Technical Evaluation of the COBAS EGFR Semiquantitative Index (SQI) for Plasma cfDNA Testing in NSCLC Patients with EGFR Exon 19 Deletions

Abstract

The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p < 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p < 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p < 0.00001) compared to samples collected during TKI treatment: r2 = 0.76; p < 0.00001 for SQI and VAF and r2 = 0.75; p < 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.

Variant Allele Frequency Analysis of Circulating Tumor DNA as a Promising Tool in Assessing the Effectiveness of Treatment in Non-Small Cell Lung Carcinoma Patients

“…However, it is important to consider that ddPCR enables the detection of fewer mutations than NGS approaches. Further validation of methods based on NGS could improve its sensitivity and expand the possibility of VAF analysis in clinical practice [100,112,113].…”

Patient-centric thresholding of Cobas® EGFR mutation Test v2 for surveillance of EGFR- mutated metastatic Non-Small Cell Lung Cancer

“…SQI is a measure of cycle threshold of the multiplex PCR and shows a positive, exponential correlation with actual mutant EGFR allele concentrations or ratios of mutated allele to wild type allele (variant allele frequency, VAF). Several (9)(10)(11) but not all (12,13) studies con rmed this quantitative power of SQI. Cohort analyses established SQI as surrogate indicator for therapeutic response to TKI (10)(11)(12).…”

“…Since our statistical approach with dichotomization for absence/presence of progression within 12 weeks is theoretically applicable to any quantitative ctDNA data, we here challenged it in another advanced cancer type, using another liquid biopsy assay: Cobas® EGFR Mutation Test v2 (hence Cobas EGFR test) in EGFR-mutated metastatic non-small cell lung cancer (NSCLC). In 2016, this test was FDA approved for qualitative detection of EGFR variants actionable by tyrosine kinase inhibitors (TKI) (8,9). It uses allele (mutation)-speci c PCR, with automated calculation of a semiquantitative index (SQI) value.…”