Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.

中文翻译：

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使用线粒体基因组超深度测序发现体细胞线粒体突变揭示了头颈鳞状细胞癌的空间肿瘤异质性。


线粒体 DNA (mtDNA) 突变与头颈鳞状细胞癌 (HNSCC) 的风险、进展和治疗反应有关。由于其克隆性质和高拷贝数，线粒体突变可以作为强大的分子标记，用于检测体液、手术切缘、活检和淋巴结（LN）转移中的癌细胞，特别是在组织学上肿瘤受累不明显的部位。尽管迫切需要高通量、经济高效的 mtDNA 突变分析系统，但目前的文库制备方法对于检测低流行异质突变仍然不完善。为此，我们设计了一种基于超深扩增子的测序文库制备方法，覆盖整个线粒体基因组。我们对 28 个 HNSCC 的 mtDNA 进行了测序，匹配了淋巴结、手术切缘和体液，并对 14 个原发性肿瘤应用了多区域测序方法。我们的结果表明，这种快速、灵敏且经济高效的方法可以获得线粒体异质性的快照，并且可用于检测 LN、痰和血清样本中与肿瘤相关的低频 mtDNA 突变。这些发现为利用线粒体测序进行风险评估、早期检测和肿瘤监测奠定了基础。