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Breast Marker StudiesDecember 21, 2009: PhenoPath Laboratories Retests Nearly 3,000 Breast Cancer Specimens from Quebec Breast Cancer Inquiry. See the press release. The role of the pathologist in the evaluation of tumors now transcends that of determining the correct 'cell type' diagnosis, and this is best illustrated in the context of breast cancer. Breast oncologists want to know not only the standard histologic features of the breast cancer, such as its type and grade, but also make treatment decisions in breast cancer patients based upon the phenotypic and/or genotypic characteristics of the tumor, such as the presence of hormone receptors and the HER2 oncogene status. The most clinically relevant prognostic and treatment-guiding markers include the following:
ER/PREstrogen and progesterone receptor status of a primary breast tumor strongly predicts response to tamoxifen. ER and PR determination by immunohistochemistry in the setting of formalin fixed, deparaffinized tissue is the most reliable method to determine hormone receptor status in breast cancer.
HER2 (c-erbB-2) oncogeneThis oncogene, a member of the epidermal growth factor receptor family, has been found to be altered in approximately 20% of breast cancers. The underlying alteration is an amplification of the gene, resulting in more than the usual 2 copies of HER2 gene present (one on each copy of chromosome 17) in both normal breast epithelium and the majority of breast cancers without HER2 alterations. (Gene amplification is defined as the ratio of HER2 to chromosome 17 signals of greater than 2.) Amplification of the HER2 gene is almost always associated with overexpression of the HER2 protein, as assessed by immunohistochemistry. The data that PhenoPath Laboratories have accumulated over the past few years, the HER2 status of more than 4,000 breast cancer cases simultaneously evaluated with FISH as well as IHC, demonstrate that the positive predictive value of a 3+ IHC study for amplification by FISH is 94%, and the negative predictive value for a negative IHC study (i.e., a score of 0 or 1) for negative amplification by FISH is 96%. A disproportionate fraction of the cases with discordant IHC and FISH results are cases of aneuploid tumors showing excess copies of chromosome 17, which may lead to overexpression of the protein, but without concomitant amplification (as the number of copies of the HER2 gene may not be more than two-fold that of chromosome 17). We recommend the use of IHC as an initial screen for HER2 status, with FISH studies performed on cases that show 2+ levels of immunostaining; such cases are indicated as showing an 'indeterminate' HER2 status by IHC. An altered HER2 status is an independent marker of adverse clinical outcome in breast cancer. More recently, however, interest has focused on the demonstration that HER2 overexpression can predict response to Adriamycin-based adjuvant chemotherapy, as well as resistance to tamoxifen, even in the setting of ER/PR expression. Furthermore, the recent introduction of immunotherapy with a 'humanized' monoclonal antibody, Trastuzumab (Herceptin™) directed at the HER2 protein, has resulted in increased screening of breast cancers for HER2 overexpression for determination of optimal treatment. p53 tumor suppressor genep53 appears to normally function as a 'brake' on cell proliferation, and loss of function can lead to loss of cell proliferation control. Mutation of the p53 gene usually leads to overexpression of the protein, which is immunohistologically detectable as nuclear positivity with anti-p53 antibodies. Approximately 10-20% of primary breast cancers overexpress p53, and several studies suggest that this is a powerful predictor of long-term outcome.
Myoepithelial markersNormal breast ducts and lobules are comprised of two epithelial cell layers. Loss of the outer myoepithelial layer is a hallmark of infiltrating carcinomas in the breast. The outer myoepithelial layer is retained in all benign proliferative processes as well as ductal carcinoma in situ. Consequently, identification of the presence or loss of myoepithelium using antibodies to the myoepithelial-specific proteins can help in a number of important problems in breast pathology: (a) distinguishing in situ vs. infiltrating carcinoma, e.g., ruling in or out the presence of microinvasion; (b) distinguishing sclerosing adenosis and other benign sclerosing lesions from infiltrating carcinoma; (c) distinguishing benign papillomas from papillary carcinoma, among others. These studies lend themselves well to core needle biopsies, where the larger 'architecture' is often not apparent. Published studies performed at PhenoPath Laboratories and elsewhere point to the proteins, smooth muscle myosin heavy chain (SMM-HC) and p63, as the most sensitive and specific markers of myoepithelium for this purpose, although other markers (cytokeratin 5, CD10, calponin, etc.) can be of help in some cases.
Markers of cell proliferationImmunohistochemical studies can be used to quantify cell proliferation in breast cancers by using an antibody that specifically identifies proliferating cells. MIB-1 is a 'second generation' antibody to the Ki-67 antigen, a protein complex expressed during all non-GO (i.e., non-resting) phases of the cycle. Note that the fraction of tumor cells which are Ki-67- positive is generally much higher than that fraction in S-phase, as determined by, for example, flow cytometric analysis. In general, 'Ki-67 indices' correlate with histologic grade and, in selected studies where follow-up has been obtained, have been predictive of clinical outcome. Which antibodies to run in selected clinicopathologic settingsWe believe that antibodies are best used in a selective manner, with the exact choice of antibodies determined by the clinical setting. It is through careful initial review of a given case, including the H&E stained section and the clinical history, that the years of experience of PhenoPath Laboratories' pathologists can be brought to bear to select the most appropriate antibodies. For example, in the setting of a carcinoma in the lung presenting in a patient with a history of breast cancer, the principal differential diagnosis is metastatic breast vs. primary lung carcinoma, and the antibodies most useful will be those to: estrogen and progesterone receptors (assuming the tumor was ER and/or PR positive); GCDFP-15 and mammaglobin; TTF-1 and surfactant ApoA. In this setting antibodies to CK7 and CK20 may not be informative. Additional markers however, will prove useful if all the above markers are negative and other organs are entertained as possible primary sites. |
