Kumar Manish, Srivastava Shilpee, Singh Seram Anil, Das Anup Kumar, Das Ganesh Chandra, Dhar Bishal, Ghosh Sankar Kumar, Mondal Rosy
1 Molecular Medicine Laboratory, Department of Biotechnology, Assam University, Silchar, India.
2 Department of Applied Biology, University of Science and Technology, Meghalaya, India.
Tumour Biol. 2017 Oct;39(10):1010428317736643. doi: 10.1177/1010428317736643.
Head and neck squamous cell carcinoma is the most commonly diagnosed cancer worldwide. The lifestyle, food habits, and customary practices manifest the Northeast Indian population toward higher susceptibility to develop head and neck squamous cell carcinoma. Here, we have investigated the association of smoke and smokeless tobacco, and alcohol with copy number variation of cell-free mitochondrial DNA and cell-free nuclear DNA in cases and controls. Cell-free DNA from plasma was isolated from 50 head and neck squamous cell carcinoma cases and 50 controls with informed written consent using QIAamp Circulating Nucleic Acid Kit. Real-time polymerase chain reaction was done for copy number variation in cell-free mitochondrial DNA and cell-free nuclear DNA. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic application between the two study groups using clinicopathological parameters. The levels of cell-free nuclear DNA and cell-free mitochondrial DNA of cases in association with smoke and smokeless tobacco, alcohol with smoking (p < 0.05) were significantly higher (p < 0.01 and p < 0.001, respectively) than controls. Using receiver operating characteristic curve analysis between head and neck squamous cell carcinoma cases and controls, we distinguished cell-free mitochondrial DNA (cutoff: 19.84 raw Ct; sensitivity: 84%; specificity: 100%; p < 0.001) and cell-free nuclear DNA (cutoff: 463,282 genomic equivalent/mL; sensitivity: 53%; specificity: 87%; p < 0.001). The copy number variation in cases (cell-free nuclear DNA: 5451.66 genomic equivalent/mL and cell-free mitochondrial DNA: 29,103,476.15 genomic equivalent/mL) and controls (cell-free nuclear DNA: 1650.9 genomic equivalent/mL and cell-free mitochondrial DNA: 9,189,312.54 genomic equivalent/mL), respectively. Our result indicates that the cell-free mitochondrial DNA content is highly associated with smoke and smokeless tobacco, betel quid chewing, and alcohol which shows greater promises, holding the key characteristics of diagnostic biomarkers, that is, minimal invasiveness, high specificity, and sensitivity.
头颈鳞状细胞癌是全球最常被诊断出的癌症。生活方式、饮食习惯和习俗使印度东北部人群更易患头颈鳞状细胞癌。在此，我们研究了吸烟和无烟烟草以及酒精与病例组和对照组中游离线粒体DNA和游离核DNA拷贝数变异之间的关联。在获得知情书面同意后，使用QIAamp循环核酸试剂盒从50名头颈鳞状细胞癌病例和50名对照的血浆中分离游离DNA。对游离线粒体DNA和游离核DNA的拷贝数变异进行实时聚合酶链反应。使用临床病理参数进行受试者工作特征曲线分析，以评估两个研究组之间的诊断应用。病例组中与吸烟和无烟烟草、饮酒相关的游离核DNA和游离线粒体DNA水平（p < 0.05）显著高于对照组（分别为p < 0.01和p < 0.001）。通过对头颈鳞状细胞癌病例和对照进行受试者工作特征曲线分析，我们区分了游离线粒体DNA（临界值：19.84原始Ct；敏感性：84%；特异性：100%；p < 0.001）和游离核DNA（临界值：463,282基因组当量/mL；敏感性：53%；特异性：87%；p < 0.001）。病例组（游离核DNA：5451.66基因组当量/mL，游离线粒体DNA：29,103,476.15基因组当量/mL）和对照组（游离核DNA：1650.9基因组当量/mL，游离线粒体DNA：9,189,312.54基因组当量/mL）的拷贝数变异情况。我们的结果表明，游离线粒体DNA含量与吸烟和无烟烟草、嚼槟榔以及饮酒高度相关，具有更大的前景，具备诊断生物标志物的关键特征，即微创性、高特异性和敏感性。
