Patrick H. Lizotte

<h2>Summary</h2> Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8⁺ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, <i>TCF7</i>, was visualized within CD8⁺ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.

Abstract Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216–33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Jenkins et al., p. 196. This article is highlighted in the In This Issue feature, p. 127

A comprehensive understanding of the molecular vulnerabilities of every type of cancer will provide a powerful roadmap to guide therapeutic approaches. Efforts such as The Cancer Genome Atlas Project will identify genes with aberrant copy number, sequence, or expression in various cancer types, providing a survey of the genes that may have a causal role in cancer. A complementary approach is to perform systematic loss-of-function studies to identify essential genes in particular cancer cell types. We have begun a systematic effort, termed Project Achilles, aimed at identifying genetic vulnerabilities across large numbers of cancer cell lines. Here, we report the assessment of the essentiality of 11,194 genes in 102 human cancer cell lines. We show that the integration of these functional data with information derived from surveying cancer genomes pinpoints known and previously undescribed lineage-specific dependencies across a wide spectrum of cancers. In particular, we found 54 genes that are specifically essential for the proliferation and viability of ovarian cancer cells and also amplified in primary tumors or differentially overexpressed in ovarian cancer cell lines. One such gene, PAX8, is focally amplified in 16% of high-grade serous ovarian cancers and expressed at higher levels in ovarian tumors. Suppression of PAX8 selectively induces apoptotic cell death of ovarian cancer cells. These results identify PAX8 as an ovarian lineage-specific dependency. More generally, these observations demonstrate that the integration of genome-scale functional and structural studies provides an efficient path to identify dependencies of specific cancer types on particular genes and pathways.

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The ‘in situ vaccination’ immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic antitumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from cowpea mosaic virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic antitumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the antitumour immune response. CPMV also exhibited clear treatment efficacy and systemic antitumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer. Virus-like nanoparticles such as the cowpea mosaic virus, known to have inherent immunogenic properties, are now used to suppress metastatic cancer in various mouse models.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Abstract Using a genome-scale, lentivirally delivered shRNA library, we performed massively parallel pooled shRNA screens in 216 cancer cell lines to identify genes that are required for cell proliferation and/or viability. Cell line dependencies on 11,000 genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 11M cells with one shRNA-virus per cell and determining the relative enrichment or depletion of each of the 54,000 shRNAs after 16 population doublings using Next Generation Sequencing. All the cell lines were screened using standardized conditions to best assess differential genetic dependencies across cell lines. When combined with genomic characterization of these cell lines, this dataset facilitates the linkage of genetic dependencies with specific cellular contexts (e.g., gene mutations or cell lineage). To enable such comparisons, we developed and provided a bioinformatics tool to identify linear and nonlinear correlations between these features.

Abstract Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping. Experimental Design: We identified patients with advanced non–small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations. Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH. Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437–43. ©2018 AACR. See related commentary by Bauml and Levy, p. 4352

<h2>Summary</h2> Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of <i>EGFR</i>-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic <i>BMF</i>, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of <i>YAP1</i>, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.

(Cell 175, 998–1013.e1–e20; November 1, 2018) In the originally published version of this article, the text associated with the vertical axis in Figure 6E mistakenly referred to the CD39−TIM3−/ CD39+TIM3+ ratio, but the values correspond to the inverse ratio, i.e., CD39+TIM3+/CD39−TIM3−. The corrected Figure 6E is shown here, and this error has now been corrected in the article online. The authors apologize for any confusion this error may have caused.Figure 6EDifferential Chromatin Accessibility in CD39+TIM3+ and CD39−TIM3− cells (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Defining T Cell States Associated with Response to Checkpoint Immunotherapy in MelanomaSade-Feldman et al.CellNovember 01, 2018In BriefSingle-cell analysis of immune cells from melanoma patients treated with immune checkpoint therapy uncovers a TCF7+ memory-like state in the cytotoxic T cell population and demonstrates its association with a positive outcome. Full-Text PDF Open Archive

Abstract Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. Significance: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection. This article is highlighted in the In This Issue feature, p. 1861

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and is often linked to loss of chromosome 3p, which harbors the VHL tumor suppressor gene, loss of chromosome 14q, which includes HIF1A, and gain of chromosome 5q. The relevant target(s) on chromosome 5q is not known. Here, we show that 5q amplification leads to overexpression of the SQSTM1 oncogene in ccRCC lines and tumors. Overexpression of SQSTM1 in ccRCC lines promoted resistance to redox stress and increased soft agar growth, while downregulation of SQSTM1 decreased resistance to redox stress, impaired cellular fitness, and decreased tumor formation. Therefore, the selection pressure to amplify 5q in ccRCC is driven, at least partly, by SQSTM1.

Abstract Effective therapies for non–small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein, we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell–mediated antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC. Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852–67. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 783

BACKGROUND.Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking.Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS.We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically "hot" cluster with abundant CD8 + T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8 + T cells and expression of inhibitory markers.The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC.There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster.Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS.Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.

Abstract Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8+ T cell–mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8+ T cells and NK cells, but did not require CD4+ T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity. Two degraders targeting zinc finger transcription factor IKZF2 (Helios) were developed by reprogramming CRL4CRBN E3 ligase, and the pharmacologic degradation of Helios results in Treg destabilization.

Abstract Purpose: Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti–PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS). Patients and Methods: In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling. Results: Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8–71.7) and 1-year OS was 85.7% (95% CI, 66.3–94.4). Two-year DFS and OS were 64% and 80% among pathologic responders. Conclusions: (Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

Abstract Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist–induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Importance Proliferative verrucous leukoplakia (PVL) is an aggressive oral precancerous disease characterized by a high risk of transformation to invasive oral squamous cell carcinoma (OSCC), and no therapies have been shown to affect its natural history. A recent study of the PVL immune landscape revealed a cytotoxic T-cell–rich microenvironment, providing strong rationale to investigate immune checkpoint therapy. Objective To determine the safety and clinical activity of anti–programmed cell death 1 protein (PD-1) therapy to treat high-risk PVL. Design, Setting, and Participants This nonrandomized, open-label, phase 2 clinical trial was conducted from January 2019 to December 2021 at a single academic medical center; median (range) follow-up was 21.1 (5.4-43.6) months. Participants were a population-based sample of patients with PVL (multifocal, contiguous, or a single lesion ≥4 cm with any degree of dysplasia). Intervention Patients underwent pretreatment biopsy (1-3 sites) and then received 4 doses of nivolumab (480 mg intravenously) every 28 days, followed by rebiopsy and intraoral photographs at each visit. Main Outcomes and Measures The primary end point was the change in composite score (size and degree of dysplasia) from before to after treatment (major response [MR]: &amp;amp;gt;80% decrease in score; partial response: 40%-80% decrease). Secondary analyses included immune-related adverse events, cancer-free survival (CFS), PD-1 ligand 1 (PD-L1) expression, 9p21.3 deletion, and other exploratory immunologic and genomic associations of response. Results A total of 33 patients were enrolled (median [range] age, 63 [32-80] years; 18 [55%] were female), including 8 (24%) with previously resected early-stage OSCC. Twelve patients (36%) (95% CI, 20.4%-54.8%) had a response by composite score (3 MRs [9%]), 4 had progressive disease (&amp;amp;gt;10% composite score increase, or cancer). Nine patients (27%) developed OSCC during the trial, with a 2-year CFS of 73% (95% CI, 53%-86%). Two patients (6%) discontinued because of toxic effects; 7 (21%) experienced grade 3 to 4 immune-related adverse events. PD-L1 combined positive scores were not associated with response or CFS. Of 20 whole-exome sequenced patients, all 6 patients who had progression to OSCC after nivolumab treatment exhibited 9p21.3 somatic copy-number loss on pretreatment biopsy, while only 4 of the 14 patients (29%) who did not develop OSCC had 9p21.3 loss. Conclusions and Relevance This immune checkpoint therapy precancer nonrandomized clinical trial met its prespecified response end point, suggesting potential clinical activity for nivolumab in high-risk PVL. Findings identified immunogenomic associations to inform future trials in this precancerous disease with unmet medical need that has been difficult to study. Trial Registration ClinicalTrials.gov Identifier: NCT03692325

Cemiplimab is approved for treating locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC). Solid organ transplant recipients have been excluded from immunotherapy trials, given concern for allograft rejection despite their increased risk of skin cancers. Chronic immunosuppression is necessary to prevent organ rejection but may attenuate antitumor response with PD-1 inhibitors.We report a phase I study of cemiplimab for kidney transplant recipients (KTRs) with advanced CSCC. After cross-taper to a mammalian target of rapamycin (mTOR) inhibitor and pulsed dose corticosteroids (prednisone 40 mg once daily, the day before and on days 1-3 of each cycle, followed by 20 mg once daily on days 4-6, then 10 mg once daily until the day before each subsequent cycle), patients received cemiplimab 350 mg intravenously once every 3 weeks for up to 2 years and were assessed for response every 8 weeks. The primary end point was the rate of kidney rejection, with key secondary end points including rate and duration of response, and survival.Twelve patients were treated. No kidney rejection or loss was observed. A response to cemiplimab was observed in five of 11 evaluable patients (46%; 90% CI, 22 to 73), including two with durable responses beyond a year. Median follow-up was 6.8 months (range, 0.7-29.8). Treatment-related grade 3 or greater adverse events occurred in five patients (42%), including diarrhea, infection, and metabolic disturbances. One patient died of angioedema and anaphylaxis attributed to mTOR inhibitor cross-taper.mTOR inhibitor and corticosteroids represent a favorable immunosuppressive regimen for KTRs with advanced CSCC receiving immunotherapy. This combination resulted in durable antitumor responses with no kidney rejection events (funded by Regeneron Pharmaceuticals [ClinicalTrials.gov identifier: NCT04339062]).

Abstract Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c+ antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c+ cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c+ cells regained the ability to efficiently cross-present antigen and prime CD8+ T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8+ T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8+ T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors. Cancer Res; 73(13); 3842–51. ©2013 AACR.

A principal mechanism by which tumors evade immune-mediated elimination is through immunosuppression. Previous approaches to tumor immunotherapy have focused on modifying the immunosuppressive environment with immune checkpoint inhibitors, cytokine therapy, and other modalities with the intent to generate T-cell based anti-tumor immunity. We hypothesized that transformation of the suppressive ovarian cancer microenvironment could be achieved by introduction of the attenuated ΔactA/ΔinlB strain of Listeria monocytogenes. ΔactA/ΔinlB introduced into the microenvironment of the aggressive ID8-Defb29/Vegf-A murine ovarian carcinoma is preferentially phagocytosed by tumor-associated macrophages (TAMs) and reprograms that population from one of suppression to immunostimulation. TAMs in the peritoneum upregulated their co-stimulatory molecules CD80 and CD86, increased transcription of inflammatory cytokines, and downregulated transcription of suppressive effector molecules. Surprisingly, therapeutic benefit was not mediated by T- or NK-cell activity. ΔactA/ΔinlB-induced repolarization of TAMs activated direct tumor cell lysis via Nos2 production of nitric oxide. Modulation of the immunosuppressive nature of the ID8-Defb29/Vegf-A microenvironment, specifically by reprogramming of the TAM suppressive population from M2 to M1 polarization, is critical for our observed immune-mediated survival benefit.

PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45+ immune cells, a significantly higher proportion of infiltrating CD3+ T cells, and a significantly higher percentage of CD3+ cells displaying the activated HLA-DR+/CD38+ phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8+ T cells, a higher fraction of FOXP3+/CD4+ Tregs, and increased expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells. Double-positive PD-1+/TIM-3+ CD8+ T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3+ T cells and PD-1+/TIM-3+ CD8+ T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR.

Abstract We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b–restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti–PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti–PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti–PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/– mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed.We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines.We show that multiplex gene editing of mouse lung organoids using the CRISPR-Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell-mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti-PD-1 therapy.We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage-inducing therapies in the treatment of LSCC.

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0 months, p = 0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher Tregs. Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.

9519 Background: Solid organ transplant recipients are often excluded from immunotherapy trials given the risk of allograft rejection and loss. We report the results of the first prospective study using the PD-1 inhibitor Cemiplimab (Cemi) for kidney transplant recipients (KTR) with advanced, incurable cutaneous squamous cell carcinoma (cSCC), adopting a standardized approach to immunosuppression (IS) with mTOR inhibition and dynamic prednisone (NCT04339062). Methods: This single-arm, open-label prospective clinical trial enrolled KTRs (eGFR ≥30 mL/min without proteinuria) with advanced cSCC, ECOG ≤2, measurable disease (RECIST v1.1), with no prior immunotherapy exposure. KTRs received mTOR inhibition (target trough 4-6 ng/mL) with a prednisone taper each cycle (40 mg on day -1 to 3, 20 mg days 4-6, 10 mg days 7-20) along with Cemi 350 mg IV every 21-days. Primary endpoint: rate of rejection (futility defined as ≥2/3 or 4/6 KTRs with rejection events). Secondary endpoints: overall response rate (ORR), duration of response, progression-free survival (PFS), overall survival (OS), infection rates. Exploratory: baseline tumor PD-L1 score, molecular and immunologic predictors of response. Results: From 11/2020 to 1/2023, 10 KTRs (median years from transplant: 8, range: 3-31) enrolled including 8 (80%) men, median age 64 (range: 43-86), median eGFR 48 (range: 32-60) often with head and neck primaries (9, 90%) and distant metastases (7, 70%). Six (60%) had prior systemic therapy. For mTOR inhibition, 7 (70%) received sirolimus and 3 (30%) everolimus. At a median follow-up of 6.3 months (range: &lt; 1-24.9), no patients experienced kidney allograft rejection or loss. Of 8 evaluable patients, ORR was 50% (2 CR, 2 PRs), while 4 had PD. At data cutoff no responder had progressed, with 2/4 in response &gt; 18 months (range: &lt; 1-22.7+). One patient is pending first restaging; 1 was unevaluable (died before first restaging). One KTR with initial PD experienced a subsequent durable response to cetuximab. Fatigue (40%) and limb edema (30%) were the most common treatment-related adverse events (TRAEs). Grade 3+ TRAEs occurred in 5 (50%) patients including diarrhea, infections (n = 3), and electrolyte derangements; there were no Cemi-related deaths. Median PFS was 7.9 mos (95%CI: 1.2-not reached [NR]); the 3-month OS estimate was 61% (95%CI: 27-83). Baseline tumor PD-L1 scores ranged from 0-5%; median TMB was 49 muts/Mb (range: 10-97). Tumor mutations in TP53, CDKN2A, and NOTCH1 were common. Exploratory tumor/circulating multiparametric immune profiling and circulating tumor (ct)DNA findings will be presented. Conclusions: Using IS with mTOR inhibition and dynamic prednisone resulted in no kidney allograft rejection among KTRs treated with Cemi for advanced cSCC. Durable anti-tumor efficacy was observed. mTOR inhibition with prednisone should be the preferred IS regimen when treating KTRs with anti-PD-1 therapy. Clinical trial information: NCT04339062 .

Histone deacetylase (HDAC) overexpression has been documented in various cancers and may be associated with worse outcomes. Data from early-phase studies of advanced non-small cell lung cancer (NSCLC) suggest encouraging antitumor activity with the combination of an HDAC inhibitor and either platinum-based chemotherapy or an EGFR inhibitor; however, toxicity is a limiting factor in the use of pan-HDAC inhibitors. Selective inhibition of HDAC6 may represent a potential therapeutic target and preclinical studies revealed immunomodulatory effects with HDAC6 inhibition, suggesting the potential for combination with immune checkpoint inhibitors. This phase Ib, multicenter, single-arm, open-label, dose-escalation study investigated the HDAC6 inhibitor ACY-241 (citarinostat) plus nivolumab in patients with previously treated advanced NSCLC who had not received a prior HDAC or immune checkpoint inhibitor.The orally administered ACY-241 dose was escalated (180, 360, or 480 mg once daily). Nivolumab was administered at 240 mg (day 15 of cycle 1, then every 2 weeks thereafter). The primary endpoint was to determine the maximum tolerated dose (MTD) of ACY-241 plus nivolumab. Secondary endpoints included safety, tolerability, and preliminary antitumor activity. Pharmacodynamics was an exploratory endpoint.A total of 18 patients were enrolled, with 17 patients treated. No dose-limiting toxicities (DLTs) occurred with ACY-241 at 180 or 360 mg; 2 DLTs occurred at 480 mg. The MTD of ACY-241 was 360 mg. The most common grade ≥ 3 treatment-emergent adverse events were dyspnea (n = 3; 18%) and pneumonia (n = 3; 18%). At the 180-mg dose, 1 complete response and 2 partial responses (PRs) were observed. At the 360-mg dose, 3 PRs were observed; 1 patient achieved stable disease (SD) and 1 experienced progressive disease (PD). At the 480-mg dose, no responses were observed; 1 patient achieved SD and 3 experienced PD. Acetylation analyses revealed transient increases in histone and tubulin acetylation levels following treatment. An increase in infiltrating total CD3+ T cells was observed following treatment.The study identified an MTD for ACY-241 plus nivolumab and the data suggest that the combination may be feasible in patients with advanced NSCLC. Responses were observed in patients with advanced NSCLC.https://clinicaltrials.gov/ct2/show/NCT02635061 (identifier, NCT02635061).

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1–4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the αβ T cell lineage5,6. PreTCRs arrayed on CD4−CD8− double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αβ T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7–10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αβ T cell differentiation, the absence of preTCR–pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR–pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities. Aberrant thymocyte developmental programming results when interactions between thymic stroma and pre-T cell receptors occur in the absence of major histocompatibility complex bound to antigen peptide.

Abstract With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.

Background Immune checkpoint inhibitors have revolutionized cancer treatment, but the benefits in refractory patients with esophageal cancer have been modest. Predictors of response as well as new targets for novel therapeutic combinations are needed. In this phase 2 clinical trial, we tested single-agent pembrolizumab in patients with advanced esophageal cancer, who received at least one prior line of therapy. Methods Pembrolizumab 200 mg every 3 weeks was tested in 49 patients with refractory esophageal cancer: 39 with adenocarcinoma and 10 with esophageal squamous cell carcinoma. Major endpoints were radiological response by Immune-related Response Evaluation Criteria In Solid Tumors and survival. Tumor samples were evaluated for programmed cell death ligand 1 (PD-L1) expression, tumor mutational burden (TMB), and immune contexture by both NanoString mRNA expression analysis and flow cytometry. Peripheral blood mononuclear cells and a panel of circulating chemokines were also analyzed. Results The overall response rate (ORR) was 8% (4 of 49 patients; 95% CI 2.3% to 19.6%). Median overall survival (OS) was 5.8 months (95% CI 4.0 to 9.5). ORR and OS were not associated with histology. For PD-L1-positive patients, ORR was 13.3% (95% CI 1.7% to 40.5%) and median OS was 7.9 months (95% CI 4.7 to 15.5). A trend toward improved OS was observed in seven patients with a TMB ≥10 mut/Mb (p=0.086). Tumors with a PD-L1 Combined Positive Score ≥1 showed enrichment of LAG3 (p=0.005) and IDO1 (p=0.04) gene expression. Baseline levels of circulating CXCL10, interleukin 2 (IL2) receptor α (IL2RA) and IL6 were associated with survival: CXCL10 favorably, (HR 0.37, p=0.002 (progression-free survival); HR 0.55, p=0.018 (OS)); IL2RA and IL6 unfavorably (HR 1.57, p=0.020 for IL6 (OS); HR 2.36, p=0.025 for IL2RA (OS)). Conclusions Pembrolizumab monotherapy was modestly effective in refractory esophageal cancer. Circulating CXCL10 at baseline appeared to be a robust predictor of response. Other T cell exhaustion markers are upregulated in PD-L1-positive patients, suggesting that immunotherapy combinations such as anti-LAG3/programmed cell death protein 1 (PD-1) or anti-IDO1/PD-1 may be of promise in refractory esophageal cancer.

Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI-resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI-resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI-resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity.MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

<h2>Summary</h2> Despite small cell lung cancers (SCLCs) having a high mutational burden, programmed death-ligand 1 (PD-L1) immunotherapy only modestly increases survival. A subset of SCLCs that lose their ASCL1 neuroendocrine phenotype and restore innate immune signaling (termed the "inflammatory" subtype) have durable responses to PD-L1. Some SCLCs are highly sensitive to Aurora kinase inhibitors, but early-phase trials show short-lived responses, suggesting effective therapeutic combinations are needed to increase their durability. Using immunocompetent SCLC genetically engineered mouse models (GEMMs) and syngeneic xenografts, we show durable efficacy with the combination of a highly specific Aurora A kinase inhibitor (LSN3321213) and PD-L1. LSN3321213 causes accumulation of tumor cells in mitosis with lower ASCL1 expression and higher expression of interferon target genes and antigen-presentation genes mimicking the inflammatory subtype in a cell-cycle-dependent manner. These data demonstrate that inflammatory gene expression is restored in mitosis in SCLC, which can be exploited by Aurora A kinase inhibition.

Abstract A variety of strategies, have been applied to cancer treatment and the most recent one to become prominent is immunotherapy. This interest has been fostered by the demonstration that the immune system does recognize and often eliminate small tumors but tumors that become clinical problems block antitumor immune responses with immunosuppression orchestrated by the tumor cells. Methods to reverse this tumor‐mediated immunosuppression will improve cancer immunotherapy outcomes. The immunostimulatory potential of nanoparticles ( NPs ), holds promise for cancer treatment. Phagocytes of various types are an important component of both immunosuppression and immunostimulation and phagocytes actively take up NPs of various sorts, so NPs are a natural system to manipulate these key immune regulatory cells. NPs can be engineered with multiple useful therapeutic features, such as various payloads such as antigens and/or immunomodulatory agents including cytokines, ligands for immunostimulatory receptors or antagonists for immunosuppressive receptors. As more is learned about how tumors suppress antitumor immune responses the payload options expand further. Here we review multiple approaches of NP ‐based cancer therapies to modify the tumor microenvironment and stimulate innate and adaptive immune systems to obtain effective antitumor immune responses. This article is categorized under: Therapeutic Approaches and Drug Discovery &gt; Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology &gt; Nanoscale Systems in Biology

The high sensitivity of metaiodobenzylguanidine (MIBG) scintigraphy for sympathomedullary tumors such as neuroblastoma and pheochromocytoma is well documented. The specificity of MIBG scintigraphy for these tumors is also high but has been incompletely characterized for other neural crest tumors and non-neural crest tumors of childhood.The medical records and MIBG scans of all children who had undergone MIBG scintigraphy for known or suspected neuroblastoma or pheochromocytoma were retrospectively reviewed at five major referral centers. Those patients found to have pathologies other than neuroblastoma or pheochromocytoma form the basis of this study.One hundred children with a total of 110 lesions met the inclusion criteria. All had negative MIBG scans except 1 of 2 children with infantile myofibromatosis, 1 of 2 with neuroendocrine carcinomas, 1 of 2 with pancreaticoblastomas and 1 of 10 with primitive neuroectodermal tumors.MIBG scintigraphy is highly specific for neuroblastoma and pheochromocytoma. Only 4% (4/100) of nonsympathomedullary tumors (non-pheochromocytoma and non-neuroblastoma) in childhood showed MIBG uptake, of which only 2% (2/100) were of non-neural crest origin.

Neoadjuvant anti-PD1 (aPD1) therapies are being explored in surgically resectable head and neck squamous cell carcinoma (HNSCC). Encouraging responses have been observed but further insights into the mechanisms underlying resistance and approaches to improve responses are needed.We integrated data from syngeneic mouse oral carcinoma (MOC) models and neoadjuvant pembrolizumab HNSCC patient tumor RNA-Seq data to explore the mechanism of aPD1 resistance. Tumors and tumor draining lymph nodes (DLN) from MOC models were analyzed for antigen specific priming. CCL5 expression was enforced in an aPD1 resistant model.An aPD1 resistant mouse model showed poor priming in the tumor DLN due to type 1 conventional dendritic cell (cDC1) dysfunction, which correlated with exhausted and poorly responsive antigen specific T cells. Tumor microenvironment analysis also showed decreased cDC1 in aPD1 resistant tumors compared to sensitive tumors. Following neoadjuvant aPD1 therapy, pathologic responses in patients also positively correlated with baseline transcriptomic cDC1 signatures. In an aPD1 resistant model, intra-tumoral cDC1 vaccine was sufficient to restore aPD1 response by enhancing T cell infiltration and increasing antigen-specific responses with improved tumor control. Mechanistically, CCL5 expression significantly correlated with neoadjuvant aPD1 response and enforced expression of CCL5 in an aPD1 resistant model enhanced cDC1 tumor infiltration, restored antigen specific responses, and recovered sensitivity to aPD1 treatment.These data highlight the contribution of tumor infiltrating cDC1s in HNSCC aPD1 response, and approaches to enhance cDC1 infiltration and function that may circumvent aPD1 resistance in HNSCC patients.

Abstract Background: Antibody drug conjugates (ADCs) have demonstrated significant antitumor activity against multiple treatment refractory cancers. While Trastuzumab deruxtecan, a HER2 directed ADC, has shown impressive clinical activity in HER2-expressing cancers, the ability to predict responses of ADCs by IHC has been limited. Dato-DXd is a TROP2 directed ADC that is currently being studied in a number of registrational phase 3 trial incld. TROPION-Lung01. In the phase 1b TROPION-Pan tumor 01 trial (TP01) response to Dato-DXd did not correlate to TROP2 IHC expression. Here we investigate molecular determinants of sensitivity to Dato-DXd using short-term microfluidic culture of patient derived organotypic tumor spheroids (PDOTS). Methods: Surgical NSCLC cases collected from Brigham and Women’s Hospital and St. Elizabeth’s Medical Center under an IRB approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by live/dead imaging, TROP2 antigen density on EPCAM+ cells by flow cytometry (FCM) and by immunofluorescence (IF). T cell and myeloid cell populations were analyzed by FCM of CD45+ cells isolated during tumor preparation. For a subset of samples single cell RNA sequencing (scRNAseq) was performed. Results:Eleven NSCLC explants were studied. Two specimens were excluded due to excess of fibrotic material. Response to Dato-DXd was measured by change in raw live cell area compared to control samples. Median response to Dato-DXd was -48.5% (ranging from +33.9% to -77.8%). Using a cut-off of -40%, four cases were characterized as non-responders (NR) and 5 as responders (R). Tumor TROP2 expression by IF ranged from 0 to 3+ with no relationship between TROP2 expression and response being observed. TROP2 staining positivity by IF didn’t correlate to Dato-DXd sensitivity ex vivo. TROP2 FCM, on the other hand discriminated between responsive and non-responsive models in 100% of the cases, using a cutoff of MFI &amp;gt;13000. Furthermore, we observed an increase of tumor CD8 abundance in Dato-DXd R compared to NR (median %CD8 in R 10.3% (range 5.1%-62.5%); median %CD8 of live cells in NR 2.3% (range 1.2%-3.7%) p-value 0.029). Conversely, we also observed a trend of higher granulocytic myeloid-derived suppressor cells abundance in NR compared to R (NR median %gMDSC of CD15+ cells 84.1% (range 73.4%-90.2%) ; R median %gMDSC 61.7% (range 54.3%-90.6%); p-value 0.069). In 2 cases with high and low TROP2 MFI available scRNAseq data showed TROP2 expression exclusively in tumor cells, with further analysis ongoing. Conclusion: By functionally profiling NSCLC PDOTS that retain immune cells, we correlated multiple parameters with ex vivo response to Dato-DXd. Our results have important implications for precision deployment of ADCs and suggest that target tumor antigen density in conjunction with addition immune cell features may refine patient selection strategy. Citation Format: Satoru Yasuda, Elena Ivanova, Ha V. Vo, Sophie Kivlehan, Iliana Gjeci, Minh Ha, Patrick Lizotte, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Functional profiling in lung tumor explants show contributions of the tumor microenvironment to response of Dato-DXdex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5086.

Abstract Background: Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable anti-tumor immunity. Dazostinag (TAK-676) is a novel systemically delivered STING agonist that ignites the innate immune system and mobilizes adaptive immunity. Preclinical studies with dazostinag demonstrated a strong, persistent CD8+ T cell anti-tumor response driven by innate immune derived type I IFN and pro-inflammatory cytokines. The role of CD8+ T cells in response to dazostinag was further supported by a previously demonstrated correlation between response and CD8+ T cell frequency in a patient-derived organotypic tumor spheroids (PDOTS) model of mesothelioma. Within the current work, we sought to determine the extent to which response to dazostinag in mesothelioma depends on CD8+ T cells versus the contribution of other immune cells. Methods: Surgical mesothelioma cases were collected along with clinicopathological variables at Brigham and Women’s Hospital under an IRB-approved protocol. PDOTS were generated as described previously. Ex vivo response was determined by fluorescent life/dead imaging, and orthogonally evaluated by multiplexed cytokine array, immunofluorescence, and flow cytometry; subset of tumors was studied using single cell RNA sequencing (scRNAseq). Results: Dazostinag-driven tumor cell killing was evaluated in 20 mesothelioma PDOTS. 11 samples were characterized as responders (R) with a cut-off of &amp;gt;30% reduction in live cell area; 9 as non-responders (NR). In 12 out of 20 PDOTS, CD8 neutralization effect was assessed: four were TAK-676 NR, and out of 8 R 4 had at least 20% killing abrogated by aCD8. Effect did not depend on the baseline amount of CD8+ T cells in the sample. scRNAseq analysis showed that R and NR T cells have different activation/exhaustion phenotypes and varying levels of interferon stimulated genes (ISG) in response to STING agonism. Other cell populations emerged as candidates for dazostinag resistance: NR cases carried remodeled immunosuppressive myeloid cells or matrix metalloproteinases-expressing fibroblasts. In general, myeloid cell abundance at baseline characterized by flow had a trend in negatively correlating with dazostinag response. Intrinsic expression of type I interferons shown by multiplexed cytokine array was linked to dazostinag sensitivity (R vs. NR IFN-β p=0.028; IFNα2a p=0.016) and can potentially be used as a predictor for response. Conclusion: We evaluated immune modulations in mesothelioma after treatment with a systemic STING agonist dazostinag using human tumor ex vivo culture. Innate interferon type I expression was higher in responding samples. TAK-676 efficacy was partially reduced in absence of effector lymphocytes, however other cells, such as immunosuppressive myeloid cells and fibroblasts, can have a negative impact on dazostinag response in mesothelioma. Citation Format: Elena Ivanova, Sung R. Park, Ari P. Zlota, Nathaniel Spicer, Minh Ha, Sophie Kivlehan, Iliana Gjeci, Simona Innocenti, Lauren Zasadil, Patrick Lizotte, Michael Y. Tolstorukov, Kai Ding, Adnan O. Abu-Yousif, Jeffrey Raizer, Vicky A. Appleman, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6733.

Abstract Background: Increasing the immunogenicity of solid tumors may potentiate durable anti-tumor immunity. Targeting innate pattern recognition receptor signaling is a promising strategy to repolarizing the suppressive tumor immune microenvironment (TME). A potential synergistic approach is the addition of cellular therapy. Tumor models expressing high levels of endogenous STING, but where the pathway is not active, were considered for evaluating STING agonism as a potential intervention. Additionally, prior work has demonstrated that STING induction is toxic to T cells; however, natural killer (NK) cells are unaffected, due to their differential regulation of autophagy. Here we investigate the immunomodulatory properties of STING agonism alone and in combination with NK cells using short-term microfluidic culture of fresh patient-derived organotypic tumor spheroids (PDOTS). Methods: Surgical cases from Brigham and Women’s Hospital under an IRB-approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by fluorescent live/dead imaging, immunofluorescence (IF), multiplexed cytokine array, and single cell RNA sequencing (scRNAseq). Baseline immune phenotypes were analyzed by FACS from single cells isolated during tumor sample preparation. Results: Twelve explants were studied. Two specimens were excluded from viability assessment due to excess of fibrotic material and low viability. Using a cut-off of -40%, three cases were characterized as responders (R) and seven as non-responders (NR). We observed significant induction of CXCL10 in all samples treated with STING agonist. IFN-β, IFN-γ, TNF-α, and MIP1-α were also consistently induced by STING agonist treatment. More detailed analysis of two samples by scRNAseq revealed a strong interferon stimulated genes (ISG) signature in STING agonist conditions; the cellular source of CXCL10 secretion in STING agonist treatment was tumor cells and fibroblasts. We also observed profound changes in myeloid biology, indicating that these cells polarized to a more pro-inflammatory and antigen-presenting phenotype with STING agonism. Interestingly, NK-based cell therapy added to PDOTS cultures displayed better persistence and enhanced effector function relative to endogenous NK cells, with and without STING agonism. Conclusions: Using tumor explants and a variety of orthogonal techniques, we investigated the response to STING agonism and NK cellular therapy ex vivo. Our results indicate that STING agonism remodels the TME, creating a more immune-permissive environment. STING agonism induces secretion of chemoattractant CXCL10 by tumor cells and fibroblasts. STING agonism does not abrogate NK cell effector function and therefore, may synergize with NK cell therapies. Citation Format: Patrick H. Lizotte, Elena Ivanova, Sung Park, Nathaniel Spicer, Sophie Kivlehan, Iliana Gjeci, Stefan Kiesgen, Vicky Appleman, LeeAnn Talarico, Michael Y. Tolstorukov, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5235.

&lt;p&gt;Figure S5 A, B. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 tumors harvested on day 16 post tumor inoculation. Number of cells per tumor mg are shown. (n=6 for esc1_Ctrl, n=8 for esc1_CCL5, representative data of two independent experiments.) C. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 DLN harvested on day 16 post tumor inoculation. (Left panel, n=12 for Ctrl, n=14 for esc1_CCL5, pooled data from two independent experiments. Right panel, n=18 for Ctrl, n=20 for esc1_CCL5. Pooled data from three independent experiments.) D, E. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 DLN harvested on day 16 post tumor inoculation. (n= 6 for Ctrl, n=8 for CCL5, representative data of two independent experiments.) F. Total cells (5*105cells/well) isolated from MOC1esc1_Ctrl or MOC1esc1_CCL5 DLNs were stimulated with indicated peptides for 48 hours and evaluated by IFN-γ ELISA. PC; (positive control (PMA + ionomycin)), NC; negative control (no peptides). (Left panel, n=8. Right panel, n=3, representative data of two independent experiments.) G. Flow cytometric analysis of MOC2_Ctrl and MOC2_CCL5 tumors harvested on day 10 post tumor inoculation. (n=8.)&lt;/p&gt;

&lt;p&gt;Figure S2 A. Bulk RNA-sequencing data from MOC22 tumors harvested on day 17, and MOC1 and MOC1-esc1 tumors harvested on day 14 post implantation are shown for indicated genes as z-score. (n=3 for each model.) B. qPCR data of Xcr1 from in vivo established tumors harvested on day 12 post-inoculation. n=16, pooled data from two independent experiments. C. Bulk RNA-seq data of indicated genes in pretreatment tumor samples from patients who received subsequent aPD1 therapy shown as z-score. “NK signature” represents the average z-score of GNLY, KLRC3, KLRD1, KLRF1, and NCR1 (10). (n=8 responders, n=15 non-responders.) D, E. Flow cytometric analysis of MOC22, MOC1P and MOC1esc1 tumors harvested on day 12 post-inoculation. (n=8 tumors each, representative data of two independent experiments.) F, G. Representative gating strategies for T and NK cells (F) and DCs (G) used throughout the paper&lt;/p&gt;

&lt;p&gt;Figure S3 A. Representative flow cytometry results of isolated Xcr1+ cDC1s used for DC vaccine experiments. B. Histogram of indicated marker expression gated on Xcr1+ cDC1s analyzed by flow cytometry. Xcr1+ cDCs were isolated and cultured with (=activated) or without (=no stimulation) PolyI:C (20 μg/ml) for 4 hours before staining. C, D. IFN-γ ELISA testing the ability of Xcr1+ DCs to activate CD8+ T cells. CD8+ cells isolated from OT-1 mouse splenocyte (OT1) were co-cultured with Xcr1+ cDC1s (DC), with or without stimulation of ovalbumin short peptide (short), ovalbumin synthetic long peptide (SLP) in Figure S3C and MOC1esc1-OVA (full length) cell lysate (MOC1esc1OVA) in Figure S3D. n=2-3. E, F. Flow cytometric analysis of MOC1esc1 tumors (E) and DLNs (F) treated with intra-tumoral PBS or DC vaccine on days 1/4/7 post inoculation, and harvested on day 14 post tumor inoculation. (n=8, representative data of two independent experiments.) G. Tumor weight measured on last day of experiment in Figure 3E. H. Photo of tumors harvested in experiment shown in Figure 3E&lt;/p&gt;

&lt;p&gt;Figure S1 A. Xcr1+ DCs in DLNs of MOC1P-ova/MOC1esc1-ova harvested 10 days post inoculation were analyzed by flow cytometry. Total number of cells per LN and number of cells per 10,000 CD45+ cells are shown. (n=5, representative data of two independent experiments.) B. Representative gating strategies for Figure 1B, C. Individual data and mean ± SD are plotted in Figure S1A. Data were analyzed using the Mann–Whitney U Test to generate two-tailed P values in Figure S1A&lt;/p&gt;

&lt;p&gt;Figure S4. A. Heatmap of CXC chemokines ligands (CXCL) based on pretreatment bulk RNA-seq data between responders and non-responders. B. CCL5 expression in each MOC model cultured media with or without IFN-γ stimulation (100U/ml for 48 hours) were analyzed by CCL5-ELISA. n=2-3. C. p value and r squared value calculated from Pearson correlation coefficient of cDC score and chemokine RNA expression are plotted. Broken line represents a p value of 0.05. D. Number of Xcr1+ DCs migrated using the migration assay described in Methods. E. Distribution plots showing cumulative expression levels of CCL5 in various immune subtypes other than CD8+ T cells, based on scRNA-seq of HNSCC patients prior to pembrolizumab. Left panel responders, right panel non-responders. F, G. Comparison of CCL5 expression in bulk RNA-seq data from pre and post treatment samples in responders (F) and non-responders (G), shown as z-score. n=6. H. Violin plot showing CCL5 expression level in human dendritic cells between responders pre- and post-treatment based on scRNA-seq data&lt;/p&gt;

&lt;p&gt;Figure S5 A, B. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 tumors harvested on day 16 post tumor inoculation. Number of cells per tumor mg are shown. (n=6 for esc1_Ctrl, n=8 for esc1_CCL5, representative data of two independent experiments.) C. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 DLN harvested on day 16 post tumor inoculation. (Left panel, n=12 for Ctrl, n=14 for esc1_CCL5, pooled data from two independent experiments. Right panel, n=18 for Ctrl, n=20 for esc1_CCL5. Pooled data from three independent experiments.) D, E. Flow cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 DLN harvested on day 16 post tumor inoculation. (n= 6 for Ctrl, n=8 for CCL5, representative data of two independent experiments.) F. Total cells (5*105cells/well) isolated from MOC1esc1_Ctrl or MOC1esc1_CCL5 DLNs were stimulated with indicated peptides for 48 hours and evaluated by IFN-γ ELISA. PC; (positive control (PMA + ionomycin)), NC; negative control (no peptides). (Left panel, n=8. Right panel, n=3, representative data of two independent experiments.) G. Flow cytometric analysis of MOC2_Ctrl and MOC2_CCL5 tumors harvested on day 10 post tumor inoculation. (n=8.)&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Neoadjuvant anti-PD1 (aPD1) therapies are being explored in surgically resectable head and neck squamous cell carcinoma (HNSCC). Encouraging responses have been observed, but further insights into the mechanisms underlying resistance and approaches to improve responses are needed.&lt;/p&gt;Experimental Design:&lt;p&gt;We integrated data from syngeneic mouse oral carcinoma (MOC) models and neoadjuvant pembrolizumab HNSCC patient tumor RNA-sequencing data to explore the mechanism of aPD1 resistance. Tumors and tumor-draining lymph nodes (DLN) from MOC models were analyzed for antigen-specific priming. CCL5 expression was enforced in an aPD1-resistant model.&lt;/p&gt;Results:&lt;p&gt;An aPD1-resistant mouse model showed poor priming in the tumor DLN due to type 1 conventional dendritic cell (cDC1) dysfunction, which correlated with exhausted and poorly responsive antigen-specific T cells. Tumor microenvironment analysis also showed decreased cDC1 in aPD1-resistant tumors compared with sensitive tumors. Following neoadjuvant aPD1 therapy, pathologic responses in patients also positively correlated with baseline transcriptomic cDC1 signatures. In an aPD1-resistant model, intratumoral cDC1 vaccine was sufficient to restore aPD1 response by enhancing T-cell infiltration and increasing antigen-specific responses with improved tumor control. Mechanistically, CCL5 expression significantly correlated with neoadjuvant aPD1 response and enforced expression of CCL5 in an aPD1-resistant model, enhanced cDC1 tumor infiltration, restored antigen-specific responses, and recovered sensitivity to aPD1 treatment.&lt;/p&gt;Conclusions:&lt;p&gt;These data highlight the contribution of tumor-infiltrating cDC1 in HNSCC aPD1 response and approaches to enhance cDC1 infiltration and function that may circumvent aPD1 resistance in patients with HNSCC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Figure S1 A. Xcr1+ DCs in DLNs of MOC1P-ova/MOC1esc1-ova harvested 10 days post inoculation were analyzed by flow cytometry. Total number of cells per LN and number of cells per 10,000 CD45+ cells are shown. (n=5, representative data of two independent experiments.) B. Representative gating strategies for Figure 1B, C. Individual data and mean ± SD are plotted in Figure S1A. Data were analyzed using the Mann–Whitney U Test to generate two-tailed P values in Figure S1A&lt;/p&gt;

&lt;p&gt;Figure S4. A. Heatmap of CXC chemokines ligands (CXCL) based on pretreatment bulk RNA-seq data between responders and non-responders. B. CCL5 expression in each MOC model cultured media with or without IFN-γ stimulation (100U/ml for 48 hours) were analyzed by CCL5-ELISA. n=2-3. C. p value and r squared value calculated from Pearson correlation coefficient of cDC score and chemokine RNA expression are plotted. Broken line represents a p value of 0.05. D. Number of Xcr1+ DCs migrated using the migration assay described in Methods. E. Distribution plots showing cumulative expression levels of CCL5 in various immune subtypes other than CD8+ T cells, based on scRNA-seq of HNSCC patients prior to pembrolizumab. Left panel responders, right panel non-responders. F, G. Comparison of CCL5 expression in bulk RNA-seq data from pre and post treatment samples in responders (F) and non-responders (G), shown as z-score. n=6. H. Violin plot showing CCL5 expression level in human dendritic cells between responders pre- and post-treatment based on scRNA-seq data&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Neoadjuvant anti-PD1 (aPD1) therapies are being explored in surgically resectable head and neck squamous cell carcinoma (HNSCC). Encouraging responses have been observed, but further insights into the mechanisms underlying resistance and approaches to improve responses are needed.&lt;/p&gt;Experimental Design:&lt;p&gt;We integrated data from syngeneic mouse oral carcinoma (MOC) models and neoadjuvant pembrolizumab HNSCC patient tumor RNA-sequencing data to explore the mechanism of aPD1 resistance. Tumors and tumor-draining lymph nodes (DLN) from MOC models were analyzed for antigen-specific priming. CCL5 expression was enforced in an aPD1-resistant model.&lt;/p&gt;Results:&lt;p&gt;An aPD1-resistant mouse model showed poor priming in the tumor DLN due to type 1 conventional dendritic cell (cDC1) dysfunction, which correlated with exhausted and poorly responsive antigen-specific T cells. Tumor microenvironment analysis also showed decreased cDC1 in aPD1-resistant tumors compared with sensitive tumors. Following neoadjuvant aPD1 therapy, pathologic responses in patients also positively correlated with baseline transcriptomic cDC1 signatures. In an aPD1-resistant model, intratumoral cDC1 vaccine was sufficient to restore aPD1 response by enhancing T-cell infiltration and increasing antigen-specific responses with improved tumor control. Mechanistically, CCL5 expression significantly correlated with neoadjuvant aPD1 response and enforced expression of CCL5 in an aPD1-resistant model, enhanced cDC1 tumor infiltration, restored antigen-specific responses, and recovered sensitivity to aPD1 treatment.&lt;/p&gt;Conclusions:&lt;p&gt;These data highlight the contribution of tumor-infiltrating cDC1 in HNSCC aPD1 response and approaches to enhance cDC1 infiltration and function that may circumvent aPD1 resistance in patients with HNSCC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Figure S3 A. Representative flow cytometry results of isolated Xcr1+ cDC1s used for DC vaccine experiments. B. Histogram of indicated marker expression gated on Xcr1+ cDC1s analyzed by flow cytometry. Xcr1+ cDCs were isolated and cultured with (=activated) or without (=no stimulation) PolyI:C (20 μg/ml) for 4 hours before staining. C, D. IFN-γ ELISA testing the ability of Xcr1+ DCs to activate CD8+ T cells. CD8+ cells isolated from OT-1 mouse splenocyte (OT1) were co-cultured with Xcr1+ cDC1s (DC), with or without stimulation of ovalbumin short peptide (short), ovalbumin synthetic long peptide (SLP) in Figure S3C and MOC1esc1-OVA (full length) cell lysate (MOC1esc1OVA) in Figure S3D. n=2-3. E, F. Flow cytometric analysis of MOC1esc1 tumors (E) and DLNs (F) treated with intra-tumoral PBS or DC vaccine on days 1/4/7 post inoculation, and harvested on day 14 post tumor inoculation. (n=8, representative data of two independent experiments.) G. Tumor weight measured on last day of experiment in Figure 3E. H. Photo of tumors harvested in experiment shown in Figure 3E&lt;/p&gt;

&lt;p&gt;Figure S2 A. Bulk RNA-sequencing data from MOC22 tumors harvested on day 17, and MOC1 and MOC1-esc1 tumors harvested on day 14 post implantation are shown for indicated genes as z-score. (n=3 for each model.) B. qPCR data of Xcr1 from in vivo established tumors harvested on day 12 post-inoculation. n=16, pooled data from two independent experiments. C. Bulk RNA-seq data of indicated genes in pretreatment tumor samples from patients who received subsequent aPD1 therapy shown as z-score. “NK signature” represents the average z-score of GNLY, KLRC3, KLRD1, KLRF1, and NCR1 (10). (n=8 responders, n=15 non-responders.) D, E. Flow cytometric analysis of MOC22, MOC1P and MOC1esc1 tumors harvested on day 12 post-inoculation. (n=8 tumors each, representative data of two independent experiments.) F, G. Representative gating strategies for T and NK cells (F) and DCs (G) used throughout the paper&lt;/p&gt;

8555 Background: Defactinib is an oral Focal Adhesion Kinase (FAK) inhibitor with preclinical activity in MPM. We assessed responses to defactinib treatment prior to planned surgical resection in naive patients with MPM. Methods: Three cohorts of 10 participants each received defactinib 400mg BID for 12, 35 and 21 days. Pre- and post-treatment blood, tumor biopsies and imaging were obtained for biomarker, immune cell and tumor response (modified RECIST, Tumor volume and SUV max) assessment. Toxicity was monitored for 30 days post treatment. Results: Between 12/2013 and 12/2017, 31 participants were registered at our center; 1 withdrew prior to intervention. Among 30 treated, 24 (80%) were male; median age 70 (47-83) years; surgery was EPP 7%, complete pleurectomy decortication (PD) 10%, extended PD 60%, partial PD 10%, unresectable 13%; MPM subtype was epithelioid 67%, biphasic 17%, sarcomatoid 17%. Expected complications of FAK inhibition, diagnostic/staging/operative procedures occurred in 83% (grade 1, 30%; grade 2, 43%; grade 3, 10%). Unexpected adverse events occurred in 77% (grade 1, 63%; grade 2, 20%; grade 3, 17% [wound-infection, prolonged QT interval, and hyperglycemia in 3% each; increased INR in 7%]; grade 5, 7% [due to progressive disease in 3%, intraoperative anaphylactoid reaction unrelated to the drug in 3%]). Objective partial response was observed in 13%, stable disease in 67%, progression in 17%. Tumor volume decreased 3-72% in 47% patients and increased 1-82% in 53%. SUV max decreased 3-69% in 50% and increased 1-61% in 50%. Biological correlates of treatment included target inhibition (75% pFAK reduction); tumor immune microenvironment changes: increased naïve (CD45RA+PD-1+CD69+) CD4 and CD8 T cells, reduced myeloid and Treg immuno-suppressive cells, reduced exhausted T cells (PD-1+CD69+), reduced peripheral MDSCs; and histological subtype change (pleomorphic or biphasic to epithelioid) in 13% of cases. Conclusions: Brief preoperative defactinib exposure was well tolerated, did not alter resectability or mortality compared to prior series, and showed evidence of therapeutic and immunomodulatory effects. Clinical trial information: NCT02004028.

Scientific Data 1:140035 doi: 10.1038/sdata.2014.35 (2014); Published 30 September 2014; Updated 11 November 2014 The original version of this Data Descriptor contained a typographical error in the spelling of the author Terence C. Wong, which was incorrectly given as Terrence C. Wong. This has now been corrected in the PDF and HTML versions of the Data Descriptor.

6053 Background: Locoregional recurrence (LRR) is a major cause of death for patients (pts) with squamous cell carcinoma of the head and neck (SCCHN). With therapy options limited by prior treatment, surgery often represents the best chance for disease control. Emerging data suggests a role for neoadjuvant immunotherapy in upfront resectable SCCHN and the importance of NK cells in the tumor microenvironment. We hypothesized that dual immune checkpoint inhibition (anti-PD-1, nivolumab [N] and anti-KIR, lirilumab [L]) before and after salvage surgery would improve 1-year disease-free survival (DFS). Methods: Pts with operable LRR of SCCHN (any HPV or smoking status) with a disease-free interval of &gt; 8 weeks after curative intent therapy were eligible for this phase II trial. Pts received a single dose of pre-op N (240 mg) + L (240 mg) 7-21 days before surgery, followed by 6-cycles of adjuvant N+L on days 1, 15 (N alone) of a 28-day cycle (C) for C1-3; and on day 1 for C4-6. Primary endpoint was 1-year DFS; 37 DFS events among N = 54 pts provided 81% power to detect improvement in 1-year DFS from 57% to 67.5% (one-sided 10% Wald’s test). Secondary endpoints: safety, radiologic response (RECIST v1.1) to pre-op N+L, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 status, and immunoprofiling. Results: Between 3/15/18 and 5/29/20, N = 29 enrolled (stopped due to expiration of drug supply). Among 28 treated pts, median age: 66, 18% (5/28) women, 83% smokers; primary site: 10 oral cavity, 8 oropharynx (5/8 HPV+), and 10 larynx/hypopharynx. 96% (27/28) had prior HN radiation; 71% (20/28) prior chemotherapy. There were no delays to surgery. Grade 3+ adverse events: 11% (3/28); no deaths from treatment. At time of surgery, 96% (27/28) had stable disease radiologically with 3 showing regression, 4% (1/28) had disease progression. Pathologic response to N+L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤10%); 8/28 (29%) partial (TV ≤50%). PD-L1 CPS at surgery was similar regardless of pathologic response (p = 0.63). 68% (19/28) completed all 6-cycles of adjuvant N+L; N = 1 came off for toxicity. Ten pts (36%) recurred (local = 8, distant = 2). 5/28 (18%) had positive margins, of which 4 (80%) recurred; 4/28 (14%) declined to start adjuvant N+L, of which 3 (75%) later recurred. At median follow-up of 20.2 months, 1-year DFS70% (95%CI, 48-84%) and 1-year OS: 85% (95%CI, 65-94%). Median tumor mutational burden was 4 (range, 1-11). TP53 was the most frequent alteration (78%, 21/27). CD39 expression by TILs and CD38 expression by circulating CD4/8+ T cells increased after N+L exposure (p &lt; 0.05). Conclusions: Neoadjuvant and adjuvant N+L was safe and well tolerated. We observed a 43% pathologic response rate prior to salvage surgery, with a favorable 1-year DFS of 70% and 1-year OS &gt; 80% among previously irradiated pts. Further evaluation of this strategy is warranted (NCT03341936).

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

8549 Background: Treatment options are limited for patients (pts) with MPM who experience disease progression after first-line pemetrexed-based chemotherapy. This study was designed to explore the activity of combined CTLA-4 + PD-L1 immune checkpoint inhibition using tremelimumab plus durvalumab in previously-treated MPM. Methods: We conducted a phase 2 study of tremelimumab 75 mg plus durvalumab 1500 mg administered intravenously every 4 weeks for four cycles followed by durvalumab maintenance every 4 weeks. Eligible pts had previously received pemetrexed-based platinum doublet chemotherapy and had measurable disease using modified RECIST criteria for mesothelioma. The primary endpoint was overall response rate (ORR) and secondary endpoints were progression-free survival (PFS), overall survival (OS), and duration of response (DoR) as well as safety and tolerability of this combination. A Simon two-stage design was employed to enroll up to 40 patients if 4 or more responses were observed among the first 19 study patients. Pre-treatment, on-treatment, and optional post-progression biopsies underwent flow cytometric immunoprofiling for correlative studies. Results: Among 19 pts enrolled in this study, the best objective response was a confirmed partial response in one patient (5%), stable disease in 9 pts (47%), progressive disease in 8 pts (42%), and not evaluable in one patient. At a median follow-up of 7.1 months, the median PFS was 2.8 months (95% CI 2.04-5.72), and the median OS was 7.8 months (95% CI 6.24-not reached). Of 17 PD-L1 evaluable cases, 10 (59%) were PD-L1 negative, and 7 (41%) had a PD-L1 tumor proportion score of ≥1%. Treatment was generally well-tolerated and there were no treatment-related study discontinuations or deaths. Flow cytometric immunologic changes over the course of treatment associated with disease control will be presented. Conclusions: Tremelimumab + durvalumab was well-tolerated in unselected pts with previously-treated MPM. This study did not meet its primary endpoint. Additional strategies are necessary to develop novel immunotherapeutics and biomarkers of response in MPM. Clinical trial information: NCT03075527.

Abstract Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA (dsDNA) sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, NK therapies or immune checkpoint blockade. Significance statement Modulation of the immune microenvironment is critical for immunosuppressive and therapy refractory tumors like glioblastoma. Activation of the STING pathway deeply remodels the brain tumor environment and attracts innate immune cells and natural killer cell populations, producing a robust antitumor effect with long-term immune memory. We further show that human glioblastoma tissue can respond to the therapy and lay the foundations for combined intracranial immunotherapies by using crosslinked biodegradable brain implants.

Summary paragraph Programming T lymphocytes to distinguish self from non-self is a vital, multi-step process arising in the thymus 1–4 . Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, constitutes a critical early checkpoint in thymocyte development within the αβ T-cell lineage 5, 6 . Recent work demonstrates that preTCRs arrayed on double negative (DN) thymocytes, like αβ TCRs appearing on double positive (DP) thymocytes, ligate peptides bound to MHC molecules (pMHC) on thymic stroma but via a different molecular docking strategy 7–10 . Here we show the consequences of those distinctive interactions for thymocyte progression, using synchronized fetal thymic progenitor cultures differing in the presence or absence of pMHC on support stroma, determining single cell transcriptomes at key thymocyte developmental transitions. Although MHC negative stroma fosters αβ T lymphocyte differentiation, the absence of pMHC-preTCR interplay leads to deviant thymocyte transcriptional programming associated with de-differentiation. Highly proliferative DN and DP subsets with antecedent characteristics of T cell lymphoblastic and myeloid malignancies emerge. Thus, at least in vitro , beyond fostering β chain repertoire broadening for subsequent αβ TCR utilization, preTCR-pMHC interaction limits cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduces significant developmental vulnerabilities.

Abstract As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells. Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4935.

Abstract Immune checkpoint blockade improves survival in a subset of patients with non-small cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. To develop a more comprehensive knowledge of the NSCLC tumor immune landscape, we performed comprehensive flow-cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). Cytometric profiling identified two main tumor immunophenotypes: an immunologically “hot” cluster with high abundance of CD8+ T cells expressing high levels of the inhibitory receptors PD-1 and TIM-3, and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function by mRNA analysis, and high PD-L1 expression by IHC. Within the “hot” cluster there was a small subgroup with high granulocytic infiltrates and lower relative T cell abundance, but with high expression of inhibitory receptors by T cells. There was no correlation between immunophenotype and KRAS or EGFR mutation status, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, ∼20% of cases had high B cell infiltrates and we demonstrate that a subset of these cells display characteristics of immunosuppressive IL-10-producing cells. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. Citation Format: Patrick Lizotte, Elena Ivanova, Mark Bittinger, Kwok-Kin Wong. Multi-parametric profiling of non-small cell lung cancers reveals distinct immunophenotypes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A132.

&lt;p&gt;PDF file - 256K, Supplementary Figure S3. Gating strategy for figure 2.&lt;/p&gt;

&lt;p&gt;PDF file - 167K, Supplementary Figure S4. Th17 cells do not play a significant role in the treatment by cps of ID8-Defb29/Vegf-A ovarian cancer model.&lt;/p&gt;

&lt;p&gt;PDF file - 135K, Supplementary Figure S1. CD8a+CD11c+DEC205+MHCII+ are the predominant CD45+ MHC II+ APC's in terminal ascites and can be stimulated to efficiently present antigens.&lt;/p&gt;

&lt;p&gt;PDF file - 140K, Supplementary Figure S2. Ex vivo application of cps to terminal ascites increases expression of CD80 and CD86 on CD11c+ cells and increases expression of CD69 on T cell.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using &lt;i&gt;cps&lt;/i&gt;, an avirulent, nonreplicating uracil auxotroph strain of the parasite &lt;i&gt;Toxoplasma gondii&lt;/i&gt; (&lt;i&gt;T. gondii&lt;/i&gt;), which preferentially invades immunosuppressive CD11c&lt;sup&gt;+&lt;/sup&gt; antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c&lt;sup&gt;+&lt;/sup&gt; cells invaded by &lt;i&gt;cps&lt;/i&gt; were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to &lt;i&gt;cps&lt;/i&gt; treatment of the immunosuppressive ovarian tumor environment, CD11c&lt;sup&gt;+&lt;/sup&gt; cells regained the ability to efficiently cross-present antigen and prime CD8&lt;sup&gt;+&lt;/sup&gt; T-cell responses. Correspondingly, &lt;i&gt;cps&lt;/i&gt; treatment markedly increased tumor antigen-specific responses by CD8&lt;sup&gt;+&lt;/sup&gt; T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal &lt;i&gt;cps&lt;/i&gt; treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of &lt;i&gt;cps&lt;/i&gt; treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with &lt;i&gt;T. gondii&lt;/i&gt;. Taken together, our results establish that &lt;i&gt;cps&lt;/i&gt; preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8&lt;sup&gt;+&lt;/sup&gt; T-cell responses. Immunochemotherapeutic applications of &lt;i&gt;cps&lt;/i&gt; might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors. &lt;i&gt;Cancer Res; 73(13); 3842–51. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using &lt;i&gt;cps&lt;/i&gt;, an avirulent, nonreplicating uracil auxotroph strain of the parasite &lt;i&gt;Toxoplasma gondii&lt;/i&gt; (&lt;i&gt;T. gondii&lt;/i&gt;), which preferentially invades immunosuppressive CD11c&lt;sup&gt;+&lt;/sup&gt; antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c&lt;sup&gt;+&lt;/sup&gt; cells invaded by &lt;i&gt;cps&lt;/i&gt; were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to &lt;i&gt;cps&lt;/i&gt; treatment of the immunosuppressive ovarian tumor environment, CD11c&lt;sup&gt;+&lt;/sup&gt; cells regained the ability to efficiently cross-present antigen and prime CD8&lt;sup&gt;+&lt;/sup&gt; T-cell responses. Correspondingly, &lt;i&gt;cps&lt;/i&gt; treatment markedly increased tumor antigen-specific responses by CD8&lt;sup&gt;+&lt;/sup&gt; T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal &lt;i&gt;cps&lt;/i&gt; treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of &lt;i&gt;cps&lt;/i&gt; treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with &lt;i&gt;T. gondii&lt;/i&gt;. Taken together, our results establish that &lt;i&gt;cps&lt;/i&gt; preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8&lt;sup&gt;+&lt;/sup&gt; T-cell responses. Immunochemotherapeutic applications of &lt;i&gt;cps&lt;/i&gt; might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors. &lt;i&gt;Cancer Res; 73(13); 3842–51. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 256K, Supplementary Figure S3. Gating strategy for figure 2.&lt;/p&gt;

&lt;p&gt;PDF file - 135K, Supplementary Figure S1. CD8a+CD11c+DEC205+MHCII+ are the predominant CD45+ MHC II+ APC's in terminal ascites and can be stimulated to efficiently present antigens.&lt;/p&gt;

&lt;p&gt;PDF file - 167K, Supplementary Figure S4. Th17 cells do not play a significant role in the treatment by cps of ID8-Defb29/Vegf-A ovarian cancer model.&lt;/p&gt;

&lt;p&gt;PDF file - 140K, Supplementary Figure S2. Ex vivo application of cps to terminal ascites increases expression of CD80 and CD86 on CD11c+ cells and increases expression of CD69 on T cell.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Immunotherapy has shown limited efficacy in patients with &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancer. Efforts to enhance the immunogenicity of &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (&lt;a href="#bib1" target="_blank"&gt;1&lt;/a&gt;). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of &lt;i&gt;FOSL1&lt;/i&gt;. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8&lt;sup&gt;+&lt;/sup&gt; T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancers and promote immunogenicity.&lt;/p&gt;Significance:&lt;p&gt;MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Immunotherapy has shown limited efficacy in patients with &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancer. Efforts to enhance the immunogenicity of &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (&lt;a href="#bib1" target="_blank"&gt;1&lt;/a&gt;). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of &lt;i&gt;FOSL1&lt;/i&gt;. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8&lt;sup&gt;+&lt;/sup&gt; T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant &lt;i&gt;EGFR&lt;/i&gt;-mutated lung cancers and promote immunogenicity.&lt;/p&gt;Significance:&lt;p&gt;MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.&lt;/p&gt;&lt;/div&gt;

Supplementary Data from MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer

Supplementary Data from MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer

&lt;p&gt;Droplet digital PCR (ddPCR) detected the KRAS mutation in peripheral blood cells (PBC, A) but did not detect the EGFR driver mutation (B). Highly sensitive next-generation sequencing (NGS, C) confirmed the presence of the KRAS G12D mutation in PBC and cellular subpopulations (D) at a similar allelic fraction (AF) as found with ddPCR.&lt;/p&gt;

&lt;p&gt;Figures S1-9&lt;/p&gt;

&lt;p&gt;Cell lines models&lt;/p&gt;

&lt;p&gt;CRISPR/Cas9 guides, primers and sequencing results&lt;/p&gt;

&lt;p&gt;Antibodies used for Immunohistochemistry, Immunofluroesence and immunblotting&lt;/p&gt;

&lt;p&gt;Pathway enrichment of differentially expressed genes (DEG) between mouse LSCC and LADC.&lt;/p&gt;

&lt;p&gt;qRT-PCR primer sequences&lt;/p&gt;

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

&lt;p&gt;Droplet digital PCR (ddPCR) of the peripheral blood cell DNA from 4 cases with JAK2 V617F found on plasma next-generation sequencing (NGS). In each, the JAK2 mutation was detected (top), consistent with clonal hematopoiesis, while not detected in a control sample.&lt;/p&gt;

&lt;p&gt;Droplet digital PCR (ddPCR) detected the KRAS mutation in peripheral blood cells (PBC, A) but did not detect the EGFR driver mutation (B). Highly sensitive next-generation sequencing (NGS, C) confirmed the presence of the KRAS G12D mutation in PBC and cellular subpopulations (D) at a similar allelic fraction (AF) as found with ddPCR.&lt;/p&gt;

&lt;p&gt;qRT-PCR primer sequences&lt;/p&gt;

&lt;p&gt;Cell lines models&lt;/p&gt;

&lt;p&gt;Pathway enrichment of differentially expressed genes (DEG) between mouse LSCC and LADC.&lt;/p&gt;

&lt;p&gt;Antibodies used for Immunohistochemistry, Immunofluroesence and immunblotting&lt;/p&gt;

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck