Rui Zhang1,2, Rongxue Peng1,3,2, Ziyang Li1,3,2, Peng Gao1,3,2, Shiyu Jia1,2,4, Xin Yang1,3,2, Jiansheng Ding1,3,2, Yanxi Han1,2, Jiehong Xie1,2, Jinming Li5,3,2. 1. National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China. 2. Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China. 3. Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China. 4. Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China. 5. National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; jmli@nccl.org.cn.
Abstract
BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS: The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS: The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.
BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS: The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFRT790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS: The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.
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: J Remon; R García-Campelo; E de Álava; R Vera; J L Rodríguez-Peralto; Á Rodríguez-Lescure; B Bellosillo; P Garrido; F Rojo; R Álvarez-Alegret Journal: Clin Transl Oncol Date: 2019-09-26 Impact factor: 3.405
Authors: Luka de Vos; Heidrun Gevensleben; Andreas Schröck; Alina Franzen; Glen Kristiansen; Friedrich Bootz; Dimo Dietrich Journal: Clin Epigenetics Date: 2017-12-01 Impact factor: 6.551