Anton Safonov

New Strategies in Breast Cancer: Immunotherapy

More than 70% of breast cancers contain lymphocytic infiltration in the stroma, and preclinical studies suggest that immunoediting and partial control of cancer progression by the local immune microenvironment operate in most breast cancers. Consistent with this hypothesis, a large number of studies demonstrated a favorable prognostic and chemotherapy response predictive role for immune infiltration in breast cancer. The evidence is particularly strong for triple-negative and HER2-positive cancers. The development of clinically effective immune checkpoint inhibitors now provides an opportunity to test the therapeutic potential of augmenting the local antitumor immune response. Several phase I clinical trials using single-agent anti–PD-1 and anti–PD-L1 antibodies demonstrated objective tumor response rates, with remarkably durable responses, in heavily pretreated, metastatic, triple-negative cancers and somewhat lower responses in estrogen receptor–positive cancers. Currently, close to 50 ongoing, or soon to open, clinical trials evaluate the role of this new treatment modality in breast cancer. Clin Cancer Res; 22(9); 2105–10. ©2016 AACR.

Systematic drug screening identifies tractable targeted combination therapies in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains an aggressive disease without effective targeted therapies. In this study, we addressed this challenge by testing 128 FDA-approved or investigational drugs as either single agents or in 768 pairwise drug combinations in TNBC cell lines to identify synergistic combinations tractable to clinical translation. Medium-throughput results were scrutinized and extensively analyzed for sensitivity patterns, synergy, anticancer activity, and were validated in low-throughput experiments. Principal component analysis revealed that a fraction of all upregulated or downregulated genes of a particular targeted pathway could partly explain cell sensitivity toward agents targeting that pathway. Combination therapies deemed immediately tractable to translation included ABT-263/crizotinib, ABT-263/paclitaxel, paclitaxel/JQ1, ABT-263/XL-184, and paclitaxel/nutlin-3, all of which exhibited synergistic antiproliferative and apoptotic activity in multiple TNBC backgrounds. Mechanistic investigations of the ABT-263/crizotinib combination offering a potentially rapid path to clinic demonstrated RTK blockade, inhibition of mitogenic signaling, and proapoptotic signal induction in basal and mesenchymal stem-like TNBC. Our findings provide preclinical proof of concept for several combination treatments of TNBC, which offer near-term prospects for clinical translation. Cancer Res; 77(2); 566-78. ©2016 AACR.

Immune Gene Expression Is Associated with Genomic Aberrations in Breast Cancer

The presence of tumor-infiltrating lymphocytes (TIL) is a favorable prognostic factor in breast cancer, but what drives immune infiltration remains unknown. Here we examine if clonal heterogeneity, total mutation load, neoantigen load, copy number variations (CNV), gene- or pathway-level somatic mutations, or germline polymorphisms (SNP) are associated with immune metagene expression in breast cancer subtypes. Thirteen published immune metagenes correlated separately with genomic metrics in the three major breast cancer subtypes. We analyzed RNA-Seq, DNA copy number, mutation and germline SNP data of 627 ER+, 207 HER2+, and 191 triple-negative (TNBC) cancers from The Cancer Genome Atlas. P-values were adjusted for multiple comparisons, and permutation testing was used to assess false discovery rates. Increased immune metagene expression associated significantly with lower clonal heterogeneity estimated by MATH score in all subtypes and with a trend for lower overall mutation, neoantigen, and CNV loads in TNBC and HER2+ cancers. In ER+ cancers, mutation load, neoantigen load, and CNV load weakly but positively associated with immune infiltration, which reached significance for overall mutation load only. No highly recurrent single gene or pathway level mutations associated with immune infiltration. High immune gene expression and lower clonal heterogeneity in TNBC and HER2+ cancers suggest an immune pruning effect and equilibrium between immune surveillance and clonal expansion. Thus, immune checkpoint inhibitors may tip the balance in favor of immune surveillance in these cancers. Cancer Res; 77(12); 3317-24. ©2017 AACR.

Methods for detecting co-mutated pathways in cancer samples to inform treatment selection

Tumor genomes evolve through a selection of mutations. These mutations may complement each other to promote tumorigenesis. To better understand the functional interactions of different processes in cancer, we studied mutation data of a set of tumors and identified significantly co-mutated pathways. Fisher’s exact test is a standard approach that can be used to assess the significance of the joint dysregulation of pathways pairs across a patient population. We developed a robust test to identify co-occurrence using DNA mutations, which overcomes deficiencies of the Fisher’s exact test by taking into account the large variability in overall mutation load and sequencing depth. Applying our method to a study of six common cancer types, we identify enrichment of co-mutated signal transduction pathways such as IP3 synthesis and PI3K and pairs of co-mutated pathways involving other processes such as immunity and development. We observed enrichment of clonal co-mutation of the proteasome and apoptosis pathways in colorectal cancer, which suggests potential mechanisms for immune evasion.

Abstract P4-07-01: DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer

Background: Mutations or epigenetic silencing of BRCA1/2 genes result in DNA repair deficiency in a large proportion of TNBC cases. Yet it is unclear whether this deficiency is associated with increased chemosensitivity and improved benefit from standard-of-care chemotherapy. We systematically evaluated BRCA deficiency in TNBC using integrated DNA and RNA sequencing data and its association clinical outcome, exploring the potential role of tumor immune response. Patients and Methods: Whole-exome, DNA methylation, copy number variation, and RNA sequencing data from 102 stage I-III TNBCs were retrieved from The Cancer Genome Atlas (TCGA). Almost all patients received adjuvant taxane-anthracycline-cyclophosphamide (T-AC) chemotherapy and had >30 days of follow-up. The number of predicted neoantigens and an estimate of the level of immune cell activity for 77 of these tumors were previously published. Deleterious germline or somatic BRCA1/2 mutations were identified by majority voting on predictions of 5 variant scoring algorithms. Definition of BRCA1/2 deficiency (BRCA-D) included carrier of deleterious BRCA1/2 mutations or BRACA1/2 normal (BRCA-N) with wild type BRCA1/2 expression less than the maximum observed in mutation carriers. Normalized genomic mutation rate and mutant allele tumor heterogeneity (MATH) were computed as broad measures of genomic instability. Characteristics of BRCA-D vs N tumors were compared using the Wilcoxon rank test. Results: Twenty tumors (19.6%) had mutations in BRCA1, 6 (5.8%) in BRCA2, and 2 (1.9%) in both. Based on the expanded definition, 39 cases (38%) were characterized as BRCA1 deficient, 5 (4.9%) as BRCA2 deficient and 4 (3.9%) as deficient in both. BRCA-D tumors (47%) were associated with a significantly higher mutation rate (P=8x10-4) but had similar clonal heterogeneity (P=0.55) as BRCA-N tumors. BRCA-D tumors had excellent 4-year overall survival (100%) compared to 79.5% (95%CI: 66.6- 94.9) for BRCA-N tumors (log-rank P=0.02). BRCA-D tumors also presented a significantly higher number of predicted neoantigents (P=0.003), which resulted in increased level of immune cell activity. In contrast, low immunogenic TNBC tumors were underrepresented in BRCA-D (p=0.05) and showed potential signs of immunoediting (observed/expected number of predicted neoantigens Conclusions: Deleterious mutations in BRCA1/2 genes occur in 25% of TNBC tumors, but parallel quantification of wild-type BRCA1/2 expression identifies 47% of TNBC samples with double strand break DNA repair deficiency. These BRCA-D TNBC tumors are characterized by a significantly higher mutation rate and present a significantly greater number of neoantigens that result in increased immune cell activity. Our analysis suggested that enhanced immune activation could explain to a large extent the excellent clinical outcome in patients with BRCA-D tumors treated with standard-of-care T-AC chemotherapy. Citation Format: Jiang T, Safonov A, Bianchini G, Shi W, Wali VB, Pusztai L, Hatzis C. DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-01.

Abstract P6-11-03: A cost effectiveness analysis of baseline left ventricular function assessment for breast cancer patients undergoing anthracycline chemotherapy

Background: It is unclear if all breast cancer (BC) patients require baseline left ventricular function (LVEF) assessment prior to anthracycline based chemotherapy (ABC), and the approach is variable in clinical practice. Our objective is to determine the cost effectiveness of obtaining a baseline LVEF assessment prior to (neo) adjuvant ABC in clinical practice. Methods: We performed a retrospective analysis of the Yale Equilibrium Radionuclide Angiography (ERNA) database for 701 breast cancer patients who had a baseline ERNA scan prior to systemic therapy for an initial diagnosis of stages I-IV BC between July 2003 and May 2013. We found that 14 of 701 (2%) patients had a baseline LVEF Results: Assuming that 20% of the unscreened patients with a LVEF Conclusion: Baseline LVEF assessment was found to be cost-effective under a willingness-to-pay threshold of

Abstract P4-04-20: Subtype specific differential expression and immunogenicity of endogenous retrovirus elements in breast cancer

50,000/QALY. Our sensitivity analysis suggests that risk factor-guided LVEF baseline LVEF screening may increase the number of high-risk patients in the treatment population, thus further increasing the cost-effectiveness of baseline LVEF assessment. Citation Format: Safonov A, Hatzis C, Stratton J, Gross CP, Russell R, Pusztai L, Abu-Khalaf MM. A cost effectiveness analysis of baseline left ventricular function assessment for breast cancer patients undergoing anthracycline chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-11-03.

Assessing cost-utility of predictive biomarkers in oncology: a streamlined approach

Background: Endogenous retroviruses (ERVs) are germline encoded DNA sequences that entered the human genome millions of years ago. While they are mostly inactivated due to accumulated termination codons and deletions, previous studies have demonstrated overexpression and antibody-targeted immunotherapeutic potential, of ERV-related env proteins in breast cancer. We sought to elucidate subtype specificity, immunogenicity, and correlation with innate immunity related gene signatures of ERVs in breast cancer. Methods: We utilized publically available RNASeq gene expression data of breast cancer samples along and corresponding matched normals from TCGA. The dataset included 191 ER-/HER2- (TN), 197 HER2+, and 627 ER+/HER2- (Luminal) breast cancers. ERV expression was obtained by mapping bowtie2-aligned reads of recently annotated to be transcriptionally active to the RNAseq bam files (Rooney et al 2015, Mayer et al 2011). ERVs preferentially expressed in tumors compared normal tissue were identified as those for which the 5th percentile of ERV expression in the tumors exceeded the 95th percentile of ERV expression in the normal samples. A gene signature involving GZMB, PRF1, CXCL13, IRF1, IKZF1, and HLA-E was used as a measure of immune activity. To assess the immunogenic potential of the tumor-specific ERVs, we compared the expression level of the ERV within the lower and higher immune signature tertiles using the Wilcoxon signed-rank test. To elucidate mechanism of potential immune response, ERVs found to be significantly associated with immune response at a false discovery rate of Results: Out of the 66 original annotated ERVs, 47 were found to be expressed at significantly higher levels in breast cancer compared to normal tissue and 22 were immunogenic. Examples include members of the ERV-K family, as have also been previously detected by flow cytometry and IHC. Subtype-specific immunogenic potential was demonstrated in 4 ERVs in TNBC (ERVK10, ERVK17, ERVFRD.1, ERVPABLB.1) and in 7 ERVs in the luminal subtype (ERV3.1, ERVE.4, ERVFRD.2, ERVK.15, ERVK.19, ERVK.20, ERVK.25, ERVW.3). Twelve of the 22 immunogenic ERVs were significantly correlated with expression of all ten TLR evaluated, while four ERVs showed more specific correlation patterns with TLRs. High ERV3.1 expression was associated with high TLR3, TLR8, TLR9 that specifically target double stranded or single stranded RNA, suggesting a potential mechanism for mediation of ERV related immune response. Conclusion: Our results suggest breast cancer subtype specific ERV dysregulation and immunogenicity. The potentially immunogenic ERVs were generally not self-correlated or located in the same amplicon as HLA genes, suggesting an independent immune response pathway. Furthermore, ERV expression correlates with specific endosomal nucleic-acid recognizing toll-like receptors, which may prompt further investigation into subtype-specific TLR-targeted therapy. Citation Format: Safonov A, Bianchini G, Jiang T, Pusztai L, Hatzis C. Subtype specific differential expression and immunogenicity of endogenous retrovirus elements in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-20.

Effect of DNA hypermethylation on immune escape through downregulation of antigen presentation genes in breast cancer.

Abstract Evaluation of cost-utility is critical in assessing the medical utility of predictive or prognostic biomarkers. Current methods involve complex state-transition models, requiring comprehensive data inputs. We propose a simplified decision-analytic tool to explore the relative effect of factors contributing to the cost-utility of a biomarker. We derived a cost-utility metric, the “test incremental cost-effectiveness ratio” (TICER) for biomarker-guided treatment compared to no biomarker use. This method uses data inputs readily accessible through clinical literature. We compared our results with traditional cost-effectiveness analysis of predictive biomarkers for established (HER2-guided trastuzumab, ALK-guided crizotinib, OncotypeDX-guided adjuvant chemotherapy) and emerging (ROS1-guided crizotinib) targeted treatments. We conducted sensitivity analysis to determine which factors had the greatest impact on TICER estimates. Base case TICER for HER2 was

Association between DNA level aberrations and immune cell infiltration in breast cancer.

149,600/quality-adjusted life year (QALY), for ALK was