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

Mariella Dono,Manlio Ferrarini,Maria Giovanna Dal Bello,Giuseppa De Luca,Jean Louis Ravetti,Antonella Vigani,Carlo Genova,Irene Vanni,Giorgia Anselmi,Francesco Grossi,Sonia Lastraioli,Simona Zupo,Simona Coco

doi:10.1186/s10020-019-0082-5

Abstract

BackgroundThe 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.MethodsA 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.ResultsCompared 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%).ConclusionsTag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

Highlights

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
Analytical validation of OncomineTM Lung cell-free DNA (cfDNA) assay Sensitivity testing was initially performed starting from 30 ng cfDNA of each Multiplex I cfDNA Reference Standard at 5, 1, 0.1 and 0% mutation frequencies and analyzing 4 different EGFR hotspots, that is E746_A750del, L858R, T790M and V769_D770ins (Table 1)
Since the mutant DNA copies for L858R resulted underestimated compared to mutant copies found for the other three variants (Table 1), we checked reproducibility of L858R variant call in critical samples and tested the assay using 20 ng of the reference standard at 0.1% mutated allele frequency

Summary

Introduction

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. The cfDNA is becoming a reliable alternative source to tumor DNA, the sensitivity of methods using cfDNA is generally lower (Ramalingam et al 2018; Vanni et al 2015; Luo et al 2014; Oxnard et al 2016) This approach is non-invasive, does not pose limitations to repeated sampling, and provides a sufficiently accurate assessment of intra and inter-tumor heterogeneity (Sundaresan et al 2016; Murtaza et al 2013; Diaz and Bardelli, 2014). We have studied a commercial NGS panel using molecular tagging of the DNA alleles present in the plasma, and compared the results obtained with those by Real Time PCR

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Discussion

Conclusion