Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Development, validation, and comparison of gene analysis methods for detecting <i>EGFR</i> mutation from non-small cell lung cancer patients-derived circulating free DNA.

Author(s): Hanibuchi M, Kanoh A, Kuramoto T, Saito T, Tobiume M, Saijo A, Kozai H, Kondo M, Morizumi S, Yoneda H, Kagawa K, Ogino H, Sato S, Kawano H, Otsuka K, Toyoda Y, Nokihara H, Goto H, Nishioka Y

Publication: Oncotarget, 2019, Vol. 10, Page 3654-3666

PubMed ID: 31217900 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the results of EGFR mutation detection in plasma cfDNA and FFPE biopsy specimens using four different platforms. The authors also investigated the effects of patient age, gender, smoking status, and prior EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment on the amount of cfDNA isolated. A total of 44 patients with lung adenocarcinoma and one with adenosquamous carcinoma were selected based on having stage III or IV or recurrent disease and a known activating EGFR mutation as determined by the PNA-LNA PCR clamp method. Twenty-seven of the patients had been treated with EGFR-TKI while the remaining 18 had not. Blood was collected in EDTA tubes and centrifuged within three hours to obtain plasma. DNA was extracted from plasma using the QIAamp circulating nucleic acids kit and quantified by PicoGreen. DNA was extracted from the FFPE sections of the initial biopsy for 37 of the cases and a second biopsy for eight cases using the QIAamp DNA FFPE tissue kit and mutations detected using Therscreen. EGFR mutations were detected in both cfDNA and genomic DNA using the BNA-clamped fluorescence resonance energy transfer-based preferential homoduplex formation assay (F-PHFA), BNA-clamped real-time PCR, ddPCR, and by ultra-deep (>100,000 reads/amplicon) NGS using the Ion PGM Template OT2 200 Kit.

   Summary of Findings:

   The authors report no effect of patient age (44-82), gender (36% male), smoking status, or prior EGFR-TKI treatment on the amount of cfDNA isolated; however, the amount of cfDNA isolated was correlated with the sum of the diameter of the targeted legion (P<0.01), negatively correlated with progression-free survival with all regimes (P<0.01), and EGFR-TK1 treatment (P<0.01). Using each of the four technologies, the limit of detection for EGFR mutations was 0.05% and the concordance with tissue-based analysis 56-64%. The specificity of the four mutation detection methods in cfDNA was high (96% for Del 19 by NGS, 100% for all others). In contrast, the sensitivity was much lower and depended on the mutation with higher sensitivity noted for Del 19 than L858R (74-80% versus 35-57%). Further, the sensitivity was method-dependent with overall sensitivities of 56%, 60%, 63%, and 67% for F-PHFA, ddPCR, real-time PCR, and NGS respectively. Among patients with disease progression following EGFR-TKI treatment (27 patients), the T790M mutation was detected in 44%, 41%, 37%, and 46% of plasma specimens by F-PHFA, NGS, ddPCR, and real-time PCR; respectively, but the T790M mutation was detected in plasma from the 18 patients not treated with EGFR-TKI only by NGS (11%). The concordance between methods for T790M was 83% (ddPCR and real-time PCR), 84% (NGS and F-PHFA or ddPCR), and 86% (F-PHFA and ddPCR or real-time PCR, or real-time PCR and NGS). The concordance between methods was slightly higher for L858R than for T790M at 89% (NGS and F-PHFA), 91% (ddPCR and NGS), 93% (F-PHFA and ddPCR) or 95% (F-PHFA and real-time PCR, ddPCR and real-time PCR or real-time PCR and NGS). The concordance in detecting exon 19 deletions was 93% (F-PHFA and ddPCR or NGS), 95% (F-PHFA and real-time PCR), 96% (ddPCR and NGS), 98% (ddPCR and real-time PCR) or 100% (real-time PCR and NGS). For the 8 cases in which a second biopsy was performed, the agreement between tissue and plasma for activating EGFR mutations ranged between 63 and 100%, but the agreement was lower when only T790M mutations were considered (50-63%). In contrast, DNA extracted from the initial biopsy showed 94-100% concordance to plasma when analyzed using real-time PCR or NGS but 69-100% using F-PHFA, which the authors state indicates a need to adjust F-PHFA to increase sensitivity.

   Biospecimens

   • Tissue - Lung
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	PHFA
   DNA	Real-time qPCR
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient diet	44-82 years
   Preaquisition	Patient gender	Female Male
   Preaquisition	Other drugs	EGFR-TKI treated Not treated with EGFR-TKI Smoker Non-smoker
   Preaquisition	Prognostic factor	Effects of lesion size investigated
   Next generation sequencing Specific	Technology platform	Real-time PCR ddPCR F-PHFA
   Biospecimen Acquisition	Biospecimen location	Plasma Tissue
   Next generation sequencing Specific	Targeted nucleic acid	EGFR T790M EGFR L858R EGFR Del 19 EGFR G719X
   Digital PCR Specific	Targeted nucleic acid	EGFR G719X EGFR T790M EGFR Del19 EGFR L858R
   Real-time qPCR Specific	Targeted nucleic acid	EGFR L858R EGFR Del19 EGFR T790M EGFR G719X
   PHFA Specific	Targeted nucleic acid	EGFR T790M EGFR L858R EGFR Del19 EGFR G719X

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