基于前哨淋巴结活检术的乳腺癌淋巴结转移“滞留期”相关肿瘤标记物的探寻与验证

Reseaches on lympn node metastasis in breast cancer patients are relatively limited because of the lacking of acceptable markers and research models. Sentinel lymph node biopsy is one of the routine surgeries for breast cancer patients. About 40-70 percent of patients with positive sentinel lymph nodes are free from non-sentinel lymph nodes metastases, which is a relatively special phage in the progression of the carcinoma. It is a meaningful "retension stage", in which tumor cells from primary carcinoma have obtained the ability of invasion and metastasis, and have successfully arrived in the regional lymph nodes. In the present study, the technique of sentinel lymph node biopsy is employed as the research platform to find out the "retention phase" of lymph node metastasis in breast cancer patients. Fresh tissue will be obtained intraoperatively. Laser capture microdissection is introduced to purify the tissue, and to pick up the tumor cells of the primary carcinoma, the tumor cells in sentinel lymph nodes, the microenvironment around the tumor cells in sentinel lymph nodes, and the microenvironment within negative non-sentinel lymph nodes. We will apply the iTRAQ-labelled, high performance liquid chromatography electrospray-mass spectrometry to find out the differentially expressed protain betweem the tumor cells in the primary carcinoma and in the sentinel lymph nodes, to discuss the new traits that the tumor cells obtained among the period of lymph node metastasis. The same technique would be applied to find out the differentially expressed protain in the microenvironment of the positive sentinel lymph nodes and in the negative non-sentienl lymph nodes. "Retention phase" related markers would be discovered and verified. Also, we may include the new discovered "retention phase" related markers in the previous established non-sentinel lymph node metastasis prognostic nomogram to increase the accuracy. In addition, further research would be focus on the verification of the biological function of the "retension phase" related markers.

肿瘤淋巴结转移机制的研究因缺乏良好的标记物及研究模型而较少。前哨淋巴结活检术是乳腺癌常规手术方式之一。在前哨淋巴结阳性的患者中，有40-70%的患者其非前哨淋巴结均为阴性- - 转移淋巴结仅限于前哨淋巴结，这是肿瘤发展过程中所出现的一个比较特殊的时期，是原发灶肿瘤细胞获得侵袭转移能力且成功进入淋巴系统并抵达区域淋巴结后，与淋巴组织微环境发生相互作用的早期制衡阶段，是肿瘤淋巴结转移过程中具有特殊意义的"滞留期"。本课题拟利用前哨淋巴结活检术筛选淋巴结转移"滞留期"的患者，以此为研究平台，在激光捕获显微切割技术的支持下，通过稳定同位素iTRAQ标记/高效液相色谱-质谱联用技术，探讨肿瘤细胞获得相关恶性特征的时间节点，以及淋巴结微环境发生改变的时间节点；对筛选所得差异表达蛋白质进行验证，建立非前哨淋巴结病理学状态预测模型；同时进行生物学功能验证，以期为进一步完整描绘乳腺癌淋巴结转移机制图谱搭建框架。

肿瘤淋巴结转移机制的研究因缺乏良好的标记物及研究模型而较少。前哨淋巴 结活检术是乳腺癌常规手术方式之一。在前哨淋巴结阳性的患者中，有 40-70%的患者其非前 哨淋巴结均为阴性——转移淋巴结仅限于前哨淋巴结，这是肿瘤发展过程中所出现的一个比 较特殊的时期，是原发灶肿瘤细胞获得侵袭转移能力且成功进入淋巴系统并抵达区域淋巴结 后，与淋巴组织微环境发生相互作用的早期制衡阶段，是肿瘤淋巴结转移过程中具有特殊意 义的“滞留期”。本课题拟利用前哨淋巴结活检术筛选淋巴结转移“滞留期”的患者，以此为研究平台，通过稳定同位素 iTRAQ 标记/高效液相色谱-质谱联用技术以及比较基因组学技术，探讨肿瘤细胞原发灶与淋巴结转移灶中差异基因表达与差异蛋白质表达；同时进行生物学功能验证，以期为进一步完整描绘乳腺癌淋巴结转移机制图谱搭建框架。作为一个探索性的研究，本课题发现了与淋巴结转移相关靶点，包括miRNA如miR-3163、miR-23a-3p等；mRNA如SNAI1、PRKC1等；TF如CPBP、NFATC2等；蛋白质如PCOLCE、TBCA；lncRNA 如CASC15、LINC00478等。所发现的靶点拟进一步进行机制研究。

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