Mutational signatures of ionizing radiation in second malignancies

Abstract

Ionizing radiation is a potent carcinogen, inducing cancer through DNA damage. The signatures of mutations arising in human tissues following in vivo exposure to ionizing radiation have not been documented. Here, we searched for signatures of ionizing radiation in 12 radiation-associated second malignancies of different tumour types. Two signatures of somatic mutation characterize ionizing radiation exposure irrespective of tumour type. Compared with 319 radiation-naive tumours, radiation-associated tumours carry a median extra 201 deletions genome-wide, sized 1-100 base pairs often with microhomology at the junction. Unlike deletions of radiation-naive tumours, these show no variation in density across the genome or correlation with sequence context, replication timing or chromatin structure. Furthermore, we observe a significant increase in balanced inversions in radiation-associated tumours. Both small deletions and inversions generate driver mutations. Thus, ionizing radiation generates distinctive mutational signatures that explain its carcinogenic potential.

Figure 1. Indels in radiation-associated tumours.

(a) Indel/substitution ratio. Shown is the indel/substitution ratio for each tumour. The ratio was significantly increased in radiation-associated second malignancies (0.0003, linear mixed effects model). Each dot represents a tumour. Different colours represent different tumour types (see legend, top right). Boxplots: vertical line – median; whiskers – minimum and maximum without outliers. (b) Deletion/insertion ratio. Shown is the deletion/insertion ratio of every tumour. Deletions were significantly (*) enriched in radiation-associated second malignancies (P<2.2 × 10⁻¹⁶, linear mixed effects model) and in breast tumours with germline BRCA1 or BRCA2 deficiency. Symbols, boxplots as per a. (c) Clonal versus subclonal indels in radiation-associated second malignancies. Shown are the absolute clonal (early) and subclonal (late) indel burdens of each tumour, by indel type. Amongst clonal indels, deletions were significantly enriched. P-values refer to the comparison of proportion of deletions/other indels in clonal versus subclonal indels (Fisher's exact test). (d) Indel likelihood across the genome. Shown is the probability of deletion or insertion to occur (vertical axis) across different regions of the genome (horizontal axis). The probability was modelled on the basis of associations between indels and genomic properties (see Methods). Chromosome 14 is shown as a representative chromosome. Radiation-associated indels were compared to indels of 35 non-radiation-associated osteosarcomas. Radiation-associated deletions, but not insertions, followed a more uniform distribution across the genome than in radiation-naive samples. (e) Distribution of indels in relation to genomic features. Comparison of the mutation density of radiation versus non-radiation indels in relation to genomic features. X axis: ratio of mutation density of non-radiation-associated indels or radiation-associated indels over background density. Y axis: genomic feature. The distribution of insertions in both radiation-associated and radiation-naïve tumours correlated with several genomic features, with few significant differences (asterisk) between the two. In contrast, the distribution of deletions in radiation-induced cancers, but not in radiation-naive tumours, showed little variability and resembled the background distribution more closely. Thus, significant differences (asterisk) were seen in the deletion density in relation to genomic features comparing radiation-associated and radiation-naïve tumours. P-values are detailed in Supplementary Data 4.

Figure 2. Balanced inversions in radiation-associated tumours.

(a) Overview of rearrangements. Tumours exhibited tumour-type specific features. Balanced inversions (black bars) were found in every tumour, except PD7530a. (b) Example of a balanced inversion in PD7188a. A 0.9 Mb inversion. The inversion was validated by PCR across the breakpoint (gel image) and by split reads. Note that the split reads carried a heterozygous SNP at the head end.

Figure 3. Indels in prostate tumours.

(a) Indels in radiation-naive versus radiation-exposed prostate tumours. Shown is the indel burden, by indel subtype, found in radiation-naive and in radiation-exposed prostate tumours. In radiation-exposed tumours radiotherapy had been administered to the primary tumour before formation of metastases. Deletions were significantly enriched in radiation-exposed tumours (P=0.0002, generalized linear model). Note that radiation-associated tumours with confounding BRCA1 or BRCA2 deficiency were excluded from the statistical analysis (cases PD13412 and PD11335). (b) Indels in tumours from a patient whose primary lesion was treated with ionizing radiation after formation of metastases. Shown are indels that were found exclusively in the primary lesion and indels found in all other lesions. Deletions were significantly enriched amongst indels exclusive to the primary lesion. Comparison by Fisher's exact test, of the ratio of deletions over other indels.