Department of Pathology, Medicum, Faculty of Medicine, University of Helsinki and HUSLAB, HUS Diagnostic Center, Helsinki University Hospital, Helsinki, Finland.
Applied Tumor Genomics Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
PLoS One. 2020 Nov 25;15(11):e0239819. doi: 10.1371/journal.pone.0239819. eCollection 2020.
Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45-63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.
循环肿瘤 DNA（ctDNA）从癌细胞中释放出来，ctDNA 中的致癌突变可以从血浆样本中测量到。液滴数字 PCR（ddPCR）是一种用于检测 ctDNA 突变的敏感和特异的方法。我们分析了 10 名转移性结直肠癌（mCRC）患者的连续血浆样本（n=80），这些患者的原发性肿瘤中存在已知的 KRAS 突变。这些患者正在接受贝伐单抗联合交替卡培他滨和奥沙利铂或伊立替康的肿瘤治疗。基线 ddPCR KRAS 突变等位基因频率（MAF）值范围为 0%至 63%。在治疗开始后 45-63 天进行了首次实体瘤反应评估标准（RECIST）评估，此时有 3 名患者的 KRAS 突变检测不到，1 名患者的 MAF 值为 0.5%，1 名患者的 MAF 值为 3%，与基线值相比降低了 95%。在这 3 名患者中，RECIST 评估为稳定疾病，2 名为部分缓解。在 7 名患者中，ddPCR MAF 值在影像学疾病进展或死亡之前增加，而 1 名患者仍保持无疾病状态且 KRAS 突变水平检测不到。接下来，我们分析了所有可用的 Idylla ctKRAS 系统的血浆样本（n=60），发现 ddPCR 和 Idylla 之间的总体一致性几乎是完美的（kappa 值=0.860）。我们使用下一代测序（NGS）检测了每位患者最后一个连续血浆样本中的治疗诱导突变，但与原发性肿瘤相比，没有发现任何新的突变。这项研究表明，ddPCR 和 Idylla 在检测 mCRC 患者液体活检中的 KRAS 突变方面同样有效，ctDNA 可能预示着治疗反应性 KRAS 阳性 mCRC 克隆的消失，并作为疾病进展的早期迹象。
