Explore The New Method Of Preoperative Diagnosis Of Axillary Lymph Nodes In Breast Cancer Patients And Study On The Function Of CD8~+T Cells And The Relationship Between Its PD-1 Expression And The Clinical Stage

Breast cancer is a common malignant tumor in women,its prevalence rate ranks the first place among the female malignant tumors in China.The treatment of breast cancer mainly depends on the tumor type and the tumor stage.Whether breast cancer metastases to axillary lymph nodes or not directly defines the disease stage and is also an important basis for determining the treatment approach.This study focused on the samples of axillary lymph node fine-needle aspirate eluent from breast cancer patients,aiming to find a new auxiliary detection method to improve the preoperative diagnosis efficiency of suspicious lymph node.The analysis of the surface PD-1 expression and the intracellular factor expression of CD8⁺T cells helps to provide the basis for evaluating the immune response.This study also explored a new indicator to assess the severity of breast cancer.The lymph node fine-needle aspirate eluent and blood samples of the patients with initial breast cancer were collected from Ruijin hospital affiliated to Shanghai Jiao Tong University School of Medicine.First,a new analytical method,called“ratio method”（detection value of tumor markers in eluent/detection value of tumor markers in serum）,was established to diagnose the suspicious axillary lymph nodes of breast cancer patients.The levels of carcinoembryonic antigen（CEA）,carbohydrate antigen 15-3（CA15-3）and cytokeratin 19 fragment（CYFRA21-1）in serum and puncture eluent were measured respectively to evaluate the diagnostic value of tumor markers in metastatic lymph nodes of breast cancer;and to explore the best combined diagnosis method with ultrasound-guided fine-needle aspiration cytology（US-FNAC）.Second,the proportion of CD8⁺T cells,the distribution pattern of CD8⁺T subsets,the surface PD-1 expression,and the intracellular IFN-γ,TNF-αand Granzyme B expression of CD8⁺T cells were analyzed by flow cytometry to reflect the function of CD8⁺T cells in metastatic or negative lymph nodes.The relationship between the expression level of PD-1 on the surface of CD8⁺T cells in metastatic nodes and the pathological parameters of breast cancer patients was analyzed to explore whether PD-1 in metastatic lymph node puncture eluent could be a reference marker for evaluating the severity of breast cancer.The frist part results showed as follows:（1）The sensitivity,specificity and accuracy of US-FNAC for the diagnosis of lymph nodes metastasis of breast cancer were 83.7%,100%and 88.4%,respectively.（2）The detection rates of CEA,CA15-3and CYFRA21-1 from serum samples for the diagnosis of lymph nodes metastasis were17.4%,21.1%and 47.7%,respectively.（3）The sensitivity of CEA,CA15-3 and CYFRA21-1 detected in eluent for the diagnosis of lymph nodes metastasis in breast cancer were 50.1%,44.8%and 95.3%,respectively,with specificity of 92.9%,91.4%and 92.9%,and accuracy of 62.8%,58.3%and 94.6%,respectively.（4）The sensitivity of the ratio method of CEA,CA15-3 and CYFRA21-1 in the diagnosis of lymph nodes metastasis in breast cancer were 47.7%,40.1%and 94.2%,respectively,with specificity of 100%all and accuracy of 62.8%,57.4%and 95.9%,respectively.（5）The results of US-FNAC combined with the above methods showed that the detection efficiency of CYFRA21-1 by the combination ratio method,was optimized which could improve the detection sensitivity to 97.1%,specificity to 100%and accuracy to97.9%.The second part results showed as follows:（1）Compared to the negative lymph node group,the percentage of T_Naive cells in CD8⁺T cells in the metastatic axillary node group was much lower,the percent of T_EM cells was much higher,and no significant difference was found in T_CMcells and T_EMRAcells.（2）Compared to the negative lymph node,the PD-1 expression on the surface of CD8⁺T cells and its intracellular IFN-γand TNF-αexpression in metastatic lymph node were increased.And the expression of IFN-γ,TNF-αand Granzyme B in PD-1⁺CD8⁺T cells was decreased,but the expression of these factors in PD-1^-CD8⁺T cells was increased.（3）The expression level of PD-1on the surface of CD8⁺T cells in metastatic lymph node is positive related to the TNM stage of breast cancer and the amounts of metastatic lymph nodes.We found that US-FNAC eluent is a novel source of specimens.The tumor marker CYFRA21-1 was better than CEA and CA15-3 for diagnosing breast cancer.The combination of CYFRA21-1 ratio method and combined with US-FNAC could greatly improve the diagnostic sensitivity of lymph node metastasis without methodological specificity lost.The method could reduce the sentinel lymph node biopsy rate in breast cancer patients.Using flow cytometry to analyze lymphocyte population in eluent,we found that CD8⁺T subsets in metastatic lymph nodes were differentiated from T_Naive to T_EM cells and its function was enhanced,which revealed that the function of CD8⁺T cells in metastatic lymph nodes was changed.The proportion of PD-1⁺CD8⁺T cells was increased and their function was decreased in the metastatic lymph node group;the high expression of PD-1 on CD8⁺T cell surface was associated with the increased stage level of breast cancer and number of metastatic lymph nodes.These data suggested that the proportion of PD-1 expression on the surface of CD8⁺T cells in metastatic lymph node eluent could be used as a reference marker to evaluate the severity of breast cancer patients.To sum up,this study provided new ideas for diagnosis of breast cancer lymph metastatic and targeted therapy of breast cancer.