Diagnostic implication of a circulating serum-based three-microRNA signature in hepatocellular carcinoma.

Owing to the diagnostic dilemma, the prognosis of hepatocellular carcinoma (HCC) remains impoverished, contributing to the globally high mortality rate. Currently, HCC diagnosis depends on the combination of imaging modalities and the measurement of serum alpha-fetoprotein (AFP) levels. Nevertheless, these conventional modalities exhibit poor performance in detecting HCC at early stages. Thus, there is a pressing need to identify novel circulating biomarkers to promote diagnostic accuracy and surveillance. Circulating miRNAs are emerging as promising diagnostic tools in screening various cancers, including HCC. However, because of heterogenous and, at times, contradictory reports, the universality of miRNAs in clinical settings remains elusive. Consequently, we proposed to explore the diagnostic potential of ten miRNAs selected on a candidate-based approach in HCC diagnosis. The expression of ten candidate miRNAs (Let-7a, miR-15a, miR-26a, miR-124, miR-126, miR-155, miR-219, miR-221, miR-222, and miR-340) was investigated in serum and tissue of 66 subjects, including 33 HCC patients and 33 healthy controls (HC), by rt-PCR. Receiver operating characteristic curve (ROC) analysis was used to determine the diagnostic accuracy of the prospective serum miRNA panel. To anticipate the potential biological roles of a three-miRNA signature, the target genes were evaluated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway. The serum and tissue expression of miRNAs (Let-7a, miR-26a, miR-124, miR-155, miR-221, miR-222, and miR-340) were differentially expressed in HCC patients (p < 0.05). The ROC analysis revealed promising diagnostic performance of Let-7a (AUC = 0.801), miR-221 (AUC = 0.786), and miR-2 (AUC = 0.758) in discriminating HCC from HC. Furthermore, in a logistic regression equation, we identified a three-miRNA panel (Let-7a, miR-221, and miR-222; AUC = 0.932) with improved diagnostic efficiency in differentiating HCC from HC. Remarkably, the combination of AFP and a three-miRNA panel offered a higher accuracy of HCC diagnosis (AUC = 0.961) than AFP alone. The functional enrichment analysis demonstrated that target genes may contribute to pathways associated with HCC and cell-cycle regulation, indicating possible crosstalk of miRNAs with HCC development. To conclude, the combined classifier of a three-miRNA panel and AFP could be indispensable circulating biomarkers for HCC diagnosis. Furthermore, targeting predicted genes may provide new therapeutic clues for the treatment of aggressive HCC.

Telomere Attrition With Concomitant hTERT Overexpression Involved in the Progression of Gastric Cancer May Have Prognostic and Clinical Implications in High-Risk Population Group From North India.

Genetic instabilities exacerbated by the dysfunction of telomeres can lead to the development of cancer. Nearly 90% of all human malignancies are linked with telomere dysregulation and overexpression of telomerase, an enzyme that catalyzes the synthesis of telomeric DNA repeats at the ends of chromosomes. The burden of gastric cancer continues to inflict a deterring impact on the global health scenario, accounting for over one million new cases in 2020. The disease is asymptomatic in its early stages of progression, which is attributed to the poor prognosis and overall surge in mortality rate worldwide. Exploiting telomere physiology can provide extensive mechanistic insight into telomere-associated gastric cancer progression and its use as a target in a variety of therapeutic interventions. In this study, we aimed to evaluate the clinical implications of c-Myc, human telomerase reverse transcriptase (hTERT) expression, and telomere length in patients with gastric cancer. A total of 57 gastric cancer cases and adjacent controls were included in the study. RT-PCR and immunohistochemistry were used to assess the expression levels of c-Myc and hTERT. The relative telomere length was measured by MMQPCR using the Cawthon method. Our results indicated that the shorter telomere and increased hTERT expression were associated with gastric cancer progression. The study also highlighted the role of short telomeres and increased expression of hTERT in gastric cancer progression and its association with various etiological risk factors, transcriptional activators, and overall survival among the ethnic Kashmiri population of North India.

JAK/STAT Signaling: Molecular Targets, Therapeutic Opportunities, and Limitations of Targeted Inhibitions in Solid Malignancies.

JAK/STAT signaling pathway is one of the important regulatory signaling cascades for the myriad of cellular processes initiated by various types of ligands such as growth factors, hormones, and cytokines. The physiological processes regulated by JAK/STAT signaling are immune regulation, cell proliferation, cell survival, apoptosis and hematopoiesis of myeloid and non-myeloid cells. Dysregulation of JAK/STAT signaling is reported in various immunological disorders, hematological and other solid malignancies through various oncogenic activation mutations in receptors, downstream mediators, and associated transcriptional factors such as STATs. STATs typically have a dual role when explored in the context of cancer. While several members of the STAT family are involved in malignancies, however, a few members which include STAT3 and STAT5 are linked to tumor initiation and progression. Other STAT members such as STAT1 and STAT2 are pivotal for antitumor defense and maintenance of an effective and long-term immune response through evolutionarily conserved programs. The effects of JAK/STAT signaling and the persistent activation of STATs in tumor cell survival; proliferation and invasion have made the JAK/STAT pathway an ideal target for drug development and cancer therapy. Therefore, understanding the intricate JAK/STAT signaling in the pathogenesis of solid malignancies needs extensive research. A better understanding of the functionally redundant roles of JAKs and STATs may provide a rationale for improving existing cancer therapies which have deleterious effects on normal cells and to identifying novel targets for therapeutic intervention in solid malignancies.

Transforming Growth Factor-Beta (TGF-ß) Signaling in Cancer-A Betrayal Within.

A ubiquitously expressed cytokine, transforming growth factor-beta (TGF-ß) plays a significant role in various ongoing cellular mechanisms. The gain or loss-of-function of TGF-ß and its downstream mediators could lead to a plethora of diseases includes tumorigenesis. Specifically, at the early onset of malignancy TGF-ß act as tumour suppressor and plays a key role in clearing malignant cells by reducing the cellular proliferation and differentiation thus triggers the process of apoptosis. Subsequently, TGF-ß at an advanced stage of malignancy promotes tumorigenesis by augmenting cellular transformation, epithelial-mesenchymal-transition invasion, and metastasis. Besides playing the dual roles, depending upon the stage of malignancy, TGF-ß also regulates cell fate through immune and stroma components. This oscillatory role of TGF-ß to fight against cancer or act as a traitor to collaborate and crosstalk with other tumorigenic signaling pathways and its betrayal within the cell depends upon the cellular context. Therefore, the current review highlights and understands the dual role of TGF-ß under different cellular conditions and its crosstalk with other signaling pathways in modulating cell fate.

Evaluation of biomarkers, genetic mutations, and epigenetic modifications in early diagnosis of pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) is one of the deadliest malignancies with an alarming mortality rate. Despite significant advancement in diagnostics and therapeutics, early diagnosis remains elusive causing poor prognosis, marred by mutations and epigenetic modifications in key genes which contribute to disease progression. AIM: To evaluate the various biological tumor markers collectively for early diagnosis which could act as prognostic biomarkers and helps in future therapeutics of PC in Kashmir valley. METHODS: A total of 50 confirmed PC cases were included in the study to evaluate the levels of carbohydrate antigen 19-9 (CA 19-9), tissue polypeptide specific antigen (TPS), carcinoembryonic antigen (CEA), vascular endothelial growth factor-A (VEGF-A), and epidermal growth factor receptor (EGFR). Mutational analysis was performed to evaluate the mutations in Kirsten rat sarcoma (KRAS), Breast cancer type 2 (BRCA-2), and deleted in pancreatic cancer-4 (DPC-4) genes. However, epigenetic modifications (methylation of CpG islands) were performed in the promoter regions of cyclin-dependent kinase inhibitor 2A (p16; CDKN2A), MutL homolog 1 (hMLH1), and Ras association domain-containing protein 1(RASSF1A) genes. RESULTS: We found significantly elevated levels of biological markers CA 19-9 (P ≤ 0.05), TPS (P ≤ 0.05), CEA (P ≤ 0.001), and VEGF (P ≤ 0.001). Molecular genetic analysis revealed that KRAS gene mutation is predominant in codon 12 (16 subjects, P ≤ 0.05), and 13 (12 subjects, P ≤ 0.05). However, we did not find a mutation in DPC-4 (1203G > T) and BRCA-2 (617delT) genes. Furthermore, epigenetic modification revealed that CpG methylation in 21 (P ≤ 0.05) and 4 subjects in the promoter regions of the p16 and hMLH1 gene, respectively. CONCLUSION: In conclusion, CA 19-9, TPS, CEA, and VEGF levels were significantly elevated and collectively have potential as diagnostic and prognostic markers in PC. Global data of mutation in the KRAS gene commonly in codon 12 and rare in codon 13 could augment the predisposition towards PC. Additionally, methylation of the p16 gene could also modulate transcription of genes thereby increasing the predisposition and susceptibility towards PC.

Implications of VEGF gene sequence variations and its expression in recurrent pregnancy loss.

RESEARCH QUESTION: What is the association between VEGF gene sequence variants and its mRNA expression in recurrent pregnancy loss (RPL)? Vascular endothelial growth factor (VEGF) has a prominent role in pregnancy and affects pregnancy outcome. The association of VEGF gene 1154G>A, 634G>C and 583C>T polymorphic variations with cases of RPL and full-term fertile women as controls was investigated. DESIGN: Two hundred women with RPL and 240 women healthy controls were included. The restriction fragment length polymorphism method was used for genotyping and quantitative real-time polymerase chain reaction was used for analysis of mRNA expression. RESULTS: In VEGF 1154G>A, significant differences were found in homozygous AA genotype between case and control participants. The variant allele A frequency was significantly more abundant in RPL cases (0.41) than controls (0.19) (P < 0.0001). Only RPL cases with the multi-generation family history of miscarriages and those without any history showed significant differences of combined genotype GA+AA (P < 0.0001). In VEGF 634 G>C, CC genotype and allele C showed significantly increased frequency in RPL cases compared with healthy controls (P < 0.0001). The association between VEGF-1154 G>A SNP and VEGF-A mRNA expression levels was significant in RPL cases (P = 0.004). Also in VEGF-583 C>T, CT genotypes were seen significantly associated with cases (P = 0.003). The heterozygous genotype GA was significantly (P = 0.03) associated with upregulation and downregulation of VEGF mRNA, whereas the homozygous variant genotype AA only leads to low expression levels of VEGF mRNA in patients with RPL. CONCLUSIONS: All the variants of VEGF play a vital role in an increased susceptibility to RPL. Also, VEGF-1154, AA genotypes are associated with its altered low mRNA expression in women with RPL and seem to affect pregnancy outcome.

Single nucleotide polymorphisms (SNPs) in prostate cancer: its implications in diagnostics and therapeutics.

Prostate cancer is one of the most frequently diagnosed malignancies in developed countries and approximately 248,530 new cases of prostate cancer are likely to be diagnosed in the United States in 2021. During the late 1990s and 2000s, the prostate cancer-related death rate has decreased by 4% per year on average because of advancements in prostate-specific antigen (PSA) testing. However, the non-specificity of PSA to distinguish between benign and malignant forms of cancer is a major concern in the management of prostate cancer. Despite other risk factors in the pathogenesis of prostate cancer, recent advancement in molecular genetics suggests that genetic heredity plays a crucial role in prostate carcinogenesis. Approximately, 60% of heritability and more than 100 well-recognized single-nucleotide-polymorphisms (SNPs) have been found to be associated with prostate cancer and constitute a major risk factor in the development of prostate cancer. Recent findings revealed that a low to moderate effect on the progression of prostate cancer of individual SNPs was observed compared to a strong progressive effect when SNPs were in combination. Here, in this review, we made an attempt to critically analyze the role of SNPs and associated genes in the development of prostate cancer and their implications in diagnostics and therapeutics. A better understanding of the role of SNPs in prostate cancer susceptibility may improve risk prediction, enhance fine-mapping, and furnish new insights into the underlying pathophysiology of prostate cancer.

Association of strong risk of hTERT gene polymorphic variants to malignant glioma and its prognostic implications with respect to different histological types and survival of glioma cases.

BACKGROUND: Germline genetic variants of human telomerase reverse transcriptase (hTERT) are known to predispose for various malignancies, including glioma. The present study investigated genetic variation of hTERT T/G (rs2736100) and hTERT G/A (rs2736098) with respect to glioma risk. METHODS: Confirmed cases (n = 106) were tested against 210 cancer-free healthy controls by the polymerase chain reaction-restriction fragment length polymorphism technique for genotyping. RESULTS: Homozygous variant 'GG' genotype of rs2736100 frequency was > 4-fold significantly different in cases versus controls (39.6% 17.2%; p < 0.0001). Furthermore, variant 'G' allele was found to be significantly associated with cases (0.5 versus 0.2 in controls; p < 0.0001). Homozygous variant rs2736098 'AA' genotype (35.8% versus 23.8%) and allele 'A' (0.49 versus 0.34) showed a marked significant difference in cases and controls, respectively (p < 0.05). In hTERT rs2736100, the GG genotype significantly presented more in higher grades and GBM (p < 0.0001). Furthermore, the GG variant of hTERT rs2736100 had a poor probability with respect to the overall survival of patients compared to TG and TT genotypes (log rank p = 0.03). Interestingly, two haplotypes of hTERT rs2736100/rs2736098 were identified as GG and GA that conferred a > 3- and 5-fold risk to glioma patients respectively, where variant G/A haplotype was observed to have the highest impact with respect to glioma risk (p < 0.0001). CONCLUSIONS: The results of the present study indicate that hTERT rs2736098 and rs2736100 variants play an important role in conferring a strong risk of developing glioma. Furthermore, hTERT rs2736100 GG variant appears to play a role in the bad prognosis of glioma patients. Haplotypes GG and GA could prove to be vital tools for monitoring risk in glioma patients.

Cancer immunotherapy: a promising dawn in cancer research.

Cancer is a highly proliferative disease, which is caused due to the loss of regulation of cell cycle and apoptosis, DNA damage, faulty repair system etc. The cancer microenvironment plays a pivotal role in disease progression as they contain different types of innate and adaptive immune cells. The most important molecules that establish a correlation between inflammation, innate immunity, adaptive immunity, and cancer are the molecules released by inflammatory cells in cancer microenvironment. These molecules secreted by the immune cells, which might activate a pro-tumorigenic and anti-tumorigenic response in cancer. In inflammatory microenvironment, the equilibrium state of immunosuppressive and immunostimulatory signals are important in tumor suppression. The immunotherapeutic approaches could be more effective in cancer treatment. However, advancement in immunobiology and cancer are improving the prospects of immunotherapy alone and/or in combination with the conventional therapies. Thus, the review attempts to highlight a promising and futuristic immunotherapeutic approach in combination with conventional treatment modalities.

Helicobacter pylori Subdues Cytokine Signaling to Alter Mucosal Inflammation via Hypermethylation of Suppressor of Cytokine Signaling 1 Gene During Gastric Carcinogenesis.

Helicobacter pylori infection has been associated with the onset of gastric mucosal inflammation and is known to perturb the balance between T-regulatory (Treg) and T-helper 17 (Th17) cells which causes a spurt of interleukin 17 (IL17) and transforming growth factor-ß (TGF-ß) from Th17 and Treg cells within the gastric milieu. IL17 instigates a surge of interleukin 6 (IL6) from T-helper 1 (Th1) and T-helper 2 (Th2) cells. Further, H. pylori infection is known to stimulate the atypical DNA methylation in gastric mucosa. However, the precise role of cytokine signaling in induction of epigenetic modifications during gastric carcinogenesis is vaguely understood. In this study, patient samples from were examined using real-time polymerase chain reaction (qPCR), PCR, methylation-specific (MS)-PCR, and enzyme-linked immunosorbent assays. We found that H. pylori infection augments the production of interleukin 10 (IL10), IL6, and TGF-ß in the gastric milieu and systemic circulation. Together with the IL6/IL10 mediated hyperactivation of the JAK/STAT pathway, H. pylori infection causes the inactivation of suppressor of cytokine signaling 1 (SOCS1) gene through the hypermethylation of the promoter region. This study signifies that H. pylori-mediated epigenetic silencing of SOCS1 in concert with inflammatory cytokines miffs hyperactivation of the JAK/STAT cascade during gastric carcinogenesis.

Favorable role of IDH1/2 mutations aided with MGMT promoter gene methylation in the outcome of patients with malignant glioma.

AIM: The implications of molecular biomarkers IDH1/2 mutations and MGMT gene promoter methylation were evaluated for prognostic outcome of glioma patients. MATERIALS & METHODS: Glioma cases were analyzed for IDH1/2 mutations and MGMT promoter methylation by DNA sequencing and methylation-specific PCR, respectively. RESULTS: Mutations found in IDH1/2 genes totaled 63.4% (N = 40) wherein IDH1 mutations were significantly associated with oligidendrioglioma (p = 0.005) and astrocytoma (p = 0.0002). IDH1 mutants presented more, 60.5% in MGMT promoter-methylated cases (p = 0.03). IDH1 mutant cases had better survival for glioblastoma and oligodendrioglioma (log-rank p = 0.01). Multivariate analysis confirmed better survival in MGMT methylation carriers (hazard ratio [HR]: 0.59; p = 0.031). Combination of both biomarkers showed better prognosis on temozolomide (p < 0.05). CONCLUSION: IDH1/2 mutations proved independent prognostic factors in glioma and associated with MGMT methylation for better survival.

Significant Pattern of Promoter Hypermethylation of UNC5C Gene in Colorectal Cancer and Its Implication in Late Stage Disease

Background:The development of Colorectal Cancer (CRC) is a complex multistep process involving an accumulation of multiple genetic and epigenetic alterations. Epigenetic modifications, particularly DNA methylation in selected gene are recognized as common molecular alterations in human tumors. Netrin-1 receptors are aberrantly methylated in primary colorectal cancer. Epigenetic alterations in the netrin-1 receptors have been found to be related with the malignant potential of CRC. Purpose: In the present study, we evaluated the role of promoter hypermethylation of UNC5C gene (one of the netrin-1 receptors) in colorectal cancer patients of Kashmiri population (North India). Hypermethylation in tumour tissue was detected by Methylation- Specific Polymerase Chain Reaction (MS-PCR). Results: UNC5C promoter hypermethylation was significantly found to be associated with colorectal cancer cases where frequency was 62% (31 of 50) and 38% (19 of 50) patients were unmethylated (p<0.0001).UNC5C methylation was significantly higher in CRCs with a frequency of 62% than 10% in corresponding normal mucosa of (p<0.0001). Further, UNC5C hypermethylation was found to be significantly associated with stage-III/IV as compared to stage I/II with a frequency of 75.8% and 42.8% respectively(p>0.05). Conclusion: We conclude that UNC5C hypermethylation is implicated in CRC which plays a role in its tumorigenesis and may predict the late stage disease.

Role of the Functional Polymorphism of Survivin Gene (-31G/C) and Risk of Breast Cancer in a North Indian Population.

INTRODUCTION: Survivin is an apoptosis inhibitor and plays a primary role in cancer development and progression. One of the most common polymorphism of the survivin promoter -31G/C (rs9904341) influences its expression and is associated with the risk of cancer development. This study was conducted to explore survivin promoter gene -31G/C (rs9904341) polymorphism and the risk of breast cancer. PATIENTS AND METHODS: The study group included 190 pathologically confirmed breast cancer patients, in addition to 200 distinct cancer-free controls from Jammu and Kashmir region of India, where breast cancer is the most common cancer in women. Single nucleotide polymorphism genotyping for -31G/C polymorphism in the survivin promoter region was done using a polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The variant genotype/allele was found in 54.1% of the cases compared with 46.5% of controls. The combined prevalence of genotype GC+CC was significantly higher in patients compared with the control group (P = .02). Analyses of odds ratios (ORs) in the patient and control groups indicated that the presence of homozygous CC genotype was associated with increased risk for development of breast cancer (OR, 2.04; 95% confidence interval [CI], 1.07-2.98). The gene frequencies for G and C alleles were statistically different between patient and control groups (OR, 1.37; 95% CI, 1.03-1.84). CONCLUSION: The results suggest the association of -31G/C survivin polymorphism at a genotypic and allelic level in breast cancer.

SNP and Haplotype Analysis of Vascular Endothelial Growth Factor (VEGF) Gene in Lung Cancer Patients of Kashmir

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis involving tumor growth and metastasis. In this large case-control study, we investigated whether functional polymorphisms (+405C>G, +936C>T) in the VEGF gene are associated with the risk of lung cancer. The study investigates the association between variants of VEGF gene and lung cancer. We performed single nucleotide polymorphism (SNP), haplotype and linkage disequilibrium studies on 100 patients and 128 healthy controls with 2 SNPs in the VEGF gene. The results were analyzed using logistic regression models, adjusted for age and sex. No Significant association was detected between individual SNPs and lung cancer using all the models of inheritance (codominant, dominant, recessive, over dominant and additive) for finding an association between genotypes and the cancer risk. The P values obtained for two markers were nonsignificant (P>0.05). Haplotype analysis produced additional support for the non-association of individual haplotypes/ all haplotypes with the cancer risk (Global association P=0.56). Our findings suggest the non-involvement of genetic variants (+405C>G, +936C>T) of the VEGF gene in the etiology of lung cancer.

Hypermethylation Status of E-Cadherin Gene in Gastric Cancer Patients in a High Incidence Area.

Gastric cancer (GC) is the fourth most prevalant cancer and the second leading cause of cancer-related mortality worldwide. As in other cancers gastric carcinogenesis is multifactorial involving environmental, genetic and epigenetic components. Epigenetic silencing due to hypermethylation of tumour suppressor genes is one of the key events in gastric carcinogenesis. This study was aimed to analyse the hypermethylation status of the E-Cadherin (CDH1) gene promoter in GCs in the ethnic Kashmiri population. In this study a total of 80 GC patients were recruited. Hypermethylation in tumour tissue was detected by methylation specific PCR (MS-PCR). Hypermethylation of CDH1 promoter was observed in 52 (65%) of gastric carcinoma cases which was significantly much higher than adjacent normal tissue [p≤0.0001]. Further the frequency of CDH1 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [55.7% vs 82.1%; <0.05]. Moreover females and cases with lymph node invasion had higher frequencies of CDH1 hypermethylation [P≤0.05]. Thus the current data indicate a vital role of epigenetic alteration of CDH1 in the causation and development of gastric cancer, particularly of diffuse type, in our population.

PHA-Induced Peripheral Blood Cytogenetics and Molecular Analysis: a Valid Diagnostic and Follow-up Modality for Acute Promyelocytic Leukemia Patients Treated with ATRA and/or Arsenic Tri-oxide.

BACKGROUND: Acute promyelocytic leukemia (APML) is characterized by the reciprocal translocation t(15;17) (q22;q12) resulting in the PML-RARα fusion gene. A dual diagnostic and follow up approach was applied including cytogenetic demonstration of the t(15;17) translocation and detection of PML-RARα chimeric transcripts by molecular means. PURPOSE: Conventional cytogenetics involving bone marrow is beset with high probability of poor metaphase index and was substituted with phytohemagglutinin (PHA)-induced peripheral blood culture based cytogenetic analysis as a diagnostic and follow up modality in APML patients of Kashmir (North India). Both qualitative (RT-PCR) and quantitative (Q-PCR) tests were simultaneously carried out to authenticate the modified cytogenetics. MATERIALS AND METHODS: Patient samples were subjected to the said techniques to establish their baseline as well as follow-up status. RESULTS: Initial cytogenetics revealed 30 patients (81%) positive for t(15;17) whereas 7 (19%) had either cryptic translocation or were negative for t(15;17). Two cases had chromosome 16q deletion and no hallmark translocation t(15;17). Q-PCR status for PML-RARα was found to be positive for all patients. All the APML patients were reassessed at the end of consolidation phase and during maintenance phase of chemotherapy where 6 patients had molecular relapse, wherein 4 also demonstrated cytogenetic relapse. CONCLUSIONS: It was found that PHA-induced peripheral blood cytogenetics along with molecular analysis could prove a reliable modality in the diagnosis and assessment of follow up response of APML patients.

Promoter hypermethylation of KLOTHO; an anti-senescence related gene in colorectal cancer patients of Kashmir valley.

Hypermethylation of CpG islands located in the promoter regions of genes is a major event in the development of the majority of cancer types, due to the subsequent aberrant silencing of important tumor suppressor genes. KLOTHO; a novel gene associated primarily with suppressing senescence has been shown to contribute to tumorigenesis as a result of its impaired function. Recently the relevance of KLOTHO promoter hypermethylation in colorectal carcinoma in humans has been reported. We analyzed the promoter hypermethylation of KLOTHO gene in 50 histopathologically confirmed tumor and adjacent normal tissues of colorectal cancer patients. Methylation was assessed by bisulfite conversion of DNA followed by methylation specific-polymerase chain reaction. Methylation status was compared with gender, smoking status and histopathological parameters of patients. Promoter hypermethylation in KLOTHO gene was detected in 86% (43/50) of tumor tissues and 14% (7/50) of adjacent normal tissues. The methylation pattern differed significantly between tumor and adjacent normal tissues (P<0.0001). However, no association was found between promoter hypermethylation status and gender (P=0.68), smoking status (P=0.64) or other histopathological parameters (P>0.05) of colorectal cancer patients. We conclude that this novel tumor suppressor gene is epigenetically inactivated in colorectal cancer in our population paving way towards the potential of KLOTHO promoter hypermethylation as a predictor of the prognosis in colorectal cancer patients.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

PURPOSE: Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. METHODS: In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. RESULTS: Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). CONCLUSIONS: Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

CagA subtyping in Helicobacter pylori isolates from gastric cancer patients in an ethnic Kashmiri population.

UNLABELLED: The association between gastric cancer and Helicobacter pylori has been well established. Among H. pylori virulence genes the most important determinant is the cytotoxin associated antigen gene (cagA) which is characterized by the presence of repeated EPIYA motifs at the C terminus of the protein. From the alignment and number of these EPIYA motifs, two major types of CagA protein have been identified. AIMS: The aim of this study was to classify the CagA into eastern or western type and to determine the number and type of motifs present. METHODS: The CagA subtyping was done by PCR and multiplex PCR for eastern/western classification and determination of EPIYA motifs respectively. RESULTS: All the isolates studied were of the western type, with 70% of the isolates having more than one EPIYA-C motifs. No statistically significant association was found between the presence of CagA and more than one EPIYA-C motifs with the clinical outcome (differentiation status of the tumour).

Study of TLR4 and IL-8 gene polymorphisms in H.pylori-induced inflammation in gastric cancer in an ethnic Kashmiri population.

BACKGROUND: TLRs play an essential role in the initial handling of H. pylori and determine the clinical outcomes that range from simple asymptomatic gastritis to peptic ulcer disease and gastric cancer. Asp299Gly and Thr399Ile polymorphisms in TLR4 have been associated with a variety of inflammatory and infectious conditions including gastric cancer. The T-251A polymorphism in the promoter region of IL-8 gene has been found to be associated with changing the in vitro levels of IL-8 production. IL-8 exhibits angiogenic activity and is responsible for tumor-associated angiogenesis in several cancers. MATERIALS AND METHODS: 130 gastric cancer patients and 200 healthy controls were included in this study. DNA extraction was followed by PCR detection of H. pylori infection, PCR-RFLP for the TLR 4 polymorphism and PCR-CTPP for IL-8 gene polymorphism. RESULTS: The adjusted OR for gastric cancer risk was 1.15 (95% CI, 0.8357-1.3463); 1.39 (0.6964-2.781) and 1.43 (0.954-2.1515) for Asp299Gly, Thr399Ile and IL-8 T_251A respectively. Odds Ratio analysis showed CT genotype and AT and AA genotypes as risk factors for the development of gastric cancer. We found the Asp299Gly polymorphism carrier to be significantly associated (p value 0.03)with the development of tumours in the distal part of the stomach and Thr399Ile polymorphism to be significantly associated(p value 0.008) with the development of well-differentiated gastric adenocarcinoma.The IL-8 T-251A polymorphism was not found to be associated with any of the clinicopathological characteristics. DISCUSSION: No correlation was found between the appearance of disease and HP infection or the presence of TLR4 and IL-8 gene polymorphisms and HP infection.