EGFR T790M detection rate in lung adenocarcinomas at baseline using droplet digital PCR and validation by ultra-deep next generation sequencing

Abstract

Background: Routine testing of baseline EGFR T790M mutation may have important clinical impact but many discordant data have been reported regarding the diagnostic, prognostic and predictive role of this marker. In this study we aimed to assess T790M frequency in 164 untreated EGFR-mutated NSCLCs using methods with different sensitivity as well as to analyze the relationship between baseline T790M mutation status, patient's clinicopathologic features and tyrosine kinase inhibitors (TKI) treatment outcomes.

Methods: We compared the diagnostic performance, sensitivity and specificity of three methods, namely MALDI-TOF mass spectrometry (MS), Allele-Specific Real Time PCR (AS-PCR), droplet digital PCR (ddPCR). Ultra-deep next generation sequencing (NGS) validation of T790M-mutant NSCLCs was performed using SiRe^® panel.

Results: Baseline T790M occurred in 17% of the tumors. Intermediately sensitive techniques such as MALDI-TOF MS (detection limit of T790M ≥5%) allow to detect T790M in 2% of cases exhibiting mutant-allele fractions ranging from 11.5% to 17%. Median overall survival (OS) in these patients was poor (7.3 months) and progression free survival (PFS) was of 3.3 months in patients treated with a 1st generation EGFR TKI. The remaining T790M-positive cases showed very low mutant-allele fractions ranging from 0.07% to 0.38% and required highly sensitive methods such as ddPCR and NGS to be identified. All these cases showed a concurrent sensitizing EGFR mutation (mainly exon 19 deletion), and clinicopathological features similar to those observed in EGFR mutant cancers. Median OS of these patients was 27 months while median PFS after TKI treatment was 20 months.

Conclusions: Routine test of baseline EGFR T790M may have an important role in the prediction to EGFR TKI therapy response and should be performed using highly sensitive and quantitative methods, such as ddPCR and NGS, in order to reliably distinguish NSCLCs with high or very low T790M mutant-allele fraction.

Keywords: EGFR T790M mutation; MALDI-TOF mass spectrometry; droplet digital PCR (ddPCR); real time quantitative PCR (AS-PCR); ultra-deep next generation sequencing (NGS).

Figure 1

Flow diagram of the study population. NSCLCs, non-small cell lung cancer; AS-PCR, real time quantitative PCR; ddPCR, droplet digital PCR.

Figure 2

Frequency of EGFR T790M comparing results obtained with MALDI-TOF MS, AS-PCR and ddPCR. T790M alone was observed only in case 3 (AF>10%) while the remaining 19 NSCLCs showed the coexistence of T790M with an activating mutation in exon 18, 19 or 21. T790M allelic fraction ranged from 0.07% to 0.38% in the tumors tested negative for T790M using MALDI-TOF-MS. NGS confirmed all the sensitizing mutations detected by MALDI-TOF MS and validated the presence of T790M in 15 out of 16 mutation-positive NSCLCs, reporting mAFs very close to those quantified by ddPCR. ne: not evaluable.

Figure 3

Progression-free survival (A), time to treatment failure (B) and overall survival (C) at fixed times during follow-up according to EGFR T790M status.

Figure S1

Sensitivity of MALDI-TOF MS, AS-PCR and ddPCR evaluated by serial dilutions of control reference DNA. 1: MS of EGFR T790M assay at fractional abundance (FA) of 5% (A), 1% (B), 0.1% (C) and wild type (D). Only the 5% mutated sample was identified as positive (arrow in A). 2: the same analysis in real time PCR identified as positive only samples with 5% and 1% mutation (E and F, respectively) while samples with 0.1% (G) and 0% (H) were scored as negative. 3: ddPCR identified and quantified all the mutated samples, down to a FA of 0.1%.

Figure S2

Scatter plot showing the number of false positive droplets for the EGFR T790M assay in ddPCR in wild type controls from WBC and from FFPE normal lung samples. Limit of blank (LoB) for whole blood cells (WBC) and Formalin Fixed Paraffin Embedded (FFPE) samples were respectively 4 and 7 droplets, as shown in red on the graph. We set the limit of detection (LOD) for FFPE ddPCR assay at 10 positive droplets.