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Cytogenetic fingerprint of heavy ion exposure detected with mBAND in vitro and in prostate cancer patients treated with IMRT and C-ions

Identification of a chromosomal biomarker that allows to determine if an individual has been exposed to high or low LET radiation is a long-standing goal in radiation biology and several chromosome aberration (CA) types have been proposed as fingerprints. Low LET radiation is characterized by a homogenous distribution of the ionization events in the target, while for high LET exposure the energy is released along the particle track resulting in an inhomogeneous ionization pattern. Based on this difference in the energy deposition it has been suggested, for example, that high LET radiation induces a higher rate of intrachromosomal exchanges in comparison to sparsely ionizing radiation. In other words the likelihood to hit several regions belonging to the same chromosome domain is higher when ionization events are densely distributed. In vitro studies have partially confirmed this hypothesis, showing that complex intrachanges (CAs arising from 3 or more breaks and where at least two of them occur on the hybridized chromosome) are more common after exposure to heavy ions than after exposure to sparsely ionizing radiation, while the yield of simple intrachanges was not radiation quality dependent [1]. Yet, it is still not clear, if a similar pattern is observable after in vivo exposure. To address this question we measured CAs in peripheral blood lymphocytes (PBLs) from patients treated with photon IMRT (Intensity Modulated Radiation Therapy) alone or in combination with a C-ion boost. CAs were visualized by means of the mBAND technique, which allows the detection of both interand intrachromosomal aberrations. Two patient groups were selected for the cytogenetic investigation: prostate cancer patients treated with Photon-IMRT alone (IMRT*, 38x2 Gy, mean target volume 936 cm) and patients exposed at GSI to a C-ion boost (6x3 GyE) followed by IMRT (30x2 Gy, mean target volume 119 cm). PBLs were obtained at the end of the therapy treatment from 2 patients per group. Chromosome spreads were collected 48h after stimulation in vitro following standard procedures. Chromosome preparations were hybridized with the mBAND kit for chromosome 2 and images were captured using an Olympus microscope equipped with UV lamp and appropriate filter sets. Processing and image analysis were performed with the ISIS software (MetaSystems). For comparison, PBLs obtained from a healthy donor were exposed in vitro to X-rays (4 Gy) and to C-ions (2 Gy, 11.4 MeV/n). In total more than 2500 spreads were analysed. The frequency of aberrant cells in patients treated with IMRT alone (0.11±0.01) was significantly higher (p <0.01) than the rate observed in patients treated with the combined approach (0.018±0.003). This outcome can be explained by the different target volumes, i.e. in the IMRT* group the target volume was larger than in the group receiving C-ion boost and IMRT, resulting in a higher number of exposed lymphocytes and subsequently in a higher frequency of aberrations. This result confirms previous data obtained with the mFISH technique [2]. The yield of simple interchanges did not change significantly for any radiation quality, both from in vivo and from in vitro exposures (Fisher exact test p> 0.1). However, after in vitro exposure a higher (p< 0.05) incidence of complex intrachanges was found after high LET in comparison to low LET radiation in line with previous finding [1]. The frequency of simple intrachanges showed no radiation quality dependence (Fig.1).