Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations

化学 对偶(语法数字) 下降(电信) 计算生物学 色谱法 生物 计算机科学 电信 文学类 艺术
作者
Dimitra Stergiopoulou,Stavroula Smilkou,Vasilis Georgoulias,Loukas Kaklamanis,Evi Lianidou,Athina Markou
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:95 (37): 14068-14076 被引量:3
标识
DOI:10.1021/acs.analchem.3c02692
摘要

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.
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