In recent years, the fixed RBE value of 1.1 is being questioned with respect to its safety, because if the dose to the tumor is too low, the risk of tumor recurrence increases. On the other hand, if the dose is too high, the chances for acute and last side effects will increase. Disregarding this RBE and LET variations could have negative clinical implications, especially when an organ at risk is located near the distal end of a tumor [
35]. A fixed RBE during fractionated exposures disregards any effects due to the variation of dose per fraction and the total number of fractions delivered in relation to the LET. However, a number of recent in vitro studies have reported that the RBE within the SOBP is not constant and the RBE increases at the distal end of the SOBP. Table
1 summarizes these in vitro studies. The study of Britten et al., demonstrated that the RBE of the proton beam at certain depths is greater than 1.1 and therefore there is an increased potential for cell killing and normal tissue damage in the distal regions of the Bragg peak [
10]. Proton beam therapy has a higher LET rate, particularly toward the distal edge of the SOBP, compared with conventional X-ray radiation. An enhanced efficiency in the induction of cell inactivation can be measured at different positions along the SOBP [
49,
50]. Differences in the RBE which are depending on the position along the SOBP were reported in several studies. The study of Petrovic et al., found an increased killing ability at the SOBP distal edge, which was the consequence of increasing proton LET [
51]. Another study reported on the variation of the RBE with depth in the SOBP of the 76 MeV proton beams, where they found that, despite a homogeneous physical dose, the tumor cells at the distal end receives a higher biologically equivalent dose than at the proximal end [
16]. More recent, the study of Hojo et al., demonstrated that the RBE using an high-energy proton beam, differed according to the position on the SOBP in two human esophageal cancer cell lines with differing radiosensitivities [
52]. Also the number of unrepaired double-stranded DNA breaks, as assessed by the number of γ-H2AX foci assay 24 h after irradiation was higher for irradiation at the distal end of the SOBP. In a theoretical study of Carante and Ballarini, a biophysical model of radiation-induced cell death and chromosome aberrations called Biophysical Analysis of Cell death and chromosome Aberrations (BIANCA) was used in order to predict the cell death and the yield of dicentric chromosomes at different depth positions along a SOBP dose profile of therapeutic protons [
53]. These simulation data are consistent with the experimental cell survival data as reported in Chaudhary et al. [
11] and for both investigating endpoints an increased beam effectiveness was shown along the plateau, implying that the assumption of a constant RBE along a proton SOBP may be sub-optimal [
53]. The results of an ex vivo study, where the intestine of mice was irradiated with 200 MeV clinical proton beam are consistent with in vitro data showing an increased proton RBE with depth in an SOBP for both investigated biological endpoints, the intestinal crypt regeneration and lethal dose 50% (LD
50) [
54]. The study of Marshall et al. have analyzed clinical implications of a variable RBE on proton dose fractionation in human skin fibroblast (AG01522) cells using pencil scanned proton clinical beam of maximum energy 219.65 MeV. Their findings have shown significant variations in the cell killing RBE for both acute and fractionated exposures along the proton dose profile, with a sharp increase in RBE toward the distal position [
55]. The study of Chaudhary et al. used the same cell line and investigated the DNA damage response after irradiation with a modulated SOBP and a pristine proton beam, as this new delivery technique was applied in form of intensity-modulated particle therapy (IMPT) in more and more proton therapy centers worldwide [
56]. A significantly higher frequency of persistent DNA damage foci was observed at the distal end of the SOBP, whereas the irradiation with a monoenergetic proton beam resulted in significantly increased number of foci at Bragg peak position 24 h after irradiation [
56]. In the study of Guan et al. clonogenic cell survival has been mapped as a function of LET along pristine scanned proton beam and the findings indicated that the measured biological effects are greater than reported in previous studies [
57]. Furthermore a non-linear RBE for cell survival as a function of LET near and beyond the Bragg peak was observed in this study.
Calugaru et al., 2011 [ 16] | Human cervix cancer cells HeLa/Head and neck squamous cancer cells SQ20B | Cell survival SF = 0.37 | 76 201 | 3 20 | 1.07/1.09 (entrance), 1.14/1.17 (mid-SOBP), 1.33/1.30 (distal) No variation with depth along SOBP for 201-MeV energy beam | 137Cs γ-rays |
Wouters et al., 2015 [ 24] | Chinese hamster cells V-79 | Cell survival A) SF = 0.34 B) SF = 0.71 | 160 230 | 10 | A) 1.07 (entrance), 1.10 (prox. half), 1.17 (distal half) and 1.21 (distal edge) Similar effects also for 230 MeV beam B) 1.13 (entrance), 1.15 (prox. half), 1.26 (distal half), 1.30 (distal edge) | 60Co γ-rays |
| U2OS | DNA damage repair A) 3 h B) 24 h | 152 | 10 | RBE increases as a function of depth along the Bragg peak A) RBE > 2 (entrance), RBE > 4.0 (distal) B) RBE > 2 (entrance), RBE > 6.0 (distal) | 6 MV X-rays |
Britten et al., 2013 [ 10] | Human laryngeal cancer cells Hep2/ Chinese hamster cells V79 | Cell survival SF = 0.10 | 87 200 | | 1.46/1.23 (mid), 2.1/1.46 (distal), 2.3/1.78 (dose fall-off) Similar D0.1 isoeffect RBE values as for 200 MeV proton beam irradiation | 60Co γ-rays |
Chaudhary et al., 2014 [ 11] | Human fibroblasts AG01522 and glioma cells U87 | Cell survival | 62 | | RBE increases for both cell lines and SF = 0.50, SF = 0.10 and SF = 0.01 as a function of depth of the SOBP | 225 kVp X-rays |
Matsumoto et al., 2014 [ 13] | Human salivary gland tumor cells HSG | Cell survival A) SF = 0.10 B) SF = 0.60 | 190 | 5 | A) 1.24 (150 mm- middle), 1.5 (180 mm - distal) B) 1.20 (150 mm- middle), 1.86 (180 mm - distal) | 6 MV X-rays |
Bettega et al., 2000 [ 50] | Human squamous cell carcinoma of the tongue SCC25 | Cell survival SF = 0.10 | 65 | | 0.99 (2 mm) – entrance 1.04 (15.6 mm) and 1.22 (25 mm) – in the SOBP 1.34 (27.2 mm) and 1.98 (27.8 mm) – distal declining edge | 60Co γ-rays |
Petrovic et al., 2010 [ 51] | HTB140 melanoma | Cell survival | 62 | | 1.68–2.84 at the distal end of SOBP 7.14 at its distal declining edge | Middle of the SOBP |
| Human esophageal cancer cell lines OE21/KYSE450 | Cell survival A) SF = 0.10 B) SF = 0.37 | 235 | | A) 1.06/1.03 (entrance), 1.17/1.06 (proximal), 1.22/1.20 (middle), 1.24/1.24 (distal) B) 1.16/1.02 (entrance), 1.33/1.09 (proximal), 1.31/1.21 (middle), 1.40/1.27 (distal) | 6 MV X-rays |
Slabbert et al., 2015 [ 54] | Ex vivo murine jejunum | Regeneration of intestinal crypts | 200 | A) 3 B) 7 | A) RBE increase of 5% ± 3% from the middle to the intermediate position, and an RBE increase of 9% ± 4% from the middle to the end of the SOBP B) RBE increase of 10% ± 4% from the middle to the end of the SOBP | 60Co γ-rays |
Marshall et al., 2016 [ 55] | Human skin fibroblasts AG01522 | Cell survival as a function of total dose delivered in a single (A) and triple exposure (B) SF = 0.10 | 219.65 | | A: 1.02 (entrance), 1.13 (proximal), 1.25 (center), 1.40 (distal) B: 1.11 (entrance), 1.31 (proximal), 1.40 (center), 2.01 (distal) | 225 kVp X-rays |
Chaudhary et al. 2016 [ 56] | Human skin fibroblasts AG01522 | DNA damage repair | 60 | Modulated SOBP and monoenergetic proton beam | Modulated SOBP: increased complexity of DNA lesions at the distal end of SOBP and slower repair kinetics Pristine beam: Significantly increased number of foci at Bragg peak position 24 h after irradiation | 225 kVp X-rays |
| Non-small cell lung cancer cells H460 and H1437 (p53 mutant) | Cell survival SF = 0.10 | 79.7 | Monoenergetic scanning beam with 4.8 cm range in water | Increased RBE at and beyond the Bragg peak. Non-linear relationship between RBE and LET for both cell lines, RBE scaled in a biphasic maner | 137Cs γ-rays |