NEW YORK (GenomeWeb News) – An international research group led by investigators in the US and Italy reported in the Journal of Experimental Medicine today that it has tracked down recurrent coding mutations linked to patient outcomes in a form of leukemia known as chronic lymphocytic leukemia.
The team did exome sequencing and copy number analyses of matched tumor-normal samples from five individuals with CLL. While the exomes contained relatively modest numbers of mutations overall, the team found an over-representation of mutations in the transmembrane protein coding gene NOTCH1. Their subsequent screens of CLL tumors, corresponding normal samples, and clinical samples indicate that activating mutations in NOTCH1 tend to coincide with particularly aggressive forms of CLL and poor survival rates.
"The present study documents a high frequency of NOTCH1 mutations in aggressive clinical phases of CLL … and identifies NOTCH1 alterations as an independent predictor of poor prognosis," co-corresponding author Gianluca Gaidano, a hematology researcher at the University of Eastern Piedmont, and co-authors wrote.
In general, CLL, also known as B-cell chronic lymphocytic leukemia, is linked to a wide range of outcomes, the researchers explained, with some patients surviving many years and others quickly progressing to life-threatening forms of the disease. For example, they noted, patients with Richter syndrome, whose CLL transforms to diffuse large B-cell lymphoma, tend to have much worse outcomes than other individuals with CLL.
Although some mutations — including a deletion on chromosome 13 and alterations affecting TP53, ATM, and MYC — have been tied to CLL, many of the genetic hiccups contributing to the disease and to Richter syndrome are still unidentified.
In an effort to explore this in more detail, Gaidano and his colleagues used the NimbleGen Sequence Capture 2.1M capture human exome array to capture protein-coding regions for five CLL tumor and matched normal genomes from individuals who had been diagnosed with the disease but had not yet received any treatment. They then sequenced these regions with the Roche 454 GS FLX.
Overall, the team identified between six and 10 tumor-specific coding mutations per CLL case. Of the 39 genes containing somatic, non-silent coding mutations in these samples, they reported, NOTCH1 was the only one that was mutated in more than one of the tumor exomes.
Meanwhile, the researchers' copy number analyses using the Affymetrix SNP 6.0 chip uncovered a dozen copy number changes in the five tumors, including a handful of deletions and duplications reported for CLL in the past.
In general, they found that deletions were more common than duplications, though only one deletion, which fell on chromosome 13, turned up in more than one of the five tumors.
Together, their findings suggest that CLL genomes contain between seven and 13 alterations apiece. Taking into account mutations that may have been missed by the current coverage level, they estimated that each tumor contains fewer than 20 coding changes.
Next, the team did follow-up testing in 48 CLL tumor-normal samples, including CLL cases with or without mutations in IGHV, a gene whose mutation status appears to influence CLL outcomes.
When they assessed the genes identified in the first phase of the study in these additional tumor samples, the team found recurrent mutations in NOTCH1 and in three other genes: TGM7, BIRC3, and PLEKHG5. Whereas most of the genes were mutated in fewer than five percent of cases, they noted, NOTCH1 was mutated in around 15 percent.
Moreover, when they turned their attention to clinical samples, the researchers found evidence that NOTCH1 mutation status tended to coincide with everything from five year treatment-free survival and chemotherapy outcomes to Richter syndrome occurrence, with the presence of activating NOTCH1 mutations at the time of disease diagnosis corresponding to worse outcomes.
Based on their findings, those involved in the study argue that NOTCH1 mutation status may provide insights into disease progression and open the door to new, targeted treatments for some CLL cases.
"NOTCH1 represents a well-established therapeutic target with some drugs already available … and others under active development," they wrote. "Our results add CLL to the cadre of common diseases in which these drugs should be tested for their efficacy alone or in combination with available therapeutic regimens."
Recurrent mutations in NOTCH1 and several other genes were also reported in a whole-genome sequencing study of four CLL patients and follow-up work involving hundreds more cases that was published recently in Nature by investigators from Spain and the UK.
At the Biology of Genomes meeting last month, University of Oxford hematologist Anna Schuh presented findings from an effort to characterize the genetics behind B-cell CLL treatment response, remission, and relapse using a whole-genome sequencing strategy.
In a study appearing online in the New England Journal of Medicine this weekend, meanwhile, an Italian-led research group reported that it used whole-exome sequencing and follow-up screening to find mutations — including BRAF V600E mutations — in individuals with a form of B-cell lymphocyte cancer known as hairy cell leukemia.