Rapid Clonal Evolution Of Dormant Micrometastatic Disease In Head And Neck Cancer

Abstract Head and neck cancer (HNC) is prone to locoregional recurrence which results in poor clinical outcome. Recent HNC clinical studies demonstrated that cervical lymph node dissection results in improved patient outcome even in early disease. These results suggest that dormant micrometastatic cancer occurs early in head and neck carcinogenesis. We used our established in vivo chemical carcinogenesis model of head and neck cancer to characterize dormant micrometastatic disease using genetic lineage tracing and null mutant mice. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by immunofluorescent 53BP1 localization at telomeres, and by ATM/ATR western blotting. Keratin expression and proliferation index were determined by immunohistochemistry. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres, telomere circular DNA analysis, and chromosome orientation fluorescence in situ hybridization. Genomic instability was determined by high throughput sequencing. Cancer stem cells were sorted by flow cytometry and transplanted subcutaneously in Matrigel. Advanced HNC exhibited significant metastatic disease in the majority of lymph nodes. Early HNC and even premalignant lesions exhibited micrometastatic disease in most lymph nodes. These micrometastatic lesions contained multiple HNC clones that were dependent for proliferation on paracrine chemokine signaling from a tumorigenic keratin 15+ cancer stem cell population. In situ, the micrometastatic K15+ cancer stem cell population exhibited increased telomere DNA damage response including activated ATM and ATR which correlated with low proliferation index. Clonal evolution of K15+ micrometastatic cancer stem cells was dependent on extremely short telomeres. These cells expressed both telomerase and recombination-based alternative lengthening of telomeres (ALT) activities. K15+ micrometastatic cancer stem cells exhibited high levels of genomic instability resulting from telomere fusions and expressed metastasis-specific gene expression programs. These patterns of micrometastatic gene expression revealed targeted therapeutic vulnerabilities. These results indicate that dormant micrometastatic disease develops rapidly during head and neck carcinogenesis from normal epithelial cells with short telomeres and is sensitive to targeted therapies. Citation Format: Angelica M. Lagunas, Jianchun Wu, David L. Crowe. Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1993.