NEW YORK (GenomeWeb) – Separate research teams from Memorial Sloan Kettering Cancer Center and Dartmouth Hitchcock Medical Center (DHMC) last week presented unpublished validation studies on Biocartis' Idylla microsatellite instability assay, demonstrating its ability to detect traces of tumor mutations in a variety of cancers such as endometrial and colorectal carcinoma.
The posters were part of a larger group of studies presented by Biocartis collaborators at the Association for Molecular Pathology annual meeting last week that examined the use of multiple Idylla assays in detecting mutations in genes including EGFR, BRAF, and KRAS for a variety of oncology applications.
Microsatellite instability (MSI) occurs when mismatch repair proteins (MMR) fail to fix a DNA replication error, leading to frame-shift mutations in a person's genetic code and non-functioning proteins. MSI has acted as a strong marker for genetic conditions such as Lynch syndrome that can lead to a variety of cancers.
As such, multiple companies have developed MSI assays to detect a variety of early-stage cancers. The US Food and Drug Administration has granted Breakthrough Device designation to both Personal Genome Diagnostics and Foundation Medicine for their existing next-generation sequencing RUO liquid biopsy assays to include MSI detection. Under the program, the FDA works with a test developer to reduce the time and cost from development to approval.
Promega's RUO MSI assay applies fluorescent multiplex PCR to co-amplify seven markers for analysis of the MSI-high (MSI-H) phenotype.
In July, Biocartis launched its automated MSI assay for research use only, which runs on the firm's Idylla PCR-based platform. The tool analyzes seven biomarkers located in the ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2 genes.
In one study presented at a Biocartis-sponsored workshop at AMP, MSKCC molecular geneticist Khedoudja Nafa and her team validated use of the Idylla MSI assay for ultra-rapid assessment of MSI status on formalin-fixed, paraffin-embedded tissue sections without the need for prior DNA extraction or concurrent testing with a normal control.
Nafa's team examined 52 tumor samples — 38 FFPE and 14 extracted DNA — with known MSI status and ran them on the Idylla MSI assay.
Nafa's team labeled the samples based on the presence of instability in the seven markers: if the samples had two or more unstable biomarkers, they were considered MSI-H; if the samples had fewer than two unstable biomarkers, they were considered MSI stable (MSS). Nafa and her team then compared the results to previously determined MSI status based on NGS data generated by MSK-IMPACT, Promega's MSI-PCR tool, and an MMR immunohistochemistry assay.
The team found concordant results for 24 of 25 MSI-H and 26 of 27 of the MSS cases between Biocartis and Promega's assays, for an overall concordance of 96 percent. The researchers required four minutes of hands-on time to run the assay, with about 150 minutes from setup to report generation.
Nafa's team concluded that the Idylla MSI test is a simple and fully-automated solution for MSI status determination, providing quick results that are concordant with other MSI testing approaches.
In another study presented in a poster at AMP, lead author Catherine Nicka and her colleagues at DHMC's Laboratory for Clinical Genomics and Advanced Technology (CGAT) used the Idylla MSI assay to detect cases of microsatellite instability caused by deficiencies in the mismatch repair gene in endometrial carcinoma.
In a follow-up email after the meeting, Nicka explained that her team chose endometrial carcinoma because the condition is the second most common type of cancer associated with Lynch syndrome.
Nicka's group collected DNA from 12 deficient mismatch repair (dMMR) endometrial cancer samples that had been previously screened by IHC for deficiency or MMR proteins. Adding one microliter of DNA to the Idylla MSI cartridges, the researchers then used the Idylla platform to perform DNA preparation, PCR, and melt curve analysis on the samples.
Nicka's team found that MRE11 popped up as the most commonly mutated marker in the samples.
Comparing the MSI status to known MMR protein status by IHC, the researchers found that 75 percent of the MSI cases were classified as MSI-H, while 25 percent were classified as MSS. In the MSI-H samples, they saw that the number of gene mutations ranged from two to six biomarkers.
Nicka noted that her team's main challenge using the Idylla system was optimizing the proper amount of input tumor tissue for the assay. However, she highlighted that the Idylla assay does not require normal control tissue, and researchers can instead use extracted DNA or FFPE tissue sections.
While Idylla results showed concordance with mismatch repair status in most of the samples, Nicka's team noted that additional studies will need to identify reasons for discrepant results. Nicka said her team plans to routinely analyze MSI status in endometrial cancers.
In another CGAT-led validation study, researchers investigated whether the Idylla MSI assay could serve as an alternative method to determine MMR status by comparing it to IHC results for colorectal cancer.
Nolan Maloney, lead author and chief resident at DHMC's department of pathology and laboratory medicine, explained that his team selected 42 retrospective samples from DHMC collected between 2011 and 2018, including 10 MMR-proficient carcinomas and 34 MMR-deficient carcinomas. The team collected two 5-micrometer thick sections of tissue from FFPE samples for each tumor and ran the samples on the MSI assay.
The researchers found an overall agreement of 95 percent between the two techniques. All MMR-proficient tumors by IHC were MSI stable with the Idylla assay, while 32 of 34 MMR-deficient tumors by IHC showed high MSI by Idylla.
Maloney and his team also found that two MMR-deficient tumors by IHC were also microsatellite stable using the Idylla assay. The researchers further analyzed the two tumors with a Promega MSI analysis system, which revelated microsatellite stability for both tumors and confirmed the Idylla results.
The team concluded that the Idylla assay showed high concordance with IHC in terms of determining MMR status in CRC, indicating that it could be used an alternative to find MMR status in CRC.
"We will definitely be going live with the MSI assay and have plans to further evaluate the KRAS cartridge for CRCs," Maloney said. "In addition, others in the lab have been experimenting with novel sample types, including a 'molecular' touch prep method."
Additional assays
While multiple studies examined applications for Biocartis' MSI assay, the firm also presented promising validation data regarding its EGFR assay. During the company workshop, Maria Arcila, director of MSKCC's diagnostic molecular pathology laboratory, discussed her team's adoption of Idylla for EGFR mutation assessment.
While the Idylla system has been validated for FFPE samples, Arcila explained that her team examined the EGFR mutation status in lung adenocarcinoma, comparing the Idylla EGFR mutation assay to the MSKCC's IMPACT sequencing platform on a variety of tissue types.
Arcila's team collected 62 tissue samples — 50 mutation positive and 12 negative samples — that encompassed stained cytology smears and touch preparations, residual minimal cytology material, extracted DNA, and aliquots of pre-capture NGS libraries prepared for the 486-gene IMPACT large panel NGS assay.
The researchers concurrently tested the samples with IMPACT and a sizing assay for rapid detection of indels in EGFR and ERRB2.
According to Arcila, the researchers had specific requirements for a tool before transitioning to the Idylla platform.
"We needed a platform that could allow rapid testing of high-impact genetic variants, which was simple, fast, and easy," Arcila explained. Also important were a fast turnaround time and use of minimal tissue so her team could use the samples with next-generation sequencing.
Arcila and her team found that the mutations detected with Idylla had a perfect correlation with the IMPACT system at MSKCC. They noted that sensitivity studies established a limit of detection of between 2.5 percent and 5 percent variant frequency. The team also achieved 100 percent accuracy in mutation detection compared to the reference methods with "excellent reproducibility."
According to Arcila, the overall process required four minutes of hands-on time and about 150 minutes from setup to report generation.
In addition to lung carcinoma, Arcila noted that her team also validated Biocartis' BRAF and KRAS mutation assays for melanoma.
"Sometimes when you have melanomas that are highly pigmented, you can get a failure," Arcila said. "Here, we confirmed that even if you have highly pigmented [cells], you can use it and not get a failure, [since] there is a clean-up step within the cartridges, as well."