| Objective:To research the young breast cancer patients (?35 years) resection margin status of breast-conserving surgery for the effect of clinical treatment and prognosis. Detect the expression of the Indoleamine 2,3-Dioxygenase(IDO), regulatory T cells (Tregs) and CD 105 in the resection margin of breast-conserving surgery, then analyze the immune microenvironment of the breast-conserving surgery cutting edge, study the effect of IDO on the prognosis of breast conservation. Further more study the role of IDO, Tregs and microvessel density(MVD) in tumor metastasis, and analyze the clinical significance of the breast-conserving immune microenvironment state in patients.Methods:1.Retrospective analysis the clinical and pathological data of 71 cases of breast cancer patients in Tianjin Medical University Cancer Hospital 1995.1?2006.12 who received the breast-conserving surgery therapy and 70 patients with modified radical mastectomy at the same period, compare the survival state of the two groups, and analyze the prognostic factors of young breast cancer with breast-conserving surgery.2. The expression of IDO, Tregs and CD 105 in cancer of breast-conserving surgery, resection margin and normal breast tissue were evaluated by immunohistochemical staining method, and analysis the correlation between the IDO and Tregs, CD105, detect the status of the immune microenvironment in breast-conserving surgery resection margin, then analyze the correlation between the expression of IDO and prognosis of young patients with breast-conserving surgery.3. Established CHO which was high expression of IDO gene by transfection. semi-quantitative RT-PCR and Western blot were used to detect the expression of IDO in transfected CHO. study the apoptosis of small spleen T cell after transfection the CHO by flow cytometry; using Transwell chamber, detect the effect of IDO on the ability of HUVEC into tube; animal studies to detect the expression of IDO in not transfer and the transfer of breast cancer in mice., study the detection of cancer infiltratior of CD4+CD25+CD127-T cells and the proportion by flow cytometry. Results:1. Young breast cancer patients who received breast-conserving surgery were followed up for 5 years, and found that 5 patients had local recurrence,8 patients had distant metastasis,7 cases died,3-year survival rate was 94.4%,5-year survival rate was 78.9%, and overall survival was 90.1%; in breast cancer patients who received modified radical mastectomy,3 cases had local recurrence,6 cases had distant metastases,5 cases died,3-year survival rate was 95.7%,5-year survival rate was 82.9%, overall survival was 92.9%; it found no statistically significant difference in prognosis of young breast cancer patients with two different surgical (P all> 0.05).2. Analysis of prognostic factor in Young patients with breast conserving surgery,it found that positive margins had correlation with local control rate, disease-free survival and overall survival rate (P all<0.05); lymph node metastasis had correlation with disease-free survival and overall survival rate(P all<0.05).3. The expression of IDO, Foxp3 and CD 105 in cancer was higher than that among breast-conserving surgery resection margin and normal breast tissue, and it had significant differences, and the expression of peripheral tissues were also higher than among normal breast tissue, but it had no significant statistical difference (P> 0.05).4. The expression of IDO had positive correlation with Tregs and MVD-CD105 in breast cancer; the IDO expression in conserving surgery peripheral tissues and the distribution of Tregs was also positively correlated, but had no correlation with the expression of CD 105.5. The 5-year disease-free survival and overall survival in the IDO-positive group of breast cancer were lower than IDO-negative group (P<0.05), and the patients had distant metastasis in the IDO-positive group were more than IDO-negative group; and the 5-year disease-free survival and overall survival in the group with the IDO-positive group were lower than IDO-negative group in peripheral tissues, but it had no significant difference (P>0.05)6. IDO inhibitor 1-MT was used to cytological experiments, it found that IDO can promote the number of the formation of HUVEC with tube-like structure.7. There was an obvious cell apoptosis in the CD3+T cells after co-cultureing with CHO/IDO, and its apoptosis rate was (79.07±8.13)%,while the apoptosis rate of pIRES2-EGFP transfer group and normal CD3+T cells were respectively (59.80±11.46)% and (32.40±6.40)%,and it had statistically significant between the groups comparative (P<0.05).8.Immunohistochemistry results showed that the percentage of IDO+cells in primary [(12.40±5.15)%] and secondary [(14.28±6.46)%] cancer tissues of metastasis mouse were all higher than the cancer that didn't have metastasized [(3.16±1.63)%] (P<0.05).The Flow cytometer results showed that the percentage of CD4+CD25+ CD127-T cells in primary [(4.70±1.10)%] and secondary [(5.10±1.18)%] cancer tissues of metastasis mouse were all higher than the cancer that didn't have metastasized [(2.27±0.83)%] (P<0.05). The percentage of IDO+ cells was positively correlated with the percentage of CD4+CD25+CD127- T cells in breast cancer (r=0.475,P<0.05)Conclusion:1. Young breast cancer patients who received breast-conserving surgery or radical mastectomy had no significant difference prognosis, it suggested that age is not the basis for surgical selection. Margin status, axillary lymph node metastasis was the important prognostic factors in young breast cancer patients, only the negative margin on pathological can not accurately assess the the risk of the recurrence and metastasis with breast conserving therapy, it need to look for more indicators of molecular pathology as clinical reference.2. The expression levels of IDO in breast cancer tissue had correlation with clinical stage and axillary lymph node metastasis, the disease-free survival of IDO-positive group was higher than IDO-negative group, it suggested that the positive expression of IDO may be associated with breast cancer progression and metastasis.3. The expression of IDO, Foxp3 and CD 105 in cancer was higher than that among breast-conserving surgery resection margin and normal breast tissue, and it had significant differences, the expression of peripheral tissues were also higher than among normal breast tissue, which infer that the immune status in local tumor may be different. 4. The expression of IDO had positive correlation with Tregs and MVD-CD105 in breast cancer; the IDO expression in conserving surgery peripheral tissues and the distribution of Tregs was also positively correlated, it prompted that IDO may affect the immune status of local tumor by induce the Tregs and promote the local activation of tumor angiogenesis.5. In vitro experiments, IDO can facilitate the formation of HUVEC tube-like structure,T cell proliferation and induced its apoptosis, it demonstrate that in the mechanisms of immune tolerance which IDO induced in tumors may include the accelerated formation of new blood vessels and lead to T-cell apoptosis.6. The percentage of IDO+ cells and CD4+CD25+CD127-T cells in primary and secondary cancer of metastasis mouse were all higher than the tumor that didn't have metastasized. Meanwhile, the percentage of IDO+ cells was positively correlated with the percentage of CD4+CD25+CD127-T cells in breast cancer, which suggested that IDO maybe induced the differentiation of Tregs, then to mediate immune tolerance and promote cancer cells metastasis.In conclusion, this study analyzed the prognostic factors of the young breast cancer patients with breast-conserving surgery, and it promoted that the immune status of surgical margins may be have clinical value to assess the prognosis of patients and establishing the means of individual follow-up. It had a detailed analysis the expression of IDO, Tregs and CD 105 in breast-conserving surgery samples, and the relationship between them. To further validate our inference, we had made it in vitro experiments and animal studies. The results show that, IDO may affect local tumor immune microenvironment which affects the prognosis of breast cancer patients through a number of ways, such as promoting the formation of new blood vessels, leading to T cell apoptosis and cell differentiation induced Tregs. |
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