2564 Background: Denileukin diftitox-cxdl (E7777) is an FDA-approved direct cytotoxic agent that depletes T-regulatory cells by targeting the IL-2 receptor. Pre-clinical studies have demonstrated synergistic activity between the immunomodulator E7777 and PD-1 immune checkpoint inhibitor (ICI). Here, we report positive results from a phase I study of E7777 + pembrolizumab (P) in r/r gynecological (GYN) malignancies. Methods: Phase I dose-escalation trial of E7777 given at 4 IV dose levels (DL): 3, 6, 9, and 12 mg/kg on days 1-3, combined with P (IV 200 mg, day 1) in a 21-day cycle x8, followed by maintenance P until progression. Dose-limiting toxicities (DLTs) were assessed during cycle 1 according to CTCAE v5 criteria. Responses were measured using the RECIST 1.1 criteria. Blood samples were collected for translational studies. Results: Pt demographics included: median age 64y (43, 88); race, 88% white; histology 40% endometrial, 36% ovarian. Of the 24/25 pts evaluable for DLTs, only 1 case of reversible capillary leak syndrome (CLS) was seen at DL4. Anemia, fatigue, chills, and hypoalbuminemia were the most common AEs. A comprehensive overview of AEs, SAEs, and immune-related adverse events (irAEs) will be presented at the annual meeting. Table 1 describes the 4 DLs and their responses in the 21 pts evaluable for efficacy. An ORR of 24% (5 PRs) was reported in this heavily pretreated patient population with a mDOR of 21.1m (4.2-35.0). E7777 + P responses were seen in patients who progressed on prior ICI therapy, and across different GYN histologies. The median Progression-free survival (mPFS) was 5.8 m (1.1 – 37), with 5 pts having a PFS of ≥20 months. The clinical benefit rate (CR; PR; SD ≥ 6 m) was demonstrated in 48% of pts (n=10), who achieved a mPFS = 17.4m (6.2 – 37); patients with documented PD had a mPFS = 2.1 m (1.1 – 4.7). Conclusions: The results from this Phase I study of two immunomodulatory agents in combination, E7777 + P, in patients with r/r GYN tumors was well tolerated and demonstrated promising efficacy. A max tolerated dose wasn't established. No new significant safety signals or irAEs were reported. ORR of 24 % was demonstrated. Responders achieved a mDOR of 21.1m (4.2 - 35.0) and a mPFS = 23 m (10.4 – 37.0). Thus, E7777 + P showed prolonged responses and clinical benefit in pts with r/r GYN malignancies and limited options. These results will inform a Phase II study. Clinical trial information: NCT05200559 . Response- evaluable patients (RECIST v1.1) DL1 (N=3) DL2 (N=2) DL3 (N=6) DL4 (N=10) Total (N=21) Best response CR 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) PR 1 (33.3%) 0 (0%) 1 (16.7%) 3 (30.0%) 5 (23.8%) SD 1 (33.3%) 1 (50.0%) 2 (33.3%) 2 (20.0%) 6 (28.6%) PD 1 (33.3%) 1 (50.0%) 3 (50.0%) 5 (50.0%) 10 (47.6%) CBR w/ Durable SD (CR/PR/SD≥ 6m) 2 (66.7%) 1 (50.0%) 2 (33.3%) 5 (50.0%) 10 (47.6%) PD 1 (33.3%) 1 (50.0%) 4 (66.7%) 5 (50.0%) 11 (52.4%)

Immunotherapy is currently effective in less than half of patients with solid tumors, and most responders develop secondary progression. High infiltration of the tumor microenvironment (TME) with CD8+ cytotoxic T cells (CTLs) and low infiltration with regulatory T cells (Treg) predicts the patients’ responses to immunotherapy and long-term outcomes. To identify the mechanisms regulating long-term stability of CTL infiltration, we analyzed the impact of CTL-produced cytokines on the TME by co-culturing patient-isolated ascites cells with activated T cells. Unexpectedly, we observed that activated CTLs selectively induce cytotoxic T cell-attracting chemokines but not chemokines that attract T regulatory cells in ovarian cancer TME and tumor-associated myeloid cells, resulting in recruitment of additional CTLs without Tregs. This selectivity resulted from the unique dependence of CCL22 induction on both canonical and alternative NF-κB and the suppression of alternative NF-κB signaling by T cell-released IFNγ. Our data demonstrate that T cell-produced IFNγ suppresses alternative NF-κB signaling in TME-associated myeloid cells, allowing for the induction of CTL-attracting chemokines with the concomitant suppression of Treg-attracting CCL22. These novel functions of IFNγ and activated T cells in regulating the balance between canonical and alternative NF-κB signaling in myeloid cells provide new opportunities to enhance and stabilize the selective CTL influx in the TME.

PURPOSE:We observed that the tumor microenvironment (TME) in metastatic epithelial ovarian cancer (EOC) and in other solid tumors can reprogram normal neutrophils to acquire a complement-dependent suppressor phenotype characterized by inhibition of stimulated T cell activation. This study aims to evaluate whether serum markers of neutrophil activation and complement at diagnosis of EOC would be associated with clinical outcomes. EXPERIMENTAL DESIGN:We conducted a two-center prospective study of patients with newly diagnosed EOC (N = 188). Blood and ascites fluid were collected at diagnosis for biomarker analysis. Patients were evaluated for progression-free survival (PFS) and overall survival (OS). RESULTS:The median OS was 47 months (95 % CI: 34-58) and the median PFS was 12 months (95 % CI: 11-15). Pre-treatment serum levels of genomic DNA (gDNA), markers of neutrophil degranulation (myeloperoxidase [MPO]) and neutrophil extracellular traps (NETs) (citrullinated histone H3 [CitH3]), and complement activation (C3b/c) were each associated with worse OS in univariate analysis. In multivariate analyses controlling for age, stage, and optimal debulking, serum gDNA, MPO, and CitH3 remained associated with worse OS, while C3b/c levels were not. In an exploratory analysis, the largest magnitude of difference in 2-year OS occurred in patients with low C3b/c and low CitH3 compared to all other patients (87 % vs 46 % survival, respectively). In ascites fluid, increased factor H, a negative regulator of complement activation, was associated with improved OS in univariate analysis. CONCLUSIONS:These results point to serum gDNA, NETs, and complement activation as potential prognostic biomarkers in patients with newly diagnosed EOC.

INTRODUCTION:Several factors of template-based interstitial brachytherapy in gynecologic cancers, including large tumor size, invasion into adjacent organs or fistula, dose heterogeneity, and twice daily fractionation cause inherent dose-escalation effects, potentially increasing toxicity. This study reports a single-institutional dose escalation experience in twice daily template-based interstitial brachytherapy treatments to demonstrate tumor control and toxicity outcomes, with the hypothesis that with image-based planning dose-escalation with interstitial brachytherapy is safe and efficacious. METHODS:Patients treated with template-based interstitial brachytherapy at our institution from 2006 to 2022 were identified. Over time, HDR brachytherapy boost dose at our institution has been dose-escalated from 18.75 Gy in 5 fractions to 27.5 Gy in 5 fractions. Local control and survival outcomes were analyzed using the Kaplan-Meier method and log-rank test to compare between groups. Formal tumor control probability (TCP) analysis was performed using logistic dose-response modeling. RESULTS:214 patients were identified with median follow-up of 28.1 months (IQR 8.2-58.7). Total HDR dose correlated significantly with local and locoregional control when analyzed as a continuous variable, and when dichotomized around median dose of 25 Gy (p = 0.024). TCP analysis showed a dose-response effect between HR CTV D90 and local control in the entire cohort, and separately in cervical and vaginal cancer subsets. The actuarial 5-year incidence of grade 3 or worse toxicity was 6.1%, and there was no significant association between toxicity and total HDR dose or HR CTV D90. CONCLUSION:In patient treated with twice-daily template-based interstitial brachytherapy for gynecologic cancers brachytherapy dose correlates with local control with no significant association between brachytherapy dose and toxicity, thus suggesting room for dose-escalation.

OBJECTIVE:Neighborhood-level social determinants of health (N-SDoH) impact cancer survival. However, the relationship between N-SDoH and epithelial ovarian cancer (EOC) survival remains understudied. METHODS:We used data on all Pennsylvania residents diagnosed with EOC from 2000 to 2023 throughout the University of Pittsburgh Medical Center to assess the impact of N-SDoH on survival. We used the Social Vulnerability Index (SVI) to characterize four N-SDoH themes and overall N-SDoH vulnerability based on each case's census tract at diagnosis. High-SVI overall and by N-SDoH theme was defined as being in the 75th percentile in Pennsylvania for that metric. Cox proportional hazard models assessed the association between high-SVI and overall mortality. RESULTS:Among 4970 EOC cases, high-SVI overall was associated with later stage at diagnosis, greater residual disease, and a lower likelihood of receiving standard-of-care platinum-based therapy. High-SVI was also associated with a 13 % increased mortality hazard (adjusted-HR:1.13 95 %CI:1.02-1.25). The Household Characteristics, Racial and Ethnic Minority Status, and Housing Type and Transportation themes were also associated with increased mortality hazards (adjusted-HR[95 %CI]: 1.10[1.01-1.21], 1.23[1.08-1.39], 1.09[1.00-1.18], respectively). The Socioeconomic Status theme was associated with an increased mortality hazard of borderline significance (adjusted-HR 1.10, 95 %CI:0.99-1.23). The overall high-SVI association appeared similar when stratifying by race, although the number of Black cases was small (n = 168). CONCLUSION:Higher neighborhood social vulnerability is associated with worse EOC survival. Replicating study findings in more diverse populations can help illuminate the neighborhood factors most influencing survival and support the design and testing of programs to reduce poor EOC outcome, especially within marginalized communities.

Preclinical modeling of epithelial ovarian cancer in immune-competent mice progressing to orthotopic, spontaneous tumors is challenging, requiring multiple genetic modifications in the host. Transplantable models using cell lines are easier to implement than spontaneous animal models, given that they reproduce the key disease characteristics. To create new in vivo ovarian tumor models, we generated 28 murine ovarian cancer cell lines with distinct genetic traits, such as deletion of Trp53, activation of KrasG12D, or deletion of Pten or KrasG12D/Pten-/- combination. Two distinct Trp53 null cell lines recapitulate high-grade serous histology when orthotopically injected into immune-competent, syngeneic hosts. Cells with Pten deletion trigger high-grade endometrioid tumors, and cells with dual KrasG12D activation and Pten deletion model carcinosarcoma. The cells express different tumor antigens, secrete varying levels of cytokines and chemokines, and trigger tumors with diverse inflammation profiles and various intratumoral T- and B-lymphocyte infiltration patterns. RNA-sequencing data from 16 cell lines reveal the gene expression profile across distinct models with different histotypes. This versatile collection of murine cell lines supports translationally relevant studies in ovarian cancer.

Approximately 15% of women with epithelial ovarian cancer (EOC) have a germline BRCA1/2 pathogenic variant. Genetic testing for BRCA is recommended for all EOC patients, but not routinely performed. This study estimates the cost-effectiveness of BRCA screening with primary tumor testing versus routine germline testing. The model used literature-based probability estimates and published cost data. Effectiveness was the probability of testing completion for each strategy, providing cost per additional woman tested. A strategy was favored if it cost ≤$5000 per additional woman tested, reflecting costs of 100% receiving germline testing and 85% subsequently receiving tumor testing. In the base case, primary tumor testing costs $3057 per additional woman tested. While more costly, primary tumor testing increased efficacy 2.67-fold with an incremental cost of $1500. In sensitivity analyses, results were most sensitive to varying testing costs. Tumor testing costs ≤$5000 per additional woman tested when individually varying all parameters through clinically plausible ranges. Primary germline testing was favored in >60% of cases (base case 30%) when it occurred. In probabilistic sensitivity analysis, varying all parameters simultaneously over plausible ranges 5000 times, tumor testing cost ≤$5000 per additional woman tested in 100% of model iterations. Cost effectiveness data already support BRCA1/2 screening for EOC with clear implications for cancer prevention. On the basis of this model, primary tumor testing leads to a 2.67-fold increase in testing with an incremental cost of $1500, supporting this strategy as a cost-effective way to improve BRCA testing.

Under chronic conditions, NLRP3 inflammasome acts as instigator of pathological inflammation causing inflammation-induced carcinogenesis. Despite killing the cancer cells and being primary treatment modalities, platinum and PARPi regimen activate NLRP3 inflammasome that drives the cancer-promoting inflammatory and immunosuppressive environment and promotes inflammation-associated tumor progression ultimately contributing to resistance and therapy failure. We hypothesize that combining NLRP3 inhibitor (NLRP3i)/IL-1β inhibitor (IL-1βi) to platinum agent and PARPi can improve their anti-tumor activity by modulating inflammation associated ovarian cancer (OvCa) and overcoming resistance in pre-clinical OvCa models. The cytotoxicity of the drugs, platinum agent (cisplatin), PARPi (olaparib), NLRP3i (MCC950) and IL-1βi (AS101) as single agent and in combination against human OvCa cells (UWB1.289.BRCA1, OVCAR-3, PEO-1 and PEO-4) was analyzed by MTT assay and drug synergism was evaluated by combination index. qPCR, flow cytometry, multiplex immunoassay and immunoblot analysis were performed in human OvCa cells co-cultured with primary human blood monocytes and the human monocytic cell line (THP-1). Statistical significance was determined by two-tailed Student’s t test. p<0.05 were considered statistically significant. The drug combination showed synergistic enhanced anticancer activity by apoptosis induction and cancer cell proliferation inhibition. Platinum agent and PARPi was associated with upregulation of inflammasome downstream genes, IL-1β, IL-6 and CCL2 in OvCa cells indicating NLRP3 activation. There was a significant decrease in NLRP3, IL-1β, IL-6 and CCL2 levels in combination groups indicating that NLRP3 blockade can delay OvCa cell growth by alleviating immunosuppressive and inflammatory microenvironment. Combination treatment polarized M2 macrophages by downregulating M2-related markers, CCR2, ARG1, CD163 and CD206 towards the M1 phenotype by upregulating M1-related markers, iNOS, CD86 and CXCL10 suggesting that NLRP3 regulates OvCa cell function by modulating recruitment and polarization of tumor associated macrophages. Enhanced anti-tumor immunity was evidenced by increased IFN-β level in combination groups. Our study evaluated the effects of inflammasome modulation on anti-tumor efficacy of platinum agent and PARPi highlighting the interconnection between inflammatory mechanisms and carcinogenesis. Targeting NLRP3 inflammasome synergized and enhanced the anti-tumor activity of platinum agent and PARPi potentially by reshaping the tumor microenvironment (TME) and increasing the anti-tumor immunity and apoptosis. Overall, NLRP3 blockade can be a novel strategy for overcoming therapy resistance and for modulating TME in OvCa. Shaheen Mahira, Mackenzy Radolec, Philip Jones, Santiago Panesso, Siddharth Krishnan, Fernando Munguia, Sibel Yilmaz, Robert P. Edwards, Alexander B. Olawaiye, Haider Mahdi. NLRP3 inflammasome targeting to overcome platinum and PARPi resistance by modulating cancer related inflammation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2172.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. ONC212 is a second-generation imipridone with antitumor effects in human PDAC cell lines. ONC212 has been shown to bind to mitochondrial protease ClpP, suppress ClpX, and impair oxidative phosphorylation by decreasing ATP production. While ONC212 has been evaluated in immunocompromised mice xenografted with human pancreatic tumors, ONC212 has not been evaluated, either alone or in combination with MEK and/or immune checkpoint inhibition (ICI), in immunocompetent mice bearing notoriously aggressive KrasLSL.G12D/+; Tp53LSL.R172H/+; Pdx1Cretg/+ (KPC) murine PDAC tumors. We hypothesized that, like human PDAC cells, KPC cells would demonstrate sensitivity to ONC212 both in vitro and in vivo. Our group has previously shown that ONC212 synergizes with trametinib to induce tumor cell death in human PDAC cells. We therefore hypothesized that this combination, together with ICI, would enhance KPC tumor cell death in vivo. Methods: We determined in vitro sensitivity of the KPC cells to ONC212 alone and in combination with trametinib using CellTiter-Glo® luminescent cell viability assays. Results were analyzed after 72 hours of incubation using Compusyn and Combenefit. In vivo experiments involved C57BL/6 mice that were injected subcutaneously with 3 × 105 KPCy cells in 100 mL of PBS/matrigel. To determine the ideal ONC212 dose, we tested five different doses/dosing frequencies (50 mg/kg; 25 mg/kg; 12.5 mg/kg given by oral gavage weekly or twice weekly) in tumor-bearing (50-75 mm3) mice. Mouse weight and tumor size was measured every four days. Treatment was stopped once tumor volumes reached 3,000 mm3 or if ulceration occurred. Once the ideal dose of ONC212 was determined, a study treating KPC tumor-bearing C57BL/6 mice with ONC212, trametinib, and ICI (anti-PD-1 mAb) alone and all possible doublet/triplet combinations was performed using the same methods. Results: We found that the combination of ONC212 and trametinib exhibited synergy in the KPC cell line in vitro. Our in vivo experiments revealed that ONC212 controlled KPC tumor growth in a dose-dependent manner, however, toxicity was also noted at higher, more frequent doses. While both 25 mg/kg and 50 mg/kg twice weekly were equally effective, 25 mg/kg was better tolerated and determined to be the ideal dose. In the combination therapy study, all treatments resulted in tumor reduction, as compared to the vehicle control; however, the triple therapy group had the lowest average tumor size at day twenty. Toxicity was noted in mice receiving at least two treatments, reflected by reduced weights and mobility. Further analysis of the tumor immune microenvironment using multiplex cytokine and immunofluorescence are ongoing. Citation Format: Jasper Chan, Alexis J. Lannigan, Grace Sun, Varun V. Prabhu, Robert Edwards, Leiqing Zhang, Lanlan Zhou, Wafik S. El-Deiry, Alexander Grenander Raufi. The imipridone ONC212 cooperates with MEK and immune checkpoint inhibition to elicit in vivo regression of KPC mouse pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2078.

Abstract Background: Responsiveness or ignorance of antigens presented to naïve T cells by dendritic cells (DC) involves the CD28-driven “signal 2”, but it is unclear how activated CTLs respond to the same non-mutated tumor-associated antigens (TAAs) presented by cancer versus healthy cells to avoid autoimmunity. Since CD8+ T cells express activating NK receptors (NKRs) and NKR ligands are commonly expressed by cancer cells but not healthy tissues, we evaluated the crosstalk between NKRs and TCR during recognition of cancer cells by DC-primed TAA-specific CTLs. Methods: Associations between DNAM-1, NKG2D, CTL markers, and patients’ survival was analyzed using TCGA. In vitro sensitization was used to induce human TAA-specific CTLs by TAA-loaded DCs. The roles of DNAM-1 and NKG2D in CTL recognition and killing were tested using IFN-γ ELISpot, conjugate formation, degranulation, and cytotoxicity assays. The mechanisms and molecular effects of NKR-mediated costimulation were analyzed using IsoPlexis, calcium flux, western blot, and RNA sequencing. Double-transduction of T cells with TCR and NKR constructs or pre-treatment of cancer cells by chemotherapy was used to manipulate the levels of NKRs on T cells or their ligands on cancer cells. Results: TCGA analysis revealed that DNAM-1 and NKG2D are strongly associated with intratumoral CD8+ T cells rather than NK cells, being critical for the long-term survival of melanoma patients. Human DC-primed CTLs significantly upregulated DNAM-1 and NKG2D compared to naïve CD8+ T cells, but retained strict dependence on TCR in cancer cell recognition. Unexpectedly, blockade of DNAM-1 and NKG2D prevented the TCR-mediated CTL recognition and killing of cancer cells expressing low-TAA levels, but were redundant in the recognition of high-TAA-expressing cancer cells. DNAM-1, and to a lesser extent NKG2D, lowered TCR activation threshold and enhanced proximal TCR signaling and CTL polyfunctionality. NKR overexpression in TCR-transgenic T cells or chemotherapy-driven elevation of NKR ligands on cancer cells allowed effective recognition and killing of weakly immunogenic cancer cells. Conclusions: Reduced TCR activation threshold in the presence of NKR costimulation enables CTL activation by low levels of cognate MHC I-peptide complexes on cancer cells. Our data helps explain the ability of non-mutated “self” antigens to mediate tumor rejection in the absence of autoimmunity, and provides new tools to enhance the effectiveness of cancer therapies. Citation Format: Bowen Dong, Nataša Obermajer, Takemasa Tsuji, Junko Matsuzaki, Cindy Bonura, Henry Withers, Mark Long, Colin Chavel, Scott H. Olejniczak, Hans Minderman, Robert P. Edwards, Walter J. Storkus, Pedro Romero, Pawel Kalinski. NK receptors enable TCR-mediated CTL recognition and killing of cancer cells expressing non-mutated tumor-associated antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3966.

CTL recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells but also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM-1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes (TIL) and long-term survival of melanoma patients. Using MART-1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM-1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior costimulatory effects of DNAM-1 over CD28 involved enhanced TCR signaling, CTL killer function and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in the antigen specificity and selectivity of killer function. In addition, the elevation of NKR-Ligand expression on cancer cells by chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAA. Our data help to explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.

5555 Background: High grade serous ovarian cancer (HGSC) is a complex deadly disease. Due to the lack of symptoms at early stages, vast majority patients present at stage III/IV. Frontline chemotherapy and surgery is effective with nearly all patients experiencing a remission, though over 80% patients will recur in the first 1-2 years and ultimately die of the disease in the next few years. Approximately 20% will be long-term survivors, but no current clinical data can predict who may be a short- or long-term survivor. Clinical management, monitoring, and systemic treatment decisions could be optimally individualized to patients if able to better define the prognosis. The goal of this study is to employ artificial intelligence multi-omic machine learning models to predict survival outcomes. Methods: Clinical data and tumor specimens from University of Pittsburgh Center from 2010-2016 were analyzed. Patient data, whole exome sequencing (WES), whole transcriptome sequencing (WTS), drug response profile, and digital pathology profile were used as input feature sets for training the multi-omic machine learning (ML) models. Hypothesis-free training of the ML models was utilized to classify patient survival at 2yr and 5yr threshold. Model performance was estimated using AUROC (area under the receiver operating characteristic curve) metric, scores >0.5 having higher prediction potential. Validation of the prediction results of each ML model with top performing models meeting statistical significance threshold of α=0.01. Results: 160 HGSC model builds were completed. ML models achieved high prediction accuracy for both short-term (2y) and long-term (5y) patient cohorts, identifying 7 models with ≥ 0.7 AUROC model performance for 2yr OS threshold and 13 models with ≥ 0.7 AUROC model performance for 5yr OS threshold. Addition of multi-omic feature sets to clinical profile improved the model’s ability to predict OS for both short-term and long-term thresholds. Multi-omic feature set inputs led to superior prediction and improved performance over clinical profile information alone, and top performing multi-omic models predicted better than any feature set in isolation. Comparison of top features identified by 2y and 5y OS threshold models, we found that molecular features (WTS followed by WES feature sets) predominantly drove 2y cohort while digital pathology imaging features were the driver the 5y cohort top performing models. Conclusions: Current clinical data does not accurately predict prognosis. Utilizing multi-omic machine learning models, superior prediction of short- and long-term survivors was achieved. The specific drivers of the top performing models were different for the short- and long-term cohorts, identifying future research opportunities as well as development potential of a clinical decision tool.

We previously reported that dendritic cell (DC)-based vaccines targeting antigens expressed by tumor-associated vascular endothelial cells (VECs) and pericytes effectively control tumor growth in translational mouse tumor models. In the current report, we examined whether the therapeutic benefits of such tumor blood vessel antigen (TBVA)-targeted vaccines could be improved by the cotargeting of tumor antigens in the s.c. B16 melanoma model. We also evaluated whether combination vaccines incorporating anti-PD-L1 checkpoint blockade and/or a chemokine-modulating (CKM; IFNα + TLR3-L [rintatolimod] + Celecoxib) regimen would improve T cell infiltration/functionality in tumors yielding enhanced treatment benefits. We report that DC–peptide or DC–tumor lysate vaccines coordinately targeting melanoma antigens and TBVAs were effective in slowing B16 growth in vivo and extending survival, with superior outcomes observed for DC–peptide-based vaccines. Peptide-based vaccines that selectively target either melanoma antigens or TBVAs elicited a CD8+ T cell repertoire recognizing both tumor cells and tumor-associated VECs and pericytes in vitro, consistent with a treatment-induced epitope spreading mechanism. Notably, combination vaccines including anti-PD-L1 + CKM yielded superior therapeutic effects on tumor growth and animal survival, in association with the potentiation of polyfunctional CD8+ T cell reactivity against both tumor cells and tumor-associated vascular cells and a pro-inflammatory TME.

Objective. Elevated allostatic load (AL), an integrated, cumulative marker of physiologic damage due to socioenvironmental stress, is associated with increased mortality in patients with breast, lung, and other cancers. The relationship between allostatic load and mortality in ovarian cancer patients remains unknown. We examined the relationship between allostatic load and overall survival in ovarian cancer patients. Methods. This cross-sectional study used data from 201 patients enrolled in a prospective observational ovarian cancer cohort study at a National Cancer Institute -designated Comprehensive Cancer Center from October 2012 through June 2022. All patients underwent debulking surgery and completed a full course of standardof -care platinum -based chemotherapy. Follow-up was completed through January 2024. Allostatic load was calculated as a summary score by assigning one point to the worst sample quartile for each of ten biomarkers measured within 45 days before the ovarian cancer diagnosis. High allostatic load was de fined as having an allostatic load in the top quartile of the summary score. A Cox proportional hazard model with robust variance tested the association between allostatic load and overall survival. Results. There were no associations between allostatic load and ovarian cancer clinical characteristics. After accounting for demographic, clinical, and treatment factors, high allostatic load was associated with a signi ficant increase in mortality (hazard ratio 2.17 [95%CI, 1.13 -4.15]; P = 0.02). Conclusion. Higher allostatic load is associated with worse survival among ovarian cancer patients. Allostatic load could help identify patients at risk for poorer outcomes who may benefit from greater socioenvironmental support during treatment. (c) 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Purpose Antithrombotic therapy is considered by some as a contraindication to template-based interstitial brachytherapy (IS-BT) in patients with gynecologic cancers. IS-BT in these patients requires hospital admission with prolonged immobilization, creating competing risks of hemorrhage from treatment of the tumor versus thrombotic events from prolonged immobilization. A prospective phase II trial studied the feasibility stereotactic body radiotherapy (SBRT) as an alternative to IS-BT in patients with locally advanced cervical cancers who were not candidates for brachytherapy, including patients on antithrombotic therapy. The study was terminated early due to unacceptable toxicity and worse local control using SBRT. We retrospectively report our institutional experience treating patients on antithrombotic therapy with gynecologic cancers using template-based IS-BT to demonstrate the safety and feasibility of this approach. Materials and Methods Patients treated with template-based IS-BT for gynecologic cancers from 2007-2022 while on antithrombotic therapy were retrospectively reviewed. The peri-procedural management of antithrombotic therapy was based on patient's indication and risk. In general, antithrombotic therapy was held for the brachytherapy procedure. Those at higher risk due to active venous thromboembolism were bridged to heparin, which was held for applicator insertion and removal, but otherwise continued during hospital admission. Data regarding the patients’ clinical features, antithrombotic therapy, clinical outcomes, and toxicities were collected. Disease control, survival outcomes, and toxicities were analyzed with time-to-event analysis using Kaplan-Meier method. Results From 2007-2022, 13 patients treated with template-based IS-BT while on antithrombotic therapy were identified, and 12 had available follow-up data. Median follow-up was 14.7 months (IQR 9.5-20.3). Out of 12 patients, 11 (91.7%) were treated with chemoradiation followed by IS-BT, while 1 (8.3%) proceeded directly to IS-BT due to prior history of pelvic radiation. Four patients (33.3%) were on antiplatelet therapy and simply held their medication peri-procedurally. The remaining 8 patients (66.7%) were on full-dose anticoagulation therapy. Three patients were deemed high-risk and bridged to heparin for the duration of the procedure, 3 had an inferior vena cava filter placed allowing them to hold anticoagulation, and 2 simply held anticoagulation during the procedure. No bleeding or clotting complications occurred during admission, and no clotting complications occurred within 3 months after the procedure. Two patients had vaginal hemorrhage within a month of the procedure (one grade 1 and one grade 3). Actuarial rate of any vaginal bleeding within 3 months of the procedure was 16.7% (95% CI 0.0-37.8%). No grade 3 or worse gastrointestinal or genitourinary toxicities were reported. The actuarial rates of one-year LFFS, LRFFS, DMFS, and OS were 88.9% (95% CI 68.4-100%), 88.9% (95% CI 68.4-100%), 85.7% (95% CI 59.8-100%), and 71.3% (95% CI 47.3-98.9%). Conclusions IS-BT for gynecologic cancers on antithrombotic therapy is associated with low rate of toxicity, including low rates of bleeding and/or clotting complications during or after the procedure. Disease control and survival outcomes are comparable to those in other studies and significantly improved relative to reported SBRT experiences. Interstitial brachytherapy in patients with gynecologic cancers on antithrombotic therapy is safe and feasible. Given that SBRT for these tumors results in unacceptably high toxicities and worse local control, interstitial brachytherapy should remain the standard of care and be offered to these patients.

Purpose/Objective(s) Vulvar cancer (VC) has a heterogeneous microenvironment with complex cellular and molecular interactions, which poses a challenge to predict clinical outcomes. VC is commonly known to associated with 2 pathways: HPV dependent, and independent pathway. Little is known about the combined impact of p53 and PD-L1 expression on VC prognosis. We aimed to examine the combined impact of p53 status and PD-L1 expression on treatment outcomes for vulvar cancer. Materials/Methods A single institutional retrospective study of 90 VC patients treated from 2010 to 2021 was conducted. P53 status was evaluated for wild-type (p53wt) versus aberrant (p53a) by immunohistochemical (IHC) expression. Positive PD-L1 status (PD-L1+) was defined as a Combined Positive score of ≥ 1. The VCs were classified into a 4-category scheme based on PD-L1 and p53 status (PD-L1/p53): PD-L1+/p53a; PD-L1+/p53wt; PD-L1-/p53a; and PD-L1-/p53wt. Associations with outcomes including overall survival (OS), disease-free survival (DFS), local control (LC), and regional control (RC) were assessed via log-rank tests and multivariable Cox regression analyses. Results The median age was 72 years (IOR = 62–80 years). Most cases (n = 88) were squamous cell carcinoma, 48% of FIGO I-II, 35% were positive for p16, and close to half (44%) were PD-L1+/p53a (see Table 1). Most (74%) received as initial treatment surgery± postoperative radio± chemotherapy (S+POT), versus 26% received upfront definitive chemoradiotherapy (DCRT). Median follow-up: 38 months (IQR = 17–66). The p53a VC showed a statistically significantly worse 5-yr OS (45%, 95 CI = 27–63) than the p53wt VCs (85%, 95 CI = 72–97, P = 0.01) as with DFS, LC, and RC. We observed a significant difference in 5yr OS when patients were stratified by initial treatments received (Table 1) and with 5-yr DFS, and RC as outcomes of measures. On multivariable analysis, PD-L1-/p53a status was associated with worse OS (HR = 3.6, 95% CI = 1.1–11.3) after controlling for p16 status, age, FIGO stage, and other clinicopathologic factors. Other PD-L1/p53 status groups were not found to be independent predictors of clinical outcomes. Conclusion These results suggest that PD-L1-/p53a VCs as a subtype are prognostic for worse OS. This classification suggests the complex interaction between p53 and PD-L1 and the potential prognostic significance for oncological outcomes. The PD-L1/p53 classification could help risk-stratify VCs in routine practice and potentially guide personalized therapy.

IntroductionIntra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells.MethodsB lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry.ResultsAntibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients’ sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells.DiscussionThese studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.