Spread-out Bragg peak FLASH: quantifying normal tissue toxicity in a murine model.

Objective: A favorable effect of ultra-high dose rate (FLASH) radiation on normal tissue-sparing has been indicated in several preclinical studies. In these studies, the adverse effects of radiation damage were reduced without compromising tumor control. Most studies of proton FLASH investigate these effects within the entrance of a proton beam. However, the real advantage of proton therapy lies in the Spread-out Bragg Peak (SOBP), which allows for giving a high dose to a target with a limited dose to healthy tissue at the entrance of the beam. Therefore, a clinically relevant investigation of the FLASH effect would be of healthy tissues within a SOBP. Our study quantified the tissue-sparing effect of FLASH radiation on acute and late toxicity within an SOBP in a murine model. Material/Methods: Radiation-induced damage was assessed for acute and late toxicity in the same mice following irradiation with FLASH (Field dose rate of 60 Gy/s) or conventional (CONV, 0.34 Gy/s) dose rates. The right hindleg of unanesthetized female CDF1 mice was irradiated with single-fraction doses between 19.9-49.7 Gy for CONV and 30.4-65.9 Gy for FLASH with 5-8 mice per dose. The leg was placed in the middle of a 5 cm SOBP generated from a mono-energetic beam using a 2D range modulator. Acute skin toxicity quantified by hair loss, moist desquamation and toe separation was monitored daily within 29 days post-treatment. Late toxicity of fibrotic development measured by leg extendibility was monitored biweekly until 30 weeks post-treatment. Results: Comparison of acute skin toxicity following radiation indicated a tissue-sparing effect of FLASH compared to conventional single-fraction radiation with a mean protection ratio of 1.40 (1.35-1.46). Fibrotic development similarly indicated normal tissue sparing with a 1.18 (1.17-1.18) protection ratio. The acute skin toxicity tissue sparing was similar to data from entrance-beam irradiations of Sørensen et al. (4). Conclusion: Full dose-response curves for acute and late toxicity after CONV and FLASH radiation were obtained. Radiation within the SOBP retains the normal-tissue-sparing effect of FLASH with a dose-modifying factor of 40% for acute skin damage and 18% for fibrotic development.

Proton FLASH: Impact of Dose Rate and Split Dose on Acute Skin Toxicity in a Murine Model.

PURPOSE: Preclinical studies have shown a preferential normal tissue sparing effect of FLASH radiation therapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biologic effect of varying dose rates, time structure, and introducing pauses in the dose delivery. METHODS AND MATERIALS: The right hind limbs of nonanaesthetized mice were irradiated in the entrance plateau of a pencil beam scanning proton beam with 39.3 Gy. Experiment 1 was with varying field dose rates (0.7-80 Gy/s) without repainting, experiment 2 was with varying field dose rates (0.37-80 Gy/s) with repainting, and in experiment 3, the dose was split into 2, 3, 4, or 6 identical deliveries with 2-minute pauses. In total, 320 mice were included, with 6 to 25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation. RESULTS: The dose rate50, which is the dose rate to induce a response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into 2 deliveries reduced the FLASH effect, and for 3 or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity. CONCLUSIONS: The dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where 2 or more FLASH fields overlap within the same treatment session.

Acute normal tissue responses in a murine model following fractionated irradiation of the head and neck with protons or X-rays.

BACKGROUND: The purpose of this study was to investigate acute normal tissue responses in the head and neck region following proton- or X-irradiation of a murine model. MATERIALS AND METHODS: Female C57BL/6J mice were irradiated with protons (25 or 60 MeV) or X-rays (100 kV). The radiation field covered the oral cavity and the major salivary glands. For protons, two different treatment plans were used, either with the Bragg Peak in the middle of the mouse (BP) or outside the mouse (transmission mode; TM). Delivered physical doses were 41, 45, and 65 Gy given in 6, 7, and 10 fractions for BP, TM, and X-rays, respectively. Alanine dosimetry was used to assess delivered doses. Oral mucositis and dermatitis were scored using CTC v.2.0-based tables. Saliva was collected at baseline, right after end of irradiation, and at day 35. RESULTS: The measured dose distribution for protons (TM) and X-rays was very similar. Oral mucositis appeared earlier, had a higher score and was found in a higher percentage of mice after proton irradiation compared to X-irradiation. Dermatitis, on the other hand, had a similar appearance after protons and X-rays. Compared to controls, saliva production was lower right after termination of proton- and X-irradiation. The BP group demonstrated saliva recovery compared to the TM and X-ray group at day 35. CONCLUSION: With lower delivered doses, proton irradiation resulted in similar skin reactions and increased oral mucositis compared to X-irradiation. This indicates that the relative biological effectiveness of protons for acute tissue responses in the mouse head and neck is greater than the clinical standard of 1.1. Thus, there is a need for further investigations of the biological effect of protons in normal tissues.

Kilovoltage X-ray beam quality effect on the relative response of alanine pellet dosemeters.

Determination of beam quality correction factors is crucial for performing accurate alanine pellet dosimetry in non-reference fields. For some complex irradiation geometries, interpolation from literature data is more convenient than an experimental approach to establish these factors. Here we investigate the validity of extracting quality correction factors from literature data based on information on beam qualifiers such as half-value layer (HVL) or effective energy ${E}_{\text{eff}}$. A combination of Monte Carlo calculated dose ratios and a microdosimetric assessment of the relative efficiency allows for numerical evaluation of quality correction factors for a wide array of X-ray qualities. The computational analysis demonstrates that the average energy of the X-ray beam is optimal for characterizing the relative response. Special care should be taken when using the common X-ray beam qualifiers HVL or ${E}_{\text{eff}}$ to determine quality correction factors from literature data.

Chitooligosaccharides Improve the Efficacy of Checkpoint Inhibitors in a Mouse Model of Lung Cancer.

YKL-40 (also named chitinase 3 like-1 protein [CHI3L1]) is a secreted chitinase-like protein which is upregulated in cancers and suggested to have pro-tumorigenic activity. YKL-40 lacks enzymatic function, but it can bind carbohydrates such as chitin. Chitooligosaccharides (COS) derived from deacetylation and hydrolysis of chitin might be used for the blockade of YKL-40 function. Here, public single-cell RNA sequencing datasets were used to elucidate the cellular source of YKL-40 gene expression in human tumors. Fibroblasts and myeloid cells were the primary sources of YKL-40. Screening of YKL-40 gene expression in syngeneic mouse cancer models showed the highest expression in the Lewis lung carcinoma (LL2) model. LL2 was used to investigate COS monotherapy and combinations with immune checkpoint inhibitors (anti-PD-L1 and anti-CTLA-4) (ICIs) and radiotherapy (8 Gy × 3) (RT). COS tended to reduce plasma YKL-40 levels, but it did not affect tumor growth. LL2 showed minimal responses to ICIs, or to RT alone. Interestingly, ICIs combined with COS led to delayed tumor growth. RT also enhanced the efficacy of ICIs; however, the addition of COS did not further delay the tumor growth. COS may exert their anti-tumorigenic effects through the inhibition of YKL-40, but additional functions of COS should be investigated.

Pencil beam scanning proton FLASH maintains tumor control while normal tissue damage is reduced in a mouse model.

PURPOSE: Preclinical studies indicate a normal tissue sparing effect when ultra-high dose rate (FLASH) radiation is used, while tumor response is maintained. This differential response has promising perspectives for improved clinical outcome. This study investigates tumor control and normal tissue toxicity of pencil beam scanning (PBS) proton FLASH in a mouse model. METHODS AND MATERIALS: Tumor bearing hind limbs of non-anaesthetized CDF1 mice were irradiated in a single fraction with a PBS proton beam using either conventional (CONV) dose rate (0.33-0.63 Gy/s field dose rate, 244 MeV) or FLASH (71-89 Gy/s field dose rate, 250 MeV). 162 mice with a C3H mouse mammary carcinoma subcutaneously implanted in the foot were irradiated with physical doses of 40-60 Gy (8-14 mice per dose point). The endpoints were tumor control (TC) assessed as no recurrent tumor at 90 days after treatment, the level of acute moist desquamation (MD) to the skin of the foot within 25 days post irradiation, and radiation induced fibrosis (RIF) within 24 weeks post irradiation. RESULTS: TCD50 (dose for 50% tumor control) was similar for CONV and FLASH with values (and 95% confidence intervals) of 49.1 (47.0-51.4) Gy for CONV and 51.3 (48.6-54.2) Gy for FLASH. RIF analysis was restricted to mice with tumor control. Both endpoints showed distinct normal tissue sparing effect of proton FLASH with MDD50 (dose for 50% of mice displaying moist desquamation) of <40.1 Gy for CONV and 52.3 (50.0-54.6) Gy for FLASH, (dose modifying factor at least 1.3) and FD50 (dose for 50% of mice displaying fibrosis) of 48.6 (43.2-50.8) Gy for CONV and 55.6 (52.5-60.1) Gy for FLASH (dose modifying factor of 1.14). CONCLUSIONS: FLASH had the same tumor control as CONV, but reduced normal tissue damage assessed as acute skin damage and radiation induced fibrosis.

Characterization of a Radiofluorogenic Polymer for Low-Energy Electron Beam Penetration Depth Visualization.

Low-energy (80-300 keV) electron beam accelerators are gaining in importance in the radiation processing industry due to their ease of use and wide range of applications (e.g. product surface sterilizations or polymer curing and cross-linking). Due to their very low penetration depth (tens to hundreds of microns), currently used film dosimeters exhibit dose gradients over their thickness and do not resolve the dose response in the first microns of the irradiated material. Hence, the surface dose, defined as the dose in the first micron Dµ, cannot be measured directly. This study presents a polymer material as a dosimeter candidate for high-dose low-energy electron beam irradiations. The readout of the dose-dependent fluorescence intensity, originating from a pararosaniline dye reaction when irradiated, is measured using fluorescence microscopy. So far, no in-depth characterization of the material has been performed, leaving the stability and fluorescence properties of the material not fully optimized. We describe the improvements in polymer composition and the fabrication method, and characterize the material properties in terms of the thermal stability, glass transition temperature, refractive index, hardness, rheological behavior, and water affinity. All of these create a complex set of requirements a polymer needs to fulfill to become an effective dosimeter when measuring using confocal microscopy. The fluorescence readout procedure will be addressed in further studies.

In vivo validation and tissue sparing factor for acute damage of pencil beam scanning proton FLASH.

BACKGROUND AND PURPOSE: Preclinical studies indicate a normal tissue sparing effect using ultra-high dose rate (FLASH) radiation with comparable tumor response. Most data so far are based on electron beams with limited utility for human treatments. This study validates the effect of proton FLASH delivered with pencil beam scanning (PBS) in a mouse leg model of acute skin damage and quantifies the normal tissue sparing factor, the FLASH factor, through full dose response curves. MATERIALS AND METHODS: The right hind limb of CDF1 mice was irradiated with a single fraction of proton PBS in the entrance plateau of either a 244 MeV conventional dose rate field or a 250 MeV FLASH field. In total, 301 mice were irradiated in four separate experiments, with 7-21 mice per dose point. The endpoints were the level of acute moist desquamation to the skin of the foot within 25 days post irradiation. RESULTS: The field duration and field dose rate were 61-107 s and 0.35-0.40 Gy/s for conventional dose rate and 0.35-0.73 s and 65-92 Gy/s for FLASH. Full dose response curves for five levels of acute skin damage for both conventional and FLASH dose rate revealed a distinct normal tissue sparing effect with FLASH: across all scoring levels, a 44-58% higher dose was required to give the same biological response with FLASH as compared to the conventional dose rate. CONCLUSIONS: The normal tissue sparing effect of PBS proton FLASH was validated. The FLASH factor was quantified through full dose response curves.

Homo erectus at Trinil on Java used shells for tool production and engraving.

The manufacture of geometric engravings is generally interpreted as indicative of modern cognition and behaviour. Key questions in the debate on the origin of such behaviour are whether this innovation is restricted to Homo sapiens, and whether it has a uniquely African origin. Here we report on a fossil freshwater shell assemblage from the Hauptknochenschicht ('main bone layer') of Trinil (Java, Indonesia), the type locality of Homo erectus discovered by Eugène Dubois in 1891 (refs 2 and 3). In the Dubois collection (in the Naturalis museum, Leiden, The Netherlands) we found evidence for freshwater shellfish consumption by hominins, one unambiguous shell tool, and a shell with a geometric engraving. We dated sediment contained in the shells with (40)Ar/(39)Ar and luminescence dating methods, obtaining a maximum age of 0.54 ± 0.10 million years and a minimum age of 0.43 ± 0.05 million years. This implies that the Trinil Hauptknochenschicht is younger than previously estimated. Together, our data indicate that the engraving was made by Homo erectus, and that it is considerably older than the oldest geometric engravings described so far. Although it is at present not possible to assess the function or meaning of the engraved shell, this discovery suggests that engraving abstract patterns was in the realm of Asian Homo erectus cognition and neuromotor control.