Background:Few models exist for studying experimental therapeutics in inflammatory breast cancer (IBC). Our study objective was to characterize a novel patient-derived xenograft (PDX) from a HER2 positive IBC patient refractory to neoadjuvant chemotherapy. Methods:We derived a novel PDX from a patient with hormone receptor negative, HER2-positive IBC refractory to neoadjuvant chemotherapy with Docetaxel, Carboplatin, Trastuzumab, and Pertuzumab (TCHP). Tumor was implanted into NOD/SCID/γ mice (NSG) and used for serial propagation of PDX. We performed short-tandem repeat (STR) profiling, plotted tumor growth curves for mice treated with alpelisib/everolimus vs. untreated, and immunohistochemistry (IHC), and performed clinical genomic assays. Paired Student's t-tests were used to compare tumor growth curves. We used 10X Genomics for single cell transcriptome analysis of 1000 cells derived from the PDX. Results:ctDNA sequencing revealed amplifications in MYC, ERBB2 (HER2), androgen receptor (AR), PIK3CA, vMYB and loss of CDKN2A. Tumor sequencing found a H1047R mutation in PIK3CA. STR profiling showed the propagated PDX tumor matched the original tumor. The engraftment rate was 12/15 (80%) and median tumor volume doubling was 24.5 days (range 9.2-175 days) for n = 15 untreated controls. Alpelisib plus everolimus decreased tumor growth in our PDX (p = 0.006). TCHP-resistant tumor cells downregulated HER2 expression, which was re-expressed after treatment with alpelisib and everolimus. Conclusion:We established a PDX of a HER2-positive IBC tumor with a PIK3CA hotspot mutation (H1047R) refractory to TCHP. Targeting the PI3K/mTOR pathway may be useful to overcome resistance in HER2-positive IBC with a H1047R mutation in PIK3CA.

Curative immune checkpoint blockade (ICB)-based multimodal therapy is now widely used across oncology; yet drivers of efficacy and resistance in most cancer types, including squamous cell carcinoma of the head and neck (SCCHN), are not well understood. To address this issue, we comprehensively characterized the tumor genome, microenvironment, microbiome, and TCR repertoire in a phase III international randomized trial to identify factors that shape outcomes to avelumab plus chemoradiotherapy versus chemoradiotherapy alone. Patients receiving avelumab whose tumors contained distinct immunologic and genetic features had superior outcomes compared to patients in the control arm that did not receive immunotherapy. In contrast, avelumab-treated patients with increased myeloid/neutrophil signatures had poorer outcomes compared to placebo. Strikingly, these tumors possessed telltale intratumoral bacteria, elevated tumor-associated neutrophils, high systemic neutrophil-to-lymphocyte ratio, suppressed levels of adaptive immunity, and were the least likely to respond to therapy. We use these findings to define tumor ecosystem classes that associate with response and resistance to combination chemoimmunotherapy. Importantly, our data demonstrate for the first time, the effects of intratumoral bacteria on ICB response within the context of a randomized trial. These discoveries enhance our understanding of combination immunotherapy response, provide a highly useful multi-omic resource, and identify unanticipated interactions between ICB and CRT that may guide future therapeutic strategies. Tyler Alban, Nadeem Riaz, Robert Haddad, Michelle Saul, Vladamir Makarov, Yingjie Zhu, Ezra Cohen, Robert Ferris, Peter Chang, Jin-Ching Lin, Amanda Pyrri, Prerana Parthasarathy, Ardijana Novaj, Mruniya Gawali, Jennifer Ko, Phineas Hamilton, Natalie Silver, Ivan Juric, Daniel Chawla, Ana Gradissimo, Daniel McGrail, Craig Davis, Nancy Lee, Timothy Chan. Tumor Ecosystem and Microbiome Features Associated with Efficacy from Avelumab-based Multimodal Therapy in a Phase III Randomized Trial [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C064.

Immune checkpoint blockade-based multimodal therapy is widely used across oncology; yet drivers of resistance in most cancer types are not well understood. Here, we comprehensively characterized the tumor genome, microenvironment and microbiome in a phase 3 international randomized trial ( NCT02952586 ) to identify factors that shape outcomes to anti-PD-L1 avelumab plus standard-of-care chemoradiotherapy versus placebo/chemoradiotherapy in individuals with locally advanced head and neck cancer. Patients receiving avelumab whose tumors contained distinct immunologic and genetic features had superior outcomes compared to those receiving placebo. By contrast, patients with increased myeloid/neutrophil activities had worse outcomes with avelumab than those treated with placebo. Strikingly, these tumors possessed telltale intratumoral bacteria, elevated tumor-associated neutrophils, high systemic neutrophil-to-lymphocyte ratios and suppressed adaptive immunity. We define tumor ecosystems associated with benefit to chemoimmunotherapy. Our data demonstrate how intratumoral bacteria affect immune checkpoint blockade response within a randomized trial. These discoveries enhance our understanding of combination immunotherapy, provide a useful multiomic resource and identify unanticipated interactions that may guide future therapeutic strategies. Chan and colleagues report that tumor ecosystem and microbiome features are associated with response to anti-PD-L1 avelumab plus chemoradiotherapy in patients with locally advanced head and neck cancer.

T cell receptors (TCRs) are critical for immune surveillance and successful adaptive immune response against foreign antigens. TCRs drive this key arm of the immune system through recognition of peptide epitopes presented on MHC complexes. However, they are limited due to their stochastic nature and generation via genetic recombination. In silico design of functional TCRs that target defined peptide epitopes would be of considerable utility but has up until now been unsuccessful. Here, we develop an artificial intelligence (AI)-powered approach using a hybrid physics-based simulation and generative AI that successfully engineers TCRs against defined epitopes presented by MHC-I. We use this approach to design TCRs against two cancer antigens, a HERC1 neoantigen and an immunogenic neoepitope in mutant EGFR. We engineer multiple TCRs against the HERC1 neoantigen which activate T cells in response to exposure to peptide-MHC I and kill cancer cells more effectively than a patient-derived TCR. In addition, we used generative AI to design functional TCRs that target the EGFR T790M neoantigen, engineering greater specificity against the mutant sequence. We present an AI-based approach to TCR design with broad utility for efforts to engineer TCRs and for the development of new cell therapies. One sentence summary:Artificial intelligence-based approach enables the directed engineering of functional TCRs with enhanced features that target cancer neoantigens.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint protein that impairs antitumor T cell responses. While broadly expressed on myeloid cells and T cells, the specific contribution of T cell-intrinsic VISTA to antitumor immunity remains undefined. This study investigated the phenotypic and functional consequences of T cell-specific VISTA deletion in tumor-specific CD8+ T cells. Single-cell transcriptomic analysis, TCR repertoire profiling, and flow cytometry revealed that loss of T cell-intrinsic VISTA enhanced early priming and short-term expansion of CD8+ T cells, yet this initial advantage failed to confer durable tumor control. Persistent dysfunction in VISTA-deficient T cells was in part driven by trans-VISTA on myeloid cells, while CTLA-4 upregulation further constrained T cell responses. T cell-intrinsic VISTA deficiency cooperated with CTLA-4 blockade to improve T cell survival and broaden TCR repertoire diversity, resulting in more robust tumor regression than CTLA-4 inhibition alone. A transcriptional signature enriched in VISTA-deficient cytotoxic T cells correlated with favorable outcomes in cancer patients treated with existing immune checkpoint inhibitors. These findings collectively define T cell-intrinsic mechanisms by which VISTA enforces T cell dysfunction and underscore its potential as both a therapeutic target and a biomarker of resistance to current immunotherapies.

Bacillus Calmette-Guérin (BCG) is the mainstay of treatment for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC), yet recurrence rates remain high. To improve the efficacy of BCG, a better understanding of the immune landscape underlying BCG resistance is critical. Here, we performed single-cell RNA-sequencing (scRNA-seq) and whole-exome sequencing on tumors from NMIBC patients before and after BCG treatment. Our analysis revealed a marked increase in CD6/ALCAM interactions between T cells and urothelial cells in BCG recurrent tumors. CD6-high T cells were enriched in recurrent tumors and exhibited downregulation of activation-related genes, indicative of functional impairment. These observations were supported by analysis of an independent BCG-treated NMIBC cohort, in which CD6/ALCAM signaling was correlated with shorter recurrence-free survival (p = 0.00059). Our findings reveal a previously unrecognized association between CD6/ALCAM signaling and BCG resistance in NMIBC patients and highlight this pathway as a potential therapeutic target to enhance response to BCG.

BACKGROUND:Given the scarcity of effective therapeutic targets, metastatic triple negative breast cancer (mTNBC) has shorter survival times compared to other advanced breast cancer subtypes. Although chemo-immunotherapy with immune checkpoint inhibitors (ICIs) in PD-L1+ mTNBC has shown promise, survival benefit remains modest. Therefore, it is crucial to gain improved insight into the mechanisms underlying response and resistance to checkpoint inhibition in mTNBC. METHODS:We employed single cell RNA sequencing (scRNAseq), single cell secretomics, and flow cytometry to identify transcriptomic and proteomic peripheral immune cell signatures associated with response and non-response to anti-PD-1/PD-L1 therapy and chemotherapy in mTNBC. RESULTS:Transcriptomic analysis reveal divergent transcriptional programming of CD33+ myeloid cells between responders and non-responders, even in pretreatment PBMC samples. This divergence, in responders, is characterized by an immune-promoting CD33+ cell phenotype involving IL1b signaling compared to non-responders, where an immunosuppressive phenotype marked by IL1b inhibition is observed. These baseline differences become more pronounced during the course of chemo-immunotherapy. Differences in CD33+ cell phenotype result in functional differences in lymphocyte activities between responders and non-responders. Depletion of CD33+ cells in pre-treatment samples from non-responders, restores T cell effector function. CONCLUSION:Our findings highlight CD33+ cell phenotype as a key determinant of response to chemo-immunotherapy, which can be assessed from peripheral blood. This offers a valuable tool in the context of metastatic TNBC, in which tissue sampling is often challenging.

Glioblastoma (GBM) is the most common primary malignant brain tumor, and current therapies provide only palliation, not a cure. GBM is difficult to treat due to its complex, multi-faceted development involving various cell types in the tumor microenvironment (TME). Recent observations highlight sex differences in these interactions, influenced by biological, hormonal, and immune factors affecting disease progression, treatment responses, survival, and the TME. Platelets, key regulators of immune responses and tumor progression, may contribute to these sex-based differences by altering TME dynamics, though precise molecular mechanisms remain unclear. Our previous work showed that thrombin, a major platelet activator via protease-activated receptor 1 (PAR1) and protease-activated receptor 4 (PAR4), is secreted by cancer stem cells into the tumor microenvironment. We now demonstrate that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, targeting PAR4 with BMS986120 and genetic inhibition of PAR4 prolong survival in females but not males. This survival advantage is estrogen-dependent and TME-specific, driven by enhanced CD8+ T cell infiltration regulated by an estrogen receptor response element on the PAR4 promoter. Inhibiting platelet PAR4 signaling decreases alpha-granule secretion in female tumor-bearing mice while enriching alternative exocytosis pathways, thereby influencing CD8+ T cell activity. PAR4-activated platelets within the TME suppress CD8+ T cell function, and CD8+ T cell depletion eliminates tumor-induced platelet reactivity and the survival benefit of PAR4 inhibition. These findings demonstrate how activated platelets interact and regulate immune cell populations in GBM. These results suggest that the hyper-thrombotic state seen in many GBM patients, which serves as a major risk of death, simultaneously contributes to the immunosuppressive TME. In addition, these results identify therapeutic strategies to leverage the platelet hyperactivity seen in GBM by hyperactive thrombin-PAR4 for sex-dependent therapeutic purposes.

Summary: Myeloid-derived suppressor cells (MDSCs) impair antitumor immune responses. Identifying regulatory circuits during MDSC development may bring new opportunities for therapeutic interventions. We report that the V-domain suppressor of T cell activation (VISTA) functions as a key enabler of MDSC differentiation. VISTA deficiency reduced STAT3 activation and STAT3-dependent production of polyamines, which causally impaired mitochondrial respiration and MDSC expansion. In both mixed bone marrow (BM) chimera mice and myeloid-specific VISTA conditional knockout mice, VISTA deficiency significantly reduced tumor-associated MDSCs but expanded monocyte-derived dendritic cells (DCs) and enhanced T cell-mediated tumor control. Correlated expression of VISTA and arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival. Taken together, these findings unveil the VISTA/polyamine axis as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy.

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell–specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1 + CD62L hi PD-1 low ) and a reciprocal increase in progenitor and memory-like CTLs (TCF1 + PD-1 + ). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8 + CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

Background Mononuclear and polymorphonuclear myeloid-derived suppressor cells (M-MDSC and PMN-MDSC) accumulate in many cancer types and impair anti-tumor immune response and reduce cancer immunotherapy efficacy.1 2 The role of immune checkpoint protein V-domain Suppressor of T cell Activation (VISTA) in the differentiation and function of MDSC remains incompletely understood.3–10 Methods Flow analysis was applied for cell surface makers analysis. Gene expression was determined by RT-qPCR. Intracellular proteins and signaling molecules were detected by Western Blotting. Multiplex immunohistochemistry was used to stain the tissue slides. Mitochondrial functions were detected by seahorse metabolic flux analysis. Results Here, by studying BM-derived MDSCs, we identified that ablation of VISTA significantly reduces the expression of arginase1 (Arg1), iNOS and diminishes the inhibitory effects of MDSC on T cell proliferation. In bone marrow (BM) chimera mouse tumor model, depletion of VISTA reduces the frequency of MDSC and expression of Arg1 and iNOS. Transcriptomic analysis of BM-MDSC indicates that oxidative phosphorylation, Myc-targets, mTORC1, UPR pathways are down-regulated in VISTA-deficient MSDC cells. The deregulation of oxidative and phosphorylation pathway in VISTA knockout MDSC cells correlates the diminished mitochondrial function with significant lower basal oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and lower spare respiratory capacity (SRC). Myeloid specific deletion of VISTA mice demonstrated a durable better tumor control through reduced MDSC differentiation and enhanced T-cell mediated cytotoxicity. Mechanistically, VISTA directly regulates the signaling of MDSC in response to inflammatory stimuli including GM-CSF and IL-6 by augmenting the activation of STAT3. Phosphorylated STAT3 positively regulates the expression of Arg1 and iNOS and modulates the mitochondrial function. Correlated expression of VISTA and Arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival. Conclusions These studies unveil the role of VISTA as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy. Acknowledgements National Institute of Health/National Cancer Institute R01CA164225 (L.L.W), National Institute of Health/National Cancer Institute R01CA223804 (L.L.W), National Institute of Health/National Cancer Institute R21CA258618 (L.L.W), Department of Defense CDMRP W81XWH-21-MRP-MCAA ME210229 (L.L.W), Department of Defense CDMRP W81XWH-21-LCRP-IITRA LC210336 (L.L.W), American Cancer Society RSG-18–045-01-LIB (L.L.W), The Norma C. and Albert I. Geller Professorship in Ovarian Cancer Research (S.A.), Case Comprehensive Cancer Center, Genomics Pilot Grant (S.A.), The V Foundation Scholar award V2020–011 (A.A.C), Department of Defense Early Career Investigator grant KCRP AKCI-ECI, W81XWH-20–1-0804 (A.A.C), NCCN Young Investigator Award (A.A.C), American Cancer Society Research Scholar Grant (RSG-22–067-01-TBE) (A.A.C), American Cancer Society RSG-22–135-01-IBCD (S.C.-C.H) Melanoma Research Foundation Career Development Award (S.C.-C.H) Andrew McDonough B+ Foundation Grant Award (S.C.-C.H) Case GI SPORE DRP grant 5P50CA150964–08 (S.C.-C.H) Cancer Research Institute CLIP Investigator Award (S.C.-C.H References Barry ST, DI Gabrilovich, OJ Sansom, AD Campbell, JP Morton. Therapeutic targeting of tumour myeloid cells. Nat Rev Cancer. 2023. Davidov V, G Jensen, S Mai, SH Chen, PY Pan. Analyzing One Cell at a TIME: Analysis of Myeloid Cell Contributions in the Tumor Immune Microenvironment. Front Immunol. 2020;11:1842. Deng J, J Li, A Sarde, JL Lines, YC Lee, DC Qian, DA Pechenick, R Manivanh, I Le Mercier, CH Lowrey, FS Varn, C Cheng, DA Leib, RJ Noelle, R Mabaera. Hypoxia-Induced VISTA Promotes the Suppressive Function of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment. Cancer Immunol Res. 2019;7:1079–1090. Johnston RJ, LJ Su, J Pinckney, D Critton, E Boyer, A Krishnakumar, M Corbett, AL Rankin, R Dibella, L Campbell, GH Martin, H Lemar, T Cayton, RY Huang, X Deng, A Nayeem, H Chen, B Ergel, JM Rizzo, AP Yamniuk, S Dutta, J Ngo, AO Shorts, R Ramakrishnan, A Kozhich, J Holloway, H Fang, YK Wang, Z Yang, K Thiam, G Rakestraw, A Rajpal, P Sheppard, M Quigley, KS Bahjat, AJ Korman. VISTA is an acidic pH-selective ligand for PSGL-1. Nature. 2019;574:565–570. Kumar V, S Patel, E Tcyganov, DI Gabrilovich. The Nature of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment. Trends Immunol. 2016;37:208–220. Rodriguez PC, DG Quiceno, J Zabaleta, B Ortiz, AH Zea, MB Piazuelo, A Delgado, P Correa, J Brayer, EM Sotomayor, S Antonia, JB Ochoa, AC Ochoa. Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses. Cancer Res. 2004;64:5839–5849. Schaafsma E, W Croteau, M ElTanbouly, EC Nowak, NC Smits, J Deng, A Sarde, CA Webber, D Rabadi, C Cheng, R Noelle, JL Lines. VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance. Cancer Immunol Res 2023;11:38–55. Wang L, R Rubinstein, JL Lines, A Wasiuk, C Ahonen, Y Guo, LF Lu, D Gondek, Y Wang, RA Fava, A Fiser, S Almo, RJ Noelle. VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J Exp Med 2011;208:577–592. Xu W, J Dong, Y Zheng, J Zhou, Y Yuan, HM Ta, HE Miller, M Olson, K Rajasekaran, MS Ernstoff, D Wang, S Malarkannan, L Wang. Immune-Checkpoint Protein VISTA Regulates Antitumor Immunity by Controlling Myeloid Cell-Mediated Inflammation and Immunosuppression. Cancer Immunol Res. 2019;7:1497–1510. Xu W, T Hieu, S Malarkannan, L Wang. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol. 2018;15:438–446.

Introduction Triple negative breast cancer (TNBC) is a heterogeneous breast cancer subtype which continues to portend a particularly poor prognosis compared to other breast cancer subtypes. Immune checkpoint inhibitor (ICI) therapies have emerged as promising options for locally advanced and metastatic TNBC. However, clinical trials have demonstrated mixed results with respect to ICI response. Given varied outcomes and the potential for immune-related adverse events associated with ICIs, there is critical need for identification of accurate biomarkers of response and improved strategies to counteract ICI resistance and/or toxicity. Methods Advanced immunoprofiling of peripheral blood mononuclear cells (PBMCs) and plasma from 7 metastatic TNBC patients with variable responses to anti-PD1 or anti-PDL1 therapy was performed. Samples were analyzed from blood draws obtained: (i) prior to first administration, (ii) while receiving ICI treatment, and (iii) at the time of conformed clinical progression or response to therapy. Response was determined by standard radiological assessment. Immunoprofiling included high parameter flow cytometry, single cell transcriptomics (10x genomics) and secretome analysis (Isoplexis). Single cell RNA profiles from 63,984 cells were analyzed. Results High parameter flow cytometry identified higher circulating levels of a subpopulation of activated CD4+ T cells with a phenotype of CXCR3low CD62Llow CD45RAhigh and CD57high expression in responders versus non-responders. Higher effector function of CD4+ T cells was corroborated by significantly elevated plasma concentrations of IL-2 (p=0.02) and IL-5 (p< 0.0001) among responders. Single cell transcriptomic analysis revealed clusters of B and T cells with distinct activation patterns that were associated with radiographic response. Strikingly, genes involved in B cell activation and T cell-B cell conjugation such as CD81 were found to be highly upregulated among CD4+ T cells from responders. Conclusions Our results are consistent with previous reports describing an association of increased B cell activity in TNBC with improved overall survival. We identified a subpopulation of CD4+ T cells with effector functions consistent with type 2 helper T cells that may not only target cancer cells by direct cytotoxic function, but also promote increased B cell anti-tumor activity. Our study provides insight into specific mechanisms of immune cell interplay that may drive response to ICI therapy. These cell populations and their associated pathways may represent potential biomarkers of response and/or targets for resistance reversal. Citation Format: Avia D. Wilkerson, Patricia A. Rayman, Paul G. Pavicic Jr, Hana Husic, Vladimir Makarov, Ivan Juric, Timothy Chan, Alberto J. Montero, Marcela Diaz-Montero. A multiomic approach to the identification of immune signatures of anti-PD1/PDL1 therapy responders in metastatic triple negative breast cancer: new implications in the role of helper T-cell and B-cell interplay [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-04.

526 Background: Myeloid derived suppressors cells (MDSC) are immune cells that create an immunosuppressive microenvironment. Increased expression of MDSC subsets is associated with worse overall survival in ICI-treated mUC pts, but their role in immune-related adverse events (irAE) is unknown. Immune profiles associated with irAE are also unknown. We investigated associations of MDSC and –omics profiles with response and irAE in ICI-treated mUC pts. Methods: Baseline (B) and on-treatment (Tx) blood samples were collected from ICI-treated mUC pts. MDSC were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSC were identified by flow cytometry in WB, defined as LinloCD33+/HLADR-, and subclassified as polymorphonuclear (PMN)-MDSC (CD15+/CD14-), monocytic (M)-MDSC (CD15-/CD14+), and uncommitted (UC)-MDSC (CD15-/CD14-). MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. irAE severity was graded by CTCAE v5. In a subcohort of 17 pts, proteomics and transcriptomics were analyzed via Olink and Bulk RNAseq, respectively. Wilcoxon rank sum test compared MDSC and –omics among response and irAE groups. Kruskal-Wallis test compared –omics results between irAE responders (irAE-R), irAE non-responders (irAE-NR), and no irAE/non-responders (noAE-NR). Results: 41 ICI-treated mUC pts (25 anti-PD-L1, 16 anti-PD-1) had at least 1 MDSC sample: 28 pts at B, 30 pts at Tx, and 17 pts at both B and Tx. Primary UC sites were bladder (78%) and upper tract (22%); 73% male; median age 72 (range, 28-82); 85% had KPS > 80%; 51% had visceral metastasis. ICI was first and second-line therapy in 37% and 63% of pts, respectively. 13 pts were responders (R); 26 pts were non-responders (NR); 2 pts were not evaluable. 22 pts developed irAE. Median time to irAE was 84 days (range, 21-145); 10 pts required steroids; 3 required ICI discontinuation. UC-MDSC was predominant in WB and PMN-MDSC in PBMC in both B and Tx. Between B and Tx, WB UC-MDSC and PB UC-MDSC increased in R (n = 13; p = 0.04), but decreased in NR (n = 26; p = 0.02). In the subcohort of 17 pts, 11 had irAE (7 irAE-R; 4 irAE-NR), 6 had noAE-NR. Proteomic analysis showed increased expression of CXCL12 in noAE-NR pts (p = 0.006) and increased expression of IL-8 (p = 0.016), IL-18 (p = 0.012), and IL-18R1 (p = 0.016) in all irAE pts. At the transcriptome level, upregulation of IFN-γ was associated with response, whereas upregulation of both IFN-γ and IFN-α differentiated irAE-R from irAE-NR. Conclusions: In ICI-treated mUC pts, WB & PB UC-MDSC increased in R and decreased in NR between B and Tx. Increased expression of pro-inflammatory chemokines was observed in irAE pts, independent of response. A distinct inflammatory pathway was observed in irAE-R. Prospective investigation of blood-based biomarkers of response and irAE development is warranted.

Defects in pathways governing genomic fidelity have been linked to improved response to immune checkpoint blockade therapy (ICB). Pathogenic POLE/POLD1 mutations can cause hypermutation, yet how diverse mutations in POLE/POLD1 influence antitumor immunity following ICB is unclear. Here, we comprehensively determined the effect of POLE/POLD1 mutations in ICB and elucidated the mechanistic impact of these mutations on tumor immunity. Murine syngeneic tumors harboring Pole/Pold1 functional mutations displayed enhanced antitumor immunity and were sensitive to ICB. Patients with POLE/POLD1 mutated tumors harboring telltale mutational signatures respond better to ICB than patients harboring wild-type or signature-negative tumors. A mutant POLE/D1 function-associated signature-based model outperformed several traditional approaches for identifying POLE/POLD1 mutated patients that benefit from ICB. Strikingly, the spectrum of mutational signatures correlates with the biochemical features of neoantigens. Alterations that cause POLE/POLD1 function-associated signatures generate T cell receptor (TCR)-contact residues with increased hydrophobicity, potentially facilitating T cell recognition. Altogether, the functional landscapes of POLE/POLD1 mutations shape immunotherapy efficacy.