Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification

Abstract

INTRODUCTION: Targeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecular strategies are now available for molecular testing of non-small cell lung cancers. However, almost all of them are cost-intensive and laborious and require high-end advanced equipment. Thus, assays that are rapid, simple, and cost-effective, yet sensitive, are most ideal in clinical settings for screening such therapeutically relevant mutations. MATERIALS AND METHODS: Allele-specific loop-mediated isothermal amplification assay (AS-LAMP), which is a variant of the original LAMP assay, is a promising diagnostic technique for screening single-nucleotide polymorphisms. Using commercially available plasmid constructs as template DNA, AS-LAMP assay for EGFR T790M mutation was optimized with six different sets of reaction mixture containing varying concentrations of buffer and primers. The results of AS-LAMP assay were further validated by ultrasensitive droplet digital polymerase chain reaction. RESULTS: Only one of the six sets of reaction mixture could accurately distinguish between wild type and mutated DNA, indicating that the primers and buffer are the two most critical components that determine the accuracy of AS-LAMP. The optimized AS-LAMP assay was further used to screen germ line and somatic T790M mutations in non-small cell lung cancer using blood and tissue samples collected from patients. CONCLUSION: Development of an accurate and rapid diagnostic assay that can detect resistant mutations without the need for sequencing is highly useful for clinicians in deciding on the eligibility of patients for TKI therapy. Considering its several inherent advantages, AS-LAMP assay could become an effective molecular tool for screening baseline or acquired EGFR T790M mutations in non-small cell lung cancer patients.

Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

“…Here we demonstrate a compact AS-Mini-LAMP + USS mutant speci c reaction that consists of 6-primers (FIP, BIP, F3, B3, USS FB, USS BB) spanning 155 bp, within the typical range of plasma cfDNA. In contrast to alternative mutation-selective LAMP design strategies (PNA-LNA-LAMP 11 and AS-LAMP/CF-LAMP 13,14 ) selectivity is encoded at the terminal 5' position of both the FIP and BIP primers, promoting selective self-primed exponential ampli cation upon self-hybridisation and loop formation from the FIP and BIP initiated reactions. Using synthetic template models, the mutant selective reaction (AS-Mini-LAMP Mut + USS WT ) demonstrated analytical sensitivity of 500 copies of mutant DNA within 25 minutes, > 20 minutes ahead of non-selective WT template.…”

“…With only a single base pair difference at their 5' terminal FIP and BIP primers, our data reinforce the intricacies of LAMP and requirement for design and testing of multiple primer sets. Arjuna et al, 13 similarly observed that only one of six primer sets could accurately distinguish between wild type and mutated DNA for detection of the EGFR T790M point mutation.…”

“…Kumasaka et al, 12 developed mutation-oriented LAMP (MO-LAMP), a 5-primer reaction (FIP, BIP, F3, B3 plus loop backwards primer) plus a PNA for the sensitive detection of KRAS gene mutations. Arjuna et al, 13 developed allele-speci c LAMP (AS-LAMP), a typical 6-primer LAMP reaction, incorporating the T790M EGFR mutation or wild type counterpart in the backward inner primer (BIP). Subsequent application on cfDNA template material (termed CF-LAMP) included a pre-LAMP conventional PCR using LAMP outer primers (F3 and B3) to enrich for copies of target cfDNA prior to CF-LAMP 14 .…”