Beijing Children’s Hospital guidelines on the design and conduction of the first standardized database for medulloblastoma

Medulloblastoma (MB) is one of the most common malignant childhood brain tumors (WHO grade IV). Its high degree of malignancy leads to an unsatisfactory prognosis, requiring more precise and personalized treatment in the near future. Multi-omics and artificial intelligence have been playing a significant role in precise medical research, but their implementation needs a large amount of clinical information and biomaterials. For these reasons, it is urgent for current MB researchers to establish a large sample-size database of MB that contains complete clinical data and sufficient biomaterials such as blood, cerebrospinal fluid (CSF), cancer tissue, and urine. Unfortunately, there are few biobanks of pediatric central nervous system (CNS) tumors throughout the world for limited specimens, scarce funds, different standards collecting methods and et cl. Even though, China falls behind western countries in this area. The present research set up a standard workflow to construct the Beijing Children’s Hospital Medulloblastoma (BCH-MB) biobank. Clinical data from children with MB and for collecting and storing biomaterials, along with regular follow-up has been collected and recorded in this database. In the future, the BCH-MB biobank could make it possible to validate the promising biomarkers already identified, discover unrevealed MB biomarkers, develop novel therapies, and establish personalized prognostic models for children with MB upon the support of its sufficient data and biomaterials, laying the foundation for individualized therapies of children with MB.


Introduction
Medulloblastoma (MB) is among the most common malignant childhood brain tumors (WHO grade IV) and approximately accounts for 25% of childhood brain tumors (Cavalli et al. 2017;Roussel and Hatten 2011).However, it is uncommon in adults, accounting for only 1% of adult intracranial tumors (de Oliveira et al. 2018).Its high degree of malignancy leads to an unsatisfactory prognosis which makes it a significant cause of childhood death from tumors.The knowledge of medulloblastoma has progressed from pathological classification (Orr 2020) to molecular typing (Waszak et al. 2018;Wang et al. 2018;Northcott et al. 2019).The treatment paradigm for MB has also progressed toward individualization and precision.Despite the combination of multiple therapeutic approaches, the worst five-year survival of MB patients could only reach about 60% (Sh et al. 2020).
Wei Yang, Wenping Ma and Jiansong Huang contributed equally to this work.Multi-omics (including genomics, transcriptomics, epigenomics, proteomics, metabolomics, and other omics areas) and artificial intelligence technologies can not only facilitate our understanding of the epidemiology and pathogenesis of MB but also contribute to strengthening the personalized therapeutic approach and identifying early diagnostic markers.Meanwhile, on account of the particularity of pediatric CNS tumors, hazards in these immature and developing brains through different therapeutic approaches should be paid more attention to.To accomplish these goals, not only a large amount of clinical data is needed, but also biomaterials, including cancer tissue, blood, cerebrospinal fluid, urine, etcl should be provided.
The concept of biobank was first introduced in 1996(S and He 1996), and the term "biobank" has been gradually adopted to describe any collections of biospecimens or human genetic data suitable for research purposes.The current mainstream Biobank is broadly comprised of population-based and disease-oriented, with the disease-oriented Biobanks mainly focusing on the collection of diseaserelated tissue samples and clinical data (Annaratone et al. 2021).Starting from cancer, biobanks were linked to the ambitious chance of screening and treating any disease (Patil et al. 2018).
Currently, only a few centers have been conducted regarding biobanking of pediatric CNS tumors globally (van Rooij et al. 2016;Hermansen et al. 2022) and there is a lack of experience in establishing biobanking of pediatric CNS tumors in China.Therefore, establishing a standardized biobank workflow for medulloblastoma, which contains the collection and storage of clinical data and biomaterials, is an urgent issue to be addressed.
In this study, we propose a standard workflow to collect clinical data from children with medulloblastoma in Beijing Children's Hospital and store the corresponding biomaterials to build the BCH-MB biobank.Through this biobank, we could further explore the pathogenesis of medulloblastoma, validate the promising biomarkers already identified, identity unrevealed diagnostic MB biomarkers, develop precise therapeutic targets (Schakelaar et al. 2023;Chaemin et al. 2023), and investigate the prognostic impact of different treatment modalities on children with MB. it will provide sufficient data and samples for further basic and clinical research.

Design
The legal and administrative aspects of the BCH-MB biobank project in the National children's cancer monitoring center are supported by Beijing Children's Hospital, Capital Medical University, National Center for Children's Health.The BCH-MB biobank aims to establish a pediatric tumor registration and monitoring system to improve the standardized diagnosis and treatment of MB by exhaustively collecting relevant patient information and collecting biological samples for storage.Patients are enrolled since 2015 and followed up regularly.Currently, 102 patients have been initially included, and the goal is to include children with MB as many as possible until 2030.

Ethics statement
The research was approved by the ethics committee of Beijing Children's Hospital, Capital Medical University, National Center for Children's Health and we confirm that the procedures involving experiments on human subjects met the ethical standards of the Helsinki Declaration in 1975.Patients and families are fully aware and signed the informed consent.The collection and storage of all biological materials passed the ISO9000 certification.

Patients
All patients under 19 years old with a confirmed clinical diagnosis of medulloblastoma will be included to obtain a sufficient sample size for the pediatric medulloblastoma database.Confirmed clinical diagnosis refers to having sufficient clinical manifestations, imaging manifestations, and laboratory findings, and also confirmed by pathological examination.

Baseline information
We collected baseline demographic information including age, sex, height, weight, geography (province), and ethnicity.

Medical history and family history
Data on past medical conditions were collected, such as nevoid basal cell carcinoma syndrome, familial adenomatous polyposis, and Li-Fraumeni syndrome.We also recorded whether there was a family history of medulloblastoma.

Syndrome and physical examination
We divided the clinical symptoms of medulloblastoma into three main categories: intracranial hypertension, motor system involvement, and cranial nerve involvement.The intracranial hypertension symptoms were recorded by "yes or no", the cranial nerve involvement was recorded by which brain nerve was damaged, and the motor system involvement symptoms were scored by ECOG/WHO score, modified Rankin scale (mRS), and Friedreich's Ataxia Rating Scale (FARS) three scoring methods.

Blood
Plasma extraction 10 ml of blood was collected into a streck blood collection tube, and then centrifuged at 3000 rpm for 10 min at room temperature.The supernatant was divided into 1.5 mL EP tubes at -80 ℃.Serum extraction 10 ml of blood was collected in centrifuge tubes at room temperature and stayed for 1 h for coagulation and stratification.Then centrifuge at 3000 rpm for 10 min, and transfer the supernatant to a clean centrifuge tube.The supernatant was then centrifuged at 12,000 rpm for 10 min at 4 ℃, and the supernatant was divided into 1.5 mL centrifuge tubes of 0.2 mL each and stored at -80 ℃.PBMC extraction 10 ml of blood was collected in a 50 ml centrifuge tube, add 10 ml of PBS solution to dilute and mix gently.Then add 7.5ml of the diluted blood to the upper layer of the 15ml centrifuge tube containing 7.5ml of Ficoll solution, without mixing the two solutions.Centrifuge the 15ml tube at 2000 rpm for 30 min and adjust the speed reduction setting as braking to 0. After centrifugation, the liquid in the tube was split into three layers and the white film in the middle was PBMC.Aspire it with a pipette, dispense it into 1.5 ml centrifuge tubes, and store it at -80 °C.Urine Take 8-12ml of urine, centrifuge at 4000 g for 10 min at 4℃, take the supernatant and dispense into 4-6 2ml EP tubes and store at -80℃.Cerebrospinal fluid (CSF) CSF was extracted from the subarachnoid space during surgery, but before tumor resection, then ≥ 5 ml of cerebrospinal fluid was collected into a streck collection tube, mixed thoroughly, followed by centrifugation at 1600 g for 10 min at 4 °C.The supernatant 1.5 ml was centrifuged in an EP tube for 10 min at 16,000 g, after which the supernatant and lower cells in the EP tube were taken separately and stored at -80 °C.Tumor specimens Tumor specimens resected during surgery were washed by PBS and directly snapped frozen in liquid nitrogen or -80 °C refrigerator.A formalin-fixed, paraffin-embedded (FFPE) block of MB tissue is also prepared and stored at room temperature.

Pathology classification
The medulloblastoma pathology type was classified by HE staining method: classic Medulloblastoma, Desmoplastic/nodular medulloblastoma (DN), Medulloblastoma with extensive nodularity (MBEN), Large cell/Anaplastic medulloblastoma (LCA), non-specific (NOS).When the diagnoses were inconsistent, a third authoritative pathological center were referred for adjudication.

Molecular classification
The RNA expression level of medulloblastoma is measured by subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology.According to the 2021 WHO Classification of Tumors of the Central Nervous System, medulloblastoma was classified into WNT, SHH-p53 mutant, SHH-p53 wild type, and non-WNT/non-SHH type.The non-SHH/non-WNT types were further classified into Group 3 and Group 4.

Chang staging system
Chang staging system was used to grade the degree of tumoral metastasis, where M0 stands for no evidence of metastasis in MRI and cerebrospinal fluid (CSF) studies, M1 stands for the presence of microscopic tumor cells in CSF cytology preparations, M2 stands for intracranial metastatic deposits observed beyond the primary site using MRI, M3 stands for metastatic deposits in the spinal subarachnoid space, and M4 stands for metastasis outside the central nerve system.

Surgical treatment
The different surgery styles are distinguished in terms of the extent of surgical resection and the different surgical approaches.The extent of surgical resection is subdivided into GTR (gross total resection), NTR (near-total resection), and STR (sub-total resection).Surgical approaches can be divided into the following five categories (1) telovelar, not transvermian; (2) transvermian, not telovelar; (3) telovelar and transvermian; (4) cerebellar hemispheric approach, without telovelar, transvermian, or subvermian; and (5) other surgical approach.diagnostic markers, establish individualized prognostic models, and gradually achieve precision medicine (Cai et al. 2023;Rausch et al. 2023).Multi-omics as well as artificial intelligence technologies, needing a large amount of clinical data and biomaterials, can assist in accomplishing these goals.Construction of the BCH-MB biobank to provide support for subsequent research on MB is in great urgency.Therefore, we propose the first version a protocol for establishing a biobank for pediatric central nervous system tumors in China.

Rationales for acquiring biological specimens and clinical data
This biobank collects baseline demographic information, MB classification, clinical data, treatment modalities, and follow-up information in detail according to the principles of practicality and generalizability.These clinical data include the patients' medical history, clinical symptoms, MR images.We also collected four biomaterials of patients' blood, urine, CSF, and cancer tissue.The workflow is shown in Fig. 1.
Currently, 102 children have been included in the BCH-MB biobank and their baseline information is shown in Table 1.In the future, we will include more patients and encourage the participation of other medical centers.About 5%~ 6% of children with medulloblastoma have germline mutations in specific genes that predispose them to medulloblastoma and other cancers (Waszak et al. 2018).We carefully recorded the diagnosis of typical hereditary syndromes.We also inquiry whether they have a family history of medulloblastoma, for there's still a few children have a family history of the disease (Moschovi et al. 1998;von Koch et al. 2002).The clinical manifestations of MB are mainly intracranial hypertension, motor system involvement, and, rarely, cerebral nerve involvement.There are several ways to quantify motor system involvement, and we used the ECOG/WHO score, mRS, and FARS, which are commonly used in clinical practice.We collected MRI data from patients with fixed parameters, and the MRI sequences are shown in Table 2.In the future, these imaging data can be used in artificial intelligent diagnosis and prognosis under anonymous situation.Surgical treatment is imperative for children with MB, but surgeries vary depending on the size of the cancer, and the location of the cancer growth.The different surgery styles will be distinguished in terms of the extent of surgical resection and the different surgical approaches.GTR, NTR, and STR are defined as no residual tumor, less than 1.5 cm² residual tumor, and 1.5 cm² or more of residual tumor respectively( (Thompson et al. 2016a).Surgical approaches can be subdivided into five aforementioned categories (Grønbaek et al. 2021).

Therapeutic irradiation
The children with MB are divided into three groups: those who received radiotherapy for whole brain and whole spinal cord, those who received radiotherapy but only local radiotherapy, and those who did not receive radiation therapy.

Chemotherapy
The children with MB are divided into two groups according to "whether or not receiving chemotherapy".

Follow-up assessment
Post-operative patients are followed up every six months for the first three years, at least once a year for the next 3-5 years, and once every 2-3 years for 5-10 years postoperatively.In the case of recurrence and treatment-seeking children, we collect the above-mentioned biological samples and relevant medical information from the children.The five-year survival period and the five-year relapse-free survival period are recorded through regular follow-up.Meanwhile, whether the child has complication of treatment would be recorded, such as cerebellar mutism, neurocognitive impairment, hearing loss, skeletal problems, endocrine abnormalities, etc.As regard to cerebellar mutism syndrome, motor assessment and duration of mutism are also recorded.

Data collection, procession and storage
Based on the available data, the collection method described above will be used to regularly follow up each included child and record the data.A standardized biobank data preservation system will be used to organize these data.

Basis for establishing the BCH-MB Biobank
As the most common childhood cancer, the treatment of medulloblastoma remains a great achievement company with a lot of challenges.At present, the treatment of medulloblastoma is still mainly surgical treatment combined with post operative adjuvant therapy.However, because of the immature central nervous system and the great individual risk hierarchy among children, the individualized treatment regimen is imperative.
In order to progress toward individualized and precise treatment, we need to further study epidemiology and pathogenesis of MB, based on which we can identity early are collected from each case and processed, transported, and preserved through a standardized process (Rutkowski et al. 2018).Table 3 lists the biomaterials-processing procedure and the purpose for storage.

Current data and prospective research agenda
We have currently enrolled more than 100 patients (Table 1) and have regular follow-up.In the future, we plan to enroll children with MB as many as possible, encourage the participation of other medical centers, and strictly follow the current workflow.Also, considering that we are the first biobank for medulloblastoma in China and have proposed a detailed protocol, we are also considering the inclusion of multiple centers in the near future.

The BCH-MB biobank in advancing Future Research
The large amount of clinical data, biomaterials, and follow-up data collected will provide the original information for future research such as multi-omics studies combined genomics, transcriptomics, epigenomics, proteomics, metabolomics, other omics areas, and artificial intelligence studies with various algorithm (Kojic et al. 2023;Luo et al. 2023), with the aim of (1) validating the promising markers Each kind of treatment may be associated with delayed complications in children with MB that severely affect the survival as well as the quality of medulloblastoma patients' lives (Ning et al. 2015;(Salloum et al. 2019a).These complications include cerebellar mutism syndrome (Albazron et al. 2019), neurocognitive impairment (King et al. 2017), hearing loss (King et al. 2017;(Salloum et al. 2019b), skeletal problems (Goldwein et al. 1996), and endocrine abnormalities (Vatner et al. 2018).Among them, we pay more attention to the evaluation of cerebellar mutism syndrome, for it is a unique postoperative complication, worthy of further investigation.Tumor tissue, blood, CSF, and urine exactly comprehensive, and there may be a small number of specimens that were not collected or not well preserved.However, since the establishment of the workflow, the collection of the data and preservation of biological samples as well as MR images will be conducted more formally.
already identified, or search for new unrevealed diagnostic biomarkers as a way to improve the diagnostic accuracy of pediatric medulloblastoma to reduce the burden on patients' families (Hovestadt et al. 2019(Hovestadt et al. , 2020)).( 2) the storage of tumor specimens can support sequencing, culturing, and establishing organoid models (Ballard et al. 2019).It will lay the foundation for the study of medulloblastoma pathogenesis, and the molecular mechanisms to facilitate the discovery of potential therapies in the future.(3) new prognostic models can be established, based on clinical data, biological information, in order to facilitate the assessment of impact of treatments on patients' quality of life.In the future, we will be able to analyze the effects of different treatments and combine therapies on different medulloblastoma subgroups( (Thompson et al. 2016b) and to further personalize the therapeutic regimen to Chinese children.
Regarding the shortcomings of this study, the collection of retrospective data at the preliminary stage was not

Table 1
The current baseline data of included patients

Table 2
Type of biomaterial to be collected and the purpose for storage FFPE = formalin-fixed, paraffin-embedded.IHC = immunohistochemistry.FISH = fluorescence in situ hybridization.PDX = patientderived xenograft.*This has not yet been done and is planned for the future