Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

Abstract

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Comparison of Circulating Cell-Free DNA Extraction Methods for Downstream Analysis in Cancer Patients

“…For multigene predictive testing using NGS a high input of ccfDNA is required for optimal and sensitive detection of ctDNA. Thus, processing high volumes of plasma in highly concentrated eluates is required [36]. Using six cancer patient-derived DLA samples, the numbers of copies of 137, 420, and 1950 bp fragments per mL of plasma were slightly (not significant) higher for all three fragment sizes with the CNA kit, while a higher total ccfDNA yield per mL of plasma was detected using the CNA kit as determined by Qubit.…”

“…However, as the citrate and BCT plasmas were not drawn from the same patients, factors such as stage of disease or response to therapy might explain these differences. Nevertheless, we have recently compared plasmas collected by DLA in citrate and from peripheral blood in Streck BCT-tubes from the same patients at the same time and using NGS analysis revealed a very high concordance between the VAFs of various tumor-specific variants [36].…”

Mass Spectrometry as a Highly Sensitive Method for Specific Circulating Tumor DNA Analysis in NSCLC: A Comparison Study

“…The accuracy of the detection method is not only dependent on the tumor-to-wildtype DNA ratio in cell-free plasma as determined by the VAF, but also by the total amount of ccfDNA input. We have recently reported that decreasing DNA input lowers the accuracy of VAF detection using next-generation sequencing, droplet digital PCR (ddPCR) as well as the UltraSEEK™ approaches [ 15 ]. The Liquid IQ ® Panel determines DNA quantity (determination of the number of amplifiable ccfDNA copies) and DNA quality (identification of long DNA fragments (>340 bp) from cell necrosis and WBC contamination).…”

“…Thus, the ctDNA fraction might account for less than 1% in many plasmas, and therefore it requires highly sensitive detection methods [ 13 , 14 ]. A major drawback in the implementation of liquid biopsy approaches is that preanalytical and analytical factors have not been harmonized while their effects on performance and results of ctDNA assays are major [ 13 , 14 , 15 , 16 , 17 ]. Variation of white blood cell (WBC)-derived DNA in cell-free plasma due to hemolysis during the collection, transport and processing of blood is an essential factor that can affect PCR and sequencing results by dilution of ctDNA with wild-type or non-cancerous DNA, increasing false-negative results, and erode reliable quantification of the variant allelic frequency (VAF).…”

Comparison of three commercial decision support platforms for matching of next-generation sequencing results with therapies in patients with cancer

“…The AVENIO platform was previously extensively validated in our lab using commercially available highly multiplexed reference standards with distinct mutations at defined allele frequencies, which enabled variant detection down to a variant allele frequency (VAF) of 0.125%. 40 Libraries were sequenced 150 bp paired-end on an Illumina NextSeq, obtaining between 30 and 40 million paired-end reads per sample. Data analysis was performed using the AVENIO ctDNA Analysis Software (Roche) with customised somatic variant filtration settings.…”