The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias

Abstract

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Figure 1

Somatic mutations detected by whole genome sequencing of infant MLL-R ALL. (a–c) CIRCOS plots of somatic non-silent mutations in three infant MLL-R ALL cases. (a) INF006 contained a balanced t(4;11)(q21;q23) encoding the MLL-AFF1 and gain of chromosome X. (b) INF009 contained a balanced t(11;19)(q23;p13.3) with the break on chromosome 19 occurring 22.7kb 5` of MLLT1 resulting in a spliced in-frame MLL-MLLT1 chimeric gene. The translocation had an inverted intrachromosomal duplication spanning 0.3kb at the breakpoint on chromosome 19 that resulted in an out-of-frame RFX2-MLL fusion. In addition, an unrelated intrachromosomal deletion of 46bp was detected on chromosome 6. (c) INF004 contained a complex three-way translocation involving chromosomes 9, 10, and 11 that encoded the MLL-MLLT10 on the derivative chromosome 11, an out-of-frame MLLT10-CNTNAP3B chimeric gene on chromosome 9, and a truncated 3` MLL on chromosome 10. The case also contained a t(8;14)(q24;q11.2) that resulted in the juxtaposition of MYC to the T-cell antigen receptor alpha (TRA) gene as denoted by the ∧. In addition, a non-silent SNV in COL13A1 and a deletion on chromosome 9 disrupted PAX5. (d) Structure of the INF004 complex translocation involving chromosomes 9p11 (blue), 10p12 (yellow), and 11q14–23 (burgundy). The genomic coordinates (hg18) are indicated above each genomic segment. The MLL-MLLT10 fusion gene is depicted by the solid arrow and all other rearrangements by dotted arrows. Copy number gains (red) and losses (blue) at the respective break points are shown. The final genomic products on chromosomes 9, 10 and 11 are shown in Supplementary Fig. 9. Ter, terminus; Cen, centromere.

Figure 2

Recurrently mutated genes detected in 47 cases of infant MLL-R ALL. WGS was performed on 22 leukemic samples (discovery cohort) and targeted capture sequencing of the coding exons of 232 genes on an additional 25 infant MLL-R ALL samples (validation cohort). (a) Recurrent mutations were identified in 21 genes/loci in the combined infant MLL-R ALL cohorts. The * indicates that more than two genes were targeted in this locus (see Supplementary Tables 7, 8, 14, 19 and 24). (b) Distribution of mutated genes across the 47 infant MLL-R cases. For mutations in kinases or in genes in the PI3K/RAS pathway, only those known to confer activation of the pathway are shown. * indicates that more than two genes were targeted within the locus in one or more of the cases with only the first gene in the locus listed. † indicates that the sample lacked a matched non-leukemic sample. ∧ indicates that this sample is an identical twin to 060; the matched non-leukemic sample from 060 was used. # indicates that this sample has a novel MLL-USP2 fusion identified through RNA sequencing. A SNV marked with an open circle designates that the mutant allele is expressed as determined by RNA sequencing and a dash indicate that it is not expressed at the level of detection for our analysis.

Figure 3

Clonal evolution from diagnosis to relapse in infant INF002. (a,b) CIRCOS plots showing the non-silent SNVs and SVs detected by WGS at (a) diagnosis and (b) relapse. (c) Five mutation clusters identified at diagnosis with MAF at 0.426 (red), 0.236 (blue), 0.114 (purple), 0.058 (green), and 0.024 (orange), respectively. The diagnostic mutations with a MAF of 0.426 are consistent with heterozygous mutations present in every leukemic cell, whereas clusters with lower MAF represent mutations present in minor leukemic sub-clones. (d) Evolutionary trajectory from diagnosis to relapse. Five clones were determined from diagnosis. Only a subclone with estimated population frequency of 6.25% survived therapy and persisted to relapse. It contained 3 mutation clusters (red, purple orange) from diagnosis but also acquired additional mutations at relapse. The relapsed tumor is likely to contain only a single clone as there is only 1 major MAF cluster (Supplementary Fig. 6b). “X” indicates that these clones also have an extra copy of “X”.

Figure 4

Mutational profiles of infant and non-infant MLL-R leukemia. (a) The number of non-silent SNVs and indels in the dominant leukemia clone affecting annotated genes in infant MLL-R ALL and non-infant MLL-R leukemia. (b) Distribution of somatic SNVs, indels, and CNAs in epigenetic regulatory genes in infant MLL-R ALL and non-infant MLL-R leukemias showing that these genes are significantly more often mutated in non-infant MLL-R leukemia (two-sided Fishers exact test, P=0.04). The black line between SJMLL002 and SJMLL021 indicate the separation between the lymphoblastic and myeloid non-infant leukemias. The only gene mutation that was found not to be expressed at the detection limit of our RNAseq analysis was the one in L3MBTL3.