Undifferentiated sarcomas (USrc) are aggressive mesenchymal tumors that do not show characteristic features and therefore remain a diagnosis of exclusion. USrc typically have a low tumor mutational burden but are genomically complex, carrying many large structural variants (SVs) and copy number aberrations (CNAs). Improving diagnostic practice by adding novel whole genome-technologies based on high molecular weight DNA could increase the sensitivity for detecting genetic alterations in USrc, allowing identification of recurrent aberrations or patterns with clinical implications. Snap-frozen tissue from 41 USrc in 38 patients were analyzed by both conventional karyotyping and Optical Genome Mapping (OGM). Chromothripsis was determined based on OGM data and defined as more than eight copy number segments with oscillating copy number in co-occurrence with more than eight chromosomal rearrangements within 50 Mb, or more than 20 rearrangements within 50 Mb. These rearrangements should be interleaved and approximately half of them should be inversion-type aberrations. In addition, we identify known cancer genes (COSMIC Cancer Gene Census) which are amplified/lost or show evidence of disruptive SVs. DNA methylation profiling using EPIC arrays was done on 15 USrc followed by sarcoma classification with the Deutsches Krebsforschungszentrum (DKFZ) sarcoma classifier (v12.3). 32 out of 41 USrc karyotypes showed at least subclonal whole genome-doubling with complex patterns of SVs and CNAs, including double minutes, ring chromosomes and giant markers. OGM was performed on all USrc biopsies yielding an average coverage of 352X. 25 out of 38 patient samples presented with chromothripsis with an average of 4.3 chromosomes affected (1-13 chromosomes). 70% of amplifications (CN greater than or equal to 5) and 83% of high, focal amplifications (CN greater than or equal to 10) occurred in regions with chromothripsis. Aberrations in 590 COSMIC cancer genes were detected. The most frequently altered cancer genes were CDKN2A (42%), RB1 (34%), PTPRD (29%), MGMT (26%), and ARHGEF12 (24%). For 3 patients, biopsies from two different time points were profiled, showing a high proportion of shared aberrations. DNA methylation patterns classified 8 out of 15 analyzed samples as USrc, and the remaining 7 showed no match to any reference sarcoma group. Copy number profiles derived from OGM and DNA methylation arrays were near identical. MGMT promotor hypermethylation was observed in 5 out of 16 cases. Implementation of OGM improves the detection of SVs and CNAs over standard-of-care. This is demonstrated by the detection of chromothripsis regions and novel recurrent aberrations in USrc. Further investigation to confirm these findings and to identify single nucleotide variants and allele-specific copy numbers are currently ongoing using long-read HiFi sequencing (PacBio). Jef Baelen, Raphael Sciot, Ruben Cools, Jonas Demeulemeester, Isabelle Vanden Bempt. Deep genomic analysis of undifferentiated sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7177.

Soft-tissue and bone tumors represent a heterogeneous group of tumors encompassing more than 100 histologic subtypes today. Identifying genetic aberrations increasingly is important in these tumors for accurate diagnosis. Although gene mutations typically are detected by second-generation sequencing, the identification of structural variants (SVs) and copy number alterations (CNAs) remains challenging and requires various cytogenetic techniques including karyotyping, fluorescence in situ hybridization, and arrays, each with important limitations. Optical Genome Mapping (OGM), a non-sequencing-based technique for high-resolution detection of SVs and CNAs, was applied in a retrospective series of diagnostic soft-tissue and bone tumor samples. Sample preparation was successful in 38 of 53 cases, with the highest success rate in nonadipocytic soft-tissue tumors (24 of 27 cases; 89%). In 32 of 35 cases carrying a diagnostic SV or CNA, OGM identified the aberration (91%), including a POU2AF3::EWSR1 fusion in a round cell sarcoma and a translocation t(1;5)(p22;p15) in a myxoinflammatory fibroblastic sarcoma. Interestingly, OGM shed light on the genomic complexity underlying the various aberrations. In five samples, OGM showed that chains of rearrangements generated the diagnostic fusion, three of which involved chromoplexy. In addition, in nine samples, chromothripsis was causal to the formation of giant marker/ring/double-minute chromosomes. Finally, compared with standard-ofcare cytogenetics, OGM revealed additional aberrations, requiring further investigation of their potential clinical relevance. (J Mol Diagn 2024, 26: 374-386; https://doi.org/10.1016/ j.jmoldx.2024.02.003)

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA-based sequencing (targeted RNA-seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA-seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA-seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA-seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA-seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH-positive analyses showed a lower percentage of rearrangement-positive nuclei (range 15-41%) compared to the concordant FISH-positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA-seq findings. For the EWSR1 FISH probe, we observed a gene-dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA-seq (22.9%). This study demonstrates an added value of targeted RNA-seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA-seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

Introduction The ETV6::NTRK3 fusion is the most common gene alteration in infantile fibrosarcoma, a soft tissue tumor affecting patients under two years of age. Less frequently, these tumors harbor fusions of genes encoding other kinases, such as BRAF, which activates MEK in the mitogen-activated protein kinase pathway. The identification and characterization of these oncogenes are crucial to facilitate diagnosis, validate new treatments, and better understand the pathophysiology of these neoplasms. Methods Herein, we analyzed an ETV6::NTRK3-negative infantile fibrosarcoma from a 5-day-old patient by RNA-sequencing to identify new fusion transcripts. Functional exploration of the fusion of interest was performed by in vitro assays to study its activity, oncogenicity, and sensitivity to the MEK inhibitor trametinib. Results We identified a novel fusion involving the PHIP and BRAF genes. The corresponding fusion protein constitutively activated the mitogen-activated protein kinase pathway, resulting in fibroblast transformation. Treatment of transfected cells with trametinib effectively inhibited signaling by PHIP::BRAF. Conclusion PHIP::BRAF is a novel fusion oncogene that can be targeted by trametinib in infantile fibrosarcoma.

AbstractIntroductionTheETV6::NTRK3fusion is the most common gene alteration in infantile fibrosarcoma, a soft tissue tumor affecting patients under two years of age. Less frequently, these tumors harbor fusions of genes encoding other kinases, such asBRAF, which activates MEK in the mitogen‐activated protein kinase pathway. The identification and characterization of these oncogenes are crucial to facilitate diagnosis, validate new treatments, and better understand the pathophysiology of these neoplasms.MethodsHerein, we analyzed anETV6::NTRK3‐negative infantile fibrosarcoma from a 5‐day‐old patient by RNA‐sequencing to identify new fusion transcripts. Functional exploration of the fusion of interest was performed byin vitroassays to study its activity, oncogenicity, and sensitivity to the MEK inhibitor trametinib.ResultsWe identified a novel fusion involving thePHIPandBRAFgenes. The corresponding fusion protein constitutively activated the mitogen‐activated protein kinase pathway, resulting in fibroblast transformation. Treatment of transfected cells with trametinib effectively inhibited signaling byPHIP::BRAF.ConclusionPHIP::BRAFis a novel fusion oncogene that can be targeted by trametinib in infantile fibrosarcoma.

The etiology of Langerhans cell histiocytosis (LCH), a disease characterized by uncontrolled proliferation of Langerhans cells, is unknown. Although some believe that LCH is reactive, others support a neoplastic origin. We tested the hypothesis that LCH is neoplastic by investigating potential consistent chromosomal aberrations in LCH cells. We used multiparameter DNA flow cytometry to analyze the DNA ploidy LCH cells in 20 cases, performed karyotype analysis in 31 cases, array-based comparative genomic hybridization (arrayCGH) and single nucleotide polymorphism (SNP) arrays with DNA from flow-sorted CD1a-positive and CD1a-negative cells in 19 cases. Ploidy analysis revealed diploid DNA content in all cases. The karyotype of all patients analyzed was normal, excluding the presence of balanced translocations. ArrayCGH and SNP arrays did not show genome abnormalities. Despite positive TP53 protein immunohistochemical staining, sequencing of exon 5 to 8 of p53 gene showed no alterations in 7 cases. This study strongly suggests that gross chromosomal abnormalities do not cause LCH. Although we cannot exclude cryptic point mutations in as yet unidentified genes, this study of 72 LCH cases shows that LCH may be the result of restricted oligoclonal stimulation rather than unlimited neoplastic proliferation. (c) 2008 Wiley-Liss, Inc.

Myxofibrosarcoma is one of the most frequent soft tissue tumours in elderly patients, mostly arising in the extremities. Grade I lesions are only locally aggressive whereas grade II and grade III lesions have metastatic potential. The differential diagnosis contains several other (benign) myxoid soft tissue tumours. A number of sarcomas are characterised by specific cytogenetic aberrations, giving not only insight in their biological pathways; they also serve as molecular markers in difficult diagnoses. Cytogenetic data on myxofibrosarcoma are scarce with only few isolated cases described in the literature. No specific chromosomal aberrations have been detected so far. Moreover, molecular pathways in tumorigenesis and progression of myxofibrosarcoma are barely understood. We studied the clinicopathologic data and karyotypes of 32 myxofibrosarcomas using conventional banding and multicolour COmbined Binary RAtio labelling fluorescence in situ hybridisation technique. We included eight grade I, eight grade II and 16 grade III lesions. In all, 22 were primary tumours, nine were local recurrences and one a lymph node metastasis. The myxofibrosarcomas showed equal sex distribution, were mostly located at the extremities with two thirds deep-seated and had an average age of occurrence of 66 years. We found normal karyotypes in eight cases and clonal beside nonclonal aberrations in 22 cases. Complex cytogenetic anomalies were found in all grades. However, no tumour-specific chromosomal abnormalities could be withdrawn. Local recurrences showed increase in grade compared to their primary lesions. Interestingly, these recurrences showed more complex cytogenetic aberrations. Increase in grade seems to parallel increase in cytogenetic aberrations and malignant potential. Since the chromosomal aberrations found were not tumour type specific, they seem to be rather the result of secondary events in tumour progression and tumour genetic instability. Based on these findings, we suggest that tumorigenesis of myxofibrosarcoma is mainly a multistep genetic process, probably ruled by genetic instability caused by targeted checkpoint genes.

Infection of hamsters with the murine flavivirus Modoc results in (meningo) encephalitis, which is, during the acute phase, frequently associated with flaccid paralysis, as also observed in patients with West Nile virus encephalitis. Twenty percent of the hamsters that recover from the acute encephalitis develop life-long neurological sequelae, reminiscent of those observed, for example, in survivors of Japanese encephalitis. Magnetic resonance imaging and histology revealed severe lesions predominantly located in the olfactory-limbic system, both in hamsters with acute encephalitis as in survivors. Prominent pathology was also detected in the spinal cord of hamsters with paralysis. Modoc virus infections in hamsters provide a unique model for the study of encephalitis, a poliomyelitis-like syndrome and neurological sequelae following flavivirus infection.

Intravascular lymphomatosis is a rare variant of non-Hodgkin's lymphoma with an unusual predilection for the central nervous system. Most cases are not diagnosed until postmortem because of variable clinical presentation and non-specific laboratory findings. We studied a 41-year-old lady who presented with progressive neurological symptoms. MR showed multifocal grey and white matter lesions. Brain biopsy showed an intravascular lymphoma of the B-cell lineage. This report illustrates the ischaemic origin of the radiological lesions, as they are all hyperintense on the diffusion-weighted images. This has, to our knowledge, never been published before. MR did not show any enhancement after intravenous Gadolinium-DTPA (parenchymal or meningeal)which is a very uncommon finding in this entity. Intravascular lymphomatosis should be taken into account in the differential diagnosis of repeated cerebral ischemia of unclear aetiology.

Immunohistochemical demonstration of the MIC2 gene product (CD99) represents a valuable diagnostic tool that makes identification of peripheral primitive neuroectodermal tumors easier. Different commercially available antibodies have been raised that show a high sensitivity along with a specificity that is widely regarded as acceptable although not absolute. MIC2 expression has been reported in lymphoblastic lymphoma, occasionally in several other types of sarcoma, and in some neuroendocrine tumors and ependymomas. The recent personal observation of immunopositivity for MIC2 gene product in some synovial sarcomas that had been difficult to diagnose prompted us to undertake a systematic study of a larger series of 50 synovial sarcomas, of which 62% showed positive immunoreactivity for either 013 (Signet) or 12E7 (Dako MIC2). This is the highest incidence of CD99 positivity reported to date in any type of sarcoma other than the Ewing's/primitive neuroectodermal tumor group. Immunopositivity for CD99 was common in all morphologic subtypes of synovial sarcoma. As poorly differentiated synovial sarcoma may overlap morphologically with primitive neuroectodermal tumor, careful histologic examination along with evaluation of a wider panel of differentiation markers is mandatory to avoid this potential diagnostic pitfall, which has both therapeutic and prognostic implications.

Molecular analysis of a new series of synovial sarcomas confirms that t(X;18)(p11.2;q11.2) breakpoints occur at two distinct regions on Xp designated SS1 and SS2. Breakpoint position correlates with tumor phenotype. Monophasic tumors with no evidence of glandular components have breakpoints within the SS2 region in Xp11.21, and biphasic tumors with a focal poorly differentiated or extensive glandular structure have breakpoints within the SS1 region in Xp11.23.

Abtract We report on the case of a young woman, presenting with a left lateral neck lump, which turned out to be a bony callus originating from the thyroid cartilage; similar but smaller lesions were found on the same side in the hyoid and cricoid cartilages. The CT, MRI and pathological findings are described.