Expansion of preexisting cancer driver mutant clones induced by benzo[b]fluoranthene in MutaMouse lung
Clonal expansion of cancer driver mutant cells is being developed as an early biomarker of future cancer risk. The goal is to improve carcinogenicity testing through the molecular detection of carcinogen-induced, cancer driver mutant cell proliferation before clonal expansion is observable through the analysis of histopathological lesions. Toward this goal, this study investigated the carcinogenic impact of treatment with the genotoxic carcinogen benzo[b]fluoranthene (B[b]F). Specifically, MutaMouse males were treated with 0, 6.25, 12.5, and 25 mg/kg/day B[b]F by gavage for 90 and 180 days. Mutation levels in hotspot containing regions of cancer driver genes (Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Trp53) were measured in the lung DNAs of treated mice using CarcSeq. The CarcSeq method incorporates unique molecular identifier sequences during PCR amplification of cancer driver genes segments, followed by creation of single-strand consensus sequences (SSCSs) for error correction by consensus. Mutation levels greater than or equal to 10-4 were measured as mutant fraction (MF, the ratio of mutant SSCSs to all SSCSs observed at a single base position). Mutations were stratified based on their occurrence in lung-specific or non-lung driver gene sequences and the clonal expansion biomarker was assessed as median absolute deviation in MF (MAD). A significant, dose-dependent increase in MAD was observed for lung-specific drivers after 180 days of B[b]F-treatment but not after 90 days of treatment. After 180 days of treatment, dose and treatment duration related increases in MF and numbers of mutations recovered were observed for select lung-specific drivers (Egfr, Pik3ca, and Stk11). Also, at this timepoint, MF and numbers of mutations recovered were significantly decreased for non-lung drivers. Histopathological analysis of lung tissue samples from mice treated for 180 days did not reveal a significant increase in lung lesions. Importantly, the normalized trinucleotide mutation spectrum observed in the cancer driver sequences was distinct from the known B[b]F mutational signature (primarily C:G > A:T) and did not vary with dose. This result shows that exposure to B[b]F caused amplification of preexisting, spontaneously arising cancer driver gene mutations. These results indicate CarcSeq and the MAD biomarker of clonal expansion were able to detect the carcinogenic effect of treatment with the genotoxic carcinogen B[b]F in mouse lung (the known tumor target in mouse) at a time preceding the development of a histopathologically-detectable effect. The results of the study, therefore, indicate analysis of clonal expansion using CarcSeq is a promising approach for obtaining mechanistic information that can be used in cancer risk assessment.