Performance evaluation of the Biocartis Idylla EGFR Mutation Test using pre-extracted DNA from a cohort of highly characterised mutation positive samples

Abstract

AimsTargeted therapies for non-small cell lung carcinoma (NSCLC) rely on the detection of specific genomic lesions, such as mutations within the epidermal growth factor receptor (EGFR) gene. The Biocartis Idylla platform and single-use EGFR mutation test cartridge is CE-IVD for use with formalin-fixed paraffin embedded (FFPE) tumour material, but can also function off-scope using extracted DNA as input material. This can expand the utility of the platform and potentially conserve valuable tissue.MethodsWe sought to evaluate the performance of this system to detect known EGFR mutations using extracted DNA at different input levels. 130 next generation sequencing-characterised NSCLC cases possessing EGFR mutations that were theoretically detectable by the Idylla system were selected. Replicate analyses were performed using the Idylla EGFR test with up to three different DNA input levels (20 ng, 50 ng and 250 ng).ResultsConsidering only variants within the test manufacturer’s specified scope, the Idylla EGFR test generated concordant findings for 90.77% of cases at 20 ng DNA input, 98.46% at 50 ng input and 100% at 250 ng input. Analyses with discordant findings all generated control quantification cycle (CQ) values greater than 23. Very low CQ values were associated with EGFR gene amplification.ConclusionsThe Idylla EGFR Mutation Test can be used at least as well with pre-extracted DNA than with direct FFPE input. In cases with control CQ >23, reanalysis with an increased DNA input should ideally be undertaken. If this is not possible, the risk of false negative calls may be mitigated by manual review of the quantitative PCR data and/or by reflexing to alternative analysis options.

IdyllaEGFRassay on extracted DNA: advantages, limits and place in molecular screening according to the latest guidelines for non-small-cell lung cancer (NSCLC) patients

“…In the Dijon CHU cohort, although no Idylla false negatives were reported within the repertoire of the pyrosequencing technique, the two invalid Idylla results were associated with inputs below 50 ng (table 2). Samples tested with inputs lower than 50 ng were mostly characterised by a total Cq EGFR >23, considered by Grant et al 10 as a critical value. Conversely, this cycle quantification (Cq) value was never exceeded with inputs of at least 50 ng.…”

Detection of EGFR Mutations From Plasma of NSCLC Patients Using an Automatic Cartridge-Based PCR System

“…However, adjusting the threshold always poses the risk of reducing specificity and thus would require evaluation in a larger prospective study to not impair the performance of the assay. Interestingly, based on a study made with previously isolated DNA from FFPE tissue specimen, the CQ value of the internal control of the cartridge-based system could indicate when a reanalysis of the results would be recommended, which could overcome uncertainties when analyzing mutations which were below the predefined threshold (Grant et al, 2021). The recent development of NGS systems with fast turn-around times increasingly challenges the use of single-gene assays (Low et al, 2020).…”

Contribution of the IdyllaTM System to Improving the Therapeutic Care of Patients with NSCLC through Early Screening of EGFR Mutations

“…The off-label use of CE-IVD methods was thoroughly validated before being used in routine testing during a retrospective study not reported here. The risk to exhaust the tumor sample could, however, be lifted by the reuse of H&E, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) diagnostic slides [ 36 ], by the use of plasma from patients with lung cancer [ 37 , 38 ] or of the DNA extracted from FFPE sections for NGS analysis [ 14 , 19 , 26 , 28 , 39 ]. Indeed, using the same DNA for Idylla TM and NGS assays could discard divergent results linked to tumor heterogeneity and to the fact that the two analyses are not carried out on strictly the same part of the tumor sample.…”