Fazlur Rahman Talukdar

Epigenetic, Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes. Methodology/Principal Findings The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models. Conclusion Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.

Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

Background Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients. Methodology/Principal Findings The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC. Conclusion Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.

Association of mitochondrial D-loop mutations with GSTM1 and GSTT1 polymorphisms in oral carcinoma: A case control study from Northeast India

OBJECTIVES Mitochondrial dysfunction is a hallmark of cancer cells. Tobacco consumption in various forms is one of the major risk factors for the development of oral squamous cell carcinoma which makes the mitochondrial DNA susceptible to damage by reactive oxygen species. The GSTT1 and GSTM1 members of the glutathione S-transferase multigene family are candidate carcinogen metabolizing genes. Here we determined the hot spot mutations in the D-loop region and revealing correlation if any, with clinical parameters, along with analysing the genetic polymorphism of GSTT1 and GSTM1 and its susceptibility towards oral cancer. MATERIALS AND METHODS To determine the hot spot mutations 25 matched tissue samples of OSCC patients with 25 control subjects were used for PCR and direct sequencing. Analysis for GSTM1 and GSTT1 gene polymorphism was done by multiplex PCR. RESULTS Several mutations were found within the D-loop region among which mutations at nt146, nt152 and nt196 are found to be hot spot (P<0.0001, P<0.0001 and P<0.001 respectively). A significant association was found between the numbers of D-loop mutation and GSTM1 (OR=2.03; 95% CI, 1.04-3.96, P=0.003), GSTT1 (OR=1.73; 95% CI, 1.10-2.71, P=0.0027) null genotypes respectively. We observed a significant correlation between the increased number of D-loop mutations with the advancement in tumour stage of the patients (P=0.009, r=0.48). CONCLUSION The association of null genotypes and mutations can be used as a possible biomarker for early detection and preventive measure of oral cancer for those habituated to tobacco consumption.

Tobacco carcinogen-metabolizing genes CYP1A1, GSTM1, and GSTT1 polymorphisms and their interaction with tobacco exposure influence the risk of head and neck cancer in Northeast Indian population

Genetic polymorphisms in tobacco-metabolizing genes may modulate the risk of head and neck cancer (HNC). In Northeast India, head and neck cancers and tobacco consumption remains most prevalent. The aim of the study was to investigate the combined effect of cytochrome P450 1A1 (CYP1A1) T3801C, glutathione S-transferases (GSTs) genes polymorphisms and smoking and tobacco–betel quid chewing in the risk of HNC. The study included 420 subjects (180 cases and 240 controls) from Northeast Indian population. Polymorphisms of CYP1A1 T3801C and GST (M1 & T1) were studied by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR, respectively. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approach were applied for statistical analysis. LR analysis revealed that subjects carrying CYP1A1 TC/CC + GSTM1 null genotypes had 3.52-fold (P < 0.001) increase the risk of head and neck squamous cell carcinoma (HNSCC). Smokers carrying CYP1A1 TC/CC + GSTM1 null and CYP1A1 TC/CC + GSTT1 null genotypes showed significant association with HNC risk (odds ratio [OR] = 6.42; P < 0.001 and 3.86; P = 0.005, respectively). Similarly, tobacco–betel quid chewers carrying CYP1A1 TC/CC + GSTM1 null genotypes also had several fold increased risk of HNC (P < 0.001). In MDR analysis, the best model for HNSCC risk was the four-factor model of tobacco–betel quid chewing, smoking, CYP1A1 TC/CC, and GSTM1 null genotypes (testing balance accuracy [TBA] = 0.6292; cross-validation consistency [CVC] = 9/10 and P < 0.0001). These findings suggest that interaction of combined genotypes of carcinogen-metabolizing genes with environmental factors might modulate susceptibility of HNC in Northeast Indian population.

TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors

Summary Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

Rectal cancer profiling identifies distinct subtypes in India based on age at onset, genetic, epigenetic and clinicopathological characteristics

Rectal cancer is a heterogeneous disease that develops through multiple pathways characterized by genetic and epigenetic alterations. India has a comparatively higher proportion of rectal cancers and early‐onset cases. We analyzed genetic (KRAS, TP53 and BRAF mutations, and MSI), epigenetic alterations (CpG island methylation detection of 10 tumor‐related genes/loci), the associated clinicopathological features and survival trend in 80 primary rectal cancer patients from India. MSI was detected using BAT 25 and BAT 26 mononucleotide markers and mutation of KRAS, TP53, and BRAF V600E was detected by direct sequencing. Methyl specific polymerase chain reaction was used to determine promoter methylation status of the classic CIMP panel markers (P16, hMLH1, MINT1, MINT2, and MINT31) as well as other tumor specific genes (DAPK, RASSF1, BRCA1, and GSTP1). MSI and BRAF mutations were uncommon but high frequencies of overall KRAS mutations (67.5%); low KRAS codon 12 and a novel KRAS G15S mutation with concomitant RASSF1 methylation in early onset cases were remarkable. Hierarchical clustering as well as principal component analysis identified three distinct subgroups of patients having discrete age at onset, clinicopathological, molecular and survival characteristics: (i) a KRAS associated CIMP‐high subgroup; (ii) a significantly younger MSS, CIMP low, TP53 mutant group having differential KRAS mutation patterns, and (iii) a CIMP‐negative, TP53 mutated group. The early onset subgroup exhibited the most unfavorable disease characteristics with advanced stage, poorly differentiated tumors and had the poorest survival compared to the other subgroups. Genetic and epigenetic profiling of rectal cancer patients identified distinct subtypes in Indian population.

Epigenetic pathogenesis of human papillomavirus in upper aerodigestive tract cancers

Human papillomavirus (HPV) has been recently associated with squamous cell carcinoma of upper aerodigestive tract (SCC of UADT), but its possible role in promoting aberrant methylation in these tumors has largely remained unexplored. Herein, we investigated the association of HPV with aberrant methylation in tumor‐related genes/loci consisting of the classical CpG Island Methylator Phenotype (CIMP) panel markers (p16, MLH1, MINT1, MINT2, and MINT31) and other frequently methylated cancer‐related genes (DAPK1, GSTP1, BRCA1, ECAD, and RASSF1) and survival of UDAT cancers. The study includes 219 SCC of UADT patients from different hospitals of Northeast India. Detection of HPV and aberrant promoter methylation was performed by PCR and Methylation Specific PCR respectively. Association study was conducted by Logistic regression analysis and overall survival analysis was done by Kaplan–Meier plot. HPV was detected in 37% of cases, with HPV‐18 as the major high‐risk sub‐type. Although HPV presence did not seem to affect survival in overall UADT cancers, but was associated with a favourable prognosis in head and neck squamous cell carcinoma. Hierarchical clustering revealed three distinct clusters with different methylation profile and HPV presence. Among these, the CIMP‐high subgroup exhibited the highest HPV positive cases (66%). Furthermore, multivariate analysis revealed a strong synergistic association of HPV and tobacco towards modulating promoter hypermethylation in UADT cancer (OR = 27.50 [95% CI = 11.51–88.03] for CIMP‐high vs. CIMP‐low). The present study proposes a potential role of HPV in impelling aberrant methylation in specific tumor related loci, which might contribute in the initiation and progression of SCC of UADT.

Molecular landscape of esophageal cancer: implications for early detection and personalized therapy

Esophageal cancer (EC) is one of the most lethal cancers and a public health concern worldwide, owing to late diagnosis and lack of efficient treatment. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are main histopathological subtypes of EC that show striking differences in geographical distribution, possibly due to differences in exposure to risk factors and lifestyles. ESCC and EAC are distinct diseases in terms of cell of origin, epidemiology, and molecular architecture of tumor cells. Past efforts aimed at translating potential molecular candidates into clinical practice proved to be challenging, underscoring the need for identifying novel candidates for early diagnosis and therapy of EC. Several major international efforts have brought about important advances in identifying molecular landscapes of ESCC and EAC toward understanding molecular mechanisms and critical molecular events driving the progression and pathological features of the disease. In our review, we summarize recent advances in the areas of genomics and epigenomics of ESCC and EAC, their mutational signatures and immunotherapy. We also discuss implications of recent advances in characterizing the genome and epigenome of EC for the discovery of diagnostic/prognostic biomarkers and development of new targets for personalized treatment and prevention.