A comparison of Roche's Cobas 4800 BRAF V600 Mutation Test and Sanger sequencing has demonstrated that the latter platform may be more sensitive in detecting BRAF mutations in melanoma patients.
The study, conducted by clinical laboratory testing services provider Quest Diagnostics and presented at the American Society of Clinical Oncology's annual meeting earlier this month, analyzed 125 formalin-fixed paraffin-embedded tissue samples from melanoma patients by the PCR-based Cobas test and bi-directional Sanger sequencing. The analysis revealed that the two platforms agreed approximately 83 percent of the time.
The US Food and Drug Administration approved Roche's BRAF inhibitor Zelboraf (vemurafenib) last year as a treatment for metastatic melanoma patients with BRAF-mutated tumors. At the same time, the agency also approved Roche's Cobas BRAF V600 Mutation Test as a companion diagnostic for assessing which patients should receive Zelboraf.
The drug's label states that doctors should use an FDA-cleared BRAF mutation test to figure out which patients should receive Zelboraf. Roche's Cobas test is not mentioned by name in the drug label, but it is currently the only test that the agency has approved for use as a companion diagnostic for the treatment. Most insurers have put in place prior-authorization schemes to ensure that BRAF testing to administer Zelboraf is covered only when an FDA-approved diagnostic is performed.
A Quest spokesperson told PGx Reporter that the company conducted the study in order to meet federal requirements for running the FDA-cleared Cobas platform in its lab. "We do not plan to gain FDA clearance for the Quest Diagnostics laboratory-developed test used in the study or otherwise offer it as an alternative to the Roche assay," the spokesperson added.
Quest provides BRAF testing in various clinical settings, using a number of technology platforms, and was offering such testing to gauge BRAF mutations in melanoma patients prior to the approval of Zelboraf and the Roche companion test.
After the drug/test combination came to market, however, the company started offering Roche's test "to help ensure that our physician clients had access to the FDA-cleared companion diagnostic for Zelboraf." Quest still provides BRAF mutation testing on different technologies for other tumor types, the company spokesperson said.
The Quest analysis showed that Sanger sequencing detected V600 dinucleotide mutations in nine samples that the Cobas test deemed negative. Sanger sequencing, on the other hand, yielded no results for 10 samples due to "suboptimal PCR," Quest researchers reported in the abstract. Of these samples, two were found to be BRAF mutation-positive by the Cobas test.
Meanwhile, the Cobas assay had two invalid results, but Sanger sequencing reported one of these samples to be positive for the V600E mutation. The Cobas assay also detected seven out of 11 V600K mutations.
The PCR-based Cobas test gauges BRAF V600E and V600K mutations. Melanoma patients who harbor these mutations respond to BRAF inhibitors such as Zelboraf.
Based on these findings, researchers from Quest concluded that "the Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by Cobas compared to the two seen by Cobas but not Sanger sequencing." Overall, out of 57 BRAF mutation-positive patients in the study, Sanger sequencing was able to gauge 16 percent more, or nine additional patients, as candidates for Zelboraf treatment than did the Cobas platform.
The findings are somewhat in contrast to earlier data presented by Roche comparing its Cobas test against Sanger sequencing.
At the European Multidisciplinary Cancer Congress last year, Roche presented data showing that after 433 evaluable samples were analyzed by Cobas and Sanger sequencing, there was 96.4 percent agreement between the two tests for cases that were BRAF mutation positive and 80 percent agreement for those that were mutation negative. The Cobas test "had a lower failure rate than Sanger; was more sensitive in the detection of V600E mutations than Sanger, and detected a majority of V600K mutations in the cohort," concluded the authors of the study, which was funded by Roche (PGx Reporter 9/28/2011).
In samples for which the two tests yielded discordant results, Roche tested them again with Roche 454 sequencing. The analysis revealed that for "42 samples that were mutation-positive by the Cobas test and negative for the V600E mutation by Sanger, 17 samples were wild-type (15) or non-V600E (2) by Sanger but V600E-positive by 454 sequencing," the authors reported. Meanwhile, 24 of those samples were V600K positive by both Sanger and 454 sequencing, and Sanger sequencing found one sample harbored a rare GTG-to-GAC mutation.
Since the Cobas test also detects V600K mutations, around 10 patients with V600K mutations were included in the Zelboraf arm in the study that led to the drug's approval, and these patients were shown to have some response to the drug.
Based on the results of last year's study, Roche researchers concluded that more than 13 percent of patients who would benefit from Zelboraf treatment would have been denied the drug if they were tested with Sanger sequencing. In comparison, the Cobas test would have denied treatment to less than 1 percent of patients who would have responded to the drug.
Roche conducted the head-to-head comparison shortly after the launch of Zelboraf last year in an effort to convince labs to switch to the FDA-approved platform. Industry observers had notably pointed out at the time that some large hospitals weren't using Roche's test when it came to deciding which patients should be treated with Zelboraf, choosing instead to maintain testing methods they had already adopted.
Roche said then that its sales team was conducting educational programs and seminars to gain more customers for the Cobas BRAF test and that it was disseminating information about the test to doctors.
Anthony Sferruzza, senior scientific director of oncology/coagulation at Quest and one of the investigators of the new study presented at ASCO, told PGx Reporter that "several factors" may explain why the two comparisons arrived at opposite conclusions. According to Sferruzza, extraction technology, the age of the sample, specimen fixation, and the nature of the tumor itself might impact the study results.
For Roche's test, the Quest researchers extracted DNA from 5 μm tissue sections without macrodissection using the Cobas DNA extraction kit; while for Sanger sequencing, they used Agencourt kits from Beckman Coulter to extract DNA from 5 μm to 10 μm tissue after macrodissection.
"It is well known that melanin will interfere with PCR reactions, and therefore the PCR reaction is highly dependent on thorough removal of the melanin," Sferruzza explained. "Residual melanin may inhibit the PCR reaction."
The FDA-approved labeling for the Cobas BRAF test notes this weakness of the PCR-based platform. "Melanin is a known inhibitor of PCR reactions. The DNA sample preparation kit removes melanin from the specimen during extraction; however, melanin in a specimen may still cause invalid results," the test label states.
Similarly, there are also challenges with Sanger sequencing. For example, this type of technology has difficulty analyzing highly heterogeneous tumors, so there is a risk of false negatives if the tissue sample isn't correctly dissected in order to enrich for the tumor population.