NEW YORK (GenomeWeb) – A team from the University of Washington's Department of Laboratory Medicine has developed a novel method for combined mutation and microsatellite instability testing in colorectal cancer using next-generation sequencing.
The results, which appeared last month in the Journal of Molecular Diagnostics, showed that the approach, which the investigators call MSIplus, has high accuracy in detection mutations in cancer-associated genes, and slightly superior sensitivity and specificity in determining MSI phenotype compared to current methods.
Stephen Salipante, the study's senior author, told GenomeWeb that UW has been using MSIplus clinically for a few months now.
The development of the NGS-based assay began with an earlier publication in Clinical Chemistry in which Salipante and his colleagues first demonstrated the possibility of measuring MSI status from NGS data.
"That was a little bit different, in that we were retrospectively inferring MSI status from targeted gene capture data — so either exome sequencing at the extreme, or smaller gene panels that we're offering now for clinical diagnosis," Salipante said. "But that was the origin … where we really put forth this idea of inferring MSI status from NGS data.
For their latest work, the researchers created a more targeted and cost-effective way of analyzing MSI and a few other loci that are important in colorectal cancers, he said.
"The current methods for looking at MSI are really, really focused. They look at a small number of markers and they do it using a really trailing edge, analog technology, capillary electrophoresis. So our goal here was to sort of upgrade the classic MSI assay … and then also to enable a way of looking at far more markers than the previous assays allowed," Salipante explained.
The result, MSIplus, includes a set of microsatellite markers selected based on the group's earlier study, as well as others that reflect currently used MSI-PCR strategies. It also includes primers designed to span exons containing mutational hotspots in the cancer-associated genes KRAS, NRAS, and BRAF.
According to the UW authors, the assay involves a first PCR stage that is performed in two separate reactions — one using microsatellite primers, the other using primers targeting mutational hotspots. This is followed by a second PCR stage and sequencing of the resulting amplicons on the Illumina MiSeq.
In their study, Salipante and his colleagues evaluated the sensitivity and specificity of MSIplus, both in evaluating MSI phenotype and in detecting the presence of KRAS, NRAS, and BRAF mutations.
In a set of 61 patient samples with known mutation status, based on prior clinical testing, the assay demonstrated 100 percent sensitivity in identifying cancer-associated mutations with a detection limit of a two percent mutant allele fraction for the three target genes, the authors wrote.
In a separate set of 78 tumor specimens, the investigators determined that the approach was 97 percent sensitive and 100 percent specific for ascertaining MSI phenotype — a similar or slightly better performance than that reported for conventional multiplex PCR- and capillary electrophoresis-based MSI testing.
While other research groups have published methods to infer MSI status from NGS data, Salipante said that to the best of his knowledge, UW is alone so far in combining NGS-based MSI and cancer mutation testing, or in offering this type of assay clinically.
Clinical use of the test has been going very well, he said. "One [primary] advantage is that the interpretation is automated by a computer, so it's much less subjective than doing capillary electrophoresis … Sign out of this test takes significantly less effort and time ... and as our paper provides evidence, the calls are actually slightly better overall than capillary electrophoresis."
According to the study authors, the assay is compatible with a two- to three-day turnaround time, and requires only about 50 nanograms of input DNA.
Also, unlike current conventional MSI testing, the approach does not require matched normal patient samples.
Initially targeted at MSI status and mutational hotspots in KRAS, NRAS, and BRAF, the assay is designed to expand to include additional microsatellite loci and other gene targets as they become clinically relevant, Salipante said.