微滴数字PCR与Super-ARMS PCR检测非小细胞肺癌患者表皮生长因子受体(EGFR)酪氨酸激酶抑制剂治疗耐药后血浆游离DNA EGFR基因T790M突变的对比分析

Comparison of epidermal growth factor receptor (EGFR)gene T790M mutation by droplet digital PCR and Super-ARMS PCR in plasma ctDNA samples of non-small cell lung cancer patients with the resistance to EGFR-tyrosine kinase inhibitor

目的 比较微滴数字PCR(ddPCR)与Super-扩增阻滞突变系统( ARMS)法检测表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(TKI)治疗后耐药非小细胞肺癌患者血浆中EGFR基因耐药位点T790M突变的差异,并探讨ddPCR的临床应用价值.方法 收集同济大学附属上海市肺科医院2017年5至11月间37例经EGFR-TKI治疗后耐药的非小细胞肺癌患者血浆标本;分别采用ddPCR和Super-ARMS法检测血浆标本中EGFR 基因T790M突变情况.结果 EGFR-TKI治疗耐药后非小细胞肺癌患者,男性17例,女性20 例,年龄范围40~83岁,中位年龄 64岁.治疗前,37例患者肿瘤组织均检测出EGFR基因敏感突变,无 T790M 突变.靶向治疗耐药后,ddPCR检测患者血浆游离DNA EGFR 基因T790M突变,阳性率为45. 9%(17/37),Super-ARMS检测阳性率为35. 1%(13/37),统计学分析发现,两种方法检测 EGFR T790M 突变阳性率差异具有统计学意义(P<0. 05). 13例Super-ARMS法T790M突变病例(ΔCt值<8),经ddPCR法检测均为阳性; Super-ARMS法10例翘尾病例(ΔCt值≥8,T790M突变阴性),ddPCR法检测显示有3例T790M突变;Super-ARMS法14例完全阴性病例(无ΔCt值,T790M突变阴性),ddPCR法检测显示有1例T790M突变弱阳性. ddPCR法检测T790M突变结果与 Super-ARMS法检测判读的 ΔCt 值之间有显著相关性.在 ddPCR 检测出的17例T790M阳性的患者中,其中9例患者接受了奥西替尼治疗,7 例患者都有良好的获益.结论ddPCR检测EGFR-TKI治疗耐药后非小细胞肺癌患者血浆EGFR基因T790M突变,比Super-ARMS法具有更高的灵敏度,这可能会在指导临床靶向药物治疗上具有广泛的应用价值.

Objective To compare droplet digital PCR (ddPCR) and Super-amplification refractory mutation system (ARMS) in the detection of T790M mutation of epidermal growth factor receptor ( EGFR) in the plasma of non-small cell lung cancer patients who had developed resistance to EGFR tyrosine kinase inhibitor (TKI) , and to investigate the clinical application of ddPCR. Methods Plasma samples were collected from non-small cell lung cancer patients who had acquired EGFR-TKI resistance at Shanghai Pulmonary Hospital, Tongji University,from May 2017 to November 2017. Extracted ctDNA was analyzed by ddPCR and Super-ARMS to evaluate the T790M mutation status of EGFR gene. Results A total of 37 patients with activating EGFR mutation that acquired resistance to EGFR-TKI were selected in the study, including 17 male and 20 female with a median age of 64 years (range 40-83 years). Before TKI treatment, all the patients harbored EGFR inhibitor sensitive mutations but without T790M mutation. After acquiring resistance to EGFR-TKI treatment, the T790M mutation rate detectable by ddPCR was 45. 9%(17/37). In contrast, the mutation rate of T790M detectable by Super-ARMS was 35. 1%(13/37, P<0. 05). For the 13 positive cases detected by Super-ARMS (ΔCt<8), they were all positive by ddPCR assay; Among the 10 negative cases detected by Super-ARMS (ΔCt≥8), there were 3 cases positive by ddPCR assay. For patients without ΔCt by Super-ARMS assay, there was one weak positive case detectable by ddPCR assay. Among 17 EGFR T790M positive patients, 9 received EGFR inhibitor Osimertinib treatment, and 7 of them had good therapeutic response after the treatment. Conclusions While a significant correlation between the two methods is shown. ddPCR is more sensitive than Super-ARMS in the detection of EGFR T790M mutation, indicating that it is a better method in guiding target drug therapy of non-small cell lung cancer patients after acquiring the resistance to EGFR-TKI.