Brandon Wenz

T-cell invigoration to tumour burden ratio associated with anti-PD-1 response

Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.

Evaluation of ACMG-Guideline-Based Variant Classification of Cancer Susceptibility and Non-Cancer-Associated Genes in Families Affected by Breast Cancer.

Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of a variant-classification methodology based on American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG-designated reportable cancer and non-cancer-associated genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole-exome sequencing in 404 individuals in 253 families and classified 1,640 variants. Potentially clinically actionable (likely pathogenic [LP] or pathogenic [P]) versus nonactionable (VUS, likely benign, or benign) calls were 95% concordant with locus-specific databases and Clinvar. LP or P mutations were identified in 12 of 25 breast cancer susceptibility genes in 26 families without identified BRCA1/2 mutations (11%). Evaluation of 84 additional genes associated with autosomal-dominant cancer susceptibility identified LP or P mutations in only two additional families (0.8%). However, individuals from 10 of 253 families (3.9%) had incidental LP or P mutations in 32 non-cancer-associated genes, and 9% of individuals were monoallelic carriers of a rare LP or P mutation in 39 genes associated with autosomal-recessive cancer susceptibility. Furthermore, 95% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant-classification guidelines. Additionally, evaluation of extended panels of cancer-associated genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results.

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P < 0.0001, two-tailed Student’s t-test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy (P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.Most tumours associated with germline BRCA1/BRCA2 loss of function mutations respond to DNA damaging agents, however, some do not. Herein, the authors identify that a subset of breast/ovarian tumors retain a normal allele, which is associated with decreased overall survival after DNA damage-inducing platinum chemotherapy.

The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk

Understanding the gene-specific risks for development of breast cancer will lead to improved clinical care for those carrying germline mutations in cancer predisposition genes. We sought to detail the spectrum of mutations and refine risk estimates for known and proposed breast cancer susceptibility genes. Targeted massively-parallel sequencing was performed to identify mutations and copy number variants in 26 known or proposed breast cancer susceptibility genes in 2134 BRCA1/2-negative women with familial breast cancer (proband with breast cancer and a family history of breast or ovarian cancer) from a largely European–Caucasian multi-institutional cohort. Case–control analysis was performed comparing the frequency of internally classified mutations identified in familial breast cancer women to Exome Aggregation Consortium controls. Mutations were identified in 8.2% of familial breast cancer women, including mutations in high-risk (odds ratio > 5) (1.4%) and moderate-risk genes (2 < odds ratio < 5) (2.9%). The remaining familial breast cancer women had mutations in proposed breast cancer genes (1.7%), Lynch syndrome genes (0.5%), and six cases had two mutations (0.3%). Case–control analysis demonstrated associations with familial breast cancer for ATM, PALB2, and TP53 mutations (odds ratio > 3.0, p < 10−4), BARD1 mutations (odds ratio = 3.2, p = 0.012), and CHEK2 truncating mutations (odds ratio = 1.6, p = 0.041). Our results demonstrate that approximately 4.7% of BRCA1/2 negative familial breast cancer women have mutations in genes statistically associated with breast cancer. We classified PALB2 and TP53 as high-risk, ATM and BARD1 as moderate risk, and CHEK2 truncating mutations as low risk breast cancer predisposition genes. This study demonstrates that large case–control studies are needed to fully evaluate the breast cancer risks associated with mutations in moderate-risk and proposed susceptibility genes.Familial breast cancer: Pinning down susceptibility genes beyond BRCAWomen with the heritable form of breast cancer often harbor mutations in cancer-linked genes other than the usual suspects, BRCA1 and BRCA2. Slavin, Maxwell, Lilyquist, Joseph, and colleagues from major national and international cancer centers studied 2134 women with familial breast cancer who tested negative for BRCA1/2 gene mutations. The researchers sequenced 26 known or proposed breast cancer susceptibility genes and found mutations in approximately 1 in every 12 of the study subjects. They then further broke down the susceptibility genes into those that confer high-, moderate- or low-risk—although not all the proposed breast cancer genes reached statistical significance and, as such, their clinical importance remains unclear. The results support adding some of the high- and moderate-risk genes to multi-panel diagnostic tests that aim to determine the likelihood of a women developing heritable breast cancer.

A patient-derived-xenograft platform to study BRCA-deficient ovarian cancers

Approximately 50% of high-grade serous ovarian cancers (HGSOCs) have defects in genes involved in homologous recombination (HR) (i.e., BRCA1/2). Preclinical models to optimize therapeutic strategies for HR-deficient (HRD) HGSOC are lacking. We developed a preclinical platform for HRD HGSOCs that includes primary tumor cultures, patient-derived xenografts (PDXs), and molecular imaging. Models were characterized by immunohistochemistry, targeted sequencing, and reverse-phase protein array analysis. We also tested PDX tumor response to PARP, CHK1, and ATR inhibitors. Fourteen orthotopic HGSOC PDX models with BRCA mutations (BRCAMUT) were established with a 93% success rate. The orthotopic PDX model emulates the natural progression of HGSOC, including development of a primary ovarian tumor and metastasis to abdominal viscera. PDX response to standard chemotherapy correlated to that demonstrated in the patient. Pathogenic mutations and HGSOC markers were preserved after multiple mouse passages, indicating retention of underlying molecular mechanisms of carcinogenesis. A BRCA2MUT PDX with high p-CHK1 demonstrated a similar delay of tumor growth in response to PARP, CHK1, and ATR inhibitors. A poly (ADP-ribose) polymerase (PARP) inhibitor radiotracer correlated with PARP1 activity and showed response to PARP inhibition in the BRCA2MUT PDX model. In summary, the orthotopic HGSOC PDX represents a robust and reliable model to optimize therapeutic strategies for BRCAMUT HGSOC.

A Recurrent ERCC3 Truncating Mutation Confers Moderate Risk for Breast Cancer.

Known gene mutations account for approximately 50% of the hereditary risk for breast cancer. Moderate and low penetrance variants, discovered by genomic approaches, account for an as-yet-unknown proportion of the remaining heritability. A truncating mutation c.325C>T:p.Arg109* (R109X) in the ATP-dependent helicase ERCC3 was observed recurrently among exomes sequenced in BRCA wild-type, breast cancer-affected individuals of Ashkenazi Jewish ancestry. Modeling of the mutation in ERCC3-deficient or CRISPR/Cas9-edited cell lines showed a consistent pattern of reduced expression of the protein and concomitant hypomorphic functionality when challenged with UVC exposure or treatment with the DNA alkylating agent IlludinS. Overexpressing the mutant protein in ERCC3-deficient cells only partially rescued their DNA repair-deficient phenotype. Comparison of frequency of this recurrent mutation in over 6,500 chromosomes of breast cancer cases and 6,800 Ashkenazi controls showed significant association with breast cancer risk (ORBC = 1.53, ORER+ = 1.73), particularly for the estrogen receptor-positive subset (P < 0.007). SIGNIFICANCE A functionally significant recurrent ERCC3 mutation increased the risk for breast cancer in a genetic isolate. Mutated cell lines showed lower survival after in vitro exposure to DNA-damaging agents. Thus, similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC3 could constitute potential therapeutic targets in a subset of hereditary breast cancers. Cancer Discov; 6(11); 1267-75. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1197.

Abstract A22: Homologous recombination deficiency negatively predicts for immune infiltration and antitumor immune activity in breast tumors with BRCA1/2 alterations

Tumors with germline or somatic BRCA1 or BRCA2 alterations are sensitive to DNA-damaging agents but often develop therapeutic resistance. Breast cancers associated with BRCA1/2 alterations have a relatively high mutational load, defined as the number of nonsynonymous single nucleotide mutations, relative to BRCA1/2 wild-type tumors, leading to suggestions that patients with BRCA1/2 mutations are possible candidates for immune checkpoint blockade. However, immune infiltration can range widely in tumors with BRCA1/2 alterations, and molecular determinants that account for this variability remain unclear. Our goal was to delineate the molecular features associated with immunogenicity in breast cancers associated with germline or somatic BRCA1/2 alterations through genetic/genomic and histopathologic analyses in 115 tumors from the TCGA (n=89) and Penn (n=26). In the TCGA dataset, we found that the level of homologous recombination deficiency (HRD), as measured by the copy number-based HRD score (high vs. low, dichotomized by median), correlated positively with neoantigen load in BRCA1/2 tumors (p=0.02), but unexpectedly inversely with immunogenicity as measured by cytolytic index (p=0.04) (geometric mean of PRF1 and GZMA expression) and immune ESTIMATE (p=0.002), an RNA-seq-based measure of immune cell infiltration. In prior studies, we had found that a surprisingly high percentage of BRCA1 and BRCA2 germline mutation-associated breast cancers lacked allele-specific loss of heterozygosity (LOH), 10% and 46%, respectively. We thus evaluated whether allele-specific LOH of germline BRCA2 mutations and somatic BRCA1/2 mutation clonality were drivers of the negative association between HRD level and cytolytic index/immune ESTIMATE. We found that allele-specific LOH was associated with higher HRD score (p=0.015) but lower enrichment of the Type II IFN signaling immune metagene (p=0.01), lower cytolytic index (p=0.04), and lower immune ESTIMATE score (p=0.01); tumors with clonal somatic BRCA1/2 mutations had significantly higher HRD score (p=2.4E-07) and mutational burden (p=0.05) but lower cytolytic index (p=0.003) and immune ESTIMATE scores (p=5E-05) than tumors with subclonal somatic BRCA1/2 mutations. Stratifying by both hormone receptor expression and median HRD score, we found that triple-negative breast cancers with low HRD were the most immunogenic and hormone receptor-positive tumors with high HRD the least (p=0.001). We confirmed these TCGA findings by histopathology in tumors associated with germline mutations in BRCA1/2 from Penn. We found that CD45+ leukocyte (p=0.03) and CD8+ cytotoxic T-cell infiltration (p=0.03) as well as expression of the immune effector PRF1 (p=0.048) were significantly lower with high HRD. Further, tumors with allele-specific LOH of the germline BRCA1/2 mutation had significantly lower PRF1 staining (p=0.004), lower membrane levels of the immune checkpoint protein PDL1 (p=0.05), and lower CD8+ (p=0.001), FoxP3+ (p=0.03), and CD20+ (p=0.04) immune cells, suggesting an immunosuppressive role for allele-specific LOH leading to high HRD. These data are consistent with a prior pan-TCGA analysis demonstrating that increased tumor chromosome/arm level copy number alterations suppress immunogenicity. We have found, for the first time, that genomic instability in BRCA1/2 tumors is associated with lower antitumor immune activity, and that the immunogenicity of BRCA1/2 mutation-associated breast cancers is directly related to their HRD level (driven by allele-specific LOH and clonality) and receptor status, both clinically evaluable biomarkers. These findings have immediate implications for the stratification of patients with BRCA1/2 mutation-associated breast cancer for checkpoint blockade therapy. Citation Format: Adam A. Kraya, Kara N. Maxwell, Bradley Wubbenhorst, Brandon M. Wenz, John Pluta, Andrew J. Rech, Nicole Lunceford, Amanda Barrett, Nandita Mitra, Jennifer J.D. Morrissette, Anupma Nayak, Michael Feldman, Susan M. Domchek, Robert H. Vonderheide, Katherine L. Nathanson. Homologous recombination deficiency negatively predicts for immune infiltration and antitumor immune activity in breast tumors with BRCA1/2 alterations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A22.

Author Correction: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk

A correction to this article has been published and is linked from the HTML version of this article.

Abstract A12: Analysis of tumor immunogenicity in germline BRCA1/2 mutation associated breast and ovarian cancers

Germline mutations in BRCA1 or BRCA 2 can lead to increased genomic instability and mutational burden in mutation-associated breast and ovarian cancer. Given the association of tumor neoepitope load and clinical response to immune checkpoint therapy, we used whole exome sequencing (WES) to predict missense mutational burden and potential neoepitope expression in 34 breast and 71 ovarian tumors with germline BRCA1/2 mutations (combining Penn and TCGA cohorts). Analysis of WES data found that tumors from patients with BRCA1/2 mutations had significantly higher mutational burden than non-BRCA1/2 tumors (p Citation Format: Adam Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Daniel De Sloover, Nicole Lunceford, Amanda Barrett, Jennifer D. Morrissette, Michael Feldman, Robert H. Vonderheide, Susan M. Domcheck, Katherine L. Nathanson. Analysis of tumor immunogenicity in germline BRCA1/2 mutation associated breast and ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A12.

Abstract PR05: Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response

Despite the clinical success of PD-1 based therapies in human melanoma patients, the majority of patients do not have durable clinical benefit from anti-PD-1 monotherapy. A major challenge remains identifying which patients will respond to anti-PD-1 therapy and defining the underlying reasons for successful response versus treatment failure. Pre-existing T cell infiltration and/or PD-L1 expression in tumors may predict clinical responses; however, the use of blood-based profiling to understand the immunologic mechanism of PD-1 blockade has been less explored. Here we used detailed immune profiling of peripheral blood from stage IV melanoma patients before and after pembrolizumab (pembro), and identified pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (TEX). Robust induction of Ki67 in this subset of circulating CD8 T cells post-therapy (re-invigoration) occurred in 78% of patients indicating strong, on target immunological effects of PD-1 blockade in most patients studied here. Despite this high immunological response rate, the objective clinical response rate in this cohort was less than 40%. Ki67 in CD8 T cells alone did not predict clinical outcomes and, in fact, higher systemic immune activation at baseline was associated with lower overall survival. Rather, the magnitude of re-invigoration of circulating TEX in relation to pre-treatment tumor burden correlated with clinical response. We identified a TEX re-invigoration to tumor burden ratio which could be used to predict clinical response and overall survival as early as 6 weeks post therapy. Consistent observations were found in a second independent cohort and suggest that clinical failure of PD-1 blockade in many patients may not solely be due to an inability to induce immune re-invigoration but rather, an imbalance between T cell re-invigoration and tumor burden. Thus, by focused profiling of a mechanistically relevant circulating T cell subpopulation calibrated to pre-treatment disease burden, we identify a clinically accessible predictor of response to PD-1 blockade. These findings also provide a framework for dissecting distinct types of treatment failures in melanoma and have implications for stratifying patients into additional immunotherapeutic treatment approaches. Citation Format: Alexander Huang, Michael A. Postow, Robert J. Orlowski, Rosemarie Mick, Bertram Bengsch, Sasi Manne, Wei Xu, Shannon Harmon, Matthew Adamow, Deborah Kuk, Katherine Panangeas, Cristina Carerra, Phillip Wong, Felix Quagliarello, Kristen E. Pauken, Ramin S. Herati, Suzanne McGettigan, Shawn Kothari, Sangeeth M. George, Brandon Wenz, Kurt D9Andrea, Xiaowei Xu, Ravi K. Amaravadi, Giorgos Karakousis, Lynn M. Schuchter, Katherine L. Nathanson, Jedd D. Wolchok, Tara C. Gangadhar, John Wherry. Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR05.