SF3B1 and other novel cancer genes in chronic lymphocytic leukemia

Abstract

Background: The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood.

Methods: We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease.

Results: Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing.

Conclusions: Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

Figure 1. Genes with Significant Mutation Frequencies in 91 Patients with Chronic Lymphocytic Leukemia

The figure shows the number of mutations per gene that were found at a significant frequency, the percentage of patients who had each mutated gene, and for each gene, the total territory in numbers of base pairs with sufficient sequencing coverage across normal and malignant samples from the 91 patients. The P values and Q values were calculated by comparing the probability of the observed constellation of mutations with the background mutation rates across the data set.

Figure 2. Core Signaling Pathways in Chronic Lymphocytic Leukemia

The nine genes with significant mutation frequencies fall into five core signaling pathways, in which the genes play well-established roles: DNA damage repair and cell-cycle control (Panel A), Notch signaling (Panel B), inflammatory pathways (Panel C), Wnt signaling (Panel D), and RNA splicing and processing (Panel E). Genes with significant mutation frequencies are shown in red, and genes with mutations that are in a signaling pathway related to chronic lymphocytic leukemia are shown in pink. A list of additional pathway-associated genes with mutations is provided in Table 6 in the Supplementary Appendix. Co-A denotes coenzyme A, TLR toll-like receptor, and TNFR tumor necrosis factor receptor.

Figure 3. Associations between Gene Mutations and Clinical Characteristics

Samples from the 91 patients in the study were sorted by means of fluorescence in situ hybridization (FISH), with the use of a model of hierarchical risk established by Döhner et al. Samples were scored for the presence or absence of mutations in the nine genes with significant mutation frequencies (darker colors), as well as for mutations in additional pathway-associated genes (lighter colors) and in the immunoglobulin heavy-chain variable (IGHV) mutation status (blue indicates mutation, white no mutation, and hatched unknown status). (A list of additional mutated pathway-associated genes is provided in Table 6 in the Supplementary Appendix.) Associations between gene mutation status and FISH cytogenetic features or IGHV status were calculated with the use of Fisher’s exact test and corrected for multiple hypothesis testing (Q≤0.1 for all comparisons shown).

Figure 4. Mutations in SF3B1 and Altered mRNA Splicing

In Panel A, a Cox multivariable regression model designed to test for clinical factors contributing to the need for earlier initiation of treatment showed that an SF3B1 mutation was an independent predictor of a shorter time to treatment, regardless of the status of several other independent predictive markers. Panel B shows the relative amounts of spliced and unspliced spliceosome target messenger RNA (mRNA) in BRD2 and RIOK3 in normal CD19+ B cells and chronic lymphocytic leukemia B cells with wild-type or mutated SF3B1, as measured by means of a quantitative polymerase-chain-reaction assay. The ratios of unspliced to spliced mRNA were normalized to the percentage of leukemia cells per sample, and comparisons were calculated with the use of the Wilcoxon rank-sum test. CI denotes confidence interval.