Tag-based next generation sequencing: a feasible and reliable assay for EGFR T790M mutation detection in circulating tumor DNA of non small cell lung cancer patients

Abstract

Background The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure. Methods A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients. Results Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07–0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06–0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%). Conclusions Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients. Electronic supplementary material The online version of this article (10.1186/s10020-019-0082-5) contains supplementary material, which is available to authorized users.

Liquid Biopsy in Non-Small Cell Lung Cancer: Highlights and Challenges

“…However, except for PD-L1 tissue expression (>50%) that is used to select advanced NSCLC patients to receive pembrolizumab in the first line setting, no reliable prognostic or predictive marker is presently acknowledged for other ICIs. In this context, in preclinical studies, we found that circulating markers may have significant results [29,95,96]. Moreover, the correlation between PD-L1 expression on CTCs and prognosis of advanced patient cohorts treated with immunotherapy has been explored.…”

“…ctDNA levels can vary greatly, ranging from 0.01% to more than 90% of total cfDNA [26]. Different high-sensitivity approaches including beads, emulsion, amplification, and magnetics (BEAMing), droplet digital PCR (ddPCR), next generation sequencing (NGS), and dedicated protocols are available to detect even a few copies of ctDNA [27][28][29].…”

Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

“…In depth, 30/61 carried only one mutation, 30/61 two mutations, whereas only one patient (pt.43) had three mutations (Gly719Cys, Set768Ile and Thr790Met). In all the patients whose plasma resulted in being informative, i.e., positive for the sensitizing EGFR mutations, the variants matched between primary tissue and the corresponding cfDNA (data not shown; see [16]).…”

“…Reproducibility of the Oncomine Lung cfDNA panel was already evaluated in our previous paper [16]. Specifically, a high sensitivity tool such as the dPCR was tested on 26 cfDNA samples: 10 T790M-negative and 16 T790M-positive, with a 100% of concordance in both samples demonstrated.…”

“…The Oncomine TM Lung cell-free DNA Assay (OLcfA, ThermoFisher Scientific) is an NGS panel based on molecular tagging technical method, that we have already wet-lab tested with the main goal to detect low MAFs of EGFR Thr790Met mutation in plasma samples from tyrosine kinase inhibitors (TKIs)-treated NSCLC patients [16]. We also showed that the NGS results for the search of Thr790Met mutation were comparable with those obtained with a digital PCR (dPCR) approach, achieving a 100% of concordance in terms of sensitivity and specificity [16].…”