Deep sequencing reveals recurrent somatic mutations and distinct molecular subgroups in gastric cancer in Mizo population, North East India.

In India, Mizoram has the highest incidence of gastric cancer (GC) which might be associated with environmental factors such as diet, Helicobacter pylori (H.pylori) and Epstein-Barr virus (EBV) infections, and somatic genomic alterations. We performed PCR cum sequencing and fragment analysis for detection of H. pylori/EBV infection and microsatellite Instability (MSI) in GC patients (N = 68). Somatic mutations were identified by targeted and exome sequencing. We found 87% of GC patients infected with H. pylori and or EBV. Pathogenic infections were mostly mutually exclusive with only 16% of coinfection. TP53, MUC6, and ARID1A were significantly mutated. Two molecular subgroups with distinctive mutational profiles were identified: (1) patients harboring mutations in TP53 and (2) patients harboring mutations in RTK/RAS/PI3-K signaling pathway and chromatin-remodeling genes. Therefore, EBV and H. pylori infections and somatic mutations in the genes involved in RTK/RAS/PI3K signaling pathway, chromatin-remodeling, and TP53 might drive GC development and progression in Mizo patients.

Development, systematic optimisation and biofilm disruption activity of eugenol-based nanosized emulsions stabilised with Tween 80.

The aims of this study were to systematically optimise a formula for eugenol emulsions via face-centered central composite design and to assess the activity against two-different bacterial strains (Staphylococcus aureus and Propionibacterium acnes) present at planktonic and biofilm forms. The molecular interaction of excipients, mean particle size (MPS) including zeta potential (ZP), drug entrapment efficiency (DEE) and in vitro drug release of optimised emulsions was done using FT-IR, Malvern Zetasizer, ultracentrifugation technique and membrane-free dissolution model, respectively. The emulsions consisted of 151.3 ± 1.45 nm MPS, -21.3 ± 1.25 mV ZP and 93.98 ± 1.41% DEE values. On storage of emulsions at 25 °C for 3 months, the value of DEE was found to be 72.12 ± 2.82%. The Tween 80 emulsifier film coverage onto the dispersed eugenol droplets of emulsions delayed significantly the drug release (12%-19%) compared to the drug release occurred from pure eugenol. The treatment of planktonic S. aureus and P. acnes with diluted eugenol emulsions showed the minimum inhibitory concentration and minimum bactericidal concentration values at 1.25-2.5 mg/ml whereas it occurred at 10 mg/ml for pure eugenol. Treating the biofilms with eugenol emulsions (1-2 mg/ml) yielded 59-70% minimum biofilm eradication concentration but 10 mg/ml pure eugenol showed 60%. Hence, the eugenol emulsions displayed antibacterial activity and could be projected as an antibiofilm or biofilm disruption agent.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer.

BACKGROUND: The role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer. METHODS: We investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue. RESULTS: APC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases. CONCLUSION: The two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.

Xenobiotic Pathway Gene Polymorphisms Associated with Gastric Cancer in High Risk Mizo-Mongoloid Population, Northeast India.

BACKGROUND: The aim of this study was to evaluate the risk of gastric cancer associated with individual or combined glutathione S-transferases (GSTs) polymorphism and their interaction with environmental factors. MATERIALS AND METHODS: Genotyping by PCR was carried out for 80 cases and controls each for GSTM1, GSTT1, and GSTP1 polymorphism and mapped for gene-environment association studies. The samples were subjected to pathogen detection and GSTP1 expression for analyzing their association with different genotypes. Logistic regression analyses were conducted to compute the influence of both genetic and environmental factors for gastric cancer. MDR analysis was performed to assess the risk of gastric cancer by studying the gene-gene and gene-environment effect on the basis of GST genotyping and GSTP1 gene expression. RESULTS: Infection with Helicobacter pylori and CagA+ strains was more frequent in patients with GSTM1/T1 null genotype. Intake of high fermented fat and smoked meat was found to be significantly associated with gastric cancer. The G/G, A/G (rs1695), and T/T (rs1138272) were found to be significantly associated with low expression of GSTP1 gene in cancer tissue. CONCLUSION: Presence of H. pylori with CagA genotype showed significant individual effect with GSTT1 polymorphism as well as strong synergistic effect in gastric cancer risk. Majority of the gastric cancer samples showed significant negative expression in G/G, A/G (rs1695), and T/T (rs1138272) genotypes. This study shows that GST gene polymorphism was significantly relevant for determining the individual susceptibility to gastric cancer.

LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer.

WNT/ß-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair ß-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of ß-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as ß-catenin activity and its target gene VEGFA. Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed ß-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since ß-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and ß-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients.

Panel of significant risk factors predicts early stage gastric cancer and indication of poor prognostic association with pathogens and microsatellite stability.

BACKGROUND: There are very few studies covering the epidemiological risk factors associated with Epstein Barr Virus (EBV) and Microsatellite stability for Gastric Cancer (GC) cases. Early diagnosis of GC through epidemiological risk factors is very necessary for the clinical assessment of GC. The aim of this study was to find out the major risk factors to predict GC in early stage and the impact of pathogen infection and MSI on survival rate of patients. GC samples were screened for Helicobacter pylori, Epstein Barr Virus, and Mismatch repair (MMR) gene status (microsatellite stable or instable). Chi-square and logistic regression analysis of Odd ratio and 95% confidence interval (OR, 95% CI) were performed to find out the association between epidemiological factors and the risk of gastric cancer. The pathogen and MMR gene status were analysed to predict their effect on overall survival and the risk score and hazard ratio was calculated for prognostic assessment. RESULTS: Excess body weight, consumption of extra salt, smoked food, alcohol, and smoking were the major risk factors for GC development. This study achieved a high area under the curve (AUC 0.94) for the probable GC patients in early-stage using the five-panel epidemiological risk factors. H. pylori infected cases were significant with smoked food, while EBV was found to be associated with tuibur intake and smoked food. In overall survival analysis EBV infected and microsatellite stable (HR: 1.32 and 1.34 respectively) GC cases were showing poor prognosis. CONCLUSION: This study might provide new opportunities for personalized treatment options using this epidemiological factor risk score and clinicopathological factors assessment for early detection and prognosis in high-risk GC populations.

Lifestyle chemical carcinogens associated with mutations in cell cycle regulatory genes increases the susceptibility to gastric cancer risk.

In the present study, we correlated the various lifestyle habits and their associated mutations in cell cycle (P21 and MDM2) and DNA damage repair (MLH1) genes to investigate their role in gastric cancer (GC). Multifactor dimensionality reduction (MDR) analysis revealed the two-factor model of oral snuff and smoked meat as the significant model for GC risk. The interaction analysis between identified mutations and the significant demographic factors predicted that oral snuff is significantly associated with P21 3'UTR mutations. A total of five mutations in P21 gene, including three novel mutations in intron 2 (36651738G > A, 36651804A > T, 36651825G > T), were identified. In MLH1 gene, two variants were identified viz. one in exon 8 (37053568A > G; 219I > V) and a novel 37088831C > G in intron 16. Flow cytometric analysis predicted DNA aneuploidy in 07 (17.5%) and diploidy in 33 (82.5%) tumor samples. The G2/M phase was significantly arrested in aneuploid gastric tumor samples whereas high S-phase fraction was observed in all the gastric tumor samples. This study demonstrated that environmental chemical carcinogens along with alteration in cell cycle regulatory (P21) and mismatch repair (MLH1) genes may be stimulating the susceptibility of GC by altering the DNA content level abnormally in tumors in the Mizo ethic population.