结直肠癌前哨淋巴结多通道荧光检测技术的发展 - 探索和临床意义

Tumor metastasis of lymph nodes is correlated with prognosis of cancer patients. Thus, the histochemistry dissection is an very important detection technology for routine pathology diagnosis. However, there are high rate of false positive or negative results due to the limitations of the pathological dissection technology, influenced by small size of the sample, variation of tumor marker expressing and objective factors, etc. The time consuming also could cause the delay of the diagnosis and treatment. By using FACS, We will develop a new technology platform for metastasis of lymph node detection. The tumor antigens, related to colorectal cancer will be identified and screened by different fluofores for selection of a tumor markers for the study; Three markers will be labeled by multiple fluofores and read by FACS simultaneously for detection of tumor cell draining of the nodes; Finally, the application of the technology in clinic will be validated. More than 100 samples from 30 patients at least suffer from colorectal cancer will be evaluated by the two technologies, routine histochemisty and multiple color FACS. Apparently, The multiple parameter FACS assay has higher sensitivity, capacity and reliability, which could be a new diagnostic technology in addition to the routine pathology detection. It also can be used for better prediction of the prognosis by the assay of the micro metastasis quantitatively. The Multiple parameter FACS technology can be also applied in other solid tumor diagnosis. The diagnostic kits further developed could have wide clinic applications and market significance.

淋巴结的转移，与肿瘤患者的预后有重要的关系。所以，前哨淋巴结的病理组化是非常重要的常规检测。由于病理切片取材的局限性，肿瘤标志物的表达变异型性，除了有较高的漏检，假阳性或假阴性报告率；且检测耗时长，耽误患者的诊断和治疗。本课题应用流式细胞仪，探索和发展新的病理检测技术平台。用荧光标记EpCAM，CEA,CA19-9，CK20,Keratin等抗原，鉴定和筛选标志物；建立多通道流式检测，选择其中三种标志物的组合，多色标记，同时测定信号，检测淋巴结的浸润；扩大样本，入组30个以上结直肠癌患者，选择100个以上的前哨淋巴结，平行比较常规组化检测结果，证实多通道流式检测临床应用的可行性。 作为常规病理组化检测的补充或替代，该技术不但提高了可靠性，灵敏性，快速性，还能量化微转移细胞和预后的关系。多通道流式诊断技术的平台，也可以应用于其他实体肿瘤的诊断，是临床病理诊断技术的创新，有广阔的临床应用前景

结直肠癌是常见的恶性肿瘤，淋巴结肿瘤细胞微转移是结直肠癌预后的重要因素之一。病理组化是淋巴结肿瘤细胞转移非常重要的常规检测方法，该方法有较高的漏检率和假阳性。本项目意在发展诊断结直肠癌淋巴结微转移的多色流式技术，通过本课题研究，我们筛选出CK20，Epcam和Pan-CK三个抗体组合能够识别淋巴结中转移的肿瘤细胞。与金标准病理检测比较，该方法的敏感性为100%，特异性为68.75%，病理需连续切片才检查到淋巴结微转移肿瘤细胞，流式检测一次就能检测到微转移的肿瘤细胞，且检测结果与患者临床指征相符。流式细胞学检测可以将检测时间缩短至6个小时以内，有利于及时为患者制定治疗方案。. 结果显示流式细胞学多参数检测方案具有高灵敏性、高准确性和快速检测的特点，是一种新的淋巴结诊断技术，能辅助常规淋巴结活检，提高肿瘤淋巴结转移的检出率。同时，该检测方法也可以设计为检测试剂盒，有进一步拓展应用范围及市场化推广的潜力。课题组在研究中获得2项专利国际授权，1项中国发明专利授予，发表SCI文章2篇。自主研发的淋巴结示踪剂已投入生产。