基於循環腫瘤細胞的應用於晚期非小細胞肺癌患者其預後和基因檢測

The Circulating Tumor Cell-based Application for Prognosis and Genetic Testing in Advanced Non-small Cell Lung Cancer Patients

徐臣緯(Chen-Wei Hsu)

指導教授 ： 俞松良

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200771

摘要

肺癌是全球癌症相關死亡的主要原因，其抗藥性和癌細胞轉移時常發生在晚期非小細胞肺癌(NSCLC)。非侵入性液態活檢包括循環腫瘤細胞(CTC)在內，提供了另一種方法用以監測疾病進展與檢測用藥基因突變。因此我們建立了一個可以同時有效進行臨床監控和基因檢測的高純度細胞分離系統。此分離系統是基於免疫抗體磁珠(immunomagnetic bead)來完成CTC富集並透過介電泳(dielectrophoretic)分離系統達到單細胞純化。下一步利用質譜儀(MASS)和次世代定序(NGS)分析測得循環腫瘤細胞的基因突變。首先，我們通過客製化的上皮細胞粘附分子(EpCAM)抗體優化了免疫抗體磁珠的富集效率，並使用外加H1975細胞株於全血的方式來評估目標細胞的回收率和純度，接著進一步評估基因檢測需要的最小細胞數極限，也在晚期非小細胞肺癌患者中觀察到了CTC數量的動態變化。在體外測試結果中免疫抗體磁珠富集系統裡客製化的EpCAM抗體能將富集回收率從70%提高到88%。最後純化成功的單細胞全基因擴增成功率為60%，質譜儀檢測單細胞表皮生長因子接受器(EGFR) L858R和T790M基因突變的一致性為100%，變異係數(CV)分別為4%和5%。在NGS中，純化後的H1975細胞株一顆細胞、五顆細胞和十顆細胞的十倍深度覆蓋率(coverage)分別為59.92%、61.68%和79.27%。臨床測試結果中從NSCLC患者周邊血分離的 10 顆CTC裡鑑定出了EGFR del 19基因突變。我們也同時觀察到了CTC計數與其他臨床治療的臨床相關性，發現中性粒細胞與淋巴細胞的比率(NLR)與CTC計數結合可能能作為預後不良的潛在指標。值得注意的是，CTC數量的動態變化有潛力可以預測多發性腦轉移和肺到肺轉移。另外我們還發現了結合CTC和循環游離核酸(cfDNA)的NGS分析結果可以提高基因檢測的檢測率。綜合以上，我們建立了基於免疫抗體磁珠介電泳的方法來純化高純度CTC，且可同時監測CTC數量和基因突變，並展現CTC有潛力在肺癌病患預測與預後中可以當作生物標誌物用於測量癌症主導基因(driver gene)的表現，顯示了CTC應用於肺癌患者個人化治療的可行性。

關鍵字

循環腫瘤細胞 ； CTC ； 細胞分離 ； 肺癌 ； NGS ； 動態變化

並列摘要

Lung cancer is the leading cause of cancer-related deaths worldwide. The drug resistance and metastasis eventually occurred in advanced non-small cell lung cancer (NSCLC). Non-invasive liquid biopsy including circulating tumor cell (CTC) provides an alternative solution to monitor disease progress and identify druggable mutations once progressive disease occurred. We establish an effective system for high-purity CTC isolation for monitoring and gene testing simultaneously. An immunomagnetic bead-based enrichment system was used for CTC enrichment and a dielectrophoretic separation system was used for single cell sorting. Gene mutations of resulting CTCs were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and next-generation sequencing (NGS). First, we optimized the efficiency of immunomagnetic beads-based CTC enrichment by customized EpCAM antibodies and spiked H1975 cells were used for the evaluation of recovery rate and purity. The minimal number of CTCs for gene testing was evaluated and the dynamic change of CTC number was determined in advanced NSCLC. The customized EpCAM pooled antibodies improved the recovery rates of immunomagnetic beads-based enrichment system from 70% to 88% in three spiked cell lines. The successful rate of single cell whole genome amplification is 60% and the concordance of both L858R and T790M EGFR mutations in a single cell assayed by MALDI-TOP MS is 100% and coefficient variations are 4% and 5%, respectively. The coverages of 10X depth of NGS in 1-cell, 5-cell and 10-cell groups of H1975 cells are 59.92%, 61.68% and 79.27%, respectively. EGFR del 19 mutation was identified in 10 CTCs isolated from NSCLC patients. We demonstrated the clinical correlation of CTC count with other clinical treatment and peripheral blood cells, neutrophil-to-lymphocyte ratio (NLR), might serve as a potential indicator of poor prognosis Notably, the dynamic changes of CTC number may predict multiple brain and lung-to-lung metastases. We also found that combining CTC and circulating cell free DNA (cfDNA) may improve the detection rate on the NGS result of gene detection. Taken together, We established the magnetic beads-based dielectrophoretic sorting method for high-purity CTC capture which can enumerate CTC number and determine gene mutations simultaneously and evaluate the feasibility of serial CTC detections and gene testing for personalized therapy of lung cancer patients, evaluate the driver gene expression for predictive and prognostic biomarkers in lung cancer patients.

並列關鍵字

circulating tumor cell ； CTC ； cell isolation ； lung cancer ； NGS ； dynamic change

參考文獻

1. Siegel, R.L., et al., Cancer statistics, 2022. CA: a cancer journal for clinicians, 2022.

2. Ferlay, J., et al., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer, 2015. 136(5): p. E359-86.

3. Hirsch, F.R. and P.A. Bunn, Jr., EGFR testing in lung cancer is ready for prime time. Lancet Oncol, 2009. 10(5): p. 432-3.

4. Langer, C.J., Epidermal growth factor receptor inhibition in mutation-positive non-small-cell lung cancer: is afatinib better or simply newer? J Clin Oncol, 2013. 31(27): p. 3303-6.

5. O'Kane, G.M., et al., Uncommon EGFR mutations in advanced non-small cell lung cancer. Lung Cancer, 2017. 109: p. 137-144.