Bamlet, William R.

Abstract Purpose: We have previously identified tissue methylated DNA markers (MDMs) associated with pancreatic ductal adenocarcinoma (PDAC). In this case–control study, we aimed to assess the diagnostic performance of plasma MDMs for PDAC. Experimental Design: Thirteen MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, GH05J042948, HOXA1, PRKCB, SHISA9, and NTRK3) were identified on the basis of selection criteria applied to results of prior tissue experiments and assays were optimized in plasma. Next, 340 plasma samples (170 PDAC cases and 170 controls) were assayed using target enrichment long-probe quantitative amplified signal method. Initially, 120 advanced-stage PDAC cases and 120 healthy controls were used to train a prediction algorithm at 97.5% specificity using random forest modeling. Subsequently, the locked algorithm derived from the training set was applied to an independent blinded test set of 50 early-stage PDAC cases and 50 controls. Finally, data from all 340 patients were combined, and cross-validated. Results: The cross-validated area under the receiver operating characteristic curve (AUC) for the training set was 0.93 (0.89–0.96) for the MDM panel alone, 0.91 (95% confidence interval, 0.87–0.96) for carbohydrate antigen 19-9 (CA19-9) alone, and 0.99 (0.98–1) for the combined MDM-CA19-9 panel. In the test set of early-stage PDAC, the AUC for MDMs alone was 0.84 (0.76–0.92), CA19-9 alone was 0.87 (0.79–0.94), and combined MDM-CA19-9 panel was 0.90 (0.84–0.97) significantly better compared with either MDMs alone or CA19-9 alone (P = 0.0382 and 0.0490, respectively). At a preset specificity of 97.5%, the sensitivity for the combined panel in the test set was 80% (28%–99%) for stage I disease and 82% (68%–92%) for stage II disease. Using the combined datasets, the cross-validated AUC was 0.9 (0.86–0.94) for the MDM panel alone and 0.89 for CA19-9 alone (0.84–0.93) versus 0.97 (0.94–0.99) for the combined MDM-CA19-9 panel (P ≤ 0.0001). Overall, cross-validated sensitivity of MDM-CA19-9 panel was 92% (83%–98%), with an observed specificity of 92% at the preset specificity of 97.5%. Conclusions: Plasma MDMs in combination with CA19-9 detect PDAC with significantly higher accuracy compared with either biomarker individually.

Background: Hypoplastic left heart syndrome (HLHS) with risk of poor outcome has been linked to MYH6 variants, implicating overlap in genetic etiologies of structural and myopathic heart disease. Methods: Whole genome sequencing was performed in 197 probands with HLHS, 43 family members, and 813 controls. Data were filtered for rare, segregating variants in 3 index families comprised of an HLHS proband and relative(s) with cardiomyopathy. Whole genome sequencing data from cases and controls were compared for rare variant burden across 56 cardiomyopathy genes utilizing a weighted burden test approach, accounting for multiple testing using a Bonferroni correction. Results: A pathogenic MYBPC3 nonsense variant was identified in the first proband who underwent cardiac transplantation for diastolic heart failure, her father with left ventricular noncompaction, and 2 fourth-degree relatives with hypertrophic cardiomyopathy. A likely pathogenic RYR2 missense variant was identified in the second proband, a second-degree relative with aortic dilation, and a fourth-degree relative with dilated cardiomyopathy. A pathogenic RYR2 exon 3 in-frame deletion was identified in the third proband diagnosed with catecholaminergic polymorphic ventricular tachycardia and his father with left ventricular noncompaction and catecholaminergic polymorphic ventricular tachycardia. To further investigate HLHS-cardiomyopathy gene associations in cases versus controls, rare variant burden testing of 56 genes revealed enrichment in MYH6 ( P =0.000068). Rare, predicted-damaging MYH6 variants were identified in 10% of probands in our cohort—4 with familial congenital heart disease, 4 with compound heterozygosity (3 with systolic ventricular dysfunction), and 4 with MYH6 – FLNC synergistic heterozygosity. Conclusions: Whole genome sequencing in multiplex families, proband-parent trios, and case-control cohorts revealed defects in cardiomyopathy-associated genes in patients with HLHS, which may portend impaired functional reserve of the single-ventricle circulation.

Abstract Background: Critically shortened telomeres contribute to chromosomal instability and neoplastic transformation and are associated with early death of patients with certain cancer types. Shorter leukocyte telomere length (LTL) has been associated with higher risk for pancreatic ductal adenocarcinoma (PDAC) and might be associated also with survival of patients with PDAC. We investigated the association between treatment-naïve LTL and overall survival of patients with incident PDAC. Methods: The study included 642 consecutively enrolled PDAC patients in the Mayo Clinic Biospecimen Resource for Pancreas Research. Blood samples were obtained at the time of diagnosis, before the start of cancer treatment, from which LTL was assayed by qRT-PCR. LTL was first modeled as a continuous variable (per-interquartile range decrease in LTL) and then as a categorized variable (short, medium, long). Multivariable-adjusted HRs and 95% confidence intervals (CI) were calculated for overall mortality using Cox proportional hazard models. Results: Shorter treatment-naïve LTL was associated with higher mortality among patients with PDAC (HRcontinuous = 1.13, 95% CI: 1.01–1.28, P = 0.03; HRshortest vs. longest LTL = 1.29, 95% CI: 1.05–1.59, Ptrend = 0.01). There was a difference in the association between LTL and overall mortality by tumor stage at diagnosis; resectable tumors (HRcontinuous = 0.91; 95% CI: 0.73–1.12), locally advanced tumors (HRcontinuous = 1.29; 95% CI: 1.07–1.56), and metastatic tumors (HRcontinuous = 1.17; 95% CI: 0.96–1.42), Pinteraction = 0.04. Conclusion: Shorter treatment-naïve LTL is associated with poorer overall survival of patients with incident PDAC. Impact: Peripheral blood LTL might be a prognostic marker for PDAC.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed too late for effective therapy. The classic strategy for early detection biomarker advancement consists of initial retrospective phases of discovery and validation with tissue samples taken from individuals diagnosed with disease, compared with controls. Using this approach, we previously reported the discovery of a blood biomarker panel consisting of thrombospondin-2 (THBS2) and CA19-9 that together could discriminate resectable stage I and IIa PDAC as well as stages III and IV PDAC, with c-statistic values in the range of 0.96 to 0.97 in two phase II studies. We now report that in two studies of blood samples prospectively collected from 1 to 15 years prior to a PDAC diagnosis (Mayo Clinic and PLCO cohorts), THBS2 and/or CA19-9 failed to discriminate cases from healthy controls at the AUC = 0.8 needed. We conclude that PDAC progression may be heterogeneous and for some individuals can be more rapid than generally appreciated. It is important that PDAC early-detection studies incorporate high-risk, prospective prediagnostic cohorts into discovery and validation studies. Prevention Relevance: A blood biomarker panel of THBS2 and CA19-9 detects early stages of pancreatic ductal adenocarcinoma at diagnosis, but not when tested across a population up to 1 year earlier. Our findings suggest serial sampling over time, using prospectively collected samples for biomarker discovery, and more frequent screening of high-risk individuals.

Abstract Purpose: To compare the clinical characteristics and overall survival (OS) of germline mutation carriers in homologous recombination repair (HRR) genes and noncarriers with pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Germline DNA from 3,078 patients with PDAC enrolled in a prospective registry at Mayo Clinic between 2000 and 2017 was analyzed for mutations in 37 cancer predisposition genes. Characteristics and OS of patients with mutations in eight genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, PALB2, RAD51C, and RAD51D) involved in HRR were compared with patients testing negative for mutations in all 37 genes. Results: The 175 HRR mutation carriers and 2,730 noncarriers in the study had a median duration of follow-up of 9.9 years. HRR mutation carriers were younger (median age at diagnosis: 63 vs. 66 years, P < 0.001) and more likely to have metastatic disease at diagnosis (46% vs. 36%, P = 0.004). In a multivariable model adjusting for sex, age at diagnosis, and tumor staging, patients with germline HRR mutations had a significantly longer OS compared with noncarriers [HR, 0.83; 95% confidence interval (CI), 0.70–0.97; P = 0.02]. Further gene-level analysis demonstrated that germline ATM mutation carriers had longer OS compared with patients without germline mutations in any of the 37 genes (HR, 0.72; 95% CI, 0.55–0.94; P = 0.01). Conclusions: This study demonstrates that germline mutation carrier status in PDAC is associated with longer OS compared with noncarriers. Further research into tumor biology and response to platinum-based chemotherapy in germline mutation carriers with PDAC are needed to better understand the association with longer OS.

Abstract Background: Whether circulating polyunsaturated fatty acid (PUFA) levels are associated with pancreatic cancer risk is uncertain. Mendelian randomization (MR) represents a study design using genetic instruments to better characterize the relationship between exposure and outcome. Methods: We utilized data from genome-wide association studies within the Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case–Control Consortium, involving approximately 9,269 cases and 12,530 controls of European descent, to evaluate associations between pancreatic cancer risk and genetically predicted plasma n-6 PUFA levels. Conventional MR analyses were performed using individual-level and summary-level data. Results: Using genetic instruments, we did not find evidence of associations between genetically predicted plasma n-6 PUFA levels and pancreatic cancer risk [estimates per one SD increase in each PUFA-specific weighted genetic score using summary statistics: linoleic acid odds ratio (OR) = 1.00, 95% confidence interval (CI) = 0.98–1.02; arachidonic acid OR = 1.00, 95% CI = 0.99–1.01; and dihomo-gamma-linolenic acid OR = 0.95, 95% CI = 0.87–1.02]. The OR estimates remained virtually unchanged after adjustment for covariates, using individual-level data or summary statistics, or stratification by age and sex. Conclusions: Our results suggest that variations of genetically determined plasma n-6 PUFA levels are not associated with pancreatic cancer risk. Impact: These results suggest that modifying n-6 PUFA levels through food sources or supplementation may not influence risk of pancreatic cancer.

Abstract Background: Discovery of methylated DNA markers (MDM) of esophageal squamous cell carcinoma (ESCC) has sparked interest in assessing these markers in tissue. We evaluated MDMs in ESCC from three geographically and ethnically distinct populations, and explored the feasibility of assaying MDMs from DNA obtained by swallowed balloon devices. Methods: MDMs were assayed in ESCC and normal tissues obtained from the populations of United States, Iran, and China, and from exfoliative cytology specimens obtained by balloons in a Chinese population. Areas under the receiver operating curve (AUC) of MDMs discriminating ESCC from normal tissues were calculated. Random forest prediction models were built, trained on U.S. cases and controls, and calibrated to U.S.-only controls (model 1) and three-country controls (model 2). Statistical tests were used to assess the relationship between dysplasia and MDM levels in balloons. Results: Extracted DNA from 333 ESCC and 322 normal tissues was analyzed, in addition to archival DNA from 98 balloons. For ESCC, model 1 validated in Iranian and Chinese tissues with AUCs of 0.90 and 0.87, and model 2 yielded AUCs of 0.99, 0.96, and 0.94 in tissues from the United States, Iran, and China, respectively. In Chinese balloons, MDMs showed a statistically significant trend of increasing levels with increasing grades of dysplasia (P < 0.004). Conclusions: MDMs accurately discriminate ESCC from normal esophagus in tissues obtained from high- and low-incidence countries. Preliminary data suggest that levels of MDMs assayed in DNA from swallowed balloon devices increase with dysplasia grade. Larger studies are needed to validate these results. Impact: MDMs coupled with minimally invasive collection methods have the potential for worldwide application in ESCC screening.

Abstract Registry-based epidemiologic studies suggest associations between chronic inflammatory intestinal diseases and pancreatic ductal adenocarcinoma (PDAC). As genetic susceptibility contributes to a large proportion of chronic inflammatory intestinal diseases, we hypothesize that the genomic regions surrounding established genome-wide associated variants for these chronic inflammatory diseases are associated with PDAC. We examined the association between PDAC and genomic regions (±500 kb) surrounding established common susceptibility variants for ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, chronic pancreatitis, and primary sclerosing cholangitis. We analyzed summary statistics from genome-wide association studies data for 8,384 cases and 11,955 controls of European descent from two large consortium studies using the summary data-based adaptive rank truncated product method to examine the overall association of combined genomic regions for each inflammatory disease group. Combined genomic susceptibility regions for ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis were associated with PDAC at P values < 0.05 (0.0040, 0.0057, 0.011, and 3.4 × 10−6, respectively). After excluding the 20 PDAC susceptibility regions (±500 kb) previously identified by GWAS, the genomic regions for ulcerative colitis, Crohn disease, and inflammatory bowel disease remained associated with PDAC (P = 0.0029, 0.0057, and 0.0098, respectively). Genomic regions for celiac disease (P = 0.22) and primary sclerosing cholangitis (P = 0.078) were not associated with PDAC. Our results support the hypothesis that genomic regions surrounding variants associated with inflammatory intestinal diseases, particularly, ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis are associated with PDAC. Significance: The joint effects of common variants in genomic regions containing susceptibility loci for inflammatory bowel disease and chronic pancreatitis are associated with PDAC and may provide insights to understanding pancreatic cancer etiology.

Abstract Background: Obesity and diabetes are major modifiable risk factors for pancreatic cancer. Interactions between genetic variants and diabetes/obesity have not previously been comprehensively investigated in pancreatic cancer at the genome-wide level. Methods: We conducted a gene–environment interaction (GxE) analysis including 8,255 cases and 11,900 controls from four pancreatic cancer genome-wide association study (GWAS) datasets (Pancreatic Cancer Cohort Consortium I–III and Pancreatic Cancer Case Control Consortium). Obesity (body mass index ≥30 kg/m2) and diabetes (duration ≥3 years) were the environmental variables of interest. Approximately 870,000 SNPs (minor allele frequency ≥0.005, genotyped in at least one dataset) were analyzed. Case–control (CC), case-only (CO), and joint-effect test methods were used for SNP-level GxE analysis. As a complementary approach, gene-based GxE analysis was also performed. Age, sex, study site, and principal components accounting for population substructure were included as covariates. Meta-analysis was applied to combine individual GWAS summary statistics. Results: No genome-wide significant interactions (departures from a log-additive odds model) with diabetes or obesity were detected at the SNP level by the CC or CO approaches. The joint-effect test detected numerous genome-wide significant GxE signals in the GWAS main effects top hit regions, but the significance diminished after adjusting for the GWAS top hits. In the gene-based analysis, a significant interaction of diabetes with variants in the FAM63A (family with sequence similarity 63 member A) gene (significance threshold P < 1.25 × 10−6) was observed in the meta-analysis (PGxE = 1.2 ×10−6, PJoint = 4.2 ×10−7). Conclusions: This analysis did not find significant GxE interactions at the SNP level but found one significant interaction with diabetes at the gene level. A larger sample size might unveil additional genetic factors via GxE scans. Impact: This study may contribute to discovering the mechanism of diabetes-associated pancreatic cancer.

e22024 Background: Disease surveillance in high risk melanoma patients using PET/CT is recommended every 6 months, an algorithm that is extremely expensive and often difficult to access. Biomarkers capable of detecting recurrence and low volume metastasis have application in tools to improve the cost effectiveness of surveillance. Towards this long term goal, our aim was to discover melanoma-specific methylated DNA markers (MDMs). Methods: Candidate MDMs were identified by reduced representation bisulfite sequencing of selected age- and sex-balanced snap frozen metastatic melanoma, benign control skin and white blood cells. Sequences were filtered for highest methylation difference, lowest control methylation background ( < 1%), and highest discrimination by variance inflated logistic regression. Using formalin-fixed tissues, the top 32 MDMs were further evaluated in metastatic melanoma from 35 patients, primary melanoma from 26 patients, and 73 control samples (47 benign precursor lesions or normal skin, and 26 healthy buffy coat samples to assess for background DNA in circulation). Candidate markers were blindly assayed by quantitative methylation specific PCR. After normalization by within sample ACTB level, random forest modeling with cross-validation identified marker combinations with greatest predictive accuracy. Results: On receiver operator characteristics analyses of individual marker candidates, areas under the curve (AUCs) ranged from 0.43 to 0.97. Median AUC was 0.835. At 100% specificity, a cross-validated panel of five MDMs (see Table), yielded a sensitivity of 33/35 cases (94.3%; 95% CI, 86.6-100%) for metastatic melanoma and 22/26 cases (84.6%; 95% CI, 70.7-98.5%) for primary melanoma. Furthermore, none of the 5 markers were detected in normal buffy coat samples. Conclusions: A panel of five novel MDMs assayed on tissue and undetectable in normal buffy coat achieves very high discrimination between melanoma and benign control tissues. This panel can be assessed in blood or other bodily fluids for application in melanoma surveillance. [Table: see text]