Verena Haselmann1, Parviz Ahmad-Nejad2, Wolf J Geilenkeuser3, Angelika Duda1, Merle Gabor1, Romy Eichner1, Simon Patton4, Michael Neumaier1. 1. Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany. 2. Institute for Medical Laboratory Diagnostics, Centre for Clinical and Translational Research (CCTR), HELIOS Hospital, Witten/Herdecke University, Wuppertal, Germany. 3. Reference-Institute for Bioanalytics, German Society for Clinical Chemistry and Laboratory Medicine (DGKL), Bonn, Germany. 4. European Molecular Genetic Quality Network (EMQN), Manchester Centre for Genomic Medicine, St Mary's Hospital, Manchester, UK.
Abstract
BACKGROUND: Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. METHODS: The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. RESULTS: Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. CONCLUSIONS: This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.
BACKGROUND: Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. METHODS: The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAFV600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. RESULTS: Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. CONCLUSIONS: This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.
Authors: Rongxue Peng; Rui Zhang; Martin P Horan; Li Zhou; Sze Yee Chai; Nalishia Pillay; Kwang Hong Tay; Tony Badrick; Jinming Li Journal: Oncologist Date: 2019-08-30
Authors: Rongxue Peng; Rui Zhang; Martin P Horan; Li Zhou; Sze Yee Chai; Nalishia Pillay; Kwang Hong Tay; Tony Badrick; Jinming Li Journal: Oncologist Date: 2019-08-30
Authors: Sarah R Greytak; Kelly B Engel; Sonya Parpart-Li; Muhammed Murtaza; Abel J Bronkhorst; Mark D Pertile; Helen M Moore Journal: Clin Cancer Res Date: 2020-03-02 Impact factor: 12.531
Authors: Cleo Keppens; Elisabeth M C Dequeker; Simon J Patton; Nicola Normanno; Francesca Fenizia; Rachel Butler; Melanie Cheetham; Jennifer A Fairley; Hannah Williams; Jacqueline A Hall; Ed Schuuring; Zandra C Deans Journal: BMC Cancer Date: 2018-08-09 Impact factor: 4.430
Authors: Lisanne F van Dessel; Silvia R Vitale; Jean C A Helmijr; Saskia M Wilting; Michelle van der Vlugt-Daane; Esther Oomen-de Hoop; Stefan Sleijfer; John W M Martens; Maurice P H M Jansen; Martijn P Lolkema Journal: Mol Oncol Date: 2018-12-22 Impact factor: 6.603