Mutation analysis of circulating tumor DNA (ctDNA) has applications in monitoring of colorectal cancer (CRC) patients for recurrence. Considering the low tumor fraction of ctDNA in cell-free DNA (cfDNA) isolated from blood plasma, the sensitivity of the detection method is important. Here, plasma DNA collected at diagnosis and follow-up from 25 CRC patients was analyzed using a multiplex superRCA mutation detection assay. The assay was also performed on genomic DNA (gDNA) from tumor and normal tissue from 20 of these patients. The lower limit of detection for most sequence variants was in the range of 10−5, while when analyzing cfDNA from plasma with a typical input of 33 ng, the practical detection limit was ~10−4or 0.01% mutant allele frequency (MAF). In 17 of 19 patients with identified hotspot mutations in tumor gDNA, at least one hotspot mutation could be detected in plasma DNA at the time of diagnosis. The MAF increased at subsequent time points in four of the patients who experienced a clinical relapse. Multiplex superRCA analysis of the remaining six patients did not reveal any hotspot mutations. In conclusion, multiplex superRCA assays proved suitable for monitoring CRC patients by analyzing hotspot mutations in cfDNA, and dynamic changes in MAF were observed in patients with clinical relapse.
循环肿瘤DNA（ctDNA）的突变分析在监测结直肠癌（CRC）患者复发方面具有应用价值。考虑到从血浆中分离出的游离DNA（cfDNA）中ctDNA的肿瘤比例较低，检测方法的灵敏度至关重要。本研究采用多重超级滚环扩增（superRCA）突变检测方法，对25例CRC患者诊断时及随访期间采集的血浆DNA进行了分析。同时，对其中20例患者的肿瘤组织和正常组织基因组DNA（gDNA）也进行了相同检测。大多数序列变异的检测下限在10⁻⁵范围内，而在分析典型投入量为33 ng的血浆cfDNA时，实际检测限约为10⁻⁴或0.01%的突变等位基因频率（MAF）。在19例肿瘤gDNA中检出热点突变的患者中，有17例在诊断时的血浆DNA中至少能检测到一个热点突变。在经历临床复发的四例患者中，其MAF在后续时间点呈现上升趋势。其余六例患者的多重superRCA分析未发现任何热点突变。综上所述，多重superRCA检测方法通过分析cfDNA中的热点突变，被证明适用于CRC患者的监测，并在临床复发患者中观察到MAF的动态变化。
肿瘤(癌症)患者之家