循环肿瘤DNA检测在转移性结直肠癌表皮生长因子受体单克隆抗体治疗效果监测中的应用价值

Application value of circulating tumor DNA monitoring effect of epithelial growth factor receptor monoclonai antibody therapy for metastatic colorectal cancer

目的：探讨循环肿瘤DNA（ctDNA）检测在转移性结直肠癌表皮生长因子受体（EGFR）单克隆抗体治疗效果监测中的应用价值。方法：采用回顾性横断面研究方法。收集2012年3月至2013年12月北京大学肿瘤医院收治的9例转移性结直肠癌患者的临床病理资料。患者均行EGFR单克隆抗体（西妥昔单克隆抗体或帕尼单克隆抗体）单药治疗。运用ctDNA和高通量测序技术，检测ctDNA浓度以及靶向捕获测序及分析22个EGFR通路相关基因。观察指标：（1）治疗情况。（2）治疗前后血浆ctDNA浓度。（3）血浆ctDNA中EGFR通路相关基因变异情况。（4）随访和生存情况。采用门诊和电话方式进行随访，了解患者生存情况。随访时间截至2015年8月。结果：（1）治疗情况：9例患者中，6例行西妥昔单克隆抗体单药治疗，3例行帕尼单克隆抗体单药治疗。9例患者行EGFR单克隆抗体治疗后，1例最佳疗效为部分缓解，6例最佳疗效为疾病稳定，2例治疗后疾病进展。（2）治疗前后血浆ctDNA浓度： 9例患者治疗前ctDNA浓度为1.7-25.0 μg/L，个体差异较大。9例患者ctDNA浓度在治疗过程中均发生变化：1例部分缓解时ctDNA浓度升高；6例疾病稳定时5例ctDNA浓度降低，1例升高；9例疾病进展时4例ctDNA浓度降低，5例升高。（3）血浆ctDNA中EGFR通路相关基因变异情况：①9例患者中，2例治疗后疾病进展时RAS/RAF基因突变，1例NRAS Q61H和BRAF V600E基因突变，突变丰度分别为17.8%和5.2%；1例KRAS G13D基因突变（治疗前ctDNA检测出突变丰度为5.1%），疾病进展时突变丰度达到16.7%。②7例患者治疗前ctDNA中检测出TP53基因突变，治疗后5例部分缓解或疾病稳定，其中4例TP53基因突变丰度下降，1例TP53基因突变丰度升高；5例由部分缓解或疾病稳定发展为疾病进展时，其中4例TP53基因突变丰度升高，1例TP53基因突变丰度下降；2例治疗后疾病直接进展时TP53基因突变丰度下降。③2例患者存在SMAD4基因突变。④EGFR、PIK3CA、AKT1、ERBB2、PTEN、STK11、MAP2K1、ALK、DDR2、CTNNB1、MET、FBX7、FGFR3、NOTCH1、ERBB4、FGFR1、FGFR2基因为内含子突变，未引起氨基酸的改变，未纳入分析。（4）随访和生存情况：9例患者均获得治疗后随访，随访时间为1-31个月，中位随访时间为7个月。9例患者至随访结束均死亡。结论：转移性结直肠癌行EGFR单克隆抗体治疗时监测ctDNA，发现RAS/RAF基因存在变异现象。

Objective：To investigate the application value of circulating tumor DNA （ctDNA） monitoring effect of epithelial growth factor receptor （EGFR） monoclonal antibody therapy for metastatic colorectal cancer （mCRC）. Methods：The retrospective cross-sectional study was conducted. The clinicopathological data of 9 mCRC patients who were admitted to the Peking University Cancer Hospital between March 2012 and December 2013 were collected. Patients received single EGFR monoclonal antibody treatment （cetuximab or parni monoclonal antibody）. The ctDNA concentration and variations of 22 genes in EGFR pathway of ctDNA were analyzed using high-throughput sequencing and ctDNA. Observation indicators： （1） treatment situations; （2） ctDNA concentration in plasma before and after treatment; （3） related gene mutations of EGFR pathway of ctDNA; （4） follow-up and survival situations. Follow-up using outpatient examination and telephone interview was performed to detect survival up to August 2015. Results：（1） Treatment situations： of 9 patients, 6 and 3 underwent respectively single drug treatment using cetuximab monoclonal antibody or parni monoclonal antibody. After EGFR monoclonal antibody treatment, best response was partial remission in 1 patient, stable disease in 6 patients and disease progression in 2 patients. （2） CtDNA concentration in plasma before and after treatment： ctDNA concentration of 9 patients was 1.7-25.0 μg/L before treatment, showing a significantly individual difference. There were some changes in ctDNA concentration of 9 patients during treatment： ctDNA concentration was increased in 1 patient during partial remission; of 6 patients during stable disease, ctDNA concentration was decreased in 5 patients and increased in 1 patient; of 9 patients during disease progression, ctDNA concentration was decreased in 4 patients and increased in 5 patients. （3） Related gene mutations of EGFR pathway of ctDNA： ① Of 9 patients, 2 possessed RAS/RAF genetic mutations after treatment during disease progression, NRAS Q61H and BRAF V600E mutations were found in 1 patient, and mutation abundances were respectively 17.8% and 5.2%; 1 possessed KRAS G13D genetic mutation, a mutation abundance was 5.1% before treatment and then was increased to 16.7% during disease progression. ② Seven patients possessed TP53 genetic mutation before treatment, 5 had partial response or stable disease after treatment, including 4 with a reduced mutation abundance and 1 with an increased mutation abundance. Of 5 patients with partial response or stable disease to disease progression, mutation abundance of TP53 gene was increased in 4 patients and reduced in 1 patient; 2 patients had disease progression after treatment and mutation abundances of TP53 gene were reduced. ③ Two patients possessed SMAD4 genetic mutation. ④ The intron mutations were found in EGFR, PIK3CA, AKT1, ERBB2, PTEN, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, FBX7, FGFR3, NOTCH1, ERBB4、FGFR1 and FGFR2 genes, without changes of amino acids, these were not included in analysis. （4） Follow-up and survival situations： 9 patients were followed up for 1-31 months after treatment, with a median time of 7 months, and died at end of follow-up. Conclusions：RAS/RAF gene mutations are detected by monitoring ctDNA when mCRC patients underwent EGFR monoclonal antibody therapy.