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Purpose:Mistletoe extract (ME) is widely used for patients with cancer to support therapy and to improve quality of life (QoL). However, its use is controversial due to suboptimal trials and a lack of data supporting its intravenous administration.Materials and Methods:This phase I trial of intravenous mistletoe (Helixor M) aimed to determine the recommended phase II dosing and to evaluate safety. Patients with solid tumor progressing on at least one line of chemotherapy received escalating doses of Helixor M three times a week. Assessments were also made of tumor marker kinetics and QoL.Results:Twenty-one patients were recruited. The median follow-up duration was 15.3 weeks. The MTD was 600 mg. Treatment-related adverse events (AE) occurred in 13 patients (61.9%), with the most common being fatigue (28.6%), nausea (9.5%), and chills (9.5%). Grade 3+ treatment-related AEs were noted in 3 patients (14.8%). Stable disease was observed in 5 patients who had one to six prior therapies. Reductions in baseline target lesions were observed in 3 patients who had two to six prior therapies. Objective responses were not observed. The disease control rate (percentage of complete/partial response and stable disease) was 23.8%. The median stable disease was 15 weeks. Serum cancer antigen-125 or carcinoembryonic antigen showed a slower rate of increase at higher dose levels. The median QoL by Functional Assessment of Cancer Therapy-General increased from 79.7 at week 1 to 93 at week 4.Conclusions:Intravenous mistletoe demonstrated manageable toxicities with disease control and improved QoL in a heavily pretreated solid tumor population. Future phase II trials are warranted.Significance:Although ME is widely used for cancers, its efficacy and safety are uncertain. This first phase I trial of intravenous mistletoe (Helixor M) aimed to determine phase II dosing and to evaluate safety. We recruited 21 patients with relapsed/refractory metastatic solid tumor. Intravenous mistletoe (600 mg, 3/week) demonstrated manageable toxicities (fatigue, nausea, and chills) with disease control and improved QoL. Future research can examine ME's effect on survival and chemotherapy tolerability.

In a prospective multicenter study, gemcitabine monotherapy followed by stereotactic body radiation therapy (SBRT) was well tolerated with outcomes comparable to chemoradiation for locally advanced pancreatic cancer (LAPC). Recent trials have reported improved survival with multiagent chemotherapy (MA-CTX) alone. This prospective trial explored whether SBRT could be safely delivered after MA-CTX. Herein, we report the long-term outcomes of adding SBRT after MA-CTX in LAPC patients and evaluate whether genetic profiles of specimens obtained before SBRT influence outcomes.This prospective nonrandomized controlled phase 2 trial enrolled 44 LAPC and 4 locally recurrent patients after multidisciplinary evaluation between 2012 and 2015 at a high-volume pancreatic cancer center. For induction CTX, most received modified FOLFIRINOX (mFFX), or gemcitabine and nab-paclitaxel (GnP) followed by 5-fraction SBRT for all. During fiducial placement, biopsies were obtained with DNA extracted for targeted sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform.Median induction CTX duration was ≥4 months, and 31 patients received mFFX (65%). Among 44 LAPC patients, 17 (39%) were surgically explored, and 12 of 16 (75%) achieved a R0 resection. Median overall survival (mOS) was 20.2 and 14.6 months from diagnosis and SBRT, respectively. One- and 2-year OS from SBRT was 58% and 28%. The mOS after resection was 28.6 and 22.4 months from diagnosis and SBRT, respectively. Median local progression-free survival was 23.9 and 15.8 months from diagnosis and SBRT, respectively. The mOS for pre-SBRT CA 19-9 ≤180 U/mL versus180 was 23.1 and 11.3 months, respectively (hazard ratio, 0.53; P = .04). Only 1 patient (2.1%) had late grade ≥2 gastrointestinal toxic effects attributable to SBRT. Despite significant pretreatment with chemotherapy, 88% of tumor specimens were effectively sequenced; survival outcomes were not significantly associated with specific mutational patterns. Quality of life was prospectively collected pre- and post-SBRT with the EORTC QLQ-C30 and PAN26 questionnaires showing no significant change.SBRT was safely administered with MA-CTX with minimal toxicity. A high proportion of LAPC patients underwent R0 resection with favorable survival outcomes.

Surgical resection is the only potential curative treatment for patients with pancreatic ductal adenocarcinoma (PDAC), but unfortunately most patients recur within 5 years of surgery. This article aims to assess the practice patterns across major academic institutions and develop consensus recommendations for postoperative imaging and interpretation in patients with PDAC.The consensus recommendations for postoperative imaging surveillance following PDAC resection were developed using the Delphi method. Members of the Society of Abdominal Radiology (SAR) PDAC Disease Focused Panel (DFP) underwent three rounds of surveys followed by live webinar group discussions to develop consensus recommendations.Significant variations currently exist in the postoperative surveillance of PDAC, even among academic institutions. Differentiating common postoperative inflammatory and fibrotic changes from tumor recurrence remains a diagnostic challenge, and there is no reliable size threshold or growth rate of imaging findings that can provide differentiation. A new liver lesion or peritoneal nodule should be considered suspicious for tumor recurrence, and the imaging features should be interpreted in the appropriate clinical context (e.g., CA 19-9, clinical presentation, pathologic staging).Postoperative imaging following PDAC resection is challenging to interpret due to the presence of confounding postoperative inflammatory changes. A standardized reporting template for locoregional findings and report impression may improve communication of relaying risk of recurrence with referring providers, which merits validation in future studies.

Purpose: To investigate the role of pre- and post-stereotactic body radiation therapy (SBRT) neutrophil-to-lymphocyte ratio (NLR) in patients with localized pancreatic cancer treated with anti-PD-1 (programmed cell death protein-1) antibody and SBRT.Materials and Methods: This was a retrospective review of 68 patients with borderline resectable or locally advanced pancreatic cancer treated with anti-PD-1 antibody and SBRT after multi-agent chemotherapy. Immunotherapy was administered with 5-fraction SBRT in the neoadjuvant, concurrent, or adjuvant/maintenance setting. Clinical outcomes included overall survival (OS), local progression-free survival, distant metastasis-free survival, and progression-free survival. Median pre- and post-SBRT peripheral blood markers were compared with the Mann-Whitney U test. Univariate and multivariable analyses (UVA and MVA) were performed to identify variables associated with clinical outcomes. Linear regression was performed to determine correlations between variables and peripheral blood markers. Results: A total of 68 patients were included in the study. The percent change between median pre- and post-SBRT absolute lymphocyte count (ALC), absolute neutrophil count, and NLR were -36.0% (p < 0.001), -5.6% (p = 0.190), and +35.7% (p = 0.003), respectively. Median OS after SBRT was 22.4 months. On UVA, pre-SBRT CA19-9 (hazard ratio [HR] = 1.001; 95% confidence interval [CI], 1.000–1.001; p = 0.031), post-SBRT ALC (HR = 0.33; 95% CI, 0.11–0.91; p = 0.031), and post-SBRT NLR (HR = 1.13; 95% CI, 1.04–1.22; p = 0.009) were associated with OS. On MVA, induction chemotherapy duration (HR = 0.75; 95% CI, 0.57–0.99; p = 0.048) and post-SBRT NLR (HR = 1.14; 95% CI, 1.04–1.23; p = 0.002) predicted for OS. Patients with post-SBRT NLR ≥3.2 had a median OS of 15.6 months versus 27.6 months in patients with post-SBRT NLR

Table S3 shows reasons for discontinuation with patient charateristics

Figure S3 shows functional assessment of cancer therapy - general over time

Supplementary Materials and Methods show the trial protocol

Purpose:Mistletoe extract (ME) is widely used for patients with cancer to support therapy and to improve quality of life (QoL). However, its use is controversial due to suboptimal trials and a lack of data supporting its intravenous administration.Materials and Methods:This phase I trial of intravenous mistletoe (Helixor M) aimed to determine the recommended phase II dosing and to evaluate safety. Patients with solid tumor progressing on at least one line of chemotherapy received escalating doses of Helixor M three times a week. Assessments were also made of tumor marker kinetics and QoL.Results:Twenty-one patients were recruited. The median follow-up duration was 15.3 weeks. The MTD was 600 mg. Treatment-related adverse events (AE) occurred in 13 patients (61.9%), with the most common being fatigue (28.6%), nausea (9.5%), and chills (9.5%). Grade 3+ treatment-related AEs were noted in 3 patients (14.8%). Stable disease was observed in 5 patients who had one to six prior therapies. Reductions in baseline target lesions were observed in 3 patients who had two to six prior therapies. Objective responses were not observed. The disease control rate (percentage of complete/partial response and stable disease) was 23.8%. The median stable disease was 15 weeks. Serum cancer antigen-125 or carcinoembryonic antigen showed a slower rate of increase at higher dose levels. The median QoL by Functional Assessment of Cancer Therapy-General increased from 79.7 at week 1 to 93 at week 4.Conclusions:Intravenous mistletoe demonstrated manageable toxicities with disease control and improved QoL in a heavily pretreated solid tumor population. Future phase II trials are warranted.Significance:Although ME is widely used for cancers, its efficacy and safety are uncertain. This first phase I trial of intravenous mistletoe (Helixor M) aimed to determine phase II dosing and to evaluate safety. We recruited 21 patients with relapsed/refractory metastatic solid tumor. Intravenous mistletoe (600 mg, 3/week) demonstrated manageable toxicities (fatigue, nausea, and chills) with disease control and improved QoL. Future research can examine ME's effect on survival and chemotherapy tolerability.

Immune-checkpoint therapy has failed to demonstrate meaningful clinical benefit in unselected cases of pancreatic adenocarcinoma (PDAC), but a subset of PDACs are known to upregulate pathways involved in acquired immune suppression. Further delineation of immunologic subtypes of PDAC is necessary to improve clinical trial designs and identify patients who might benefit from immune-checkpoint therapy. We used clinical survival and RNA expression data from The Cancer Genome Atlas (TCGA) to investigate the relationship between immune-modulating pathways and immune subset markers and their impact on survival in PDAC patients. Of the adaptive immune-resistance pathways, expression of PD-L1 and IDO1 was individually associated with poor survival. Although CD8 expression alone was not correlated with survival, the combination of PD-L1− and high CD8 expression identified a subtype with favorable survival. We further extended these observations using an independent PDAC cohort from our institution via IHC, again observing that the PD-L1−/CD8high subtype was associated with positive prognosis. Although PDAC is regarded as a poorly immunogenic cancer type, these findings infer that T-cell infiltration in the absence of adaptive immune-resistance pathways is a feature of long-term survival in PDAC and imply the importance of developing future immunotherapeutic strategies based on data-supported biomarkers to refine patient selection.

Supplemental Figure S1-S4

Supplemental Tables

MYPT1 and PSMC5 expression in 3 different subtypes (basal, HER-2 positive and ER positive) of breast cancer.

Western blots showing the antibody responses for PSMC5, MYPT1 and TFRC in patients with greater than and less than 3 years disease free survival

Selection criteria used to narrow the proteins identified in the screen to those shown in this study.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Less than 6% of patients survive beyond the fifth year due to inadequate early diagnostics and ineffective treatment options. Our laboratory has developed an allogeneic, granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting pancreatic cancer vaccine (GVAX) that has been tested in phase II clinical trials. Here, we employed a serum antibodies–based SILAC immunoprecipitation (SASI) approach to identify proteins that elicit an antibody response after vaccination. The SASI approach uses immunoprecipitation with patient-derived antibodies that is coupled to quantitative stable isotope–labeled amino acids in cell culture (SILAC). Using mass spectrometric analysis, we identified more than 150 different proteins that induce an antibody response after vaccination. The regulatory subunit 12A of protein phosphatase 1 (MYPT1 or PPP1R12A), regulatory subunit 8 of the 26S proteasome (PSMC5), and the transferrin receptor (TFRC) were shown to be pancreatic cancer–associated antigens recognized by postvaccination antibodies in the sera of patients with favorable disease-free survival after GVAX therapy. We further interrogated these proteins in over 80 GVAX-treated patients' pancreases and uniformly found a significant increase in the expression of MYPT1, PSMC5, and TFRC in neoplastic compared with non-neoplastic pancreatic ductal epithelium. We show that the novel SASI approach can identify antibody targets specifically expressed in patients with improved disease-free survival after cancer vaccine therapy. These targets need further validation to be considered as possible pancreatic cancer biomarkers. Cancer Immunol Res; 4(3); 225–33. ©2016 AACR.

Venn diagram showing overlap of proteins identified in 3 patients after the SASI screen.

Additional immunohistochemical validation showing PSMC5, MYPT1 and TFRC expression in an independent set of pancreatic cancers.

Purpose: Genetic alterations of KRAS, CDKN2A, TP53, and SMAD4 are the most frequent events in pancreatic cancer. We determined the extent to which these 4 alterations are coexistent in the same carcinoma, and their impact on patient outcome.Experimental Design: Pancreatic cancer patients who underwent an autopsy were studied (n = 79). Matched primary and metastasis tissues were evaluated for intragenic mutations in KRAS, CDKN2A, and TP53 and immunolabeled for CDKN2A, TP53, and SMAD4 protein products. The number of altered driver genes in each carcinoma was correlated to clinicopathologic features. Kaplan–Meier estimates were used to determine median disease free and overall survival, and a Cox proportional hazards model used to compare risk factors.Results: The number of genetically altered driver genes in a carcinoma was variable, with only 29 patients (37%) having an alteration in all 4 genes analyzed. The number of altered driver genes was significantly correlated with disease free survival (P = 0.008), overall survival (P = 0.041), and metastatic burden at autopsy (P = 0.002). On multivariate analysis, the number of driver gene alterations in a pancreatic carcinoma remained independently associated with overall survival (P = 0.046). Carcinomas with only 1 to 2 driver alterations were enriched for those patients with the longest survival (median 23 months, range 1 to 53).Conclusions: Determinations of the status of the 4 major driver genes in pancreatic cancer, and specifically the extent to which they are coexistent in an individual patients cancer, provides distinct information regarding disease progression and survival that is independent of clinical stage and treatment status. Clin Cancer Res; 18(22); 6339–47. ©2012 AACR.

PDF file - 77K, Primers used for amplification and sequencing of pancreatic cancer driver genes

Serial Dilution Data

Supplementary fig 2 Schematic overview of 10-color Multiplex IHC and Image visualization.

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Supplementary fig 4 Changes in the functional markers within each cluster with GVAX + IPI.

Purpose:Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF–secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated Listeria monocytogenes–expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B).Patients and Methods:Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates.Results:Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7–8.6] and 6.1 (95% CI, 3.5–7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55–1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8+ T cells and a decrease in CD68+ myeloid cells, were observed in long-term survivors in Arm A only.Conclusions:Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.

PDF file - 94K, Complete sequencing and IHC data for all 79 Patients

Supplementary fig 1 Schematic for defining the immune composition of peripheral blood lymphocytes from Arm A patients using CyTOF.

Purpose:Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses and activation and recruitment of natural killer (NK) and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively.Experimental Design: A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1 × 106, 3 × 107, or 3 × 108 colony-forming units (cfu). CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic, or ovarian cancers. Seventeen subjects received up to 4 doses of 1 × 108, 3 × 108, 1 × 109, or 1 × 1010 cfu.Results: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1 × 109 cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, listeriolysin O and mesothelin-specific T-cell responses were detected and 37% of subjects lived ≥15 months.Conclusions: ANZ-100 and CRS-207 administration was safe and resulted in immune activation. Clin Cancer Res; 18(3); 858–68. ©2011 AACR.

Purpose:In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology–certified clinical laboratory.Experimental Design:Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed.Results:ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%–98%), specificity 88% (95% CI, 62%–98%)] with a median lead time of 84 days (interquartile range, 25–146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011).Conclusions:Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

Supplement: eTables 1-4, eFigures 1-7

Supplementary Tables S1 to S6 clean version.

Purpose:Tumor mutational burden (TMB) has emerged as a potential predictive biomarker for clinical response to ICI therapy, but whether TMB also predicts toxicity remains unknown. We investigated the relationship between TMB, objective response rate (ORR), overall survival (OS), and toxicity for ICI therapy across multiple cancer types.Experimental Design:We searched MEDLINE, PubMed, and ASCO/ESMO/AACR meetings for clinical trials of anti-PD(L)1, CTLA-4, or combination in 29 cancer types. We assessed ICI administered, responses (complete or partial response), median OS, OS HR, and grade 3/4 toxicity. We conducted a systematic review, meta-analysis and meta-regression using tumor level TMB data from Foundation Medicine.Results:One hundred seventeen clinical trials, which included 12,450 patients treated with ICI therapy were analyzed. Meta-regression analysis revealed that TMB was significantly associated with ORR for anti-PD(L)1, anti–CTLA-4, and combination (P < 0.0001 for all), but not associated with toxicity in all treatment groups. OS data were unavailable for most studies included in our meta-analysis, and the relationship between TMB and OS in this subset was not significant (P = 0.26). In high TMB tumor types (≥10 mut/megabase) the improvement of ORR and increase in grade 3/4 toxicity with combination ICI therapy as compared with PD(L)1 monotherapy were 21.13% and 25.41%, respectively, as compared with 3.73% and 18.78% in low TMB tumor types (Conclusions:There is a positive association between TMB and clinical response with anti-PD(L)1, anti–CTLA-4, and combination ICIs, but no association between TMB and toxicity. These results imply a favorable risk/benefit ratio for ICIs in tumors with a higher TMB.

Supplementary Data

Purpose:This phase II study tested granulocyte-macrophage colony-stimulating factor (GM-CSF)-allogeneic pancreatic tumor cells (GVAX) and ipilimumab in metastatic pancreatic ductal adenocarcinoma (PDA) in the maintenance setting.Patients and Methods:Patients with PDA who were treated with front-line chemotherapy consisting of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) in the metastatic setting and had ongoing response or stable disease after 8–12 doses were eligible. Patients were randomized 1:1 to treatment with GVAX and ipilimumab given every 3 weeks for four doses then every 8 weeks (Arm A) or to FOLFIRINOX continuation (Arm B). The primary objective was to compare overall survival (OS) between the two arms.Results:Eighty-two patients were included in the final analysis (Arm A: 40; Arm B: 42). The study was stopped for futility after interim analysis. Median OS was 9.38 months [95% confidence interval (CI), 5.0–12.2] for Arm A and 14.7 months (95% CI, 11.6–20.0) for Arm B (HR, 1.75; P = 0.019). Using immune-related response criteria, two partial responses (5.7%) were observed in Arm A and four (13.8%) in Arm B. GVAX + ipilimumab promoted T-cell differentiation into effector memory phenotypes both in the periphery and in the tumor microenvironment and increased M1 macrophages in the tumor.Conclusions:GVAX and ipilimumab maintenance therapy did not improve OS over continuation of chemotherapy and resulted in a numerically inferior survival in metastatic PDA. However, clinical responses and biological effects on immune cells were observed. Further study of novel combinations in the maintenance treatment of metastatic PDA is feasible.

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Supplemental Methods, Figures and Tables

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Background Patients with borderline resectable pancreatic cancer (BRPC) or locally advanced pancreatic cancer (LAPC) are at high risk of margin-positive resection. Neoadjuvant stereotactic body radiation therapy (SBRT) may help sterilize margins, but its additive benefit beyond neoadjuvant chemotherapy (nCT) is unclear. The authors report long-term outcomes for BRPC/LAPC patients explored after treatment with either nCT alone or nCT followed by five-fraction SBRT (nCT-SBRT). Methods Patients with BRPC or LAPC from 2011 to 2016 who underwent resection after nCT alone or nCT-SBRT were retrospectively reviewed. Baseline characteristics were compared, and the propensity score with inverse probability weighting (IPW) was used to compare pathologic/survival outcomes. Results Of 198 patients, 76 received nCT, and 122 received nCT-SBRT. The nCT-SBRT cohort had a higher proportion of LAPC (53% vs 22%; p p = 0.03), but adjuvant chemotherapy was less frequently administered (53% vs 67.1%; p p p p = 0.02). In the multivariate analysis, nCT-SBRT remained associated with R0 resection (p p = 0.81). Conclusions Despite having more advanced disease, the nCT-SBRT cohort was still more likely to undergo an R0 resection and experienced similar survival outcomes compared with the nCT alone cohort.

Gene variants that encode pancreatic enzymes with impaired secretion can induce pancreatic acinar endoplasmic reticulum (ER) stress, cellular injury and pancreatitis. The role of such variants in pancreatic cancer risk has received little attention. We compared the prevalence of ER stress-inducing variants in CPA1 and CPB1 in patients with pancreatic ductal adenocarcinoma (PDAC cases), enrolled in the National Familial Pancreas Tumor Registry, to their prevalence in non-cancer controls in the Genome Aggregation Database (gnomAD). Variants of unknown significance were expressed and variants with reduced secretion assessed for ER stress induction. In vitro assessments were compared with software predictions of variant function. Protein variant software was used to assess variants found in only one gnomAD control ("n-of-one" variants). A meta-analysis of prior PDAC case/control studies was also performed. Of the 1385 patients with PDAC, 0.65% were found to harbor an ER stress-inducing variant in CPA1 or CPB1, compared to 0.17% of the 64,026 controls (Odds Ratio (OR): 3.80 [1.92-7.51] P=0.0001). ER stress-inducing variants in the CPA1 gene were identified in 4 of 1385 PDAC cases versus 77 of 64,026 gnomAD controls (OR: 2.4 [0.88-6.58], p=0.087), and variants in CPB1 were detected in 5 of 1385 cases versus 33 of 64,026 controls (OR: 7.02 [2.74-18.01], p=0.0001). Meta-analysis demonstrated strong associations for pancreatic cancer and ER-stress inducing variants for both CPA1 (OR: 3.65 [1.58, 8.39], p

Background Pancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation. Methods We measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR. Results No evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (∼50%). Conclusions We found no evidence of germline promoter methylation for the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.

PURPOSE Genetic alterations in many components of the homologous recombination, DNA damage response, and repair (HR-DDR) pathway are involved in the hereditary cancer syndromes, including familial pancreatic cancer. HR-DDR genes beyond BRCA1, BRCA2, ATM, and PALB2 may also mutate and confer the HR-DDR deficiency in pancreatic ductal adenocarcinoma (PDAC). METHODS We conducted a study to examine the genetic alterations using a companion diagnostic 15-gene HR-DDR panel in PDACs. HR-DDR gene mutations were identified and characterized by whole-exome sequencing and whole-genome sequencing. Different HR-DDR gene mutations are associated with variable homologous recombination deficiency (HRD) scores. RESULTS Eight of 50 PDACs with at least one HR-DDR gene mutation were identified. One tumor with BRCA2 mutations is associated with a high HRD score. However, another tumor with a CHEK2 mutation is associated with a zero HRD score. Notably, four of eight PDACs in this study harbor a RAD51B gene mutation. All four RAD51B gene mutations were germline mutations. However, currently, RAD51B is not the gene panel for germline tests. CONCLUSION The finding in this study thus supports including RAD51B in the germline test of HR-DDR pathway genes.

Purpose: Immunotherapy is currently ineffective for nearly all pancreatic ductal adenocarcinomas (PDAC), largely due to its tumor microenvironment (TME) that lacks antigen-experienced T effector cells (Teff). Vaccine-based immunotherapies are known to activate antigen-specific Teffs in the peripheral blood. To evaluate the effect of vaccine therapy on the PDAC TME, we designed a neoadjuvant and adjuvant clinical trial of an irradiated, GM-CSF-secreting, allogeneic PDAC vaccine (GVAX). Patients and Methods: Eighty-seven eligible patients with resectable PDAC were randomly assigned (1:1:1) to receive GVAX alone or in combination with two forms of low-dose cyclophosphamide. Resected tumors following neoadjuvant immunotherapy were assessed for the formation of tertiary lymphoid aggregates (TLA) in response to treatment. The clinical endpoints are disease-free survival (DFS) and overall survival (OS). Results: The neoadjuvant treatment with GVAX either alone or with two forms of low-dose cyclophosphamide is safe and feasible without adversely increasing the surgical complication rate. Patients in Arm A who received neoadjuvant and adjuvant GVAX alone had a trend toward longer median OS (35.0 months) than that (24.8 months) in the historical controls who received adjuvant GVAX alone. However, Arm C, who received low-dose oral cyclophosphamide in addition to GVAX, had a significantly shorter DFS than Arm A. When comparing patients with OS > 24 months to those with OS < 15 months, longer OS was found to be associated with higher density of intratumoral TLA. Conclusions: It is safe and feasible to use a neoadjuvant immunotherapy approach for PDACs to evaluate early biologic responses. In-depth analysis of TLAs is warranted in future neoadjuvant immunotherapy clinical trials.

Background: Novel strategies are needed to decrease the high recurrence rates of pancreatic ductal adenocarcinoma (PDAC) after curative-intent surgery. Mutant KRAS (mKRAS) is an oncogenic driver found in approximately 90% of PDAC that has emerged as a target for neoantigen-specific vaccination. Methods: This is a single-arm, open-label, first-in-human phase I study of a pooled synthetic long peptide (SLP) vaccine targeting six mKRAS subtypes (G12D, G12R, G12V, G12A, G12C, G13D) combined with ipilimumab/nivolumab (ipi/nivo) in patients with resected PDAC (NCT04117087). Key inclusion criteria: presence of a vaccine-targeted KRAS mutation, disease-free status after completing adjuvant chemotherapy within 6 months of enrollment. Priming phase: mKRAS vaccine given on days 1, 8, 15, and 22 along with ipi 1 mg/kg every 6 weeks for 2 doses and nivo 3 mg/kg every 3 weeks for 4 doses. Boost phase: mKRAS vaccine given on weeks 13, 21, 29, 37, and 45 along with nivo 480 mg every 4 weeks for 10 doses. Co-primary endpoints: safety, mKRAS-specific T cell response. Secondary endpoints: disease-free survival (DFS), overall survival (OS). Results: At the time of data cutoff (December 1, 2022), 11 patients were treated with a median follow-up time of 10.7 months. 8/11 patients achieved a mKRAS-specific T cell response, defined as >5-fold change in IFN-gamma-producing mKRAS-specific T cells within 17 weeks post-vaccination, as assessed by serial ELISpot. The median fold change in IFN-gamma-producing mKRAS-specific T cells within 17 weeks post-vaccination was 10.2 (range: 1.5-686.3). The majority of all adverse events were grade 1 (79.5%) or grade 2 (15.2%) in severity per NCI CTCAE v5.0. Four grade 3 immune-related adverse events (pneumonitis, adrenal insufficiency, arthralgias, myalgias) and discontinuation of checkpoint blockade occurred in 2/11 patients. The median DFS for the entire cohort was 6.4 months and not reached for OS. Patients who mounted a mKRAS-specific T cell response demonstrated a significant improvement in median DFS compared to immune non-responders (not reached vs 2.8 months, p = 0.045). Conclusions: The combination of a pooled SLP mKRAS vaccine and dual checkpoint blockade is tolerable and immunogenic in patients with resected PDAC. Induction of an mKRAS-specific T cell response is associated with improved DFS in this cohort. Ongoing correlative studies will apply multi-omic approaches to identify novel biomarkers of immune response and resistance. Citation Format: Saurav D. Haldar, Amanda Huff, Thatcher R. Heumann, Maureen Berg, Anna Ferguson, Su Jin Lim, Hao Wang, Amy M. Thomas, Julie M. Nauroth, Marina Baretti, Katherine M. Bever, Mark Yarchoan, Daniel A. Laheru, Elizabeth M. Jaffee, Nilofer S. Azad, Neeha Zaidi. Safety and immunogenicity of a first-in-human mutant KRAS long peptide vaccine combined with ipilimumab/nivolumab in resected pancreatic cancer: Preliminary analysis from a phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT036.

719 Background: Outcomes in BR and LA PDAC remain historically poor, in part due to low rates of R0 resection. A prior phase II study demonstrated that losartan (L) as a TGF-beta inhibitor combined with FOLFIRINOX (FFX) and radiation in LA PDAC led to a 61% R0 resection rate. Additionally, prior phase II studies suggest potential synergy with SBRT and nivolumab (N) in PDAC. We conducted a multi-center, randomized phase II trial to evaluate the effect of L and L+N in combination with TNT using FFX and SBRT. Methods: Patients with BR or LA PDAC by NCCN criteria, pathologically confirmed, ACE/ARB naïve, were randomized to TNT with FFX and SBRT (Arm 1), TNT + L (Arm 2), and TNT+L+N (Arm 3), stratified by BR/LA. Patients already on an ACE or ARB were enrolled on an exploratory arm of TNT+N (Arm 4) and will be reported separately. TNT consisted of FFX x 8 followed by SBRT (6.6 Gy x 5). L was given at 50 mg qd throughout TNT and for 6 mo after surgery. N was given at 240 mg flat dosing q2 wks concurrent with SBRT and for 12 doses postoperatively. All patients were recommended for surgical exploration after TNT. The study was designed to compare the R0 resection rate on each of Arms 2 and 3 independently versus Arm 1 at a one-sided 0.10 level. Secondary endpoints were PFS, OS, and pCR rates and analyzed using two-sided tests with Arm 1 as the control arm. Intent-to-treat analysis was based on eligible patients who started therapy on protocol. Results: Patients with BR or LA PDAC by NCCN criteria, pathologically confirmed, ACE/ARB naïve, were randomized to TNT with FFX and SBRT (Arm 1), TNT + L (Arm 2), and TNT+L+N (Arm 3), stratified by BR/LA. Patients already on an ACE or ARB were enrolled on an exploratory arm of TNT+N (Arm 4) and will be reported separately. TNT consisted of FFX x 8 followed by SBRT (6.6 Gy x 5). L was given at 50 mg qd throughout TNT and for 6 mo after surgery. N was given at 240 mg flat dosing q2 wks concurrent with SBRT and for 12 doses postoperatively. All patients were recommended for surgical exploration after TNT. The study was designed to compare the R0 resection rate on each of Arms 2 and 3 independently versus Arm 1 at a one-sided 0.10 level. Secondary endpoints were PFS, OS, and pCR rates and analyzed using two-sided tests with Arm 1 as the control arm. Intent-to-treat analysis was based on eligible patients who started therapy on protocol. Conclusions: We did not observe effects of L and L+N on the R0 resection rate, PFS, OS, and pCR rate when added to TNT with FFX and SBRT for BR or LA PDAC. The lack of differences may reflect heterogeneity in surgical opinion as the decision for proceeding to surgery following TNT tends to be highly variable in a population with historically low resection rates. Clinical trial information: NCT03563248 .

A 26-year-old male with a two-year history of FLC developed progressive somnolence and disorientation. Treatment history for FLC had included cytotoxic chemotherapy, lenvatinib, and immunotherapy. A CT scan confirmed extensive stage FLC with numerous liver, lung, and pelvic metastasis. Laboratory results showed bilirubin 0.3 mg/dL, creatinine 0.4 mg/dl, leukocytes of 9.5x109 /L, hemoglobin 11.2 g/dL, platelets 369x109 /L, and ammonia 247 µmol/L (reference range: 0-32 µmol/L). Plasma amino acid analysis revealed relatively low citrulline (14 µmol/L), arginine (32 µmol/L), and ornithine (35 µmol/L). Urinary orotic acid excretion was markedly elevated at 149 mmol/mol creat (reference range: 0.68-3.52 mmol/mol creat).

Recent research on genomic profiling of pancreatic ductal adenocarcinoma (PDAC) has identified many potentially actionable alterations. However, the feasibility of using genomic profiling to guide routine clinical decision making for PDAC patients remains unclear. We retrospectively reviewed PDAC patients between October 2013 and December 2017, who underwent treatment at the Johns Hopkins Hospital and had clinical tumor next-generation sequencing (NGS) through commercial resources. Ninety-two patients with 93 tumors tested were included. Forty-eight (52%) patients had potentially curative surgeries. The median time from the tissue available to the NGS testing ordered was 229 days (interquartile range 62–415). A total of three (3%) patients had matched targeted therapies based on genomic profiling results. Genomic profiling guided personalized treatment for PDAC patients is feasible, but the percentage of patients who receive targeted therapy is low. The main challenges are ordering NGS testing early in the clinical course of the disease and the limited evidence of using a targeted approach in these patients. A real-time department level genomic testing ordering system in combination with an evidence-based flagging system for potentially actionable alterations could help address these shortcomings.

Objective We evaluated survival outcomes in patients with distal pancreatic ductal adenocarcinoma (D-PDAC) after distal pancreatectomy (DP) and adjuvant chemotherapy or chemoradiation. Methods A retrospective analysis of patients who underwent DP for D-PDAC from 2000 to 2015 at the Johns Hopkins Hospital was performed. Demographics, baseline risk factors, and type of adjuvant treatment were assessed for associations with overall survival (OS) and disease-free survival (DFS). Comparisons were made with log-rank tests and Cox proportional hazards regression models. Results A total of 294 patients underwent DP for D-PDAC. Of these, 105 patients were followed at the Johns Hopkins Hospital. Forty-five patients received chemotherapy only and 60 patients received chemoradiation. The median OS with chemoradiation was 33.6 months and 27.9 months (P = 0.54) with chemotherapy only. The median DFS was 15.3 months with chemoradiation and 19.8 months with chemotherapy only (P = 0.89). Elevated carbohydrate antigen 19-9, stage II to III disease, splenic vein involvement, and vascular invasion were significant risk factors in multivariate analyses. Conclusions In this retrospective analysis, there were no significant differences in OS or DFS with chemoradiation compared with chemotherapy alone after DP in patients with D-PDAC.