探討非小細胞肺癌病人循環腫瘤DNA基因圖譜及其與標靶治療反應之關係

Investigation of the Mutation Profiles of Circulating Tumor DNAs from Patients with Non-Small Cell Lung Cancer and its Correlation with Targeted Therapy Response

王智亮(Chih-Liang Wang) ； 王美嘉(Mei-Chia Wang) ； 張仕政(Shih-Cheng Chang) ； 劉秀玲(Hsiu-Ling Liu) ； 張璧月(Pi-Yueh Chang)

《Journal of Biomedical & Laboratory Sciences》 33卷3期 (2021/09) Pp. 143-152

摘要

檢測循環腫瘤DNA（ctDNA）可即時反應治療有效性及監控抗藥基因，本研究收集了17位上皮細胞生長因子受體（EGFR）突變陽性接受酪胺酸激酶抑制劑（TKI）標靶治療的末期肺腺癌病人，於用藥前及用藥後常規回診日收集其周邊血，在平均約1年的追蹤期中，有5位發生復發。我們使用含有12個肺癌治療相關基因且敏感度可達0.1%的次世代定序（NGS）套組，檢測4位復發及1位未復發病人的ctDNA，其餘12位病人，則以胜肽核酸箝制（PNA）PCR檢測7種EGFR的突變。NGS結果顯示，治療前ctDNA中可100%（5/5）檢出與癌組織完全一致的EGFR基因型，血流中癌基因突變比率與原始癌組織大小無關，但與復發的時間成反比，且ctDNA可於治療後一個月後完全測不到，但在復發前3個月又可再度檢測到突變。PNA檢測敏感度則只有25%（3/12），突變型與癌組織相同，治療後皆測不到ctDNA。綜上，以NGS檢測ctDNA的正確性及敏感度可達100%，此非侵入性的周邊血採檢方式是未來臨床應用的利基。

關鍵字

肺腺癌 ； 上皮細胞生長因子受體 ； 標靶治療 ； 循環腫瘤DNA ； 次世代定序

並列摘要

Detection of circulating tumor DNA (ctDNA) can dynamically reflect the effectiveness of targeted therapy and monitor the resistant genes. In this study, 17 advanced non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations in their tumors and receiving tyrosine kinase inhibitor (TKI) treatment were enrolled. Peripheral blood specimen before treatment and at follow-up interval after TKI treatment were serially collected till disease progress. In this average one-year follow-up period, five of them developed progressive disease (PD) and the rest were in stable disease (SD) or partial response (PR) status. We selected 5 paired before and after treatment blood specimen from four PD and one PR patients to comprehensively investigate the mutation panel by a next generation sequencing (NGS) test which can simultaneously detect alterations in 12 lung cancer associated genes with 0.1% of allele detection sensitivity. The rest of specimen were investigated by a multiplex peptide nucleic acid (PNA) clamping PCR to detect 7 types of EGFR mutations in ctDNA. The results showed: 1. For NGS platform, variants pattern detected in circulation in all five patients are the same as those in their local tumors. The detection sensitivity is 100%(5/5). Two of them also harbored mutations on TP53 other than EGFR in ctDNA. Variant frequency in circulation has no relation with the size of local tumor. However, progression free period is reversely correlated with the variant frequency in ctDNA before treatment. Vanish speed of specific variants in circulation is also correlated with the original tumor size and the same mutation or new mutations can be detected 3 months before relapse. 2. For PNA platform, only three out of 12 patients can detect EGFR mutations in their pre-treatment blood. The detection sensitivity is only 25% (3/12). The variant pattern is the same as that in original tumors. No mutation can be detected in their follow up blood. In conclusion, the accuracy and sensitivity are 100% for NGS platform to detect mutations in ctDNA before treatment. The non-invasive circulating tumor DNA detection test is promising for future clinical applications.

並列關鍵字

Lung adenocarcinoma ； epidermal growth factor receptor (EGFR) ； targeted therapy ； circulating tumor DNA ； next generation sequencing (NGS)