γ-H2AX level in peripheral blood lymphocytes as a risk predictor for bladder cancer

Abstract

Identification of susceptibility to double-strand breaks (DSBs) may provide valuable information about individual bladder cancer (BC) risk. The formation of γ-H2AX foci is a highly sensitive marker for DNA DSBs induction. We assessed whether levels of γ-H2AX in peripheral blood lymphocytes (PBL) obtained after stimulation by ionizing radiation (IR) are able to predict BC risk. Patients were enrolled from an ongoing BC case-control study. Baseline- and IR-induced H2AX phosphorylation was assessed in PBL from 174 newly diagnosed and untreated BC patients and from 174 matched control subjects by a novel, image-based, high-throughput phenotypic assay. The ratio of γ-H2AX level of IR-treated cells to that of non-treated cells (baseline) was used as the parameter to assess the sensitivity to the mutagen. The mean γ-H2AX ratios were significantly higher for cases than for controls (1.43±0.14 versus 1.35±0.12; P = 8.45×10(-8)). This trend was irrespective of age, sex and smoking status. The risk estimates of BC for induced DSBs by tertile distributions in controls showed also a significant trend for increased risk at the highest tertile for the whole cohort (odds ratio = 5.16; 95% confidence interval = 2.69, 9.89; P = 7.78 × 10(-7)) as well as for each category. Our findings suggest that a higher susceptibility to induction of DSBs as measured by the γ-H2AX assay is significantly associated with an increased risk for BC. This might help to identify individuals at high risk for this cancer, adding new perspectives to established epidemiological and genetic risk factors. Further research of the role of γ-H2AX in biological processes of BC is warranted.

Fig. 1.

Fluorescein γ-H2AX signals in PBLs as measured using the standard filter settings with 488 nm argon laser. Nuclei were counterstained with propidium iodide. (A) Low-intensity staining (baseline). (B) High-intensity staining (after γ-radiation).

Fig. 2.

Dot plot charts of γ-H2AX ratios according to host characteristics comparing cases and controls among (A) overall subjects; (B) subjects age ≤66 years; (C) age >66 years; (D) males; (E) females; (F) never smokers and (G) ever smokers. Horizontal line with crosses indicates mean value with upper and lower dotted lines marking the upper and lower values of mean ± SD. The mean ± SD values for cases and controls and the P values for each comparison are shown.