Abstract PD9-03: Genomic analysis of the CALGB 40603 (Alliance) neoadjuvant trial in TNBC identifies immune features associated with pathological complete response and event-free survival
Authors: Shepherd Jonathan H , Hyslop Terry , Fan Cheng , Martinez Aranzazu Fernandez , Parker Joel , Hoadley Katherine , Hu Zhiyuan , Li Yun , Soloway Matthew , Spears Patricia , Partridge Ann , Sikov William , Carey Lisa A , Perou Charles M 
Published: February 2021

Abstract
Purpose: CALGB (now part of the Alliance for Clinical Trials in Oncology) 40603 was a randomized Phase II study investigating pathological complete response rates (pCR) in triple-negative breast cancer (TNBC) patients receiving neoadjuvant weekly paclitaxel followed by doxorubicin and cyclophosphamide +/- bevacizumab and/or carboplatin for which 5-year event-free survival (EFS) results are now available. This is a valuable resource to characterize clinical and genomic features associated with response and survival. Methods: Clinical parameters and pre-treatment tumor biopsy RNA-sequencing (RNAseq) from 295 TNBC patients were analyzed to identify features associated with pCR and/or EFS. A panel of 689 previously published gene expression signatures were evaluated to provide insights regarding potential associations between clinical endpoints and genomically determined cell types and signaling pathway activities. Additionally, B cell receptor (BCR) and T cell receptor (TCR) sequences were examined, and repertoire abundance, richness, and diversity measures were calculated to investigate correlations with pCR and EFS status. Univariate Mann-Whitney U-Tests were used for continuous variables, and Fisher’s test was used for categorical features, with unadjusted p-values &lt; 0.05 designated as significant. Results: While the addition of bevacizumab and carboplatin each significantly improved pCR rates, neither improved EFS. We examined outcomes according to race and no differences were seen for either pCR rate or EFS. 131 features, including high proliferation and multiple interferon signatures were significantly associated with pCR, but not with EFS. Alternatively, 69 features, including clinical factors for T stage and node status, were prognostic for EFS, but not significantly associated with pCR. Nevertheless, pCR itself was the strongest predictor of EFS, and was the only feature significantly associated with EFS after adjusting for multiple comparisons (Benjamini-Hochberg False Discovery Rate = 6.7e-3). In total, 52 genomic features were significantly correlated with both pCR and EFS, 44 of which were features of the immune microenvironment. Immune associated features included signatures of T cells, B cells and NK cells, immune checkpoint pathways (PD-1, PD-L1, CTLA4), and antigen presentation (dendritic cells, MHC-I, MHC-II). In particular, low BCR evenness, which is a measure of uniformity of unique BCR sequence abundance, was strongly associated with both pCR and EFS, suggesting that an antigen-specific adaptive immune response with clonally selected B cells is occurring in patients that have improved response and survival. Furthermore, a multivariate Cox Proportional Hazards model assessing BCR evenness along with age, T stage, N stage, grade and pCR found BCR evenness to be an independent prognostic feature for EFS in TNBC. Conclusions: Evidence of distinct predictors of pCR and EFS in TNBC patients treated with neoadjuvant chemotherapy suggests that, while pCR is still the strongest prognostic feature, high expression of many immune related gene expression signatures in pretreatment tumor samples are promising biomarkers of improved EFS. In addition to the important role of T cells in an anti-tumor response, these data show high IgG expression and evidence of B cell clonal selection associates with improved response and survival, supporting an important role for B cells in the adaptive response that portends a long-term benefit of chemotherapy in TNBC. Support: U10CA180821, U10CA180882, U24CA196171, P50-CA58223, Genentech, and The Breast Cancer Research Foundation. https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifier: NCT00861705
Citation Format: Jonathan H Shepherd, Terry Hyslop, Cheng Fan, Aranzazu Fernandez Martinez, Joel Parker, Katherine Hoadley, Zhiyuan Hu, Yun Li, Matthew Soloway, Patricia Spears, Ann Partridge, William Sikov, Lisa A Carey, Charles M Perou. Genomic analysis of the CALGB 40603 (Alliance) neoadjuvant trial in TNBC identifies immune features associated with pathological complete response and event-free survival [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-03.