Breast Cancer Subtypes and Screening Mammography Sensitivity

Transcription

1 Breast Cancer Subtypes and Screening Mammography Sensitivity Mémoire Sue-Ling Chang Maîtrise en Épidémiologie Maître ès sciences (M.Sc.) Québec, Canada Sue-Ling Chang, 2014

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3 Résumé Les cancers du sein peuvent être classifiés selon le statut de récepteur d estrogène (RE), de récepteur de progestérone (RP), de récepteur HER2, ou selon quatre sous-types (Luminal A, Luminal B, HER2-enrichi, Triple-négatif) ayant des propriétés biologiques et cliniques différentes. La sensibilité du dépistage par mammographie pourrait varier selon ces types de cancers mais ceci n est pas encore clair. L agressivité de la tumeur, mesurée par le grade histologique pourrait expliquer cette association. Les types de cancers d intervalle ont été comparés à ceux de cancers détectés par dépistage parmi 1536 cas infiltrants provenant d un centre de référence de Québec. Les tumeurs RE-négatif, RP-négatif, HER2-positif, Luminal B, HER2- enrichi et TPN étaient tous plus fréquentes chez les femmes avec cancers d intervalle que chez celles avec cancers détectés par dépistage. À l exception des tumeurs HER2-positif et HER2-enrichi, le grade histologique expliquait en grande partie la variabilité observée entre les types de cancer et la sensibilité. iii

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5 Abstract Breast cancers can be classified according to tumour estrogen (ER) and progesterone (PR) receptors, human epidermal growth factor receptor 2 (HER2), and according to four subtypes (Luminal A, Luminal B, HER2- enriched, Triple-negative), each with different biological and clinical profiles. These tumour types may also influence screening mammography sensitivity but this is still not clear. Tumour aggressiveness, measured by the histological grade, may also play a role in explaining this association. Interval cancer types were compared to screen-detected cancer types in 1536 invasive cases obtained from a reference center in Quebec. ERnegative, PR-negative and HER2-positive, Luminal B, HER2-enriched and TPN tumours were all more frequent in women with interval cancers than in women with screen-detected cancers. Except for HER2- positive and HER2-enriched tumours, histological grade explained most of the variability observed between tumour receptor status, subtypes and sensitivity. v

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7 Table of Contents Résumé... iii Abstract... v Table of Contents... vii List of Tables... ix List of Figures... xi List of Abbreviations... xiii Acknowledgments... xv Foreword... xvii Introduction... 1 Literature Review BREAST CANCER INTRINSIC SUBTYPES Description of intrinsic breast cancer subtypes Classification of breast cancer subtypes by immunohistochemistry Section summary BREAST CANCER TYPES AND AGGRESSIVENESS Markers of aggressiveness Breast cancer types and aggressiveness Section summary BREAST CANCER TYPES AND RADIOLOGIC FEATURES Definition and description of radiologic features Breast cancer types and radiologic features Section summary SCREENING SENSITIVITY Definition Characteristics of women Aggressiveness and sensitivity Radiologic features at screening and sensitivity Breast cancer types and sensitivity Section summary Objectives and Conceptual Framework OBJECTIVES CONCEPTUAL FRAMEWORK Breast Cancer Subtypes and Screening Mammography Sensitivity RÉSUMÉ ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION vii

8 4.7 REFERENCES... ERREUR! SIGNET NON DEFINI. Conclusion References Appendix APPENDIX A. COMPLETE-CASE RESULTS OF TUMOUR TYPE AND HISTOLOGICAL GRADE OF BREAST CANCERS (N=806) APPENDIX B. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN-DETECTED CANCERS (N=858) APPENDIX C. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE CLINICAL BREAST CANCERS RELATIVE TO SCREENED BREAST CANCERS (N=1536) APPENDIX D. TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN-DETECTED BREAST CANCERS, ADJUSTED FOR VASCULAR INVASION (N=858) APPENDIX E. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN-DETECTED BREAST CANCERS, ADJUSTED FOR VASCULAR INVASION (N=858) viii

9 List of Tables TABLE 1. BREAST CANCER SUBTYPES DETERMINED BY IHC ASSESSMENT OF ER, PR, AND HER2 STATUS TABLE 2. FEATURES AND RELATED SCORES TO EVALUATE HISTOLOGICAL GRADE ACCORDING TO THE ELSTON-ELLIS GRADING METHOD. 11 TABLE 3. SUMMARY AND DESCRIPTION OF GRADE 1, 2, AND TABLE 4. CHARACTERISTICS OF STUDIES REPORTING ADJUSTED RESULTS FOR THE RELATIONSHIP BETWEEN SUBTYPES AND HISTOLOGICAL GRADE TABLE 5. COMPARISON OF STUDIES EXAMINING BREAST CANCER SUBTYPES AND SENSITIVITY Article tables TABLE 1. CHARACTERISTICS OF STUDY POPULATION (N=1536) TABLE 2. TUMOUR TYPE AND HISTOLOGICAL GRADE OF BREAST CANCERS (N=806) TABLE 3. TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN-DETECTED BREAST CANCERS (N=858). 62 TABLE 4. TUMOUR TYPE OF INVASIVE CLINICAL BREAST CANCERS RELATIVE TO SCREENED BREAST CANCERS (N=1536) Appendix tables ADDITIONAL TABLE 1. COMPLETE-CASE RESULTS OF TUMOUR TYPE AND HISTOLOGICAL GRADE OF BREAST CANCERS (N=806) ADDITIONAL TABLE 2. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN- DETECTED CANCERS (N=858) ADDITIONAL TABLE 3. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE CLINICAL BREAST CANCERS RELATIVE TO SCREENED BREAST CANCERS (N=1536) ADDITIONAL TABLE 4. TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN-DETECTED BREAST CANCERS, ADJUSTED FOR VASCULAR INVASION (N=858) ADDITIONAL TABLE 5. COMPLETE-CASE RESULTS OF TUMOUR TYPE OF INVASIVE INTERVAL BREAST CANCERS RELATIVE TO SCREEN- DETECTED BREAST CANCERS, ADJUSTED FOR VASCULAR INVASION (N=858) ix

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11 List of Figures FIGURE 1. BREAST ANATOMY AND HISTOLOGY FIGURE 2. DENDROGRAM FROM HIERARCHICAL CLUSTERING OF 115 TUMOUR SAMPLES FIGURE 3. TYPICAL HISTOLOGICAL VIEWS OF TUMOURS BY GRADE SCORES RELATIVE TO NORMAL BREAST TISSUE FIGURE 4. RADIOLOGIC ABNORMALITIES OF THE BREAST FIGURE 5. DRAWN REPRESENTATION OF MASS SHAPES AND MARGINS, AND EXAMPLES OF MICROCALCIFICATION MORPHOLOGIES. 18 FIGURE 6. REPRESENTATION OF THE NATURAL HISTORY OF BREAST CANCER: THE NON-DETECTABLE PHASE, THE PRECLINICAL DETECTABLE PHASE AND THE CLINICAL PHASE FIGURE 7. MODIFIED 2X2 TABLE FEATURING RESULTS OF SCREENING MAMMOGRAPHY, ASSESSMENT, AND BREAST CANCER DIAGNOSIS. 23 FIGURE 8. REPRESENTATION OF CASE-CONTROL AND STUDY DESIGN FIGURE SENSITIVITY ODDS RATIOS CALCULATED IN POPULATION AND STUDY SAMPLE FIGURE 10. REPRESENTATION OF TUMOUR DETECTION BY SCREENING ACCORDING TO LENGTH OF PRECLINICAL PHASE FIGURE 11. CONCEPTUAL FRAMEWORK OF STUDY Article figure FIGURE 1. CAUSAL DIAGRAM ILLUSTRATING THE POSSIBLE PATHWAYS BY WHICH BREAST CANCER TYPES MAY INFLUENCE SCREENING SENSITIVITY xi

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13 List of Abbreviations ADJ Adjustment BMI Body Mass Index CCNB1 Cyclin B1 CEP17 Chromosome 17 centromere CI Confidence interval CK Cytokeratins CK2 Cytokeratin 2 CK5/6 Cytokeratins 5 and 6 CMSDF Centre des Maladies du Sein Deschênes-Fabia DCIS Ductal Carcinoma in Situ ER Estrogen Receptor ERBB2 V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog FISH Fluorescent in situ hybridization FOXA1 Forkhead Box Protein 1 GATA3 GATA-Binding Protein 3 GRB7 Growth Factor Receptor-Bound Protein 7 HER1 Epidermal Growth Factor Receptor HER2 Human Epidermal Growth Factor Receptor 2 HRT Hormone Replacement Therapy IC Interval Cancer IDC Invasive Ductal Carcinoma IHC Immunohistochemistry ILC Invasive Lobular Carcinoma INSPQ Institut national de santé publique du Québec KRT5 Cytokeratin 5 KRT6 Cytokeratin 6 KRT17 Cytokeratin 17 LUM Luminal MKI67 Ki-67 MYBL2 Myb-Related Protein OR Odds Ratio PQDCS Programme québécois de dépistage du cancer du sein PR Progesterone Receptor RC Rapport de cotes RE Récepteur d estrogène RP Récepteur de progestérone SD Screen-Detected Cancer Se Sensitivity SPF S-Phase Fraction TPN Triple-negative WHO World Health Organization XBP1 X-Box Binding Protein xiii

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15 Acknowledgments I would like to thank foremost my thesis director Dr. Jacques Brisson for graciously accepting me as his student, and offering me an interesting and exciting project. Not having any prior experience in epidemiology, he gave me the opportunity to obtain real-life epidemiology experience by working alongside epidemiologists and breast cancer screening experts, and to participate in two publications. His rigorous approach to research and thinking about epidemiological problems motivated me to improve my own work, and I am certain that all the skills I have gained as his student will help me in my future endeavours. I would be remiss if I didn t thank Dr. Caroline Diorio for introducing me to the field of molecular epidemiology and for helping me further my knowledge in molecular biology by inviting me to interact with her team. Her knowledge of these fields helped me further understand certain concepts of this present work. I will always be grateful for her encouragement, support, and words of wisdom that she gave me during my studies. Her approachable and welcoming disposition helped me become more at ease with the research environment. I would like to thank Caty Blanchette for her patience and support during my earlier ordeals with SAS and for assembling the database used during this research. In addition, I thank Sylvie Bérubé for helping me understand the CMSDF database. I also would like to extend my gratitude to the members of the PQDCS Evaluation team and other colleagues at the INSPQ notably Isabelle Théberge, Marie-Hélène Guertin, Éric Pelletier, André Langlois and Roxanne Gagnon. Thank you for helping me in various ways throughout this work either by helping me understand epidemiological concepts, working through problems, resolving SAS bugs or simply by extending your friendship. I am also grateful to Daniela Furrer-Soliz for helping me understand immunohistochemistry and some aspects of HER2. On a personal note, I thank my mother Elena, my step-father Serge, and my husband Kayne, for their patience and support throughout this endeavour. xv

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17 Foreword This project culminated in the drafting of an article entitled Breast cancer intrinsic subtypes and screening mammography sensitivity which will be submitted for publication. In this study, I participated in the discussion leading to the formulation of the study objectives and conducted the literature review. I was involved in assembling the data with the support of Sylvie Bérubé who, along with Julie Lemieux, contributed valuable knowledge of the Centre des Maladies du Sein Deschênes-Fabia (CMSDF) database. I was responsible for matching the data from the CMSDF to the database of the Programme Québécois de Dépistage du Cancer du Sein (PQDCS), preparing the data for analyses, conducting the analyses, drafting and editing the article. Éric Pelletier helped me navigate the PQDCS database and also contributed his knowledge of the screening program to the article. François Sanschagrin was responsible for the pathology and laboratory information. Dr. Jacques Brisson supervised my work throughout the entire project. During my training, I also had the chance to participate in the writing of two other articles that have now been published. I was also closely involved with the data collection of an on-going project of the effect of mammography quality on the sensitivity of mammography screening. These complementary experiences enriched my research training considerably although it does not appear in this document. 1. Fontenoy AM, Langlois A, Chang SL, Daigle JM, Pelletier É, Guertin M, Théberge I, Brisson J. Contribution and performance of mobile units in an organized mammography screening program. Can J Public Health. 2013; 104(3): Théberge I, Chang SL, Vandal, N, Daigle JM, Guertin M, Pelletier É, Brisson J. Radiologist interpretive volume and breast cancer screening accuracy in a Canadian organized screening program. J Natl Cancer Inst. 2014; 106 (3): 1-9. xvii

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19 Introduction Sensitivity is the property that enables screening mammography to detect breast cancer early in its preclinical phase which provides an opportunity to advance treatment and thus, reduce mortality. Sensitivity is therefore an important determinant of the disease control value of a screening program. 1 Molecular studies have shown that breast cancers can be classified into four unique subtypes by virtue of their distinctive gene-expression profiles (Luminal A, Luminal B, HER2-enriched, and Basal-like) 2-5. Luminal A and Luminal B subtypes have gene expression patterns that resemble the gene expression profiles of luminal epithelial breast cells, and are characterized by the expression of the estrogen receptor (ER), progesterone receptor (PR) and genes associated with ER activation. 6,7 Luminal B tumours are different from Luminal A in that they also express the human epidermal growth factor receptor 2 (HER2) and HER2-associated genes. 3,8-10 In contrast to the luminal subtypes, HER2-enriched tumours lack expression of ER, PR and ER-associated genes. However, like Luminal B, HER2- enriched tumours overexpress HER2 and HER2-associated genes. Lastly, the Basal-like subtype is characterized by the lack of expression of ER, PR, ER-associated genes and HER2 but expresses genes encoding for epithelial cytokeratins (CK) 5, 6, 14, and The four breast cancer subtypes have clinical relevancy as they have been shown to have different treatment responses and survival. 5,7,8,11 For instance, endocrine therapy such as Tamoxifen is used for Luminal A, while biological therapy like Transtuzumab, is used for HER2-enriched tumours, and a combination of both may be used for Luminal B. 5 There are no targeted treatments for Basal-like tumours to date. Since breast cancers represent different types of tumours with unique biological and clinical characteristics, these differences may also influence the way they appear on a mammogram. For instance, tumour appearance may affect sensitivity if a tumour type has a more conspicuous appearance at screening which would make it more detectable than another that blends in with mammary tissue. The literature suggests that breast tumour types (ER, PR, HER2 and subtype) may differ in radiologic appearance. ER-positive tumours generally tend to occur more frequently as spiculated masses than ER-negative tumours with only one study not reporting significant findings. 22 PR and HER2 were not generally associated with masses. A few studies observed that PR-positive tumours 12,20 were more frequent in spiculated masses but most findings were not significant. 14,15,17,18,22 HER2-positivivity was not associated with masses. 12,13,18,22-26 ER-negative tumours were also more frequent in microcalcifications than ER-positive tumours 14,15,19,20,27 but not consistently ,24,26,28 PR-negative tumours also showed frequently microcalcifications 20,27 but generally findings were not statistically significant. 15,17,18,24,28 Conversely, HER2-positive tumours were more likely than HER2-negative to appear as microcalcifications 22,23,25,26,28-32 with few disagreeing studies. 18,24 Subtype studies were not consistent. 13,31,33-37 Basal-likes were more frequent in ill-defined masses than non-basal-like subtypes 33,34 and this was similarly reported for Triple-negatives when compared to HER-positive and ER-positive tumours. 13 However, others reported 33 that non-basal tumours (grouped into one group) were more frequent in spiculated 1

20 masses than Basal-like tumours, another used a non-conventional subtype classification 35, and still another did not provide statistics for all comparisons. 36 Two studies comparing all subtypes reported that Triple-negative tumours were more frequently masses than Luminal A but no margin descriptors were used. 31,37 These studies suggested that HER2-enriched 31,37 and Luminal B 37 frequently appeared as microcalcifications when compared to other subtypes. An association with Luminal A and architectural distortion is reported which has not been described elsewhere. 37 Screening sensitivity may also vary according to tumour aggressiveness. Aggressive cancers progress rapidly through the preclinical phase, therefore, narrowing the window of opportunity to detect them, before they become symptomatic. Conversely, non-aggressive cancers progress slowly (low grade (I)) through the preclinical phase and so, there is greater opportunity to detect them. Tumour receptor status and grade have been extensively studied in the past and generally suggest that ER-positive and PR-positive tumours are more frequently grade I than grade 3 tumours. These findings concur with more recent studies of this subject The association of HER2 with grade has been less studied and is less consistent than ER and PR although some suggest HER2-positive tumours are more frequently high grade. 38,40-42 Molecular studies revealed that the four subtypes varied in the expression of aggressive genes such as MKI67, CCNB1, and MYBL2 which are known to be associated with cellular proliferation. 3,10 Studies examining the association between subtype and grade reporting both crude and adjusted results were consistent 7,21,38,43-53 with few disagreeing studies and suggested that compared to Luminal A, Basal-like and HER2- enriched subtypes were strongly associated with high-grade. An association between Luminal B and higher grade was also reported in one study. 45 Since breast cancer types vary in radiologic appearance at screening and tumour aggressiveness, it is possible they may also vary in screening sensitivity. To date, studies of tumour types and sensitivity compared the proportion of subtypes in interval cancers to that in screen-detected cancers. In general, ER-negative tumours are more frequent in interval cancers than in screen-detected cancers with few studies not reporting similar findings The evidence for an association of PR status with sensitivity is less consistent than for ER but, in general; studies do not suggest a strong association of PR with sensitivity. While some studies 59,61,62,66 suggest that PR-negative tumours are more frequent in interval cancers than in screen-detected cancers, many do not report significant findings. 55,56,58,60,63,64,67-69 Like studies of PR status and sensitivity, studies of HER2 do not suggest an association with sensitivity. Studies of HER2 and sensitivity are inconsistent with a few reporting an association between HER2-positivity and decreased sensitivity 55,63,70 but many more not reporting any significant findings. 40,58-61,64,68 There are few studies of tumour subtypes and sensitivity. 60,61,68,71 Most suggest that Triple-negative/Basal-like subtypes are found more frequently in interval cancers than in screen-detected cancers but, in these studies, all non- Triple-negative and non-basal-like are used as comparison groups. 60,61,68 Thus, apart from Triple- negative, the relation of other individual subtypes to sensitivity was not examined. Only one small study 71 of 277 cases from 2

21 Florence compared interval and screen-detected cancers distinguishing all subtypes; however, this study did not report any significant findings but its statistical power was limited. Given the goal of screening to reduce breast cancer mortality, it is imperative to understand the factors that may influence screening mammography sensitivity. Since the evidence suggests breast cancer types vary in aggressiveness and in radiological appearance, they may also influence screening sensitivity. Therefore, the main objective of this study is to assess the relation of breast cancer types (including tumour subtypes as well as ER, PR and HER2 status) and screening sensitivity. A second objective is to determine if tumour aggressiveness can explain the variability in sensitivity across tumour type if any such variability is observed. 3

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23 Literature Review 2.1 Breast cancer intrinsic subtypes Description of intrinsic breast cancer subtypes According to the World Health Organization (WHO), twenty major histological types of breast cancers are known which highlights their rich phenotypic diversity. 2 Most of all diagnosed breast cancers, approximately 70-80%, will be classified either as invasive ductal carcinoma (IDC) or invasive lobular carcinoma (ILC). 5,72 Invasive breast tumours are those that penetrate all or part of the basement membrane of the epithelial site of origin. 73 These tumours are mostly adenocarcinomas that are thought to be derived from mammary epithelial cells of the terminal duct lobular units (Figure 1). 73 Invasive cancers represent a mixture of cancers with different biological and clinical profiles. 5,72 For instance, two invasive ductal carcinomas will differ in their propensity to metastasize and in their response to treatment. 73,74 These observations set the stage for more in-depth characterization of breast cancers to assess if biological differences could also be found at the molecular level. Figure 1. Breast anatomy and histology. The basic unit in the breast (left) is the terminal duct lobular unit (middle). Cross-section of the terminal duct lobular unit is represented (right) with luminal epithelial cells lining the lumen (triangular-shaped cells in blue). Myoepithelial cells line the basal membrane (elongated pink cells). Source: Breast development and anatomy 75 Given that histological diversity could not explain all the differences in breast cancer behaviour, Perou et al. 2 sought to characterize breast cancers at the molecular level. They speculated that breast cancers histological diversity could generate an equally rich molecular diversity 2 which in turn, could help further understand breast cancer behaviour. In their landmark study 2, the authors characterized the gene expression from 8102 human genes and identified 496 discriminating genes that varied little in duplicate samples of the same tumour but exhibited marked variability 5

24 between different tumours in 65 breast cancers obtained from 42 patients. Tumours with similar gene-expression patterns were grouped using a hierarchical clustering method 76 (Figure 2). Based on this method, two distinctive groups of breast cancers emerged based on the expression of the estrogen receptor (ER) and ER-associated genes. 2 This finding was compatible with breast cancer biology since estrogen is believed to play a crucial role in breast cancer aetiology. 5 Its role in aetiology is consistent with the fact that estrogen binds to the nuclear ER and controls cell proliferation, 5,77 moreover, hormone therapy such as Tamoxifen treatment, which targets ER, has been found to increase survival. 78,79 The cluster expressing ER (ER-positive) comprised tumours that had a gene expression profile similar to the gene profile of luminal cells found in the normal breast (Figure 1) 80 and was aptly named Luminal A subtype. 4 Within this same ER-positive cluster, a group of tumours were also found to overexpress HER2 and was named Luminal B to distinguish it from the Luminal A group which did not overexpress this receptor. In the cluster not expressing ER (ERnegative), two groups of tumours were observed which also were differentiated by the expression of HER2. The cluster of ER-negative tumours overexpressing HER2 and HER2-associated genes such as GRB7 was named HER2-enriched. 3,4 The role of HER2 in breast cancer can be inferred from the biological therapy with humanized antibody Transtuzumab that target HER2. 70 The remaining group of tumours within the ER-negative cluster did not overexpress HER2. However, this group was rich in cells expressing cytokeratins (CK), which are usually expressed in normal breast myoepithelial, or basal cells that underlie the breast luminal cells (Figure 1), and was thus termed Basal-like subtype. 4 All of these studies inspired a new molecular taxonomy for breast cancers, 2-4,6,74,81 and the term "intrinsic subtypes" was adopted to reflect each of the subtype s unique biological properties. 3 The four studied intrinsic subtypes ab are Luminal A, Luminal B, HER2-enriched, and Basal-like. 2-4,6,74,81 Luminal A The Luminal A subtype has a gene expression pattern resembling the gene expression of the cells of the luminal epithelial layer of the breast and is characterized by the expression of ER, PR and genes associated with ER activation. 5,6 In particular, Luminal A breast cancers have high expression of ER and ER-regulated genes such as GATA-binding protein 3 (GATA3), X-box binding protein (XBP1), and Forkhead box protein A1 (FOXA1), and low expression of proliferative genes such as proliferation associated antigen Ki-67 (MKI67), cyclin B1 (CCNB1), and Myb-related protein 2 (MYBL2). 3,8-10,82 Luminal A is the most frequent tumour subtype representing about 55-65% of a Perou et al. 2 originally also reported a normal-like subtype. However, this last subtype is believed to be laboratory contamination and will not be studied in this work. 3 b Additional studies have been published since alluding to the possible existence of other subtypes. However, this work will focus on subtypes which have been extensively studied and which have been found to have clinical relevancy. 6

25 total breast cancers diagnosed. 5 Patients diagnosed with this subtype are treated with Tamoxifen, and have a better prognosis than all other subtypes with an average 5-year survival of %. 5,7,8,11 Luminal B Like the Luminal A subtype, this subtype is associated with the expression of ER, PR and genes involved in ERregulation. In contrast to Luminal A, the Luminal B subtype also expresses HER2 and HER2-associated genes (ERBB2 and GRB7). In addition, it is also rich in genes associated with proliferation like MKI67, CCNB1, and MYBL2. 3,82 This subtype is not very frequent compared to Luminal A, and represents about 7-12% of total diagnosed cancers. Patients diagnosed with this subtype can benefit from Tamoxifen, and possibly Transtuzumab, but the average 5-year survival can range anywhere from 45-90%. 5,7,8,11 HER2-enriched Unlike the Luminal A and B subtypes, the HER2-enriched subtype does not express of ER, PR and ER-associated genes but overexpresses HER2 and HER2-associated genes (ERBB2 and GRB7). Human growth factor receptor 2, or HER2, is a transmembrane receptor tyrosine kinase and the amplification of the gene or protein overexpression is associated with accelerated cell growth and proliferation. 77 GRB7 is a gene encoding for growth factor receptor-bound protein 7, which is an adapter protein that interacts with tyrosine kinases, and signalling molecules. 83 This subtype highly expresses genes involved in cellular proliferation such as MKI67, CCNB1, and MYBL2. 3,82 It is the least common of all subtypes and represents about 6-10% of total breast cancers diagnosed. Although patients diagnosed with this subtype can benefit from targeted therapy such as Transtuzumab, the average 5-year survival is poorer than for Luminal subtypes, and ranges from 20-75%. 5,7,8,11 Basal-like Like the HER2-enriched subtype, Basal-like does not express ER, PR and ER-associated genes. But it also does not express HER2. It is characterized by high level expression of genes KRT5, KRT6, and KRT17 encoding for epithelial cytokeratins 5, 6, and This subtype also highly expresses genes involved in cellular proliferation MKI67, CCNB1, and MYBL2. 3,82 It represents approximately 10-15% of total breast cancers diagnosed, and also has a poorer prognosis than Luminal subtypes with an average 5-year survival of 30-80%. 5,7,8,11 Unlike the other subtypes, women diagnosed with this subtype of breast cancer do not benefit from a targeted therapy. 7

26 A B Figure 2. Dendrogram from hierarchical clustering of 115 tumour samples. Columns represent tumour samples while rows are representative of specific genes (genes shown ER, HER2, CK2, HER1). Gene overexpression compared to a reference standard sample is represented in red while low expression of the gene against the standard is in green. A Molecular-signatures specific to each breast cancer subtype is shown (left to right): Luminal A (purple branches), Luminal B (blue branches), and HER2-enriched (pink branches), Basal-like (orange branches) and Normal-like (green branches). B Corresponding immunohistochemistry classification according to ER, PR, and HER2 status is shown at bottom of the dendrogram. Source: Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study Classification of breast cancer subtypes by immunohistochemistry Identification of breast cancer subtypes by immunohistochemistry The use of gene-expression based assays while very informative is both laborious and expensive, and a simpler method to recreate the molecular-derived subtypes was required for routine clinical work. Immunohistochemistry (IHC), a protein-based assay, is a relatively simple laboratory method that is widely used in pathology laboratory and proved a practical alternative to approximate the subtypes. 7,84 IHC takes advantage of the capacity of antibodies to bind to very specific antigens. 85 Sections of breast tumours are incubated with specific antibodies directed at ER, PR, HER2, and CK5/6 proteins. 86 For tumours expressing one of these markers, the specific antibody will bind it and form a complex. A secondary antibody, linked to an enzyme will target this complex. 85 A substrate c is added and an enzymatic reaction occurs leaving a colour deposit at the site of the complex. Under the microscope, areas that contain bound antibody will appear darker than other areas. By this method, it is then possible to estimate the number of positive staining cells for a specific marker from a section of c A substrate is a substance that reacts with an enzyme. 82 8

27 tumour. According to current guidelines, if more than 1% of tumour cells are stained then the tumour sample is considered ER- and PR-positive. 87,88 To ascertain HER2 receptor status, IHC is also used to assess protein expression. Tumours are classified as HER2- positive if the IHC score is 3+, meaning intense membranous staining of > 30% of tumour cells. 89 Scores of 0 or 1+ are considered HER2-negative. 89 Tumours classified, as 2+ are considered equivocal and need to be analyzed by fluorescent in situ hybridization (FISH). FISH is a laboratory procedure that uses fluorescent probes to detect DNA sequences on a chromosome. 90,91 These detected areas light up when viewed under a fluorescent microscope and show whether or not there are many copies of the HER2 gene in cancer cells. 92 HER2 is considered amplified if on average more than six copies of the HER2 gene are detected per cell, or if more than 2.2 HER2 genes are counted for every copy of chromosome 17 (or CEP 17, the reference gene). 89 A result is considered equivocal if on average four to six copies of HER2 are counted per cell, or if 1.8 to 2.2 HER2 genes are counted per copy of chromosome If there are less than four copies of HER2 per cell, or if the HER2 to CEP 17 ratio is less than 1.8, then the result is negative. 89 Unlike ER, PR, and HER2, assessment of CK 5/6 by IHC to approximate the Basal-like subtype is not routinely performed and is considered insufficiently reproducible for general use. 31,34,93 In standard practice, the absence of ER, PR and HER2 receptor expression is considered a proxy for Basal-like, but because it doesn t include results for cytokeratins, it is called Triple-negative In the literature both terms are used depending if cytokeratins were assessed but Triple-negative is the most common terminology. d Combinations of IHC-derived markers (ER, PR, and HER2) were subsequently validated 7 to determine the classification that best matched the original gene expression patterns (Table 1). ef Table 1. Breast cancer subtypes determined by IHC assessment of ER, PR, and HER2 status. Breast cancer subtypes IHC classification Luminal A ER + and/or PR + HER2- Luminal B ER + and/or PR + HER2+ HER2 enriched ER- PR - HER2+ Triple negative ER- PR - HER2- d Triple-negative will be used in this work while the term Basal-like will be used when cited in the literature. e Some studies examine tumour in terms of ER, PR and HER2 status separately. In this work, we will use both these individual receptors and the subtypes. For ease of interpretation, we refer to both these groups as types. f In this work, we denote HER2-enriched when referring to the subtype and HER2-positive when referring to the individual receptor status. 9

28 2.1.3 Section summary Molecular studies allowed us to gain a better understanding of breast cancer biology. They allowed classifying breast cancers according to four distinct intrinsic subtypes (Luminal A, Luminal B, HER2-enriched and Triple-negative). These subtypes can be approximated by determining the status of the ER, PR, and HER2 receptors using IHC. Therefore, researchers can now study all breast cancer types meaning in terms of each individual receptor or combine them to reproduce the subtypes. Classifying breast cancers according to types has allowed clinicians to better treat patients, and has paved the way for further studies concerning breast cancer aetiology, detection and progression Breast cancer types and aggressiveness Aggressiveness can be thought of, as the potential of the cancer to grow and spread. It is a broad term encompassing cellular proliferation, cellular differentiation, and tumour dissemination. 96,97 The ability of a tumour to proliferate, differentiate and disseminate is dictated by its aggressiveness, which is a biological characteristic of the tumour Markers of aggressiveness Molecular studies not only showed that breast cancers could be classified according to distinct subtypes but also revealed that they differed in the expression of aggressive genes such as MKI67, CCNB1, and MYBL2 3,82 Aggressiveness in breast cancers can be assessed by evaluating histological grade, determining KI-67 expression and quantifying the S-phase fraction. Determining the histological grade, which measures the degree of cellular differentiation, 97,98 is an ideal method to infer tumour aggressiveness since it was reported to correlate well with proliferative genes identified in earlier molecular studies. 2 Since proliferative genes MKI67, CCNB1, and MYBL2 encode for cell cycle proteins, the Ki-67 index, which measures the expression of the nuclear protein Ki-67, and the S-phase fraction, which measures the rate of cell turnover or proliferation 97 can also be used to infer aggressiveness. Studies of these three markers suggest that the Ki-67 index correlates with the S-phase fraction and the mitotic index, an integral component of the histological grade. 99 Histological grade Differentiation is the process, or is the result of the process, where during development an organ or a body part is modified into a special form or for a specific function. 100 Fully differentiated tissue is normal tissue with normal structures and function; all tumours are less differentiated in one of these characteristics. 100 The degree of differentiation will be inversely related to the extent to which these characteristics have been lost. 100 There is a relationship between the extent of differentiation of a tumour and its biological behaviour for instance; poorly differentiated tumours tend to be more aggressive than well-differentiated tumours. 101 The difference in differentiation between the tumours relative to normal tissue can be assessed by the histological grade. 5,98 Various methods to evaluate grade have been described based on assessment of cellular, architectural and nuclear structures. Its 10

29 evaluation has been improved over the years by adding standardized criteria to reduce subjectivity and improve concordance. 73 Bloom and Richardson first improved the method by adding numerical scoring but clear criteria for cut-offs was still lacking. 102,103 Elston and Ellis then modified the Bloom Richardson system g by establishing criteria, and were able to achieve greater objectivity and concordance. 73,102 The method, also known as Nottingham Grading System, is considered reproducible and is recommended internationally as the reference method. 98, Grade is assessed under microscopy for three morphologic features: 1) degree of tubule or gland formation, 2) degree of nuclear pleomorphism which is the change in size and shape of nuclei 98,105 and 3) mitotic count which is used to measure the degree of cellular proliferation by counting the number of mitotic bodies per 10 high power fields of view. 98,107 These three features are scored individually on a scale from 1 to 3 (Table 2), and the three scores are then added to obtain a total score ranging from 3 to 9. This total score is used to assign a grade ranging from Grade 1 to Grade 3. 73,106 (Table 3) Table 2. Features and related scores to evaluate histological grade according to the Elston-Ellis* grading method. Features Tubule and gland formation Score Majority of tumour (>75%) 1 Moderate degree (10-75%) 2 Little or none (<10%) 3 Nuclear pleomorphism Small, regular, uniform cells 1 Moderate increase in size and variation 2 Marked variation 3 Mitotic count 7 mitoses per 10 high power fields mitoses per 10 high power fields 2 15 mitoses per 10 high power fields 3 * Elston-Ellis method 102 Mitotic count criteria will vary depending on the field diameter of the microscope of the pathologist; criteria given for 0.50 mm field diameter. 108 g Also known as Nottingham Grading System (NGS) or Nottingham modification of the Scarff-Bloom-Richardson grading system. 11

30 Table 3. Summary and description of Grade 1, 2, and 3. Grade Overall Elston-Ellis Score 102 Description 1 (I) 3-5 Well differentiated 2 (II) 6-7 Moderately differentiated 3 (III) 8-9 Poorly differentiated A grade 1 tumour is considered well-differentiated meaning that it retains features of normal breast tissue (Figure 3, Box B vs. Box A). Tumour cells grow following a slow and organized pattern. 109 Grade 1 tumours are not considered aggressive. Grade 2 tumours are moderately differentiated in that they show some tissue changes relative to normal breast tissue (Figure 3, Box C vs. Box A). They are considered moderately aggressive. Grade 3 tumours are poorly differentiated in that the tissue has lost any semblance to normal breast tissue. These tumour cells grow following a rapid and disorganized cell pattern (Figure 3, Box D vs. Box A), and the tumours are considered aggressive. Normal Breast Grade I Grade II Grade III A B C D Figure 3. Typical histological views of tumours by grade scores relative to normal breast tissue. Grade I (B) tumour still possesses features of normal tissue (A) and is considered well-differentiated. Grade II (C) tumour shows some tissue changes relative to normal tissue (A). Grade III tumour (D) does not resemble normal tissue, and is considered less differentiated than normal breast tissue (A). Sources: Diagnosis of breast cancer 110 (A) Breast cancer prognostic classification in the molecular era: the role of histological grade. 98 (B, C, D) Ki-67 Ki-67 is a protein expressed in the nucleus in proliferating cells during all cell cycle phases, except at the resting state (G0). 107 The function of the protein is not well known but seems to play a role in cell proliferation. 111 The Ki-67 protein can be assessed by IHC but may be difficult to interpret since its expression may be heterogeneous in tumours. 99 It is estimated as the percentage of tumour cells positively staining for Ki-67 protein. 111 A consensus has yet to be reached on relevant reference values but IHC staining of <10% of tumour cells generally represents a tumour that is low proliferative, 10-20% represents a borderline proliferative tumour, and >20% is considered highly proliferative

31 Although both the Ki-67 and S-phase fraction give an indication of cellular proliferation, the assessment of Ki-67 is less cumbersome than quantifying the S-phase fraction. 96 S-phase fraction S-phase fraction (SPF) is a measure of proliferation and refers to the fraction of cells engaged in DNA synthesis 107 and is determined by flow cytometry. 96,97 This method allows obtaining information on the number of cells synthesizing DNA and their DNA content 113 but the analysis can be hampered by pronounced intratumour heterogeneity. 99 Like Ki- 67, there is no current agreement on relevant reference values. Tumours with SPF results of <6% are considered low proliferative, 6-10% moderately proliferative, and >10% highly proliferative Breast cancer types and aggressiveness Molecular studies revealed that breast cancers were heterogeneous diseases and could be classified according to subtypes each with distinctive gene profiles. These subtypes can be reproduced with immunohistochemistry by assessing the status of ER, PR and HER2 receptor expression. Molecular studies also showed that these subtypes had different levels of expression of proliferative genes; hinting that they could also vary in aggressiveness. ER, PR, HER2 and Histological Grade The relationship between ER, PR and histological grade is well established and has been the subject of numerous studies dating as far back as In two recent studies, 38,39 ER-positive tumours were found to be more frequently Grade I than Grade III. Indeed, Chen et al. 38, reported that ER-positive were more frequently Grade I than Grade II and Grade III tumours (90.4% vs. 71.9% vs. 44.2%, p<0.0001, respectively). Similarly, PR-positive tumours were more frequently Grade I (78.9%) than Grade II (69.3%) and Grade III tumours (46.2%), (P<0.0001), respectively. This study had a large sample size and also assessed histological grade according to the recommended Nottingham Grading System. Sarode et al. 39 reported similar results despite using a smaller sample size and using lower threshold of positivity for ER and PR (5%) compared to Chen et al. 38 who used 10% as a threshold. They similarly report that ER and PR expression levels decrease with increasing grade, P< and P=0.0051, respectively. However, none of these studies adjusted for possible confounders. But even adjusted studies 40 reported that ER and PR-positive tumours were inversely associated with high grade compared to ER and PR-negative tumours. However, although this study adjusted for tumour size, vascular invasion, lymph node status and mode of detection no point estimates were provided. HER2-positive tumours have been generally associated with higher grade 115 but the evidence is not as consistent as ER and PR. In a recent study, 38 HER2-positive tumours were more often Grade III than Grade II or Grade I tumours (29.2% vs. 25.5% vs. 4.2%, P<0.0001, respectively). Lesser quality studies also reported similar observations despite not providing quantitative information 41 and classifying HER2 2+ as negative without FISH confirmation. 42 Moreover, a study adjusting for tumour size, vascular invasion, lymph node status and mode of detection, reported that HER2-13

32 positivity was associated with higher grade when compared to HER2-negative tumours, but no point estimates were provided. 40 On the other hand, Sarode et al. 39 reported no statistical difference in mean HER2 amplification ratios between Grades I, II and III tumours (mean FISH ratios 3.3, 5.2, and 4.3, respectively, P-value not provided). However, the authors performed FISH on tumour samples only scoring 2+ and 3+ in IHC. Since this pool of samples will be biased to HER2-positivity, it may explain the non-significance of the results. Subtypes and Histological Grade Studies (reporting both crude and adjusted results) examining the association between subtype and grade were consistent. 7,21,38,43-53 Compared to Luminal A, Basal-like and HER2-enriched subtypes were strongly associated with high-grade tumours. An association between Luminal B and higher grade was also reported in one study when compared to Luminal A. 45 In studies reporting adjusted results (Table 4), strong associations were reported between grade and subtype, notably for HER2-enriched and Basal-like. In a well-cited study by Carey et al., 7 HER2-enriched tumours and Basal-like were more likely high (III) than low/moderate grade (I/II) (adjusted OR= 6.2, 95% CI= and OR=8.3, 95% CI= , respectively), compared to Luminal A but no difference was noted for Luminal B (adjusted OR 1.0, 95% CI= ). A subsequent study using a larger sample size of 1018 cases, reported that compared to Luminal A, HER2-enriched and Basal-like were more likely high (III) than low/moderate (I/II) grade (adjusted OR=3.6, 95% CI= and OR=5.3, 95% CI= , respectively) and again no difference was noted for Luminal B (adjusted OR=1.4, 95% CI= ). 46 In a following study that included slightly more cases (1082 cases), results concurred with those of Carey et al. 7 and Tamimi et al. 46 except for the Luminal B subtype. Luminal B, HER2- enriched and Triple-negative were more likely poorly differentiated than well/differentiated tumours (adjusted OR= 3.1, 95% CI= , OR 10.7, 95% CI= , and OR=14.1, 95% CI= , respectively) compared to Luminal A. 45 The difference in these results may be due, at least in part, to the IHC criteria used to determine HER2 positivity since both Carey et al. 7 and Tamini et al. 46 used a lower threshold (2+ and 3+ are positive), than Onitilo et al. 45 (3+ positive, 2+ negative unless verified by FISH) and did not confirm amplification of the HER2 gene by FISH. Therefore cases that Onitilo et al. 45 would have considered HER2-negative, Carey et al. 7, and Tamini et al. 46, would have considered HER2-positive, and their Luminal B category may represent a group which includes Luminal A, a less aggressive subtype. 14

33 Table 4. Characteristics of studies reporting adjusted results for the relationship between subtypes and histological grade. * Author Year Subtypes IHC Thresholds N Adjusted Adj. Grade OR (95% CI) method Grade III vs. I/II Carey et al. 7 Luminal A ER+, PR+ if >5% stained (Referent) Age, Nottingham 2006 Luminal B HER2+ if 10% stained ( ) race, I-III HER2-enriched Cytokeratin+ if any stained ( ) stage Basal-like ( ) Tamimi et al. 46 Luminal A ER+, PR+ if >10% stained (Referent) Age Nottingham 2008 Luminal B HER2+ if >10% stained ( ) I-III HER2-enriched (2+, 3+) ( ) Basal-like Cytokeratin + if any stained ( ) Onitilo et al. 45 ǁ Luminal A ER+, PR+ if >20% stained (Referent) Age Not available 2009 Luminal B HER2+ if >10% stained or ( ) HER2-enriched FISH if ( ) TPN ( ) * OR= odds ratio, CI=confidence interval, Adj=adjustments. Bold results are statistically significant. Luminal A: ER+ and/or PR+ HER2-; Luminal B: ER+ and/or PR+ HER2+; HER2-enriched: ER- PR- HER2+; Basal-like: ER- PR- HER2- CK5/6+ and/or HER1+ Luminal A: ER+ and/or PR+ HER2-; Luminal B: ER+ and/or PR+ HER2+; HER2-enriched: ER- PR- HER2+; Basal-like: ER- PR- HER2- CK5/6+ ǁ Luminal A: ER+ and/or PR+ HER2-; Luminal B: ER+ and/or PR+ HER2+; HER2-enriched: ER- PR- HER2+; TPN: ER- PR- HER2- ER, PR, HER2 and S-phase Fraction and Ki-67 The S-phase fraction (SPF) has now been largely replaced by the histological grade to infer tumour aggressiveness, and much of the literature on this topic is not very recent. In a systematic review conducted in the authors retrieved 19 articles published in the span of 10 years ( ) examining the association of SPF and hormone receptors. In general, most studies reported that hormone receptor positive (ER and PR) tumours were most likely low SPF tumours, despite using different techniques and cut points, with few studies refuting the findings. In the most recent study in 2001, 117 ER-negative tumours had higher levels of SPF (defined as the third SPF tertile) (62.7%) than ER-positive tumours (27.5%) (P< ). Similarly, PR-negative tumours had higher levels of SPF (55.6%) than PRpositive tumours (26.0%) (P< ). These results agree with past studies. All studies to date have only reported crude results. Studies examining HER2 and SPF are scarce In the most recent study published in 2001, which also included the largest number of cases (295 cases), HER2-positive tended to have a high SPF (defined as 6.1%) while HER2- negative tumours tended toward lower SPF (P<0.001). This result concurs with others, and although it is difficult to draw conclusions based on these few studies, results are suggestive of a more aggressive profile for HER2-positive tumours. However, like ER and PR studies, these studies also only reported crude results. 15

34 Studies have also focused on ER, PR, HER2 and Ki-67. In a recent literature review conducted in , the majority of studies (9 of 12 studies) suggested that ER and PR-positivity were associated with lower levels of Ki-67. The results for HER2-positivity and Ki-67, however, were less consistent than ER and PR. In five studies, three suggested an association between HER2-positivity and high Ki-67 expression. According to the author, discordances in results may be due mostly to differences in laboratory protocols between studies. 111 Subtypes and S-phase fraction and Ki-67 Given the infrequent use of the SPF as a routine proliferative marker, there is a paucity of literature examining SPF and subtypes. However, there is some evidence to suggest that higher levels of SPF are more frequent in HER2- enriched and Triple-negative subtypes than in Luminal subtypes. In the only study retrieved, Del Casar et al. 43 reported in 2008, luminal tumours (Luminal A and Luminal B tumours) had more frequently lower SPF values (<7.69%) than Basal-like and HER2-enriched tumours (P=0.03). However, there was a considerable amount of missing data for SPF that exceeded 35% and the results were limited to a crude analysis; therefore, they need to be interpreted carefully. There were more studies of subtypes and Ki-67 but comparing results is difficult due to different definitions of subtypes used and lack of standardized Ki-67 cut-off. 39,48,49,51 In the study classifying subtypes according to recommended criteria using ER, PR, and HER2 receptor status, 51 Luminal A tumours were more frequent in the low ( 5%) and middle (5-20%) Ki-67 category while Luminal B, Triple-negative, and HER2-enriched tumours were more frequent in the high (>20%) Ki-67 category (P<0.0001) Section summary Aggressiveness when assessed by histological grade, SPF, and Ki-67 seems to vary by receptor status and subtype. In general, ER-positive and PR-positive tumours are less aggressive than their negative counterparts while HER2- positive tumours are more aggressive than HER2-negative tumours. Luminal A seems to be the least aggressive subtype when compared to Luminal B, HER2-enriched and Basal-like/Triple-negative. Luminal B seems to be moderately aggressive; it is more aggressive than Luminal A but less aggressive than HER2-enriched and Basallike/Triple-negative tumours. Conversely, compared to luminal tumours, HER2-enriched and Basal-like/Triplenegative are very aggressive. 2.3 Breast cancer types and radiologic features Since breast cancer subtypes have unique biological properties, these differences may influence the radiological appearance of the tumour on the screening mammogram. 16