一种通过三色彩色数字 PCR 分析在单个标本中快速检测 PIK3CA 热点突变的多重分析方法。

A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis.

机构信息

Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Centre Rotterdam, Rotterdam, the Netherlands.

Department of Clinical and Experimental Medicine, Center for Experimental Oncology and Hematology, University of Catania, Catania, Italy.

出版信息

J Appl Lab Med. 2024 Sep 3;9(5):913-925. doi: 10.1093/jalm/jfae064.

DOI:10.1093/jalm/jfae064

PMID:39012846

Abstract

BACKGROUND

Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies.

METHODS

A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy.

RESULTS

The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA.

CONCLUSIONS

Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.

摘要

背景

磷脂酰肌醇-4,5-二磷酸 3-激酶催化亚单位 α（PIK3CA）基因的激活突变在实体瘤中经常被检测到。目前临床上已有针对突变 PIK3CA 的靶向治疗方法，因此分子诊断至关重要。我们的目的是设计一种多重数字 PCR（dPCR）检测方法，同时评估 4 种最常见的 PIK3CA 热点突变，以对其进行特征分析和定量检测。

方法

使用 Stilla 3 色 dPCR Naica 系统开发了一种多重检测方法，用于检测外显子 9 的 p.E542K 和 p.E545K 突变，以及外显子 20 的 p.H1047L 和 p.H1047R 突变。该检测方法在含有上述突变的细胞系的原始和预扩增 DNA 作为单一和混合样本，以及在健康献血者（HBD）和乳腺癌患者的无细胞 DNA（cfDNA）上进行了评估，以确定检测阈值和诊断准确性。

结果

该检测方法可区分 cfDNA 中的所有 4 种 PIK3CA 突变，当存在双重突变时也可区分。原始和预扩增 cfDNA 样本的检测阈值分别为突变拷贝浓度的 0.11 和 0.40 拷贝/μL（cp/μL），以及变异等位基因频率（VAF）的 0.003%和 0.68%。该检测方法在 96 例 PIK3CA 突变的患者中，82 例与靶向下一代测序（NGS）定义的 PIK3CA（突变）状态一致，在 12 例 PIK3CA 野生型患者中，11 例与 NGS 定义的 PIK3CA（野生型）状态一致。