BREAST CANCER IN PTEN HAMARTOMA TUMOR SYNDROME: CAN A PREDICTIVE FINGERPRINT BE IDENTIFIED? AGNIESZKA MACHAJ. Master of Science

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1 BREAST CANCER IN PTEN HAMARTOMA TUMOR SYNDROME: CAN A PREDICTIVE FINGERPRINT BE IDENTIFIED? By AGNIESZKA MACHAJ Submitted in partial fulfillment of the requirements for the degree Master of Science Thesis Advisor: Anne Matthews, RN, PhD Committee Members: Anna L Mitchell, MD, PhD Michelle Merrill, MS, CGC Jessica Mester, MS, CGC Charis Eng, MD, PhD Genetic Counseling Department of Genetics & Genome Sciences CASE WESTERN RESERVE UNIVERSITY May 2014

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Agnieszka Machaj Candidate for the Master of Science degree*. (signed) Anne Matthews, RN, PHD (chair of the committee) Anna L Mitchell, MD, PhD Michelle Merrill, MS, CGC Jessica Mester, MS, CGC Charis Eng, MD, PhD (date) April 2014 *We also certify that written approval has been obtained for any Proprietary material contained therein 2

3 TABLE OF CONTENTS PREFACE LISTS... 5 ACKNOWLEDGMENTS 8 ABSTRACT. 9 CHAPTER ONE: PROJECT OVERVIEW, PURPOSE, AND SPECIFIC AIMS.. 10 CHAPTER TWO: LITERATURE REVIEW.. 19 Breast Cancer. 19 Breast Carcinoma Histologies 21 Molecular Classifications of Breast Carcinomas Molecular Apocrine Breast Cancer 26 Apocrine Carcinomas. 27 Genotype-Phenotype Correlation of Breast Cancer Susceptibility Syndromes.. 28 PTEN.. 31 PI3K Signaling Pathway 32 PTEN/pAKT Pathway in Breast Cancer 33 PTEN Hamartoma Tumor Syndrome (PHTS) PHTS-Associated Breast Cancer. 37 Screening for Germline PTEN Mutations.. 40 The Clinical Management of PHTS Patients. 42 Patient-tailored Treatment for Individuals with PHTS.. 44 CHAPTER 3: AIM 1 46 Aim 1: Material and Methods 46 Aim 1: Results 51 Aim 1: Discussion.. 58 CHAPTER 4: AIM 2 61 Aim 2: Material and Methods. 61 Aim 2: Results. 68 Aim 2: Discussion CHAPTER 5: AIM 3 83 Aim 3: Material and Methods 83 Aim 3: Results 84 Aim 3: Discussion

4 CHAPTER 6: AIM 4 89 Aim 4: Material and Methods 89 Aim 4: Results 90 Aim 4: Discussion.. 92 CHAPTER 7: CONCLUSION. 93 Implications for Genetic Counseling.. 94 Limitations of the Study. 95 Directions for Future Research.. 96 CHAPTER 7: APPENDIX Appendix A1. Cowden Syndrome Testing Criteria Guidelines Appendix B1. Authorization for Release of Medical Information Appendix B2. Table of All Tissue Specimens Obtained Appendix B3. Frequencies of Histopathologic Features in PHTS Appendix C1. Protocol for PTEN Immunohistochemistry Appendix C2. Protocol for pakt Immunohistochemistry Appendix D1. PTEN Mutation and Detection Analysis CHAPTER 8: REFERENCES

5 LIST OF TABLES Table 1. Differences in Histopathology and Their Associated Therapies.. 14 Table 2. Risk Factors and Their Associated Relative Risk for Breast Cancer Table 3. Histologic Grading: Nuclear Grading System. 22 Table 4. Table 5. Molecular Classification of Breast Cancer Based on Gene Expression Hypothesized Association Between Molecular Subtype Classifications with Histological Assignment and Immunohistochemical Biomarkers.. 26 Table 6. Breast Cancer-Associated Cancer Predisposition Syndromes. 29 Table 7. Table 8. Expression of Immunohistochemical Markers in BRCA1- and BRCA2-Related Breast Cancers Relative to Sporadic Breast Cancers. 30 List of Cancers, Benign Growths and CNS Involvements Associated with PHTS 36 Table 9. Summary of Carcinomas Occurring in PHTS.. 39 Table 10. Recommendations for Diagnostic Workup and Cancer Surveillance in Patients with PTEN Mutations. 38 Table 11. Types of Histological Samples Received per Specimen Collection 49 Table 12. Patient Demographics of Women With PHTS and Invasive Carcinoma 52 Table 13. Comparison of the Malignant Lesions Observed in 25 PHTS- Associated Invasive Carcinomas to Those of the General Population. 53 Table 14. Frequencies of Histopathologic Features Seen in Association With PHTS-Associated Invasive Breast Carcinomas 55 Table 15. Comparison of Distinctive Histopathological Features Observed in PHTS-associated Invasive Carcinomas to Those of the General Population. 56 5

6 Table 16. Descriptive Statistical Analysis of the PR, ER, and HER2 Biomarker Expression Status in PHTS-associated Invasive Breast Carcinomas Per Original Pathology Reports 68 Table 17. Hormone Receptor Status Observed in PHTS-associated Carcinomas are Similar to Those Observed in the General Population. 70 Table 18. Informative pakt and PTEN expression in PHTS-associated Carcinomas and Those Observed in the General Population Table 19. Analysis of Correlation between Blood-PTEN Expression Levels in the Lowest Quartile Versus Remaining Quartiles in Women With and Without Germline PTEN mutations.. 84 Table 20. Analysis of Correlation between Blood-pAKT Expression Levels in the Highest Quartile Versus Remaining Quartiles in Women With and Without Germline PTEN mutations.. 85 Table 21. Comparison of Blood-PTEN and Blood-pAKT Expression Levels in Women With and Without PHTS. 86 Table 22. Multivariate Analysis of Predictive Factors for Pathogenic Germline PTEN Mutations in CS and CS-like Patients Presenting with Breast Carcinoma. 90 6

7 LIST OF FIGURES Figure 1. The PTEN/PI3K/AKT Pathway.. 11 Figure 2. PTEN Loss Leads to PI3K/AKT Activation Figure 3. Schematic of Specimen Collection. 48 Figure 4. Control PTEN IHC in a Normal Breast Lobule and Control PTEN IHC in an Invasive Carcinoma 62 Figure 5. Control pakt IHC in Normal Breast Lobule 63 Figure 6. Control pakt IHC in an Invasive Carcinoma Figure 7. PTEN IHC in PHTS-associated Invasive Carcinoma and pakt IHC in PHTS-Associated Invasive Carcinoma 65 Figure 8. AR IHC in PHTS-Associated Invasive Carcinoma. 67 Figure 9. Descriptive Analysis of PTEN and pakt IHC Staining Patterns in PHTS-Associated Invasive Carcinoma, DCIS, and Invasive Carcinoma Controls 73 7

8 ACKNOWLEDGEMENTS I would like to extend my appreciation to the wonderful women and families in the PTEN Study and in the PTEN-breast Study for their participation in this study. Thank you to my advisor, Dr. Anne Matthews for her advice and encouragement, as well as my committee members Dr. Anna Mitchell, Michelle Merrill and Jessica Mester for their interest, input and commitment to the success of this project. A sincere thank you to Dr. Charis Eng for granting me the opportunity to contribute to this collaborative project. I am grateful to Dr. Eng and the entire GMB and GMI Laboratory for their help and endless patience throughout these past two years. Thank you to Dr. Erinn Downs-Kelly for contributing her time and expertise in breast pathology and immunohistochemical analysis. Finally, I am thankful to my classmates, friends, and family for their love, support, and memories over the past three years. 8

9 Breast Cancer in PTEN Hamartoma Tumor Syndrome: Can a Predicative Fingerprint Be Identified? Abstract by AGNIESZKA MACHAJ Breast carcinoma is a primary malignant tumor occurring in PTEN Hamartoma Tumor syndrome (PHTS). PHTS is caused by germline mutations in the tumor suppressor gene PTEN. Little is known about the histopathologic features and molecular profile of PHTS-related breast cancers. Furthermore, no data have been published on blood-pten/pakt protein expression in individuals with germline PTEN mutations or other potential predictors of PTEN mutation status. Histopathological review of original Hematoxylin and Eosin slides demonstrated that apocrine features, atypical apocrine adenosis, and atypical ductal hyperplasia are distinctive histopathological features of PHTS-related breast tumors. A distinct PHTSassociated molecular profile was not identified; the association of PHTS-related breast cancers with a molecular apocrine profile was not confirmed. Furthermore, we determined that low blood-pten protein and/or high blood-pakt expression, as well as the Cleveland Clinic PTEN risk score, may be used as predictors of germline PTEN mutations in Cowden syndrome/cs-like presentations of breast cancer. 9

10 INTRODUCTION, PURPOSE, AND SPECIFIC AIMS In 2013, it is estimated that nearly a quarter of a million new cases of breast cancer will be diagnosed among American women (Siegel et al., 2013). The risk of any woman developing breast cancer over her lifetime is 12% (SEER). In addition to several known risk factors (Table 1), genetic background plays a significant role in the etiology of breast cancer. Depending on how strong the genetic influence is, breast cancer can be divided into somewhat overlapping subtypes: sporadic, familial or hereditary. The majority of breast cancer is sporadic, which seems to primarily be influenced by environmental factors but may include a small risk contribution from multiple genetic factors. In familial breast cancer, there are more cases of cancer within a family than statistically expected, but no inherited syndrome can be identified. Approximately 5-10% of breast cancer diagnoses are due to the presence of an inherited cancer syndrome, in which the patient has a germline mutation in a cancer susceptibility gene, with most syndromes inherited in an autosomal dominant manner (Berliner et al., 2007). There are several known susceptibility genes that confer an increased risk for breast cancer. One such gene is the tumor suppressor gene PTEN (Marsh et al., 1999; Eng 2000; Eng 2003). Due to its molecular functions, PTEN plays a significant role in a number of important regulatory molecular pathways that involve cellular growth, proliferation, migration and apoptosis (Tamguney & Stokoe, 2007). One of the molecular pathways that PTEN is involved in is the PI3K (phosphatidylinositol 3-kinase)/AKT pathway, a serine/threonine kinase pathway. 10

11 Figure 1. The PTEN/PI3K/AKT Pathway 1 1 Zbuk and Eng 2007 The PI3K/AKT pathway is a potent oncogenic signaling cascade that promotes cell transformation, proliferation, migration, angiogenesis and genomic instability (Figure 1). From a molecular angle, PTEN is the key negative regulator of the PI3K/AKT pathway because it dephosphorylates the activator of AKT, PIP3 (phosphatidylinositol-3, 4, 5- triphosphate) into PIP2 (phosphatidylinositol-4, 5-phosphate); by catalyzing this reaction, PTEN is suppressing the functions of PI3K/AKT pathway by inhibiting the downstream signaling of AKT (Baker, 2007). 11

12 Figure 2. PTEN Loss Leads to AKT Activation 1 1 Zhou et al Consequently, the loss of PTEN results in the loss of regulation of the PI3K/AKT pathway, leading to cell proliferation and cancer cell survival (Figure 2). One of the causes of PTEN loss is a germline mutation of the PTEN gene. A germline mutation in PTEN causes a hereditary cancer syndrome known as PTEN Hamartoma Tumor syndrome (PHTS). PHTS is an umbrella term used to describe 12

13 individuals with variable phenotypes, including Cowden syndrome (CS) and Bannayan- Riley-Ruvalcaba syndrome who have a germline mutation of the PTEN gene. Early studies suggest that up to 85% of individuals who meet the diagnostic criteria for CS have a detectable pathogenic germline PTEN mutation (Zbuk and Eng 2007). More recently, other research found that approximately 25% of individuals meeting diagnostic criteria for CS have a detectable pathogenic germline PTEN mutation (Tan et al., 2012). The features that are seen in PHTS is summarized in Table 8 on page 36. The clinical signs of PHTS resemble what would be expected from the changes in the pathways mentioned above characterized by an increased risk of benign and malignant neoplasias or neoplasms including an increased risk of developing breast cancer. In comparison to the 12% (SEER) lifetime risk of any woman developing breast cancer, current research estimates that the lifetime breast cancer risk for any women with PHTS is up to 85% (Tan et al., 2012). Moreover, adenocarcinoma of the breast is the highest malignancy risk in individuals with PHTS (Tan et al., 2012). Breast cancer can be divided into biologically and clinically meaningful subgroups based on histology and/or by a biomarker profile defined by a specific pattern of immunohistochemical (IHC) markers (Rakha et al., 2010). Interestingly, several studies have evaluated the hisopathological features and/or the biomarker profile associated with other breast cancer predisposition syndromes (Table 1). 13

14 Table 1. Differences in Histopathology and Their Associated Therapies Gene-associated breast cancer BRCA2-associated breast cancer BRCA1-associated breast cancer Germline E-cadherin mutations Histopathology Expresses Estrogen and Progesterone receptors 1,2 Higher than expected frequencies of medullary histology, high histolgic grade and the triple-negative breast phenotype 3,4,1 Lobular breast cancer Therapy Use of aromatase inhibitors, or the use of selective estrogen receptor modulators (SERM) in tumors that express the estrogen receptor Triple negative breast cancers can benefit from the possible use of Poly(ADP-ribose) polymerase inhibitors alone 5 or with PI3K inhibitors cotargeting the PI3K pathway 6 or cisplatin-targeted therapy 10 Possible use of ADP- Ribosylation Factor 6 Mediated chemical chaperons to restore in vitro mutant E-cadherin 7 Leads to the loss of E- cadherin expression PHTS-associated Possible molecular apocrine Possible use of Androgen and breast cancer subtype 8 ERRB2 pathway inhibitors 7 1 Breast Cancer Linkage Consortium et al Marcus et al Eisinger et al Peshkin et al Sandhu et al Kimbung et al., Figueiredo et al Banneau et al Naderi et al Byrski et al However, little is known about the histopathologic features and/or the biomarker profile associated with PHTS-associated breast carcinomas. Much of the published literature at the time of this present study did not distinigush patients based on their germline PTEN status. Rather, the available phenotypic data are based on studies performed on study populations comprising of CS patients (Schrager et al., 1998; Banneau et al., 2010). Schrager et al. (1998) concluded that certain histopathologic features, including apocrine features, were commonly seen in their population of 19 women with CS. Banneau et al. (2010) concluded that activation of the HER2/PI3K/AKT pathway by the loss of PTEN 14

15 promotes the development of tumors with apocrine features. However, Banneau et al. (2010) study restricted their population study to individuals with known germline PTEN mutations (n = 15). Thus, whether or not these CS patients had germline PTEN mutations and represent the PHTS population in the Schrager et al. (1998) study is unclear. Moreover, due to the small sample populations of both of these studies, the genotypic-phenotypic correlations of breast tumors arising in women with PHTS, were not well-defined in the literature available at the time of the present study. Thus, the present study will attempt to better characterize the histopathological features arising in women with PHTS as well as confirm the previously associated immunohistochemical profile of breast cancer in patients with germline PTEN mutations. In addition to breast cancer, individuals with PHTS are also at increased risk for a number of other malignancies. Making timely a diagnosis and identifying all at-risk germline PTEN mutation carriers is critical for risk management. Currently, a PHTS diagnosis may be suspected based on the evaluation of a patient s personal and family history for the subtle clinical signs seen in PHTS. In comparison to other known cancer syndromes, family history may not be as helpful of a predictor due to the fact that PTEN has an estimated de novo mutation rate of up to 47% (Mester and Eng, 2012). Consequently, due to the variable and often subtle manifestation of PHTS and the possibility of absent PHTS features within a family history, many individuals remain untested and therefore undiagnosed. Due to the current inherent difficulties associated with identifying individuals who may benefit from PTEN mutation analysis and the importance of a timely diagnosis, 15

16 it becomes increasingly important to identify predictors of germline PTEN mutation status. By building upon the study conclusions derived from Schrager et al and Banneau et al this study attempted to provide an accurate histopathological description of PHTS-associated breast carcinomas and attempted to identify defining characteristics and attempted to identify defining characteristics and a biomarker profile that may potentially be used as a predictor of germline PTEN mutations within the breast cancer population. Furthermore, this study explored the potential use of blood-pten and blood-pakt protein levels (Ngeow et al., 2010) as well as tested the utility of an established risk assessment screening tool as potential predictors of germline PTEN mutations in CS and CS-like presentations of breast cancer. Individuals who are CS-like have similar clinical features as individuals with CS, but do not fulfill the full CS clinical diagnostic criteria. Finally, the identification of defining histopathologic differences may elucidate targeted treatment options (Table 1) specific to PHTS-associated breast carcinomas. Identifying a specific breast cancer fingerprint as a predictor of germline PTEN mutations may also aid genetic counselors and other health care professionals in identifying individuals who should be referred to genetics for consideration of PTEN testing. Purpose of Study The purpose of this study was to characterize the histopathological features and immunohistochemical profile of breast cancers in patients with germline PTEN mutations. Original Hematoxylin and Eosin stained slides of PHTS-related breast cancers were used to explore the histological features of PHTS-related breast cancer. An 16

17 immunohistochemical approach was used to determine whether PHTS-related breast cancer is associated with a molecular apocrine profile and attempted to define a biomarker profile unique to PHTS-related breast cancers. Blood-PTEN and blood-pakt protein expression levels between breast cancer patients with and without germline PTEN mutations were compared to determine whether either protein value was predictive of PTEN mutation status. Finally, the utility of the PTEN risk assessment tool in combination with other findings as predictors of germline PTEN mutations in CS and CSL presentations of breast cancer was tested. Specific Aims The specific aims of the study were: 1. To use a histopathologic approach to review original Hematoxylin and Eosin stained slides to identify distinctive features of PHTS-related breast cancer and determine whether PHTS-associated tumors consist of apocrine features. 2. To use immunohistochemistry to identify a biomarker profile that is unique to PHTS-related breast cancers, as well as to determine whether PHTS-related breast cancers are associated with a molecular apocrine profile. 3. To compare blood-pten and blood-pakt protein levels data of women with invasive breast cancer with and without germline PTEN mutations, to determine if protein expression levels could serve as a screening tool to predict germline PTEN mutations in CS and CS-like presentations of breast cancer. 17

18 4. To test the utility of the Cleveland Clinic (CC) PTEN risk assessment tool along with other findings as predictors of germline PTEN mutations in PHTS and PHTS-like presentations of breast cancer. 18

19 BACKGROUND AND REVIEW OF THE LITERATURE Breast Cancer: Statistics and Risk Factors Among women, cancer is the second leading cause of death in the United States. Of the estimated 805,500 new cancer diagnoses, breast cancer will account for approximately 232,340 (29%) of these cases. According to the Surveillance Epidemiology and End Results (SEER) Program of the National Cancer Institute, based on rates from , the risk of any woman developing breast cancer over her lifetime is 12%. The incidence of breast cancer tends to increases with a woman s age; the median age at the time of cancer diagnosis is 61 years (Siegel et al., 2013). There are several risk factors that are known to modify a woman s risk of developing breast cancer. These risk factors include past history of breast cancer, current age, ionizing radiation exposure, age of first childbirth, hormone replacement therapy, body mass index, alcohol intake and biopsy history (Table 2). Additionally, family history is a risk factor with risk rising with the increased number of relatives and degree of relatedness.. 19

20 Table 2. Risk Factors and Their Associated Relative Risk for Breast Cancer 1 Risk factor Category at Risk Comparison Group Relative Risk Alcohol Intake 2 drinks per day Nondrinker 1.2 Body Mass Index 80 th percentile, age 55 or 20 th percentile 1.2 older BMI among non- Baseline BMI 31.1 Baseline BMI HRT 2 users HRT with estrogen and progesterone Current user for at least 5 years Never used 1.3 Ionizing radiation Treatment for Hodgkin s No exposure 5.2 exposure disease Repeated fluoroscopy No exposure 1.6 Early menarche Younger than 12 years Older than 15 years 1.3 Late menopause Older than 55 years Younger than Age at risk Nulliparous or 1 st child 1 st child before age Childbirth after age 30 Current age 65 or older Less than Past history of BC Invasive breast cancer No history of invasive 5.8 breast carcinoma Other histological Lobular carcinoma in situ No abnormalities detected 16.4 findings Ductal carcinoma in situ No abnormalities detected 17.3 Breast Biopsy Hyperplasia without atypia No hyperplasia 1.9 Hyperplasia with atypia No hyperplasia 5.3 Hyperplasia with atypia and a positive family history No hyperplasia, negative family history 11 Family History First degree relative with pre-menopausal BC No 1 st or 2 nd degree relatives with BC First degree relative with No 1 st or 2 nd degree post-menopausal BC relatives with BC 2 nd degree relative with BC No 1 st or 2 nd degree relatives with BC Two first degree relatives No 1 st or 2 nd degree with BC relatives with BC 1 Adopted from Singletary et al Morimoto et al HRT, Hormone Replacement Therapy BC, Breast Cancer

21 Breast Carcinoma Histologies Breast cancer is a heterogeneous disease compromising numerous distinct entities. Clinically, malignant breast tumors present as ill-defined masses, variable in size, arising anywhere in the breast parenchyma or accessory breast tissue (Moinfar, 2007). Once diagnosed, invasive breast cancers may be classified in a number of different ways. One of the ways to classify breast carcinoma is based on histopathologic features. Invasive breast carcinomas may be classified into biologically and clinically meaningful subgroups by histology types and histologic (nuclear) grades according to their growth patterns and degree of differentiation (how closely they reflect normal breast epithelial cells) (Rkha et al., 2010; Weigelt et al., 2010). Among all the tumor types, invasive ductal carcinomas (IDC) of no special type or not otherwise specified (NOS) are the most common type of invasive breast cancers. IDC represent approximately 60 75% of all breast cancers and constitute a diagnosis of exclusion (e.g. a malignant tumor that cannot be classified as any special type of breast carcinoma) (Li et al., 2003; Li et al. 2005a; Moinfar 2007). The histomorphology of the IDC tumors are highly variable, ranging from low- to high-grade carcinomas (Moinfar, 2007). Most IDC are histologically graded based on a modified Bloom and Richardson system (Nottingham (Elston and Ellis) Grading System (NGS)) which is based on three morphologic features: the degree of tubular formation, nuclear atypia, and mitotic activity/count (Moinfar, 2007; Elsten and Elis, 1991) (Table 3). 21

22 Table 3. Histologic Grading: Nuclear Scoring System 1,2 Score Description Tubule formation 1 Tumors show tubule formation with visible lumens in a majority (more than 75%) of the lesion. 2 Tumors show a moderate degree of tubule arrangement (10 75%) in areas of solid tumor growth. 3 Tumors show little (less than 10%) or no tubules formation. Cells appear to grow in sheets or cords. Nuclear 1 Tumors have uniform or regular, small nuclei or pleomorphism exhibit minimal variations. (nuclear atypia) 2 Tumors show a moderate degree of variation in nuclear size and shape and occasional nucleoli. 3 Tumors show marked variation in nuclear size and shape or contain bizarre nuclei, often with irregular chromatin distribution and one or more prominent nucleoli. Mitotic count 1 In the most mitotically active areas, tumor has a count of 0-9 mitotic figures per 10 high-power fields. 2 In the most mitotically active areas, tumor has a count of mitotic figures per 10 high-power fields. 3 In the most mitotically active areas, tumor has a count 20 or more mitotic figures per 10 highpower fields. 1 Elston and Ellis, Page and Anderson, 1989 Degree of differentiation Proliferative activity Special subtype carcinomas make up the remaining approximately 25% (Weigelt et al., 2010) of all invasive breast carcinomas. Each special subtype is identified by distinct morphological and cytologic patterns (Li et al., 2005; Weigelt et al., 2010). According to the latest edition of the World Health Organization (WHO) classification several distinct entities are recognized and include invasive lobular carcinoma, apocrine carcinoma, and other less frequent types. For this project, we will focus on the apocrine subtype and it will be further discussed in a subsequent section of this text. 22

23 Molecular Classification of Breast Carcinoma In addition to being classified by breast cancer histology, each breast cancer can also be classified by the expression of various biomarkers. The use of predictive markers such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor type 2 (HER2), among others, has been utilized to identify high-risk phenotypes and to select the most effective therapy for a particular patient (Weigelt et al., 2010; Presson et al., 2011). Earlier studies utilizing microarray-based gene expression profiling (Sorlie et al., 2001; Sorlie et al., 2003; Farmer et al. 2005; Hu et al., 2006; Sortiriou et al., 2006) analyzed human breast carcinomas at the molecular level. These studies revealed that each breast tumor has its own unique molecular profile. Almost all of these molecular studies showed distinct differences in hormone receptor-positive and hormone receptornegative diseases. The molecular classification of breast cancer often begins with classifying each individual breast tumor into one of the two major breast cancer subtype profiles: ER-positive or ER-negative groups. These can then be further subdivided into additional subgroups with distinct biological and clinical significance (Sorlie et al., 2001) (Table 4). Additionally, breast cancers are also often described in terms of luminal and basal subgroups corresponding to their likely origin based on hormone receptor status. Approximately 75% of breast tumors express ER (Niemeier et al., 2010). ERpositive tumors typically express PR, ER-responsive genes, and other genes that encode typical proteins of luminal epithelial cells. (Niemeier et al., 2010; Sorlie et al., 2001). Due to the expression of luminal epithelial proteins ER-positive tumors may also be referred 23

24 to as the luminal subgroup. This group can be further divided into a luminal A and luminal B subgroup, depending on the expression of proliferation-related genes. Table 4. Molecular Classification of Breast Cancer Based on Gene Expression ER positive ER status Molecular Subtype Gene Expression Grade Luminal A 1,3 ERα Low GATA binding protein 3 B-cell CLL/lymphoma 2 luminal CK8 luminal CK18 X-box binding factor 3 hapatocyte nuclear factor 3α estrogen-regulated LIV-1 ERBB3 ERBB4 Luminal B 1,3 Moderate / CK 8 Moderate / CK 18 High ER negative HER2-positve 2,3 HER2 HER2-related genes BLBC 2,4 basal CK 5 basal CK 6 basal CK14 basal CK 17 fatty acid binding protein 7 EGFR p-cadherin fascin caveolins 1 and 2 αβ-crystallin Normal-breast like 5,6 CK, cytokeratin ERRB, receptor tyrosine-protein kinase EGFR, Epidermal Growth Factor Receptor adipose-related genes non-epithelial cell type-related genes luminal marker 1 Niemeier et al., 2010, 2 Rakha et al., 2007, 3 Sorlie et al., 2001, 4 Green and Lin Nielsen et al., 2004, 6 Perou et al.,

25 The remaining 25% of breast tumors lack ER expression. ER-negative tumors can be further subdivided into three groups: HER2-positive, Basal-like Breast Cancer (BLBC), and normal breast-like (Sorlie et al., 2001; Niemeir et al., 2010). Due to the expression of basal/myoepithelial markers (Green and Lin 2012) BLBC are also referred to as the basal subgroup. In addition to the use of microarray-based gene expression profiling to define molecular classes, several studies (Farmer et al., 2005; Carey et al., 2006; Parker et al. 2009) have also explored the use of immunohistochemistry-based biomarkers to define various molecular classes (Table 5). Biomarkers, rather than traditional prognostic factors, have become an important factor in determining a patient s clinical management (Goldhirsh et al., 2006). This may be because the differences in biomarkers, such as hormone receptor status and protein expression, are thought to be reflective of differences in gene expression of breast tumor cells (Table 4, Table 5). Thus, the differences in gene expression patterns among breast cancer subtypes are also likely to reflect basic differences in the cell biology of the tumor (Sorlie et al., 2003). The identification of morphimmunohistologic biomarker correlates (between molecular subtypes and histology) may not only expand our knowledge of breast carcinoma but may provide useful clinical information for application in routine practice. 25

26 Table 5. Hypothesized Association Between Molecular Subtype Classifications with Histological Assignment and Immunohistochemical Biomarkers ER and/or PR positive subtype ER negative subtype Molecular subtype 2 Luminal A Lumina B ER, PR, HER2 ER + (-) PR+ / - HER2 (+) HER-positive ER - PR- HER2+ BLBC ER - PR- HER2- Normal breast-like ER - PR unknown HER2- Molecular Apocrine ER - PR- HER2+/- 1 Adapted from Weigelt et al Perou et al Additional markers CK5/6 +/- EGFR+/- CK5/6 + EGFR+ CK5/6 + EGFR+ AR+ CK5/6 +/- EGFR+/- Histological type Apocrine Lobular Micropapillary Mucinous Neuroendocrine Pleomorphic lobular Tubular Apocrine Lobular Micropapillary Pleomorphic Acinic cell Adenoid cystic Medullary Metaplastic Pleomorphic lobular Secretory Medullary Metaplastic Apocrine Pleomorphic lobular Molecular Apocrine Breast Cancer A new class of breast cancer called molecular apocrine tumors has been suggested as a breast cancer subtype based on increased expression of androgen receptor (AR) (Farmer et al., 2005; Lopez-Garcia et al., 2010). Immunohistologically, molecular apocrine breast tumors are typically ER- and PR-negative and AR-positive and share some features with the HER2-positive type (Table 3, Table 4). HER2 gene amplification 26

27 is more common in this group in comparison to the other breast carcinoma subtypes (Farmer et al., 2005). In general, it is this HER2 amplification in apocrine carcinomas that is the single most significant genetic alteration (Farmer et al., 2005; Banneau et al., 2010). Therefore, apocrine carcinomas are thought to be of either a molecular apocrine or HER2-positive molecular subtype (Weigelt et al., 2010). Upon histological examination, these tumors have some morphological hallmarks of apocrine tumors. A 2012 study done by Francini de Mattos et al. demonstrated that the apocrine molecular profile was strongly associated with apocrine morphological features identified in their series. They concluded that apocrine features are associated with a molecular apocrine molecular profile and a higher malignancy grade. Apocrine Carcinomas Apocrine carcinomas are rare, corresponding to 0.3 4% of all invasive ductal carcinomas. Apocrine carcinomas are thought to evolve from epithelial cells in terminal ductal-lobular units in a stepwise manner, going through different stages that may involve metaplasia, hyperplasia, atypia and carcinoma in situ (Celis et al., 2009). In addition, apocrine carcinomas are characterized by epithelium with apocrine differentiation (Monifar 2007). Apocrine differentiation describes the process of when normal breast epithelial cells begin to differentiate and resemble the androgen-dependent scent/sweat gland cells normally found in the axilla and perineum (Farmer et al., 2005). The identification of special subtype carcinomas based on histologic morphology can provide valuable prognostic information. Literature data indicate that apocrine 27

28 carcinomas display a prognosis similar to IDC, with outcome influenced by the expression of biological features such as HER2, EGFR, and hormone receptor status (ER,PR,AR) (Vranic et al., 2010). Therefore, treatment for apocrine tumors should be tailored according to tumor biology and disease extension, analogous to the approach used for IDC (Goldhirsch et al., 2011). Genotype-Phenotype Correlation of Breast Cancer Susceptiblity Syndromes Approximately 5 10% of all breast cancers are thought to be a result of an underlying hereditary breast cancer syndrome, in which the patient has a germline mutation in a cancer susceptibility gene (Berliner et al., 2007). Mutations in the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) genes (as seen in Hereditary Breast and Ovarian Cancer syndrome (HBOC)) account for the largest portion (approximately 30%- 50%) of hereditary breast cancer (Verhoog et al., 2000). Other low-prevalence-highpenetrance genes that are related to other hereditary cancer syndromes include TP53 (Li- Fraumeni syndrome), STK11 (Peuz-Jeghers syndrome), CDH1 (Hereditary Diffuse Gastric Cancer syndrome), and PTEN (PTEN Hamartoma Tumor syndrome, PHTS) (Table 6). Research has been done exploring the potential of special subtype carcinomas being characterized by germline alterations. Furthermore, several studies have also evaluated the histopathologic features and the immunohistochemical markers of several of these genetic syndromes (Table 1, Table 7) such as BRCA1 and BRCA2 associated breast cancers (Table 7). 28

29 Table 6. Breast Cancer-Associated Cancer Predisposition Syndromes Syndrome Gene Estimated breast cancer risk Hereditary BRCA1 Up to 65% risk by the Breast & age of 70 8 Ovarian BRCA2 Up to 47% risk by the syndrome age of 70 9 Li- Fraumeni syndrome Peutz- Jeghers syndrome PTEN Hamartoma Tumor syndrome (PHTS) Hereditary Diffuse Gastric Cancer TP53 STK11 PTEN CDH1 50% - 60% risk by the age of 45 1 Other associated characteristics/cancers Ovarian cancer, pancreatic cancer, prostate cancer, melanoma Soft tissue sarcoma, osteosarcoma, brain tumors, adrenocortical carcinoma, leukemia, lymphoma 45% risk by the age of Melanocytic macules of the lips, buccal 70 2 mucosa, and digits; gastrointestinal hamartomatous polyps, sex cord tumors, sertoli cells tumors of the testes, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer. 85% lifetime risk by the age of 85, with 50% penetrance by the age of 50 4 Epithelial thyroid cancer, endometrial cancer, colorectal cancer, kidney cancer, melanoma 4 Lifetime risk Diffuse gastric cancer, signet ring colon 39 52% 5,6 cancer 7 syndrome 1 Bradbury et al., Hearle et al., Starink et al., Tan et al., Pharoah et al., Kaurah et al., Brooks-Wilson et al., Antoniou, Chen et al., 2007 Mutations in breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) confer Hereditary Breast and Ovarian Syndrome (HBOC). Immunohistochemical (Foulkes et al., 2003; Lakhani et al., 2005) and expression array (Sorlie et al., 2003) studies on BRCA1- associated breast cancers demonstrate the great majority (more than 75%) of these tumors display a triple-negative phenotype, a basal-like phenotype, or both. Literature data indicates that patients with triple-negative tumors have an approximate 10% risk of 29

30 having a germline BRCA1 mutation (Kandel et al., 2008). This established genotypephenotype correlate is an example of how the characterization of specific histopathologic features and molecular profiles with specific germline alterations impacts cancer genetics risk assessment, clinical risk discussion, and counseling. Table 7. Expression of Immunohistochemical Markers in BRCA1- and BRCA2- Related Breast Cancers Relative to Sporadic Breast Cancers 1 BRCA1 related cancers BRCA2 related cancers 2 Cytokeratin 5/6 Cyotkeratin 14 Cytokeratin8/18 ER = PR = Her2/neu = EGFR Laminin? P-cadherin? Caveolin 1 = Bax BCL2 1 Adapted from van der Groep et al., Few data available The identification of genotype-phenotype correlates may aid genetic counselors and health care professionals in identifying individuals from the general population who would benefit from genetic testing. However, little is known about the histopathologic features and molecular profiles of breast cancer caused by germline muations in genes other than BRCA1 and BRCA2, such as PTEN. Even though germline mutations in PTEN account for less than 1% of hereditary breast cancer, germline PTEN mutations confer a significant increased risk in a number of malignancies including breast cancer (Tan et al., 2012) (Table 9). The limited information that is known about the histopathologic features 30

31 and molecular profiles associated with PHTS-associated carcinomas will be discussed in a subsequent section of this text. PTEN The PTEN (Phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene, also known as MMAC1 (mutated in multiple advanced cancers) or TEP1 (TGFβ-regulated and epithelial cell-enriched phosphatase) is located on chromosome 10q23.3, a genomic region that undergoes loss of heterozygosity (LOH) in many human cancers (Cantley and Neel, 1999). Somatic deletions or mutations of this gene, which result in its inactivation, occur in multiple primary sporadic tumor types including glioblastomas, endometrial carcinomas, melanoma, advanced prostate cancer, breast cancer and other assorted malignancies. As previously mentioned, germline mutations of PTEN have been found to occur in a cancer susceptibility syndrome referred to as the PTEN Hamartoma Tumor syndrome, abbreviated as PHTS (Eng, 2003). PTEN acts as a tumor suppressor gene through the action of its dual-specificity phosphatase protein product. It is both a lipid and protein phosphatase and contains a protein tyrosine phosphatase domain which is able to phosphorylate protein substrates on serine, threonine and tyrosine residues (Myers et al., 1997). PTEN targets and binds to a number of different substrates (Tamura et al., 1998; Liliental et al., 2000). Potential targets of PTEN range from membrane bound receptor tyrosine kinases and cytoplasmic signaling molecules to transcription factors in the nucleus (Yin and Shen, 2008). 31

32 However, the PI3K/AKT pathway is regarded as the primary physiological target of PTEN (Leslie and Downes, 2004). Specifically, as a lipid phosphatase, PTEN dephosphorylates phosphoinositol- 3,4,5-triphosphate (PIP3) to phosphoinositol-4,5-triphosphate (PIP2). PIP3 is a key signaling component of the phosphoinositol-3-kinase (PI3K) pathway (Maehama and Dixon, 1998). PTEN is one central negative regulator of the PI3K signal transduction cascade. PI3K Signaling Pathway The PI3Ks are members of a family of intracellular lipid kinases that phosphorylate the 3 -hydroxyl group of phosphatidylinositol and phosphoinositides (Engelman et al., 2006). The PI3K holoenzyme, in response to signals transmitted via growth factor receptor tyrosine kinases or G-protein coupled receptors phosphorylates phosphatidylinositol-4, 5-bisphosphate (PIP2) at the 3 OH position of the inositol ring to form PIP3. This reaction leads to the activation of intracellular signaling pathways that regulate many cellular functions, many of which are associated with cell proliferation, survival, and migration. The principal target of PIP3 is the protein serine/threonine kinase AKT. Binding of PIP3 to AKT leads to the membrane recruitment of AKT and subsequent phosphorylation by the mtor (mammalian target of rapamycin)-rictor kinase complex (Sarbassov et al., 2005) and by PDK1 (3-phosphoinostide-dependent kinase) (Mora et al., 2004). This leads to the full activation of AKT, which in turn phosphorylates many substrate targets thereby regulating a range of cellular functions (Figure 1). 32

33 PTEN/pAKT Pathway in Breast Cancer PTEN is a powerful and distinct tumor suppressor. PTEN exerts tumor suppressor functions by the regulation of proliferation and survival, cell migration, invasion, and angiogenesis, and maintains genomic stability through multiple pathways (Chen et al., 2007; Trotman et al., 2007). One of these pathways is the PTEN/PI3K/AKT pathway. PTEN negatively regulates this pro-survival pathway through its lipid phosphatase activity by converting PIP3 to PIP2. Thus, the loss or inactivation of PTEN would result in a net increase of PIP3 and effective oncogenic activation of the PI3K/AKT pathway. Loss of PTEN expression occurs in a large percentage of human cancers. In breast cancer, loss of PTEN function occurs in up to half of sporadic breast tumors (Nassiri et al., 2009; Jones et al., 2012). Although a very small percentage of breast tumors harbor PTEN mutations (Feilotter et al., 1999), about 40% display loss of heterozgosity at 10q23 (Bose et al., 1998; Singh et al., 1998; Feilotter et al., 1999), and nearly half display aberrant promoter methylation (Garcia et al. 2005). Furthermore, approximately 40% exhibit a loss of PTEN expression upon immunohistochemistry studies (Depowski et al., 2001; Perez-Tenorio et al., 2007). Research done by He and colleagues (2012) indicated that the loss of PTEN leads to elevated pakt in both the cytoplasm and the nucleus of the cell. These authors demonstrated that in breast cancer, PTEN suppresses nuclear pakt mainly through decreasing pakt nuclear translocation by reducing the PIP3/pAKT reservoir in the cytoplasm and thereby preventing the activation of the associated pro-survival pathway. Furthermore, data from He et al., (2012) suggested that patients with germline PTEN 33

34 mutations had naturally elevated pakt due to PTEN s inability to down-regulate cytoplasmic and nuclear pakt levels in the cell. Given the established interactions within the PTEN/PIK3/AKT pathway, these data support the concept that cells with diminished or absent PTEN activity would be unable to suppress the PI3K pathway, resulting in elevated pakt activity. Thus, the up-regulation of AKT is a plausible explanation for many component phenotypes of PHTS. Taken together, individuals with germline PTEN mutations would be predisposed to various malignancies due to the loss of the PI3K pathway regulator, PTEN. PTEN Hamartoma Tumor Syndrome (PHTS) PTEN Hamartoma Tumor syndrome (PHTS) describes any patient with a germline mutation in the tumor suppressor gene PTEN. It was historically thought that several syndromes including Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), and Proteus-like syndrome (PLS) were genetically unrelated to one another. However, after the discovery of germline PTEN mutations in most families with CS and the subsequent identification of PTEN mutations in patients with BRRS and PLS, it is now common knowledge that these conditions are allelic. While Proteus syndrome (PS) was also once included in this umbrella, it has since been found that most patients with PS have an activating somatic mutation in AKT1, the gene immediately downstream of and normally suppressed by PTEN (Marjoirie et al., 2011). As discussed previously, PTEN is the central negative regulator of the PI3K/AKT signal cascade; it suppresses growth and proliferation by suppressing AKT. Although the specific genes involved differ, the major disease pathway involved is the same between PHTS and PS. 34

35 PHTS is now well-recognized as a highly variable, autosomal dominant disorder with age- and gender-related penetrance characterized by malignant and benign lesions. A diagnosis of PHTS may be suspected based on a number of clinical signs (Table 8). However, hallmark features of PHTS, such as macrocephaly and mucocutaneous features, are under-recognized within the medical community (Liaw et al., 1997). Thus the subtlety of findings and the variable manifestation of PHTS (many of which can occur in isolation in the general population) often makes the recognition of these features a challenge. 35

36 Table 8. List of Cancers, Benign Growths and CNS Involvements Associated with PHTS Benign growths Hamartomas 1 Acral keratoses 3 Trichilemmomas 1,3 Mucocutaneous papillomatous papules 1,3 Early onset uterine leimyoma 1 Other benign findings Fibrocystic breast disease 1 Horseshoe kidney Bicornuate uterus Thyroid lesion Associated malignancies Breast cancer 4 Thyroid cancer 4 Endometrial cancer 4 Colorectal cancer 4 Kidney cancer 4 Melanoma 4 The hamartomatous lesions observed in PHTS can arise in any of the three embryonic germ cell layers. 1 Thus, hamartomas can be found in virtually every organ, but most commonly in the skin and gastrointestinal (GI) tract. Defined as flesh-colored or slightly pigmented smooth or warty papules on the upper surface of hands and feet. Defined as flesh-colored hamartomas of the outer sheath of the hair follicle. Defined as mucocutaneous lesions, or raised areas of the skin, found on the face, oral mucosa, and acral surfaces. Approximately half of women with PHTS develop uterine leiomyomas or fibroids, typically by the third decade of life 5 Benign changes that occur in the breast tissue. Also known as benign breast disease. A congenital partial fusion of the kidneys resulting in a horseshoe shape A congenital uterine malformation resulting in a heartshaped uterus Have been noted to occur up to 75% of PHTS patients including adenomas, hamartomas, multinodular goiter, and Hashimoto s thyroiditis 6,7 CNS involvement Lhermitte-Duclos disease(ldd) Macrocephaly A rare, slow growing, benign hamartomatous lesion of the brain, characterized by dysplastic gangliocytomas of the cerebellum, which can lead to hydrocephalus, increased intracranial pressure, ataxia, and seizures An abnormally large head and/or brain 1 Eng Eng Jornayvaz and Philippe, Tan et al Gustafson et al., Harach et al., Starink et al

37 Before the identification of the cancer susceptibility gene PTEN as the PHTS gene, the estimated incidence of CS was estimated to 1 in 1,000,000 (Nelen et al., 1996). After its identification, molecular-based studies revealed the incidence of germline PTEN mutations in the Dutch population to be approximately 1 in 250,000 (Nelen et al. 1999). It is most likely that this reflects a general underestimation, as these early studies identified patients due to hallmark dermatologic features, and it is now recognized that PHTS is associated with a high degree of phenotypic variability and an expanding spectrum of clinical features. In addition to various mucocutaneous findings, germline PTEN mutations also confer an increased risk for a number of malignancies (Table 8). As discussed earlier, many features in PHTS are common or may be easily overlooked by healthcare professionals involved in the care of the general population. As a result, individuals with potential PTEN mutations may not receive referral for cancer genetic counseling and germline testing prior to a cancer diagnosis. Thus, these individuals remain unaware of their increased risk for cancer. PHTS-Associated Breast Cancer All patients with a germline PTEN mutation, and thus a PHTS diagnosis, are at increased risk for a number of malignancies (Table 9). The most significant association of PHTS with organ-specific cancer susceptibility is with the breast (Tan et al., 2012). 37

38 Table 9. Summary of Carcinomas Occurring in PHTS Cancer type Breast (female) Epithelial thyroid 2 GP risk 1 Life time cancer risks in patients with PHTS Median age of onset in general population cases 1 Median age of onset of presentation in patients with PHTS Specific histopathology associated with PHTS ~ 12% ~85% 5 61y Late 30s to early 40s 5 IDC 7 Molecular apocrine 8 ~ 1.0% 35% 5 50y 37y 2 Follicular and papillary 2,4? Endometrial <2.64% 28.2% 5 61y Late 30s to early 40s 5 CRC 4.96% 9.0% 5 69y ~ 40y 5? Kidney 1.60% Kidney & renal pelvis ~34% 5 64y for kidney & renal pelvis ~ 40y 5 Papillary and chromophobe 6 2:1 M:F ratio Melanoma 1.99% 6% 3 61y ~35y 5? 1 SEER data 2 Ngeow et al., Starink et al., Tan et al., Mester et al., Schrager et al., Banneau et al Heald et al., 2010 IDC, invasive ductal carcinoma CRC, colorectal cancer GP, general population Breast cancer was first clearly recognized to be a component feature of CS in 1978 (Brownstein et al. 1978), and was felt to be the most common PHTS malignancy. Previously, lifetime breast cancer risks was estimated to range from 25-50%, (Starink et al., 1986; Schrager et al.,1998; Longy and Lacombe, 1996) with those women without cancer found to have benign breast disease which was both extensive and bilateral (Schrager et al., 1998). Current research estimates that the lifetime breast cancer risk for 38

39 women with PHTS approaches 85% (Tan et al., 2012). Consequently, breast cancer carries the highest malignancy risk in individuals with PHTS (Tan et al. 2012). As with other hereditary breast cancer syndromes, the diagnosis of breast cancer usually occurs at younger ages compared to the general population, with the average age diagnosis between the late 30s to the early 40s (Tan et al. 2012). Schrager and colleagues (1998) is one of the only known studies that performed a systematic study looking at the histology of CS-related breast tumors in a series of nineteen women with CS (59 cases) and unknown PTEN mutation status. These authors described a spectrum of extensive benign histopathological findings including proliferative and non-proliferative fibrocystic change, intraductal papillomatosis, fibroadenomas, and hamartomas. Benign breast disease or FCC were often noted to be bilateral and extensive. These authors noted that common lesions such as fibroadenomas, apocrine metaplasia, microcysts and adenosis, as well as a form of mammary hamartomalike lesions were the characteristic lesions of their series. These authors also described a spectrum of malignant breast lesions. In contrast to the extensive range of benign lesions observed, the malignant lesions observed in this series showed a usual spectrum of pathology similar to the general population, characterized by the predominant type of breast cancer, ductal carcinoma. However, lobular and tubular carcinomas histology subtypes were also observed. A more recent study by Banneau et al also evaluated the histological features of 15 tumors from patients with CS, all of whom had germline PTEN mutation and thus PHTS. This study also utilized RNA microarray and immunohistochemistry 39

40 (IHC) to define a CS-associated molecular profile. This histological study showed that 7 of 15 (47%) of tumors in their series had apocrine features and 3 of 15 (20%) had an IHC profile consistent with a molecular apocrine tumor. The RNA microarray data demonstrated that the gene expression profile of tumors from CS patients showed considerable overlap with the molecular apocrine molecular profile. In addition, these authors hypothesized that the activation of the HER2/PI3K/AKT pathway by the loss of PTEN promotes breast tumors with apocrine features. Taken together, these studies suggest that certain histopathologic features in CS patients as well as a molecular apocrine molecular profile may be distinct to PHTSassociated carcinomas. However, the identification of distinct PHTS-associated histopathologic features and the confirmation of the association between a molecular apocrine profile with PHTS-associated breast carcinomas may help identify individuals with potential germline PTEN mutations. Moreover research findings could also impact cancer genetics risk assessment, clinical risk discussion, and counseling for PHTS. Screening for Germline PTEN Mutations It was not until recently that there were criteria based on large prospective patient cohorts that could be used to select candidate individuals for germline PTEN testing. In 2011, Tan et al. (2011) presented the first evidence-based clinical practice model to select patients for genetics referral and PTEN germline testing. By evaluating the correlation of clinical phenotype with PTEN genotype the authors were able to create an easily accessible web-based PTEN risk calculator. 40

41 This particular study performed PTEN mutation scanning including promoter and large deletion analysis on 3042 samples that satisfied relaxed CS clinical criteria (Tan et al., 2011). Utilizing these data, Tan et al., (2011) developed an optimized clinical practice model that would estimate a pretest probability of a germline PTEN mutation based on a semi-quantitative score known as the Cleveland Clinic (CC) score. For adults, a clinical threshold score of 10 or greater (corresponding to an estimated >3% pretest probability of a pathogenic PTEN mutation) led to the recommendation for referral to a genetics professional to consider PHTS (Tan et al., 2011). At this threshold, the projected sensitivity for detecting a patient with a PTEN mutation is 90% (Tan et al., 2011). The performance of the CC weighted scoring system exceeded that of the NCCN 2010 testing criteria (Appendix A1) demonstrating improved sensitivity and positive predictive value for germline PTEN mutations. In addition to its improved accuracy, the CC scoring system provides an easily obtained individualized probability estimate for a patient s risk of being a germline PTEN mutation carrier (Tan et al., 2011). Such a quantified single-numbered clinical scoring system can clearly communicate the probability of a PTEN mutation in a given individual, and can be easily incorporated into a more simplified risk assessment discussion between patients and healthcare providers (Tan et al., 2011). Thus, the CC score or the PTEN risk calculator represents a tool that can be utilized to appropriately select patients for genetic referral and PTEN germline testing among patients who may express a wide variety of phenotypes within the PHTS spectrum. 41

42 In addition to the CC score, blood-pten and blood-pakt protein expression levels can potentially be used as a screening tool to assess the likelihood for germline PTEN mutations. Based on their findings, Ngeow et al., (2010) hypothesized that blood- PTEN protein expression could serve as a screening tool to predict a germline mutation. These authors demonstrated that low blood-pten protein expression correlates with positive germline PTEN mutation status in individuals with thyroid cancer (Ngeow et al., 2010). It could be possible that, like thyroid cancer, PTEN and pakt protein expression levels may also be used as predictors of a germline PTEN mutation in CS and CS-like presentations of breast cancer. The Clinical Management of PHTS Patients Once a germline PTEN mutation is found in an individual, appropriate management and screening recommendations can be made. Tan and colleagues (2011) proposed a comprehensive approach to surveillance of patients with PTEN mutations taking into account new data about age-related cancer risks (Table 10). 42

43 Table 10. Recommendations for Diagnostic Workup and Cancer Surveillance in Patients With PTEN Mutations 1 Baseline workup Cancer surveillance from diagnosis from the age of 30 a years Pediatric (<18y) Adult male Adult female Target history and Target history and physical examination physical examination Baseline thyroid ultrasound Dermatologic examination Neurologic & psychologic testing Annual thyroid ultrasound and skin evaluation As per adult recommendation Target history and physical examination Baseline thyroid ultrasound Dermatologic examination Annual thyroid ultrasound and skin evaluation Baseline thyroid ultrasound Dermatologic examination Annual thyroid ultrasound and skin evaluation Annual mammogram (consider breast MRI instead of mammography if dense breast) Annual endometrial sampling or transvagenial ultrasound ( or from 5 yrs before age of earliest endometrial cancer) From the age of 40 * years Prophylactic surgery 1 Tan et al., 2011 As per adult recommendation Biannual Biannual colonoscopy ** colonoscopy ** Biannual renal Biannual renal ultrasound/mri ultrasound/mri Nil Nil Individual discussion of prophylactic mastectomy or hysterectomy * Surveillance may begin 5 years before the earliest onset of a specific cancer in the family, but no later than the recommended age cutoff point. ** The presence of multiple nonmalignant polyps in patients with PTEN mutations may complicate noninvasive methods of colon evaluation. More frequent colonoscopy should be considered for patients with a heavy polyp burden. 43

44 Patient-Tailored Treatment for Individuals with PHTS As previously discussed, PTEN is a lipid phosphatase that acts as a negative regulator of the PI3K/AKT/mTOR pathway, thus it is an important regulator of cell growth and survival. As such, pharmacological inhibitors of this pathway may be exploited for therapy. Current hypothesized therapies of tumors with altered PTEN and/or for individuals with PHTS include pakt and mtor inhibitors. Several compounds that have been designed to inhibit the PI3K/AKT/mTOR pathway are in clinical development (Hollander et al., 2010). Murine studies have demonstrated that inhibitors of mtor, such as rapamycin, and its analogues can prevent tumorigenesis (Milam et al., 2007; Blando et al., 2009). Newer pathway inhibitors currently in research include dual PI3K-mTOR inhibits, AKT inhibitors and mtor complex catalytic site inhibitors (Yuan et al., 2009; Cortney et al., 2010; Pal et al., 2010). These compounds target more upstream components and may be better able to compensate for PTEN loss by circumventing AKT feedback activation (Hollander et al. 2010). However, these drug therapies are likely to be more toxic than pure mtor inhibitors (Hollander et al., 2010) and because the PI3K signaling pathway is an important regulator of numerous normal cell processes, agents that target this enzyme could potentially cause a number of side effects (Cully et al., 2006). The efficacy and safety of PI3K pathway inhibitors will depend on our understanding of the role of PTEN, PI3K, AKT and mtor in normal cell function and in tumor formation. In addition to PI3K/AKT/mTOR pathway inhibitors, another therapeutic approach may include the identification and inhibition of additional targets in a cancer specific 44

45 manner. Farmer et al., (2005) proposed the question as to whether molecular apocrine tumors (defined as being ER-, PR-, AR+ and HER2 amplified) would benefit from androgen blockade and/or targeted drug therapies against various androgen and lipid metabolism genes that differ in expression in the apocrine breast cancer compared to other breast cancer subtypes. Other studies proposed the consideration of androgen as a biomarker for treatment decision making in breast cancer (Ogawa et al., 2008), especially in those that fall under the apocrine subtype (Suzuki et al., 2010, Miller et al., 1985). Interestingly, Naderi et al., (2008) demonstrated that there is a functional crosstalk between the AR and HER2 pathways in ER-negative breast cancer. These authors noted that testosterone stimulates the proliferation of molecular apocrine breast cell lines, and that this effect can be reversed using anti-androgen agent flutamide and an ErbB2 inhibitor, AG825. Furthermore, Naderi et al., (2008) demonstrated that the combined application of AR and Cdc25A inhibitors is a promising therapeutic strategy in molecular apocrine breast cancer. By expanding our knowledge of breast cancer arising in women with PHTS, we will be able to identify potentially prognostic biomarkers and histopathologic features of PHTS-associated breast cancer. But more importantly, research results may aid in the identification of potential PTEN germline mutation carriers and the subsequent referral for genetic counseling, molecular testing, and medical management. 45

46 AIM 1: MATERIALS AND METHODS Specimen Collection Research participants provided informed consent (Appendix B1) for the Cleveland Clinic Institutional Review Board protocol 8458 (IRB#8458 protocol) titled Molecular Mechanisms Involved in Cancer Predisposition nicknamed The PTEN Study. The purpose of the study was to look at the frequency of the particular characteristics such as cancer diagnoses among individuals with PTEN alterations by combining clinical patient information with DNA, RNA, and protein studies of the PTEN gene and related pathways ( Adult probands (> 18 years of age) who met relaxed International Cowden Consortium criteria for the diagnosis of Cowden syndrome, had a score of 10 or greater per the Cleveland Clinic PTEN Risk Calculator, or had a known PTEN mutation or variant of uncertain significance were eligible. Pediatric patients (< 18 years of age) with macrocephaly plus at least one of the following were eligible: Autism/mental retardation/developmental delay Lipoma, biopsy-proven trichlemmomas, oral papilloma, or hemangioma Arteriovenous malformation One or more gastrointestinal polyp Once accrued, germline DNA was extracted from each individual s leukocytes and subjected to comprehensive PTEN mutation analysis of the PTEN coding regions, 46

47 exon/intron boundaries, flanking intronic sequences of up to approximately +/- 40 bases, and the core promoter region. For this project, enrolled adult research participants with a deleterious germline PTEN mutation and personal history of invasive breast carcinoma were selected. This led to the identification of 43 patients eligible for inclusion in this project. Each eligible participant s medical records were examined and primary documentation of medical records/pathology reports were obtained by the researcher for confirmation of the breast cancer and precise histology. After reviewing pathology reports and records, the researcher requested hematoxylin and eosin stained (H&E) slides for morphological diagnosis and pathology re-review. Original tissue blocks or 5-7 routine paraffin-embedded 4-6 micron slides of the area(s) of interest were requested from the respective housing institution. During this request process some participants specimens were discovered to be unavailable, as they had been destroyed or were unattainable due to missing or expired consent forms and/or legal documentation. Thus, of the 44 eligible participants, specimen materials representative of 25 participants were obtained. Figure 3 is a schematic of the specimen collection process. 47

48 Figure 3. Schematic of Specimen Collection Control Group Control study participants were gathered from a separate protocol, Frequency and Clinical Spectrum of Germline PTEN Mutations in a Hospital Population Based Series of Incident Invasive Breast Cancer Cases Study (IRB# Case2107-CC302) nicknamed The PTEN-breast study. The purpose of this study was to determine the frequency of germline PTEN mutations in a population-based series of incident invasive breast cancer cases, to link clinical features at presentation with mutation positive versus negative status, and to study additional genetic causes of susceptibility to breast cancer. ( All participant samples underwent comprehensive PTEN mutation analysis as previously discussed for the patients in The PTEN Study. Participants with a germline PTEN mutation were excluded from the pool of eligible control participants. 48

49 Each eligible participant s Cleveland Clinic medical record and primary documentation of medical records/pathology reports were reviewed for confirmation of the histology and grade of the invasive breast cancer. Eligible participants were categorized on the variables of mean age of cancer diagnosis, race, and breast cancer histology. Similar to the study population, after the review of pathology reports and records, original tissue blocks of the area(s) of interest were requested and obtained from within the Cleveland Clinic; a total of four control specimen samples representative of the PTEN mutation positive study population were selected based on age of diagnosis, race, and breast cancer histology. Breast Tissue Pathology In total, 25 histological samples representative of 25 women with PTEN Hamartoma Tumor syndrome (PHTS) and invasive breast carcinoma were examined by the researcher and an independent Cleveland Clinic pathologist (EDK) with a scholarly interest in breast pathology. The number of slides received per case ranged from 1 to 51, with an average of approximately 8 slides per case. Table 11. Types of Histological Samples Received per Specimen Collection Type of Specimen Percentage (n/25) Biopsies 32% (8/25) Lumpectomies 12% (3/25) Mastectomies 40% (10/25) Re-section for new surgical margins 8% (2/25) Fine Needle aspiration (FNA) 4% (1/25) Lactiferous duct excision 4% (1/25) 49

50 In addition one biopsy and three mastectomy tissue blocks were received without corresponding H&E slides, resulting in a total of 29 PHTS-associated invasive carcinoma diagnoses. All obtained H&E slides were reviewed by an independent pathologist (EDK) with the researcher present. H&E slides were examined and detailed descriptions of staging and/or grading category, invasive carcinoma histology, in situ carcinoma histology, and histopathologic features of the background breast epithelium were documented. These data were organized, coded and analyzed utilizing nominal descriptive statistics. Histopathologic findings were compiled and percentages were calculated for the entire study group. The IBM SPSS Statistics 21 Data Editor was used for all statistical analysis. 50

51 RESULTS AIM 1 Patient Demographics The 25 patients in this study included 25 unrelated women with PHTS and invasive carcinoma. Over half (14/25, 56%) of these women had mutations resulting in truncation of the protein, making it the most common PTEN mutation type of this series. The majority (23/25, 92%) of patients were Caucasian, with a mean age of 46 years (range, 27 to 57 years) at the time of their initial breast cancer diagnosis. The mean age of diagnoses for received breast cancer specimens was 48 years (range, 41 to 71). Of note, of these 25 women, two had bilateral disease, three had invasive carcinoma recurrences, and two were post-treatment when specimen samples were collected. Additional patient demographic information is summarized in Table 12. For additional clinical information, please see Appendix B2. 51

52 Table 12. Patient Demographics of Women With PHTS and Invasive Carcinoma Percentage (n/25) Gender Female 100% (25/25) Ethnicity Caucasian 92% (23/25) American Indian/Alaska Native and Caucasian 4% (1/25) American Indian/Alaska Native, African American and Caucasian 4% (1/25) PTEN Germline Mutation Type Missense 32% (8/25) Truncation 56% (14/25) Whole gene deletion 4% (1/25) Promoter 4% (1/25) Unknown 4% (1/25) Invasive Carcinoma Laterality Unilateral 92% (23/25) Bilateral 8% (2/25) Recurrence 12% (3/25) Specimen collection Pre-treatment 92% (23/25) Post-treatment 8% (2/25) 52

53 Pathology Re-review PHTS-Associated Invasive Components Based on Histology and Grade Of the 25 cases of PHTS-associated invasive tumors evaluated for histology and grade. The most common type of infiltrating carcinoma was ductal (with and without apocrine features), present in 80% of cases (20/25).The degree of differentiation ranged from G1 (well-differentiated) to G3 (poorly differentiated) and had a distribution comparable to the general population (Table 13). Table 13. Comparison of the Malignant Lesions Observed in 25 PHTS-Associated Invasive Carcinomas to Those of the General Population Invasive Component Histology General Population (Sporadic BC) 1 Ductal carcinomas constitutes up to 75% of all infiltrating breast carcinomas PHTS-associated Carcinomas 80% of cases were invasive ductal carcinoma Conclusions Similar with the exception that 24% of PHTS cases had apocrine features Grade G1: 23% G2: 38.5% G3: 38.6% 1 D Eredita et al., 2011 G1: 29% G2: 32% G3: 29% Similar Of the 25 cases with invasive carcinoma, 19 also had an in situ component. We noted that the most common type of in situ carcinoma was ductal. The degree of differentiation of the Ductal Carcinoma In situ (DCIS) ranged from well to poor; of the cases for which grade was assessed, the majority of tumors appeared to be less differentiated (moderate to poorly differentiated). 53

54 Histopathologic Features Seen in Association with PHTS-Associated Breast Carcinomas In contrast to the malignant lesions, which contained a more usual spectrum of pathology, the benign lesions showed a larger range of both typical and rare lesions. Upon examination of the total 25 H&E cases, eight cases lacked normal breast epithelium. Consequently histopathologic features around the tumor could not be accurately assessed. Histopathologic features evaluated included apocrine changes observed in the background tissue and in the invasive and in situ carcinomas; fibrocystic changes (FCC); proliferative epithelial changes including FCC, non-obligate precursor lesions and additional high risk lesions; and other benign lesions. Table 12 summarizes the frequencies of the histopathologic features seen in association with PHTS-associated invasive breast carcinomas. Thirteen of these 17 cases were assessed on the degree of FCC involvement. FCC composed of 0% to 60% of the total cellularity observed, with FCC accounting for an average of 13% of the breast epithelium. 54

55 Table 14. Frequencies of Histopathologic Features Seen in Association With PHTS- Associated Invasive Breast Carcinomas histopathologic feature Present Study Pathology Re-review # Cases (n) * Percentage (n/17) Apocrine features 13 77% Apocrine features as part of FCC or proliferative 12 71% epithelial changes Apocrine DCIS ** 5 30% Apocrine features observed within the cancer 4 24% FCC and/or Proliferative Epithelial Changes 13 77% Non-proliferative FCC 13 77% Stromal fibrosis 1 6% Apocrine metaplasia 12 71% Usual Ductal Hyperplasia (UDH) 9 53% Adenosis 0 0% Cyst(s) 1 6% Proliferative Epithelial Changes 10 59% Columnar cell changes 1 6% Florid UDH 1 6% Atypical Ductal Hyperplasia (ADH) 5 30% ADH with apocrine features 3 18% Atypical apocrine hyperplasia 2 12% Sclerosing adenosis 7 41% Apocrine adenosis 5 30% Atypical apocrine adenosis 3 18% Radial scar 1 6% Complex sclerosis lesion 1 6% Atypical lobular hyperplasia 0 0% Other benign Lesions 10 59% Papilloma(s) 9 53% Papilloma (s) with UDH 1 6% Fibroadenoma(s) 1 6% * Histopathologic features of the cases (8) that lacked normal breast epithelium were not included in this analysis. Of note, one of these patients had apocrine DCIS and another had apocrine features observed within the invasive carcinoma ** 2 cases from this series did not have a DCIS component to evaluate 55

56 Of all of the histopathologic features evaluated, three specific histopathological features seemed to be distinctive to breast carcinomas arising in women with germline PTEN mutation in comparison to the general population: atypical apocrine adenosis, atypical ductal hyperplasia, and apocrine features (Table 15). Table 15. Comparison of Distinctive Histopathological Features Observed in PHTS- Associated Invasive Carcinomas to Those of the General Population Feature General Population (Sporadic BC) PHTS-associated Carcinomas Atypical Apocrine Adenosis Percentage 0.4% (37/9340) 1 18% (3/17) P=0.001 Atypical Ductal Hyperplasia Percentage 9% (25/264) 2 30% (5/17) P=0.02 Apocrine features Percentage common 3 77% (13/17) 1 Fuehrer et al., Liberman et al., Wells and El-Ayat, 2007 Lastly, although it was not the focus of the present study, we also examined a total of 15 histological samples representative of 12 women with PTEN Hamartoma Tumor Syndrome (PHTS) who were not diagnosed with an invasive carcinoma at the time of their procedure. For data on the frequencies of histopathologic features seen in association with these prophylactic specimens please see Appendix B2. 56

57 AIM 1: DISCUSSION The histologic features of the PHTS-associated breast carcinomas in this current study are very similar to what is observed within the general population. The predominant type of breast cancer identified is ductal, observed in 80% of cases assessed for histology (included cases of IDC, residual IDC and IDC with apocrine features). In addition, of the cases for which grade was assessed, the distribution of grade also appeared to be no different than that seen in sporadic cancer (D Eredita et al., 2011). Interestingly, none of the carcinomas were classified as apocrine carcinomas although 24% of cases with invasive carcinomas had apocrine features observed within the invasive component. Furthermore, of the total cases with DCIS, 30% had DCIS with apocrine features. Although the invasive histological type and grade observed in PHTSassociated carcinomas were of the common type, many had apocrine features within the invasive and/or in the in situ component. Thus, the presence of apocrine features may be suggestive of a germline PTEN mutation. In this study apocrine features were observed in 77% (13/17) of specimens associated with an invasive component and 87% (13/15) of specimens obtained from prophylactic specimens. This includes apocrine features as part of fibrocystic changes and/or proliferative epithelial changes within the DCIS component and/or within the invasive carcinoma component. Of note, these apocrine features were observed more often in the normal background breast epithelium than in the invasive or in situ components (Table 12 and Appendix B3). This observation supports the conclusion made by Banneau et al., (2010) that germline PTEN mutations predispose individuals to 57

58 develop breast tumors with apocrine features. In addition, these data suggest that mammary tissue from PHTS patients may be more likely to demonstrate apocrine features compared to sporadic breast cancer. The systematic review of hematoxylin-eosin (H&E) stained slides in the present study may provide a more accurate description of the histopathological features observed in the PHTS population than other studies. In addition to apocrine features, the presence of fibrocystic changes (FCC) is a common feature of PHTS-associated breast carcinomas. Our histopathologic findings in the breast tissue of women with PHTS showed extensive fibrocystic changes. This observation supports the conclusion made by Schrager and others (1998) that women with CS-related breast carcinomas typically demonstrated a spectrum of severe or extensive benign histopathologic findings with a range of malignant disease. Of all the cases examined (including specimens associated with an invasive carcinoma component and prophylactic specimens), 84% (27/32) had fibrocystic changes (FCC) that included a range of non-proliferative and proliferative lesions. These changes included a spectrum of common and rare architectural alterations, and, on average, made up 13% (range 0 60) of the total cellularity observed in specimens containing an invasive component and 30% (range 0 60) of the total cellularity observed in prophylactic specimens. Interestingly, individuals without an invasive component, on average, had a larger component of FCC observed than those with invasive carcinoma and/or carcinoma in situ. These data provide more evidence that women with PHTS, regardless of their breast tumor status, have extensive FCC and/or proliferative epithelial changes (Table 12 and Appendix B3). 58

59 Although FCCs are observed clinically in up to 50% and histologically in 90% of breast cancer patients (Bartow et al., 1897; Nielson et al., 1984; Santen and Mansel, 2005; Guray and Sahin, 2006), we found that certain rare FCCs were more commonly observed in our population than in the general population. While FCC such as fibroadenomas, cysts and ductal ectasias, mild hyperplasia and non-sclerosing adenosis are common (Sandison, 1962; Goehring and Moraia, 1997), we noted two rare FCCs that may be unique to PHTS-associated breast carcinomas; those features include apocrine adenosis (AAA) and atypical ductal hyperplasia (ADH). AAA is an uncommon proliferative epithelial breast lesion. In a series of 9340 women, AAA was identified in 37 cases; representing less that 0.4% of all benign breast diagnoses in this series (Fuehrer et al., 2012). Interestingly, our present study observed AAA in 18% (n = 3) of specimens associated with an invasive component and 7% (n =1) of prophylactic specimens. This rate is statistically significant and is higher compared to the reported incidence rate in the general population (Fuehrer et al., 2012). These data suggest that AAA may be a distinctive feature of PHTS-associated breast tumors. ADH is another uncommon proliferative epithelial breast lesion. It is observed in up to 4% of symptomatic benign biopsies (Page et al., 1985; Stomper et al., 1993). Our present study observed ADH in 30% (n = 5) of specimens associated with an invasive component and 27% (n = 4) of prophylactic specimens. Furthermore, we observed three additional cases (18%) of ADH with apocrine features in specimens associated with an invasive component and 3 additional cases (20%) of ADH with apocrine features in prophylactic specimens. The rate of ADH with and without apocrine features is 59

60 statistically significant and is higher compared to the reported incidence rate of ADH in the general population (Page et al., 1985; Stomper et al., 1993). These data suggest that ADH with and without apocrine features may be another distinctive feature of PHTSassociated breast tumors, further strengthening the hypothesis that apocrine features, in general, are characteristic of PHTS-associated breast tissue. In summary, these data provide evidence that PHTS may be more difficult to recognize based on the histology type alone given that the invasive carcinomas arising in women with PHTS are of a common type. Therefore, it becomes more important to also pay attention to the fibrocystic changes that surround the invasive and in situ components. Specifically, we described in detail the histopathological findings in breasts of women with PHTS. Our study results indicate that the finding AAA, ADH with and without apocrine features, and the presence of apocrine features suggest a diagnosis of PHTS. However, because of the rarity of germline PTEN mutations in the breast cancer population as well as the study s small sample size, definitive conclusions cannot be made on disease pathology alone. Despite this recognized limitation, our series provides a detailed characterization of histopathologic features observed in both PHTS-associated carcinomas and PHTS-associated prophylactic samples. The recognition of the clinical features and/or the distinctive types of breast lesions associated with PHTS may assist pathologists in the identification of individuals in whom genetic assessment and testing may be warranted. 60

61 AIM 2: MATERIALS AND METHODS Biomarker Analysis by Immunohistochemical Study The researcher performed PTEN and pakt immunohistochemistry (IHC) on both PHTS-associated breast cancer and control breast cancer tissues. PTEN and pakt IHC protocols were piloted on formalin-fixed paraffin-embedded sections of randomly chosen invasive ductal carcinoma tissue sections. Four to six micrometer thick formalin-fixed paraffin-embedded tissue sections were cut from previously obtained tissue blocks. For description of IHC protocols, please see Appendix C1 and Appendix C2. PTEN and pakt expression of the ductal carcinoma in situ component (if applicable) and the invasive carcinoma component of each tumor were evaluated under a light microscope by an independent pathologist (EDK). PTEN and pakt expression were scored using the scoring system proposed by Perren et al., (1999), which compares the amount of staining observed in the in situ/invasive component with that of the normal breast epithelial tissue. Each component was graded as either 0, 1+ or 2+ where 0 signified no trace of staining, 1+ demonstrated decreased but present staining intensity and 2+ showed increased or equal staining. In addition, the location of staining, whether it was nuclear, cytoplasmic or both was also noted. Figures 4 through 7 demonstrate differences in staining patterns of PTEN and pakt IHC between normal breast epithelium and invasive carcinoma. 61

62 Figure 4. Control PTEN IHC in a Normal Breast Lobule and a Control PTEN IHC in an Invasive Carcinoma The left photograph shows pattern of normal breast epithelium staining for the PTEN antibody on a control sample (patient c.1) at 20X magnification. The staining intensity observed in the normal breast epithelium serves as the comparison group for the invasive and in situ components. The right photograph shows the pattern of staining on an invasive breast cancer control sample (patient c.1) for the PTEN antibody at 20X magnification. This is an example of a sample that would be scored 2+ on the basis that the staining intensity is equal to or greater than that observed in the normal breast epithelium. 62

63 Figure 5. Control pakt IHC in Normal Breast Lobule. Photograph shows pattern of normal breast epithelium staining for the pakt antibody on a control sample (patient c.3) at 20X magnification. The staining intensity observed in the normal breast epithelium serves as the comparison group for the invasive and in situ components. 63

64 Figure 6. Control pakt IHC in an Invasive Carcinoma. Photograph shows pattern of normal breast epithelium staining for the pakt antibody on a control sample (patient c.3) at 20X magnification. This is an example of a sample that would be scored 1+ on the basis that the staining intensity is less than that observed in the normal breast epithelium. 64

65 Figure 7. PTEN IHC in PHTS-associated Invasive Carcinoma and pakt IHC in PHTS-associated Invasive Carcinoma The left photograph shows pattern of normal breast epithelium and invasive staining for the PTEN antibody on a PHTS-associated sample at 20X magnification. A normal breast lobule is present within the upper left corner denoted with arrow marks. The invasive carcinoma is present in the lower right corner of the image, denoted with asterisks. This is an example of a sample that would be scored 1+ on the basis that the staining intensity is less than that observed in the normal breast epithelium. The right photograph shows pattern of normal breast epithelium and invasive staining for the pakt antibody on a PHTS-associated sample at 20X magnification. A normal breast lobule is present within the upper left corner denoted with arrow marks. The invasive carcinoma is present in the lower right corner of the image, denoted with asterisks. This is an example of a sample that would be scored 2+ on the basis that the staining intensity is equal to that observed in the normal breast epithelium. 65

66 Androgen receptor (AR) IHC was performed independently by the Cleveland Clinic s pathology department on PHTS-associated carcinomas. AR expression of the ductal carcinoma in situ component (if applicable) and of the invasive carcinoma component of the tumor were evaluated under a light microscope by an independent pathologist (EDK). AR expression was evaluated based on percentage of nuclear staining (range: negative to greater than 95%) and staining intensity (range: none, weak, moderate to strong nuclear staining). An immunohistochemical percentage greater than 1% was considered a positive result for AR. Figure 8 is a photograph of AR-positive moderately stained PHTS-associated invasive carcinoma. The estrogen receptor (ER), progesterone receptor (PR) and HER2 status for PHTS-associated carcinomas were derived from original tumor pathology reports. Original positive and negative results were used in subsequent analyses. These data were organized, coded and analyzed utilizing nominal descriptive statistics and univariate analysis. Due to small sample sizes, a two-tailed Fisher s exact test was used to examine the significance of association between categorical data. In the instances in which the Fisher s exact test was not available Pearson chi-square was utilized. The Fisher s exact test was also used to compare the observed distribution of data into discrete categories with the distribution expected by theory. Lastly, the McNemar's test was utilized to determine if associations between certain variables existed. 66

67 Figure 8. AR IHC in PHTS-associated Invasive Carcinoma. Photograph shows pattern of normal breast epithelium and invasive staining for the AR antibody on a PHTSassociated sample at 20X magnification. This is an example of a positive AR sample demonstrating moderate staining intensity; with 60% of the invasive component staining positively for AR.. 67