Biological effectiveness of high-energy proton transmission beams: <i>in vitro</i> evaluation of cell survival and viability

OBJECTIVE: Clinical proton therapy assumes a constant relative biological effectiveness (RBE) of 1.1, although proton RBE varies with linear energy transfer (LET). High-energy transmission beams (TBs) deposit dose entirely within the low-LET entrance plateau, suggesting a potentially distinct radiobiological behavior compared with conventional spread-out Bragg peaks (SOBP). APPROACH: We performed in vitro clonogenic survival experiments with four cell lines (A431, CRL2189, V79, A253) irradiated with TB, SOBP, mixed fields (SOBP+TB), and reference photons. Early post-irradiation metabolic viability was also assessed using the MTT assay. MAIN RESULTS: TB irradiation produced RBE values close to unity at 2 Gy (median 1.04-1.09), comparable to photons, whereas SOBP beams showed higher RBE values (≈1.10-1.35). In the tested 40% TB / 60% SOBP configuration, the mixed-field response remained biologically indistinguishable from conventional SOBP and was well described by the prediction of the theory of dual radiation action (TDRA) derived from the independently measured TB and SOBP survival parameters. MTT viability measurements showed the same overall hierarchy, with TB responses close to photons and stronger effects observed for SOBP and SOBP+TB. SIGNIFICANCE: High-energy proton TBs operate in a near-photon radiobiological regime. A mixed field containing 40% TB remained biologically indistinguishable from conventional SOBP and was well described by the TDRA. Complementary viability measurements showed the same modality-dependent pattern.