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Summary Functional PET imaging using a PARP inhibitor (PARPi) analog may serve as an early, dynamic biomarker of response to PARPi therapy in high-grade serous ovarian cancer. Clinically, this could help to rapidly identify PARPi nonresponders, thereby maximizing efficacy and avoiding toxicities of futile treatments. See related article by Pantel et al., p. 1515

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN.Significance:This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer.See related commentary by Conejo-Garcia and Curiel, p. 1841.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Figure from Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer

Human papillomavirus (HPV) infection drives tumorigenesis in the majority of cervical, oropharyngeal, anal, and vulvar cancers. Genetic and epidemiologic evidence has highlighted the role of immunosuppression in the oncogenesis of HPV-related malignancies. Here we review how HPV modulates the immune microenvironment and subsequent therapeutic implications. We describe the landscape of immunotherapies for these cancers with a focus on findings from early-phase studies exploring antigen-specific treatments, and discuss future directions. Although responses across these studies have been modest to date, a deeper understanding of HPV-related tumor biology and immunology may prove instrumental for the development of more efficacious immunotherapeutic approaches.Significance:HPV modulates the microenvironment to create a protumorigenic state of immune suppression and evasion. Our understanding of these mechanisms has led to the development of immunomodulatory treatments that have shown early clinical promise in patients with HPV-related malignancies. This review summarizes our current understanding of the interactions of HPV and its microenvironment and provides insight into the progress and challenges of developing immunotherapies for HPV-related malignancies.

Supplementary data (Table S1 and Figure S1)

Supplementary Data from Microsatellite Instability–High Endometrial Cancers with MLH1 Promoter Hypermethylation Have Distinct Molecular and Clinical Profiles

Purpose:Microsatellite instability–high (MSI-H) endometrial carcinomas are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H endometrial cancers harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph).Experimental Design:Of > 1,100 patients with endometrial cancer that underwent clinical tumor-normal sequencing, 184 had MSI-H endometrial cancers due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor-infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using nonparametric tests. Log-rank tests were used for categorical associations; Kaplan–Meier method and Wald test based on Cox proportional hazards models were employed for continuous variables and survival analyses.Results:Compared with patients with germline (n = 25) and somatic (n = 39) mutations, patients with MLH1ph endometrial cancers (n = 120) were older (P < 0.001), more obese (P = 0.001) and had more advanced disease at diagnosis (P = 0.025). MLH1ph endometrial cancers were enriched for JAK1 somatic mutations as opposed to germline MMR-D endometrial cancers which showed enrichment for pathogenic ERBB2 mutations. MLH1ph endometrial cancers exhibited lower tumor mutational burden and TIL scores compared with endometrial cancers harboring germline or somatic MMR mutations (P < 0.01). MLH1ph endometrial cancer patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models, stage at diagnosis remained the only predictor of survival. For stage I/II endometrial cancer, two-year PFS was inferior for patients with MLH1ph endometrial cancers compared with germline and somatic MMR groups (70% vs. 100%, respectively).Conclusions:MLH1ph endometrial cancers likely constitute a distinct clinicopathologic entity compared with germline and somatic MMR-D ECs with potential treatment implications.

Progression-free survival and hazard ratios by histology

Purpose:Advanced-stage endometrial cancers have limited treatment options and poor prognosis, highlighting the need to understand genetic drivers of therapeutic vulnerabilities and/or prognostic predictors. We examined whether prospective molecular characterization of recurrent and metastatic disease can reveal grade and histology-specific differences, facilitating enrollment onto clinical trials.Experimental Design:We integrated prospective clinical sequencing and IHC data with detailed clinical and treatment histories for 197 tumors, profiled by MSK-IMPACT from 189 patients treated at Memorial Sloan Kettering Cancer Center.Results:Patients had advanced disease and high-grade histologies, with poor progression-free survival on first-line therapy (PFS1). When matched for histology and grade, the genomic landscape was similar to that of primary untreated disease profiled by TCGA. Using multiple complementary genomic and mutational signature-based methods, we identified patients with microsatellite instability (MSI), even when standard MMR protein IHC staining failed. Tumor and matched normal DNA sequencing identified rare pathogenic germline mutations in BRCA2 and MLH1. Clustering the pattern of DNA copy-number alterations revealed a novel subset characterized by heterozygous losses across the genome and significantly worse outcomes compared with other clusters (median PFS1 9.6 months vs. 17.0 and 17.4 months; P = 0.006). Of the 68% of patients harboring potentially actionable mutations, 27% were enrolled to matched clinical trials, of which 47% of these achieved clinical benefit.Conclusions:Prospective clinical sequencing of advanced endometrial cancer can help refine prognosis and aid treatment decision making by simultaneously detecting microsatellite status, germline predisposition syndromes, and potentially actionable mutations. A small overall proportion of all patients tested received investigational, genomically matched therapy as part of clinical trials.

Additional clinical information for patients in MSKCC cohort

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC.Significance:A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Purpose:The study goal was to determine safety, antitumor activity, and pharmacodynamic profile of mogamulizumab, an anti–C-C chemokine receptor 4 (anti-CCR4) mAb targeting effector regulatory T cells (eTreg), in combination with mAb checkpoint inhibitors durvalumab or tremelimumab.Patients and Methods:This was a multicenter, phase I, dose escalation study, followed by disease-specific cohort expansion (NCT02301130). Mogamulizumab dose escalation proceeded with concurrent dose escalation of durvalumab or tremelimumab in patients with advanced solid tumors. Cohort expansion occurred with mogamulizumab 1 mg/kg plus durvalumab 10 mg/kg or tremelimumab 10 mg/kg in patients with advanced pancreatic cancer.Results:Forty patients were enrolled during dose escalation, followed by 24 patients during dose expansion. No dose-limiting toxicities occurred during dose escalation. No new or unexpected toxicities were seen. Tolerability, the primary endpoint, was acceptable utilizing mogamulizumab 1 mg/kg plus durvalumab or tremelimumab 10 mg/kg in the combined dose escalation and dose expansion cohorts (each n = 19). At these doses, the objective response rate was 5.3% (95% confidence interval, 0.1%–26.0%; one partial response) with each combination treatment. At all doses, mogamulizumab treatment led to almost complete depletion of peripheral eTregs, as well as reduction of intratumoral Tregs in the majority of patients. There was no clear correlation of clinical response with peripheral or intratumoral reduction in CCR4+ eTregs or with baseline degree of CCR4+ expression.Conclusions:Mogamulizumab in combination with durvalumab or tremelimumab did not result in potent antitumor efficacy in patients with advanced solid tumors. Tolerability of mogamulizumab 1 mg/kg combined with durvalumab or tremelimumab 10 mg/kg was acceptable.

Supplementary Figure from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Supplementary Table and Figure Legends

Supplementary Data from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Supplementary Information

Frequent relapse and chemoresistance cause poor outcome in ovarian cancer (OC) and cancer stem cells (CSCs) are important contributors. While most studies focus exclusively on CSCs, the role of the microenvironment in providing optimal conditions to maintain their tumor-initiating potential remains poorly understood. Cancer associated fibroblasts (CAFs) are a major constituent of the OC tumor microenvironment and we show that CAFs and CSCs are enriched following chemotherapy in patient tumors. CAFs significantly increased OC cell resistance to carboplatin. Using heterotypic CAF-OC cocultures andin vivolimiting dilution assay, we confirmed that the CAFs act by enriching the CSC population. CAFs were found to increase the symmetric division of CSCs as well as the dedifferentiation of bulk OC cells into CSCs. The effect of CAFs was limited to OC cells in their immediate neighborhood, which could be prevented by inhibiting Wnt. Analysis of single cell RNA-seq data from OC patients revealed that Wnt5a as the highest expressed Wnt in CAFs and that certain subpopulations of CAFs express higher levels of Wnt5a. We found that Wnt5a from CAFs activated a noncanonical Wnt signaling pathway involving the ROR2/PKC/CERB1 axis in the neighboring CSCs. While canonical Wnt signaling was predominant in interactions between cancer cells in patients, non-canonical Wnt pathway was activated by CAF-OC crosstalk. Treatment with a Wnt5a inhibitor sensitized tumors to carboplatinin vivo. Together, our findings demonstrate a novel mechanism of CSC maintenance by signals from the microenvironmental CAFs, which can be targeted to treat OC chemoresistance and relapse.Statement of significanceCAFs serve as CSC niche through a Wnt5a mediated noncanonical Wnt signaling. Disease relapse and development of chemoresistance is a major problem in OC, which can be potentially addressed by targeting Wnt5a.

Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3–6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical ‘free fitness’ framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.

OBJECTIVE: To determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. METHODS: Normal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. RESULTS: RNA-sequencing revealed 4,231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value

Multiplexed imaging of 8 molecular targets with SERS nanoprobes in mice reveals spatially heterogeneous tumor responses to immunotherapy. Multichannel image segmentation enables classification of the tumor regions into responders and naïve.

We examined the safety and immunogenicity of sequential administration of a tetravalent, non-HLA (human leukocyte antigen) restricted, heteroclitic Wilms’ Tumor 1 (WT1) peptide vaccine (galinpepimut-S) with anti–PD-1 (programmed cell death protein 1) nivolumab. This open-label, non-randomized phase I study enrolled patients with WT1-expressing ovarian cancer in second or third remission from June 2016 to July 2017. Therapy included six (every two weeks) subcutaneous inoculations of galinpepimut-S vaccine adjuvanted with Montanide, low-dose subcutaneous sargramostim at the injection site, with intravenous nivolumab over 12 weeks, and up to six additional doses until disease progression or toxicity. One-year progression-free survival (PFS) was correlated to T-cell responses and WT1-specific immunoglobulin (Ig)G levels. Eleven patients were enrolled; seven experienced a grade 1 adverse event, and one experienced a grade ≥3 adverse event considered a dose-limiting toxicity. Ten (91%) of eleven patients had T-cell responses to WT1 peptides. Seven (88%) of eight evaluable patients had IgG against WT1 antigen and full-length protein. In evaluable patients who received >2 treatments of galinpepimut-S and nivolumab, the 1-year PFS rate was 70%. Coadministration of galinpepimut-S and nivolumab demonstrated a tolerable toxicity profile and induced immune responses, as indicated by immunophenotyping and WT1-specific IgG production. Exploratory analysis for efficacy yielded a promising 1-year PFS rate.

Single-cell T cell repertoire sequencing can pair both T cell receptor (TCR) and gene expression sequence data, providing an enriched view of T cell behavior. This powerful tool can identify and characterize specific clonotypes and phenotypes as well as track their changes in response to therapy, such as immune checkpoint blockade (ICB). We present a novel information theoretic framework called TCRi for characterizing single cell T cell repertoires by formalizing the relationship between clonotype and phenotype in a joint probability distribution. Our strategy allows for the identification of subpopulations of T cells and jointly quantifies their TCR and expression profiles in response to stimuli, in addition the framework tracks the phenotypic changes in individual T cell clones over time. We applied this framework to four datasets of T cells sequenced from cancer patients treated with anti-PD-(L)1 ICB immunotherapies and examined evolution of T cell responses pre- and post-treatment. Quantitative of phenotypic and clonotypic entropy analysis with TCRi demonstrated improvements in characterization of the transcriptional signature of clonotypes. Furthermore, TCRi highlighted the importance of phenotypic flux and specific T-cell phenotypes as determinants of therapeutic response.

Purpose: Microsatellite instability–high (MSI-H) endometrial carcinomas are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H endometrial cancers harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph). Experimental Design: Of > 1,100 patients with endometrial cancer that underwent clinical tumor-normal sequencing, 184 had MSI-H endometrial cancers due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor-infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using nonparametric tests. Log-rank tests were used for categorical associations; Kaplan–Meier method and Wald test based on Cox proportional hazards models were employed for continuous variables and survival analyses. Results: Compared with patients with germline (n = 25) and somatic (n = 39) mutations, patients with MLH1ph endometrial cancers (n = 120) were older (P < 0.001), more obese (P = 0.001) and had more advanced disease at diagnosis (P = 0.025). MLH1ph endometrial cancers were enriched for JAK1 somatic mutations as opposed to germline MMR-D endometrial cancers which showed enrichment for pathogenic ERBB2 mutations. MLH1ph endometrial cancers exhibited lower tumor mutational burden and TIL scores compared with endometrial cancers harboring germline or somatic MMR mutations (P < 0.01). MLH1ph endometrial cancer patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models, stage at diagnosis remained the only predictor of survival. For stage I/II endometrial cancer, two-year PFS was inferior for patients with MLH1ph endometrial cancers compared with germline and somatic MMR groups (70% vs. 100%, respectively). Conclusions: MLH1ph endometrial cancers likely constitute a distinct clinicopathologic entity compared with germline and somatic MMR-D ECs with potential treatment implications.

PURPOSE Despite therapeutic advances in the treatment of ovarian cancer (OC), 5-year survival remains low, and patients eventually die from recurrent, chemotherapy-resistant disease. The National Cancer Gynecologic Cancer Steering Committee identified the integration of scientifically defined subgroups as a top strategic priority in clinical trial planning. METHODS A group of experts was convened to review the scientific literature in OC to identify validated predictive biomarkers that could inform patient selection and treatment stratification. Here, we report on these findings and their potential for use in future clinical trial design on the basis of hierarchal evidence grading. RESULTS The biomarkers were classified on the basis of mechanistic targeting, including DNA repair and replication stress, immunotherapy and tumor microenvironment, oncogenic signaling, and angiogenesis. Currently, BRCA mutations and homologous recombination deficiency to predict poly (ADP-ribose) polymerase inhibitor response are supported in OC by the highest level of evidence. Additional biomarkers of response to agents targeting the pathways above have been identified but require prospective validation. CONCLUSION Although a number of biomarkers of response to various agents in OC have been described in the literature, high-level evidence for the majority is lacking. This report highlights the unmet need for identification and validation of predictive biomarkers to guide therapy and future trial design in OC.