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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.

Outcomes for patients with advanced pancreatic cancer have improved in the past 12 years, mainly because of progress made in systemic therapies. New treatment strategies for advanced pancreatic cancer include switch maintenance with cytotoxic therapies, induction maintenance, and the utilization of targeted agents for patients with actionable variants, as well as ongoing development of cytotoxic regimens, such as NALIRIFOX. The activity of immunotherapy has been disappointing to date, but novel combinations and identifying appropriate patient populations may further unlock its potential.

Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC (NCT03214250). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.

Figure S1. Sample cohort of this study, which included 204 subjects in total. Workflow shows patient cohorts involved in each classification. * indicated 8 patients repeated in the discovery set and the training set. ** indicated 32 locally advanced patients that were not used in the occult metastases detection as training set did not have locally advanced PDAC patients.

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

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.

Introduction The use of total neoadjuvant therapy (TNT) for locally advanced rectal cancer has been increasing in recent years, but the long-term overall survival characteristics of this approach is currently unknown. Methods We performed a retrospective study of patients with clinical stage II/III rectal cancer within the National Cancer Database. Patients who received TNT (defined as chemotherapy, followed by CRT, followed by surgery) were propensity score matched to patients who received adjuvant therapy (defined as CRT, followed by surgery, followed by chemotherapy). We compared overall survival (OS) and rates of pathologic complete response (pCR) between the two arms. Results Of the 4,300 patients in our cohort, 3,502 (81%) received adjuvant therapy and 798 (19%) received TNT. At baseline, patients who received TNT were more likely to have higher clinical T and N stages (p Conclusion In this observational study, we found TNT was not associated with a lower OS compared to standard adjuvant chemotherapy. This finding potentially reassures clinicians choosing TNT as an alternative to adjuvant chemotherapy. However, future prospective data are needed to confirm these findings. Microabstract The long-term overall survival characteristics of total neoadjuvant therapy (TNT) for locally advanced rectal cancer has not been well-defined despite its increasing popularity. In a large National Cancer Database cohort, we found that TNT was not associated with worse survival compared to historical controls.

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.

Purpose: This was an open-label phase 1a study assessing the maximum tolerated dose (MTD), safety, and tolerability of CXCR4 peptide antagonist, LY2510924, administered in combination with durvalumab in patients with advanced refractory solid tumors. Methods: Patients received LY2510924 at 20, 30, or 40 mg subcutaneous (SC) once daily in combination with durvalumab at 1500 mg intravenously (IV) on day 1 of each 28-day cycle. The primary objective was to assess the MTD and safety of LY2510924 SC daily in combination with durvalumab in patients with advanced (metastatic and/or unresectable) solid tumors. Secondary objectives included pharmacokinetics (PK) and the antitumor activity of LY2510924 in combination with durvalumab. Exploratory objectives were biomarker analysis, including pharmacodynamic markers, relevant to LY2510924 and durvalumab, including immune functioning, drug targets, cancer-related pathways, and the disease state. Results: Nine patients (three each at 20, 30, and 40 mg) were enrolled in the study (eight patients with pancreatic cancer and one patient with rectal cancer). The majority of patients completed one or two cycles (100.0% ≥ 1 cycle; 88.9% ≥ 2 cycles) of LY2510924 and durvalumab. No dose limiting toxicities were reported. Most common (>10%) treatment-emergent adverse events were injection-site reaction (44.4%), fatigue (33.3%), and increased white blood cell count (33.3%). PK parameters for combination were similar to those reported in previous studies when given as monotherapy. Best overall response of stable disease was observed in four (44.4%) patients and one patient had unconfirmed partial response. Conclusion: The recommended phase 2 dose is 40 mg SC once-daily LY2510924 in combination with durvalumab 1500 mg IV and showed acceptable safety and tolerability in patients with advanced refractory tumors.

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.

Patients newly diagnosed with metastatic pancreatic ductal adenocarcinoma generally have poor survival, with heterogeneous rates of progression. Biomarkers that could predict progression and/or survival would help inform patients and providers as they make care decisions. In a previous retrospective study, we discovered that circulating thrombospondin-2 (THBS2) could, in combination with CA19-9, better distinguish patients with PDAC versus healthy controls. Here we evaluated whether THBS2 levels, previously not known to be prognostic, were associated with outcome in 68 patients at time of diagnosis of metastatic PDAC. Specifically, we interrogated the association of THBS2 level, alone or in combination with CA19-9, with progression by 90 days and/or survival to 180 days. The results indicate that elevated THBS2 levels alone, at the time of a metastatic PDAC diagnosis, can identify patients with a shorter time to death and thus help patients and providers when planning treatment.

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.

Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi), is approved as maintenance therapy for patients with advanced pancreatic cancer (PC) and a germlineEligible patients had advanced PC; germline (g) or somatic (s) PVs inOf 46 enrolled patients, 42 were evaluable (27 gMaintenance rucaparib is a safe and effective therapy for platinum-sensitive, advanced PC with a PV in

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 Background Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. Methods This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0–1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov , NCT03214250 and is ongoing. Findings Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0–19·4; cohort B1 22·0 months [21·4–22·7], cohort B2 18·2 months [17·0–18·9], cohort C1 17·9 months [14·3–19·7], cohort C2 15·9 months [12·7–16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3–4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). Interpretation APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. Funding Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.

This economic evaluation uses Markov modeling to identify and compare the cost and utility of doublet and standard treatments for patients with BRAF V600E variant colorectal cancer., Key Points Question Is the use of doublet therapy (encorafenib and cetuximab) compared with standard chemotherapy (with cetuximab and irinotecan-based regimens) a cost-effective treatment strategy in patients with metastatic BRAF variant colorectal cancer? Findings In this economic evaluation of the cost-effectiveness of 2 treatment strategies, doublet therapy was associated with an incremental cost of $78 233, an incremental effectiveness of 0.15 quality-adjusted life years, and an incremental cost-effectiveness ratio of $523 374 per quality-adjusted life year gained. Meaning Results of this study suggest that doublet therapy for metastatic BRAF variant colorectal cancer is unlikely to be a cost-effective treatment under current pricing., Importance The BEACON trial showed that combination therapy with encorafenib (BRAF inhibitor) and cetuximab (EGFR inhibitor) was associated with prolonged overall survival compared with standard chemotherapy in patients with metastatic BRAF variant colorectal cancer. However, the cost-effectiveness of using these agents in this clinical setting is unknown. Objective To create a cost-effectiveness model to compare doublet therapy (encorafenib plus cetuximab) with standard chemotherapy (cetuximab plus irinotecan or cetuximab plus folinic acid, fluorouracil, and irinotecan) in treating patients with metastatic BRAF variant colorectal cancer. Design, Setting, and Participants This economic evaluation constructed a Markov model to compare the lifetime cost and utility of doublet therapy and standard chemotherapy. Parametric survival modeling was used to extrapolate the effectiveness of each line of therapy from large clinical trials. One-way and probabilistic sensitivity analyses assessed the uncertainty in the model. Patients mirrored the cohorts in the BEACON trial: they had metastatic BRAF variant colorectal cancer and were followed up as they progressed through multiple lines of therapy, best supportive care, and death. Data collection and data analysis were performed from November 15, 2019, to July 14, 2020. Main Outcomes and Measures The main outcome was the incremental cost-effectiveness ratio, which was calculated using the cumulative cost and effectiveness in quality-adjusted life years (QALYs), of doublet therapy compared with standard chemotherapy. Results The model patient cohort had a mean age of 61 years, and 53% of the patients were women, 66% had 1 previous line of therapy, and 8% had high microsatellite instability. Doublet therapy was associated with an improvement of 0.15 QALYs compared with standard chemotherapy. However, the incremental cost of doublet therapy was $78 233, leading to an incremental cost-effectiveness ratio of $523 374 per QALY gained. Concomitant decreases in the price of encorafenib and cetuximab are needed to achieve cost-effectiveness at a willingness-to-pay threshold of $150 000 per QALY gained. Conclusions and Relevance This study found that doublet therapy for metastatic BRAF variant colorectal cancer was unlikely to be cost-effective under current pricing. Cost-effectiveness needs to be considered in clinical trial design, particularly when combining new therapies with non-cost-effective treatments that are coadministered without a fixed duration.

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.

Background CTLA-4 pathway blockade with ipilimumab (IPI) ± nivolumab (NIVO; anti–PD-1) is an effective treatment for several cancers. A nonfucosylated version of IPI, BMS-986218, was developed to increase the effects of CTLA-4 blockade and enhance intratumoral regulatory T-cell depletion via its increased affinity for Fcγ receptors (FcγR, CD16) on natural killer T cells and macrophages, resulting in enhancement of antibody-dependent cellular cytotoxicity. Preclinical data supported the mechanism of action of BMS-986218 and demonstrated greater antitumor activity in an MC38 tumor model vs IPI.1 Here, we present initial results from the first-in-human phase 1/2a trial of BMS-986218 ± NIVO in previously treated patients with advanced cancer (NCT03110107). Methods Patients with ≥1 prior therapy received BMS-986218 2–70 mg intravenously Q4W. Safety, tolerability, pharmacokinetics, and pharmacodynamics were evaluated. Results As of December 12, 2019, 65 patients (median age, 61 years [range, 24–85 years]) received BMS-986218 monotherapy. TRAEs occurred in 52% of patients; most were grade 1–2. The most common (≥10%) TRAEs (any grade; grade 3) were pruritus (12%; 0%) and diarrhea (11%; 3%). Eight patients (12%) had grade 3 TRAEs; most resolved with protocol-defined management. No grade 4 TRAEs were reported; 1 grade 5 TRAE (pneumonitis; 2 mg) occurred. Seven patients (11%) discontinued treatment due to TRAEs; 4 dose-limiting toxicities occurred. The maximum tolerated dose has not been reached. BMS-986218 exposure increased dose proportionally, and the half-life was ≈2 weeks. Increased levels of serum chemokine ligands 9 and 10 and interferon-γ indicate that pharmacodynamic changes occurred at the lowest dose tested (2 mg [≈0.03 mg/kg]), similar to previous findings with IPI 3 mg/kg, and at higher doses (40–70 mg [≈0.06–1 mg/kg]), consistent with findings with IPI 10 mg/kg. In a subset of patients with paired biopsies, BMS-986218 was associated with an increased gene signature linked to CD8+ T-cell infiltration and inflammation. In a highly heterogeneous population, as part of dose escalation, BMS-986218 monotherapy treatment was associated with clinical activity in some patients. Updated data based on a September 2020 data cutoff will be presented. Conclusions BMS-986218 monotherapy demonstrated an acceptable safety profile and signs of clinical benefit in this heterogeneous patient population with select advanced cancers. Preliminary pharmacodynamic activity was consistent with enhanced effects of CTLA-4 blockade. Data from continuing dose escalation of BMS-986218 ± NIVO along with preclinical results provide support for ongoing monotherapy expansions and for BMS-986218 + NIVO expansions in patients with advanced cancer. Acknowledgements The authors acknowledge Dr Charles Drake while at Columbia University Medical Center, New York, NY, USA, for his contributions to the study. Trial Registration NCT03110107 Ethics Approval This study was approved by the WCG Independent Review Board, approval number 20170464 Reference Engelhardt J, Akter R, Valle J, et al. Preclinical characterization of BMS-986218, a novel nonfucosylated anti–CTLA-4 antibody designed to enhance antitumor activity. Presented at the American Association for Cancer Research (AACR) Virtual Annual Meeting II; June 22–24, 2020 [poster 4552].

Background CDX-1140 is an agonist anti-CD40 mAb selected to optimize systemic exposure and hence tumor microenvironment (TME) ingress. CDX-1140 activity may be enhanced by combining with CDX-301 (recombinant Flt3L), a dendritic cell growth factor, or with pembrolizumab, an anti-PD-1 mAb. Methods Patients with advanced solid or hematologic (Part 1 only) tumors are enrolled. Part 1 dose-escalation results have been presented (SITC 2019). In Part 2, CDX-1140 dose-escalation (0.09–1.5 mg/kg q4w) is in combination with CDX-301 (75 mcg/kg sc QD x 5 for 2 cycles). In Part 3, CDX-1140 dose-escalation (0.72–1.5 mg/kg q3w) is in combination with pembrolizumab 200 mg q3w. Part 1 and 2 expansion cohorts are dosed at the CDX-1140 MTD, 1.5 mg/kg q4w. Part 3 expansion cohorts are planned. Peripheral blood and tumor biomarkers analysis are ongoing. Results 92 patients have been treated (Part 1 n=57, Part 2 n=31, Part 3 n=4). Part 1 expansion cohorts in SCCHN (n=7) and RCC (n=5) are fully enrolled. Part 2 dose-escalation completed to the highest CDX-1140 dose and a SCCHN expansion cohort is ongoing. Part 3 dose-escalation recently initiated. Safety data is available for 23 and 10 patients at the MTD in Part 1 and 2, respectively. In general, the safety profiles were similar, with arthralgia (52% vs. 50%), pyrexia (44% vs 50%), fatigue (30% vs. 50%), chills (39% vs. 40%), vomiting (30% vs. 20%), nausea (26% vs 40%), myalgia (22% vs. 30%), increased ALT (22% vs. 20%), and increased AST (22% vs. 30%) being the most common drug related AEs at the MTD in Part 1 and 2, respectively. Most AEs were low grade. Across all cohorts, cytokine release syndrome (CRS) (G2 n=4, G3 n=2) occurred in 6 (Part 1 n=2; Part 2 n=4) and pneumonitis (G3) occurred in 5 (Part 1 n=4; Part 2 n=1) patients. Immune activation in the TME consistent with CD40 agonism and increases serum inflammatory cytokines were observed. Evidence of anti-tumor activity/clinical benefit include SD (n=13), tumor cavitation (n=2) and a uPR in solid tumors. A patient with follicular lymphoma has an ongoing durable complete metabolic response. Conclusions The CDX-1140 MTD dose of 1.5 mg/kg, a dose level expected to provide good systemic exposure and TME penetration, is generally well tolerated alone and with CDX-301. Transaminitis and CRS have generally been low grade and infrequent. A cohort combining CDX-1140 with chemotherapy will be initiated in patients with previously untreated metastatic pancreatic adenocarcinoma. Trial Registration NCT03329950 Ethics Approval The study was approved by the following: Providence St. Joseph Health IRB, approval number MOD2020001128; WIRB, approval number 1188814 (Hauke, Gabrail, Bordoni & Gordon); University of Pennsylvania IRB, approval number UPCC 18917; Mount Sinai School of Medicine IRB, approval number 18-00202; Memorial Sloan Kettering Cancer Center IRB, approval number 18-225A; Houston Methodist IRB, approval number MOD00000836

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]

Mesothelin is a promising target for immune-based therapy, specifically for mesothelioma and pancreatic and ovarian cancers that have high levels of mesothelin expression. Many preclinical and clinical studies that target tumors with high mesothelin expression with antibodies, immunotoxins, antibody–drug conjugates and vaccines have shown the potential of mesothelin as a target. Studies of T cells genetically modified with chimeric antigen receptors (CAR) report significant efficacy in hematologic malignancies, and antimesothelin CAR T cells are currently being investigated in clinical studies. Here we outline the rationale for using mesothelin as a target for immunotherapy, review the clinical and preclinical studies evaluating mesothelin-directed therapies and explore the promise of CAR T cells directed against mesothelin for immunotherapy in the future.

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

Background: Chemotherapy-induced oral thermal hyperalgesia (OTH) is a common and debilitating side effect of platinum-based anticancer agents. This study evaluated the efficacy of oral cryotherapy in preventing OTH during oxaliplatin chemotherapy infusion. Methods: Patients with gastrointestinal cancer treated with biweekly oxaliplatin (85 mg/m2 over 120 minutes) at Abramson Cancer Center at the University of Pennsylvania were randomized to receive oral cryotherapy (ice chips) during oxaliplatin infusion or standard-of-care treatment. All patients completed baseline questionnaires regarding oral and peripheral symptoms and on-treatment questionnaires on day 1 of each subsequent chemotherapy cycle. Those in the treatment arm were asked to document how long they kept the ice chips in their mouths (0, Results: Of 62 randomized patients with a variety of gastrointestinal malignancies, 50 (25 per treatment arm) were evaluable for efficacy. The rate of patients with oral symptoms after the first treatment cycle was significantly lower in the intervention arm (n=8; 32%) than in the control arm (n=18; 72%), meeting the primary study objective (P=.01). The magnitude of difference in symptom scores before versus after the first treatment cycle was significantly less in the intervention versus control arm (P=.001). No difference in oral symptoms over time was seen between the intervention and control groups (P=.20), although a high attrition rate was noted. Duration of ice chip exposure was associated with improved oral symptoms over time (P=.02). Conclusions: Oral cryotherapy is a tolerable and cost-effective method of diminishing OTH in patients receiving oxaliplatin chemotherapy, and seems to be most effective in the early stages of treatment.

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.