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

Abstract

AimsIdylla epidermal growth factor receptor (EGFR) is a fast and fully automated mutation assay that is easy to implement. However, under the Biocartis-recommended technical conditions, tissue sections are directly introduced into the cartridge, at the risk of exhausting the tumour sample. In this study, we evaluate the performance of Idylla EGFR on extracted DNA and discuss its place within the global non-small-cell lung cancer (NSCLC) screening strategy.Methods577 comparative tests between Idylla EGFR on extracted DNA and next-generation sequencing (NGS) were performed across two centres.ResultsPreanalytical thresholds were established (20% tumour cell content, 50 ng DNA input) and challenged prospectively in routine practice. 16.8% of samples referred for screening were considered non eligible for Idylla EGFR testing. Due to discordant by design cases, Idylla EGFR sensitivity was 86.9% for currently actionable EGFR mutations. Idylla EGFR specificity was 100% in first-line screening. NGS was always feasible on the same DNA.ConclusionIdylla EGFR on extracted DNA is feasible and enables tumour material to be saved compared with tissue section use. It is not necessary to replace the analytical thresholds of the Biocartis algorithm. Due to both the limits of the mutational repertoire and the high increase of targetable genes in NSCLC, the use of Idylla EGFR should be restricted to clinical emergency situations accompanied by NGS.

Validation of the Idylla GeneFusion assay to detect fusions and MET exon-skipping in non-small cell lung cancers

“…1 A). Our evaluation of the Idylla cartridges using commercial RNA standards highlighted the fact that extracted RNA can be directly pipetted into the Idylla GeneFusion cartridges for analysis, as previously observed for other Idylla PCR-based assays 24 – 29 . This could be of particular interest for samples rejected for NGS analysis due to low RNA quality, as archival extracted RNA could be reused for Idylla assay instead of collecting supplementary FFPE sections.…”

Comparison and Validation of Rapid Molecular Testing Methods for Theranostic Epidermal Growth Factor Receptor Alterations in Lung Cancer: Idylla versus Digital Droplet PCR

“…Khalifa et al reported 13 FN in a cohort of 65 pre-extracted DNA samples from NSCLC, all with Cq ref above 23. In particular, low DNA input (5 ng) resulted in FN with Cq ref above 26 [25]. The authors recommend reducing the threshold to 23, as do Grant et al [27], who also used pre-extracted DNA from FFPE.…”

“…Grant et al demonstrated that analytical sensitivity remains stable from 100% to 98.46% but drops to 90.77% when DNA input decreases from 250 ng to 50 ng and 20 ng [27]. Khalifa et al also reported a sensitivity drop from 88.6% to 47.4% when using more or less than 50 ng [25]. Based on these findings, we recommend using 50 ng as input and not interpreting results with less than 10 ng (assuming tumor content is above 30%).…”

“…Comparing FFPE ribbons to extracted DNA from other sections of the same sample is unsatisfactory. To overcome these biases, DNA was extracted from the same sections and partitioned between the two techniques at a fixed ratio of one per five, corresponding to 10 ng for ddPCR and 50 ng for Idylla, as commonly recommended by the manufacturer's instructions and reference publication [25].…”

“…We pr posed to lower this threshold only for mutated samples. Among our samples identified mutated by NGS, twelve (n°12, 22,25,28,30,31,32,47,51,57,58,63) demonstrated less th 200 TPDs (ranging between 62 and 194), with VAF ranging from 20.2% to 76.9% (with M > 4; see Table S2). Consequently, the technical validation threshold was set at 200 TPDs f WT samples and 50 TPDs for mutated samples (Table 1).…”