CNS tumors with PLAGL1-fusion: beyond ZFTA and YAP1 in the genetic spectrum of supratentorial ependymomas.

A novel methylation class, "neuroepithelial tumor, with PLAGL1 fusion" (NET-PLAGL1), has recently been described, based on epigenetic features, as a supratentorial pediatric brain tumor with recurrent histopathological features suggesting an ependymal differentiation. Because of the recent identification of this neoplastic entity, few histopathological, radiological and clinical data are available. Herein, we present a detailed series of nine cases of PLAGL1-fused supratentorial tumors, reclassified from a series of supratentorial ependymomas, non-ZFTA/non-YAP1 fusion-positive and subependymomas of the young. This study included extensive clinical, radiological, histopathological, ultrastructural, immunohistochemical, genetic and epigenetic (DNA methylation profiling) data for characterization. An important aim of this work was to evaluate the sensitivity and specificity of a novel fluorescent in situ hybridization (FISH) targeting the PLAGL1 gene. Using histopathology, immunohistochemistry and electron microscopy, we confirmed the ependymal differentiation of this new neoplastic entity. Indeed, the cases histopathologically presented as "mixed subependymomas-ependymomas" with well-circumscribed tumors exhibiting a diffuse immunoreactivity for GFAP, without expression of Olig2 or SOX10. Ultrastructurally, they also harbored features reminiscent of ependymal differentiation, such as cilia. Different gene partners were fused with PLAGL1: FOXO1, EWSR1 and for the first time MAML2. The PLAGL1 FISH presented a 100% sensitivity and specificity according to RNA sequencing and DNA methylation profiling results. This cohort of supratentorial PLAGL1-fused tumors highlights: 1/ the ependymal cell origin of this new neoplastic entity; 2/ benefit of looking for a PLAGL1 fusion in supratentorial cases of non-ZFTA/non-YAP1 ependymomas; and 3/ the usefulness of PLAGL1 FISH.

FGFR1 fusions as a novel molecular driver in rhabdomyosarcoma.

The wide application of RNA sequencing in clinical practice has allowed the discovery of novel fusion genes, which have contributed to a refined molecular classification of rhabdomyosarcoma (RMS). Most fusions in RMS result in aberrant transcription factors, such as PAX3/7::FOXO1 in alveolar RMS (ARMS) and fusions involving VGLL2 or NCOA2 in infantile spindle cell RMS. However, recurrent fusions driving oncogenic kinase activation have not been reported in RMS. Triggered by an index case of an unclassified RMS (overlapping features between ARMS and sclerosing RMS) with a novel FGFR1::ANK1 fusion, we reviewed our molecular files for cases harboring FGFR1-related fusions. One additional case with an FGFR1::TACC1 fusion was identified in a tumor resembling embryonal RMS (ERMS) with anaplasia, but with no pathogenic variants in TP53 or DICER1 on germline testing. Both cases occurred in males, aged 7 and 24, and in the pelvis. The 2nd case also harbored additional alterations, including somatic TP53 and TET2 mutations. Two additional RMS cases (one unclassified, one ERMS) with FGFR1 overexpression but lacking FGFR1 fusions were identified by RNA sequencing. These two cases and the FGFR1::TACC1-positive case clustered together with the ERMS group by RNAseq. This is the first report of RMS harboring recurrent FGFR1 fusions. However, it remains unclear if FGFR1 fusions define a novel subset of RMS or alternatively, whether this alteration can sporadically drive the pathogenesis of known RMS subtypes, such as ERMS. Additional larger series with integrated genomic and epigenetic datasets are needed for better subclassification, as the resulting oncogenic kinase activation underscores the potential for targeted therapy.

Imaging characterization of paediatric tumours with the neurotrophic tyrosine receptor kinase fusion transcript.

OBJECTIVES: The neurotrophic tyrosine receptor kinase (NTRK) fusion transcript (FT) is a major genetic landmark of infantile fibrosarcoma (IFS) and cellular congenital mesoblastic nephroma (cCMN) but is also described in other tumours. The recent availability of NTRK-targeted drugs enhances the need for better identification. We aimed to describe the anatomic locations and imaging features of tumours with NTRK-FT in children. CASE SERIES: Imaging characteristics of NTRK-FT tumours of 41 children (median age: 4 months; 63% <1 year old; range: 0-188) managed between 2001 and 2019 were retrospectively analysed. The tumours were located in the soft tissues (n = 24, including 19 IFS), kidneys (n = 9, including 8 cCMN), central nervous system (CNS) (n = 5), lung (n = 2), and bone (n = 1). The tumours were frequently deep-located (93%) and heterogeneous (71%) with necrotic (53%) or haemorrhagic components (29%). Although inconstant, enlarged intratumoural vessels were a recurrent finding (70%) with an irregular distribution (63%) in the most frequent anatomical locations. CONCLUSION: Paediatric NTRK-FT tumours mainly occur in infants with very variable histotypes and locations. Rich and irregular intra-tumoural vascularization are recurrent findings. ADVANCES IN KNOWLEDGE: Apart from IFS of soft tissues and cCMN of the kidneys, others NTRK-FT tumours locations have to be known, as CNS tumours. Better knowledge of the imaging characteristics may help guide the pathological and biological identification.

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups.

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.

Sequential genomic analysis using a multisample/multiplatform approach to better define rhabdomyosarcoma progression and relapse.

The genomic spectrum of rhabdomyosarcoma (RMS) progression from primary to relapse is not fully understood. In this pilot study, we explore the sensitivity of various targeted and whole-genome NGS platforms in order to assess the best genomic approach of using liquid biopsy in future prospective clinical trials. Moreover, we investigate 35 paired primary/relapsed RMS from two contributing institutions, 18 fusion-positive (FP-RMS) and 17 fusion-negative RMS (FN-RMS) by either targeted DNA or whole exome sequencing (WES). In 10 cases, circulating tumor DNA (ctDNA) from multiple timepoints through clinical care and progression was analyzed for feasibility of liquid biopsy in monitoring treatment response/relapse. ctDNA alterations were evaluated using a targeted 36-gene custom RMS panel at high coverage for single-nucleotide variation and fusion detection, and a shallow whole-genome sequencing for copy number variation. FP-RMS have a stable genome with relapse, with common secondary alterations CDKN2A/B, MYCN, and CDK4 present at diagnosis and impacting survival. FP-RMS lacking major secondary events at baseline acquire recurrent MYCN and AKT1 alterations. FN-RMS acquire a higher number of new alterations, most commonly SMARCA2 missense mutations. ctDNA analyses detect pathognomonic variants in all RMS patients within our collection at diagnosis, regardless of type of alterations, and confirmed at relapse in 86% of FP-RMS and 100% FN-RMS. Moreover, a higher number of fusion reads is detected with increased disease burden and at relapse in patients following a fatal outcome. These results underscore patterns of tumor progression and provide rationale for using liquid biopsy to monitor treatment response.

Neurotrophic tropomyosin receptor kinase (NTRK) fusion positive tumors: a historical cohort analysis.

BACKGROUND: NTRK gene fusions have been identified in various tumors; some requiring aggressive therapy and sometimes new TRK inhibitors (TRKi). We aimed to describe a national, unselected, retrospective, multicenter cohort. RESEARCH DESIGN AND METHODS: Patients were identified through the French sarcoma diagnostic laboratory at Institut Curie through samples analyzed by RT-qPCR or whole-transcriptome sequencing. RESULTS: From 2001 to 2019, 65 NTRK fusion tumors were identified within 2120 analyses (3.1%): 58 by RNA sequencing (including 20 after RT-qPCR analysis) and 7 exclusively by RT-qPCR. Of the 61 patients identified, 37 patients had infantile soft tissue or kidney fibrosarcomas (IFS), 15 other mesenchymal (Other-MT) and nine central nervous system (CNS) tumors. They encompassed 14 different tumor types with variable behaviors. Overall, 53 patients had surgery (3 mutilating), 38 chemotherapy (20 alkylating agents/anthracycline), 11 radiotherapy, two 'observation strategy' and 13 received TRKi. After a median follow-up of 61.0 months [range, 2.5-226.0], 10 patients died. Five-year overall survival is, respectively, 91.9% [95%CI, 83.5-100.0], 61.1% [95%CI, 34.2-100.0] and 64.8% [95%CI, 39.3-100.0] for IFS, Other-MT, and CNS groups. CONCLUSIONS: NTRK-fusion positive tumors are rare but detection is improved through RNA sequencing. TRKi could be considered at diagnosis for CNS NTRK-fusion positive tumors, some IFS, and Other-MT. TRIAL REGISTRATION: Not adapted.

CNS tumor with EP300::BCOR fusion: discussing its prevalence in adult population.

The Central Nervous System (CNS) tumor with BCOR internal tandem duplication (ITD) has recently been added as a novel embryonal histomolecular tumor type to the 2021 World Health Organization (WHO) Classification of CNS Tumors. In addition, other CNS tumors harboring a BCOR/BCORL1 fusion, which are defined by a distinct DNA-methylation profile, have been recently identified in the literature but clinical, radiological and histopathological data remain scarce. Herein, we present two adult cases of CNS tumors with EP300::BCOR fusion. These two cases presented radiological, histopathological, and immunohistochemical homologies with CNS tumors having BCOR ITD in children. To compare these tumors with different BCOR alterations, we performed a literature review with a meta-analysis. CNS tumors with EP300::BCOR fusion seem to be distinct from their BCOR ITD counterparts in terms of age, location, progression-free survival, tumor growth pattern, and immunopositivity for the BCOR protein. CNS tumors from the EP300::BCOR fusion methylation class in adults may be added to the future WHO classification.

NTRK-rearranged spindle cell neoplasms are ubiquitous tumours of myofibroblastic lineage with a distinct methylation class.

AIMS: NTRK gene fusions have been described in a wide variety of central nervous system (CNS) and soft tissue tumours, including the provisional tumour type 'spindle cell neoplasm, NTRK-rearranged' (SCN-NTRK), added to the 2020 World Health Organisation Classification of Soft Tissue Tumours. Because of histopathological and molecular overlaps with other soft tissue entities, controversy remains concerning the lineage and terminology of SCN-NTRK. METHODS AND RESULTS: This study included 16 mesenchymal tumours displaying kinase gene fusions (NTRK fusions and one MET fusion) initially diagnosed as infantile fibrosarcomas (IFS), SCN-NTRK and adult-type fibrosarcomas from the soft tissue, viscera and CNS. We used immunohistochemistry, DNA methylation profiling, whole RNA-sequencing and ultrastructural analysis to characterise them. Unsupervised t-distributed stochastic neighbour embedding analysis showed that 11 cases (two CNS tumours and nine extra-CNS) formed a unique and new methylation cluster, while all tumours but one, initially diagnosed as IFS, clustered in a distinct methylation class. All the tumours except one formed a single cluster within the hierarchical clustering of whole RNA-sequencing data. Tumours from the novel methylation class co-expressed CD34 and S100, had variable histopathological grades and frequently displayed a CDKN2A deletion. Ultrastructural analyses evidenced a myofibroblastic differentiation. CONCLUSIONS: Our findings confirm that SCN-NTRK share similar features in adults and children and in all locations combine an infiltrative pattern, distinct epigenetic and transcriptomic profiles, and ultrastructural evidence of a myofibroblastic lineage. Further studies may support the use of new terminology to better describe their myofibroblastic nature.

An extracranial CNS presentation of the emerging "intracranial" mesenchymal tumor, FET: CREB-fusion positive.

A novel histomolecular tumor, the "intracranial mesenchymal tumor (IMT), FET::CREB fusion-positive", has recently been identified and added to the 2021 World Health Organization Classification of Tumors of the Central Nervous System. One of the essential diagnostic criteria defined in this classification is the intracranial location of the tumor. Herein, we report a spinal case of IMT with a classical EWSR1::CREM fusion. We compare its clinical, histopathological, immunophenotypical, genetic and epigenetic features with those previously described in IMT, FET::CREB fusion-positive. The current case presented histopathological (epithelioid morphology with mucin-rich stroma, and expression of EMA and desmin), radiological (an extraparenchymal lobulated mass without dural tail), genetic (fusion implicating the EWSR1 and CREM genes), and epigenetic (DNA-methylation profiling) similarities to previously reported cases. This case constitutes the third "extracranial" observation of an IMT. Our results added data suggesting that the terminology "IMT, FET::CREB fusion-positive" is provisional and that further series of cases are needed to better characterize them.

Intra- and extra-cranial BCOR-ITD tumours are separate entities within the BCOR-rearranged family.

BCOR-ITD tumours form an emerging family of aggressive entities with an internal tandem duplication (ITD) in the last exon of the BCOR gene. The family includes cerebral tumours, termed central nervous system BCOR-ITD (CNS BCOR-ITD), and sarcomatous types described in the kidney as clear cell sarcoma of the kidney (CCSK), in the endometrium as high-grade endometrial stromal sarcoma, and in the bone and soft tissue as undifferentiated round cell sarcoma or primitive myxoid mesenchymal tumour of infancy. Based on a series of 33 retrospective cases, including 10 CNS BCOR-ITD and 23 BCOR-ITD sarcomas, we interrogated the homogeneity of the entity regarding clinical, radiological, and histopathological findings, and molecular signatures. Whole-transcriptomic sequencing and DNA methylation profiling were used for unsupervised clustering. BCOR-ITD tumours mostly affected young children with a median age at diagnosis of 2.1 years (range 0-62.4). Median overall survival was 3.9 years and progression-free survival was 1.4 years. This dismal prognosis is shared among tumours in all locations except CCSK. Histopathological review revealed marked differences between CNS BCOR-ITD and BCOR-ITD sarcomas. These two groups were consistently segregated by unsupervised clustering of expression (n = 22) and DNA methylation (n = 21) data. Proximity between the two groups may result from common somatic changes within key pathways directly related to the novel activity of the ITD itself. Conversely, comparison of gene signatures with single-cell RNA-Seq atlases suggests that the distinction between BCOR-ITD sarcomas and CNS BCOR-ITD may result from differences in cells of origin.

Novel EWSR1::UBP1 fusion expands the spectrum of spindle cell rhabdomyosarcomas.

Over the last decade, the development of next-generation sequencing techniques has led to the molecular dismantlement of adult and pediatric sarcoma, with the identification of multiple gene fusions associated with specific subtypes and currently integrated into diagnostic classifications. In this report, we describe and discuss the identification of a novel EWSR1-UBP1 gene fusion in an adult patient presenting with multi-metastatic sarcoma. Extensive pathological, transcriptomic, and genomic characterization of this tumor in comparison with a cohort of different subtypes of pediatric and adult sarcoma revealed that this fusion represents a novel variant of spindle cell rhabdomyosarcoma with features of TFCP2-rearranged subfamily.

An integrative histopathological and epigenetic characterization of primary intracranial mesenchymal tumors, FET:CREB-fused broadening the spectrum of tumor entities in comparison with their soft tissue counterparts.

FET:CREB fusions have been described in a variety of tumors from various phenotypes. Recently, these fusion transcripts were reported in intracranial tumors, variably named intracranial mesenchymal myxoid tumors or angiomatoid fibrous histiocytomas. Controversy remains concerning the terminology for these tumors. Here, we report 11 cases of central nervous system mesenchymal tumors with proven FET:CREB fusion. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor or Sarcoma Classifier (v11b4/v12.2). However, by using unsupervised t-SNE and hierarchical clustering analyses, six of the cases constituted a distinct cluster. The remaining four tumors showed no obvious relation to any of the other referenced classes but were close to the clusters of extra-CNS angiomatoid fibrous histiocytomas (n = 1), clear cell sarcomas (n = 1), or solitary fibrous tumors (n = 2). Our findings confirm that intracranial FET:CREB-fused tumors do not represent a single molecular tumor entity, although most samples clustered close to each other, indicating the existence of a distinct epigenetic group that could potentially be partially masked by the low number of cases included. Further analyses are needed to characterize intracranial FET:CREB fused-defined tumors in more detail.

A novel SMARCA2-CREM fusion: expanding the molecular spectrum of intracranial mesenchymal tumors beyond the FET genes.

A novel histomolecular tumor of the central nervous system, the "intracranial mesenchymal tumor (IMT), FET-CREB fusion-positive" has recently been identified in the literature and will be added to the 2021 World Health Organization Classification of Tumors of the Central Nervous System. However, our latest study using DNA-methylation analyses has revealed that intracranial FET-CREB fused tumors do not represent a single molecular tumor entity. Among them, the main subgroup presented classical features of angiomatoid fibrous histiocytoma, having ultrastructural features of arachnoidal cells, for. Another tumor type with clear cell component and histopathological signs of aggressivity clustered in close vicinity with clear cell sarcoma of soft tissue. Herein, we report one case of IMT with a novel SMARCA2-CREM fusion which has until now never been described in soft tissue or the central nervous system. We compare its clinical, histopathological, immunophenotypic, genetic and epigenetic features with those previously described in IMT, FET-CREB fusion-positive. Interestingly, the current case did not cluster with IMT, FET-CREB fusion-positive but rather presented histopathological (clear cell morphology with signs of malignancy), clinical (with a dismal course with several recurrences, metastases and finally the patient's death), genetic (fusion implicating the CREM gene), and epigenetic (DNA-methylation profiling) similarities with our previously reported clear cell sarcoma-like tumor of the central nervous system. Our results added data suggesting that different clinical and histomolecular tumor subtypes or grades seem to be included within the terminology "IMT, FET-CREB fusion-positive", and that further series of cases are needed to better characterize them.

Molecular description of meningeal solitary fibrous tumors/hemangiopericytomas compared to meningiomas: two completely separate entities.

INTRODUCTION: Meningeal solitary fibrous tumors (SFT), like all SFT, are defined by NAB2-STAT6 fusion and share clinicopathologic similarities with meningiomas, the most frequent meningeal tumors. Our aim is to establish the molecular identity of meningeal SFT and seek molecular prognostic factors. METHODS: RNA sequencing and whole exome sequencing were performed in STAT6-positive SFT and grade 2-3 meningiomas, and data concerning other soft tissues tumors was obtained from the local database. Uniform manifold approximation and projection, individual gene expression and Gene Set Enrichment Analysis were performed. RESULTS: RNA clustering shows that SFT share a common molecular signature, different from any other type of tumoral tissue. Meningeal SFT aggregate with other SFT, with no clinical or histological subgroup. Comparison of genes expressions suggests significant over-expressions of ZIC2, ZIC3, ZIC5, GABBR2, TP53 in CNS-SFT. The pathogenic TP53 c.743G>T variant, previously undescribed in SFT, was found in one sample of meningeal SFT during malignant progression. CONCLUSIONS: Meningeal SFT are molecular counterparts of extra-meningeal SFT, completely separate from meningiomas. They might develop from the same tissues and benefit from the same treatments as SFT.

Identification of Tissue of Origin and Guided Therapeutic Applications in Cancers of Unknown Primary Using Deep Learning and RNA Sequencing (TransCUPtomics).

Cancers of unknown primary (CUP) are metastatic cancers for which the primary tumor is not found despite thorough diagnostic investigations. Multiple molecular assays have been proposed to identify the tissue of origin (TOO) and inform clinical care; however, none has been able to combine accuracy, interpretability, and easy access for routine use. We developed a classifier tool based on the training of a variational autoencoder to predict tissue of origin based on RNA-sequencing data. We used as training data 20,918 samples corresponding to 94 different categories, including 39 cancer types and 55 normal tissues. The TransCUPtomics classifier was applied to a retrospective cohort of 37 CUP patients and 11 prospective patients. TransCUPtomics exhibited an overall accuracy of 96% on reference data for TOO prediction. The TOO could be identified in 38 (79%) of 48 CUP patients. Eight of 11 prospective CUP patients (73%) could receive first-line therapy guided by TransCUPtomics prediction, with responses observed in most patients. The variational autoencoder added further utility by enabling prediction interpretability, and diagnostic predictions could be matched to detection of gene fusions and expressed variants. TransCUPtomics confidently predicted TOO for CUP and enabled tailored treatments leading to significant clinical responses. The interpretability of our approach is a powerful addition to improve the management of CUP patients.