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.
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。.
Keywords: Carcinoma, non-small-cell lung; Drug resistance, neoplasm; Liquid biopsy; Molecular targeted therapy; Polymerase chain reaction; Receptor, epidermal growth factor.