Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

Laure Sorber,Patrick Pauwels,Filip Lardon,Christian Rolfo,Pablo Reclusa,Elien Augustus,Koen De Winne,Kaat Van Casteren,Karen Zwaenepoel,Sofie Goethals,Geert Roeyen,Marc Peeters,Jan Van Meerbeeck,Julie Jacobs

doi:10.3390/cancers11040458

Laure Sorber, Patrick Pauwels + Show 12 more

Open Access

https://doi.org/10.3390/cancers11040458

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Abstract

The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

Highlights

The analysis of circulating cell-free DNA as a liquid biopsy has shown tremendous potential in many aspects of oncology, ranging from minimally invasive molecular profiling and treatment monitoring to the detection of resistance mutations [1]
Five centrifugation protocols were compared for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA in either either BD Vacutainer K2EDTA (EDTA) or Streck tubes (Table 1)
No significant differences in KRAS ctDNA concentration or allele frequency (AF) could be observed between the other centrifugation protocols

Summary

Introduction

The analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) as a liquid biopsy has shown tremendous potential in many aspects of oncology, ranging from minimally invasive molecular profiling and treatment monitoring to the detection of resistance mutations [1]. Several limitations of ctDNA-based liquid biopsies have been addressed over the last few years. CtDNA has been established as an excellent source of mutation and methylation analysis [4], it might be less appropriate for the detection of gene rearrangements. Even though the group of Russo et al were successful in detecting gene-fusions in ctDNA [6], extensive deep sequencing or break-point encompassing targeted assays are necessary to detect rearrangements. Circulating cell-free (tumor) RNA (cfRNA/ctRNA) analysis could complement ctDNA in determining the molecular profile

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