Search

Table S1. Antibodies used in immunofluorescence and immunohistochemistry Table S2. qPCR primer sequences

Supplemental Microarray Data

Supplemental Materials and Methods

Carcinoma-associated fibroblasts (CAF) represent a significant component of pancreatic cancer stroma and are biologically implicated in tumor progression. However, evidence of both cancer-promoting and -restraining properties amongst CAFs suggests the possibility of multiple phenotypic subtypes. Here, it is demonstrated that senescent CAFs promote pancreatic cancer invasion and metastasis compared with nonsenescent control CAFs using in vitro Transwell invasion models and in vivo xenograft mouse models. Screening by gene expression microarray and cytokine ELISA assays revealed IL8 to be upregulated in senescent CAFs. Experimental modulation through IL8 overexpression or receptor inhibition implicates the IL8 pathway as a mediator of the proinvasive effects of senescent CAFs. In a cohort of human pancreatic cancer cases, more abundant stromal senescence as indicated by p16 immunohistochemistry correlated with decreased survival in patients with early-stage disease. These data support senescent fibroblasts as a pathologically and clinically relevant feature of pancreatic cancer. The inhibition of senescent stroma–cancer signaling pathways has the potential to restrain pancreatic cancer progression.Implications: Findings show that senescent cancer-associated fibroblasts secret excess IL8 to promote pancreatic cancer invasion and metastasis; thus, senescent CAFs represent a phenotypic subtype, challenging conventional assumptions that CAFs are a homogeneous population. Mol Cancer Res; 15(1); 3–14. ©2016 AACR.

Figure S1. MiaPaca2-YFP flow sort parameters. Figure S2. Immunofluorescence of primary CAF cell lines. Figure S3. Immunohistochemistry of Thy-1 and PDGFR-β on frozen pancreatic cancer tissues. Figure S4. Flow cytometry of CAF5 cells and cell cycle analysis. Figure S5. β-galactosidase staining of five CAF lines. Figure S6. Liver (A) and lung (E) metastasis from orthotopically implanted PDAC cells. Figure S7. Effect of IL8 on pancreatic cancer cell growth rates. Figure S8. Cell count and toxicity assays for SB225002. Figure S9. Effect of reparixin on transwell cancer migration. Figure S10. p16 and Ki67 immunohistochemistry in human PDAC. Figure S11. IHC of human PDAC showing low (A) vs. high (B) IL8 staining.

Supplementary Figure 1 shows the results of the Disambiguate algorithm used to assign the PDAC xenograft RNAseq reads to individual species

Supplementary Figure 9 shows the Cox proportional hazards model, integrating Moffitt transcriptomic subtype, tumour ploidy, stage and age

Supplementary Figure 5 shows the immunohistochemical analysis of apoptotic activity in xenografts treated with gemcitabine mono- and combination therapies

Supplementary Figure 7 shows the comparison of COSMIC SBS mutational signatures between untreated parent xenografts vs. xenografts treated with gemcitabine-cisplatin

Supplementary Figure 3 shows the histological analysis of the xenografts evaluated in the preclinical trial

Supplementary Figure 6 shows the tumour ploidy analysis of xenografts evaluated in the preclinical trial that were not whole genome sequenced

Supplementary Figure 2 shows the results of a pilot monotherapy trial to evaluate regimen-related toxicity

Supplementary Table 3 shows the establishment of primary cell cultures from HRD PDAC xenografts

Supplementary Table 1 shows the clinical characteristics of the patients from whom patient-derived xenografts were generated and evaluated in the preclinical trial

Supplementary Figure 8 shows the principal component analysis followed by k-means clustering to group xenografts based on their sensitivity to cisplatin, talazoparib and cisplatin-talazoparib

Purpose:Pancreatic ductal adenocarcinoma (PDAC) arising in patients with a germline BRCA1 or BRCA2 (gBRCA) mutation may be sensitive to platinum and PARP inhibitors (PARPi). However, treatment stratification based on gBRCA mutational status alone is associated with heterogeneous responses.Experimental Design:We performed a seven-arm preclinical trial consisting of 471 mice, representing 12 unique PDAC patient-derived xenografts, of which nine were gBRCA mutated. From 179 patients whose PDAC was whole-genome and transcriptome sequenced, we identified 21 cases with homologous recombination deficiency (HRD), and investigated prognostic biomarkers.Results:We found that biallelic inactivation of BRCA1/BRCA2 is associated with genomic hallmarks of HRD and required for cisplatin and talazoparib (PARPi) sensitivity. However, HRD genomic hallmarks persisted in xenografts despite the emergence of therapy resistance, indicating the presence of a genomic scar. We identified tumor polyploidy and a low Ki67 index as predictors of poor cisplatin and talazoparib response. In patients with HRD PDAC, tumor polyploidy and a basal-like transcriptomic subtype were independent predictors of shorter survival. To facilitate clinical assignment of transcriptomic subtype, we developed a novel pragmatic two-marker assay (GATA6:KRT17).Conclusions:In summary, we propose a predictive and prognostic model of gBRCA-mutated PDAC on the basis of HRD genomic hallmarks, Ki67 index, tumor ploidy, and transcriptomic subtype.

Supplementary Table 2 shows the clinical characteristics of 21 HRD PDAC patients whose tumours were whole genome and whole transcriptome sequenced

In resectable pancreatic ductal adenocarcinoma (PDAC), few pre-operative prognostic biomarkers are available. Radiomics has demonstrated potential but lacks external validation. We aimed to develop and externally validate a pre-operative clinical-radiomic prognostic model. Retrospective international, multi-center study in resectable PDAC. The training cohort included 352 patients (pre-operative CTs from five Canadian hospitals). Cox models incorporated (a) pre-operative clinical variables (clinical), (b) clinical plus CT-radiomics, and (c) post-operative TNM model, which served as the reference. Outcomes were overall (OS)/disease-free survival (DFS). Models were assessed in the validation cohort from Ireland (n = 215, CTs from 34 hospitals), using C-statistic, calibration, and decision curve analyses. The radiomic signature was predictive of OS/DFS in the validation cohort, with adjusted hazard ratios (HR) 2.87 (95% CI: 1.40–5.87, p

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.

We investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes (One hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing.The FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified. The mutation prevalence among patients without FC/AJ ancestry was 4.9% (95% CI, 2.6% to 8.8%). Mutations were more frequent in patients diagnosed at ≤ 50 years of age (Considering these results, we recommend reflex founder mutation testing of patients with FC/AJ ancestry and full gene sequencing of patients who are ≤ 50 years or meet the identified family history criteria. Reflex testing of all incident patients for these four genes may become justified as full gene sequencing costs decline.

Purpose:Pancreatic ductal adenocarcinoma (PDAC) arising in patients with a germline BRCA1 or BRCA2 (gBRCA) mutation may be sensitive to platinum and PARP inhibitors (PARPi). However, treatment stratification based on gBRCA mutational status alone is associated with heterogeneous responses.Experimental Design:We performed a seven-arm preclinical trial consisting of 471 mice, representing 12 unique PDAC patient-derived xenografts, of which nine were gBRCA mutated. From 179 patients whose PDAC was whole-genome and transcriptome sequenced, we identified 21 cases with homologous recombination deficiency (HRD), and investigated prognostic biomarkers.Results:We found that biallelic inactivation of BRCA1/BRCA2 is associated with genomic hallmarks of HRD and required for cisplatin and talazoparib (PARPi) sensitivity. However, HRD genomic hallmarks persisted in xenografts despite the emergence of therapy resistance, indicating the presence of a genomic scar. We identified tumor polyploidy and a low Ki67 index as predictors of poor cisplatin and talazoparib response. In patients with HRD PDAC, tumor polyploidy and a basal-like transcriptomic subtype were independent predictors of shorter survival. To facilitate clinical assignment of transcriptomic subtype, we developed a novel pragmatic two-marker assay (GATA6:KRT17).Conclusions:In summary, we propose a predictive and prognostic model of gBRCA-mutated PDAC on the basis of HRD genomic hallmarks, Ki67 index, tumor ploidy, and transcriptomic subtype.

ObjectiveTo describe the clinical, pathological and genomic characteristics of pancreatic cancer with DNA mismatch repair deficiency (MMRD) and proficiency (MMRP).DesignWe identified patients with MMRD and MMRP pancreatic cancer in a clinical cohort (N=1213, 519 with genetic testing, 53 with immunohistochemistry (IHC)) and a genomic cohort (N=288 with whole-genome sequencing (WGS)).Results12 out of 1213 (1.0%) in the clinical cohort were MMRD by IHC or WGS. Of the 14 patients with Lynch syndrome, 3 (21.4%) had an MMRP pancreatic cancer by IHC, and 4 (28.6%) were excluded because tissue was unavailable for testing. MMRD cancers had longer overall survival after surgery (weighted HR after coarsened exact matching 0.11, 95% CI 0.02 to 0.78, p=0.001). One patient with an unresectable MMRD cancer has an ongoing partial response 3 years after starting treatment with PD-L1/CTLA-4 inhibition. This tumour showed none of the classical histopathological features of MMRD. 9 out of 288 (3.1%) tumours with WGS were MMRD. Despite markedly higher tumour mutational burden and neoantigen loads, MMRD cancers were significantly less likely to have mutations in usual pancreatic cancer driver genes like KRAS and SMAD4, but more likely to have mutations in genes that drive cancers with microsatellite instability like ACV2RA and JAK1. MMRD tumours were significantly more likely to have a basal-like transcriptional programme and elevated transcriptional markers of immunogenicity.ConclusionsMMRD pancreatic cancers have distinct clinical, pathological and genomic profiles. Patients with MMRD pancreatic cancer should be considered for basket trials targeting enhanced immunogenicity or the unique genomic drivers in these malignancies.

Background There is inconsistent evidence on the association between physical activity and pancreatic cancer risk and few studies have investigated early life or life-course physical activity. The objective of this study was to evaluate the association between trajectories of physical activity across the life-course and pancreatic cancer risk. Methods A population-based case-control study was conducted (2011–2013) using cases (n = 315) from the Ontario Pancreas Cancer Study and controls (n = 1254) from the Ontario Cancer Risk Factor Study. Self-reported recall of moderate and vigorous physical activity was measured at three time points: young adulthood (20s–30s), mid-adulthood (40s–50s) and older-adulthood (1 year prior to questionnaire completion). Physical activity trajectories were identified using latent class analysis. Odds ratios (OR) and 95% confidence intervals (CI) were estimated from multivariable logistic regression adjusted for covariates: age, sex, race, alcohol, smoking, vegetable, fruit and meat consumption, and family history of pancreatic cancer. Results Six life-course physical activity trajectories were identified: inactive at all ages (41.2%), low activity at all ages (31.9%), increasingly active (3.6%), high activity in young adulthood with substantial decrease (13.0%), high activity in young adulthood with slight decrease (5.0%), and persistent high activity (5.3%). Compared to the inactive at all ages trajectory, the associations between each trajectory and pancreatic cancer after confounder adjustment were: low activity at all ages (OR: 1.11; 95% CI: 0.75, 1.66), increasingly active (OR: 1.11; 95% CI: 0.56, 2.21), high activity in young adulthood with substantial decrease in older adulthood (OR: 0.76; 95% CI: 0.47, 1.23), high activity in young adulthood with slight decrease in older adulthood (OR: 0.98; 95% CI: 0.62, 1.53), and persistently high activity (OR: 1.50; 95% CI: 0.86, 2.62). When time periods were evaluated separately, the OR for the association between high moderate activity in the 20s–30s and pancreatic cancer was 0.89 (95% CI: 0.64, 1.25) and some sex differences were observed. Conclusion Distinct life-course physical activity trajectories were identified, but there was no evidence that any of the trajectories were associated with pancreatic cancer. Future studies with larger sample sizes are needed to understand the associations between physical activity trajectories over the life-course and pancreatic cancer risk.

IMPORTANCE: Pathogenic germline variants in the ATM gene have been associated with pancreatic cancer risk. Although genetic testing identifies these variants in approximately 1% to 3% of unselected patients with pancreatic cancer, the lifetime risk of pancreatic cancer among individuals with pathogenic ATM variants has not been well estimated. OBJECTIVE: To estimate age-specific penetrance of pancreatic cancer in individuals with a pathogenic variant in the ATM gene. DESIGN, SETTING, AND PARTICIPANTS: This was a multicenter cohort study of pancreatic cancer family registries in the US and Canada using pedigree data from 130 pancreatic cancer kindreds with a pathogenic germline ATM variant. Data analyses were performed from January 2020 to February 2021. MAIN OUTCOMES AND MEASURES: Observational age-specific risk of pancreatic cancer. Penetrance was estimated using modified segregation analysis. RESULTS: The study population of 130 families (123 [95%] White families) comprised 2227 family members (mean age [SD], 58 [22] years; 1096 [49%] women) with complete records (ie, including familial relationships, pancreatic cancer diagnosis, ATM status, proband status, and age), of which 155 individuals had positive results for an ATM pathogenic variant, 16 had a negative result, and the remainder did not have a test result. In these 130 families, 217 individuals had pancreatic cancer: 78 families had 1 such member; 34 families had 2 such members; and 18 families had 3 or more members with pancreatic cancer. The average (range) age at diagnosis was 64 (31-98) years. The cumulative risk of pancreatic cancer among individuals with a germline pathogenic ATM variant was estimated to be 1.1% (95% CI, 0.8%-1.3%) by age 50 years; 6.3% (95% CI, 3.9%-8.7%) by age 70 years; and 9.5% (95% CI, 5.0%-14.0%) by age 80 years. Overall, the relative risk of pancreatic cancer was 6.5 (95% CI, 4.5-9.5) in ATM variant carriers compared with noncarriers. CONCLUSIONS AND RELEVANCE: This multicenter cohort study found that individuals with a germline pathogenic ATM variant were at an increased lifetime risk of pancreatic cancer. These risk estimates can help guide decision-making when evaluating the risks and benefits of enhanced early detection surveillance.

In resectable pancreatic ductal adenocarcinoma (PDAC), few pre-operative prognostic biomarkers are available. Radiomics has demonstrated potential but lacks external validation. We aimed to develop and externally validate a pre-operative clinical-radiomic prognostic model.Retrospective international, multi-center study in resectable PDAC. The training cohort included 352 patients (pre-operative CTs from five Canadian hospitals). Cox models incorporated (a) pre-operative clinical variables (clinical), (b) clinical plus CT-radiomics, and (c) post-operative TNM model, which served as the reference. Outcomes were overall (OS)/disease-free survival (DFS). Models were assessed in the validation cohort from Ireland (n = 215, CTs from 34 hospitals), using C-statistic, calibration, and decision curve analyses.The radiomic signature was predictive of OS/DFS in the validation cohort, with adjusted hazard ratios (HR) 2.87 (95% CI: 1.40-5.87, p 0.001)/5.28 (95% CI 2.35-11.86, p 0.001), respectively, along with age 1.02 (1.01-1.04, p = 0.01)/1.02 (1.00-1.04, p = 0.03). In the validation cohort, median OS was 22.9/37 months (p = 0.0092) and DFS 14.2/29.8 (p = 0.0023) for high-/low-risk groups and calibration was moderate (mean absolute errors 7%/13% for OS at 3/5 years). The clinical-radiomic model discrimination (C = 0.545, 95%: 0.543-0.546) was higher than the clinical model alone (C = 0.497, 95% CI 0.496-0.499, p 0.001) or TNM (C = 0.525, 95% CI: 0.524-0.526, p 0.001). Despite superior net benefit compared to the clinical model, the clinical-radiomic model was not clinically useful for most threshold probabilities.A multi-institutional pre-operative clinical-radiomic model for resectable PDAC prognostication demonstrated superior net benefit compared to a clinical model but limited clinical utility at external validation. This reflects inherent limitations of radiomics for PDAC prognostication, when deployed in real-world settings.• At external validation, a pre-operative clinical-radiomics prognostic model for pancreatic ductal adenocarcinoma (PDAC) outperformed pre-operative clinical variables alone or pathological TNM staging. • Discrimination and clinical utility of the clinical-radiomic model for treatment decisions remained low, likely due to heterogeneity of CT acquisition parameters. • Despite small improvements, prognosis in PDAC using state-of-the-art radiomics methodology remains challenging, mostly owing to its low discriminative ability. Future research should focus on standardization of CT protocols and acquisition parameters.

Introduction Identifying families with an underlying inherited cancer predisposition is a major goal of cancer prevention efforts. Mendelian risk models have been developed to better predict the risk associated with a pathogenic variant of developing breast/ovarian cancer (with BRCAPRO) and the risk of developing pancreatic cancer (PANCPRO). Given that pathogenic variants involving BRCA2 and BRCA1 predispose to all three of these cancers, we developed a joint risk model to capture shared susceptibility. Methods We expanded the existing framework for PANCPRO and BRCAPRO to jointly model risk of pancreatic, breast, and ovarian cancer and validated this new model, BRCAPANCPRO on three data sets each reflecting the common target populations. Results BRCAPANCPRO outperformed the prior BRCAPRO and PANCPRO models and yielded good discrimination for differentiating BRCA1 and BRCA2 carriers from non-carriers (AUCs 0.79, 95% CI: 0.73-0.84 and 0.70, 95% CI: 0.60-0.80) in families seen in high-risk clinics and pancreatic cancer family registries, respectively. In addition, BRCAPANCPRO was reasonably well calibrated for predicting future risk of pancreatic cancer (observed-to-expected (O/E) ratio = 0.81 [0.69, 0.94]). Discussion The BRCAPANCPRO model provides improved risk assessment over our previous risk models, particularly for pedigrees with a co-occurrence of pancreatic cancer and breast and/or ovarian cancer.

Purpose Pancreatic cancer has the highest fatality rate of all cancers. Adulthood obesity is an established risk factor for pancreatic cancer; however, life-course obesity is not well understood. The aim of this study was to evaluate the association between body mass index (BMI) trajectories throughout the life-course and pancreatic cancer risk. Methods A population-based case–control study was conducted (2011–2013) in Ontario, Canada. Cases were recruited from the Ontario pancreas cancer study (n = 310) and controls from the Ontario cancer risk factor study (n = 1258). Questionnaires captured self-reported height and weight at four timepoints (adolescence, 20 s, 30–40 s, 50–60 s). BMI trajectories were identified using latent class growth mixture modeling. Odds ratios (OR) and 95% confidence intervals (CI) were estimated from multivariable logistic regression. Results Five BMI trajectories were identified: stable-normal weight (38.9%), progressively overweight (42.2%), persistent overweight (12.6%), progressive obesity (4.2%), and persistent obesity (2.1%). The persistent overweight (OR = 1.55; 95% CI 1.02, 2.39) and progressive obesity trajectories (OR = 1.49; 95% CI 0.77, 2.87) compared to stable-normal weight were associated with increased odds of pancreatic cancer. When BMI was evaluated separately the strongest associations with pancreatic cancer emerged in young and mid-adulthood. Conclusion BMI trajectories characterized by overweight in early adulthood were associated with increased pancreatic cancer risk suggesting a life-course approach to disease risk. Electronic supplementary material The online version of this article (10.1007/s10552-019-01197-9) contains supplementary material, which is available to authorized users.

Background Stage 3 pancreatic ductal adenocarcinoma (PDAC) is defined by arterial involvement. This study objective was to evaluate outcomes for patients with stage 3 PDAC with potentially reconstructable arterial involvement, considered for neoadjuvant therapy (NAT) and pancreatic resection, and to compare outcomes following arterial (AR) and non-arterial resection (NAR). Methods This study included patients from 2009 to 2016 with biopsy-proven stage 3 PDAC who were offered NAT before surgical exploration. AR was performed if required to achieve R0 resection. Time to event outcomes were analysed from diagnosis date. Results 87/89 patients (97.8%) received NAT (chemotherapy 41.6%, chemotherapy/radiotherapy 56.2%). 46/89 (51.7%) underwent exploration; 31 underwent resection (AR n = 20, NAR n = 11). AR patients had longer operative time (681 vs. 563 min, p = 0.006) and more blood loss (1600 vs. 575 mL, p = 0.0004), with no difference for blood transfusion, pancreatic fistula, length of stay, reoperation, or mortality. R0 rate was 30/31. Post-resection 90-day mortality was 3.2%. Median overall survival was statistically comparable between the AR and NAR groups (19.7 vs. 28.4 months, p = 0.41). Conclusions AR had comparable clinical and oncologic outcomes to NAR. Following careful selection and non-progression after NAT, major AR may cautiously be considered if required to obtain a negative resection margin.

Background: Individuals with a family history of pancreatic adenocarcinoma (PC) or with a germline mutation in a PC susceptibility gene are at increased risk of developing PC. These high-risk individuals (HRIs) may benefit from PC surveillance. Methods: A PC surveillance program was developed to evaluate the detection of premalignant lesions and early-stage PCs using biannual imaging and to determine whether locally advanced or metastatic PCs develop despite biannual surveillance. From January 2013 to April 2020, asymptomatic HRIs were enrolled and followed with alternating MRI and endoscopic ultrasound every 6 months. Results: Of 75 HRIs, 43 (57.3%) had a germline mutation in a PC susceptibility gene and 32 (42.7%) had a familial pancreatic cancer (FPC) pedigree. Branch-duct intraductal papillary mucinous neoplasms (BD-IPMNs) were identified in 26 individuals (34.7%), but only 2 developed progressive lesions. One patient with Peutz-Jeghers syndrome (PJS) developed locally advanced PC arising from a BD-IPMN. Whole-genome sequencing of this patient’s PC and of a second patient with PJS-associated PC from the same kindred revealed biallelic inactivation of STK11 in a KRAS-independent manner. A review of 3,853 patients from 2 PC registries identified an additional patient with PJS-associated PC. All 3 patients with PJS developed advanced PC consistent with the malignant transformation of an underlying BD-IPMN in KRAS mutations. Conclusions: PC surveillance identifies a high prevalence of BD-IPMNs in HRIs. Patients with PJS with BD-IPMNs may be at risk for accelerated malignant transformation.

Background: Genome-wide association studies (GWAS) identify associations of individual single-nucleotide polymorphisms (SNPs) with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants is a powerful tool to identify networks of susceptibility genes. Methods: We conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product method to identify gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDAC) in 9040 cases and 12 496 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes contributing to the top associated pathways and gene sets. All statistical tests were two-sided. Results: We identified 14 pathways and gene sets associated with PDAC at a false discovery rate of less than 0.05. After Bonferroni correction (P

Purpose We investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes ( BRCA1, BRCA2, PALB2, and ATM) in consecutive patients with pancreatic adenocarcinoma. Methods One hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing. Results The FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified. The mutation prevalence among patients without FC/AJ ancestry was 4.9% (95% CI, 2.6% to 8.8%). Mutations were more frequent in patients diagnosed at ≤ 50 years of age ( P = .03) and in patients with either two or more first- or second-degree relatives with pancreas, breast, ovarian or prostate cancer, or one such relative and a second primary of one of these cancer types ( P < .001). BRCA1, BRCA2, and PALB2 carriers with late-stage (III or IV) disease had an overall survival advantage ( P = .049), particularly if treated with platinum-based chemotherapies ( P = .030). Conclusion Considering these results, we recommend reflex founder mutation testing of patients with FC/AJ ancestry and full gene sequencing of patients who are ≤ 50 years or meet the identified family history criteria. Reflex testing of all incident patients for these four genes may become justified as full gene sequencing costs decline.

Background and Aims Homologous recombination deficiency (HRD) in pancreatic ductal adenocarcinoma (PDAC), remains poorly defined beyond germline (g) alterations in BRCA1, BRCA2, and PALB2. Methods We interrogated whole genome sequencing (WGS) data on 391 patients, including 49 carriers of pathogenic variants (PVs) in gBRCA and PALB2. HRD classifiers were applied to the dataset and included (1) the genomic instability score (GIS) used by Myriad's MyChoice HRD assay; (2) substitution base signature 3 (SBS3); (3) HRDetect; and (4) structural variant (SV) burden. Clinical outcomes and responses to chemotherapy were correlated with HRD status. Results Biallelic tumor inactivation of gBRCA or PALB2 was evident in 43 of 49 germline carriers identifying HRD-PDAC. HRDetect (score ≥0.7) predicted gBRCA1/PALB2 deficiency with highest sensitivity (98%) and specificity (100%). HRD genomic tumor classifiers suggested that 7% to 10% of PDACs that do not harbor gBRCA/PALB2 have features of HRD. Of the somatic HRDetecthi cases, 69% were attributed to alterations in BRCA1/2, PALB2, RAD51C/D, and XRCC2, and a tandem duplicator phenotype. TP53 loss was more common in BRCA1- compared with BRCA2-associated HRD-PDAC. HRD status was not prognostic in resected PDAC; however in advanced disease the GIS (P = .02), SBS3 (P = .03), and HRDetect score (P = .005) were predictive of platinum response and superior survival. PVs in gATM (n = 6) or gCHEK2 (n = 2) did not result in HRD-PDAC by any of the classifiers. In 4 patients, BRCA2 reversion mutations associated with platinum resistance. Conclusions Germline and parallel somatic profiling of PDAC outperforms germline testing alone in identifying HRD-PDAC. An additional 7% to 10% of patients without gBRCA/PALB2 mutations may benefit from DNA damage response agents.

Background Although 20 pancreatic cancer susceptibility loci have been identified through genome-wide association studies in individuals of European ancestry, much of its heritability remains unexplained and the genes responsible largely unknown. Methods To discover novel pancreatic cancer risk loci and possible causal genes, we performed a pancreatic cancer transcriptome-wide association study in Europeans using three approaches: FUSION, MetaXcan, and Summary-MulTiXcan. We integrated genome-wide association studies summary statistics from 9040 pancreatic cancer cases and 12 496 controls, with gene expression prediction models built using transcriptome data from histologically normal pancreatic tissue samples (NCI Laboratory of Translational Genomics [n = 95] and Genotype-Tissue Expression v7 [n = 174] datasets) and data from 48 different tissues (Genotype-Tissue Expression v7, n = 74–421 samples). Results We identified 25 genes whose genetically predicted expression was statistically significantly associated with pancreatic cancer risk (false discovery rate < .05), including 14 candidate genes at 11 novel loci (1p36.12: CELA3B; 9q31.1: SMC2, SMC2-AS1; 10q23.31: RP11-80H5.9; 12q13.13: SMUG1; 14q32.33: BTBD6; 15q23: HEXA; 15q26.1: RCCD1; 17q12: PNMT, CDK12, PGAP3; 17q22: SUPT4H1; 18q11.22: RP11-888D10.3; and 19p13.11: PGPEP1) and 11 at six known risk loci (5p15.33: TERT, CLPTM1L, ZDHHC11B; 7p14.1: INHBA; 9q34.2: ABO; 13q12.2: PDX1; 13q22.1: KLF5; and 16q23.1: WDR59, CFDP1, BCAR1, TMEM170A). The association for 12 of these genes (CELA3B, SMC2, and PNMT at novel risk loci and TERT, CLPTM1L, INHBA, ABO, PDX1, KLF5, WDR59, CFDP1, and BCAR1 at known loci) remained statistically significant after Bonferroni correction. Conclusions By integrating gene expression and genotype data, we identified novel pancreatic cancer risk loci and candidate functional genes that warrant further investigation.

Introduction: Identifying families with an underlying inherited cancer predisposition is a major goal of cancer prevention efforts. Mendelian risk models have been developed to better predict the risk associated with a pathogenic variant of developing breast/ovarian cancer (with BRCAPRO), and the risk of developing pancreatic cancer (PANCPRO). Given that pathogenic variants involving BRCA2 and BRCA1 predispose to all three of these cancers, we developed a joint risk model to capture shared susceptibility. Methods: We expanded the existing framework for PANCPRO and BRCAPRO to model underlying risk of pancreatic, breast, and ovarian cancer. We then validated this model on three datasets each reflecting the target populations for these models, including patients referred to a high-risk genetics clinic, patients with multiple family members with pancreatic cancer who underwent genetic testing for BRCA1/2, as well as a prospective registry of pancreatic cancer families. The new model is designated BRCAPANCPRO. Results: BRCAPANCPRO yielded good discrimination for differentiating BRCA1 and BRCA2 carriers from non-carriers (AUC = 0.84, 95% CI: 0.78, 0.87) and was reasonably well-calibrated for predicting future risk of pancreatic cancer (observed-to-expected (O/E) ratio = 0.82 [0.69, 0.94]). In these families, BRCAPANCPRO outperformed both the prior BRCAPRO and PANCPRO models. Discussion: The BRCAPANCPRO model improves the identification of families with a pathogenic variant in BRCA2 or BRCA1, particularly for families where there is the co-occurrence of pancreatic cancer in addition to breast and/or ovarian cancer. The model also improves the prediction of pancreatic cancer among families with a pancreatic cancer family history, with or without breast and/or ovarian cancer. Citation Format: Amanda L. Blackford, Erica J. Childs, Nancy Porter, Gloria Petersen, Kari Rabe, Steven Gallinger, Ayelet Borgida, Sapna Syngal, Ann Schwartz, Michele Cote, Ralph Hruban, Michael Goggins, Alison P. Klein, Giovanni Parmigiani. A risk prediction tool for individuals with a family history of breast, ovarian, or pancreatic cancer: BRCAPANCPRO [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 885.

Background Biliary tract malignancies, in particular cholangiocarcinomas (CCA), are rare tumors that carry a poor prognosis. BRCA2 mutation carriers have an increased risk of developing CCA with a reported relative risk of ∼5 according to the Breast Cancer Linkage Consortium. In addition to this risk, there are potential therapeutic implications in those harboring somatic and/or germline (GL) BRCA mutations. Therefore, it is important to define the clinical characteristics of GL/somatic BRCA1/2 variants in CCA patients. Materials and methods We performed a multicenter retrospective analysis of CCA patients diagnosed between January 2000 and December 2013 with GL or somatic variants in BRCA1/2 genes detected by GL mutations testing and/or by tumor next generation sequencing. Cases were identified from clinical databases at participating institutions. Data including demographics, clinical history, surgical procedures, and systemic chemotherapy or radiation were extracted from patients' records. Results Overall, 18 cases were identified: 5 carriers of GL BRCA1/2 mutations (4 BRCA2; 1 BRCA1) and 13 harboring somatic variations (7 BRCA1; 6 BRCA2). Mean age at diagnosis was 60, SD ± 10 years (range 36-75 years), with male and female prevalence rates of 61.2% and 38.8%, respectively. Stage at diagnosis was I (n = 4), II (n = 3), III (n = 3), and IV (n = 8). Six patients had extrahepatic CCA and the rest intrahepatic CCA. Thirteen patients received platinum-based therapy and four were treated with poly ADP ribose polymerase inhibitors, of whom one experienced sustained disease response with a progression-free survival of 42.6 months. Median overall survival from diagnosis for patients with stage I/II in this study was 40.3 months (95% confidence interval [CI], 6.73-108.15) and with stages III/IV was 25 months (95% CI, 15.23-40.57). Conclusion BRCA-associated CCA is uncommon. This multicenter retrospective study provides a thorough clinical analysis of a BRCA-associated CCA cohort, which can serve as a benchmark for future development and design of expanded analyses and clinical trials. Implications for practice BRCA-associated CCA is uncommon but a very important subtype of hepatic malignancies, due to its rising prevalence. Better clinical characterization of this subtype might allow application of targeted therapy for CCA patients with germline or somatic mutations in BRCA1/2 genes, especially due to previously reported success of such therapies in other BRCA-associated malignancies. Thus this study, first of its kind, provides a basis for future multi-centered analyses in larger cohorts, as well as clinical trials. Additionally, this study emphasizes the importance of both germline and somatic genotyping for all CCA patients.

Background: BRCA1/BRCA2 germ line (GL) mutation carriers with pancreatic adenocarcinoma (PDAC) may have distinct outcomes. We recently described an apparent more favourable prognosis of surgically resected BRCA-associated PDAC patients in a single-arm, uncontrolled, retrospective study. However, the prognostic impact of GL BRCA1/2 mutations in surgically resected PDAC has not been compared with a matched control population. Methods: A larger multi-centre, case–control retrospective analysis was performed. Cases were patients with surgically resected, BRCA1/2-associated PDAC from 2004 to 2013. Controls included surgically resected PDAC cases treated during the same time period that were either BRCA non-carriers, or had no family history of breast, ovarian or pancreatic cancers. Cases and controls were matched by: age at diagnosis (within ±5-year period) and institution. Demographics, clinical history, overall survival (OS) and disease-free survival (DFS) were abstracted from patient records. Statistical comparisons were assessed using χ2- and Fisher's exact test, and median DFS/OS using Kaplan–Meier method and log-rank testing. Results: Twenty-five patients with BRCA1-(n=4) or BRCA2 (N=21)-associated resectable PDAC were identified. Mean age was 55.7 years (range, 34–78 years), 48% (n=12) were females and 76% (n=19) were Jewish. Cases were compared (1 : 2) with 49 resectable PDAC controls, and were balanced for age, ethnicity and other relevant clinical and pathological features. BRCA-associated PDAC patients received neoadjuvant, or adjuvant platinum-based treatment more frequently than controls (7 out of 8 vs 6 out of 14) and (7 out of 21 vs 3 out of 44), respectively. No significant difference in median OS (37.06 vs 38.77 months, P=0.838) and in DFS (14.3 vs 12.0 months, P=0.303) could be demonstrated between cases and controls. A trend to increased DFS was observed among BRCA-positive cases treated with neoadjuvant/adjuvant platinum-containing regimens (n=10) compared with similarly treated controls (n=7) (39.1 vs 12.4 months, P=0.255). Conclusions: In this retrospective analysis, the prognosis of surgically resectable BRCA-associated PDAC is no different than that of sporadic PDAC from the same institution. The role of platinum-based adjuvant therapy in this setting requires prospective investigation.

BackgroundWith a dismal 8% median 5-year overall survival (OS), pancreatic ductal adenocarcinoma (PDAC) is highly lethal. Only 10-20% of patients are eligible for surgery, and over 50% of these will die within a year of surgery. Identify molecular predictors of early death would enable the selection of PDAC patients at high risk.MethodsWe developed the Pancreatic Cancer Overall Survival Predictor (PCOSP), a prognostic model built from a unique set of 89 PDAC tumors where gene expression was profiled using both microarray and sequencing platforms. We used a meta-analysis framework based on the binary gene pair method to create gene expression barcodes robust to biases arising from heterogeneous profiling platforms and batch effects. Leveraging the largest compendium of PDAC transcriptomic datasets to date, we show that PCOSP is a robust single-sample predictor of early death (≤1 yr) after surgery in a subset of 823 samples with available transcriptomics and survival data.ResultsThe PCOSP model was strongly and significantly prognostic with a meta-estimate of the area under the receiver operating curve (AUROC) of 0.70 (P=1.9e-18) and hazard ratio (HR) of 1.95(1.6-2.3) (P=2.6e-16) for binary and survival predictions, respectively. The prognostic value of PCOSP was independent of clinicopathological parameters and molecular subtypes. Over-representation analysis of the PCOSP 2619 gene-pairs (1070 unique genes) unveiled pathways associated with Hedgehog signalling, epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) signalling.ConclusionPCOSP could improve treatment decision by identifying patients who will not benefit from standard surgery/chemotherapy and may benefit from alternate approaches.AbbreviationsAUROCArea under the receiver operating curveGOGene annotationOSOverall survivalPCOSPPancreatic cancer overall survival predictorPDACPancreatic ductal adenocarcinomaTSPTop scoring pairs.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that is clinically asymptomatic in its early stages of development. Non-invasive testing for pancreatic cancer biomarkers would significantly improve early detection and patient care. Extracellular vesicles (EVs) are circulating tumor fragments present in the blood and may express cancer specific biomarkers that would enable early detection of pancreatic cancer. We tested the utility of a blood test enumerating EVs positive for the pancreas-specific marker Glycoprotein 2 (GP2) and the putative pancreatic cancer marker Glypican-1 (GPC1) in patients with PDAC. Various levels of GPC1-positive and GP2/GPC1-positive EVs were detected in PDAC patients but were not significantly higher than benign pancreatic disease (BPD) patients. The sensitivity and specificity of the GPC1 EV test was 26.67% and 87.50% respectively, whereas the sensitivity and specificity for the GPC1+GP2 EV test was 23.33% and 90.00% respectively. Immunohistochemistry of GPC1 expression in a tissue microarray of PDAC and various controls also did not demonstrate specificity of GPC1 to PDAC. Hence, enumeration of GPC1-positive EVs, solely or in conjunction with GP2, was unable to effectively distinguish between BPD and pancreatic cancer.

The use of liquid biopsies for cancer detection and management is rapidly gaining prominence1. Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations2–5. By contrast, large-scale epigenetic alterations—which are tissue- and cancer-type specific—are not similarly constrained6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns. An immunoprecipitation-based protocol is developed to analyse DNA methylation in small quantities of circulating cell-free DNA, and can detect and classify cancers in plasma samples from several tumour types.

430 Background: Familial pancreatic cancer (FPC) is broadly defined as kindreds with at least a pair of first-degree relatives with pancreatic ductal adenocarcinoma (PDA). The role of DNA damage response agents, including platinum has not been well studied in this patient population. Methods: In this retrospective analysis, treatment details and clinical outcomes were analyzed in pts with FPC with advanced, unresectable or recurrent disease enrolled in the Ontario Pancreatic Cancer Study database. The primary outcome, overall survival (OS) was calculated from the initial diagnosis of advanced disease until death. 179 non-FPC patients from the COMPASS trial [NCT02750657] served as a control cohort all of whom had full molecular profiling and family history documented. OS between pts that received platinum-based therapy, and those that did not was compared using multivariable Cox proportional hazards model adjusting for age, sex, diagnosis year and FPC status. Interaction between FPC status and platinum was evaluated. Results: A total of 205 FPC pts were identified, 71% of pts had full germline testing and 16 (8%) had germline pathogenic variants in BRCA1/2. 104 (51%) were female and 101 (49%) male. Median age was 63 years (20-93) and 58 (28%) received platinum-based chemotherapy. Within the control arm (n=179), 71 (40%) were female, and 108 (60%) male; the median age was 64 years (29-84) and 106 (59%) received platinum-based therapy. In univariable analysis, median OS in pts with FPC was 16.9 months compared to 9.6 months (HR 0.46 [95% CI 0.37-0.58]). FPC patients receiving platinum had a superior median OS of 19 months compared to 15.5 months without platinum. In a multivariable analysis, both FPC (HR 0.33 [95% CI 0.21-0.51]) and receipt of platinum HR 0.53 [95% CI 0.38-0.73]) were prognostic. No interaction was seen with FPC and receipt of platinum (p=0.15). Conclusions: FPC status is prognostic but not predictive of platinum response in this study. Further molecular profiling of this unique cohort of patients will provide insights into putative predisposing germline alterations, and novel treatment strategies.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours. Funding sources for this study include grants to the Pancreatic Cancer Sequencing Initiative program from the Ontario Institute for Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation, the Canada Foundation for Innovation; research award to F.N. from the OICR and the Canadian Institutes for Health Research (CIHR); Canadian Friends of the Hebrew University, the SMGS Family Foundation, NCI grant P50 CA102701 (Mayo Clinic SPORE in Pancreatic Cancer) and NCI grant R01 CA97075 (Pancreatic Cancer Genetic Epidemiology Consortium). F.N. is supported by a fellowship award from CIHR and is a recipient of a scholar’s research award from the Ontario Institute of Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation. G.Z. is a Clinician–Scientist of the Fonds de la Recherche en Sante du Quebec. P.J.C. is a Wellcome Trust Senior Clinical Fellow. T.J.H., L.D.S., J.D.M. and S.G. are recipients of Senior or Clinician–Scientist Awards from the Ontario Institute for Cancer Research

Carcinoma-associated fibroblasts (CAF) represent a significant component of pancreatic cancer stroma and are biologically implicated in tumor progression. However, evidence of both cancer-promoting and -restraining properties amongst CAFs suggests the possibility of multiple phenotypic subtypes. Here, it is demonstrated that senescent CAFs promote pancreatic cancer invasion and metastasis compared with nonsenescent control CAFs using in vitro Transwell invasion models and in vivo xenograft mouse models. Screening by gene expression microarray and cytokine ELISA assays revealed IL8 to be upregulated in senescent CAFs. Experimental modulation through IL8 overexpression or receptor inhibition implicates the IL8 pathway as a mediator of the proinvasive effects of senescent CAFs. In a cohort of human pancreatic cancer cases, more abundant stromal senescence as indicated by p16 immunohistochemistry correlated with decreased survival in patients with early-stage disease. These data support senescent fibroblasts as a pathologically and clinically relevant feature of pancreatic cancer. The inhibition of senescent stroma–cancer signaling pathways has the potential to restrain pancreatic cancer progression. Implications: Findings show that senescent cancer-associated fibroblasts secret excess IL8 to promote pancreatic cancer invasion and metastasis; thus, senescent CAFs represent a phenotypic subtype, challenging conventional assumptions that CAFs are a homogeneous population. Mol Cancer Res; 15(1); 3–14. ©2016 AACR .