Whole genome sequencing identifies rare genetic variants in familial pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most lethal malignancies with very high mortality and short survival time. About 5-10% of the PDAC patients have a familial predisposition to the disease designated as familial pancreatic cancer (FPC), suggesting genetic modulation of FPC pathogenesis. It is estimated that currently identified sequence variants account for less than 20% of the genetic basis of FPC leaving the majority of the genetic architecture unclarified. We performed whole genome sequencing (WGS) analysis on benign formalin-fixed paraffin-embedded (FFPE) tissues from 35 FPC patients focusing on genes enriched by rare and functional sequence variants. We identified 40 genes hosting at least 2 protein truncating variants (PTVs). Significant overlaps of the 40 genes were found (p < 1 × 10-22 ) with cancer genes, cancer driver genes and genes found in previous studies on cancer, including ATM, POLE, BRCA2, TYR03, PABPC1 and SSC5D. The PTV genes are significantly overrepresented in biological pathways in cancer development and progression including extracellular matrix organization, signaling by RHO GTPases and RHO GTPase cycle. Association analysis using external controls detected 6 genes with p < 0.05. The WGS analysis revealed high heterogeneity in the detected rare variants among FPC patients and provides novel genes harboring potential mutational hotspots for future validation and replication.

Whole genome sequencing identifies rare germline variants enriched in cancer related genes in first degree relatives of familial pancreatic cancer patients.

First-degree relatives (FDRs) of familial pancreatic cancer (FPC) patients have increased risk of developing pancreatic ductal adenocarcinoma (PDAC). Investigating and understanding the genetic basis for PDAC susceptibility in FPC predisposed families may contribute toward future risk-assessment and management of high-risk individuals. Using a Danish cohort of 27 FPC families, we performed whole-genome sequencing of 61 FDRs of FPC patients focusing on rare genetic variants that may contribute to familial aggregation of PDAC. Statistical analysis was performed using the gnomAD database as external controls. Through analysis of heterozygous premature truncating variants (PTV), we identified cancer-related genes and cancer-driver genes harboring multiple germline mutations. Association analysis detected 20 significant genes with false discovery rate, q < 0.05 including: PALD1, LRP1B, COL4A2, CYLC2, ZFYVE9, BRD3, AHDC1, etc. Functional annotation showed that the significant genes were enriched by gene clusters encoding for extracellular matrix and associated proteins. PTV genes were over-represented by functions related to transport of small molecules, innate immune system, ion channel transport, and stimuli-sensing channels. In conclusion, FDRs of FPC patients carry rare germline variants related to cancer pathogenesis that may contribute to increased susceptibility to PDAC. The identified variants may potentially be useful for risk prediction of high-risk individuals in predisposed families.

Cohort profile and heritability assessment of familial pancreatic cancer: a nation-wide study.

BACKGROUND: Familial Pancreatic Cancer (FPC) is responsible for up to 10% of all cases of pancreatic ductal adenocarcinoma (PDAC). Individuals predisposed for FPC have an estimated lifetime risk of 16-39% of developing PDAC. While heritability of PDAC has been estimated to be 36% in a Nordic twin study, no heritability estimate specific on FPC has been reported. METHODS: A national cohort of Danish families with predisposition for FPC is currently included in a screening program for PDAC at Odense University Hospital. Family members included in the screening program were interviewed for pedigree data including: cases of PDAC among first-degree relatives (FDRs) and number of affected/unaffected siblings. Heritability for FPC in the predisposed families was assessed by doubling the estimated intra-class correlation coefficient (ICC) from a random intercept logistic model fitted to data on FDRs. RESULTS: Among families with predisposition for FPC, 83 cases of PDAC were identified. The median age at diagnosis of PDAC was 66 years, and median time from diagnosis to death was 7.6 months. A total of 359 individuals were found as unaffected FDRs of the 83 PDAC cases. The retrieved FDRs included a total of 247 individuals in sibship and 317 individuals in parent-offspring relatedness. We estimated an ICC of 0.25, corresponding to a narrow sense additive heritability estimate of 0.51 in the FPC family cohort. CONCLUSION: We have established a nation-wide cohort of FPC families to facilitate clinical and genetic studies on FPC. The estimated heritability of 51% prominently underlines a strong genetic background of FPC.

A Plasma Protein Biomarker Strategy for Detection of Small Intestinal Neuroendocrine Tumors.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) are difficult to diagnose in the early stage of disease. Current blood biomarkers such as chromogranin A (CgA) and 5-hydroxyindolacetic acid have low sensitivity (SEN) and specificity (SPE). This is a first preplanned interim analysis (Nordic non-interventional, prospective, exploratory, EXPLAIN study [NCT02630654]). Its objective is to investigate if a plasma protein multi-biomarker strategy can improve diagnostic accuracy (ACC) in SI-NETs. METHODS: At the time of diagnosis, before any disease-specific treatment was initiated, blood was collected from patients with advanced SI-NETs and 92 putative cancer-related plasma proteins from 135 patients were analyzed and compared with the results of age- and sex-matched controls (n = 143), using multiplex proximity extension assay and machine learning techniques. RESULTS: Using a random forest model including 12 top ranked plasma proteins in patients with SI-NETs, the multi-biomarker strategy showed SEN and SPE of 89 and 91%, respectively, with negative predictive value (NPV) and positive predictive value (PPV) of 90 and 91%, respectively, to identify patients with regional or metastatic disease with an area under the receiver operator characteristic curve (AUROC) of 99%. In 30 patients with normal CgA concentrations, the model provided a diagnostic SPE of 98%, SEN of 56%, and NPV 90%, PPV of 90%, and AUROC 97%, regardless of proton pump inhibitor intake. CONCLUSION: This interim analysis demonstrates that a multi-biomarker/machine learning strategy improves diagnostic ACC of patients with SI-NET at the time of diagnosis, especially in patients with normal CgA levels. The results indicate that this multi-biomarker strategy can be useful for early detection of SI-NETs at presentation and conceivably detect recurrence after radical primary resection.

Gene Expression Network Analysis of Precursor Lesions in Familial Pancreatic Cancer.

Purpose: High-grade pancreatic intraepithelial neoplasia (PanIN) are aggressive premalignant lesions, associated with risk of progression to pancreatic ductal adenocarcinoma (PDAC). A depiction of co-dysregulated gene activity in high-grade familial pancreatic cancer (FPC)-related PanIN lesions may characterize the molecular events during the progression from familial PanIN to PDAC. Materials and Methods: We performed weighted gene coexpression network analysis (WGCNA) to identify clusters of coexpressed genes associated with FPC-related PanIN lesions in 13 samples with PanIN-2/3 from FPC predisposed individuals, 6 samples with PDAC from sporadic pancreatic cancer (SPC) patients, and 4 samples of normal donor pancreatic tissue. Results: WGCNA identified seven differentially expressed gene (DEG) modules and two commonly expressed gene (CEG) modules with significant enrichment for Gene Ontology (GO) terms in FPC and SPC, including three upregulated (p < 5e-05) and four downregulated (p < 6e-04) gene modules in FPC compared to SPC. Among the DEG modules, the upregulated modules include 14 significant genes (p < 1e-06): ALOX12-AS1, BCL2L11, EHD4, C4B, BTN3A3, NDUFA11, RBM4B, MYOC, ZBTB47, TTTY15, NAPRT, LOC102606465, LOC100505711, and PTK2. The downregulated modules include 170 genes (p < 1e-06), among them 13 highly significant genes (p < 1e-10): COL10A1, SAMD9, PLPP4, COMP, POSTN, IGHV4-31, THBS2, MMP9, FNDC1, HOPX, TMEM200A, INHBA, and SULF1. The DEG modules are enriched for GO terms related to mitochondrial structure and adenosine triphosphate metabolic processes, extracellular structure and binding properties, humoral and complement mediated immune response, ligand-gated ion channel activity, and transmembrane receptor activity. Among the CEG modules, IL22RA1, DPEP1, and BCAT1 were found as highly connective hub genes associated with both FPC and SPC. Conclusion: FPC-related PanIN lesions exhibit a common molecular basis with SPC as shown by gene network activities and commonly expressed high-connectivity hub genes. The differential molecular pathology of FPC and SPC involves multiple coexpressed gene clusters enriched for GO terms including extracellular activities and mitochondrion function.

Microscopic findings in EUS-guided fine needle (SharkCore) biopsies with type 1 and type 2 autoimmune pancreatitis.

The International Consensus Diagnostic Criteria (ICDC) for the diagnosis of autoimmune pancreatitis (AIP) include the histological criterion that is based on either pancreatic core needle biopsies (CNBs) or surgical specimens. However, CNBs are difficult to obtain by endoscopic ultrasound (EUS). EUS fine-needle aspiration (EUS-FNA) cytology is usually not sufficient for the diagnosis of AIP, but may sometimes contain tissue microfragments. Another approach is EUS-guided histological fine-needle biopsy (EUS-FNB), using needles such as the SharkCore or ProCore needle. Published data regarding EUS-guided SharkCore FNB for the diagnosis of AIP are lacking. We aimed to describe our histological findings in one type 1 and two type 2 AIP patients who underwent EUS SharkCore FNB. The EUS-FNBs of two patients fulfilled the histological level 2 ICDC for type 1 AIP or type 2 AIP. The EUS-FNB of one patient fulfilled the histological level 1 ICDC for type 2 AIP. The tissue cylinders and fragments measured 55, 28 and 17 mm in total. At least histological level 2 ICDC were fulfilled in all cases, and our findings regarding the utility of EUS SharkCore FNB for the diagnosis of AIP are therefore promising, but further studies based on larger numbers of patients are warranted.

Is screening for pancreatic cancer in high-risk groups cost-effective? - Experience from a Danish national screening program.

OBJECTIVE: Pancreatic cancer (PC) is the fourth leading cause of cancer death worldwide, symptoms are few and diffuse, and when the diagnosis has been made only 10-15% would benefit from resection. Surgery is the only potentially curable treatment for pancreatic cancer, and the prognosis seems to improve with early detection. A hereditary component has been identified in 1-10% of the PC cases. To comply with this, screening for PC in high-risk groups with a genetic disposition for PC has been recommended in research settings. DESIGN: Between January 2006 and February 2014 31 patients with Hereditary pancreatitis or with a disposition of HP and 40 first-degree relatives of patients with Familial Pancreatic Cancer (FPC) were screened for development of Pancreatic Ductal Adenocarcinoma (PDAC) with yearly endoscopic ultrasound. The cost-effectiveness of screening in comparison with no-screening was assessed by the incremental cost-utility ratio (ICER). RESULTS: By screening the FPC group we identified 2 patients with PDAC who were treated by total pancreatectomy. One patient is still alive, while the other died after 7 months due to cardiac surgery complications. Stratified analysis of patients with HP and FPC provided ICERs of 47,156 US$ vs. 35,493 US$ per life-year and 58,647 US$ vs. 47,867 US$ per QALY. Including only PDAC related death changed the ICER to 31,722 US$ per life-year and 42,128 US$ per QALY. The ICER for patients with FPC was estimated at 28,834 US$ per life-year and 38,785 US$ per QALY. CONCLUSIONS: With a threshold value of 50,000 US$ per QALY this screening program appears to constitute a cost-effective intervention although screening of HP patients appears to be less cost-effective than FPC patients.

Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls.

OBJECTIVES: There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles and PDAC in a disease and disease-control cohort. METHODS: We performed a microarray profiling of 847 different mature miRNAs from plasma in an exploratory cohort of 20 patients with PDAC or other pancreatic diseases, profiling of 45 miRNAs in plasma samples from PDAC (n = 48) and disease controls (n = 47), and evaluation of associations of data with diagnosis, survival, and CA-19-9. RESULTS: We find 7 significantly deregulated miRNAs in PDAC using univariate statistics. At a false-discovery rate of 5%, miRNA-375 remained significantly elevated in PDAC. MicroRNA-375 did not improve diagnosis of PDAC in this cohort (70% accuracy) and did not correlate with survival. However, 3 controls (other gastrointestinal cancers) with increased CA-19-9 did not show increased miRNA-375. CONCLUSIONS: In the plasma-miRNA population, we find miRNA-375, which is selectively expressed in the endocrine pancreas under normal conditions, increased in PDAC cases compared with patients with other pancreatic or gastrointestinal diseases. The miRNA-375 does not outperform CA-19-9 diagnostically in the present cohort. However, it shows promising specificity and should be examined in larger prospective studies.

Is the SPINK1 variant p.N34S overrepresented in patients with acute pancreatitis?

OBJECTIVES: Serine Protease Inhibitor Kazal type 1 (SPINK1) protects against premature intracellular activation of trypsinogen and development of acute pancreatitis. Our aim was to determine the prevalence of SPINK1 mutations (a) in unselected patients with first-time acute pancreatitis and (b) in the Danish background population (c) in a meta-analysis to combine the results with findings in similar investigations worldwide and (d) to evaluate whether patients with SPINK1 mutations had a more severe clinical course. METHODS: A total of 75 consecutive patients admitted to a surgical department with first-time acute pancreatitis were prospectively included. In addition, 188 healthy controls were tested for the SPINK1 variants: p.N34S, p.P55S, p.R65Q, p.R67C, and IVS3+2 T>C, in order to calculate the prevalence of SPINK1 mutations in the Danish background population. A meta-analysis was conducted on previous studies on acute pancreatitis and SPINK1 mutations. RESULTS: Two patients (2.7%) and two controls (1.1%) were heterozygous for the p.N34S variant. The meta-analysis confirmed that the p.N34S variant is overrepresented in patients with acute pancreatitis compared with the background population (OR=3.16, P<0.001). But this analysis did not clarify whether this was only true for patients with first-time acute pancreatitis or recurrent pancreatitis as the present studies do not provide this information, and those who do not have enough patients to reach levels of statistic significance, even if data are pooled. CONCLUSION: The SPINK1 variant p.N34S is overrepresented in patients with acute pancreatitis, but more studies distinguishing between first-time and recurrent acute pancreatitis have to be done to determine whether this is only true for patients with recurrent acute pancreatitis.

Genetic, epidemiological, and clinical aspects of hereditary pancreatitis: a population-based cohort study in Denmark.

OBJECTIVES: In a population-based, well-defined group of patients first regarded as having pancreatitis of unknown origin (PUO), we identified, described, and compared the clinical and genetic aspects of patients with hereditary pancreatitis (HP) and with cystic fibrosis transmembrane conductance regulator gene (CFTR) and serine protease inhibitor Kazal type 1 gene (SPINK1) mutations with patients who retained the diagnosis of true idiopathic pancreatitis (tIP) after genetic testing for HP, SPINK1, and CFTR mutations. METHODS: Patients with PUO were identified in the Danish National Registry of Patients or were referred by clinicians. DNA from blood was analyzed for cationic trypsinogen (PRSS1), SPINK1, and CFTR mutations. Considering the diagnosis of HP, a pedigree was drawn for each patient. RESULTS: A genetic mutation was found in 40% of 122 patients with PUO. After testing first-degree relatives of the 18 initially identified HP patients, 38 HP patients in total were identified, and 28 patients had SPINK1-CFTR mutations. Among HP patients, no p.N29I mutations were found and the p.A16V mutation was more frequent than previously reported, 45 and 32% had exocrine and endocrine insufficiency, respectively, and among tIP patients 9 and 12%, respectively. Pancreatic cancer was diagnosed in 5% of the HP families. CONCLUSIONS: The genotype of the Danish population with HP differs from that of previously described cohorts. The occurrence of exocrine and endocrine insufficiency is higher among patients with HP than in patients with SPINK1-CFTR mutations and tIP, and more HP families develop pancreatic cancer. Genetic testing thus helps to predict the prognosis of the pancreatitis.

Comparison of circulating MMP-9, TIMP-1 and CA19-9 in the detection of pancreatic cancer.

BACKGROUND/AIM: The performance of the circulating tumor markers carbohydrate antigen 19-9 (CA19-9), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were evaluated separately and in combination for their potential value in detecting pancreatic ductal adenocarcinoma. PATIENTS AND METHODS: The patients had symptoms of pancreatic cancer. The discriminative strength of MMP-9 and TIMP-1 were compared to that of CA19-9 using receiver operating characteristics curves, area under the curves (AUC), specificity and sensitivity. RESULTS: The sensitivities of MMP-9, TIMP-1 and CA19-9 in detecting pancreatic ductal adenocarcinoma were 58.82%, 47.1% and 86%, respectively, with specificities of 34.6%, 69.2% and 73%. The AUCs of MMP-9, TIMP-1 and CA19-9 were 0.50, 0.64 and 0.84, respectively. Combining the three markers did not significantly improve detection of pancreatic ductal adenocarcinoma compared to CA19-9 used alone. CONCLUSION: Circulating MMP-9 and TIMP-1 were inferior to CA19-9 as markers for detecting pancreatic ductal adenocarcinoma and did not improve the diagnostic accuracy when combined with CA19-9.

Comparison of plasma Tu-M2-PK and CA19-9 in pancreatic cancer.

OBJECTIVES: The performance of the 2 tumor markers carbohydrate antigen 19-9 (CA19-9) and tumor M2 pyruvate kinase (Tu-M2-PK) separately and in combination detecting pancreatic ductal adenocarcinoma (PDAC) was evaluated in a prospective study. METHODS: The study comprised 103 patients referred because of suspicion of pancreatic cancer. Of these, 51 patients had their conditions diagnosed as PDAC, whereas this diagnosis was ruled out in 52 after 12 months of follow-up. The performance of Tu-M2-PK was compared with that of CA19-9 using cutoff values 15 and 37 U/mL, respectively. RESULTS: The sensitivity of Tu-M2-PK and CA19-9 in detecting PDAC was 55% and 86% at specificities of 52% and 73%, respectively. The area under the curve (AUC) of Tu-M2-PK was 0.55 and that of CA19-9 was 0.84. Combining the 2 markers did not significantly improve AUC (AUC = 0.85, P = 0.72) compared with CA19-9 when used alone. The presence of chronic pancreatitis or jaundice causes increased levels of CA19-9 but does not influence Tu-M2-PK. CONCLUSIONS: Tu-M2-PK was inferior to CA19-9 as marker of PDAC. Tu-M2-PK may have a role in diagnosing PDAC because it is not affected by cholestasis or Lewis phenotype. Neither tumor marker can stand alone in the diagnosis of PDAC.