NEW YORK (GenomeWeb) – For metastatic melanoma, BRAF genotyping is typically performed to determine the best pharmacotherapy, but different genotyping methods can have differing efficiencies and ideal uses, particularly in the case of formalin-fixed, paraffin-embedded samples.
According to a new head-to-head comparison of five BRAF genotyping methods performed at the University of Lorraine and Lorraine Cancer Institute in France, certain PCR-based tests are more suitable than others while immunohistochemistry should only be used as a screening tool.
The research, published last month in Molecular Diagnosis and Therapy, examined FFPE tumor samples from 59 patients. It compared next-generation sequencing and immunohistochemistry using a Val600Glu-specific antibody to three assays that use reagents from Roche Diagnostics — namely, the Cobas 4800 BRAF V600 Mutation Test kit, high-resolution melting PCR using Roche's LC 480 HRM Master kit, and the LC480 SybrGreen Master kit and thermocycler for multiplex real-time allele specific amplification, or multiplexed RT-ASA.
The researchers undertook the comparison because they were looking to determine the best BRAF assay for FFPE samples in terms of cost, time, and overall effectiveness, corresponding author Alexandre Harlé said in an email.
In the study, 31 samples were found to have BRAF mutations using NGS. The multiplexed RT-ASA assay detected 29, while the Cobas V600 kit and IHC detected 28 and 27 mutations respectively. The NGS data revealed that 26 of the mutations were c.1799 T-to-A mutations of Val600Glu, or V600E. Others included three c.1798-1799 GT to AA Val600Lys, one c.1789-1790 CT to TC Leu597Ser, and two complex mutations.
Compared to NGS, the IHC and HRM assays had a sensitivity of about 87 percent, while the Cobas kit and multiplex RT-ASA had sensitivities of 90 percent and 93 percent, respectively. Meanwhile, specificity was 100 percent for the Cobas test, IHC, and multiplex RT-ASA and 96 percent for HRM.
The researchers concluded that the IHC test should be considered as a screening tool. They also concluded that the BRAF Cobas assay is Val600Glu-specific and that it is therefore important to use other assays, such as multiplexed RT-ASA or NGS, to avoid false-negative results and "deprive the patient of an important treatment option."
The multiplex RT-ASA assay was a SybrGreen-based PCR that consisted specifically of primers for the detection of BRAF p.Val600Glu (c.1799 T>A), p.Val600Lys (c.1798_1799 GT>AA), p.Val600Arg (c.1798_1799 GT>AG) and p.Val600Asp (c. 1799_1800 TG>AT), plus positive controls. "This assay is really simple, takes 90 minutes, and requires less than 10 ng of DNA," Harlé said.
John Osiecki, director of medical and scientific affairs at Roche Diagnostics, highlighted in an email interview that the study authors also noted that the Cobas 4800 BRAF V600 Mutation Test is indicated as a companion diagnostic for Zelboraf (vemurafenib). The test is used to guide treatment with the drug because of its activity in the V600E mutation-positive melanoma patient population, as demonstrated in the BRIM-3 clinical trial.
The labeling of the test indicates that it is "an in vitro diagnostic device intended for the qualitative detection of BRAF V600E mutations in DNA extracted from formalin-fixed, paraffin-embedded human melanoma tissue," Osiecki said. "The Cobas test was not designed to detect all BRAF mutations."
More specifically, the Cobas BRAF test uses primers that define a 116-base pair sequence of human genomic DNA containing the BRAF codon 600 site in exon 15, Osiecki said, and the entire BRAF gene is not amplified. "The test is designed to detect the T1799A nucleotide change in the BRAF gene which results in a valine-to-glutamic acid substitution at codon 600", he said.
However, Harlé noted that it has been previously described that vemurafenib is also effective in all BRAF V600 mutated melanomas, and that is why the researchers decided to include other BRAF mutations in the study. Vemurafenib is used in Europe for all BRAF V600 mutated melanomas, he said, and it has also been shown that patients with melanomas bearing a mutation close to codon 600 of BRAF were also responders to this drug, but the drug is used off-label in those cases.
Osiecki pointed out that it is not clear how frequently the rare and complex mutations that were detected via sequencing occur. "Therefore, additional, larger research and clinical trial studies are needed to determine the impact diagnostic methods such as sequencing may contribute as an aid in selecting melanoma patients for treatment with targeted therapies," he said.
Although sequencing did detect more mutations, Harlé said the lab's routine use of sequencing would depend on the application. "Some of our oncologists want to have a quick answer for the BRAF status of their patients," he said, and for a quick determination, real-time PCR is "clearly the best assay." NGS is often more expensive and requires at least two to three days from sample extraction to final results, he added.
Harlé's group published a study in PLoS One last year using the Idylla system from Biocartis, a fully-automated platform that is able to detect all the V600 mutations of BRAF in 90 minutes with a hands-on time of about two minutes, he said, compared to one hour of hands-on time with multiplex RT-ASA. That study found the Idylla test was suitable for routine molecular diagnostics. However, the lab still often uses NGS, "because complex mutations are not detected by real-time PCR assays."
Still, the future of oncology diagnostics may yet be in NGS, Harlé said, noting that he is convinced that use of NGS will one day be mandatory, particularly in cases where a large number of potential targets need to be accurately assessed for personalized treatment determination. Harlé imagines that sequencing technologies of the future will uncover more information about tumor genes with quick, cost-effective assays.
In the meantime, Harlé's group has also just finished a clinical trial to study the impact of a rapid determination of BRAF mutations in patients with melanoma and expects to publish the results soon.