高陽張景春康熙雄

1 首都醫(yī)科大學附屬北京天壇醫(yī)院 首都醫(yī)科大學檢驗診斷學系 （北京 100020）

2 杭州華得森生物技術有限公司 （浙江 杭州 310051）

3 吉林大學附屬中日聯(lián)誼醫(yī)院 （吉林 長春 130033）

精準醫(yī)學背景下CTC檢測的應用

高陽1,2張景春3康熙雄1

1 首都醫(yī)科大學附屬北京天壇醫(yī)院 首都醫(yī)科大學檢驗診斷學系 （北京 100020）

2 杭州華得森生物技術有限公司 （浙江 杭州 310051）

3 吉林大學附屬中日聯(lián)誼醫(yī)院 （吉林 長春 130033）

精準醫(yī)學是隨著科學技術不斷發(fā)展，人們對健康需求的不斷提高，而逐漸興起的新的醫(yī)療理念和模式，主要基于分子病理醫(yī)學和最先進的醫(yī)學檢測技術，通過對患者進行基因組、轉錄組、蛋白組、表觀遺傳學、分子影像學和分子病理學檢測，結合患者病例病理信息，為患者制定針對個體疾病特征的最優(yōu)化治療方案，以追求最大的治療效果和最低毒副作用。腫瘤精準醫(yī)學的發(fā)展方向之一就是無創(chuàng)診療，其中液體活檢愈來愈受醫(yī)療診斷業(yè)界關注，液體活檢通過無創(chuàng)方式檢測體液（主要為外周血、唾液、尿液、胸腹腔積液等）中的循環(huán)腫瘤細胞（CTC）、循環(huán)腫瘤DNA（ctDNA）、外泌體（Exsome）等腫瘤來源的生物標志物，動態(tài)反應腫瘤的疾病進展，為腫瘤的早期診斷、預后評估、療效監(jiān)測、復發(fā)轉移檢測、實時動態(tài)監(jiān)測和個體化診療等，為腫瘤診療提供了一種簡便快捷的手段。

精準醫(yī)學 液體活檢 循環(huán)腫瘤細胞

隨著基因檢測等新技術的飛速發(fā)展，越來越多的精準醫(yī)學、精準醫(yī)療診斷產品涌入臨床檢驗，這種通過各種高端檢測技術，實現(xiàn)個性化診療的手段，已經被大家所認識并接受。但在全民提倡的精準醫(yī)學背景下，所謂的精準技術、精準服務到底體現(xiàn)在哪里，如何去實現(xiàn)，是每個醫(yī)療專業(yè)人員十分關心的問題。精準醫(yī)療是一個被賦予了很多期望并可能改變醫(yī)療認知的新興領域，雖然探索之路還面臨很多困難，但希望研究者可以利用現(xiàn)有迅速發(fā)展的技術平臺，不斷積累健康數據，覆蓋更多的醫(yī)療和健康領域。

1.精準醫(yī)療應用的主要領域

根據應用范圍來看，可以總結為四大領域：①與新生兒相關的疾病領域，如體外受精的卵母細胞選擇、孕婦產前診斷、新生兒先天性代謝、先天性免疫缺陷、單基因遺傳等疾病的篩查；②對人的分型，過去用ABO血型和肝臟移植的HLA分型，現(xiàn)在可以通過基因測序進行藥物代謝分型，把人群分為快、中、慢幾種代謝類型等；③生物藥物、可提前篩查抗體藥物的適用人物；④在腫瘤的篩查，診斷和評價上進行精細的指導治療。

2.循環(huán)腫瘤細胞（CTC）檢測起源及現(xiàn)狀

醫(yī)學檢驗已成為一門學科，在臨床診斷中起著決定性的指導作用。一些精準醫(yī)療產品也層出不窮。目前，臨床應用最多的是基于循環(huán)腫瘤細胞（Circulating Tumor Cell，CTC）和ctDNA的精準診療檢測產品。在1896年，澳大利亞學者Ashworth發(fā)現(xiàn)血液中存在的一些血細胞同尸檢發(fā)現(xiàn)的腫瘤細胞相似，首次提出CTC的概念[1]。CTC目前定義為自發(fā)或因診療操作由實體瘤或轉移灶釋放進入外周血循環(huán)的腫瘤細胞。進入循環(huán)的腫瘤細胞絕大多數在短期內死亡，只有極少數具有高度活力、高度轉移潛能的腫瘤細胞在循環(huán)系統(tǒng)中存活下來，相互聚集形成微小癌栓，并在一定條件下發(fā)展為轉移灶，也就是腫瘤可怕的血行轉移和復發(fā)。

很多研究證明，外周血CTC存在于乳腺癌、肺癌、前列腺癌、結直腸癌、胃癌和卵巢癌等多種惡性腫瘤中，與患者的臨床分期、無進展生存期、總生存期、藥物療效以及早期復發(fā)和轉移均密切相關，對外周血中CTC的檢測有助于早期發(fā)現(xiàn)腫瘤的微轉移、監(jiān)測術后復發(fā)、評估療效及預后，以及選擇合適的個體化治療[2-39]。因而這種檢測技術有望成為實時的“液體活檢”，已備受關注。

由于CTC在血液中的比例極少（幾十萬分之一），則對于CTC富集方面是需要攻克的難題。目前CTC富集方法主要有免疫磁性分離法和基于形態(tài)學的富集法。前者的白血球污染問題，而后者由于腫瘤細胞的大小沒有定義，所以其分離靈敏性受到質疑。采用流式分選也可得到CTC，但由于細胞在流式分選系統(tǒng)中承受的各種外力導致細胞損傷，沒有活性。如何有效地富集到高純度、有活性的CTC成為了一個重大挑戰(zhàn)！

美國FDA于2004年認證了CTC檢測技術的第一款上市設備美國上市公司強生的CellSearch分析系統(tǒng)，并分別在2004年、2007年和2008年批準采用CellSearch檢測系統(tǒng)用于轉移性乳腺癌、轉移性結直腸癌及轉移性前列腺癌的預后評估，無進展生存（PFS）期和總生存（OS）期的預測，拉開了全球CTC的臨床研究和應用的序幕。其中CTC細胞計數是目前唯一獲得FDA認證的CTC臨床應用領域，可以作為臨床上乳腺癌、前列腺癌以及腸癌的輔助診斷指標之一。

2012年8月，中國國家食品藥品監(jiān)督管理局（CFDA）正式批準CellSearch CTC檢測與分析系統(tǒng)用于轉移性乳腺癌患者的預后分析，這是中國首次批準CTC檢測進入臨床應用。目前，CTC檢測在基礎臨床研究及臨床應用方面得到越來越多的重視。

國內各省腫瘤醫(yī)院、大型綜合性三甲醫(yī)院和部分二甲以上的醫(yī)院都已經開展CTC檢測，或正籌備開展CTC臨床檢測服務和各種科研應用。CTC檢測是一種安全可靠的被美國FDA批準的癌癥臨床輔助診斷手段。CellSearch CTC檢測儀器和試劑雖搶占了先機，但隨著臨床單位對CTC檢測方法及結果的更高要求，對后續(xù)的精準治療方面有著更深的需求下，中國國內其他相關檢測儀器和試劑隨著注冊臨床試驗的進行，已經陸續(xù)取得了CFDA的注冊認可。未來更先進和小巧的CTC檢測儀器將踴躍出現(xiàn)，后來居上，搶占市場。CTC檢測將會在中國得到越來越多的重視，并迎來更加廣闊的臨床及科研應用前景，

雖然CTC已被FDA批準應用于轉移性乳腺癌、轉移性直結腸癌、轉移性前列腺癌的檢測，其臨床應用仍然面臨諸多挑戰(zhàn)。各實驗室CTC分析方法和結果解讀的標準化是亟需解決的問題。

3.國內市場主要CTC檢測平臺

在國內市場上，已有多家公司推廣了CTC檢測平臺，見表1。

目前國內已有上百家醫(yī)院開展了CTC檢測項目，如中國人民解放軍第三〇七醫(yī)院，復旦大學附屬腫瘤醫(yī)院，中山大學附屬孫逸仙紀念醫(yī)院，天津腫瘤醫(yī)院等醫(yī)院，以及湖北省同濟醫(yī)院、協(xié)和醫(yī)院、武漢大學中南醫(yī)院、江漢大學實驗室等。

4.CTC檢測的價值及前景

CTC檢測具有良好的社會效益和經濟效益，是腫瘤?？漆t(yī)院不可或缺的檢驗檢測項目。該技術的全球市場2012年達到近28億美元的規(guī)模，2013年達到37億美元。今后該市場以年復合成長率18.9%的速度增長，預計2018年將達到89億美元的規(guī)模。

目前，國內各省市對于CTC檢測尚無明確醫(yī)療服務收費標準，現(xiàn)階段各省多數定價無法統(tǒng)一，而且收費較高。據悉，浙江省已開展CTC檢測的醫(yī)療檢測機構的服務收費為6240元/測試，上海CTC檢測服務收費為4800元/測試左右，個別省市CTC檢測服務收費為4600元/測試左右。未來CTC在臨床應用還需要多方面的努力才能順利普及開展。

從宏觀的角度分析精準醫(yī)學，應該體現(xiàn)在檢測平臺、醫(yī)療數據平臺、分子病理平臺、臨床科研平臺多方面發(fā)展，發(fā)現(xiàn)更多的如KRAS/EGFR等個體化基因標志物，希望通過臨床的開展，大數據的算法，對疾病的發(fā)生發(fā)展規(guī)律或者個體健康和疾病進行預測，甚至開拓新的醫(yī)學預測學科建設。隨著國家政策對精準醫(yī)學的扶持，希望新的檢測技術能為精準醫(yī)學在臨床的應用提供更多更廣的空間。

表1. 國內宣傳CTC項目及平臺的部分廠家

表2. 部分已開展CTC檢測的醫(yī)療檢測機構

參考文獻

[1] Ghossein RA, Carusone L, Bhattacharya S. Review:polymerase chain reaction detection of micrometastase and circulating tumor cells:application to melanoma, prostate, and thyroid carcinomas[J]. Diagn Mol Pathol, 1999,8(4):165-175.

[2] Meng S, Tripathy D, Shete S, et al. HER-2 gene amplifcation can be acquired as breast cancer progresses[J]. Proc Natl Acad Sci U S A, 2004,101(25):9393-9398.

[3] Georgoulias V, Bozionelou V, Agelaki S, et. al. Trastuzumab decreases the incidence of clinical relapses in patients with early breast cancer presenting chemotherapy-resistant CK-19mRNA-positive circulating tumor cells:results of a randomized phase II study[J]. Ann Oncol, 2012,23(7):1744-1750.

[4] Liu Y, Liu Q, Wang T, et al. Circulating tumor cells in HER2-positive metastatic breast cancer patients:a valuable prognostic and predictive biomarker[J]. BMC cancer, 2013,23;13:1-8.

[5] Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer[J]. Engl J Med, 2004,351(8):781-791.

[6] Jiang ZF , Cristofanilli M, Shao ZM, et. al. Circulating tumor cells predict progression-free and overall survival in Chinese patients with metastatic breast cancer, HER2-positive or triple-negative (CBCSG004):a multicenter, double-blind, prospective trial[J]. Ann Oncol, 2013,24(11):2766-2772.

[7] Pierga JY, Bidard FC, Mathiot C, et al. Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial[J]. Clin Cancer Res, 2008,14(21):7004-7010.

[8] Franken B, de Groot MR, Mastboom WJ, et al. Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer[J]. Breast Cancer Research 2012,14 (5):R133.

[9] Zhang L, Ridgway LD, Wetzel MD, et al. The Identifcation and Characterization of Breast Cancer CTC Competent for Brain Metastasis[J]. Sci Transl Med, 2013,5(180):120-128.

[10] Jordan NV, Bardia A, Wittner BS, et al. HER2 expression identifes dynamic functional states within circulating breast cancer cells[J]. Nature, 2016,537(7618):102-106.

[11] De Luca F, Rotunno G, Salvianti F, et al. Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer[J]. Oncotarget, 2016,7(18):26107-26119.

[12] Krebs MG, Metcalf RL, Carter L, et al. Molecular analysis of circulating tumour cells-biology and biomarkers[J]. Nature Reviews Clinical Oncology, 2014,11(3):129-144.

[13] Maheswaran S, Sequist LV, Nagrath S, et al. Detection of mutations in EGFR in circulating lung-cancer cells[J]. N Engl J Med, 2008,359(4) :366-377.

[14] Das M, Riess JW, Frankel P, et al. ERCC1 expression in circulating tumor cells (CTC) using a novel detection platform correlates with progression-free survival (PFS) in patients with metastatic non-small-cell lung cancer (NSCLC) receiving platinum chemotherapy[J]. Lung Cancer, 2012,77(2):421-426.

[15] Pailler E, Adam J, Barthélémy A, et al. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer[J]. J Clin Oncol, 2013,31(18):2273-2281.

[16] Ni X, Zhuo M, Su Z, et al. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients[J]. PNAS, 2013,110(52):21083-21088.

[17] Punnloose EA, Atwal S, Liu W, et al. Evaluation of circulating tumor cells and circulating tumor DNA in non-small cell lung cancer:association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib[J]. Clin Cancer Res, 2012,18(8):2391-2401.

[18] Reid AH, Attard G, Danila DC, et al. Signifcant and sustained antitumor activity in post-docetaxel, castration-resistant prostate cancer with the CYP17 inhibitor abiraterone acetate[J]. J Clin Oncol, 2010,28(9):1489-1495.

[19] de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival beneft from treatment in metastatic castration-resistant prostate cancer[J]. Clinical Cancer Research, 2008,14(19):6302-6309.

[20] Lohr JG, Adalsteinsson VA, Cibulskis K, et al. Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer[J]. Nature biotechnology, 2014,32(5):479-484.

[21] Cohen SJ, Punt CJ, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer[J]. J Clin Oncol, 2008,26(19):3213-3221.

[22] Uen YH, Lu CY, Tsai HL, et al. Persistent presence of postoperative circulating tumor cells is a poor prognostic factor for patients with stage I-III colorectal cancer after curative resection[J]. Ann Surg Oncol, 2008,15 (8):2120-2128.

[23] de Albuquerque A, Kubisch I, St?lzel U, et al. Prognostic and predictive value of circulating tumor cell analysis in colorectal cancer patients[J]. J Transl Med, 2012,10(1):222.

[24] Lu CY, Tsai HL, Uen YH, et al. Circulating tumor cells as a surrogate marker for determining clinical outcome to mFOLFOX chemotherapy in patients with stage III colon cancer[J]. Br J Cancer, 2013,108(4):791-797.

[25] Van Dalum G, Stam GJ, Scholten LF, et al. Importance of circulating tumor cells in newly diagnosed colorectal cancer[J]. Int J Oncol, 2015,46(3):1361-1368.

[26] Iinuma H, Watanabe T, Mimori K, et al. Clinical signifcance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes' stage B and C colorectal cancer[J]. J Clin Oncol, 2011,29(12):1547-1555.

[27] Yen LC, Yeh YS, Chen CW, et al. Detection of KRAS oncogene in peripheral blood as a predictor of the response to cetuximab plus chemotherapy in patients with metastatic colorectal cancer[J]. Clin Cancer Res, 2009,15(13):4508-4513.

[28] Abdallah EA, Fanelli MF, Buim ME, et al. Thymidylate synthase expression in circulating tumor cells:a new tool to predict 5-fuorouracil resistance in metastatic colorectal cancer patients[J]. Int J Cancer, 2015,137(6):1397-1405.

[29] Zou K, Yang S, Zheng L, et al. Prognostic Role of the Circulating Tumor Cells Detected by Cytological Methods in Gastric Cancer:A Meta-Analysis[J]. BioMed Research International, 2016,2016:2765464.

[30] Uenosono Y, Arigami T, Kozono T, et al. Clinical signifcance of circulating tumor cells in peripheral blood from patients with gastric cancer[J]. Cancer, 2013,119(22):3984-3991.

[31] Huang X, Gao P, Sun J, et al. Clinicopathological and prognostic signifcance of circulating tumor cells in patients with gastric cancer:a meta-analysis[J]. International Journal of Cancer, 2015,136(1):21-33.

[32] Li TT, Liu H, Li FP, et al. Evaluation of epithelial-mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer:Relevance to therapy response[J]. World journal of gastroenterology, 2015,21(47):13259.

[33] Li Y, Zhang X, Ge S, et al. Clinical signifcance of phenotyping and karyotyping of circulating tumor cells in patients with advanced gastric cancer[J]. Oncotarget, 2014,5(16):6594.

[34] Aktas B, Kasimir-Bauer S, Heubner M, et al. Molecular profling and prognostic relevance of circulating tumor cells in the blood of ovarian cancer patients at primary diagnosis and after platinum-based chemotherapy[J]. Gynecological Cancer, 2011,21(5):822-830.

[35] Poveda A, Kaye SB, McCormack R, et al. Circulating tumor cells predict progression free survival and overall survival in patients with relapsed/recurrent advanced ovarian cancer[J]. Gynecologic oncology, 2011,122(3):567-572.

[36] Kuhlmann JD, Wimberger P, Bankfalvi A, et al. ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance[J]. Clinical chemistry, 2014,60(10):1282-1289.

[37] Fehm T, Banys M, Rack B, et al. Pooled analysis of the prognostic relevance of disseminated tumor cells in the bone marrow of patients with ovarian cancer[J]. Gynecological Cancer, 2013,23(5):839-845.

[38] Behbakht K, Sill MW, Darcy KM, et al. Phase II trial of the mTOR inhibitor, temsirolimus and evaluation of circulating tumor cells and tumor biomarkers in persistent and recurrent epithelial ovarian and primary peritoneal malignancies:a Gynecologic Oncology Group study[J]. ynecologic oncology, 2011,123(1):19-26.

[39] Obermayr E, Maritschnegg E, Speiser P, et al. Circulating rare cells enable highly effcient cancer detection[J]. BMC cancer, 2010,10(1):666.

Application of CTC Detection in Precision Medical Background

GAO Yang1,2ZHANG Jing-chun3KANG Xi-xiong1
1 Capital Medical University Beijing Tiantan Hospital Laboratory Diagnosis Department (Beijing 100020)
2 Hangzhou Waston Biotech. INC (Zhejiang Hangzhou 310051)
3 Jilin Univercity China-Japan Fellowship Hospital (Jilin Changchun 130033)

Precision medicine is a new medical concepts and models, which gradual rise with the continuous development of science and technology, and the people's health needs. It mainly based on molecular pathology and the most advanced medical testing technology, through the genomics, proteomics, epigenetics, molecular imaging and molecular pathology, combined with patient case and pathology information, for patients with the development of individual disease for the characteristics of the optimal treatment program, to pursue the greatest therapeutic effect and the lowest side effects. One of the directions of development of tumor precision medicine is noninvasive diagnosis, in which liquid biopsy is increasingly concerned by the medical diagnosis. Liquid biopsy provides a simple and quick means for cancer diagnosis and treatment, by detecting the circulating tumor cell (CTC), circulating tumor DNA (ctDNA), exosomes and other tumor-derived biomarkers, which come from body fuids (mainly peripheral blood, saliva, urine, pleural effusion, etc.), in a relatively noninvasive way, dynamic response of tumor disease progression, for the early diagnosis of cancer, prognosis evaluation, effcacy monitoring, recurrence and metastasis detection, real-time dynamic monitoring and individual diagnosis and treatment.

precision medicine, liquid biopsy, circulating tumor cell (CTC)

1006-6586(2017)11-0014-04

R197.39

A

2017-04-18