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Tissue-based Biomarkers in Breast Cancer
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Assessment of biomarkers in tissue obtained from the patient's breast tumor is routinely performed to obtain prognostic information and to guide therapy.
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Estrogen Receptor (ER) and Progesterone Receptor (PR)
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ER and PR are intracellular receptors that bind to lipid-soluble steroid hormones that diffuse into target cells. Following ligand binding, 2 receptor subunits dimerize to form a single, functional DNA-binding unit that binds to specific DNA target sequences to induce transcription of target genes. There are 2 different forms of the ER, termed ER-α and ER-β, which are encoded by separate genes. Clinical assays assess ER-α, the classical form of the receptor. PR has 2 isoforms that differ in molecular weight but are encoded by a single gene.
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Assessment of biomarkers in tissue obtained from the patient's breast tumor is routinely performed to obtain prognostic information and to guide therapy.
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Measurement of the ER and PR status of the tumor is recommended in all patients with breast cancer. ER expression is present in approximately 70% of breast cancers, is associated with a favorable prognosis, and suggests that the growth of the tumor may be estrogen-dependent. The primary purpose of determining ER and PR status in breast cancers is to identify those patients, in both the adjuvant and metastatic settings, who are likely to respond to endocrine treatments. These treatments act by either preventing the formation of estrogen from its precursors or blocking estrogen from binding to its receptors. Endocrine treatments include tamoxifen, ovarian ablation (surgical or chemical), aromatase inhibitors (anastrozole, letrozole, and exemestane), and irreversible ER inhibitors (eg, fulvestrant). In patients with ER-positive tumors, 5 years of adjuvant treatment with tamoxifen significantly reduces annual death rates from breast cancer, while in patients with ER-negative tumors, tamoxifen shows little effect on recurrence or death, and it does not significantly modify the effects of polychemotherapy.
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ER/PR status is routinely assessed by immunohistochemistry (IHC) in the clinical setting. IHC evaluates the percentage of cells with nuclear ER/PR. The intensity of staining is also recorded as a measure of assay quality. The use of validated antibodies is required, and a positive control (ie, a control tissue with tumor cells known to express the respective receptor) must be examined in parallel. A tumor is scored as positive for either ER or PR if ≥1% of tumor cell nuclei are immunoreactive. A tumor is scored as negative for ER or PR if <1% of tumor cell nuclei are immunoreactive in the presence of demonstrable staining in adjacent normal breast epithelial cells, which serves as an internal positive control. If tumor cells are not found to be immunoreactive and the specimen lacks an appropriately stained internal control, the tumor is scored as uninterpretable for ER or PR. For optimal results, breast resection specimens should be fixed within 1 hour. Fixation should be performed in 10% neutral buffered formalin for at least 6 hours and for not more than 72 hours in order to preserve ER and PR epitope recognition and thus avoid false-negative results.
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HER2 (also known as ERBB2 and NEU) is a proto-oncogene located at chromosome 17q11 that is a member of the epidermal growth factor receptor (EGFR) family. Like other EGFR family members, HER2 is a transmembrane receptor with cytoplasmic tyrosine kinase activity. Dimerization of the receptor leads to phosphorylation of a variety of substrates, resulting in the activation of intracellular signaling pathways important for cell proliferation and survival.
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While normal cells contain 2 copies of the HER2 gene (1 copy on each chromosome 17), in approximately 10% to 25% of breast cancers HER2 gene copy number is increased at least 2-fold relative to the number of copies of chromosome 17, a phenomenon termed gene amplification. Gene amplification results in overexpression of the HER2 protein at the cell surface, which in turn promotes tumor cell proliferation and survival. Tumors that overexpress HER2 behave more aggressively than those lacking overexpression, and they are associated with poorer clinical outcomes.
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Assessment of HER2 status of the tumor is recommended in all patients with invasive breast cancer. The primary purpose of HER2 testing is to identify those patients with early or advanced breast cancer who are eligible for treatment with trastuzumab, a recombinant monoclonal antibody that recognizes HER2. Although its exact mechanism of action remains to be fully elucidated, trastuzumab has been shown in both in vitro assays and animal studies to inhibit proliferation of human tumor cells that overexpress HER2. In patients with HER2-positive early stage breast cancer, the addition of trastuzumab to adjuvant chemotherapy significantly improves disease-free and overall survival. Additionally, in patients with HER2-positive metastatic breast cancer, the addition of trastuzumab to adjuvant chemotherapy significantly increases the time until disease progression. Because a small percentage of patients treated with trastuzumab develop cardiotoxicity, the elimination of false-positive HER2 results is important so that patients are not exposed to this risk unnecessarily.
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The primary purpose of HER2 testing is to identify those patients with early or advanced breast cancer who are eligible for treatment with trastuzumab, a recombinant monoclonal antibody that recognizes HER2.
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HER2 status is routinely assessed in formalin-fixed tissues by either fluorescence in situ hybridization (FISH) or IHC. FISH assesses HER2 status at the DNA level. A fluorescent-labeled nucleic acid probe that recognizes the HER2 gene on chromosome 17 is hybridized on tissue sections, and the average number of HER2 signals per nucleus is determined in areas of invasive tumor. In some assay systems, an additional probe that recognizes the centromeric region of chromosome 17 (CEP17) (and which is labeled with a different fluorophore) is included to allow the ratio of the average number of copies of HER2:CEP17 (the “FISH ratio”) to be calculated. Tumors with intermediate results are considered equivocal for gene amplification; in these cases, IHC for HER2 protein may be performed to resolve HER2 status. Chromogenic in situ hybridization (CISH) may be performed as an alternative to FISH. In CISH, the HER2 probe is visualized by an immunoperoxidase reaction. This enables CISH results to be scored using a conventional light microscope rather than the fluorescence microscope that is required for FISH.
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In contrast to the FISH assay, IHC assesses HER2 status at the protein level. The level of HER2 protein expression is scored on a semiquantitative scale. Tumors with 3+ protein expression are scored as positive for HER2 protein expression, while tumors with 0 or 1+ protein expression are scored as negative. Tumors with intermediate staining patterns (eg, cases showing complete membrane staining that is weak in intensity) are considered equivocal; in these cases, FISH may be performed to resolve HER2 status.
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Multigene Prognostic Assays
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Recently, clinical assays that utilize expression information gathered across a panel of genes have been developed to predict recurrence risk and guide adjuvant chemotherapy decisions in patients with early stage breast cancer. Several assays, which examine different sets of genes, are currently available. Two of the more widely validated testing platforms involve examination of 21 and 70 genes, respectively. Depending on the particular testing platform, fresh frozen tissue or formalin-fixed, paraffin-embedded tissue may be required. While methods used to quantify gene expression vary by platform, 1 approach involves using the enzyme reverse transcriptase to convert messenger RNA into complementary cDNA; the resulting cDNA then serves as a template for assays such as quantitative polymerase chain reaction or microarray gene expression profiling. While the role for these multigene prognostic assays in the routine clinical management of patients with breast cancer remains to be fully established, several of these assays have been validated in retrospective studies, and their utilities are now being examined in prospective studies.
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Serum-based Biomarkers in Breast Cancer
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Serum-based tumor markers may be useful in the identification and management of patients with breast cancer. The ideal breast cancer marker would be both specific for breast cancer and sufficiently sensitive for screening purposes. Unfortunately, no marker identified to date meets these criteria. However, some markers may have utility in evaluating the progression of disease after initial therapy and for monitoring subsequent treatment.
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When considering the use of serum tumor markers, several points should be kept in mind: 1) none of the currently available markers is elevated in all patients with breast cancer, even in the setting of advanced disease, so that a normal tumor marker level does not exclude a malignancy; 2) these markers are most sensitive for detecting metastatic disease and have little value in the diagnosis of local or regional recurrences; 3) the magnitude of change in marker levels that correlates with disease progression or regression has not been firmly established; 4) tumor marker levels may paradoxically rise after initiation of chemotherapy, a phenomenon attributed to therapy-mediated apoptosis or necrosis of tumor cells; 5) tumor marker levels may be increased in the setting of certain benign diseases.
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The ideal breast cancer marker would be both specific for breast cancer and sufficiently sensitive for screening purposes. Unfortunately, no marker identified to date meets these criteria. However, some markers may have utility in evaluating the progression of disease after initial therapy and for monitoring subsequent treatment.
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CA 15-3 and CA 27.29 represent different but overlapping epitopes of the MUC1 protein, a large, complex glycoprotein expressed at the luminal surface of glandular epithelial cells. In malignant cells, MUC1 may be overexpressed, and increased amounts of MUC1 may be shed into the circulation. MUC1 levels in serum may be assessed by immunoassays employing distinct monoclonal antibodies that recognize either the CA 15-3 or CA 27.29 epitopes. Results obtained from assays assessing CA 15-3 and CA 27.29 are highly correlated. Importantly, CA 15-3 elevations have been shown to occur in other malignancies and, to a lesser extent, in nonneoplastic disease. These pathologic conditions include adenocarcinomas of the colon, lung, ovary, and pancreas, as well as chronic hepatitis, cirrhosis, sarcoidosis, tuberculosis, and systemic lupus erythematosus.
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In early stage breast cancer, elevated CA 15-3 levels are associated with a worse prognosis. However, because CA 15-3 and CA 27.29 show fairly low sensitivity for detection of early disease, the role of these markers in the management of early stage breast cancer remains unclear. Measurement of either CA 15-3 or CA 27.29 is therefore not recommended for screening, diagnosis, or staging of breast cancer. After primary and/or adjuvant therapy, increases in CA 15-3 or CA 27.29 can predict recurrence several months before other testing modalities or the development of symptoms. However, because prospective, randomized clinical trials have yet to demonstrate if such early detection of occult or asymptomatic metastases impacts disease-free or overall survival, the routine use of CA 15-3 and CA 27.29 for this application is not currently recommended. In patients with metastatic disease who are undergoing active therapy, CA 15-3 or CA 27.29 testing may be used in conjunction with history, physical exam, and diagnostic imaging to monitor the response to treatment. While the use of CA 15-3 or CA 27.29 alone to monitor response to treatment is not recommended, in the absence of readily measurable disease, an increasing CA 15-3 or CA 27.29 may be used nonetheless to identify treatment failure. In most clinical trials to date, a significant alteration in CA 15-3 has been defined as a concentration change of at least 25%.
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Carcinoembryonic Antigen (CEA)
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CEA is a cell-surface glycoprotein involved in cell adhesion that is normally expressed in the developing fetus. CEA levels in the blood decrease to very low levels after birth, although levels may be elevated slightly in smokers. CEA expression may be elevated in several types of cancer, including cancers of the breast, colon, pancreas, lung, and ovary, because of antigen shedding into the circulation. CEA may also be elevated in nonneoplastic diseases, including inflammatory bowel disease, pancreatitis, and liver disease. CEA is detected by immunoassay.
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In early stage breast cancer, elevated CEA levels are associated with a worse prognosis. CEA is not recommended for screening, diagnosis, staging, or routine surveillance of breast cancer patients after primary therapy. Like CA 15-3 and CA 27.29, CEA testing may be used in conjunction with history, physical exam, and diagnostic imaging to monitor the response to treatment in patients with metastatic disease who are undergoing active therapy. However, CEA should not be used alone for this purpose. Compared with CA 15-3, CEA is generally a less sensitive marker for breast cancer. However, in some patients with breast cancer, elevations of CEA may occur in the setting of normal CA 15-3 or CA 27.29 levels.