Validation of Cell-Free DNA Collection Tubes for Determination of EGFR Mutation Status in Liquid Biopsy from NSCLC Patients.
Author(s): Sesé M, Somoza R, Maestu I, Ureste MM, Sanchez A, Cordoba JF, Sansano I, Venturas G, Ramón Y Cajal S, Hernández-Losa J
Publication: Oncol Ther, 2019, Vol. 7, Page 131-139
PubMed ID: 32699985 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare plasma cell-free DNA (cfDNA) epidermal growth factor receptor (EGFR) genotyping data from matched plasma obtained from blood shipped in Roche cell-free DNA tubes at room temperature to plasma obtained from EDTA blood processed immediately and shipped on dry ice.
Conclusion of Paper
Invalid genotyping results were found for three specimens shipped as blood in Roche cell-free DNA tubes at room temperature (BCT cfDNA) and two specimens shipped as K2EDTA plasma on dry ice (gold-standard). Of the remaining 44 matched pairs, EGFR genotypes were concordant between “gold-standard” plasma and BCT cfDNA plasma in 42 cases. The discordant cases both had a mutation detected in the BCT cfDNA plasma but not the “gold-standard” plasma. The matched FFPE specimen confirmed the mutation identified in one of the two BCT cfDNA plasma specimens but not the other. Sensitivity and specificity of BCT cfDNA plasma for detection of mutations identified in the “gold-standard” plasma were 100% and 94.6%, respectively. The cfDNA BCTs were found to have a positive predictive value (PPV) of 81.8% and negative predictive value (NPV) of 100%.
Studies
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Study Purpose
The purpose of this study was to compare plasma cfDNA genotyping data from matched plasma obtained from blood shipped in Roche cell-free DNA tubes at room temperature to plasma obtained from EDTA blood processed immediately and shipped on dry ice. Blood from 49 patients with advanced non-small cell lung cancer was collected into matched Roche cell-free DNA BCT tubes and K2EDTA tubes. Plasma was obtained from blood in K2EDTA tubes by double centrifugation of blood at 3000 rpm within 20 min of blood collection and subsequently frozen at -80°C. EDTA plasma was shipped frozen on dry ice while blood in Roches BCTs was shipped at room temperature. After arrival, plasma was obtained from the cell-free DNA blood collection tube by centrifugation at 3000 x g for 10 min followed by 16,000 x g for 1 min. cfDNA was extracted from plasma using QIAsymphony DSP Virus/Pathogen Kit. EGFR mutations were detected using the real-time PCR based Cobas EGFR Mutation Test v2 kit. EGFR mutation positive was defined as a specimen with at least one activating mutation.
Summary of Findings:
Invalid genotyping results were found for three specimens shipped as blood in Roche cell-free DNA tubes at room temperature (BCT cfDNA) and two specimens shipped as K2EDTA plasma on dry ice (gold-standard). Of the remaining 44 matched pairs, EGFR genotypes were concordant between “gold-standard” plasma and BCT cfDNA plasma in 42 cases (95.4% concordance; 33 wild-type and 9 mutant). The discordant cases both had a mutation detected in the BCT cfDNA plasma but not the “gold-standard” plasma. The matched FFPE specimen confirmed the mutation identified in one of the two BCT cfDNA plasma specimens but not the other. Sensitivity and specificity of BCT cfDNA plasma for detection of mutations identified in the “gold-standard” plasma were 100% and 94.6%, respectively. The cfDNA BCTs were found to have a PPV of 81.8% and NPV of 100%.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Specimen transport duration/condition Room temperature as blood in Roche BCT
On dry ice as plasma
Biospecimen Acquisition Type of collection container/solution Roche cell-free DNA BCT
K2EDTA tube