The detectability of the pretreatment EGFR T790M mutations in lung adenocarcinoma using CAST-PCR and digital PCR

Abstract

Background: A gatekeeper T790M mutation is thought to cause resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment. The detection of a 2nd mutation is important for planning the next therapy when patients acquire resistance to the first line EGFR-TKI.

Methods: We used a competitive allele-specific polymerase chain reaction (CAST-PCR) to analyze the incidence and clinical significance of T790M mutations in 153 lung adenocarcinomas with EGFR-activating mutations. To increase the sensitivity and specificity of the detection of T790M mutations, we subjected 20 of the 153 cases to a digital PCR. The genomic DNAs were extracted from frozen, surgically resected tumor tissue specimens.

Results: The CAST-PCR detected T790M mutations in 45 (29.4%) of the 153 cases. The analytical sensitivity in the detection T790M mutations was 0.13-2.65% (average 0.27%, median 0.20%). In contrast, the digital PCR, detected T790M mutations in 8 (40%) out of 20 cases.

Conclusions: Our study shows that the pretreatment incidence of T790M mutation was less than that reported in previous studies. In order to clinically use pretreatment EGFR T790M mutation identification method, we should clarify the adequate methods and tissue preserved status.

Keywords: CAST-PCR; EGFR mutation; Non-small cell lung cancer (NSCLC); T790M mutation; digital PCR.

Figure 1

Overall survival (A) and recurrence-free survival (B) stratified according to the T790M mutation status.

Figure 2

The T790M mutation status was investigated using a digital PCR. Blue plot (T790-positive): high FAM fluorescence intensity was only observed in T790M mutations. Green plot (T790M-negative): the T790M mutation + wild-type showed a high FAM fluorescence intensity. Red plot (T790M-negative): only the wild-type showed a high FAM fluorescence intensity. PCR, polymerase chain reaction.

Figure 3

Four cases in which a digital PCR was performed to detect T790M mutations. Blue plot (T790-positive): High FAM fluorescence intensity was only observed in T790M mutations. Green plot (T790M-negative): the T790M mutation + wild-type showed a high FAM fluorescence intensity. Red plot (T790M-negative): only the wild-type showed a high FAM fluorescence intensity. A (case 3) and B (case 13) show T790M mutation-negative cases. C (case 14) and D (case 15) show T790M mutation-positive cases. PCR, polymerase chain reaction.