| Objective: Based on previous research results, in this study we hypothesized that the abscopal effect of high intensity focused ultrasound (HIFU) would correlate with the increased apoptosis of tumor cells and the activation of cytolytic T-lymphocyte cytotoxicity mediated through perforin (Pf) - granzyme B (GzB) and Fas ligand (FasL) pathways. The purpose of this study was to observe the changes in both apoptotic tumor cell and lymphocyte cytotoxicity at primary cancer and axillary lymph node after HIFU treatment for breast cancer, and to explore the effect of HIFU on apoptosis of tumor cells, especially in the abscopal apoptosis, and the mechanism of the abscopal effect induced by HIFU.Methods: A total of 48 female patients with biopsy-proven breast cancer were divided into HIFU group and control group. Among them, 25 patients received modified radical mastectomy alone (control group), and the remaining 23 patients underwent HIFU treatment 1-2 weeks before surgery (HIFU group). After the operation, the specimens of both primary cancer and axillary lymph node were collected, fixed in 10% neutralbuffered formalin, embedded in paraffin, and cut into 0.5μm thick slices. Using HE staining, histological changes were observed; terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) and SP immunohistochemical stain were used to assess the apoptotic tumor cells and the Pf, GzB, and FasL expressions of cytolytic T-lymphocyte at primary cancer and axillary lymph node in both groups, respectively. The data obtained in both groups were analyzed by using Student t test, product moment correlation, and multiple regression analysis.Results: (1) HIFU caused the coagulative necrosis of primary cancer, and no apoptotic tumor cells were detected in HIFU group; but the number of apoptotic tumor cells was 1.10±0.76/HPF in control group. The number of apoptotic tumor cells at axillary lymph node was 0.90±0.74/HPF in control group, and 2.66±0.57/HPF in HIFU group, respectively. There was a significant difference of apoptotic tumor cells between two groups (P<0.001). (2) The number of positive cells of FasL, GzB, and Pf in primary cancer was 29.6±12.3/mm~2, 32.2±11.3/mm~2, and 12.2±8.2/mm~2 in HIFU group, respectively; while in control group the number was 21.2±9.8/mm~2, 17.5±6.3/mm~2, and 6.8±5.2/mm~2, respectively. The positive cells of FasL, GzB, and Pf were significantly more in HIFU group than those in control group (P<0.05). (3) The number of positive cells of FasL, GzB, and Pf in metastasis-negativeaxillary lymph node was 71.1±36.0/mm~2, 70.5±31.2/mm~2, and 30.6±16.8/mm~2 in HIFU group, respectively; while in control group the number was 21.2±9.8/mm~2, 17.5±6.3/mm~2, and 6.8±5.2/mm~2, respectively. The number of FasL, GzB, and Pf positive cells were significantly higher in HIFU group than those in control group (P<0.05). (4) The number of positive cells of FasL, GzB, and Pf in metastasis-positive axillary lymph node was 55.9±15.8/mm~2, 52.0±15.5/mm~2, and 25.4±8.1/mm~2 in HIFU group, respectively; while in control group the number was 32.5±13.1/mm~2, 32.1±16.6/mm~2, and 12.2±5.4/mm~2, respectively. There were significant differences of these data between two groups (P<0.05). (5) The coefficient of product moment correlation between apoptotic tumor cells and FasL, GzB, and Pf was 0.967, 0.919, and 0.983 in metastasis-positive axillary lymph node, respectively. There was a significant positive-correlation between them (P<0.05); The linear equation analyzed by multiple regression analysis was as follows: the number of apoptotic tumor cells=0.012×FasL+0.010×GzB+0.032×Pf+0.700. There was a statistical significance of the multiple linear regression equation (P<0.05). Conclusions: (1) A significant increase of apoptotic tumor cells was detected at metastasis-positive axillary lymph node in HIFU group, indicating that there was a strong evidence of abscopal effect induced by HIFU treatment for primary breast cancer. (2) The positive cells of FasL,Gz... |
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