Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay

Abstract

Background: The epidermal growth factor receptor (EGFR) mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/sequencing assay. MET amplification was also analyzed.

Methods: Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFR mutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH.

Results: Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37).

Conclusions: The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients.

Figure 1

Genomic sequence of EGFR exon 20 (highlighted in blue, between arrows) with flanking intronic sequences. The sequences and location of the PCR primers are highlighted in yellow. The green bar, above positions 789–791, specifies the target area of the LNA probe with the site of the T790M (EGFR*2369C>T) located at the center of the locked portion (dark green = locked residues, light green = unmodified). The nearby common germline SNP 2361G/A (Q787Q) is also indicated.

Figure 2

Flow chart of patients consented to rebiopsy: of 144 patients consented, 130 met strict criteria for rebiopsy; 121 underwent sampling procedures; 104 were molecularly profiled for sensitizing mutations and 99 for the T790M mutation.

Figure 3

Representative electropherograms of the sensitivity study for EGFR T790M. A. Sequencing after standard PCR of undiluted mutant DNA (100%) shows both a mutant peak (T) and a WT peak (C) at position 2369 (arrow) at roughly the same proportions. Serial dilutions of mutant DNA with normal control DNA show a sequential decrease in size of the mutant peak. The mutation is clearly seen at dilutions of 12.5% or higher in both the forward and reverse directions. Below this level, the mutant peak is at the level of the background or completely absent. % M denotes percent of mutant DNA; () denotes SNP at position 2361. B. LNA-PCR/sequencing. With the introduction of the LNA probe, only the mutant peak (T) is seen at position 2369 (arrow) at all dilutions of 0.8% and above. At progressively lower concentrations, the WT peak becomes visible but the mutant peak remains predominant down to 0.1% (percentages have been rounded). At the SNP position () only G is seen with complete suppression of the WT allele.