Small cell osteosarcoma versus fusion-driven round cell sarcomas of bone: retrospective clinical, radiological, pathological, and (epi)genetic comparison with clinical implications.

Small cell osteosarcoma (SCOS), a variant of conventional high-grade osteosarcoma (COS), may mimic fusion-driven round cell sarcomas (FDRCS) by overlapping clinico-radiological and histomorphological/immunohistochemical characteristics, hampering accurate diagnosis and consequently proper therapy. We retrospectively analyzed decalcified formalin-fixed paraffin-embedded (FFPE) samples of 18 bone tumors primarily diagnosed as SCOS by methylation profiling, fusion gene analysis, and immunohistochemistry.In eight cases, the diagnosis of SCOS was maintained, and in 10 cases it was changed into FDRCS, including three Ewing sarcomas (EWSR1::FLI1 in two cases and no identified fusion gene in the third case), two sarcomas with BCOR alterations (KMT2D::BCOR, CCNB3::BCOR, respectively), three mesenchymal chondrosarcomas (HEY1::NCOA2 in two cases and one case with insufficient RNA quality), and two sclerosing epithelioid fibrosarcomas (FUS::CREBL3 and EWSR1 rearrangement, respectively).Histologically, SCOS usually possessed more pleomorphic cells in contrast to the FDRCS showing mainly monomorphic cellular features. However, osteoid was seen in the latter tumors as well, often associated with slight pleomorphism. Also, the immunohistochemical profile (CD99, SATB2, and BCOR) overlapped.Clinically and radiologically, similarities between SCOS and FDRCS were observed, with by imaging only minimal presence or lack of (mineralized) osteoid in most of the SCOSs.In conclusion, discrimination of SCOS, epigenetically related to COS, versus FDRCS of bone can be challenging but is important due to different biology and therefore therapeutic strategies. Methylation profiling is a reliable and robust diagnostic test especially on decalcified FFPE material. Subsequent fusion gene analysis and/or use of specific immunohistochemical surrogate markers can be used to substantiate the diagnosis.

Molecular analysis of cancer genomes in children with Lynch syndrome: Exploring causal associations.

Lynch syndrome (LS) predisposes to cancer in adulthood and is caused by heterozygous germline variants in a mismatch repair (MMR) gene. Recent studies show an increased prevalence of LS among children with cancer, suggesting a causal relationship. For LS-spectrum (LSS) cancers, including high-grade gliomas and colorectal cancer, causality has been supported by typical MMR-related tumor characteristics, but for non-LSS cancers, causality is unclear. We characterized 20 malignant tumors of 18 children with LS, including 16 non-LSS tumors. We investigated second hits, tumor mutational load, mutational signatures and MMR protein expression. In all LSS tumors and three non-LSS tumors, we detected MMR deficiency caused by second hit somatic alterations. Furthermore, these MMR-deficient tumors carried driver variants that likely originated as a consequence of MMR deficiency. However, in 13 non-LSS tumors (81%), a second hit and MMR deficiency were absent, thus a causal link between LS and cancer development in these children is lacking. These findings demonstrate that causality of LS in children with cancer, which can be determined by molecular tumor characterization, seems to be restricted to specific tumor types. Large molecular and epidemiological studies are needed to further refine the tumor spectrum in children with LS.

Case series on clinical applications of liquid biopsy in pediatric solid tumors: towards improved diagnostics and disease monitoring.

Background and aims: Solid tumors account for about 30% of all pediatric cancers. The diagnosis is typically based on histological and molecular analysis of a primary tumor biopsy. Liquid biopsies carry several advantages over conventional tissue biopsy. However, their use for genomic analysis and response monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. In this case series we discuss six clinical cases of children with a solid tumor for whom a liquid biopsy assay was performed and demonstrate the potential of liquid biopsy for future clinical decision making. Methods: We performed quantitative real-time PCR (RT-qPCR), droplet digital PCR (ddPCR) or reduced representation bisulphite sequencing of cell-free DNA (cfRRBS) on liquid biopsies collected from six pediatric patients with a solid tumor treated between 2017 and 2023 at the Princess Máxima Center for Pediatric Oncology in the Netherlands. Results were used to aid in clinical decision making by contribution to establish a diagnosis, by prognostication and response to therapy monitoring. Results: In three patients cfRRBS helped to establish the diagnosis of a rhabdomyosarcoma, an Ewing sarcoma and a neuroblastoma (case 1-3). In two patients, liquid biopsies were used for prognostication, by MYCN ddPCR in a patient with neuroblastoma and by RT-qPCR testing rhabdomyosarcoma-specific mRNA in bone marrow of a patient with a rhabdomyosarcoma (case 4 and 5). In case 6, mRNA testing demonstrated disease progression and assisted clinical decision making. Conclusion: This case series illustrates the value of liquid biopsy. We further demonstrate and recommend the use of liquid biopsies to be used in conjunction with conventional methods for the determination of metastatic status, prognostication and monitoring of treatment response in patients with pediatric solid tumors.

Evaluation of Potential Targets for Fluorescence-Guided Surgery in Pediatric Ewing Sarcoma: A Preclinical Proof-of-Concept Study.

Fluorescence-guided surgery (FGS), based on fluorescent tracers binding to tumor-specific biomarkers, could assist surgeons to achieve complete tumor resections. This study evaluated potential biomarkers for FGS in pediatric Ewing sarcoma (ES). Immunohistochemistry (IHC) was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. LINGO-1 and GD2 evaluation did not work on the acquired tissue. Based on the immunoreactive scores, anti-CD99 and anti-CD117 were evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. Anti-GD2, a tracer in the developmental phase, was also tested. These three tracers were topically applied to a freshly resected ES tumor and adjacent healthy tissue. IHC demonstrated moderate/strong CD99 and CD117 expression in ES tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117 and low GD2 expression, in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient. In conclusion, CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Genomic characterization of DICER1-associated neoplasms uncovers molecular classes.

DICER1 syndrome is a tumor predisposition syndrome that is associated with up to 30 different neoplastic lesions, usually affecting children and adolescents. Here we identify a group of mesenchymal tumors which is highly associated with DICER1 syndrome, and molecularly distinct from other DICER1-associated tumors. This group of DICER1-associated mesenchymal tumors encompasses multiple well-established clinicopathological tumor entities and can be further divided into three clinically meaningful classes designated "low-grade mesenchymal tumor with DICER1 alteration" (LGMT DICER1), "sarcoma with DICER1 alteration" (SARC DICER1), and primary intracranial sarcoma with DICER1 alteration (PIS DICER1). Our study not only provides a combined approach to classify DICER1-associated neoplasms for improved clinical management but also suggests a role for global hypomethylation and other recurrent molecular events in sarcomatous differentiation in mesenchymal tumors with DICER1 alteration. Our results will facilitate future investigations into prognostication and therapeutic approaches for affected patients.

Imaging in rhabdomyosarcoma: a patient journey.

Rhabdomyosarcoma, although rare, is the most frequent soft tissue sarcoma in children and adolescents. It can present as a mass at nearly any site in the body, with most common presentations in the head and neck, genitourinary tract and extremities. The optimal diagnostic approach and management of rhabdomyosarcoma require a multidisciplinary team with multimodal treatment, including chemotherapy and local therapy. Survival has improved over the last decades; however, further improvement in management is essential with current 5-year overall survival ranging from 35% to 100%, depending on disease and patient characteristics. In the full patient journey, from diagnosis, staging, management to follow-up after therapy, the paediatric radiologist and nuclear physician are essential members of the multidisciplinary team. Recently, guidelines of the European paediatric Soft tissue sarcoma Study Group, the Cooperative Weichteilsarkom Studiengruppe and the Oncology Task Force of the European Society of Paediatric Radiology (ESPR), in an ongoing collaboration with the International Soft-Tissue Sarcoma Database Consortium, provided guidance for high-quality imaging. In this educational paper, given as a lecture during the 2022 postgraduate ESPR course, the multi-disciplinary team of our national paediatric oncology centre presents the journey of two patients with rhabdomyosarcoma and discusses the impact on and considerations for the clinical (paediatric) radiologist and nuclear physician. The key learning points of the guidelines and their implementation in clinical practice are highlighted and up-to-date insights provided for all aspects from clinical suspicion of rhabdomyosarcoma and its differential diagnosis, to biopsy, staging, risk stratification, treatment response assessment and follow-up.

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program 'iTHER'.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Improved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing.

PURPOSE: Gene fusions play a significant role in cancer etiology, making their detection crucial for accurate diagnosis, prognosis, and determining therapeutic targets. Current diagnostic methods largely focus on either targeted or low-resolution genome-wide techniques, which may be unable to capture rare events or both fusion partners. We investigate if RNA sequencing can overcome current limitations with traditional diagnostic techniques to identify gene fusion events. METHODS: We first performed RNA sequencing on a validation cohort of 24 samples with a known gene fusion event, after which a prospective pan-pediatric cancer cohort (n = 244) was tested by RNA sequencing in parallel to existing diagnostic procedures. This cohort included hematologic malignancies, tumors of the CNS, solid tumors, and suspected neoplastic samples. All samples were processed in the routine diagnostic workflow and analyzed for gene fusions using standard-of-care methods and RNA sequencing. RESULTS: We identified a clinically relevant gene fusion in 83 of 244 cases in the prospective cohort. Sixty fusions were detected by both routine diagnostic techniques and RNA sequencing, and one fusion was detected only in routine diagnostics, but an additional 24 fusions were detected solely by RNA sequencing. RNA sequencing, therefore, increased the diagnostic yield by 38%-39%. In addition, RNA sequencing identified both gene partners involved in the gene fusion, in contrast to most routine techniques. For two patients, the newly identified fusion by RNA sequencing resulted in treatment with targeted agents. CONCLUSION: We show that RNA sequencing is sufficiently robust for gene fusion detection in routine diagnostics of childhood cancers and can make a difference in treatment decisions.

EWSR1-The Most Common Rearranged Gene in Soft Tissue Lesions, Which Also Occurs in Different Bone Lesions: An Updated Review.

EWSR1 belongs to the FET family of RNA-binding proteins including also Fused in Sarcoma (FUS), and TATA-box binding protein Associated Factor 15 (TAF15). As consequence of the multifunctional role of EWSR1 leading to a high frequency of transcription of the chromosomal region where the gene is located, EWSR1 is exposed to aberrations such as rearrangements. Consecutive binding to other genes leads to chimeric proteins inducing oncogenesis. The other TET family members are homologous. With the advent of widely used modern molecular techniques during the last decades, it has become obvious that EWSR1 is involved in the development of diverse benign and malignant tumors with mesenchymal, neuroectodermal, and epithelial/myoepithelial features. As oncogenic transformation mediated by EWSR1-fusion proteins leads to such diverse tumor types, there must be a selection on the multipotent stem cell level. In this review, we will focus on the wide variety of soft tissue and bone entities, including benign and malignant lesions, harboring EWSR1 rearrangement. Fusion gene analysis is the diagnostic gold standard in most of these tumors. We present clinicopathologic, immunohistochemical, and molecular features and discuss differential diagnoses.

Loss of H3K27me3 occurs in a large subset of embryonal rhabdomyosarcomas: Immunohistochemical and molecular analysis of 25 cases.

Loss of histone 3 lysine 27 trimethylation (H3K27me3) has been described as a diagnostic marker for malignant peripheral nerve sheath tumor (MPNST), also discriminating MPNST with rhabdomyoblastic differentiation (malignant Triton tumor) from rhabdomyosarcoma (RMS). We studied the immunohistochemical expression of H3K27me3 in embryonal RMSs (ERMSs), performed methylation profiling in order to support the diagnosis and RNA-sequencing for comparison of the transcriptome of H3K27me3-positive and -negative cases. Of the 25 ERMS patients, 17 were males and 8 were females with an age range from 1 to 67 years (median, 6 years). None were known with neurofibromatosis type 1. One patient had Li-Fraumeni syndrome. Tumor localization included paratesticular (n = 9), genitourinary (n = 6), head/neck (n = 5), retroperitoneal (n = 4) and lower arm (n = 1). Five MPNSTs served as reference group. All ERMS had classical features including a variable spindle cell component. Immunohistochemical loss (partial or complete) of H3K27me3 was detected in 18/25 cases (72%). Based on methylation profiling, 22/22 cases were classified as ERMS. Using RNA sequencing, the ERMS group (n = 14) had a distinct gene expression profile in contrast to MPNSTs, confirming that the H3K27me3 negative ERMS cases do not represent malignant Triton tumors. When comparing H3K27me3-negative and -positive ERMSs, gene set enrichment analysis revealed differential expression of genes related to histone acetylation and normal muscle function with H3K27me3 negative ERMSs being associated with acetylation. Conclusion: Loss of H3K27me3 frequently occurs in ERMSs and correlates with H3K27 acetylation. H3K27me3 is not a suitable marker to differentiate ERMS (with spindle cell features) from malignant Triton tumor.

Nodular Fasciitis With Malignant Morphology and a COL6A2-USP6 Fusion: A Case Report (of a 10-Year-old Boy).

Nodular fasciitis is usually a benign lesion genetically characterized by ubiquitin-specific protease 6 (USP6) rearrangements. We present a case of a 10-year-old boy with a 1.5-week history of a painless mass on the right chest wall, which was excised. A histomorphologically malignant tumor with pronounced pleomorphism, atypical mitotic figures, and a myoid immunophenotype was observed. The methylation profile was consistent with nodular fasciitis and fluorescence in situ hybridization confirmed USP6 rearrangement. Using Archer Fusion Plex (Sarcoma Panel) and RNA sequencing, a collagen, type VI, alpha 2 (COL6A2)-USP6 gene fusion was subsequently identified. Furthermore, DNA clustering analysis also showed a match with nodular fasciitis. During the follow-up of 22 months, no recurrence or metastasis occurred. In conclusion, we describe a clinically benign, histomorphologically malignant mesenchymal neoplasm with a myoid immunophenotype, and a genetic and epigenetic profile consistent with nodular fasciitis. In such cases, molecular analysis is a useful adjunct to avoid unnecessary overtreatment.

USP6-Associated Neoplasms: A Rapidly Expanding Family of Lesions.

Nearly 20 years ago, the first description of a translocation involving chromosome 17 on which USP6 resides was described. Since then, not only the culprit gene but also many fusion partners, leading to transcriptional activation of USP6, have been detected. The first neoplasm known to harbor USP6 rearrangements was aneurysmal bone cyst. Since then, other entities like nodular fasciitis, myositis ossificans, fibro-osseous pseudotumor of digits, and a subgroup of fibromas of tendon sheath, probably representing tenosynovial nodular fasciitis, have been added to the list of USP6-rearranged lesions. Remarkably, all of them share clinical as well as morphological characteristics, and authors have suggested that these entities actually belong to the same spectrum. This review summarizes the current knowledge regarding USP6-rearranged lesions and further elaborates on how these neoplasms relate to one another. We propose to call these lesions UAN (Usp6-associated neoplasm).

Potential Diagnosis of Vitreoretinal Lymphoma by Detection of MYD88 Mutation in Aqueous Humor With Ultrasensitive Droplet Digital Polymerase Chain Reaction.

Importance: The diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. Objective: To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. Design, Setting, and Participants: This cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. Main Outcomes and Measures: The presence of MYD88 L265P mutation detected by ddPCR in AH and VF. Results: The study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. Conclusions and Relevance: The high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.