Supplementary Figure 1 from Pancreatic Cancers Epigenetically Silence SIP1 and Hypomethylate and Overexpress miR-200a/200b in Association with Elevated Circulating miR-200a and miR-200b Levels

Variants of unknown significance

PDF file, 186KB, Supplemental Figure 1. Copy number alterations in case 1 (PanIN-1). For A-D: The upper, middle and lower panels are the allele ratio, the copy number and the allele-specific ratio, respectively. Each red and blue dot in the lower panel represents allele and the dots are displayed on the panel to indicate the ratio of each allele. A) Lesion 6, PanIN-1, chromosome 1. B) Endocrine neoplasm, chromosome 11.

PDF file - 123KB, PCR and sequencing primer sequences.

Supplementary Figures 2-4 from Pancreatic Cancers Epigenetically Silence SIP1 and Hypomethylate and Overexpress miR-200a/200b in Association with Elevated Circulating miR-200a and miR-200b Levels

Supplementary Figures S1-S3 and Supplementary Tables S1-S2.

BACKGROUND AND AIMS: Serum diagnostic markers of early-stage pancreatic ductal adenocarcinoma (PDAC) are needed, especially for stage I disease. As tumors grow and cause pancreatic atrophy, markers derived from pancreatic parenchyma such as serum carboxypeptidase A (CPA) activity lose diagnostic performance. We evaluated, with CA19-9, serum CPA as a marker of early pancreatic cancer. METHODS: Serum CPA activity levels were measured in 345 controls undergoing pancreatic surveillance, divided into 2 sets, set 1 being used to establish a reference range. Variants within the CPA1 locus were sought for their association with pancreatic CPA1 expression to determine if such variants associated with serum CPA levels. A total of 190 patients with resectable PDAC were evaluated. RESULTS: Among controls, those having 1 or more minor alleles of CPA1 variants rs6955723 or rs2284682 had significantly higher serum CPA levels than did those without (P=.001). None of the PDAC cases with pancreatic atrophy had an elevated CPA. Among 122 PDAC cases without atrophy, defining serum CPA diagnostic cutoffs by a subject's CPA1 variants yielded a diagnostic sensitivity of 18% at 99% specificity (95% confidence interval [CI], 11.7-26) (vs 11.1% sensitivity using a uniform diagnostic cutoff); combining CPA with variant-stratified CA19-9 yielded a sensitivity of 68.0% (95% CI, 59.0-76.2) vs 63.1% (95% CI, 53.9- 71.7) for CA19-9 alone; and among stage I PDAC cases, diagnostic sensitivity was 51.9% (95% CI, 31.9-71.3) vs 37.0% (95% CI, 19.4-57.6) for CA19-9 alone. In the validation control set, the variant-stratified diagnostic cutoff yielded a specificity of 98.2%. CONCLUSION: Serum CPA activity has diagnostic utility before the emergence of pancreatic atrophy as a marker of localized PDAC, including stage I disease.

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 noncancer 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 = .0001). ER stress-inducing variants in the CPA1 gene were identified in 4 of 1385 PDAC cases vs 77 of 64 026 gnomAD controls (OR: 2.4 [0.88-6.58], P = .087), and variants in CPB1 were detected in 5 of 1385 cases vs 33 of 64 026 controls (OR: 7.02 [2.74-18.01], P = .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 < .023) and CPB1 (OR: 9.51 [3.46-26.15], P < .001). Rare variants in CPB1 and CPA1 that induce ER stress are associated with increased odds of developing pancreatic cancer.

BACKGROUND & AIMS: The tumor microbiome of patients with pancreas ductal adenocarcinoma (PDAC) includes bacteria normally present in the upper gastrointestinal tract. If the predominant source of intratumoral bacteria in patients with PDAC is retrograde migration from the duodenum, duodenal fluid could be a representative biospecimen for determining microbiome profiles of patients with PDAC or at risk of developing PDAC. METHODS: We performed a case-control study comparing bacterial and fungal (16S and 18S rRNA) profiles of secretin-stimulated duodenal fluid collections from 308 patients undergoing duodenal endoscopy including 134 normal pancreas control subjects, 98 patients with pancreatic cyst(s) and 74 patients with PDAC. RESULTS: Alterations in duodenal fluid microbiomes with diminished alpha diversity were significantly associated with age >70 and proton pump inhibitor use. Patients with PDAC had significantly decreased duodenal microbial alpha diversity compared with age-matched control subjects with normal pancreata and those with pancreatic cyst(s). There was evidence of enrichment of Bifidobacterium genera in the duodenal fluid of patients with PDAC compared with control subjects and those with pancreatic cyst(s). There were also enrichment of duodenal fluid Fusobacteria and Rothia bacteria among patients with PDAC with short-term survival. Duodenal fluid microbiome profiles were not significantly different between control subjects and patients with pancreatic cyst(s). CONCLUSION: Patients with PDAC have alterations in their duodenal fluid microbiome profiles compared with patients with pancreatic cysts and those with normal pancreata.

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.

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normal-range digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.

BACKGROUND & AIMS: Levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and cancer antigen 125 (CA-125) in blood are used as markers to determine the response of patients with cancer to therapy, but are not used to identify patients with pancreatic cancer. METHODS: We obtained blood samples from 504 patients undergoing pancreatic surveillance from 2002 through 2018 who did not develop pancreatic cancer and measured levels of the tumor markers CA19-9, CEA, CA-125, and thrombospondin-2. Single-nucleotide polymorphisms (SNPs) in FUT3, FUT2, ABO, and GAL3ST2 that have been associated with levels of tumor markers were used to establish SNP-defined ranges for each tumor marker. We also tested the association between additional SNPs (in FUT6, MUC16, B3GNT3, FAM3B, and THBS2) with levels of tumor markers. To calculate the diagnostic specificity of each SNP-defined range, we assigned the patients under surveillance into training and validation sets. After determining the SNP-defined ranges, we determined the sensitivity of SNP-adjusted tests for the tumor markers, measuring levels in blood samples from 245 patients who underwent resection for pancreatic ductal adenocarcinoma (PDAC) from 2010 through 2017. RESULTS: A level of CA19-9 that identified patients with PDAC with 99% specificity had 52.7% sensitivity. When we set the cut-off levels of CA19-9 based on each SNP, the test for CA19-9 identified patients with PDAC with 60.8% sensitivity and 98.8% specificity. Among patients with FUT3 alleles that encode a functional protein, levels of CA19-9 greater than the SNP-determined cutoff values identified 66.4% of patients with PDAC, with 99.3% specificity. In the validation set, levels of CEA varied among patients with vs without SNP in FUT2, by blood group, and among smokers vs nonsmokers; levels of CA-125 varied among patients with vs without the SNP in GAL3ST2. The use of the SNPs to define the ranges of CEA and CA-125 did not significantly increase the diagnostic accuracy of the assays for these proteins. Combining data on levels of CA19-9 and CEA, CA19-9 and CA-125, or CA19-9 and thrombospondin-2 increased the sensitivity of detection of PDAC, but slightly reduced specificity. CONCLUSIONS: Including information on SNPs associated with levels of CA19-9, CEA, and CA-125 can improve the diagnostic accuracy of assays for these tumor markers in the identification of patients with PDAC.

Background: Pancreatic cancer is the fourth-leading cause of cancer death in both men and women in the United States. The currently identified common susceptibility loci account for a small fraction of estimated heritability. We sought to estimate overall heritability of pancreatic cancer and partition the heritability by variant frequencies and functional annotations. Methods: Analysis using the genome-based restricted maximum likelihood method (GREML) was conducted on Pancreatic Cancer Case-Control Consortium (PanC4) genome-wide association study (GWAS) data from 3,568 pancreatic cancer cases and 3,363 controls of European Ancestry. Results: Applying linkage disequilibrium- and minor allele frequency-stratified GREML (GREML-LDMS) method to imputed GWAS data, we estimated the overall heritability of pancreatic cancer to be 21.2% (SE = 4.8%). Across the functional groups (intronic, intergenic, coding, and regulatory variants), intronic variants account for most of the estimated heritability (12.4%). Previously identified GWAS loci explained 4.1% of the total phenotypic variation of pancreatic cancer. Mutations in hereditary pancreatic cancer susceptibility genes are present in 4% to 10% of patients with pancreatic cancer, yet our GREML-LDMS results suggested these regions explain only 0.4% of total phenotypic variance for pancreatic cancer. Conclusions: Although higher than previous studies, our estimated 21.2% overall heritability may still be downwardly biased due to the inherent limitation that the contribution of rare variants in genes with a substantive overall impact on disease are not captured when applying these commonly used methods to imputed GWAS data. Impact: Our work demonstrated the importance of rare and common variants in pancreatic cancer risk.

PURPOSE To compare the risk of neoplastic progression by germline mutation status versus family history without a known germline mutation (familial risk) among individuals with an increased risk for pancreatic cancer who are undergoing surveillance. METHODS Of 464 high-risk individuals in the Cancer of the Pancreas Screening program at Johns Hopkins Hospital who were undergoing pancreatic surveillance, 119 had a known deleterious germline mutation in a pancreatic cancer susceptibility gene; 345 met family history criteria for pancreatic surveillance but were not known to harbor a germline mutation. We used next-generation sequencing to identify previously unrecognized germline mutations among these 345 individuals. We compared the development of pancreatic cancer, high-grade dysplasia, or clinically worrisome features, adjusting for competing mortality, among all germline mutation carriers with the risk of progression in a cohort without a known germline mutation. RESULTS Fifteen (4.3%) of 345 individuals classified as having familial risk had a previously unrecognized pancreatic cancer susceptibility gene mutation (nine that involved ATM, two BRCA2, one BRCA1, one PALB2, one TP53, and one CPA1). The cumulative incidence of pancreatic cancer, high-grade dysplasia, or worrisome features on pancreatic imaging was significantly higher in the germline mutation risk group (n = 134) than in the familial risk group (n = 330 [for pancreatic cancer, hazard ratio, 2.85; 95% CI, 1.0 to 8.18; P = .05]). CONCLUSION The cumulative incidence of pancreatic cancer is significantly higher among individuals with an identifiable deleterious germline mutation in a pancreatic cancer susceptibility gene than it is among individuals with a strong family history but no identified mutation. Gene testing of individuals who meet criteria for pancreatic surveillance on the basis of their family history may better define those most at risk for neoplastic progression.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

To evaluate whether germline variants in genes encoding pancreatic secretory enzymes contribute to pancreatic cancer susceptibility, we sequenced the coding regions of CPB1 and other genes encoding pancreatic secretory enzymes and known pancreatitis susceptibility genes (PRSS1, CPA1, CTRC, and SPINK1) in a hospital series of pancreatic cancer cases and controls. Variants in CPB1, CPA1 (encoding carboxypeptidase B1 and A1), and CTRC were evaluated in a second set of cases with familial pancreatic cancer and controls. More deleterious CPB1 variants, defined as having impaired protein secretion and induction of endoplasmic reticulum (ER) stress in transfected HEK 293T cells, were found in the hospital series of pancreatic cancer cases (5/986, 0.5%) than in controls (0/1,045, P = 0.027). Among familial pancreatic cancer cases, ER stress-inducing CPB1 variants were found in 4 of 593 (0.67%) vs. 0 of 967 additional controls (P = 0.020), with a combined prevalence in pancreatic cancer cases of 9/1,579 vs. 0/2,012 controls (P < 0.01). More ER stress-inducing CPA1 variants were also found in the combined set of hospital and familial cases with pancreatic cancer than in controls [7/1,546 vs. 1/2,012; P = 0.025; odds ratio, 9.36 (95% CI, 1.15-76.02)]. Overall, 16 (1%) of 1,579 pancreatic cancer cases had an ER stress-inducing CPA1 or CPB1 variant, compared with 1 of 2,068 controls (P < 0.00001). No other candidate genes had statistically significant differences in variant prevalence between cases and controls. Our study indicates ER stress-inducing variants in CPB1 and CPA1 are associated with pancreatic cancer susceptibility and implicate ER stress in pancreatic acinar cells in pancreatic cancer development.

Abstract The accurate diagnosis and monitoring of cancer, using circulating tumor DNA (ctDNA), is a major challenge, given the low concentration and complexity of the target molecules. Suitable reference materials are required for clinical laboratories to achieve the high levels of measurement assurance and reproducibility. The methods and instruments for ctDNA assays are rapidly evolving to improve the sensitivity and specificity, resulting in a challenge to compare these different assays for the measurement of the diverse classes of ctDNA cancer biomarkers. To address this goal, a new reference material was developed consisting of 40 cancer mutations in a background of wild-type DNA at six different allele fractions (2%, 1%, 0.5%, 0.25%, 0.125, and 0 %). The cancer mutations include single nucleotide variants, insertions, deletions, and two structural variations that were selected for their clinical importance and challenges for next generation sequencing (NGS) methods. The reference material is formulated in a synthetic plasma matrix at a concentration of 25 ng/mL, levels that are consistent with patient samples. The DNA has been processed to accurately simulate the size of patient ctDNA, resulting in a similar conversion rate of the DNA for next generation sequencing methods. The samples were sent to five different laboratories involved in ctDNA measurements. Digital PCR assays for the variants were developed to confirm the allele fractions by several laboratories. When absorbance, fluorescent dye binding, and PCR methods were used to quantify the extracted ctDNA reference material, the results were significantly different, indicating the need for standard methods and reference materials to ensure reproducible results of this critical step. Different NGS methods (including targeted amplicons, hybrid selection, and SiMSen-seq) and instruments were used by the labs to analyze the refence materials at the different allele fractions. The results showed that the assays had significant differences in the ability to detect classes of variants at different concentrations. This new reference material was shown to be useful to benchmark the sensitivity and selectivity of different NGS methods and bioinformatic pipelines. This multi-laboratory assessment clearly demonstrates that the new reference material is highly valuable tool to ensure the reproducibility of ctDNA measurements including the sample extraction, analytical, and bioinformatic steps. Citation Format: Erica Stein, Hua-Jun He, Kenneth D. Cole, Russell K. Garlick, Yves Konigshofer, Tony E. Godfrey, Michael G. Goggins, Michael Borges, Margaret Gulley, Mickey Williams, Chris Karlovich, Corinne Camalier, Lynn Sorbara, Matthew R. Young, Sudhir Srivastava. Multi-laboratory assessment of a new reference material for quality assurance of circulating tumor DNA measurements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3657.

In 2020, 146,063 deaths due to pancreatic cancer are estimated to occur in Europe and the United States combined. To identify common susceptibility alleles, we performed the largest pancreatic cancer GWAS to date, including 9040 patients and 12,496 controls of European ancestry from the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4). Here, we find significant evidence of a novel association at rs78417682 (7p12/ TNS3 , P = 4.35 × 10 −8 ). Replication of 10 promising signals in up to 2737 patients and 4752 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium yields new genome-wide significant loci: rs13303010 at 1p36.33 ( NOC2L , P = 8.36 × 10 −14 ), rs2941471 at 8q21.11 ( HNF4G , P = 6.60 × 10 −10 ), rs4795218 at 17q12 ( HNF1B , P = 1.32 × 10 −8 ), and rs1517037 at 18q21.32 ( GRP , P = 3.28 × 10 −8 ). rs78417682 is not statistically significantly associated with pancreatic cancer in PANDoRA. Expression quantitative trait locus analysis in three independent pancreatic data sets provides molecular support of NOC2L as a pancreatic cancer susceptibility gene.

Objective Secretin-stimulated pancreatic juice is collected from the duodenum and analyzed to identify biomarkers of pancreatic neoplasia, but the optimal duration of pancreatic juice collection is not known. Methods We compared the yield of KRAS mutations detected in pancreatic juice samples aspirated from near the duodenal papilla at 1 to 5, 6 to 10, and 11 to 15 minutes after secretin infusion, and from the third part of the duodenum (at 15 minutes) from 45 patients undergoing endoscopic ultrasound pancreatic surveillance. KRAS mutation concentrations were measured by using droplet digital polymerase chain reaction. Results Forty of 45 patients had KRAS mutations detected in their pancreatic juice, and most patients' juice samples had more than 1 KRAS mutation. Of 106 KRAS mutations detected in 171 pancreatic juice samples, 58 were detected in the 5-minute samples, 70 mutations were detected in the 10-minute samples, and 65 were detected in the 15-minute samples. Nine patients who did not have KRAS mutations detected in their 5-minute sample had mutations detected in samples collected at later time points. Ninety-percent of all pancreatic juice mutations detected in any sample were detected in the 5- or 10-minute samples. Conclusions Collecting pancreatic juice for 10 minutes after secretin infusion increases the likelihood of detecting pancreatic juice mutations over shorter collections.

Abstract Purpose: The measurement of mutations in pancreatic juice samples collected from the duodenum during endoscopic ultrasound (EUS) may improve the diagnostic evaluation of patients undergoing pancreatic surveillance. Our aim was to evaluate the accuracy of using pancreatic juice mutation concentrations to predict the presence and histologic grade of neoplasia in the pancreas. Experimental Design: Digital next-generation sequencing (NGS) of pancreatic juice DNA using a targeted 12-gene panel was performed on 67 patients undergoing pancreatic evaluation during EUS, including patients with pancreatic ductal adenocarcinoma, patients who subsequently underwent pancreatic resection for precursor lesions, patients undergoing surveillance for their familial/inherited susceptibility to pancreatic cancer, and normal pancreas disease controls. Results: Patients with pancreatic cancer or high-grade dysplasia as their highest grade lesion had significantly higher pancreatic juice mutation concentrations than all other subjects (mean/SD digital NGS score; 46.6 ± 69.7 vs. 6.2 ± 11.6, P = 0.02). Pancreatic juice mutation concentrations distinguished patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 72.2% sensitivity and 89.4% specificity [area under the curve (AUC) = 0.872]. Mutant TP53/SMAD4 concentrations could distinguish patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 61.1% sensitivity and 95.7% specificity (AUC = 0.819). Among 31 high-risk individuals under surveillance, 2 of the 3 individuals with most abnormal pancreatic juice mutation profiles also had the most abnormalities on pancreatic imaging. Conclusions: Pancreatic juice mutation analysis using digital NGS has potential diagnostic utility in the evaluation of patients undergoing pancreatic surveillance. Clin Cancer Res; 24(12); 2963–74. ©2018 AACR. See related commentary by Lipner and Yeh, p. 2713