The main findings of this study are the initial elucidation of the complexity of the DLBCL coding genome, and the discovery of a novel set of recurrent lesions that may be of relevance for the understanding of the pathogenesis of this malignancy.
The combined set of mutations and CNAs detected in the six DLBCL discovery cases does not allow a final assessment of the precise number of genomic alterations affecting coding genes, but provides information about the order of magnitude of the lesions associated with this malignancy. The estimate of >30 alterations/case emerging from this study reflects only those events with abundant to complete clonal representation, i.e. changes that were likely present during the initial phases of tumor expansion, and thus promoted malignant transformation. Furthermore, this estimate does not include an additional 40% of mutations that may have been missed by the WES approach due to the relatively low depth of coverage (see Methods
), as well as chromosomal translocations other than those affecting BCL6, BCL2
(not detectable by the two methodologies used). Thus, it can be concluded that the coding genome of DLBCL contains <100 lesions on average. Although very approximate, this figure is informative for future studies and may serve as an initial database for the determination of recurrence in additional panels of cases.
When compared to other malignancies, the order of magnitude of lesions detected in DLBCL appears lower than that reported for certain epithelial cancers49,50
. Among hematologic malignancies, the complexity of the DLBCL genome is not significantly different from multiple myeloma51
, while it appears much more complex than acute myeloid leukemia52–54
and chronic lymphocytic leukemia23,27
in terms of both CNAs and mutational load. On the other hand, the observed predominance of transitions over transversions, the preferential targeting of C:G and G:C basepairs and the significant bias toward alterations at CpG dinucleotides is emerging as a common feature shared by most malignancies studied so far, including those of epithelial derivation. This pattern of alterations is generally derived from endogenous biochemical processes, such as the spontaneous deamination of 5-methylcytosine residues. However, it cannot be excluded that the abnormal and/or ectopic activity of activation-induced cytidine deaminase (AICDA) has a role in the generation of DNA lesions affecting these residues; indeed, AICDA has been shown to target the 5’ portion of multiple genes –mostly noncoding– in DLBCL, and some of the mutations detected in this study may in fact reflect the activity of aberrant somatic hypermutation (for example, PIM1)55
Among the vast array of genetic lesions identified in the DLBCL genome, alterations of MLL family members, and in particular MLL2
, appear especially frequent. MLL2
encodes a trimethyltransferase with well-documented influence on the expression of a large number of genes, including homeobox genes. The pattern of monoallelic somatic inactivation observed in DLBCL suggests a role for MLL2
as a haploinsufficient tumor suppressor, consistent with the observation that monoallelic MLL2
truncating mutations leading to descreased gene dosage have a pathogenic effect in Kabuki syndrome, a congenital disorder characterized by developmental and intellectual abnormalities56
. A role for MLL
genes in malignant transformation is supported by the involvement of MLL
in the pathogenesis of acute leukemia57
, although by a distinct and partially unclear mechanism, and by the recent finding of somatic inactivating mutations in both MLL2
in various cancers26,51,58
. Collectively, alterations of chromatin modifying enzymes, including HMT and HAT, emerge as the most frequent alterations associated with DLBCL pathogenesis, being present in over one third of cases independent of disease subtype (GCB-DLBCL, ~50%; ABC-DLBCL, ~30%).
A second novel finding in the DLBCL coding genome is the high frequency of alterations in genes that are involved in immune recognition by T cells. In particular, the observation that bi-allelic inactivation of B2M
is more common than originally implicated based on only a few cases analyzed59
suggests a major role for these lesions in causing the loss of HLA class I expression, which constitutes a frequent event in DLBCL60
. The findings herein provide a mechanistic explanation for a fraction of these cases, and suggest the existence of additional genetic or epigenetic mechanisms preventing the expression of HLA class I molecules. Since it is well established that the lack of these molecules makes cells insensitive to cytotoxic T cell-mediated killing, loss of B2M
may represent a major mechanism of tumor escape from immune surveillance.
The distribution of recurrent lesions among DLBCL subtypes defined by cell of origin supports the existence of both common and subtype-specific genetic lesions. The former are represented by those inactivating chromatin-modifying functions, including HAT and HMT genes, as well as the newly discovered alterations of immune recognition functions. These alterations may represent common pathways necessary for the development of the DLBCL phenotype, independent of cell of origin. Conversely our results confirm the preferential association of BCL2
alterations with GCB-DLBCL7
, and of alterations in the NF-κB and BCL6/BLIMP1 axis with ABC-DLBCL10
. The observed distribution has immediate clinical implications since it suggests the development of therapies that combine drugs targeting commonly altered pathways with those targeting pathways selectively disrupted in DLBCL subtypes.