Abstract 4523: Comparison of pre-analytical methods using liquid biopsy NSCLC samples for molecular diagnosis
Authors: Marinho Feliciana , Pereira Michele , Machado Cythia , Freire Maíra , Mateo Elvis , Zalis Mariano , Cirillo Priscila 
Published: August 2018

Introduction: Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer types with 5% of survival rate. The development of strategies to improve the treatment of these patients is important. EGFR druggable-specific mutations can be detected in plasma from circulating-free tumor DNA (cfDNA) in NSCLC patients through liquid biopsy. In plasma, cfDNA has short half-life then, it is necessary to ensure that sample collection and processing does not interfere in the yield and quality of the cfDNA.

Objective: The aim of this study was to compare three commercial blood collection tubes, acid nucleic acid purification kits and four different time-point of plasma processing after collection of NSCLC samples from different regions of Brazil.

Methods: Blood samples were collected from 10 NSCLC patients in EDTA, PAXgene and Streck tubes. EDTA samples were processed immediately after collection and plasma was frozen at -20°C. PAXgene and Streck samples were processed at room temperature within 24h, 72h or 168h after collection. Plasma were aliquoted (1mL) and frozen at -80°C before cfDNA extraction. cfDNA of all samples were extracted using QIAamp Circulating Nucleic Acid or QIAamp MinElute Virus Vacuum kits. cfDNA concentration and integrity were evaluated by fluorometry (Qubit) and Bioanalyzer (Agilent), respectively. In addition, patients also were evaluated by Cobas® EGFR Mutation Test v2 (Roche) and just T790M results were confirmed by Droplet Digital PCR (ddPCR). All samples were submitted to Next-Generation Sequencing (NGS) using Oncomine Lung cfDNA Assay (Thermo Fisher). Statistical analysis was performed by IBM SPSS Statistics v19.0 software.

Results: cfDNA concentration did not show strong variation regardless of the tubes collection and time-point processing (p=0.57) and no statistically significant differences were found between extraction kits (p=0.80). However, the cfDNA yield were 10x higher after virus (mean 0.49±0.10) compared to circulating (mean 0.03±0.01) kits. Bioanalyzer results showed cfDNA size among 100 and 200bp, but there was no observed difference between the cfDNA integrity regardless of all conditions. Cobas EGFR results showed 7 patients wild-type, 2 had exon 19 deletion (Del19) and 1 had G719X mutations. ddPCR confirmed the absence of T790M mutation among these patients in all cfDNA sample conditions. NGS data will be presented in the poster session.

Conclusion: Plasma collection using virus kit shown higher cfDNA yield, compared to circulating kit. Regardless of the tube and processing time, the cfDNA samples showed satisfactory yield and integrity for identification of EGFR mutations as observed by Cobas and ddPCR concordant results. NGS analysis will allow us to investigate whether the cfDNA quality could interfere in the amplification of the target regions (11 genes panel). This is a pilot study and will be replicated for a greater number of cases.

Citation Format: Feliciana Marinho, Michele Pereira, Cythia Machado, Maíra Freire, Elvis Mateo, Mariano Zalis, Priscila Cirillo. Comparison of pre-analytical methods using liquid biopsy NSCLC samples for molecular diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4523.