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5583 Background: EC and a subset of OVCA are associated with high rates of dMMR and are responsive to PD-1 blockade. It is unknown what additional biomarkers beyond dMMR may enrich for benefit in these patients (pts). Methods: This was an investigator-initiated, single-arm, phase II study. Eligible pts had recurrent EC or OVCA that met one of the following criteria: 1) dMMR, as determined by immunohistochemical loss of expression of 1+ MMR genes; 2) MSI-H, as determined by next generation sequencing (MSK-IMPACT); or 3) hypermutated, defined as 20+ non-synonymous somatic mutations. Pts received nivo 240mg IV every 2 weeks or 480mg IV every 4 weeks until toxicity or progression. The co-primary endpoints were 1) the progression-free survival (PFS) rate at 24 weeks (PFS24) and 2) the objective response rate (ORR) by RECIST v1.1. The study was designed using Simon’s two-stage design, with a sample size of 40 pts based on a promising ORR of 25% with a type I error rate of 0.025 and a type II error rate of 0.05. Overall survival (OS), PFS and duration of response (DOR) were calculated using the method of Kaplan-Meier. Adverse events (AEs) were graded per CTCAE and tabulated. Biomarker analyses on the available archival tissue were performed using multiplex immunofluorescence (mIF) labeling for CD8, PD-1, TOX, PD-1, PD-L1, and FoxP3. Quantification of immune phenotypes and interaction studies between CD8+ T cells and PD-L1+ cells was performed in HALO. Results: Between 9/2017 and 5/2021, 35 pts were enrolled; the study closed early due to slow accrual. The median duration of follow-up was 33.2 months. The median age was 64 years (range 36-87); 82% of pts were white, 54% had high grade EC, and 65% had confirmed MLH1 hypermethylation. The ORR was 57.1% (97.5% CI 39.4-100%) [37% PR, 20% CR]. The PFS24 was 62.9% and median PFS was 26.7 months (95% CI 4.9-NE). Neither median DOR nor OS was reached. OS at 1 year was 76.4% (95% CI 58.2-87.4%). The ORR in patients with MLH1 hypermethylation was 52%; 4 of 5 patients with confirmed germline MMR alterations had a response by RECIST. AEs were consistent with the reported literature. Notable treatment related AEs included Grade 4 myocarditis with associated grade 4 AV block, grade 2 extraocular paresis, grade 3 Type 1 diabetes mellitus, and grade 3 elevations in AST/ALT. On mIF analysis, PD-L1 expression did not distinguish responders from non-responders, though interaction between CD8+ T cells and PD-L1+ cells was associated with CR/PR. Increase in relative fraction of dysfunctional CD8+ T cells (characterized by CD8+TOX+PD-1+ phenotype) was also associated with CR/PR. Conclusions: Nivo is an effective and tolerable treatment option for patients with MMR-D/MSI-H or hypermutated EC or OVCA. Presence of dysfunctional CD8+ T cells in the tumors was associated with response, while expression of PD-L1 was not predictive. Clinical trial information: NCT03241745.

Cervical cancer is a socially and scientifically distinguishable disease. Its pathogenesis, sexual transmission of high-risk HPV to a metaplastic portion of the uterine cervix, makes cervical cancer preventable by safe and effective HPV vaccines commercially available since 2006. Despite this, cervical cancer remains the deadliest gynecologic cancer in the world. Regrettably, global incidence and mortality rates disproportionately affect populations where women are marginalized, where HIV infection is endemic, and where access to preventive vaccination and screening for preinvasive disease are limited. In the United States, cervical cancer incidence has gradually declined over the last 25 years, but mortality rates remain both constant and disparately higher among communities of color because of the adverse roles that racism and poverty play in outcome. Until these conditions improve and widespread prevention is possible, treatment innovations are warranted. The last standard-of-care treatment changes occurred in 1999 for locally advanced disease and in 2014 for metastatic and recurrent disease. The viral and immunologic nature of HPV-induced cervical cancer creates opportunities for both radiation and immunotherapy to improve outcomes. With the advent of T cell–directed therapy, immune checkpoint inhibition, and techniques to increase the therapeutic window of radiation treatment, an overdue wave of innovation is currently emerging in cervical cancer treatment. The purpose of this review is to describe the contemporary developmental therapeutic landscape for cervical cancer that applies to most tumors and to discuss notable rare histologic subtypes that will not be adequately addressed with these treatment innovations.

Radiation therapy exerts a tumoricidal local effect as well as both local and systemic immunomodulation. Immune checkpoint blockade has become a widely used treatment modality across cancer types with a rapidly growing list of agents and US Food and Drug Administration-approved indications. Moreover, there may be synergy between radiation therapy and immune checkpoint blockade. Various strategies have been used, but the optimal sequencing of these therapies is unclear. In this review, the authors discuss the major mechanisms of available immune checkpoint inhibitors and explore the available preclinical and clinical evidence regarding treatment sequencing. They also review safety considerations and conclude with possible future directions.

OBJECTIVE: To characterize and compare the molecular subtypes and profiles of prospectively-accrued newly-diagnosed early- and advanced-stage endometrial cancers (ECs). METHODS: EC patients consented to an IRB-approved protocol of massively parallel sequencing of 410–468 cancer-related genes; 175 ECs of 7 histologic types (n=135 FIGO stages I/II, n=40 FIGO stages III/IV) were included. Previously reported sequencing data from 99 additional advanced-stage ECs were retrieved for comparisons. RESULTS: Irrespective of histologic type, all 175 ECs could be stratified into the molecular subtypes, with 75 (43%) being of p53 wild-type, 49 (28%) MMR-deficient, 39 (22%) p53 abnormal and 12 (7%) of POLE molecular subtypes. Subtype distribution, mutational and copy number profiles varied according to histologic type. In endometrioid ECs, genetic alterations varied according to histologic grade. Potential therapeutic targets, including high tumor mutational burden, ERBB2 amplification and PIK3CA hotspot mutations, were found across histologic types in 63% (n=110) of all ECs. Compared to their early-stage counterparts, advanced-stage endometrioid ECs had a significantly higher fraction of genome altered (median 0.1% vs 12%, p0.05) and PIK3CA mutations (46% vs 27%, p>0.05). Whole-genome doubling was found in advanced- but not early-stage carcinosarcomas and clear cell carcinomas. CONCLUSIONS: Our findings demonstrate the molecular heterogeneity within and across histologic types of EC and the increased genomic complexity of advanced-stage ECs. Molecular subtypes are present across EC histologic types and may help stratify EC patients for prognostic and therapeutic purposes.

Missense driver mutations in cancer are concentrated in a few hotspots

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

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.

Background Although immune checkpoint blockade revolutionized cancer therapy, response rates have been mixed in gynecological malignancies. While uterine endometrial cancer with high microsatellite instability (MSIHI) and high tumor mutational burden (TMB) respond robustly to checkpoint blockade, high-grade serous ovarian cancer (HGSOC) with low TMB respond modestly. Currently, there has been no known immune signature or T cell phenotype that predicts clinical response in gynecological tumors. Methods To dissect the immune landscape and T cell phenotypes in gynecological cancer patients receiving PD-1 blockade, we used high-dimensional cytometry (flow cytometry and mass cytometry (CyTOF)). We performed longitudinal deep immune profiling of PBMC from patients with recurrent uterine endometrial cancer receiving single-arm nivolumab, and HSGOC patients receiving neoadjuvant nivolumab plus platinum-based chemotherapy prior to debulking surgery. Results Chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab had a proliferative T cell response 2–4 weeks post PD-1 blockade, consistent with responses seen in high TMB melanoma and lung cancer. The responding Ki67+ CD8 T cell population was largely CD45RAloCD27hi or CD45RAloCD27lo and highly expressed PD1, CTLA-4, and CD39, consistent with the phenotype of exhausted T cells (TEX). These exhausted-like cells are enriched in responders, whereas early expansion Tregs are enriched in non-responders. Unlike patients with uterine endometrial cancer, patients with TMBlo ovarian cancer did not have a clear proliferative CD8 T cell response after neoadjuvant nivolumab plus chemotherapy treatment, suggesting systemic immune suppression. At baseline, ovarian cancer without recurrence have more terminally differentiated effector-like CD8 T cells, and patients with recurrence have more naive-like cells. Thus, both high and low TMB gynecological tumors have distinct immune landscapes associated with clinical response. Additionally, in MSI-H uterine endometrial cancer patients, the length of time between the prior chemotherapy and the initiation of immunotherapy was negatively correlated with T cell reinvigoration post immunotherapy and clinical response. This suggests the importance of optimize therapeutic timing to maximize the therapeutic efficacy when combining immunotherapy and chemotherapy. Conclusions Collectively, our immune profiling revealed the distinct immune signatures associated with clinical response to PD-1 blockade in gynecological cancers. Our results also suggest that TMBhi inflamed versus TMBlo cold tumor microenvironment, and timing of chemo/immunotherapy could impact differentiation and functions of T cells. Ethics Approval The study was approved by MSKCC Ethics Board, approval number 17–180 and 17–182.

Background The development of oncolytic viruses for the treatment of cancer has been significantly hampered by their rapid clearance in circulation due to complement and antibody-mediated neutralization. In our recent first-in-human Phase I clinical trial, we evaluated the safety and feasibility of our approach to enhance virus delivery and improve tumor targeting by utilizing an autologous stromal vascular fraction (SVF) based cell delivery system. Patient sample analysis demonstrated that patients could be stratified based on the level of vaccinia virus amplification in vivo, as evidenced by analysis of persistent viral DNA in the blood. Methods In the current study, we evaluated the immunomodulatory potential of vaccinia virus delivered by autologous stromal vascular fraction (SVF)-derived cells and attempted to identify immunological correlates of successful vaccinia virus amplification in vivo. To this end, we performed an extensive time-course analysis of cytokines in patients‘ plasma as well as various peripheral blood immune subpopulations using Luminex multi-analyte profiling and multiparameter flow cytometry, respectively. We also analyzed the impact of this therapeutic approach on the innate and adaptive immune subpopulations, including NK cells, myeloid cells, as well as effector, regulatory and memory T cells. Results Therapy with SFV-delivered oncolytic vaccinia virus induced a coordinated activation of cytokine, T cell and NK cell responses in patients as early as 1 day after treatment, which peaked around 1-week and lasted for up to 1-month post treatment. The ability of the oncolytic virus to effectively amplify in cancer patients correlated with significant changes of multiple innate (NK) and adaptive (T cell) immunological parameters. Interestingly, patient stratification into groups with transient versus persistent viral DNA was linked to opposing and mutually exclusive patterns of robust activation of NK versus T cell responses, respectively. Our study also identified intriguing cytokine and immune subset frequency signatures present at baseline and associated with successful amplification and persistence of oncolytic vaccinia virus in vivo. Conclusions Overall, this study establishes the timeline of treatment-related immunological changes and identifies biomarkers present at baseline and potential immunological correlates associated with the persistence of virus amplification in vivo. Therefore, our findings provide new insights into the role of interpatient immunological variability and will contribute to the proper evaluation of the therapeutic potency of oncolytic virotherapy in future clinical trials.

BackgroundThere are limited treatment options for patients with metastatic or recurrent cervical cancer. Platinum-based chemotherapy plus the anti-vascular endothelial growth factor antibody bevacizumab remains the mainstay of advanced treatment. Pembrolizumab is Food and Drug Agency approved for programmed death ligand 1 (PD-L1) positive cervical cancer with a modest response rate. This is the first study to report the efficacy and safety of the PD-L1 antibody atezolizumab in combination with bevacizumab in advanced cervical cancer.MethodsWe report the results from a phase II, open-label, multicenter study (NCT02921269). Patients with advanced cervical cancer were treated with bevacizumab 15 mg/kg intravenous every 3 weeks and atezolizumab 1200 mg intravenous every 3 weeks. The primary objective was to measure the objective response rate (ORR). Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety.ResultsIn the total evaluable population (n=10), zero patients achieved an objective response as assessed by Response Evaluation Criteria In Solid Tumors (RECIST) V.1.1, resulting in a confirmed ORR of 0%. Of note, there were two patients who achieved an unconfirmed PR. The DCR by RECIST V.1.1 was 60% (0% complete response, 0% partial response, 60% stable disease). Median PFS was 2.9 months (95% CI, 1.8 to 6) and median OS was 8.9 months (95% CI, 3.4 to 21.9). Safety results were generally consistent with the known safety profile of both drugs, notably with two high-grade neurologic events.ConclusionsThe combination of bevacizumab and atezolizumab did not meet the predefined efficacy endpoint, as addition of bevacizumab to PD-L1 blockade did not appear to enhance the ORR in cervical cancer.

The clinical spectrum of coronavirus disease 2019 (COVID-19) is still not fully understood. Cancer patients are uniquely vulnerable to COVID-19, and many have been or will be infected. Although an unfortunate minority will die from the infection, most will recover. This poses a challenge in which clinicians must weigh the benefits of initiation or resumption of antineoplastic therapy against the risks that antineoplastic treatment may worsen outcomes related to COVID-19 infection. A recent study of 423 patients at our institution found that patients in active cancer treatment who develop COVID-19 infection did not fare any worse than other hospitalized patients, yet guidance as to who requires testing prior to antineoplastic therapy and when to resume therapy post–COVID-19 diagnosis remains unknown. Our institution, therefore, commissioned a task force to help create guidelines for treating oncologists using available published literature. The task force focused on the ambulatory care testing guidelines only, because all inpatients receiving antineoplastic therapy are tested for COVID-19 prior to hospital admission. The guidelines focus solely on the safety and well-being of the individual patient undergoing antineoplastic therapy and are not designed to address infection control issues.

Purpose Selinexor, a selective inhibitor of nuclear export, monotherapy causes nuclear accumulation of tumor-suppressor proteins and has anti-tumor activity in ovarian and endometrial cancers. The safety and tolerability of oral selinexor plus intravenous carboplatin and paclitaxel chemotherapy (selinexor + CP) was evaluated in this population. Patients and methods This phase I, 3 + 3 dose-escalation study assessed 4 selinexor + CP regimens. Patients in cohorts of 3, regardless of disease type, were administered 1 of 4 alternating regimens (selinexor at 30 mg/m2 or 60 mg plus CP at AUC 5 and 175 mg/m2 or 80 mg/m2, respectively) for 6–10 cycles (1 cycle = 21 days), followed by selinexor maintenance. Enrolled patients with ovarian cancer had received 1 prior platinum-based therapy. Patients with endometrial cancer were chemotherapy-naive or had received 1 prior platinum-based therapy. Response was evaluated every 9 weeks. Results Twenty-three patients were treated (5 serous ovarian cancer; 18 endometrial cancer, including 6 carcinosarcomas). The most common treatment-related adverse events (TRAEs) were thrombocytopenia (100%), leukopenia (91%), and hyperglycemia (87%). The most common grade 3/4 TRAEs were leukopenia (70%), neutropenia (70%), lymphopenia (61%), anemia (57%), and alanine transaminase increase (43%). One treatment-related dose-limiting toxicity (grade 3 syncope) occurred. Twelve patients achieved a partial response and 1 achieved a complete response. Responses to all four regimens were observed in ovarian and endometrial cancers. Conclusions Combination selinexor + CP was safe and tolerated in advanced ovarian and endometrial cancers.

PURPOSE Single-agent PD-1 blockade exhibits limited efficacy in epithelial ovarian cancer (EOC). We evaluated ipilimumab plus nivolumab compared with nivolumab alone in women with persistent or recurrent EOC. METHODS Eligibility criteria included measurable disease, 1-3 prior regimens, and platinum-free interval (PFI) < 12 months. Participants were randomly allocated to intravenous nivolumab (every 2 weeks) or induction with nivolumab plus ipilimumab for 4 doses (every 3 weeks), followed by every-2-week maintenance nivolumab for a maximum of 42 doses. The primary null hypothesis was equal probability of objective response within 6 months of random allocation in each arm. RESULTS One hundred patients were allocated to receive either nivolumab (n = 49), or nivolumab plus ipilimumab (n = 51), with PFI of < 6 months in 62%. Six (12.2%) responses occurred within 6 months in the nivolumab group and 16 (31.4%) in the nivolumab plus ipilimumab group (odds ratio, 3.28; 85% CI, 1.54 to infinity; P = .034). The median progression-free survival (PFS) was 2 and 3.9 months in the nivolumab and nivolumab plus ipilimumab groups, respectively, with a PFI-stratified hazard ratio of 0.53 (95% CI, 0.34 to 0.82); the respective hazard ratio for death was 0.79 (95% CI, 0.44 to 1.42). Grade ≥ 3 related adverse events occurred in 33% of patients in the nivolumab group and 49% in the combination group, with no treatment-related deaths. PD-L1 expression was not significantly associated with response in either treatment group. CONCLUSION Compared with nivolumab alone, the combination of nivolumab and ipilimumab in EOC resulted in superior response rate and longer, albeit limited, PFS, with toxicity of the combination regimen comparable to prior reports. Additional combination studies to enhance durability of the dual regimen are warranted.

The lectin, galectin-3 (Gal3), has been implicated in a variety of inflammatory and oncogenic processes, including tumor growth, invasion, and metastasis. The interactions of Gal3 and MUC16 represent a potential targetable pathway for the treatment of MUC16-expressing malignancies. We found that the silencing of Gal3 in MUC16-expressing breast and ovarian cancer cells in vitro inhibited tumor cell invasion and led to attenuated tumor growth in murine models. We therefore developed an inhibitory murine monoclonal anti–Gal3 carbohydrate-binding domain antibody, 14D11, which bound human and mouse Gal3 but did not bind human Galectins-1, -7, -8 or -9. Competition studies and a docking model suggest that the 14D11 antibody competes with lactose for the carbohydrate binding pocket of Gal3. In MUC16-expressing cancer cells, 14D11 treatment blocked AKT and ERK1/2 phosphorylation, and led to inhibition of cancer cell Matrigel invasion. Finally, in experimental animal tumor models, 14D11 treatment led to prolongation of overall survival in animals bearing flank tumors, and retarded lung specific metastatic growth by MUC16 expressing breast cancer cells. Our results provide evidence that antibody based Gal3 blockade may be a viable therapeutic strategy in patients with MUC16-expressing tumors, supporting further development of human blocking antibodies against Gal3 as potential cancer therapeutics.

Intratumoral heterogeneity of cancer cells and tumor-infiltrating immune cells is increasingly being viewed as a key factor driving tumor progression and response to therapy. Over the past several years, technological advances have created powerful tools to analyze the tumor microenvironment on a single-cell level, including mass cytometry and single-cell RNA sequencing, which is particularly pertinent to tumor immunology and cancer immunotherapy. The integrity and reliability of the data generated from these single-cell technologies, however, are highly influenced by the process and quality of sample preparation, which, if carried out inappropriately, has a potential to produce misleading results. In this chapter, we describe a protocol for the generation of single cell suspensions from human tumor samples that has been optimized for single-cell RNA sequencing. This protocol can be easily adapted for other single cell applications such as mass and flow cytometry. Throughout the entire workflow, we aim to maximize viability and minimize factors contributing to cellular stress that could affect downstream analyses.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation. ispartof: JOURNAL FOR IMMUNOTHERAPY OF CANCER vol:8 issue:1 ispartof: location:England status: published

Intratumoral heterogeneity of cancer cells and tumor-infiltrating immune cells is increasingly being viewed as a key factor driving tumor progression and response to therapy. Over the past several years, technological advances have created powerful tools to analyze the tumor microenvironment on a single-cell level, including mass cytometry and single-cell RNA sequencing, which is particularly pertinent to tumor immunology and cancer immunotherapy. The integrity and reliability of the data generated from these single-cell technologies, however, are highly influenced by the process and quality of sample preparation, which, if carried out inappropriately, has a potential to produce misleading results. In this chapter, we describe a protocol for the generation of single cell suspensions from human tumor samples that has been optimized for single-cell RNA sequencing. This protocol can be easily adapted for other single cell applications such as mass and flow cytometry. Throughout the entire workflow, we aim to maximize viability and minimize factors contributing to cellular stress that could affect downstream analyses.

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic–phenotypic correlation in ovarian neoplasms., Little is known about the genetics of sclerosing stromal tumor of the ovary, a rare type of sex cord-stromal tumor. Here, the authors use sequencing strategies to show that in a cohort of 26 tumor samples 65% carry a FHL2-GLI2 fusion gene and demonstrate in vitro that the fusion gene has oncogenic properties.

Intratumoral therapy with oncolytic viruses is increasingly being explored as a strategy to potentiate an immune response against cancer, but it remains unknown whether such therapy should be restricted to cancers sensitive to virus-mediated lysis. Using Newcastle Disease Virus (NDV) as a model, we explore immunogenic potential of an oncolytic virus in bladder cancer, where existing immunotherapy with PD-1 and PD-L1-targeting antibodies to date has shown suboptimal response rates. Infection of human and mouse bladder cancer cells with NDV resulted in immunogenic cell death, activation of innate immune pathways, and upregulation of MHC and PD-L1 in all tested cell lines, including the cell lines completely resistant to NDV-mediated lysis. In a bilateral flank NDV-lysis-resistant syngeneic murine bladder cancer model, intratumoral therapy with NDV led to an increase of immune infiltration in both treated and distant tumors and a shift from an inhibitory to effector T cell phenotype. Consequently, combination of intratumoral NDV with systemic PD-1 or CTLA-4 blockade led to improved local and abscopal tumor control and overall survival. These findings encourage future clinical trials combining intratumoral NDV therapy with systemic immunomodulatory agents and underscore the rationale for such treatments irrespective of tumor cell sensitivity to NDV-mediated lysis.

Anti-viral immunity presents a major hurdle for systemically administered oncolytic viruses (OV). Intratumoral OV therapy has a potential to overcome this problem through activation of anti-tumor immune response, with local and abscopal effects. However, the effects of anti-viral immunity in such a setting are still not well defined. Using Newcastle Disease Virus (NDV) as a model, we explore the effects of pre-existing anti-viral immunity on therapeutic efficacy in syngeneic mouse tumor models. Unexpectedly, we find that while pre-existing immunity to NDV limits its replication in tumors, tumor clearance, abscopal anti-tumor immune effects, and survival are not compromised and, on the contrary, are superior in NDV-immunized mice. These findings demonstrate that pre-existing immunity to NDV may increase its therapeutic efficacy through potentiation of systemic anti-tumor immunity, which provides clinical rationale for repeated therapeutic dosing and prompts investigation of such effects with other OVs.

Adjuvant chemotherapy is recommended for patients with resected high‐risk adult granulosa cell tumors (GCT), although strong data to support this are lacking. The objective of this study was to assess the outcomes of GCT patients, with the specific focus on patients that received adjuvant chemotherapy with curative intent (stage I‐III), reported in a large national cancer registry. Data from the Surveillance, Epidemiology, and End Results (SEER) database between 2000 and 2013 were used for analysis. Patient and disease characteristics were extracted and analyzed for association with administration of chemotherapy. Impact on disease‐specific survival (DSS) was analyzed using log‐rank test. A total of 739 patients with surgically treated adult GCT were identified. Median age was 51 years. 570 (77%) patients were stage I, 87 (12%) were stage II, and 82 (11%) were stage III. Adjuvant chemotherapy was administered to 176 (24%) patients. Young age, higher stage, and hysterectomy were associated with chemotherapy administration. Higher disease stage was associated with decreased five‐year DSS (IA/B 98.5%, IC 95.1%, II 86.1%, III 83.5%, P

Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in tumor cells. However, the efficacy of OVs as single agents remains limited. We introduce a strategy that boosts the therapeutic efficacy of OVs by combining their activity with immuno-modulating, small molecule protein tyrosine phosphatase inhibitors. We report that vanadium-based phosphatase inhibitors enhance OV infection in vitro and ex vivo, in resistant tumor cell lines. Furthermore, vanadium compounds increase antitumor efficacy in combination with OV in several syngeneic tumor models, leading to systemic and durable responses, even in models otherwise refractory to OV and drug alone. Mechanistically, this involves subverting the antiviral type I IFN response toward a death-inducing and pro-inflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced antitumor immune stimulation. Overall, we showcase a new ability of vanadium compounds to simultaneously maximize viral oncolysis and systemic anticancer immunity, offering new avenues for the development of improved immunotherapy strategies.

Objective Since the approval of pembrolizumab for advanced or recurrent PD-L1 positive (CPS > 1%) cervical cancer, the clinical characteristics associated with response have remained undefined. We sought to characterize the clinicopathologic features of patients with advanced cervical cancer at our institution who derived durable clinical benefit from treatment with pembrolizumab. Methods We conducted a retrospective cohort study of 14 patients with recurrent or metastatic cervical cancer who received pembrolizumab monotherapy from August 2017 to November 2019 and were followed until November 1, 2020. Reviewed clinical data included age, histology, tumor molecular profiling results, stage at diagnosis, treatment history, baseline pattern of metastatic disease at initiation of anti-PD-1 therapy, and outcomes. Treatment response was evaluated by computed tomography using RECIST v1.1 criteria. Results The objective response rate was 21% (n = 3), including two partial responses and one complete response. Two patients (14%) had stable disease of six months or greater, for an observed durable clinical benefit rate of 36%. When stratified by those who derived clinical benefit, metastatic spread to lung and/or lymph node only at baseline was associated with improved response to pembrolizumab (n = 7, p = 0.02) and associated with significantly improved PFS and OS. Tumor mutational burden was higher in those with durable clinical benefit compared to non-responders (median 12.7 vs. 3.5 mutations/megabase, p = 0.03). Conclusions Our findings highlight clinical features that may select for a population most likely to benefit from pembrolizumab monotherapy and underscores the need for identification of additional biomarkers of response.

Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade., Oncolytic viruses induce a variety of immune targets in the infected tumours. Here, the authors show that Newcastle Disease Virus (NDV) upregulates the inducible co-stimulator (ICOS) on T cells and that intratumoral targeting of ICOS with engineered NDV in combination with CTLA-4 blockade induces systemic anti-tumour immunity in mice.

PURPOSE Adrenocortical carcinomas (ACC) are rare and aggressive malignancies with limited treatment options. This study was undertaken to evaluate the immunogenicity of ACC. PATIENTS AND METHODS Patients with advanced ACC were enrolled in a phase II study to evaluate the clinical activity of pembrolizumab 200 mg every 3 weeks, without restriction on prior therapy. The primary end point was objective response rate. Efficacy was correlated with tumor programmed death-ligand 1 expression, microsatellite-high and/or mismatch repair deficient (MSI-H/MMR-D) status, and somatic and germline genomic correlates. RESULTS We enrolled 39 patients with advanced ACC and herein report after a median follow-up of 17.8 months (range, 5.4 months to 34.7 months). The objective response rate to pembrolizumab was 23% (nine patients; 95% CI, 11% to 39%), and the disease control rate was 52% (16 patients; 95% CI, 33% to 69%). The median duration of response was not reached (lower 95% CI, 4.1 months). Two of six patients with MSI-H/MMR-D tumors responded. The other seven patients with objective responses had microsatellite stable tumors. The median progression-free survival was 2.1 months (95% CI, 2.0 months to 10.7 months), and the median overall survival was 24.9 months (95% CI, 4.2 months to not reached). Thirteen percent of patients (n = 5) had treatment-related grade 3 or 4 adverse events. Tumor programmed death-ligand 1 expression and MSI-H/MMR-D status were not associated with objective response. CONCLUSION MSI-H/MMR-D tumors, for which pembrolizumab is a standard therapy, are more common in ACC than has been recognized. In advanced ACC that is microsatellite stable, pembrolizumab provided clinically meaningful and durable antitumor activity with a manageable safety profile.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.

Newcastle disease virus (NDV) is an avian paramyxovirus that has been extensively studied as an oncolytic agent, in addition to being an economically important pathogen in the poultry industry. The establishment of a reverse genetics system for this virus has enabled the development of genetically modified recombinant NDV viruses with improved oncolytic and immunotherapeutic properties. In this chapter, we describe the materials and methods involved in the in vitro cloning and rescue of NDV expressing murine 4-1BBL as well as the in vivo evaluation of NDV expressing 4-1BBL in a B16-F10 murine melanoma model.

Objectives This was a single institution, phase II, Simon 2-stage with safety lead-in study of the oral androgen-receptor antagonist, enzalutamide, in patients with recurrent AR+ ovarian cancer with measurable disease and 1–3 prior lines of chemotherapy. The primary objective was to determine the proportion of patients surviving progression free for 6 months (PFS6) and overall response rate by RECIST 1.1 Criteria; with 7/58 responses or PFS6 of 13 being considered a positive study. Methods Following consent, archival tissue was screened for AR+ by IHC with ≥5% considered positive. Enrolled patients were treated with enzalutamide 160mg po daily until progression of disease or treatment discontinuation. A cycle was 28 days. Adverse events were graded by CTCAE V 4.0. Results Between 11/2013–7/2018 160 patients were screened and 59 patients [45 high grade serous(HGS), 14 low grade serous(LGS)] consented to treatment on the study (1 patient was replaced; efficacy cohort=58, safety cohort=59). There was 1 confirmed and 1 unconfirmed partial response (PR), PFS6 was 22% (90% CI: 15.1–100%) with PFS6 for those with HGS 19.8% (90% CI: 12.7–100%) and for LGS 38.5% (21.7%-100%). Median PFS was 3.5 months. There were no toxicities >grade 3 related to study drug. Related grade 3 toxicities occurred in 6 patients [fatigue (1), rash (2), hypertension (1), anemia (1), and transaminase elevation (1)]. Conclusions The study met its primary endpoint, with 13 patients (22%) remaining progression free at 6 months, however the response rate was low. Enzalutamide was well tolerated and may offer a good treatment option in select patients.

Frequent relapse and development of chemoresistance are major causes of poor survival in ovarian cancer. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are capable of initiation and maintenance. CSCs are typically resistant to cytotoxic chemotherapy and are a potential cause of relapse and chemoresistance. Most studies on CSCs focus on cancer cells alone while CSCs exist in a complex microenvironment in tumors. This microenvironment or CSC niche provides optimal conditions to maintain their tumor initiating potential. Hence, understanding the crosstalk between CSCs and the tumor microenvironment can potentially provide effective therapeutic targets to prevent chemoresistance and relapse. Cancer associated fibroblasts (CAFs) are a major constituent of the ovarian cancer tumor microenvironment and are highly enriched in the residual tumors following chemotherapy. Therefore, we studied the role of CAFs in promoting ovarian cancer chemoresistance and disease relapse through providing an optimal microenvironment for CSCs. CAFs isolated from ovarian cancer patient tumors were used in heterotypic 3D or 2D coculture systems with high grade serous ovarian cancer cell lines or with patient-derived ovarian cancer cells to study their effect on CSCs and chemoresistance. Matched pre- and post-chemotherapy patient tumors were used to confirm our findings. CAFs significantly increased resistance to carboplatin and enriched CSCs by increasing their symmetric division as well as dedifferentiation of bulk ovarian cancer cells. Pre-coculture with CAFs increased in vivo tumor initiation capacity of the ovarian cancer cells 10-fold. The CSC-CAF crosstalk responsible for CSC induction was found to be mediated by Wnt signaling. Inhibition of Wnt secretion by CAFs could block their effect on CSCs. Wnt5a was the most highly expressed Wnt in CAFs, which was further induced by ovarian cancer cells. CRISPR knockdown of Wnt5a in CAFs or treatment with a specific Wnt5a inhibitor abrogated the induction of CSCs by CAFs. Wnt5a was found to signal through a non-canonical Wnt pathway in the CSCs involving the coreceptor ROR2, protein kinase C (PKC), and CAMP Responsive Element Binding Protein 1 (CREB1). Inhibition of each of them prevented CSC induction and functional rescue experiments confirmed the sequence of the Wnt5a-ROR2-PKC-CREB1 axis. Treatment of mouse xenografts established by co-injection of CAFs and ovarian cancer cells, with the Wnt5a inhibitor sensitized them to carboplatin, and eliminated the CSCs in the residual tumors. Our results indicate that CAF-derived Wnt5a is instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of the mechanism of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Xue Xiao, Ji Wang, Subramanyam Dasari, Kenneth P. Nephew, Dmitriy Zamarin, Anirban K. Mitra. Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT020.

Although immune checkpoint blockade revolutionized cancer therapy, response rates have been mixed in gynecological malignancies. While uterine endometrial cancer with high microsatellite instability (MSI-HI) and high tumor mutational burden (TMB) respond robustly to checkpoint blockade, high-grade serous ovarian cancer (HGSOC) with low TMB respond modestly. Currently, there has been no known immune signature or T cell phenotype that predicts clinical response in gynecological tumors. To dissect the immune landscape and T cell phenotypes in gynecological cancer patients receiving PD-1 blockade, we used high-dimensional cytometry (flow cytometry and mass cytometry (CyTOF)). We performed longitudinal deep immune profiling of PBMC from patients with recurrent uterine endometrial cancer receiving single-arm nivolumab, and HSGOC patients receiving neoadjuvant nivolumab plus platinum-based chemotherapy prior to debulking surgery. Chemotherapy-resistant MSI-HI uterine cancer patients treated with nivolumab had a proliferative T cell response 2-4 weeks post PD-1 blockade, consistent with responses seen in high TMB melanoma and lung cancer. The responding Ki67+ CD8 T cell population was largely CD45RAloCD27hi or CD45RAloCD27lo and highly expressed PD1, CTLA-4, and CD39, consistent with the phenotype of exhausted T cells (TEX). These exhausted-like cells, especially expansion of T-bet+ subset, were enriched in responders, whereas early expansion of regulatory T cells (Treg) and enrichment of terminally differentiated effector/senescence-like T cells were observed in non-responders. Unlike patients with uterine endometrial cancer, patients with TMB-low ovarian cancer did not have a clear proliferative CD8 T cell response, but had an uncoupled proliferation of Treg, after neoadjuvant nivolumab plus chemotherapy treatment, suggesting systemic immune suppression in ovarian cancer patients. At baseline, ovarian cancer without recurrence had more terminally differentiated effector-like CD8 T cells, in contrast to TMB-high uterine cancer. Furthermore, in both cohorts, non-responders or patients with recurrence, had more naive-like cell population. Thus, both high and low TMB gynecological tumors have distinct immune landscapes associated with clinical response. Additionally, in MSI-HI uterine endometrial cancer patients, the length of time between the prior chemotherapy and the initiation of immunotherapy was negatively correlated with T cell reinvigoration and clinical response. This suggests that therapeutic timing and optimized strategies for combining chemotherapy and immunotherapy are still needed. Collectively, our immune profiling revealed the distinct immune signatures associated with clinical response to PD-1 blockade in gynecological cancers. Our results also suggest that high TMB (inflamed) versus low TMB (cold) tumor microenvironment, and timing of chemo/immunotherapy could impact differentiation and functions of T cells. Citation Format: Yuki Muroyama, Sasikanth Manne, Alexander C. Huang, Divij Mathew, Lakshmi Chilukuri, Allison R. Greenplate, Takuya Ohtani, Dmitriy Zamarin, Claire F. Friedman, E. John Wherry. Distinct immune signatures predicting clinical response to PD-1 blockade therapy in gynecological cancers revealed by high-dimensional immune profiling [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO068.

Immune checkpoint blockade therapy targeting the T cell immune inhibitory receptors (e.g. PD1 and CTLA4) has reinvigorated the field of cancer immunotherapy and demonstrated the power of harnessing the adaptive immune system to fight cancers. However, fundamental challenges and questions remain. Endometrial cancers specifically, are largely resistant to immune checkpoint blockade, while the underlying mechanisms of why T cell-mediated tumor elimination largely fails in EC are not well understood. Endometrial cancer encompasses a number of genetically-distinct diseases characterized by recurrent molecular alterations driving each disease subtype. Specifically, four major molecular subtypes have been identified, including POLE-ultramutated, MSI-high, and microsatellite stable copy-number high (serous-like) and copy number-low (endometrioid) cancers. In MSI-H endometrial cancers specifically, response rates of over 50% to single agent PD-1/PD-L1 blockade have been reported across a number of studies. Unfortunately, in non-MSI-H tumors, responses to immune checkpoint inhibitors have been rather limited, calling for novel combinations. Importantly, a trial of combination of multi-targeted tyrosine kinase inhibitor (TKI) lenvatinib with pembrolizumab (anti-PD1) demonstrated response rates of close to 40% irrespective of MMR status, which established this combination as a new standard of care treatment in metastatic endometrial cancer. Nevertheless, even with these promising findings: 1) A large proportion of patients does not benefit; 2) Predictive biomarkers and mechanisms defining immunotherapy response or resistance remain unknown; and 3) Insights into the mechanistic basis underlying the TKI/anti-PD-1 synergy are desperately needed. There is a growing appreciation that intrinsic tumor genomic features are critical in shaping the composition and phenotype of the immune microenvironment, including the recruitment, infiltration, phenotypes and functional states of tumor infiltrating lymphocytes. Understanding how these features predict response to immune checkpoint blockade will be key to development of novel biomarkers and therapeutic combinations. Citation Format: Dmitriy Zamarin. Immunology of endometrial cancers: Tumor genetics, immune recognition, and immunotherapy response [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA010.

Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors., Highlights • Tumor-infiltrating MDSCs promote accelerated outgrowth of IDO-expressing tumors. • MDSCs infiltrating IDO-expressing tumors mediate resistance to immunotherapy. • Combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. • CSF-1R blockade sensitizes tumors to the effects of immune checkpoint blockade. Our data demonstrate that therapy with CSF-1R-blocking agents offers therapeutic benefit as a single agent and potentiates the effect of immunotherapeutic agents in IDO-expressing tumors infiltrated with CSF-1R-expressing MDSCs. These findings provide important insights into basic mechanisms underlying IDO mediated immune suppression and resistance to immunotherapies. In addition, it provides a strong rationale for therapeutic combinations with CSF-1R inhibitors in tumors expressing elevated IDO and highly infiltrated with MDSCs as predictive biomarkers.

Simple Summary Newcastle disease virus (NDV) is an RNA virus belonging to the Paramyxoviridae family. In nature, NDV primarily infects birds, but poses no threat to human health. Multiple studies have demonstrated that NDV caries oncolytic potential due to its predilection for infection and replication in human cancer cells while sparing normal cells. In addition to its direct lytic effects, the virus triggers both innate and adaptive immune responses. In animal models, NDV injection into a tumor has been demonstrated to result in local inflammation and the recruitment of tumor-specific T cells, an effect that can be further potentiated through the use of viruses encoding immunomodulatory ligands and through combinations with immune checkpoint blockade. Initial clinical trials with naturally occurring NDV administered intravenously demonstrated durable responses across a number of cancer types. Clinical studies utilizing recombinant NDV in combination with immune checkpoint inhibitors are ongoing. Abstract Preclinical and clinical studies dating back to the 1950s have demonstrated that Newcastle disease virus (NDV) has oncolytic properties and can potently stimulate antitumor immune responses. NDV selectively infects, replicates within, and lyses cancer cells by exploiting defective antiviral defenses in cancer cells. Inflammation within the tumor microenvironment in response to NDV leads to the recruitment of innate and adaptive immune effector cells, presentation of tumor antigens, and induction of immune checkpoints. In animal models, intratumoral injection of NDV results in T cell infiltration of both local and distant non-injected tumors, demonstrating the potential of NDV to activate systemic adaptive antitumor immunity. The combination of intratumoral NDV with systemic immune checkpoint blockade leads to regression of both injected and distant tumors, an effect further potentiated by introduction of immunomodulatory transgenes into the viral genome. Clinical trials with naturally occurring NDV administered intravenously demonstrated durable responses across numerous cancer types. Based on these studies, further exploration of NDV is warranted, and clinical studies using recombinant NDV in combination with immune checkpoint blockade have been initiated.

BackgroundThis phase Ib study evaluated the safety, clinical activity, pharmacokinetics, and pharmacodynamics (PD) of emactuzumab (anti-colony stimulating factor 1 receptor monoclonal antibody (mAb)) in combination with selicrelumab (agonistic cluster of differentiation 40 mAb) in patients with advanced solid tumors.MethodsBoth emactuzumab and selicrelumab were administered intravenously every 3 weeks and doses were concomitantly escalated (emactuzumab: 500 to 1000 mg flat; selicrelumab: 2 to 16 mg flat). Dose escalation was conducted using the product of independent beta probabilities dose-escalation design. PD analyzes were performed on peripheral blood samples and tumor/skin biopsies at baseline and on treatment. Clinical activity was evaluated using investigator-based and Response Evaluation Criteria In Solid Tumors V.1.1-based tumor assessments.ResultsThree dose-limiting toxicities (all infusion-related reactions (IRRs)) were observed at 8, 12 and 16 mg of selicrelumab together with 1000 mg of emactuzumab. The maximum tolerated dose was not reached at the predefined top doses of emactuzumab (1000 mg) and selicrelumab (16 mg). The most common adverse events were IRRs (75.7%), fatigue (54.1%), facial edema (37.8%), and increase in aspartate aminotransferase and creatinine phosphokinase (35.1% both). PD analyzes demonstrated an increase of Ki67+-activated CD8+ T cells accompanied by a decrease of B cells and the reduction of CD14Dim CD16bright monocytes in peripheral blood. The best objective clinical response was stable disease in 40.5% of patients.ConclusionEmactuzumab in combination with selicrelumab demonstrated a manageable safety profile and evidence of PD activity but did not translate into objective clinical responses.Trialregistration numberNCT02760797.

Background: Oncolytic viruses selectively infect tumors and activate antitumor immune responses via innate and adaptive pathways, which may potentiate the efficacy of immune checkpoint inhibitors. MEDI5395 is a recombinant Newcastle disease virus incorporating a granulocyte macrophage colony-stimulating factor (GM-CSF) transgene that exhibits tumor cell-specific viral replication, stimulates PD-L1 expression, and induces tumor regression in murine and patient-derived xenograft models. Increased T cell recruitment and expression of IFNγ-inducible genes observed in vitro suggest immune activation in the tumor microenvironment (TME). Antitumor activity is enhanced by concurrent PD-L1 blockade. This clinical study is evaluating IV MEDI5395 in combination with the anti-PD-L1 antibody durvalumab in patients with advanced solid tumors. Methods: This is a Phase 1, first-in-human, multicenter, open-label trial (NCT03889275) enrolling patients with breast cancer, colorectal cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, renal cell carcinoma, non-small-cell lung cancer or melanoma. Eligibility criteria include relapsed/refractory disease or intolerance to standard therapy after ≥1 prior line of treatment for recurrent/metastatic disease, and ≥1 tumor measurable by RECIST v1.1. Exclusion criteria include rapidly progressing disease precluding a break of ≥8 weeks from systemic therapy, life expectancy ≥12 weeks by GRIm Score, uncontrolled metastatic central nervous system disease, and concomitant immunosuppressive therapy. The initial dose escalation phase will enroll up to 84 patients across all eligible tumor types. Three sequentially ascending dose levels of MEDI5395 will be evaluated. Durvalumab is administered either sequentially or concurrently with MEDI5395, for up to 2 years or until radiologically confirmed disease progression, clinical deterioration, withdrawal of consent, or unacceptable toxicity. The subsequent dose expansion phase will include 2 cohorts of ~40 patients, each enrolling a single tumor type. The tumor types, MEDI5395 dose level and schedule will be selected based on dose escalation data. On-treatment biopsies of the primary tumor or a metastatic site are required in the dose escalation phase and optional in the dose expansion phase. Primary objectives are evaluating the safety, tolerability and incidence of dose-limiting toxicities of MEDI5395, and identifying the optimal dose/schedule in combination with durvalumab. Secondary objectives include assessing MEDI5395 pharmacokinetics (viremia and GM-CSF transgene expression), pharmacodynamics in the TME, immunogenicity, and initial efficacy in combination with durvalumab. The trial is actively accruing. Citation Format: Grace K. Dy, Diwakar Davar, Evanthia Galanis, Danielle Townsley, Djuro Karanovic, Maria Schwaederle, Beth Kelly, Dmitriy Zamarin, Mitesh Borad, Kevin Harrington. A phase 1 study of IV MEDI5395, an oncolytic virus, in combination with durvalumab in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT244.

The development of oncolytic viruses for the treatment of cancer has been significantly hampered by their rapid clearance in circulation due to complement and antibody-mediated neutralization. In our recent first-in-human Phase I clinical trial, we confirmed the safety and feasibility of our approach to enhance virus delivery and improve tumor targeting by utilizing an autologous stromal vascular fraction (SVF) based cell delivery system. Patient sample analysis demonstrated that patients could be stratified based on the level of vaccinia virus amplification in vivo, as evidenced by analysis of persistent viral DNA in the blood. In the current study, we evaluated the immunomodulatory potential of vaccinia virus delivered by autologous stromal vascular fraction (SVF)-derived cells and attempted to identify immunological correlates of successful vaccinia virus amplification in vivo. To this end, we performed an extensive time-course analysis of cytokines in patients' plasma as well as various peripheral blood immune subpopulations using Luminex multi-analyte profiling and multiparameter flow cytometry, respectively. We also analyzed the impact of this therapeutic approach on the innate and adaptive immune subpopulations, including NK cells, myeloid cells, as well as effector, regulatory and memory T cells. Therapy with SFV-delivered oncolytic vaccinia virus induced a coordinated activation of cytokine, T cell and NK responses in patients as early as 1 day after treatment, which peaked around 1-week and lasted for up to 1-month post treatment. The ability of the oncolytic virus to effectively amplify in cancer patients correlated with significant changes of multiple innate (NK) and adaptive (T cell) immunological parameters. Interestingly, patient stratification into groups with transient versus persistent viral DNA was linked to opposing and mutually exclusive patterns of robust activation of NK versus T cell responses, respectively. Overall, this study establishes the timeline of treatment-related immunological changes and identifies potential immunological correlates associated with the persistence of virus amplification in vivo. Therefore, our findings provide new insights into the role of interpatient immunological variability and will contribute to the proper evaluation of the therapeutic potency of oncolytic virotherapy in future clinical trials. Citation Format: Dobrin Draganov, Antonio Santidrian, Ivelina Minev, Duong Nguyen, Dmitriy Zamarin, Francesco Marincola, Lisa Butterfield, Boris Minev. Evaluation of the potential of oncolytic vaccinia virus delivered by autologous SVF to modulate innate and adaptive immunity in patients with diverse solid and hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4473.

Within the ovarian cancer tumor microenvironment, cancer stem-like cells (CSC) interact with carcinoma associated mesenchymal stem/stromal cells (CA-MSC) through multiple secreted cytokines and growth factors. These paracrine interactions have been revealed to cause enrichment of CSC and their chemoprotection, however, it is still not known if platelet-derived growth factor (PDGF) signaling is involved in facilitating these responses. In order to probe this undiscovered bidirectional communication, we created a model of ovarian malignant ascites in the three-dimensional (3D) hanging drop heterospheroid array, with CSC and CA-MSC. We hypothesized that PDGF secretion by CA-MSC increases self-renewal, migration, epithelial to mesenchymal transition (EMT) and chemoresistance in ovarian CSC. Our results indicate that PDGF signaling in the CSC-MSC heterospheroids significantly increased stemness, metastatic potential and chemoresistance of CSC. Knockdown of PDGFB in MSC resulted in abrogation of these phenotypes in the heterospheroids. Our studies also reveal a cross-talk between PDGF and Hedgehog signaling in ovarian cancer. Overall, our data suggest that when the stromal signaling via PDGF to ovarian CSC is blocked in addition to chemotherapy pressure, the tumor cells are significantly more sensitive to chemotherapy. Our results emphasize the importance of disrupting the signals from the microenvironment to the tumor cells, in order to improve response rates. These findings may lead to the development of combination therapies targeting stromal signaling (such as PDGF and Hedgehog) that can abrogate the tumorigenic, metastatic and platinum resistant phenotypes of ovarian CSC through additional investigations.

Breakthroughs in tumor immunology have led to development of novel therapies targeting the mechanisms governing tumor resistance to the immune system, such as programmed death receptor -1 (PD-1) and its ligand PD-L1. Based on these findings, therapy with PD-1 and PD-L1 blocking antibodies has been evaluated in several trials in epithelial ovarian cancer. Unfortunately, to date these drugs have demonstrated rather limited single-agent activity, while predictors of response and resistance as well as general mechanisms of immune recognition in ovarian cancer remain poorly characterized. Ovarian cancer is clinically and biologically distinct from cancers such as lung cancer and melanoma, where immune checkpoint blockade has had more success. These differences bear direct impact on patients’ clinical experience, utility of known biomarkers, and rationale for specific therapeutic combinations. Below, we highlight some of the clinical and biologic features of epithelial ovarian cancer that present challenges for therapeutic implementation of immune checkpoint blockade and provide rationale for combination strategies that may help overcome some of the challenges. 1. Ovarian cancer patients frequently discontinue immune checkpoint blockade early due to symptomatic disease progression. Due to the unique pattern of metastatic disease, characterized by peritoneal carcinomatosis and/or ascites, ovarian cancer patients are uniquely vulnerable to symptomatic disease progression and may often be unable to tolerate early disease growth for the treatment duration required to allow time for immune response. We have identified several pretreatment clinical factors, including bulky peritoneal disease, liver metastases, and high neutrophil-to-lymphocyte ratio, to be associated with early discontinuation of therapy. Overall, it is likely that immune therapy should not be reserved as a late-line therapy for patients with refractory and bulky disease, but rather should be used earlier in patients with lower disease burden, allowing for the potential delayed benefit in the patients who can tolerate early tumor growth. 2. Combination therapies may extend the benefit of immune checkpoint blockade to a larger percentage of ovarian cancer patients. Several early studies have highlighted that combinations of PD-1/PD-L1 blockade with other agents may extend the benefit of immunotherapy to a larger percentage of ovarian cancer patients. These include combinations with chemotherapy, CTLA-4 blockade, and PARP inhibitors. However, even with these early signals of activity, biomarkers of response remain unknown. 3. Biomarkers predictive of response in other cancer types have poor predictive value in ovarian cancer. Studies in other cancers have identified the overall tumor mutational burden (TMB) and tumor PD-L1 expression to be predictive of response to immune checkpoint blockade. However, unlike melanoma and lung cancer, ovarian cancer is in general characterized by low TMB, and is driven by complex genetic alterations resulting in several prognostic mutational processes (e.g., HRD, fold-back inversions, and tandem duplications). Our analyses to date indicate that TMB is not predictive of response to immune checkpoint blockade. We also failed to find any association between the presence of deleterious germline or somatic BRCA mutations and response to immune checkpoint blockade. Lastly, reports from the largest studies of immune checkpoint blockade in ovarian cancer to date have failed to identify significant enrichment of responses in patients whose tumors exhibit positivity for PD-L1 expression. These findings once again highlight the ongoing unmet need to identify the mechanisms of response and resistance to immunotherapy and suggest that the unique biology of ovarian cancer may require testing of alternative hypotheses. 4. Tumor heterogeneity poses a challenge to development of valid biomarkers and effective immunotherapies in ovarian cancer. Epithelial ovarian cancer is subject to marked inter- and intratumoral heterogeneity with divergent tumor clonal composition in different intraperitoneal tumor sites, which has been demonstrated to be associated with marked heterogeneity in the tumor microenvironment. On the transcriptional level, even within the same patient, tumor cells isolated from multiple tumor sites exhibit marked tumor cell-intrinsic transcriptional heterogeneity, which is in turn associated with immune heterogeneity in the tumor microenvironment. This creates challenges to development of effective tissue-based biomarkers for cancer immunotherapy, as sampling of multiple tumor sites is logistically not feasible. Radiomics measures provide a potential noninvasive solution to measure tumor heterogeneity. Our recent findings indeed demonstrate that low intra- and intertumor heterogeneity are associated with durable clinical benefit from immunotherapy in ovarian cancer. 5. Novel combination strategies to overcome tumor heterogeneity may be required to make immune checkpoint blockade effective in all ovarian cancer patients. Tumor microenvironment heterogeneity thus presents a significant challenge for development of effective immunotherapies in ovarian cancer. To overcome the TME heterogeneity problem, successful immunotherapies in ovarian cancer will likely require combinations of agents that could drive T-cell infiltration into all tumors. To this end, we are exploring regional therapeutic approaches using oncolytic viruses. Using preclinical tumor models, we have demonstrated that intratumoral therapy with oncolytic viruses can drive infiltration of T cells into distant, noninfected tumors and potentiate responses to immune checkpoint blockade. Our studies have highlighted that mechanistically, intratumoral therapy with oncolytic virus acts as an “in situ” vaccine and induces/expands tumor-specific T cells that can carry out their activity on a systemic level. Based on these findings, we have initiated a trial of intraperitoneally administered oncolytic virus in combination with systemic PD-L1 blockade in patients with advanced ovarian cancer. In conclusion, single-agent immune checkpoint blockade in ovarian cancer to date has yielded poor efficacy results, while biomarkers of response are yet to be defined. Understanding of the relationship between ovarian cancer genetics, tumor heterogeneity, and anti-immune response will be key to development of novel biomarkers and therapeutic combinations. Citation Format: Dmitriy Zamarin. Immunotherapy in ovarian cancer: Challenges and novel opportunities [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA19.

BackgroundImmune checkpoint inhibitors (ICIs) to date have demonstrated limited activity in advanced ovarian cancer (OC). Folate receptor alpha (FRα) is overexpressed in the majority of OCs and presents an attractive target for a combination immunotherapy to potentially overcome resistance to ICI in OCs. The current study sought to examine clinical and immunologic responses to TPIV200, a multiepitope FRα vaccine administered with programmed death ligand 1 (PD-L1) inhibitor durvalumab in patients with advanced platinum-resistant OC.MethodsFollowing Simon two-stage phase II trial design, 27 patients were enrolled. Treatment was administered in 28-day cycles (intradermal TPIV200 and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 cycles and intravenous durvalumab for 12 cycles). Primary endpoints included overall response rate and progression-free survival at 24 weeks. Translational parameters focused on tumor microenvironment, PD-L1 and FRα expression, and peripheral vaccine-specific immune responses.ResultsTreatment was well tolerated, with related grade 3 toxicity rate of 18.5%. Increased T cell responses to the majority of peptides were observed in all patients at 6 weeks (pConclusionsCombination of TPIV200 and durvalumab was safe and elicited robust FRα-specific T cell responses in all patients. Unexpectedly durable survival in this heavily pretreated population highlights the need to investigate the impact of FRα vaccination on the OC biology post-treatment.

e15271 Background: Clinical trials with oncolytic viruses for cancer treatment have shown limited efficacy due to viruses’ rapid clearance by patients’ innate and adaptive immune systems. In a recent first-in-human clinical trial, we confirmed the safety and feasibility of our approach to enhance oncolytic vaccinia virus (OVV) delivery and improve tumor targeting by utilizing an autologous cell-based cell delivery system (auto-OVV). We also developed an allogeneic cell-based platform (SuperNova1c - SNV1c) aiming to protect and potentiate OVV’s antitumor effects in large patient populations. Methods: We evaluated the immunomodulatory potential of auto-OVV by an extensive time-course analysis of cytokines in patients’ plasma (Luminex profiling) and peripheral blood immune cells (flow cytometry). We also analyzed the ability of SNV1c to protect the OVV from antibody/complement inactivation in vitro and in vivo following intratumoral injection in various mouse tumors. The immune cell infiltrations of the injected tumors were also analyzed. Results: Therapy with auto-OVV induced a coordinated activation of cytokine, T cell and NK responses in patients as early as 1 day, peaking around 1-week and lasting for up to 1-month post treatment. Effective OVV amplification in cancer patients correlated with significant changes of multiple innate and adaptive immune parameters. Patient stratification into groups with transient versus persistent viral DNA was linked to opposing and mutually exclusive patterns of robust activation of NK versus T cell responses, respectively. SNV1c showed significantly enhanced protection of OVV in vitro and led to statistically significant tumor growth inhibition as compared to control non-treated tumors or to naked OVV-treated tumors. Importantly, local administration of SNV1c induced systemic therapeutic effects. Five days after SNV1c administration, tumor infiltrating lymphocytes from both treated and untreated tumors showed increased CD4 and CD8 T-cell infiltrations, decreased Tregs, and improved effector to Treg ratios, associated with tumor growth inhibition at both treated and untreated tumor sites. Conclusions: This study establishes the timeline of treatment-related immunological changes and identifies potential immunological correlates associated with the OVV persistence in vivo. We also demonstrate the ability of our cell-based platforms to protect and potentiate OVV by circumventing innate and adaptive immune barriers, resulting in enhanced oncolytic virotherapy.

6030 Background: Cervical Gastric-type adenocarcinoma (CGA) is a non-HPV-associated adenocarcinoma, comprising 10% of all cervical adenocarcinomas (Park et al, 2019). The optimal management approach is unclear, given that most data in advanced cervical cancer is driven by HPV-positive disease. We summarize our experience with this rare tumor type at a large cancer center. Methods: A retrospective review was performed for all women diagnosed with CGA 6/1/2002- 7/1/2019. Patients who did not follow up after a single visit were excluded. Kaplan-Meier survival analysis was performed to determine progression-free survival (PFS) and overall survival (OS) from date of diagnosis. Tumors from a subset of patients were subjected to MSK-IMPACT targeted sequencing and analysis (Zehir et al, 2017). Results: A total of 68 women were identified; 47 met inclusion criteria. The median age at diagnosis was 52 years (range 27-83). The majority of patients were white (70%), an additional 19% were Asian. The majority of patients (60%, n=28) presented with advanced disease (FIGO 2018, stage II-IV), while 40% (n=19) were Stage I. Of note, 26% (n=12) had positive pelvic lymph nodes and 13% (n=6) had ovarian metastases at time of surgical resection. For upfront treatment: 13% (n=6) had surgery alone of whom 83% had stage 1 disease, 36% (n=17) had surgery followed by adjuvant therapy, 30% (n =14) received definitive chemo-radiation (CRT). All patients with stage IV disease 15% (n=7) received chemotherapy alone. At completion of primary treatment, 19% (n=9) of patients had persistent disease. In patients who received CCRT, 65% (n=22) recurred, the majority (64%) within 12 months of completion of upfront therapy. Pelvic recurrence was the most common site (n=14, 64%). With a median follow up time of 30 months (range 1-159), the median PFS for Stage I was 34.4 months, compared to 17.5 months in patients with Stage II-IV disease (p= 0.29). Of the 24 patients that had MSK-IMPACT, the most common mutation was TP53 (n=16, 64%) followed by mutations in the RAS pathway (n=8, 33%), PIK3CA (n=3, 12.5%), STK11 (n=3, 12.5%), and ERBB2 alterations (n=2, 8.3 %). 2 (8.3%) women enrolled on a clinical trial based on their NGS results, one targeting ERBB2 and one targeting PIK3CA. Conclusions: Consistent with prior published literature, CGA is an aggressive form of cervical cancer with poor median OS in the advanced setting. With universal HPV vaccination, HPV negative cervical cancer will represent a larger percentage of newly diagnosed cancers and further research is needed to identify the optimal management approach.

TPS3171 Background: Human Papillomavirus 16 (HPV16) is linked to several cancer types; treatment options are limited for patients with HPV16+ recurrent or metastatic cancers. The generation and maintenance of the HPV16+ malignant state requires the stable expression of HPV16-specific E7 and E6 oncogenes, which can also serve as immunogenic tumor-associated antigens. HB-201 is a replication-competent live-attenuated vector based on the arenavirus LCMV encoding a non-oncogenic E7 and E6 fusion protein. In preclinical models, both intravenously (IV) and intratumorally (IT) administered HB-201 demonstrate potent immunogenicity by induction of HPV16-specific cytotoxic T cells and associated efficacy. Methods: This is a first in human, Phase I/II study of HB-201 monotherapy or in combination with PD-1 immune checkpoint inhibitor (anti-PD-1) in HPV16+ confirmed recurrent/metastatic cancers. Phase I consists of 2 treatment groups, each conducted with a 3+3 dose escalation design. Group 1 is enrolling patients with HPV16+ head and neck squamous cell carcinoma who will receive HB-201 IV only. Group 2 is enrolling HPV16+ cancer patients with a safely accessible tumor site who will receive HB-201 IT for the first dose, followed by HB-201 IV for subsequent doses (IT-IV). HB-201 will be administered every 21 days. The Phase II component will be conducted with the recommended Phase II doses (RP2Ds) defined in Phase I and will consist of 3 groups: Group A (HB-201 IV only), Group B (HB-201 IV plus anti-PD-1), and Group C (HB-201 IT-IV). Key eligibility criteria include age > 18, ECOG performance status 0-1, confirmed HPV16+ recurrent or metastatic cancer, disease progression from at least 1 systemic standard of care therapy, and measurable disease per RECIST v1.1. The Phase I primary objective is to determine RP2Ds for IV and IT HB-201. The Phase II primary objective is to assess antitumor activity. Secondary endpoints for both phases include safety, tolerability, overall survival, progression-free survival, and duration of response. Exploratory objectives include characterization of immunogenicity of HB-201 and biomarkers associated with immune or antitumor response. Enrollment to Groups 1 and 2 began in December 2019. Clinical trial information: NCT04180215 .

3017 Background: Metastasis to the peritoneal cavity is associated with end-stage disease in many cancers, including OC and CRC, both of which exhibit poor responses to checkpoint inhibitors. Locoregional treatment with oncolytic viruses may be used to improve the efficacy of checkpoint inhibitors at both treated and distant tumor sites. This study evaluates the combination of IP-administered ONCOS-102, an oncolytic adenovirus encoding for granulocyte macrophage colony stimulating factor (GMCSF), with systemic durvalumab, an anti PD-L1 antibody, in patients with peritoneal disease who have histologically confirmed OC or metastatic CRC and have failed prior standard therapies. Methods: This ongoing Phase 1/2, open-label study (NCT02963831) evaluates safety and antitumor/biologic activity of durvalumab (1500 mg IV, every 4 weeks x 12) + ONCOS-102 (IP, weekly x 6); cyclophosphamide is given pre first ONCOS-102 dose. Phase 1 uses a 3+3 design to evaluate the ONCOS-102 dose (1 or 3 x 1011 VP) to be given with durvalumab. Phase 2 evaluates the activity of the combination using Simon’s 2-stage MINIMAX design. Safety, response rate by RECIST 1.1, and immunological effects in tumors were evaluated for Phase 1; the current abstract reports on the phase 1 results. Results: Enrollment opened 7 Sep 2017; data cutoff, 1 Nov 2019. There were 17 patients treated in Phase 1: 8 CRC, 9 ovarian; 94% female; median age, 56 [37-77] years; ECOG PS0, 47%; ECOG PS1, 53%. There were no DLTs. Grade 3 treatment-related AEs included hypokalemia (n = 2); anemia, myocarditis, increased GGT, and influenza like illness (n = 1 each). There were 4 deaths due to PD. One patient had durable confirmed partial response and remains on treatment > 1 year; 4 patients had stable disease as best overall response. Two patients remained on treatment at data cutoff. Analysis of pre- and on-treatment tumor biopsies revealed changes in the tumor-infiltrating immune cells and PD-L1 expression, including an increase in tumor-infiltrating CD8 T cells in 5 of 11 evaluable patients. Conclusions: Combination of durvalumab and IP ONCOS-102 was safe, and no DLTs were observed. Preliminary analyses demonstrate evidence of biologic and clinical activity. Phase 2 enrollment is ongoing. Clinical trial information: NCT02963831 .

e18006 Background: Pembrolizumab received accelerated FDA approval for treatment of metastatic or recurrent PD-L1 positive cervical cancer in 6/2018. Since then, the clinical characteristics of patients who best respond to this immunotherapy have remained undefined. We sought to describe patients with advanced cervical cancer who derived durable clinical benefit (DCB) from pembrolizumab therapy. Methods: Patients with advanced or recurrent cervical cancer who received pembrolizumab from 8/2017-12/2019 at our institution were assessed. Reviewed data included patient demographics, treatment history, baseline pattern of metastatic disease at initiation of anti-PD-1 treatment, and outcomes. Kaplan-Meier survival analysis was performed to determine progression-free survival (PFS). Treatment response was evaluated by computed tomography using RECIST 1.1 criteria. Results: 14 patients with recurrent cervical cancer received pembrolizumab. Median age was 55 years (range, 21-76); 79% (n = 11) had squamous cell carcinoma (SCC). The 3 non-SCC histologies included endocervical adenocarcinoma, high-grade endometroid adenocarcinoma, and mesonephric adenocarcinoma. 93% (n = 13) expressed PD-L1; 1 patient whose tumor was MSI-H did not undergo PD-L1 testing. 93% (n = 13) had received prior radiation, and 86% (n = 12) had received 1 or more prior lines of chemotherapy. Median duration of treatment was 4.9 months (range, 1.4-15.2), and median follow-up was 7.9 months (range, 2.1-29.9). On RECIST review, the overall response rate was 21% (n = 3), including 2 partial responses and 1 complete response. Two patients (14%) had stable disease of ≥6 months for an observed DCB of 36%. Of the 5 patients with DCB, 4 remain on pembrolizumab, with observed antitumor activity of up to 15.4 months. On univariable analysis, a lymph node and/or lung only baseline pattern of metastasis was associated with improved response to pembrolizumab (n = 7, P= 0.02). On survival analysis, patients with lymph node and/or lung pattern of disease had a 6-month PFS rate of 86%, compared to a 6-month PFS rate of 0% in patients with visceral or multi-site disease ( P< 0.001). Conclusions: Pembrolizumab showed promising activity in our patient cohort, with a DCB rate of 33%. Patients with lymph node and/or lung only patterns of metastasis at baseline had the greatest radiologic response compared to patients with visceral or multi-site disease. Our results suggest that there may be an underlying pathophysiology of metastatic patterns that confer improved response to anti-PD-1 therapy in advanced cervical cancer.

Despite the high tumor mutational burden (TMB), immune checkpoint blockade (ICB) still fails in some microsatellite instability-high (MSI-H) tumors, suggesting the underlying T cell dysfunction. Immune profiling of PBMC from chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab identified the proliferative T cell response 2–4 weeks post PD-1 blockade. However, this “immunological response” to ICB was observed regardless of clinical response, suggesting the additional determinants of clinical outcome beyond the proliferative response or high TMB. To test the hypothesis that the gap between immunological and clinical response to ICB was related to T celI-intrinsic response to genotoxic and/or proliferative stress, a novel high dimensional cytometry platform was developed. The platform enables simultaneous analysis of T cell differentiation state and interrogation of multiple DNA damage and repair response (DDR) pathways at single cell resolution. This DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage. Application of the DDR-Immune platform to this cohort revealed that, in clinical responders, Ki67+ CD8 T cells had rapid increase of pATM, presumably in response to proliferative stress induced by PD-1 blockade. This DDR was not clearly observed in clinical non-responders. In addition, Tregs from clinical non-responders had elevated DDRs compared to responders, suggesting their greater resistance to genotoxic/proliferative stress. Collectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome.