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Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients.
Yu, Qian; Jiang, Huiqin; Su, Xi; Jiang, Zhengxin; Liang, Xue; Zhang, Chunyan; Shang, Wu; Zhang, Yiliang; Chen, Hao; Yang, Zhijie; Shen, Minna; Huang, Fei; Chen, Xinning; Yang, Yihui; Pan, Baishen; Wang, Beili; Lu, Daru; Guo, Wei.
Affiliation
  • Yu Q; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China; Department of Laboratory Medicine, Xiamen Branch, Zhongshan Hospital, Fudan University, Xiamen, China.
  • Jiang H; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Su X; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Jiang Z; Nanjing Pregene Biotechnology, Nanjing, China.
  • Liang X; State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
  • Zhang C; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China; Department of Laboratory Medicine, Xiamen Branch, Zhongshan Hospital, Fudan University, Xiamen, China.
  • Shang W; Nanjing Pregene Biotechnology, Nanjing, China.
  • Zhang Y; Nanjing Pregene Biotechnology, Nanjing, China.
  • Chen H; Nanjing Pregene Biotechnology, Nanjing, China.
  • Yang Z; Nanjing Pregene Biotechnology, Nanjing, China.
  • Shen M; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Huang F; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Chen X; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Yang Y; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Pan B; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Wang B; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
  • Lu D; State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China. Electronic address: drlu@fudan.edu.cn.
  • Guo W; Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China; Department of Laboratory Medicine, Xiamen Branch, Zhongshan Hospital, Fudan University, Xiamen, China. Electronic address: guo.wei@zs-hospital.sh.cn.
J Mol Diagn ; 25(6): 388-402, 2023 06.
Article in En | MEDLINE | ID: mdl-36963484
ABSTRACT
The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Colorectal Neoplasms / Proto-Oncogene Proteins B-raf Type of study: Diagnostic_studies / Prognostic_studies Limits: Humans Language: En Journal: J Mol Diagn Journal subject: BIOLOGIA MOLECULAR Year: 2023 Document type: Article Affiliation country: China

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Colorectal Neoplasms / Proto-Oncogene Proteins B-raf Type of study: Diagnostic_studies / Prognostic_studies Limits: Humans Language: En Journal: J Mol Diagn Journal subject: BIOLOGIA MOLECULAR Year: 2023 Document type: Article Affiliation country: China
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