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A. Agraz-Doblas et al.
stratification according to the Interfant-06 protocol (based on age at diagnosis, white blood cell count and response to prednisone) was performed with the Cox model and the Wald test. All tests were two-sided. Analyses were performed using SAS 9.2.
DNA, RNA and B-cell receptor (VDJ) repertoire genome-wide analyses and data analysis
Preparation and analysis of all DNA and RNA genome-wide high-throughput sequencing is detailed in the Online Supplementary Methods, Online Supplementary Figure S1 and Online Supplementary Table S2.
Results
At diagnosis infant B-cell precursor acute lymphoblastic leukemia shows a silent mutational landscape irrespective of MLL gene status
Whole-exome sequencing and whole-genome sequenc- ing analyses showed a silent mutational landscape in the three iBCP-ALL subtypes studied here: MLL-AF4+, MLL- AF9+ and non-MLL (n=42 patients, Online Supplementary Table S1). Our study revealed an average of one genomic rearrangement and 2.5 non-silent single nucleotide vari- ants, a 2-fold higher number than that reported by
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Andersson et al.,20 likely reflecting the 3-fold larger sequencing coverage (Figure 1A, Online Supplementary Figure S1 and Online Supplementary Table S3). All mutations found at diagnosis were validated using orthogonal meth- ods. This mutational frequency is the lowest described for any other pediatric tumor type according to recent reports34 (Online Supplementary Figure S2). Intriguingly, one third of the mutations validated showed a mutant allele frequency (MAF) <20% indicating that iBCP-ALL contains genetically different intratumoral subclones despite its genomic stability, likely explaining the higher mutational load than that reported by Andersson et al.20 (Figure 1A and Online Supplementary Table S3). Despite the paucity of mutations, ~80% of the validated protein-coding muta- tions (90/116) are predicted to produce deleterious effects on the protein (Online Supplementary Figure S3A) which might support a strong selective pressure in iBCP-ALL. To gain insights into the molecular mechanisms underlying the accumulation of mutations, we analyzed the enrich- ment of specific mutational signatures as described by Alexandrov et al.35 In the MLL-AF4+ iBCP-ALL subgroup we identified a significant enrichment of signature 1 char- acterized by the accumulation of C>T/G>A transitions, linked to a spontaneous deamination of 5-methylcytosine (Online Supplementary Figure S3B,C).35 This mutational sig-
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Figure 1. Somatic mutations detected by whole-exome sequencing in the discovery cohort of infant B-cell precursor acute lymphoblastic leukemia. (A) Total num- ber of mutations identified in each individual patient. The total number of non-synonymous mutations (yellow area, right Y axis) and mutant allele frequency (MAF) for each mutation (individual dots, left Y axis) are represented. (B) Oncodrive software identified the PI3K-RAS pathway as the only recurrently mutated pathway in infant B-cell precursor acute lymphoblastic leukemia. The distribution of mutations in genes of the PI3K-RAS pathway is shown for all patients within the three iBCP- ALL subgroups: [total 42 patients: 27 t(4;11)+, 5 t(9;11)+ and 10 MLLwt].
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haematologica | 2019; 104(6)