This chapter focuses on laboratory testing relevant to breast cancer. Infections of the breast are included in Chapter 5.

Description

Cancers of the breast constitute a major cause of mortality in women of Western countries. In the United States, the lifetime probability that a woman will develop breast cancer is 1 in 8. Breast cancer accounts for 29% of new cancer cases and 14% of cancer deaths in American women. About 1% of breast cancers occur in males. The risk of developing breast cancer is influenced by several factors. These factors include increased age, family history of breast cancer (especially in a first-degree relative), hormonal factors (early age at menarche, older age of menopause, older age at first full-term pregnancy, fewer number of pregnancies, and use of hormone replacement therapy), clinical factors (high breast tissue density and benign breast diseases associated with atypical hyperplasia), obesity, and alcohol consumption. Since 1990, the mortality rate associated with female breast cancer has decreased in the United States, a decline that has been attributed to both therapeutic advances and early detection.

For localized breast cancer, primary treatment typically consists of either breast-conserving surgery and radiation or mastectomy. Most patients with invasive breast cancer subsequently receive systemic adjuvant chemotherapy and/or hormone therapy, both of which have been shown to reduce systemic recurrence and breast cancer-related mortality. However, the fact that some patients who lack lymph node involvement are cured by the combination of surgery and radiotherapy suggests that adjuvant treatment may not be necessary in all cases. Therefore, to rationally administer adjuvant therapy to patients with local disease, several prognostic factors are considered to assess the risk for recurrence. These prognostic factors include tumor size, axillary node involvement, histological type, cytological grade, lymphatic and vascular invasion, and certain biomarkers associated with breast cancer.

While adjuvant therapy improves patient outcomes, 25% to 30% of women with lymph node-negative and at least 50% to 60% of women with node-positive disease develop recurrent or metastatic disease. Metastatic breast cancer is currently regarded as incurable. Therapeutic options for metastatic disease include chemotherapy, hormone therapy, and molecularly targeted therapies. In the context of metastatic disease, information gained from serial monitoring of tumor markers detected in the serum may contribute to decisions to continue or terminate a particular treatment.

Tissue-based Biomarkers in Breast Cancer

Assessment of biomarkers in tissue obtained from the patient’s breast tumor is routinely performed to obtain prognostic information and to guide therapy.

Estrogen Receptor (ER) and Progesterone Receptor (PR)

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.

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.

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.

HER2

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.

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.

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.

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.