Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients

Abstract

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.

Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma

“…Recently, an implementation and technical validation of additional screening strategies for EGFR mutational status—such as the droplet digital PCR (ddPCR) assays or, more in general, the ultrasensitive PCR approaches as well as the NGS-based analytical assessment of the allele frequency of gene variants (VAF) [ 48 , 49 , 50 ]—using liquid biopsies or minimal cytological specimens from NSCLC patients may be helpful to identify a higher number of mutation-positive patients, providing predictive biomarkers of clinical behaviour.…”

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for More Personalized Medicine in Patients’ Care

“…To identify single base changes or short deletion, amplification-refractory mutation system (ARMS-PCR) exploits sequence-specific PCR primers that allow amplification of DNA only when the target is contained within the sample, thus lowing the limit of detection in comparison with conventional PCR [84,85]. The same results can be obtained by peptide nucleic acid (PNA) clamp PCR, which prevent nucleic acid amplification of wild-type DNA, increasing the amplification of the mutant DNA [86,87]. Another alternative is the co-amplification at lower denaturation temperature-based PCR (COLD-PCR), which results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position.…”

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for Personalized Medicine in Patients’ Care

“…To identify single base changes or short deletion, the amplification refractory mutation system (ARMS-PCR) exploits sequence-specific PCR primers that allow amplification of DNA only when the target is contained within the sample, thus lowering the limit of detection in comparison with conventional PCR [71,72]. The same results can be obtained by peptide nucleic acid (PNA) clamp PCR, which prevents the nucleic acid amplification of wild-type DNA, increasing the amplification of the mutant DNA [73,74]. Another alternative is the co-amplification at lower denaturation temperaturebased PCR (COLD-PCR), which results in the enhancement of both known and unknown minority alleles during PCR, irrespective of the mutation type and position.…”