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/day benzo[b]fluoranthene (B[b]F) by gavage for 90 and 180 days 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 non-lung 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 days 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 non-lung 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 suggests a CE endpoint may be useful for evaluating cancer risk associated with tumor promoters or complete carcinogens.