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.

Keywords: C797S; Circulating tumor DNA; EGFR TKIs; Liquid biopsy; Molecular tag; NSCLC; Next generation sequencing; T790M resistance mutation.

Fig. 1

EGFR mutations in 42 post-TKI NSCLC patients. (a) Distribution of the various EGFR mutations types in the 42 patients according to Real Time PCR (PCR) and tag-based NGS (NGS). The y-axis shows patients count according to different mutation patterns detected by the two platforms. (b) Coincident Rate between Real Time PCR (grey bars) and tag-based NGS (black bars) according to the different EGFR mutations types found. The y-axis indicated the number of cases concordant for the specific variation with both Real Time PCR and tag-based NGS and corresponding percentages are indicated within the histogram. PCR, Real Time PCR; NGS, tag-based NGS; del, deletions; Sens, sensitizing; pos, positive; neg, negative

Fig. 2

Variant allele frequency (%) in plasma. Sensitizing EGFR mutations (n = 36 cases) and T790M mutations (n = 18 cases) were determined in plasma by tag-based NGS and are reported as variant allele frequency percentage (%) (a) Variant allele frequency (%) for EGFR mutations determined by tag-based NGS in two patients groups classified as positive or negative for the sensitizing mutation of EGFR based on both (black circles) or one (black squares) the methods employed in the study (b) Results of tests similar to those in b except that the L858R and T790M mutations were measured in (c) and (d), respectively. Each dot represents one patient. Solid lines represent median values. Statistical P values were derived from a Mann-Whitney test. PCR, Real Time PCR; NGS, tag-based NGS; sens mut, sensitizing mutations; pos, positive; neg, negative

Fig. 3

T790M detection comparison between tag-based NGS and ddPCR. Variant Allele Frequencies detected by tag-based NGS (black line) and ddPCR (dashed line) for 23 plasma samples are shown. NGS, tag-based NGS; ddPCR, droplet digital polymerase chain reaction; VAF, Variant Allele Frequency

Fig. 4

Workflow for the identification of EGFR T790M in TKI progressed patients with advanced NSCLC. Procedures and timing of cfDNA EGFR testing from sample arrival is represented together with the decision algorithm suggested. *Patients resulting T790M negative after NGS analysis on cfDNA can undergone tumor biopsy, when feasible. Alternatively, the T790M negative patient can be retested on a second cfDNA after 2–4 weeks following the National Scientific Society recommendations on liquid biopsy (https://www.aiom.it/wp-content/uploads/2018/09/2018_biopsialiquida.pdf)