positive ones, with mean survival of 46 months and 42 months respectively (P = 0.0367; Fig 34). When c-erbB2 and estrogen receptor (ER) status were combined, c-erbB2 status had a negative effect on the prognosis of the estrogen receptor (ER) positive group with OS curves of c-erbB2 positive and negative patients within the ER positive subset differing significantly, with a mean survival period of 40 months and 47 months respectively (P = 0.0092; Fig 35). C-erbB2 expression did not have any effect on OS in the estrogen receptor negative group of cases and in women aged ≤ 50 years. In those > 50 years of age, the mean survival was 41 months for c-erbB2 positive patients and 47 months for cerbB2 negative ones, a statistically significant difference (P = 0.0096; Fig 36). 1.1 1.0 c-erbB2 -ve Overall Survival (Proportion) .9 c-erbB2 +ve .8 .7 .6 .5 .4 .3 P = .0166 .2 .1 0 6 12 18 24 30 36 42 48 54 60 Survival time in months Fig. 32 Kaplan-Meier curves for overall survival (OS) with regard to c-erbB2 overexpression (n = 290). 1.1 1.0 c-erbB2 -ve Overall Survival (Proportion) .9 c-erbB2 +ve .8 .7 .6 .5 .4 .3 P = .0047 .2 .1 0 6 12 18 24 30 36 42 48 54 60 Survival time in months Fig. 33 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with node-positive tumors (n = 118). 1.1 Overall Survival (Proportion) 1.0 .9 c-erbB2 -ve c-erbB2 +ve .8 .7 .6 .5 .4 .3 P = .0367 .2 .1 0 6 12 18 24 30 36 42 48 54 60 Survival time in months Fig. 34 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with histologic grade 1 and 2 tumors (n = 109). 1.1 1.0 Overall Survival (Proportion) c-erbB2 -ve .9 .8 c-erbB2 +ve .7 .6 .5 .4 .3 P = .0092 .2 .1 0 6 12 18 24 30 36 42 48 54 60 Survival time in months Fig. 35 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients with ER positive tumors (n = 185). 1.1 Overall Survival (Proportion) 1.0 c-erbB2 -ve .9 .8 c-erbB2 +ve .7 .6 .5 .4 .3 P = .0096 .2 .1 0 6 12 18 24 30 36 42 48 54 60 Survival time in months Fig. 36 Kaplan-Meier curves for overall survival (OS) stratified by c-erbB2 status, in patients in age group > 50 years (n = 159). 3.6.2 Multivariate Cox regression analysis Multivariate analysis by Cox regression model showed c-erbB2 overexpression to be an independent predictor of overall survival (P = 0.029; Table 28) when histologic grade, nodal and ER status were considered. However, when tumor size and pathologic stage were included in the multivariate analysis, c-erbB2 lost its independent prognostic power. Table 28 Multivariate analysis of important prognostic factors for their relationship with overall survival Prognostic variables P-value C-erbB2 0.029 ER 0.443 Histologic Grade 0.831 Nodal status 0.995 CHAPTER 4 DISCUSSION 4.1 C-erbB2 (HER2/neu) status in invasive breast carcinoma 4.1.1 Brief overview of c-erbB2 assessment A wide range of assay methods have been used to detect Her2/neu or c-erbB2 status in breast cancer. Each technique has its own advantages and disadvantages, some of which are summarized in Table 29. Table 29 Comparison of methods used to detect c-erbB2 status (modified from Perez et al., 2002; Schnitt, 2001; Dowsett et al., 2000; Molina et al., 1996) Methods Advantages Disadvantages Detects protein overexpression. Not a standardized technique. Relatively simple method that can be performed on sections cut from paraffin embedded or frozen tissues. Sensitivity and specificity may vary for different antibodies. Controls need to be used for every run. IHC Procedure time is very short and can be accomplished in any laboratory. No standardized scoring system. No expert training required. Automation is applicable. FDA approved for therapy. Cost effective. Detects gene amplification. Longer procedure time. Can be applied to paraffin embedded or frozen tissues. FISH Needs expert training for the procedures. Internal controls. Special instruments and reagents needed to interpret and proceed. Quantitative assessment. Difficult to accomplish in all laboratories. Expensive and no automation. Southern blot Detects gene amplification. Expert training needed. Good sensitivity and specificity. Not an easy procedure. No FDA approval. Detects serum). ELISA (serum) protein overexpression (shed in No automation Cannot perform on slides. No FDA approval. Highly sensitive and specific. No automation. Standardized technique. 4.1.2 C-erbB2 protein overexpression by Immunohistochemistry (IHC) Immunohistochemistry is one of the most widely used tools for research and diagnosis. The practice of immunohistchemistry originated with Albert H. Coons and his colleagues in the early 1940’s (Polak and Van Noorden, 1997). One of the main diagnostic uses of IHC is to determine the nature of tumors. IHC has been specifically adapted and the most practical method to perform in the routine practice of pathology for detection of c-erbB2 protein overexpression using specific antibodies (Schnitt, 2001; Slamon et al., 1989; van de Vijver et al., 1988). IHC has been used in the past to identify overexpression of c-erbB2 protein on the cell membrane of breast cancer cells in fixed tissues. C-erbB2 protein detection in archival paraffin-embedded tissues is highly variable or even compromised due to certain factors. Factors which have to be considered are time and nature of tissue fixation; method of tissue processing; temperature of paraffin embedding; duration of storage (especially unstained slides); type of antibody used; and the staining procedure used (Dowsett et al., 2000). Although different methods of antigen retrieval are used, it should be optimized because high antigen retrieval may result in high rates of positivity and even benign cells are also highlighted, whereas inadequate antigen retrieval may yield false negative results (Lian and Tan, 2002). Standardization of the procedures may be preferable to obtain reliable results. Many antibodies for example CB-11 (monoclonal antibody, Ventana), A0485 (polyclonal antibody, DAKO), Mab-1/Pab 1 (cocktail antibody) and HercepTest are used by many laboratories world wide. Each antibody has different levels of sensitivity and specificity. The HercepTest has high sensitivity and specificity, and has been approved by FDA for therapeutic applications. There is still debate on the reliability of IHC. The quality of IHC results can be improved by following a few steps such as: (1) standardizing tissue fixation and processing; (2) use of appropriate positive and negative controls; and (3) participation is external quality assurance programmes. The role of c-erbB2 oncoprotein overexpression in invasive breast cancer has been widely investigated. C-erbB2 overexpression occurs in about a third of breast tumors and is linked to an adverse prognosis (Fitzgibbons et al., 2000). There is still controversy in the determination of immunopositivity of c-erbB2 status in invasive breast carcinoma. One researcher has considered membrane staining of any intensity in any malignant cell as indicating protein overexpression (Sjogren et al., 1998); yet another has defined c-erbB2 positivity as only when “tumor cells showed intense circumferential cell membrane staining easily identified with a 10X objective” (Jacobs et al., 2000). Ellis and co-workers have recommended that if > 10% of breast tumor cells reveal moderate or strong complete membrane reactivity, c-erbB2 expression is considered as positive on immunohistochemistry using the HercepTest scoring method (Ellis et al., 2000). In this present study, we have considered moderate (2+) or strong (3+) membrane staining intensity in 10% or more of invasive carcinoma cells as positive. Amplification or overexpression of c-erbB2 gene or protein has been reported by several studies to occur in 10-40% of primary breast carcinomas (Cho et al., 2003; Konigshoff et al., 2003; Hoff et al., 2002; Tsai et al., 2001; Hoang et al., 2000; Ross and Fletcher, 1999; Ross et al., 1998; Odagiri et al., 1994; Slamon et al., 1987;). The rate of cerbB2 immunoexpression in our study was 34.3%, which is within the range reported for breast cancers in the literature. Also in this present study, invasive ductal carcinoma exhibited higher c-erbB2 immunopositivity (36.1%) compared to other types like invasive lobular, invasive tubular, and others (25.5%). This is also in accord with that reported by Smith et al (1994). 4.1.3 C-erbB2 gene amplification by Fluorescence in situ hybridization (FISH) Fluorescence in situ hybridization is one of the most convenient assays currently used to assess c-erbB2 gene amplification. There are many factors which may affect the detection of fluorescent signals by FISH analysis. Technical limitations include the type of fixative used, artifacts caused by sectioning of tissue blocks, thickness of sections, storage of cut sections and probe penetration (Ellis et al., 2000; Gozetti et al., 2000; Thomson et al., 1993). In our surgical pathology laboratory, specimens are generally fixed in 10% buffered formalin for a duration that varies from approximately 8 to 32 hours due to inherent vagaries of surgical schedules. It would appear from our study that a fixation period ranging from 2 hrs to 1 week would not affect FISH results. Beyond one week however, no signals could be detected, with the exception of the highly amplified case 1 in group 3 (refer to “Results” chapter). Formalin fixation increases the complexity of cellular structure and chromatin condensation making it difficult for the probe to penetrate and interact with the target DNA (Thompson et al., 1994). It has been suggested that generally, the longer the primary fixation, the permeabilization step of pretreatment and enzyme digestion needs to be more aggressive (Pauletti and Slamon, 1999). However, we obtained similar results for the different fixation protocols in all 3 groups in this study even though we did not vary the permeabilization step, with the exception of 4 cases in the extended fixation protocol where no signals were obtained. Our finding is at variance with that of Goelz et al (1985) who reported previously that DNA isolated from formaldehyde fixed and embedded tissues was not affected by the length of time in fixation. FISH is a cytogenetic technique widely used to evaluate gene amplification and other genetic aberrations in fresh tumor tissues. However, if FISH analyses could be easily applied to archival paraffin-embedded tumor tissues from patients with known clinical outcome, valuable information regarding the diagnostic and prognostic significance of such aberrations would be facilitated (Hyytinen et al., 1994). FISH analyses in archival paraffin wax embedded tissues have been performed for colon, prostate, lung and breast cancer (Persons et al., 1994; Kim et al., 1993; Steiner et al., 1993 Zitzelsberger et al., 1993; Arnoldus et al., 1991). Despite methodological and technical advances, there are limitations to obtaining signals by FISH in archival paraffin-embedded tissues as compared with fresh tumor tissues (Arnoldus et al., 1991). Although several methods have been used for analysis of archival paraffin-embedded tissues by FISH, they were only partially successful in many instances (Matsumura et al., 1992; Hopman et al., 1991). The primary problems encountered are background tissue fluorescence and the relatively weak intensity of the signals obtained (Dhingra et al., 1992). Furthermore, the formalin fixation period, sample pretreatments, protein digestion and denaturation have to be individually optimized for each sample. It would appear that for the FISH procedure to be successful in archival paraffinembedded breast cancer tissues stored for more than 12 months, sections have to undergo optimization of the pretreatment regime. For good results, pretreatment methods like protease digestion and NaSCN incubations have to be optimized for each individual case. Too short a digestion time yielded poor hybridization signals in most specimens, possibly because of inaccessibility of the probe to the cell nuclei. On the other hand, excessive protease digestion before hybridization may yield sufficient chromosome signals but with distortion of the nuclear morphology. Despite the extensive variations in the pretreatment protocol, we were not able to get clear hybridization signals that could be evaluated in a reproducible and reliable way for all tissues that had been stored for more than 12 months. There was also a tendency for auto-fluorescence in the archival paraffin-embedded sections. As microwave oven tissue fixation, which has been used in many laboratories to shorten fixation time, was routinely used for fixation in many of our archived breast cancer specimens, the effect of this fixation method on the retrieval of FISH signals was also analyzed. The result shows that microwave oven fixation did not affect FISH signals. Targeting c-erbB2 by antibody (Herceptin) as a cancer therapeutic modality is of clinical importance. Integration of this assay into routine testing should help in rationalization of the most appropriate therapy for individuals with breast cancer. 4.1.4 Concordance between c-erbB2 gene amplification and protein overexpression The observation of a good concordance between FISH and immunohistochemistry detection of c-erbB2 is in accord with those of other investigators (Couturier et al., 2000; Hoang et al., 2000; Vang et al., 2000). The concordance rate was as high as > 80% reported in literature (Perez et al., 2002; Ratcliffe et al., 1997). In this present study, 2+ IHC staining cases in the routine fixation protocol group showed no c-erbB2 (HER2/neu) gene amplification but in the other groups, 2+ IHC staining cases showed gene amplification. This is concordant with other studies done with larger samples. The c-erbB2 (HER2/neu) gene was reportedly amplified between 12% to 35% of such (2+ IHC) cases (Perez et al., 2002; Kakar et al., 2000; Ridolfi et al., 2000). Possible reasons for the disparity in results obtained by FISH and IHC analyses include transcriptional or posttranscriptional regulation for increased surface receptor expression in the absence of gene amplification (Hoang et al., 2000) and intrinsic variability of the IHC assay in terms of specimen processing and antigen retrieval (Wang et al., 2000). Studies show that only distinct membranous immunostaining correlates with gene amplification and bears prognostic value in breast cancer and the cytoplasmic immunostaining is without such relevance (Lehr et al., 2001; Pegram and Slamon, 1999). Investigation in one study, which compared the immunohistochemical and FISH methods for evaluation of c-erbB2 in breast cancer, found that both techniques were equally good and therefore, recommended immunohistochemistry as a less time-consuming and expensive method for routine use in laboratories (Jacobs et al., 1999). However, in view of problems associated with IHC, particularly in the interpretation of results, FISH may provide additional useful information for classification of tumors as c-erbB2 positive or negative (Jimenez et al., 2000). 4.2 C-erbB2 status and clinicopathological parameters 4.2.1 C-erbB2 overexpression and hormonal receptor status Estrogen and progesterone play major roles in normal breast development, in addition to breast carcinogenesis, by modifying the expression of a variety of genes through their receptors (Jin et al., 2000; Celentano et al., 1998). Estrogen and progesterone receptor status (both positive and negative) has been established as a predictive factor for breast cancer treatment, recurrence and prognosis (Li et al., 2003; Chu et al., 2001). Estrogen receptor (ER) and progesterone receptor (PR) status has also been validated as factors which predict response to hormonal therapy (Ring and Ellis, 2002; Yokota et al., 1999). Reports have revealed an inverse relationship between the presence of ER and PR and c-erbB2 overexpression (Schroeder et al., 1997; Descotes et al., 1993; Tsuda et al., 1993)). This is in agreement with findings in this present study, where c-erbB2 overexpressed cases showed an inverse association with ER and PR positivity; ER positive tumors being more likely to be c-erbB2 negative than were ER negative tumors (74.1% versus 48.1%). A related finding of high levels of c-erbB2 being associated with ER and PR negativity was reported by Pous et al (2000). On the other hand, there have also been studies which do not show any correlation of hormone receptor positivity with c-erbB2 overexpression (Gullick et al., 1991). There are studies which suggest that ER-positive breast cancers that overexpress c-erbB2 may be less responsive to tamoxifen (a selective estrogen receptor modulator [SERM] which binds to ER and partially inhibits its activity, and is effective in the treatment and prevention of breast cancer), than breast cancer with low or no c-erbB2 overexpression (Osborne et al., 2003; Benz et al., 1993). Differences found in c-erbB2 amplification between steroid receptor positive and negative tumors could be helpful to define a specific subset of women in whom adjuvant therapy should be directed. Controversy exists between studies in relation to tamoxifen adjuvant therapy in cerbB2 positive and negative groups. One shows that overall survival in the cerbB2 positive group treated with tamoxifen was significantly worse than the c-erbB2 negative group (Carlomagno et al., 1996). Another study showed no difference between c-erbB2 positive and negative groups (Constantino et al., 1994). A recent study showed that AIB-1, also called SRC-3, is an ER co-activator that plays a role in development of breast cancer. It has been found that high levels of activated AIB-1 could reduce the antagonist effects of tamoxifen especially in tumors that overexpress the c-erbB2 gene (Bouras et al., 2001). The expression of c-erbB2 and hormone receptors are not always mutually exclusive but there are studies which showed that amplified c-erbB2 overexpression might precede the loss of hormone-dependence (Kurokawa et al., 2000; Schroeder at al., 1997). Overexpression of c-erbB2 confers anti-estrogen resistance to breast cancer. This has been well documented in multiple studies. A full-length cerbB2 cDNA transfected into ER positive MCF-7 human breast cancer cells causes loss of sensitivity to tamoxifen or estrogen dependence (Dowsett et al., 2001; Kurokawa et al., 2000; Miller et al., 1994). It is still unclear how c-erbB2 overexpression potentially mediates tamoxifen resistance in breast cancers (Chung et al., 2002). However, c-erbB2 overexpression results in activation of the Ras/MAPK signaling pathway in breast cancers (Tzahar et al., 1998). This leads to phosphorylation of Ser-118 in ER, leading to ligand-independent ER activation with loss of the inhibitory effect of tamoxifen on ER-mediated transcription (Kurokawa et al., 2000; Bunone et al., 1996). This shows that anti-Her2/neu therapy facilitates the inhibitory effect of tamoxifen on c-erbB2 overexpressing hormone dependent breast cancers (Witters et al., 1997). There are also contradictory studies which do not support the hypothesis that overexpression of c-erbB2 is associated with tamoxifen unresponsiveness (Elledge et al., 1998). 4.2.2 C-erbB2 overexpression with histological grade and nuclear morphometry Histological grading is an essential component of the pathologic assessment of invasive breast carcinomas and its prognostic significance has been amply demonstrated (Richer et al., 1998; Periera et al., 1995; Ellis et al., 1991). Histological grade is regarded as a marker of cancer cell differentiation, and is determined by a combination of factors: tubule formation, nuclear pleomorphism and mitotic activity (Elston and Ellis, 1998). It is well known that cell differentiation is a complicated process, involving regulation of gene transcription, differential RNA processing and translation as well as intra- and intercellular biomolecular regulation (Lin et al., 2003). Histologic grading of breast carcinoma is potentially of great clinical value in determining the prognosis. Studies of the relationship between histologic grade per se and prognosis show a strong correlation. C-erbB2 expression was found to correlate with histological grade in Caucasian and Japanese women with breast cancer (Hoff et al., 2002; Hung and Lau, 1999; Tsuda et al., 1993). In a limited study of 167 Taiwanese cases, c-erbB2 overexpression was also correlated with the histological grade of infiltrative ductal breast carcinoma (Tsai et al., 2001). In this current Singapore study, comprising a larger group of predominantly Chinese women, c-erbB2 overexpression was found in 26.1% of grade 1 carcinomas, 28 % of grade 2 carcinomas and 43.3% of grade 3 carcinomas. This study also supports a positive correlation between c-erbB2 expression and histologic grade of breast cancer, with overexpression being less frequent in grade 1 and 2 than in grade 3 carcinomas. This is in concordance with many other studies (Hoff et al., 2002; Pous et al., 2000; Elstone and Ellis, 1991; Rilke et al., 1991; Tsuda et al., 1990). On the other hand, there are also studies which found no correlation between histologic grade and c-erbB2 overexpression (van de Vijver et al., 1988). Other than c-erbB2, progesterone hormone also has influence on histologic differentiation. PR negative tumors more often belong to the less differentiated, histological grade 3 invasive ductal breast cancers than the more differentiated, histological grade 2 tumors (Ruibal et al., 2001). This has been proven in an ‘in vivo’ study showing that progesterone hormone also has an impact over the histologic grade (Lin et al., 2003). Until now, the relationship between malignancy associated nuclear morphological features and functional properties of tumor cells remain unclear (Huang et al., 2000). Nuclear morphometry has been shown to be of prognostic utility in various cancers (Ikeguchi et al., 1998) as well as in invasive breast carcinoma (Tan et al., 2001; Kronqvist et al., 2000). In fact, increasing nuclear size has been postulated to be the result of accumulation of abnormal genetic material during carcinogenesis (Tan et al., 2001). In this study of 96 cases of breast carcinoma from Singapore women, there was no significant association between nuclear area and perimeter with c-erbB2 expression per se but tumors with positive c-erbB2 immunostaining had less round nuclei as compared to those defined as c-erbB2 negative. Variations in nuclear size and shape have been used as defining features of nuclear pleomorphism, a parameter in the designation of histologic grade. That c-erbB2 positive tumors comprised cancer cells with less round nuclei corroborates its link with poorer histologic grade. There are studies which postulate that oncogene amplification could be related to the morphology of a tumor (Poller et al., 1991; Cardiff et al., 1988), although our study did not show any association between nuclear size and overexpression of the c-erbB2 oncoprotein. Among c-erbB2 positive tumors, the nuclear size was significantly larger in histological grade 3 compared to histological grade 1 or 2 tumors, supporting the notion of increased amounts of abnormal genetic material in higher grade cancers. In addition, c-erbB2 positive tumors larger than 20 mm were associated with increased nuclear perimeter of the malignant cells. Though the nuclear size appeared larger for tumors which had lympho-vascular invasion and lymph node positivity, the results were not statistically significant. Quantitation of nuclear parameters is a powerful tool that may be an adjunct to other cytologic and molecular indicators of cancer diagnosis and prognosis (Pienta and Coffey, 1991). Nuclear morphometry of malignant tumors can reveal features that may otherwise escape subjective analysis of cellular morphology. Further studies with larger number of patients will be required to determine the importance of quantitative morphometry. 4.2.3 C-erbB2 status and other clinicopathological parameters The relationship between c-erbB2 overexpression and other clinicopathological parameters such as patient age, tumor size, axillary lymph node status, pathologic stage and lymphovascular invasion have remained controversial. One study found a statistically significant relationship between c-erbB2 and age at diagnosis, with a more pronounced cerbB2 overexpression in younger patients (Sjogren et al., 1998). Interestingly, in this present study, it was found that the frequency of c-erbB2 immuno-positivity decreased with older age at diagnosis. There were more c-erbB2 immuno-positive cases in the women aged ≤ 50 years as compared to those > 50 years i.e., 37.3% vs 33.2%. The findings are similar to that reported by Sjogren et al (1998). Most of the recent studies, as well as this present one, failed to attain any statistically significant difference between the age at diagnosis and c-erbB2 overexpression (Tsutsui et al., 2003; Pous et al., 2000; Bebenek et al., 1998; Sjogren et al., 1998; Chariyalertsak et al., 1996; Horiguchi et al., 1994). There were variable results observed when cases were stratified as premenopausal versus post-menopausal groups. No significant difference was observed in a study between these groups (Yokota et al., 1999). In another study, statistically significantly worse OS was observed for c-erbB2 positive patients than negative ones (Sjogren et al., 1998). Tumor size by itself is one of the most powerful predictors of tumor behaviour in breast cancer (Fitzgibbons et al., 2000). However, its role as a prognostic marker combined with other prognostic factors is still not well investigated. The vast majority of breast cancers overexpressing c-erbB2, regardless of nodal status, are > 20mm in diameter (24.3 vs 20.7% in > 20mm and ≤ 20mm respectively). However, no statistical significance noted between c-erbB2 overexpression and tumor diameter in this present study. Several studies have not reported any association between c-erbB2 overexpression and tumor size though several authors did (Korkolis et al., 2001; Pous et al., 2000; Ceccarelli et al., 1995; Rilke et al., 1991; van de Vijver et al., 1988). On the other hand, Giai et al (1994) and Rilke et al (1991) found that c-erbB2 overexpression was present more frequently in tumors larger than 20 mm when compared to smaller ones, while Helal et al (2000) observed a similar trend for T3 and T4 tumors. Axillary lymph node dissection is a routine staging procedure in the management of invasive breast cancer. The use of adjuvant systemic treatment is dependent on the axillary nodal status. Even though c-erbB2 overexpression by itself is potentially prognostic in breast cancer, uncertainty still remains regarding its prognostic effect with reference to axillary lymph node metastasis (Mittra et al., 1995). Most studies have found no association between c-erbB2 overexpression and axillary node status (Helal et al., 2000; Sjogren et al., 1998; Barbati et al., 1997; Clarke and McGuire, 1991). Similarly, no statistically significant association was noted between c-erbB2 overexpression and nodal status in this present study. Positive nodes have been observed to be more often present when the primary tumor overexpresses c-erbB2 (Seshadri et al., 1993). The prognostic influence of c-erbB2 overexpression has been shown to increase arithmetically with increasing number of involved axillary lymph nodes (Mittra et al., 1995). In this present study, there was no significant association between c-erbB2 overexpression and pathological staging, although breast cancer patients who showed cerbB2 positivity showed a trend towards more advanced stages; that is 42.9% of stage IV patients, 39.3% of stage III patients, 36.5% of stage II patients and 22.1% of stage I patients expressed c-erbB2 immunopositivity in their tumors. C-erbB2 overexpression has been observed in about 40% to 60% of breast DCIS (Poller et al., 1991) as compared to about 10% to 40% only in invasive breast cancer. Plausible reasons for the relatively less frequent expression of c-erbB2 in invasive breast carcinoma include: overexpression of c-erbB2 diminishes during evolution from an in situ state to invasive breast carcinoma; or that the occurrence of invasive disease did not involve the c-erbB2 gene (Allred et al., 1992). The clinical usefulness of c-erbB2 overexpression in DCIS is currently uncertain. As c-erbB2 plays an important role in tumor cell motility and distant spread (Bobrow et al., 1995), its expression may be useful in predicting local recurrence in women who have undergone conservative resection of breast DCIS; or be indicative of the need to have wider resection margins during excision of these tumors. Lympho-vascular invasion is predictive of local failure and reduced overall survival (Pinder et al., 1994). In this present study, even though there was no statistically significant difference between c-erbB2 overexpression and lympho-vascular invasion, more c-erbB2 immuno-positive cases showed lympho-vascular invasion compared to cerbB2 negative cases (i.e.) 38% vs 33% respectively. This again implies c-erbB2 may play a role in cell motility in invasive disease. 4.3 C-erbB2 status and biological markers Besides the conventional clinicopathological parameters, biological markers also play role in deciding the behavioral variability of invasive breast cancers. Recently, there has been much interest in the biological markers of breast cancer, as they may provide useful information on the clinical outcome and may also help in deciding the treatment modality. The biological markers evaluated in this study were Ki-67, iNOS and c-myc. 4.3.1 C-erbB2 status and cell proliferation (Ki-67) Increased cell proliferation or decreased cell death (apoptosis or programmed cell death) are essential processes vital to carcinogenesis. There are many methods to evaluate cell proliferation in tissue samples; of all, Ki-67 immunostaining is a reliable, reproducible and easy method of estimating cell proliferative activity (Scholzen and gerdes, 2000). The Ki-67 index has been widely used in breast cancer research (Thor et al., 1999). There are studies which showed correlation between c-erbB2 overexpression and Ki-67 proliferation index (Rehman et al., 2000; Lee et al., 1992). De Potter et al (1995) suggested that increased cell proliferation and enhanced cell motility are associated with overexpression of c-erbB2 in invasive breast cancers. In this present study, there was no statistically significant association between Ki67 proliferative activity and c-erbB2 overexpression, in keeping with studies which also did not find such a relationship (Val et al., 2002; Pavelic et al., 1992). It remains to be clarified why c-erbB2 overexpressed cancer cell clones are preferentially selected for outgrowth if they do not experience a proliferative advantage from this genomic alteration. 4.3.2 C-erbB2 status and iNOS Nitric oxide (NO) is a potentially toxic molecule, which has been implicated in a wide range of diverse patho-physiological processes. Inducible nitric oxide synthase (iNOS) has been the primary enzyme for the production of large amounts of NO in human tissues. Large amounts of NO formation have been shown to initiate apoptosis, helping to kill cancer cells and intracellular pathogens (Brune et al., 1998). iNOS expression has been widely studied in many cancers including breast cancer (Franchi et al., 2002; Kaguora et al., 2001; Vakkala et al., 2000; Klotz et al., 1998;). The c-erbB2 overexpresion in relation to iNOS has not been much studied previously in breast cancers. This present study did not find any significant association between c-erbB2 overexpression and iNOS expression, though there was a trend observed. Patients whose tumors stained positive for c-erbB2 protein were more likely to express iNOS compared to c-erbB2 negative patients (58.8% vs 42.6% respectively). One in vivo study showed a lowered NO producing breast cancer cell line to have reduced EGFR expression, which is a component of the c-erbB2 growth factor family (Martin et al., 1999). Further exploration is needed to assess the association between iNOS and c-erbB2 in invasive breast carcinoma. 4.3.3 C-erbB2 status and c-myc The proto-oncogene c-myc encodes a transcription factor which plays a major role in early embryogenesis, as well as regulates normal cellular proliferation and differentiation. Aberrant c-myc expression is seen in many breast cancers (Pavelic et al., 1991). In a normal cell, c-myc expression levels are under stringent control. C-myc is known to collaborate with other oncogenes to promote malignant transformation (Hynes et al., 2001). Hynes et al., in their study, further showed that deregulated c-erbB2 activity effectively stimulated cytoplasmic signaling pathways which in turn impinged on c-myc at various levels, causing its deregulated expression. C-myc amplification has been found to be a strong indicator of poor prognosis (Chrzan et al., 2001; Scorilas et al., 1999). In this present study, there was no significant association between c-erbB2 overexpression and c-myc expression. This is concordant with that reported by Spandidos and co-workers (Spandidos et al., 1989). The small sample size could have been a factor which influenced the statistical analysis of the correlation between c-erbB2 overexpression and c-myc expression in this present study. 4.3.4 Clinicopathological parameters and biological markers: Ki-67, iNOS and c-myc In this study, significant associations were obtained between proliferation marker Ki-67 and histologic subtype and histologic grade. Ki-67 has been positively correlated with mitotic counts, which is a component of histologic grade (Lee et al., 1992; Isola et al., 1990). Invasive ductal carcinoma subtype and histologic grade 3 tumors showed high proliferative activity in this study. Ki-67 has been shown to play a major role in cancer behavior and aggressiveness. It is also believed to predict DFS (Imamura et al., 1999). It has been documented that Ki-67 has good association with S-phase fraction (SPF) and aneuploidy, which reflect the biological behavior and aggressiveness of the cancer. These imply that determination of Ki-67 in invasive breast cancers may become useful in future. In this present study, iNOS expression is strongly positively correlated with the axillary lymph nodal status. This is in accordance with another study, where they found a very strong correlation between the presence of NOS and axillary lymph node metastasis (Duenas-Gonzalez et al., 1997). This may be due to its major role in promoting angiogenesis. Vascularization is an absolute requirement for sustained tumor growth and its extent correlates positively with tumor metastasis (Tschugguel et al., 1999). Another report did not find any correlation between lymph node status and iNOS (Vakkala et al., 2000). The other clinicopathological parameters do not seem to produce any correlation with iNOS immunopositivity. Some studies reported a negative correlation between iNOS activity versus proliferation and grade, emphasized in one in vitro study, which showed inhibition of proliferation of human breast cancer cells by NO (Reveneau et al., 1999). C-myc expression did not correlate with any of the clinicopathological parameters. Its true impact needs further study in larger samples. 4.4 Follow up and survival analysis C-erbB2 gene amplification or protein overexpression independently predicts the DFS and OS in univariate and multivariate analysis. (Kobayashi et al., 2002; Dowsett et al., 2000; Gancberg et al., 2000; Ross & Fletcher, 1998; Hawkins et al., 1993; Slamon et al., 1987). C-erbB2 overexpression has been reported to contribute to increased metastatic potential of cancer cells and to enhance malignancy (Ceccarelli et al., 1995). Upregulation of MMP-9 and MMP-2 protease activities by c-erbB2 causes increased invasiveness and VEGF expression thereby leading to a stronger angiogenic response and conferring apoptosis resistance in breast cancer cells (Yu and Hung, 2000; Tan et al., 1997). Slamon et al (1987) found that c-erbB2 gene amplification independently predicted OS and DFS in node-positive breast cancer patients. It has been reported that overexpression of cerbB2 protein is an unfavorable prognostic indicator in breast cancer, especially in patients with axillary nodal metastases. The influence of c-erbB2 on prognosis among node-negative patients appears uncertain (Agrup et al., 2000). A small series of mainland China breast cancers concluded that c-erbB2 overexpression was weakly associated with poor prognosis (Suo et al., 2001). In this present study, c-erbB2 status generated statistically significant differences in OS but not in DFS, though there was a trend observed for a shorter DFS in women with c-erbB2 positive breast tumors. Besides the significant difference in prognosis between patients with c-erbB2 positive and negative breast cancer in this study, a c-erbB2 positive status also adversely affected women with ER positive, node positive, histologic grade 1 and 2 tumors, and women aged over 50 years at diagnosis. It has been suggested that c-erbB2 amplification promotes estrogen independence and tamoxifen resistance in ER positive tumors, leading to a poorer outlook for this group (Dowsett et al., 2001). In contrast, Elledge et al (1998) found no evidence of a poorer response to tamoxifen in ER positive metastatic breast cancer in spite of cerbB2 expression. The latter study, however, defined c-erbB2 positivity when > 1% of cells stained immunohistochemically, and it is possible that differences in assignment of what constitutes c-erbB2 immunopositivity may have contributed to variability in the findings. Its adverse effect on histologic grade 1 and 2 tumors is not unexpected, and may be used to identify poor performers in this subset. The poorer prognosis of women older than 50 years with breast cancer that overexpressed c-erbB2 has been documented by Ferrero-Pous et al (2000), though they did not elaborate on the possible reasons for this observation. There was no prognostic significance of c-erbB2 overexpression in node negative or estrogen receptor negative patients and women ≤ 50 years at diagnosis. Multivariate analyses applied to the overall population identified c-erbB2 overexpression as an independent prognostic factor when compared with ER and nodal status, and histologic grade; but lost its independent prognostic power when tumor size and pathologic stage were incorporated, confirming the latter two parameters as unequivocally powerful prognostic factors. 4.5 Conclusion In conclusion, the salient findings in this study are: (1) C-erbB2 overexpression in invasive breast cancer in Singapore women has a similar pattern and trend as that reported in studies from other countries with mostly western populations. (2) C-erbB2 gene amplification by the FISH procedure can be compromised by the duration of fixation of tissues, storage of tissue blocks and influenced by pretreatment protocols. It is to be expected that in future, assessment of oncogene alterations in many tumor types will play a critical role in guiding diagnosis and treatment. Standardization of the assays to assess these gene alterations will be necessary before these assays can be used for routine clinical evaluation. (3) Strong correlation is found in between c-erbB2 gene amplification and protein overexpression. (4) C-erbB2 overexpression is strongly associated with poorly differentiated breast carcinoma and inversely correlated with hormone receptor status (5) Image cytometry analysis found correlation between nuclear roundness and histological grade and tumor size in c-erbB2 positive tumors. (6) Overexpression of c-erbB2 in invasive breast cancer is associated with poor overall survival. This study supports the hypothesis that c-erbB2 overexpression in breast tumors from Singapore women is associated with poor prognosis and overall survival (Fig. 37). C-erbB2 overexpression Associated with higher histologic grade & Hormonal receptor status Poor prognosis & Diminished overall survival Fig. 37 Flow chart of significance of c-erbB2 overexpression/amplification in invasive breast cancer in Singapore women. 4.6 Future study Although there is increasing information on the role of c-erbB2 as a prognostic factor and as a factor predicting response to systemic therapy, there is a pressing need to develop a comprehensive profile of the biologic and molecular characteristics of a tumor, rather than assessing one marker at a time. In future, the tools of molecular biology, such as microarray technology will permit such an assessment and will dramatically alter the manner in which breast cancers are classified and the way in which prognostic and predictive factors are determined. However, the new molecular biology methods will need to be integrated with standard methods of pathologic evaluation. Expanding this study further by comparing c-erbB2 overexpression with other tumor markers like EGFR, bcl-2, pS2, Cathepsin D, p53, may help in establishing its significance in terms of prognosis. It is likely that in the near future, assessment of tumor suppressor genes and oncogene alterations in many tumor types will play a critical role in managing treatment modalities. Standardization of the assays to assess gene alterations will be necessary before these assays can be used for routine clinical evaluation. Assessment of c-erbB2 status can serve as a model for the standardization of such assays and interpretation of results. REFERENCES Agrup M, Stal O, Olsen K, Wingren S. C-erbB-2 overexpression and survival in early onset breast cancer. Breast Cancer Res Treat 63:23-29, 2000 Akiyama T, Sudo, C, Ogawara H, Toyoshima K, Yamamoto T. The product of the CerbB2 gene: a 185 – kilodalton glycoprotein with tyrosine kinase activity. Science 232: 1644-1646, 1986 Allan SM, Dean CJ, Eccles S, Sacks NP. Clinical radioimmunolocalization with a rat monoclonal antibody directed against c-erbB-2. Cell Biophys 24-25:93-98, 1994 Allred DC, Clark GM, Molina R, Tandon AK, Schnitt SJ, Gilchrist KW, Osborne CK, Tormey DC, McGuire WL. Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer. Hum Pathol 23:974-979, 1992 Allred DC, Clark GM, Tandon AK, Molina R, Tormey DC, Osborne CK, Gilchrist KW, Mansour EG, Abeloff M, Eudey L, McGuire WL. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 10:599-605, 1992 Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 410:83-86, 1997 Ambs S, Hussain SP, Harris CC. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. FASEB J 11:443-8, 1997 Arnoldus EP, Dreef EJ, Noordermeer IA, Verheggen MM, Thierry RF, Peters AC, Cornelisse CJ, Van der Ploeg M, Raap AK. Feasibility of in situ hybridisation with chromosome specific DNA probes on paraffin wax embedded tissue. J Clin Pathol 44:900-904, 1991 Azzopardi JG, Chepick OF, Hartmann WH, Jafarey NA, Bosch AL, Ozzello L, Rilke F, Sasano N, Sobin LH, Sommers SC, Stalsberg H, Sugar J, Willams AO. Histologic typing of breast tumors, 2nd ed. World Health Organization, Geneva, 1981 Baly DL, Wirth CM, Allison DA, Gilkerson E, Hotaling TE, Fox JA. Development and characterization of rhuMAb HER2 antibody assay for clinical evaluation of cytoxic potency. Proc Am Assoc Cancer Res 38:181, 1997 Barbati A, Cosmi EV, Sidoni A, Collini P, Porpora MG, Ferri I, Luthy M, Lauro V, Bucciarelli E. Value of c-erbB-2 and p53 oncoprotein co-overexpression in human breast cancer. Anticancer Res 17:401-405, 1997 Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319:226-230, 1986 Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J. Recombinant humanized antiHER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 58:28252831, 1998 Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, Sklarin NT, Seidman AD, Hudis CA, Moore J, Rosen PP, Twaddell T, Henderson IC, Norton L. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol 14:737-744, 1996 Beahrs OH. Staging of cancer of the breast as a guide to therapy. Cancer 53:592-594, 1984 Bebenek M, Bar JK, Harlozinska A, Sedlaczek P. Prospective studies of p53 and c-erbB-2 expression in relation to clinicopathological parameters of human ductal breast cancer in the second stage of clinical advancement. Anticancer Res 18:619-623, 1998 Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK. Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with Her2/neu. Breast Cancer Res Treat 24: 85-95, 1993 Bergstein I. Molecular alterations in breast cancer. In: Breast Cancer: Molecular Genetics, Pathogenesis and Therapeutics. Bowcock AM (eds). Humana Press Inc., Totowa, NJ,1999 Bloom HJG, Richardson WW. Histological grading and prognosis in breast cancer. Br J Cancer 11:359-377, 1957 Bobrow LG, Millis RR. The pathology of ductal carcinoma in situ of the breast. Clin Oncol (R Coll Radiol) 7:232-235, 1995 Bouras T, Southey MC, Venter DJ. Overexpression of the steroid receptor coactivator AIB1 in breast cancer correlates with the absence of estrogen and progesterone receptors and positivity for p53 and HER2/neu. Cancer Res 61:903-917, 2001 Brenner AJ, Aldaz CM. The genetics of sporadic breast cancer. In: Etiology of Breast and Gynecological Cancers. Aldaz CM, GouldMN, McLauchlan J, Slaga TJ eds. WileyLiss, New York, NY. pp 63-82, 1997 Brown JM, Benson EA, Jones M. Confirmation of a long-term prognostic index in breast cancer. The Breast 2:144-147, 1993 Brown RW, Allred CD, Clark GM, Osborne CK, Hilsenbeck SG. Prognostic value of Ki67 compared to S-phase fraction in axillary node-negative breast cancer. Clin Cancer Res 2:585-592, 1996 Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacol 351:261-272, 1998 Bunone G, Briand PA, Miksicek RJ, Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174-2183, 1996 Burden S, Yarden Y. Neuregulins and their receptors: a versatile signaling module in organogenesis and oncogenesis. Neuron 18:847-855, 1997 Cardiff RD. Cellular and molecular aspects of neoplastic progression in the mammary gland. Eur J Cancer Clin Oncol 24:15-20, 1988 Carlomagno C, Perrone F, Gallo C, De Laurentiis M, Lauria R, Morabito A, Pettinato G, Panico L, D'Antonio A, Bianco AR, De Placido S. c-erb B2 overexpression decreases the benefit of adjuvant tamoxifen in early-stage breast cancer without axillary lymph node metastases. J Clin Oncol 14:2702-2708, 1996 Ceccarelli C, Santini D, Chieco P, Taffurelli M, Marrano D, Mancini,AM. Multiple expression patterns of biopathological markers in primary invasive breast carcinoma: a useful tool for elucidating its biological behaviour. Ann Oncol 6: 275-282, 1995 Celentano E, Montella M, Bonelli P, Cecco L, De Marco M, Di Cintio P, Iannuzzo M, Tuccillo F, Botti G, D'Aiuto G. Does a relationship exist between trends in estrogen receptor levels and breast cancer incidence and mortality? Int J Oncol 13:129-135, 1998 Chariyalertsak S, Chariyalertsak S, Cheirsilpa A, Sugano K, Ohkura H. Immunohistochemical detection of c-erbB-2 oncoprotein in patients with breast cancer. J Med Assoc Thai 79:715-721, 1996 Charpin C, Andrac L, Vacheret H, Habib MC, Devictor B, Lavaut MN, Toga M. Multiparametric evaluation (SAMBA) of growth fraction (monoclonal Ki67) in breast carcinoma tissue sections. Cancer Res 48:4368-4374, 1988 Chia KS, Du WB, Sankaranarayanan R, Sankila R, Seow A, Lee HP. Population-based cancer survival in Singapore, 1968 to 1992: an overview. Int J Cancer 93:142-147, 2001 Chia KS, Lee JJ, Wong JL, Gao W, Lee HP, Shanmugaratnam K. Cancer incidence in Singapore, 1998 to 1999. Ann Acad Med Singapore 31:745-750, 2002 Chia KS, Seow A, Lee HP, Shanmugaratnam K. Cancer incidence in Singapore 19931997. Singapore Cancer Registry Report No.5, 2000 Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney Jr DW, Leahy DJ. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature 421:756-760, 2003 Chrzan P, Skokowski J, Karmolinski A, Pawelczyk T. Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue. Clin Biochem 34:557-562, 2001 Chu KC, Anderson WF, Fritz A, Ries LA, Brawley OW. Frequency distributions of breast cancer characteristics classified by estrogen receptor and progesterone receptor status for eight racial/ethnic groups. Cancer 92:37-45, 2001 Chung YL, Sheu ML, Yang SC, Lin CH, Yen SH. Resistance to tamoxifen-induced apoptosis is associated with direct interaction between Her2/neu and cell membrane estrogen receptor in breast cancer. Int J Cancer 97:306-312, 2002 Clarke GM, McGuire WL. Follow-up study of HER-2/neu amplification in primary breast cancer. Cancer Res 51:944-948, 1991 Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, Francke U, Levinson A, Ullrich A. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230:1132-1139, 1985 Couturier J, Vincent-Salomon A, Nicolas A, Beuzeboc P, Mouret E, Zafrani B, SastreGarau X. Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the erbB2 (HER2/neu) gene status in breast carcinoma. Mod Pathol 13:1238-43, 2000 DAKO HercepTestTM for Immunoenzymatic Staining . Package insert, March 1999. Dalton LW, Page DL, Dupont WD. Histologic grading of breast carcinoma. A reproducibility study. Cancer 73:2765-2770, 1994 Dawkins HJ, Robbins PD, Sarna M, Carrello S, Harvey JM, Sterrett GF. c-erbB-2 amplification and overexpression in breast cancer: evaluation and comparison of Southern blot, slot blot, ELISA and immunohistochemistry. Pathology 25:124-132, 1993 De Potter CR, Schelfhout AM. The neu-protein and breast cancer. Virchows Arch 426:107-115, 1995 Deapen D, Liu L, Perkins C, Bernstein L, Ross RK. Rapidly rising breast cancer incidence rates among Asian-American women. Int J Cancer 99:747-750, 2002 Descotes F, Pavy JJ, Adessi GL. Human breast cancer: correlation study between HER2/neu amplification and prognostic factors in an unselected population. Anticancer Res 13:119-124, 1993 Dhingra K, Sahin A, Supak J, Kim SY, Hortobagyi G, Hittelman WN. Chromosome in situ hybridization on formalin-fixed mammary tissue using non-isotopic, non-fluorescent probes: technical considerations and biological implications. Breast Cancer Res Treat 23:201-210, 1992 Dimmeler S, Zeiher AM. Nitric oxide and apoptosis: another paradigm for the doubleedged role of nitric oxide. Nitric Oxide 1:275-281, 1997 Dixon JM, Anderson TJ, Page DL, Lee D, Duffy SW. Infiltrating lobular carcinoma of the breast. Histopathology 6:149-161, 1982 Dixon JM, Page DL, Anderson TJ, Lee D, Elton RA, Stewart HJ, Forrest AP. Long-term survivors after breast cancer. Br J Surg 72:445-448, 1985 Dowsett M, Cooke T, Ellis I, Gullick WJ, Gusterson B, Mallon E, Walker R. Assessment of HER2 status in breast cancer: why, when and how? Eur J Cancer 36:170-176, 2000 Dowsett M, Harper-Wynne C, Boeddinghaus I, Salter J, Hills M, Dixon M, Ebbs S, Gui G, Sacks N, Smith I. HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer. Cancer Res 61:8452-8458, 2001 du Toit RS, Locker AP, Ellis IO, Elston CW, Nicholson RI, Blamey RW. Invasive lobular carcinomas of the breast--the prognosis of histopathological subtypes. Br J Cancer 60:605-609, 1989 Duenas-Gonzalez A, Isales CM, del Mar Abad-Hernandez M, Gonzalez-Sarmiento R, Sangueza O, Rodriguez-Commes J. Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease. Mod Pathol 10:645-649, 1997 Eisenman RN, Tachibana CY, Abrams HD, Hann SR:v-myc and c-myc encoded proteins are associated with the nuclear matris. Mol Cell Biol 5: 114-126, 1985 Eissa S, Khalifa A, el-Gharib A, Salah N, Mohamed MK. Multivariate analysis of DNA ploidy, p53, c-erbB-2 proteins, EGFR, and steroid hormone receptors for short-term prognosis in breast cancer. Anticancer Res 17:3091-3097, 1997 el-A Helal T, Khalifa A, Kamel AS. Immunohistochemical expression of p53 and c-erbB2 proteins in breast cancer in Egypt. Anticancer Res 20:2145-2150, 2000 Elledge RM, Green S, Ciocca D, Pugh R, Allred DC, Clark GM, Hill J, Ravdin P, O'Sullivan J, Martino S, Osborne CK. HER-2 expression and response to tamoxifen in estrogen receptor-positive breast cancer: a Southwest Oncology Group Study. Clin Cancer Res 4:7-12, 1998 Ellis IO, Dowsett M, Bartlett J, Walker R, Cooke T, Gullick W, Gusterson B, Mallon E, Barrett Lee P. Recommendations for HER2 testing in the UK. J Clin Pathol 53:890-892, 2000 Ellis IO, Elston CW, Poller DN. Ductal carcinoma in situ. In: The breast.3rd edition. Elston CW and Ellis IO eds. Churchill Livingstone, Edinburgh, UK. pp 249-281, 1998 Ellis IO, Galea M, Broughton N, Locker A, Blamey RW, Elston CW. Pathological prognostic factors in breast cancer. II. Histological type. Relationship with survival in a large study with long-term follow-up. Histopathology 20: 479-489, 1992 Elston CW, Ellis IO. Assessment of histological grade. In: The breast. 3rd edition. Elston CW and Ellis IO eds. Churchill Livingstone, Edinburgh, UK. pp 365-384, 1998 Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term followup. Histopathology 19:403-410, 1991 Escot C, Theillet C, Lidereau R, Spyratos F, Champeme MH, Gest J, Callahan R. Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas. Proc Natl Acad Sci U S A 83:4834-4838, 1986 Ferrero-Pous M, Hacene K, Bouchet C, Le Doussal V, Tubiana-Hulin M, Spyratos F. Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis. Clin Cancer Res 2000, 6, 4745-4754. Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, Ruby SG, O'Malley F, Simpson JF, Connolly JL, Hayes DF, Edge SB, Lichter A, Schnitt SJ. Prognostic factors in breast cancer. Review. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 124:966-978, 2000 Franchi A, Gallo O, Paglierani M, Sardi I, Magnelli L, Masini E, Santucci M. Inducible nitric oxide synthase expression in laryngeal neoplasia: correlation with angiogenesis. Head Neck 24:16-23, 2002 Frykberg ER, Bland KI. Overview of the biology and management of ductal carcinoma in situ of the breast. Cancer 74:350-361, 1994 Gancberg D, Lespagnard L, Rouas G, Paesmans M, Piccart M, Di Leo A, Nogaret JM, Hertens D, Verhest A, Larsimont D. Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases. Am J Clin Pathol 113:675-682, 2000 Garau X. Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 (HER-2/neu) gene status in breast carcinoma. Mod Pathol 13:1238-1243, 2000 Gasparini G, Dal Fior S, Pozza F, Bevilacqua P. Correlation of growth fraction by Ki-67 immunohistochemistry with histologic factors and hormone receptors in operable breast carcinoma. Breast Cancer Res Treat 14:329-336, 1989 Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13-20, 1983 Giai M, Roagna R, Ponzone R, De Bortoli M, Dati C, Sismondi P. Prognostic and predictive relevance of c-erbB-2 and ras expression in node positive and negative breast cancer. Anticancer Res 14:1441-1450, 1994 Goelz SE, Hamilton SR, Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 130:118-126, 1985 Gozzetti A, Le Beau MM. Fluorescence in situ hybridization: uses and limitations. Semin Hematol 37: 320-333, 2000 Graus-Porta D, Beerli RR, Daly JM, Hynes NE. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16:1647-1655, 1997 Gullick WJ, Love SB, Wright C, Barnes DM, Gusterson B, Harris AL, Altman DG. cerbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes. Br J Cancer 63:434-448, 1991 Hansen RK, Fuqua SAW. The estrogen receptor and breast cancer. In. Breast Cancer: Molecular genetics, pathogenesis and therapeutics. Bowcock AM eds. Humana press Totawa, New Jersey. pp 1-30, 1999 Harwerth IM, Wels W, Schlegel J, Muller M, Hynes NE. Monoclonal antibodies directed to the erbB-2 receptor inhibit in vivo tumour cell growth. Br J Cancer 68:1140-1145, 1993 HER2 Monograph: Roche. Gardiner-Caldwell, Cheshire, UK, 1999 Hermanek P, Hutter RVP, Scheibe O, Sobin LH, Wagner G, Wittekind Ch. (eds.) International Union Against Cancer. TNM-Atlas: Illustrated guide to the TNN/pTNMclassification of malignant tumors. 4th ed. Berlin Heidelberg: Springer-Verlag, 201-212, 1997 Hoang MP, Sahin AA, Ordonez NG, Sneige N. HER-2/neu gene amplification compared with HER-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol 113: 852-859, 2000 Hoff ER, Tubbs RR, Myles JL, Procop GW. HER2/neu amplification in breast cancer: stratification by tumor type and grade. Am J Clin Pathol 117:916-921, 2002 Hopman AH, Ramaekers FC, Raap AK, Beck JL, Devilee P, van der Ploeg M, Vooijs GP. In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry 89:307-316, 1988 Horiguchi J, Iino Y, Takei H, Yokoe T, Ishida T, Morishita Y. Immunohistochemical study on the expression of c-erbB-2 oncoprotein in breast cancer. Oncology 51:47-51, 1994 Horwitz K, McGuire W. Predicting response to endocrine thereapy in human breast cancer: a hypothesis. Science 189:726-727, 1975 Howe HL, Wingo PA, Thun MJ, Ries LA, Rosenberg HM, Feigal EG, Edwards BK. Annual report to the nation on the status of cancer (1973 through 1998), featuring cancers with recent increasing trends. J Natl Cancer Inst 93:824-842, 2001 Huang J, Bay BH. Tan PH. Nuclear morphometry and glutathione S- transferase π expression in breast cancer. Onco Rep 7:609-613, 2000 Hung MC, Lau YK. Basic science of HER-2/neu: a Review. Sem Oncol 12:51-59, 1999 Hynes NE, Lane HA. Myc and mammary cancer: Myc is a downstream effector of the ErbB2 receptor tyrosine kinase. J Mammary Gland Biol Neoplasia 6:141-150, 2001 Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi OP, Isola JJ. Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 16:93-99, 1994 Ikeguchi M, Sato N, Hirooka Y, Kaibara N. Computerized nuclear morphometry of hepatocellular carcinoma and its relation to proliferative activity. J Surg Oncol 68:225230, 1998 Imamura H, Haga S, Shimizu T, Watanabe O, Kajiwara T, Aiba M. Prognostic significance of MIB1-determined proliferative activities in intraductal components and invasive foci associated with invasive ductal breast carcinoma. Br J Cancer 79:172-178, 1999 Isola JJ, Helin HJ, Helle MJ, Kallioniemi OP. Evaluation of cell proliferation in breast carcinoma. Comparison of Ki-67 immunohistochemical study, DNA flow cytometric analysis, and mitotic count. Cancer 65:1180-1184, 1990 Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement. Am J Clin Pathol 113:251-258, 2000 Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol 17:1983-1987, 1999 Jenkins DC, Charles IG, Thomsen LL, Moss DW, Holmes LS, Baylis SA, Rhodes P, Westmore K, Emson PC, Moncada S. Roles of nitric oxide in tumor growth. Proc Natl Acad Sci U S A 92:4392-4406, 1995 Jeziorski A, Blonski JZ, Niewiadomska H. The expression of products of oncogens cerbB2 and EGFR and proliferating antigens Ki67 and PCNA in primary invasive ductal cancer of female breast. J Exp Clin Cancer Res 19:61-67, 2000 Jimenez RE, Wallis T, Tabasczka P, Visscher DW. Determination of Her-2/Neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 13:37-45, 2000 Jin R, Bay BH, Chow VT, Tan PH, Lin VC. Metallothionein 1E mRNA is highly expressed in oestrogen receptor-negative human invasive ductal breast cancer. Br J Cancer 83:319-323, 2000 Kagoura M, Matsui C, Toyoda M, Morohashi M. Immunohistochemical study of inducible nitric oxide synthase in skin cancers. J Cutan Pathol 28:476-481, 2001 Kakar S, Puangsuvan N, Stevens JM, Serenas R, Mangan G, Sahai S, Mihalov ML. HER2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: comparison of results and correlation with survival. Mol Diagn 5:199-207, 2000 Kim SY, Lee JS, Ro JY, Gay ML, Hong WK, Hittelman WN. Interphase cytogenetics in paraffin sections of lung tumors by non-isotopic in situ hybridization. Mapping genotype/phenotype heterogeneity. Am J Pathol 142:307-317, 1993 Klotz T, Bloch W, Volberg C, Engelmann U, Addicks K. Selective expression of inducible nitric oxide synthase in human prostate carcinoma. Cancer 82:1897-1903, 1998 Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 298:249-258, 1994 Kobayashi M, Ooi A, Oda Y, Nakanishi I. Protein overexpression and gene amplification of c-erbB-2 in breast carcinomas: a comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffin-embedded tissues. Hum Pathol 33:21-28, 2002 Konigshoff M, Wilhelm J, Bohle RM, Pingoud A, Hahn M. HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification, heterozygosity, and immunohistochemical status in breast cancer tissue. Clin Chem 49:219-229, 2003 Konturek SJ, Brzozowski T, Majka J, Pytko-Polonczyk J, Stachura J. Inhibition of nitric oxide synthase delays healing of chronic gastric ulcers. Eur J Pharmacol 239:215-217, 1993 Korkolis D, Ardavanis A, Yotis J, Kyroudi A, Gorgoulis V, Kittas C. HER-2/neu overexpression in breast cancer: an immunohistochemical study including correlations with clinicopathologic parameters, p53 oncoprotein and cathepsin-D. Anticancer Res 21:2207-2212, 2001 Krag D, Weaver D, Ashikaga T, Moffat F, Klimberg VS, Shriver C, Feldman S, Kusminsky R, Gadd M, Kuhn J, Harlow S, Beitsch P. The sentinel node in breast cancer-a multicenter validation study. N Engl J Med 339:941-946, 1998 Kreipe H, Feist H, Fischer L, Felgner J, Heidorn K, Mettler L, Parwaresch R. Amplification of c-myc but not of c-erbB-2 is associated with high proliferative capacity in breast cancer. Cancer Res 53:1956-1961, 1993 Kronqvist P, Kuopio T, Collan Y. Morphometric grading of breast cancer: thresholds for tubular differentiation. Br J Cancer 82:1656-1661, 2000 Kurokawa H, Lenferink AE, Simpson JF, Pisacane PI, Sliwkowski MX, Forbes JT, Arteaga CL. Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res 60:5887-5894, 2000 Lagio MD. Ductal carcinoma in situ: pathology and treatment. Surg Clin North Am 70:853-871, 1990 Lala PK, Orucevic A. Role of nitric oxide in tumor progression: lessons from experimental tumors. Cancer Metastasis Rev 17:91-106, 1998 Lee AK, Wiley B, Loda M, Bosari S, Dugan JM, Hamilton W, Heatley GJ, Cook L, Silverman ML. DNA ploidy, proliferation, and neu-oncogene protein overexpression in breast carcinoma. Mod Pathol 5:61-67, 1992 Lehr HA, Jacobs TW, Yaziji H, Schnitt SJ, Gown AM. Quantitative evaluation of HER2/neu status in breast cancer by fluorescence in situ hybridization and by immunohistochemistry with image analysis. Am J Clin Pathol 115:814-822, 2001 Lemmon MA, Schlessinger J. Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem Sci 19:459-463, 1994 Leung GM, Thach TQ, Lam TH, Hedley AJ, Foo W, Fielding R, Yip PS, Lau EM, Wong CM. Trends in breast cancer incidence in Hong Kong between 1973 and 1999: an ageperiod-cohort analysis. Br J Cancer 87:982-988, 2002 Li CI, Anderson BO, Daling JR, Moe RE. Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA 289:1421-1424, 2003 Li CI, Daling JR, Malone KE. Incidence of invasive breast cancer by hormone receptor status from 1992 to 1998. J Clin Oncol 21:28-34, 2003 Lian SC, Tan PH. CerbB2 status in breast cancer: pathologic issues. Ann Acad Med Singapore 31:793-798, 2002 Lin VC, Jin R, Tan PH, Aw SE, Woon CT, Bay BH. Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA. Am J Pathol 162:1781-1787, 2003 Lindahl T, Engel G, Ahlgren J, Klaar S, Bjohle J, Lindman H, Andersson J, von Schoultz E, Bergh J. Can axillary dissection be avoided by improved molecular biological diagnosis? Acta Oncol 39:319-326, 2000 Lishman SC, Lakhani SR. Atypical lobular hyperplasia and lobular carcinoma in situ: surgical and molecular pathology. Histopathology 35:195-200, 1999 Mariani-Costantini R, Escot C, Theillet C, Gentile A, Merlo G, Lidereau R, Callahan R. In situ c-myc expression and genomic status of the c-myc locus in infiltrating ductal carcinomas of the breast. Cancer Res 48:199-205, 1988 Martin JH, Alalami O, van den Berg HW. Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence. Cancer Lett 144:65-74, 1999 Mass R. The role of HER-2 expression in predicting response to therapy in breast cancer. Semin Oncol 27:46-52, 2000 Matsumura K, Kallioniemi A, Kallioniemi O, Chen L, Smith HS, Pinkel D, Gray J, Waldman FM. Deletion of chromosome 17p loci in breast cancer cells detected by fluorescence in situ hybridization. Cancer Res 52:3474-3477, 1992 Menard S, Valagussa P, Pilotti S, Biganzoli E, Casalini P, Tomasic G, Marubini E, Colnaghi MI, Cascinelli N, Bonadonna G. Benefit of CMF treatment in lymph-node positive breast cancer overexpressing HER2. Proc Am Soc Clin Oncol 18:69, 1999 Midulla C, Pisani T, De Iorio P, Cenci M, Divizia E, Nofroni I, Vecchione A. Cytological analysis and immunocytochemical expression of Ki67 and Bcl-2 in breast proliferative lesions. Anticancer Res 22:1341-1345, 2002 Miller DL, el-Ashry D, Cheville AL, Liu Y, McLeskey SW, Kern FG. Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor (EGFR) under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression. Cell Growth Differ 5:1263-1274, 1994 Mittra I, Redkar AA. Badwe RA. Prognosis of breast cancer: evidence for interaction between c-erbB-2 overexpression and number of involved axillary lymph nodes. J Surg Oncol 60:106-111, 1995 Molina R, Jo J, Filella X, Zanon G, Pahisa J, Munoz M, Farrus B, Latre ML, Gimenez N, Hage M, Estape J, Ballesta AM. C-erbB-2 oncoprotein in the sera and tissue of patients with breast cancer. Utility in prognosis. Anticancer Res 16:2295-2300, 1996 Nguyen MQ, Nguyen CH, Parkin DM. Cancer incidence in Ho Chi Minh City, Viet Nam, 1995-1996. Int J Cancer 76:472-479, 1998 Normanno N, Ciardiello F. EGF-related peptides in the pathophysiology of the mammary gland. J Mammary Gland Biol Neoplasia 2:143-151, 1997 Odagiri E, Kanada N, Jibiki K, Demura R, Aikawa E, Demura H. Reduction of telomeric length and c-erbB-2 gene amplification in human breast cancer, fibroadenoma, and gynecomastia. Relationship to histologic grade and clinical parameters. Cancer 73:29782984, 1994 Oka T, Tsutsumi O, Kurachi and Okamoto S. The role of epidermal growth factor in normal and neoplastic growth of mouse mammary epitheial cells, In:Breast Cancer: Cellular and Molecular Biology, Lippman ME, and Dickson RB eds. Kluwer Press, Boston, MA. pp. 343-362, 1988 Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, Wong J, Allred DC, Clark GM, Schiff R. Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst 95:353-361, 2003 Padhy LC, Shih C, Cowing D, Finkelstein R, Weinberg RA. Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas. Cell 28:865-871, 1982 Page DL, Anderson TJ. Diagnostic Histopathology of the Breast. Churchill Livingstone, London, 1987 Pauletti G, Slamon DJ. Interphase cytogenetics of the HER2/neu oncogene in breast cancer. In: Introduction to fluorescence in situ hybridization: principles and clinical applications. Andreeff M, Pinkel D, eds. New York, Wiley-Liss, pp 153-171, 1999 Pavelic ZP, Pavelic L, Lower EE, Gapany M, Gapany S, Barker EA, Preisler HD. c-myc, c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and noninvasive breast carcinoma. Cancer Res 52:2597-2602, 1992 Pavelic ZP, Steele P, Preisler HD. Evaluation of c-myc proto-oncogene in primary human breast carcinomas. Anticancer Res 11:1421-1428, 1991 Pegram MD, Slamon DJ. Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastatic breast cancer: evidence for receptor-enhanced chemosensitivity. Semin Oncol 26:89-95, 1999 Pereira H, Pinder SE, Sibbering DM, Galea MH, Elston CW, Blamey RW, Robertson JF, Ellis IO. Pathological prognostic factors in breast cancer. IV: Should you be a typer or a grader? A comparative study of two histological prognostic features in operable breast carcinoma. Histopathology 27:219-226, 1995 Perez EA, Roche PC, Jenkins RB, Reynolds CA, Halling KC, Ingle JN, Wold LE. HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clin Proc 77:148-154, 2002 Persons DL, Takai K, Gibney DJ, Katzmann JA, Lieber MM, Jenkins RB. Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma. Hum Pathol 25:678-683, 1994 Pienta KJ, Coffey DS. Correlation of nuclear morphometry with progression of breast cancer. Cancer 68:2012-2016, 1991 Pietras RJ, Arboleda J, Reese DM, Wongvipat N, Pegram MD, Ramos L, Gorman CM, Parker MG, Sliwkowski MX, Slamon DJ. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 10:2435-2446, 1995 Pinder SE, Ellis IO, Galea M, O'Rouke S, Blamey RW, Elston CW. Pathological prognostic factors in breast cancer. III. Vascular invasion: relationship with recurrence and survival in a large study with long-term follow-up. Histopathology 24:41-47, 1994 Pinkas-Kramarski R, Eilam R, Alroy I, Levkowitz G, Lonai P, Yarden Y. Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation. Oncogene 15:2803-2815, 1997 Polak JM, Van Noorden S. Introduction to immunocytochemistry. 2nd edition. BIOS Scientific Publishers, 1997 Poller DN, Galea M, Pearson D, Bell J, Gullick WJ, Elston CW, Blamey RW, Ellis IO. Nuclear and flow cytometric characteristics associated with overexpression of the cerbB-2 oncoprotein in breast carcinoma. Breast Cancer Res Treat 20:3-10, 1991 Popescu NC, King CR, Kraus MH. Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32. Genomics. 4:362-366, 1989 Press MF, Bernstein L, Thomas PA, Meisner LF, Zhou JY, Ma Y, Hung G, Robinson RA, Harris C, El-Naggar A, Slamon DJ, Phillips RN, Ross JS, Wolman SR, Flom KJ. HER2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas. J Clin Oncol 15:2894-2904, 1997 Press MF, Hung G, Godolphin W, Slamon DJ. Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res 54:2771-2777, 1994 Ratcliffe N, Wells W, Wheeler K, Memoli V. The combination of in situ hybridization and immunohistochemical analysis: an evaluation of Her2/neu expression in paraffinembedded breast carcinomas and adjacent normal-appearing breast epithelium. Mod Pathol 10:1247-1252, 1997 Rehman S, Crow J, Revell PA. Bax protein expression in DCIS of the breast in relation to invasive ductal carcinoma and other molecular markers. Pathol Oncol Res 6:256-263, 2000 Reveneau S, Arnould L, Jolimoy G, Hilpert S, Lejeune P, Saint-Giorgio V, Belichard C, Jeannin JF. Nitric oxide synthase in human breast cancer is associated with tumor grade, proliferation rate, and expression of progesterone receptors. Lab Invest 79:1215-1225, 1999 Richer JK, Lange C, Manning G, Owen G, Powell R, Horwitz KB. Convergence of progesterone with growth factor and cytokine signaling in breast cancer. J Biol Chem 273, 31317-31326, 1998 Ridolfi RL, Jamehdor MR, Arber JM. HER-2/neu testing in breast carcinoma: A combined immunohistochemical and fluorescence in situ hybridization approach. Mod Pathol 13:866-873, 2000 Rilke F, Colnaghi MI, Cascinelli N, Andreola S, Baldini MT, Bufalino R, Della Porta G, Menard S, Pierotti MA, Testori A. Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors. Int J Cancer 49:44-49, 1991 Ring AE, Ellis PA. Predictors of response to systemic therapy in breast cancer. Forum (Genova) 12:19-32, 2002 Robbins RA, Barnes PJ, Springall DR, Warren JB, Kwon OJ, Buttery LD, Wilson AJ, Geller DA, Polak JM. Expression of inducible nitric oxide in human lung epithelial cells. Biochem Biophys Res Commun 203:209-2018, 1994 Rose DS, Maddox PH, Brown DC. Which proliferation markers for routine immunohistology? A comparison of five antibodies. J Clin Pathol 47:1010-1014, 1994 Rosen PP, Fracchia AA, Urban JA, Schottenfeld D, Robbins GF. “Residual” mammary carcinoma following simulated partial mastectomy. Cancer 35:739-747, 1975 Ross JS, Fletcher JA. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells 16:413-428, 1998 Rudas M, Neumayer R, Gnant MF, Mittelbock M, Jakesz R, Reiner A. p53 protein expression, cell proliferation and steroid hormone receptors in ductal and lobular in situ carcinomas of the breast. Eur J Cancer 33:39-44, 1997 Ruibal A, Arias JI, Delrio MC, Lapena G, Schnieider J, Tejerina A. Histological grade in breast cancer:association with clinical and biological features in a series of 229 patients. Int J Biol Markers 16:56-61, 2001 Schnitt JS, Connolly JL. Pathology of Benign Breast Disorders. In: Diseases of the Breast. 2nd eds. Harris JR, Lippman ME, Morrow M, Osborne CK eds. Lippincott Willams & Wilkins, Philadelphia, PA, USA, pp 75-93, 2000 Schnitt SJ, Silen W, Saowsky NL, Connolly JL, Harris JR. Ductal carcinoma in situ (intraductal carcinoma) of the breast. N Engl J Med 318:853-871, 1988 Schnitt SJ. Breast cancer in the 21st century: neu opportunities and neu challenges. Mod Pathol 14:213-218, 2001 Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311-322, 2000 Schroeder W, Biesterfeld S, Zillessen S, Rath W. Epidermal growth factor receptorimmunohistochemical detection and clinical significance for treatment of primary breast cancer. Anticancer Res 17:2799-2802, 1997 Scorilas A, Trangas T, Yotis J, Pateras C, Talieri M. Determination of c-myc amplification and overexpression in breast cancer patients: evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathological characteristics using univariate and multivariate analysis. Br J Cancer 81:1385-1391, 1999 Scott GK, Dodson JM, Montgomery PA, Johnson RM, Sarup JC, Wong WL, Ullrich A, Shepard HM, Benz CC. p185HER2 signal transduction in breast cancer cells. J Biol Chem 266:14300-14305, 1991 Seow A, Duffy SW, McGee MA, Lee J, Lee HP. Breast cancer in Singapore: trends in incidence 1968-1992. Int J Epidemiol 25:40-45, 1996 Seshadri R, Firgaira FA, Horsfall DJ, McCaul K, Setlur V, Kitchen P. Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group. J Clin Oncol 11:1936-1942, 1993 Sharkey FE, Allred DC, Valente PT. Breast. In: Anderson’s Pathology. Damjanov I, Linder J, Mosby eds. pp 2354-2385, 1996 Shih C, Padhy LC, Murray M, Weinberg RA. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature 290:261-264. 1981 Shoker BS, Sloane JP. DCIS grading schemes and clinical implications. Histopathology 35:393-400, 1999 Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark G, Edge SB, Hayes DF, Hughes LL, Hutter RV, Morrow M, Page DL, Recht A, Theriault RL, Thor A, Weaver DL, Wieand HS, Greene FL. Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol 20:3628-3636, 2002 Sjogren S, Inganas M, Lindgren A, Holmberg L, Bergh J. Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers. J Clin Oncol 16:462-469, 1998 Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, Press MF. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989 Sloane JP, Anderson TJ, Blamey RW. Pathology reporting in breast cancer screening. 2nd Ed. National coordinating group for breast screening pathology NHSBSP publication no 3, 19-21, 1995 Smith KL, Robbins PD, Dawkins HJ, Papadimitriou JM, Redmond SL, Carrello S, Harvey JM, Sterrett GF. c-erbB-2 amplification in breast cancer: detection in formalinfixed, paraffin-embedded tissue by in situ hybridization. Hum Pathol 25:413-418, 1994 Soomro S, Shousha S, Taylor P, Shepard HM, Feldmann M. c-erbB-2 expression in different histological types of invasive breast carcinoma. J Clin Pathol 44:211-214, 1991 Spandidos DA, Yiagnisis M, Papadimitriou K, Field JK. ras, c-myc and c-erbB-2 oncoproteins in human breast cancer. Anticancer Res 9:1385-1393, 1989 Spiessl B, Beahrs OH, Hermanek P, Hutter RVP, Scheibe O, Sobin LH, Wagner G. (eds.) International Union Against Cancer. TNM-Atlas: Illustrated guide to the TNN/pTNMclassification of malignant tumors. 3rd ed. Berlin Heidelberg: Springer-Verlag, 174-183, 1989 Steiner MG, Harlow SP, Colombo E, Bauer KD. Chromosomes 8, 12, and 17 copy number in Astler-Coller stage C colon cancer in relation to proliferative activity and DNA ploidy. Cancer Res 53:681-686, 1993 Stewart TA, Pattengale PK, Leder P. Spontaneous mammary adenocarcinomas in transgenic mic that carry and express MTV/myc fusion genes. Cell 38:627-637, 1984 Suo Z, Yang H, Mei Q, Skovlund E, Cui J, Nesland JM. Type 1 protein tyrosine kinases in Chinese breast carcinomas: a clinicopathologic study. Int J Surg Pathol 9:177-187, 2001 Tan M, Yao J, Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities. Cancer Res 57:1199-1205, 1997 Tan PH, Chiang GSC, Ng EH, Low SC, Ng FC. Screen detected breast cancer in an Asian population:pathological findings of the Singapore breast screening project. The Breast 8:120-125, 1999 Tan PH, Goh BB, Chiang G, Bay BH. Correlation of nuclear morphometry with pathologic parameters in ductal carcinoma in situ of the breast. Mod Pathol 14:937-941, 2001 The Research Group for Population-based Cancer Registration in Japan. Cancer incidence and incidence rates in Japan in 1997: estimates based on data from 12 population-based cancer registries. Jpn J Clin Oncol 32:318-322, 2002 Thompson CT, LeBoit PE, Nederlof PM, Gray JW. Thick-section fluorescence in situ hybridization on formalin-fixed, paraffin-embedded archival tissue provides a histogenetic profile. Am J Pathol 144: 237-243, 1994 Thor AD, Liu S, Moore DH 2nd, Edgerton SM. Comparison of mitotic index, in vitro bromodeoxyuridine labeling, and MIB-1 assays to quantitate proliferation in breast cancer. J Clin Oncol 17:470-477, 1999 Tsai KB, Hou MF, Lin HJ, Chai CY, Liu CS, Huang TJ. Expression of HER-2/NEU oncoprotein in familial and non-familial breast cancer. Kaohsiung J Med Sci 17:64-76, 2001 Tschugguel W, Schneeberger C, Unfried G, Czerwenka K, Weninger W, Mildner M, Gruber DM, Sator MO, Waldhor T, Huber JC. Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade. Breast Cancer Res Treat 56:145-151, 1999 Tsuda H, Hirohashi S, Shimosato Y, Hirota T, Tsugane S, Watanabe S, Terada M, Yamamoto H. Correlation between histologic grade of malignancy and copy number of cerbB-2 gene in breast carcinoma. A retrospective analysis of 176 cases. Cancer 65:17941800, 1990 Tsuda H, Iwaya K, Fukutomi T, Hirohashi S. p53 mutations and c-erbB-2 amplification in intraductal and invasive breast carcinomas of high histologic grade. Jpn J Cancer Res 84:394-401, 1993 Tsuda H, Tsugane S, Fukutomi T, Nanasawa T, Yamamoto H, Hirohashi S. Prognostic factors for recurrent breast cancer: univariate and multivariate analyses including histologic grade and amplification of the c-erbB-2 proto-oncogene. Jpn J Clin Oncol 22:244-249, 1992 Tsutsui S, Ohno S, Murakam S, Hachitanda Y, Oda S. Prognostic value of p53 protein expression in breast cancer; an immunohistochemical analysis of frozen sections in 514 Japanese women. Breast Cancer 8:194-201, 2001 Tsutsui S, Ohno S, Murakami S, Hachitanda Y, Oda S. Prognostic value of c-erbB2 expression in breast cancer. J Surg Oncol 79:216-223, 2002 Tsutsui S, Ohno S, Murakami S, Kataoka A, Kinoshita J, Hachitanda Y. Prognostic significance of the coexpression of p53 protein and c-erbB2 in breast cancer. Am J Surg 185:165-167, 2003 Tzahar E, Yarden Y.The ErbB-2/HER2 oncogenic receptor of adenocarcinomas: from orphanhood to multiple stromal ligands. Biochim Biophys Acta 1377:M25-M37, 1998 Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res 6:2408-2416, 2000 Val JF, Losada J, Murua MA, Sarria R. Cell proliferation, nuclear ploidy, and EGFr and HER2/neu tyrosine kinase oncoproteins in infiltrating ductal breast carcinoma. Cancer Genet Cytogenet 138:69-72, 2002 van de Vijver MJ, Peterse JL, Mooi WJ, Wisman P, Lomans J, Dalesio O, Nusse R. Neuprotein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 319:1239-1245, 1988 van der Geer P, Hunter T, Lindberg RA. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10:251-337, 1994 Vang R, Cooley LD, Harrison WR, Reese T, Abrams J. Immunohistochemical determination of HER-2/neu expression in invasive breast carcinoma. Am J Clin Pathol 113:669-674, 2000 Varley JM, Swallow JE, Brammar WJ, Whittaker JL, Walker RA. Alterations to either cerbB2(neu) or c-myc proto-oncogenes in breast carcinomas correlate with poor short term prognosis. Oncogene 1: 423-430, 1987 Vlastos G, Rubio IT, Mirza NQ, Newman LA, Aurora R, Alderfer J, Buzdar AU, Singletary SE. Impact of multicentricity on clinical outcome in patients with T1-2, N0-1, M0 breast cancer. Ann Surg Oncol 7:581-587, 2000 Walker RA, Camplejohn RS. Comparison of monoclonal antibody Ki-67 reactivity with grade and DNA flow cytometry of breast carcinomas. Br J Cancer 57:281-283, 1988 Walker RA, Senior PV, Jones JL, Critchley DR, Varley JM. An immunohistochemical and in situ hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinomas. J Pathol 158:97-105, 1989 Wang S, Saboorian MH, Frenkel E, Hynan L, Gokaslan ST, Ashfaq R. Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays. J Clin Pathol 53:374-81, 2000 Whitten TM, Fraser HR, Christensen WN, Turk PS. Axillary lymph node metastasis in stage T1a breast cancer: a pathologic review of 82 patients. Am Surg 63:144-149, 1997 Wingo PA, Ries LA, Rosenberg HM, Miller DS, Edwards BK. Cancer incidence and mortality, 1973-1995: a report card for the U.S. Cancer 82:1197-1207, 1998 Witters LM, Kumar R, Chinchilli VM, Lipton A. Enhanced anti-proliferative activity of the combination of tamoxifen plus HER-2-neu antibody. Breast Cancer Res Treat 42:15, 1997 Xie K, Huang S, Dong Z, Gutman M, Fidler IJ. Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362.Cancer Res 55:3123-3131, 1995 Xie K, Huang S, Dong Z, Juang SH, Gutman M, Xie QW, Nathan C, Fidler IJ Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J Exp Med 181:1333-1343, 1995 Yarbro JW, Page DL, Fielding LP, Partridge EE, Murphy GP. American Joint Committee on Cancer prognostic factors consensus conference. Cancer 86:2436-2446, 1999 Yip CH, Ng EH. Breast cancer-a comparative study between Malaysian and Singaporean women. Singapore Med J 37:264-267, 1996 Yokota T, Imamura M, Teshima S, Suzuki H, Tezuka F, Kikuchi S, Kunii Y, Yamauchi H. c-erbB-2, p53 protein expression and steroid hormone receptors in breast carcinomas: an immunohistochemical study. Anticancer Res 19:4007-4012, 1999 Yu D, Hung MC. Overexpression of ErbB2 in cancer and ErbB2-targeting strategies. Oncogene 19:6115-6121, 2000 Zhou DJ, Ahuja H, Cline MJ. Proto-oncogene abnormalities in human breast cancer: cERBB-2 amplification does not correlate with recurrence of disease. Oncogene 4:105108, 1989 Zitzelsberger H, Szucs S, Weier HU, Lehmann L, Braselmann H, Enders S, Schilling A, Breul J, Hofler H, Bauchinger M. Numerical abnormalities of chromosome 7 in human prostate cancer detected by fluorescence in situ hybridization (FISH) on paraffinembedded tissue sections with centromere-specific DNA probes. J Pathol 172:325-335, 1994 APPENDIX RECIPES Citrate buffer – Antigen retrieval (pH 6.0) 0.01M sodium citrate adjusted to pH 6 by 0.01M citric acid DAB reaction solution DAB TBS H2O2 50mg 100ml 33µl DAPI solution DAPI 0.1µg/ml in 2x SSC Harris Hematoxylin Hematoxylin Absolute alcohol Ammonium alum (or potassium alum) Mercuric oxide dH2O 1g 10ml 20g 0.5g 200ml Hydrogen peroxide/Methanol 30% Hydrogen peroxide Methanol 100µl 5.9ml Protease solution (0.25mg/ml) Protease Protease buffer 25mg 50ml Silane coated slides 2% solution of silane in absolute alcohol for 2 min Tris Buffered Saline (TBS) 0.5M Tris stock solution, pH 7.4 9% Sodium chloride Tween 20 Water up to 100ml 100ml 0.5ml 1 litre 0.5% Tween 20 Tween 20 Wash buffer (2x SSC) 0.5ml 100ml 10% Buffered formalin (neutral) 37-40% formaldehyde solution Water Sodium phosphate monobasic Sodium phosphate dibasic 5 litres 45 litres 200g 325g 0.2N HCl Concentrated Hcl (37%) DEPC water 0.41ml 25ml 20x SSC (pH 7.4) Tri sodium citrate Sodium chloride Water 88.2g 175.4g 1 litre 2x SSC 20x SSC Distilled water 100ml 900ml [...]... 9 Intensity of c- erbB2 immunostaining in all patients 52 Fig 10 C- erbB2 immunostaining of invasive ductal breast cancer tissues Negative control 53 C- erbB2 immunostaining of invasive ductal breast carcinoma 1+ staining (considered negative) 54 C- erbB2 immunostaining of invasive ductal breast carcinoma 2+ positive staining 54 C- erbB2 immunostaining of invasive ductal breast carcinoma 3+ positive staining... positivity of the cancer cells of invasive breast carcinoma 77 Negative control showed no iNOS immunostaining of the cancer cells of invasive breast carcinoma 77 Positive c- myc immunostaining showed nuclear as well as cytoplasmic positivity of the cancer cells of invasive breast carcinoma 79 Negative control showed no c- myc immunostaining of the cancer cells of invasive breast carcinoma 79 Fig 22 Fig 23 Fig... origin Non -invasive Ductal carcinoma in situ Microinvasive carcinoma Lobular carcinoma in situ Invasive Ductal no special type (NST) Lobular Medullary Tubular Invasive cribriform Mucinous Metaplastic Mixed types Uncommon types Secretory Adenoid cystic Mucoepidermoid Invasive papillary Tubulolobular Inflammatory Rare types Signet ring Lipid rich Clear cell Myoepithelioma Carcinoid Mesenchymal origin... negative invasive ductal carcinoma digitally outlined using a computer mouse 69 Positive Ki67 immunostaining showed nuclear positivity of the proliferating cancer cells of invasive breast carcinoma 75 Negative control (omission of primary antibody) showed no Ki67 immunostaining ofthe cancer cells of invasive breast carcinoma 75 iNOS immunostaining showed strong cytoplasmic positivity of the cancer cells... Sarcomas Miscellaneous origin Hematopoietic Metastatic carcinoma 1.2.2.1 Ductal carcinoma in situ (DCIS) Ductal carcinoma in situ (DCIS) originates from the terminal duct-lobular unit (TDLU), and implies malignant transformation of lining epithelial cells restricted within the basement membrane Myoepithelial cells are seen in DCIS, which is a distinct feature that differentiates it from invasive carcinoma. .. apocrine changes, calcifications 1.2.2 Malignant breast disease (Histologic subtypes) Breast carcinoma presents in a great variety of histological patterns, including specific types which have useful clinical correlates and prognostic implications Morphological classification of invasive breast carcinoma has existed for several decades The classification system currently followed is based on a descriptive... c- erbB2 in invasive breast cancer is associated with poor overall survival Strong correlation is found in c- erbB2 expression at the genetic and protein expression level CHAPTER 1 INTRODUCTION 1.1 Epidemiology of breast cancer 1.1.1 Breast cancer around the world Breast cancer is the second leading cause of cancer deaths in women today (after lung cancer) and is the most common cancer among women, excluding...FISH Fluorescence in situ hybridization GAP GTPase activating protein GFR Growth factor receptor H&E Hematoxylin and Eosin H2O2 Hydrogen peroxide HCl Hydrochloric acid HER2 Human epidermal growth receptor 2 hr/hrs Hour/hours IDC Invasive ductal carcinoma IGF-1 Insulin like growth factor IgG Immunoglobulin G IHC Immunohistochemistry ILC Invasive lobular carcinoma iNOS Inducible nitric oxide synthase... affecting the lobular units (Lishman and Lakhani, 1999) It is now regarded as risk indicator, rather than as a true forerunner of invasive breast cancer Low nuclear grade solid DCIS may mimic LCIS, and pose diagnostic difficulty The relationship between DCIS, LCIS and invasive breast cancer needs further elucidation 1.2.2.3 Invasive ductal carcinoma (IDC) Invasive ductal carcinoma (IDC) is the most common... invasive breast carcinoma The 7th World Congress on Advances in Oncology and 5th International Symposium on Molecular Medicine, Hersonissos, Crete, Greece 2002 (Abstract) 3 Selvarajan S, Bay BH, Tan PH Nuclear morphometry in c- erbB2 positive invasive ductal breast carcinoma The 7th World Congress on Advances in Oncology and 5th International Symposium on Molecular Medicine, Hersonissos, Crete, Greece 2002 ... immunostaining of invasive ductal breast carcinoma 2+ positive staining 54 C- erbB2 immunostaining of invasive ductal breast carcinoma 3+ positive staining 55 Ductal Carcinoma in situ component of invasive. .. cell Myoepithelioma Carcinoid Mesenchymal origin Sarcomas Miscellaneous origin Hematopoietic Metastatic carcinoma 1.2.2.1 Ductal carcinoma in situ (DCIS) Ductal carcinoma in situ (DCIS) originates... cytoplasmic positivity of the cancer cells of invasive breast carcinoma 77 Negative control showed no iNOS immunostaining of the cancer cells of invasive breast carcinoma 77 Positive c- myc immunostaining