Center for Digital Services - Healthcare, Research & Development Group, Hitachi, Ltd., 1-280, Higashi-Koigakubo, Kokubunji, Tokyo, 185-8601, Japan.
Department of Hepatobiliary and Pancreatic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.
Sci Rep. 2023 Feb 21;13(1):3039. doi: 10.1038/s41598-023-30131-y.
A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.
需要一种高度敏感且高度多重化的核酸定量技术，以便通过液体活检来预测和评估癌症治疗效果。数字 PCR（dPCR）是一种高度敏感的定量技术，但传统的 dPCR 通过探针荧光染料的颜色来区分多个靶标，这限制了多重化的程度，无法超过荧光染料颜色的数量。我们之前开发了一种结合熔解曲线分析的高度多重化 dPCR 技术。在此，我们通过缩短扩增子大小，提高了熔解曲线分析的多重化 dPCR 的检测效率和准确性，以检测从临床样本中制备的循环肿瘤 DNA（ctDNA）中的 KRAS 突变。通过改变 G12A 的突变类型确定算法，突变检测效率从输入 DNA 的 25.9%提高到 45.2%，突变检测的下限从 0.41%提高到 0.06%，使得所有目标突变的检测下限均低于 0.2%。然后，测量了来自胰腺癌患者血浆中的 ctDNA 并进行了基因分型。测量的突变频率与传统 dPCR 测量的频率非常吻合，传统 dPCR 只能测量 KRAS 突变体的总频率。在有肝或肺转移的患者中，82.3%检测到了 KRAS 突变，与其他报道一致。因此，本研究证明了熔解曲线分析的多重 dPCR 具有足够的灵敏度，可用于检测和分析来自血浆的 ctDNA 的临床实用性。
