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PURPOSE Pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. Multianalyte signatures, including liquid biopsy and traditional clinical variables, have shown promise for improving prognostication in other solid tumors but have not yet been rigorously assessed for PDAC. MATERIALS AND METHODS We performed a prospective cohort study of patients with newly diagnosed locally advanced pancreatic cancer (LAPC) or metastatic PDAC (mPDAC) who were planned to undergo systemic therapy. We collected peripheral blood before systemic therapy and assessed circulating tumor cells (CTCs), cell-free DNA concentration (cfDNA), and circulating tumor KRAS (ctKRAS)–variant allele fraction (VAF). Association of variables with overall survival (OS) was assessed in univariate and multivariate survival analysis, and comparisons were made between models containing liquid biopsy variables combined with traditional clinical prognostic variables versus models containing traditional clinical prognostic variables alone. RESULTS One hundred four patients, 40 with LAPC and 64 with mPDAC, were enrolled. CTCs, cfDNA concentration, and ctKRAS VAF were all significantly higher in patients with mPDAC than patients with LAPC. ctKRAS VAF (cube root; 0.05 unit increments; hazard ratio, 1.11; 95% CI, 1.03 to 1.21; P = .01), and CTCs ≥ 1/mL (hazard ratio, 2.22; 95% CI, 1.34 to 3.69; P = .002) were significantly associated with worse OS in multivariate analysis while cfDNA concentration was not. A model selected by backward selection containing traditional clinical variables plus liquid biopsy variables had better discrimination of OS compared with a model containing traditional clinical variables alone (optimism-corrected Harrell's C-statistic 0.725 v 0.681). CONCLUSION A multianalyte prognostic signature containing CTCs, ctKRAS, and cfDNA concentration outperformed a model containing traditional clinical variables alone suggesting that CTCs, ctKRAS, and cfDNA provide prognostic information complementary to traditional clinical variables in advanced PDAC.

PURPOSE Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi), is approved as maintenance therapy for patients with advanced pancreatic cancer (PC) and a germline BRCA1 or BRCA2 pathogenic variant (PV). This investigator-initiated, single-arm phase II study assessed the role of the PARPi rucaparib as maintenance therapy in advanced PC with germline or somatic PV in BRCA1, BRCA2, or PALB2. PATIENTS AND METHODS Eligible patients had advanced PC; germline (g) or somatic (s) PVs in BRCA1, BRCA2, or PALB2, and received at least 16 weeks of platinum-based chemotherapy without evidence of platinum resistance. Chemotherapy was discontinued and patients received rucaparib 600 mg orally twice a day until progression. The primary end point was the progression-free survival (PFS) rate at 6 months (PFS6). Secondary end points included safety, ORR, disease control rate, duration of response, and overall survival. RESULTS Of 46 enrolled patients, 42 were evaluable (27 g BRCA2, seven g BRCA1, six g PALB2, and two s BRCA2). PFS6 was 59.5% (95% CI, 44.6 to 74.4), median PFS was 13.1 months (95% CI, 4.4 to 21.8), and median overall survival was 23.5 months (95% CI, 20 to 27). The PFS at 12 months was 54.8%. ORR of the 36 patients with measurable disease was 41.7% (3 complete responses; 12 partial responses; 95% CI, 25.5 to 59.2), and disease control rate was 66.7% (95% CI, 49.0 to 81.4). Median duration of response was 17.3 months (95% CI, 8.8 to 25.8). Responses occurred in patients with gBRCA2 (41%, 11 out of 27), gPALB2 (50%, 3 out of 6), and sBRCA2 (50%, 1 out of 2). No new safety signals were noted. CONCLUSION Maintenance rucaparib is a safe and effective therapy for platinum-sensitive, advanced PC with a PV in BRCA1, BRCA2, or PALB2. The finding of efficacy in patients with g PALB2 and s BRCA2 PVs expands the population likely to benefit from PARPi beyond g BRCA1/ 2 PV carriers.

Cancer immunotherapy tools include antibodies, vaccines, cytokines, oncolytic viruses, bispecific molecules, and cellular therapies. This review will focus on adoptive cellular therapy, which involves the isolation of a patient’s own immune cells followed by their ex vivo expansion and reinfusion. The majority of adoptive cellular therapy strategies utilize T cells isolated from tumor or peripheral blood, but may utilize other immune cell subsets. T-cell therapies in the form of tumor-infiltrating lymphocytes, T-cell receptor T cells, and CAR T cells may act as “living drugs” as these infused cells expand, engraft, and persist in vivo, allowing adaptability over time and enabling durable remissions in subsets of patients. Adoptive cellular therapy has been less successful in the management of solid tumors because of poor homing, proliferation, and survival of transferred cells. Strategies are discussed, including expression of transgenes to address these hurdles. Additionally, advances in gene editing using CRISPR/Cas9 and similar technologies are described, which allow for clinically translatable gene-editing strategies to enhance the antitumor activity and to surmount the hostilities advanced by the host and the tumor. Finally, the common toxicities and approaches to mitigate these are reviewed.

Summary Pancreatic ductal adenocarcinoma (PDA) is among the most immune-resistant tumor types. Its unique genomic landscape shaped by oncogenic drivers promotes immune suppression from the earliest stages of tumor inception to subvert adaptive T cell immunity. Single-agent immune modulators have thus far proven clinically ineffective, and multi-modal therapies targeting mechanisms of immunotherapy resistance are likely needed. Here, we review novel immunotherapy strategies currently under investigation to (1) confer antigen specificity, (2) enhance T cell effector function, and (3) neutralize immunosuppressive elements within the tumor microenvironment that may be rationally combined to untangle the web of immune resistance in PDA and other tumors.

Background Palbociclib is highly active in oestrogen-receptor positive (ER+) metastatic breast cancer, but neutropenia is dose limiting. The goal of this study was to determine whether early neutropenia is associated with disease response to single-agent palbociclib. Methods Blood count and disease-response data were analysed from two Phase 2 clinical trials at different institutions using single-agent palbociclib: advanced solid tumours positive for retinoblastoma protein and advanced liposarcoma. The primary endpoint was PFS. The primary exposure variable was the nadir absolute neutrophil count (ANC) during the first two cycles of treatment. Results One hundred and ninety-six patients (61 breast, 135 non-breast) were evaluated between the two trials. Development of any grade neutropenia was significantly associated with longer median PFS in both the breast cancer (HR 0.29, 95% CI 0.11–0.74, p = 0.010) and non-breast cancer (HR 0.57, 95% CI 0.38–0.85, p = 0.006) cohorts. Grade 3–4 neutropenia was significantly associated with prolonged PFS in the non-breast cohort (HR 0.57, 95% CI 0.38–0.85, p = 0.006) but not in the breast cohort (HR 0.87, 95% CI 0.51–1.47, p = 0.596). Multivariate analysis yielded similar results. Conclusions Treatment-related neutropenia in the first two cycles was significantly and independently associated with prolonged PFS, suggesting that neutropenia may be a useful pharmacodynamic marker to guide individualised palbociclib dosing. Clinical trials registration information Basket Trial: NCT01037790; Sarcoma Trial: NCT01209598.

e15580 Background: The antiangiogenic tyrosine kinase inhibitor regorafenib provides a survival benefit in patients with previously treated metastatic colorectal cancer. Antiangiogenic therapy causes hypoxic stress within tumor cells, which activate autophagy as a survival mechanism. Entinostat, a histone deacetylase (HDAC) inhibitor, increases dependence on autophagy through epigenetic mechanisms. Hydroxychloroquine (HCQ) blocks autophagy by blunting lysosomal acidification and is synergistic with antiangiogenic therapies. We hypothesized that HCQ and entinostat would be tolerable with regorafenib and potentiate the antitumor response. Methods: This was a 3+3 phase I trial to find the recommended phase II dose (RP2D) of HCQ and entinostat with regorafenib in patients with metastatic colorectal cancer previously treated with a fluoropyrimidine, oxaliplatin, and irinotecan. No prior regorafenib or HDAC inhibitor therapy was permitted. Regorafenib was dosed at 160mg daily on days 1-21 of 28-day cycles, with provision to lower the starting dose to 80mg if toxicity was excessive. Entinostat was dosed at 3mg weekly in dose level 1 and at 5mg weekly in dose levels 2 and 3 while HCQ was dosed at 200mg qAM and 400mg qPM in dose levels 1 and 2 and at 600mg BID at dose level 3. Expansion was planned at the RP2D with a primary endpoint of objective response rate. Results: Twenty-eight patients were screened, and 20 patients were enrolled from November 2017 to January 2020. Six patients were treated at dose level 1 with no dose-limiting toxicity. The starting regorafenib dose was reduced to 80mg after 3 patients discontinued therapy early due to fatigue or rash due to regorafenib. At dose level 2, 7 patients were enrolled to achieve 6 evaluable patients. One DLT (G3 fatigue) was noted and one patient withdrew consent after 14 days due to fever and tumor pain flare possibly related to treatment. Six patients enrolled at dose level 3; no DLTs were seen. One additional patient received HCQ 400mg BID instead of 600mg BID due to a clerical error. Weight loss (60%), fatigue (50%), and anorexia (50%) were the most common toxicities. Thirteen grade 3 toxicities were noted, with rash (15%), fatigue (10%), and alkaline phosphatase elevation (10%) the most common. No grade 4 toxicities were observed. Seven patients discontinued therapy early due to toxicity. Nearly all patients experienced rapid weight loss, with a range of 1.5 lbs to 27.1 lbs and a median weight loss of 9.5 lbs at two weeks. No objective responses were observed. The median PFS was 1.8 months, the median OS was 5.2 months, and no patient remained on study longer than 4 months. Expansion was not pursued due to toxicity and lack of efficacy. Conclusions: The combination of regorafenib, HCQ, and entinostat was poorly tolerated without evident activity in metastatic colorectal cancer. The substantial weight loss suggests a potential adverse metabolic interaction between these drugs. Clinical trial information: NCT03215264.

Introduction: In the setting of metastatic pancreatic adenocarcinoma (mPDAC), lower baseline plasma KRAS mutation levels have been associated with improved survival. While tissue-agnostic, plasma-based copy number instability (CNI) has been demonstrated as an early indicator of response to immunotherapy for some solid tumors, it has not been assessed for patients with mPDAC, nor in combination with KRAS mutations for patients receiving standard of care chemo/radiotherapy. Here we evaluate the combination of mutant KRAS (mKRAS) and CNI detection in plasma as a predictor of overall and progression-free survival (OS/PFS) in mPDAC patients who received standard of care therapy. Methods: Cell-free DNA was extracted from plasma and libraries prepared at baseline (Week 0) and weeks 8, 16 and 24 on therapy, and analyzed by next-generation sequencing (CNI) and droplet digital PCR (mKRAS). Descriptive statistics were computed for variables including CNI (score is a measure of circulating tumor DNA) and mKRAS variant allele fraction. Detection was defined as above the limit of detection (mKRAS=0.13%) and above the 95th percentile of the value in normal individuals (CNI=24). Therapy response was assessed by OS and PFS. Results: 196 plasma samples from 64 mPDAC patients were analyzed. When dichotomized as detectable vs undetectable, CNI alone was significantly associated with OS at all on-therapy timepoints but not baseline, whereas mKRAS was significantly associated with OS for all 4 timepoints (Table 1). Detection of both CNI and mKRAS in combination was strongly associated with worse OS at all timepoints, yielding the highest HR. Similar results were obtained when mKRAS and CNI were dichotomized at their respective median values or with PFS as the clinical endpoint. Conclusions: Combined CNI and mKRAS detection at baseline and on-therapy may provide a strong and early indication of worse prognosis for patients with mPDAC. Table 1. Association of CNI and mKRAS with Overall Survival (HazardRatio [95% CI], log-rank p-value) Timepoint CNI mKRAS CNI and KRAS Baseline/Week 0 1.54 [0.89-2.68], 0.1 2.05 [1.12-3.78], 0.02 2.50 [1.46-4.28], 0.0006 Week 8 1.78 [0.99-3.18], 0.05 2.21 [1.19-4.08], 0.01 9.81 [3.40-28.28], Citation Format: Samuele Cannas, Jacob E. Till, Kristine Kim, Michael J. LaRiviere, Charles M. Vollmer, Jennifer R. Eads, Thomas B. Karasic, Peter J. O'Dwyer, Charles J. Schneider, Ursina R. Teitelbaum, Kim A. Reiss Binder, Mark H. O'Hara, Douglas T. Ross, Kim McGregor, Kirsten Bornemann-Kolatzki, Ekkehard Schütz, Julia Beck, Erica L. Carpenter. Liquid biopsy signature combining copy number instability and mutant KRAS detection is associated with survival for patients with metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1043.

Immune checkpoint inhibitors have limited clinical activity in pancreatic cancer. Based on preclinical data, we hypothesized that hypofractionated radiation may cooperate with dual checkpoint inhibition in patients (Rech AJ, et al, Cancer Research, 2018). We therefore designed a phase 1 study to evaluate the safety and feasibility of two schedules of hypofractionated radiation with durvalumab (anti-PDL1) and tremelimumab (anti-CTLA-4) in patients with metastatic pancreatic, lung, and breast cancers and melanoma. Here, we present the data for pancreatic adenocarcinoma. Methods: Patients with metastatic pancreatic cancer treated with at least one prior line of therapy with measurable disease by RECIST in addition to an index lesion amenable to hypofractionated radiation were enrolled sequentially to two cohorts – cohort A evaluating 3 fractions of 8 Gy or cohort B evaluating 1 fraction of 17 Gy. Patients received 4 cycles of tremelimumab 1 mg/kg IV and durvalumab 20 mg/kg IV every 4 weeks for 4 doses followed by durvalumab 10 mg/kg IV every 2 weeks until progression. Radiation was given in week 2 of treatment. Patients were replaced if they did not receive week 5 of therapy on trial, but were included in safety/feasibility analysis. Blood and, when feasible, baseline and on-treatment biopsies were obtained for exploratory biomarker evaluation. Results: 10 patients were treated in cohort A and 21 patients in cohort B. All patients were included in the safety and feasibility assessment. Overall, treatment was well tolerated in both cohorts. The most common adverse events were grade 1 or 2 fatigue (A 30%, B 23.8%), diarrhea (A 10%, B 14.3%), pruritis (A 10%, B 14.3%), AST/ALT elevation and constipation (each A 10%, B 9.5%). Grade 3 diarrhea, elevated bilirubin, pneumonitis, and syncope were noted in 1 patient each, all in cohort B. Grade 5 pneumothorax occurred after baseline biopsy in 1 patient. Grade 2 hyperthyroidism (A) and pneumonitis (B) were noted each in 1 patient. 8 patients in cohort A and 13 patients in cohort B were evaluable for response. In cohort A, 50% of patients had stable disease as best response, and median overall survival was 4.9 months. In cohort B, 23.1% had PR and 30.8% had SD as best response and mOS was 5.2 months. Responses occurred more frequently when metastatic lung nodules were radiated – SD in 3/5 (60%) patients in cohort A, PR in 3/10 (30%) and SD in 4/10 (40%) patients in cohort B, compared to SD in 1/3 (33%) patients in cohort A and 0/3 (0%) patients in cohort B who underwent radiation to a liver lesion. Biomarker analysis will be presented. Conclusions: The combination of durvalumab, tremelimumab with hypofractionated radiation is safe and feasible in a refractory pancreatic adenocarcinoma patient population. Encouraging clinical activity warrants further evaluation, especially when hypofractionated radiation is delivered to lung nodules. Citation Format: Mark H. O'Hara, Adham S. Bear, Max M. Wattenberg, Ursina R. Teitelbaum, Kim A. Reiss, Thomas B. Karasic, Charles J. Schneider, Peter J. O'Dwyer, Edgar H. Ben-Josef, Andrzej P. Wojcieszynski, Amit H. Maity, Rosemarie H. Mick, Robert H. Vonderheide. Phase 1 study of hypofractionated radiation in combination with tremelimumab and durvalumab in refractory metastatic pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A016.

Up to 85% of patients with resectable pancreatic ductal adenocarcinoma (PDAC) experience metastatic relapse after curative intent surgery with many recurring early. Detection of such occult metastases (those thought to be below the level of detection of standard of care imaging at the time of resection) could steer patients to a different treatment course rather than delaying systemic therapy until after recovery from inappropriate surgery. Here we investigate the potential of pancreas-specific circulating cell-free DNA (cfDNA) as a biomarker for the detection of occult metastatic disease and as a prognostic biomarker. Blood specimens were collected from 53 patients (15 negative control healthy subjects, 11 positive control metastatic PDAC patients, and 27 resectable PDAC patients prior to resection), processed to plasma, and banked. Plasma cfDNA was extracted, quantified, treated with bisulfite, and used as template for PCR amplification of 9 marker loci that are uniquely unmethylated in DNA of pancreatic acinar or duct cells. Following deep sequencing of PCR products, the fraction of cfDNA molecules derived from the pancreas was determined and multiplied by the total cfDNA concentration to yield pancreas-specific cfDNA. Recurrence and survival data were abstracted from the medical record and receiver operator curve analysis was utilized to determine statistical significance. Metastases were categorized as overt (present at diagnosis), occult (discovered during or within 4 months of curative intent surgery), or two-year (discovered during or within two years of curative intent surgery). Pancreas-derived cfDNA was significant for the detection of occult or overt metastases in our full cohort (18 of 52 evaluable subjects) with an area under the curve (AUC) of 0.86 (95% Confidence Interval, 0.74-0.80) and 0.91 (0.83-1.00) for liver-specific occult or overt metastases (15 of 52). It was borderline significant for the detection of occult metastases in the resectable sub-cohort (7 of 27) with an AUC of 0.71 (0.47-0.96) but significant for the detection of occult liver-specific metastases (5 of 27) with an AUC of 0.79 (0.62-0.96). Further, detection of overt metastases or two-year metastases (28 of 50) was significant with an AUC of 0.85 (0.74-0.96). In the resectable sub-cohort, it was also significant for the detection of two-year metastases (17 of 25) with an AUC of 0.79 (0.60-0.97) and prognostic for 2-year overall survival (12 of 24) with an AUC of 0.81 (0.62-1.00) in the resectable sub-cohort. Liver-derived cfDNA was also analyzed and was always outperformed by pancreas-specific cfDNA. In this pilot cohort, enumeration of pancreas-specific cfDNA shows promise as biomarker of occult metastatic disease, two-year metastatic progression, and two-year overall survival in resectable PDAC. Further investigation of a larger cohort and potential combination with other known markers like CA19-9 and tumor size is underway; results for an additional ~40 patients will be available by the time of the meeting. Citation Format: Jacob E. Till, Roni Ben-Ami, Ruth Shemer, Kristine Kim, Aseel Abdalla, Samuele Cannas, Charles M. Vollmer, Mark H. O'Hara, Ben Z. Stanger, Yuval Dor, Erica L. Carpenter. Pancreas-specific circulating cell-free DNA for detection of occult metastases and prognosis in resectable pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A030.

This phase I study investigated the safety and activity of lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin (CART-meso) in patients with malignant pleural mesothelioma, ovarian carcinoma, and pancreatic ductal adenocarcinoma. Fifteen patients with chemotherapy-refractory cancer (n = 5 per indication) were treated with a single CART-meso cell infusion. CART-meso cells were engineered by lentiviral transduction with a construct composed of the anti-mesothelin single-chain variable fragment derived from the mouse monoclonal antibody SS1 fused to intracellular signaling domains of 4-1BB and CD3zeta. Patients received 1–3 × 10(7) or 1–3 × 10(8) CART-meso cells/m(2) with or without 1.5 g/m(2) cyclophosphamide. Lentiviral-transduced CART-meso cells were well tolerated; one dose-limiting toxicity (grade 4, sepsis) occurred at 1–3 × 10(7)/m(2) CART-meso without cyclophosphamide. The best overall response was stable disease (11/15 patients). CART-meso cells expanded in the blood and reached peak levels by days 6–14 but persisted transiently. Cyclophosphamide pre-treatment enhanced CART-meso expansion but did not improve persistence beyond 28 days. CART-meso DNA was detected in 7/10 tumor biopsies. Human anti-chimeric antibodies (HACA) were detected in the blood of 8/14 patients. CART-meso cells were well tolerated and expanded in the blood of all patients but showed limited clinical activity. Studies evaluating a fully human anti-mesothelin CAR are ongoing.

The liver is the most common site of metastatic disease1. Although this metastatic tropism may reflect the mechanical trapping of circulating tumour cells, liver metastasis is also dependent, at least in part, on the formation of a 'pro-metastatic' niche that supports the spread of tumour cells to the liver2,3. The mechanisms that direct the formation of this niche are poorly understood. Here we show that hepatocytes coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. During early pancreatic tumorigenesis in mice, hepatocytes show activation of signal transducer and activator of transcription 3 (STAT3) signalling and increased production of serum amyloid A1 and A2 (referred to collectively as SAA). Overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and many patients with locally advanced and metastatic disease show increases in circulating SAA. Activation of STAT3 in hepatocytes and the subsequent production of SAA depend on the release of interleukin 6 (IL-6) into the circulation by non-malignant cells. Genetic ablation or blockade of components of IL-6-STAT3-SAA signalling prevents the establishment of a pro-metastatic niche and inhibits liver metastasis. Our data identify an intercellular network underpinned by hepatocytes that forms the basis of a pro-metastatic niche in the liver, and identify new therapeutic targets.

Purpose: This study evaluates the toxicity and tumor response with concurrent nab-paclitaxel chemoradiotherapy (CRT) compared with standard (5-fluorouracil or gemcitabine) CRT. Materials and Methods: Fifty patients with borderline resectable or unresectable pancreatic adenocarcinoma from 2014 to 2017 were divided into 2 groups: concurrent nab-paclitaxel (100 to 125 mg/m2 weekly) CRT (median: 2.1 Gy fraction size and 52.5 Gy total) or standard CRT (median: 1.8 Gy fraction size, 54.5 Gy total). The primary endpoint was toxicity, and secondary endpoints were local failure and conversion to resectability. Comparisons were made using rank-sum or Fisher exact test and multivariable competing risk regression for the cumulative incidence of local failure. Results: There were 28 patients in the nab-paclitaxel CRT group and 22 in the standard CRT group; 88% had the unresectable disease. The median follow-up was 18 months. The median duration of chemotherapy before concurrent CRT was 1.9 and 2.3 months in the nab-paclitaxel and standard CRT groups (P=0.337), and radiotherapy dose was 52.5 Gy (range, 52.5 to 59.4 Gy) and 54.5 Gy (range, 45.0 to 59.4 Gy), respectively. There were no statistically significant grade ≥2 toxicities. The nab-paclitaxel CRT group experienced a nonstatistically significant lower incidence of local failure (hazard ratio=0.91, 95% confidence interval: 0.27-3.03, P=0.536). More patients in the nab-paclitaxel CRT group proceeded to surgery (9/28 compared with 3/22 in the standard CRT, P=0.186); of which 6 (25%) in the nab-paclitaxel CRT and 2 (10%) in the standard CRT groups were initially unresectable. Conclusions: Nab-paclitaxel CRT had similar toxicity compared with standard CRT in the treatment of borderline resectable or unresectable pancreatic cancer. Its use was associated with an arithmetically lower cumulative incidence of local failure and an arithmetically higher conversion to resectability, both of which were not statistically significant.

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαβ isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein., KRAS is commonly mutated at codon 12 in several cancer types, offering a unique opportunity for the development of neoantigen-targeted immunotherapy. Here the authors present a pipeline for the prediction, identification and validation of HLA class-I restricted mutant KRAS G12 peptides, leading to the generation of mutant KRAS-specific T cell receptors for adoptive T cell immunotherapy.

Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of a subset of B cell malignancies but has shown limited clinical success in solid tumor therapy. T cell exhaustion is an important factor involved in treatment failure, and can, in part, result from continuous stimulation of the CAR by tumor cells expressing the cognate antigen. To gain deeper understanding of CAR T cell exhaustion induced by chronic antigen exposure (CAE), we developed and validated a robust in vitro model, in which mesothelin-redirected CAR T cells (M5CAR) were continuously stimulated with mesothelin-expressing AsPC-1 pancreatic tumor cells such that tumor cells were never cleared, and we characterized these CAE CAR T cells by gene expression at population and single-cell levels, and by epigenetic analyses. CAE M5CAR T cells recapitulate the hallmarks of T cell exhaustion, including reduced proliferation, down-modulation of surface CAR, decreased cytokine production, and reduced cytotoxicity. In addition, CAR T cells undergoing CAE have a transcriptional signature and an epigenetic landscape consistent with exhaustion. Further, transcriptomic analysis revealed that CAE M5CAR T cells undergo a transition from T cells to a post-thymic NK-like T cell phenotype. This plasticity was confirmed by TCR lineage tracing and was also detected in CD19 CAR T cells analyzed from post-infusion blood from DLBCL patients and in M5CAR T cells infiltrating relapsed tumors derived from a xenograft NSG/AsPC-1 mouse model. The dysfunctional signature and the NK phenotype were further detected in vivo by transcriptomic analysis in Ly95 TCR-specific TILs infiltrating NY-ESO-1 tumors. Among the genes included in the dysfunctional signature, the transcription factors SOX4 and ID3 were identified as potential regulators of dysfunction by differential gene expression and pathway analyses. To determine their role in the establishment of a dysfunctional phenotype, we generated ID3KO.M5 and SOX4KO.M5 CAR T cells using CRISPR technology in primary human lymphocytes. The cytotoxic potential of the M5 CAR T cell product generated was not modified by the genetic disruption of the transcription factors. However, when CAR T cells were challenged with chronic antigen stimulation, KO CAR T cells showed a significant reduction of the dysfunctional signature and the NK-like T cell gene expression. Importantly, CAE KO CAR T cells exhibit improved cytotoxicity as compared to Mock.M5CAR T cells. In summary, we have developed a robust in vitro model that recapitulates the hallmarks of T cell exhaustion and that facilitated the identification of a gene signature defining CAR dysregulation, a T-to-NK-like-T cell transition as a novel feature of CAR T cell dysfunction and the transcription factors SOX4 and ID3 as key regulators of CAR T cell exhaustion. Citation Format: M. Angela Aznar, Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Sangya Agarwal, Greg Donahue, Kenichi Ishiyama, Nils Wellhausen, Austin K. Rennels, Yujie Ma, Lifeng Tian, Sonia Guedan, Katherine A. Alexander, Zhen Zhang, Philipp C. Rommel, Nathan Singh, Karl M. Glastad, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O’Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3602.

2548 Background: Circulating tumor DNA (ctDNA) is increasingly used as a prognostic marker with high ctDNA shedding associated with poor survival. Gene-, but not variant-specific, differences in ctDNA shedding have been reported. Tumor burden, mitotic rate, and cell death rate have been proposed as contributors to ctDNA shedding. Here we investigate associations of ctDNA shedding for the two most common mPDAC KRAS variants, G12D and G12V, with tumor burden, mitotic score, and overall survival (OS). Methods: Pretreatment (baseline) ctDNA was analyzed by droplet digital PCR for 86 (including 44 G12D, 30 G12V) patients with mPDAC receiving front-line chemoimmunotherapy in a randomized open-label Phase II study (NCT03214250). Baseline tumor burden in total, within the pancreas, and distally, was assessed by sum of RECIST target lesion diameters. Tumor tissue variant allele fraction (tVAF) and mitotic score (geometric mean expression of 65 mitosis-associated genes) were calculated from DNA and RNA sequencing. Results: ctKRAS shedding (dichotomized at median mutant copies/mL) was associated with OS for G12D bearing tumors (p = 0.03) but not G12V (p = 0.17, log-rank test). To identify variant-specific features of shedding, we examined the Spearman correlation for total tumor burden and ctKRAS shedding; G12D but not G12V shedding was correlated with tumor burden (p = 0.01 and p = 0.22 respectively). However, the higher tVAF in G12V compared to G12D tumors (p = 0.048, Mann-Whitney test) could result from differences in purity, ploidy, and KRAS copy number. Thus, we used tVAF as a scalar to calculate an adjusted tumor burden which was significantly correlated with both G12D and G12V ctDNA shedding (p = 0.004 and 0.02, respectively). When a patient’s distal vs. pancreatic lesions were analyzed separately, pancreatic tumor burden was not correlated with G12D or G12V shedding (p = 0.10 and 0.33, respectively) but distal burden was correlated with both (p = 0.001 and 0.02, respectively). While there was no difference by KRAS variant for the correlation between adjusted tumor burden and shedding, these results do suggest that, in patients with mPDAC, distal rather than primary tumor burden may drive ctDNA shedding. Finally, tumor mitotic rate was combined with adjusted total tumor burden in a linear regression model; both were significant for predicting G12D shedding (p = 0.007 and p < 0.0001, respectively) but not for G12V (p = 0.045 and p = 0.16, respectively). Conclusions: These data suggest that ctDNA shedding and survival associations may be KRAS variant-specific in mPDAC. Tumor mitotic score and location of tumors may explain some variant-specific differences in shedding. As clinical ctDNA tests become more widely used, further investigation of variant-specific shedding in KRAS and other genes may be key for proper interpretation of ctDNA tests.

4131 Background: We recently reported the results of a single arm phase II study of maintenance rucaparib in patients with platinum-sensitive advanced pancreatic cancer and a pathogenic variant in BRCA1, BRCA2, or PALB2 (NCT 03140670; Reiss, JCO 2021). However, optimal treatment following progression on PARP inhibitors (PARPi) has not been defined. Here, we report a descriptive study of post-progression treatment and outcomes of this patient population. Methods: Patients with advanced pancreatic cancer and germline or somatic BRCA1, BRCA2, or PALB2mutations treated with at least 16 weeks of platinum-based chemotherapy without progression were enrolled and treated with rucaparib until progression or unacceptable toxicity. At the time of progression, patients were treated with physician-choice chemotherapy. Here we evaluate the objective response rates (ORR) by RECIST 1.1. Overall survival (OS) and time to second progression (PFS2) calculated from trial enrollment and progression free survival on chemotherapy (PFS) by regimen were secondary endpoints. Time-to-event was analyzed by the Kaplan-Meier method and censored at date of last clinic visit, with a cutoff date of 12/10/21. Results: The trial enrolled 42 patients; 31 patients had progressed. Of these, 22 received second-line chemotherapy: nine were treated with an oxaliplatin-based regimen, nine were treated with a cisplatin-based regimen, and four were treated with non-platinum regimens. Demographics were balanced between those who received platinum versus non-platinum. All patients who received second-line chemotherapy regimens met the PFS2 endpoint and all but one patient had died at time of data cutoff. No patients had a complete response, five patients had a partial response (PR). By regimen, 1/9 patients treated with cisplatin had a PR, 3/9 treated with oxaliplatin had a PR, and 1/4 patients treated with non-platinum had a PR. OS, PFS2, PFS, and ORR results by regimen are shown in the table. Conclusions: In this small sample of patients with advanced pancreatic cancer with progressive disease on PARPi, chemotherapy retains some activity. Further study to identify predictors of response and/or resistance to post-PARPi treatment are underway. [Table: see text]

4021 Background: Establishing alternatives to perpetual chemotherapy for pts with aPDAC has been proposed to address inevitable chemotherapy resistance and cumulative toxicity. Poly (ADP ribose) polymerase (PARP) inhibitors have shown clinical efficacy in this setting, and preclinical data suggest that the addition of immune checkpoint blockade (ICB) may offer synergistic tumor control. We performed a randomized, phase Ib/II study of nira plus anti-PD-1 (nivo) or nira plus anti-CTLA-4 (ipi) maintenance for pts with aPDAC who had not progressed after >4 mo of platinum-based therapy. Methods: After discontinuation of chemotherapy, pts were randomized 1:1 to nira 200mg PO daily plus either nivo 240mg IV q2 weeks (later amended to 480mg IV q4 weeks) or ipi 3mg/kg IV q3 weeks for four doses. Nira could be escalated to 300mg PO daily if tolerated. Pts were treated until progression or unacceptable toxicity. The primary endpoint was progression free survival at six months (PFS6) in each arm. Secondary endpoints included safety, OS, ORR, and outcomes by DNA damage repair (DDR) deficiencies. Pts were evaluable for safety if they had received > 1 dose of study treatment and for efficacy if they had also received > 1 follow-up imaging study. Based on historical data, the null hypothesis of PFS6 = 44% vs a 2-sided alternative hypothesis of PFS ≠ 44% was tested. 42 pts per arm provided 81% power for testing at a 5% significance level to detect inferior PFS6 < 27% or superior PFS6 > 61%. Results: As of Oct 2021, 91 pts were enrolled, of whom 84 were evaluable for efficacy (44 nira/nivo; 40 nira/ipi). The median potential follow-up was 23 mos. Most common treatment-related AEs were nira/nivo: thrombocytopenia (28%), arthralgia (25%), nausea (23%), and fatigue (23%) and nira/ipi: thrombocytopenia (45%), anemia (43%), fatigue (43%), nausea, (41%), AST increase (36%), rash (34%) and ALT increase (29%). 88% of AEs were grade 1-2. Efficacy results were: nira/nivo: PFS6 20.6% (95% CI 8.3-32.9, p = 0.0002), mPFS 1.9 mo and nira/ipi: PFS6 59.6% (95% CI 44.3-74.9, p = 0.045), mPFS 8.1 mo. Fifteen pts (8 nira/nivo; 7 nira/ipi) had pathogenic variants in BRCA or PALB2 . Excluding these: mPFS on nira/nivo was 1.9 mo (95% CI 1.8-1.9) and mPFS on nira/ipi was 7.6 mo (95% CI 4.0 – 11.1). Conclusions: In a randomized phase 1B/II study, nira/ipi as maintenance therapy met the primary endpoint of superior PFS6 while nira/nivo yielded inferior PFS6 for pts with aPDAC who had not progressed on first-line platinum-based chemotherapy. The benefit of nira/ipi maintenance persisted in pts without known DDR variants. Clinical trial information: NCT03404960. [Table: see text]

4010 Background: Preclinical and small clinical studies of chemoimmunotherapy for metastatic pancreatic ductal adenocarcinoma (mPDAC) point to a yet unrealized potential of clinically significant immune activation. In our phase II study of the CD40 agonist antibody sotigalimab (sotiga) and/or nivolumab (nivo) with gemcitabine and nab-paclitaxel (chemo), we observed promising improvements in overall survival (OS) in 105 patients with newly diagnosed mPDAC (NCT03214250); the primary endpoint of 1-year OS rate was 57.7% (p = 0.006) in the nivo/chemo arm, 48.1% (p = 0.062) in the sotiga/chemo arm and 41.3% (p = 0.233) in the nivo/sotiga/chemo arm (O’Hara, ASCO 2021) as compared to a historical control of 35%. Here, we report results of multi-omic translational analyses designed to identify signatures predictive of OS benefit. Methods: Longitudinal blood and tumor tissue samples were collected for immune and tumor biomarker analysis. Tumor samples underwent RNA sequencing and multiplex immunofluorescence (mIF). Peripheral blood was analyzed by mass cytometry time of flight (CyTOF), high parameter flow cytometry, and proteomics. Machine learning (ML) algorithms were applied to the data to identify biosignatures related to OS in each arm. Results: Comprehensive multi-omic, multi-parameter immune and tumor biomarker analyses identified distinct pretreatment immune signatures predictive of longer OS specific to nivo/chemo or sotiga/chemo (Table, representative examples). Because patients in each arm received chemotherapy, these and other arm-unique biomarkers suggest a relationship to the immunotherapy rather than chemotherapy in this randomized study. There was evidence of immune exhaustion in the sotiga/nivo/chemo arm that may explain the lack of survival benefit. Conclusions: From in-depth translational and ML analyses of randomized phase II trial of first-line chemoimmunotherapy in mPDAC patients, we identified novel biomarkers that associated with OS distinctly in each arm. Clinical trials in first-line mPDAC exploiting these previously unappreciated biomarkers and aiming to enrich patients for response, are warranted to further advance chemoimmunotherapy in this disease. Clinical trial information: NCT03214250. [Table: see text]

Summary Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Purpose: To determine whether a multianalyte liquid biopsy can improve the detection and staging of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: We analyzed plasma from 204 subjects (71 healthy, 44 non-PDAC pancreatic disease, and 89 PDAC) for the following biomarkers: tumor-associated extracellular vesicle miRNA and mRNA isolated on a nanomagnetic platform that we developed and measured by next-generation sequencing or qPCR, circulating cell-free DNA (ccfDNA) concentration measured by qPCR, ccfDNA KRAS G12D/V/R mutations detected by droplet digital PCR, and CA19-9 measured by electrochemiluminescence immunoassay. We applied machine learning to training sets and subsequently evaluated model performance in independent, user-blinded test sets. Results: To identify patients with PDAC versus those without, we generated a classification model using a training set of 47 subjects (20 PDAC and 27 noncancer). When applied to a blinded test set (N = 136), the model achieved an AUC of 0.95 and accuracy of 92%, superior to the best individual biomarker, CA19-9 (89%). We next used a cohort of 20 patients with PDAC to train our model for disease staging and applied it to a blinded test set of 25 patients clinically staged by imaging as metastasis-free, including 9 subsequently determined to have had occult metastasis. Our workflow achieved significantly higher accuracy for disease staging (84%) than imaging alone (accuracy = 64%; P < 0.05). Conclusions: Algorithmically combining blood-based biomarkers may improve PDAC diagnostic accuracy and preoperative identification of nonmetastatic patients best suited for surgery, although larger validation studies are necessary.

We conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers. There were two strata (12 patients each): (i) NSCLC/melanoma progressing on prior anti-PD-1 therapy, (ii) other cancer types; anti-PD-1-naive. Patients received 6 cycles of pembrolizumab, starting 1 week before HFRT. Patients had ≥2 lesions; only one was irradiated (8 Gy × 3 for first half; 17 Gy × 1 for second half in each stratum) and the other(s) followed for response. Of the 24 patients, 20 (83%) had treatment-related adverse events (AEs) (all grade 1 or 2). There were eight grade 3 AEs, none treatment related. There were no dose-limiting toxicities or grade 4/5 AEs. Stratum 1: two patients (of 12) with progression on prior PD-1 blockade experienced prolonged responses (9.2 and 28.1 months). Stratum 2: one patient experienced a complete response and two had prolonged stable disease (7.4 and 7.0 months). Immune profiling demonstrated that anti-PD-1 therapy and radiation induced a consistent increase in the proliferation marker Ki67 in PD-1-expressing CD8 T cells. HFRT was well tolerated with pembrolizumab, and in some patients with metastatic NSCLC or melanoma, it reinvigorated a systemic response despite previous progression on anti-PD-1 therapy. Clinical Trial Registration: NCT02303990 ( www.clinicaltrials.gov ).

Pancreatic ductal adenocarcinoma (PDAC) is resistant to T-cell mediated immunotherapy. We engineered T cells to transiently express an mRNA encoding a chimeric antigen receptor (CAR) specific for mesothelin—a protein that is over-expressed by PDAC cells. We performed a phase 1 study to evaluate the safety and efficacy of adoptive cell therapy with autologous mesothelin-specific CAR T cells (CARTmeso cells) in 6 patients with chemotherapy-refractory metastatic PDAC. Patients were given intravenous CARTmeso cells 3 times weekly for 3 weeks. None of the patients developed cytokine release syndrome or neurologic symptoms and there were no dose limiting toxicities. Disease stabilized in 2 patients, with progression-free survival times of 3.8 and 5.4 months. We used FDG-positron emission tomography/computed tomography imaging to monitor the metabolic active volume (MAV) of individual tumor lesions. The total MAV remained stable in 3 patients and decreased by 69.2% in 1 patient with biopsy-proven mesothelin expression; in this patient, all liver lesions had a complete reduction in FDG uptake at 1 month compared to baseline, although there was no effect on the primary PDAC. Transient CAR expression was detected in patients’ blood after infusion and led to expansion of new immunoglobulin G proteins. Our results provide evidence for the potential anti-tumor activity of mRNA CARTmeso cells, as well as PDAC resistance to the immune response. Keywords: immune response heterogeneity, immune therapy, anti-tumor immunity, pancreatic cancer treatment

BackgroundGemcitabine/nab-Paclitaxel (GnP) is a standard of care regimen for first-line metastatic pancreatic ductal adenocarcinoma (PDAC) and has a 1-year overall survival (OS) rate of approximately 35%. There is an urgent need for novel therapeutics and precision medicine approaches in PDAC. PRINCE, a randomized phase 2 trial, reported an increased 1-year OS relative to historical data, for patients treated with nivolumab (nivo)/GnP (57.3%, p = 0.007, n=34) and sotigalimab (sotiga) (APX005M; CD40 agonist)/GnP (48.1%, p = 0.062, n= 36).MethodsTo investigate immune modulatory and pharmacodynamic (PD) effects of nivo or sotiga in combination with GnP we used several orthogonal minimally invasive, blood-based biomarker technologies. Immune population profiles were evaluated by CyTOF and features of T cell phenotype and function by multicolor flow cytometry. Soluble proteins were evaluated with predefined panels using the Olink platform (Immuno-oncology (IO) and Immune Response) along with an unbiased mass spectrometry proteomic approach (Biognosys) that identified circulating soluble proteins of significance.ResultsRelative to baseline, patients who received nivo/GnP had numerically increased frequencies of proliferating, activated CD8+ and CD4+ effector memory T cells in circulation across multiple timepoints. These patients also had significantly increased levels of soluble proteins associated with type II interferon responses and immune cell migration and T cell activation, as well as significantly decreased levels of immunomodulatory proteins.Patients who received sotiga/GnP had increased expression of the co-stimulatory molecule CD86 on conventional dendritic cells. These patients also had significantly increased concentrations of soluble proteins associated with mature antigen presenting cells, and the activation of helper CD4+ T cells, B cells, and monocytes. Significant increases in soluble proteins associated with type-1 cell-mediated effector immunity and decreases in immunosuppressive factors were observed in both arms. Significant proteins were defined as p ≤ 0.05, log2 expression fold change ≥ 0.5 (Olink) and Sparse PLS discriminant analysis was used with zero as a threshold (Biognosys).ConclusionsThis study is a first to use multiomic minimally invasive biomarker approaches in PDAC to demonstrate PD effects and immune modulation with immunotherapy/chemotherapy combinations. Orthogonal assays demonstrate that nivo/GnP and sotiga/GnP elicit unique immune responses and the observed effects are consistent with their distinct mechanisms of action. These data suggest that multiomic biomarker signatures may identify subsets of patients who may benefit from an immunotherapy/chemo approach in PDAC. Moreover, results from these analyses will support early phase clinical study development decisions.AcknowledgementsWe extend our gratitude to the patients, their families, the clinical investigators, and their site staff members who are making this trial possible. We would also like to thank Sultan Nawabi at Parker Institute for Cancer Immunotherapy (PICI) for operations leadership of the trial. We thank Bristol Myers Squibb (BMS) and Apexigen for collaboration and study drugs. The study was funded by Cancer Research Institute, BMS and PICI.Trial RegistrationNCT03214250Ethics ApprovalThis study was approved by University of Pennsylvania Institutional Review Board; Federalwide assurance #00004028.

Importance Autophagy is a mechanism of treatment resistance to chemotherapy that has a role in the maintenance of pancreatic cancer. Hydroxychloroquine sulfate (HCQ) is an inhibitor of autophagy that inhibits the fusion of the autophagosome to the lysosome. Objective To determine whether HCQ improves overall survival at 1 year in combination with gemcitabine hydrochloride and nab-paclitaxel (GA) among patients with metastatic pancreatic cancer. Design, Setting, and Participants Open-label, phase 2 randomized clinical trial conducted between March 18, 2013, and November 16, 2017, at the University of Pennsylvania, HonorHealth, and The Johns Hopkins University among 112 patients with previously untreated metastatic or advanced pancreatic ductal adenocarcinoma, Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate marrow and organ function. All efficacy analyses were performed for the intention-to-treat population. Interventions Patients were randomized in a 1:1 ratio to receive GA with or without HCQ. All patients received standard doses of GA, and those randomized to receive HCQ were treated continuously with 600 mg orally twice daily. Main Outcome and Measure Overall survival at 1 year. Results A total of 112 patients (45 women and 67 men; median age, 65 years; range, 43-86 years) were enrolled; 55 were randomized to receive GA plus HCQ, and 57 to receive GA. Overall survival at 12 months was 41% (95% CI, 27%-53%) in the HCQ group and 49% (95% CI, 35%-61%) in the non-HCQ group. Median progression-free survival was 5.7 months (95% CI, 4.0-9.3 months) in the HCQ group and 6.4 months (95% CI, 4.5-7.6 months) in the non-HCQ group. Median overall survival was 11.1 months (95% CI, 9.0-14.2 months) in the HCQ group and 12.1 months (95% CI, 9.3-15.5 months) in the non-HCQ group. Overall response rate was 38.2% (n = 21) in the HCQ group and 21.1% (n = 12) in the non-HCQ group (P = .047). Treatment-related grade 3 or 4 adverse events that differed between the HCQ and non-HCQ groups were neutropenia (23 of 54 [42.6%] vs 12 of 53 [22.6%]), anemia (2 of 54 [3.7%] vs 9 of 53 [17.0%]), fatigue (4 of 54 [7.4%] vs 0), nausea (5 of 54 [9.3%] vs 0), peripheral neuropathy (7 of 54 [13.0%] vs 3 of 53 [5.7%]), visual changes (3 of 54 [5.6%] vs 0), and neuropsychiatric symptoms (3 of 54 [5.6%] vs 0). Conclusions and Relevance The addition of HCQ to block autophagy did not improve the primary end point of overall survival at 12 months. These data do not support the routine use of GA plus HCQ for metastatic pancreatic cancer in the absence of a biomarker. However, improvement seen in the overall response rate with HCQ may indicate a role for HCQ in the locally advanced setting, where tumor response may permit resection. Trial Registration ClinicalTrials.gov identifier:NCT01506973

Activating missense mutations within RAS oncogenes are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here, we employ multi-omic approaches to characterize optimal HLA class I-restricted mKRAS epitopes. Using in silico epitope prediction, we identify candidate mKRAS G12 epitopes with high predicted binding affinity to prevalent HLA class I alleles. We utilize biochemical approaches to identify mKRAS epitopes with high measured binding affinity and stability to HLA-A*03:01, -A*11:01 and -B*07:02. Furthermore, we provide proteomic validation of epitope processing and presentation by these alleles. We report mKRAS G12V and G12R epitopes are immunogenic as evidenced by the isolation of mKRAS G12V- and G12R-specific TCRαβ from healthy donors. The transfer of mKRAS-TCRs to primary CD8+ T cells serves as sensitive probes to validate mKRAS epitope processing and presentation by tumor cells and confers cytotoxicity against mKRAS human tumor cell lines of various histologies. The kinetics of lytic activity correlate with measured TCR avidity and mKRAS/HLA class I complex abundance expressed on the tumor cell surface. Finally, the adoptive transfer of mKRAS-TCR engineered primary CD8+ T cells to NOD scid gamma mice leads to tumor eradication in an orthotopic mKRAS lung cancer model. This study validates mKRAS as a bona fide tumor neoantigen and supports strategies to identify recurrent driver alterations for immunological targeting. Based on this work, clinical studies targeting mKRAS in cancer patients using vaccines and adoptive T cell therapy are under active investigation. Citation Format: Adham S. Bear, Tatiana Blanchard, Mark H. O'Hara, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting [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 1899.

Deploying CD40 activation to stimulate T cell responses upstream of immune checkpoint molecules is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological treatment effects especially treatment-related modulation of the tumor microenvironment (TME), remain poorly understood. Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy (gemcitabine and nab-paclitaxel) to 16 resectable patients with pancreatic ductal adenocarcinoma (PDAC) prior to surgery followed by adjuvant chemotherapy and CD40 mAb. The toxicity profile was acceptable, including only grade 1 or 2 cytokine release syndrome and expected toxicities from chemotherapy. Disease-free survival was 13.8 months (95% CI 2.9 - 24.8 months) and median overall survival was 23.4 months (95% CI 18.0 - 28.8), with 8 patients alive at a median of 20.0 months after surgery (follow-up range 12.2 to 34.8 months). Neoadjuvant selicrelumab induced major pharmacodynamic differences in the TME, as revealed by a multiplex imaging platform auditing the immune ecosystem, compared to resection specimens from PDAC patient previously untreated or given neoadjuvant chemotherapy/chemoradiotherapy only. For tumors resected after selicrelumab, 82% (9/11) were T-cell enriched, compared to 37% (38/104) (p=0.004) of untreated tumors and 23% (93/13) of chemotherapy/chemoradiation-treated tumors (p=0.012). Moreover, for selicrelumab tumors, tumor-associated fibrosis was less, “M2” macrophages were fewer, dendritic cells were more mature, and T cells were activated and proliferative, compared to the non-selicrelumab groups. In the periphery, CD8+ and CD4+ T cells were more activated and proliferative, and serum inflammatory cytokines CXCL10 and CCL22 increased after treatment. This study provides proof-of-concept in patients that agonistic CD40 mAb alters the TME, enhances T-cell infiltration, and modulates systemic inflammatory responses. These findings inform design of next-generation CD40 clinical trials. Citation Format: Katelyn T. Byrne, Courtney B. Betts, Rosemarie Mick, Shamilene Sivagnanam, David L. Bajor, Daniel A. Laheru, E. Gabriela Chiorean, Mark H. O'Hara, Shannon M. Liudahl, Craig Newcomb, Cécile Alanio, Ana P. Ferreira, Byung S. Park, Takuya Ohtani, Austin P. Huffman, Sara A. Väyrynen, Andressa Dias Costa, Judith C. Kaiser, Andreanne M. Lacroix, Colleen Redlinger, Martin Stern, Jonathan A. Nowak, E. John Wherry, Martin A. Cheever, Brian M. Wolpin, Emma E. Furth, Elizabeth M. Jaffee, Lisa M. Coussens, Robert H. Vonderheide. T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial [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 CT005.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust model to study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after continuous antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of NK-like CAR T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study shed light on the plasticity of human CAR T cells and suggest new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction. Citation Format: Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Greg Donahue, Kenichi Ishiyama, Yujie Ma, Nils Wellhausen, Lifeng Tian, Sangya Agarwal, Sonia Guedan, M. Angela Aznar, Katherine A. Alexander, Zhen Zhang, Nathan Singh, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O'Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [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 60.

4019 Background: Results from a ph1b trial evaluating gem/NP with CD40 agonistic monoclonal antibody APX005M ± nivo demonstrated promising clinical activity in pts with untreated mPDAC (O’Hara 2021). Herein, we report results from the follow-on, randomized (rand) ph2 trial evaluating gem/NP ± nivo ± APX005M. Methods: Pts with untreated mPDAC were rand to 1 of 3 open-label arms: gem/NP/nivo (A), gem/NP/APX005M (B), gem/NP/nivo/APX005M (C). All pts were treated with 1000 mg/m2 gem and 125 mg/m2 NP. Patients received 240 mg nivo in arms A and C and 0.3 mg/kg APX005M (RP2D) IV in arms B and C. Ph1b pts were included in ph2 analyses. 1° endpoint: 1-year OS rate of each arm, compared to a 35% historical OS rate for gem/NP (Von Hoff 2013). Key 2° endpoints: ORR, DCR, DOR, PFS and safety. Tumor and blood were collected for biomarker analysis. Planned enrollment of 35 pts/arm provided 81% power for testing the alternative of 58% OS rate vs 35%, using a 1-sided, 1-sample Z test with 5% type I error. Trial was not powered for cross-arm comparison. Results: 93 pts were rand in ph2 (N = 34, 30, 29 to A, B, C); when ph1b pts included, a total of 105 pts (34, 36, 35) were analyzed for efficacy and 108 pts (36, 37, 35) for safety. Min follow-up was 14 months (mos). Baseline characteristics were balanced across arms, inclusive of tumor burden, presence of liver metastases and stage at initial diagnosis (stage 1-3 vs 4). 1-year OS rate was 57% (1-sided p = 0.007 vs 35% historical rate, 95% lower CI bound = 41%) for A, 51% (p = 0.029, 95% bound = 36%) for B and 41% (p = 0.236, 95% bound = 27%) for C. Median OS and secondary endpoints are listed in Table. TRAE rates were similar across arms and to ph1b. 8 (7%) pts experienced an AE leading to tx discontinuation (6, 1, 1 in A, B, C), 40 (37%) pts experienced a serious TRAE (14, 15, 11 in A, B, C) and 2 pts died due to TRAEs; 1 each in B (acute hepatic failure) and C (intracranial hemorrhage). Conclusions: In this ongoing, seamless ph1b/2 trial of gem/NP ± nivo ± APX005M in pts with mPDAC, antitumor activity was observed in all arms. 1° endpoint of 1-year OS > 35% was met when combining gem/NP with either nivo or APX005M; however, not the combination. Safety was manageable; consistent with ph1b. Detailed multiomic immune and tumor biomarker analyses are underway to elucidate mechanisms of action and inform pt subsets that benefit most from these combinations. Clinical trial information: NCT03214250. [Table: see text]

4122 Background: Traditional imaging-guided therapeutic decision-making for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) may lag and, on occasion, be misleading. The concept of liquid biopsy-based molecular response holds promise for proximate and accurate therapy monitoring and assessment of emerging resistance to therapy. Here we investigate the association between baseline (pre-treatment) level and early, on-treatment changes in plasma circulating cell-free DNA (ccfDNA) mutant KRAS (ctKRAS) with progression-free survival (PFS) and overall survival (OS) in mPDAC. Methods: 189 plasma samples were analyzed from 123 total patients with mPDAC. An initial cohort included 54 patients treated at the University of Pennsylvania who received first-line standard of care (SOC) regimens and had a baseline plasma sample. Of these, 21 also had an on-therapy sample collected at ̃8 weeks. We also analyzed an independent cohort of 69 patients enrolled in the PRINCE trial (NCT03214250) who had a baseline sample, of which 45 also had an on-treatment sample at ̃8 weeks. PRINCE trial patients received gemcitabine/nab-paclitaxel with immunotherapy (I/O) agents (APX005M and/or nivolumab). ctKRAS variant allele fraction (VAF) was quantified by droplet digital PCR on pre-amplified ccfDNA. Baseline ctKRAS was dichotomized at 5% VAF. ctKRAS clearance was defined as detectable ctKRAS at baseline followed by ctKRAS becoming undetectable in the on-treatment sample. Results: Baseline ctKRAS (above/below 5% VAF) and ctKRAS clearance were associated with PFS and OS in both cohorts (Table). Further, in a multivariate cox regression model, ctKRAS clearance associated with improved PFS (HR 3.8, 1.4-10.9 or 3.6, 1.8-7.2) in both the SOC and I/O cohorts, respectively, and OS in the SOC cohort (HR 5.5, 1.5-20.8) after adjusting for baseline VAF. Conclusions: Baseline ctKRAS is significantly associated with OS and PFS in mPDAC in both independent cohorts. Further, early on-treatment ctKRAS clearance is strongly associated with improved PFS and OS, independent of baseline ctKRAS VAF. These data strongly support further investigation of ccfDNA as a biomarker of response and resistance to therapy.[Table: see text]

4108 Background: Circulating tumor DNA (ctDNA) testing can be used for the assessment of molecular residual disease (MRD) in patients with early-stage or advanced colorectal cancer (CRC). Prospective evaluation of this methodology in clinical practice has been limited to-date. Methods: A personalized and tumor-informed multiplex PCR assay (Signatera 16-plex bespoke mPCR NGS assay) was used for the detection and quantification of ctDNA for MRD assessment. We analyze and present results from an ongoing early adopter program of ctDNA testing across the spectrum of CRC management. Results: Here we present a total of 250 patients with colon (n=200), rectal (n=40), and other lower gastrointestinal cancers (n =10; anal, appendiceal, small bowel). MRD positivity rates and ctDNA quantification (mean tumor molecules/mL) are shown in Table. ctDNA detection was significantly associated with stage of disease (p

Colorectal cancer was a model for the early discovery of processes of carcinogenesis at a molecular level, yet despite this, therapeutic advances have been slow. A gradual appreciation of the influence of signaling through the Ras/Raf/MEK pathway showed that tumors carrying mutations in this pathway were resistant to EGFR inhibition, but it has not been until recently that targeting this pathway has yielded therapeutic efficacy. Patients with tumors carrying a mutant BRAF are responsive to combined approaches to the pathway. In 2015, reports have also been presented to show the effect of targeting HER2-amplified tumors with combinations and microsatellite unstable tumors with anti-PD1 immune checkpoint modifiers. Studies have now been initiated in Europe, and are planned in the USA, to allocate patients to trials of specific therapies based on the molecular characteristics of their tumors, and accelerated development of treatments can be predicted.

Patients with locally advanced pancreatic cancer typically have poor outcomes, with a median survival of approximately 16 months. Novel methods to improve outcomes are needed. Nab-paclitaxel (Abraxane) has shown efficacy in pancreatic cancer and is FDA-approved for metastatic disease in combination with gemcitabine. Nab-paclitaxel is also a promising radiosensitizer based on laboratory studies, but it has never been clinically tested with definitive radiotherapy for locally advanced pancreatic carcinoma. We performed a phase 1 study using a 3 + 3 dose escalation strategy to determine the safety and tolerability of dose-escalated nab-paclitaxel with fractionated radiotherapy for patients with unresectable or borderline resectable pancreatic cancer. Following induction chemotherapy with two cycles of nab-paclitaxel and gemcitabine, patients were treated with weekly nab-paclitaxel and daily radiotherapy to a dose of 52.5 Gy in 25 fractions. Final dose-limiting toxicity (DLT) determination was performed at day 65 after the start of radiotherapy. Nine patients received nab-paclitaxel at a dose level of either 100 mg/m2 (n = 3) or 125 mg/m2 (n = 6). There were no observed grade 3 gastrointestinal toxicities. One DLT (grade 3 neuropathy) was observed in a patient who received 125 mg/m2 of nab-paclitaxel. Other grade 3 toxicities included fatigue (11%), anemia (11%) and neutropenia (11%). No grade 4 toxicities were observed. Following chemoradiotherapy, four patients (borderline resectable, n = 2 and unresectable, n = 2) underwent surgical resection, all with negative margins and with significant treatment effect with limited tumor viability. The combination of fractionated radiation and weekly full dose nab-paclitaxel was safe and well-tolerated.

The use of microtechnology for the highly selective isolation and sensitive detection of circulating tumor cells has shown enormous promise. One challenge for this technology is that the small feature sizes – which are the key to this technology's performance – can result in low sample throughput and susceptibility to clogging. Additionally, conventional molecular analysis of CTCs often requires cells to be taken off-chip for sample preparation and purification before analysis, leading to the loss of rare cells. To address these challenges, we have developed a microchip platform that combines fast, magnetic micropore based negative immunomagnetic selection (>10 mL h−1) with rapid on-chip in situ RNA profiling (>100× faster than conventional RNA labeling). This integrated chip can isolate both rare circulating cells and cell clusters directly from whole blood and allow individual cells to be profiled for multiple RNA cancer biomarkers, achieving sample-to-answer in less than 1 hour for 10 mL of whole blood. To demonstrate the power of this approach, we applied our device to the circulating tumor cell based diagnosis of pancreatic cancer. We used a genetically engineered lineage-labeled mouse model of pancreatic cancer (KPCY) to validate the performance of our chip. We show that in a cohort of patient samples (N = 25) that this device can detect and perform in situ RNA analysis on circulating tumor cells in patients with pancreatic cancer, even in those with extremely sparse CTCs (

Pathologic somatic gene mutations within the KRAS proto-oncogene occur in greater than 90% of pancreatic ductal adenocarcinomas (PDA). These mutations are often conserved involving the codon 12 position and most frequently result in G12D, G12V, or G12R transitions. Unfortunately, no mutant KRAS (mKRAS)-targeted therapies exist to date. We hypothesize conserved hot-spot KRAS mutations may serve as cancer neoantigens for targeted immune-based therapeutic approaches. Using a validated in silico epitope prediction pipeline, we identified mKRAS epitopes with predicted strong binding affinity to the highly prevalent MHC class I alleles HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. We then confirmed the processing, presentation, and binding of putative mKRAS epitopes using HPLC-tandem mass spectrometry and competitive peptide-binding assays. The immunogenicity of bona fide mKRAS epitopes was confirmed by the in vitro generation of mKRAS-specific CD8+ T cell responses using healthy donor blood samples that were restricted to HLA-A*03:01, -A*11:01, or -B*07:02 as determined by cytokine release assay and peptide/MHC multimer staining. Following T-cell receptor (TCR) sequencing, transgenic expression of mKRAS-specific TCRs on primary CD8+ T cells conferred cytotoxic potential against KRAS-mutated tumor cell lines expressing target HLA/mKRAS combinations. Importantly, mKRAS-specific CD8+ T cells did not cross-react with wild-type or alternatively mutated KRAS epitopes as measured by cytokine release or cytotoxicity assays. Based on these data, we initiated an adjuvant vaccine clinical study at the University of Pennsylvania Abramson Cancer Center targeting mKRAS in patients with resected PDA. The objective of this clinical study is to identify further mKRAS-specific TCR sequences for future adoptive T-cell therapy applications. To date, a total of 7 subjects have been enrolled, and 3 subjects have been vaccinated against targeted mKRAS short peptides utilizing a dendritic cell-based platform. Results from the first vaccinated subject have demonstrated the generation of a KRAS G12V-specific CD8+ T-cell response restricted to HLA-A*11:01 following vaccination with >10% mKRAS-specific T cells isolated following primary in vitro expansion of CD8+ T cells using antigen-pulsed dendritic cells as measured by peptide/MHC multimer staining. TCR sequencing has been completed and functional studies to validate antigen specificity and tumor cytotoxicity are in process. The results of these studies will serve to develop a widely applicable T-cell therapy for PDA patients, and will have implications for the large number of patients with other KRAS-mutated cancers. Citation Format: Adham S. Bear, Andrew J. Rech, Lee P. Richman, Mark H. O'Hara, Gerald P. Linette, Beatriz M. Carreno, Robert H. Vonderheide. Identification of T-cell receptors targeting mutant KRAS in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B04.

CDX-1140 is an agonist CD40 mAb intended to promote priming of CD8+ effector T cells through activation of dendritic cells (DC), enhance macrophage tumoricidal activity, and mediate direct killing of CD40-expressing tumor cells. CDX-1140 is a human IgG2 mAb with Fc receptor independent activity and is synergistic with sCD40L, suggesting a potential to enhance CD40L-mediated immune activation. CDX-1140 has a broad dose-dependent activity that may achieve maximal agonist activity at doses that provide significant tumor penetration with minimal toxicities from systemic CD40 activation. CDX-301 (rhFLT3L) is a DC growth factor that markedly increases CD141+ DCs, including in the tumor microenvironment (TME). CD141+ DCs are critical for initiating anti-tumor immune responses and are often depleted within the TME. Preclinical studies have shown that treatment with CDX-301 synergizes with CD40 signaling to enhance anti-tumor immune responses, suggesting that combining CDX-1140 and CDX-301 could result in enhanced clinical benefit. CDX1140-01(NCT03329950) is a Phase I dose-escalation study of CDX-1140 as monotherapy or in combination with CDX-301 evaluating safety, PK, PD, and preliminary clinical activity in patients (pts) with refractory solid tumors and non-Hodgkin lymphoma. CDX-1140 is given iv q 4 weeks at doses from 0.01 to 3 mg/kg utilizing a 3+3 design (first 2 cohorts are single-pts). In combination cohorts (solid tumors only), CDX-301 at 75 μg/kg daily x 5 is administered sc prior to cycles 1 and 2 of escalating doses of CDX-1140 starting at 0.09 mg/kg. Tumor-specific expansion cohorts are planned to further explore the activity of monotherapy or combination treatment. To date, 18 pts across 10 tumor types have been dosed in CDX-1140 monotherapy cohorts with doses ranging from 0.01 to 0.72 mg/kg and without identification of an MTD. There has been one DLT (0.18 mg/kg): grade 3 pneumonitis and hypoxia. One pt (0.72 mg/kg) experienced grade 3 fatigue requiring hospitalization 48 hrs after the first infusion. There have been 3 additional grade 3 treatment-related adverse events: dyspnea (0.18 mg/kg), nausea (0.36 mg/kg), and vomiting (0.36 mg/kg). The first combination cohort (n=5) has been completed. In monotherapy and combination cohorts there have been no significant drug-related changes in liver function tests to date. CDX-1140 PK is quantifiable at doses ≥ 0.09 mg/kg and exposure appears dose proportional. Expected dose-dependent PD effects have shown activation of peripheral blood lymphocytes and increases in pro-inflammatory cytokines and chemokines. Combination with CDX-301 has shown enhanced cytokine responses. The data suggest that CDX-1140 has the expected biologic and safety profile predicted from preclinical studies and may achieve dose levels optimal for systemic exposure. The addition of CDX-301 may enhance the activity of CDX-1140. Citation Format: Rachel E. Sanborn, Nashat Y. Gabrail, Nina Bhardwaj, Michael S. Gordon, Mark O'Hara, Danny Khalil, Thomas Hawthorne, Richard Gedrich, Laura Vitale, Mark Rogalski, Tianshu Li, Tracey Rawls, Tibor Keler, Michael Yellin. First-in-human Phase I study of the CD40 agonist mAb CDX-1140 and in combination with CDX-301 (rhFLT3L) in patients with advanced cancers: Interim results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-194.

Background: Checkpoint inhibitors such as anti-PD-1 are ineffective as single agents for patients (pts) with PDAC. Preclinical data suggest that chemotherapy with agonistic CD40 antibodies can be combined with anti-PD-1 to trigger effective T cell immunity. We conducted a multi-center, open label clinical trial to evaluate the combination of APX005M with nivo and standard chemotherapy (gem and NP). Herein, we report safety and efficacy from the ongoing study. Methods: Pts with previously untreated PDAC were enrolled in 4 cohorts: Gem/NP/APX005M 0.1 mg/kg (B1), Gem/NP/APX005M 0.3 mg/kg (B2), Gem/NP/Nivo/APX005M 0.1 mg/kg (C1) and Gem/NP/Nivo/APX005M 0.3 mg/kg (C2). Primary objectives were to evaluate safety and determine the recommended Phase II dose (RP2D) of APX005M. Secondary objectives included tumor response and immune pharmacodynamics. Analyses were performed on dose-limiting toxicity (DLT)-evaluable subjects, defined as receiving 1 dose of APX005M and ≥ 2 doses of gem/NP during Cycle 1 and remaining on study through Cycle 2 Day 1. Results: N=30 pts dosed; 24 DLT-evaluable (6 per cohort). Median follow up is 32.2 weeks. N=13 (54%) pts experienced an AE leading to discontinuation, and 10 (42%) pts experienced a treatment-related serious AE. Two DLTs were observed, 1 each in cohorts B2 and C1 (grade 3 and 4 febrile neutropenia, respectively). AE rates were similar across cohorts. Four (17%) subjects died (2 each in Cohorts B1 and C1) due to disease progression (n=2) and AE (n=2, sepsis and septic shock in the setting of neutropenia). Within the DLT-evaluable population, best overall responses included 14 (58%) PR (11 confirmed, 3 unconfirmed), 8 (33%) SD, 1 (4%) PD and 1 (4%) NE. Post-baseline tumor assessments were available for 2 of the 6 DLT-inevaluable pts (best overall responses of SD and confirmed PR). Multiplexed IHC analysis of baseline tumors revealed a low CD8 T cell and high macrophage infiltrate. Analysis of circulating mutant KRAS DNA showed marked and rapid decrease with therapy in some pts. Immune profiling of PBMCs at baseline and on-treatment by CyTOF revealed remodeling of the myeloid compartment in response to treatment, with rapid activation of dendritic cells in most pts. Conclusions: Gem/NP/APX005M ± nivo demonstrated manageable safety profiles and promising antitumor activity in untreated metastatic PDAC pts. APX005M 0.3 mg/kg was selected as the dose for a randomized Phase II study in which the primary endpoint is 1-year overall survival. Clinical trial information: NCT02482168. Citation Format: Mark H. O'Hara, Eileen M. O'Reilly, Mick Rosemarie, Gauri Varadhachary, Zev A. Wainberg, Andrew Ko, George A. Fisher, Osama Rahma, Jaclyn P. Lyman, Christopher R. Cabanski, Erica L. Carpenter, Travis Hollmann, Pier Federico Gherardini, Lacey Kitch, Cheryl Selinsky, Theresa LaVallee, Ovid C. Trifan, Ute Dugan, Vanessa M. Hubbard-Lucey, Robert H. Vonderheide. A Phase Ib study of CD40 agonistic monoclonal antibody APX005M together with gemcitabine (Gem) and nab-paclitaxel (NP) with or without nivolumab (Nivo) in untreated metastatic ductal pancreatic adenocarcinoma (PDAC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT004.

Background: PARP inhibitors have activity in multiple BRCA-related malignancies and have recently demonstrated dramatic efficacy as a maintenance strategy for platinum-sensitive ovarian cancer. Between 5-8% of patients with pancreatic cancer (PC) have a pathogenic mutation in BRCA1, BRCA2 or PALB2. Therefore, we initiated a single arm phase II clinical trial of maintenance monotherapy rucaparib in patients with advanced PC and a pathogenic germline or somatic BRCA or PALB2 mutation, whose cancer had not progressed following at least four months of platinum-based chemotherapy (NCT 03140670). Methods: Patients were enrolled and treated with rucaparib 600mg PO BID until disease progression or unacceptable toxicity. The primary endpoint is progression free survival (PFS). Patients have previously received >4 months of platinum-based chemotherapy without evidence of disease progression. However, patients with a medical contraindication to receiving the full four months of platinum have been permitted to enroll at the discretion of the primary investigator. Responses were determined using RECIST v1.1. Results: As of December 31st, 2018, we have enrolled 24 of the planned 42 patients, of which 19 are evaluable for PFS at the time of this interim analysis. For these patients, the mutational distribution includes: 13 germline BRCA2, 3 germline BRCA1, 2 germline PALB2, 1 somatic BRCA2. Patients were predominantly female (84.2%) with a median age of 61 years (range: 35-81). Patients had received a median of four months (range 0.5-32 months) of prior platinum therapy for advanced disease. All patients were evaluable for toxicity. Overall, treatment with rucaparib was well tolerated without dose limiting toxicities. The most common adverse events that were at least possibly related to treatment included nausea (grade 1, 41.6%; grade 2, 4.2%), dysgeusia (grade 1, 33.3%) and fatigue (grade 1, 25%). One patient required dose reduction for nausea. The median PFS was 9.1 months from the start of rucaparib therapy with an ORR of 36.8% (six PRs; one CR). Disease control rate (CR + PR + SD) was 89.5% for at least eight weeks. Two patients (10.5%) had progressive disease at first follow-up scan two months after beginning treatment. Eight patients have been on rucaparib for >6 months and two patients remain on treatment for >1 year (13 months and 15 months). The seven responding patients include those with germline BRCA2 mutations (4 patients), germline PALB2 mutations (2 patients) and somatic BRCA2 mutation (1 patient). Conclusions: Based on these early data, maintenance rucaparib following induction with platinum-based chemotherapy shows encouraging disease control with minimal toxicity in patients with platinum-sensitive advanced PC and a pathogenic mutation in BRCA1, BRCA2 or PALB2. Citation Format: Kim A. Reiss Binder, Rosemarie Mick, Mark O'Hara, Ursina Teitelbaum, Thomas Karasic, Charles Schneider, Peter J. O'Dwyer, Erica Carpenter, Austin Pantel, Mehran Makvandi, David Mankoff, Katherine Nathanson, Kara Maxwell, Stacy Cowden, Mary Jane Fuhrer, Janae Romeo, Gregory L. Beatty, Susan Domchek. A Phase II, single arm study of maintenance rucaparib in patients with platinum-sensitive advanced pancreatic cancer and a pathogenic germline or somatic mutation in BRCA1, BRCA2 or PALB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT234.

e15125 Background: Autophagy is a resistance mechanism to chemotherapy that is inhibited by hydroxychloroquine (HCQ). We have reported a phase II trial of FOLFOX/bevacizumab/HCQ in 28 evaluable patients with untreated metastatic colorectal cancer (mCRC). Overall response rate was 68%, with 11% complete response (CR) and 57% partial response (PR), while stable disease (SD) was seen in 30%. We hypothesize that analysis of CT imaging features via machine learning (ML) will enhance subtle yet important radiographic characteristics, and reveal imaging signatures determinant of outcome and mutational status. Methods: Baseline CT images were collected and 1265 quantitative imaging (QI) features extracted across all the metastatic sites – liver, lung, and lymph nodes – including descriptors of size, morphology, texture, and intensity. Cross-validated sequential feature selection coupled with support vector machine (SVM) was used to determine the most discriminative QI features for our integrative predictor of response and mutational status, and support vector regression (SVR) was used to derive imaging predictors of overall survival. The model predictions were compared with actual clinical results, including response, survival, and genomic aberrations. Results: Various QI features, primarily descriptive of texture and tumor volume, were determined as most important by the ML predictor. Using this signature, our predictor classified “Responder (PR+CR) vs Non-responder (SD)” with an accuracy of 85% [sensitivity(se) = 0.84, specificity(sp) = 0.71, AUC = 0.85]. The QI features were also able to detect KRAS and TP53 mutational status with an accuracy of 88% [se = 0.92, sp = 0.85, AUC = 0.87] and 86% [se = 0.80, sp = 0.90, AUC = 0.84], respectively. The SVM model predicted overall survival greater than the median (32 mo) with an accuracy of 86% [se = 0.93, sp = 0.79, AUC = 0.87]. The Pearson correlation coefficient between the SVR score and overall survival was estimated to be 0.73 (p < 0.0001). Conclusions: Radiomic analysis of baseline CT imaging features analyzed by ML yielded an imaging signature predictive of response, survival, and KRAS and TP53 mutational status. If validated in a larger clinical data set, machine learning may offer a predictive biomarker to aid clinical decision making for mCRC patients.

TPS4161 Background: The treatment paradigm for advanced pancreatic ductal adenocarcinoma (PDAC) typically involves ongoing chemotherapy until either disease progression or clinical deterioration. A subset of patients with advanced PDAC have exceptional responses to platinum-based chemotherapy. We hypothesized that durable platinum sensitivity in patients with advanced PDAC might be indicative of a DNA repair deficiency, and that these patients may respond to a combination of niraparib, a PARP inhibitor, plus immune checkpoint blockade. Methods: We have enrolled 25 of 84 planned patients on study NCT 03404960. Eligibility criteria include inoperable PDAC and stability on platinum-based chemotherapy for ≥16 weeks without evidence of progressive disease. Patients who have progressed on platinum-based treatment or who have received prior therapy with PARP inhibitors are excluded. Patients are randomized to receive oral niraparib 200mg PO daily plus nivolumab 240mg IV every two weeks in continuous 28 day cycles or oral niraparib 200mg PO daily plus ipilimumab 3mg/kg IV every three weeks for four doses in continuous 21 day cycles. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include response rate, duration of response and overall survival. Paired biopsies are obtained, as well as serial blood collections for circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and peripheral blood mononuclear cells (PBMCs). Correlative assays will include germline whole exome sequencing and analyses of serially collected PBMCs, CTCs and ctDNA to identify genomic and immunologic innate and adaptive resistance mechanisms. Clinical trial information: NCT 03404960.

To understand potential immune system alterations in newly diagnosed, untreated, pancreatic cancer patients and provide a foundation for immunotherapy, we profiled PBMC from pancreatic ductal adenocarcinoma (PDA) patients and age-matched healthy controls using high-dimensional CyTOF analysis. We developed two immune profiling panels: a broad profiling panel that includes 45 phenotypic markers that together permit the identification and enumeration of the main innate and adaptive immune cell subsets in humans, and a deep profiling panel that includes 45 features focusing on T-cell phenotype and biology. We report a 2-fold increase in monocytes, more regulatory T-cells, and more plasmocytes in circulation in pancreatic cancer patients as compared to age-matched controls, as well as a bias towards cytokine-producing NK cells. Using high-dimensional approaches, we observed skewed T-cell differentiation in pancreatic cancer patients, with peripheral blood CD8 T-cells being biased towards more CD45RA-positive CD27-positive CCR7-positive CD95-positive CD49d-positive stem cell memory cells (P=7x10-5), more CD45RA-negative CD27-positive CCR7-negative effector memory cells (P=0.002) and less CD45RA-positive CD27-negative CCR7-negative late effector memory cells (P=0.003) as compared to age-matched controls. Strikingly, when examinating T-cell differentiation in human spleens, we found increased proportions of late effector memory T-cells in the splenic CD8 T-cell compartment of pancreatic cancer patients as compared to age-matched controls. These results suggest an impaired T-cell trafficking in pancreatic cancer patients, with late memory T-cells being retained in the spleen. We are now investigating the mechanisms underlying these observations, as well as their impact on T-cell immunity of the cancer patients. Our goal is to understand the nature of the skewing and how any changes in baseline immune health of the T-cell compartment relate to disease progression and/or response to therapy. These studies should provide a foundation for improving therapy in pancreatic cancer patients. Citation Format: Cecile Alanio, Bertram Bengsch, Josephine R. Gies, Sarah Henrickson, Nan Ping Weng, Janáe A Ritz-Romeo, Mark O'Hara, Joseph J Melenhorst, Simon Lacey, Regina M Young, Carl H June, E. John Wherry. Skewed CD4 and CD8 T-cell differentiation in pancreatic cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A123.

TPS465 Background: Treatment options for PDAC are limited; thus, new therapies that can improve outcomes and extend survival are needed. PDAC is associated with high infiltration by tumor-associated macrophages (TAMs) that inhibit antitumor T-cell activity. Blocking colony-stimulating factor 1 receptor (CSF-1R) signaling—which supports the recruitment, differentiation, and maintenance of immunosupressive macrophages in tumors—may lead to depletion of TAMs and upregulation of T-cell checkpoints. Cabira, a humanized IgG4 monoclonal antibody, binds to CSF-1R and blocks its signaling, a key determinant of TAM activation and survival. By reducing TAMs and promoting a proinflammatory microenvironment, cabira may stimulate T-cell responses, thereby sensitizing PDAC to therapy with nivo (anti‒PD-1). In a phase 1a/b study cabira + nivo was tolerable and showed evidence of on-target tumor immune modulation and durable clinical benefit in heavily pretreated patients (pts) with advanced PDAC (Wainberg et al. J Immunother Cancer. 2017 [abst O42]; Carleton et al. J Clin Oncol. 2018 [abst 3020]). Here we describe a randomized, open-label, phase 2 study evaluating the safety and efficacy of cabira + nivo ± chemo in advanced PDAC. Methods: Pts aged ≥18 y with locally advanced/metastatic PDAC that progressed on/after first-line chemo (gemcitabine [gem] or 5-fluorouracil [5-FU] based) will be enrolled. Pts with active/suspected autoimmune disease, uncontrolled/significant cardiovascular disease, or prior exposure to select immune cell–modulating antibodies are not eligible. Depending on prior chemo received, pts will be randomized to 1 of 4 arms (n≈40 each): cabira + nivo; cabira + nivo + gem/nab-paclitaxel; cabira + nivo + oxaliplatin/5-FU/leucovorin; or investigator’s choice of standard-of-care chemo. Endpoints include median progression-free survival (primary), overall survival rate, objective response rate, median duration of response, pharmacokinetics, and safety. In a completed preliminary safety cohort, 12 pts were treated with cabira + nivo + chemo and monitored for 4 wk; competitive enrollment is open, with 32 pts enrolled. (NCT03336216, NCT02526017) Clinical trial information: NCT03336216.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in CD19 expressing hematologic malignancies, but how to translate this success to solid malignancies remains elusive. Effective translation of CAR T cells to solid tumors will require an understanding of potential therapeutic barriers, including factors that regulate CAR T cells expansion, persistence, trafficking, and fate within tumors. Herein, we describe the current state of CAR T cells in solid tumors; define key barriers to CAR T cell efficacy and mechanisms underlying these barriers, outline potential avenues for overcoming these therapeutic obstacles, and discuss the future of translating CAR T cells for the treatment of patients with solid malignancies.

TPS531 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a median overall survival of < 1 year. A subset of PDAC is characterized by a homologous recombination deficiency (HRD). The most well-defined patients within this group are those with deleterious mutations in BRCA1, BRCA2 and PALB2. This subset of tumors respond exceptionally well to treatment with platinum agents, leading to durable responses. However, cumulative toxicity can complicate or even prevent continued therapy, and there is an unmet need to establish maintenance strategies for such patients.Prior studies have shown that BRCA1 and BRCA2 associated PDACs respond to PARP inhibitors. Cross-resistance to platinum and PARP inhibitors exists, so initiating PARP inhibitor therapy after the development of platinum-resistance is an inferior approach. In this setting, we have designed a phase II trial of rucaparib as maintenance therapy for patients with deleterious BRCA1, BRCA2 or PALB2 who have sustained stability on platinum-based treatment. Methods: We have enrolled 2 of 42 planned patients on study NCT03140670. Eligibility criteria include inoperable PDAC, a known somatic or germline deleterious mutation in BRCA1, BRCA2 or PALB2 and stability on platinum-based chemotherapy for ≥16 weeks. Patients who have progressed on platinum-based treatment or who have received prior therapy with PARP inhibitors are excluded. Patients will receive oral rucaparib twice daily continuously in 28-day cycles. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include response rate, duration of response and overall survival. A pre-treatment tumor biopsy and biopsy at progression will be obtained, as well as serial blood collections for circulating tumor material. Correlative assays will include tumor and circulating tumor DNA sequencing to identify genomic predictors of outcome and study resistance mechanisms. Clinical trial information: NCT03140670.

Lessons Learned Palbociclib monotherapy demonstrated minimal clinical activity in patients with previously treated gastroesophageal cancers. Further clinical evaluation of palbociclib monotherapy is not warranted in gastroesophageal cancers, but improved understanding of resistance mechanisms may permit rational combination approaches. Background Dysregulation of the cell cycle is a hallmark of cancer. Progression through the G1/S transition requires phosphorylation of retinoblastoma (RB) by cyclin-dependent kinases (CDKs) 4 and 6, which are regulated by cyclins D and E. Amplifications of cyclin D loci and activating mutations in CDKs are frequent molecular aberrations in gastroesophageal malignancies. We conducted a phase II trial of the CDK4/6 inhibitor palbociclib as an initial test of efficacy. Methods Patients with previously treated metastatic gastroesophageal cancers with intact RB nuclear expression by immunohistochemistry were treated with 125 mg daily of palbociclib for days 1–21 of 28-day cycles. The primary endpoint was overall response rate. Results We screened 29 patients and enrolled 21 patients: 5 with gastric adenocarcinoma, 3 with gastroesophageal junction adenocarcinoma, 8 with esophageal adenocarcinoma, and 5 with esophageal squamous cell carcinoma. All 29 tumors screened had intact nuclear RB expression, and four treated patients tested positive for CCND1 overexpression. No objective responses were seen. Median progression-free survival was 1.8 months, and median overall survival was 3.0 months. All recurrent grade 3 or 4 toxicities were hematologic, with neutropenia in eight patients (38%), anemia in four patients (19%), and thrombocytopenia in two patients (10%). Conclusion Palbociclib has limited single-agent activity in gastroesophageal tumors.

TPS528 Background: Hepatocellular carcinoma (HCC) is a common and lethal malignancy with few effective treatment options. Inherently aggressive disease biology combined with the immunosuppressive hepatic microenvironment creates a unique therapeutic challenge. TGF-β, the strongest known immunosuppressive cytokine [1, 2], modulates the hepatic immune response to various antigens and to ionizing radiation [3, 4]. TGF-β is constitutively released by liver cells and plays a key role in the early and late pathogenesis of HCC [5-7] by dampening the local T-cell response to the oncogenic hepatitis B and –C viruses [4, 8-11]. TGF-β is activated by ionizing radiation, where it blocks the effector T-cell response to cellular destruction and the release of tumor-specific antigens [4]. Preclinical data demonstrate that neutralizing TGF-β during radiation therapy effectively generates a CD8+ T-cell response to multiple endogenous tumor antigens [4], thereby generating an in-situ vaccine against a tumor [12-17]. We hypothesize that the combination of TGF-β receptor inhibition plus radiation therapy will produce a potent and clinically effective antitumor immune response against HCC. Methods: We have enrolled 9 of 15 planned patients on study NCT02906397. Eligibility criteria include inoperable HCC, Childs Pugh score of ≤7, and either failure of or refusal to take sorafenib. Patients must be 4 weeks from prior therapy and may not be taking immunosuppressants. Patients with major cardiac disease or abnormalities or a predisposition toward aneurysm development are excluded. Patients receive galunisertib on days 1-14 of 28 day cycles. SBRT will be delivered in a single fraction of 18 Gy between days 15-28 of C1. Pre-treatment and on-treatment biopsies are obtained, as well as serial blood collections for circulating tumor material. Immunologic evaluation will include TCR deep sequencing to track T-cell receptor clones, analysis of serum inflammatory cytokines, analysis of myeloid and B cell activation, multiplex flow cytometry of PBMCs to measure percentages and absolute counts of T-cell subsets and tissue assessment of immune markers. Peripheral and tissue levels of TGF-β will be assessed. Clinical trial information: NCT02906397.

An increasing appreciation for the role of the immune system in targeting cancer cells over the last decade has led to the development of several immunomodulatory agents aimed at enhancing the systemic antitumor immune response. One such method is the use of T cells that are genetically engineered to express chimeric antigen receptors (CARs). The remarkable success of this approach in advanced hematologic malignancies has garnered much enthusiasm for using this novel tool in treating other cancers. However, multiple challenges have hampered the application of this therapy to a broader set of solid tumors, most notably lung cancer. Immunotherapy has already shown great success in lung cancer, and is now the first-line treatment in PD-L1 expressing metastatic disease. Given the mounting evidence that radiation therapy plays a crucial role in amplifying the immune response elicited by immunomodulatory agents, there is potential for radiation to help in overcoming some of these challenges. In this review, we describe the basic principles of CAR T cell therapy and examine its successes and challenges to date. We then discuss the preclinical and clinical data supporting the use of radiation with immunomodulatory agents with a focus on preclinical rationale for combining CAR T cells and radiation therapy in future experiments with a focus on lung cancer.

Importance Palbociclib (PD0332991) is a newly developed drug that received breakthrough designation and recent US Food and Drug Administration approval in combination with endocrine therapy in the treatment of hormone receptor positive, ERBB2-negative (formerly HER2 or HER2/neu) breast cancer in the first-line metastatic setting. Objective Herein we describe the preclinical and translational data and early- and late-phase clinical trials in which palbociclib has been investigated in a broad array of tumor types. We discuss the pharmacodynamics, pharmacokinetics, toxic effects, and clinical response rates. Evidence Review On March 1, 2015, we conducted a review of the literature describing the development of palbociclib. We used the PubMed search terms “PD0332991,” “palbociclib,” and “CDK4/6 inhibitor” to find all published articles of interest, without limitation as to publication date. Findings Palbociclib is a potent and specific oral cyclin-dependent kinase (CDK) 4/6 inhibitor that has strong preclinical data to support its activity in retinoblastoma protein–expressing tumors. Phase 1 trials have demonstrated safety, and phase 2 trials have shown single-agent activity in mantle-cell lymphoma, breast cancer, liposarcoma, and teratoma with reversible neutropenia as the main toxic effect. Addition of palbociclib to endocrine therapy improves progression-free survival in endocrine therapy–naive and endocrine therapy–resistant metastatic settings. Conclusions and Relevance Palbociclib is well tolerated and has therapeutic potential for multiple cancers, including breast cancer, where its efficacy has been demonstrated alone and in combination with endocrine therapy. Additional combinations of palbociclib with endocrine therapy, chemotherapy, and targeted therapy have potential in various tumors, and phase 3 trials are under way.