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Radiographic imaging is the standard approach for evaluating the disease involvement of lymph nodes in patients with operable NSCLC although the impact of neoadjuvant immune checkpoint inhibitors (ICIs) on lymph nodes has not yet been characterized. Herein, we present an ad hoc analysis of the NEOSTAR trial (NCT03158129) where we observed a phenomenon we refer to as “nodal immune flare” (NIF) in which patients treated with neoadjuvant ICIs demonstrate radiologically abnormal nodes post-therapy that upon pathological evaluation are devoid of cancer and demonstrate de novo non-caseating granulomas. Abnormal lymph nodes are analyzed by computed tomography and 18F-fluorodeoxyglucose positron emission tomography/computer tomography to evaluate the size and the maximum standard uptake value post- and pre-therapy in NEOSTAR and an independent neoadjuvant chemotherapy cohort. NIF occurs in 16% (7/44) of patients treated with ICIs but in 0% (0/28) of patients after neoadjuvant chemotherapy. NIF is associated with an inflamed nodal immune microenvironment and with fecal abundance of genera belonging to the family Coriobacteriaceae of phylum Actinobacteria, but not with tumor responses or treatment-related toxicity. Our findings suggest that this apparent radiological cancer progression in lymph nodes may occur due to an inflammatory response after neoadjuvant immunotherapy, and such cases should be evaluated by pathological examination to distinguish NIF from true nodal progression and to ensure appropriate clinical treatment planning., Granulomatous/sarcoid-like lesions have been reported in patients treated with immune checkpoint inhibitors (ICIs). Here the authors report the occurrence of “nodal immune flare”, an apparent radiological cancer progression in the nodes characterized by the absence of cancer and the presence of non-caseating granulomas, in patients with non-small cell lung cancer following neoadjuvant ICI treatment.

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rβ). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies., Interleukin-2 can induce an anti-tumour response, but is associated with toxicity. Here, the authors demonstrate that an engineered interleukin-2 promotes intratumoral T regulatory cell depletion while enhancing effective anti-tumour CD8+ T cell responses that result in potent tumor suppression.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

This single-arm, phase I dose-escalation trial (NCT02983045) evaluated bempegaldesleukin (NKTR-214/BEMPEG), a CD122-preferential IL2 pathway agonist, plus nivolumab in 38 patients with selected immunotherapy-naïve advanced solid tumors (melanoma, renal cell carcinoma, and non–small cell lung cancer). Three dose-limiting toxicities were reported in 2 of 17 patients during dose escalation [hypotension (n = 1), hyperglycemia (n = 1), metabolic acidosis (n = 1)]. The most common treatment-related adverse events (TRAE) were flu-like symptoms (86.8%), rash (78.9%), fatigue (73.7%), and pruritus (52.6%). Eight patients (21.1%) experienced grade 3/4 TRAEs; there were no treatment-related deaths. Total objective response rate across tumor types and dose cohorts was 59.5% (22/37), with 7 complete responses (18.9%). Cellular and gene expression analysis of longitudinal tumor biopsies revealed increased infiltration, activation, and cytotoxicity of CD8+ T cells, without regulatory T-cell enhancement. At the recommended phase II dose, BEMPEG 0.006 mg/kg plus nivolumab 360 mg every 3 weeks, the combination was well tolerated and demonstrated encouraging clinical activity irrespective of baseline PD-L1 status. Significance: These data show that BEMPEG can be successfully combined with a checkpoint inhibitor as dual immunotherapy for a range of advanced solid tumors. Efficacy was observed regardless of baseline PD-L1 status and baseline levels of tumor-infiltrating lymphocytes, suggesting therapeutic potential for patients with poor prognostic risk factors for response to PD-1/PD-L1 blockade. See related commentary by Rouanne et al., p. 1097. This article is highlighted in the In This Issue feature, p. 1079

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy. Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL). Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti–PD-1–treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3′ untranslated region (UTR) of HLA-A to destabilize the mRNA. Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3′ UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366–76. ©2018 AACR. See related commentary by Kalbasi and Ribas, p. 3239

Background: The ImmunogenomiC prOfiling of Non-small cell lung cancer (NSCLC) Project (ICON) represents an ambitious undertaking to comprehensively characterize immuno-genomic diversity in NSCLC across diverse platforms. The depth and breadth of this cohort presented a unique opportunity to develop a specialized method for multi-platform data integration and exploration, which can be broadly applied to forthcoming large-scale patient profiling studies. Such a holistic approach can unlock insights for therapeutic targets, biomarkers, and treatment plans by providing a more complete view of phenomena driving disease pathogenesis and evolution. Purpose: We developed a novel shared nearest neighbors (SNN) approach to create an integrated network of ICON’s multi-platform data and identified collections of closely related measurements within the resulting network tied to noteworthy patient characteristics, including recurrence and oncogenotype. Methods: The ICON dataset is derived from tumor and normal lung tissue samples collected from 150 patients at time of resection as well as blood samples collected then and at intervals during the year following. Tissue samples underwent RNA-sequencing (RNA-seq), whole exome sequencing, T-cell receptor sequencing, multiplex immunofluorescence for immune cells, and reverse phase protein array profiling; flow cytometry for immune cells was performed on tissue and blood samples. From these data, the ICON data network was built using an integrative approach based on the SNN algorithm in which genes were linked on the basis of their shared top correlates in orthogonal datasets. Results: The ICON data network currently includes over 20,000 genes linked by over 500,000 connections derived from correlations between RNA-seq and orthogonal platforms. We captured established associations between cancer-related genes and examined these along with new ones in the network. To do so, we used the InfoMap algorithm to extract more interpretable sub-networks, termed modules, from the ICON data network. Single sample gene set enrichment scores for each module were used in multivariate analysis to highlight modules linked to clinical characteristics of interest. As an example, we found modules significantly tied to disease recurrence. The most notable of these was strongly associated with metabolic pathways, and other modules associated with platelets and ion channels were also identified. The metabolic pathway module is being explored as a prognostic biomarker, underscoring the opportunites enabled by mining the network. Conclusions: Through the framework developed, we identified modules in the ICON data network significantly associated with important patient characteristics like recurrence and oncogenotype. We are validating the gene sets identified as potential biomarkers and are developing an interactive application to facilitate further mining of the network. Taken together, our SNN network-building approach enables the integration and exploration of patient data from diverse platforms. Citation Format: Stephanie T. Schmidt, Neal Akhave, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Natalie Vokes, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Timothy P. Heffernan, Marcelo V. Negrao, Don L. Gibbons. A shared nearest neighbors approach for integrated, multi-platform networks and its application to the exploration of multiomics data from early-stage non-small cell lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P009.

BackgroundPancreatic cancer (PaC) is immune-quiescent and resistant to single-agent checkpoint inhibitor (CPI). NT-I7 (efineptakin alfa) is the first-in-class long-acting IL-7 that can increase T-cell infiltration in the tumor microenvironment (TME) and may enhance tumor responsiveness to CPI therapy. We hypothesize that the combination of NT-I7 and pembrolizumab may result in enhanced efficacy in CPI-naïve advanced PaC.MethodsThis is an open-label, phase 2a, study in subjects with relapsed/refractory (R/R) tumors, including CPI-naïve R/R PaC. Subjects received NT-I7 intramuscularly at 1200 µg/kg every 6 weeks (Q6W) plus pembro 200 mg intravenously Q3W. Antitumor activity based on Overall Response Rate (ORR) was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Biomarker analyses of peripheral blood and tumor biopsies were performed.ResultsAs of 15-July-2021, 26 subjects were enrolled in the CPI naïve R/R PaC cohort. Median age 69 years [31–81], ECOG PS 0 (35%), 1 (65%). Twenty-one (81%) subjects had ≥ 2 prior therapies All subjects had metastatic or locally advanced disease at enrollment. The median duration of follow-up was 3.3 months. Among 10 subjects with at least 1 post-treatment tumor assessment, the RECIST1.1-based ORR and disease-control rate (DCR) were 10% and 50%, respectively. One subject with MSS and TMB of 1, achieved a confirmed partial response (cPR) with 65% tumor reduction and drastically improving CA19-9. Treatment-related adverse events (AEs) occurred in 14 (53.8%) subjects, 9 (34.6%) G1–2, 3 (11.5%) G3; 2 (7.7%) G4; no G5 AEs were reported. No subjects discontinued from treatment due to AE. NT-I7 + pembro elicited a significant increase in the peripheral absolute lymphocyte count that peaked at week 3 (>3X from baseline, p15X, pConclusionsThe chemo-free combination of NT-I7 + pembro was well tolerated and showed promising anti-tumor activity in subjects with CPI-naïve R/R PaC. Increased TSCM and CD8+ T-cell infiltration within TME may be the underlying mechanisms of action for the observed efficacy. These results support continued evaluation of NT-I7 + pembro in subjects with CPI-naïve R/R PaC.AcknowledgementsThe authors thank ICON for their partnership in conducting this trial.Trial RegistrationNCT04332653Ethics ApprovalThe trial was approved by MD Anderson IRB (#2020–0008_MOD001), Mary Crowley IRB (#20–13) and Advarra IRB (#Pro00042639)All participant gave informed consent prior to study enrollment.

BackgroundWhile immune checkpoint blockade is regarded as standard of care for treatment of non-small cell lung cancer (NSCLC), up to 50% of patients with metastatic NSCLC do not achieve an optimal response.1–3 Previous work by our group and others in adoptive cell therapy (ACT) of metastatic melanoma (MM) has shown that infusion of a CD8+-rich TIL product significantly improved clinical outcomes, yet traditional IL-2 expansion methods have resulted in a predominantly CD4+ NSCLC TIL expansion product.7–12 This preclinical study explores the feasibility of producing a tumor-specific, CD8+-enriched NSCLC TIL product for ACT with an improved culture method.MethodsTIL from resected NSCLC tumors were cultured using 1) the traditional method using IL-2 alone in 24-well plates (TIL 1.0) or 2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). Expanded TIL were phenotyped using flow cytometry for CD4 and CD8 subset assessment and the CDR3-beta variable region of the T-cell receptor (TCR) involved in antigen binding was sequenced to assess the T-cell repertoire.ResultsIn a shorter manufacturing time (median of 14 days vs 27.5 days), TIL 3.0 expanded on average 5.3-times more NSCLC TIL (95% CI= 4.3–6.2, pConclusionsThis study reports the feasibility of using the TIL 3.0 methodology to robustly expand a CD8+ T-cell repertoire which maintains the respective clonal hierarchy in NSCLC tumors and enriches for putative tumor-specific TIL clones. The robustness and speed of the new process may facilitate testing and implementing effective TIL ACT in NSCLC.ReferencesGaron EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372(21):2018–28.Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373(17):1627–39.Gettinger S, Horn L, Jackman D, Spigel D, Antonia S, Hellmann M, et al. Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results from the CA209–003 Study. J Clin Oncol 2018;36(17):1675–84.Melioli G, Ratto G, Guastella M, Meta M, Biassoni R, Semino C, et al. Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy. Eur J Cancer 1994;30A(1):97–102.McGranahan N, Furness AJ, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351(6280):1463–9.Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 2011;17(13):4550–7.Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Hershkovitz L, et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res 2010;16(9):2646–55.Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S, et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J Immunother 2012;35(8):615–20.Radvanyi LG, Bernatchez C, Zhang M, Fox PS, Miller P, Chacon J, et al. Specific Lymphocyte Subsets Predict Response to Adoptive Cell Therapy Using Expanded Autologous Tumor-Infiltrating Lymphocytes in Metastatic Melanoma Patients. Clinical Cancer Research 2012;18(24):6758–70.Forget MA, Haymaker C, Hess KR, Meng YJ, Creasy C, Karpinets T, et al. Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome. Clin Cancer Res 2018;24(18):4416–28.Ben-Avi R, Farhi R, Ben-Nun A, Gorodner M, Greenberg E, Markel G, et al. Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients. Cancer Immunol Immunother 2018;67(8):1221–30.Ma Y, Ou J, Lin T, Chen L, Wang J, Qiao D, et al. Phenotypic analysis of tumor-infiltrating lymphocytes from non-small cell lung cancer and their potential application for adoptive cell therapy. Immunopharmacol Immunotoxicol 2020;42(4):319–29Ethics ApprovalThis study was performed on NSCLC tumor tissue resected from 16 patients enrolled, following informed consent, in the ImmunogenomiC prOfiling of early-stage NSCLC (ICON) project. This study was approved by the University of Texas MD Anderson Cancer Center‘s Institutional Review Board (protocol number PA15-1112_MODCR001).

BackgroundMalignant pleural mesothelioma (MPM) is a rare and aggressive cancer associated with exposure to asbestos with limited treatment strategies and poor prognosis.1 There is no curative treatment for MPM, and patients follow standard treatment options.2,3 Radiographic tumor thickness (TT) has recently emerged as a surrogate marker associated with overall survival,4 however, the influence of TT on the complexity of the immune component within tumor microenvironment is unknown. Our aim was to perform comprehensive profiling of the tumor immune microenvironment (TIME) relative to the TT to understand of this relationship to guide the design of novel treatment strategies.MethodsTumor specimens (n=29) were obtained from surgically managed MPM patients. Analysis was performed based on the median tumor thickness of 80mm, which defined thick vs thin groups. Additional analysis used TT as a continuous variable for correlations with immune features. The immune cell composition in the TIME was determined by flow cytometry (n=23). Transcriptomic profiling (n=20) was assessed using Nanostring nCounter Tumor Signaling 360 panel. Expanded tumor-infiltrating lymphocytes (TIL) were utilized for further characterization by 10x Genomics single cell RNA-seq profiling (n=8) and cytotoxicity assays (n=18). Level of significance was determined using unpaired non-parametric statistical test.ResultsFresh tumor tissue cytometry showed substantial differences in the immune profiles for thick versus thin tumors, highlighting a relationship of the TT and its immune composition. Thin tumors contained more Tregs, and higher OX40 expression (p=0.081 for non-Treg CD4+ and pConclusionsUtilizing multi-platform immune profiling approaches, we observed a distinct relationship between the TT and immune signatures. Understanding underlying immune signatures underpinning the biology of thick versus thin MPM tumors provides insights to potential responsiveness to immune-based therapies and may inform on the design for future novel strategies relative to the disease extent based on TT.ReferencesBibby AC, Tsim S, Kanellakis N, Ball H, Talbot DC, Blyth KG, Maskell NA, Psallidas I. Malignant pleural mesothelioma: an update on investigation, diagnosis and treatment. Eur Respir Rev 2016;25(142):472–86.Faig J, Howard S, Levine EA, Casselman G, Hesdorffer M, Ohar JA. Changing pattern in malignant mesothelioma survival. Transl Oncol 2015;8(1):35–9.Alpert N, van Gerwen M, Taioli E. Epidemiology of mesothelioma in the 21st century in Europe and the United States, 40 years after restricted/banned asbestos use. Transl Lung Cancer R 2020;9:S28-S38.de Perrot M, Dong Z, Bradbury P, Patsios D, Keshavjee S, Leighl NB, Hope A, Feld R, Cho J. Impact of tumour thickness on survival after radical radiation and surgery in malignant pleural mesothelioma. Eur Respir J 2017;49(3).Ethics ApprovalThis study was approved by the University of Texas MD Anderson Cancer Center‘s IRB; approval number LAB08-0380; participants gave informed consent before taking part.

BackgroundAdoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TIL) have shown durable responses in metastatic melanoma, yet these responses are unpredictable. Bioenergetics dictates the function and fate of adoptively transferred human T cells within the tumor microenvironment but the nature of metabolic competition leading to T-cell function and dysfunction are incompletely understood.MethodsWe integrated the profiling of TIL co-cultured with their autologous primary tumor cells with the aid of a suite of high-throughput single-cell functional assays, transcriptional, and proteomic assays. We validated the results of the model using flow cytometry and confocal microscopy. Association of functional features with clinical outcome was assessed.ResultsTimelapse Imaging Microscopy In Nanowell Grids (TIMING) demonstrated that while TIL frequencies in killing autologous tumor cells are equivalent across the donors, R-TIL had a significantly higher survival rate than NR-TILs. Tumor cells from NR patients had higher motility and showed increased elongation compared to R-tumors. RNA-sequencing (RNA-seq) and proteomics showed that NR-tumors were enriched in pathways associated with utilization of fatty acids (FAs) and adipogenesis, as well as cancer cell metastasis, cellular motility, adhesion, and migration. Candidate genes associated with ameboidal migration (MYH9, MYH2; WNT5B and SERPINE1) and FA utilization (CD36 and PPARG) were enriched in the NR-tumors. Flow cytometry and confocal microscopy confirmed that NR-tumors showed increased CD36 expression and FA uptake compared to R-tumors. To simulate metabolic competition, we co-cultured the TIL with autologous tumors and sorted TIL for RNAseq. The R-TIL were enriched in pathways related to mitochondrial and carbohydrate metabolism; fatty acid oxidation (FAO), and long-chain FAs with a direct enrichment in fatty acyl CoA biosynthesis and both peroxisomal and mitochondrial FAO. Since patient-derived TILs were limiting for metabolomics type assays, we utilized genome-scale metabolic models to infer relevant metabolic pathways by comparison to the human metabolic Atlas (HMR2). At the level of individual metabolites, the significantly enriched metabolites within R TILs were dominated by peroxisome and mitochondria derived fatty acyl-CoA: e.g. palmitoyl-CoA, linoleoyl-CoA, and oleoyl-CoA. We utilized flow cytometry and confocal microscopy to perform pulse-chase assays with FAs for validation. R-TILs showed an increased accumulation of FA into the mitochondria confirming a direct role for TIL FAO.ConclusionsEfficient competition for FAs is a key attribute of T-cell efficacy in ACT. R-TILs are able to utilize FAs via FAO when in competition with autologous tumor cells whereas NR tumors effectively uptake and store FAs preventing T-cell function.AcknowledgementsThis abstract was supported by the NIH (R01CA174385); CPRIT (RP180466); MRA Established Investigator Award to NV (509800), Welch Foundation (E1774); NSF (1705464); CDMRP (CA160591); Owens Foundation (NV). We would like to acknowledge the MDACC Flow Cytometry and Cellular Imaging Core facility for the FACS sorting (NCI P30CA16672), UH Seq-n-edit core for RNA-seq, Intel for the loan of computing cluster, and the UH Center for Advanced Computing and Data Systems (CACDS) for high-performance computing facilities.Trial Registration protocol (2004–0069)Ethics ApprovalApproved by the Institutional Review Board (IRB) of the MD Anderson Cancer Center (Houston, TX) and an FDA- approved Investigational New Drug (IND) application (NCT00338377)

Background Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett’s test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor–treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey’s test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

Purpose: Survival for pancreatic ductal adenocarcinoma (PDAC) patients is extremely poor and improved therapies are urgently needed. Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) has shown great promise in other tumor types, such as metastatic melanoma where overall response rates of 50% have been seen. Given this success and the evidence showing that T-cell presence positively correlates with overall survival in PDAC, we sought to enrich for CD8+ TILs capable of autologous tumor recognition. In addition, we explored the phenotype and T-cell receptor repertoire of the CD8+ TILs in the tumor microenvironment. Experimental Design: We used an agonistic 4-1BB mAb during the initial tumor fragment culture to provide 4-1BB costimulation and assessed changes in TIL growth, phenotype, repertoire, and antitumor function. Results: Increased CD8+ TIL growth from PDAC tumors was achieved with the aid of an agonistic 4-1BB mAb. Expanded TILs were characterized by an activated but not terminally differentiated phenotype. Moreover, 4-1BB stimulation expanded a more clonal and distinct CD8+ TIL repertoire than IL2 alone. TILs from both culture conditions displayed MHC class I-restricted recognition of autologous tumor targets. Conclusions: Costimulation with an anti-4-1BB mAb increases the feasibility of TIL therapy by producing greater numbers of these tumor-reactive T cells. These results suggest that TIL ACT for PDAC is a potential treatment avenue worth further investigation for a patient population in dire need of improved therapy. Clin Cancer Res; 23(23); 7263–75. ©2017 AACR.

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation. Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs. Results: CD8+BTLA− TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA− T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151–64. ©2017 AACR.

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity. Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088–97. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Introduction: Differences in the host immune environment are thought to mediate heterogeneous treatment responses in non-small cell lung cancer (NSCLC). Unlike individual platform analyses, integrative analysis of multi-platform profiling allows for the discovery of novel interactions that expand our understanding of the disease. Utilizing the ImmunogenomiC prOfiling of NSCLC patient cohort (ICON), a prospective multi-omics protocol of operable early-stage NSCLC tumors with integrated immune, genomic, and clinical data, we hypothesized that multi-platform analyses would identify differences in the immune-genomic landscape that are associated with disease recurrence. Methods: Tumor and tumor-adjacent uninvolved lung was collected at resection; blood was collected before and after surgery. Tissue samples underwent WES, RNAseq, TCR sequencing (TCRseq), multiplex immunofluorescence (mIF), and RPPA profiling; tissue and blood (PBMC) samples were analyzed by flow cytometry. An integrated, inter-modality network was built using Spearman correlations between measurement pairs from different data modalities. Multivariate analysis was performed to adjust for stage and histology. Results: A total of 89 treatment-naïve patients with Stage 1-3 resected NSCLC (Squamous: 19; Non-squamous: 70) and 24 months of follow-up were analyzed (recurrence N = 24; no recurrence N = 65). The data network includes over 4,000 measurements linked by over 50,000 correlations. InfoMap, a community detection approach, was used to extract sub-network modules, which were used to contextualize the results of multivariate analysis. Tumors from patients with recurrence demonstrated decreased immune cell infiltration and activation including decreased cytotoxic CD8 T-cells (CD8+PD1+; fold-change (FC) = 0.898, p = 0.018; flow cytometry), decreased T-cell clonality (FC = 0.954, p = 0.017; TCRseq), and decreased tumor-associated macrophages (CD68+PD-L1+; FC = 0.426, p = 0.011; mIF). Furthermore, circulating CD8+ICOS+ activated T cells were decreased in patients with recurrence suggesting an impaired systemic anti-tumor immune response (FC = 0.552, p = 0.042; PBMC Flow). Finally, tumor-adjacent uninvolved lungs showed distinct T-cell phenotypes with accumulation of inactive CD8 T-cells (CD8+PD1-TIM3-) in patients with recurrence and increased populations of activated CD8 T-cells (CD8+PD1+) in patients without recurrence. Conclusion: Integrative multi-omic analysis suggests preserved anti-tumor immune surveillance in patients who are disease-free after 2 years from surgical resection with curative intent for treatment of NSCLC relative to patients with disease recurrence. Further analysis is ongoing to interrogate genomic and immune variables that are associated with disease recurrence. Citation Format: Neal Akhave, Stephanie Schmidt, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J. McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Marcelo V. Negrao, Don L. Gibbons. Integrated multi-platform profiling of early-stage non-small cell lung cancer identifies relationship between disease recurrence and decreased native immune response in treatment-naïve resected NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 619.

Sarcomas encompass a rare but highly diverse set of tumor malignancies, contributing disproportionately to years of life lost. Immune checkpoint blockade (ICB) has been successful across various tumor types; however, their efficacy and predictability in sarcomas remain unknown. We conducted a translational study using pre- and on-treatment tumor biopsies collected prospectively on a Phase II clinical trial (NCT02815995) evaluating the role of combination anti-PD-L1 and anti-CTLA-4 in 57 patients, enrolled across multiple histologies of metastatic sarcoma. We obtained tumor biopsies at baseline and after 6 weeks of treatment and performed whole-exome, T-cell repertoire (TCR) and RNA-sequencing, along with multiplexed-immunofluorescence (mIF).We deconvoluted substantial variability present in the tumor microenvironment (TME) within sarcomas and found instances of relatively inflamed tumors which failed to respond to ICB. However, amongst those potential molecular correlates of response analyzed, elevated levels of B-cells both at the transcriptome and through validation staining (p = 0.047 and p = 0.022) were most significantly correlated with response. In order to gain more insight into the phenotype and function of B-cells in contributing to response, we inferred BCR-templates and found higher levels of both IGH and IGL diversity (p = 0.0276 and p = 0. 0889 respectively) in responders to ICB. Additionally, we detected increased levels of hyperexpanded IGH clones at the on-treatment time point in patients that responded to therapy (p = 0.048). This B-cell enrichment was validated and found to be predictive of response (p = 0.043) in an independent sarcoma anti-PD-1 treated cohort with matched molecular data. Responsive tumors were also associated with higher levels of TCR richness indicating a strong association of diversity in the TCR of responders (p = 0.047). This work demonstrates the potential for multi-lineage immune cell enrichment and frames the potential molecular features of the TME that may influence response in ICB treated sarcomas. Citation Format: Akash Mitra, Neeta Somaiah, Anthony P. Conley, Behrang Amini, Heather Lin, Beatriz E. Sanchez, Celia Garcia-Prieto, Grace Mathew, Chantale Bernatchez, Vinod Ravi, Dejka Araujo, Maria A. Zarzour, John A. Livingston, Christina L. Roland, Najat Daw, Joshua Baguley, Wei-Lien Wang, Hannah Beird, Taylor Tate, Cara Haymaker, Latasha D. Little, Curtis Gumbs, Xingshi Song, Emily Z. Keung, Shaojun Zhang, Swati Gite, Jianhua Zhang, Luisa Solis, Hussein Tawbi, Linghua Wang, Shreyaskumar Patel, Robert S. Benjamin, Alexander J. Lazar, Ignacio I. Wistuba, Andrew Futreal. Immunogenomic correlates of response to combination immune checkpoint blockade in advanced sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 518.

Background: We have previously demonstrated that the presence of a proinflammatory peripheral cytokine milieu correlates with high levels of circulating neutrophils at the time of surgery and reduced overall survival and recurrence-free survival. In this study, we hypothesized that functional immune features or states in circulation may indicate early tumor recurrence when assessed longitudinally. We also investigated whether potential deficiencies in peripheral immune functionality at the time of lung cancer resection could identify correlates with subsequent outcome. Methods: We performed flow cytometry and luminex profiling of blood samples collected from patients with stage I-IIIA resected NSCLC (n=150) and enrolled on the Immunogenomic profiling of NSCLC (ICON) prospective protocol. Patient characteristics include 75 adenocarcinomas, 30 squamous and 12 mixed or other histologies. Only patients who underwent primary cancer resection without neoadjuvant therapy were included. At a median follow up of 18.2 months, 37 patients had disease recurrence. Blood was collected at the time of primary lung cancer resection, at 4 weeks, and 4 months thereafter with PBMCs utilized for flow cytometry and plasma for cytokine assessment. Changes in cytokines were assessed by normalizing to baseline levels. Results: Larger tumors as well as advanced clinical and pathological stages were associated with higher frequencies of proliferating Ki67+CD4+ and Ki67+CD8+ T cells in circulation at time of resection, suggesting an activated circulating immune response. We identified novel strong correlations in the plasma between soluble BTLA and Tim3 (r= 0.87, p=1.74e-140), PD1 and CD80 (r=0.72, p=6.41e-74) and moderate correlations between soluble PD1 and PDL1 (r=0.39, p=8.41e-18). Efforts are ongoing to determine the association of specific circulating immune states with the presence of these soluble receptors. CD8+Tim3+ T cells (FC=5.2%, p=0.028) and CTLA4+NK cells (FC=47.7%, p=0.002) as well as CTLA4+Tregs (FC=25.2%, p=0.05) were found to be increased in circulation at pre-recurrence time points (either 4 weeks or 4 months) relative to resection. This suggests the emergence of a suppressive cell type as well as induction of specific checkpoint receptors on effector cells that correlate with tumor recurrence. Finally, we identified a cytokine signature associated with recurrence by testing three sets within the ICON cohort with a training set AUC = 0.76 and the test set AUC=0.72, which was validated in a third set of patients yielding an AUC = 0.76. Conclusions: We identified circulating immune features associated with initial tumor size and overall stage as well as unique associations among soluble proteins. Increased presence of potentially inhibited or suppressed CD8+ T cells and NK cells as well as Tregs are suggestive of mechanisms of immune suppression relative to tumor recurrence. Citation Format: Younghee Lee, Daniel McGrail, Hai Tran, Mayra E. Vasquez, Carlos Ramos, Alexandre Reuben, Ara A. Vaporciyan, Annikka Weissferdt, Chantale Bernatchez, Tina Cascone, Ignacio I. Wistuba, Jianjun Zhang, John Heymach, Marcelo V. Negrao, Don L. Gibbons, Boris Sepesi, Cara L. Haymaker. Circulating biomarkers are associated with recurrence following complete resection of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1670.

9519 Background: MM pts with LMD have a dismal prognosis, with median overall survival (OS) < 3 months, no approved therapies and extremely limited clinical trial options. We previously reported initial safety findings from an open label, single arm, single center phase I/IB trial (NCT03025256), in which IT and IV N were well tolerated, without any CNS-specific or unexpected toxicity. Here we report an update on safety and maximum tolerated dose (MTD) for all patients enrolled, and efficacy for the completed dose cohorts. Methods: MM patients aged >18 with evidence of LMD by MRI and/or CSF cytology, ECOG PS ≤2 were treated with IT and IV N. Dexamethasone ≤4mg/daily and concurrent BRAF/MEK inhibitor(i) treatment was allowed. For cycle 1, IT N was administered via intraventricular reservoir on day (D)1. For subsequent cycles (every 14 days), pts received IT N on D1, followed by IV N 240 mg on D2. IT N doses evaluated were 5, 10, 20 mg and 50 mg. Blood and CSF were collected at multiple time points for translational research. The primary objectives of this first-in-human study were to determine the safety and MTD of IT N given with IV N in MM pts with LMD. Bayesian mTPI methodology was used to define the MTD. Results: To date, 23 pts have been treated: two at 5, three at 10, fourteen at 20 mg and four at 50 mg IT N. Median age at LMD diagnosis was 42 (28-73); 12 pts are male. All pts had radiographic evidence of LMD and neurological symptoms; 14 pts had positive CSF cytology at baseline. 21 pts received prior therapies for their metastatic melanoma: anti-PD1 (n = 19), BRAFi/MEKi (n = 14), chemo (n = 2), IT IL2 (n = 4) other (n = 2). 19 pts had prior XRT, including whole brain RT (n = 7). Two pts were treatment-naïve. The median number of IT N doses was five (1- 66). The combination regimen was well tolerated by all evaluable pts (n=23), with only five pts (22%) experiencing gr 3 AEs, and no reported gr 4 or 5 toxicities. Nausea (30%), diarrhea (26%), and rash (22%) were the most common AEs. Eight pts (23%) experienced AEs after IT N administration, all gr 1. Initial efficacy analysis included only pts (n=19) treated with first three dose levels (5-20mg). Median follow-up for these pts is 4.5 months (mos) (1.1, 31.5 mos) and median OS is 63 % at 3 mos, 42 % at 6 mos and 30% at 12 mos. Conclusions: The trial demonstrates the feasibility and safety of IT administration of modern immunotherapy for MM pts with LMD. No unexpected systemic or neurological toxicity was observed with 20mg IT N. 2 additional patients are required to complete the 50mg IT N cohort. OS rates at 6 and 12 mos are encouraging and support further evaluation of IT administration of immunotherapy agents for pts with MM and LMD. Final presentation will include results of LMD for all dose cohorts, composite response assessment and comparative analysis of longitudinal CSF samples to assess immunologic effects. Clinical trial information: NCT03025256.

8557 Background: MPeM is a rare and aggressive cancer with very limited treatment options. Lack of dedicated research has impeded improvements in outcomes. Defining prevalent GAs is a critical unmet need for use of targeted therapies in these patients. Although MPeM is notably distinct from MPM vis-à-vis epidemiologic and clinical attributes, the genomic underpinings of these differences have yet to be established. We aimed at describing a comprehensive genomic profile (CGP) of MPeM in comparison to MPM. Methods: We performed a retrospective comparative analysis between 89 patients with MPeM and 241 patients with MPM (N = 330) who underwent CGP using CLIA certified next-generation sequencing assays. The cohort was generated using mesothelioma patients at MD Anderson Cancer Center (N = 223) and supplemented by additional mesothelioma patients (N = 107) from a publicly available database from Memorial Sloan Kettering Cancer Center, the MSK-IMPACT database. Essential clinicopathological variables were collected. Descriptive statistics, Fisher’s exact and Mann-Whitney tests were used for comparison. Kaplan-meier method and log rank tests were used for overall survival (OS) estimates. Results: MPeM cohort (vs. MPM) had more women (54% vs. 31%, P < 0.001) and younger age at diagnosis (56 vs. 69 years, P < 0.001). Histology was epithelioid, biphasic and sarcomatoid in 86%, 7% and 7% cases, a distribution similar to MPM cohort. At least 1 GA was found in 64 (72% vs. 82% in MPM, P = 0.044) of MPeM patients with a median of 1 (range 1 – 12) (vs. a median of 2, range 1 – 24, P < 0.001) GA per patient. A significantly lower proportion of MPeM patients had ≥ 3 mutations (14% vs. 26%, OR 2.1, P = 0.028) per patient. The most frequent mutations were present in the following genes: TP53 (24%), BAP1 (16%), NF2 (15%), MET (9%) and TRAF7, KIT and PIK3CA (each 6%). MPeM patients harbored more mutations in MET (9% vs. < 1%, P < 0.001) and TRAF7 (6% vs. < 1%, P = 0.02) but fewer mutations in BAP1 (16% vs. 32%, P = 0.003) and CDKN2B (0% vs. 5%, P = 0.041). The most common copy number variations (CNVs: amplifications or deletions) were seen in BAP1, MCL1, SETD2, WT1 (each 2%) and AURKA (1%) genes. Among genes with CNVs, MPeM had a lower rate of deletions in CDKN2A (1% vs. 6%, P = 0.040). Among more common GAs, only BAP1 mutations appeared to be associated with poor OS (45.7 vs. 127.1 months, HR 2.5, 95%CI: 0.6 – 10.1, P = 0.050) in patients with MPeM. Conclusions: In this large cohort with CGP, we identified potential molecular drivers in MPeM and demonstrated key genomic differences between MPeM and MPM. MPeM is frequently driven by GAs involved in cell cycle control, a potentially targetable pathway. Despite this insight from CGP, a large subset of patients do not have actionable GAs and for these patients, further collaborative trans-“omic” research efforts are needed to advance potential therapeutic options.

We report here on a patient with metastatic melanoma who had extensive brain metastases. After being treated with the sequential combination of whole brain radiation therapy followed by the PD-1–inhibitory antibody, pembrolizumab, the patient had a durable complete response. Retrospective laboratory studies of T cells revealed that, after treatment with anti-PD-1 commenced, effector CD8+ T cells in the blood expanded and the ratio of CD8+:Treg T cells increased. A CD8+ T-cell clone present in the initial brain metastases was expanded in the blood after anti-PD-1 treatment, which suggested an antitumor role for this clone. Immunohistochemical analysis confirmed the presence of CD8+ T cells and low PD-L1 expression in the brain metastases before immunotherapy initiation. This sequence of therapy may provide an option for melanoma patients with unresponsive brain metastases. Cancer Immunol Res; 5(2); 100–5. ©2017 AACR.

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8(+) T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8(+) T cells into central memory-like T cells. De-differentiation of CD8(+) T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T cell populations that can augment the efficacy of adoptively transferred T cells.

BACKGROUND: The tumor immune microenvironment (TIME) of lung cancer brain metastasis is largely unexplored. We carried out immune profiling and sequencing analysis of paired resected primary tumors and brain metastases of non-small-cell lung carcinoma (NSCLC). PATIENTS AND METHODS: TIME profiling of archival formalin-fixed and paraffin-embedded specimens of paired primary tumors and brain metastases from 39 patients with surgically resected NSCLCs was carried out using a 770 immune gene expression panel and by T-cell receptor beta repertoire (TCRβ) sequencing. Immunohistochemistry was carried out for validation. Targeted sequencing was carried out to catalog hot spot mutations in cancer genes. RESULTS: Somatic hot spot mutations were mostly shared between both tumor sites (28/39 patients; 71%). We identified 161 differentially expressed genes, indicating inhibition of dendritic cell maturation, Th1, and leukocyte extravasation signaling pathways, in brain metastases compared with primary tumors (P

Importance In recurrent human papilloma virus (HPV)–driven cancer, immune checkpoint blockade with anti–programmed cell death 1 (PD-1) antibodies produces tumor regression in only a minority of patients. Therapeutic HPV vaccines have produced strong immune responses to HPV-16, but vaccination alone has been ineffective for invasive cancer. Objective To determine whether the efficacy of nivolumab, an anti–PD-1 immune checkpoint antibody, is amplified through treatment with ISA 101, a synthetic long-peptide HPV-16 vaccine inducing HPV-specific T cells, in patients with incurable HPV-16–positive cancer. Design, Setting, and Participants In this single-arm, single-center phase 2 clinical trial, 24 patients with incurable HPV-16–positive cancer were enrolled from December 23, 2015, to December 12, 2016. Duration of follow-up for censored patients was 12.2 months through August 31, 2017. Interventions The vaccine ISA101, 100 μg/peptide, was given subcutaneously on days 1, 22, and 50. Nivolumab, 3 mg/kg, was given intravenously every 2 weeks beginning day 8 for up to 1 year. Main Outcomes and Measures Assessment of efficacy reflected in the overall response rate (per Response Evaluation Criteria in Solid Tumors, version 1.1). Results Of the 24 patients (4 women and 20 men; 22 with oropharyngeal cancer; median age, 60 years [range, 36-73 years]), the overall response rate was 33% (8 patients; 90% CI, 19%-50%). Median duration of response was 10.3 months (95% CI, 10.3 months to inestimable). Five of 8 patients remain in response. Median progression-free survival was 2.7 months (95% CI, 2.5-9.4 months). Median overall survival was 17.5 months (95% CI, 17.5 months to inestimable). Grades 3 to 4 toxicity occurred in 2 patients (asymptomatic grade 3 transaminase level elevation in 1 patient and grade 4 lipase elevation in 1 patient), requiring discontinuation of nivolumab therapy. Conclusions and Relevance The overall response rate of 33% and median overall survival of 17.5 months is promising compared with PD-1 inhibition alone in similar patients. A randomized clinical trial to confirm the contribution of HPV-16 vaccination to tumoricidal effects of PD-1 inhibition is warranted for further study. Trial Registration ClinicalTrials.gov identifier:NCT02426892

NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor βγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated patients. Immune activation and increased numbers of immune cells were observed in the periphery across all doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional analysis of tumor biopsies showed that NKTR-214 engaged the IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks. Significance: We believe that IL2- and IL2 pathway–targeted agents such as NKTR-214 are key components to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors. See related commentary by Sullivan, p. 694. This article is highlighted in the In This Issue feature, p. 681

312 Background: Sitra is a spectrum-selective receptor tyrosine kinase inhibitor (TKI) that targets TAM receptors (TYRO3, AXL, MERTK), VEGFR2, c-Kit, and MET. These receptors regulate several immune suppressive cell types in the tumor microenvironment, including M2-polarized macrophages, MDSCs, and T regulatory cells, which are implicated in resistance to checkpoint inhibitors. ccRCC is characterized by upregulation of VEGF and overexpression of MET and AXL. Sitra may combine effectively with immune checkpoint inhibition to augment antitumor activity in ccRCC. About 39% of patients with accRCC who receive surgery with curative intent relapse representing an unmet need in this setting. Together these data support the evaluation of neoadjuvant sitra with nivo in accRCC. Methods: This phase II study (NCT03680521) evaluated sitra and nivo in pts with locally- advanced ccRCC who were candidates for curative nephrectomy. Single-agent sitra (120 mg) was administered daily (QD) for 2 weeks, with nivo (240 mg intravenously Q2W) added to sitra for 4-6 weeks. A plan for potential dose de-escalation was implemented using a modified toxicity probability interval method with a maximum toxicity of 20% at the tolerated dose. Pts underwent pathology/tissue evaluation at 3 timepoints: biopsy prior to treatment, biopsy prior to the addition of nivo, and nephrectomy specimen evaluation at time of nephrectomy. The primary endpoint was objective response (RECIST 1.1); secondary endpoints included safety, PK, and correlative immune effects (selected protein and gene expression and immune cell populations). Results: A total of 20 pts were evaluated for safety (95% had T3 or higher stage tumors, 65% with baseline hypertension). Dose-limiting toxicities (DLTs) led to a dose de-escalation, resulting in 7 pts treated at 120 mg QD sitra and 13 pts treated at 80 mg QD. DLTs included grade 3 (Gr3) hypertension (n=6); deep vein thrombosis and pulmonary embolism (Gr3) were observed in 1 additional pt. Median duration of sitra treatment was 6.3 weeks at the 80 mg dose and 7.1 weeks at the 120 mg dose. With a median follow-up of 9.4 months after initiation of systemic therapy, no pts have relapsed. In 17 pts evaluable for efficacy, the investigator-assessed confirmed ORR was 11.8%, including 2 PRs (33.3% ORR in pts who received 120 mg sitra). No pts experienced progressive disease while on therapy. Median DFS was not reached. There was 1 delayed surgery due to nivo-related thyroiditis that resolved. Reported TRAEs: Gr1/Gr2 in 55% of pts (dysphonia 50%, fatigue 45%, diarrhea 40%, hypertension 30%, increased ALT 30%), Gr3 in 45% of pts (hypertension 30%). There were no Gr4/Gr5 TRAEs. Correlative blood and tissue analyses will be presented. Conclusions: The combination of sitra and nivo is clinically active with a manageable safety profile as a neoadjuvant therapy for accRCC. Clinical trial information: NCT03680521 .

The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

T cell–mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell–mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell–mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti–PD-1 and anti–CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K–AKT pathway inhibitors. Significance: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K–AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K–AKT pathway to increase the efficacy of immunotherapy. Cancer Discov; 6(2); 202–16. ©2015 AACR. See related commentary by Rizvi and Chan, p. 128. This article is highlighted in the In This Issue feature, p. 109

Background: Tilsotolimod, an investigational Toll-like receptor 9 (TLR9) agonist, modulates the tumor immune microenvironment and has single-agent antitumor activity in preclinical models. The ILLUMINATE-101 phase 1b study (NCT03052205) explored the safety, efficacy, and immune effects of intratumoral tilsotolimod in multiple solid tumors. Methods: Adults with a histologically- or cytologically-confirmed diagnosis of cancer not amenable to curative therapies received intratumoral tilsotolimod 8, 16, 23, or 32 mg into a single lesion on Days 1, 8, and 15 of Cycle 1 and Day 1 of each subsequent 3-week cycle, for up to 17 cycles. Additionally, patients with advanced melanoma were enrolled into an expansion cohort at the recommended phase 2 dose of 8 mg. The primary objective was to characterize safety (dose escalation cohort) and efficacy (expansion cohort). Secondary objectives included pharmacokinetics of tilsotolimod. Immunological assessment of injected and non-injected tumors was an exploratory objective. Blood samples and tumor biopsies of injected lesions were obtained at baseline and 24 hours post treatment for immune analyses. Results: A total of 54 patients were enrolled. Of the 38 patients in the dose escalation cohort, 35 had metastatic disease. Patients in this cohort had a median of 7 prior lines of treatment, and the most common cancer types were pancreatic (12 patients) and colorectal (7 patients). All 16 patients in the melanoma cohort had metastatic disease with a median of 3 lines of prior therapy, and 10 patients had elevated LDH. Injected lesions were deep and required interventional radiology in 52 of 54 patients. No dose-limiting toxicities were observed. The most common treatment-related adverse events were pyrexia, fatigue, chills, nausea, and vomiting. Compared to pretreatment, biopsies of injected tumors at 24 hours showed increased activation of the Type-I IFN pathway, upregulation of MHC class I/II, IFNγ expression, and expression of multiple immune checkpoints (i.e. PD-1, LAG3). Of the 35 evaluable patients in the dose escalation cohort, 12 (34%) achieved a best overall response of stable disease (SD). Of the 16 evaluable patients in the melanoma cohort, 3 had SD, 1 who had a 35% tumor reduction with no confirmatory scan. Conclusions: Tilsotolimod was generally well tolerated and induced alterations in the tumor microenvironment, including immune checkpoint upregulation, activation of dendritic cells, and induction of Type-I IFN signaling. Additional clinical studies of tilsotolimod in combination with checkpoint inhibitors are underway (NCT03445533, NCT03865082, and NCT02644967). Citation Format: Hani M. Babiker, Vivek Subbiah, Asim Ali, Alain Algazi, Jacob Schachter, Michal Lotem, Corinne Maurice-Dror, Daniel Hendler, Shah Rahimian, Hans Minderman, Cara Haymaker, Chantale Bernatchez, Ravi Murthy, Rolf Hultsch, Nadia Caplan, Gregory Woodhead, Charles Hennemeyer, Sri Chunduru, Peter Anderson, Adi Diab, Erkut Borazanci, Igor Puzanov. Tilsotolimod engages the TLR9 pathway to promote antigen presentation and Type-I IFN signaling in solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT134.

Background: Acral melanoma (ACML), the rarest form of melanoma (MEL) represents 4-6% of all MEL cases however it is the most common subtype darker pigmented individuals accounting for up to 70% of all cases. Checkpoint inhibitors (CPI) have emerged as an efficacious treatment option. In cutaneous melanoma (CtMEL), tumor mutational burden (TMB) correlates with response rate. Despite low TMB in AMEL, responses to CPI parallel those in CtMEL. Given the differences in ethnicity among ACML patients compared CtMEL patients we aimed to study: (i) the correlation between response and TMB, (ii) the relationship between ethnicity and response and (iii) the underlying immune-biology accounting for differential responses. Method: All advanced and metastatic ACML patients (pts) treated with anti-CTLA4 (ipilimumab) or anti-PD1 (pembrolizumab or nivolumab) immunotherapy between March 2011 and January 2019 at MD Anderson Cancer Center, Texas, were included in this retrospective analysis. Clinical response, progression free survival and overall survival (PFS and OS) and their correlation to ethnicity and TMB were evaluated. Objective response was measured using RECIST 1.1 and analyzed using logistic regression. PFS and OS were assessed using the Kaplan Meir method and log-rank test. TMB was predicted using validated algorithm based on a defined gene mutation set obtained using next generation sequencing. Results: 44 pts with Stages III-IV ACML (IIIA: 2%, IIIB 9%, IIIC 27%, IV-M1A: 16%, IV-M1B: 25%, IV-M1C: 20%, IV-M1D2%) were included in the analysis. Median age was 63 years old (39-88) and 60% were men. Of 44 patients 12 (27%) self-identified as Hispanic, 2 as (5%) Asian and 30 (68%) as White. The overall response rate was nearly 5 times times higher in Hispanic compared to White pts (OR 4.60, p-value 0.04). The median TMB in Hispanic patients was 16 mutations/mb compared to 7 mutations/mb in White pts. The median PFS and OS for White pts were 7.2 months and 34.3 months respectively while for Hispanic pts the median PFS and OS were 6.7 (log-rank p=0.69) and 26.1 months (log-rank p=0.38) respectively. In addition to this data deep immune analysis of tumor tissue including nanostring, gene expression and TCR sequencing will be assessed and reported. Conclusions: The data from this small retrospective study suggests that White pts with ACML had low response rates to CPI presumably due to low TMB while interestingly, Hispanic pts, despite relatively low TMB have high response rates paralleling those seen in the overall CtMEL population. Despite higher response rates in Hispanic patients, there was no significant difference in OS. This data together with further immune analysis provides the rationale to design prospective studies to investigate how tumor micro-environment varies with ethnicity. Additionally we will investigate the social and biological determinants that limit survival in Hispanic pts despite higher response rates. Citation Format: Kaysia Ludford, Chantal Saberian, Sara K Nabhan, Stephen Gruschkus, Chantale Bernatchez, Natalie Jackson, Cara L Haymaker, Patrick Hwu, Adi Diab. Investigating the immuno-biology underlying differential response to immunotherapy in White and non-White patients with metastatic acral melanoma [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D075.

e15242 Background: The Cancer Immune Monitoring and Analysis Centers – Cancer Immunology Data Commons (CIMAC-CIDC) network is a National Cancer Institute-funded initiative to identify biomarkers of mechanisms and response to cancer immunotherapy clinical trials, using state-of-the-art assay technologies. A primary platform for CIMAC-CIDC biomarker studies is CyTOF mass cytometry, which is performed at all four CIMAC laboratories. Methods: To test the ability to generate comparable data across labs, a cross-site harmonization effort was undertaken. We first harmonized SOPs between centers. Because of a new acquisition protocol introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed a cross-site assay harmonization experiment, using 5 shared cryopreserved PBMC samples and one lyophilized control cell preparation, along with a shared lyophilized antibody cocktail consisting of 14 markers, as validated in the HIPC consortium, plus CD45. These reagents and samples were distributed to the four sites, and FCS files were centrally analyzed by both manual gating and automated methods (Astrolabe). Results: Average CVs across sites for each cell population were reported and compared to a previous multisite CyTOF study. Once a cell recovery issue at two sites was resolved, this experiment resulted in inter-site reproducibility of under 20% CV for most cell subsets, very similar to the previous study. Conclusions: These results emphasize the ability to reproduce CyTOF across sites, and also highlights procedures, such as use of spike-in control samples, useful for tracking variability in this assay.

3027 Background: Metastatic UM is a rare cancer with poor response rates to systemic therapy. Adoptive transfer of patient-specific TIL may represent the best strategy for treatment. TIL are harvested from primary or metastatic tumors and initially expanded in culture with high dose IL-2 prior to undergoing rapid expansion protocol and therapeutic administration. Here, we report improved rates of initial expansion using a previously described TIL 3.0 method which utilizes dual agonistic antibodies to TCR and 4-1BB (Urelumab) for stimulation, respectively, with high dose IL-2, compared to the traditional method. Methods: Between 2006 and 2019, patients were consented for TIL harvest from either primary or metastatic UM tumors. Demographics, clinical features, and outcomes of the TIL initial expansion were collected. Success rates, number of cells expanded, and days in culture for the two methods were analyzed using partially overlapping samples t-tests and z-tests. Results: There were 85 harvests and expansions from 76 patients using the traditional method and 32 expansions from 30 patients using TIL 3.0. Initial TIL expansion was successful in 97% of TIL 3.0 harvests compared to 35% for the traditional method (p < 0.001). More TIL were expanded with TIL 3.0 compared to the traditional method (291.3 million cells vs. 88.6 million cells, p < 0.001), and fewer days were required in culture (18.5 vs. 29.0, p < 0.001). Both primary UM harvests and metastatic harvests were more successful with TIL 3.0 (90% vs. 12% for primary, p < 0.001, and 100% vs. 42% for metastatic, p < 0.001). Conclusions: Expansion of UM tumors via the TIL 3.0 method led to successful growth in 97% of harvests. Therapeutic administration to patients with TIL 3.0 is under active investigation. [Table: see text]

10008 Background: MM pts with LMD have a dismal prognosis, with a median overall survival (OS) < 3 months and no approved therapies. IT administration of interleukin-2 (IL2) achieves survival in ~15% of MM LMD pts, but at cost of severe toxicities. Given the favorable clinical activity and safety of systemic anti-PD1, we hypothesized that IT N administration is safe and can achieve clinical benefit in pts with LMD. Methods: The primary objectives of this first-in-human study (NCT03025256) were to determine the safety and the maximum tolerated dose (MTD) of IT N given with IV N in MM pts with LMD. Eligible pts had MM, ECOG PS < / = 2, and evidence of LMD by MRI and/or CSF cytology. Dexamethasone < / = 4mg/daily was allowed. For cycle 1, IT N is administered via intraventricular reservoir on day (D) 1; Blood and CSF is collected at multiple time points for translational research. For subsequent cycles (every 14 days), pts receive IT N on D1, followed by IV N 240 mg on D2. IT N doses evaluated were 5, 10, and 20 mg. Bayesian mTPI methodology was used to define the MTD. The study was recently amended to allow for concurrent BRAF/MEK inhibitor(i) treatment. Results: To date, 15 pts have been treated: two at 5, three at 10, and 10 at 20 mg IT N. Median age at LMD diagnosis was 41.8 (30.9-73.2) years; 6 pts are male. All pts had radiographic evidence of LMD and neurological symptoms; 8 pts had positive CSF cytology. 12 pts received prior therapies for their MM: anti-PD1 (n = 11), BRAFi/MEKi (n = 9), chemo (n = 2), IT IL2 (n = 4) other (n = 2). 11 pts had prior XRT, including whole brain RT (n = 7). 1 pt was treatment-naïve. The median numbers of IT N doses was 4 (1-42). No grade (Gr) 4-5 AEs were attributed to IT N or IV N; only 4 events (Gr 1, n = 2; Gr2, n = 2) were possibly related to the IT N. With a median follow-up of 18.7 weeks (1-83.3 wks), the median OS is 46.1 weeks (0.1-83.3). Clinical response data, translational research endpoints, including changes in CSF cytokines and cfDNA, will be reported. Conclusions: The trial demonstrates the feasibility of prospective clinical trials in MM patients with LMD. The combination of IT/ IV N was safe and well-tolerated, with no unexpected systemic or neurological toxicity. Final presentation will include results of LMD composite response assessment, comparative analysis of longitudinal CSF/blood samples to assess immunologic effects. Finally, the interim OS of the patients is encouraging, and supports further evaluation of IT administration of immunotherapy agents for pts with MM and LMD. Clinical trial information: NCT03025256.

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

Background Tilsotolimod, a synthetic toll-like receptor 9 (TLR9) agonist, modulates the tumor immune microenvironment and has antitumor activity as monotherapy in preclinical models. In the ILLUMINATE-204 phase I/II study of tilsotolimod in combination with ipilimumab in patients with refractory melanoma, increased antitumor immune activity was observed in injected and uninjected tumors at 24 hours following tilsotolimod treatment. The ILLUMINATE-101 phase Ib study explored the safety, efficacy, and immune effects of tilsotolimod monotherapy in multiple solid tumors. Methods Adults with histologically or cytologically confirmed diagnosis of cancer not amenable to curative therapies received intratumoral tilsotolimod in doses escalating from 8 mg to 32 mg into a single lesion at weeks 1, 2, 3, 5, 8, and 11. Additional patients with advanced melanoma were enrolled into an expansion cohort at the 8-mg dose, the recommended phase II dose established for melanoma. Objectives included characterizing safety and efficacy, and immunological assessment. Blood samples and tumor biopsies of injected and distal lesions were obtained at baseline and 24 hours post treatment for immune analyses. Results As of May 6 2019, 54 patients have been enrolled, including 38 patients into the dose evaluation portion and 16 patients into the melanoma expansion cohort. No dose-limiting toxicities were observed. The most common treatment-related adverse events were fever, fatigue, and chills. Within 24 hours, fresh tumor biopsies showed increased gene expression for multiple immune checkpoint pathways, increased IFN gamma levels, activation of the type 1 IFN pathway, and upregulation of MHC class I and II, compared to pretreatment biopsies. Of 43 evaluable patients, 15 (35%) had a RECIST v1.1 disease assessment of stable disease (duration 1.2 to 11.1 months, 1 patient ongoing). Conclusions Tilsotolimod was well tolerated and induced robust alterations in the tumor microenvironment, including immune checkpoint upregulation, activation of dendritic cells, and induction of Type 1 IFN signaling. A phase II study of tilsotolimod in combination with nivolumab and ipilimumab has been initiated for the treatment of multiple solid tumors (ILLUMINATE-206; NCT03865082). Clinical trial identification NCT03052205. Editorial acknowledgement Ted Everson, Idera Pharmaceuticals, Inc. Legal entity responsible for the study Idera Pharmaceuticals, Inc. Funding Idera Pharmaceuticals, Inc. Disclosure H. Babiker: Advisory / Consultancy: Endocyte; Advisory / Consultancy: Celgene. V. Subbiah: Advisory / Consultancy: Idera Pharmaceuticals; Advisory / Consultancy: Novartis; Advisory / Consultancy: Bayer; Advisory / Consultancy: Blueprint Medicines; Advisory / Consultancy: Loxo Oncology. S. Rahimian: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. C. Haymaker: Research grant / Funding (institution): Idera Pharmaceuticals. C. Bernatchez: Research grant / Funding (institution): Idera Pharmaceuticals. G. Bindra: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. I. Iverson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. S. Chunduru: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. A. Diab: Advisory / Consultancy: Array Biopharma; Advisory / Consultancy: Nektar Therapeutics; Honoraria (self): Bristol-Myers Squibb; Advisory / Consultancy: Jounce Therapeutics; Honoraria (self): Novartis; Advisory / Consultancy: Idera. All other authors have declared no conflicts of interest.

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence. Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated. Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome. Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416–28. ©2018 AACR.

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies. Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366–75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders. Experimental Design: We used aAPC or g-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed on the basis of the pattern of T-cell receptor usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. Results: The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, whereas increasing the frequency of CD8 + T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an upregulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity, and CD8 + T cells showed comparable antitumor function as those expanded with PBMC feeders. Conclusions: TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical application of aAPC to manufacture TIL for the treatment of melanoma.

Following the clinical success achieved with the first generation of adoptive cell therapy (ACT) utilizing in vitro expanded tumor-infiltrating lymphocytes (TILs), the second and third generations of TIL ACT are evolving toward the use of genetically modified TIL. TIL therapy generally involves the transfer of a high number of TIL, ranging from 109 to 1011 cells. One of the technical difficulties in genetically modifying TIL, using a retroviral vector, is the ability to achieve large expansion of transduced TIL, while keeping the technique suitable to a Good Manufacturing Practices (GMP) environment. Consequently, we developed and optimized a novel method for the efficient production of large numbers of GMP-grade, gene-modified TIL for the treatment of patients with ACT. The chemokine receptor CXCR2 was used as the gene of interest for methodology development. The optimized procedure is currently used in the production of gene-modified TIL for two clinical trials for the treatment of metastatic melanoma at MD Anderson Cancer Center.

T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity., Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.

The low response rates to immunotherapy in uveal melanoma (UM) sharply contrast with reputable response rates in cutaneous melanoma (CM) patients. To characterize the mechanisms responsible for resistance to immunotherapy in UM, we performed immune profiling in tumors from 10 metastatic UM patients and 10 metastatic CM patients by immunohistochemistry (IHC). Although there is no difference in infiltrating CD8+ T cells between UM and CM, a significant decrease in programmed death-1 (PD-1)-positive lymphocytes was observed and lower levels of programmed death ligand-1 (PD-L1) in UM metastases compared with CM metastases. Tumors from metastatic UM patients showed a lower success rate of tumor-infiltrating lymphocyte (TIL) growth compared with metastatic CM (45% vs. 64% success), with a significantly lower quantity of UM TIL expanded overall. These studies suggest that UM and CM are immunologically distinct, and provide potential explanation for the impaired success of immunotherapy in UM.

Triple-negative breast cancer (TNBC) highly infiltrated with CD8+ tumor-infiltrating lymphocytes (TIL) has been associated with improved prognosis. This observation led us to hypothesize that CD8+ TIL could be utilized in autologous adoptive cell therapy for TNBC, although this concept has proven to be challenging, given the difficulty in expanding CD8+ TILs in solid cancers other than in melanoma. To overcome this obstacle, we used an agonistic antibody (urelumab) to a TNFR family member, 4-1BB/CD137, which is expressed by recently activated CD8+ T cells. This approach was first utilized in melanoma and, in this study, led to advantageous growth of TILs for the majority of TNBC tumors tested. The agonistic antibody was only added in the initial setting of the culture and yet favored the propagation of CD8+ TILs from TNBC tumors. These expanded CD8+ TILs were capable of cytotoxic functions and were successfully utilized to demonstrate the presence of immunogenic mutations in autologous TNBC tumor tissue without recognition of the wild-type counterpart. Our findings open the way for a successful adoptive immunotherapy for TNBC. Cancer Immunol Res; 5(6); 439–45. ©2017 AACR.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (, Melanoma: Tumor differences within a patient may explain heterogeneous responses Patients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

Background Immune related adverse events (irAEs) are common side effects of checkpoint inhibitory (CPI) therapies targeting CTLA-4 and PD-1/PD-L1. Grover’s disease is an uncommon dermatologic condition with unclear pathogenesis previously reported as an irAE with ipilimumab. Case Presentation We report an additional case of ipilimumab-induced Grover’s disease. Interestingly, this dermatologic side effect did not appear with use of anti-PD-1 therapy in our patient. Immune analysis was performed and suggests a possible role of Th2 cells in its patholgenesis. Conclusion This case suggests that Grover's disease is an irAE induced by Ipilimumab. Our immune analysis suggests that Th2 cells may be pathogenic mediators which warrants further study.