Cancer of the Breast

Diet

The best-established dietary factor associated with increased risk for breast cancer is higher consumption of alcohol. Initial studies correlated higher fat intake and decreased intake of fruit and vegetables with an increased risk for breast cancer, but more recent prospective studies have failed to confirm those observations. One randomized study (Women's Healthy Eating and Living [WHEL] Study) in breast cancer patients who consumed a low-fat and high–fruit-and-vegetable diet did not demonstrate a decreased risk of breast cancer recurrence. However, a second randomized study (Women's Intervention Nutrition Study [WINS]) demonstrated a lower risk of recurrence with a decreased intake of dietary fat associated with modest weight loss in breast cancer survivors.

Dietary phytoestrogens, such as those found in soybeans, have a chemical structure that is similar to that of 17β-estradiol and can bind to the ER to compete with estrogens. Their consumption may have a weak protective effect against breast cancer. More recent studies have suggested an inverse association of soy intake and breast cancer in Asian but not non-Asian populations. Although there is significant interest in the association of vitamin D intake and breast cancer risk, there are insufficient data to draw conclusions regarding potential benefit at this time; some data suggest that there may be an association in postmenopausal women.

Ionizing Radiation

The accumulated knowledge about radiation-related breast cancer risk in women derives mainly from epidemiologic studies of patients exposed to diagnostic or therapeutic radiation and of Japanese atomic bomb survivors. Therapeutic radiation, particularly extended-field radiation therapy for Hodgkin lymphoma, has been associated with an increased risk of breast cancer. The age of exposure is important, with the highest breast cancer risk seen in women undergoing mantle irradiation around puberty, and is minimal for women exposed after menopause.

Exogenous Hormones

The NHS originally was designed to determine whether oral contraceptive use was associated with an increase in breast cancer. Analysis of data from that study and many additional studies has confirmed that current users of exogenous hormones have a slightly increased risk of developing breast cancer, although this risk resolves within 5 years of discontinuation of therapy. In addition, use of exogenous hormones is associated with a decreased risk of colon and ovarian cancer; there is no increase in overall cancer mortality associated with their use. The Women's Health Initiative (WHI) randomized study of HRT in postmenopausal women demonstrated an increased risk of invasive breast cancer with the combination of estrogen plus progesterone therapy, but not with estrogen alone. Other studies have also demonstrated that breast cancer risk is affected by type of HRT and time of initiation of HRT, with an increased risk seen with therapy started soon after menopause. After initial publication of the WHI results and widespread discontinuation of HRT, age-adjusted incidences of invasive breast cancer decreased in the United States between 2002 and 2003 by 7%, primarily in ER-positive tumors in women 50 years of age or older.

Reproductive Factors and Endogenous Hormones

Breast cancer risk consistently has been correlated with an earlier age of menarche, later age of menopause, nulliparity, and late age of first birth, all of which determine the cumulative number of ovarian cycles. This finding is consistent with the correlation between estrogen levels and breast cancer risk. In postmenopausal women, an increased risk of breast cancer has been demonstrated in those with estradiol levels in the highest quartile.

Obesity and Body Habitus

There is a clear association between obesity and breast cancer risk. Women with a body mass index (BMI) in the overweight (25–30) or obese (>30) range have been shown to have an elevated risk of breast cancer. In addition, increased BMI is associated with an increased risk of breast cancer death. The increased risk of breast cancer with obesity in postmenopausal women appears to be mediated, at least in part, by elevated endogenous estrogen and, possibly, insulin levels. An increased risk of breast cancer has also been associated with increasing height, with a relative risk (RR) of 1.17 (95% confidence interval [CI], 1.15–1.19) for every 10 cm of additional height.

Prior Breast Biopsy

Finding a nonproliferative lesion such as a cyst or mild hyperplasia of the usual type with a breast biopsy is generally not associated with an increased risk of breast cancer ( Table 88.2 ). Proliferative lesions without evidence of atypia, such as usual ductal hyperplasia, sclerosing adenosis, and intraductal papillomas, are associated with a 1.5- to 2-fold increased risk of developing breast cancer. In contrast, the risk of developing breast cancer is substantially increased in patients with a prior breast biopsy with evidence of atypical hyperplasia, with an RR of breast cancer of 3.7 to 5.3. In contrast, the risk of developing breast cancer after a finding of flat epithelial atypia (FEA) was only marginally increased compared with the risk from a nonproliferative lesion.

BRCA1, BRCA2, TP53, and Hereditary Susceptibility to Breast Cancer

Germline mutations in BRCA1 and BRCA2 represent the most common identifiable cause of hereditary breast cancer. BRCA1 (chromosome 17q) and BRCA2 (chromosome 13q) are tumor suppressor genes; inherited mutations in BRCA1/2 confer impaired homologous recombination repair of DNA and are highly penetrant for hereditary breast and ovarian cancer syndrome. Mutations are particularly prevalent in the Ashkenazi Jewish population. Depending on multiple factors including the specific gene mutation, modifiers, and the population studied, the cumulative cancer risks by age 70 are estimated for a female BRCA1 mutation carrier at approximately 60% to 70% for breast cancer and 20% to 50% for ovarian cancer, and for a female BRCA2 mutation carrier, 50% to 70% for breast cancer and 10% to 30% for ovarian cancer, with higher estimates in earlier studies due to ascertainment bias and the early examination of families with multiple breast and ovarian cancer cases. Individuals with pathogenic BRCA mutations may also be at increased risk for male breast cancer, prostate cancer, pancreatic cancer, and melanoma. Multiple other genes interact with BRCA1 and/or BRCA2 and play a role in homologous recombination repair, such as PALB2, ATM, and CHEK2 ; mutations in several of these genes also confer risks of breast and other cancers, although the magnitude of these risks are lower than the those conferred by BRCA . Why these genes predispose primarily to breast and ovarian cancers remains unclear. Important to note, prognosis of BRCA mutation carriers with breast cancer appears similar to prognosis for sporadic breast cancer patients.

Inheritance of a germline pathogenic p53 mutant allele causes the rare high-penetrance familial Li-Fraumeni syndrome, characterized by multiple early-onset cancers, including sarcoma, leukemia, adrenocortical tumors, and breast cancer in women who survive childhood cancers. Thirty percent to 50% of sporadic cases of breast cancers harbor somatic mutations in p53, based primarily on sequencing of exons 5 through 8 (the DNA-binding core), where approximately 90% of mutations are found. Currently, p53 mutations that have been observed in breast cancers are listed in the p53 database maintained by the International Agency for Research on Cancer.

Myriad personal and family history factors should prompt a clinician to refer a breast cancer patient for genetic evaluation, including diagnosis before the age of 45, Ashkenazi Jewish ancestry, or diagnosis of triple-negative disease before age 60. Genetic evaluation begins with a comprehensive assessment of risk factors. Genetic testing, if indicated, should be preceded by a thorough informed consent that includes potential issues of uninformative results or variants of uncertain significance, and general implications of a positive result.

Genetic testing for hereditary breast and ovarian cancer syndrome mutations can occur in a targeted fashion or with full gene sequencing and evaluation of large genomic rearrangements. In addition, testing can be conducted with multigene panels using massively parallel sequencing that evaluates BRCA1/2 and multiple other genes simultaneously. The clinical usefulness of testing for mutations in moderate- and low-penetrance genes, particularly in the setting of phenotypic discordance, is often unclear. When testing with a multiplex panel, pretest counseling should also include discussion of the possibilities of unanticipated or phenotypically discordant findings in high-penetrance genes and low- or moderate-penetrance mutations that may have undefined clinical validity. The most recent Genetic/Familial High-Risk Assessment: Breast and Ovarian National Comprehensive Cancer Network (NCCN) Guidelines (Version 2.2017) provides recommendation for management of breast cancer risk in the context of pathogenic mutations in multiple genes, including ATM, BRCA1/2, CDH1, CHEK2, NBN, PALB2, PTEN, STK11, and TP53. Testing should be followed by posttest counseling including a discussion of the known and unknown data regarding cancer risks and management strategies.

Invasive Breast Carcinoma

The majority of invasive breast cancers are epithelial in origin and are histologically heterogeneous. Most are adenocarcinomas arising from the terminal ducts. Invasive ductal carcinoma accounts for approximately 85% of breast cancers and can form ductal structures ( Fig. 88.2 ). In contrast, invasive lobular carcinoma, which is characterized by small, regular epithelial cells that tend to align in single file and grow around ducts and lobules, accounts for 5% to 15% of breast cancers ( Fig. 88.3 ). The overall prognosis of classic invasive lobular cancer is thought to be similar to that of the invasive ductal subtype, although there may be some differences in likelihood of response to chemotherapy and endocrine therapy compared with ER-positive, HER2-negative invasive ductal carcinomas.

There are a number of less common types of breast carcinoma, including mucinous, tubular, and papillary, which are generally hormone receptor positive. Tubular cancers often resemble normal mammary ducts ( Fig. 88.4 ), whereas mucinous (colloid) carcinoma is characterized microscopically by abundant accumulation of extracellular mucin around tumor cells ( Fig. 88.5 ). In contrast, a frond-forming growth pattern characterizes papillary carcinoma ( Fig. 88.6 ). All three subtypes usually have a more favorable prognosis compared with hormone receptor–positive invasive ductal carcinomas of similar size and nodal status. In fact, according to the NCCN guidelines, adjuvant endocrine therapy is not strongly recommended for node-negative favorable-histology tumors measuring up to 3 centimeters in size, whereas treatment is recommended for invasive ductal or lobular carcinomas that are larger than 1 cm.

Although medullary carcinoma is typically ER, PR, and HER2 negative, which generally portends a poor prognosis, when it meets all the histologic criteria defined hereafter it too has a more favorable prognosis. The required histopathologic features include a well-circumscribed border, intense reaction with lymphocytes and plasma cells, poorly differentiated nuclei, a syncytial growth pattern, and little or no intraductal carcinoma ( Fig. 88.7 ). On the other hand, atypical medullary tumors, which do not meet all of these criteria, do not have the same excellent outcome, and care must be taken to avoid an incorrect diagnosis.

Adenoid cystic carcinoma and apocrine carcinoma are other rare subtypes that are typically ER, PR, and HER2 negative. In addition, apocrine carcinomas often express androgen receptor. Despite these characteristics, these tumors also typically have more indolent behavior. In contrast, some uncommon histologic types are associated with a poor prognosis and poor response to standard treatment. For example, metaplastic carcinomas generally have differentiation of neoplastic epithelial cells into other phenotypes, including squamous, spindle cell, or mesenchymal. Although they are less likely to have nodal involvement at the time of diagnosis, relapse rates are high.

BRCA- Associated Breast Cancers

BRCA1- and BRCA2- associated tumors have typical pathologic features. BRCA1 -associated breast cancers are usually of the basal-like subtype and are more likely to be negative for hormone receptors and HER2 overexpression. Conversely, BRCA2- associated breast cancers have more variable pathologic features and are often phenotypically similar to sporadic cancers, with a predominance of hormone receptor–positive tumors. Once cancer has been diagnosed, standard therapy is dictated by similar prognostic and predictive features to sporadic breast cancer, and no specific regimens are indicated on the basis of genetic predisposition at this time. The involvement of BRCA proteins in DNA repair by homologous recombination suggests that these tumors would be particularly sensitive to chemotherapies that induce DNA interstrand cross-links such as platinum salts, and there are clinical data supportive of this hypothesis. Furthermore, data suggest that BRCA -associated breast cancers may have higher 21-gene recurrence scores (RSs), potentially consistent with chemosensitivity of these tumors. Finally, poly (ADP-ribose) polymerase 1 (PARP1) is involved in the base excision repair pathway; when this repair pathway is pharmacologically inhibited in the setting of BRCA deficiency, synthetic lethality may result in cytotoxicity. The sensitivity of BRCA -associated ovarian cancer to the PARP inhibitor olaparib is demonstrative of this principle ; studies in BRCA- associated breast cancer have not yet demonstrated the same magnitude or consistency of response to date.

Noninvasive Breast Carcinomas

Much evidence supports the view that the development of malignancy is a multistep process and that invasive breast cancer has a preinvasive phase. During the carcinoma in situ (CIS) phase, normal epithelial cells undergo enough genetic alterations to result in malignant transformation. Transformed epithelial cells proliferate and pile up within lobules or ducts but lack the additional required genetic alteration that enables cells to penetrate the investing basement membrane. Two types of noninvasive breast cancers have been described: lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS).

The original description of LCIS characterized the lesion as a lobular unit with a cluster of ductules or acini filled, distorted, and distended by proliferating epithelial cells ( Fig. 88.8 ). LCIS cells have a fairly uniform pattern of clear cytoplasm containing rounded, bland nuclei. Intercellular spaces are preserved, and clear vacuoles within the cytoplasm may displace the nucleus. This often is referred to as classic LCIS. In contrast, pleomorphic LCIS is a morphologic variant that is more aggressive. Pleomorphic LCIS elicits a similar pattern of infiltrative growth as invasive lobular carcinoma, yet neoplastic cells have marked nuclear atypia and pleomorphism.

The terminal ductal lobular unit has been proposed as the site of origin of most breast cancer, including DCIS ( Fig. 88.9 ). In contrast to LCIS, which tends to be multicentric, DCIS is generally confined to one branching ductal system in the breast. DCIS, by definition, has intact basement membrane on light microscopy. Unlike pure DCIS, when microinvasion is present, type IV collagen and lamina are lost from the basement membrane in association with loss of membrane continuity. Such observations lend weight to the argument that DCIS is a precursor of invasive ductal cancer. Not all cases of DCIS progress to invasive cancer. Identifying the predictors of this process is a major challenge in understanding the biology of this disease.

The pathologic classification of DCIS previously was based primarily on the architectural patterns of DCIS as seen microscopically, including solid, cribriform ( Fig. 88.10 ), comedo, micropapillary, and papillary variants. The features of DCIS that are currently routinely used to classify lesions as low or high risk for clinical care include nuclear grade, size, presence of comedo or necrosis, and, to a lesser extent, the architectural patterns.

Estrogen and Progesterone Receptors

Estrogen plays a key role in the development of both normal breast epithelium and breast cancer, and the modulation of estrogen concentrations and ER signaling are key therapeutic modalities for the majority of breast cancers. Estrogens interact with mammary epithelial cells via specific ERs that function as nuclear transcription factors. The two known receptors, ERα and ERβ, are encoded by different genes and share an overall sequence homology of approximately 30%, although the homology is higher in the DNA- and hormone-binding regions. ERα is the receptor most closely associated with breast cancer; it is expressed mainly in the breast, uterus, and ovary. ERβ is more widely expressed, and its relationship to breast cancer is less clear. Binding of estrogen to the receptor results in a conformational change, displacement of heat shock proteins, and homodimerization. This homodimer then binds to the estrogen-responsive elements of the target genes and recruits coactivators or corepressors that further influence transcription. This participation in a multimeric signaling process likely contributes to the differential biologic effects that estrogen and estrogen-related compounds such as the selective estrogen receptor modulator (SERM) tamoxifen can have in different target organs. ER also has a minor ligand-independent transcriptional activating function, which is modified by phosphorylation and can facilitate “cross talk” between ER activity and receptor tyrosine kinase (RTK) signal transduction pathways. Approximately 70% of breast cancers express ERα (i.e., they are ER positive); these tumors tend to grow more slowly and appear better differentiated than ER-negative tumors. Moreover, antiestrogen therapy is effective in preventing development of breast cancer, reducing the likelihood of recurrence in the adjuvant setting, and prolonging survival once metastases have developed.

PR, itself encoded by an estrogen-regulated gene, gives rise to two distinct isoforms, PRA and PRB, by alternative splicing. PRB appears to be more specific to breast cancer. Interesting to note, a polymorphism in the PR promoter (+331 G/A) that increases transcription of PRB is associated with an elevated risk of endometrial and breast cancer. PR is variably expressed in ER-positive tumors, and this variability has prognostic relevance. ER-positive/PR-negative tumors occur more commonly in women older than 50 years, tend to be more aneuploid, and present as larger tumors with more frequent nodal involvement than ER- and PR-positive tumors. Furthermore, ER- and PR-positive tumors may be more likely to respond to antiestrogen therapy than ER-positive/PR-negative tumors, although more recent data suggest this likely reflects the better prognosis of the dual positive tumors as opposed to increased responsiveness to tamoxifen.

ERBB2 (HER2)

Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 20% of breast cancers as a result of amplification of the HER2/ neu gene on chromosome 17q. HER2 functions as a transmembrane RTK, activated on dimerization with another member of the epidermal growth factor family of receptors, including EGFR (ERBB1), ERBB3 (HER3), and ERBB4 (HER4). The dimerization domain of a partner receptor, such as EGFR or HER3, is exposed on binding of a ligand such as heregulin or, alternatively, through ligand-independent mechanisms. Of note, HER2 itself does not have ligand-binding capacity. The HER2-HER3 heterodimer is the most potent of these dimer pairs, based on strength of interaction and downstream signaling. The oncogenic effects of this dimer pair are predominantly exerted through activation of intracellular signaling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway, which in turn promotes proliferation and cell survival. In contrast to EGFR, in which amplifications in lung cancers frequently harbor activating mutations in the kinase domain, such activating mutations have not been found frequently in HER2 in breast cancer according to the Sanger Centre's COSMIC database, and HER2 oncogenic activity is exerted primarily through amplification.

Clinically, breast cancers marked by HER2 amplification have a more aggressive clinical course and decreased survival as compared with HER2-nonamplified tumors. HER2-amplified tumors can be associated with intracranial metastases. The identification of HER2 as an oncogene in breast cancer facilitated the development of therapies directed against it. The development and incorporation of HER2-targeted therapies into the adjuvant and metastatic settings have altered the natural history of HER2-amplified breast cancer dramatically. Trastuzumab is a HER2-targeted humanized monoclonal antibody that forms the cornerstone of HER2 targeted treatment, with proposed mechanisms of action including downregulation of HER2 dimerization and growth factor signaling cascades and induction of antibody-dependent cytotoxicity. Trastuzumab and other anti-HER2–directed therapies for treatment of both early-stage and metastatic breast cancer are described in more detail later.

Although the advent of HER2-targeted therapy has dramatically improved survival for patients with HER2-amplified breast cancer, de novo and acquired resistance to these treatments contributes to mortality. Studies aimed at elucidating mechanisms of resistance are providing further insight into the pathophysiology of HER2-positive breast cancer; two proteins, HER3 and PI3K, have emerged as key mediators of this resistance, including mutational activation of the latter. HER3 can promote oncogenic PI3K signaling, even in the presence of HER2 inhibition with trastuzumab. Furthermore, the CLEOPATRA phase III trial demonstrated that patients whose tumors harbored somatic activating mutations in the PIK3CA gene had shorter survival than those with wild-type disease. Data in the preoperative systemic therapy setting similarly identified an association between PIK3CA mutations and decreased pathologic complete response (pCR). However, these findings, while intriguing, do not yet have sufficient clinical usefulness to affect selection of therapy for individual patients. Increased levels of insulin-like growth factor–1 receptor (IGF1R) may bypass HER2 blockade by trastuzumab and allow growth factor activation of AKT, and a similar outcome may result from PTEN loss.

In addition to alterations that may circumvent HER2 signaling, changes in HER2 itself or effects of genes located nearby on chromosome 17 may also influence response to therapy. A truncated form of the HER2 protein itself that lacks the extracellular domain but retains the kinase activity has been described to correlate with trastuzumab resistance. Activating mutations in the HER2 gene in non-HER2-amplified breast cancer have been identified that may confer sensitivity to anti-HER2 directed therapy. Finally, the amplicon containing HER2 frequently contains additional genes that influence therapeutic efficacy. Although initially it was thought that HER2-positive disease is more sensitive to anthracyclines, recent data suggest that this sensitivity is conferred by the topoisomerase II gene ( TOP2A ) when it is present in the amplicon. Other cellular mechanisms of anti-HER2 therapy resistance are under investigation.

PI3K and PTEN

The PI3K pathway includes the PI3K holoenzyme and the downstream effector kinases AKT and mTOR; this pathway is critical for cancer cell growth, proliferation, and survival. This pathway is aberrantly activated through various mechanisms including activation of upstream RTKs including HER2, as described earlier, and activating mutations in pathway components such as PIK3CA kinase domain mutations and loss of functional regulatory components such as PTEN . Activating mutations in the PI3K catalytic subunit are common in breast cancer, particularly in ER-positive, HER2-nonamplified tumors, with an incidence in these tumors of approximately 30% to 40%.

PI3K pathway signaling is initiated by membrane-bound RTKs that undergo activation, for example, by dimerization. These activated RTKs bind to the regulatory subunit of PI3K, p85. This binding relieves inhibitory interactions and allows activation of the kinase subunit of PI3K, called p110, which is encoded for by the PIK3CA gene. Activated PI3K phosphorylates PIP2 to the second messenger PIP3, which then ultimately results in activation of AKT and mTOR. Ultimately, cell survival and proliferation result. The tumor suppressor phosphatase and tensin homolog (PTEN) counteracts PI3K activity by dephosphorylating PIP3 to PIP2.

Germline PTEN mutations cause a hereditary cancer predisposition syndrome known as Cowden syndrome, characterized by a high incidence of breast, uterine, thyroid, and skin neoplasms. PTEN is inactivated in a wide variety of human tumors as a result of either mutation or, more commonly, epigenetic silencing through methylation, including in breast cancer. PTEN loss is associated with genetic instability, and primary breast tumors that lack PTEN have increased aneuploidy. In addition, PTEN loss has been associated with a decreased likelihood of response to anti-HER2 therapy with trastuzumab. Data elucidating the relationship between presence of PIK3CA mutations and PTEN status show that they are inversely correlated.

The observed frequency of PI3K activation in breast cancer has led to clinical investigation of inhibitors of PI3K in the context of hormone receptor–positive and HER2-amplified disease (NCT02167854). These agents have demonstrated some efficacy but are also associated with substantial toxicity including rash, diarrhea, hyperglycemia, and depression. Their role in breast cancer remains to be elucidated.

TP53

The tumor suppressor TP53 (p53), also termed the “guardian of the genome,” is the most frequently mutated gene in human cancer. It plays a central role in sensing genotoxic and nongenotoxic stresses and transducing an antiproliferative effect (cell cycle arrest or apoptosis) in response. It is activated and regulated by posttranslational modifications to the N-terminal region, including phosphorylation and ubiquitination. In addition, by binding as a tetramer to specific DNA sequences via a central DNA-binding core region, it exerts its primary biologic function by modulating the transcription of dozens of genes. In transgenic mice, loss of p53 is associated with multiple spontaneous tumors, although not of the mammary glands. However, p53 loss accelerates the appearance of mammary tumors in murine mammary tissue that also overexpresses MYC, HER2, IGF1, and/or WNT1. These genetic studies are consistent with a role for p53 loss late in tumor development. Indeed, the transgenic restoration of p53 function leads to tumor regressions.

Mutant p53 accumulates in the nucleus of neoplastic cells. Thus initial studies that described only a weak association between aberrant p53 and adverse prognosis in breast cancer relied on immunohistochemistry (IHC) detection; more recent analyses based on mutation detection have confirmed a strong association. Mutations in exons 5 through 8 are more common in ductal and medullary tumors, tumors with an aggressive phenotype (high grade, large size, node-positive cases, and low hormone receptor content), and in women younger than 60 years. Furthermore, the presence of a mutation conferred an overall 2.27-fold increased RR of breast cancer–specific mortality, independent of other known prognostic markers (e.g., tumor size, node status, and estrogen receptor and PR expression). Finally, although not all mutations confer the same biologic properties (e.g., missense versus nonmissense), all have similar prognostic usefulness.

Breast cancers in individuals with germline p53 mutations are frequently ER and HER2 positive. Adjuvant radiation therapy is generally avoided in breast cancer patients with Li-Fraumeni syndrome owing to the risk of radiation-induced sarcomas ; mastectomy, often bilateral mastectomy, is more common. As noted earlier, many tumors harbor sporadic p53 mutations; p53 is an obvious target for cancer therapy, despite the apparent intractability of a loss-of-function genotype. Multiple approaches are being studied to overcome this limitation, including small molecules to restore p53 function or inhibit its interaction with MDM2, and adenoviral-mediated gene delivery. There are now multiple compounds in clinical trials, although no results are yet available in breast cancer.

Molecular Profiling in Breast Cancer

Genome-wide RNA transcriptional profiling in combination with novel bioinformatic approaches has led to the development of molecular classifications based on gene signatures comprising the quantitative expression of thousands of genes. Tumors can be classified either according to transcripts that tend to segregate together according to underlying biologic differences (unsupervised clustering) or sorted according to a given end point such as prognosis or response to therapy (supervised clustering).

Initial studies with array-based expression profiling showed the ability of the technology to classify breast cancer according to five gene clusters: luminal subtype A, luminal subtype B, HER2-positive, basal, and normal breast–like subtypes ( Fig. 88.11 ). The ER-positive group was characterized by high expression of many genes expressed by breast luminal cells, and the ER-negative group showed gene expression characteristic of basal epithelial cells. However, a third group showed genes related to HER2 overexpression, suggesting that this molecular characteristic may have equal or greater weight than ER expression in subclassifying breast cancers. Finally, the normal breast-like group of breast cancers clustered with normal breast epithelium. These distinct subtypes of breast tumors show distinctive molecular signatures and appear to represent diverse biologic entities associated with distinct clinical outcomes, and the comparison of several independently developed gene signatures appears to show similar prognostic information, suggesting the existence of a common set of biologic phenotypes. Luminal A–type cancers have the most favorable long-term survival, whereas HER2-positive and basal-like cancers may be more sensitive to chemotherapy but have the worst overall prognosis. These patterns of gene expression appear to provide more specific information than identification of a single gene with a specific effect. Thus breast cancer is not a single disease with heterogeneous ER and HER2 expression but appears to comprise three to five molecularly and clinically distinct subtypes.

A similar approach can be taken by examining DNA copy number abnormalities, including amplifications and deletions, across the genome. Mapping these abnormalities against both the biologic subtypes described earlier and clinical outcome data demonstrates the potential to identify high-level DNA amplification that, similar to HER2, may be useful in identifying therapeutic targets. Emerging knowledge now aims to integrate patterns of gene expression, methylation, copy number variation, and mutation. This approach, using The Cancer Genome Atlas (TCGA) Network, holds significant potential but will require independent validation using data sets that include treatment and outcome.

In the meantime, although these classifications are robust across a population, they have not yet been shown to be clinically applicable for individual treatment decision making. Furthermore, because much of the molecular heterogeneity overlaps with conventional histopathology and is captured by determination of ER, PR, and HER2 status and tumor grade, use of these molecular tools adds little to current standard pathologic information.

Comprehensive Genomic Analysis

More recently, more comprehensive approaches have been undertaken to examine the molecular profiles of breast cancer. A comprehensive and integrated analysis of 825 primary breast cancers using genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing, and reverse-phase protein arrays was conducted by TCGA and confirmed the existence of four main subtypes of breast cancer. In addition, it provided a wealth of data regarding genetic and epigenetic information for potential therapeutic targeting. Complementary to TCGA, the ENCODE project (Encyclopedia of DNA Elements) mapped regions in non–protein-coding DNA of transcription factor association, transcription, chromatin structure, and histone modification with the goal of determining biochemical functions for many areas of the genome, including those mutated in cancer. As technology advances and becomes more inexpensive, even more comprehensive analysis of breast cancer genomes will likely continue to increase our understanding of both breast cancer and how to use the information for both treatment and prevention.

Disseminated Tumor Cells

More than 90% of patients diagnosed with breast cancer have no evidence of disease outside of the breast and ipsilateral axillary lymph nodes. However, approximately 30% of patients with primary operable breast cancer have disseminated tumor cells (DTCs) that can be detected in bone marrow at the time of diagnosis. In a follow-up study, 15.5% of patients had detectable DTCs 3 years after diagnosis, a finding that was associated with increased risk of relapse and death. Despite these data, however, assessment of DTCs has not been incorporated into routine practice, in part because of the need for a relatively invasive procedure.

Liquid Biopsies

Tumor markers are proteins, such as those derived from MUC1 (e.g., CA15-3, CA27.29) and carcinoembryonic antigen, that are shed from tumors and identified in the circulation. However, their usefulness is limited to monitoring of response to therapy or progression of disease. More recently there have been considerable methodologic advances in the assessment of circulating cancer biomarkers, including circulating tumor cells (CTC) and circulating nucleic acids, which may play a role in diagnosis, monitoring, and treatment of disease.

It is possible to measure CTCs using an automated immunobead enrichment followed by pancytokeratin staining. With this approach, presence of five or more CTCs per 7.5 mL of blood is prognostic, with significantly worse overall survival (OS; 8.2 months versus 18 months) compared with those patients with fewer CTCs. The clinical usefulness of CTCs for treatment decision making in metastatic breast cancer was prospectively tested in a randomized phase III trial; however, use of the assay was not shown to result in improved disease outcomes and therefore it is unclear how best to incorporate it into routine clinical care. The immunobead method lacks sensitivity owing to the use of epithelial cell adhesion molecule (EpCAM)–based detection, because less differentiated cells often lack EpCAM expression and will avoid detection, so newer methodologies including isolation of CTCs based on size or other properties are being developed. Furthermore, the ability to isolate CTCs permits single-cell analysis, including assessment of molecular markers such as ER expression and HER2 amplification in real time.

Technologic advances have also resulted in the ability to detect nucleic acids, including circulating tumor DNA (ctDNA) and microRNA (miRNA), that are shed from dying tumor cells into the blood and may be more sensitive and specific than standard biomarkers. Because analysis of circulating nucleic acids has the potential to identify emerging mutations that could lead to altered treatment options, their use has potential to improve disease outcomes. ctDNA and can be specifically identified by the presence of tumor-specific mutations, including in more than 90% of patients with metastatic breast cancer. Serial assessment of ctDNA via detection of these mutations may be useful for monitoring of patients for disease recurrence or progression ; relative levels have been shown to correlate with tumor burden. Similar findings have been demonstrated with miRNAs.

More recently, it has become apparent that in addition to detection of cancer, assessment of circulating biomarkers may be useful for monitoring changes in tumors that could affect treatment. For example, multiple groups have identified activating mutations in the ligand-binding domain of ESR1, the gene that encodes ERα, which are present in up to 30% of endocrine therapy–resistant patients but which are only rarely detected in primary tumor specimens. Studies have also demonstrated the use of massively parallel sequencing of ctDNA for examining intratumor heterogeneity, which could also potentially be used to identify targetable mutations. Companies are now capitalizing on the new technology and providing assays for monitoring patients, although the clinical usefulness of these commercial assays is currently still in question. As the sensitivity of detection of these circulating biomarkers improves with advances in technology, it is likely that liquid biopsies will become routinely used for monitoring patients with metastatic breast cancer. However, use of this technology for diagnosis or detection of disease recurrence remains questionable.