Chapter 21: Breast

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.

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.

Multigene Prognostic Assays

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.

Serum-based Biomarkers in Breast Cancer

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.

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.

CA 15-3 and CA 27.29

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.

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%.

Carcinoembryonic Antigen (CEA)

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.

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.

Description

While most cases of breast cancer are caused by acquired somatic mutations, approximately 5% to 10% of breast cancer cases are attributed to a germline mutation in a highly penetrant cancer predisposition gene. A large proportion of these hereditary cases are associated with mutations in 2 genes, BRCA1 and BRCA2. Mutations in BRCA1 and BRCA2 cause the hereditary breast and ovarian cancer syndrome, an autosomal dominant disorder in which the risk of both breast and ovarian cancer is significantly increased compared with the general population. BRCA1 and BRCA2, which are located at chromosome 17q21 and 13q12, respectively, are tumor suppressor genes that play essential roles in the repair of double-stranded DNA breaks and thus in the maintenance of genome stability. Accordingly, in tumors from individuals with hereditary breast and ovarian cancer syndrome, the wild-type BRCA1 or BRCA2 allele is deleted, consistent with a tumor suppressor function for BRCA1 and BRCA2.

A woman's risk for harboring either a BRCA1 or BRCA2 mutation is increased by certain factors related to her personal and family medical history. Personal factors include: 1) early onset breast cancer (ie, diagnosis before 50 years of age); 2) bilateral or multifocal breast cancers; and 3) a history of both breast and ovarian cancer. Factors from the family history include: 1) breast cancer or breast and ovarian cancer in a pattern consistent with autosomal dominant transmission; and 2) breast cancer in a male relative.

Hereditary breast and ovarian cancer syndrome shows incomplete penetrance. Among women with either a BRCA1 or BRCA2 mutation, the lifetime risk of developing breast cancer is 60% to 80%. The lifetime risk of developing ovarian cancer is 15% to 60% for women with BRCA1 mutations and 10% to 27% for women with BRCA2 mutations. Mutations in BRCA1 and BRCA2 also increase the risk of male breast cancer. Individuals with hereditary breast and ovarian cancer syndrome are also at risk for other tumors, including melanoma and cancers of the prostate (in males) and pancreas.

Determination of BRCA1 and BRCA2 mutation status is an important clinical assessment, as the identification of a deleterious mutation may alter clinical management. Interventions available to BRCA1 and BRCA2 mutation carriers include intensive screening, chemoprevention, prophylactic mastectomy, and prophylactic oophorectomy. Prophylactic oophorectomy, which reduces the risk of breast cancer as well as ovarian cancer, is recommended for all mutation carriers at the completion of childbearing.

Determination of BRCA1 and BRCA2 Mutations

Genetic testing for BRCA1 and BRCA2 mutations should be offered to individuals with a personal or family history suspicious for hereditary breast and ovarian cancer syndrome, and to women at risk because of a family member known to harbor a deleterious mutation in 1 of these genes. It is critical that testing be offered in the setting of appropriate genetic counseling, so that individuals are provided appropriate information regarding the risks, benefits, and limitations of genetic testing. Such counseling should also include consideration of how the results of such testing might affect other family members.

Study of kindreds with hereditary breast and ovarian cancer syndrome has identified hundreds of different deleterious mutations in the BRCA1 and BRCA2 genes. Most consist of frameshift or nonsense mutations, which are predicted to result in a loss of function of the encoded gene product. Due to the large number of different mutations described, genetic testing to assess for most of these involves examination of the DNA sequence of the entire coding region of each gene. Additional molecular testing may also be employed to assess for certain large genomic rearrangements that cannot be detected by routine DNA sequencing. In cases where a known deleterious mutation has already been identified in a family member, targeted mutation analysis is performed to specifically assess for the familial mutation.

When possible, genetic testing should be performed on an individual who has been diagnosed with breast or ovarian cancer because this strategy provides the most information for other members of the family. Genetic testing can lead to 4 possible outcomes: a true-positive result, a true-negative result, an uninformative result, and a variant of uncertain significance. A true-positive result occurs when a deleterious mutation known to be associated with increased cancer risk is identified; such a result confirms the diagnosis of hereditary breast and ovarian cancer syndrome. A true-negative result occurs only when the tested individual is found to lack a specific deleterious mutation already known to run in the family; a true-negative result thus reduces the tested individual's risk of developing breast and/or ovarian cancer to that of general population. An uninformative result occurs when a mutation is not identified in an individual from a family in which a deleterious mutation has not yet been identified; an uninformative result does not exclude the possibility of a BRCA1 or BRCA2 mutation that cannot be detected by current testing methodologies, nor does it exclude the possibility of a mutation in another cancer susceptibility gene. Genetic variants of uncertain significance are typically missense variants of unknown functional significance or intronic variants not predicted to disrupt mRNA processing; individuals harboring variants of unknown significance may still be at increased risk for cancer, and their medical management should be based on the known family history. In all cases, posttest genetic counseling should be performed to ensure that individuals fully comprehend the implications of their testing results.

In addition to BRCA1 and BRCA2, highly penetrant forms of hereditary breast cancer have also been linked to mutations in the tumor suppressor genes TP53, PTEN (phosphatase tensin homolog), and STK11. While mutations in these genes comprise a smaller proportion of hereditary breast cancer cases than BRCA1 and BRCA2, identification of these mutations similarly has profound implications for clinical management and for genetic risks of family members. Genetic testing for mutations in these genes is conducted using molecular approaches similar to those used for BRCA1 and BRCA2 and can also lead to the same 4 possible test outcomes described above. Genetic testing for these mutations should be offered in the setting of appropriate genetic counseling to at-risk family members of an individual with a known deleterious mutation and to individuals with personal or family histories suspicious for these particular syndromes, which are described in detailed below.

Li-Fraumeni Syndrome

Germline mutations in TP53 cause Li-Fraumeni syndrome, a rare autosomal dominant condition associated with the development of a variety of tumor types throughout life, including in childhood. TP53, located at chromosome 17p13, encodes the p53 protein, which plays key roles in DNA repair, cell cycle control, and the initiation of apoptosis. In addition to breast cancer, tumors associated with Li-Fraumeni syndrome include osteosarcomas, soft tissue sarcomas, brain tumors, leukemias, and adrenocortical carcinomas. In families with Li-Fraumeni syndrome, breast cancer is common and may account for one third of all cancers.

Cowden Syndrome

Germline mutations in PTEN cause Cowden syndrome, a rare autosomal dominant condition characterized by an increased risk of certain cancers and the development of multiple skin and mucosal hamartomas (focal malformations that resemble neoplasms but result from faulty development of the tissue). PTEN, located at chromosome 10q23, encodes a phosphatase that downregulates the phosphatidylinositol-3-kinase (PI3K) signal transduction pathway, thereby contributing to the regulation of cell growth. The spectrum of cancers seen in Cowden syndrome includes thyroid cancer, uterine cancer, renal cell cancer, and breast cancer. Females with Cowden syndrome have a 25% to 50% lifetime risk of developing breast cancer.

Peutz-Jeghers Syndrome

Germline mutations in STK11 cause Peutz-Jeghers syndrome, a rare autosomal dominant condition characterized by gastrointestinal polyps and mucocutaneous pigmentation. STK11, located at chromosome 19p13, encodes serine/threonine-protein kinase 11 and functions to regulate cell polarity, energy utilization, and apoptosis. Individuals with Peutz-Jeghers syndrome are at increased risk for the development of a variety of cancers, including colorectal, gastric, pancreatic, ovarian, and breast. The lifetime risk of breast cancer reported for females with Peutz-Jeghers syndrome has been estimated at 30% to 50%.