Cancer Res Treat.  2024 Jan;56(1):81-91. 10.4143/crt.2023.408.

Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology–Based EGFR Mutation Assay in Patients with Operable Non–Small Cell Lung Cancer

• Park IH
• Son DS
• Choi YL
• Choi JH
• Park JE
• Jeon YJ
• Cho M
• Kim HK
• Choi YS
• Shim YM
• Kang JH
• Park S
• Lee J
• Kim SH
• Lee BC
• Kim J

• KMID: 2550325
• DOI: http://doi.org/10.4143/crt.2023.408

Abstract

Purpose
Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens.
Materials and Methods
In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non–small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing–based CancerSCAN was utilized as a referee.
Results
The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients.
Conclusion
The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Keyword

Non-small-cell lung carcinoma; mutation; EGFR-TKI treatment; ADPS Mutation Test Kit; Companion diagnostics

Figure

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  Fig. 1. Multiplex strategy to detect multiple epidermal growth factor receptor (EGFR) mutations. (A) To detect Ex19Del, Ex20Ins, G719X and L858R that have multiple variant alleles, as many allele-specific primers were designed, but with a common florescent probe. (B) EGFR mutations are grouped into seven mutation groups and the four reaction master mixes with internal controls. CFO 560, CAL Flour Orange 560 (compatible dye of HEX); F, fluorophore (red: CFO 560; green: FAM); IC, internal control; MMX, master mix; Q, quencher; QS 670, Quasar 670 (Compatible dye of CY5).

• 

  Fig. 2. Sensitivity using genomic DNA from epidermal growth factor receptor (EGFR)–mutant cell lines. (A) Seven standard amplification curves of EGFR mutation groups. As templates, 99 ng of gDNA blend with mutant allele fraction (MAF) ranging from 25.6% to 0.1% were used. △Rn is the difference of Rn (fluorescence signal of reporter probe normalized to that of reference dye) between the experimental versus baseline signal. (B) Linearity shown in the ranges from MAF 0.1%, using the Ct value as y axis, and log MAF as x axis.

Reference

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