Highly sensitive mtDNA sequencing in Head and neck squamous cell carcinoma as a marker of intratumoral heterogeneity and lymph node metastasis

The prognosis and treatment regiments vary dramatically upon the manifestation of lymph node (LN) metastasis in Head and neck squamous cell carcinoma (HNSCC). Current tools for detection of tumor cells in resected LN may miss the presence of micrometastasis. Novel strategies based on the analysis of genetic aberrations by NGS offer new hope for improved risk assessment and selection of the treatment regimen. By virtue of their clonal nature, higher mutation rate and copy number, assessing mitochondrial DNA (mtDNA) mutations in histologically clean LN may provide a sensitive diagnostic tool. Additionally, due to its circular configuration, mtDNA thought to be more stable than genomic DNA, and may also be suitable for sequencing formalin-fixed paraffin-embedded derived genomic material, an invaluable resource for clinical research. We have developed a novel amplicon-based approach for deep sequencing of the entire mtDNA specifically suited for micro-sized specimens. We sequenced mtDNA from 28 HNSCCs and multiple matched metastatic or histologically clean LN. We also sequenced saliva, serum and histological clean surgical margins. Furthermore, to assess intra-tumor heterogeneity, a multiregional sequencing approach was applied. Overall, we obtained over 97% coverage with a median average depth of 4179X. Although our analysis revealed large intra-tumor heterogeneity, clonal events were detected in 8 out of 16 patients with multi-regional collections. Interestingly, 6 patients carried mtDNA mutations in LNs that were histologically free of atypical cells. Taken together, this quick, sensitive and cost-efficient method may be used for detection of low frequency tumor-associated mtDNA mutations as a measurer of possible metastatic processes in histologically clean LN.

Publication History

Publication Date:
18 April 2018 (online)