第三类体外诊断试剂-循环血活性肿瘤细胞检测试剂盒（绿色荧光蛋白法）

type创新产品

详细说明

　　循环血活性肿瘤细胞检测试剂盒（绿色荧光蛋白法），是由锤特生物创新开发的一款肿瘤体外诊断产品，能定性、定量检测循环血中具有活性的肿瘤细胞，可用于结直肠癌等实体肿瘤的辅助诊断的。

　　该试剂盒利用腺病毒感染肿瘤细胞的特性，通过分子生物学手段，创新构建成对肿瘤细胞具有靶向性、感染能力强的重组腺病毒载体Ad11-5ETel-GFP，进而简单、快速地定量判断血液样品中是否含有存活的肿瘤细胞。

　　本产品最大的优势在于，能特异性检测具有高活力、高转移潜能的肿瘤细胞。此外，检测方法流程简便、快捷、高效，可直接观察到肿瘤细胞的形态且不依赖肿瘤细胞表面标识物，更为本产品的临床推广提供了便利。

　　循环血活性肿瘤细胞检测试剂盒（绿色荧光蛋白法），是一款新型的肿瘤体外诊断（液体活检）产品，能定性、定量检测循环血中具有活性的肿瘤细胞，可用于各种恶性肿瘤（如肺癌、乳腺癌、胰腺癌、黑色素瘤、神经胶质瘤、结直肠癌等）的早期诊断、疗效预后评估、复发转移检测等。

　　该试剂盒利用腺病毒感染肿瘤细胞的特性，创新构建成对肿瘤细胞具有靶向性、复制能力及感染能力强的重组腺病毒载体，能特异性检测具有高活力、高转移潜能的肿瘤细胞。此外，本产品检测方法快捷、简单，可直接观察到肿瘤细胞的形态且不依赖肿瘤细胞表面标识物，利于临床应用。

　　Detection kit for viable circulating tumor cells (Green fluorescent protein assay), which provides a specific and sensitive method for detecting viable circulating tumor cells (CTCs) among more than hundred million of peripheral blood, is composed primarily of a BioTTT patented replication-selective oncolytic adenovirus expressing GFP (Ad11-5ETel-GFP). Ad11-5ETel-GFP is very sensitive to cancer cells and is capable of infecting a wide spectrum of cancer cells, thereby being specific, sensitive, and economic to apply in cancer cells detection in circulaiton blood.

　　产品介绍：

　　中文名：循环血活性肿瘤细胞检测试剂盒（绿色荧光蛋白法）

　　英文名：Detection kit for viable circulating tumor cells (Green fluorescent protein assay)

　　作用原理：

　　腺病毒Ad11对肿瘤细胞比Ad5具有更高的感染能力。用调节Ad5早期基因表达的增强子及在肿瘤细胞内高表达的人端粒酶逆转录酶基因的启动子（hTERT-p）替换控制Ad11腺病毒早期基因表达的相应元件，同时在Ad11E3区基因中插入绿色荧光蛋白（GFP）基因，构建成对肿瘤细胞具有靶向性、感染能力强、并表达GFP的重组腺病毒载体Ad11-5ETel-GFP。

　　本试剂盒使用Ad11-5ETel-GFP与去除红细胞的新鲜血液样品在37℃共同孵育24h后，端粒酶活性极高的活细胞能表达GFP，荧光显微镜下观察细胞中GFP的表达情况，并结合细胞核特异性DAPI染色，即可判断血液样品中是否含有存活的肿瘤细胞，并可计数。

　　Circulating tumor cells (CTCs) are cells that have shed into the vasculature from a primary tumor and circulate in the bloodstream. CTCs thus could be considered a “liquid biopsy” which reveals metastasis in action, providing live information about the patient’s disease status. Analysis of blood samples found a propensity for increased CTC detection as the disease progressed in individual patients.

　　To explore the application of the new adenovirus vector and improve the sensitivity to detect circulating tumor cells in the blood using replication-selective adenovirus, the Ad5 promoter of Ad11-5EP is substituted by a promoter of human telomerase gene, a reporter gene GFP was inserted into E3gp18.5 K of Ad11, and a replication-selective adenovirus (Ad11-5ETel-GFP) capable of expressing reporter genes was created by homologous recombination. As telomerase is highly expressed in 95% of human tumor cells, Ad11-5ETel-GFP selectively replicates and expresses GFP in tumor cells but has no activity in normal epithelial cells.

临床前研究：

　　Circulation Tumor Cells (CTCs) Detection Using Ad11-5ETel-GFP

　　Peripheral blood cells were mixed with 0, 10, 100, and 1000 human pancreatic cancer cell line SUIT-2, respectively (an infection concentration of 100 pfu/cell). The samples were processed as described above. GFP positive cells were observed under the immunofluorescence microscope after 24 h of culturing and it demonstrated that the cancer cell line SUIT-2 is Ad11-5ETel-GFP-sensitive. The GFP-positive cells were correlated to the number of tumor cells mixed with the blood cells.

临床应用：

　　本产品可广泛应用于正常人群的肿瘤筛查、早期诊断，及肿瘤患者的疗效评价、敏感药物筛选、肿瘤术后复发和转移监测：

　　1、肿瘤筛查及早期诊断：肿瘤发生的早期即有肿瘤细胞进入血液循环，故本产品可以作为液体活检方法用于正常人群的肿瘤筛查及早期诊断；

　　2、敏感药物筛选：个体化治疗的体外肿瘤药物筛选，帮助患者确定有效治疗方案；

　　3、疗效评价：目前，国内外通常在病人结束化疗后12周才开始评估治疗效果。而使用本产品，只需监测3周内循环血中肿瘤细胞（CTCs）数目的变化，即可快速指导临床医生调整用药方案，提高治疗效果和减轻治疗药物对病人所产生的毒副作用；

　　4、评估预后：本产品可通过检测样本中存活CTCs的数量，判断患者预后及存活时间；

　　5、监测肿瘤复发和转移：肿瘤的复发是肿瘤持续释放入血向远端转移的缓慢的过程，可以通过检测CTCs直接监测病人是否肿瘤复发。CTCs的增多很有可能是肿瘤复发的前兆或复发的过程。

Clinical application:

　　This product can be widely used in the tearly diagnosis of tumor, the screening of sensitive drugs, the evaluation of therapeutic effect, and the monitoring of tumor relapse and metastasis.

　　1. Screening and early diagnosis of tumor: The importance of CTC's in modern cancer research began in the mid 1990's with the demonstration that CTC's exist early on in the course of the disease.

　　2. Screening sensitive drugs and the evaluation of therapeutic effect and prognosis: CTCs level in peripheral blood in tumor patient is taken as the basis for monitoring, adjusting the treatment, and anticipating the results. The ability to monitor disease progression over time could facilitate appropriate modification to a patient's therapy, potentially improving their prognosis and quality of life.

　　3. Monitoring of tumor relapse and metastasis: CTCs constitute seeds for subsequent growth of additional tumors (metastasis) in vital distant organs. Increasing of CTCs is likely to be an early sign or process of tumor relapse and metastasis.

　　文献资料：

　　1. Wong HH, Jiang G,Gangerswaran R, et al. Modification of the early gene enhancer-promoterimproves the oncolytic potency of adenovirus 11. Molecular Therapy 2012, 20(2):3016-16. (doi: 10.1038/mt.2011.242)

　　2. Toru K, Yuuri H,Yuichi W, et al. A simple biological imaging system for detecting viable humancirculating tumor cells. J Clin Invest 2009, 119(10): 3172-81. (doi:10.1172/JCI38609)

　　3. 王尧河，姜国忠，黄汉熙，曹风雨，尼克莱蒙。溶肿瘤能力增强的B型人腺病毒AD11突变体的构建和应用。中国专利，2011，专利号：CN102260712.

　　4. Kim SJ, Masago A,Tamaki Y, et al. A novel approach using telomerase-specificreplication-selective adenovirus for detection of circulating tumor cells inbreast cancer patients. Breast Cancer Res Treat 2011; 128: 765–73. (doi:10.1007/s10549-011-1603-2)

　　5. Paterlini-Brechot P,Benali NL. Circulating tumor cells (CTC) detection: clinical impact and futuredirections. Cancer Lett 2007; 253: 180-204. (doi: 10.1016/j.canlet.2006.12.014)

　　6. 肖瑶, 刘双, 王增智等.重组病毒特异性检测肺癌患者外周血活性循环肿瘤细胞的临床意义[J].中华结核和呼吸杂志2019 年2月第42 卷第2期. 101-105.（doi: 10. 3760/cma.j.issn.1001?0939.2019.02.005）

　　经典案例：

　　患者男，50岁，从临床表现提示高度可疑癌骨转移，但入院数日未得到明确病理诊断。患者入院时行走困难。查出CTC后按照腺癌转移给予药物治疗后，自主行走迅速得到改善。

　　-CTC检测结果

　　? 首日：五个细胞及细胞团

　　? 隔三日：15个细胞及细胞团

　　-15日后骨髓图片证实为癌转移

　　-未找到原发灶

　　Male, 50. Clinical symptom indicated carcinoma with bone metastasis.

　　--CTCs detection reports

　　Day 0: 5 cells or cell masses in 6ml blood

　　Day3: 15 cells or cell masses in 6ml blood