Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays

Circulating tumor DNA (ctDNA) samples reflect the total tumor burden and allow longitudinal monitoring of mutational sensitizing alterations in routine use for advanced non-small cell lung cancer (NSCLC). Various assays are developed at high sensitivity and specificity. To drive the choice of the best assay at diagnosis, we compared the clinical performance of an ultra-sensitive Plasma-SeqSensei (TM) SOLID CANCER IVD kit with the Plasma OncoBEAM (TM) EGFR V2 assay, or with our custom validated NGS routine assay. Global clinical concordances rates of 75% and 68% were found between the Plasma-SeqSensei (TM) SOLID CANCER IVD assay with the Plasma OncoBEAM (TM) EGFR V2 assay, and our custom validated NGS assays. The Plasma-SeqSensei (TM) SOLID CANCER IVD tool enables the identification of the maximum number of patients bearing sensitizing alterations for a tyrosine kinase inhibitor indication at diagnosis, while the custom NGS assay, with weaker clinical sensitivity, is dedicated to the exploration of resistance mechanisms and co-mutations during clinical progression. Introduction: Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. Patients and Methods: Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei (TM) SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAM (TM) EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. Results: In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei (TM) SOLID CANCER IVD Kit. In comparison with the OncoBEAM (TM) EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAM (TM) EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei (TM) SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei (TM) SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei (TM) SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. Conclusions: The Plasma-SeqSensei (TM) SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.