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Exome Sequencing Points to V600E BRAF Amplification in Some BRAF Inhibitor-Resistant Melanomas

NEW YORK (GenomeWeb News) – A University of California at Los Angeles-led team has identified a copy number change to the BRAF gene that coincides with drug resistance in a form of metastatic melanoma involving BRAF mutation.

The BRAF inhibitor vemurafenib, marketed by Roche subsidiary Genentech as Zelboraf, is used to treat forms of melanoma that harbor a characteristic V600E mutation in BRAF. The US Food and Drug Administration approved the drug for treating metastatic melanoma last August, along with a companion diagnostic test for determining BRAF mutation status.

In an effort to better understand how resistance to this targeted treatment can develop, researchers used exome sequencing to compare matched normal samples from individuals with V600E melanoma to tumor samples taken before and after vemurafenib treatment.

As they reported online today in Nature Communications, investigators found copy number gains in the mutated V600E gene that corresponded to the development of drug resistance in the exomes — a gain of function mutation that they ultimately saw in four of the 20 cases of BRAF inhibitor resistance that were tested.

"For the first time, we were able to see in actual patient tissue samples how the cancer gets around this drug by altering the target," corresponding author Roger Lo, a dermatology, molecular and medical pharmacology, and cancer researcher at UCLA, said in a statement. "It appears that the drug target is not only mutated and hyper-activated, but it's also massively over-produced in some cases of clinical relapse."

Lo and his colleagues used Agilent arrays to capture coding sequences from normal, pre-treatment, and treatment-resistant samples for two melanoma patients. The exomes were subsequently sequenced using the Illumina GAII or Illumina HiSeq2000.

Analyses of the exomes revealed mutations that increased the V600E BRAF gene copy number by two to almost 13 times — a finding that the team verified using quantitative PCR.

When researchers tested additional samples from their collection of 20 matched pre-and post-treatment melanoma samples, they saw similar V600E BRAF amplification in two more melanomas that had become resistant to the BRAF inhibitor.

"[I]n some patients, the cancer simply makes more of the target, the mutated BRAF gene, so that the drug dose becomes too weak to fight the cancer," Lo explained. "[W]e get more of the drug target, which has the same effect as dropping the drug level."

The team's follow-up experiments supported these results, showing that enhanced V600E BRAF expression can produce resistance to BRAF inhibition, while knocking down the mutated gene makes cells more sensitive to such treatment.

The new findings add to the tally of BRAF inhibitor resistance mechanisms found so far, researchers noted, though the genetic roots of drug resistance remain to be discovered for some 30 or 40 percent of relapse cases.

"If we know what happens in every relapse, we can have a plan in place that will help us avoid or overcome resistance," Lo said.

For instance, results of the current study indicate that BRAF inhibitor-resistant tumors that contain additional copies of V600E BRAF reactivate downstream signaling pathways via somewhat different routes than those used by drug resistant tumors harboring NRAS gene mutations, they reported, suggesting "alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired BRAF inhibitor resistance in melanoma."

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