Roxanne Nelson

December 12, 2011

December 12, 2011 (San Diego, California) — A number of mutated genes in chronic lymphocytic leukemia (CLL) have been identified, leading to a better understanding of the mechanism of the disease. The new findings will help to refine the molecular classification of the disease and will help physicians estimate prognosis, experts predict.

The new findings were presented here at the 53rd annual meeting of the American Society of Hematology and are simultaneously published online December 12 in the New England Journal of Medicine (NEJM).

The study identified 9 significantly mutated genes that occurred in 5 core signaling pathways in which the genes play established roles: DNA damage repair and cell-cycle control (TP53, ATM), Notch signaling (FBXW7, NOTCH1), inflammatory pathways (MYD88, DDX3X, MAPK1), and RNA splicing/processing (SF3B1, DDX3X).

Of these mutations, 5 of the mutated genes have been implicated in CLL for the first time, according to study coauthor Youzhong Wan, PhD, who presented the data at ASH.

"We sequenced 3 whole genomes and 88 whole exomes, of all CLL tumors and paired the germline DNA," said Dr. Wan, who is from the Department of Medical Oncology at the Dana-Farber Cancer Center in Boston, Massachusetts.

Sequencing a large sample set increases the chance of detecting the full range of mutated pathways, he explained, adding that "it can enable reconstruction of gene pathways underlying disease pathogenesis, and reveals associations between genetic events and the clinically important disease features."

Raises Provocative Possibilities

CLL is a common and clinically heterogenous leukemia, and the somatic genetic basis of this disease has been poorly understood, note the study authors. However, they point out that the use of massively parallel sequencing technology provides a way of identifying genetic alterations that underlie disease, as well as uncovering new therapeutic targets and biomarkers.

"The identification of mutations in genes involved in RNA splicing was highly unexpected," note the authors of an editorial accompanying the NEJM paper, Benjamin Ebert, MD, PhD, from Brigham and Women's Hospital in Boston and Olivier A. Bernard, PhD, from the Institut Gustave Roussy in Villejuf, France. However, "it converges remarkably with recently published studies making use of genome sequencing in myelodysplastic syndromes," they add.

The editorialists also point out that finding SF3B1 mutations in both CLL and myelodysplastic syndromes "resonates with the recent finding of TET2 mutations in both lymphoid and myeloid cancers."

"These developments raise the provocative possibility that SF3B1 mutations might in some cases occur initially in hematopoietic stem cells," they write, "with additional mutations then being acquired in either the lymphoid or the myeloid lineages and causing chronic lymphocytic leukemia or myelodysplastic syndromes, respectively."

Consistent with this hypothesis is the finding that stem cells from patients with CLL have "recently been reported to be abnormally lymphoid-primed, a finding that suggests that chronic lymphocytic leukemia could also derive from a stem-cell defect."

A Big Surprise

In this study, Dr. Wan and colleagues sequenced DNA samples obtained from the tumors and tissue of 91 patients with CLL (discovery cohort). Additional DNA samples were obtained from 101 patients with CLL (extension cohort). The samples were collected from patients who displayed a characteristic representative of the broad clinical spectrum of CLL, including those with del(11q) and del(17p), and with unmutated or mutated status of the variable region of the immunoglobulin heavy chain gene.

Massive parallel sequencing was conducted for 88 exomes and 3 genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in CLL. The authors also constructed libraries, which were sequenced on an Illumina GA-II sequencer to deep coverage.

A total of 1828 nonsilent mutations in protein-coding sequences were identified, and these corresponded to an average somatic mutation rate of 0.72/Mb (range, 0.075 - 2.14/Mb). Within this group, 9 cancer genes were subsequently identified. These 9 genes were mutated significantly more often than the background rate, given their sequence composition across all 91 CLL/normal pairs, note the researchers.

Of these 9 genes, 4 have already been described in CLL (TP53, ATM, MYD88, and NOTCH1). However, the remaining 5 are novel and have not been described previously in CLL (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X).

Dr. Wan noted that a "big surprise in our sequencing" was the discovery that SFB31 was the second most frequently mutated gene, associated with mutations in 15% (n = 14) of patients with CLL. SFB31 mutations occurred primarily in tumors with deletions in chromosomes 11q, tumors associated with a poor prognosis. Mutations in SFB31 have also been reported in myelodysplastic syndromes, he added.

"SF3B1 mutation independently predicts poor prognosis," said Dr. Wan, "And is independent from other clinical factors of CLL."

The authors also found that all 14 mutations in these patients were localized to a discrete region (exons 12-18), and 7 were recurrent (K700E). They then validated the SF3B1-K700E mutation, along with 2 other frequently occurring mutations — MYD88-L265P and NOTCH1-P2514fs — through genotyping of the 101 independent CLL samples.

These common cytogenetic aberrations in CLL carry strong prognostic significance, the authors write, and imply that they reflect distinct pathogenesis. In support of this hypothesis, they point out that they also discovered "strong associations between different driver mutations and key FISH [fluorescence in situ hybridization] abnormalities."

"Understanding the mutational landscape of CLL provides a starting point for systematic analyses to address fundamental questions in CLL, including how mutated genes alter cellular networks and phenotypes, and thereby contribute to disease heterogeneity," concluded the study authors at the meeting.

The study was supported by grants from the National Human Genome Research Institute, National Cancer Institute, the Blavatnik Family Foundation, and National Institutes of Health. Author disclosures are available with the full text of this article at the journal's Web site.

Annual Meeting of the American Society of Hematology. Abstract 463. Presented December 12, 2011.

N Engl J Med. Published online December 12. Study full text, Editorial full text

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