Cell-free mitochondrial DNA copy number variation in head and neck squamous cell carcinoma: A study of non-invasive biomarker from Northeast India

Abstract

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.

Review of emerging biomarkers in head and neck squamous cell carcinoma in the era of immunotherapy and targeted therapy

“…More recently, markers from liquid biopsies that are directly related to cancer cells or cancer‐produced molecules have been employed, including circulating tumor cells (CTCs), cell‐free DNA (cfDNA)/circulating tumor DNA (ctDNA), cfRNA, and exosomes . It is generally accepted that CTCs may play an important role in cancer metastasis .…”

“…In addition, PD‐L1‐positive CTCs may play a role in predicting response to immunotherapy . With regard to cfDNA, mitochondrial cfDNA content appears to be strongly associated with HNSCC in patients with lifestyle risk factors for the disease …”

“…188 However, some are not always cancer-specific, giving rise to problems in distinguishing between malignant and inflammatory disease. [189][190][191][192] More recently, markers from liquid biopsies that are directly related to cancer cells or cancer-produced molecules have been employed, including circulating tumor cells (CTCs), 147,[150][151][152][153][154][155][156][157][158][159][160][161][162][163][164]193,194 cell-free DNA (cfDNA)/ circulating tumor DNA (ctDNA), [187][188][189][190][191][192]195,196 cfRNA, 184 and exosomes. [196][197][198] It is generally accepted that CTCs may play an important role in cancer metastasis.…”

“…163,182 With regard to cfDNA, mitochondrial cfDNA content appears to be strongly associated with HNSCC in patients with lifestyle risk factors for the disease. 155 Various other liquid biomarkers may provide additional diagnostic and prognostic information in HNSCC. Examples include elevated serum miR-21, 179 circulating PD-L1 + Note: To calculate a CPS, the pathologist must score the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages), divide that total by the number of viable tumor cells, and multiply by 100.…”

Life and death of circulating cell-free DNA

“…Some studies have also described small extrachromosomal circular DNA (eccDNA), microDNA, and single-stranded (ss), viral, bacterial, or food-derived (plant and meat) DNAs. [64][65][66][67] Most cfDNA that originates from the nucleus is packaged in the form of mono-or oligonucleosomes. 68 cfDNA is present both on the surface and in the lumen of vesicles.…”