CarcSeq detection of lorcaserin-induced clonal expansion of Pik3ca H1047R mutants in rat mammary tissue.

ABSTRACT

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a non-genotoxic rat carcinogen, which induced mammary tumors in male and female rats in a two-year bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 weeks. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary and liver samples using ultra high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as non-genotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations (CDMs), namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, p < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 weeks as compared to 12 weeks. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a non-genotoxic carcinogen using a treatment duration as short as 3 months.