Predictive Significance of an Optimized Panel for Basal-like Breast Cancer: Results from the Canadian Cancer Trials Group MA.5 and MA.12 Phase III Clinical Trials.

PURPOSE: Accurate IHC biomarkers incorporating nestin positivity or inositol polyphosphate-4-phosphate (INPP4B) loss have recently been optimized to identify the basal-like intrinsic breast cancer subtype regardless of estrogen, progesterone, or Her2 status. We examined the predictive capacity of these basal biomarkers in the CCTG MA.5 chemotherapy and MA.12 endocrine therapy trials. EXPERIMENTAL DESIGN: Formalin-fixed paraffin embedded blocks of primary tumors from patients randomized in the two trials were used to build tissue microarrays. IHC staining for nestin and INPP4B followed published methods and REMARK criteria. A prespecified statistical plan tested the hypothesis that patients with basal breast cancer (nestin+ or INPP4B-) would not benefit from anthracycline substitution in MA.5 or from tamoxifen in MA.12. RESULTS: Nestin positivity or INPP4B loss was observed in 110/453 (24%) interpretable samples from MA.5 and 47/366 (13%) from MA.12, and was associated with high grade, younger age, estrogen receptor negativity, triple-negative, core basal, and PAM50 basal-like subtypes. In the MA.5 trial, patients assigned as basal experienced lower benefit from anthracycline versus nonanthracycline adjuvant chemotherapy [HR, 1.49; 95% confidence interval (CI), 0.72-3.10] when compared with non-basal (nestin- and INPP4B+) cases where there was a higher benefit from anthracyclines (HR, 0.75; 95% CI, 0.54-1.04; P interaction = 0.01). In the MA.12 trial, patients assigned as basal did not demonstrate a benefit from adjuvant tamoxifen versus placebo (HR, 0.48; 95% CI, 0.12-1.86; P = 0.29), whereas nonbasal cases displayed significant benefit (HR, 0.66; 95% CI, 0.45-0.98; P = 0.04), although the interaction test was not significant. CONCLUSIONS: The nestin/INPP4B IHC panel identifies women with basal breast cancers who benefit from nonanthracycline chemotherapy but not endocrine adjuvant treatments.

Predictive Biomarkers for Adjuvant Capecitabine Benefit in Early-Stage Triple-Negative Breast Cancer in the FinXX Clinical Trial.

PURPOSE: Recent studies have demonstrated a benefit of adjuvant capecitabine in early breast cancer, particularly in patients with triple-negative breast cancer (TNBC). However, TNBC is heterogeneous and more precise predictive biomarkers are needed. EXPERIMENTAL DESIGN: Tumor tissues collected from TNBC patients in the FinXX trial, randomized to adjuvant anthracycline-taxane-based chemotherapy with or without capecitabine, were analyzed using a 770-gene panel targeting multiple biological mechanisms and additional 30-custom genes related to capecitabine metabolism. Hypothesis-generating exploratory analyses were performed to assess biomarker expression in relation to treatment effect using the Cox regression model and interaction tests adjusted for multiplicity. RESULTS: One hundred eleven TNBC samples were evaluable (57 without capecitabine and 54 with capecitabine). The median follow-up was 10.2 years. Multivariate analysis showed significant improvement in recurrence-free survival (RFS) favoring capecitabine in four biologically important genes and metagenes, including cytotoxic cells [hazard ratio (HR) = 0.38; 95% confidence intervals (CI), 0.16-0.86, P-interaction = 0.01], endothelial (HR = 0.67; 95% CI, 0.20-2.22, P-interaction = 0.02), mast cells (HR = 0.78; 95% CI, 0.49-1.27, P-interaction = 0.04), and PDL2 (HR = 0.31; 95% CI, 0.12-0.81, P-interaction = 0.03). Furthermore, we identified 38 single genes that were significantly associated with capecitabine benefit, and these were dominated by immune response pathway and enzymes involved in activating capecitabine to fluorouracil, including TYMP. However, these results were not significant when adjusted for multiple testing. CONCLUSIONS: Genes and metagenes related to antitumor immunity, immune response, and capecitabine activation could identify TNBC patients who are more likely to benefit from adjuvant capecitabine. Given the reduced power to observe significant findings when correcting for multiplicity, our findings provide the basis for future hypothesis-testing validation studies on larger clinical trials.

The Prosigna gene expression assay and responsiveness to adjuvant cyclophosphamide-based chemotherapy in premenopausal high-risk patients with breast cancer.

BACKGROUND: The PAM50-based (Prosigna) risk of recurrence (ROR) score and intrinsic subtypes are prognostic for women with high-risk breast cancer. We investigate the predictive ability of Prosigna regarding the effectiveness of cyclophosphamide-based adjuvant chemotherapy in premenopausal patients with high-risk breast cancer. METHODS: Prosigna assays were performed on the NanoString platform in tumors from participants in Danish Breast Cancer Group (DBCG) 77B, a four-arm trial that randomized premenopausal women with high-risk early breast cancer to no systemic treatment, levamisole, oral cyclophosphamide (C) or cyclophosphamide, methotrexate and fluorouracil (CMF). RESULTS: In total, this retrospective analysis included 460 women (40% of the 1146 randomized patients). The continuous Prosigna ROR score was prognostic in the no systemic treatment group (unadjusted P < 0.001 for disease-free survival (DFS), P = 0.001 for overall survival (OS)). No statistically significant interaction of continuous ROR score and treatment on DFS and OS was found. A highly significant association was observed between intrinsic subtypes and C/CMF treatment for DFS (Pinteraction = 0.003 unadjusted, P = 0.001 adjusted) and OS (Pinteraction = 0.04). In the adjusted analysis treatment with C/CMF was associated with a reduced risk of DFS events in patients with basal-like (hazard ratio (HR) 0.14; 95% CI 0.06; 0.32) and luminal B (HR 0.48; 95% CI 0.27; 0.84) subtypes but not in patients with Human epidermal growth factor receptor-enriched (HR 1.05; 95% CI 0.56; 1.95) or luminal A (HR 0.61; 95% CI 0.32; 1.16) subtypes. CONCLUSION: The Prosigna ROR score and intrinsic subtypes were prognostic in high-risk premenopausal patients with breast cancer, and intrinsic subtypes identify high-risk patients with or without major benefit from adjuvant C/CMF treatment.

Effect of continuous statistically standardized measures of estrogen and progesterone receptors on disease-free survival in NCIC CTG MA.12 Trial and BC Cohort.

INTRODUCTION: We hypothesized improved inter-laboratory comparability of estrogen receptor (ER) and progesterone receptor (PgR) across different assay methodologies with adjunctive statistical standardization, akin to bone mineral density (BMD) z-scores. We examined statistical standardization in MA.12, a placebo-controlled pre-menopausal trial of adjuvant tamoxifen with locally assessed hormone receptor +/- tumours, and in a cohort of post-menopausal British Columbia (BC) tamoxifen-treated patients. METHODS: ER and PgR were centrally assessed for both patient groups with real time quantitative reverse transcription polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Effects on disease-free survival (DFS) were investigated separately for 345 MA.12 and 673 BC patients who had both qPCR and IHC assessments. Comparisons utilized continuous laboratory units and statistically standardized z-scores. Univariate categorization of ER/PgR was by number of standard deviations (SD) above or below the mean (z-score ≥1.0 SD below mean; z-score <1.0 SD below mean; z-score ≤1.0 SD above mean; z-score >1.0 SD above mean). Exploratory multivariate examinations utilized step-wise Cox regression. RESULTS: Median follow-up for MA.12 was 9.7 years; for BC patients, 11.8 years. For MA.12, 101 of 345 (29%) patients were IHC ER-PgR-. ER was not univariately associated with DFS (qPCR, P = 0.19; IHC, P = 0.08), while PgR was (qPCR, P = 0.09; IHC, P = 0.04). For BC patients, neither receptor was univariately associated with DFS: for ER, PCR, P = 0.36, IHC, P = 0.24; while for PgR, qPCR, P = 0.17, IHC, P = 0.31. Multivariately, MA.12 patients randomized to tamoxifen had significantly better DFS (P = 0.002 to 0.005) than placebo. Meanwhile, jointly ER and PgR were not associated with DFS whether assessed by qPCR or by IHC in all patients, or in the subgroup of patients with IHC positive stain, for pooled or separate treatment arms. Different results by type of continuous unit supported the concept of ER level being relevant for medical decision-making. For postmenopausal BC tamoxifen patients, higher qPCR PgR was weakly associated with better DFS (P = 0.06). CONCLUSIONS: MA.12 pre-menopausal patients in a placebo-controlled tamoxifen trial had similar multivariate prognostic effects with statistically standardized hormone receptors when tumours were assayed by qPCR or IHC, for hormone receptor +/- and + tumours. The BC post-menopausal tamoxifen cohort did not exhibit a significant prognostic association of ER or PgR with DFS. Adjunctive statistical standardization is currently under investigation in other NCIC CTG endocrine trials.

Responsiveness of intrinsic subtypes to adjuvant anthracycline substitution in the NCIC.CTG MA.5 randomized trial.

PURPOSE: Recent studies suggest that intrinsic breast cancer subtypes may differ in their responsiveness to specific chemotherapy regimens. We examined this hypothesis on NCIC.CTG MA.5, a clinical trial randomizing premenopausal women with node-positive breast cancer to adjuvant CMF (cyclophosphamide-methotrexate-fluorouracil) versus CEF (cyclophosphamide-epirubicin-fluorouracil) chemotherapy. EXPERIMENTAL DESIGN: Intrinsic subtype was determined for 476 tumors using the quantitative reverse transcriptase PCR PAM50 gene expression test. Luminal A, luminal B, HER2-enriched (HER2-E), and basal-like subtypes were correlated with relapse-free survival (RFS) and overall survival (OS), estimated using Kaplan-Meier plots and log-rank testing. Multivariable Cox regression analyses determined significance of interaction between treatment and intrinsic subtypes. RESULTS: Intrinsic subtypes were associated with RFS (P = 0.0005) and OS (P < 0.0001) on the combined cohort. The HER2-E showed the greatest benefit from CEF versus CMF, with absolute 5-year RFS and OS differences exceeding 20%, whereas there was a less than 2% difference for non-HER2-E tumors (interaction test P = 0.03 for RFS and 0.03 for OS). Within clinically defined Her2(+) tumors, 79% (72 of 91) were classified as the HER2-E subtype by gene expression and this subset was strongly associated with better response to CEF versus CMF (62% vs. 22%, P = 0.0006). There was no significant difference in benefit between CEF and CMF in basal-like tumors [n = 94; HR, 1.1; 95% confidence interval (CI), 0.6-2.1 for RFS and HR, 1.3; 95% CI, 0.7-2.5 for OS]. CONCLUSION: HER2-E strongly predicted anthracycline sensitivity. The chemotherapy-sensitive basal-like tumors showed no added benefit for CEF over CMF, suggesting that nonanthracycline regimens may be adequate in this subtype although further investigation is required.

TIMP-1 in combination with HER2 and TOP2A for prediction of benefit from adjuvant anthracyclines in high-risk breast cancer patients.

HER2 amplification, TOP2A aberrations, and absence of tissue inhibitor of metalloproteinase (TIMP-1) expression in breast carcinomas have been shown to be associated with incremental benefit from anthracycline-containing adjuvant chemotherapy, and this study was undertaken to validate these findings in a similar, but independent, randomized clinical trial. TIMP-1 was examined by immunohistochemistry in archival tumor tissue from 403 of 716 premenopausal high-risk patients with known HER2 and TOP2A status who were randomized to cyclophosphamide, epirubicin, and fluorouracil (CEF) or cyclophosphamide, methotrexate, and fluorouracil (CMF) in the MA.5 trial. Ninety-eight (24%) patients had no TIMP-1 staining of tumor cells, 27% were HER2 amplified, and 18% were TOP2A aberrant. Forty-four percentage was classified as HT responsive (HER2 amplified and/or TIMP-1 negative) and 37% as 2T responsive (TOP2A aberrant and/or TIMP-1 negative). There was no heterogeneity in treatment effect of CEF versus CMF according to TIMP-1. In HT-responsive patients, CEF was superior to CMF with an improved RFS (adjusted HR, 0.64; 95% CI, 0.42-0.97), but this was not significant for OS (adjusted HR, 0.66; 95% CI, 0.42-1.04). A significant HT profile versus treatment interaction was detected for OS (P = 0.03). In 2T-responsive patients, CEF seemed to improve RFS compared to CMF (adjusted HR, 0.67; 95% CI, 0.43-1.03) and improved OS (adjusted HR, 0.58; 95% CI, 0.36-0.93). A significant 2T profile versus treatment interaction was detected for OS (P = 0.01). With this study, we validate a more substantial reduction in mortality by CEF compared to CMF in patients with an HT- or 2T-responsive profile; however, we could not show a similarly significant reduction in RFS events, where a benefit of CEF over CMF was found irrespective of TIMP-1 status. Further studies are necessary before the HT and 2T profiles may be used to direct the use of anthracyclines.