Phase II Clinical Trial of Pembrolizumab and Chemotherapy Reveals Distinct Transcriptomic Profiles by Radiologic Response in Metastatic Triple-Negative Breast Cancer.

PURPOSE: A single arm, phase II trial of carboplatin, nab-paclitaxel, and pembrolizumab (CNP) in metastatic triple-negative breast cancer (mTNBC) was designed to evaluate overall response rate (ORR), progression-free survival (PFS), duration of response (DOR), safety/tolerability, overall survival (OS), and identify pathologic and transcriptomic correlates of response to therapy. PATIENTS AND METHODS: Patients with ≤2 prior therapies for metastatic disease were treated with CNP regardless of tumor programmed cell death-ligand 1 status. Core tissue biopsies were obtained prior to treatment initiation. ORR was assessed using a binomial distribution. Survival was analyzed via the Kaplan-Meier method. Bulk RNA sequencing was employed for correlative studies. RESULTS: Thirty patients were enrolled. The ORR was 48.0%: 2 (7%) complete responses (CR), 11 (41%) partial responses (PR), and 8 (30%) stable disease (SD). The median DOR for patients with CR or PR was 6.4 months [95% confidence interval (CI), 4-8.5 months]. For patients with CR, DOR was >24 months. Overall median PFS and OS were 5.8 (95% CI, 4.7-8.5 months) and 13.4 months (8.9-17.3 months), respectively. We identified unique transcriptomic landscapes associated with each RECIST category of radiographic treatment response. In CR and durable PR, IGHG1 expression was enriched. IGHG1high tumors were associated with improved OS (P = 0.045) and were concurrently enriched with B cells and follicular helper T cells, indicating IGHG1 as a promising marker for lymphocytic infiltration and robust response to chemo-immunotherapy. CONCLUSIONS: Pretreatment tissue sampling in mTNBC treated with CNP reveals transcriptomic signatures that may predict radiographic responses to chemo-immunotherapy.

Immunosurveillance in clinical cancer management.

The progression of cancer involves a critical step in which malignant cells escape from control by the immune system. Antineoplastic agents are particularly efficient when they succeed in restoring such control (immunosurveillance) or at least establish an equilibrium state that slows down disease progression. This is true not only for immunotherapies, such as immune checkpoint inhibitors (ICIs), but also for conventional chemotherapy, targeted anticancer agents, and radiation therapy. Thus, therapeutics that stress and kill cancer cells while provoking a tumor-targeting immune response, referred to as immunogenic cell death, are particularly useful in combination with ICIs. Modern oncology regimens are increasingly using such combinations, which are referred to as chemoimmunotherapy, as well as combinations of multiple ICIs. However, the latter are generally associated with severe side effects compared with single-agent ICIs. Of note, the success of these combinatorial strategies against locally advanced or metastatic cancers is now spurring successful attempts to move them past the postoperative (adjuvant) setting to the preoperative (neoadjuvant) setting, even for patients with operable cancers. Here, the authors critically discuss the importance of immunosurveillance in modern clinical cancer management.

Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Radiopharmaceuticals as combinatorial partners for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of multiple cancer types. However, only a fraction of patients with cancer responds to ICIs employed as stand-alone therapeutics, calling for the development of safe and effective combinatorial regimens to extend the benefits of ICIs to a larger patient population. In addition to exhibiting a good safety and efficacy profile, targeted radionuclide therapy (TRT) with radiopharmaceuticals that specifically accumulate in the tumor microenvironment has been associated with promising immunostimulatory effects that (at least in preclinical cancer models) provide a robust platform for the development of TRT/ICI combinations. We discuss preclinical and clinical findings suggesting that TRT stands out as a promising partner for the development of safe and efficient combinatorial regimens involving ICIs.

Characterizing the Immune Microenvironment and Neoantigen Landscape of Hürthle Cell Carcinoma to Identify Potential Immunologic Vulnerabilities.

Hürthle cell carcinoma (HCC) is a rare type of thyroid cancer with high rates of distant metastasis and recurrence. Along with the scarcity of effective systemic therapies for HCC, these factors contribute to poor clinical outcomes. The immunologic features of HCC are poorly defined and response rates with immune checkpoint blockade have not been reported. A more comprehensive understanding of the immune landscape and factors that predict response to checkpoint inhibitors is needed. We performed RNA sequencing on 40 tumors to characterize the neoantigen landscape and immune microenvironment of HCC. We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction and correlated these to genetic features such as tumor mutation burden, neoantigen burden, mitochondrial mutations, and LOH from chromosomal uniparental disomy. Finally, immune profiles of patients with recurrence were compared with those of patients without recurrence. HCC tumors exhibited low levels of immune infiltration, with the more aggressive widely invasive phenotype associated with more immune depletion. There was a negative correlation between tumor mutation burden, neoantigen burden, programmed cell death ligand 1 (PD-L1) expression, and the immune infiltration score. HCC tumors that exhibited a global LOH from chromosomal uniparental disomy or haploidization had the lowest level of immune infiltration. HCC tumors that recurred displayed an immune-depleted microenvironment associated with global LOH and aerobic glycolysis. These findings offer new insights into the functional immune landscapes and immune microenvironment of HCC. Our data identify potential immunologic vulnerabilities for these understudied and often fatal cancers. Significance: The immune landscape of HCC is poorly defined and response rates to immunotherapy have not been reported. The authors found the immune microenvironment in HCC to be depleted. This immunosuppression is associated with a global LOH from haploidization and uniparental disomy, resulting in whole chromosome losses across the genome.

Nivolumab plus ipilimumab in advanced salivary gland cancer: a phase 2 trial.

Salivary gland cancers (SGCs) are rare, aggressive cancers without effective treatments when metastasized. We conducted a phase 2 trial evaluating nivolumab (nivo, anti-PD-1) and ipilimumab (ipi, anti-CTLA-4) in 64 patients with metastatic SGC enrolled in two histology-based cohorts (32 patients each): adenoid cystic carcinoma (ACC; cohort 1) and other SGCs (cohort 2). The primary efficacy endpoint (≥4 objective responses) was met in cohort 2 (5/32, 16%) but not in cohort 1 (2/32, 6%). Treatment safety/tolerability and progression-free survival (PFS) were secondary endpoints. Treatment-related adverse events grade ≥3 occurred in 24 of 64 (38%) patients across both cohorts, and median PFS was 4.4 months (95% confidence interval (CI): 2.4, 8.3) and 2.2 months (95% CI: 1.8, 5.3) for cohorts 1 and 2, respectively. We present whole-exome, RNA and T cell receptor (TCR) sequencing data from pre-treatment and on-treatment tumors and immune cell flow cytometry and TCR sequencing from peripheral blood at serial timepoints. Responding tumors universally demonstrated clonal expansion of pre-existing T cells and mutational contraction. Responding ACCs harbored neoantigens, including fusion-derived neoepitopes, that induced T cell responses ex vivo. This study shows that nivo+ipi has limited efficacy in ACC, albeit with infrequent, exceptional responses, and that it could be promising for non-ACC SGCs, particularly salivary duct carcinomas. ClinicalTrials.gov identifier: NCT03172624 .

Clinical-genomic determinants of immune checkpoint blockade response in head and neck squamous cell carcinoma.

BACKGROUNDRecurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) is generally an incurable disease, with patients experiencing median survival of under 10 months and significant morbidity. While immune checkpoint blockade (ICB) drugs are effective in approximately 20% of patients, the remaining experience limited clinical benefit and are exposed to potential adverse effects and financial costs. Clinically approved biomarkers, such as tumor mutational burden (TMB), have a modest predictive value in HNSCC.METHODSWe analyzed clinical and genomic features, generated using whole-exome sequencing, in 133 ICB-treated patients with R/M HNSCC, of whom 69 had virus-associated and 64 had non-virus-associated tumors.RESULTSHierarchical clustering of genomic data revealed 6 molecular subtypes characterized by a wide range of objective response rates and survival after ICB therapy. The prognostic importance of these 6 subtypes was validated in an external cohort. A random forest-based predictive model, using several clinical and genomic features, predicted progression-free survival (PFS), overall survival (OS), and response with greater accuracy than did a model based on TMB alone. Recursive partitioning analysis identified 3 features (systemic inflammatory response index, TMB, and smoking signature) that classified patients into risk groups with accurate discrimination of PFS and OS.CONCLUSIONThese findings shed light on the immunogenomic characteristics of HNSCC tumors that drive differential responses to ICB and identify a clinical-genomic classifier that outperformed the current clinically approved biomarker of TMB. This validated predictive tool may help with clinical risk stratification in patients with R/M HNSCC for whom ICB is being considered.FUNDINGFundación Alfonso Martín Escudero, NIH R01 DE027738, US Department of Defense CA210784, The Geoffrey Beene Cancer Research Center, The MSKCC Population Science Research Program, the Jayme Flowers Fund, the Sebastian Nativo Fund, and the NIH/NCI Cancer Center Support Grant P30 CA008748.

Antigen presentation in cancer - mechanisms and clinical implications for immunotherapy.

Over the past decade, the emergence of effective immunotherapies has revolutionized the clinical management of many types of cancers. However, long-term durable tumour control is only achieved in a fraction of patients who receive these therapies. Understanding the mechanisms underlying clinical response and resistance to treatment is therefore essential to expanding the level of clinical benefit obtained from immunotherapies. In this Review, we describe the molecular mechanisms of antigen processing and presentation in tumours and their clinical consequences. We examine how various aspects of the antigen-presentation machinery (APM) shape tumour immunity. In particular, we discuss genomic variants in HLA alleles and other APM components, highlighting their influence on the immunopeptidomes of both malignant cells and immune cells. Understanding the APM, how it is regulated and how it changes in tumour cells is crucial for determining which patients will respond to immunotherapy and why some patients develop resistance. We focus on recently discovered molecular and genomic alterations that drive the clinical outcomes of patients receiving immune-checkpoint inhibitors. An improved understanding of how these variables mediate tumour-immune interactions is expected to guide the more precise administration of immunotherapies and reveal potentially promising directions for the development of new immunotherapeutic approaches.

Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas.

Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. Here, we demonstrate that haploinsufficient deletions of chromatin-bound tumor suppressor NFKB inhibitor alpha (NFKBIA) display distinct patterns of occurrence in relation to other genetic markers and are disproportionately present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation and induce a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wild-type tumors. An externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas confirms that NFKBIA deletions predict comparatively brief survival. Thus, NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into models predicting the disease fate of diffuse gliomas.

The MIF promoter SNP rs755622 is associated with immune activation in glioblastoma.

Intratumoral heterogeneity is a defining hallmark of glioblastoma, driving drug resistance and ultimately recurrence. Many somatic drivers of microenvironmental change have been shown to affect this heterogeneity and, ultimately, the treatment response. However, little is known about how germline mutations affect the tumoral microenvironment. Here, we find that the single-nucleotide polymorphism (SNP) rs755622 in the promoter of the cytokine macrophage migration inhibitory factor (MIF) is associated with increased leukocyte infiltration in glioblastoma. Furthermore, we identified an association between rs755622 and lactotransferrin expression, which could also be used as a biomarker for immune-infiltrated tumors. These findings demonstrate that a germline SNP in the promoter region of MIF may affect the immune microenvironment and further reveal a link between lactotransferrin and immune activation.

Immune selection determines tumor antigenicity and influences response to checkpoint inhibitors.

In cancer, evolutionary forces select for clones that evade the immune system. Here we analyzed >10,000 primary tumors and 356 immune-checkpoint-treated metastases using immune dN/dS, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome, to measure immune selection in cohorts and individuals. We classified tumors as immune edited when antigenic mutations were removed by negative selection and immune escaped when antigenicity was covered up by aberrant immune modulation. Only in immune-edited tumors was immune predation linked to CD8 T cell infiltration. Immune-escaped metastases experienced the best response to immunotherapy, whereas immune-edited patients did not benefit, suggesting a preexisting resistance mechanism. Similarly, in a longitudinal cohort, nivolumab treatment removes neoantigens exclusively in the immunopeptidome of nonimmune-edited patients, the group with the best overall survival response. Our work uses dN/dS to differentiate between immune-edited and immune-escaped tumors, measuring potential antigenicity and ultimately helping predict response to treatment.

Novel Genetic Subtypes of Urothelial Carcinoma With Differential Outcomes on Immune Checkpoint Blockade.

PURPOSE: Immune checkpoint blockade (ICB) therapy has significantly improved clinical outcomes in bladder cancer. Identification of correlates of benefit is critical to select appropriate therapy for individual patients. METHODS: To reveal genetic variables associated with benefit from ICB, we performed whole-exome sequencing on tumor specimens from 88 patients with advanced bladder cancer treated with ICB. RESULTS: We identified several genetic factors that correlated with progression-free and overall survival after ICB therapy including ARID1A mutation, tumor mutational burden, intratumoral heterogeneity, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome (immune dN/dS), and tumor cell purity. In addition, we noted that neutrophil-to-lymphocyte ratio and smoking history were negatively associated with overall survival. These genetic characteristics define four molecular subtypes demonstrating differential sensitivity to ICB. We validated the association of these four subtypes with clinical benefit from ICB in an independent cohort (IMvigor210). Finally, we showed that these molecular subtypes also correlate with outcome, although with distinct relationships, among patients not treated with ICB using The Cancer Genome Atlas (TCGA) bladder cancer cohort. Using parallel RNA sequencing data, the subtypes were also shown to correlate with immune infiltration and inflammation, respectively, in the IMvigor210 and TCGA cohorts. CONCLUSION: Together, our study defines molecular subgroups of bladder cancer that influence benefit from ICB.

KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability. SIGNIFICANCE: A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

FAT1 expression in T-cell acute lymphoblastic leukemia (T-ALL) modulates proliferation and WNT signaling.

FAT atypical cadherin 1 (FAT1), a transmembrane protein, is frequently mutated in various cancer types and has been described as context-dependent tumor suppressor or oncogene. The FAT1 gene is mutated in 12-16% of T-cell acute leukemia (T-ALL) and aberrantly expressed in about 54% of T-ALL cases contrasted with absent expression in normal T-cells. Here, we characterized FAT1 expression and profiled the methylation status from T-ALL patients. In our T-ALL cohort, 53% of patient samples were FAT1 positive (FAT1pos) compared to only 16% FAT1 positivity in early T-ALL patient samples. Aberrant expression of FAT1 was strongly associated with FAT1 promotor hypomethylation, yet a subset, mainly consisting of TLX1-driven T-ALL patient samples showed methylation-independent high FAT1 expression. Genes correlating with FAT1 expression revealed enrichment in WNT signaling genes representing the most enriched single pathway. FAT1 knockdown or knockout led to impaired proliferation and downregulation of WNT pathway target genes (CCND1, MYC, LEF1), while FAT1 overexpressing conveyed a proliferative advantage. To conclude, we characterized a subtype pattern of FAT1 gene expression in adult T-ALL patients correlating with promotor methylation status. FAT1 dependent proliferation and WNT signaling discloses an impact on deeper understanding of T-ALL leukemogenesis as a fundament for prospective therapeutic strategies.

Unsupervised and supervised AI on molecular dynamics simulations reveals complex characteristics of HLA-A2-peptide immunogenicity.

Immunologic recognition of peptide antigens bound to class I major histocompatibility complex (MHC) molecules is essential to both novel immunotherapeutic development and human health at large. Current methods for predicting antigen peptide immunogenicity rely primarily on simple sequence representations, which allow for some understanding of immunogenic features but provide inadequate consideration of the full scale of molecular mechanisms tied to peptide recognition. We here characterize contributions that unsupervised and supervised artificial intelligence (AI) methods can make toward understanding and predicting MHC(HLA-A2)-peptide complex immunogenicity when applied to large ensembles of molecular dynamics simulations. We first show that an unsupervised AI method allows us to identify subtle features that drive immunogenicity differences between a cancer neoantigen and its wild-type peptide counterpart. Next, we demonstrate that a supervised AI method for class I MHC(HLA-A2)-peptide complex classification significantly outperforms a sequence model on small datasets corrected for trivial sequence correlations. Furthermore, we show that both unsupervised and supervised approaches reveal determinants of immunogenicity based on time-dependent molecular fluctuations and anchor position dynamics outside the MHC binding groove. We discuss implications of these structural and dynamic immunogenicity correlates for the induction of T cell responses and therapeutic T cell receptor design.

Spatiotemporal evolution of the clear cell renal cell carcinoma microenvironment links intra-tumoral heterogeneity to immune escape.

BACKGROUND: Intratumoral heterogeneity (ITH) is a hallmark of clear cell renal cell carcinoma (ccRCC) that reflects the trajectory of evolution and influences clinical prognosis. Here, we seek to elucidate how ITH and tumor evolution during immune checkpoint inhibitor (ICI) treatment can lead to therapy resistance. METHODS: Here, we completed a single-arm pilot study to examine the safety and feasibility of neoadjuvant nivolumab in patients with localized RCC. Primary endpoints were safety and feasibility of neoadjuvant nivolumab. Then, we spatiotemporally profiled the genomic and immunophenotypic characteristics of 29 ccRCC patients, including pre- and post-therapy samples from 17 ICI-treated patients. Deep multi-regional whole-exome and transcriptome sequencing were performed on 29 patients at different time points before and after ICI therapy. T cell repertoire was also monitored from tissue and peripheral blood collected from a subset of patients to study T cell clonal expansion during ICI therapy. RESULTS: Angiogenesis, lymphocytic infiltration, and myeloid infiltration varied significantly across regions of the same patient, potentially confounding their utility as biomarkers of ICI response. Elevated ITH associated with a constellation of both genomic features (HLA LOH, CDKN2A/B loss) and microenvironmental features, including elevated myeloid expression, reduced peripheral T cell receptor (TCR) diversity, and putative neoantigen depletion. Hypothesizing that ITH may itself play a role in shaping ICI response, we derived a transcriptomic signature associated with neoantigen depletion that strongly associated with response to ICI and targeted therapy treatment in several independent clinical trial cohorts. CONCLUSIONS: These results argue that genetic and immune heterogeneity jointly co-evolve and influence response to ICI in ccRCC. Our findings have implications for future biomarker development for ICI response across ccRCC and other solid tumors and highlight important features of tumor evolution under ICI treatment. TRIAL REGISTRATION: The study was registered on ClinicalTrial.gov (NCT02595918) on November 4, 2015.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T cell responses to MCPyV, which have typically been CD8, but little is known about the T cell response to UV-induced neoantigens. METHODS: A patient in her mid-50s with virus-negative (VN) MCC developed large liver metastases after a brief initial response to chemotherapy. She received anti-PD-L1 (avelumab) and had a partial response within 4 weeks. Whole exome sequencing (WES) was performed to determine potential neoantigen peptides. Characterization of peripheral blood neoantigen T cell responses was evaluated via interferon-gamma (IFNγ) ELISpot, flow cytometry and single-cell RNA sequencing. Tumor-resident T cells were characterized by multiplexed immunohistochemistry. RESULTS: WES identified 1027 tumor-specific somatic mutations, similar to the published average of 1121 for VN-MCCs. Peptide prediction with a binding cut-off of ≤100 nM resulted in 77 peptides that were synthesized for T cell assays. Although peptides were predicted based on class I HLAs, we identified circulating CD4 T cells targeting 5 of 77 neoantigens. In contrast, no neoantigen-specific CD8 T cell responses were detected. Neoantigen-specific CD4 T cells were undetectable in blood before anti-PD-L1 therapy but became readily detectible shortly after starting therapy. T cells produced robust IFNγ when stimulated by neoantigen (mutant) peptides but not by the normal (wild-type) peptides. Single cell RNAseq showed neoantigen-reactive T cells expressed the Th1-associated transcription factor (T-bet) and associated cytokines. These CD4 T cells did not significantly exhibit cytotoxicity or non-Th1 markers. Within the pretreatment tumor, resident CD4 T cells were also Th1-skewed and expressed T-bet. CONCLUSIONS: We identified and characterized tumor-specific Th1-skewed CD4 T cells targeting multiple neoantigens in a patient who experienced a profound and durable partial response to anti-PD-L1 therapy. To our knowledge, this is the first report of neoantigen-specific T cell responses in MCC. Although CD4 and CD8 T cells recognizing viral tumor antigens are often detectible in virus-positive MCC, only CD4 T cells recognizing neoantigens were detected in this patient. These findings suggest that CD4 T cells can play an important role in the response to anti-PD-(L)1 therapy.