Specific BRCA and immune configurations determine optimal response to platinum-based chemotherapy in triple negative breast and ovarian carcinomas

Loss of homologous recombination repair (HRR) via germline and somatic BRCA1 or BRCA2 gene mutations and via BRCA1 promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers with BRCA1 promoter methylation ( BRCA1 meth) respond more poorly as compared to those bearing mutations in BRCA1 and BRCA2 ( BRCA mut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts, BRCA1 mut and BRCA1 meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients with BRCA mut cancers, but not those with BRCA1 meth cancers, had significantly better response outcomes when compared to those with BRCA proficient cancers. When fully promoter methylated BRCA1 PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylated BRCA1 promoter allele emerged in resultant tumors associated with an increase in BRCA1 expression. A separate analysis of PDXs derived from treatment naïve TNBCs featured complete methylation of the BRCA1 promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates to in vivo platinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partially BRCA1 meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of the BRCA1 promoter alleles and not to the outgrowth of a non-methylated clone. Clinically, analysis of primary OvCas confirmed that high levels of BRCA1 methylation were significantly associated with reduced BRCA1 gene expression whereas cancers with lower levels of BRCA1 methylation had expression levels approaching those found in BRCA1 proficient cancers. These data suggest that unlike BRCA mut cancers, where HRR deficiency is achieved via mutations that are genetically ‘fixed’, BRCA1 meth cancers are highly adaptive to genotoxin exposure and more likely to recover BRCA1 expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients with BRCA proficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa. ### Competing Interest Statement E.T.L. reported being on the Scientific Advisory Board of Veracyte, a molecular diagnostic company that focuses on RNA profiles in thyroid nodules and non-malignant lung disease with no impact on the current manuscript, and being a consultant for Temasek Holdings, a sovereign fund for the country of Singapore with holdings in the biotech space, but none are pertinent to the current manuscript. Y.Y. reported receiving grants/research funding from Eisai, Genentech, Merck, Novartis, Pfizer and Puma, and has received compensation as a consultant to Eisai, Immunomedics, Novartis and Pfizer. Y.Y. also reported receiving personal fees for speaking at Eisai, Genentech, AstraZeneca, Daiichi, Gilead, Merck, Immunomedics, Novartis and Pfizer. M.T.L is a Founder of, and an uncompensated Manager in StemMed Holdings L.L.C., an uncompensated Limited Partner in StemMed Ltd., and is a Founder of and equity stake holder in Tvardi Therapeutics. L.D. is a compensated employee of StemMed Ltd. Selected BCM PDX models described herein are exclusively licensed to StemMed Ltd. resulting in tangible property royalties to M.T.L. and L.D. E.T.L. and F.M. are listed as inventors on the following patent applications, which are directly associated with the findings presented in this study: J0227.70099US00/J0227.70099US01 (Prognostic methods for platinum-based chemotherapeutics) and U.S. provisional application number 62/613,256, filed January 3, 2018 J0227.70031US01 (Gene mutations associated with Tandem Duplicator Phenotype). All other authors declare no competing interests.