Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Abstract

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Figure 1. Profile of somatic mutations in four CLL genomes

a, Distribution of somatic alterations. For each tumour genome, copy number (solid lines), density of mutations per 5-Mb window (bars) and protein-coding mutations (dots) are shown. The shaded rectangle indicates the location of the 13q14 deletion that was present in three of the four CLL cases. Chromosome numbers are listed below the four profiles. b, Frequency of substitutions in each CLL tumour for the six possible classes of mutation. c, Distribution of the four possible NpA dinucleotides for the A to C transversion in each tumour genome, compared with the expected distribution across the genome. The total number of A to C substitutions per case is indicated at the top (**, P < 0.001).

Figure 2. Mutational and functional analysis of NOTCH1 in CLL

a, Schematic representation of human NOTCH1, showing the main domains and locations of the three different somatic mutations identified in CLL. NEC, NOTCH1 extracellular subunit; NTM, NOTCH1 transmembrane subunit; ICN, intracellular domain of NOTCH1; LNR, Lin-12 NOTCH repeats; RAM, RAM domain; ANK, ankyrin repeat domain; PEST, PEST domain. b, Electropherogram showing the heterozygous CT deletion recurrently identified in CLL. c, Western blot showing NOTCH1 protein levels in CLL cases with or without the NOTCH1 p.P2515Rfs*4 mutation, and in Jurkat cells as a control. The arrow indicates the band corresponding to the NTM; the large arrowhead indicates the smaller band corresponding to the mutant form. d, Heat map showing the 23 genes of the NOTCH1 pathway that are differentially expressed in NOTCH1-mutated versus non-mutated CLL. e, Distribution of disease stage (Binet), ZAP-70 expression status, CD38 expression status and IGHV mutational status (UM, unmutated IGHV) in patients with or without mutations in NOTCH1 (*, P < 0.02; **, P < 0.01). f, Actuarial probability of overall survival of CLL patients with mutated or unmutated NOTCH1 (*, P = 0.03).

Figure 3. Mutational and functional analysis of MYD88 in CLL

a, Multiple sequence alignment of MyD88 around the mutated residue (arrow) in different species. Cons., degree of conservation. b, Electropherogram showing the recurrent heterozygous p.L265P MYD88 mutation (arrow) detected in CLL. c, Cell extracts from a MYD88-mutated CLL (L265P) and a MYD88-unmutated CLL (WT) were immunoprecipitated with anti-MyD88 antibody. The immunoprecipitated and unbound fractions were analysed by western blot using anti-IRAK1 and anti-MyD88 antibodies. d, Western blot analysis of phosphorylated STAT3 (p-STAT3 (Tyr 705)) and total STAT3 in cell extracts from MYD88-mutated or unmutated CLL tumour cells. β-Actin was used as a control to show equal loading. e, Western blots showing phosphorylated IκBα (p-IκBα), total IκBα, phosphorylated p65 subunit of NF-κB (p-p65) and total p65 subunit of NF-κB in cell extracts from MYD88-mutated or unmutated CLL tumour cells. f, Heat map representing the cytokine levels secreted by B cells from eight individuals after Toll-like receptor stimulation. Only the five cytokines that showed the most significant differences between MYD88-mutated and MYD88-unmutated CLL are shown. ‘E52DEL’ indicates B cells from two patients with an inactivating MYD88 mutation, ‘WT’ corresponds to tumour cells from CLL patients without MYD88 mutation and ‘L265P’ indicates tumour cells from patients carrying a mutated MYD88. The stimulation experiments for each of the Toll-like receptors (TLRs) are represented in different colours. NS, no stimulus. g, Distribution of disease stage (Binet), ZAP-70 expression status, CD38 expression status and IGHV mutational status (UM, unmutated IGHV) in patients according to the presence or absence of p.L265P MYD88 mutation (*, P < 0.03).