For assay development, the strategy was to comprehensively cover the most frequently detected, clinically significant mutations in the most relevant genes as per public databases.
The mutation assay platform is a real time PCR assay that utilizes allele specific primers and probes that are complementary to mutant variants for the gene of interest.
Each assay was developed in an ISO 13485-certified laboratory, utilizing a panel of bench-validated probes generated for real time RT-PCR detection. These assays are optimized to work under standard cycling conditions enabling a large number of assays to be analyzed simultaneously and to have intra- and inter-assay comparability.
Data are analyzed using Ct-value cutoffs established for each assay. A control reaction is performed to assess the amount of amplifiable DNA in the sample and to calculate the difference in Cycle threshold (∆Ct) between the mutation reaction and the control reaction from the same sample.
Asterand Bioscience mutation results are based on the assay results of a single 40 micron curl. These results are represented as a direct result for the remaining sections from which the curl was removed. In addition, the presence or absence of the specific mutation is extrapolated across any additional primary tumor specimens from the same case and designated as inferred. While mutation status is not inferred across to biofluids, Asterand Bioscience identifies all biofluids and metastases from cases with known mutation status for a tested primary tissue. Asterand Bioscience performs mutational analysis on sections with an estimated tumor volume (STMR) > 50 with appropriate tissue size and no known quality issues in order to allow the greatest level of mutation allele presence detection. Due to the heterogeneity in tumor volume across a tissue block and the heterogeneous nature of mutations within tumor biospecimens, the actual detection of a mutation throughout a specific biospecimen is unable to be predetermined.
The assays were validated to have a standard and reliable 1% sensitivity (i.e. able to detect 1% mutation on a wild type background). However, the sensitivity may be higher and may be able to detect mutations at a level of 0.1% sensitivity. This sensitivity depends on the extent of fragmentation and quality of the isolated DNA. Please note, Asterand Bioscience assays were developed for research thresholds and thus may have a higher limit of detection than that required for clinical diagnostic assays. For comparison, Pyromark sequencing has a sensitivity of ~5%, multiplex PCR mutation analysis has sensitivity of ~10% whereas Sanger sequencing has a sensitivity of ~20%.
Our assays are able to detect 1% mutation within a background of wild-type DNA. However, our assays require at least 20ng of DNA to be present in the PCR reaction to enable this level of detection. In order to allow inter- and intra-assay consistency, specific control and mutation Cycle threshold (Ct) levels have been bench validated for each assay. Based on the limit of detection and sensitivity levels, any sample with control Ct values falling outside of these parameters is repeated. If the Ct values for a sample fall outside these parameters a second time, the sample result is determined to be inconclusive.
EGFR Control Ct 22-32, KRAS/BRAF Control Ct 20-30
A positive mutation call is made when the ∆Ct is less than the cut-off ∆Ct for that mutation reaction. Any result above this value means the sample either contains less than the percentage of mutant DNA detectable by the assay or the sample does not contain that specific mutation.
EGFR Mutation Ct <38.5, KRAS/BRAF Mutation Ct <38.5
Each assay contains primers and probes for an endogenous control gene. The endogenous control primers amplify an unrelated gene that is used to determine the condition of reagents and whether the reaction contains sufficient amount of amplifiable DNA (quality and amount).