Obesity and Body Habitus

There is a clear association between obesity and breast cancer risk. Women with a body mass index (BMI) of 25 to 30 (overweight) or BMI greater than 30 (obese) have been shown to have an elevated risk of breast cancer. In addition, an increased risk of breast cancer death has been seen with increasing BMI.22,23 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.²⁴ An increased risk of breast cancer has also been associated with increasing height, with a relative risk of 1.17 (95% confidence interval [CI], 1.15 to 1.19) for every 10 cm of additional height.²⁵

Familial History and Predictive Models of Breast Cancer Risk

Breast cancer is approximately twice as common among first-degree relatives of breast cancer patients as in those women with no family history of the disease. The two most important breast cancer susceptibility genes, BRCA1 and BRCA2, were identified by linkage analyses in the mid-1990s but account for less than 20% of familial clustering of breast cancer.²⁶ Since then, rare germline mutations in several other genes, such as TP53 and PTEN, have been shown to confer a substantially increased risk of breast cancer. Given that linkage analyses have failed to yield additional susceptibility loci, it is likely that most of the remaining genetic susceptibility is a result of a number of moderate and low-penetrance alleles that coexist in highly penetrant combinations in a polygenic model, although some familial risk may still be a result of rare, undiscovered, high-penetrance gene mutations.²⁷

Multiple moderate penetrance alleles including CHEK2, PALB2, ATM, BRIP, BARD, and others²⁸ have been identified; mutations in these alleles are associated with a relative risk of breast cancer of 2 to 5. The clinical utility for testing for mutations in these genes is currently unclear. Recently breast cancer has been found to be a component of hereditary nonpolyposis colorectal cancer syndrome (HNPCC or Lynch) although the cumulative risk for breast cancer is less than for the classic component tumors (i.e., colon and uterine cancer).²⁹

Apart from specific genetic susceptibility alleles, an increasing understanding of relevant risk factors has led to integrated efforts at evaluating a women’s individual risk,³⁰ and several individualized risk-assessment tools are available, such as the Gail, Claus, Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA), BRCAPRO, and Tyrer Cuzick models.³¹ One such tool is available on the National Cancer Institute (NCI) website at http://www.cancer.gov/bcrisktool/. These tools are being used for clinical counseling for healthy women perceived to be at risk and to determine candidates for chemoprevention with tamoxifen, raloxifene, and, most recently, exemestane. Given that for women living in the Western hemisphere the lifetime risk of breast cancer is approximately 11%, even a small relative risk reduction could translate into many thousands of lives saved.

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 levels is a key therapeutic modality for breast cancer. Estrogens interact with mammary epithelial cells via specific ERs that function as nuclear transcription factors.³² 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 was the first ER to be isolated and 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.³³ Most of the literature that refers to ER (e.g., the staining of breast cancer cells as “ER-positive”) refers to ERα unless otherwise specified. The major ligand-dependent transcriptional activation activity (so-called AF-2) function of ER dislodges from a complex with heat shock proteins upon binding specific ligands, undergoes a conformational change, and homodimerizes. This homodimer then binds to the estrogen-responsive elements of the target genes, and recruits various coactivators (or corepressors) that further influence transcription.³⁴ The participation in a multimeric signaling process likely contributes to the differential biological effects that estrogen and estrogen-related compounds (e.g., 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 signal transduction pathways. Approximately 70% of breast cancers express ER (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 both in preventing recurrence in the adjuvant setting and in prolonging survival once metastases have developed. However, the relationship between ER expression and cancer initiation is not clear, because ER-positive cells are nonproliferating in the normal breast epithelium that is found adjacent to proliferating cells, suggesting a paracrine effect. The paracrine effect hypothesis is further supported by evidence that even in most ER-positive tumors, only a minority of cells express ER by immunohistochemistry (IHC), and breast cancer stem cells do not express ER.

The progesterone receptor (PR), itself 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, whereas PRA is more widely expressed. Interestingly, 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.³⁵ 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-/PR-positive tumors. Furthermore, ER-/PR-positive tumors may be more likely to respond to antiestrogen therapy than ER-positive/PR-negative tumors.³⁶

BRCA1, BRCA2, and Hereditary Susceptibility to Breast Cancer

It is estimated that 5% to 10% of all breast and ovarian cancer cases occur in the setting of inherited susceptibility because of a high-penetrance gene mutation, and the breast cancer susceptibility genes BRCA1 and BRCA2 have been identified as responsible for many of these cases.²⁶ Women who carry a germline mutation in BRCA1 have a cumulative lifetime incidence of 50% to 85% of breast cancer and a 40% to 60% incidence of ovarian cancer, although estimates are somewhat lower in population-based series.^26,37 There are a large number of BRCA1-interacting proteins including BARD, BRIP1, ABRAXAS, PALB2, RAD50, NBS1, and BRCA2. Germline mutations in the genes coding these proteins have also been associated with an increased risk of breast cancer, although the risk is significantly lower than with germline mutation in BRCA1 and BRCA2.³⁸

The BRCA1 complex is integral in maintenance of genome integrity through roles in DNA damage repair, cell-cycle regulation, transcriptional regulation, and chromatin remodeling.39,40 Tumors that arise in heterozygote carriers of germline mutations usually have an inactivated wild-type allele and do not express a functional protein. Cells that lack BRCA1 or BRCA2 protein have a specific deficiency in the repair of DNA double-strand breaks by the conservative and relatively error-free mechanism of homologous recombination by gene conversion.^40,41 Evidence for this defect is further supported by the finding that BRCA2 functions as part of the Fanconi anemia complex and actually is FANCD1. Fanconi anemia complementation group FA-N results from biallelic mutations in PALB2 (“partner and localizer of BRCA2”),^42,43 whereas monoallelic mutations in the same gene are associated with an increased susceptibility to familial breast cancer.⁴⁴ Moreover, several additional genes function in the same DNA repair pathway where germline mutations carriers are at increased risk of developing breast cancer—CHEK2, ATM, NBS1, RAD50, and BRIP1.⁴⁵ Why these genes predispose primarily to breast and ovarian cancers remains unclear, and somatic mutations in them are not commonly found in sporadic breast cancers.

BRCA1-associated tumors usually are of the basal type (so-called triple negative: HER2-, ER-, and PR-negative). Conversely, sporadic triple-negative breast cancers may share phenotypic similarities with cancers arising in BRCA carriers.⁴⁶ BRCA1 pathway dysregulation or “BRCAness” does also appear to be common in high-grade epithelial serous ovarian cancer.^47,48 The identification of these genes and their function has important implications for prevention, diagnosis, and therapy of breast cancer in carriers and may have implications for sporadic ovarian cancer and, potentially, certain subtypes of triple-negative breast cancers.

Clinical management of carriers with BRCA1 or BRCA2 mutations is geared primarily toward preventing both ovarian and breast cancer.⁴⁹ Prophylactic mastectomy, albeit an invasive procedure with cosmetic consequences, is the most effective strategy available; it is associated with a 90% reduction in the risk of breast cancer. Oophorectomy, in addition to reducing ovarian cancer risk by more than 80%, reduces the risk of breast cancer by 50%. Risk-reducing surgery may reduce the mortality for breast and ovarian cancer.⁵⁰ Additionally, tamoxifen has been shown to reduce the risk of contralateral breast cancer by approximately 50% in both BRCA1 and BRCA2 carriers.⁵¹

Once cancer is diagnosed, therapy is dictated by standard prognostic and predictive features 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. This hypothesis is supported by in vitro observations in both BRCA-null and FA cell lines. Significant responses to preoperative chemotherapy with cisplatin have been seen in a small number of BRCA1 mutation carriers⁵²; however, it appears that breast cancers in BRCA1 mutation carriers are generally more chemosensitive compared with sporadic tumors.⁵³ Finally, poly (ADP-ribose) polymerase 1 (PARP1) is involved in the base excision repair pathway; when this repair pathway is inhibited, double-strand breaks form at replication forks, and these are lethal to cells that cannot repair those breaks. Accordingly, BRCA-null cells are exquisitely sensitive to PARP inhibitors.^54,55 Phase I and phase II studies have demonstrated exciting activity in BRCA1/2-related breast and ovarian cancer.^56,57 It is hoped that more efficacious, less toxic treatment may be tailored to tumors that lack functional BRCA proteins.

TP53

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 (e.g., phosphorylation and ubiquitination) and, binding as a tetramer to specific DNA sequences via a central DNA-binding core region, exerts its primary biological function by modulating the transcription of dozens of genes.⁵⁸ In transgenic mice, loss of p53 is associated with multiple spontaneous tumors, although not of the mammary glands.⁵⁹ 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.⁶⁰ Indeed, the transgenic restoration of p53 function leads to tumor regressions.⁶¹

Inheritance of a p53 mutant allele causes the rare familial Li-Fraumeni syndrome, characterized by multiple early-onset cancers, including breast cancer in women who survive childhood cancers. Breast cancers in individuals with germline p53 mutations are frequently ER- and HER2-positive.62,63 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.

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 IHC detection; more recent analyses based on mutation detection have confirmed a strong association.⁶⁴ Recently, p53 mutations from 1794 European patients with at least 10 years of follow-up were analyzed. Mutations in exons 5 through 8 were 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 relative risk of breast cancer-specific mortality, independent of other known prognostic markers (e.g., tumor size, node status, and estrogen and PR expression).⁶⁵ Finally, although not all mutations confer the same biological properties (e.g., missense vs. nonmissense), all have similar prognostic utility.

Initial attempts at correlating p53 expression (by IHC) with resistance to chemotherapy have been supplanted by studies directly evaluating mutations. Some of these studies show breast cancers with p53 mutations to be associated with less benefit from both antiestrogens66,67 and anthracylines,^68,69 although conflicting results also are reported.⁷⁰ An overall analysis is complicated by different p53 assay methods, different disease settings (adjuvant, preoperative, and metastatic), and different combinations of drugs. In contrast, it appears that no clear relation between aberrant p53 and sensitivity to paclitaxel exists.^73–73

p53 is an obvious target for cancer therapy, despite the apparent intractability of a loss-of-function genotype. Two approaches are being taken to overcome this limitation—small molecules to restore p53 function⁷⁴ or inhibit its interaction with MDM2,⁷⁵ or adenoviral-mediated gene delivery. Although the latter approach is approved for therapy of head and neck tumors in China, no clear activity in breast cancer has been demonstrated to date.⁷⁶

ERBB2 (HER2)

The protooncogene ERBB2 (HER2) encodes a transmembrane tyrosine kinase receptor, one member of a family of related growth factor receptors that include EGFR (ERBB1), ERBB3 (HER3), and ERBB4 (HER4). The ligands for these receptors, termed heregulins, are a family of growth factors known to bind to ERBB3 and ERBB4, inducing heterodimerization with ERBB2 and subsequent transduction of downstream signals. Because HER2 does not bind ligand directly,⁷⁷ the ability to heterodimerize with other family members that do bind ligand is crucial for its activity. Of these, HER3 appears to be the most important heterodimerization partner.⁷⁸

The amplification and/or protein overexpression of HER2 is found in 20% of newly diagnosed breast cancers, and is associated with a more aggressive clinical course and decreased survival time compared with tumors with normal levels of HER2.⁷⁹ Amplification leads to very high levels of protein expression and constitutive activation of the receptor tyrosine kinase signal transduction pathway, namely RAS-MEK and AKT,⁸⁰ although the downstream effects may be more pleiotropic than originally believed.⁸¹ In contrast to EGFR, in which amplifications in lung cancers frequently harbor activating mutations in the kinase domain, such mutations have not been found frequently in breast cancer (see the Sanger Centre’s COSMIC database, http://www.sanger.ac.uk/genetics/CGP/cosmic/). The identification of HER2 as an oncogene in breast cancer facilitated the development of therapies directed against it. Trastuzumab, a humanized monoclonal antibody that binds to the extracellular domain of HER2, induces clinical responses as a single agent, prolongs survival in combination with chemotherapy, and decreases recurrence rates and improves survival in the adjuvant setting when given to women whose cancers overexpress HER2.^84–84 Lapatinib, an oral, small-molecule tyrosine kinase receptor inhibitor active against HER2, and pertuzumab, a monoclonal antibody that inhibits dimerization between HER family members, have also been shown to be of benefit in metastatic, HER2-positive breast cancer. However, not all women with HER2-positive breast cancer respond to these agents, and studies aimed at elucidating mechanisms of resistance are providing further insight into the pathophysiology of HER2-positive breast cancer. For example, increased levels of insulin-like growth factor–1 receptor (IGF1R) may bypass HER2 blockade by trastuzumab and allow growth factor activation of AKT,⁸⁵ and a similar outcome may result from PTEN loss, as discussed earlier. In addition, a truncated form of HER2 that lacks the extracellular domain but retains the kinase activity has been described to correlate with trastuzumab resistance.⁸⁶

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.⁸⁷ Modern genome-wide analysis methods currently are being applied to panels of cell lines,⁸⁸ as well as to tissues from patients treated with anti-HER2 agents,⁸⁹ to better understand mechanisms of resistance. The insights gained, in turn, are being exploited for further drug discovery. Both antibody and small molecule inhibitors of IGF1R as well as phosphatidylinositol 3-kinase (PI3K) and AKT kinase inhibitors currently are in clinical development for breast cancer, and synergy with anti-HER2 therapy has been demonstrated in vitro.⁹⁰

PI3K and PTEN

Phosphatidylinositol-3,4,5-triphosphate (PIP₃) is a lipid second messenger formed by PIP₃ kinase (PI3K) in response to receptor tyrosine kinase signaling. PIP₃ activates AKT and other related pathways to induce proliferation and inhibit apoptosis. Approximately 25% of human breast cancers harbor oncogenic activating mutations in the p110α catalytic subunits of PI3K (PIK3CA).⁹¹ This signal is inactivated by the opposing phosphatase action of the tumor suppressor gene PTEN.

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.⁹² 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.⁹³ PTEN loss is associated with genetic instability, and primary breast tumors that lack PTEN have increased aneuploidy.⁹⁴ In addition, PTEN loss has been associated with a decreased likelihood of response to anti-HER2 therapy with trastuzumab.⁹⁵ Recent data elucidating the relationship between presence of PIK3CA mutations and PTEN status show that they are inversely correlated. Furthermore, PIK3CA mutations are twice as common in HER2-positive or ER-positive tumors, suggesting that more than one aberration upstream of AKT may be necessary to overcome the effect of an intact PTEN.⁹⁶ The relevance of PIK3CA mutations to the clinical activity of anti-HER2-directed therapy by trastuzumab and lapatinib is under active investigation.⁹⁷

Molecular Profiling in Breast Cancer

Simultaneous genome-wide determination of the relative abundance of RNA species (i.e., transcriptional profiling) combined with novel bioinformatic approaches has led to the development of molecular classifications based on continuous expression ranges of thousands of genes (as opposed to a binary determination of discrete factors, i.e., ER/PR and HER2). Tumors can be classified either according to transcripts that tend to segregate together according to underlying biological differences (unsupervised clustering) or sorted by a given end point such as prognosis or response to therapy (supervised clustering). The first of these studies identified an “intrinsic gene list” of 534 genes that defined five biological subtypes of breast cancer including the basal type (HER2-positive, cytokeratins 5 and 17 positive), the “triple negative” type (HER2-, ER-, and PR-negative), two luminal types (so-called luminal-A and luminal-B), and the normal-like type (Fig. 91-2).⁹⁹ Studies by others have confirmed large-scale gene expression differences between ER-positive and ER-negative tumors, and have shown additional molecular subsets within these categories.¹⁰⁰ This molecular classification of breast cancer is robust in that it is observed regardless of microarray platform type, and the various subtypes correlate with overall prognosis.^101,102 Luminal-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. Thus breast cancer is not a single disease with heterogeneous ER and HER2 expression but appears to be composed of three to five molecularly and clinically distinct subtypes.

A similar approach can be taken by assaying DNA amplifications and deletions across the genome.¹⁰³ Such data recently have been mapped against both the “intrinsic gene set” and clinical outcome data, and have the potential to identify high-level DNA amplification that, similar to HER2, may be useful as therapeutic targets.¹⁰⁴ However, it should be noted that although these classifications are robust across a population, there is no standardized assay of measurement for assigning a molecular class to a new individual case. Furthermore, much of the molecular heterogeneity overlaps with conventional histopathology and is captured by determination of ER, PR, HER2 status, and tumor grade.¹⁰⁰

In addition, although the intrinsic gene set and its derivations correlate with prognosis, this correlation does not easily translate into the specificity and sensitivity required for clinical decision making. One of the greatest clinical challenges has been determining the value of adjuvant chemotherapy for individual women with early-stage breast cancer, where many patients are treated but only a few directly benefit.

Several multigene prediction scores that correlate with outcome and provide more accuracy than traditional histopathological markers have been retrospectively derived from microarray data, and, in some cases, prospectively validated on additional datasets. The best known examples are a 70-gene predictor set (Fig. 91-3)^105,106 and a 21-gene predictor set,^107,108 and both are commercially available. The 21-gene test consists of 16 target transcripts and five reference transcripts, including ER, PR, and HER2, and a “proliferation” group of five genes (which includes Ki-67 and cyclin B₁); and a carefully constructed weighted formula is used to determine the overall “recurrence score.”^107,108 Although this recurrence score provides a continuous risk variable, for clinical use it is pared down to three categories of low, intermediate, and high (of note, given the weighting schema, it is unlikely that a HER2-positive tumor would be classified as “low risk”). It is interesting that these two gene sets as well as the “intrinsic gene set” described earlier and yet another “wound healing” signature all seem to perform similarly when tested against the same clinical data set, even though they share few to none of the same genes.¹⁰⁹ The ability of the 70-gene assay to determine which ER-positive node-negative patients are most likely to benefit from adjuvant therapy is being prospectively tested in the Microarray in Node-Negative Disease May Avoid Chemotherapy (MINDACT NCT00433589) trial. Similarly, the Trial Assigning Individualized Options for Treatment (Rx) (TAILORx) NCT00310180) study, which is testing the utility of adjuvant chemotherapy in patients who have an “intermediate risk” according to the test, has completed enrollment; low-risk patients received hormonal therapy only, whereas all high-risk patients were treated with both chemotherapy and hormonal therapy. The RxPONDER study (NCT01272037) is addressing a similar question in patients with node-positive disease, where patients with a recurrence score <=25 are being randomized to endocrine therapy alone or also receive chemotherapy. In the meantime, the Oncotype assay has been incorporated into the National Comprehensive Cancer Network (NCCN) algorithm for node-090 negative, ER-positive breast cancer patients.

Comprehensive Genomic Analysis

More recently, comprehensive approaches have been undertaken to examine the molecular profiles of breast cancer. A comprehensive and integrated analysis of primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays (The Cancer Genome Atlas or TCGA) confirmed the existence of four main subtypes of breast cancer, and provided a wealth of data regarding genetic and epigenetic information for potential therapeutic targeting.¹¹⁰ In addition, the ENCODE project (Encyclopedia of DNA Elements) has recently mapped regions in non–protein-coding DNA of transcription factor association, chromatin structure, and histone modification that will no doubt enhance our understanding of breast cancer.¹¹¹