Citations

Abstract Background: Colorectal cancer (CRC) remains a leading cause of cancer-associated mortality and is difficult to treat due to therapy resistance and limited durable responses. Esophageal cancer (EC) is similarly challenging, exhibiting highly aggressive and metastatic behavior that contribute to its poor prognosis. Adoptive TCR-engineered T cell (TCR-T) therapies show promise in these settings, but their efficacy is limited by suboptimal antigen specificity and an immunosuppressive tumor microenvironment (TME). To address these barriers, we isolated high-affinity TCRs from a phage-displayed TCR library targeting KRASG12V neoantigen in CRC and CTAG1B cancer/testis antigen (CTA) in EC. Methods: KRASG12V(8-16)/HLA-A11:01-restricted and CTAG1B(157-165)/HLA-A02:01-restricted TCRs were isolated from an optimized phage-displayed TCR library generated from healthy donor CD8+ T cells through high-throughput biopanning. Binders were screened against antigen-family peptides and irrelevant controls by phage ELISA to eliminate cross-reactivity. Binding affinities were measured by SPR. Antigen-specific TCRs (asTCRs) were cloned into lentiviral vectors and transduced into primary human T cells. TCR-T function was assessed using peptide-pulsed T2 cells and KRASG12V+ or CTAG1B+ tumor cells through LDH-release cytotoxicity and cytokine secretion assays. Normal human cells were used to evaluate off-target toxicity. In vivo antitumor activity was tested in NOD/SCID-IL2rγ-/- xenografts with histological confirmation of TCR-T infiltration. Results: Next-generation sequencing confirmed that the 109-scale library contained >90% functional genes and >80% sequence diversity. Three high-affinity TCRs were identified for each antigen (KD as low as 1.07 μM). These TCRs exhibited strict antigen specificity with no detectable cross-reactivity. TCR-T cells expressing asTCRs showed potent antigen-dependent cytotoxicity against KRASG12V+/HLA-A11:01+ colorectal and CTAG1B+/HLA-A02:01+ esophageal tumor cells (up to 80% lysis, p<0.01), accompanied by robust IFN-γ and TNF-α secretion (>1000 pg/mL, p<0.01). No killing was observed against any of seven healthy cell types. TCR-T cells remained effective at low effector-to-target ratios and maintained stable proliferation and effector-memory phenotypes. In xenograft models, KRAS- and CTAG1B-targeted TCR-T cells induced significant tumor regression compared to unmodified T cell controls (p<0.05), with increased effector TCR-T infiltration, and reduced antigen escape. Conclusions: This work establishes a rapid and robust platform for isolating potent, safe, antigen-specific TCRs targeting both oncogenic driver mutation (KRASG12V) and an immunogenic CTA (CTAG1B). TCR-T cells demonstrated strong antitumor activity in vitro and in vivo, supporting the translational potential of this approach for solid tumor treatment. Citation Format: Obed Boadi Amissah, Wenfang Chen, Zhiyuan Li, Gloria Bora Kim. KRASG12V- and CTAG1B-targeted TCR-T cells from phage TCR library suppress colorectal and esophageal tumors [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 5618.

Abstract Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2+ cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm3 (VHH) vs 48mm3 (huE85) vs 211mm3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm3 vs 106mm3 vs 382 mm3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x106 SNU-C2B and 6x106 CAFs from a GI cancer patient. At 500 mm3 tumors, mice received 5x106 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm3 vs 320mm3, p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 105 vs 1.03 x 106 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors. Citation Format: Omar L. Gutierrez Ruiz, Jennifer Feigin, Brooke Kimball, R. Leo Sakemura, Elizabeth L. Siegler, Dominic A. Skeele, Ateka Saleh, Claudia Manriquez Roman, Ismail Can, Olivia Sirpilla, Kun Yun, Carli M. Stewart, Mehrdad Hefazi, Yoh Yamaguchi, Lionel Aurelien Kankeu Fonkoua, Long K. Mai, Truc Huynh, Sophia Y. Goldberg, Grace E. DeFranco, Sara Foote, Ekene Johnkennedy Newton Ogbodo, Hong Xia, Michael Redig, Matthew L. Matthew, Tamiel Nichole Turley, Skyeler Klinge, John Alton Copland, Saad J. Kenderian. Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies [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 1525.

Abstract The sentinel lymph node (SLN) is often the first site of melanoma spread and a critical immune checkpoint that determines local tumor dissemination to systemic metastasis. Although lymph nodes are central to antitumor immunity, the mechanisms that render them permissive to metastasis remain poorly understood. This study used spatial imaging to characterize immune and stromal phenotypic alteration in tumor-negative SLNs (SLN-) and tumor-positive SLNs (SLN+) from patients with cutaneous melanoma, compared to non-melanoma-associated normal lymph nodes (NLN). We performed multiplex immunofluorescence (MxIF) analysis of formalin-fixed, paraffin-embedded lymph node tissues from patients with cutaneous melanoma and control subjects, including two subjects per group (NLN, SLN-, and SLN+). Furthermore, quantitative single-cell segmentation and classification using QuPath generated median fluorescence intensities for immune, stromal, and tumor markers across LN, SLN- and SLN+ regions of interest. In addition to the initial analysis, we employed spatial neighborhood analysis to characterize markers expression profiles surrounding target immune population. This approach quantified local microenvironment based on marker normalized intensity-based co-expression pattern and stromal remodeling signatures. We grouped these markers based on the functional panels representing T-cell activation and exhaustion, myeloid polarization, tumor interaction, stromal remodeling, and apoptosis pathways providing insights into how prometastatic niche evolve within SLN. Normalized data, via centered log ratio (CLR) transformation and Gaussian Mixture Model (GMM) gating was analyzed using principal component analysis, UMAP/tSNE embedding, and hierarchical clustering in in python. Progressive immune remodeling was observed from normal NLN to SLN+, including expansion of regulatory T cells, enrichment of exhausted T cells as well as myeloid-derived suppressor cells (MDSC), and transition toward an M2-like macrophage phenotype. The spatial data from SLN- samples showed an early sign of immune suppression, with reduced cytotoxic capacity. These results show that SLN gets features of peripheral immune tolerance for melanoma early, prior to pathological dissection of metastatic spread supporting the existence of a pre-metastatic niche that facilitates lymphatic colonization of tumor cells. This work provides insights into early immune escape and may inform strategies for immunotherapy and early intervention in melanoma. Citation Format: Shankar Suman, Wendy K. Nevala, Noah A. Stueven, Raymond M. Moore, Chathu L. Atherton, Jeffrey E. Johnson, Ray Guo, James W. Jakub, Svetomir N. Markovic. Spatial remodeling of immune and stromal microenvironments in sentinel lymph nodes during melanoma progression [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 6193.

Abstract Background: PD-L1 is best known for suppressing antitumor immunity at the cell surface, yet emerging evidence suggests additional nuclear functions. Previous studies from our laboratory demonstrated that nuclear PD-L1 interacts with the cohesin complex to maintain chromosomal stability. However, the upstream regulatory mechanism governing this interaction and its immunological consequences remain unknown. Methods and Results: Using acetylation-site prediction, mutagenesis, and structural modeling, we identified lysine 178 (human) / 177 (mouse) as a conserved acetylation switch regulating PD-L1 function. Deacetylation of PD-L1 (K178R/K177R) disrupted PD-L1-PDS5B/cohesin binding without affecting subcellular localization. This led to defective sister chromatid cohesion, micronuclei formation, and cytosolic DNA accumulation. RNA-seq, qPCR, and immunoblotting demonstrated robust activation of the cGAS-STING-IFN-γ signaling axis and enhanced MHC-I antigen-presentation machinery in PD-L1 K178R cells. Functionally, K178R cells proliferated more slowly and were highly susceptible to PBMC-mediated killing in co-culture assays, with further enhancement upon anti-PD-L1 antibody treatment. In xenograft models (NSG mice), tumors expressing PD-L1 K178R grew significantly slower than WT. In immunocompetent C57BL/6 syngeneic models, PD-L1 K177R tumors showed reduced growth and heightened responsiveness to PD-L1 blockade. Structural and biochemical analyses confirmed that PD-L1 acetylation at K178/K177 stabilizes its interaction with the cohesin complex, thereby preventing chromosomal instability-induced immune activation. Conclusions: We identify a conserved PD-L1 acetylation switch that couples cohesin stability to innate immune activation. PD-L1 deacetylation promotes chromosomal instability, activates cGAS-STING-IFN-γ signaling, enhances antigen presentation, and sensitizes tumors to PD-L1 immunotherapy. These findings uncover a previously unrecognized regulatory axis linking tumor genomic integrity to immune recognition, providing a mechanistic foundation for targeting PD-L1 acetylation in cancer treatment. Citation Format: Meijie Wang, Jia Yu, Liewei Wang. A pd-l1 acetylation switch coordinates chromosome cohesion and antitumor immunity [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 7391.

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and remains difficult to treat due to the absence of targeted therapies. While recent advances in immunotherapy, particularly with immune checkpoint inhibitors (ICIs), have transformed the treatment landscape for many cancers, response rates to ICIs remain low for TNBC, largely due to tumor-intrinsic immune evasion mechanisms. Objective: We identify and characterize a novel immune evasion pathway in TNBC driven by deregulation of cancer cell-intrinsic protease-activated receptor 1 (PAR1), a thrombin receptor frequently upregulated in aggressive tumors. Methods and Results: Through integrative bioinformatic analyses, genetic perturbations, xenograft models (immunodeficient and immunocompetent), flow cytometry-based tumor immune profiling, and RNA-seq, we demonstrate that PAR1 activation enhances immune evasion in TNBC. Mechanistically, we show that the paracaspase MALT1 acts as a critical downstream effector of PAR1 signaling and promotes immune escape by driving PD-L1 expression in TNBC cells. Functional assays reveal that genetic depletion of MALT1 or PD-L1 increases TNBC cell susceptibility to T cell-mediated cytotoxicity in vitro and significantly suppresses tumor growth in vivo. Notably, through knock-in and rescue experiments using a catalytically inactive MALT1 mutant, we reveal that MALT1’s scaffolding - rather than protease - function is essential for PD-L1 regulation. Immune cell depletion assays further identify CD4+ T cells, CD8+ T cells, and natural killer (NK) cells as key mediators of the anti-tumor immune response suppressed by the PAR1-MALT1 axis. These findings are supported by human TNBC specimen analyses, where MALT1 expression inversely correlates with T cell activation and positively associates with PD-L1 in PAR1-high, but not PAR1-low, tumors. Conclusion: Our study reveals immune evasion as a novel mechanism of PAR1-driven breast cancer pathogenesis, mediated by a PAR1/MALT1/PD-L1 signaling cascade. These findings bridge a critical gap by positioning MALT1 at the interface between tumor-intrinsic signaling and the immune microenvironment - two areas of CARMA-BCL10-MALT1 (CBM) complex research that have largely evolved in parallel over the years. Importantly, targeting MALT1 may enhance immunotherapy efficacy in TNBC. Given the availability of MALT1 inhibitors in clinical development, our work supports combining MALT1 inhibition with ICIs or other immune-oncology agents as a promising strategy to overcome immune resistance in TNBC. Citation Format: Dong Hu, Prasanna Ekambaram, Zheqi Li, Linda Klei, Maria L. Beecher, Yi Liu, Zongyou Cai, John Little, Jeffrey A. Meridew, Jia-Ying Lee, E. Aubrey Thompson, Tullia C. Bruno, Lidija Covic, Seung-Oe Lim, Anushka Dongre, Heide L. Ford, Mien-Chie Hung, Adrian V. Lee, Steffi Oesterreich, Linda McAllister-Lucas, Peter C. Lucas. A cancer cell-intrinsic PAR1/MALT1/PD-L1 signaling pathway drives immune evasion in triple-negative breast 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 7392.

Abstract Background: Hepatocellular carcinoma (HCC) is largely refractory to immune checkpoint blockade due to immune exclusion, stromal barriers, and aberrant vasculature. We evaluated whether multimodal therapy - external beam radiotherapy (EBRT) for antigen release, intratumoral dendritic cell (DC) vaccination for antigen presentation, PD-L1 blockade for immune activation, and VEGF inhibition for vascular normalization - could convert “cold” HCC tumors into sustained immune-inflamed states. Methods: Serial biopsies (baseline, post-EBRT, end-of-treatment [EOT], and progression [PD] when available) from three patients enrolled in an ongoing phase II trial (NCT03942328) underwent multiplex immunofluorescence, spatial neighborhood analysis, and transcriptomic profiling. Longitudinal changes in cellular composition, immune-stromal architecture, spatial contact networks, and pathway activity were compared. Results: Baseline tumor microenvironments (TMEs) were strongly immune-excluded, dominated by tumor (PanCK+60-70%) and stroma (SMA+/Vimentin+25-30%) with sparse immune infiltration (<10-15%). Post-therapy, immune complexity and spatial connectivity increased 2-3-fold in all patients. Patient 1 showed robust cold-to-hot conversion with CD8+(3x), CD4+(2.2x), and DC (2.3x) expansion, increased CD8-DC interactions (2.5x), and reduced tumor fraction (-40%). Transcriptomics showed marked upregulation of IFN-γ (↑3.4x), HLA-DRA (↑2.7x), and reductions in VEGFR2 (↓50%) and HIF1A (↓40%), consistent with vascular normalization. Patient 2 exhibited stromal/vascular remodeling and improved T-cell infiltration but restricted immune diversity, with CD4+/CD8+ dyads (↑2.2x) and B/NK cells(↓ 60%). Neighborhood diversity rose (3→7 communities) and immune-tumor proximity improved (distance ↓ 35%). Patient 3 showed transient inflaming with CD8+(↑3x), DC(↑2x), and new CD4-CD20/CD4-CD56 interactions, followed by relapse marked by stromal/myeloid re-expansion (SMA+↑1.8x, CD14+↑2.5x), angiogenic resurgence (VEGFR2/CD34+↑2.2x), contraction of immune networks, and genomic shift from activating CTNNB1 mutation loss to ATM gain. DCs accumulated but localized mainly to DC-DC or DC-myeloid niches, indicating non-productive antigen presentation. Conclusions: Multimodal EBRT+DC+PD-L1/VEGF blockade induces four-sided synergistic TME reprogramming - antigen release, immune priming, checkpoint release, and vascular normalization - promoting cold-to-hot conversion in HCC. Durable inflaming correlates with sustained T-cell/DC expansion and vascular remodeling, whereas relapse reflects stromal/myeloid reinforcement and genomic adaptation. These findings define mechanistic trajectories of TME remodeling and highlight actionable barriers to sustained immunotherapy response in HCC. Citation Format: Lionel Aurelien Kankeu Fonkoua, Christopher L. Hallemeier, Basim Salem, Arina Tkachuk, Kaitlynn McCay, Panwen Wang, Ying Li, Thomas D. Atwell, Krishan R. Jethwa, Caitlin Conboy, Nguyen H. Tran, Leslie A. Washburn, Melody Wu, Chen Wu, Andre De Menezes Silva Corraes, Rondell P. Graham, Jose Caetano Villasboas, Kevin Regan, Zuoyi Shao, Rayaan Kamal, Nathan R. Foster, Haidong Dong, Sean Park, Yi Lin, Lewis R. Roberts. Multimodal immunotherapy remodels the tumor microenvironment in hepatocellular carcinoma: Integrative spatial and transcriptomic profiling from a Phase II trial [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 7935.

Abstract Introduction: Placental cell-free DNA (cfDNA) activates maternal monocytes to a proinflammatory phenotype through cGAS-STING signaling during pregnancy, thereby stimulating T cells. We sought to engineer DNA nanocarriers that reproduce this mechanism in human monocytes using clinically translatable materials as a strategy for tumor-associated macrophage (TAM) reprogramming. Methods: A synthetic mimic of fetal cfDNA (scfDNA) was designed, commercially synthesized, and complexed with polyethylenimine (PEI) to form scfDNA-PEI nanoparticles (scfDNA-PEI-NPs) for cytosolic delivery. THP-1 Dual reporter monocytes expressing interferon regulatory factor (IRF)-inducible luciferase were treated with scfDNA-PEI-NPs or human cytotrophoblast derived cfDNA (CTB-DNA) to quantify cGAS-STING-IRF activation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release to define a non-toxic therapeutic window. Results: scfDNA-PEI-NPs triggered robust cGAS-STING-IRF activation in THP-1 monocytes, exceeding CTB-DNA responses. This response was abolished in cGAS-knockout cells and rescued by a cGAS-independent stimulator of the IRF pathway, confirming cGAS specificity of scfDNA-PEI-NPs. Dose-response analysis showed a > 10-fold separation between cGAS-activating and cytotoxic concentrations, establishing a therapeutic window for myeloid-selective cGAS activation. Conclusions: These findings provide proof-of-concept that pregnancy-inspired DNA nanocarriers can reproduce fetal cfDNA-driven activation of a proinflammatory monocyte phenotype in vitro. This platform lays the groundwork for future work on macrophage-targeted cGAS activation and proinflammatory reprogramming for tumor immunotherapy development. Citation Format: Mathew R. Schnorenberg, Elizabeth Ann L. Enninga, Wendy K. Nevala, Noah A. Stueven, Svetomir N. Markovic, . Pregnancy-inspired DNA nanocarriers reproduce a cGAS-driven pro-inflammatory monocyte phenotype [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 4307.

Abstract Background: Patients with residual invasive disease after neoadjuvant chemotherapy (NAC) for HER2-positive breast cancer remain at high risk for recurrence even with standard adjuvant trastuzumab emtansine (T-DM1). H2NVAC, a multi-epitope HER2 peptide vaccine, has previously demonstrated the ability to elicit robust and durable immune responses. Here, we assessed the safety and early immunogenicity of H2NVAC in combination with T-DM1 in this high-risk population. Methods: Patients with stage II-III HER2-positive breast cancer and residual invasive disease following NAC and HER2-directed therapy received standard adjuvant T-DM1 alongside six priming doses of H2NVAC administered every 3 weeks. The primary endpoint was the occurrence of dose-limiting toxicity (DLT) within 21 days of the first vaccination, with adverse events graded per CTCAE v5.0. Immunogenicity assessments included HER2-specific T-cell responses measured by IFNγ ELISpot and HER-specific serum antibodies measured by ELISA at baseline and 30 days after the sixth priming dose. Results: 20 patients were enrolled. H2NVAC plus T-DM1 was well tolerated with no DLTs observed. All treatment-emergent AEs were grade 1-2, with fatigue (grade 1 60%, grade 2 5%), peripheral sensory neuropathy (grade 1 35%, grade 2 10%), elevated alkaline phosphatase (grade 1 40%), nausea (grade 1 35%, grade 2 5%), arthralgia (grade 1 30%), and injection-site reactions (grade 1 50%) being most common. Robust vaccine-specific T-cell responses by ELISpot were observed in 94% of evaluable patients, who mounted HER2-specific T-cell responses at 30 days after the sixth priming dose, with a mean 197-fold increase over baseline. Further CYTOF analysis showed a significant decrease in exhausted T-cell phenotypes, with reduced LAG3 and TIM3 expression on both CD4+ and CD8+ T cells. For antibody responses by ELISA, 26% of patients developed HER2 protein-specific antibody responses at this time point. There was no significant increase in antibodies to H2NVAC peptides or to unrelated tumor antigens (p53, IGFBP2, TERT, myoglobin) at 30 days after the sixth vaccine. Ongoing correlative analyses will assess the evolution of immune responses at later time points following completion of T-DM1 therapy. Conclusions: Concurrent administration of H2NVAC with T-DM1 was safe and did not increase toxicity beyond single-agent T-DM1. To our knowledge, this is the first study to demonstrate that a cancer vaccine can be delivered concurrently with an antibody-drug conjugate without increasing adverse effects while still eliciting robust T-cell responses. Ongoing analyses will further define the kinetics and durability of vaccine-induced antibody and T-cell responses at later time points, as well as evaluate the efficacy of H2NVAC in reducing recurrence risk in the randomized phase 2 trial. Citation Format: Saranya Chumsri, Sharmila Giri, Andy J. Ness, David W. Hillman, Nadine Norton, Davitte Cogen, Brian M. Necela, Aziza Nassar, Donald W. Northfelt, Pooja Advani, Rohit Rao, Alvaro Mareno-Aspitia, Brenda Ernst, Kathryn J. Ruddy, Matthew P. Goetz, Keith L. Knutson. Safety, tolerability, and early immune responses in the safety run-in of an HER2 multi-epitope vaccine combined with T-DM1 in residual HER2-positive breast 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 6715.

Abstract Background: HPV16 (human papillomavirus) is a leading cause of oropharyngeal cancer (OPC) in the US. Antibodies to HPV16 early antigens are detectable in serum samples up to two decades before diagnosis and are potential biomarkers for early detection. In this study, we have developed and compared three custom HPV16 serology platforms for detection of OPC: MSD (Meso Scale Discovery) electrochemiluminescent detection, RAPID ELISA, and a novel semi-quantitative lateral flow assay (LFA) using a custom android application. Methods: For the MSD platform, recombinant HPV 16 antigens (E1, E2, E6, E7) were synthesized in mammalian cells and printed on multiplexed arrays. In RAPID ELISA, GST-tagged antigens (E1, E2, E6, E7) were expressed using mammalian in vitro transcription and translation and bound IgG detected. For the LFA, His-tagged recombinant proteins (HPV16 CE2 184-365aa and HPV16 E7) were synthesized, printed, and antibodies detected with protein G conjugated nanoparticles. Archived serum samples drawn from newly-diagnosed OPC patients (n = 60), with matching saliva samples (n=54, MSD assay only) were obtained from Mayo Clinic. Unmatched controls (n = 46) were obtained from AT Still. For the lateral flow assay, colorimetric image analysis was done using a titration curve of known concentration of monoclonal antibody. We optimized the biomarker panel (E2, E6, and E7 for MSD and ELISA, and for LFA E2, and E7) by using multiparametric analysis and plotting the receiver operating curves to the test performance, and assays were compared using the Cohen’s kappa statistic. Results: At 98% specificity, the MSD HPV16 serologic assay achieved 88% sensitivity, similar to RAPID ELISA, and the LFA assay at 85% sensitivity. The signal intensities for saliva samples were weaker at 24% sensitivity. The Cohen’s kappa value ranged from 0.74 to 0.78 for the comparison between all three assays. There was only one sample among all the cases that was negative by all three serology tests. Conclusion: All three assays have strong concordance for the detection of HPV16 early antigen serology. All the tests can facilitate large scale HPV serology research. Citation Format: Akshansh Kaushik, David Routman, Tej Patel, Joshua Eger, Padhmavathy Yuvaraj, Kathleen R. Bartemes, Marisa D. Griesel, Danielle E. Hunter, Katie Van Abel, Anu Mathew, Mingyue Wang, Leonid Dzantiev, Martin Stengelin, Jacob N. Wohlstadter, Ann Spolarich, Kristina R. Dahlstrom, Jennifer Blain Christen, Erich Sturgis, Karen Anderson. HPV serology for rapid detection of HPV positive oropharyngeal 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 5099.

Abstract Breast cancer (BC) is the most common cancer among female adolescents and young adults (AYAs; 15-39 years). While advances in treatment have improved survival, cardiovascular disease (CVD) can result from systemic therapies, and CVD is a leading cause of death in AYA cancer survivors. AYAs with BC face nearly a four-fold increased risk of CVD compared to their peers without cancer. However, data on the effects of systemic treatment on CVD in this population remains limited. We estimated risk of CVD in AYAs diagnosed with invasive BC (2006-2020), who survived ≥2 years, and were diagnosed and treated in the Kaiser Permanente (KP) Northern and Southern California. Patients were categorized by receipt of anthracycline-, alkylating-, HER2-, platinum-, and taxane-based therapies within 2 years of diagnosis. We examined the cumulative incidence of CVD starting 2 years post-diagnosis and used Cox proportional hazards regression to determine factors associated with CVD. Among 3,071 AYAs, 35.1% were non-Hispanic (NH) White, 31.6% were Hispanic, 18.3% were NH Asian, and 8.2% were NH Black. Most received systemic therapy (90.6%), including anthracycline- and alkylator without HER2-targeted therapy (41.9%) and HER2-targeted therapy without anthracycline (20.9%). Fewer AYAs received a taxane and alkylator (13.0%) or anthracycline with alkylator and taxane along with a HER2-targeting agent (6.4%). Mean follow-up after cancer diagnosis was 7.2 years (range: 2.0-17.5). The 10-year cumulative incidence of CVD was highest among AYAs who received anthracycline/alkylator/taxane/HER2-targeting therapy (19.6%), intermediate for those with anthracycline/alkylator without HER2-targeting therapy (13.0%) and those who received HER2-targeted therapy without anthracycline (14.6%), and lowest for those who received taxane/alkylator (7.3%). In the multivariable model adjusted for demographic factors and radiation, compared to taxane/alkylator, anthracycline/alkylator/taxane/HER2-targeting therapy (hazard ratio (HR)=2.63, 95% confidence interval (CI) 1.56-4.43); anthracycline/alkylator without HER2-targeting therapy (HR=1.75, CI 1.13-2.71); and HER2-targeting therapy without anthracycline (HR=2.13, CI 1.11-4.09) were associated with an increased risk of CVD. CVD risk was similar for taxane/alkylator and no systemic therapy. Other factors associated with higher risk of CVD included NH Black race/ethnicity (HR=1.90, CI 1.32-2.73 vs. NH White) and public health insurance (HR=1.67, CI 1.00-2.78 vs private). This study identifies AYA BC survivors at higher risk of CVD based on treatment regimens received, with highest risks found for those receiving anthracycline/alkylator/taxane/HER2 treatment combinations. In addition, AYAs of Black race/ethnicity and those with public health insurance experienced more CVD, underscoring the need for targeted interventions to mitigate these disparities. Citation Format: Theresa H. M. Keegan, Candice A. Sauder, Ann M. Brunson, Renata Abrahao, Anne C. Kirchhoff, Eric Haupt, Mallory Casperson, Ted Wun, Chun R. Chao, Andrew B. Smitherman, Hazel B. Nichols, Jessica Chubak, Erin E. Hahn, Lawrence H. Kushi, Kathryn J. Ruddy. Systemic therapy and cardiovascular disease in adolescent and young adult breast cancer survivors [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 5219.