对其它癌的研究认为肿瘤细胞在极早期甚至是异常增生阶段即有播散，我们利用前期构建成功的4-硝基喹啉1-氧化物（4-NQO）饮水法诱发小鼠口腔癌颌下淋巴结高转移模型，亦发现在异常增生阶段即有骨髓播散肿瘤细胞（DTC）检出且渐增多。为了证明口腔癌肿瘤细胞的早期播散、平行进化假说，本研究利用免疫组化CK染色、显微切割获取从癌前病变、鳞癌形成、淋巴结转移过程中不同阶段的DTC，通过全基因组的单核苷酸多态性分析，检查点基因检测等，与正常口腔粘膜、原发灶、转移灶细胞比较，确定并比较它们的生物学年龄差异以及基因改变的异同，以确定DTC的来源，与原发灶、转移灶细胞的彼此间进化关系，以及播散细胞在转移地有否自主进化、进化停滞等现象的基因学证据，并探索淋巴结转移癌来源于早期还是晚期DTC，或者是随机的；了解转移癌的独特基因突变，初步判定导致进化停滞、转移癌形成的基因

Many research evidence increasingly indicates that single tumor cells spread to distant sites much earlier than previously believed. Single tumor cells start to spread much earlier than expected and form single-cell metastases. Such single-cell metastases remain dormant for long periods of time. We developed a 4-nitroquinoline-1-oxide (4NQO)-treated mouse model of oral squamous cell carcinoma that displayed both high-incident rates of primary cancer and spontaneous lymph node metastasis and long time intervals（at least 8 weeks）between primary and metastasis tumor, which mimic human 'tumor dormancy' perfectly. We also found disseminated tumor cell（DTC）in very early stage of oral cancer. In this rearch, the presence of DTC in bone marrow and metastatic tumor in the enlarged lymph nodes will be confirmed via the detection of tumor infiltration by cytokeratin(CK) positivity by immunostaining and the isolation of tumor cells by laser capture microdissection technology, at precancerous, primary tumor, and lymph node metastasis stage, respectively. After whole-genome amplified DNA，check point gene detection by PCR and Genome-wide detection by single-nucleotide polymorphism array, the biological age and genetically change of the primary tumor, DTC, normal tissue, and metastasis cells will be compared to confirm the origin of DTC, and the evolutionary relationship between each other. All these will further confirm the evidence of tumor cell parallel evolution, early dissemination but late metastasis, and the lymph node metastasis origin from early or late stage DTC. Besides, we will explore and evaluate the unique gene mutation of lymph node metastasis by SNP data analysis.