Abstract 4667: mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis (2017)

Abstract Head and neck squamous cell carcinoma (HNSCC), accounts for 650,000 new cases worldwide. It is notorious for poor prognosis, which reflects its propensity to present as metastatic disease upon diagnosis. The prognosis and treatment regiments vary dramatically upon the manifestation of lymph node (LN) metastasis. Detection of regional LN metastasis is usually based on the clinical examination and imaging, followed by post-operational histological analysis. Unfortunately, current tools for detection of tumor cells in surgically resected LN may, in some cases, miss the presence of micrometastasis. Novel strategies for detection of micrometastasis based on the analysis of tumor-derived genetic aberrations by next generation sequencing (NGS) offer new hope for improved risk assessment and better selection of the treatment regimen. However, detection of mutations in minuscule metastatic lesions comprised of only few neoplastic cells may still fall below the detection threshold of these highly sensitive techniques. By virtue of their clonal nature, higher mutation rate and copy number, assessing tumor-specific mitochondrial DNA (mtDNA) mutations in histologically clean LN may provide a more sensitive diagnostic tool and eventually reduce the false negative rate in patients. 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 (FFPE) -derived genomic material, an invaluable resource for clinical research. However, current methods for library preparation are still imperfect for detection of low prevalence variants and heteroplasmy with high depth of coverage, especially in highly degraded samples. To this end, we aimed to set up a novel NGS assay for mtDNA analysis in primary and metastatic samples. Amplicon-based NGS library preparation approach, consisting of 148 primers pairs that cover the entire mitochondrial genome (with a dual coverage of 86.18%) was designed in collaboration with Fluidigm to be used on Access Array™ platform. To enhance the coverage of degraded material the amplicon size was limited to ~170 base pairs. Primers for three nuclear housekeeping genes were added to the panel to allow quantitative copy number analysis. To address mitochondrial mutational patterns, heteroplasmy and heterogeneity, we have used this novel high-throughput enrichment technology to sequence mtDNA from different areas of 16 primary HNSCC tumors (fresh frozen and FFPE), and multiple matched metastatic or histologically clean lymph-nodes. We obtained over 99% coverage with a median average depth of 3000X and no difference between fresh frozen and FFPE samples. This method allows obtaining a snapshot on the extent of mitochondrial heterogeneity of these lesions and may be used for detection of low frequency tumor-associated mtDNA mutations as a measurer of possible metastatic processes in histologically clean LN. Citation Format: Adrian D. Schubert, Evgeny Izumchenko, William H. Westra, Aditi Chatterjee, Wayne M. Koch, Mohammad O. Hoque, David Sidransky. mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4667. doi:10.1158/1538-7445.AM2017-4667