Citations

Abstract Background: Cellular immunotherapy has transformed cancer treatment, but success in solid tumors remains limited by long manufacturing times, high costs, and the need for viral vectors that permanently modify DNA. To address this, we examined electroporation (EP) and mechanoporation (MP) approach to transfect T cells with mRNA to enable the development of a bedside immune cell editing platform. NKG7, natural killer cell granule-7, was previously demonstrated to play key function in activating T-cell cytotoxicity function. mRNA transfection of T cells with NKG-7 can increase these cells’ anti-tumor cell killing. We examine EP and MP methods for NKG7 transfection in primary peripheral blood mononuclear cells (PBMCs) from healthy donors. Method: Synthetic Clean Cap mRNAs encoding NKG7 or NKG7 with membrane-bound cRNA IL-2 were introduced into PBMCs by electroporation (MaxCyte) or mechanoporation (Portal Bio). T cell expression of NKG7 and IL-2 were analyzed by CytoFlex flow and Kaluza. Result: The yield at EP and MP were comparable at 0.615+/-0.09% and 0.597+/-0.16%, respectively. Viability were high at above 80% for 3 days after EP and MP. NKG7 and NKG7+cIL-2 mRNA transfection led to different NKG7 expression kinetics in T-cells and between EP and MP. With EP, CD4 T-cells showed an increase in NKG7 expressions with the highest MFI ratio (NKG7 MFI/ IgG MFI) compared to mock EP at Day 1 for the NKG7 -only EP (1.25 ± 0.38) and at Day 2 for NKG7+cIL-2 EP (1.20 ± 0.27). CD8+ T cells displayed a similar trend, with peak NKG7 expression at Day 1 for NKG7 EP (1.22 ± 0.76) and Day 2 for NKG7+cIL-2 EP (1.21 ± 0.90). However, MP did not enhance NKG7 expression in CD4+ T cells. NKG7 expression peaked at Day 1 for both NKG7 (1.28 ± 0.42) and NKG7 + cIL-2 (1.24 ± 0.20). IL2 expression peaked at Day 1 for both EP and MP: EP, CD4+ T (91.5% ± 4.4), CD8+ T (92.7% ± 12.8); MP, CD4+ T (88.9% ± 31.7), CD8+ T (84.7% ± 9.6). Functional assays for anti-tumor cell killing of these transfected cells are currently ongoing. Conclusions: We report that EP of PBMC can transfect all cell populations. MP may yield comparable yield, viability and NKG7 expression in CD8 T cells compared to EP, but without the need to isolate CD8 T cells from PBMC. EP and MP method can be used for bedside immune cell transfection depending on the target and cell populations of interest. Citation Format: Chen Wu, Christoph Schaefers, Malvika Gupta, Kevin Regan, Zuoyi Shao, Andre De Menezes Silva Corraes, Virginia Van Keulen, Jacob Hirdler, Tobias Peikert, Timothy D. Wiltshire, Haidong Dong, Yi Lin. A non-viral mRNA engineering platform for rapid, same-day immune-cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5198.

Abstract Background: Hematologic toxicity, including early and prolonged neutropenia, anemia, and thrombocytopenia, is among the most common and clinically significant complications after CD19, CD20, CD22 and BCMA-directed CAR-T therapy. Several clinical scoresmost notably CAR-HEMATOTOX (CAR-HT) and the refined ALL-HThave been developed to predict post-infusion cytopenias, but their comparative performance across diseases has not been systematically evaluated. No prior review has summarized existing models or assessed the need for AI-based predictive approaches. Methods: We conducted a systematic review of studies that developed or validated predictive models or risk scores for hematologic toxicity after CAR-T therapy in hematologic malignancies. Eligible studies included clinical, cytokine-based, or composite predictors and reported discrimination metrics such as area under the curve (AUC). Extracted variables included CAR-T product, target antigen, disease type, predictors used, model type, validation strategy, and performance statistics. Results were synthesized qualitatively due to heterogeneity in model structure and outcome definitions. Results: Studies on the CAR-HT score in B-cell lymphomas and multiple myelomas reported AUC values of 0.89 and 0.82, respectively, for predicting severe or long-term neutropenia after CD19 and BCMA-directed CAR-T therapy. Studies on the ALL-HT score (a modified/refined version of CAR-HT replacing ferritin with bone-marrow burden to improve discrimination in B-ALL) reported AUC 0.84-0.90 for predicting prolonged neutropenia and worse overall survivability (OS) after CD19/CD22 CAR-T. A multicenter study including aggressive/indolent NHL, MM/PCL, and ALL patients treated with CD19, CD20, or BCMA CAR-T reported AUC 0.87 (eIPM-Pre) and 0.88 (eIPM-Post) for predicting ICA-HT. A CD19 CAR-T study in B-ALL and LBCL reported AUC 0.85 for an early hematotoxicity nomogram using TNF- and CRP. Conclusion: Existing prediction tools for hematologic toxicity after CAR-T therapy demonstrate strong but disease-specific performance. Yet inconsistent endpoints and heterogeneous predictors limit clinical generalizability. No current model incorporates contemporary AI or machine-learning methods, underscoring a significant unmet need. Advanced predictive frameworks may allow earlier identification of patients at risk for severe or prolonged cytopenias and guide more individualized supportive care, with the potential to reduce downstream complications. Citation Format: Mansha Gupta, Abhijith Vemulapalli, Swathi Cherukuri, Rithish Nimmagadda, Akhil Jain. Predictive models for hematologic toxicity after CAR-T therapy: A systematic review of current risk scores and unmet AI needs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5202.

Abstract Annual breast cancer screening is a critical piece of women’s health. While mammography remains the gold standard, increased breast density leads to decreased sensitivity, underscoring the need for more sensitive and accessible screening methods. Liquid-based biopsies for early breast cancer detection are emerging but currently remain out of reach for clinical use. Recent data on nucleotide assessment from plasma in breast cancer have been mixed with 87% sensitivity for late-stage disease (stage 3-4) but only 20% sensitivity for early-stage disease (stage 0-2). Proteomics is an exciting area for early cancer screening where improvements in sample preparation and equipment have enabled major advances in early cancer detection. This is particularly true for a deep proteome assay as proteins can be identified which are 8-9 orders of magnitude less abundant than the most common plasma proteins. This study evaluates the presence of breast cancer using label-free shotgun mass spectrometry-based proteomics on less than 1 ml of plasma from 1,259 women classified as either healthy or newly diagnosed treatment naïve breast cancer patients with a focus on early-stage disease. We trained using a cohort of 845 women, comprising of 466 healthy and 379 with breast cancer and validated on 397 women (195 healthy and 202 breast cancer) using a protein-based machine learning classifier to distinguish these groups. All plasma samples were processed in a blinded manner coupled with semi-quantitative, label-free mass spectrometry (MS)-based analysis. The median number of proteins detected per patient across breast cancer and healthy individuals was 7,064 and 7,054, respectively. The validation performance achieved 92.3% specificity for healthy controls and an overall sensitivity of 92.6% with an AUC of 0.975 (95% CI: 0.961-0.987) for breast cancer patients. Sensitivity broken down by clinical stage (0-IV), molecular subtypes (HR+/HER2+, HR-/HER2+, HR+/HER2-, and HR-/HER2-) and pathological subtypes (LCIS, DCIS, ILC, and IDC) were ≥85% and indistinguishable across all stages and subtypes. Gene set enrichment analyses (GSEA) identified pathways including epithelial-to-mesenchymal transition (EMT) and PI3K-AKT signaling as enriched in the breast cancer samples, highlighting that our test can identify cancer-related proteins in early-stage patients. Overall, we have developed a highly sensitive blood-based assay that utilizes deep proteomic profiling to identify distinctive cancer specific signatures in women who are undergoing screening for breast cancer. This work enables us to develop a protein-based classifier from plasma for early detection of breast cancer and enhances screening strategies for women with dense breasts who are at average or high-risk based on family history, specific genetic mutations such as BRCA1/2, race, or other factors. Citation Format: Alec Horrmann, Yash Travadi, Jacob Carey, Kevin Mallery, Ella Boytim, Grant Schaap, Carissa Rungkittikhun, Kaylee Kamalanathan, Nathaniel R. Bristow, Catalina Galeano-Garces, Adam Groth, Alexa R. Hesch, Pooja Advani, Justin Hwang, Badrinath R. Konety, Justin M. Drake. Deep plasma proteomics for early-stage breast cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4073.

Abstract BACKGROUND: Tumor-specific T cells are often characterized by reduced TCR signaling due to central and peripheral tolerance pathways that limit their responsiveness. These signals are further weakened by insufficient co-stimulation and active co-inhibitory pathways, collectively establishing a high threshold for activation. The integration of antigen recognition with co-stimulatory and inhibitory cues ultimately determines whether a T cell becomes activated or remains tolerant. Early TCR signaling must therefore be precisely regulated to prevent autoreactivity while still supporting protective immunity. Although phosphorylation and ubiquitination are well-established regulators of proximal TCR signaling, additional post-translational mechanisms remain less defined. SIRT2, a cytosolic NAD+-dependent deacetylase with emerging roles in immune regulation, has not been examined in the context of TCR signaling. METHODS: We evaluated proximal TCR signaling events in wild-type and SIRT2-deficient T cells using flow cytometry, immunoblotting, calcium flux assays, and RNA-sequencing. SIRT2-associated pathways were defined by mapping its interactome and acetylated substrates through mass spectrometry and immunoprecipitation. We screened LCK post-translational modifications by mass spectrometry and assessed SIRT2 enzymatic activity using an HPLC-based deacetylase assay. Conformational effects of LCK modification were examined using fluorescence-polarization binding assays and AlphaFold structural modeling. SIRT2 was deleted in human tumor infiltrating lymphocytes (TILs) via CRISPR/Cas9, and the impact of SIRT2 targeting was tested in lung cancer patient-derived xenograft models reconstituted with autologous TILs. RESULTS: SIRT2 deficiency amplified proximal TCR signaling, leading to elevated phosphorylation of early signaling mediators and increased calcium flux in both naïve and anergic T cells. Loss of SIRT2 also altered thymic selection dynamics and expanded TCR repertoire diversity. Mechanistically, SIRT2 interacted with and deacetylated LCK, the initiating kinase of proximal TCR signaling. Mass spectrometry identified lysine K228 in the LCK linker region as a SIRT2-regulated deacetylation site that governs LCK conformation and kinase activity. Functionally, SIRT2 inhibition in exhausted mouse and human TILs restored TCR signaling capacity and improved anti-tumor responses. CONCLUSION: Here we identify SIRT2-regulated deacetylation of LCK as a previously unrecognized mechanism that sets the strength and threshold of proximal TCR signaling. Accordingly, SIRT2 targeting reverses the exhausted phenotype of tumor-reactive T cells. Citation Format: Imene Hamaidi, Pingyan Cheng, Soo Young Jun, Min-Hsuan Wang, Min Zhang, Odesha Taylor, Luis Lopez-bailon, Ismail Can, Bin Fang, Anders Berglund, Bradford Perez, Ben Creelan, Andriy Marusyk, Virginia Shapiro, Haitao Ji, Jose R. Conejo-Garcia, Sungjune Kim. Sirt2 dictates TCR activation thresholds through post-translational control of LCK conformational state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4250.

Abstract Radiation is a cornerstone of glioblastoma (GBM) treatment, however, the majority of tumors progress within the radiation field due to inherent radiotherapy (RT) resistance. The integration of highly potent radiosensitizers, such as the ATM inhibitors AZD1390 or WSD0628, could reverse resistance and significantly improve local control of these tumors. However, the achievable brain tumor exposure of these inhibitors may be limited by drug exposure in skin and mucosa, leading to enhanced radiation toxicity. To support the clinical development of WSD0628, we developed pharmacokinetic (PK)-efficacy models to help interpret ongoing PK analyses. A model based on in vitro cell studies was based on the in vitro observations that 30 nM WSD0628 provided maximum ATM inhibition and the extent of radiosensitization was directly related to the duration of drug incubation after irradiation, up to a maximum of 24 hours. The unbound WSD0628 concentration, measured by rapid equilibrium dialysis, was 66% of the total concentration in cell culture media, and was used to define an optimal radiosensitizing target of 20 nM free WSD0628 for 4 to 24 hours. Previous PK analyses showed that the free, unbound fraction (fu) of WSD0628 in plasma and brain tumor tissue are 0.02 and 0.054, respectively, and the brain tumor-to-plasma ratio is 0.32 in GBM43 patient-derived xenografts (PDXs). From these data, a total drug level of 1160 nM in plasma is predicted to achieve unbound drug levels above the 20 nM WSD0628 target unbound concentration. To validate this prediction, parallel in vivo dose-ranging studies were performed in orthotopic PDXs. Mice with orthotopic GBM43 were randomized to treatment with RT (8 Gy x1) alone or in combination with a range of WSD0628 doses. RT combined with 0.25 mg/kg and 1 mg/kg WSD0628 had no impact on survival, compared to RT alone, while median survival increased by 1.3-fold for RT combined with 2.5 mg/kg, 1.7-fold with 5 mg/kg, and 3.2-fold with 10 mg/kg WSD0628. A similar dose-response was observed in an M12 melanoma brain metastasis PDX. Based on PK modeling of these drug doses, 2.5 mg/kg dosing will maintain total WSD0628 plasma levels above 1160 nM for 11.9 hours, 5 mg/kg for 23.6 hours, and 10 mg/kg for 40 hours. Taken together, we have developed a model to predict efficacious dosing of a brain tumor based on the free drug hypothesis, which allows integration of multiple aspects of in vitro modeling with measured drug levels in animal models to predict total plasma levels associated with robust sensitizing effects. Assuming similar tissue to plasma partitioning in human GBM, this model could be directly applied to interpret the ongoing Phase 1 PK analysis of WSD0628 in recurrent GBM. Citation Format: Ann Catherine Mladek, Juhee Oh, Sneha Rathi, Lily Liu, Danielle M. Burgenske, Brett L. Carlson, Katrina K. Bakken, Lauren L. Ott, Zeng Hu, Wei Zhong, William F. Elmquist, Jann N. Sarkaria. Modeling pharmacokinetics and efficacy of the ATM inhibitor WSD0628 in GBM and melanoma metastasis PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4629.

Abstract Several allogeneic CAR T-cell therapies are in clinical trials to evaluate safety, many of which rely on CRISPR/Cas-based genome editing. However, the Cas enzyme’s random repair mechanism elevates the risk of undesired, off-target effects, posing a challenge for safe allogeneic CAR T-cell generation. To mitigate the risk of therapy-related oncogenesis, we sought to pursue an alternative safe strategy to manufacture allogeneic CAR T-cells distinct from the preexisting ones in the clinic. We have developed a CRISPR RNA (crRNA) targeting T-cell receptor beta constant (TRBC) gene, which in conjunction with AsCas12a Ultra enzyme, causes site-specific editing by eliciting a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, which mitigates off-targeting risk. Targeted amplicon sequencing was employed to evaluate off-targeting and repair mechanisms. Graft versus host alloreactivity as well as CAR T persistence was evaluated using immunocompromised mice. In vivo tumor challenge study was conducted for over 3 months. Engraftment confirmation was done using immunohistochemistry and flow cytometry. Interestingly while evaluating the edited T-cells, we sequestered a unique T-cell population that showed TCR disruption yet remained CD3-positive and did not cause in vivo alloreactivity. These CD3-Retained, Allogeneically Functioning T-cells (CRAFT-cells) showed a similar growth rate as unedited T-cells and were then used as a platform to produce CD19-targeted CAR T-cells and BAFF-R-targeted CAR T-cells. When compared to the standard CD3-disrupted allogeneic CAR T-cells, CRAFT CAR T-cells displayed an equally robust cytotoxicity while possessing a safer genomic profile. CRAFT CAR T-cells could function as effector cells for bispecific T-cell engager (BiTE) and induce CD3-dependent cytotoxicity against tumor cells. Pertinently, CRAFT CAR T-cells as opposed to CD3-disrupted CAR T-cells, were present in mouse xenografts without alloreactivity. Having the onset of MMEJ-directed repair and minimal off-targeting, positions this CRAFT crRNA to generate safer allogeneic CAR T-cells with an overall reduced genotoxic profile in a clinical setting. This unique CRAFT CAR T-cell population displays in vivo persistence. Additionally, CRAFT CAR T can be combined with BiTE therapy, offering a promising and potentially more durable alternative to preexisting ex vivo allogeneic CAR T-cell therapies. Citation Format: Tanya Hundal, Yan Luo, Yaqing Qie, Martha E. Gadd, Shaohua Guo, Mohamed A. Kharfan-Dabaja, Hong Qin. CD3-positive allogeneic CAR T-cells: A novel platform with persistence, BiTE-capture, and no alloreactivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4283.

Abstract Prostate cancer requires continual escape from immune surveillance, but mechanisms underlying this escape in the transition from androgen sensitivity to resistance and metastasis remain incompletely understood. Androgen-deprivation therapy (ADT)—the treatment backbone—induces tumor cell apoptosis but also remodels the tumor microenvironment (TME). We have assembled a single-cell RNA-seq atlas from >800,000 cells across 68 patients treated at Columbia University and Memorial Sloan Kettering. The resource includes 33 primary tumors (16 treatment-naïve, 17 post-ADT) annotated with time to PSA recurrence, and 31 metastases, including longitudinal pre- and post-ADT data in 14 castration-sensitive tumors (CSPC), 17 castration-resistant tumors (CRPC), and 4 neuroendocrine tumors (NEPC). This dataset spans the spectrum of prostate cancer disease progression.Using ARACNe and VIPER to infer activity of transcriptional regulatory and signaling proteins, we detect conserved cell state programs while minimizing batch effects. Several significant features emerged. Immune infiltration—both lymphoid and myeloid—is significantly higher in CSPC metastases than in primary or CRPC tumors and increases further after ADT. T cells constitute ∼15% of cells in primary tumors versus 30% in treatment-naive CSPC (40% post-ADT), falling to 13% in CRPC and <1% in NEPC (p<1e-32), consistent with an initially "hot" CSPC TME that progressively excludes T cells with disease progression.Among ∼350,000 epithelial/tumor cells, trajectory analysis reveals two differentiation pathways from normal epithelium. Pathway 1—dominant in primary tumors—shows androgen receptor (AR) activity with three sub-phenotypes (T1-T3): T1 exclusive to primary tumors; T2 shared by primary and CSPC; and T3 restricted to CRPC, with high ERG expression, and very strong AR activity. Pathway 2 (clusters T4-T6), present in NEPC and a subset of CRPC, lacks AR activity and displays SOX2, FOXA2, PRKD1. These aggressive states uniformly overexpress AURKA, RET, and TOP2A, suggesting druggable targets for combination therapy.We further define five cancer-associated fibroblast (CAF) subtypes. CAF1-3 track with advanced, treatment-resistant disease—enriched in NEPC versus CRPC (25% vs 18% of CAFs) and in CRPC versus CSPC (18% vs 11%)—while CAF4-5 predominate in primary tumors. We identify druggable proteins and receptor-ligand interactions linking CAF1-3 with T4-T6 tumor states; in vivo perturbations are underway.This atlas provides a resource profiling tumor and immune ecosystems across prostate cancer progression. It highlights MR programs and stromal-tumor-immune interactions that underlie immune evasion and ADT resistance, yielding actionable targets and combination-therapy hypotheses for aggressive disease. Citation Format: Aleksandar Obradovic, Kwangmin Yoo, Chang Liu, Patrick McCann, Casey R. Ager, Matthew C. Dallos, Samir Zaidi. Comprehensive profiling of primary and metastatic prostate tumors reveals distinct tumor and fibroblast cell states associated with androgen resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7419.

Abstract Background: Black men across the African Diaspora experience a disproportionate burden of prostate cancer (CaP). The Inclusive Cancer Care Research Equity (iCCaRE) Consortium aims to eliminate CaP burden through global scientific collaboration, innovative research, community-engaged interventions, and next-generation workforce development. The primary objective for this Consortium was to optimize CaP diagnosis, treatment, and survivorship through research, research training and education, community engagement and community outreach. The Consortium integrates digital health technologies, community navigation, precision communication and precision science. Methods: Five full projects and one pilot project were launched across partner institutions in North America and Africa. Supported by multiple cores and services, the research activities included (1) implementation of a Point of Prostate Cancer Diagnosis Virtual Robot Assistant (PPCD-ViRA); (3) initiation of a cancer care at home trial; (4) migrant health studies of Caribbean and Sub-Saharan African immigrants; (5) expansion of genomic and metabolomic research; and (6) digital, psychosocial, and survivorship intervention development. Training and dissemination activities were deployed through iCCaRE TV, webinars, and community engagement programs including the Global Prostate Health Dialogue (Glo-PHD). Results: In one year, accomplishments included 45 presentations, 17 publications, 16 grant submissions, and 3 awards. The consortium delivered 408 live webinar engagements and reached more than 400 men globally through Glo-PHD. PPCD-ViRA was fully developed and prepared for clinical deployment (see https://www.youtube.com/watch?v=1mI-HS5R6sk). The African cohort expansion for genomic and metabolomic profiling was activated across several countries, with biospecimen collection underway. Media dissemination generated more than 15,000 views across platforms, including high-impact educational videos (see: www.youtube.com/@iCCaREConsortium). Training achievements included 13 early-career investigators and 7 trained prostate cancer advocates. Conclusions: In just one year, the iCCaRE Consortium Phase 2 made substantial progress toward transforming CaP equity globally. Through multidisciplinary science, digital innovation, and community partnerships, the consortium is building scalable infrastructure to address diagnosis experiences, treatment quality, and survivorship among Black men. Ongoing efforts will continue to advance precision medicine, culturally grounded interventions, and global capacity building. Citation Format: Folakemi T. Odedina, Arnold Merriweather, Roxana S. Dronca, Ernest T. Kaninjing, Solomon Rotimi, Gerardo Colon-Otero, Kimlin Ashing, Ademola A. Popoola, Gladys Asiedu, Opeyemi Bolajoko, Ewan COBRAN, Emelina Asto-Flores, Floyd Willis, Christopher Williams, Monica L. Albertie, Michelle Fudge, Oluwaseyi Toye, Collisa Mahin, Jada Melton, Inclusive Cancer Care Research Equity (iCCaRE) Consortium, Prostate Cancer Transatlantic Consortium (CaPTC). Achieving prostate cancer equity globally: Multinational advances from the iCCaRE consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2382.

Abstract Our team has demonstrated that lymphatic exudate collected via surgical drains (“lymph”) is a proximal biofluid rich in ctDNA and prognostic of recurrence in HPV-independent Head and Neck Cancer (HPV- HNSCC). Other analytes such as proteins, metabolites, and cells are also abundant in lymph. To understand the liquid tumor microenvironment (TME) immediately after tumor resection, we characterized lymph at 24 hrs in HNSCC. We performed a multi-omic survey, demonstrating that lymph is distinct from plasma and represents a largely unexplored source of biomarkers relevant to immune modulation and metastasis.Metabolomics and proteomics were run on lymph supernatant and plasma. Proteomic screening was performed via Olink and normalized to internal standards for quantitative analysis. Metabolomic samples were assayed via LC-MS and normalized. 16s libraries were sequenced utilizing Illumina and analyzed in QIIME. 16s sequencing on 6 patients indicated an array of bacterial genera present in post-surgical lymph with consistency across patients. The distribution consisted primarily of microbes native to the oral cavity, suggesting post-surgical lymph may reflect the native microbiome prior to surgery. Pathogenic blooms were observed in a subset of patients. Ongoing analysis will correlate microbiome data with both cancer and surgical outcomes.Metabolite profiling was performed on post-surgical lymph and matched plasma from 4 patients, including 3 who recurred within 1 year of surgery. The metabolite profile in lymph of all 4 patients was more diverse than plasma with higher metabolite abundance per mL. 13% of identified metabolites were >2-fold enriched in lymph. Lymph from recurred patients showed high levels of saturated and monounsaturated fatty acids, reported to be associated with metastasis. Post-surgical lymph has a unique and largely uncharted metabolome to mine for biomarkers associated with recurrence.Proteomes were compared between lymph and plasma from 44 HPV-HNSCC patients. This cohort consists of 25 recurred (REC) and 19 patients with no evidence of disease (NED) with >1 year of follow up. Lymph and plasma displayed highly non-overlapping protein repertoires. The mean absolute volcano score was 25.2, indicating a strong global difference between lymph and plasma. Lymph was significantly enriched in 91 proteins (p range 4.1e-47 to 0.037), many associated with inflammation. REC lymph was also enriched in inflammatory markers compared to NED, including IL-1a, TNF, and TSLP (p = 0.002, 0.015, 0.003, respectively), which have been implicated in cancer metastasis, invasion, and angiogenesis. Lymph hits were largely non-overlapping with plasma, indicating the opportunity for novel biomarker discovery in this fluid.Analysis of the microbiome, metabolome, and proteome of post-surgical lymph reveals a rich and differentiated tumor-adjacent biofluid to study biomarkers relevant to metastasis and the TME. Citation Format: Zachary Costliow, Seka Lazare, Zhuosheng Gu, Adam Harmon, Maciej Pacula, Megan Rivera, Aadel A. Chaudhuri, Jose P. Zevallos, Jessalyn Marie Ubellacker, Wendy Winckler. Multi-omicsinpostoperative lymphatic exudate in HPV-negative head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6137.

Abstract Background: Pembrolizumab has been increasingly used off label for recurrent gliomas, yet biomarkers predicting response are poorly defined. Gliomas exhibit substantial molecular heterogeneity across Glioblastoma, IDH-wildtype (GBM), Astrocytoma, IDH-Mutant (A-IDHm), and Oligodendroglioma, 1p/19q co-deleted (OLIGO), which may influence immunotherapy efficacy. This study aimed to identify molecular predictors of progression-free survival (PFS) in recurrent glioma patients treated with pembrolizumab. Methods: Adults ≥18 years with recurrent glioma receiving ≥2 cycles of pembrolizumab between 2014 - 2024 were retrospectively identified across Mayo Clinic. Next-generation sequencing (NGS) reports were reviewed and archival tumor tissue resected prior to pembrolizumab initiation was analyzed when available. Comprehensive molecular profiling was performed using the Mayo Clinic Solid Tumor Panel which employs the Illumina Tru-Sight Oncology 500 High-Throughput NGS assay. Progression was assessed using RANO 2.0 criteria. Genomic alterations (clinically relevant sequences and/or copy-number variants) were evaluated using the Kaplan-Meier method, with differences in PFS compared using the log-rank test. Results: Thirty-three patients were included [median (range) age: 44.0 (21-76) years; 63.6% male]. The interval between tumor tissue sampling and pembrolizumab initiation was 9.9 (0.9-176.1) months, and median treatment duration was 2.8 (1.4-10.4) months. Median PFS for the overall cohort was 2.4 (0.8-15.2) months, and median overall survival from pembrolizumab initiation was 6.9 (0.9-48.2) months. Patients with OLIGO (n=8) had a longer PFS [4.6 (1.9-15.2) months] than either A-IDHm [n=11; PFS 2.1 (1.1-4.4) months] or GBM [n=14; PFS 2.3 (0.8-9.2) months]. Within OLIGO, CDKN2A/B heterozygous deletion (n=3) predicted a shorter PFS (3.9 vs 10.9 months; p=0.0462), while in A-IDHm, FANC mutation (n=2) was associated with a longer PFS (4.17 vs 1.61 months; p=0.0224). No significant associations with PFS were observed for other altered genes or pathways evaluated, including but not limited to Tumor Mutational Burden, EGFR, RB1, TP53, FUBP1, NF 1 or 2, PTEN, PDGFRA, PIK3CA, PIK3R1, CDK4, KRAS, MLH1, MLH2, MSH6 genomic alterations or CDKN2A/B homozygous deletion (all p>0.05). Conclusions: Pembrolizumab shows limited overall efficacy in recurrent gliomas, however, exploratory analyses identified several subtype-specific genomic alterations that may correlate with PFS. These hypothesis-generating findings highlight the potential influence of underlying tumor biology on immunotherapy response and warrant further validation in larger, prospective cohorts. Citation Format: Shameel Shafqat, Muhammad Asad Maqbool, Hussam Al Kateb, Terry C. Burns, Jian L. Campian, Shannon P. Fortin Ensign, Evanthia Galanis, Julie E. Hammack, Cristaine M. Ida, Mitch L. Klebig, Timothy J. Kaufmann, Autumn C. Moon, Maciej M. Mrugala, Bryan J. Neth, Alyx B. aPorter, Michael W. Ruff, Ugur T. Sener, Wendy J. Sherman, Joon H. Uhm, Rachael A. Vaubel, Sani H. Kizilbash. Molecular correlates of progression-free survival in recurrent gliomas treated with pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5253.