Mitochondrial DNA Genome as Biomarker in Oral Squamous Cell Carcinoma from Northeast India Mitochondrial DNA Genome as Biomarker in Oral Squamous Cell Carcinoma from Northeast India Rosy Mondal 1 , Jagadish Hansa 1 , R.S.Laskar 1,2 , Fazlur Rahman 1 , Rajeev Kumar 2 , Ravi Kannan 2 , and Sankar Kumar Ghosh 1 1 Department of Biotechnology, Assam University, Department of Biotechnology, Assam University, 2 2 Cachar Cancer Hospital and Research Centre Cachar Cancer Hospital and Research Centre Silchar-788011, Assam, India INTRODUCTION India has one of the world’s highest incidence of oral squamous cell carcinoma (OSCC) which is the most common malignancy among head and neck cancers (HNC) and accounts for 30-40% cancers at all sites, out of which 9.4% being oral cancer. Prevalence of tobacco related oral squamous cell carcinoma in North east India is highest among all the states which is about 33% . Among the Northeastern states of India, Assam accounts for the highest prevalence of tobacco related oral cancer. 6.92 18.46 30.77 3.85 1.54 2.31 6.15 4.62 1.54 1.54 8.46 3.08 6.15 1.54 1.54 0.77 0.77 0 5 10 15 20 25 30 35 2007 STATISTICS OF HEAD AND NECK CANCER Mitochondrial dysfunction is a hallmark of cancer cells and mitochondrial DNA (mtDNA) mutations are strewn throughout the coding and noncoding regions. 6 10 6 5 4 29 2 26 3 2 0 5 10 15 20 25 30 35 2009 STATISTICS OF HEAD AND NECK CANCER 22 1 22 5 3 1 32 26 46 3 0 5 10 15 20 25 30 35 40 45 50 2010 STATISTICS OF HEAD AND NECK CANCER MITOCHONDRIAL DNA MUTATION IN ORALCANCER It is hypothesized that mitochondrial defects are present in tumors due to damaged respiratory systems and ATP production. Tobacco consumption in various forms is the major risk factor for the development of OSCC which not only causes single strand breakage in the DNA but also makes the mitochondrial DNA susceptible to damage by reactive oxygen species (ROS). The mutations in non coding region approximately 1,122 nucleotides which is called as control region, D-loop or hypervariable region. have been reported in OSCC. The D-loop has a polycytidine stretch (C- tract) also termed the D310 region has been found to be a ‘‘hot spot’’ for somatic mutations in many cancer types . Severe mutations might be of biological significance, because the D310 region lies in a conserved sequence block that is hypothesized to be involved in some aspect of mtDNA replication and transcription. The environment-gene interaction on carcinogenesis has been well illustrated by phase I and phase II enzymes that are involved in the metabolism of carcinogenesis. The phase I enzymes are CYPs (Cytochrome P450) that are involved in activating the environmental procarcinogens adding or exposing their functional groups. Phase II enzyme like GST (Glutathione S-transferase) are involved in GENETIC AND ENVIRONMENTAL FACTORS RESULTS 1.63 30.89 4.47 0.81 14.23 1.63 0.81 0.81 18.70 21.14 2.85 0.81 1.22 0 5 10 15 20 25 30 35 2008 STATISTICS OF HEAD AND NECK CANCER Different types of mutations were observed in the D-loop region between nt 51 and 595. Out of the 25 patients 16 (64%) patients had mutation at nucleotide position 146, 17(68%) patients had mutation at nucleotide position 152 and 12(48%) patients had mutation in nt 146 and nt152 that occurred simultaneously and nucleotides 146 and 152 were significant mutational hotspots with P=0.01 and P=0.006 respectively. The frequency of GSTM1 null genotype was found to be 15(60%), GSTT1 null genotype 10(40%) and both GSTT1 and GSTM1 null genotype 6(24%) respectively.  High frequencies of somatic mutations are reported in the mtDNA for OSCC cases in our series, 44% of D-loop mutation occurs in null GSTM1 whereas 32% of D-loop mutations occur in null GSTT1. nt146 nt152 STUDY OF POPULATION MUTATION DETECTION IN D-LOOP Analyzed the case files of Cachar Cancer Hospital and Research Centre for the years 2007, 2008, 2009 and 2010 and found that there was a statistically significant association between gender and prevalence of cancer which is biased towards males (P=0.002) and most of patient’s age ranges between 50-60. From these case files we selected 25 OSCC patients randomly for our study and the age range of the patients was 28-76 where 18(72%) patients were male and 7(28%) were female. Of the males 13 were classified as betel quid chewer, tobacco chewer and 5 males as betel quid chewer, tobacco chewer and smoker where in females 6 were found to be betel quid chewer and tobacco chewer whereas 1 female was found to be tobacco chewer and smoker. 6 8 10 s ratio GSTT1& GSTM1 POLYMORPHISM The mutations found in tumour tissue excluding the D310 region, 73% of the mutation is heteroplasmy and 27% of the mutation is homoplasmy whereas in the D310 region sequence in the tissue, 76% was heteroplasmic and 24% homoplasmic . Here the majority of the mtDNA mutations in the D-loop region detected were heteroplasmic. It has been reported that heteroplasmic mitochondrial DNA mutation promotes tumorigenesis by making alteration in ROS generation and apoptosis. HETEROPLASMY & HOMOPLASMY 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Number of mutations in the d-loop region Tumor stage NO. OF OSCC PATIENTS Figure - Correlation between the increasing numbers of mutations in D-loop region of mtDNA with the increasing tumor stage (I-IV) of the patients (1-25) using Spearman Rank Correlation test (P<0.001, r =0.74). C-tract Figure-Frequency distribution of D310 polymorphisms in lymphocytes and in tumour from OSCC patients in context to homoplasmy and heteroplasmy C7 40% C8 37% C9 23% MATCHED BLOOD C6 5% C7 18% C8 46% C9 31% TUMOUR Figure-(A) Shows heteroplasmy where the blood C-tract sequence is C7 but in the matched tumour tissue is C8/C9 whereas in (B) showing homoplasmy, the blood C-tract sequence is C8 and in the matched tumour tissue is C9. BLOOD TUMOUR HOMOPLASMY HOMOPLASMY HETEROPLASMY HETEROPLASMY BLOOD TUMOUR A B The mtDNA mutations identified in our study occurred in the hypervariable D-loop region. AACR SPECIAL CONFERENCE IN CANCER RESEARCH 13-16 th DECEMBER 2011 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 Phase II enzyme like GST (Glutathione S-transferase) are involved in detoxication of the activated metabolites of the carcinogens. In phase II enzymes like GSTM1 catalyses with the conjugation of the tripeptide GSH to PAH diol epoxides whereas GSTT1 participates in detoxication of the monohalomethanes and reactive diol epoxides. A structural deletion in these genes represents a null genotype and has been associated with an increased risk to oral cancer. Polymorphisms in the genes that code for these enzymes may alter expression or function, thus increasing or decreasing the activation or detoxication of carcinogenic compounds. MATERIALS AND MATERIALS Specimens were collected from 25 cancer patients of OSCC from Cachar Cancer Hospital and Research Centre after written informed consent obtained from each patient and approval of the hospital ethics committee (Serial No: IRB/CCHRC/01/2010). Isolation of the Gnomic and mitochondrial DNA is done from the cancerous tissues using the standard Phenol Chloroform Extraction. D2 D3 D8 D9 D10 D13 D11 D12 D5 D6 D7 D4 All PCR products were purified using PCR Purification Kit (Qiagen, K),sequenced (MWGAG –BIOTECH, Banglore, India) . Any mtDNA sequences that differed between tumour sample and its matched blood mtDNA were scored as somatic mtDNA mutations specific to the tumor using nucleotide BLAST Statistical analysis for association studies were done with Spearman Rank Correlation. Relative risk was estimated using online software (http://statpages.org/logistic.html ). TABLE: RISK GENOTYPES: ANALYSIS WITH RESPECT TO D TABLE: RISK GENOTYPES: ANALYSIS WITH RESPECT TO D-LOOP MUTATIONS LOOP MUTATIONS D-loop mutations Gene Absence n (%) Presence n (%) Odds ratio (95%CI) P Value GSTM1 Null + 12 22 44 22 3.66 (1.07-12.54) 0.038 GSTT1 Null + 4 26 32 34 3.42 (0.89-13.09) 0.094 The mutations were base substitution 3(3.8%),transversions (AT),26(33.3%);transitions(CT), 27(34.6%); transitions(TC),7(9%); transitions(AG),5(6.4%);transversions(CG),5(6.4%);transversions(C A),2(2.6%);transversions(GC), 1(1.3%);transversion(TG), 1(1.3%); transversion (TA), 1 (1.3%) and transition (GA). A significant association was found between GSTM1 and GSTT1 null genotypes and presence of D-loop mutation (OR=3.66[1.07-12.54]; 95% CI, P=0.038 and OR=3.42[0.89-13.09]; 95% CI P=0.094 respectively). A significant association between GSTT1 and GSTM1 null genotypes with the increased number of D-loop mutation (OR=1.73[1.10-2.71]; 95% CI,P=0.0027 and OR=2.03[1.04-3.96]; 95% CI, P=0.003 respectively) and the risk increases further with both GSTT1 and GSTM1 null genotypes with increased number of mutations (OR=7.77[1.13-53.28]: 95% CI, P=0.036) Figure-A) Showing the hot spot mutation at nucleotide position 146, in blood T changes to C in tumour. B) hot spot at nucleotide position 152 where in blood T changes to c in tumour. nt146 nt152 BLOOD BLOOD TUMOR TUMOR A B SAMPLE COLLECTION AND DNA ISOLATION PCR AMPLIFICATION DNA SEQUENCING AND BIOINFORMATICS TOOLS OBJECTIVES The present study is aimed to investigate the prevalence of OSCC in Northeast region of India, highly prone to head and neck cancer.  The effect of betel quid and tobacco use on the D-loop regon mutation of oral cancer patients. Establishing the correlation between the D-loop mutations with that of tumour stage.  The association of D-loop mutations and GSTT1 and GSTM1 gene polymorphism to the risk towards oral cancer progression along with establishing the hot spot mutations in the D-loop region in patients of oral squamous cell carcinoma SUMMARY OF THE D-LOOP SOMATIC MUTATIONS FOUND IN THE 25 OSCC PATIENTS Sl. No. Age Sex Tobacco/smoking /alcohol consumption status 146 position 152 position 196 position 235 position 589 position Ins/del compared with normal matched tissue Tumour stage 1 60 M a, b CT TC 303-309(+C) 311-315(+C) 2 2 76 M a, b TC CT 303-309(+C) 311-315(+C)/310(-T) 3 3 45 F a, b 303-309(+1C) 311-315(+1C) 3 4 40 M a, b CT 303-309(NC) 311-315(+2C) 3 5 56 M a, b TC 303-309(NC) 311-315(+1C)/310(-T) 1 6 42 M a, b, c TC TC AT 303-309(+1C) 311-315(+2C) 3 7 60 M a, b TC CT TC AT 303-309(NC) 311-315(+2C) 4 8 28 F a, b ,c TC CT GA TA 303-309(+2C) 311-315(+1C)/310(-T) 2 9 65 F a, b CT CT AT 303-309(+1C) 311-315(+1C) 3 10 65 M a, b CT TC AG 303-309(NC) 311-315(+2C) 3 11 65 M a, b , c CT CT 303-309(NC) 311-315(+2C) 4 12 63 F a, b CT 303-309(NC) 311-315(+2C)/310(-T) 2 13 73 M a, b CT CT 303-309(+2C) 311-315(+1C) 4 14 62 M a, b ,c, d CT CT TC AG 303-309(+1C) 311-315(+1C) 3 15 70 M a, b, c CT 303-309(+1C) 311-315(+1C) 3 16 38 M a, b CT CT 303-309(+1C) 311-315(+1C)/310(-T) 1 17 36 M a, b CT CT 303-309(NC) 311-315(+2C) 2 18 60 M a, b CT 303-309(NC) 311-315(+2C) 1 19 33 F a, b, d TC 303-309(+1C) 311-315(+1C) 3 20 35 M a, b CT TC 303-309(+1C) 311-315(+2C) 3 21 75 M a, b, c TC CT TC 303-309(+1C) 311-315(+2C) 3 22 30 M a, b AG 303-309(+1C) 311-315(+2C) 3 23 45 F a, b TC CT 303-309(NC) 311-315(+2C) 3 24 40 F a, b TC CT 303-309(+2C) 311-315(+1C) 2 25 64 M a, b 303-309(+1C) 311-315(+1C) 2 Patients are identified by code number. F, female; M, male. Mutations are sectioned by column according to nucleotide number. Mutations are described according to nucleotide change, e.g. AG means A was present in sequence in normal tissue but changed to a G in sequence in tumour tissue. Betel quid chewer is denoted by alphabet- a, tobacco chewer - b, smoker - c and alcohol user - d. For insertions and deletions: +C indicates a single insertion. -T indicates a single deletion, NC indicates no change. Tumour stages denoted by the respective numerals e.g. stage II2. 0 2 4 GSTT1 Null GSTM1 Null GSTT1+GSTM1 Null odds No. of OSCC patients CONCLUSION PRIMER NAME PRIMER SEQUENCE PRODUCT SIZE (bp) PCR programme Cycle -30,RAM-10mins Denaturation Annealing Extension D-LOOP Forward-5’-CAGGGTCATAAAGCCTAAATAG-3’ 649 94 0 C/2mins 56 0 C/ 45 sec. 72 0 C/ 45 sec Reverse-5’-GAGGTAAGCTACATAAACTGTG-3’ PRIMER SEQUENCE FOR MULTIPLEX PCR GSTT1 Forward-5’ TCCTTACTGGTCCTCACATTCTC-3’ 215 94 0 C 2mins 56 0 C 45 sec 72 0 C 45 sec Reverse-5’ TCACGGGATCATGGCCAGCA-3’ GSTM1 Forward-5’ GAACTCCCTGAAAAGCTAAAGC-3’ 480 Reverse-5’GTTGGGCTCAAATATACGGTGG-3’ CYP1 Forward-5’GAACTGCCACTTCAGCTGTCT-3’ 315 Reverse-5’GCTGCATTTGGAAGTGCTC-3’ 649 D-LOOP AMPLIFIED PRODUCT We can conclude that the information about role, patterns and timing of mitochondrial mutations in HNSCC may serve to be potentially facilitate clinical applications in relation to detect mutations and also the knowledge of role of mitochondrial mutation in tumor biology and in particular, mutations present in specific type of tumor may be helpful in assessing cancer risk, distinguishing between new primary cancer and recurrence. D-loop region of mtDNA is important in the replication process of mitochondrial DNA. In this regard, our finding of high frequency of mutations in D-loop mtDNAof tumour cells is motivating.  Our study is preliminary and more investigation is required into the rate of mutations in other mitochondrial regions.  Large-scale studies are also needed to be done to address and clarify the significance of mtDNA mutation. Further research will be also needed to determine if mtDNA analysis has the specificity for detecting changes in smoke exposure or if the estimation of an individual’s risk could be improved by coupling of mitochondrial mutations to other markers for tobacco and betel quid chewing-associated disease risk or smoking-related harm. Figure Showing D-loop amplified PCR product in 1.5% agarose gel having the DNA marker on left lane from the Oral cancer patients. Figure lane D3,D10 showing Null genotype of GSTT1 and GSM1.D1,D2,D5 showing amplification of both GSTT1 and GSTM1 Odds ratio for increase in mtDNA D-loop mutation in patients with GSTT1 and GSTM1 null genotypes. ACKNOWLEDGEMENT We thank Cachar Cancer Hospital and Research Centre (CCHRC) for the biological samples and Department of Biotechnology (DBT) for infrastructural support. GSTM1 480bp GSTT1 215bp CONTROL CYP1