Prediction of clinical toxicity in localized cervical carcinoma by radio-induced apoptosis study in peripheral blood lymphocytes (PBLs)

Abstract

Background: Cervical cancer is treated mainly by surgery and radiotherapy. Toxicity due to radiation is a limiting factor for treatment success. Determination of lymphocyte radiosensitivity by radio-induced apoptosis arises as a possible method for predictive test development. The aim of this study was to analyze radio-induced apoptosis of peripheral blood lymphocytes.

Methods: Ninety four consecutive patients suffering from cervical carcinoma, diagnosed and treated in our institution, and four healthy controls were included in the study. Toxicity was evaluated using the Lent-Soma scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24, 48 and 72 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide to determine early and late apoptosis. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody.

Results: Radiation-induced apoptosis (RIA) increased with radiation dose and time of incubation. Data strongly fitted to a semi logarithmic model as follows: RIA = betaln(Gy) + alpha. This mathematical model was defined by two constants: alpha, is the origin of the curve in the Y axis and determines the percentage of spontaneous cell death and beta, is the slope of the curve and determines the percentage of cell death induced at a determined radiation dose (beta = DeltaRIA/Deltaln(Gy)). Higher beta values (increased rate of RIA at given radiation doses) were observed in patients with low sexual toxicity (Exp(B) = 0.83, C.I. 95% (0.73-0.95), p = 0.007; Exp(B) = 0.88, C.I. 95% (0.82-0.94), p = 0.001; Exp(B) = 0.93, C.I. 95% (0.88-0.99), p = 0.026 for 24, 48 and 72 hours respectively). This relation was also found with rectal (Exp(B) = 0.89, C.I. 95% (0.81-0.98), p = 0.026; Exp(B) = 0.95, C.I. 95% (0.91-0.98), p = 0.013 for 48 and 72 hours respectively) and urinary (Exp(B) = 0.83, C.I. 95% (0.71-0.97), p = 0.021 for 24 hours) toxicity.

Conclusion: Radiation induced apoptosis at different time points and radiation doses fitted to a semi logarithmic model defined by a mathematical equation that gives an individual value of radiosensitivity and could predict late toxicity due to radiotherapy. Other prospective studies with higher number of patients are needed to validate these results.

Figure 1

Determination of apoptosis. Three different cell populations were detected after the Annexin V/PI staining of isolated PBLs. Alive cells grouped in the lower left part of the panel, early apoptotic cells grouped in the lower right part of the panel and late apoptotic cells grouped in the higher right part of the panel.

Figure 2

Radio-induced apoptosis (RIA) of lymphocytes after 24, 48 and 72 hours. RIA values at 1, 2 and 8 Gy were adjusted perfectly to a semi logarithmic model where two constants were defined: α is the origin of the curve in the Y axis and determines the percentage of spontaneous cell death and β is the slope of the curve and determines the percentage of cell death induced at a determined radiation dose (β = ΔRIA/Δln(Gy)).