By Monica Heger
In what appears to be a first in cancer sequencing, exome sequencing has identified a single protein-coding mutation, for which inhibitors already exist, that was then confirmed in all cases tested.
Researchers from the University of Perugia in Italy sequenced the whole exome of a patient with hairy cell leukemia, a rare form of leukemia characterized by leukemic B cells that have hairy-looking projections, and found a variant in the BRAF gene — the V600E mutation — that was present in all 47 additional cases tested.
Since BRAF inhibitors such as Roche/Plexxicon's vemurafenib, or PLX 4032, are currently in clinical trials for melanoma patients, the authors think there could be a rapid transition of their findings to the clinic.
"The remarkable thing was not so much that [the BRAF mutation] was found, but that every single case that they tested then had that variant," said Charles Mullighan, a pathologist at St. Jude Children's Research Hospital, who is conducting targeted sequencing in ALL patients (CSN 4/5/2011).
The finding is "exceptionally striking and suggests that it is a key alteration in the development of the leukemia," he added.
The results were published last week in the New England Journal of Medicine.
Using Agilent's in-solution capture and sequencing on the Illumina Genome Analyzer, the team sequenced the tumor and matched normal genome from a patient in remission to a mean of 71-fold coverage, generating over 42 million reads from each sample.
The team identified five missense somatic mutations in the patient's tumor DNA located in the BRAF, CSMD3, SLC5A1, CNTN6, and OR8J1 genes.
Because the BRAF gene is the most frequently mutated gene that encodes a protein kinase in human cancers, the team focused their analysis on that mutation. The so-called V600E mutation causes an amino acid substitution of glutamic acid for valine at position 600, and is a mutational hotspot in melanomas and papillary thyroid cancers, but had not yet been associated with HCL, and only found in a rare number of multiple myeloma.
When the team screened an additional 47 HCL patients using Sanger sequencing, all 47 patients had the exact same mutation.
"To my knowledge, this is the first demonstration of exome sequencing revealing a discrete genetic target in a complex disease such as cancer," said Todd Druley, an assistant professor of pediatrics and genetics at Washington University, who is leading a targeted sequencing project of 450 childhood leukemia samples (IS 2/1/2011).
"The target already has functional agents in clinical practice," added Druley. "So presumably, you could take those agents and apply them to those individuals in a short amount of time."
HCL patients are typically treated with purine nucleoside analogues, which are effective in most patients. However, the genetic alterations underlying the disease have been unknown.
Despite having an effective treatment, Brunangelo Falini, senior author of the study and professor of hematology at the University of Perugia said that around 40 percent of patients relapse within five to 10 years. Additionally, the drugs are powerful immunosuppressants, so patients can acquire infections, and some experience excessive toxicity from the drugs, he said.
After testing 47 HCL patients, Falini and his team next looked for the BRAF mutation in other B-cell lymphomas and leukemias. None of the 195 samples they investigated carried the mutation.
Finally, the team analyzed the effect of a BRAF inhibitor on in vitro cells — specifically PLX 4720, an analog of PLX 4032. Targets downstream of BRAF appeared to be inactivated when leukemic hairy cells from five different patients were incubated with the BRAF inhibitor, but remained active without the drug.
Falini said that additional in vitro analyses would have to be done, but the fact that BRAF inhibitors are currently being tested in melanoma patients with good results could enable a quick transition to the clinic for HCL patients as well.
Initially, he said, the drug would be tested in patients who did not respond to conventional therapy, had multiple relapses, or experienced excessive toxicity.
Falini added that he and his team would share the results of this study with the patients' oncologists. "These patients are under treatment, so they must know that there's been a new discovery that could be a new therapeutic opportunity in the future if their present therapy fails," he said.
Mullighan said that while the finding is promising, "a lot more work needs to be done." Aside from more in vitro testing of the drug, researchers would also have to compare the drug to existing treatments since there is no guarantee that the BRAF inhibitor would produce better results. Additionally, because it is targeted to just one mutation, the BRAF inhibitor may not be effective if other mutations are also driving the cancer and could result in the emergence of resistance, he said.
The team recently finished sequencing the exomes of another nine HCL patients, all of whom have the BRAF mutation. Falini said they are now analyzing those genomes, looking for other recurrent mutations that may be cooperating with the BRAF mutation. They are also looking at the other four somatic mutations identified in the first patient.
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