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Additional insight into the biology of ER-positive breast cancers, particularly the higher risk luminal B cancers, could aid in identifying potential targets and new, effective therapies. And though the majority of triple-negative breast cancers are the “basal-like” subtype, significant proportions are in other subtypes.
The use of gene expression profiling has impacted our understanding of breast cancer biology and increasingly has played a role in guiding clinical decisions. We have used hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status for years to guide selection of therapy. More recently, gene expression analysis has facilitated the identification of at least five intrinsic subtypes of breast cancer. Potential therapeutic targets have also been identified using genomic profiling. Several tests, such as the 21-gene recurrence score assay (Oncotype DX) and the 70-gene prognosis signature (MammaPrint), have been well validated as prognostic tools for early-stage breast cancer, and have aided in adjuvant therapy decisions for early-stage, HR-positive breast cancer patients. Genomic profiling has the potential to provide additional insight into drug discovery and clinical trial design by identifying appropriate targeted therapies for subtypes of breast cancer.
Breast cancer is a heterogeneous disease comprising different subtypes defined on clinical, pathological, and molecular levels. In clinical practice, oncologists have recognized for years that the behavior of breast cancers is variable. Assessment of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status is standard of care at the time of breast cancer diagnosis, and currently is used to guide adjuvant therapy recommendations. Estrogen receptor (ER) and HER2 expression are predictive of benefit from endocrine and HER2-targeted therapies, respectively.
Our understanding of breast cancer biology has accelerated with the use of gene expression profiling. Use of gene expression microarrays has facilitated the high-throughput analysis of multiple genes within a single tumor. In 2000, Perou et al first described the use of gene expression arrays in a small cohort of breast cancer patients who were treated with neoadjuvant doxorubicin. They selected a set of approximately 500 genes, which they called the “intrinsic” gene subset since they defined intrinsic properties of the breast cancer. Breast cancers were clustered into groups based upon expression patterns of different genes. They identified clusters of genes associated with proliferation, HER2 signaling, and HR signaling, as well as a group of genes called the “basal” cluster since they shared expression patterns with breast basal epithelial cells.
Subsequent studies in larger cohorts of patients have further defined the intrinsic subtypes of breast cancer.[2-4] The two HR-positive subtypes are called “luminal A” and “luminal B” since they have an expression pattern similar to the luminal epithelial cells of the breast. Luminal A tumors typically have higher levels of ER expression, whereas luminal B tumors typically have higher levels of genes associated with proliferation and HER2. There were several subtypes with low ER expression: the “HER2-enriched” subtype, which is characterized by high expression of HER2 and other genes located in the same region of chromosome 17; the “basal” subtype; and the less common “claudin-low” subtype. The claudin-low subtype, similar to the basal subtype, is characterized by low expression of HR- and HER2-related genes. It remains unclear if the “normal-like” subtype of breast cancer is a real subtype or if it is an artifact related to contamination from tissue surrounding the tumors.
Determination of a breast cancer’s intrinsic subtype using gene expression profiling is not currently performed in routine clinical practice. Often standard immunohistochemistry (IHC) studies of ERs and progesterone receptors (PRs) and HER2 are used as surrogate markers for intrinsic subtypes. The addition of IHC testing for cytokeratin 5/6 facilitated identification of the basal subtype. Carey et al classified luminal A tumors as ER- and/or PR-positive, HER2-negative; luminal B tumors as ER- and/or PR-positive, HER2-positive; HER2-enriched as ER/PR-negative, HER2-positive; and basal-like tumors as ER/PR/HER2-negative (triple-negative), cytokeratin 5/6-positive. The correlation between this IHC-based classification and DNA-based microarray expression profiles was also observed in different studies.
Although one does not obtain the same depth of knowledge regarding tumor biology using IHC instead of DNA microarrays, this information is often readily available in the clinic. One concern in using IHC surrogates is that ER, PR, and HER2 may not accurately identify the intrinsic subtypes. There is the possibility of having false-negative results from the laboratory. In addition, not all basal-like tumors are triple-negative, and some basal-like tumors have ER, PR, or HER2 expression.[6,8] Determining the intrinsic subtype of a breast cancer has significant prognostic value and implications for outcome.
Gene expression profiling by microarray was initially used to identify unique subtypes of breast cancer, but these subtypes also have strong prognostic implications. For example, patients with luminal A tumors have consistently been shown to have a better prognosis than all other subtypes, including the luminal B tumors, which are also ER-positive. There are several assays that clinicians are currently using in their practices to assess the molecular profile of a tumor prior to making recommendations regarding adjuvant systemic therapy.
The 21-gene recurrence score (RS) assay predicts the rate of distant recurrence in patients with early-stage, ER-positive, lymph node–negative breast cancer. The 21-gene RS is performed on fixed tissue from a surgical specimen or core biopsy. Patients receive a score ranging from 0 to 100. The scores are divided into three risk groups: low (scores 0–18), intermediate (scores 19–31), and high (scores > 31). A total of 51% of patients studied in National Surgical Adjuvant Breast and Bowel Project (NSABP) B-20 had a low RS. Patients with high scores, likely due to luminal B tumors, are most likely to benefit from adjuvant chemotherapy, have lower ER expression levels, and have higher expression levels of proliferation genes. Patients with low RS did not have improved long-term outcome with chemotherapy. The potential benefit of adjuvant chemotherapy among patients with intermediate recurrence scores is not well defined, and is being evaluated in TAILORx (Trial Assigning IndividuaLized Options for Treatment [Rx]) (see Figure 1A). Patients with intermediate scores have been randomized to chemotherapy followed by hormonal therapy or to hormonal therapy alone. Based upon results from the prospective validation studies from patients enrolled on NSABP B-20, the 21-gene RS has been incorporated into the National Comprehensive Cancer Network (NCCN), American Society of Clinical Oncology (ASCO), and St. Gallen treatment guidelines for early-stage, ER-positive, lymph node–negative breast cancer.
The use of the 21-gene RS in patients has been better studied in those with lymph node–negative disease compared with node-positive disease. Analysis of node-positive patients from the phase III Southwest Oncology Group (SWOG) 8814 clinical trial, in which patients were randomized between chemotherapy followed by tamoxifen vs tamoxifen alone, showed that the RS was prognostic in this patient population. A high RS predicted for chemotherapy benefit in node-positive patients. Although node-positive patients with low RS derived less benefit from chemotherapy than those with high RS, results of the RxPONDER (Rx for Positive Node, Endocrine Responsive Breast Cancer) trial will be needed prior to recommending routine use of the 21-gene RS in the node-positive population (see Figure 1B).
Although the 21-gene RS costs about $4,000 per patient, it has been shown to be cost-effective in multiple studies.[13-15] Several studies have also shown that results from the 21-gene RS have changed the medical oncologist’s treatment recommendation.[16,17] The largest change was typically from pre-test recommendation for adjuvant chemotherapy followed by hormonal therapy to hormonal therapy alone. So, in addition to being cost-effective, the 21-gene RS also reduces the overall morbidity associated with treating early-stage, ER-positive breast cancer, since fewer patients are exposed to the short- and long-term risks of chemotherapy.
The 21-gene RS has been compared with a combined ER, PR, Ki67, and HER2 IHC score (IHC-4) in a cohort of early-stage breast cancer patients from the ATAC (Anastrozole or Tamoxifen Alone in Combination) trial who did not receive chemotherapy. In this analysis, the IHC-4 score provided prognostic information similar to that of the 21-gene RS, with modest correlation between the two. Although the IHC-4 score provided prognostic information in this study, lack of reproducibility of quantitative IHC assays across laboratories has limited its clinical application.
The 70-gene prognosis signature was initially described by van’t Veer et al in 2002 by performing DNA microarray analysis on primary breast tumors. They were able to identify a gene expression signature that predicted for development of distant metastases. The poor-prognosis signature was characterized by expression of genes associated with proliferation, angiogenesis, and invasion. The prognostic significance of the 70-gene signature was validated in a separate cohort of patients. Patients with a good-prognosis signature had significantly lower rates of distant metastasis compared with patients who had a poor-prognosis signature. A total of 36% to 39% of the patients in evaluated studies have had a good-prognosis signature.[20,21] Additional analysis has shown that nearly all basal-like, HER2-enriched, and luminal B tumors have poor-prognosis signatures.
Clinical use of the 70-gene signature has been limited by the requirement, until recently, of frozen tissue, and by limited data validating the predictive benefit of chemotherapy among good- and poor-prognosis signatures. The 70-gene signature has been well validated in prospective studies of lymph node–negative patients who did not receive adjuvant chemotherapy[21,23-26]; however, results from ongoing clinical trials are needed for prospective validation of predictive benefit from adjuvant chemotherapy. In the MINDACT (Microarray In Node-negative and 1 to 3 positive lymph node Disease may Avoid ChemoTherapy) trial, patients with ER-positive, early-stage breast cancer (node-negative or 1 to 3 positive lymph nodes) receive recommendations for adjuvant chemotherapy based upon the 70-gene signature and an online prognostic tool using clinical and pathologic features (Adjuvant! Online). Patients who are determined to be high risk by both the online assessment and the 70-gene signature will receive adjuvant chemotherapy; those who are good risk by both will receive hormonal therapy alone. Discordant cases will be randomized to adjuvant therapy based upon either the 70-gene signature or the online tool. Recruitment to this multi-institutional randomized phase III trial has been completed (see Figure 1C).
In the past year, it has become possible to perform the 70-gene signature on fixed tissue; this should facilitate using the 70-gene signature in clinical practice, where frozen tissue is not routinely collected. The 70-gene signature has also identified a subgroup of HER2-positive patients with a good prognosis. These tumors were characterized by being ER-positive and low-risk for relapse in absence of adjuvant chemotherapy. The current standard of care for HER2-positive, early-stage breast cancer is to receive adjuvant trastuzumab (Herceptin)-based chemotherapy; however, these results suggest that there may be a subgroup of good-risk HER2-positive patients for whom chemotherapy could be avoided.
In addition to the 21-gene RS and 70-gene signature, several prognostic predictors have been developed and are commercially available, and others are in development. The Predictor Analysis of Microarray (PAM) 50-gene test has been developed to classify breast cancers into intrinsic subtypes. The PAM-50 assay provides a risk of relapse score and is commercially available.[9,28] A genomic index of sensitivity to endocrine therapy (SET) has also been developed by measuring the level of transcriptional activity related to ER. A high SET index was predictive of lower risk of distant relapse with adjuvant tamoxifen. The genomic grade index (GGI) is a 97-gene measure of histologic grade, and a high GGI is associated with a lower relapse-free survival. High GGI also predicted for increased response to neoadjuvant chemotherapy, and predicted for poor prognosis among ER-positive patients, even in the setting of chemotherapy and endocrine therapy.
The Breast Cancer Index (BCI) is an assay comprising two independently developed biomarkers: a set of five cell-cycle–related genes called the molecular grade index and a two-gene expression ratio of homeobox 13 and interleukin-17B receptor which has been shown to predict recurrence and survival in women receiving adjuvant tamoxifen. The BCI stratifies patients into three risk groups that predict risk of distant recurrence. In a recently presented analysis of a cohort of patients from the ATAC trial, prognostic performance of BCI, the 21-gene recurrence score, and IHC-4 were compared with a clinical treatment score based on size of tumor, nodal status, grade, age, and treatment. All three profiles performed well in predicting recurrence in years 1 through 5; however, only the BCI predicted for late distant recurrence in years 5 through 10 after diagnosis.
The luminal A and B subtypes are both characterized by HR expression, and 5 years of adjuvant anti-estrogen therapy became the standard of care based upon results from multiple trials. The addition of aromatase inhibitors in the adjuvant setting for postmenopausal women has improved disease-free survival compared with tamoxifen alone. Aromatase inhibitors can be used as upfront continuous treatment for 5 years,[35,36] as sequential therapy after 2 to 3 years of tamoxifen,[37,38] or as extended adjuvant therapy after 5 years of tamoxifen.
Patients with HR-positive breast cancer continue to have relapse rates of 1% to 4% per year between 5 and 15 years from diagnosis, and the optimal duration of adjuvant hormonal therapy remains an important clinical question.[40,41] Long-term results of the
ATLAS trial (Adjuvant Tamoxifen: Longer Against Shorter) were recently presented, indicating that 10 years of adjuvant tamoxifen resulted in a further reduction in recurrence and mortality compared with 5 years of adjuvant tamoxifen, with continued benefit seen beyond 10 years of therapy. These results are most relevant for premenopausal patients, for whom extended adjuvant therapy with an aromatase inhibitor is not an alternative option. Molecular profiling will likely be important in determining which patients are at highest risk of late recurrence, and potentially derive benefit from extended adjuvant hormonal therapy. Patients with luminal B tumors, whose risk of recurrence is greatest in the first 5 years, may not benefit from hormonal therapy beyond 5 years.
Despite the marked success of endocrine agents in the treatment of early-stage, HR-positive breast cancers, many patients will relapse. These tumors have either intrinsic or acquired resistance to anti-estrogen therapy. The mechanisms underlying intrinsic and acquired resistance to endocrine agents are likely similar, and include activation of upstream and downstream pathways resulting in changes in co-regulators of the estrogen receptor.
The 21-gene RS can be used to determine the benefit of tamoxifen in node-negative, ER-positive breast cancers. Breast cancers with recurrence scores greater than 31 appear to derive little benefit from adjuvant tamoxifen compared with cancers that have recurrence scores of 30 or less. Concordance between luminal B and high-recurrence-score cancers has also been shown, suggesting that the poor prognosis seen in these cancers may be due in part to intrinsic resistance to endocrine therapy.
A better understanding of the differential expression of genes and proteins in luminal A and B cancers could shed significant light on the mechanisms underlying resistance to endocrine agents, which could in turn lead to novel therapeutic approaches to circumvent this resistance. There is increasing evidence to suggest that breast cancers that express both HRs and HER2 are somewhat intrinsically resistant to endocrine agents, and that these cancers are, in fact, driven by the HER2 pathway.[43,44] Support for this hypothesis comes from data on patients with metastatic HR-positive, HER2-positive breast cancers, in whom progression-free survival following treatment with single-agent anastrozole is extremely short at just over 2 months. However, there is some evidence to suggest the existence of a subset of HER2-positive cancers that express ER and PR, which may be driven more by ER than HER2. In fact, a subset of HER2-positive breast cancers that are ER-positive have been shown to have a good-prognosis signature based on assessment with the 70-gene signature.
Other growth factor pathways, including the epidermal growth factor receptor (EGFR), insulin growth factor receptor, and vascular endothelial growth factor (VEGF) receptor, have been demonstrated to play a role in resistance to endocrine agents.[44,47-49] Other agents such as the mammalian target of rapamycin (mTOR) inhibitor everolimus (Afinitor) may also play a role, based upon results showing improved response to everolimus in combination with endocrine therapy in the metastatic and neoadjuvant setting.[50-52] Going forward, it is essential that we identify novel therapies for patients with luminal B cancers, given their poor survival when treated using conventional therapies. The use of gene expression profiling can help to identify key genes that can then be exploited therapeutically.
When patients were stratified by breast tumor subtype and analyzed for time to distant metastasis and overall survival, those with the basal subtype had the worst clinical outcome. This likely reflects both the aggressive nature of basal-subtype breast tumors and the lack of targeted therapies, since these tumors do not express the ER and do not overexpress HER2. Conventional anthracycline- and taxane-based regimens are currently used to treat patients with the basal-like subtype of breast cancer.
Although women who carry BRCA1 mutations are predisposed to developing breast cancers of the basal-like subtype, expression levels of BRCA1 have not been well characterized in sporadic triple-negative tumors.[3,53] BRCA1 mediates the cellular response to DNA damage by sensing damage, preventing apoptosis, and participating in DNA repair.[54,55] The loss of BRCA1 expression in basal-like tumors may lead to selective sensitivity to DNA cross-linking chemotherapeutic agents, such as platinum analogues. Cisplatin, a platinum analogue, has demonstrated single-agent activity as neoadjuvant treatment for triple-negative breast cancers. There are multiple ongoing clinical trials investigating the addition of cisplatin or carboplatin to neoadjuvant chemotherapy; however, most of these have enrolled patients with triple-negative breast cancer, not just the basal subtype. Genomic profiling could potentially aid in identifying tumors among BRCA-negative patients with a “BRCA-like” profile for whom platinums or other agents targeting DNA repair, such as poly(ADP-ribose) polymerase (PARP) inhibitors, may be more effective.
Other potential targets for basal-like breast cancer have been identified using genomic profiling. The EGFR is part of the basal cluster, and EGFR-targeting agents have been investigated in the metastatic setting, demonstrating modest clinical activity. In a study combining carboplatin and cetuximab (Erbitux), a monoclonal antibody against EGFR, clinical benefit was seen among patients with EGFR pathway inactivation. Anti-angiogenic agents targeting VEGF have also shown promise in the metastatic setting, and studies in the adjuvant setting are ongoing. Bevacizumab (Avastin), a monoclonal antibody against VEGF, showed improved disease-free survival in the first-line[57,58] and second-line setting, but it did not show an overall survival advantage. Benefit from the addition of bevacizumab was similar among patients with HR-positive vs HR-negative breast cancer, suggesting that intrinsic subtyping might not predict anti-angiogenic benefit.
Additional gene expression analysis of triple-negative breast cancers from multiple data sets has further defined this group of cancers. Cluster analysis identified a second “basal-like” subtype in addition to immunomodulatory, mesenchymal, mesenchymal stem-like, and luminal androgen receptor subtype. Triple-negative breast cancer cell lines corresponding to these subtypes responded differently to therapies such as cisplatin, mTOR inhibitors, Src inhibitors, and an androgen receptor antagonist. This would suggest that gene expression profiling of triple-negative breast cancers should play an important role in future trial design of novel, targeted therapies.
The HER2-enriched subtype is characterized by high expression of HER2, most commonly due to amplification of the HER2 gene. Genes such as GRB7 and TOP2A, which are located in close proximity to the HER2 gene on chromosome 17, are often co-amplified. Multiple studies have been performed to correlate TOP2A gene status, topo2a expression levels, and response to anthracyclines.[62-67] The role of TOP2A amplification was examined in the Breast Cancer International Research Group (BCIRG) 006 trial in which early-stage, HER2-positive patients were randomized between three arms: standard anthracycline- and taxane-based chemotherapy with or without trastuzumab, and a third non–anthracycline-containing regimen of docetaxel, carboplatin, and trastuzumab. Patients without co-amplification derived greater benefit from the addition of trastuzumab. In patients with co-amplification of TOP2A and HER2, minimal incremental benefit was seen with the addition of trastuzumab; however, the long-term toxicity profile favored the non–anthracycline-containing regimen.
For ER-positive, early-stage breast cancer, genomic profiling using the 21-gene RS and the 70-gene signature has already impacted clinical decision-making. These tests have aided treating oncologists by differentiating patients with low- vs high-risk ER-positive tumors for whom chemotherapy is indicated. Ongoing clinical trials such as MINDACT and TAILORx have focused primarily on how to best apply these tests in the adjuvant setting, using our current standard treatments. Additional insight into the biology of ER-positive breast cancers, particularly the higher risk luminal B cancers, may also be gained from genomic profiling, and potentially could aid in identifying potential targets and new, effective therapies.
Genomic profiling of triple-negative breast cancers has revealed that this is a heterogeneous group of cancers. Although the majority of triple-negative breast cancers are the “basal-like” subtype, significant proportions are in other subtypes. Incorporation of genomic profiling into future clinical trials will have implications for drug development, where the ability to identify aberrant gene expression will help to inform one’s choice of targeted therapies. Ultimately it is hoped that the ability to better define an individual patient’s breast cancer biology will lead to improvements in therapy selection, discovery of new drug targets, and better long-term outcomes for patients.
Financial Disclosure:The authors have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.
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