| Objective: (1) To observe the number changes and distribution of tumor-infiltrating lymphocytes (TIL) in treated tumor after in situ thermal ablation of high intensity focused ultrasound (HIFU) for patients with breast cancer. (2) To study both number and phenotype changes of CD3+, CD4+, CD8+, CD20+, and CD57+ in axillary lymph nodes after in situ thermal ablation of HIFU for patients with breast cancer. (3) To explore early changes of peripheral blood various lymphocyte and T subsets in patients with malignant tumor treated by HIFU.Methods: (1) A total of 48 female patients with breast cancer were pathologically diagnosed with needle biopsy. Among them, 23 patients with breast cancer underwent HIFU treatment one to two weeks before modified radical mastectomy (HIFU group), 25 patients only underwent modifiedradical mastectomy (control group) without any intervation. After the operation, the specimens of both tumor lesions and axillary lymph node were collected, fixed in 10% formalin, embedded in paraffin, and cut into 0.5 u m-thick slices. ㊣n the local region of breast cancer: By using HE staining technique, tumor-infiltrating lymphocytes were calculated under light microscope in control group and in HIFU group, respectively. By using SP immunohistochemical staining technique and double staining technique, CD3+, CD3++HLA-DR+, CD4+, CD8+, CD20+, and CD57+ cells were also counted in both groups. (2) Within axillary lymph nodes: The immunomorphologic patterns were histologically studied according to the Cottier's system and the Di Giorgio's standard. CD3+, CD3+HLA-DR+, CD4+, CD8+, and CD20+ cells were counted in paracortical area. CD3+ CD20+ and lymphocytes were counted in medullary cords. CD57+ lymphocytes were randomly counted in the entire lymph nodes. (2) The samples of peripheral blood were collected in sixteen patients with malignant tumor before and 1 to 2 weeks after HIFU treatment. Then the percentage of various lymphocytes and T subsets including CD3+, CD4+, CD8+, CD19+, CD (16++56+), and the ratio of CD4/CD8 were analyzed with FCM by using a direct immunofluorescent staining method. All observed data were shown as means plus or minus the standard deviation. Thedifferences in statistical and percentage data of two groups were analyzed by using Student t test and Fisher exact test, respectively.Results: (1) In local region of breast cancer: Obvious lymphocytic infiltration was frequently observed in HIFU group compared with control group. The average number of CD3+, CD3+HLA-DR+, CD4+, CD8+, CD20+, and CD57+ cells is significantly higher in FflFU group than in control group (PO.01). The ratio of CD4/CD8 was significantly elevated in HIFU group (P<0.01). (2) In axillary lymph nodes, the immunomorphologic pattern in HIFU group mostly shows mixtured high-grade cellular and humoral response compared with control group. The CD3+, CD3+HLADR+, and CD4+ lymphocytes in paracortical area, and CD57+ cells are significantly higher in HIFU group than in control group (P<0.01). CD3+ cells and CD20+ cells in medullary cords are significantly higher in HIFU group than in control group. (3) After HIFU therapy, the percentage of CD4+ and the ratio of CD4/CD8 were all elevated. There is a significant difference before and after HIFU treatment.Conclusions: (1) Increased numbers of TIL and activated T lymphocyte are obviously observed within treated breast cancer in HIFU group, compared with control group. This changes indicated that after HIFU treatment specific and nonspecific anti-cancer immune responsesignificantly enhanced in the treated region. (2) The rate of immune response in axillary lymph node significantly increased in HIFU group, and almost of them show mixtured high-grade cellular and humoral response. Increased numbers of CD3+, CD4+, CD8+, CD20+, CD57+ and activated CD3+ cells are seen in HIFU group, indicating that anti-cancer immune response significantly enhanced in the TDLN after HIFU treatment. (3) Peripheral blood CD4+ level and CD4/CD8 ration are significantly elevated, i...
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