Molecular Pathology of Breast Cancer. The Journey From Traditional Practice Toward Embracing the Complexity of a Molecular Classification

Molecular Pathology of Breast Cancer The Journey From Traditional Practice Toward Embracing the Complexity of a Molecular Classification Aaron M. Gruver, MD, PhD; Bryce P. Portier, MD, PhD; Raymond R. Tubbs, DO N Context. Adenocarcinoma of the breast is the most frequent cancer affecting women in both developed and developing regions of the world. From the moment of clinical presentation until the time of pathologic diagnosis, patients affected by this disease will face daunting questions related to prognosis and treatment options. While improvements in targeted therapies have led to increased patient survival, these same advances have created the imperative to accurately stratify patients to achieve maximum therapeutic efficacy while minimizing side effects. In this evolving era of personalized medicine, there is an ever-increasing need to overcome the limitations of traditional diagnostic practice. Objective. To summarize the molecular diagnostics traditionally used to guide prognostication and treatment of breast carcinomas, to highlight published data on the molecular classification of these tumors, and to showcase Adenocarcinoma of the breast is the most frequent cancer affecting women in both developed and developing regions of the world, with an estimated 1.38 million new cancer cases diagnosed worldwide in 2008. 1 While survival is improving, breast cancer is still a frequent cause of cancer death in women. Furthermore, the burden of this disease is predicted to increase in both the United States and globally. 2,3 Men are not immune, and there are data to suggest the incidence of male breast cancer is also on the rise. 4 Multiple approaches including the dissemination of public education resources, screening programs, and advances in treatment strategies are Accepted for publication January 11, 2011. From the Department of Molecular Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic, Lerner College of Medicine, Cleveland, Ohio. This work was supported in part by federal grants to Nanoprobes (Yaphank, New York) and The Cleveland Clinic (NIH 1R43CA84875-01, NIH/NCI 1R41CA83618-01, NIH/NCI 1R41CA83618-02, NIH 1R43GM64257-01, NIH/NIGMS 1R43GM0628250-01, and NIH 1R43 CA111182-01) and an industry-sponsored grant from Ventana Medical Systems (Tucson, Arizona) to Dr Tubbs. Dr Tubbs receives research support and honoraria for speaking on behalf of Ventana Medical Systems. The other authors have no relevant financial interest in the products or companies described in this article. Reprints: Raymond R. Tubbs, DO, Department of Molecular Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic, Lerner College of Medicine, Cleveland, OH 44195 (e-mail: TubbsR@ccf.org). molecular assays that will supplement traditional methods of categorizing the disease. Data Sources. A review of the literature covering the molecular diagnostics of breast carcinomas with a focus on the gene expression and array studies used to characterize the molecular signatures of the disease. Special emphasis is placed on summarizing evolving technologies useful in the diagnosis and characterization of breast carcinoma. Conclusions. Available and emerging molecular resources will allow pathologists to provide superior diagnostic, prognostic, and predictive information about individual breast carcinomas. These advances should translate into earlier identification and tailored therapy and should ultimately improve outcome for patients affected by this disease. (Arch Pathol Lab Med. 2011;135:544 557) resulting in improved care in developed countries, while nations with limited resources face additional challenges. 5,6 The availability of increasingly complex treatment strategies, including combination chemotherapy regimens and targeted biologic therapeutics, necessitates an increasingly accurate and sophisticated means to classify carcinomas of the breast and to stratify patients to the most appropriate treatment regimen. 7 While much work has been recently published on the molecular classification of breast carcinoma, evaluation, assimilation, and application of these discoveries should be looked upon by practicing pathologists, and pathologists-in-training, as an opportunity to help shape the evolving era of personalized medicine. 8,9 Here, we review the historical, current, and developing use of molecular testing in the diagnosis, prognosis, and treatment of breast carcinoma, with an emphasis on summarizing evidence for a molecular classification of carcinoma subtypes and an advocation for incorporating molecular technologies into standard practice. MORPHOLOGIC CLASSIFICATION AND BIOMARKER ESTABLISHMENT The difficulty of predicting both the pathologic and clinical course of breast carcinoma has been long appreciated by those studying the disease. 10 Traditional methods of classifying breast carcinomas have relied upon an observer s ability to reproducibly recognize features 544 Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al

commonly occurring along the spectrum of morphology that appears as a primary breast tumor. Early classification systems, based solely upon a description of morphology, shared little agreement. 11 Much additional work had to be accomplished before reaching the consensus that currently comprises the 2003 edition of the World Health Organization (WHO) classification of tumors of the breast. 12 While most invasive breast carcinomas without a specific histologic subtype fit into the category of invasive duct carcinoma, not otherwise specified, the current WHO classification of tumors of the breast recognizes at least 17 special histologic types that compose up to 25% of all diagnosed invasive breast carcinomas. Once established, these common patterns have provided a framework for making associations between an individual breast carcinoma subtype and clinical outcome. Published reports vary to some degree on the reproducibility with which pathologists can classify the histologic subtypes, 13,14 and the prognosis found to associate with specific histologic types of breast carcinoma only applies to tumors largely composed of that specific pattern. 15 Furthermore, the challenge of making an accurate morphologic classification is complicated by the observation that approximately one-third of invasive duct carcinomas display mixed morphologic features. 16 These limitations to a morphologic classification of breast carcinoma have led to the search for additional means to assess prognosis and predict treatment response. Traditional prognostic and predictive markers used in the assessment of breast carcinomas include axillary lymph node status, tumor size, tumor grade, histologic type, surgical margin status, and lymphatic and vascular invasion. 7 Other histologic features (eg, vascular invasion, tumor necrosis, stromal lymphoplasmacytic infiltration) have been investigated and found to provide little utility in predicting prognosis. 15 Beyond a morphologic classification, clinical features and information about the physiologic status of the tumor have long been used to assess prognosis and determine treatment. 17 In an issue of the journal Lancet in 1896, Beatson 18 provided evidence that some breast cancers regressed after surgical removal of a patient s ovaries. This early association between a female patient s hormone status and prognosis of breast cancer became a seminal observation in establishing that not all breast carcinomas with a similar morphology share a similar biology. 19 Historically, the investigation of biologic markers useful for the assessment of breast carcinoma have been categorized as those present in blood; specific tissue associated markers; oncogenes; growth factors; and a variety of cytoskeletal, cell adhesion, and extracellular matrix proteins. 15 Although examination of several of these markers, including carcinoembryonic antigen, pregnancy-specific B 1 -glycoprotein, a-lactalbumin, and b human chorionic gonadotropin, has not demonstrated a significant relationship with prognosis, 15 their utility should not be prematurely dismissed. Recently, a vaccine raised against a-lactalbumin was shown to produce promising results in an autoimmune-mediated strategy for prophylactic breast cancer vaccination in mouse models of breast carcinoma. 20 Contrary to the outcome of investigations into the clinical utility of some biologic markers listed above, testing for estrogen and progesterone receptor status was shown to be of value, and the hormone receptor status of a breast tumor has demonstrated correlation with clinical outcome. 15 The first correlation between expression of estrogen and progesterone receptor and prognosis was published by Rosen et al 21 in 1975. Estrogen receptor and progesterone receptor status has subsequently been shown to be associated with a more favorable prognosis, and clinical knowledge of the estrogen receptor status of the tumor is important to determine the utility of endocrine therapy. 22 26 Investigation into the genetic changes found in human adenocarcinomas has identified amplification of the v-erbb2 erythroblastic leukemia viral oncogene homolog 2 gene ERBB2 (HER2/neu) in mammary carcinoma. 27 29 HER2/neu overexpression was subsequently shown to correlate with a poor prognosis. 30 HER2/neu is a proto-oncogene that encodes a 185-kDa protein that is a member of the ERB family of transmembrane tyrosine kinase receptors. When activated, the HER2/neu protein dimerizes and activates various signaling pathways to potentiate a variety of cellular functions including promoting cell division while inhibiting apoptosis. 31 In 1987, Slamon et al 32 published a study directly correlating relapsed disease and survival of patients affected by breast carcinoma with amplification of HER/neu. Additional experiments demonstrated that monoclonal antibodies against the p185 product of HER2/ neu inhibited the growth of HER/neu-transformed cell lines implanted in nude mice. 33 These studies, among others, laid the foundation for the use of the humanized monoclonal antibody trastuzumab against the 185-kDa protein of HER2/neu for clinical treatment of selected breast carcinomas; moreover, they brought about the necessity for accurate diagnostic testing of HER2/neu gene status. It is estimated that hundreds of thousands of validated diagnostic tests including immunohistochemistry, fluorescence, chromogenic testing, and brightfield in situ hybridization studies are used every year to determine the HER2/neu status. 34 36 In 2007, guidelines were published jointly by the American Society for Clinical Oncology (ASCO) and the College of American Pathologists (CAP) to standardize performance and interpretation of HER2/neu testing in the United States. 37 Recently, ASCO/CAP have published similar guidelines on immunohistochemistry testing of estrogen and progesterone receptors in breast cancer. 38 In Situ Hybridization Techniques for Delineation of HER2/neu Status The demonstrated clinical utility of trastuzumab has created a mandate for accurate and reliable testing of breast carcinomas to ensure that patients with HER2/neu overexpressing tumors can benefit from antibody therapy, whereas patients with nonamplified tumors can be spared the potential cardiotoxic side effects of the drug. While HER2/neu status is readily detected by immunohistochemistry (IHC), 39 concern over equivocal and falsepositive staining patterns by IHC is illustrated in the ASCO/CAP interpretation guidelines. The potential limitations of IHC testing have required development of in situ technologies to accurately delineate the HER2/neu amplification status of breast carcinoma. Early in situ hybridization approaches relied upon radiographic, rather than fluorescent, detection of the target sequence. 40,41 The labeling of probes either directly or indirectly with a fluorochrome greatly improved the ease and safety of the hybridization technique, offered increased resolution, and introduced the possibility of simultaneously identifying Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al 545

multiple targets within the same nucleus. 42 Fluorescence in situ hybridization (FISH) is not without limitations, however, including the requirements of a dedicated fluorescence imaging system, limited morphologic assessment of overall histology, reduced stability of the detection signal(s), and overall higher cost of testing in part owing to the necessity of using well-trained personnel with specific expertise. Some of these issues have been addressed through the development of alternative in situ hybridization methodologies that rely upon detection strategies that can be appreciated with brightfield rather than fluorescence microscopy. 36 Prominent and emerging brightfield in situ hybridization (BISH) techniques are briefly discussed below. Chromogenic in situ hybridization (CISH), first described by Tanner et al 43 in 2000, is an alternative to FISH detection of HER2/neu amplification in breast tissue. Chromogenic in situ hybridization uses chromogenic, rather than fluorescent, detection of the hybridization probe. Unlike CISH, enzyme metallographic in situ hybridization uses an enzymatic reaction to facilitate the deposition of metallic silver directly from an ionic silver solution to identify the target site. Early metallographic in situ methods relied upon autometallographic deposition of gold particles, coupled with a signal amplification technique. 44 Subsequently, James Hainfeld discovered that horseradish peroxidase can be used to selectively deposit metal from solution in the absence of a particulate nucleating agent such as Nanogold (Nanoprobes, Yaphank, New York). This enzyme metallographic technology, commercially known as silver in situ hybridization (SISH), produces discrete spots of metallic silver deposition from the enzymatic action of peroxidase on silver acetate in the presence of hydroquinone, allowing a superior quantitative assessment of gene copy number. 44 The use of combined forms of CISH and SISH (ie, brightfield double in situ hybridization [BDISH]) to examine the HER2/neu status of breast carcinomas allows enumeration of both chromosome 17 and the HER2/neu gene in the same nucleus with results comparable to a manual dual-colored FISH technique (Figure 1, A and B). 45 Silver in situ hybridization and the EnzMet Gene Pro assay (Nanoprobes), a form of SISH that incorporates concomitant protein detection, have demonstrated excellent interobserver interpretive reproducibility (Figure 1, C). 46,47 Brightfield in situ hybridization methods offer several advantages over FISH detection systems, including accurate quantification of gene amplification, excellent visualization of tissue morphology, and adaptability for automation. 48 Added benefits to SISH technology include very high sensitivity with excellent resolution and signal separation capabilities. Of the BISH methodologies, CISH is Communauté Européenne (CE) marked and US Food and Drug Administration (FDA) approved, while SISH is currently CE marked but has not yet been FDA approved. The published ASCO/CAP guidelines, including the equivocal range of HER2/neu gene amplification (HER2/ neu to CEP17 ratio of 1.8:2.2), are also readily applied to BISH assessment of HER2/neu. While dual-color FISH currently remains the gold standard for in situ assessment of gene copy number, it has been suggested that an automated BDISH technique might be used in replacement of manual dual-color FISH methods in the future. 45 Alternatively, techniques combining IHC and BDISH Figure 1. Examples of HER2/neu status in breast carcinoma detected by metallographic in situ hybridization. Both breast carcinomas without (A) and with (B) HER2/neu amplification are illustrated with brightfield double in situ hybridization. The chromosome 17 signal is demonstrated in red; the HER2/neu signal is represented in black. Silver in situ hybridization coupled with immunohistochemistry demonstrates both the genomic status of HER2/neu as well as overexpression of the HER2/neu protein (C) (original magnifications 3400 [A through C]). methods may become a preferred method of HER2/neu assessment. 49 EXPRESSION PROFILING AND MOLECULAR CLASSIFICATION The first molecular portraits of human breast tumors were published by Perou and colleagues, 50 who characterized the variation in gene expression patterns by using RNA derived from 65 breast tumors (42 patients) with 546 Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al

Molecular Subtype Table 1. Commonly Recognized Molecular Subtypes of Breast Carcinoma 17,90,132 Representative Gene Expression Signature Representative Immunophenotype Luminal A ESR1 (estrogen receptor 1) Estrogen receptor ER+ and/or PR+, HER2/ neu2, low Ki-67 associated transcription factors Luminal B ESR1 ER+ and/or PR+, HER2/ Estrogen receptor neu+ associated transcription factors Basal-like KRT5 (keratin 5) ER2, PR2, HER2/neu2, KRT17 (keratin 17) CK 5/6+, and/or EGFR+ LAMC2 (laminin, g 2) HER2/neu positive Associated Histologic Features Usually low grade Usually higher grade than luminal A subtype Often high grade Associated Clinical Features Tend to be sensitive to endocrine therapy, variable response to chemotherapy, overall good prognosis Tend to be sensitive to endocrine therapy, variable response to chemotherapy, prognosis poorer than that of luminal A subtype Tend to be insensitive to endocrine therapy, variable response to chemotherapy, overall poor prognosis ERBB2 (HER2/neu) ER2, PR2, HER2/neu+ Often high grade Tend to respond to biologic ERBB2 amplicon therapy (trastuzumab), variable response to chemotherapy, overall poor prognosis Abbreviations: CK, cytokeratin; EGFR, epidermal growth factor receptor; ER, estrogen receptor; PR, progesterone receptor; ERBB2(HER2/neu), v-erbb2 erythroblastic leukemia viral oncogene homolog 2 gene; +, positive staining result; 2, negative staining result. complementary DNA microarrays representing 8102 human genes. Upon data analysis it became apparent that there was great variation in the pattern of gene expression, yet there was also a pervasive order in the expression data, reflecting the relationship of specific gene expression signatures to specific tumor types. Further analysis of intrinsic gene subsets in this study allowed the identification of 4 groups with distinct molecular features of mammary cells (estrogen receptor positive/luminallike, basal-like, HER2/neu positive, and normal breast). 50 In a follow-up report using hierarchical clustering based upon a 534 intrinsic gene set, 51 115 malignant breast tumors, as well as data from 2 additional independent studies, were analyzed. 52,53 The tumors may be subgrouped into 1 basal-like, 1 HER2/neu overexpressing, 2 luminal-like types, and 1 normal breast tissue like subgroup. Furthermore, tumors from mutant BRCA1 carriers aligned with the basal-like tumor subtype. While these data are in agreement with the study by Perou et al, 50 the normal breast-like subtype originally proposed in this study has since been recognized as probably arising from normal-tissue contamination. 19,54 A third luminal type subset, luminal C, has been identified; however, the most well-characterized and widely accepted molecular subtypes are the luminal A, luminal B, HER2/neu, and basallike types 17 (Table 1). An important distinction made in the first study by Perou et al 50 is that estrogen-negative tumors comprise at least 2 biologically distinct subtypes: the basal-like and the HER2/neu-positive tumors. While breast carcinomas with a basal/myoepithelial phenotype were described in the 1960s by careful ultrastructural examinations followed by cytokeratin profiling experiments, the gene expression experiments used to define the molecular subtypes of breast carcinoma have created much interest in these basal-type tumors. 55 Although not synonymous with triple-negative breast cancer, the basallike subtype commonly displays a triple-negative phenotype often defined by a lack of estrogen receptor and progesterone receptor protein expression, with an absence of HER2/neu overexpression. The details of this heterogenous group of tumors continues to be investigated, 56 59 but it appears that most of these tumors are associated with a ductal morphology of no special type, with high histologic grade, distinctive radiographic appearance, and an aggressive clinical course that often does not respond well to standard adjuvant therapies. 60 64 The highest incidence of these tumors has been observed among premenopausal African American women. 65 Basal-like breast carcinomas and breast carcinomas arising in BRCA1 mutation carriers demonstrate a similar phenotype. 66 Interestingly, sporadic, basal-like tumors have been shown to display dysfunction in the BRCA1 pathway. 67 69 The observation of such similarities has stimulated the hypothesis that strategies targeting the BRCA1 pathway could be used as useful therapies for patients affected by basal-like tumors. 70 Work has also been done to define the molecular signature of histologic special types of breast carcinoma. 11,71 While such experiments are challenged by the low prevalence of these rare histologic special types, 11 analysis of 113 specimens representing a series of 11 histologic special types has shown that some do indeed constitute discrete entities when analyzed by a hierarchic clustering analysis (ie, micropapillary), while others (ie, lobular and tubular) are very similar at the level of the transcriptome. 71 The study by Weigelt and colleagues 71 also found that some cases of medullary and adenoid cystic carcinomas, generally associated with good prognosis, displayed a basal-like transcriptome. These data suggest that several histologic subtypes may not represent specific biologic entities and underscores the need for accurate identification of tumor type. While not defined by a special histologic type, gene expression profiling experiments using tumors from patients with hereditary breast cancer have also revealed unique expression patterns dependent upon the status of the BRCA1 and BRCA2 mutation. 72 Molecular Subtype and Prognosis After publication of the initial molecular taxonomy of breast carcinoma by Perou and colleagues, 50 several studies have described, characterized, and predicted the clinical behavior of these breast cancer subclasses. 73 77 Interestingly, concordance of the biologic behavior of breast carcinomas is maintained across a variety of gene Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al 547

Table 2. Representative Commercially Available Genomic Assays for Prognostication in Patients With Breast Carcinoma 85,133 Test Oncotype DX MammaPrint MapQuant Dx Vendor Genomic Health Inc (Redwood City, Agendia (Irvine, California) Ipsogen (Marseille, France) California) Assay type 21-gene RT-PCR 70-gene DNA microarray 97-gene DNA microarray Tissue type FFPE Fresh or frozen Fresh or frozen Indication Node2 ER+ To predict risk of recurrence Node2 ER+ ER2 Invasive, primary, ER+, grade 2 tumors To restratify grade 2 tumors into grade 1 or grade 3 tumors To aid in prognostication FDA approval No Yes No Abbreviations: ER, estrogen receptor; FDA, US Food and Drug Administration; FFPE, formalin-fixed, paraffin-embedded; RT-PCR, reverse transcription polymerase chain reaction. sets, suggesting that multiple markers can be used to track tumor phenotype. 78 While initial review of these studies cited some methodologic problems, 79,80 subsequent reports show less reluctance to emphasize the potential for improving breast cancer management through the use of gene expression signatures. 81 A large meta-analysis of publicly available breast cancer gene expression data from 2833 breast tumors 80 confirmed the presence of the molecular subtypes originally reported by Perou and colleagues; this same study concluded that gene signatures should be tested for the ability to spare adjuvant chemotherapy for some patients and also highlighted the importance of proliferation gene expression in the assessment of prognosis. While gene expression profiling is not useful in all treatment decisions, 82 numerous additional class comparison and class prediction experiments highlight the potential benefits to clinical management and biomarker discovery that these gene expression data sets provide. 83 Several commercial genomic assays are now available to aid the prognostication for patients diagnosed with breast carcinoma 84,85 (Table 2). An excellent description of the prognostic and predictive markers for breast cancer, tailored to the needs of practicing pathologists, has been recently published. 86 Of note, while the Oncotype DX (Genomic Health Inc, Redwood City, California) and the MammaPrint (Agendia, Irvine, California) assays are already in clinical use, ongoing validation studies are underway to refine their clinical utility. The Trial Assigning Individualized Options for Treatment (TAI- LORx) is a multicenter trial that assesses the ability of Oncotype DX to aid the clinical decision-making process for patients with hormone receptor positive, axillary node negative breast cancer. 87 The Microarray in Node- Negative Disease May Avoid Chemotherapy (MINDACT) trial was undertaken to prospectively determine the usefulness of MammaPrint in assessing risk of relapse for patients diagnosed with node-negative breast cancer. 88 Much of the value of these tests for aiding clinical decision-making and for appropriately limiting exposure to chemotherapy in select patients will be determined by the outcome of these trials. However, it should be noted that in a small retrospective study of breast carcinomas that had been previously evaluated by Oncotype DX, 89 the recurrence score could be approximated by integrating weighed histopathologic and immunophenotypic variables of nuclear grade, mitotic count, hormone receptor score, and HER2/neu status. These data suggest that judicious use of these expensive genomic assays may be most appropriate in select cases, when it is difficult to make clinicopathologic decisions with conventional methods alone, and may also provide the opportunity to develop integrative molecular and immunophenotypic surrogate assays for derivative-based tests. Surrogate IHC Although gene expression profiling is considered the gold standard for defining the molecular subtypes, several classification schemes, based upon use of surrogate immunohistochemical markers to approximate the subclasses described by gene array profiling, have been reviewed recently. 90 Accepted staining patterns with these markers include expression of hormone receptors and luminal cytokeratins, lack of HER2/neu overexpression, and low Ki-67 expression in luminal A tumors; expression of hormone receptors, luminal cytokeratins, and HER2/neu overexpression in luminal B tumors; absence of hormone receptor expression and lack of HER2/neu overexpression, but expression of basal cytokeratins in basal-like tumors; and absence of hormone receptor expression with HER2/ neu overexpression in HER2/neu-positive tumors. Representative images of these staining patterns with a selected panel of markers are provided (Figure 2, A through H, and Figure 3, A through H). The IHC-based classification schemes fit well with what is known about the expression patterns of 2 of the distinct cell types found in the human mammary gland and generally match the gene expression signatures of the molecular subtypes. For example, basal (myoepithelial) tumor cells demonstrate positive staining with antibodies to cytokeratins 5 and 6, while luminal types show positivity with antibodies to cytokeratins 8 and 18. 91,92 Examination of several commonly used cell lines reveals that most can be separated by surrogate molecular subclasses by using a simple panel of immunohistochemical markers similar to that used to define clinical cases. 93 These findings have implications for R Figure 2. Representative images of luminal subtypes based upon a limited immunohistochemical classification scheme. The left column (A, C, E, and G) demonstrates a luminal A type carcinoma with low-grade histology, positive expression of hormone receptors, and nonamplified HER2/neu. The right column (B, D, F, and H) demonstrates the luminal B type carcinoma with high-grade histologic features and positive expression of hormone receptors with overexpressed HER2/neu (hematoxylin-eosin, original magnifications 3400 [A and B]; estrogen receptor, original magnifications 3400 [C and D]; progesterone receptor, original magnifications 3400 [E and F]; HER2/neu, original magnifications 3400 [G and H]). 548 Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al

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Table 3. Evolving Molecular Tools for the Diagnosis and Classification of Breast Carcinomas 98,112,134 Characteristic Array CGH MALDI-IMS Epigenetic Profiling by Array Tissue type Fresh, frozen, or FFPE Fresh, frozen, or FFPE Fresh, frozen, or FFPE Potential uses Detect genomic DNA alterations in constitutional diseases and malignancies Rapid identification and characterization of malignancy Identification of reversible modification of promoter and enhancer elements Advantages Single assay, whole-genome profile Rapid, generation of unique protein Multiplex, high-resolution Disadvantages Fails to detect balanced translocations or LOH, decreased accuracy with archived FFPE tissue profile Technically challenging, protein profiles not standardized, fails to detect high-molecular-weight proteins mapping platforms available Expensive, biases and enrichment are dependent upon probe design and density across the genome Abbreviations: CGH, comparative genomic hybridization; FFPE, formalin-fixed, paraffin-embedded; LOH, loss of heterozygosity; MALDI-IMS, matrix-assisted laser desorption/ionization-imaging mass spectrometry. investigators using these cell lines for research and open up additional possibilities for laboratory proficiency testing. EMERGING MOLECULAR RESOURCES Several established and emerging technologies are looking beyond gene expression profiling and toward the status of entire chromosomal regions, the level of protein expression, or to the pattern of gene methylation to better understand the specific signatures of malignant versus benign breast epithelium (Table 3). While some of the methods described below are already in clinical use, others are less mature and will require additional effort for their translation from the research setting. Despite present limitations, use of these techniques has great potential for assisting patients and oncologists faced with the difficulty of making treatment decisions and for accurately assigning both prognosis and prediction of therapeutic response. Array Comparative Genomic Hybridization One newer technology that is looking beyond gene expression profiling and into the status of the entire genome to help classify and characterize breast neoplasms is array-based comparative genomic hybridization (acgh). This promising technology has undergone several transformations and technical improvements that have now made it a valuable test in the clinical laboratory setting. The technology of CGH that was first used in the early 1990s, referred to as conventional or chromosomal CGH, was initially very similar to traditional cytogenetics in that it was applied directly to metaphase smears. 94,95 An early modification, which significantly improved the utility of this assay, consisted of switching from metaphase smears to an array format that used bacterial artificial chromosomes placed on a solid substrate (eg, chip or slide) for hybridization. 96 The next major advancement in acgh was replacing the large bacterial artificial chromosomes with synthesized oligonucliotides. 97 This change to an oligonucleotide-based acgh offered the benefit of higher resolution, no cloning steps, and customizable array design, all enhancements that led to the commercialization of the technology, which facilitated integration into clinical laboratories. 98 Several obstacles had to be addressed before the mainstream adoption of acgh in the clinical laboratory setting could occur. The first major obstacle was overcoming poor assay sensitivity attributable to isolating DNA from tumor samples with mixed normal and tumor cell populations. This issue was resolved by incorporation of laser capture macrodissection or selective isolation of tissue for DNA extraction. 99 A second obstacle for acgh was validating assay performance with DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue. Early clinical applications of acgh were performed on fresh tissue. 94,100 However, since most pathology specimens are fixed in neutral buffered formalin and stored as paraffin blocks, it was essential that any viable acgh assay use DNA isolated from FFPE tissue. Several groups have validated the use of FFPE-isolated DNA for acgh, 101 103 although only recently has it been demonstrated that FFPE-isolated DNA may be used for HER2/neu amplification screening by acgh. 104 Currently, performing acgh with DNA from FFPE is standard practice; however, there is preliminary evidence that prolonged storage of FFPE tissue results in degradation, which compromises acgh performance. One study that clearly addressed this issue of prolonged storage of FFPE tissue revealed that samples stored for more than 7 years show a significant decrease in assay performance. 105 However, new strategies using prearray amplification might overcome time-of-storage limitations and ultimately allow acgh analysis to be performed on older archived FFPE tissue samples. 106 While acgh is not a new technology, through continual improvement it has become a viable clinical laboratory test. The underlying utility of the assay is that in a single test one can detect genome-wide chromosomal DNA copy number alterations. In addition, unlike FISH, no preassay knowledge of abnormality is required to run or identify genetic changes with acgh. One example of the utility of acgh in breast cancer is demonstrated by superior detection of HER2/neu amplification. For HER/neu detection, acgh offers advantages over conventional IHC and FISH methodologies. 107 Two main problems arise from conventional IHC and FISH methods for HER2/neu detection that are overcome by acgh. 37 The first problem r Figure 3. Representative images of basal-like and HER2/neu-positive subtypes based upon a limited immunohistochemical classification scheme. The left column (A, C, E, and G) demonstrates a basal-like type carcinoma with high-grade histology and negative expression of hormone receptors, with non-overexpressed HER2/neu. The right column (B, D, F, and H) demonstrates a HER2/neu-positive type carcinoma with negative expression of hormone receptors and HER2/neu overexpression (hematoxylin-eosin, original magnifications 3400 [A and B]; estrogen receptor, original magnifications 3400 [C and D]; progesterone receptor, original magnifications 3400 [E and F]; HER2/neu, original magnifications 3400 [G and H]). Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al 551

Figure 4. Chromosome 17 ratio plots in HER2/neu-positive cases with aneusomy and polysomy. EQ-2, EQ-18, and EQ-20 demonstrate detection of HER2/neu overexpression in cases with complex chromosome 17 rearrangements. EQ-13 shows detection of HER2/neu overexpression in a case with polysomy (false-negative by fluorescence in situ hybridization). Reproduced with permission from Gunn et al.107 is that approximately 5% of cases evaluated both by IHC and FISH for HER2/neu are found to be equivocal by both methods.108 Clinicians have no set guidelines to treat double-equivocal patients and therefore proper treatment is potentially missed in this patient subgroup. The ability of acgh to resolve the HER2/neu status in the double-equivocal population was recently demonstrated by implementation of a commercial genomic array containing 127 probes covering the HER2/neu amplicon, pericentric regions, and both chromosome arms. Through utilization of acgh, Gunn et al107 were able to resolve 100% (n 5 20) of invasive ductal carcinomas subjected to array testing with previously unresolved equivocal HER2/ neu status (Figures 4 and 5). The second problem with traditional HER2/neu testing and specifically FISH-based methods is that HER2/neu amplification is calculated as a ratio in relation to the centromere of chromosome 17.37 This ratio can be skewed in cases of aneusomy of Figure 5. Chromosome 17 ratio plots in HER2/neu-negative cases with aneusomy and monosomy. EQ-6, EQ-16, and EQ-17 demonstrate HER2/neu cases with centromeric loss. EQ-14 shows a case with monosomy 17. Reproduced with permission from Gunn et al.107 552 Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al

Figure 6. Proposed algorithm for establishing HER2/neu status in breast cancer samples by protein expression and genomic analysis. The algorithm shows utilization of an array comparative genomic hybridization (acgh) in cases in which immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results are equivocal and in cases with discordant IHC/FISH results. Reproduced with permission from Gunn et al. 107 chromosome 17, resulting in false-positive or falsenegative results. Since acgh is not based on a single point of reference (eg, the centromere of chromosome 17) as in FISH, actual resolution of anuesomy (polysomy and monosomy) is achievable with acgh (Figures 4 and 5). In fact, it was recently shown by acgh that true chromosome 17 aneusomy is rarely found in HER2/neu-amplified breast cancers; however, pseudoaneusomy or partial amplification of chromosome 17, often including the centromeric region, is common in HER2/neu-amplified breast carcinomas. 37 An algorithm for appropriate utilization of acgh in the establishment of HER2/neu status in breast carcinomas has recently been proposed by Gunn and colleagues 107 (Figure 6). While acgh is a powerful genome-wide screening method that offers several advantages over traditional HER2/neu testing, it does not provide morphologic information on chromosome structure and can not currently be used to detect balanced translocations or loss of heterozygosity. In addition, analysis of array data can be conceptually and physically challenging because chromosomes are not directly analyzed as discrete structures, as in FISH or traditional karyotyping, but rather as DNA targets distributed throughout the genome. This loss of morphologic data has to be recreated in silico via computational bioinformatics software. This software reconstitutes the fragmented data and reassembles chromosomes by using software algorithms, each with inherent biases that are software specific, and can lead to misinterpretation and lack of standardization across laboratories. 103 In addition to general software limitations, recent data have shown that the common pipeline approach to acgh analysis itself can directly result in errors or miss significant findings. 109 Finally, an area that will need to be addressed before this technology can be universally adopted in the clinical environment is validation and standardization in reporting acgh results. Currently there is no standard reporting mechanism for detected gains or losses that are of unknown clinical significance. 110 To summarize, acgh benefits include a global view of the genome, numeric results not subject to degradation with time, generation of data banks available to mine for undiscovered complex genetic markers, 111 ability to use DNA from FFPE tissue, and the convenience of running a single assay. 103 While array-based testing offers many advantages, the methodology and results can be greatly affected by factors such as probe selection, probe density, quality of starting DNA, technical expertise of the user, and software used for downstream analysis. Therefore, like other clinical laboratory tests, extensive validation must be performed before clinical application. Proteomics and Imaging Mass Spectrometry Clinical proteomic analysis is another emerging diagnostic tool that is proving useful in the molecular classification of breast carcinomas. Several studies have now confirmed that the molecular differences between malignant and benign breast lesions literally translate into amino acid sequences with unique excitation signatures detectable by mass spectroscopy. Imaging mass spectrometry is a type of proteomic analysis that directly pairs the use of mass spectrometry with morphology. To date, several studies 112 114 have demonstrated that mass spectrometry can be used to identify key molecular differences between malignant and benign breast lesions via unique excitation signatures. Some of the earliest examples of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, paired with tissue section imaging micrographs, appeared in the late 1990s. 115 117 These initial studies were followed by intense efforts to improve many facets of the technology, including sensitivity, reproducibility, informatics processing, tissue morphology, imaging, and physical preparation treatments. 112,113 With multiple improvements over firstgeneration MALDI-imaging mass spectrometry (MALDI- Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al 553

IMS), direct clinical application of this technology is now being demonstrated, and incorporation of this methodology into daily clinical practice is not far from reality. The MALDI-IMS technology allows investigation of proteins paired with label-free, morphology-driven analysis of tissue sections. 118 This technique has multiple strengths over traditional IHC or FISH. First, a unique protein profile can be paired directly with morphology, thereby allowing the unique protein signature to be verified via its presence within the diseased tissue and its absence in the surrounding stroma or benign cells. Second, MALDI-IMS offers the ability to analyze samples without the constraints of labeling compounds or antibodies. This point is significant because it allows analysis to be performed on proteins that have low expression levels, even within a diluted cellular protein background, a task that is difficult with current methodologies. Additionally, since labeling is not a constraint of this assay, mass spectrometry is also capable of detecting biologically significant molecular details from samples in an environment in which it is difficult to perform traditional immunoassays, such as in biologic fluids. Third, MALDI-IMS can simultaneously analyze the presence of drugs within tissue as well as generate a unique protein profile. This has several advantages, as this technique can be used to monitor drug effectiveness by assessing the presence or absence of a drug metabolite in diseased tissue. In addition, the generation of unique protein profiles that change with treatment may help guide therapy selection or guide required changes in therapy due to acquired drug resistance, all monitored by generation of a unique protein profile that is directly correlated with morphology. Thereby, this technique allows real-time assessment of therapeutic efficacy and toxicity, a goal of personalized medicine that is not currently possible with other methodologies. Finally, the universality of the procedure makes it highly attractive from both a laboratory management and clinical perspective. In effect, MALDI-IMS could be used as a single, disease-wide laboratory methodology for risk stratification or detection of therapeutic biomarkers. This single-platform approach would simplify and accelerate laboratory flow, decrease laboratory training, decrease laboratory cost, as well as streamline the clinician testing menu for the institution. A good illustration of the application of MALDI-IMS for clinical use in the context of breast cancer is shown by Rauser et al, 118 in a study that used MALDI-IMS to detect specific protein expression changes that strongly correlated with presence or absence of HER2/neu overexpression. Specifically, this group identified 7 unique mass to charge signatures that were found in HER2/neu-positive but not HER2/neu-negative cases. This study demonstrates the utility of MALDI-IMS for generating a unique patient protein profile, which can subsequently be used to stratify a test population into a HER2/neu-positive or HER2/neunegative breast cancer category. In the study, Rauser et al 118 were able to stratify HER2/neu-postitive from HER2/ neu-negative cases, with a sensitivity of 83% and a specificity of 92%. This example of analyzing breast carcinoma, based on a specific defined MALDI-IMS proteomic algorithm, illustrates the utility of this morphology-driven, tissue-based diagnostics technique. It also illustrates the fact that MALDI-IMS has potentially broader utility as a technique that will allow diagnostic classification of other cancers and markers. 112 114,119 The MALDI-IMS technology has advantages over other assay methods including non-ims based formats of mass spectrometry (ie, those requiring homogenization) because it is morphologically driven. Being morphology based allows the direct evaluation of tumor cells, the correlation with other morphologic features, and the ability to assay smaller tumor tissue specimens from patients, such as needle core biopsy specimens. The link between imaging and mass spectrometry makes MALDI- IMS a seemingly ideal tool for rapid tissue diagnostics and molecular histology. It is important to note that the use of MALDI-IMS in the clinical setting is in its very early stages and that this technique will certainly improve in sensitivity, speed, resolution, and biocomputational data analysis over time. However, with minor technologic improvements, this technology stands to have a great impact in the filed of diagnostic pathology. Current utility limitations of MALDI-IMS include the limited ability to ionize certain proteins and detect highermolecular-weight proteins, and the physical limitations of resolution by the ionization laser. Whereas, theoretically, proteins up to 250 kda could be resolved with this technology, current MALDI-IMS devices report detection of compounds with a molecular weight between 400 and 50 000 kda. 120,121 This size limitation means that the unique protein profile generated, as shown in the case of HER2/ neu-positive breast cancer, is not simple detection of the 185-kDa HER2/neu protein, but rather other proteins (,25 kda) upregulated in HER2/neu signaling. 118 A second area of improvement revolves around increasing the resolution of the method by decreasing the size of the irradiated area without loss of assay sensitivity. Currently, the area of irradiation is approximately 30 mm squared an area that is much larger than an individual cell. Therefore, further advancements to decrease irradiation area to the level of individual cell size without loss of sensitivity of ion detection will be essential for higherresolution analysis and for adoption in routine clinical laboratories. Ultimately, new diagnostic tests represent vehicles that allow us to derive novel data or overcome technical problems found in current methods. Whether a new diagnostic test analyzes the genome, as with next-generation sequencing, or uses the proteome via MALDI-IMS, the ultimate goal is to apply a technique that will supply pathologists and clinicians with accurate predictive patient data. In the case of MALDI-IMS, this technique offers the possibility of collecting direct personalized information that will be critical to improving our understanding of molecular pathogenesis of disease, improve therapeutic efficacy, and enhance the quality of information that is provided to clinicians. Pulling these characteristics together in a single-assay format offers the pathologist a unique opportunity to markedly improve patient therapeutic selection, risk stratification, and overall survival. Epigenetic Profiling Epigenetic changes are molecular alterations that affect gene expression by mechanisms that do not involve changes to the DNA sequence itself. In addition to the sporadic occurrence of these modifications in response to normal aging and environmental exposures, epigenetic alterations can also be heritable. These phenomena can 554 Arch Pathol Lab Med Vol 135, May 2011 Molecular Pathology of Breast Cancer Gruver et al

occur by histone modification, noncoding RNAs, and DNA methylation. Normal epigenetic patterns are aberrant in a variety of human cancers, resulting in the hypomethylation of repetitive elements and/or hypermethylation and consequent silencing of critical tumor suppressor genes. Recent reviews on the epigenetics of breast cancer 122 125 illustrate the potential role for epigenetic changes in the initiation and progression of the disease and the possibility of using the epigenetic signatures of a tumor for diagnosis, prognosis, and treatment strategies. Detection of epigenetic changes can be carried out by several modalities including combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, bisulfite sequencing, or by genearray technologies. Distinct epigenetic patterns are beginning to emerge that correlate with breast carcinoma molecular subtypes. 126 Analysis of familial breast cancers suggests that the DNA methylome of these tumors is unique from that of sporadic breast tumors and is in part defined by their mutational status. 127 The observation that these tumors have distinct methylation profiles suggests that methylomics may become another approach to understanding and predicting the biologic behavior of a specific tumor. In contrast to gene mutations, epigenetic changes are thought to be reversible. This attribute has led to the hypothesis that targeting select epigenetic changes may relieve transcriptional repression of tumor suppressor genes and allow for novel therapeutic strategies. While the selectivity of the therapy remains an unresolved issue, both DNA methyltransferase inhibitors (in the form of cytosine analogs such as 5-aza-29-deoxycytidine) and histone deacetylase inhibitors (such as trichostatin A) have been investigated in preclinical models of breast cancer, with promising results. 125 Further investigation will be needed to determine exactly how monitoring and manipulating the epigenome may contribute to the diagnosis, prognosis, and treatment of breast carcinomas. In conclusion, while pathologists may not abandon morphology and immunohistochemistry for a purely molecular approach in the near future, a concerted effort is being made to move beyond the limitations of traditional diagnostic and prognostic markers and toward the application of the molecular signatures of breast carcinoma. 128 Morphologic context remains the cornerstone of diagnosis in the molecular era, and it is stimulating to imagine what opportunities the future holds for assimilating such technologies into clinical practice. Whether it comes via next-generation sequencing technologies to help understand the complexity of intracellular signaling pathways active in HER2/neunegative tumors or by using new biomarkers to predict prognosis, 129 131 opportunities for advancement in the molecular diagnostics of breast cancer are sure to impact how diagnosis and treatment of the disease is accomplished. 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