Novel activating SNRNP70-ALK fusion in congenital infant-type hemispheric glioma displays clinical response to lorlatinib: a case-report

Cecilia Arthur, Kleopatra Georgantzi, Teresita Díaz de Ståhl, Jikui Guan, Blaz Oder, Cecilia Jylhä, Christopher Illies, Johanna Sandgren, Jan Svoboda, Jesper Eisfeldt, Gisela Barbany, Richard Rosenquist, Ulrika Sandvik, Daniel Hägerstrand, Bengt Hallberg, Ruth Palmer, Emma Tham
NPJ Precis Oncol. 2026 Feb 26;10(1):113. doi: 10.1038/s41698-026-01336-x.

Abstract. We report a child with an antenatally detected brain tumor that progressed over three years' time despite surgery, chemo- and proton therapy. Retrospective whole-genome and transcriptome sequencing with methylation analysis of primary tumor tissue led to the molecular diagnosis infant-type hemispheric glioma, and identified a novel SNRNP70::ALK fusion, providing a therapeutic target for compassionate-use precision treatment with the ALK tyrosine kinase inhibitor lorlatinib. Functional studies confirmed the fusion protein to be expressed and active in the patient's tumor. After two years of therapy, the child has sustained partial tumor regression on MRI and no new neurological symptoms. We conclude that comprehensive multi-omics analyses are required for correct molecular diagnosis in childhood CNS tumors and can radically impact patient outcome by identifying molecular targets for precision treatment.

Pediatric brain tumor classification using digital pathology and deep learning: Evaluation of SOTA methods on a multi-center Swedish cohort

Iulian Emil Tampu, Per Nyman, Christoforos Spyretos, Ida Blystad, Alia Shamikh, Gabriela Prochazka, Teresita Díaz de Ståhl, Johanna Sandgren, Peter Lundberg, Neda Haj-Hosseini.
Brain Pathol. 2026 Jan;36(1):e70029. doi: 10.1111/bpa.70029.

Abstract. Brain tumors are the most common solid tumors in children and young adults, but the scarcity of large histopathology datasets has limited the application of computational pathology in this group. This study implements two weakly supervised multiple-instance learning (MIL) approaches on patch features obtained from state-of-the-art histology-specific foundation models to classify pediatric brain tumors in hematoxylin and eosin whole slide images (WSIs) from a multi-center Swedish cohort. WSIs from 540 subjects (age 8.5 ± 4.9 years) diagnosed with brain tumors were gathered from the six Swedish university hospitals. Instance (patch)-level features were obtained from WSIs using three pre-trained feature extractors: ResNet50, UNI, and CONCH. Instances were aggregated using attention-based MIL (ABMIL) or clustering-constrained attention MIL (CLAM) for patient-level classification. Models were evaluated on three classification tasks based on the hierarchical classification of pediatric brain tumors: tumor category, family, and type. Model generalization was assessed by training on data from two of the centers and testing on data from four other centers. Model interpretability was evaluated through attention mapping. The highest classification performance was achieved using UNI features and ABMIL aggregation, with Matthew's correlation coefficient of 0.76 ± 0.04, 0.63 ± 0.04, and 0.60 ± 0.05 for tumor category, family, and type classification, respectively. When evaluating generalization, models utilizing UNI and CONCH features outperformed those using ResNet50. However, the drop in performance from the in-site to out-of-site testing was similar across feature extractors. These results show the potential of state-of-the-art computational pathology methods in diagnosing pediatric brain tumors at different hierarchical levels with fair generalizability on a multi-center national dataset.

Systems-level immunomonitoring in children with solid tumors to enable precision medicine

Qi Chen, Binbin Zhao, Ziyang Tan, Gustav Hedberg, Jun Wang, Laura Gonzalez, Constantin Habimana Mugabo, Anette Johnsson, Erika Negrini, Laura Piñero Páez, Lucie Rodriguez, Anna James, Yang Chen, Jaromír Mikeš, Anna Karin Bernhardsson, Stefan Markus Reitzner, Ferdinand von Walden, Olivia O’Neill, Hugo Barcenilla, Chunlin Wang, Mark M. Davis, Lena-Maria Carlson, Niklas Pal, Klas Blomgren, Dirk Repsilber, Nikolas Herold, Tadepally Lakshmikanth, Per Kogner, Linda Ljungblad, Petter Brodin.
Cell, Volume 188, Issue 5, 2025, Pages 1425-1440. https://doi.org/10.1016/j.cell.2024.12.014.

Abstract. Cancer is the leading cause of death from disease in children. Survival depends not only on surgery, cytostatic drugs, and radiation but also on systemic immune responses. Factors influencing these immune responses in children of different ages and tumor types are unknown. Novel immunotherapies can enhance anti-tumor immune responses, but few children have benefited, and markers of effective responses are lacking. Here, we present a systems-level analysis of immune responses in 191 children within a population-based cohort with diverse tumors and reveal that age and tumor type shape immune responses differently. Systemic inflammation and cytotoxic T cell responses correlate with tumor mutation rates and immune cell infiltration. Clonally expanded T cell responses are rarely detected in blood or tumors at diagnosis but are sometimes elicited during treatment. Expanded T cells are similarly regulated in children and adults with more immunogenic cancers. This research aims to facilitate the development of precision immunotherapies for children with cancer.

Genomic tumor evolution dictates human medulloblastoma progression

Ruchiy Y, Tsea I, Preka E, Verhoeven BM, Olsen TK, Mei S, Sinha I, Blomgren K, Carlson LM, Dyberg C, Johnsen JI, Baryawno N. Neurooncol Adv. 2024 Oct 5;6(1):vdae172. doi: 10.1093/noajnl/vdae172. PMID: 39659836; PMCID: PMC11629688.

Background: Medulloblastoma (MB) is the most common high-grade pediatric brain tumor, comprised of 4 main molecular subgroups-sonic-hedgehog (SHH), Wnt, Group 3, and Group 4. Group 3 and Group 4 tumors are the least characterized MB subgroups, despite Group 3 having the worst prognosis (~50% survival rate), and Group 4 being the most prevalent. Such poor characterization can be attributed to high levels of inter- and intratumoral heterogeneity, making it difficult to identify common therapeutic targets.

Methods: In this study, we generated single-cell sequencing data from 14 MB patients spanning all subgroups that we complemented with publicly available single-cell data from Group 3 patients. We used a ligand-receptor analysis tool (CellChat), expression- and allele-based copy-number variation (CNV) detection methods, and RNA velocity analysis to characterize tumor cell-cell interactions, established a connection between CNVs and temporal tumor progression, and unraveled tumor evolution.

Results: We show that MB tumor cells follow a temporal trajectory from those with low CNV levels to those with high CNV levels, allowing us to identify early and late markers for SHH, Group 3, and Group 4 MBs. Our study also identifies SOX4 upregulation as a major event in later tumor clones for Group 3 and Group 4 MBs, suggesting it as a potential therapeutic target for both subgroups.

Conclusions:Taken together, our findings highlight MB’s inherent tumor heterogeneity and offer promising insights into potential drivers of MB tumor evolution particularly in Group 3 and Group 4 MBs.

The potential of liquid biopsy for detection of the KIAA1549-BRAF fusion in circulating tumor DNA from children with pilocytic astrocytoma

Krynina O, de Ståhl TD, Jylhä C, Arthur C, Giraud G, Nyman P, Fritzberg A, Sandgren J, Tham E, Sandvik U.  Neurooncol Adv. 2024 Jan 24;6(1):vdae008 | https://doi.org/10.1016/j.lanepe.2024.100881

Background: Low-grade gliomas (LGGs) represent children’s most prevalent central nervous system tumor, necessitating molecular profiling to diagnose and determine the most suitable treatment. Developing highly sensitive screening techniques for liquid biopsy samples is particularly beneficial, as it enables the early detection and molecular characterization of tumors with minimally invasive samples.

Methods: We examined CSF and plasma samples from patients with pilocytic astrocytoma (PA) using custom multiplexed droplet digital polymerase chain reaction (ddPCR) assays based on whole genome sequencing data. These assays included a screening test to analyze BRAF duplication and a targeted assay for the detection of patient-specific KIAA1549::BRAF fusion junction sequences or single nucleotide variants.

Results: Our findings revealed that 5 out of 13 individual cerebrospinal fluid (CSF) samples tested positive for circulating tumor DNA (ctDNA). Among these cases, 3 exhibited the KIAA1549::BRAF fusion, which was detected through copy number variation (CNV) analysis (n = 1) or a fusion-specific probe (n = 2), while 1 case each displayed the BRAF V600E mutation and the FGFR1 N577K mutation. Additionally, a quantitative analysis of cell-free DNA (cfDNA) concentrations in PA CSF samples showed that most cases had low cfDNA levels, below the limit of detection of our assay (<1.9 ng).

Conclusions: While CNV analysis of CSF samples from LGGs still has some limitations, it has the potential to serve as a valuable complementary tool. Furthermore, it can also be multiplexed with other aberrations, for example, to the BRAF V600 test, to provide important insights into the molecular characteristics of LGGs.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors – a nationwide, prospective Swedish study

Bianca Tesi, Kristina Lagerstedt Robinson, Frida Abel,Teresita Díaz de Ståhl, Sara Orrsjö, Anna Poluha,Maria Hellberg, Sandra Wessman, Sofie Samuelsson, Tony Frisk, Hartmut Vogt, Karin Henning, Magnus Sabel, Torben Ek, Niklas Pal, Per Nyman, Geraldine Giraud et al. The Lancet Regional Health Europe, March 2024 | https://doi.org/10.1016/j.lanepe.2024.100881

Abstract. Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors.

Implementing data on targeted therapy from the INFORM registry platform for children with relapsed cancer in Sweden

Wallin S, Øra I, Prochazka G, Sandgren J, Björklund C, Ljungman G, Vogt H, Ek T, Van Tilburg C, Nilsson A. Front. Oncol. Sec. Pediatric Oncology Volume 14 – 2024 | doi: 10.3389/fonc.2024.1340099

Abstract. Advances in treatment of childhood malignancies have improved overall cure rates to 80%. Nevertheless, cancer is still the most common cause of childhood mortality in Sweden. The prognosis is particularly poor for relapse of high-risk malignancies. In the international INFORM registry, tumor tissue from patients with relapsed, refractory, or progressive pediatric cancer as well as from very-high risk primary tumors is biologically characterized using next-generation sequencing to identify possible therapeutic targets. We analyzed data from Swedish children included in the INFORM registry concerning patient characteristics, survival, sequencing results and whether targeted treatment was administered to the children based on the molecular findings.Sweden during 2016-2021.The most common diagnoses were soft tissue and bone sarcomas followed by high grade gliomas [including diffuse intrinsic pontine glioma (DIPG)]. Complete molecular analysis was successful for 203/212 samples originating from 184 patients. In 88% of the samples, at least one actionable target was identified. Highly prioritized targets, according to a preset scale, were identified in 48 (24%) samples from 40 patients and 24 of these patients received matched targeted treatment but only six children within a clinical trial. No statistically significant benefit in terms of overall survival or progression free survival was observed between children treated with matched targeted treatment compared to all others.This international collaborative study demonstrate feasibility regarding sequencing of pediatric high-risk tumors providing molecular data regarding potential actionable targets to clinicians. For a few individuals the INFORM analysis was of utmost importance and should be regarded as a new standard of care with the potential to guide targeted therapy.

Diagnostic Yield From a Nationwide Implementation of Precision Medicine for all Children With Cancer

Wadensten E, Wessman S, Abel F, Diaz De Ståhl T, Tesi B, Orsmark Pietras C, Arvidsson L, Taylan F, Fransson S, Vogt H, Poluha A, Pradhananga S, Hellberg M, Lagerstedt-Robinson K, Raj Somarajan P, Samuelsson S, Orrsjö S, Maqbool K, Henning K, Strid T, Ek T, Fagman H, Olsson Bontell T, Martinsson T, Puls F, Kogner P, Wirta V, Pronk CJ, Wille J, Rosenquist R, Nistér M, Mertens F, Sabel M, Norén-Nyström U, Grillner P, Nordgren A, Ljungman G, Sandgren J, Gisselsson D JCO Precis Oncol. 2023 Jun;7:e2300039. doi: 10.1200/PO.23.00039. PMID: 37384868; PMCID: PMC10581599.

Purpose: Several studies have indicated that broad genomic characterization of childhood cancer provides diagnostically and/or therapeutically relevant information in selected high-risk cases. However, the extent to which such characterization offers clinically actionable data in a prospective broadly inclusive setting remains largely unexplored.

Methods: We implemented prospective whole-genome sequencing (WGS) of tumor and germline, complemented by whole-transcriptome sequencing (RNA-Seq) for all children diagnosed with a primary or relapsed solid malignancy in Sweden. Multidisciplinary molecular tumor boards were set up to integrate genomic data in the clinical decision process along with a medicolegal framework enabling secondary use of sequencing data for research purposes.

Results: During the study’s first 14 months, 118 solid tumors from 117 patients were subjected to WGS, with complementary RNA-Seq for fusion gene detection in 52 tumors. There was no significant geographic bias in patient enrollment, and the included tumor types reflected the annual national incidence of pediatric solid tumor types. Of the 112 tumors with somatic mutations, 106 (95%) exhibited alterations with a clear clinical correlation. In 46 of 118 tumors (39%), sequencing only corroborated histopathological diagnoses, while in 59 cases (50%), it contributed to additional subclassification or detection of prognostic markers. Potential treatment targets were found in 31 patients (26%), most commonly ALK mutations/fusions (n = 4), RAS/RAF/MEK/ERK pathway mutations (n = 14), FGFR1 mutations/fusions (n = 5), IDH1 mutations (n = 2), and NTRK2 gene fusions (n = 2). In one patient, the tumor diagnosis was revised based on sequencing. Clinically relevant germline variants were detected in 8 of 94 patients (8.5%).

Conclusion: Up-front, large-scale genomic characterization of pediatric solid malignancies provides diagnostically valuable data in the majority of patients also in a largely unselected cohort.

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

Díaz de Ståhl T, Shamikh A, Mayrhofer M, Juhos S, Basmaci E, Prochazka G, Garcia M, Somarajan PR, Zielinska-Chomej K, Illies C, Øra I, Siesjö P, Sandström PE, Stenman J, Sabel M, Gustavsson B, Kogner P, Pfeifer S, Ljungman G, Sandgren J, Nistér M. J Transl Med. 2023 May 23;21(1):342. doi: 10.1186/s12967-023-04178-4. PMID: 37221626; PMCID: PMC10204274.

Abstract. The Swedish Childhood Tumor Biobank (BTB) is a nonprofit national infrastructure for collecting tissue samples and genomic data from pediatric patients diagnosed with central nervous system (CNS) and other solid tumors. The BTB is built on a multidisciplinary network established to provide the scientific community with standardized biospecimens and genomic data, thereby improving knowledge of the biology, treatment and outcome of childhood tumors. As of 2022, over 1100 fresh-frozen tumor samples are available for researchers. We present the workflow of the BTB from sample collection and processing to the generation of genomic data and services offered. To determine the research and clinical utility of the data, we performed bioinformatics analyses on next-generation sequencing (NGS) data obtained from a subset of 82 brain tumors and patient blood-derived DNA combined with methylation profiling to enhance the diagnostic accuracy and identified germline and somatic alterations with potential biological or clinical significance. The BTB procedures for collection, processing, sequencing, and bioinformatics deliver high-quality data. We observed that the findings could impact patient management by confirming or clarifying the diagnosis in 79 of the 82 tumors and detecting known or likely driver mutations in 68 of 79 patients. In addition to revealing known mutations in a broad spectrum of genes implicated in pediatric cancer, we discovered numerous alterations that may represent novel driver events and specific tumor entities. In summary, these examples reveal the power of NGS to identify a wide number of actionable gene alterations. Making the power of NGS available in healthcare is a challenging task requiring the integration of the work of clinical specialists and cancer biologists; this approach requires a dedicated infrastructure, as exemplified here by the BTB.

Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma

Arthur C, Jylhä C, de Ståhl TD, Shamikh A, Sandgren J, Rosenquist R, Nordenskjöld M, Harila A, Barbany G, Sandvik U, Tham E. Cancers (Basel). 2023 Mar 25;15(7):1972. doi: 10.3390/cancers15071972. PMID: 37046633; PMCID: PMC10092983.

Abstract. Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.

Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples

Mirzazadeh R, Andrusivova Z, Larsson L, Newton PT, Galicia LA, Abalo XM, Avijgan M, Kvastad L, Denadai-Souza A, Stakenborg N, Firsova AB, Shamikh A, Jurek A, Schultz N, Nistér M, Samakovlis C, Boeckxstaens G, Lundeberg J. Nat Commun. 2023 Jan 31;14(1):509.

Abstract: Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.

Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants

Garcia M, Juhos S, Larsson M, Olason PI, Martin M, Eisfeldt J, DiLorenzo S, Sandgren J, Díaz De Ståhl T, Ewels P, Wirta V, Nistér M, Käller M, Nystedt B. F1000Res. 2020 Jan 29;9:63.

Abstract. Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.

Spatially resolved clonal copy number alterations in benign and malignant tissue

Erickson A, He M, Berglund E, Marklund M, Mirzazadeh R, Schultz N, Kvastad L, Andersson A, Bergenstråhle L, Bergenstråhle J, Larsson L, Alonso Galicia L, Shamikh A, Basmaci E, Díaz De Ståhl T, Rajakumar T, Doultsinos D, Thrane K, Ji AL, Khavari PA, Tarish F, Tanoglidi A, Maaskola J, Colling R, Mirtti T, Hamdy FC, Woodcock DJ, Helleday T, Mills IG, Lamb AD, Lundeberg J. 

Nature. 2022 Aug;608(7922):360-367. doi: 10.1038/s41586-022-05023-2. Epub 2022 Aug 10. PMID: 35948708; PMCID: PMC9365699.

Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.

Amplification of ERBB2 (HER2) in embryonal rhabdomyosarcoma: A potential treatment target in rare cases?

Mitra S, Sydow S, Magnusson L, Piccinelli P, Törnudd L, Øra I, Ljungman G, Sandgren J, Gisselsson D, Mertens F. Genes Chromosomes Cancer. 2022 Jan;61(1):5-9. doi: 10.1002/gcc.22996. Epub 2021 Sep 1. PMID: 34418214.

Abstract: The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target.