Journal
MOLECULAR AND CELLULAR PROBES
Volume 58, Issue -, Pages -Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.mcp.2021.101745
Keywords
Digital PCR; EGFR mutations; Tumor biopsy; NSCLC
Categories
Funding
- Financiadora de Estudos e Projetos (FINEP)
- Fundacao Araucaria (TECNOVA-PR Project)
- Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)
- CNPq (Conselho Nacional de Ensino e Pesquisa) fellowship [CNPq314288/20180]
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This study optimized dPCR for detection of EGFR mutations in NSCLC with higher sensitivity and specificity compared to castPCR. The results demonstrated the superior performance of dPCR in detecting EGFR mutations.
Background: Digital PCR (dPCR) is proposed to replace real time PCR and Sanger sequencing for detection and quantification of rare mutations, frequently unnoticed in the mass of tumoral cells. Screening of endothelial growth factor receptor (EGFR) mutations is mandatory before treatment with EGFR-targeted therapy with smallmolecule tyrosine kinase inhibitors, which has been approved for the treatment of advanced non-small-cell lung cancer (NSCLC). Objective: In order to establish a cost-effective method for detection of mutations, we optimized dPCR identification of EGFR mutations in exons 18-21, and determined dPCR sensitivity, limits of detection (LoD) and quantification (LoQ). Methods: For clinical validation, we compared the performance of dPCR and castPCR in 57 NSCL formalin fixed paraffin embedded samples and 10 lung cancer-free formalin fixed paraffin embedded samples. Results: EGFR mutations DEL19, p.L858R, p.G719X, p.L861Q and p.T790 M were detected by dPCR in 27 samples versus 11 detected by castPCR (p = 0.014). LoD was determined as 100 molecules of DNA/uL and LoQ as 1%. Most of the samples (87%) identified by competitive Allele-Specific TaqMan (castPCR) as wild-type and by dPCR as mutated, presented less than 10% mutated DNA molecules (mean 4.57%). Accuracy of dPCR was 94.44%, as measured with the assay recommended by the College of American Pathologists. Conclusion: These results indicated higher sensibility and specificity of dPCR for screening EGFR mutations in NSCLC biopsies, compared to castPCR.
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