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WGS in MBL and ultra-stable CLL
the three entities (ranging from 11.4 to 12.6), whereas it was significantly lower in the PMN samples (3.9 for LC- MBL and CLL and 4.1 for HC-MBL, respectively) (P<0.005 for all cases) (Figure 1C).
The transition to transversion (Ti/Tv) ratio ranged from 0.99 to 1.13 in the MBL/CLL samples, while it was slightly lower in the PMN samples (0.94) (Figure 2A). No clear dif- ferences were observed between MBL/CLL and PMN samples when the distribution of mutations among the six types was examined (Figure 2B). We then evaluated the sequence context of each mutation by incorporating infor- mation on the bases immediately upstream and down- stream of the mutated base, hence leading to 96 possible mutation types in this classification16 (Online Supplementary Table S4). Almost all major differences were identified between MBL/CLL samples and PMN samples with the former group exhibiting mainly C>T mutations at NpCpG trinucleotides (P<0.05 for ultra-stable CLL and HC-MBL).
The MutationalPatterns package33 was used to delineate the mutational signatures in our cohort. Mutational pat- terns identified in the MBL/CLL samples resembled those reported by Puente et al.;9 this finding was corroborated by
calculating the pairwise similarity with the 30 previously published signatures, where signature 9 and, to some extent, signature 1 where the main contributors (Figure 2C). The same analysis in the PMN samples gave different results, with a strong impact of mutational signatures 3 and 5 (Figure 2D). Signature 3 had previously been identi- fied in solid tumors and is associated with failure of DNA double-strand break-repair by homologous recombina- tion.16 Signature 5 exhibits transcriptional strand bias for C>T and T>C mutations at ApTpN context and displays a correlation between smoking history and mutation con- tribution.16
MBL and ultra-stable CLL display a paucity of mutations in putative CLL driver genes
Whole-genome sequencing identified 186 non-synony- mous exonic variants amongst MBL/CLL samples and 15 amongst PMN samples. The average number was 8.9 for LC-MBL (range: 1-16), 14.8 for HC-MBL (range: 9-27), 11.6 for ultra-stable CLL (range: 7-19), and 0.9 for the PMN samples (range: 0-6), respectively (Figure 3A). In MBL/CLL samples, the vast majority of non-synonymous mutations were missense [LC-MBL: 47 of 53 (88.7%); HC-
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Figure 1. Somatic mutational analysis of ultra-stable chronic lymphocytic leukemia (CLL), high-count monoclonal B-cell lymphocytosis (HC-MBL), low-count mon- oclonal B-cell lymphocytosis (LC-MBL) and control polymorphonuclear (PMN) cell samples. (A) Total number of somatic mutations identified by whole-genome sequencing (WGS) in CLL cell samples from MBL, CLL and the respective PMN samples. All samples carried similar mutational loads with the exception of a single LC-MBL sample (LC-MBL_1) that displayed a very low number of mutation events; as can be seen, the corresponding PMN sample had a mutation load similar to the other PMN samples, where comparison of mutation profiles between the MBL and PMN sample showed few common hits, thus excluding the likelihood of con- tamination. Concerning PMN control samples, they were also characterized by high homogeneity regarding the mutational load. There was a single sample with a very high mutational load; detailed comparison against its respective CLL sample showed a high overlap of mutations indicating potential tumor cell contamination, hence this sample was removed from downstream analysis. (B) Average mutation rates ± Standard Deviation (SD) for LC-MBL, HC-MBL and CLL. Highly analogous mutation rates were observed in the HC-MBL (0.79 mutations per Mb) and CLL (0.74 mutations per Mb) samples, while LC-MBL samples had a slightly lower ratio (0.63 mutations per Mb). (C) Average SNV to small indels ratio ± SD for all sample groups. All 3 entities displayed similar ratios in clear contrast to the PMN samples where the ratio was much lower.
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