## 4 Discussion
More importantly, the marked increase in the frequency of complex-type CAs (Figures 3 and 4) clearly highlights the greater effectiveness of BNCT-associated reaction products at causing complex DNA damage compared to X-rays in agreement with the prevalence of highly clustered damage, whose repair is error-prone [54]. It has also to be noted that our data confirm the underestimation of CA frequency by conventional WCP, which offers the advantage of a rapid assessment of DNA damage as it paints only a limited number of pairs of homologous chromosomes (in our case, chromosomes 1 and 2), compared to the much more expensive and time-consuming m-FISH (Figure 2). This is crucial as far as complex-type aberrations are concerned as they involve more chromosomes simultaneously. In fact, m-FISH allowed us to unveil that radiation-associated complex chromosome damage was of even greater magnitude than inferred by WCP alone in BNCT samples (Figure 3) owing to the scoring of the entire karyotype (Figure 5). Furthermore, m-FISH allowed us to quantify the staggering degree of damage complexity in terms of the mean number of chromosomes (Figure 6) and of breaks (Figure 7) per complex aberration in the BNCT compared to X-ray-irradiated samples. In line with previous work on bimodal therapeutic approaches [5], these data altogether convincingly support the role of the \({}^{10}\)B(n,\(\alpha\))\({}^{7}\)Li reaction as the key factor underlying the radiobiological effectiveness of BNCT. To our knowledge, this is the first study to show that the most likely cause of the therapeutic advantages granted by BNCT is the complexity of the chromosomal damage caused by the BNCT-generated high-LET particles [54]. This damage is prone to misrepair,thereby increasing cell lethality; work by [PERSON] et al. [55] has, in fact, shown a greater occurrence of dicentrics in metaphases from cells subjected to the BNCT mixed radiation field at various doses, including 0.5 and 2 Gy as used in our study, although they report a lower aberration frequency compared to ours. Indeed, scoring of CA in mitotic cells can lead to underestimation in the case of high-LET exposure as the most heavily damaged cells will incur longer cell-cycle delays, which the use of calyculin A-induced Premature Chromosome Condensation (PCC) circumvents [56]. Moreover, it is true that dicentrics are a well-known lethal aberration as they lead to mitotic failure and their frequency well correlates with loss of clonogenicity [57]. However, dicentrics, unlike complex-type CAs, are not a specific biomarker of high-LET exposure [36], which our data prove is mechanistically the reason for the therapeutic attractiveness of BNCT. Overcoming cancer radioresistance remains a priority in any RT scenario and several novel approaches are being explored via the combination of physics- and biology-based strategies [58], natural compound-based radiosensitization [59], or even by entirely new technological advances in hadrontherapy [60]. Our results confirm that BNCT is a truly promising option to treat radioresistancet tumors due to the elevated level of poorly repairable damage induced at the DNA level. Together with ongoing studies to identify the most effective routes in boron delivery to improve cancer cell-specific toxicity and the exciting perspectives due to the new generation of accelerator-based neutron production [8; 12; 61; 62], this study may contribute to moving BNCT closer to its adoption in clinical routine in other countries that may thus follow the example recently set by Japan [63].