Juozas Kupcinskas

Macro-Role of MicroRNA in Gastric Cancer

Gastric cancer is a heterogeneous disease with currently still unknown mechanisms of development. Besides genetic and epigenetic mechanisms, microRNAs (miRNAs) have recently been discovered as one of the crucial players in gastric carcinogenesis through posttranscriptional regulation of tumor suppressor and oncogenes. A substantial number of deregulated miRNAs have been revealed in gastric cancer and the biological significance of those miRNAs has been confirmed in multiple functional experiments. A growing number of studies suggest involvement of miRNAs in various steps of gastric carcinogenesis: from gastritis toward metastatic disease. Great biological stability of miRNAs opens novel fields in biomarker research with potential clinical implementation in screening, diagnosis, prediction of prognosis and therapeutic management. In this review, we provide the basic knowledge of miRNA biogenesis and discuss extensively the role of miRNAs in gastric carcinogenesis, including Helicobacter pylori-related miRNA alterations and potential translational clinical implementations.

Common variation at 2p13.3, 3q29, 7p13 and 17q25.1 associated with susceptibility to pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer death in the developed world. Both inherited high-penetrance mutations in BRCA2 (ref. 2), ATM, PALB2 (ref. 4), BRCA1 (ref. 5), STK11 (ref. 6), CDKN2A and mismatch-repair genes and low-penetrance loci are associated with increased risk. To identify new risk loci, we performed a genome-wide association study on 9,925 pancreatic cancer cases and 11,569 controls, including 4,164 newly genotyped cases and 3,792 controls in 9 studies from North America, Central Europe and Australia. We identified three newly associated regions: 17q25.1 (LINC00673, rs11655237, odds ratio (OR) = 1.26, 95% confidence interval (CI) = 1.19–1.34, P = 1.42 × 10−14), 7p13 (SUGCT, rs17688601, OR = 0.88, 95% CI = 0.84–0.92, P = 1.41 × 10−8) and 3q29 (TP63, rs9854771, OR = 0.89, 95% CI = 0.85–0.93, P = 2.35 × 10−8). We detected significant association at 2p13.3 (ETAA1, rs1486134, OR = 1.14, 95% CI = 1.09–1.19, P = 3.36 × 10−9), a region with previous suggestive evidence in Han Chinese. We replicated previously reported associations at 9q34.2 (ABO), 13q22.1 (KLF5), 5p15.33 (TERT and CLPTM1), 13q12.2 (PDX1), 1q32.1 (NR5A2), 7q32.3 (LINC-PINT), 16q23.1 (BCAR1) and 22q12.1 (ZNRF3). Our study identifies new loci associated with pancreatic cancer risk.

Lack of association between gene polymorphisms of Angiotensin converting enzyme, Nod-like receptor 1, Toll-like receptor 4, FAS/FASL and the presence of Helicobacter pylori-induced premalignant gastric lesions and gastric cancer in Caucasians

BackgroundSeveral polymorphisms of genes involved in the immunological recognition of Helicobacter pylori and regulating apoptosis and proliferation have been linked to gastric carcinogenesis, however reported data are partially conflicting. The aim of our study was to evaluate potential associations between the presence of gastric cancer (GC) and high risk atrophic gastritis (HRAG) and polymorphisms of genes encoding Angiotensin converting enzyme (ACE), Nod-like receptor 1 (NOD1), Toll-like receptor 4 (TLR4) and FAS/FASL.MethodsGene polymorphisms were analyzed in 574 subjects (GC: n = 114; HRAG: n = 222, controls: n = 238) of Caucasian origin. ACE I/D (rs4646994), NOD1 796G>A (rs5743336), TLR4 3725G>C (rs11536889), FAS 1377G>A (rs2234767), FAS 670A>G (rs1800682) and FASL 844T>C (rs763110) were genotyped by different PCR approaches and restriction fragment length polymorphism analysis.ResultsFrequencies of genotypes in our study are similar to the data reported on subjects of Caucasian ethnicity. There was a tendency for NOD1 796G/G genotype to be associated with increased risk of HRAG (62.4% vs. 54.5% in controls, p = 0.082). FAS 670G/G genotype was more frequent in HRAG when compared to controls, 23.9% and 17.2% respectively, however it failed to reach significance level (p = 0.077). We did not find any significant associations for all polymorphisms in relation to GC or HRAG. NOD1 796G>A and TLR4 3725G>C gene polymorphisms were also not associated with Helicobacter pylori infection.ConclusionsACE, NOD1, TRL4 and FAS/FASL gene polymorphisms are not linked with gastric carcinogenesis in Caucasians, and therefore they should not be considered as potential biomarkers for identifying individuals with higher risk for GC.

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.

Detection of cancer through exhaled breath: a systematic review

Background Timely diagnosis of cancer represents a challenging task; in particular, there is a need for reliable non-invasive screening tools that could achieve high levels of adherence at virtually no risk in population-based screening. In this review, we summarize the current evidence of exhaled breath analysis for cancer detection using standard analysis techniques and electronic nose. Methods Relevant studies were identified searching Pubmed and Web of Science databases until April 30, 2015. Information on breath test performance, such as sensitivity and specificity, was extracted together with volatile compounds that were used to discriminate cancer patients from controls. Performance of different breath analysis techniques is provided for various cancers together with information on methodological issues, such as breath sampling protocol and validation of the results. Results Overall, 73 studies were included, where two-thirds of the studies were conducted on lung cancer. Good discrimination usually required a combination of multiple biomarkers, and area under the receiver operating characteristic curve or accuracy reached levels of 0.9 or higher in multiple studies. In 25% of the reported studies, classification models were built and validated on the same datasets. Huge variability was seen in different aspects among the studies. Conclusions Analyses of exhaled breath yielded promising results, although standardization of breath collection, sample storage and data handling remain critical issues. In order to foster breath analysis implementation into practice, larger studies should be implemented in true screening settings, paying particular attention to standardization in breath collection, consideration of covariates, and validation in independent population samples.

Epigenetic silencing of miR-137 is a frequent event in gastric carcinogenesis.

MicroRNAs (miRNA) are involved in posttranscriptional regulation of gene expression and are dysregulated during carcinogenesis. CpG island methylation of miR‐137 is a common event in different cancers; however, the role of miR‐137 in gastric cancer (GC) remains largely unexplored. In this study we aimed to characterize the epigenetic alterations of miR‐137 in gastric carcinogenesis. We analyzed total 295 tissues including paired primary gastric cancer (T‐GC) with corresponding adjacent gastric mucosa (N‐GC), paired primary colorectal cancer (CRC) tissues with corresponding non‐tumorous mucosa, gastric tissues from controls (N), and patients with chronic/atrophic gastritis (CG) with and without Helicobacter pylori infection. Bisulfite pyrosequencing and TaqMan RT‐PCR were used to analyze miR‐137 methylation and expression, respectively. Survival differences were evaluated using Kaplan‐Meier analyses. miR‐137 CpG island methylation was more frequent in tumorous compared to non‐tumorous conditions and higher in CRC than in GC. In comparison to N‐GC, miR 137 methylation level was lower in N and CG tissues, which correlates with Correas cascade. MiR‐137 methylation inversely correlates with global LINE‐1 methylation and miR‐137 expression. miR‐137 methylation was higher in intestinal type GC compared to diffuse one, and higher in antrum compared to cardia and corpus, however, miR‐137 methylation was associated with worse prognosis in diffuse, but not in intestinal type of GC. The expression in colon was significantly higher compared to any gastric tissues suggesting functional difference. In summary, miR‐137 methylation is a frequent event in gastrointestinal cancers which occurs early in stepwise manner during gastric carcinogenesis and inversely correlates with global methylation.

Lack of association between miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 gene polymorphisms and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide with high mortality rates. MicroRNAs (miRNAs) have an established role in the development of different cancers. Single nucleotide polymorphisms (SNPs) in miRNA related genes were linked with various gastrointestinal malignancies. However, the data on association between miRNA SNPs and CRC development are inconsistent. The aim of the present study was to evaluate the association between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of CRC in European population. Gene polymorphisms were analyzed in 621 subjects (controls: n = 428; CRC: n = 193). 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. Overall, all genotypes and alleles of miRNA SNPs were distributed equally between control and CRC groups. We observed a tendency for miR-146a C allele to be associated with lower risk of CRC when compared to G allele, however, the difference did not reach the adjusted P-value (odds ratio (OR) = 0.68, 95% confidence interval (CI) 0.49–0.95, P = 0.025). In conclusion, gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of CRC in European subjects.

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

Background MicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues. Methods The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs. Results Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients’ plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression. Conclusions Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.

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.

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 (r2 = 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.