Hypermutation and unique mutational signatures of occupational cholangiocarcinoma in printing workers exposed to haloalkanes

Abstract

Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Figure 1.

The printing workers’ cholangiocarcinomas share a high mutation burden, strand bias and a unique trinucleotide mutational signature. (A) Number of exome SNVs in the printing workers’ and control cholangiocarcinoma cases (*P < 0.05; Student’s t-test). (B) Single-nucleotide DNA substitution profiles in the printing workers’ and control cholangiocarcinoma cases. (C) Substantial strand bias in mutation counts in the sense (S) and antisense (AS) strands of the printing workers’ cholangiocarcinoma cases. Significant strand bias for C:G to T:A mutations was observed in the printing workers’ cholangiocarcinoma cases (**P < 0.01, ***P < 0.001; chi-square test). (D) Trinucleotide mutational patterns in the printing workers’ and control cholangiocarcinoma samples. The mutational signatures were normalized using the trinucleotide frequency in the genome. (E) Secondary characteristic trinucleotide mutational pattern in the printing workers’ cholangiocarcinoma samples that emerged after elimination of the prominent mutational pattern GpCpY to GpTpY. The mutational signatures were normalized using the trinucleotide frequency in the genome.

Figure 2.

Both 1,2-DCP and DCM show mutagenic activity in S.typhimurium strain TA100. The mutagenic activity levels of 1,2-DCP (closed circle) and DCM (closed square) were estimated from the number of revertant colonies.

Figure 3.

The mutational signature of 1,2-DCP in S.typhimurium TA100 partially recapitulates the printing workers’ cholangiocarcinoma signature. (A) Number of SNVs in 1,2-DCP- and DCM-exposed TA100, except for the hisG gene target site. (B) Single-nucleotide DNA substitution profiles in TA100 exposed to 1,2-DCP or DCM except for the hisG gene target site. (C) Trinucleotide mutational pattern of TA100 exposed to 1,2-DCP or DCM except for the hisG gene target site. Clones exposed to 1,2-DCP harbored NpCpC to NpTpC changes (indicated by the asterisks). This signature partially recapitulates the printing workers’ cholangiocarcinoma signature as shown below the 1,2-DCP-exposed TA100 panel. The printing workers’ signature is the mean of the frequency of secondary characteristic mutational changes in four patients shown in Figure 1E. The mutational signatures were normalized using the trinucleotide frequency in the genome. The signature of 1,2-DCP was obtained from the sum of the 3000 and 6000 ppm exposure data.