First Description of Alpha/Beta Values in Pediatric Medulloblastoma: Implications for Tailored Approaches in Radiation Oncology

First Description of Alpha/Beta Values in Pediatric Medulloblastoma: Implications for Tailored Approaches in Radiation Oncology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Description of Alpha/Beta Values in Pediatric Medulloblastoma: Implications for Tailored Approaches in Radiation Oncology Danny Jazmati, Dennis Sohn, Ronja-Linda Preugschas, Nan Qin, Edwin Bölke, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4707241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Radiation Oncology → Version 1 posted 6 You are reading this latest preprint version Abstract Background Medulloblastoma is the most common malignant pediatric brain tumor, typically treated with normofractionated craniospinal irradiation (CSI) with an additional boost over about 6 weeks in children older than 3 years [ 1 ]. This study investigates the sensitivity of pediatric medulloblastoma cell lines to different radiation fractionation schedules. While extensively studied in adult tumors, these ratios remain unknown in pediatric cases due to the rarity of the disease. Materials and Methods Five distinct medulloblastoma cell lines (ONS76, UW228-3, DAOY, D283, D425) were exposed to varying radiation doses and fractionation schemes. In addition, ONS76 and UW2283-3 stably overexpressing MYC were analyzed. Alpha/beta values, representing fractionation sensitivity, were quantified using the linear-quadratic model of radiation survival. Results The study unveiled elevated alpha/beta ratios across diverse medulloblastoma cell lines, with a weighted mean alpha/beta value of 11.01 Gy (CI: 5.23–16.79 Gy). Neither TP53 status nor the levels of MYC expression influenced fractionated radiosensitivity. Furthermore, differences cannot be correlated with molecular subgroups (p = 0,07). Conclusion These in vitro findings strongly recommend normofractionated or hyperfractionated radiotherapy for paediatric medulloblastoma cases due to consistently high Alpha/Beta values across subgroups. Conversely, hypofractionated radiotherapy is not advisable within a curative approach. This study presents significant potential by enabling the estimation of radiobiological fractionations and dose effects in young, vulnerable patients, highlighting its importance for advancing patient-specific therapeutic strategies. radiation therapy cancer paediatric tumor brain tumor Figures Figure 1 Figure 2 Figure 3 Introduction Medulloblastoma is the most common malignant pediatric brain tumor, impacting approximately 0.5 to 1 in every 100,000 children annually [ 2 ]. It is predominantly diagnosed in young patients, with a median age at onset of 5–6 years. This malignancy is characterized by its high propensity for metastasis along the neuroaxis. Additionally, approximately 30–40% of patients present with craniospinal fluid dissemination at initial diagnosis [ 3 ]. The World Health Organization classifies medulloblastoma into four distinct molecular subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4 [ 3 ]. These subgroups exhibit unique genetic alterations, demographic patterns, clinical behaviors, and prognostic outcomes, necessitating tailored therapeutic strategies for each subgroup[ 4 , 5 ]. Treatment typically involves surgical resection, radiotherapy, and chemotherapy. Patients are stratified into standard-risk and high-risk categories based on a combination of clinical, histopathological, and cytogenetic factors. High-risk patients typically undergo an intense radiotherapy protocol, consisting of 36 Gy of craniospinal irradiation (CSI) with an additional 18 Gy boost to the posterior fossa. In contrast, standard-risk patients are treated with a less intensive CSI protocol of 23.4 Gy, delivered in fractions of 1.8 Gy [ 6 ]. Despite the effectiveness of these treatment modalities, they often lead to severe long-term side effects, including cognitive, auditory, and endocrine dysfunctions [ 7 ]. These adverse effects significantly diminish the quality of life for survivors, underscoring the critical need for therapeutic strategies that balance efficacy with minimized long-term toxicities [ 8 ]. A promising approach to achieve this balance involves the reduction of the total radiation dose. Another strategy to modify the therapeutic window involves adjusting the dose per fraction[ 9 , 10 ]. However, determining the optimal fractionation scheme in medulloblastoma remains elusive due to our limited understanding of medulloblastoma cell responses to varying fractionation schemes. The linear-quadratic model, used to estimate clinical effects of different fractionation regimens, relies on the tissue's alpha/beta ratio [ 11 ]. Tumors with high alpha/beta ratios are typically treated with standard or hyperfractionated radiotherapy, while those with lower ratios may benefit from higher dose-per-fraction or hypofractionated radiotherapy. In this regard, an alpha / beta ratio is considered high if the value is above 8 Gy and low if it is below 5 Gy. A higher dose per fraction would lead to a shorter total radiation time, which could be particularly beneficial in radiation treatments for children, ultimately enhancing their quality of life during and after treatment. A comprehensive understanding of the alpha/beta ratio is crucial for optimizing the therapeutic ratio by amplifying the antitumor effect without increasing late effects. The clinical adoption of various fractionation approaches in medulloblastoma underscores a significant gap in our understanding of pediatric patients' fractionation response. In this study, we aim to present novel experimental data on the alpha-beta ratio of medulloblastoma cells, contributing to defining dose concepts for future clinical trials in this sensitive patient population. Materials and Methods Cell Line Cultivation and Conditions: The medulloblastoma cell lines ONS76, UW228-3, DAOY, D283 and D425, representing distinct TP53 and MYC alteration profiles (Table 1 ), were used in this study. In addition, previously generated ONS76 and UW228-3 cells stably expressing either mCherry as a control or MYC to mimic activation this oncogene, were employed (Table 2 ). ONS76, UW228-3 and DAOY cells were cultured in Dulbecco’s Modified Eagle's Medium containing GlutaMAX, 4.5 µg/l D-glucose and sodium pyruvate (DMEM, Gibco, Waltham, MA, USA), whereas the D283 and D425 cell lines were cultivated in Minimal Essential Medium containing GlutaMAX (MEM, Gibco) and modified Improved Minimal Essential Medium containing 2 mM L-glutamine (IMEM, Gibco), respectively. All media were supplemented with 10% fetal bovine serum (FBS Supreme, PAN-Biotech GmbH, Aldenbach, Germany). The cells were incubated at 37°C in a 5% CO2 atmosphere. Irradiations were performed with a Gulmay RS225 X-ray tube (Xstrahl GmbH, Ratingen, Germany) using 150 kV and 15 mA. Table 1 Alpha/beta values of medulloblastoma cell lines Cell line Subgroup TP53 MYC expr. α/β +/- CI [Gy] ONS76 SHH wild-type low 17.09 +/- 6.69 UW228-3 SHH mutated low 9.36 +/- 3.87 DAOY SHH mutated low 75.09 +/- 61.66 D283 Group 3/4 mutated high 15.09 +/- 16.77 D425 Group 3 mutated high 8.42 +/- 5.85 Table 2 Alpha/beta values of medulloblastoma cell lines genetically modified to overexpress MYC Cell line Stably expr. α/β +/- CI ONS76 mCherry 13.69 +/- 5.21 ONS76 MYC 10.93 +/- 5.54 UW228-3 mCherry 15.82 +/- 9.40 UW228-3 MYC 19.64 +/- 10.48 Well Control Dose Assay: The well control dose assay was adapted from experiments described previously[ 12 ]Cell lines were seeded in 24-well-plates. In one half of the plate a high cell number was seeded (2,000 cells for ONS76, UW228-3 and D425; 4,000 cells for DAOY and D283 cells), whereas 1/10 of this number was seeded in the other half (200 and 400 cells, respectively). To counter the impact of potential cellular cooperation effects, lethal one high dose-irradiated (20 Gy) feeder cells from the same cell line were added to the lower concentration wells to generate similar cell densities. After seeding, as soon as the cells became adherent, the fractionated irradiation was initiated. Over the span of four days, the samples were either irradiated twice a day (separated by 6–8 hours), once a day or once at the beginning and once at the end of the treatment schedule, resembling 8, 4 or 2 fractions, respectively. Depending on the individual radio-sensitivity of the used cell lines, each fractionation scheme featured 5–6 different doses ranging from 0.4–12 Gy single dose to 3.2–32 Gy total dose. One full 24-well-plate was used for each single irradiation and fractionation condition. Over a 60-day period, cell proliferation in each well was monitored every two to three days and scored binary for either ongoing proliferation leading to regrowth (i.e., the formation of a confluent cell monolayer) or absence of regrowth at the end of the timepoint. As an internal control for the Well Control Dose Assay, we employed the MCF-7 breast cancer cell line that was already reported to exhibit a low alpha/beta value far below < 8 Gy [ 13 ] which is also suggested to be generally the case for breast cancer [ 11 ]. Within our experimental setup, MCF-7 cells possess an alpha/beta ratio of 1.6 Gy +/- 1.2 (95% CI; data not shown), which is in concordance with the literature. Statistical Analysis and Radiobiological Modelling: The determined experimental survival probabilities were employed together with the -linear-quadratic model of radiation survival, p = e^ (-k * e^ (-n * (α ∗ d + β ∗ d^2) + γ ∗ t) (with p = probability of well control; k = number of clonogenic cells per well; n = number of fractions; α, β = radiation sensitivity parameters; d = single dose; γ = repopulation factor; t = overall treatment time), which was transformed to ‘p = e^ (-e^ (ln (k) – β * (α/β * D + D * d))‘ (with D = d * n, i.e. total dose) so that the α/β value could be determined directly by modelling. ‘γ ∗ t’ was omitted because the overall treatment time for all samples was the same. Using this formula, the determined experimental recurrence probabilities and the SPSS Statistics software (Version 29.0.0.0, IBM, Armonk, NY, USA), we performed a non-linear regression with the three parameters ‘ln(k)’, ‘β‘ and ‘alphabeta’. A maximum likelihood estimate was employed based on a classical logarithmic loss function for binary data and minimization of the negative log-likelihood. Additionally, Bootstrapping was performed to estimating confidence intervals (CI). Calculations were done with SPSS statistics software package. The weighted means of the estimated alpha/beta values were calculated for wildtype and genetically modified cells lines as well as for all cell lines by using the excel-plugin MetaXL (V5.3, EpiGear.com, EpiGear Ltd) employing the inverse variance heterogeneity method. Results The tables and charts reflect the reactions of all medulloblastoma cell lines to radiation, demonstrating the variability of radiobiological responses observed. Visualization of the determined survival curves together with the actually obtained recurrence data points demonstrates its high fidelity (Fig. 1 A-E, Fig. 2 A-D). This study revealed high alpha/beta ratios that are all above 8 Gy across a wide range of medulloblastoma cell lines, weighted mean alpha/beta value of 11.01 Gy (CI: 5.23–16.79 Gy) (Table 1 ).We were able to achieve relatively small confidence intervals. The one exception is the D283 cell line that characteristically grows in a semi-adherent manner, making it difficult to score the wells properly, which therefore resulted in high uncertainty and bigger CI of its determined alpha/beta ratio (Fig. 1 D). Interestingly, the TP53 genetic status had no influence on fractionation radiosensitivity (Table 1 , ONS76 vs all other cell lines). Although we analyzed a panel of distinct medulloblastoma cell lines, there was no relevant difference regarding low alpha/beta values detectable (p = 0,07) (Fig. 3 ). Furthermore, MYC expression also did not impact the fractionation sensitivity because neither in with aberrant baseline expression of MYC (Table 1 , D283/D425 vs all other cell lines) nor in cell lines genetically modified to overexpress MYC (Table 2 , mCherry vs MYC) in both a TP53-wild-type and a TP53-mutated setting a change from a high to a low alpha/beta value was observed (Fig. 3 ). This suggests that these genetic factors may not singularly govern the fractionation sensitivity profile of medulloblastoma. Discussion To the best of our knowledge, this study reports the first in vitro data of alpha-beta values in pediatric medulloblastoma patients. Normal and malignant tissues exhibit varying responses to fractionation, a phenomenon known as fractionation sensitivity [ 14 ]. This sensitivity is typically described using the α/β ratio. Our preclinical data provide evidence that medulloblastomas in children exhibit high alpha-beta values, indicating reduced sensitivity to fraction size. This is an observation that had been well-documented in adult head and neck malignancies, where administering radiotherapy in smaller fractions is known to protect late-responding normal tissues in comparison to the tumor [ 15 ]. In the context of medulloblastoma, analogous clinical observations have substantiated our findings. Hyperfractionated radiotherapy was implemented with the objective of maximizing the therapeutic dose to the tumor while simultaneously minimizing the risk for adverse effects on surrounding normal tissues. For low or standard-risk patients, where high cure rates are often achievable with current normofractionated concepts, the emphasis of hyperfractionated radiotherapy is on reducing long-term toxicities and thereby preserving neurological function and quality of life. In HR patients, where the prognosis is poorer and the risk of disease recurrence is higher, the aim shifts towards enhancing the efficacy of radiotherapy without disproportionately increasing toxicity. However, the clinical experiences to date have not definitively demonstrated a significant advantage for hyperfractionated radiotherapy over conventional radiotherapy in terms of survival outcomes. The HIT-SIOP PNET 4 trial (2001–2006) examined the role of hyperfractionation in comparison to conventional radiation therapy in the treatment of pediatric patients with medulloblastoma [ 16 ]. Despite the absence of a clear advantage in terms of 5-year event-free survival (78% for hyperfractionated vs. 77% for conventional) in the HIT-SIOP PNET 4 trial (2001–2006), hyperfractionated therapy did reveal a significant improvement in verbal intelligence quotient (VQ) among children under 8 years at diagnosis (mean intergroup difference 12.02, P = .02) and exhibited a noticeable trend toward enhanced processing speed (mean intergroup difference 10.90, P = .08) [ 17 ]. In the clinical application of hyperfractionated radiotherapy in young children, significant challenges exist, irrespective of radiobiological considerations [ 8 ]. For very young children, sedation for immobilization is necessary for treatment planning and delivery. The restrictions for both food and fluids before sedation pose substantial challenges for families. Although the feasibility of this approach has been demonstrated by the St. Jude Hospital group, the twice-daily sedation of young children remains a topic of controversy [ 18 ]. This approach requires substantial resources and its safety has not been conclusively established. Controversial results have been reported for metastatic medulloblastoma, previously. While the Italian group demonstrated promising outcomes with Hyperfractionated Accelerated RT (HART) combined with pre-RT chemotherapy and maintenance chemotherapy for metastatic medulloblastoma, achieving a 5-year event-free survival (EFS) of 70%, attempts by the British to replicate these results using the same approach resulted in a lower 3-year EFS of 59% [ 9 ]. These disparities may be attributed to the heterogeneous radiobiological behavior of metastatic tumors. Incorporating predictive molecular biomarkers for radiation sensitivity holds the potential to enhance the customization of radiotherapy to individualized conditions. Both the tumor suppressor TP53 as well as the proto-oncogene MYC have been described as important modulators of proliferation and cell survival after DNA damage[ 19 – 21 ]. However, irrespective on their impact on radiosensitivity, our results reveal comparable outcomes regarding the fractionation sensitivity marker alpha/beta across different sub-classifications, surprisingly regardless of the presence or absence of MYC overexpression and/or TP53 alterations. Therefore the consistency in radioresponse observed in our study suggests the potential feasibility of a standardized radiotherapeutic approach for medulloblastoma, irrespective of specific molecular profiles. While various cellular response mechanisms contributing to fractionation sensitivity have been identified in the past, the molecular biomarkers remain elusive. Our investigations heavily rely on in vitro assessments conducted on tumor cell lines and it is important to recognize that these controlled laboratory conditions may not comprehensively replicate the intricate microenvironment encountered in clinical settings. The inherent limitations of this approach lie in its inability to encompass the full spectrum of variables that influence radiation sensitivity within the complex context of real-world patient scenarios. However, our data can serve to support prior clinical observations and provide a foundation for establishing normo- to hyper-fractionated radiation protocols for young children with medulloblastoma, based on alpha-beta values. Nonetheless, we acknowledge that larger prospective randomized studies are highly desirable to further validate and refine these findings. Abbreviations CSI craniospinal irradiation DNA desoxyribonucleic acid EFS event- free survival Gy Gray RT radiotherapiy Declarations Ethical Approval and Consent to participate According to our ethics commission board, ethical approval was not deemed necessary for the irradiation of cell lines, as it is not a mandatory requirement. • Consent for publication: all authors gave consent for publication • Availability of supporting data: The supporting data can be made available by the first and last authors. • Competing interests/Authors' contributions: all authors have no competing interests Authors contributions: DJ, CM, DS, EB, WB wrote the main manuscript, EB, DS, DJ, CM, WB helped to design the study and wrote part of the manuscript, DS prepared the figures, WB, RP, NQ, JH, RS, NN, AB , FB, TB, MF, BT, PM, TB, BT, MR, SC did the literature research and prepared the data for analysis, all authors reviewed the manuscript. • Funding: Open Access funding enabled and organized by Projekt DEAL. 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Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Radiation Oncology → Version 1 posted Reviews received at journal 26 Jul, 2024 Reviewers agreed at journal 25 Jul, 2024 Reviewers invited by journal 25 Jul, 2024 Editor assigned by journal 11 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 08 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4707241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333666085,"identity":"dcbb1cb1-6ba7-4f72-830e-3ad00c655dbc","order_by":0,"name":"Danny Jazmati","email":"","orcid":"","institution":"Heinrich Heine University","correspondingAuthor":false,"prefix":"","firstName":"Danny","middleName":"","lastName":"Jazmati","suffix":""},{"id":333666086,"identity":"b36a6672-e828-408f-868f-c01e9a6b9fd6","order_by":1,"name":"Dennis Sohn","email":"","orcid":"","institution":"University Hospital Düsseldorf, Heinrich-Heine-University 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Beez","email":"","orcid":"","institution":"Heinrich-Heine-University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Beez","suffix":""},{"id":333666120,"identity":"9f337145-b704-434d-b645-d0bcb8f8669b","order_by":15,"name":"Beate Timmermann","email":"","orcid":"","institution":"University Hospital Essen","correspondingAuthor":false,"prefix":"","firstName":"Beate","middleName":"","lastName":"Timmermann","suffix":""},{"id":333666121,"identity":"f294976b-df76-4a54-865b-499b62bdeaed","order_by":16,"name":"Marc Remke","email":"","orcid":"","institution":"Saarland University","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Remke","suffix":""},{"id":333666122,"identity":"c4c04659-46d5-4d53-bb3c-270508443d50","order_by":17,"name":"Stephanie Corradini","email":"","orcid":"","institution":"LMU University","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Corradini","suffix":""},{"id":333666123,"identity":"424c5472-6561-4521-87dd-3d93a50503d3","order_by":18,"name":"Wilfried Budach","email":"","orcid":"","institution":"Heinrich Heine University","correspondingAuthor":false,"prefix":"","firstName":"Wilfried","middleName":"","lastName":"Budach","suffix":""},{"id":333666124,"identity":"dc780c7e-651b-4883-bb6d-ab1977ec9b98","order_by":19,"name":"Christiane Matuschek","email":"","orcid":"","institution":"Heinrich Heine University","correspondingAuthor":false,"prefix":"","firstName":"Christiane","middleName":"","lastName":"Matuschek","suffix":""}],"badges":[],"createdAt":"2024-07-08 17:36:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4707241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4707241/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13014-024-02566-8","type":"published","date":"2025-01-29T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62184674,"identity":"a583a8b7-5924-457b-9ef4-17e4f78311b5","added_by":"auto","created_at":"2024-08-10 11:46:19","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":370679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModelled survival curves plotted against determined recurrence data of medulloblastoma cell lines.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4707241/v1/5d5637a549accb2941f69482.jpeg"},{"id":62184675,"identity":"1b4ef5a8-3557-47ce-bf71-e6870d2c5bbe","added_by":"auto","created_at":"2024-08-10 11:46:19","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":507010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModelled survival curves plotted against determined recurrence data of medulloblastoma cell lines genetically modified to overexpress MYC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4707241/v1/2185dc6cda44111027bdcc92.jpeg"},{"id":62184673,"identity":"36ab5b97-c803-4a7e-8f8e-2b73692dac97","added_by":"auto","created_at":"2024-08-10 11:46:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of the meta-analyses of the determined alpha/beta-values in medulloblastoma by MetaXL.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4707241/v1/0caa12ed114252fe5f07fa7c.png"},{"id":75351160,"identity":"d6f442d1-6392-48ea-acf9-ba397538a533","added_by":"auto","created_at":"2025-02-03 16:06:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1665449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4707241/v1/d64276a5-895b-4cce-ad2a-e05edf9acfc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Description of Alpha/Beta Values in Pediatric Medulloblastoma: Implications for Tailored Approaches in Radiation Oncology","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedulloblastoma is the most common malignant pediatric brain tumor, impacting approximately 0.5 to 1 in every 100,000 children annually [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is predominantly diagnosed in young patients, with a median age at onset of 5\u0026ndash;6 years. This malignancy is characterized by its high propensity for metastasis along the neuroaxis. Additionally, approximately 30\u0026ndash;40% of patients present with craniospinal fluid dissemination at initial diagnosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The World Health Organization classifies medulloblastoma into four distinct molecular subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These subgroups exhibit unique genetic alterations, demographic patterns, clinical behaviors, and prognostic outcomes, necessitating tailored therapeutic strategies for each subgroup[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTreatment typically involves surgical resection, radiotherapy, and chemotherapy. Patients are stratified into standard-risk and high-risk categories based on a combination of clinical, histopathological, and cytogenetic factors. High-risk patients typically undergo an intense radiotherapy protocol, consisting of 36 Gy of craniospinal irradiation (CSI) with an additional 18 Gy boost to the posterior fossa. In contrast, standard-risk patients are treated with a less intensive CSI protocol of 23.4 Gy, delivered in fractions of 1.8 Gy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the effectiveness of these treatment modalities, they often lead to severe long-term side effects, including cognitive, auditory, and endocrine dysfunctions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These adverse effects significantly diminish the quality of life for survivors, underscoring the critical need for therapeutic strategies that balance efficacy with minimized long-term toxicities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A promising approach to achieve this balance involves the reduction of the total radiation dose.\u003c/p\u003e \u003cp\u003eAnother strategy to modify the therapeutic window involves adjusting the dose per fraction[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, determining the optimal fractionation scheme in medulloblastoma remains elusive due to our limited understanding of medulloblastoma cell responses to varying fractionation schemes. The linear-quadratic model, used to estimate clinical effects of different fractionation regimens, relies on the tissue's alpha/beta ratio [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Tumors with high alpha/beta ratios are typically treated with standard or hyperfractionated radiotherapy, while those with lower ratios may benefit from higher dose-per-fraction or hypofractionated radiotherapy. In this regard, an alpha / beta ratio is considered high if the value is above 8 Gy and low if it is below 5 Gy. A higher dose per fraction would lead to a shorter total radiation time, which could be particularly beneficial in radiation treatments for children, ultimately enhancing their quality of life during and after treatment. A comprehensive understanding of the alpha/beta ratio is crucial for optimizing the therapeutic ratio by amplifying the antitumor effect without increasing late effects. The clinical adoption of various fractionation approaches in medulloblastoma underscores a significant gap in our understanding of pediatric patients' fractionation response. In this study, we aim to present novel experimental data on the alpha-beta ratio of medulloblastoma cells, contributing to defining dose concepts for future clinical trials in this sensitive patient population.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Line Cultivation and Conditions:\u003c/h2\u003e \u003cp\u003eThe medulloblastoma cell lines ONS76, UW228-3, DAOY, D283 and D425, representing distinct TP53 and MYC alteration profiles (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), were used in this study. In addition, previously generated ONS76 and UW228-3 cells stably expressing either mCherry as a control or MYC to mimic activation this oncogene, were employed (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ONS76, UW228-3 and DAOY cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle's Medium containing GlutaMAX, 4.5 \u0026micro;g/l D-glucose and sodium pyruvate (DMEM, Gibco, Waltham, MA, USA), whereas the D283 and D425 cell lines were cultivated in Minimal Essential Medium containing GlutaMAX (MEM, Gibco) and modified Improved Minimal Essential Medium containing 2 mM L-glutamine (IMEM, Gibco), respectively. All media were supplemented with 10% fetal bovine serum (FBS Supreme, PAN-Biotech GmbH, Aldenbach, Germany). The cells were incubated at 37\u0026deg;C in a 5% CO2 atmosphere. Irradiations were performed with a Gulmay RS225 X-ray tube (Xstrahl GmbH, Ratingen, Germany) using 150 kV and 15 mA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAlpha/beta values of medulloblastoma cell lines\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubgroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTP53\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMYC expr.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eα/β +/- CI [Gy]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eONS76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewild-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003elow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e17.09 +/- 6.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUW228-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emutated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003elow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e9.36 +/- 3.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDAOY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emutated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003elow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e75.09 +/- 61.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup 3/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emutated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e15.09 +/- 16.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emutated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e8.42 +/- 5.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAlpha/beta values of medulloblastoma cell lines genetically modified to overexpress MYC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStably expr.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eα/β +/- CI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eONS76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emCherry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e13.69 +/- 5.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eONS76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e10.93 +/- 5.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUW228-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emCherry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e15.82 +/- 9.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUW228-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e19.64 +/- 10.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWell Control Dose Assay:\u003c/h2\u003e \u003cp\u003eThe well control dose assay was adapted from experiments described previously[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]Cell lines were seeded in 24-well-plates. In one half of the plate a high cell number was seeded (2,000 cells for ONS76, UW228-3 and D425; 4,000 cells for DAOY and D283 cells), whereas 1/10 of this number was seeded in the other half (200 and 400 cells, respectively). To counter the impact of potential cellular cooperation effects, lethal one high dose-irradiated (20 Gy) feeder cells from the same cell line were added to the lower concentration wells to generate similar cell densities. After seeding, as soon as the cells became adherent, the fractionated irradiation was initiated. Over the span of four days, the samples were either irradiated twice a day (separated by 6\u0026ndash;8 hours), once a day or once at the beginning and once at the end of the treatment schedule, resembling 8, 4 or 2 fractions, respectively. Depending on the individual radio-sensitivity of the used cell lines, each fractionation scheme featured 5\u0026ndash;6 different doses ranging from 0.4\u0026ndash;12 Gy single dose to 3.2\u0026ndash;32 Gy total dose. One full 24-well-plate was used for each single irradiation and fractionation condition.\u003c/p\u003e \u003cp\u003eOver a 60-day period, cell proliferation in each well was monitored every two to three days and scored binary for either ongoing proliferation leading to regrowth (i.e., the formation of a confluent cell monolayer) or absence of regrowth at the end of the timepoint. As an internal control for the Well Control Dose Assay, we employed the MCF-7 breast cancer cell line that was already reported to exhibit a low alpha/beta value far below \u0026lt;\u0026thinsp;8 Gy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] which is also suggested to be generally the case for breast cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within our experimental setup, MCF-7 cells possess an alpha/beta ratio of 1.6 Gy +/- 1.2 (95% CI; data not shown), which is in concordance with the literature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis and Radiobiological Modelling:\u003c/h2\u003e \u003cp\u003eThe determined experimental survival probabilities were employed together with the -linear-quadratic model of radiation survival,\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;e^ (-k * e^ (-n * (α \u0026lowast; d\u0026thinsp;+\u0026thinsp;β \u0026lowast; d^2)\u0026thinsp;+\u0026thinsp;γ \u0026lowast; t)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e(with p\u0026thinsp;=\u0026thinsp;probability of well control; k\u0026thinsp;=\u0026thinsp;number of clonogenic cells per well; n\u0026thinsp;=\u0026thinsp;number of fractions; α, β\u0026thinsp;=\u0026thinsp;radiation sensitivity parameters; d\u0026thinsp;=\u0026thinsp;single dose; γ\u0026thinsp;=\u0026thinsp;repopulation factor; t\u0026thinsp;=\u0026thinsp;overall treatment time), which was transformed to\u003c/p\u003e \u003cp\u003e\u0026lsquo;p\u0026thinsp;=\u0026thinsp;e^ (-e^ (ln (k) \u0026ndash; β * (α/β * D\u0026thinsp;+\u0026thinsp;D * d))\u0026lsquo; (with D\u0026thinsp;=\u0026thinsp;d * n, i.e. total dose)\u003c/p\u003e \u003cp\u003eso that the α/β value could be determined directly by modelling. \u0026lsquo;γ \u0026lowast; t\u0026rsquo; was omitted because the overall treatment time for all samples was the same. Using this formula, the determined experimental recurrence probabilities and the SPSS Statistics software (Version 29.0.0.0, IBM, Armonk, NY, USA), we performed a non-linear regression with the three parameters \u0026lsquo;ln(k)\u0026rsquo;, \u0026lsquo;β\u0026lsquo; and \u0026lsquo;alphabeta\u0026rsquo;. A maximum likelihood estimate was employed based on a classical logarithmic loss function for binary data and minimization of the negative log-likelihood. Additionally, Bootstrapping was performed to estimating confidence intervals (CI). Calculations were done with SPSS statistics software package. The weighted means of the estimated alpha/beta values were calculated for wildtype and genetically modified cells lines as well as for all cell lines by using the excel-plugin MetaXL (V5.3, EpiGear.com, EpiGear Ltd) employing the inverse variance heterogeneity method.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe tables and charts reflect the reactions of all medulloblastoma cell lines to radiation, demonstrating the variability of radiobiological responses observed. Visualization of the determined survival curves together with the actually obtained recurrence data points demonstrates its high fidelity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). This study revealed high alpha/beta ratios that are all above 8 Gy across a wide range of medulloblastoma cell lines, weighted mean alpha/beta value of 11.01 Gy (CI: 5.23\u0026ndash;16.79 Gy) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).We were able to achieve relatively small confidence intervals. The one exception is the D283 cell line that characteristically grows in a semi-adherent manner, making it difficult to score the wells properly, which therefore resulted in high uncertainty and bigger CI of its determined alpha/beta ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the TP53 genetic status had no influence on fractionation radiosensitivity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, ONS76 vs all other cell lines). Although we analyzed a panel of distinct medulloblastoma cell lines, there was no relevant difference regarding low alpha/beta values detectable (p\u0026thinsp;=\u0026thinsp;0,07) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, MYC expression also did not impact the fractionation sensitivity because neither in with aberrant baseline expression of MYC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D283/D425 vs all other cell lines) nor in cell lines genetically modified to overexpress MYC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, mCherry vs MYC) in both a TP53-wild-type and a TP53-mutated setting a change from a high to a low alpha/beta value was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that these genetic factors may not singularly govern the fractionation sensitivity profile of medulloblastoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, this study reports the first \u003cem\u003ein vitro\u003c/em\u003e data of alpha-beta values in pediatric medulloblastoma patients. Normal and malignant tissues exhibit varying responses to fractionation, a phenomenon known as fractionation sensitivity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This sensitivity is typically described using the α/β ratio. Our preclinical data provide evidence that medulloblastomas in children exhibit high alpha-beta values, indicating reduced sensitivity to fraction size. This is an observation that had been well-documented in adult head and neck malignancies, where administering radiotherapy in smaller fractions is known to protect late-responding normal tissues in comparison to the tumor [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the context of medulloblastoma, analogous clinical observations have substantiated our findings.\u003c/p\u003e \u003cp\u003eHyperfractionated radiotherapy was implemented with the objective of maximizing the therapeutic dose to the tumor while simultaneously minimizing the risk for adverse effects on surrounding normal tissues. For low or standard-risk patients, where high cure rates are often achievable with current normofractionated concepts, the emphasis of hyperfractionated radiotherapy is on reducing long-term toxicities and thereby preserving neurological function and quality of life. In HR patients, where the prognosis is poorer and the risk of disease recurrence is higher, the aim shifts towards enhancing the efficacy of radiotherapy without disproportionately increasing toxicity. However, the clinical experiences to date have not definitively demonstrated a significant advantage for hyperfractionated radiotherapy over conventional radiotherapy in terms of survival outcomes.\u003c/p\u003e \u003cp\u003eThe HIT-SIOP PNET 4 trial (2001\u0026ndash;2006) examined the role of hyperfractionation in comparison to conventional radiation therapy in the treatment of pediatric patients with medulloblastoma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite the absence of a clear advantage in terms of 5-year event-free survival (78% for hyperfractionated vs. 77% for conventional) in the HIT-SIOP PNET 4 trial (2001\u0026ndash;2006), hyperfractionated therapy did reveal a significant improvement in verbal intelligence quotient (VQ) among children under 8 years at diagnosis (mean intergroup difference 12.02, P\u0026thinsp;=\u0026thinsp;.02) and exhibited a noticeable trend toward enhanced processing speed (mean intergroup difference 10.90, P\u0026thinsp;=\u0026thinsp;.08) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the clinical application of hyperfractionated radiotherapy in young children, significant challenges exist, irrespective of radiobiological considerations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For very young children, sedation for immobilization is necessary for treatment planning and delivery. The restrictions for both food and fluids before sedation pose substantial challenges for families. Although the feasibility of this approach has been demonstrated by the St. Jude Hospital group, the twice-daily sedation of young children remains a topic of controversy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This approach requires substantial resources and its safety has not been conclusively established.\u003c/p\u003e \u003cp\u003eControversial results have been reported for metastatic medulloblastoma, previously. While the Italian group demonstrated promising outcomes with Hyperfractionated Accelerated RT (HART) combined with pre-RT chemotherapy and maintenance chemotherapy for metastatic medulloblastoma, achieving a 5-year event-free survival (EFS) of 70%, attempts by the British to replicate these results using the same approach resulted in a lower 3-year EFS of 59% [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These disparities may be attributed to the heterogeneous radiobiological behavior of metastatic tumors.\u003c/p\u003e \u003cp\u003eIncorporating predictive molecular biomarkers for radiation sensitivity holds the potential to enhance the customization of radiotherapy to individualized conditions. Both the tumor suppressor TP53 as well as the proto-oncogene MYC have been described as important modulators of proliferation and cell survival after DNA damage[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, irrespective on their impact on radiosensitivity, our results reveal comparable outcomes regarding the fractionation sensitivity marker alpha/beta across different sub-classifications, surprisingly regardless of the presence or absence of MYC overexpression and/or TP53 alterations. Therefore the consistency in radioresponse observed in our study suggests the potential feasibility of a standardized radiotherapeutic approach for medulloblastoma, irrespective of specific molecular profiles. While various cellular response mechanisms contributing to fractionation sensitivity have been identified in the past, the molecular biomarkers remain elusive.\u003c/p\u003e \u003cp\u003eOur investigations heavily rely on \u003cem\u003ein vitro\u003c/em\u003e assessments conducted on tumor cell lines and it is important to recognize that these controlled laboratory conditions may not comprehensively replicate the intricate microenvironment encountered in clinical settings. The inherent limitations of this approach lie in its inability to encompass the full spectrum of variables that influence radiation sensitivity within the complex context of real-world patient scenarios. However, our data can serve to support prior clinical observations and provide a foundation for establishing normo- to hyper-fractionated radiation protocols for young children with medulloblastoma, based on alpha-beta values. Nonetheless, we acknowledge that larger prospective randomized studies are highly desirable to further validate and refine these findings.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecraniospinal irradiation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edesoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eevent- free survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGy\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGray\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradiotherapiy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to our ethics commission board, ethical approval was not deemed necessary for the irradiation of cell lines, as it is not a mandatory requirement.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026bull; Consent for publication: \u0026nbsp;all authors gave consent for publication\u003c/p\u003e\n\u003cp\u003e\u0026bull; Availability of supporting data: \u0026nbsp;The supporting data can be made available by the first and last authors.\u003c/p\u003e\n\u003cp\u003e\u0026bull; Competing interests/Authors\u0026apos; contributions: \u0026nbsp;all authors have no competing interests\u003c/p\u003e\n\u003cp\u003eAuthors contributions:\u0026nbsp; DJ, CM, DS, EB, WB wrote the main manuscript, EB, DS, DJ, CM, WB helped to design the study and wrote part of the manuscript, DS prepared the figures, WB, RP, NQ, JH, RS, NN, AB , FB, TB, MF, BT, PM, TB, BT, MR, SC did the literature research and prepared the data for analysis, all authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026bull; Funding: \u0026nbsp;Open Access funding enabled and organized by Projekt DEAL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAchknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank C. 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Cell Death Differ. 2008;15:959\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/cdd.2008.33\u003c/span\u003e\u003cspan address=\"10.1038/cdd.2008.33\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"radiation therapy, cancer, paediatric tumor, brain tumor","lastPublishedDoi":"10.21203/rs.3.rs-4707241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4707241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMedulloblastoma is the most common malignant pediatric brain tumor, typically treated with normofractionated craniospinal irradiation (CSI) with an additional boost over about 6 weeks in children older than 3 years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This study investigates the sensitivity of pediatric medulloblastoma cell lines to different radiation fractionation schedules. While extensively studied in adult tumors, these ratios remain unknown in pediatric cases due to the rarity of the disease.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eFive distinct medulloblastoma cell lines (ONS76, UW228-3, DAOY, D283, D425) were exposed to varying radiation doses and fractionation schemes. In addition, ONS76 and UW2283-3 stably overexpressing MYC were analyzed. Alpha/beta values, representing fractionation sensitivity, were quantified using the linear-quadratic model of radiation survival.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe study unveiled elevated alpha/beta ratios across diverse medulloblastoma cell lines, with a weighted mean alpha/beta value of 11.01 Gy (CI: 5.23\u0026ndash;16.79 Gy). Neither TP53 status nor the levels of MYC expression influenced fractionated radiosensitivity. Furthermore, differences cannot be correlated with molecular subgroups (p\u0026thinsp;=\u0026thinsp;0,07).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese \u003cem\u003ein vitro\u003c/em\u003e findings strongly recommend normofractionated or hyperfractionated radiotherapy for paediatric medulloblastoma cases due to consistently high Alpha/Beta values across subgroups. Conversely, hypofractionated radiotherapy is not advisable within a curative approach. This study presents significant potential by enabling the estimation of radiobiological fractionations and dose effects in young, vulnerable patients, highlighting its importance for advancing patient-specific therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"First Description of Alpha/Beta Values in Pediatric Medulloblastoma: Implications for Tailored Approaches in Radiation Oncology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-10 11:46:15","doi":"10.21203/rs.3.rs-4707241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-07-26T16:17:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220447604874192741638169148354957168934","date":"2024-07-25T14:17:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-25T10:01:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T07:48:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T04:02:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Radiation Oncology","date":"2024-07-08T17:35:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc3f78c2-77f5-45ca-83d3-eed8c5073bf7","owner":[],"postedDate":"August 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T15:59:29+00:00","versionOfRecord":{"articleIdentity":"rs-4707241","link":"https://doi.org/10.1186/s13014-024-02566-8","journal":{"identity":"radiation-oncology","isVorOnly":false,"title":"Radiation Oncology"},"publishedOn":"2025-01-29 15:57:02","publishedOnDateReadable":"January 29th, 2025"},"versionCreatedAt":"2024-08-10 11:46:15","video":"","vorDoi":"10.1186/s13014-024-02566-8","vorDoiUrl":"https://doi.org/10.1186/s13014-024-02566-8","workflowStages":[]},"version":"v1","identity":"rs-4707241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4707241","identity":"rs-4707241","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}