Intratumor genetic heterogeneity and head and neck cancer relapse

Background: Head and neck squamous cell carcinomas are treated by surgery, radiotherapy (RT), chemoradiotherapy (CRT) or combinations thereof, but locoregional recurrences (LRs) occur in 30-40% of treated patients. We have previously shown that in approximately half of the LRs after CRT, cancer driver mutations are not shared with the index tumor. Aim: To investigate two possible explanations for these genetically unrelated relapses, treatment-induced genetic changes and intratumor genetic heterogeneity. Methods: To investigate treatment-induced clonal DNA changes, we compared copy number alterations (CNAs) and mutations between primary and recurrent xenografted tumors after treatment with (C)RT. Intratumor genetic heterogeneity was studied by multi-region sequencing on DNA from 31 biopsies of 11 surgically treated tumors. Results: Induction of clonal DNA changes by (C)RT was not observed in the xenograft models. Multi-region sequencing demonstrated variations in CNA profiles between paired biopsies of individual tumors, with copy number heterogeneity scores varying from 0.027 to 0.333. In total, 32 cancer driver mutations could be identified and were shared in all biopsies of each tumor. Remarkably, multi-clonal mutations in these same cancer driver genes were observed in 6 of 11 tumors. Genetically distinct heterogeneous cell cultures could also be established from single tumors, with different biomarker profiles and drug sensitivities. Conclusion: Intratumor genetic heterogeneity at the level of the cancer driver mutations might explain the discordant mutational profiles in LRs after CRT, while there are no indications in xenograft models that these changes are induced by CRT.