Clinical Analysis Of Radiotherapy Effect And In Vitro Radiosensitivity Study For Triple Negative Breast Cancers

Part I:Analysis in triple negative breast cancer treated with mastectomy:patterns of failure and effect of postmastectomy radiotherapyPurpose Several studies have demonstrated poor locoregional control in patients with triple-negative breast cancer (TNBC), compared with other molecular subtypes of breast cancer. Frist, we sought to evaluate whether or not postmastectomy radiotherapy (PMRT) improves locoregional recurrence-free survival (LRFS) and disease-free survival (DFS) outcomes in TNBC patients. Second, we try to identify patterns of failure and prognostic factors for locoregional recurrence (LRR) that could justify postmastectomy radiotherapy after modified radical mastectomy in patients with early stage TNBC.Materials and methods BetweenJanuary2000and July2007,553TNBC patients treated with modified radical mastectomy (MRM) from a single institution were analyzed retrospectively. Patients were categorized into three groups:low risk (stage T1-T2N0), intermediate risk (stage T1-T2N1), and high risk (stage T3-T4and/or N2-N3). Also in mediate and low groups,390patients who underwent MRM without PMRT were analyzed. Cox proportional hazards models were used to evaluate the association between PMRT and LRFS and DFS times after adjusting for other clinicopathologic covariates.Results With a median follow-up of65months (range,1-140months),51patients (9.2%) developed locoregional recurrence and135patients (24.4%) experienced disease recurrence. On multivariate analysis, PMRT was associated with significantly longer LRFS and DFS times in the entire cohort. In the intermediate-risk group, PMRT was associated with a longer DFS time but not with the LRFS interval. In the high risk group, PMRT was associated with significantly longer LRFS and DFS times. In the mediate-low without PMRT group, multivariate analysis revealed age<50years, the presence of lymphovascular invasion, grade3tumor, and3involved lymph nodes were associated significantly with an increased risk of LRR. Patients who had2and more risk factors experienced a5-year LRR rate of>25%. Conclusions PMRT is associated with longer LRFS and DFS times in high-risk TNBC patients and a longer DFS time in intermediate-risk TNBC patients. For patient with early stage TNBC, PMRT should be considered for those patients who have2or more of risk factors.Prospective randomized studies areneededto investigate the best locoregional treatment approaches for patients with this molecular subtype of breast cancer. Part II:In vitro study of humn triple negative breast cancer cells’radiosensitivity Chapter I:Radiosensitivity and molecular mechanisms of human breast cancer cell lines with different expression of ERaPurpose Breast cancer patients with different molecular subtypes benefited variously from radiotherapy in terms of locoregional control. TNBC (ER-, PR-, and HER-2-) subtype had relatively higher locoregional recurrence rates compared to luminal A subtype (ER+or/and PR+, HER-2-), indicating that radioresistance may play an important role in them. We hypothesized that ERa might count largely for these differences in radiosensitivity.Materials and methods Two breast cancer cell lines, MCF-7(ERa+, HER2-) and MDA-MB-231(ERa-, HER2-), which have different status of ERa, were tested by colony formation assay to show different radiosensitivity. ERa expression was restored in MDA-MB-231cell (ER231) by retrovirus-mediated transfer of an ERa-cDNA. The following assays were used to test the radiosensitivity between MDA-MB-231and ER231cells:colony formation assay, CCK-8cell viability assay, split-dose revovery assay, western blot assay, cell cycle analysis and cell apoptosis analysis.Results With colony formation assay, cells with TNBC cell MDA-MB-231showed higher resistance to ionizing radiation (IR) compared to those with MCF7cell. Using retrovirus-based system mediated by phoenix cells generated retrovirus, ERa expression was stably restored in MDA-MB-231. Overexpression of ERa inhibited cell proliferation and activity, and made MDA-MB-231more sensitive to IR. Split-dose recovery assays showed that ER231had lower capacity of sublethal damage repair after IR. An increase in G2-M cell cycle arrest was seen in ER231cells with compared with MDA-MB-231cells after IR. Additionally, the autophagy was suppressed in ER231cells, while the apoptosis was increased in ER231cells when compared to231cells.Conclusions We conclude that the radioresistance of TNBC cells MDA-MB-231was attenuated with the overexpression ERa. Chapter Ⅱ:Radiosensitization of human triple negative breast cancer cell lines by PARP1inhibitor PJ34Purpose Poly (ADP-ribose) polymerase-1(PARP-1) inhibitor has been shown as radiosensitisers in various cell lines. Based on our previous findings, triple negative breast cancer cells were relatively radioresistant and their enhanced ability to repair DNA damage might account a lot for the radioresistance. Since PARP-1plays a critical role in DNA repair, we investigated the effect of PJ34, a third generation of PARP-1inhibitor, on radiosensitizing triple negative breast cancer cell linesMaterials and methods Triple negative breast cancer cell lines MDA-MB-231was treated with3μM or10μM PJ34before irradiation and tested for clonogenic survival to determine their radiosensitive effect. Gamma-H2AX foci using immunofluorescence microscopy was carried out to test their capacity for repairing DNA double strand breaks (DSB). Split-dose recovery ability, cell cycle distribution and cell apoptosis were also explored.Results PJ34sensitized MDA-MB-231cells to radiation in colony formation assays. Results from y-H2AX foci assay and split-dose recovery assay showed that PJ34had a strong inhibitory effect on the repair of DSBs and attenuated cell recovery from sublethal damage after radiation. An increase in G2-M cell cycle arrest was seen in combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study.Conclusions Overall, we conclude that PJ34enhances the radioresponse of triple negative breast cancer cells by increasing G2/M cell cycle arrest, suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced double strand breaks.