Novel Biomarkers in the Diagnosis of Benign and Malignant Gastrointestinal Diseases.

BACKGROUND: Various noninvasive biomarkers have been used in the diagnosis, prognosis, and treatment of different gastrointestinal (GI) diseases for years. Novel technological developments and profound perception of molecular processes related to GI diseases over the last decade have allowed researchers to evaluate genetic, epigenetic, and many other potential molecular biomarkers in different diseases and clinical settings. Here, we present a review of recent and most relevant articles in order to summarize major findings on novel biomarkers in the diagnosis of benign and malignant GI diseases. SUMMARY: Genetic variations, noncoding RNAs (ncRNAs), cell-free DNA (cfDNA), and microbiome-based biomarkers have been extensively analyzed as potential biomarkers in benign and malignant GI diseases. Multiple single-nucleotide polymorphisms have been linked with a number of GI diseases, and these observations are further being used to build up disease-specific genetic risk scores. Micro-RNAs and long ncRNAs have a large potential as noninvasive biomarkers in the management of inflammatory bowel diseases and GI tumors. Altered microbiome profiles were observed in multiple GI diseases, but most of the findings still lack translational clinical application. As of today, cfDNA appears to be the most potent biomarker for early detection and screening of GI cancers. Key Messages: Novel noninvasive molecular biomarkers show huge potential as useful tools in the diagnostics and management of different GI diseases. However, the use of these biomarkers in real-life clinical practice still remains limited, and further large studies are needed to elucidate the ultimate role of these potential noninvasive clinical tools.

Quantifying sequencing error and effective sequencing depth of liquid biopsy NGS with UMI error correction.

Liquid biopsies are a minimally invasive method to diagnose and longitudinally monitor tumor mutations in patients when tissue biopsies are difficult (e.g., in lung cancer). The percentage of cell-free tumor DNA in blood plasma ranges from more than 65% to 0.1% or lower. To reliably diagnose tumor mutations at 0.1%, there are two options: unrealistically large volumes of patient blood or library preparation and sequencing depth optimized to low-input DNA. Here, we assess two library preparation methods and analysis workflows to determine feasibility and reliability based on standards with known allelic frequency (0 and 0.13% in PIK3CA). However, the implementation for patients is still costly and requires elaborate setups.

Association of Long Non-Coding RNA Polymorphisms with Gastric Cancer and Atrophic Gastritis.

Long non-coding RNAs (lncRNA) play an important role in the carcinogenesis of various tumours, including gastric cancer. This study aimed to assess the associations of lncRNA ANRIL, H19, MALAT1, MEG3, HOTAIR single-nucleotide polymorphisms (SNPs) with gastric cancer and atrophic gastritis. SNPs were analyzed in 613 gastric cancer patients, 118 patients with atrophic gastritis and 476 controls from three tertiary centers in Germany, Lithuania and Latvia. Genomic DNA was extracted from peripheral blood leukocytes. SNPs were genotyped by the real-time polymerase chain reaction. Results showed that carriers of MALAT1 rs3200401 CT genotype had the significantly higher odds of atrophic gastritis than those with CC genotype (OR-1.81; 95% CI 1.17-2.80, p = 0.0066). Higher odds of AG were found in a recessive model (CC vs. TT + CT) for ANRIL rs1333045 (OR-1.88; 95% CI 1.19-2.95, p = 0.0066). Carriers of ANRIL (rs17694493) GG genotype had higher odds of gastric cancer (OR-4.93; 95% CI 1.28-19.00) and atrophic gastritis (OR-5.11; 95% CI 1.10-23.80) compared with the CC genotype, and carriers of HOTAIR rs17840857 TG genotype had higher odds of atrophic gastritis (OR-1.61 95% CI 1.04-2.50) compared with the TT genotype; however, the ORs did not reach the adjusted significance threshold (p < 0.007). In summary, our data provide novel evidence for a possible link between lncRNA SNPs and premalignant condition of gastric cancer, suggesting the involvement of lncRNAs in gastric cancer development.

The Role of miR-375-3p and miR-200b-3p in Gastrointestinal Stromal Tumors.

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST-miR-375-3p and miR-200b-3p-in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA-target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3'UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively affected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST.

miR-20b and miR-451a Are Involved in Gastric Carcinogenesis through the PI3K/AKT/mTOR Signaling Pathway: Data from Gastric Cancer Patients, Cell Lines and Ins-Gas Mouse Model.

Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP, and miR-451a: CAV1 and TSC1. Loss-of-function experiments revealed that down-regulation of miR-20b and up-regulation of miR-451a expression exhibits an anti-tumor effect in vitro (miR-20b: reduced viability, colony formation, increased apoptosis rate, and miR-451a: reduced colony forming ability). To summarize, the present study identified that expression of miR-20b and miR-451a are deregulated in vitro and in vivo and have a tumor suppressive role in GC through regulation of the PI3K/AKT/mTOR signaling pathway.

Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.

TLR1 and PRKAA1 Gene Polymorphisms in the Development of Atrophic Gastritis and Gastric Cancer.

BACKGROUND AND AIMS: Previous genome-wide association studies showed that genetic polymorphisms in toll-like receptor 1 (TLR1) and protein kinase AMP-activated alpha 1 catalytic subunit (PRKAA1) genes were associated with gastric cancer (GC) or increased Helicobacter pylori (H. pylori) infection susceptibility. The aim of this study was to evaluate the association between TLR1 and PRKAA1 genes polymorphisms and H. pylori infection, atrophic gastritis (AG) or GC in the European population. METHODS: Single-nucleotide polymorphisms (SNPs) were analysed in 511 controls, 340 AG patients and 327 GC patients. TLR1 C>T (rs4833095) and PRKAA1 C>T (rs13361707) were genotyped by the real-time polymerase chain reaction. H. pylori status was determined by testing for anti-H. pylori IgG antibodies in the serum. RESULTS: The study included 697 (59.2%) H. pylori positive and 481 (40.8%) H. pylori negative cases. We observed similar distribution of TLR1 and PRKAA1 alleles and genotypes in H. pylori positive and negative cases. TLR1 and PRKAA1 SNPs were not linked with the risk of AG. TC genotype of TLR1 gene was more prevalent in GC patients compared to the control group (29.7% and 22.3% respectively, p=0.002). Carriers of TC genotype had a higher risk of GC (aOR=1.89, 95% CI: 1.26-2.83, p=0.002). A similar association was observed in a dominant inheritance model for TLR1 gene SNP, where comparison of CC+TC vs. TT genotypes showed an increased risk of GC (aOR=1.86, 95% CI: 1.26-2.75, p=0.002). No association between genetic polymorphism in PRKAA1 gene and GC was observed. CONCLUSIONS: TLR1 rs4833095 SNP was associated with an increased risk of GC in a European population, while PRKAA1 rs13361707 genetic variant was not linked with GC. Both genetic polymorphisms were not associated with H. pylori infection susceptibility or the risk of AG.

Quantifying cell free DNA in urine: comparison between commercial kits, impact of gender and inter-individual variation.

DNA can enter the blood circulation from living cells by extracellular vesicles or at cell death, and pass into urine through the kidney barrier. Urine can be collected non-invasively, making it an interesting source of cell-free DNA (cfDNA) for research studies and ultimately for clinical diagnostics. However, there is currently a lack of data on the quantity and variability of cfDNA in urine. Here, we benchmark two commercial urine cfDNA isolation kits with respect to the quantity of DNA, the labor time, and cost. The results show distinctive differences between each kit. Furthermore, the cfDNA amount from the same probands varied strongly from day to day and may be higher in female samples than in male samples (p = 0.003).

Associations of cholesteryl ester transfer protein (CETP) gene variants with predisposition to age-related macular degeneration.

PURPOSE: To determine the frequency of the genotypes of single nucleotide polymorphisms (SNPs) in the gene encoding cholesteryl ester transfer protein (CETP) and their associations with age-related macular degeneration (AMD) in the Lithuanian population. STUDY DESIGN: A total of 1264 subjects were examined: 251 patients with early AMD, 206 patients with exudative AMD, and 807 healthy controls. METHODS: The genotyping of CETP (rs5882, rs708272, rs3764261, rs1800775, rs2303790) was carried out using the RT-PCR. RESULTS: Binomial logistic regression analysis revealed that each copy of rs5882 allele A was associated with a 1.3-fold increased risk of exudative AMD (p=0.046). The G/A and A/A genotypes of the rs708272 polymorphism were associated with 1.5-fold and 1.7-fold increased risks of exudative AMD (p=0.049 and p=0.021, respectively). Combination of two genotypes (G/A+A/A) under the dominant model were associated with a 1.5-fold increased risk of exudative AMD (p=0.021). Analysis of rs708272 revealed that the G/A and A/A genotypes under the co-dominant model were associated with 1.5-fold and 1.7-fold increased risks of exudative AMD, respectively (OR=1.450, 95% CI=1.002-2.098; p=0.049 and OR=1.710, 95% CI=1.064-2.156; p=0.021, respectively). Both genotypes (G/A+A/A) under the dominant model were associated with the 1.5-fold increased risk of exudative AMD, as well (OR=1.514, 95% CI=1.064-2.156; p=0.021) and each additional copy A allele was associated with a 1.3-fold increased risk of exudative AMD (OR=1.316, 95% CI=1.051-1.646; p=0.016). The rs3764261 polymorphism was identified to be protective: the C/A genotype and the combination of two genotypes (C/A+A/A) were associated with 1.8-fold and 1.5-fold decreased risks of exudative AMD (p=0.001 and p=0.015, respectively). CONCLUSION: Our study identified two polymorphisms with a higher risk of AMD development (rs5882 and rs708272) and a protective polymorphism for AMD (rs3764261).