In-depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors

Abstract

Cisplatin reacts with DNA and thereby likely generates a characteristic pattern of somatic mutations, called a mutational signature. Despite widespread use of cisplatin in cancer treatment and its role in contributing to secondary malignancies, its mutational signature has not been delineated. We hypothesize that cisplatin's mutational signature can serve as a biomarker to identify cisplatin mutagenesis in suspected secondary malignancies. Knowledge of which tissues are at risk of developing cisplatin-induced secondary malignancies could lead to guidelines for noninvasive monitoring for secondary malignancies after cisplatin chemotherapy. We performed whole genome sequencing of 10 independent clones of cisplatin-exposed MCF-10A and HepG2 cells and delineated the patterns of single and dinucleotide mutations in terms of flanking sequence, transcription strand bias, and other characteristics. We used the mSigAct signature presence test and nonnegative matrix factorization to search for cisplatin mutagenesis in hepatocellular carcinomas and esophageal adenocarcinomas. All clones showed highly consistent patterns of single and dinucleotide substitutions. The proportion of dinucleotide substitutions was high: 8.1% of single nucleotide substitutions were part of dinucleotide substitutions, presumably due to cisplatin's propensity to form intra- and interstrand crosslinks between purine bases in DNA. We identified likely cisplatin exposure in nine hepatocellular carcinomas and three esophageal adenocarcinomas. All hepatocellular carcinomas for which clinical data were available and all esophageal cancers indeed had histories of cisplatin treatment. We experimentally delineated the single and dinucleotide mutational signature of cisplatin. This signature enabled us to detect previous cisplatin exposure in human hepatocellular carcinomas and esophageal adenocarcinomas with high confidence.

Figure 1.

Cisplatin mutational signature. Trinucleotide-context mutational spectra shown as (A) raw counts and (B) rate of mutations per million trinucleotides for all MCF-10A (top panel) and all HepG2 (bottom panel) clones combined. In A, the number of mutations per SNS type is shown above the corresponding bars. (C) Pentanucleotide sequence contexts for all samples combined, normalized by pentanucleotide occurrence in the genome. See also Supplemental Figure S2.

Figure 2.

Associations between cisplatin mutagenesis intensity and genomic features. (A) Transcription strand bias is more prominent in highly expressed genes for C > A, C > T, and T > A mutations. See also Supplemental Figure S6. (B) Transcription strand bias decreases with increasing distance from the transcription start site (TSS). See also Supplemental Figure S7. Mutations were binned per 100,000 bp, i.e., the first bars are the numbers of mutations within the first 100,000 bp from the TSS, the next bars are the numbers of mutations in the region from 100,001 to 200,000 bp from the TSS, and so on. (C) Mutation density in regions with histone modifications and in binding sites for EZH2 and CTCF. The y-axis is the mean mutation density for the given region relative to the mutation density of each respective sample; bars show standard error of the mean (Supplemental Table S1).

Figure 3.

Cisplatin-induced dinucleotide substitutions (DNSs). (A) DNS mutation spectra of all MCF-10A (top panel) and all HepG2 (bottom panel) clones combined, displayed as DNSs per million dinucleotides (i.e., normalized for dinucleotide abundance in the genome). (B) Cisplatin induces higher numbers of DNSs than other mutational processes associated with dinucleotide substitutions such as UV (melanoma) and smoking (lung). (C) ±1-bp sequence context preferences for the most prominent DNS mutation classes (CC > NN, CT > NN, TC > NN, and TG > NN). The total number of DNSs per mutation class is indicated in parentheses. The vertical axis is the preceding (5′) base, the horizontal axis is the following (3′) base. Some prominent enrichments in sequence context are indicated (GCCT > GNNT, NTCT > NNNT, and NTGG > NNNG). The full sequence context preference plots, both raw counts and normalized for tetranucleotide abundance in the genome, are shown in Supplemental Figure S10. (D) Transcription strand bias of dinucleotide substitutions. Potential intra-strand crosslink sites are shown in blue, potential inter-strand crosslink sites are shown in red.

Figure 4.

Cisplatin mutational signature in human hepatocellular carcinomas (HCCs) and esophageal adenocarcinomas (ESADs). (A) Example SNS and (B) DNS mutational spectra of a tumor that tested positive for the cisplatin signature in the SNS analysis (HK034). In A and B, numbers of mutations in each mutation class are indicated. (C) DNS cosine similarities between the experimental cisplatin signature and HCCs, grouped on whether they were negative (left) or positive (right) for cisplatin mutagenesis in the SNS analysis. Red dots represent HCCs that were found positive for cisplatin mutagenesis in the SNS analysis but did not show the cisplatin DNS signature (false-positives) and samples that were not found cisplatin-positive in the SNS analysis but were concluded to be cisplatin-positive based on the DNS analysis (false-negatives). (D) ±1-bp sequence context preferences for the most prominent DNS mutation classes in cisplatin-positive HCCs and ESADs. Total numbers of DNSs per mutation class are indicated in parentheses. The vertical axis is the preceding (5′) base, the horizontal axis is the following (3′) base. (E) DNS transcription strand bias in all cisplatin-positive tumors combined. For the individual sample plots, see Supplemental Figure S27. (F) DNS replication timing bias in cisplatin-positive HCCs. DNSs were classified as being in either early or late replicating regions as described in Methods.

Figure 5.

Comparison of proportions of cisplatin-induced substitutions and reported cisplatin-adducts. (A) Relative abundance of cisplatin-induced base substitutions in the experimental signature. TNS = trinucleotide substitutions. (B) Relative abundances of cisplatin-adducts from Eastman (1983), Fichtinger-Schepman et al. (1989), Jamieson and Lippard (1999), Baik et al. (2003), and Enoiu et al. (2012). Colors of mutations in A correspond to colors of the adducts they are expected to be caused by in B. (C) Schematic representations of adducts in B related to cisplatin-induced substitutions in A: The colors of the borders of the schematic adduct representation correspond to the colors used in the zoomed-in section of the pie-charts on the right sides of A and B.