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.
Genomic Profiling in the Clinic
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 (Oncotype DX)
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 (MammaPrint)
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.
Additional prognostic panels
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.
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