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

doi:10.21203/rs.3.rs-4707241/v1

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

https://doi.org/10.21203/rs.3.rs-4707241/v1

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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

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.

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.

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. 1A-E, Fig. 2A-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. 1D).

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.

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.

CSI

craniospinal irradiation

DNA

desoxyribonucleic acid

EFS

event- free survival

Gray

radiotherapiy

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.

Achknowledgements

We thank C. Hachmann for excellent technical assistance.

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No competing interests reported.

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You are reading this latest preprint version

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

Status:

Version 1

Abstract

Background

Materials and Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Cell Line Cultivation and Conditions:

Well Control Dose Assay:

Statistical Analysis and Radiobiological Modelling:

Results

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1