Expansion of preexisting cancer driver mutant clones is induced by the genotoxic carcinogen benzo[b]fluoranthene in MutaMouse lung.

Clonal expansion (CE) of cells carrying cancer driver mutations (CDMs) is being developed as a biomarker of cancer risk. CE in lung of MutaMouse males treated with 0, 6.25, 12.5, and 25 mg/kg/d benzo[b]fluoranthene (B[b]F) by gavage for 90 and 180 d was assessed by CarcSeq. DNA regions encompassing mouse correlates of human hotspot CDMs were PCR amplified, attaching 18-base unique molecular identifiers (UMIs) during the PCR. Following library preparation and sequencing, UMI-defined read families were assembled to produce single-strand consensus sequences (SSCSs). Recovered mutants with mutant fractions (MFs) ≥10-4 were stratified based on their occurrence in lung-specific or nonlung driver sequences and CE was assessed on a per mouse basis as median absolute deviation in mutant fraction (MAD). A significant, dose-dependent increase in MAD was observed for lung-specific MFs after 180 d of B[b]F treatment, a duration that did not cause a significant increase in lung lesions. Dose- and treatment duration-related increases in MF were observed for Egfr, the mouse correlate of a known human lung tumor driver gene. MF and mutation counts were significantly decreased in response to longer treatment duration for some nonlung drivers, suggesting negative selection. Importantly, the normalized trinucleotide mutation spectrum derived from CDMs reflects amplification of preexisting spontaneous mutations, distinct from those induced by B[b]F mutagenesis. These results show CarcSeq detects CE of preexisting cancer driver gene mutants induced by the genotoxic carcinogen B[b]F and suggest a CE endpoint may be useful for evaluating cancer risk associated with tumor promoters or complete carcinogens.