Analysis of the coding genome of diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. Although a number of structural alterations have been associated with the pathogenesis of this malignancy, the full spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains, on average, more than 30 clonally represented gene alterations per case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2; 24% of samples) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.

Figure 1. DLBCL non-silent mutation load

Number (a) and type (b) of Sanger-validated, non-silent mutations identified in the 6 DLBCL discovery patients. c, Nucleotide targeting of the DLBCL-associated point mutations (nucleotide composition of the target exome: G, 26.2%; C, 25.7%; T, 22.0%; A, 25.9%). d, Observed mutation frequencies at specific dinucleotides (red bars). The expected frequencies (grey bars) correpond to the dinucleotide sequence composition of the target exome. Asterisks denote statistically significant differences in overrepresented changes, as assessed by a Poisson distribution after correction for multiple hypotheses.

Figure 2. Copy number analysis of the 6 DLBCL discovery cases

a, dChipSNP heatmap showing median-smoothed log2 copy number ratio of all chromosomes in the 6 index DLBCL biopsies and their paired normal DNAs. In the red-blue scale, white corresponds to a normal (diploid) copy number log-ratio, blue is deletion and red is gain. b, Curated segmentation data for the 6 DLBCL samples shown in panel a, obtained as described in Methods and visualized using the Integrative Genomics Viewer (IGV) software (http://www.broadinstitute.org/igv). b, Overall number and frequency of copy number alterations.

Figure 3. DLBCL harbors a heterogeneous load of numerical and structural genomic aberrations

Combined load of genetic lesions that were present in the major tumor clone of the 6 DLBCL discovery cases (including point mutations, copy number aberrations, and chromosomal translocations at three common target loci).

Figure 4. Recurrent mutations in DLBCL

Percentage of DLBCL primary cases harboring mutations in 56 candidate genes after targeted-resequencing of an expanded screening dataset. The final number of mutated cases over total analyzed (including the 6 DLBCL discovery cases) is given for each gene; this number includes validated somatic mutations as well as variants that are not reported in any available SNP databases (see Methods). The somatic origin of the mutations was confirmed by analysis of paired normal DNA in at least one case/gene. Red asterisks denote putative tumor suppressor genes, as suggested by the presence of predominantly inactivating mutations (stop codons, frameshift mutations and experimentally validated missense mutations). Green asterisks denote genes where the oncogenic activity of the mutation was functionally proven. Black asterisks indicate genes harboring mutations of unclear pathogenic role, but predicted to truncate the encoded protein in at least one case.

Figure 5. The MLL2 gene is mutated in a large fraction of DLBCL

a, Schematic diagram of the MLL2 gene (top) and protein (bottom), with its conserved functional domains (PHD, FYRN, FYRC, SET). In black, untranslated exons. Arrows, color-coded as in (b), indicate the position of the mutations found (cell lines on top, primary cases at the bottom). b, Overall percentage of DLBCL cases carrying MLL2 mutations, according to mutation type (cell lines and biopsies). c, Prevalence of MLL2 mutated cases in major DLBCL phenotypic subtypes; numbers on top indicate the actual number of mutated samples over total analyzed. d, Allelic distribution of the mutations in 26 of 28 affected samples for which this data could be obtained. Mutations are color-coded as in (b).

Figure 6. Disruption of histone/chromatin modification genes is a major feature of DLBCL

a, Mutation frequency of five genes encoding for histone-acetyltransferases and histone-methylation modifiers in GCB- and ABC-DLBCL (primary cases only). b, dChipSNP heatmap showing median-smoothed log2 copy number ratio for DLBCL biopsies harboring KDM2B, MLL3 and MLL5 deletions; in the red-blue scale, white corresponds to a normal (diploid) copy number, blue is deletion and red is gain. Note that, due to the presence of non-tumor cells infiltrating the biopsies, the inferred copy number, and the corresponding color intensity, may vary across samples (see Supplementary Table 11). The boxed area corresponds to the commonly deleted region, which is expanded below the heatmap to show the included genes. c, Relationship between MLL2 mutations and mutations of HATs. In the heatmap, rows correspond to the indicated genes and columns represent individual samples, color-coded according to the gene status (grey, unmutated; red, mutated or deleted). Of the 3 MLL2-mutated cases showing simultaneous lesions at CREBBP, one harbored an amino acid substitution whose functional significance is presently unclear. Thus, it is possible that this change represents a passenger event or a private germline variant not annotated in currently available databases; alternatively, these mutations may have milder effects, requiring additional cooperating lesions in the same pathway. d, Overall fraction of DLBCL biopsies carrying mutations at one or more of the four histone modification genes shown in (c).