ABSTRACT: Endocrine therapy is one of the most effective treatment strategies for breast cancer. However, in the adjuvant setting, up to 40% to 50% of patients with estrogen receptor (ER)-positive breast cancers relapse despite these interventions. Although ER and HER2 analysis has increased our ability to predict which patients will benefit from endocrine therapy, further improvement is needed, most specifically for patients with ER-positive, HER2-negative disease. Recent advances in genomic technology have made it possible to classify breast cancers into risk categories with significant prognostic implications. However, the predictive value of these tests remains the subject of investigation. Long-term follow-up of neoadjuvant endocrine therapy studies suggests that the in vivo assessment of therapeutic efficacy provided by this treatment approach is also valuable in predicting outcome. Indeed, the Preoperative Endocrine Prognostic Index (PEPI), based on tumor pathologic staging and expression levels of ER and Ki67 following 3 to 4 months of neoadjuvant endocrine therapy, reproducibly predicts long-term outcomes of hormone receptor–positive breast cancer. This article reviews ongoing progress in the effort to identify predictors of endocrine therapy responsiveness for breast cancer and discusses the value of “pre-treatment” vs “on-treatment” tumor profiling for predicting outcomes.
The hormone-dependent nature of breast cancer was first described in the literature by Beatson in 1896. Since then, a number of pharmacologic agents have been developed to either modulate tumor cell estrogen receptor (ER) function or to reduce the levels of circulating estrogens(Drug information on estrogens). Among these agents are the selective estrogen receptor modulators (SERMs: tamoxifen, raloxifene(Drug information on raloxifene) [Evista], and toremifene(Drug information on toremifene) [Fareston]), pure antiestrogens (fulvestrant [Faslodex]), luteinizing hormone-releasing hormone agonists (leuprolide, goserelin(Drug information on goserelin) [Zoladex]), and third-generation selective aromatase inhibitors (anastrozole [Arimidex], letrozole(Drug information on letrozole) [Femara], exemestane(Drug information on exemestane) [Aromasin]). The widespread application of endocrine therapy with these agents has led to a significant reduction in breast cancer mortality. However, up to 50% of women with breast cancers that are hormone receptor (HR)-positive do not derive benefit from these treatments, either due to intrinsic resistance or acquired resistance following prolonged use.[3,4] Furthermore, endocrine therapy is associated with vasomotor symptoms (tamoxifen), musculoskeletal discomfort (aromatase inhibitors) and occasionally more serious side effects (thrombosis and endometrial cancer from tamoxifen(Drug information on tamoxifen) or osteoporotic fracture from aromatase inhibitors). These problems can affect the overall quality of life and even reduce life expectancy. Identifying predictors of endocrine responsiveness is therefore important to avoid unnecessary toxicities and to promote the selection of alternative treatment strategies for patients with endocrine-resistant tumors. In this review, we will discuss recent studies in this area and debate the status of these tests in current clinical practice.
Primary Tumor Biomarker Characteristic
Most studies have investigated biomarkers on primary tumors collected before endocrine treatment, with a focus on ER, progesterone(Drug information on progesterone) receptor (PR), HER2, Ki67, and, more recently, multigene profiles that incorporated additional genes.
Estrogen and Progesterone Receptors
ER and PR are well recognized predictors of response to endocrine therapy.[4,6] The prerequisite of a positive ER and/or PR test for endocrine responsiveness was initially observed for patients with advanced disease[6,7] and was further demonstrated in the early-stage disease setting.
• Role of ER Status—In the quinquennial overview of randomized adjuvant trials by the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG), the use of 5 years of tamoxifen in patients with early-stage breast cancer was associated with a 41% reduction in the annual risk of relapse, and a 34% reduction in the annual death rate for women with ER-positive disease, but little benefit was observed for those with ER-poor disease.[2,8,9] In addition, multiple studies have revealed that the degree of ER positivity is directly related to tumor responsiveness to antiestrogen therapy. In the earlier EBCTCG Overview analysis, women with tumors that had 2+ ER staining derived a significantly larger reduction in the risk of death from 5 years of tamoxifen compared to those with 1+ staining. Similarly, patients who had tumors with an Allred score of 6 and above—calculated as the sum of an intensity score (range, 1–3) and a frequency score (range, 0–5) of ER staining—are most likely to respond to treatment.
In the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 trial, a randomized phase III study of tamoxifen vs observation in women with HR-positive breast cancer, the levels of ER expression, analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, was also predictive of tamoxifen benefit. A relationship between ER expression and response to endocrine therapy was also observed in neoadjuvant endocrine studies of aromatase inhibitors including letrozole and anastrozole(Drug information on anastrozole).[12-14] Interestingly, compared with tamoxifen in the neoadjuvant setting, aromatase inhibitors may be able to induce a response in tumors with lower levels of ER, although the sample sizes in these studies do not allow for robust conclusions in this regard.
Despite the clear value of ER expression analysis, the methodologies to evaluate ER expression and the cutoffs used to determine endocrine sensitivity are not standardized in clinical trials or in clinical practice. Conventional techniques, including the ligand-binding assay, which employs a radiolabeled steroid ligand to ER, and immunohistochemistry (IHC), which involves the use of specific antibodies to ER, have many problems. Some are pre-analytical (ie, related to poor specimen processing) and others are analytic in nature (ie, lack of assay standardization, lack of robust internal controls). The accuracy and reproducibility of scoring as well as the cutoff points for ER positivity vary among different laboratories. A newer method, which measures ER mRNA levels by qRT-PCR, allows a more quantitative and objective evaluation of ER expression and may be more accurate than conventional techniques. However, this method is only routinely available in the context of the Oncotype assay, rather than as a stand-alone test.
• Role of PR Status—In ER-positive breast cancer, the contribution of PR to the prediction of endocrine therapy responsiveness has been a subject of controversy. Since PR expression is regulated by ER, it was thought that the absence of PR likely reflects a nonfunctional ER pathway. It has also been observed that PR-negative tumors are generally associated with hyperactive growth factor signaling.[16,17] Compared to ER-positive/PR-positive tumors, ER-positive/PR-negative tumors have twice as many DNA copy number gains or losses and are frequently associated with upregulation of specific oncogenic pathways, including PI3K/Akt/mTOR. As assessed by gene expression profiling, these tumors form a distinct subset of breast cancer that is associated with aggressive pathologic features and poor outcome.
Therefore, it was hypothesized that the absence of PR in ER-positive tumor likely entails endocrine resistance. Consistent with the hypothesis, patients with tumors that are ER-positive/PR-negative have been found to be much less likely to benefit from endocrine therapy than those with ER-positive/PR-positive tumors in the metastatic setting.[7,19,20] However, studies in patients with early-stage breast cancer have failed to demonstrate a relationship between PR expression and endocrine responsiveness. In the EBCTCG Overview analysis, PR played no role in ER-positive tumors in predicting benefit to adjuvant tamoxifen therapy. In the ER-positive group, PR-positive and PR-negative patients showed similar benefit from tamoxifen (relative risk [RR] = 0.81; 95% confidence interval [CI] = 0.65–1.02; and RR = 0.70; 95% CI = 0.49–0.99, respectively).
Based on data collected in case record forms, it was initially reported that the relative benefit from anastrozole was substantially higher in the PR-negative subgroup in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) adjuvant breast cancer trial. However, upon central review of the tumor blocks collected from 2,006 of the 5,880 patients enrolled in the ATAC trial, the quantitative expression of PR or HER2 did not identify patients with differential relative benefit from anastrozole over tamoxifen. Time to recurrence was longer for anastrozole than for tamoxifen in all molecular subgroups. Similarly, in the Breast International Group (BIG) 1-98 trial, letrozole was superior to tamoxifen regardless of PR status.
HER2 positivity has generally been accepted as a marker of endocrine resistance. HER2 (HER2/neu or cerbB2) is a proto-oncogene and HER2 overexpression/amplification is associated with higher histologic grade, low expression of ER and PR, and worse clinical outcome.[25,26] About 10% of ER-positive breast cancer involves HER2 gene amplification. Preclinical studies demonstrated that HER2 overexpression reduces dependence on estrogen.[27,30] The resistance to endocrine interventions was correlated with hyperactivation of MAPK and downregulation of ER.
In the metastatic setting, HER2 overexpression is associated with reduced response to tamoxifen. In the neoadjuvant setting, while the amplification of HER2 does not seem to affect the clinical response to letrozole, suppression of Ki67 is significantly less in these tumors, suggesting therapeutic resistance.
The relationship between endocrine resistance and HER2 positivity has further been demonstrated in adjuvant endocrine therapy studies. In the adjuvant tamoxifen studies, HER2-positive cancers did not derive significant benefit from tamoxifen.[21,33] In the randomized, four-arm, phase III adjuvant BIG 1-98 trial, patients with ER-positive/HER2-positive tumors experienced inferior disease-free survival (DFS) compared to those with ER-positive/HER2-negative tumors regardless of treatment assignment, underscoring the endocrine therapy–resistant properties of ER-positive/HER2-positive tumors. HER2-positive status is therefore a bona fide marker for poor outcome in ER-positive disease and warrants treatment with trastuzumab(Drug information on trastuzumab) (Herceptin), which improves outcome in HER2-positive breast cancer regardless of ER status.
An increased expression of proliferation markers has generally been accepted to be a predictor of worse clinical outcomes in breast cancer. IHC staining of Ki67, a nuclear antigen that is present only in proliferating cells, has been shown to be a reliable methodology to enumerate the growth fraction of normal or neoplastic cell populations, and the Ki67 labeling index—the percentage of cells with positive Ki67 nuclear staining—correlates well with the S phase fraction and mitotic index.
Multiple studies have shown that baseline tumor Ki67 is a prognostic factor for breast cancer.[40,41] In a meta-analysis of 46 studies of over 12,000 patients with early-stage breast cancer, Ki67 positivity, defined by individual studies (cutoff ranges from 3.5 to 34%), is associated with a higher probability of relapse (hazard ratio [HR] = 1.93; 95% CI = 1.74–2.14; P < .001) and worse survival (HR = 1.95; 95% CI = 1.70–2.24; P < .001). However, routine use of Ki67 for prognostic assessment of ER-positive breast cancers has not been considered a standard practice. Most of the studies are retrospective and differ in the type of antibody used, the cutoff value selected to define high vs low proliferative activity, and the number of cells counted. Moreover, investigators lack an internationally standardized method for antigen retrieval, staining procedures, and scoring methods.
The value of Ki67 in predicting responses to systemic therapy has also been evaluated. Several studies in the neoadjuvant setting suggest that tumors with high Ki67 expression are more likely to respond to neoadjuvant chemotherapy.[44,45] However, in a retrospective analysis of 1,924 patients who were enrolled in two randomized International Breast Cancer Study Group (IBCSG) trials of adjuvant chemoendocrine therapy vs endocrine therapy alone for node-negative breast cancer—the IBCSG VIII and IX trials—a high Ki67 labeling index was associated with a worse prognosis, but did not predict an added benefit of chemotherapy to endocrine therapy. Interestingly, in the BIG 1-98 trial—a randomized, double blind, phase III study that showed letrozole improved DFS compared to tamoxifen for postmenopausal women with hormone receptor–positive disease[47,48]—the investigators found a greater benefit from letrozole compared to tamoxifen in tumors with a higher Ki67 labeling index.[49,50] The hazard of a DFS event for letrozole was half that for tamoxifen (HR, letrozole:tamoxifen = 0.53; 95% CI 0.39–0.72).[49,50] Therefore, high Ki67 labeling index levels may identify a patient group that particularly benefits from initial letrozole adjuvant therapy, but more studies are needed to confirm these findings.