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WGS Elucidates Recurrent Noncoding Mutations in CLL, IDs Potential Treatment Strategy

NEW YORK (GenomeWeb) – Performing a combination of whole-genome and exome sequencing on hundreds of patients with either chronic lymphocyte leukemia or monoclonal B-lymphocytosis, researchers have been able to identify novel driver genes and recurrent mutations in noncoding regions.

The findings of the study, conducted by researchers from the Universidad de Oviedo and the Universitat de Barcelona, in Spain, and elsewhere, were published today in Nature and may have implications for therapy.

CLL is very heterogeneous, and patients are classified as having an indolent form or aggressive form, depending on whether their tumor B cells express B-cell receptor immunoglobulin with hypermutated immunoglobulin heavy variable (IGHV) genes or unmutated IGHV genes. However, the molecular mechanisms responsible for the heterogeneous evolution of CLL are unknown.

To get a better understanding of the molecular differences, the researchers studied 452 patients with CLL and 54 patients with monoclonal B-lymphocytosis, a precursor. Of those, 317 patients were classified as IGHV-mutated, 179 as IGHV-unmutated, and 10 were undetermined.

The team performed whole-genome sequencing of 150 tumor/normal pairs and exome sequencing of 440 cases. Eighty-four patients had both WGS and exome sequencing.

Overall, the team identified between 240 and 5,416 SNVs per tumor, with no significant differences between CLL and MBL cases. The patients classified as IGHV-mutated had a higher burden of SNVs than those with unmutated IGHV genes, even when excluding the IG loci.

Over 13,000 SNVs and 951 copy number alterations affected protein-coding genes. The team identified 36 recurrently mutated genes across both subgroups and 23 genes that were significantly mutated in either the IGHV-mutated or IGHV-unmutated subgroups, which had recurrent or truncating mutations or had known driver mutations in other malignancies. Twelve of the recurrent genes had not previously been linked to CLL.

In total, the researchers identified eight main pathways that are frequently altered in CLL: BCR signaling, cell cycle regulation, apoptosis, DNA damage response, chromatin remodeling, nuclear factor-kappa beta signaling, NOTCH1 signaling, and RNA metabolism.

Two pathways were mutated exclusively in the IGHV-mutated subgroup — the BCR and Toll-like receptor pathways — "confirming importance of the BCR and Toll-like receptor pathways both in CLL pathobiology and as therapeutic targets," the authors wrote.

Looking at structural variants, the team confirmed both known copy number alterations and also identified novel candidate CLL driver genes, including deletions involving ZNF292, SP140, SP110, SMARCC1, SETD2, and NFKB2. Translocations involving IG genes were uncommon with the exception of BCL2 rearrangements, which occurred only in the IGHV-mutated group. Whole-genome sequencing analysis identified interchromosomal translocations in 43 out of 148 cases.

The contribution of noncoding mutations to cancer pathogenesis is largely unknown. The team identified 88 mutations in noncoding regions that were present in at least two cases. The most frequent recurrent noncoding mutation was in the 3' untranslated region of NOTCH1, which was present in four out of the 150 cases with whole-genome sequencing data. Targeted sequencing of that region in 356 cases that had received only exome sequencing found an additional seven tumors with the mutation and two cases with a mutation nearby.

Follow-up RNA sequencing of six of those cases confirmed a splicing event within the last exon of NOTCH1 that was not present in 290 tumors without the mutation. The splicing event is predicted to increase stability of the NOTCH1 protein. All patients with that mutation were in the IGHV-unmuated subgroup and had "features of adverse prognosis," the authors reported. They also "behaved similarly to patients with coding mutations in NOTCH1 in terms of the time to first treatment and overall survival."

The team also identified an intergenic region on chromosome 9 that was enriched for somatic mutations in 17 tumors. The region is enriched for lymphocyte-specific transcription factor binding sites and histone marks related to enhancer elements only in a lymphoblastoid B-cell line. The enrichment of mutations in that region was subsequently confirmed in an additional 25 cases that had received exome sequencing.

Further analysis of the region uncovered evidence that it is an enhancer related to the PAX5 gene. Mutations in this region were more prevalent in the IGHV-mutated subgroup, with 84 percent of patients with those mutations belonging to that subgroup. In seven tumors, the PAX5 mutations were the only somatic alterations, "raising the possibility that PAX5 enhancer mutations might constitute driver events contributing to the development of these tumors."

The authors noted that the study could have several clinical implications. First, the data supports the hypothesis that the "genomic differences between the two major molecular subgroups of CLL might be in part responsible for their different outcome."

Irrespective of a patient's subgroup, the number of mutations in driver genes corresponded with a patient's overall prognosis.

In addition, an analysis of the druggability of the driver genes identified candidate drugs for 19 out of 59 driver genes in 42 percent of the CLL cases.