Anti-H2AFX monoclonal antibody, clone 049DU6.2.2

Purpose The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect. Materials and methods Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using 'medium transfer' and 'cell-to-cell contact' strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed. Results Various parameters (alpha/beta, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 +/- 0.06 in HeLa and 0.5 +/- 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes (Xrcc6 and H2afx) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed. Conclusions Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.

Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.