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Authors: Rosen, Paul Peter Title: Rosen's Breast Pathology, 3rd Edition Copyright 2009 Lippincott Williams & Wilkins
> Front of Book > Introduction

Introduction
The Pathologist as a Specialist in Breast Cancer Care
The development and application of a concept of localized pathology laid the groundwork for modern specialism by providing a number of foci of interest in the field of medicine. Each such focus of interest, that is, a disease or the diseases of an organ or region of the body, provided a nucleus around which could gather the results of clinical and pathological investigation. On the technological side the influences represented in specialization manifest themselves in the multiplicity of technical skills, devices, and theories applied to the achievement of human aims in the field of medicine. --From The Specialization of Medicine by George Rosen, M.D., 1944. Impressive advances have been made in the past 50 years in the effort to prevent, treat and cure breast cancer. Major milestones include the development of mammography for early detection, the shift from mastectomy to breast conservation therapy for many patients, advances in chemotherapy for primary treatment and as an adjuvant modality, the demonstration that antiestrogenic compounds can inhibit the development and progression of breast cancer, and the introduction of sentinel lymph node mapping for axillary staging. The growth of medical specialization in the last half of the 20th century has had a profound influence on these accomplishments by fostering multidisciplinary clinical practice and research.

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Specialism in all aspects of medical care has revolutionized the role of the surgical pathologist. Rather than fostering professional independence, specialization in medicine has created circumstances in which the specialist delivering a limited segment of medical care is increasingly dependent on the assistance of colleagues who have acquired complimentary expertise. This situation is epitomized by the multidisciplinary approach that is now standard for treating breast diseases. Inherent in this circumstance is the expectation that each member of the team is capable of delivering optimal specialty care. A corollary effect is growing pressure for subspecialization in diagnostic pathology, especially in academic centers. Breast pathology has largely remained in the domain of generalists except for a few referral centers. The formation of the International Society of Breast Pathology heralds recognition of subspecialization in Breast Pathology. In 2000, a European Society of Mastology position paper set forth guidelines for a clinical program devoted to providing high-quality specialist Breast Service included among the physicians a lead pathologist plus usually not more than one other nominated pathologist specializing in Breast Disease(to be) responsible for all breast pathology and cytology (1). The number of pathologists needed to staff such a service will depend upon the number of patients cared for. This process will be furthered by growing awareness on the part of patients and patient advocacy organizations that accurate and comprehensive pathology diagnosis is fundamental to effective treatment and research in breast diseases. Major advances that contributed to the role of the pathologist as a key member of the breast cancer team include: widespread use of mammography which detects nonpalpable lesions; image-guided needle core biopsy procedures, which make it possible to acquire samples from nonpalpable lesions; breast conservation therapy, which requires a more-detailed pathologic assessment of breast specimens; the availability of histologically-based methods for detecting markers used to assess prognosis and to plan therapy; and sentinel lymph node mapping and bone marrow sampling for micrometastases. Pathologists generate an important part of the information used for therapeutic decisions. The complex multifactorial description of breast

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pathology now considered to be standard practice has expanded the diagnostic report from a brief one- or two-line statement to a catalogue of data that may be several pages in length. Immunohistochemistry makes it possible to determine the presence of prognostic and therapeutic markers by microscopic examination, and these observations are part of the pathologist's report. This technology is also essential for detecting micrometastases in sentinel lymph nodes. The expanded role of pathologists in the management of breast diseases requires their active participation with the clinical care team. Pathologists who diagnose breast specimens need to be aware of how various components of their reports are relevant to treatment decisions. Optimally, there should be a procedure for correlating imaging studies with biopsy results in regard to nonpalpable lesions detected by mammography, ultrasound or MRI (1). Coincidental with these medical developments has been the growing involvement of patients in making decisions about their treatment. This, in turn, has led to a greater public awareness of the importance of information contained in pathology reports. For the untrained layperson to read and interpret a pathology report, it is necessary to learn and understand a new vocabulary. This is a daunting taskone that is even more difficult for the patient whose name appears on the document. Books and literature provided by medical and lay societies or associations are helpful, as is the bottomless well of information that appears on the Internet. The surgeon, oncologist, and radiotherapist are experts at interpreting pathology reports for their patients and at explaining the significance of the data. Nonetheless, a substantial number of patients with breast diseases want an explanation from the pathologist who issued the report or they seek out another pathologist, often with specialized expertise, for a second opinion review. Many more patients are aware that a pathology consultant is involved in their case. In this way, pathologists increasingly participate in direct patient care and patient education, a vital public service.

Consultations and Second Opinions in Breast Pathology

Surgical pathologists in general practice provide accurate diagnoses for the great majority of the breast specimens they encounter without the assistance of intramural or extramural consultation. Nonetheless, pathology departments should have a built-in mechanism for obtaining second opinions

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internally through conferencing or other quality assurance programs. In this setting, the individual pathologist or the pathology group in a department may seek an extramural opinion from an expert consultant. This typically occurs when there is a difference of interpretation among pathologists in an institution or the diagnosis is uncertain after internal review. Consultation may also be obtained when the probable diagnosis is one with which there is little or no experience. Another category of consultation results from uncertainty about the diagnosis engendered by a limited or unrepresentative sample, poor histologic preparation, or a pathologic change that appears to be on the borderline between two or more diagnoses. As noted by Leslie et al., Second opinions in anatomic pathology are an integral part of quality practicefrequent consultation between pathologists should be fostered in all practice settings and documented as part of the quality assurance process (2). Several studies have demonstrated the important contribution to patient care of second opinion pathology consultations, generally in the context of referrals seen at academic centers. A very positive aspect of this practice is the high degree to which the primary diagnosis has been confirmed by the consultant. Epstein et al. reported concordant diagnoses (cancer vs. noncancer) in 98.7% of 535 prostatic needle biopsies diagnosed as cancer (3). Nonetheless, the 6 diagnoses not sustained as cancer were critically important for the 1.3% of patients. A cost analysis of these results suggested that the saving in medical expenses for the 6 patients who did not undergo surgery substantially exceeded the cost of reviewing all 535 biopsies. A higher rate of discrepancies was found by Abt et al. (4) who compared the original and second opinion diagnoses in a broad range of pathology among 777 patients referred to an academic center. Forty-five diagnostic disagreements (6%) were regarded as clinically significant, and overall the level of agreement was 92.1%. Perkins et al. (5) estimated that diagnoses were inaccurate in 2% to 4% of breast cancer cases, including mistaking benign for malignant disease or vice versa, over- or underdiagnoses of invasive carcinoma, or misinterpretation of prognostic markers such as HER2/neu. It is unlikely that complete microscopic pathology samples will be routinely converted to electronic images in the foreseeable future given the time and cost of this undertaking and the fact that much of the information will be a record of normal or nonlesional tissue. Consequently, the need to ship glass slides for consultation is likely to be with us for some time to come. Within the United States, several factors have contributed to the growing

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number of pathology consultations. Much of the increase is generated by patients who seek multiple clinical opinions from different physicians and institutions. Some patients are primarily concerned with confirmation of their diagnosis, and one or more consultations may be obtained directly from pathologists for this reason alone. Most of the remainder of consultations are initiated by pathologists seeking opinions from their colleagues. Surgeons, medical oncologists, and other physicians generate some second opinion reviews. The review of outside pathology slides should be mandatory whenever a patient is referred to a physician for consultation or treatment at an institution other than the one where the primary diagnosis was rendered (6). Slides sent for consultation, regardless of the reason, must be accompanied by documents that: (a) confirm the identity of the specimen with the patient, (b) explain why the material has been sent, (c) provide complete information about who should receive the report, and (d) designate who will pay for the consultation and how billing should be submitted. The correspondence may take many forms, but it is essential that the information cited above be provided. This must include a copy of the pathology/cytology report for each specimen represented, clearly displaying the name of the patient and the accession number corresponding to the slides and paraffin blocks enclosed. It is unacceptable and substandard practice to withhold the pathology report previously obtained from a consultant or second opinion institution so as not to bias the second review. In addition to confirming the anatomic source and patient identity of the slides, the pathology report provides essential information such as an index of the specific location(s) of the specimen(s) in individual slides, a description of the gross appearance of the specimen(s), clinical information provided with the specimen, frozen section interpretations, and details of the pathologist's diagnosis that should be evaluated. The pathology report must be included even if the final diagnosis has not been reached and will depend upon the consultation. When the slides are sent directly from one laboratory to another in relation to a clinical consultation at the recipient institution, the correspondence should include the pathology report, the name of the clinical physician who is being consulted, and detailed billing instructions. When more than one consultant is involved, it is vital that all consultants examine the same or equivalent material.

Progress and Uncertainty in Breast Pathology

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Extraordinary progress has been made in linking anatomic pathology, the study of normal and diseased tissues, to patient care throughout the spectrum of human ailments. The 20th century has been marked by great advances in defining the pathology of breast diseases and in relating these observations to the development of more effective therapy tailored to the specific type and extent of disease in the individual patient. The stage was set in the latter half of the 19th century and first decades of the 20th century with the flowering of classical pathology based largely on postmortem examination of the gross and microscopic changes found in diseased tissues. The principle objectives of these investigations were to describe and catalogue diseases in an effort to detect clues to their pathogenesis and to better understand their clinical manifestations. Surgical pathology, the study of tissues from the living, emerged from classical anatomic pathology as advances in surgery, made possible by effective anesthesia and antisepsis, focused greater attention on a pathologic diagnosis as a critical element in the treatment of many diseases. The study of breast pathology has been a model of interdisciplinary investigation involving clinical and laboratory science. Pathologists are in a unique position to meet the challenge of developing and adapting innovative laboratory methods to better understand and to improve the treatment of breast diseases. Despite the perceptions of the public and some medical colleagues that diagnostic pathology lacks ambiguity and subtlety, pathologists are repeatedly faced with the need to deal with uncertainty. The usually blunt, seemingly black and white recitation of a final pathology report actually represents a synthesis of possibilities that constitute the differential diagnosis. Pathologists strive to reduce uncertainty by constant study, leading to the development and application of new insights or improved techniques. Yet, each advance brings with it a new horizon of uncertainty-a new confidence interval. One manifestation of uncertainty in the study of breast cancer and precancerous breast disease is our limited ability to separate the drivers from the hitchhikers, that is to distinguish between silent alterations acquired by the malignant cell and those that truly contribute to the malignant phenotype (7). In the clinical arena, the phenomenon of advances creating new uncertainty is illustrated by the procedure for axillary lymph node staging by sentinel lymph node mapping. The coincidence of improved surgical techniques to localize the lymph node or nodes most likely to harbor metastatic carcinoma and the application of immunohistochemistry to the lymph nodes by the

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pathologist makes it possible to determine whether axillary lymph nodes harbor metastases by examining selected lymph nodes without the need for more extensive axillary dissection. Sentinel lymph node staging results in reliable information about axillary nodal status with less morbidity than conventional axillary dissection. Nonetheless, the procedure has raised new questions about the prognostic significance of the micrometastases so elegantly uncovered, and uncertainty about the need for therapy based on this finding. Pathologists have unique opportunities in breast cancer research. Technical advances now make it possible to apply the extraordinarily powerful techniques of molecular and genetic analysis directly to tissues visualized with the microscope. This is truly the intersection of the classical microscopic pathology of the 19th and 20th centuries with the molecular science of the 21st century. Using microdissection, the pathologist can select small groups of cells and even individual cells from normal and abnormal tissues that are identified and diagnosed with the microscope. DNA extracted from these minute samples can be amplified and studied for molecular alterations by a variety of techniques. This approach holds great promise for furthering our understanding of precancerous and cancerous breast diseases and for finding clues to improved prevention and therapeutic strategies. Currently, microdissection is too costly and laborious for widespread clinical application. It is possible that pathologists will employ microdissection and molecular analysis in the diagnosis of breast tissues in the next 10 to 20 years. The development of robotic instrumentation will contribute substantially to making this a clinically feasible enterprise. The ability of pathologists to distinguish between structurally normal and abnormal tissues will remain a fundamental step in diagnosis in the foreseeable future, but technological advances will require greater sophistication on the part of pathologists and continue to foster the subspeciality of Breast Pathology.

Tissue Microarrays, Gene Expression Profiles, and Breast Pathology

It is widely accepted that altered gene expression is fundamental to the neoplastic process. The devil is in the details of how the exceedingly complex system of gene actions becomes disrupted, resulting in the phenotypic changes in cells and tissues employed by pathologists for diagnosis and estimating prognosis. Interest in exploring and understanding the molecular basis of breast cancer pathology has been propelled forward in the past decade by technological advances that make it possible to

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efficiently investigate very small samples from large numbers of tumors. These studies have relied on two methodologies: high-throughput tissue microarrays and microarray gene expression profiling. The former employs histologic sections of small samples of multiple tumors and the latter uses RNA extracted from diagnostic tumor tissue samples. It can be reasonably predicted that these and related technologies will eventually have a significant impact on the role of pathologists in breast cancer diagnosis and treatment. The following discussion provides illustrations from the current literature of how these studies are being used. Although largely investigational, a few procedures, such as a recurrence score (RS) based on a 21-gene RT-PCR assay, are being employed in clinical practice for selected patients (8,9).

Tissue Microarrays
In 2003, Callagy et al. (10) described a classification of breast carcinoma based on the expression of protein biomarkers detected by immunohistochemistry in tissue microarray preparations. The inherent efficiency of tissue microarray technology made it possible for these investigators to study 13 biomarkers in 107 cases with only 39 histologic slides. The authors estimated that 1,391 slides would have been needed to obtain the same data from conventional sections. Two patterns of biomarker expression were described: estrogen receptor (ER)-related (ER, PR, bcl2, cyclin-D, p27, cytokeratin 8/18, c-myc) and proliferation-related (Mcm2, MIB1, cyclin-E, p53, c-erbB2, cytokeratin 5/6). There was a statistically significant association between the biomarker expression group and the conventional prognostic markers. Tumors in the ER-related group were more likely to have a low histologic grade and negative lymph nodes, whereas tumors expressing proliferation-related markers were more likely to be high grade and have nodal metastases. A more-complex tissue microarray study published in 2005 examined the immunohistochemical expression of 25 biomarkers in 1076 previously characterized breast carcinomas (11). Six groups of protein expression were found, representing 0.4% to 31.2% of the tumors. The biomarker-defined groups were significantly related to tumor grade, tumor size, nodal status, patient age, and prognosis. Multivariate analysis revealed that biomarker clustering was a prognostically significant independently of grade, size, and nodal status.

Gene Expression Profiling

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In contrast to tissue microarray technology that describes gene activity in terms of protein products that can be detected in tissue sections by immunohistochemistry, gene expression profiling examines large numbers of genes directly using RNA extracted from tumor tissue with the reverse transcriptase polymerase chain reaction (RT-PCR) in DNA microarrays. These studies have been done with frozen (12,13) or paraffin embedded (14) tissue samples, including needle core biopsy specimens (15,16). Needle core biopsy samples provide satisfactory material for gene expression profile analysis in the majority of cases. Zanetti-Dallenbach et al. (17) reported 82% concordance in the expression profiles for 60 genes obtained from core biopsies and subsequent surgical excision biopsy specimens from 22 patients. In four cases where gene expression profiles for the two specimens differed, the surgical biopsy specimens exhibited a higher expression of genes associated with tissue injury and repair that were probably activated by the core biopsy procedure. Rody et al. (18) found greater than 90% concordance in the gene expression profiles for estrogen and progesterone receptors and for Her2/neu in core biopsy samples from patients undergoing neoadjuvant chemotherapy when compared to immunostains of the same samples. Gene profiles that were associated with prognosis, response to chemotherapy, and patterns of metastases have been described. For example, a recurrence score (RS) indicative of the risk of recurrence after 10 years of follow-up in node-negative, estrogen receptor-positive patients treated with tamoxifen was based on a 21-gene RT-PCR expression profile (19). Analysis of the 21-gene profile resulted in a quantitative assessment of recurrence risk after treatment in this selected group of patients. Three recurrence risk categories were defined: low risk (RS<18%), intermediate risk (RS 19%30%), and high risk (RS 31%). It was found that after 10 years of follow-up, patients in the high-risk group derived a significant reduction in recurrence from combined treatment with chemotherapy and tamoxifen (11% recurrence) when compared to women treated with tamoxifen alone (38.3% recurrence). Patients in the low recurrence group did not experience a significant reduction in recurrence when chemotherapy was added to tamoxifen (tamoxifen, 3.7% recurrence; tamoxifen plus chemotherapy, 5% recurrence). There was a small benefit from chemotherapy in the intermediate-risk group (tamoxifen, 17.8% recurrence; tamoxifen plus chemotherapy, 10.1% recurrence). Histologic grade was significantly related to RS but, when graded by more than one pathologist, 5% to 12% of histologically low-grade tumors had a high RS and 19% to 36% of histologically poorly differentiated carcinomas had a low RS. Lyman et al. (9) estimated a

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net savings of $2,256 per patient treated when the choice between tamoxifen alone and tamoxifen plus chemotherapy was based on the RS derived from the 21-gene RT-PCR assay. The clinical management of a patient whose tumor grade is at odds with the RS is an issue that needs further investigation. Gene expression profiling has also been applied to breast carcinoma prognosis in other patient groups. Espinosa et al. (12) investigated a 70 gene profile in 96 stage I and II patients. A patient was classified as having a poor prognosis in this study if the tumor expressed more than 47% of the previously determined poor prognosis signature, which featured upregulation of genes involved in the cell cycle, invasion and metastasis, angiogenesis, and signal transduction (20,21). The gene profile used in this study was significantly related to overall and relapse-free survival for the entire group of patients, but not when patients were stratified by nodal status. Foekens et al. (22) used a previously validated (23) 76-gene signature to assess prognosis in node negative patients who had not received chemotherapy. Patients were classified as having a low or high risk for developing a systemic recurrence. Ten-year recurrence-free survivals were 94% and 65%, respectively, in the low- and high-recurrence risk groups as determined by the 76-gene signature. In a multivariate analysis with age at diagnosis, tumor size, grade, and menopausal status, the 76-gene signature was the only significant predictor of distant recurrence-free survival. Predicting response to chemotherapy is likely to be an important application of gene expression profiling. Mina et al. (15) identified a 22-gene signature that significantly correlated with a pathologic complete response to chemotherapy (doxorubicin and docetaxel) in 45 evaluable patients treated for locally advanced breast carcinoma. Signature genes were of three types: angiogenesis-related, proliferation-related, and invasion-related. In this study, the expression of estrogen receptor-related genes and the RS determined from the previously discussed 21-gene RT-PCR assay did not correlate with a pathologic complete response. Gianni et al. (16) reported that the expression of 86 genes correlated significantly with achieving a pathologic complete response in women with locally advanced carcinoma who received neoadjuvant paclitaxel and doxorubicin. A pathologic complete response was significantly associated with a higher expression of proliferation-related and immune-related genes and with lower expression of estrogen receptor-related genes. A pathologic complete response was

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achieved significantly more often with tumors that had a high RS based on the 21-gene RT-PCR assay. Gene expression profiling has also been investigated as a method for predicting response to neoadjuvant chemotherapy in women with primary operable breast carcinoma. One such study involved 100 women with T2-4, N0-2, M0 breast carcinoma treated with gemcitabine, epirubicin, and docetaxel (24). A signature of genes associated with a pathologic complete response included prominent representation from the following categories: TGF- signaling, RAS-related, apoptosis-related, and DNA damage responserelated genes. Ayers et al. (25) reported that a gene expression profile had 78% predictive accuracy for identifying women who achieved a complete pathologic response to multiagent neoadjuvant chemotherapy when compared to a 28% overall expected response rate. Using a different gene profile, Iwao-Koizumi et al. (26) reported 80% accuracy in predicting clinical response to docetaxel. In a trial that compared doxorubicincyclophosphamide to doxorubicin-docetaxel treatment in patients with locally advanced carcinoma, Hammerman et al. (27) found changes in gene expression profiles determined before and after neoadjuvant chemotherapy in responders but not in nonresponders. However, no gene expression profile was predictive of response to either treatment. Evidence that gene expression profiling might detect signatures associated with patterns of metastases has started to emerge. A study of primary tumor tissue from 107 patients with node negative breast carcinoma identified a panel of 69 genes that were differentially expressed when patients with metastases in bone were compared to those with non-osseous metastases (13). The genes for thyroid transcription factor 1 (TTF1) and TTF3 were found to be most highly expressed in the 69-gene signature associated with bone metastases. Other categories of genes associated with bone metastases were related to cell adhesion, signaling pathways, and cell organization. Others have also investigated gene profiles associated with breast carcinoma metastases in bone (28,29), the lungs (30), and locoregional recurrence after mastectomy (31). The mechanisms by which these gene profiles predisposed to patterns of recurrence and the roles of particular genes or groups of genes in this process have not been elucidated. Nonetheless, the ability to predict likely sites of distant metastases could provide an opportunity to selectively offer adjuvant therapy designed to inhibit site-specific recurrence such as bisphosphonates for bone metastases (32) and radiotherapy for local recurrences.

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Contradictory results have been reported in attempts to find gene profiles predictive of low and high risk for axillary nodal metastases. Weigelt et al. (33) did not find a gene signature associated with axillary lymph node metastases in a study of 295 tumors. In a considerably smaller series of cases, West et al. (34) reported that gene expression profiling was predictive of axillary nodal status. The use of gene profiling as a tool to predict the risk for axillary lymph node metastases and to identify the genes involved in this process, which is fundamental to breast carcinoma staging, has not yet received the attention warranted by the clinical importance of this aspect of the disease. Gene expression profiles may be influenced by mutations associated with hereditary breast carcinoma. Hedenfalk et al. (35) described a panel of 176 genes that had different expression patterns in tumors with BRCA1- and BRCA2-associated mutations. The gene expression patterns of the BRCAassociated carcinomas also differed from those found in sporadic carcinomas. The strength of these results is limited by the small numbers of cases studied.

The Future of Tissue Microarray and Gene Expression Profiling in Breast Cancer
The foregoing examples offer an insight into the promise and complexity of tissue microarray and gene expression profiling in breast carcinoma. Many issues remain to be addressed before these techniques can be introduced into general clinical practice. There are important technical problems related to the use of formalin-fixed paraffin embedded tissue and the limited availability of unfixed frozen tissue. Most breast carcinoma gene expression profiling studies have had significant limitations that include small numbers of tumors and/or patients, heterogeneous patient groups, and nonstandardized treatment. There does not appear to have been an a priori basis for selecting genes for study, and there is little understanding of how the genes that form a particular signature are related to the outcome under investigation (e.g., site of metastases, response to therapy or to the histomorphology of breast proliferative lesions and carcinomas). It has been reported that analysis of data from two different studies (21,23) identifying prognostic gene profiles consisting, respectively, of 70 and 76 genes had only 3 genes in common (36). Disparities of a similar magnitude between other gene signatures have also been noted (37). The predictability of gene expression profiles can be substantially reduced when they are cross-tested from one series of tumors to another (36,37). On the other hand, clustering

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of tumor subtypes (e.g., basal, luminal, erb-b2) was reported in an analysis of three studies (38). A re-analysis of data from a study (21) that reported a specific 70-gene signature revealed that this gene set was not unique and that other gene signatures also present in the data set correlated with outcome (37). No single gene profile has proven to be applicable to breast carcinoma generally. At present, the 21-gene RT-PCR assay has been used clinically in the limited subset of patients with estrogen receptor positive, lymph nodenegative tumors under treatment with tamoxifen to estimate the benefit that would be obtained from adding chemotherapy. It is likely that other clinically useful gene signatures will be found by analyzing subsets of patients which are defined by established markers (e.g., ER, erb-b2) and clinicopathologic parameters (e.g., age at diagnosis, hereditary breast carcinoma, tumor grade). To develop assays that can be standardized for clinical practice, larger groups of tumors need to be evaluated and gene profiles in defined patient groups need to be cross-tested. As more studies are performed, gene signatures associated with particular end-points can be developed and modified when new information becomes available. Gene expression profiling and tissue microarrays offer great promise as tools that may revolutionize the treatment of breast carcinoma. Nonetheless, we are certainly not on the verge of abandoning conventional prognostic markers, and there is reason to believe that they will continue to play an important clinical role in the foreseeable future, as suggested by Eden et al. (39) in a provocative study titled Good old' clinical markers have similar power in breast cancer prognosis as microarray gene expression profiles. The extent to which the pathology community adapts to this new era and incorporates these technologies into clinical practice in the pathology laboratory will be an important factor in determining the continued primary role of pathology in cancer classification and diagnosis.

References
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12. Espinosa E, Fresno Vara JA, Redondo A, et al. Breast cancer prognosis determined by gene expression profiling: a quantitative reverse transcriptase polymerase chain reaction study. J Clin Oncol 2005;23: 72787286. 13. Smid M, Wang Y, Klijn JGM, et al. Genes associated with breast cancer metastatic to bone. J Clin Oncol 2006;24:22612267. 14. Cronin M, Pho M, Dutta D, et al. Measurement of gene expression in archival paraffin-embedded tissues. Development and performance of 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol 2004;164:3542. 15. Mina L, Soule SE, Badve S, et al. Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue. Breast Cancer Res Treat 2007;103:197208. 16. Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol 2005;23:726577. 17. Zanetti-Dallenbach R, Vuaroqueaux V, Wight E, et al. Comparison of gene expression profiles in core biopsies and corresponding surgical breast cancer samples. Breast Cancer Res 2006;8:R51. Available at: http://breast-cancer-research.com/content/8/4/R51. 18. Rody A, Karn T, Gatje R, et al. Gene expression profiles of breast cancer obtained from core cut biopsies before neoadjuvant docetaxel, adriamycin, and cyclophosphamide chemotherapy correlate with routine prognostic markers and could be used to identify predictive signatures. Zentralbl Gynakol 2006;128:7681. 19. Paik S, Shak S, Tang G, et al. A multi-gene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004;351:28172826.

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