Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the TTNtv+ samples. Full-length titin was reduced in TTNtv+ compared with TTNtv- samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

IntroductionDedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes.Materials and Methods131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines.ResultsOut of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant.ConclusionsWe detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

Thyroid-like follicular renal cell carcinoma (TLFRCC), an emerging subtype of renal cell carcinoma, presents diagnostic challenges due to its resemblance to normal thyroid tissue. Here, we report a rare case of TLFRCC in a pediatric patient, a demographic rarely affected by this subtype. Histologically resembling a typical TLFRCC, our case exhibited unique features including post-neuroblastoma development, occurrence in a male teenager, and diffuse MelanA expression, which has not been previously reported in TLFRCC. Comprehensive genomic profiling revealed the EWSR1::PATZ1 fusion, confirming its genetic basis. Due to the advanced tumor stage, the patient received combined immunotherapy, and after a 9-month follow-up, remains tumor-free. Our case broadens the diagnostic spectrum of pediatric renal cell carcinomas, highlighting the importance of comprehensive molecular profiling in rare subtypes such as TLFRCC. Further research is needed to better understand TLFRCC’s genetic landscape and optimize therapeutic strategies, especially in pediatric populations with evolving treatment protocols.

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the “gold standard” fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR -amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR -amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR -amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.

Background Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. Methods Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. Results CNAs were detected in 93.8% ( n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status ( IKZF1 normal , IKZF1 del and IKZF1 plus ) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low ( n = 215): 86.3%, IKAROS-medium ( n = 27): 57.4% and IKAROS-high ( n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients ( n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients ( n = 103) showed high ( n = 74) or ultra-poor ( n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). Conclusions PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the promoter region of the TERT gene (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative lengthening of telomeres. We aimed to perform the first complex genetic analysis specifically concentrating on the latter subgroup, since the molecular properties including potential clinically relevant features are poorly characterized. Comprehensive genomic profiling (CGP) of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas was performed using the Illumina TruSight Oncology 500 targeted next-generation sequencing panel. ATRX and pTERT mutations were revealed to be mutually exclusive by CGP. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. EGFR amplification was relatively rare, while alterations of the RAS-MAPK pathway, including NF1 mutations and BRAF alterations, were more characteristic in the ATRX-deficient group. Several pathogenic or likely pathogenic variants of genes related to homologous recombination repair were detected in both groups, but with different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these showed a peculiar association of oligodendroglioma-like morphology, novel fusions involving the NTRK2 and LRRFIP2 genes, POLE mutations as well as therapy-induced MLH1 and PMS2 loss. The other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Apparent, statistically significant differences in the genetic characteristics of ATRX-deficient and ATRX-intact IDH-wildtype adult high-grade gliomas were revealed by our study. These observations suggest that tumors from the former group are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA-repair inhibitors.

ObjectiveAdministration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. MethodsNext-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. ResultsAfter data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. ConclusionsIn this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments.

Significant improvements in the survival rates of paediatric cancer have been achieved over the past decade owing to recent advances in therapeutic and diagnostic strategies. However, disease progression and relapse remain a major challenge for the clinical management of paediatric angiosarcoma. Comprehensive genomic profiling of these rare tumours using high-throughput sequencing technologies may improve patient stratification and identify actionable biomarkers for therapeutic intervention. Here, we describe the clinical, histopathological, immunohistochemical and molecular profile of a novel and precision medicine-informed case where a KHDRBS1-NTRK3 fusion determined by next-generation sequencing-based comprehensive genomic profiling led to complete and sustained remission (clinical and radiological response) in an otherwise incurable disease. Our patient represents the first paediatric angiosarcoma harbouring a targetable NTRK3 fusion in the literature and demonstrates the first example of targeting this alteration in angiosarcoma using larotrectinib, an NTRK inhibitor. Clinical and radiological remission was achieved in under two months of therapy, and the patient is currently in complete remission, 4 month after stopping larotrectinib therapy, which was given over 17 months with only mild side effects reported. Therefore, this remarkable case exemplifies the true essence of precision-based care by incorporating conventional pathology with the why, when, and how to test for rare oncogenic drivers and agnostic biomarkers in paediatric angiosarcoma.

With the advancement of molecular oncology, numerous new opportunities are available for the effective and efficient treatment of patients diagnosed with childhood brain tumors. This includes gene panel analysis aiding personalized treatment used in clinical trials, and the application of targeted therapy independent of tissue type (tumor agnostic therapy). Most personalized therapies inhibit certain kinases. In our review, we present the modern pathological diagnosis of childhood brain tumors, as well as the complex intracellular regulation of signal transduction pathways important from the point of view of clinical practice, and we describe their further targets defined on the basis of pharmacological characteristics of the pathway, based on international and our own results. Despite common mutations affecting kinases, personalized therapy is not available in many types of tumors. Through the example of childhood brain tumors, we demonstrate the expected future therapeutic significance of tyrosine kinases.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter ( pTERT ) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS–MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

Background: Scrutiny of genetic heterogeneity in follicular lymphoma (FL) patients has been mainly limited to the investigation of single-site biopsies in previous studies, while the potential of circulating cell-free DNA (cfDNA) to resolve intra-patient heterogeneity has not been fully exploited in FL. Therefore, we aimed to characterize spatiotemporal heterogeneity in relapsed/refractory (R/R) FL by performing comprehensive genomic profiling of spatially separated tissue samples with matching cfDNA samples collected throughout the disease course. Methods: We performed ultra-deep targeted sequencing of 63 tumor tissue DNA (tisDNA), 28 cfDNA and paired germline DNA samples collected from 22 R/R FL patients. Sequencing libraries were prepared using a SureSelectXT HS (Agilent, USA) panel encompassing 173 genes recurrently mutated in FL, as well as rearrangement regions for immunoglobulin V(D)J repertoire profiling. Sequencing was performed on a NovaSeq6000 or NextSeq2000 (Illumina, USA) platform reaching a median deduplicated depth of 5,419x and 1,695x for cfDNA and tisDNA samples, respectively. We called short somatic variants using GATK Mutect2 (v.4.1.7.0), copy number alterations using PureCN (v.1.20.0) and CNVKit (v.0.9.9), as well as immunoglobulin repertoire using the whole-mark module of ARResT/Interrogate. Results: Median 6 and 8 non-silent variants were detected in cfDNA and tisDNA samples, respectively, affecting KMT2D (52%), CREBBP (45%) and TNFRSF14 (33%) most frequently in both sample types. Non-silent variants were detected in 21 of 24 (87.5%) pretreatment cfDNA samples. Comparing tisDNA samples with matching pretreatment cfDNA samples median 63.6% of variants were shared, yet unique variants were identified in cfDNA in 13/20 sample pairs, involving a median 14.3% of the variants. Studying the phylogenetic evolution of tumor samples pointed to early branching in majority of the patients, with ubiquitously present mutations detected in all samples constituting a median 24.3% of variants, although late branching was also observed in a subset (2/22) of the patients with at least 50% of variants detected in all samples. Most conserved variants were detectable in KMT2D and CREBBP, with notable exceptions of a lower mutation prevalence, but a high conservation rate in ATP6V1B2 and STAT6 genes. Subclonal clustering of variants and hierarchy analysis of samples from patients (n=5) with 2 tisDNA collected at separate lymphoma sites and a matching pretreatment cfDNA indicated that spatially discordant variants formed the same subclone, that could be also recovered in cfDNA in 4/5 patients (80%). An additional patient presented with almost identical tisDNA samples, but further unique mutations in cfDNA (PAT-FL-20)(Figure 1.). Investigating the prognostic significance of ctDNA level in the relapsed setting revealed that higher ctDNA levels were significantly associated with poor response to therapy (p=0.023, Kruskal-Wallis test), yet when we stratified patients by the median pretreatment ctDNA level (1.44x10 5 haploid genome equivalent/mL plasma) progression free survival in the subsequent treatment line was not substantially different between the two arms (p=0.11, log-rank test). Conclusion: Based on our results, R/R FL is characterized by remarkable intra-patient genetic heterogeneity. Although the founder clone harboring pathogenic KMT2D and CREBBP mutations can be identified in majority of the samples, expansion of tumor subclones between lymphoma sites confers divergent evolution, leaving genetic alterations unidentified during single-site based genetic testing. We also provide preliminary evidence, that multitarget high-throughput sequencing of cfDNA can uncover spatially restricted subclones that play a role in disease progression in R/R FL and that pretreatment ctDNA level might have a predictive value in the relapsed setting of FL. Figure 1. Molecular alterations in FL patients with tisDNA samples from two separate lymphoma sites and a matching cfDNA. KMT2D and CREBBP variants show high conservation, yet further variants are often spatially restricted, which can be resolved by cfDNA profiling. Copy number alterations (CNA) show lower concordance, yet recurrent CNA-s can be recovered in a subset of the cases. Identical immunoglobulin heavy and light chain usage can be detected in all samples of 3 patients and in paired tisDNA in 2 patients.

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Advances in therapeutic strategies of pediatric acute lymphoblastic leukemia (ALL) improved 5-year survival rates; however, the clinical management of disease progression and relapse remains challenging. Profiling molecular markers with prognostic and/or predictive significance in individual patients is crucial for refined risk assessment and precise patient stratification, underlining the importance of comprehensive molecular characterization. Aims: To interrogate the genomic and transcriptomic landscape of Hungarian children diagnosed with ALL in order to identify alterations with prognostic and therapeutic relevance, and to facilitate a more advanced, risk-adapted therapy selection for clinical decision-making. Methods: Diagnostic bone marrow samples with a mean blast percentage of 78.4% were investigated from 180 patients diagnosed with B-ALL (n=150) or T-ALL (n=30). Additionally, samples drawn at the time of relapse were analyzed from 19 patients. Gene fusions were identified with the TruSight RNA Pan-Cancer Panel targeting 1,385 genes, while mutation screening was performed using a custom QIASeq Targeted DNA Panel covering 102 disease-relevant genes. Copy number alterations were screened with multiplex ligation-dependent probe amplification using SALSA P335, P202 and P383 probemixes. Measurable residual disease (MRD) monitoring was performed by flow cytometry. Results: Gene fusions were identified in 34.9% of B-ALL and 46.4% of T-ALL patients, with ETV6-RUNX1, STIL-TAL1, P2RY8-CRLF2 and TCF3-PBX1 being the most common alterations. Five novel fusions involving JAK2, PAX5, KMT2A and RUNX1 genes were also observed. Targeted mutation screening revealed a higher number of aberrations in T-ALL patients (B-ALL: 2.01±1.88 vs. T-ALL: 3.86±3.00; p<0.001). In B-ALL, variants most frequently affected RAS-pathway genes (KRAS 18.4%, NRAS 17.8% and FLT3 10.2%), while NOTCH1 (58.6%), PHF6 (27.5%), PTEN (17.2%) and WT1 (17.2%) were the most commonly altered genes in T-ALL. IKZF1 deletion was observed in 18.1% of B-ALL patients, with eight of them showing IKZF1plus genotype. The vast majority (75.0%) of UBA2 mutations accompanied ETV6-RUNX1 fusion. Besides all patients with BCR-ABL1 fusion harboring IKZF1 deletion, BCR-ABL1-like subtype showed significant association with IKZF1 deletions (p=0.028). NT5C2 mutations, undetectable at diagnosis were identified at relapse in three patients. All CCND3-mutant cases (n=9) were MRD-negative with flow cytometry at day 33 and only one patient had detectable blasts at day 78. NOTCH1 and WT1 mutation frequently co-occurred with MRD-positivity at days 33 and 78. Scrutiny of the dynamics of early therapy response from diagnosis to day 15 revealed a slower decrease of MRD in the presence of RUNX1 mutation with high variant allele frequency (‘high burden’, VAF≥25%; wild type vs. ‘high burden’ p=0.004). The dynamics of MRD decline from day 15 to day 78 differed in TP53-mutant patients: a slower response to therapy was observed in high burden cases (‘high burden’ vs. wild type, ‘high burden’ vs. ‘low burden’; p<0.001). Survival analysis showed significantly shorter 3-year overall and event-free survival in RUNX1 and/or TP53 ‘high burden’ patients (OS: 92.3% vs. 50.0%, p=0.0001; EFS: 87.0% vs. 50.0%, p=0.002). Summary/Conclusion: With novel gene fusions and uncovered associations between the dynamics of MRD decline, TP53 and RUNX1 high burden mutations in this real-world cohort, our dataset provides valuable, clinically relevant insights to the genomic and transcriptomic landscape of children diagnosed with ALL. Keywords: Pediatric, ALL, Prognosis, Mutation analysis

A 5-year-old male child was diagnosed with interdigitating dendritic cell sarcoma (IDCS) during his maintenance therapy for B-cell precursor acute lymphoblastic leukemia (B-ALL). Multiplex lymph node involvements of the neck were found by positron emission tomography CT (PET-CT). Treatments, including surgical and chemotherapy, resulted in complete remission. Four years later, systemic bone infiltration was discovered. Surgical resection of the IV rib and intensive chemotherapy led to a complete morphological remission, and allogeneic bone marrow transplantation was performed. Comprehensive genomic profiling of the formalin fixed the tumor tissue, and the cryopreserved leukemic cells revealed several common alterations and divergent clonal evolution with a novel MAP2K1 mutation of the IDCS, which is responsible for the trans-differentiation of the common lymphoid-committed tumor progenitor.

There is growing body of evidence supporting the role of germline mutations in the pathogenesis of pediatric central nervous system (CNS) tumors, and the widespread use of next-generation sequencing (NGS) panels facilitates their detection. Variants of the MUTYH gene are increasingly recognized as suspected germline background of various extraintestinal malignancies, besides their well-characterized role in the polyposis syndrome associated with biallelic mutations. Using a multigene NGS panel (Illumina TruSight Oncology 500), we detected one H3 G34V- and one H3 K27M-mutant pediatric high-grade diffuse glioma, in association with c.1178G>A (p.G393D) and c.916C>T (p.R306C) MUTYH variants, respectively. Both MUTYH mutations were germline, heterozygous and inherited, according to the subsequent genetic testing of the patients and their first-degree relatives. In the H3 K27M-mutant glioma, amplifications affecting the 4q12 region were also detected, in association with KDR-PDGFRA, KIT-PDGFRA, and KDR-CHIC2 fusions, previously unreported in this entity. Among 47 other CNS tumors of various histological types tested with the same NGS panel in our institution, only one adult glioblastoma harbored MUTYH mutation. Together with a single previous report, our data raises the possibility of an association between germline MUTYH mutations and CNS malignancies, particularly in pediatric histone H3-mutant gliomas.

Soft tissue sarcomas are a heterogeneous group of malignant tumours, accounting for the fifth most common paediatric cancer. Next-generation sequencing-based (NGS) comprehensive genomic profiling (CGP) of paediatric soft tissue sarcomas tumours associated with a poor prognosis can identify actionable biomarkers and determine prognostic and therapeutic stratification of patient management. Diagnostic samples of 20 patients diagnosed with various paediatric sarcomas were investigated using a CGP assay (Illumina TruSight Oncology 500). The identified single nucleotide variants, small insertions or deletions, copy number alterations, gene fusions, microsatellite stability status, and the tumour mutation burden were analysed. Using Clinical insight (Qiagen) and PierianDx (Pierian) software, actionable variants were determined. Our study revealed potentially actionable alterations for therapeutic intervention in 75% of cases (15/20). Novel variants were detected in several tumour entities which have not previously been published. All cases were characterised by low tumour mutation burden, with none of the cases characterised by microsatellite instability. The targetable gene fusions included three NTRK fusions, TFG-ROS1, ROS1-GIT2 and EML4-ALK fusions. The most frequent copy number alterations affected the CDK4, ALK and FGFR1 genes, identified in 28.6% (4/20), 14.3% (2/14) and 14.3% (2/14) of the cases, respectively. Single nucleotide variants were detected in 7 genes, including ALK, CHEK2, ESR1, FANCL, MET, NBN and NRAS, across six cases. Based on the NGS results, targeted therapy was prescribed in 5 cases (tazemetostat in epithelioid sarcoma, entrectinib in inflammatory myofibroblastic tumour and infantile fibrosarcoma, larotrectinib in angiosarcoma and infantile fibrosarcoma). At least partial remission was achieved in all patients receiving targeted therapies. In our study, routine application of the TSO500 CPG assay led to administration of targeted therapies in 25% of the patients with at least partial clinical response in this patient cohort.