The Swedish childhood tumor biobank: systematic collection and molecular characterization of all pediatric CNS and other solid tumors in Sweden

The Swedish childhood tumor biobank (BarnTumörBanken; BTB): a combined genomics and tissue biobank for pediatric cancers

The BTB is a translational research core facility for sample and genomic data collection from pediatric patients diagnosed with solid tumors in Sweden. The BTB is financed by The Swedish Childhood Cancer Fund (BCF) (https://www.barncancerfonden.se/en/) and is located at The Karolinska University Hospital and Karolinska Institutet in Stockholm. The BTB is built on a multidisciplinary nationwide network of physicians and researchers from the six university hospitals that manage the care of affected children. The BTB operates under standard operating procedures to ensure that biospecimens are collected and processed consistently and with high quality. It also maintains strict ethical standards and adheres to national and international regulations for biobanking. The daily work, from sample collection to data generation and storage, is conducted by qualified personnel with relevant expertise. Competence in different working areas to support the administrative and legal procedures also exists. The infrastructure is governed by a steering group that oversees BTB’s activities and takes strategic decisions, including the main goals and research services offered., The members of the steering group represent multidisciplinary clinical and research specialties in the field of pediatric cancer, the patient organization and the BCF.

BTB services

The BTB offers services to childhood cancer research projects and clinical studies in compliance with current ethical and legal regulations. Samples and genomic data can be requested by approved projects undertaking meaningful research in the field of pediatric cancer.

Offering services to clinical studies, the BTB has been responsible for the handling of clinical samples for Swedish patients referred to the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood, Germany), CMS (Cerebellar Mutism Syndrome, Denmark), BIOMEDE (Biological Medicine for Diffuse Intrinsic Pontine Glioma Eradication, France), SIOP PNET5 MB (ClinicalTrials.gov Identifier: NCT02066220) and Genomic Medicine Sweden (GMS, Childhood cancer|Genomic Medicine Sweden) studies in recent years [14, 52]. The BTB´s personnel conduct sample collection, DNA and RNA extraction, sample logistics and quality control. As of March 2022, 217 cases have been submitted to INFORM, 155 to CMS, 15 to BIOMEDE, 19 to PNET5 and 145 to GMS. Customized collection or specialized processing of tissue and molecular data for prospective studies or clinical trials is also supported upon formal agreement. The BTB requests funds only to cover personnel time and consumables for the generation of research-ready specimens and datasets.

BTB sample and data collection is continuously expanding

Biospecimens from affected children are continuously collected for the BTB on a national level. Tissue sampling from CNS and other non-CNS solid tumor patients was initiated in 2013 and 2015, respectively, and since 2020, specimens from approximately 90% of all diagnosed pediatric cancer cases in Sweden have been provided to the BTB. Currently, there are 597 CNS and 407 other non-CNS solid fresh frozen primary tumors and 183 samples with known or suspected tumor relapse in the BTB repository, all sampled with patient-matched blood. Occasionally, additional samples, including Formalin-Fixed Paraffin-Embedded (FFPE) tissue, needle aspirates or cerebrospinal fluid, are obtained. Blood samples from the parents of affected children are also being collected. As of 2022, there are over 1700 registered patients from whom at least one biological sample has been collected (Fig. 2). Large-scale genomic data is generated from the samples. Currently, WGS and transcriptome sequencing are retrospectively performed on frozen tumors, complemented with DNA methylation profiling for CNS tumors to enhance diagnostic accuracy. WGS is also performed on patient-matched blood-derived DNA.

Fig. 2figure 2

A Sample collection at the BTB specifying the cumulative numbers of patients registered at the BTB from whom at least one biological sample (blood, fresh frozen or FFPE tissue) has been collected, fresh frozen primary tumors, patient blood samples, fresh frozen tumor relapses or metastasis and FFPE tumors. Distribution of diagnosis of CNS (B) and other solid (C) tumors from which samples of fresh frozen primary, relapsed or metastatic tissue and patient-matched blood were collected until March 2022

The genomic landscape of pediatric brain tumors: analysis of somatic and germline mutations in a pilot subset of BTB samples

It is fundamental that biospecimens and data collected at the BTB can successfully support basic and translational biomedical research. To evaluate the results of the whole workflow and demonstrate the collection’s value, we outline here the overall findings derived from basic bioinformatic analyses performed on omics data from a series of pediatric brain tumor samples collected and sequenced during the first stage of the project. We analyzed data generated from WGS or WES on a total of 82 brain tumors and peripheral blood samples from 79 affected children (Additional file 2: Table S1, Fig. 3, Additional file 5: Fig. S1, Additional file 6: Fig. S2). These included 19 diagnosed medulloblastomas (MB), ten ependymomas, 11 glioblastomas (GBMs)/primitive neuroectodermal tumors (PNET)/embryonal tumors, 20 pilocytic astrocytomas (PAs), five representing other forms of astrocytoma/glioma, seven atypical teratoid rhabdoid tumors (ATRT), two oligodendrogliomas, two meningiomas, two craniopharyngiomas, and one each of pineoblastoma, ganglioglioma, pituitary adenoma, choroid plexus tumor, and schwannoma. Patient age ranged from one month to 18 years (median 5.8 years).

Fig. 3figure 3

Oncoprint of genomic alterations in CNS tumors. Heatmap of genomic alterations in the series of 82 tumors. Individual genes are represented as rows, and tumors are represented as columns. The bars at the top of the figure indicate the number of cumulative events (putative drivers) identified in the corresponding sample at baseline. The colors represent the six types of underlying genomic alterations as indicated on the right. Concordance between methylation classification or NGS results and pathology diagnosis, the potential availability of targeted therapies, and the pathology diagnosis (indicated in different colors) are also shown. The genes (rows) are sorted by their alteration frequency in the cohort, as noted on the left. When two events affect the same gene in a sample, they are represented in the same slot. Different colors and sizes of rectangles distinguish the type of alterations, as indicated in the panel on the right

The DKFZ classifier was applied to the DNA methylation data. The methylation-based classification agreed with or improved the pathologic diagnosis by confidently assigning the tumors into specific classes in 78% of analyzed cases (57 of 73 profiled tumors with classification scores > 0.9; Additional file 2: Table S1). Five additional tumors were assigned to methylation class families with lower scores (values ranging from 0.49–0.74), and in seven cases, the methylation screening failed to classify the samples or scored poorly. Some of these cases represent tumors with lower tumor cell content, while others may represent rare or uncharacterized entities or subgroups that are likely underrepresented in the reference cohort used to train the classification model.

Moreover, while accounting for molecular subgroups is important in itself, to enable the development of more appropriate therapeutic strategies and meaningful clinical trials, it is essential to determine the underlying mutation pattern of disease-relevant genes. Therefore, WGS or WES was performed on the samples. Relevant germline and somatic mutations and methylation classification results are described below and summarized in Additional file 2: Table S1.

Previously recognized genomic alterations in different diagnostic entities

Medulloblastoma (MB) is the most common malignant pediatric brain tumor in children. It represents a biologically and clinically heterogeneous disease comprising several molecularly distinct subgroups with clinical and genetic differences. Since 2012, the general consensus has been that MB can be molecularly separated into at least four core subtypes, including WNT-activated, sonic hedgehog (SHH)-activated, MB group 3 and group 4 [49]. The methylation profiling of MB confidently determined the subclasses in 16 cases and in one additional case but with a lower score (P4551_203T). This included one WNT, five group 3, six group 4 and five SHH tumors. Mutations in subgroup-restricted genes were found; for example, one MB-WNT (P7708_102T) tumor presented with the typical CTNNB1 mutation, three (F0025678-0810, F0025679-0775, BTB0001) SHH-MB were driven by mutation/deletion of PTCH1, and two (FAM2T, P2233_111T) were driven by SUFU mutation/deletion in combination with TP53 or PTEN mutation. Additionally, one MB group 4 tumor (P2233_114T) presented a TBR1 mutation, another (P4551_203T) carried a tandem duplication of the SNCAIP gene, which leads to PRDM6 activation and is the most common distinctive genomic alteration described for MB group 4 [37], and one MB group 3 tumor (P1148_116T) harbored the hotspot insertion in KBTBD4 [36]. Mutations in genes known to be mutated across the subgroups, including PIK3CA and KMT2D, were also detected (Additional file 2: Table S1).

Pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine glioma (DIPG) and GBM, are among the most aggressive brain tumors. IDH wild-type HGG comprises eight molecular subtypes determined by DNA methylation profiling (K27, G34, RTK I, RTK II, RTK III, mesenchymal, midline, MYCN) that are strongly associated with clinical features (https://www.molecularneuropathology.org/mnp). Among the samples studied here, four tumors from three patients were classified as diffuse midline glioma K27 mutant (P2233_104T, P1148_113T, P7708_127T, P7708_128T) and harbored mutations in H3F3A in addition to TP53 or ATRX mutations. The fifth GBM, classified as IDH wild-type, subclass MYCN tumor (P4551_209T), carried TP53 mutations and several amplified loci, including FGFR1, CDK6, MET and PDGFRA, but not MYCN (Fig. 3, Additional file 2: Table S1, Additional file 6: Fig. S2).

Ependymal tumors are neuroepithelial malignancies of the CNS that occur in both children and adults. Despite the histopathological similarities among variants of ependymomas at different anatomical sites, the molecular biology of this tumor is heterogeneous, and nine molecular subgroups with distinct genetic and epigenetic alterations have been identified [38]. In accordance with this knowledge, no drivers were found in the three ependymoma posterior fossa group A tumors studied here (F0025588-0809, P2233_113T, P7708_111T), and all four ependymomas in the RELA fusion class (P1148_114T, P7708_114T, P7708_115T, P7708_201T) displayed copy number alterations at 11q13.1 involving C11orf95 and RELA (Table 1, Additional file 2: Table S1, Additional file 5: Fig. S1, Additional file 6: Fig. S2G).

Table 1 Somatic structural variants predicting fusion genes

ATRTs are highly aggressive embryonic tumors primarily encountered in children with mutations in SMARCB1 as the main genetic hallmark. We studied seven tumors from six ATRT patients. Methylation profiling provided additional information concerning the subclass for five of these tumors, two being subclass MYC and two subclass TYR. In accordance with previous reports [48], the ATRT-MYC subgroup tumors presented with focal SMARCB1 deletions (Additional file 2: Table S1, Additional file 5: Fig. S1, Additional file 6: Fig. S2). Furthermore, in one of these tumors (P2233_115T), an ETV6::NTRK3 fusion was detected (Table 1). The two ATR subgroup TYR presented with a somatic deletion of the whole chr22 and an identical, not previously reported, frameshift SMARCB1:p.Pro392fs mutation. In two relapsed ATRTs (P4552_102R, P4552_103R2) resected on different occasions from the same patient, no mutations were identified apart from a focal deletion within SMARCB1, an exon loss variant of germline origin (chr22:23798791–23806662). Pathological re-examination of the slides confirmed low tumor cell content (< 30%) in both cases, explaining why additional genetic abnormalities were not detected. Furthermore, one additional tumor (P2233_104T) pathologically diagnosed as ATRT was unexpectedly assigned by methylation profiling to the diffuse midline glioma, H3 K27M mutant class. NGS findings, including the detection of somatic mutations in the TP53, NF1 and H3F3A genes, strongly supported the methylation classification in this case (Additional file 2: Table S1, Additional file 5: Fig. S1).

Craniopharyngiomas are rare, benign brain tumors of the teratoma type. There are two different subtypes that differ clinically and pathologically: adamantinomatous and papillary craniopharyngioma. These subtypes harbor mutually exclusive and clonal mutations in CTNNB1 and BRAF, respectively [4, 18]. In line with this knowledge, both adamantinomatous craniopharyngiomas profiled here (P7708_123T, P7708_126T) displayed classical mutations in exon 3 of CTNNB1 (Additional file 2: Table S1).

PA is the most common pediatric brain tumor and is classified as a WHO grade I neoplasm [29]. The most common alteration in PAs is a tandem duplication at 7q34 that results in KIAA1459::BRAF fusion [21, 40]. The 7q34 tandem duplication involving the KIAA1459 and BRAF genes was the only CNV observed in 15 of the 20 PAs studied here. The exact breakpoints of these rearrangements were identified as SVs by Manta in 13 of the tumors; of these rearrangements, 12 were tandem duplications, and one (P4551_213T) was a double inversion combined with a duplication. Other mutations were rare, and among them, we detected a pathogenic somatic missense mutation in PTPN11 in one sample (P4552_105T). Three additional PAs presented with either KRAS, FGFR1 or other BRAF mutations (P4551_214T, P4552_109R, and P1148_110T, respectively), and one carried a rare QKI::RAF1 fusion (P4552_110T) [20], all leading to MAPK pathway activation.

Unique genomic findings previously reported in rare entities

One histopathologically diagnosed PA could not be assigned to a methylation class (P4551_219T). The tumor harbored an EWSR1::PATZ1 fusion, a rare alteration that appears to define a new type of glioneuronal tumor [43] (Table 1, Additional file 2: Table 1, Additional file 5: Fig. S1, Additional file 6: Fig. S2).

Fusions involving NTRK genes are clinically relevant alterations, leading to constitutionally active chimeric receptors with oncogenic potential [1]. We detected chromosomal rearrangements involving NTRK genes in ATRT, ganglioglioma and rhabdoid meningioma (one of each; samples P2233_115T, P7708_116T, and P2233_106T; Table 1, Additional file 2: Table S1). Furthermore, the cooccurrence of FGFR1 and PIK3CA hotspot mutations was observed in two tumors pathologically diagnosed as oligodendroglioma and ependymoma (P2233_109T, P7708_112T), and both were assigned to the methylation class low-grade glioma, rosette-forming glioneuronal tumors. These findings align with previous observations indicating that recurrent and combined genetic alterations affecting both MAPK and PI3K signaling pathways appear to interact synergistically during the development of these rare neoplasms [44]. Other interesting alterations involving FGFR1 detected in this series of tumors were two FGFR1::TACC1 fusions found in one anaplastic PA (P4551_218T) and one oligodendroglioma (P7708_105T). While the methylation profiling agreed with the histopathological diagnosis in the first sample, no match was found for the second sample (Table 1, Additional file 2: Table S1).

Additional findings derived from the sequencing data that also align with the molecular alterations described within the specific tumor methylation classes included an internal BCOR gene tandem duplication, identified in a CNS high-grade neuroepithelial tumor with a BCOR alteration (HGNET-BCOR), diagnosed as ependymoma (P7708_110T); SVs in the vicinity of FOXR2 in a tumor assigned to the methylation class CNS neuroblastoma with FOXR2 activation, diagnosed as PNET (P4551_206R); and focal amplifications of the C19MC locus at 19q13.42, involving TTYH1 in two embryonal tumors with multilayered rosettes (ETMR), diagnosed as PNET and ependymoblastoma, respectively (P7708_101T, P4551_201T; Additional file 2: Table S1, Additional file 5: Fig. S1, Additional file 6: Fig. S2). It is noteworthy to mention that the updated 2016 WHO classification of CNS tumors no longer recognizes PNET as a distinct entity. Molecular analysis has revealed that many tumors previously reported as PNET are now reclassified as defined CNS tumor entities with specific genetic characteristics.

Previously unreported alterations observed here

In addition to revealing somatic mutations in a broad spectrum of genes previously implicated in pediatric cancers, we discovered numerous previously unreported events, such as focal amplifications, including BORCS5 and LMO3 in an SHH-MB (FAM2T), an amplicon containing the PLAGL2 and POFUT1 genes in a sample diagnosed as MB (P4551_210T), GADL1::RBMS3 and SYNPO2::HELQ fusions in an ependymoma (P7708_109T) and a PLEKHA5::PIK3C2G fusion in a PNET (P2233_101T). These alterations might represent novel driver events (Table 1, Additional file 5: Fig. S1, Additional file 7: Fig. S3). Notably, two of these three tumors could not be classified by methylation arrays, and the third only with a low score, Additional file 2: Table S1.

Germline mutations

There is already a clinical demand for germline sequencing data for specific genes. TP53 status, for example, is of particular relevance, and patients carrying TP53 germline mutations are at high risk for developing secondary neoplasia following irradiation or DNA-damaging chemotherapy. TP53 germline mutations were observed in two patients, a splice donor variant in an SHH-MB (FAM2N) and a missense variant in a pathologically diagnosed rhabdoid meningioma (P2233_125N, with no matching methylation class). In both tumors, loss of the wild-type TP53 allele was observed (Additional file 2: Table S1).

Other relevant germline mutations included a SMARCB1 deletion in a patient with ATRT (P4551_227N, already discussed above), a novel stop gain mutation in TSC2 in a patient affected by subependymal giant cell astrocytoma (P1315_102N), a novel frameshift mutation in KDM4C accompanied by a somatic exon loss mutation in a child with MB group 3 (P2233_124N), and a DICER1 nonsense p.Arg509Ter, rs886037672 mutation in a patient with pineoblastoma (P2233_131N). The latter mutation is reported as pathogenic in ClinVar and known to predispose patients to several cancers. Somatic LOH was also observed in the tumor, indicating that DICER1 acts as a tumor suppressor gene in pineoblastoma (Additional file 2: Table S1).

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