External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium

P van der Leest, P Rozendal, J Hinrichs, CJM van Noesel, K Zwaenepoel, B Deiman, CJJ Huijsmans, R van Eijk, EJM Speel, RJ van Haastert, MJL Ligtenberg, RHN van Schaik, MPHM Jansen, HJ Dubbink, WW de Leng, MPG Leers, M Tamminga, D van den Broek, LC van Kempen, E Schuuring

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Abstract

Background
Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands).

Methods
Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18–21, and KRAS exon 2–3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance.

Results
A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately.

Conclusions
Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.
Original languageEnglish
Article numberhvae014
Pages (from-to)759-767
Number of pages9
JournalClinical Chemistry
Volume70
Issue number5
Early online date14 Mar 2024
DOIs
Publication statusPublished - 1 May 2024

Bibliographical note

Publisher Copyright:
© 2024 Association for Diagnostics & Laboratory Medicine.

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