由于其高灵敏度和简单工作流，数字PCR（dPCR）正在成为癌症基因组变异检测和追踪的理想平台。临床可操作的癌症生物标志物数量不断增长，需要快速、易用的方法，以实现高密度信息内容和高准确度。在这里，我们描述了一个概念验证的基于振幅调制的多重检测dPCR（多allele-specific digital PCR）测定，能够检测非小细胞肺癌（NSCLC）中EGFR、KRAS、BRAF和ERBB2的12个单核苷酸和插入/缺失（indel）变异，以及ALK、RET、ROS1和NTRK1的14个基因融合和MET exon 14 skipping。我们还展示了多光谱靶标信号编码的使用，以改善变异检测的特异性，通过将背景噪音减少一个数量级。在62例人类甲醛固定石蜡嵌埋（FFPE）样本队列中，与测序法相比，该测定报告了整体上100%的阳性百分比一致性（PPA）和98.5%的阴性百分比一致性（NPA）。此外，dPCR测定在10个未能测序的样本中复原了可操作信息，突显了多重检测dPCR测定作为具有挑战性的NSCLC样本潜在反应解决方案的效用。版权所有，本文受版权保护。

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1 and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared to a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in ten samples that failed to sequence, highlighting the utility of a multiplexed digital PCR assay as a potential reflex solution for challenging NSCLC samples.This article is protected by copyright. All rights reserved.