Polygenic and multifactorial scores for pancreatic ductal adenocarcinoma risk prediction.

BACKGROUND: Most cases of pancreatic ductal adenocarcinoma (PDAC) are asymptomatic in early stages, and the disease is typically diagnosed in advanced phases, resulting in very high mortality. Tools to identify individuals at high risk of developing PDAC would be useful to improve chances of early detection. OBJECTIVE: We generated a polygenic risk score (PRS) for PDAC risk prediction, combining the effect of known risk SNPs, and carried out an exploratory analysis of a multifactorial score. METHODS: We tested the associations of the individual known risk SNPs on up to 2851 PDAC cases and 4810 controls of European origin from the PANcreatic Disease ReseArch (PANDoRA) consortium. Thirty risk SNPs were included in a PRS, which was computed on the subset of subjects that had 100% call rate, consisting of 839 cases and 2040 controls in PANDoRA and 6420 cases and 4889 controls from the previously published Pancreatic Cancer Cohort Consortium I-III and Pancreatic Cancer Case-Control Consortium genome-wide association studies. Additional exploratory multifactorial scores were constructed by complementing the genetic score with smoking and diabetes. RESULTS: The scores were associated with increased PDAC risk and reached high statistical significance (OR=2.70, 95% CI 1.99 to 3.68, p=2.54×10-10 highest vs lowest quintile of the weighted PRS, and OR=14.37, 95% CI 5.57 to 37.09, p=3.64×10-8, highest vs lowest quintile of the weighted multifactorial score). CONCLUSION: We found a highly significant association between a PRS and PDAC risk, which explains more than individual SNPs and is a step forward in the direction of the construction of a tool for risk stratification in the population.

Associations between pancreatic expression quantitative traits and risk of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers. Its poor prognosis is predominantly due to the fact that most patients remain asymptomatic until the disease reaches an advanced stage, alongside the lack of early markers and screening strategies. A better understanding of PDAC risk factors is essential for the identification of groups at high risk in the population. Genome-wide association studies (GWAS) have been a powerful tool for detecting genetic variants associated with complex traits, including pancreatic cancer. By exploiting functional and GWAS data, we investigated the associations between polymorphisms affecting gene function in the pancreas (expression quantitative trait loci, eQTLs) and PDAC risk. In a two-phase approach, we analysed 13 713 PDAC cases and 43 784 controls and identified a genome-wide significant association between the A allele of the rs2035875 polymorphism and increased PDAC risk (P = 7.14 × 10-10). This allele is known to be associated with increased expression in the pancreas of the keratin genes KRT8 and KRT18, whose increased levels have been reported to correlate with various tumour cell characteristics. Additionally, the A allele of the rs789744 variant was associated with decreased risk of developing PDAC (P = 3.56 × 10-6). This single nucleotide polymorphism is situated in the SRGAP1 gene and the A allele is associated with higher expression of the gene, which in turn inactivates the cyclin-dependent protein 42 (CDC42) gene expression, thus decreasing the risk of PDAC. In conclusion, we present here a functional-based novel PDAC risk locus and an additional strong candidate supported by significant associations and plausible biological mechanisms.

Genome-wide scan of long noncoding RNA single nucleotide polymorphisms and pancreatic cancer susceptibility.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second cancer-related cause of death by 2030. Identifying novel risk factors, including genetic risk loci, could be instrumental in risk stratification and implementation of prevention strategies. Long noncoding RNAs (lncRNAs) are involved in regulation of key biological processes, and the possible role of their genetic variability has been unexplored so far. Combining genome wide association studies and functional data, we investigated the genetic variability in all lncRNAs. We analyzed 9893 PDAC cases and 9969 controls and identified a genome-wide significant association between the rs7046076 SNP and risk of developing PDAC (P = 9.73 × 10-9 ). This SNP is located in the NONHSAG053086.2 (lnc-SMC2-1) gene and the risk allele is predicted to disrupt the binding of the lncRNA with the micro-RNA (miRNA) hsa-mir-1256 that regulates several genes involved in cell cycle, such as CDKN2B. The CDKN2B region is pleiotropic and its genetic variants have been associated with several human diseases, possibly though an imperfect interaction between lncRNA and miRNA. We present a novel PDAC risk locus, supported by a genome-wide statistical significance and a plausible biological mechanism.

Genome-wide association study identifies an early onset pancreatic cancer risk locus.

Early onset pancreatic cancer (EOPC) is a rare disease with a very high mortality rate. Almost nothing is known on the genetic susceptibility of EOPC, therefore, we performed a genome-wide association study (GWAS) to identify novel genetic variants specific for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at younger ages. In the first phase, conducted on 821 cases with age of onset ≤60 years, of whom 198 with age of onset ≤50, and 3227 controls from PanScan I-II, we observed four SNPs (rs7155613, rs2328991, rs4891017 and rs12610094) showing an association with EOPC risk (P < 1 × 10-4 ). We replicated these SNPs in the PANcreatic Disease ReseArch (PANDoRA) consortium and used additional in silico data from PanScan III and PanC4. Among these four variants rs2328991 was significant in an independent set of 855 cases with age of onset ≤60 years, of whom 265 with age of onset ≤50, and 4142 controls from the PANDoRA consortium while in the in silico data, we observed no statistically significant association. However, the resulting meta-analysis supported the association (P = 1.15 × 10-4 ). In conclusion, we propose a novel variant rs2328991 to be involved in EOPC risk. Even though it was not possible to find a mechanistic link between the variant and the function, the association is supported by a solid statistical significance obtained in the largest study on EOPC genetics present so far in the literature.

TERT gene harbors multiple variants associated with pancreatic cancer susceptibility.

A small number of common susceptibility loci have been identified for pancreatic cancer, one of which is marked by rs401681 in the TERT-CLPTM1L gene region on chromosome 5p15.33. Because this region is characterized by low linkage disequilibrium, we sought to identify whether additional single nucleotide polymorphisms (SNPs) could be related to pancreatic cancer risk, independently of rs401681. We performed an in-depth analysis of genetic variability of the telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC) genes, in 5,550 subjects with pancreatic cancer and 7,585 controls from the PANcreatic Disease ReseArch (PANDoRA) and the PanScan consortia. We identified a significant association between a variant in TERT and pancreatic cancer risk (rs2853677, odds ratio = 0.85; 95% confidence interval = 0.80-0.90, p = 8.3 × 10(-8)). Additional analysis adjusting rs2853677 for rs401681 indicated that the two SNPs are independently associated with pancreatic cancer risk, as suggested by the low linkage disequilibrium between them (r(2) = 0.07, D' = 0.28). Three additional SNPs in TERT reached statistical significance after correction for multiple testing: rs2736100 (p = 3.0 × 10(-5) ), rs4583925 (p = 4.0 × 10(-5) ) and rs2735948 (p = 5.0 × 10(-5) ). In conclusion, we confirmed that the TERT locus is associated with pancreatic cancer risk, possibly through several independent variants.

Identification of Recessively Inherited Genetic Variants Potentially Linked to Pancreatic Cancer Risk.

Although 21 pancreatic cancer susceptibility loci have been identified in individuals of European ancestry through genome-wide association studies (GWASs), much of the heritability of pancreatic cancer risk remains unidentified. A recessive genetic model could be a powerful tool for identifying additional risk variants. To discover recessively inherited pancreatic cancer risk loci, we performed a re-analysis of the largest pancreatic cancer GWAS, the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4), including 8,769 cases and 7,055 controls of European ancestry. Six single nucleotide polymorphisms (SNPs) showed associations with pancreatic cancer risk according to a recessive model of inheritance. We replicated these variants in 3,212 cases and 3,470 controls collected from the PANcreatic Disease ReseArch (PANDoRA) consortium. The results of the meta-analyses confirmed that rs4626538 (7q32.2), rs7008921 (8p23.2) and rs147904962 (17q21.31) showed specific recessive effects (p<10-5) compared with the additive effects (p>10-3), although none of the six SNPs reached the conventional threshold for genome-wide significance (p < 5×10-8). Additional bioinformatic analysis explored the functional annotations of the SNPs and indicated a possible relationship between rs36018702 and expression of the BCL2L11 and BUB1 genes, which are known to be involved in pancreatic biology. Our findings, while not conclusive, indicate the importance of considering non-additive genetic models when performing GWAS analysis. The SNPs associated with pancreatic cancer in this study could be used for further meta-analysis for recessive association of SNPs and pancreatic cancer risk and might be a useful addiction to improve the performance of polygenic risk scores.

Association of Genetic Variants Affecting microRNAs and Pancreatic Cancer Risk.

Genetic factors play an important role in the susceptibility to pancreatic cancer (PC). However, established loci explain a small proportion of genetic heritability for PC; therefore, more progress is needed to find the missing ones. We aimed at identifying single nucleotide polymorphisms (SNPs) affecting PC risk through effects on micro-RNA (miRNA) function. We searched in silico the genome for SNPs in miRNA seed sequences or 3 prime untranslated regions (3'UTRs) of miRNA target genes. Genome-wide association data of PC cases and controls from the Pancreatic Cancer Cohort (PanScan) Consortium and the Pancreatic Cancer Case-Control (PanC4) Consortium were re-analyzed for discovery, and genotyping data from two additional consortia (PanGenEU and PANDoRA) were used for replication, for a total of 14,062 cases and 11,261 controls. None of the SNPs reached genome-wide significance in the meta-analysis, but for three of them the associations were in the same direction in all the study populations and showed lower value of p in the meta-analyses than in the discovery phase. Specifically, rs7985480 was consistently associated with PC risk (OR = 1.12, 95% CI 1.07-1.17, p = 3.03 × 10-6 in the meta-analysis). This SNP is in linkage disequilibrium (LD) with rs2274048, which modulates binding of various miRNAs to the 3'UTR of UCHL3, a gene involved in PC progression. In conclusion, our results expand the knowledge of the genetic PC risk through miRNA-related SNPs and show the usefulness of functional prioritization to identify genetic polymorphisms associated with PC risk.

The validity of a biomarker method for indirect detection of gastric mucosal atrophy versus standard histopathology.

BACKGROUND: Atrophy of the stomach mucosa is considered to be premalignant lesion for gastric cancer development; easy identification of this condition from a blood-sample would allow identifying the group of individuals at increased risk for cancer development. AIMS: The objective of the current study was to validate a biomarker method (pepsinogen I/II ratio and gastrin-17) for indirect detection of atrophy of the stomach mucosa versus standard histopathology in Caucasian and Asian populations. METHODS: Altogether, 241 patients aged 55 and above referred for upper endoscopy due to dyspeptic symptoms (125 from Latvia, 76 from Lithuania, and 40 from Taiwan) were enrolled. Pepsinogen I, pepsinogen II, gastrin-17 (the latter after stimulation with protein-rich meal) and IgG/IgA antibodies to Helicobacter pylori infection were determined by ELISA method; standard histopathology according to the updated Sydney classification read by two independent expert pathologists was used for the comparison. RESULTS: Pepsinogen I/II ratio below 3 was well related to atrophy (moderate to severe) in the corpus part of the stomach (P < 0.0001) with 83.3% sensitivity and 87.1% specificity. Gastrin-17 below 5 pmol/L was related to atrophy in the antral part (P = 0.007) with 36.8% sensitivity and 86.5% specificity. CONCLUSIONS: Decreased pepsinogen I/II ratio is a reliable marker for atrophy in the corpus, and may be recommended for identification of individuals with this type of atrophy. The utility of gastrin-17 for the detection of atrophy in the antral part of the stomach still requires further evaluation due to the low sensitivity.

Precancerous gastric conditions in high Helicobacter pylori prevalence areas: comparison between Eastern European (Lithuanian, Latvian) and Asian (Taiwanese) patients.

UNLABELLED: The aim of the study was to compare the prevalence and severity of precancerous condition--gastric atrophy and intestinal metaplasia (IM) between Eastern European (Lithuania and Latvia) and Asian (Taiwan) countries in population older than 55 years. METHODS: Patients aged 55 years and older, referred for upper endoscopy due to dyspeptic symptoms, were included in the study. Gastric biopsies were histological investigated according modified Sydney classification. Helicobacter pylori (H. pylori) was detected if any two of three methods (urease test, histology, and serology) were positive. RESULTS: Overall 322 patients included: 52 from Taiwan (TW), 171 from Latvia (LV) and 99 from Lithuania (LT). There were 227 (70%) females and 95 (30%) males. The mean age of TW patients was significantly lower (61.0+/-5.8 years), than of LV (68.1+/-7.3 years) and LT (66.5+/-7.5 years) patients. H. pylori was established in 224 (69.6%) patients. H. pylori positivity was established in 43 (82.7%) TW patients, in 112 (65.5%) LV patients, and in 69 (69.7%) LT patients (P>0.05). In H. pylori-infected patients, any atrophy either in the corpus or in the antrum of the stomach was detected in 26 (60.5%) TW patients, in 40 (35.7%) LV patients, and in 36 (52.2%) LT patients (between TW and LV patients P<0.005). Severe atrophy (grade 2 or 3) detected in 8 (18.6%) TW patients, in 17 (15.2%) LV patients, and in 18 (26.1%) LT patients (P>0.05). Intestinal metaplasia was detected in 22 (51.2%) TW patients, in 37 (33.0%) LV patients and in 31 (44.9%) LT patients among countries (P>0.05). There were no significant differences in proportions of different degrees of both atrophy and intestinal metaplasia among countries. Intestinal metaplasia was found in 79 (77.5%) of 102 patients with any degree of atrophy and in 11 (9.0%) of 122 patients without atrophy (P<0.0001). We found strong statistically significant correlations between atrophy and intestinal metaplasia in antrum (r=0.89), P<0.01, and corpus (r= 0.73), P<0.01. CONCLUSIONS: The prevalence of H. pylori in the elderly population is still high in LT, LV, and TW. There are no significant differences in prevalence of gastric atrophy and intestinal metaplasia among TW, LT, and LV. There is a strong correlation between gastric atrophy and intestinal metaplasia.

Functional single nucleotide polymorphisms within the cyclin-dependent kinase inhibitor 2A/2B region affect pancreatic cancer risk.

The CDKN2A (p16) gene plays a key role in pancreatic cancer etiology. It is one of the most commonly somatically mutated genes in pancreatic cancer, rare germline mutations have been found to be associated with increased risk of developing familiar pancreatic cancer and CDKN2A promoter hyper-methylation has been suggested to play a critical role both in pancreatic cancer onset and prognosis. In addition several unrelated SNPs in the 9p21.3 region, that includes the CDNK2A, CDNK2B and the CDNK2B-AS1 genes, are associated with the development of cancer in various organs. However, association between the common genetic variability in this region and pancreatic cancer risk is not clearly understood. We sought to fill this gap in a case-control study genotyping 13 single nucleotide polymorphisms (SNPs) in 2,857 pancreatic ductal adenocarcinoma (PDAC) patients and 6,111 controls in the context of the Pancreatic Disease Research (PANDoRA) consortium. We found that the A allele of the rs3217992 SNP was associated with an increased pancreatic cancer risk (ORhet=1.14, 95% CI 1.01-1.27, p=0.026, ORhom=1.30, 95% CI 1.12-1.51, p=0.00049). This pleiotropic variant is reported to be a mir-SNP that, by changing the binding site of one or more miRNAs, could influence the normal cell cycle progression and in turn increase PDAC risk. In conclusion, we observed a novel association in a pleiotropic region that has been found to be of key relevance in the susceptibility to various types of cancer and diabetes suggesting that the CDKN2A/B locus could represent a genetic link between diabetes and pancreatic cancer risk.

Gene polymorphisms of micrornas in Helicobacter pylori-induced high risk atrophic gastritis and gastric cancer.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population. METHODS: Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR. RESULTS: Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR -2.34, 95% CI 1.08-5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14-5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC. CONCLUSIONS: Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.

Performance of routine Helicobacter pylori tests in patients with atrophic gastritis.

BACKGROUND: Decreased density of H. pylori in atrophic gastritis may lead to low sensitivity of the routine tests. AIMS: To evaluate the accuracy of routinely used H. pylori tests in atrophic gastritis. METHODS: We compared 5 H. pylori diagnostic tests in 119 dyspeptic patients (28 males/91 females) with a mean age of 67 years (range 55-84). Patients with gastric cancer, peptic ulcer, previous gastric surgery, or those who have received eradication therapy were excluded. The following tests were performed: histology, rapid urease test (RUT), culture, 13C- urea breath tests (UBT), and H.pylori IgG/IgA antibody test (serology). RESULTS: Atrophic gastritis was diagnosed in 26.1% of the patients; H. pylori was present in 87.1%. In the group with atrophy, the sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy were as follows: histology (100% for all parameters); UBT (96; 100; 100; 80; 97%); serology (96; 50; 93; 67; 90%); culture (96; 100; 100; 80; 97%); and RUT (78; 100; 100; 40; 81%), respectively. CONCLUSIONS: Histology, UBT and culture were the three best tests for diagnosing H. pylori infection. We cannot recommend using serology as a single test in a case of atrophy, but it would be reasonable to combine serology with one of the above tests.

Gastric plasma biomarkers and Operative Link for Gastritis Assessment gastritis stage.

INTRODUCTION: The Operative Link for Gastritis Assessment (OLGA) staging system has been proposed as a histopathological reporting system of gastric atrophy. Noninvasive methods for indirect evaluation of gastric mucosal atrophy by biomarkers are also being introduced. OBJECTIVES: To analyze gastric mucosal atrophy by biomarkers, pepsinogen I (PgI), pepsinogen II (PgII), PgI/PgII ratio, fasting gastrin-17 (G-17), stimulated gastrin-17 (sG-17), in relation to OLGA gastritis stage. PATIENTS AND METHODS: Gastric biopsies were taken from 269 prospective patients referred for upper endoscopy because of dyspeptic problems and evaluated by two expert pathologists (D.J. and P.S.). Atrophy was assessed according to the OLGA staging system. Pg I, PgII, Pg I/II, G-17, sG-17 were determined in a plasma sample. RESULTS: The mean levels of PgI and PgI/PgII decreased significantly from 90.8 µg/l and 7.6 in stage 0 gastritis to 64.3 µg/l and 4.3 in high-stage gastritis. The mean values of G-17 and sG-17 were significantly higher among patients with stage II gastritis compared with stage 0 and high-stage gastritis.The proportion of patients with normal mucosa and nonatrophic gastritis according to biomarkers decreased from 78% in stage 0 to 22% in high-stage (III-IV) gastritis. Among the latter no case with normal mucosa, according to biomarkers, was observed. CONCLUSIONS: A significant inverse correlation between the mean levels of PgI, PgI/II ratio and the OLGA stage was observed. Percentage of dyspeptic patients with normal mucosa, by blood biomarkers, decreased with increasing OLGA gastritis stages. OLGA staging system provides a good frame for scientific analysis of gastric mucosal atrophy.

Interleukin-1B and interleukin-1 receptor antagonist gene polymorphisms are not associated with premalignant gastric conditions: a combined haplotype analysis.

OBJECTIVE: Contradictory results have been reported about the role of interleukin-1B (IL1B) and IL1 receptor antagonist (IL1RN) alleles in gastric carcinogenesis. Here, IL1B and IL1RN polymorphisms were analyzed as genotypes and haplotypes in relation to the presence of atrophic gastritis (AG) and intestinal metaplasia in the stomach. METHODS: Two hundred and seventy-eight patients (212 Caucasians and 66 Asians) aged 50 years and above, referred for upper endoscopy because of dyspeptic symptoms, were included in the study. Gastric biopsies were histologically assessed according to the updated Sydney classification. Genomic DNA was typed for polymorphisms at position -3737, -1464, -511, -31 for the IL1B gene and the allele 2 of IL1RN using restriction fragment length polymorphism of amplified PCR fragments and intron-spanning PCR analysis, respectively. RESULTS: IL1B-1464-C/C genotype was associated with higher presence of AG in antrum of the stomach in Caucasians [odds ratio: 4.8 (95% confidence interval=1.7-14.3); P=0.028]. IL1B-1464-G/C genotype was associated with lower incidence of AG in corpus of the stomach in Asians [odds ratio: 0.7 (95% confidence interval=0.5-0.8); P=0.02]. IL1RN*2 allele was not linked with AG or intestinal metaplasia in all parts of the stomach both among Asians and Caucasians. Overall, data show that none of the major four IL1B polymorphisms (IL1B-3737C>T, -1464G>C, -511C>T, -31T>C) and the IL1RN*2 is individually, or in its haplotype configuration, linked to the presence of premalignant lesions in Caucasians. CONCLUSION: The determination of these IL1-related loci does not have any predictive value for stratification of subgroups with respect to gastric cancer risk.

Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) promoter methylation has been linked to loss of TIMP-3 expression in various cancers. In this study, we analyzed TIMP-3 gene methylation using MethyLight assay and TIMP-3 mRNA expression using reverse transcription-polymerase chain reaction analysis in 22 esophageal cancers, 27 gastric carcinomas, and 7 cancer cell lines. We also analyzed TIMP-3 protein expression by immunohistochemistry and its association with clinicopathological characteristics in two cohorts of gastric cancer comprising a total of 347 patients. The TIMP-3 gene was more commonly methylated in adenocarcinomas of the esophagus (9/13) and stomach (9/15) than in the corresponding nonneoplastic mucosa of the esophagus (1/8; P = .024) and stomach (2/14; P = .021). In gastric cancer patients, TIMP-3 was decreased in a diffuse-type gastric cancer and in cancers with poor differentiation and was associated with poor survival (P = .04). In summary, we observed frequent TIMP-3 promoter methylation in adenocarcinomas of the esophagus and stomach and the loss of TIMP-3 expression seems to be of clinical and prognostic relevance in these cancers.