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Brown tumors are rare and generally self-limiting mass lesions of bone occurring in the context of hyperparathyroidism. Although commonly regarded as endocrine-driven tumor-like lesions, we detected pathogenic hotspot KRAS mutations in 10/16 brown tumors (62%) with similar frequencies found in cases affecting the peripheral and axial skeleton. Pathogenic mutations in other driver genes of the RAS-MAPK pathway were not identified. Our findings suggest brown tumors to represent true neoplasms driven by the activation of the RAS-MAPK signaling pathway. The frequent regression of brown tumors after normalization of hyperparathyroidism points to a second hit mediated by endocrine stimulation to be required for tumor development. Our findings underline the pathogenic relation of brown tumors to nonossifying fibroma and giant cell granuloma of the jaws which both appear histologically similar to brown tumors and are also driven by RAS-MAPK signaling pathway activation.

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/β-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding β-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with β-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/β-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/β-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

Osteosarcomas are aggressive primary tumors of bone that are typically detected in locally advanced stages; however, which genetic mutations drive the cancer before its clinical detection remain unknown. To identify these events, we performed longitudinal genome‐sequencing analysis of 12 patients with metastatic or refractory osteosarcoma. Phylogenetic and molecular clock analyses were carried out next to identify actionable mutations, and these were validated by integrating data from additional 153 osteosarcomas and pre‐existing functional evidence from mouse PDX models. We found that the earliest and thus clinically most promising mutations affect the cell cycle G1 transition, which is guarded by cyclins D3, E1, and cyclin‐dependent kinases 2, 4, and 6. Cell cycle G1 alterations originate no more than a year before the primary tumor is clinically detected and occur in >90% and 50% of patients of the discovery and validation cohorts, respectively. In comparison, other cancer driver mutations could be acquired at any evolutionary stage and often do not become pervasive. Consequently, our data support that the repertoire of actionable mutations present in every osteosarcoma cell is largely limited to cell cycle G1 mutations. Since they occur in mutually exclusive combinations favoring either CDK2 or CDK4/6 pathway activation, we propose a new genomically‐based algorithm to direct patients to correct clinical trial options. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Background: Mesenchymal chondrosarcoma is a rare and aggressive sarcoma subtype with high risk for distant metastases and poor prognosis. Currently NCCN- and ESMO-Guidelines recommend using Ewing sarcoma protocols as standard treatment. Nevertheless, in localised disease overall 5-year survival rates are below 50% whereas in metastatic spread median progression-free survival rates of 5 months can be expected. Here we present a patient with metastatic osseous spread of mesenchymal chondrosarcoma that showed a sustained clinical improvement and a good partial response on imaging over a period of one year when treated with the multi-tyrosine kinase inhibitor cabozantinib as the sole systemic treatment. Case presentation: Tissue samples from the primary tumour and three different metastases were subjected to methylation and copy number analysis, as well as DNA and RNA sequencing. The copy number profiles of both the primary and metastases revealed aneuploidy of chromosome 12, and a low-level copy number gain of MYC. Of note, all metastases showed homozygous loss at 9p21.3 harboring CDKN2a (p16) that was not present in the primary tumour. The Oncomine Comprehensive Panel v3 performed in one of the metastases did not reveal any point mutation within 135 cancer genes (including RB1­). Conclusion and discussion: The sustained response to cabozantinib in the case presented here is most likely explained through inhibition of a complex interplay between VEGFR, PDGFR, PI3K-AKT- and Notch signalling pathways.

Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. More than a third of patients do not respond to standard therapy and urgently require alternative treatment options. Due to a high degree of inter‐ and intra‐tumoural genomic heterogeneity and complexity, recurrent molecular alterations that could serve as prognostic predictors or therapeutic targets are still lacking in osteosarcoma. Copy number (CN) gains involving the IGF1R gene, however, have been suggested as a potential surrogate marker for treating a subset of patients with IGF1R inhibitors. In this study, we screened a large set of osteosarcomas and found specific CN gains of the IGF1R gene in 18 of 253 (7.1%) cases with corresponding IGF1R overexpression. Despite the discouraging results observed in clinical trials in other tumours so far, focusing only on selected patients with osteosarcoma that show evidence of IGF pathway activation might represent a promising new and innovative treatment approach.

Background: Mesenchymal chondrosarcoma (MCS) is a high-grade sarcoma characterized by a biphasic histological appearance. However, more often than not, only the poorly differentiated component is present on a biopsy, thus more entities enter the differential diagnosis. Since the small cell component is morphologically non-specific, the differential diagnoses are broad, such as Ewing sarcoma, rhabdomyosarcoma, poorly differentiated synovial sarcoma small cell carcinoma, lymphoma and also melanoma. Morevoer, although the HEY1::NCOA2 gene fusion is present in almost all MCS, some studies have shown alternative alterations in the absence of the HEY1::NCOA2 fusion, suggesting genomic heterogeneity in at least a small subset of MCS. Thus, new diagnostic aids would be helpful to confirm the diagnosis. While most of the immunohistochemical profile overlaps between these tumours, NKX3.1 was recently proposed as a useful diagnostic marker for MCS. Furthermore, studies using DNA-methylation profiling show that MCS forms a distinct methylation cluster. The aim of this study is to evaluate the utility of these new diagnostic tools. Methods: Slides from 45 samples with MCS were stained with NKX3.1 antibody (EP356, Cell Marque, Roche) and were also investigated by methylome analysis (Mehylation Epic BeadChips, Illumina). Depending on tissue availability, the cases were subjected to HEY1::NCOA2 gene fusion testing. Results: The methylation profile showed a distinct cluster for mesenchymal chondrosarcoma, in line with the previous studies. Moreover, these findings were reproduced even when submitting tissue solely from one component, either the cellular or the cartilaginous one. In addition, the copy number profile derived from the methylome analysis proves its utility in distinguishing MCS from its mimics. On the other hand, only 32.6% of cases were positive for NKX3.1. Four samples initially diagnosed as MCS were histologically re-evaluated due to an atypical methylome profile, a high amount of CNV and/or lack of validation of the gene fusion. Methylation profiling led to the re-classification of three cases, whereas the fourth sample could not be further classified. Conclusions: We conclude that the methylation analysis, along with the copy number profile, are reliable tools in terms of diagnosing MCS. On the contrary, the immunomarker NKX3.1 shows contradicting results, proving itself as an unreliable marker in this setting. Citation Format: Salome Glauser, Baptiste Ameline, Vanghelita Andrei, Dorothee Harder, Chantal Pauli, Marcel Trautmann, Wolfgang Hartmann, Daniel Baumhoer. NKX3.1 immunohistochemistry and methylome profiling in mesenchymal chondrosarcoma: additional diagnostic value for a well defined disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6218.

Rearrangements of the transcription factors FOS and FOSB have recently been identified as the genetic driver event underlying osteoid osteoma and osteoblastoma. Nuclear overexpression of FOS and FOSB have since then emerged as a reliable surrogate marker despite limitations in specificity and sensitivity. Indeed, osteosarcoma can infrequently show nuclear FOS expression and a small fraction of osteoblastomas seem to arise independent of FOS/FOSB rearrangements. Acid decalcification and tissue preservation are additional factors that can negatively influence immunohistochemical testing and make diagnostic decision-making challenging in individual cases. Particularly aggressive appearing osteoblastomas, also referred to as epithelioid osteoblastomas, and osteoblastoma-like osteosarcoma can be difficult to distinguish, underlining the need for additional markers to support the diagnosis. Methylation and copy number profiling, a technique well established for the classification of brain tumors, might fill this gap. Here, we set out to comprehensively characterize a series of 77 osteoblastomas by immunohistochemistry, fluorescence in-situ hybridization as well as copy number and methylation profiling and compared our findings to histologic mimics. Our results show that osteoblastomas are uniformly characterized by flat copy number profiles that can add certainty in reaching the correct diagnosis. The methylation cluster formed by osteoblastomas, however, so far lacks specificity and can be misleading in individual cases.

BackgroundAlthough considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone.Methods and resultsWe followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenic RET mutation developed an osteosarcoma 2 years after the resection of a medullary thyroid carcinoma. Sequencing analysis of additional 336 patients with osteosarcoma led to the identification of germline activating mutations in the RET proto-oncogene in three cases and somatic amplifications of the gene locus in five matched tumours (4%, n=5/124 tumours). Functional analysis of the pathogenic variants together with an integrative analysis of osteosarcoma genomes confirmed that the mutant RET proteins couple functional kinase activity to dysfunctional ligand binding. RET mutations further co-operated with alterations in TP53 and RB1, suggesting that osteosarcoma pathogenesis bears reminiscence to the stepwise model of medullary thyroid carcinoma.ConclusionsAfter Li-Fraumeni-predisposing mutations in TP53, RET becomes the second most mutated cancer-predisposing gene in the germline of patients with osteosarcoma. Hence, early identification of RET mutation carriers can help to identify at-risk family members and carry out preventive measures.

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/β-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding β-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with β-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/β-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/β-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

In the cranio-facial skeleton, a heterogeneous group of well characterized fibro-osseous lesions can be distinguished. Whereas fibrous dysplasia can affect any skeletal bone, ossifying fibroma and cemento-osseous dysplasia exclusively develop in the cranio-facial region, with most subtypes restricted to the tooth bearing areas of the jaws. Herein we present a series of 20 fibro-osseous lesions that developed mostly in the frontal bone and in the mandible, presenting as expansile intramedullary tumors with a unique histologic appearance and an indolent clinical course. We provide evidence that these tumors are distinct from the categories included in the WHO classification and are therefore currently unclassifiable. The definition of cemento-ossifying fibroma as an odontogenic neoplasm developing only in close proximity to teeth should be re-considered and incorporate also extragnathic lesions as shown here.

According to the World Health Organization (WHO) classification, tumors showing hematogenous spread in less than 2% of cases are categorized as "rarely metastasizing" and constitute a group of neoplasms of intermediate malignancy. Since its introduction in 2002, chondroblastoma has been considered one of the prototypic examples of this category of lesions. In the fifth and only recently published edition of the WHO classification of bone and soft tissue tumors, however, chondroblastoma was re-classified from rarely metastasizing to benign due to the rarity of cases with systemic spread. Here, we present a remarkable case of a 54-year-old male who presented with an expansile tumor in his left acromion that was diagnosed as chondroblastoma following biopsy. Three years later a local recurrence was noted during routine follow-up and a bone scan detected hypermetabolic lesions in a rib and the thoracic spine. Over time, he developed multifocal skeletal spread as well as soft tissue and pulmonary metastases, which histologically all revealed conventional chondroblastoma morphology and the highly specific H3-3B point mutation (p.Lys36Met). Thirteen years after initial diagnosis he is currently treated with experimental immunotherapy and shows stable but disseminated disease. Our case highlights that although metastasizing chondroblastoma is rare, potential systemic spread should be kept in mind in patients with chondroblastoma despite the new WHO classification.

Although considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone.We followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenicAfter Li-Fraumeni-predisposing mutations in

BRCAness is a phenotypical trait resembling tumors with germline BRCA1/BRCA2 mutation and homologous recombination deficiency (HRD). Recently, osteosarcoma (OS), which is the most common primary malignant bone tumor, was shown to frequently present molecular features resembling BRCAness. The inability of the HR pathway to keep the genome integrity leads to its instability, resulting in copy number variations and loss of heterozygosity. Tumors characterized by BRCAness might have an increased sensitivity to poly ADP ribose polymerase inhibitors, which leads to synthetic lethality through tumor cell apoptosis. There are several genomic methods to assess BRCAness such as calculating the HRD score based on large genomic rearrangements. We developed a new approach based on DNA-methylation patterns and gene expression signatures. Low-coverage whole-genome (lcWGS) data, DNA-methylation, and RNA-Seq data of 41 patients with relapsed OS were obtained within the scope of the INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) project. Percentage of genome changed (PGC) by large state transitions and telomeric allele imbalance was computed with Nexus CN (BioDiscovery, Inc) and open source Control-FREEC/HRDtools from lcWGS and used for BRCAness scoring. llumina Human Methylation450k and EPIC arrays were used to determine DNA methylation profiles. We selected 18 BRCA-positive and 12 BRCA-negative samples based on the PGC to train the classifier algorithm Random Forest (RF) (R-project) and set aside 11 samples as a test set. Probes were ranked according to minimum variability within groups and maximum between groups. The top 1,000 probes were selected for RF training. The importance of probes was assessed as a mean decrease in accuracy (MDA). Probes with zero MDA were removed from training set. RNA-seq differential expression analysis was performed with DESeq2. Expression signatures were extracted by gene set enrichment analysis (GSEA). Training samples were clustered unambiguously into BRCA-positive and BRCA-negative groups with unsupervised hierarchical clustering of 1,000 probes. All 11 test samples set aside before RF training were accurately classified. Excluding noninformative probes reduced the number of informative probes to 628 and correctly clustered them into two groups. Differential expression analysis of BRCA positive and negative RNA-Seq samples resulted in 1,458 differentially expressed genes. Only upregulated genes in BRCA-positive samples showed highly significant homologous recombination, nucleotide excision, and mismatch repair signatures in GSEA. We established and validated a DNA methylation-based classifier that differentiates BRCA-negative and BRCA-positive OS samples with high accuracy. The differential expression and enrichment analyses identified genome instability signatures. Our method complements existing genomic BRCAness scoring and brings new epigenomic/trancriptomic dimensions into the computational prediction of BRCAness. Citation Format: Maxim Barenboim, Michal Kovac, Baptiste Ameline, David T.W. Jones, Olaf Witt, Stefan Bielack, Stefan Burdach, Daniel Baumhoer, Michaela Nathrath. Identifying BRCAness in osteosarcoma with DNA-methylation profiling and gene expression signatures [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A24.

Non‐ossifying fibroma (NOF) has been an intriguing entity since its first description. It is the most common bone tumour, is usually asymptomatic affecting children and adolescents, is composed of a heterogeneous cell population, and undergoes spontaneous regression after puberty. In a recent article in The Journal of Pathology, Baumhoer and colleagues demonstrate mutations activating the RAS‐MAPK pathway (KRAS, FGFR1 and NF1) in ∼80% of the tumours. Activation of the RAS‐MAPK pathway by somatic mutations is found in a plethora of tumour types, both benign and malignant, while germline mutations cause a wide range of syndromes collectively termed the RASopathies. Their findings indicate that NOF, for long thought to be reactive, should be considered a true neoplasm. Moreover, their data suggest that only a subset of cells in the lesion contain the mutation. A second cell population consisting of histiocytes and osteoclast‐like giant cells appears to be reactive. This intimate relation between WT and mutant cells is also frequently encountered in other benign and locally aggressive bone tumours and seems essential for tumourigenesis. The spontaneous regression remains enigmatic and it is tempting to speculate that pubertal hormonal signalling, especially increased oestrogen levels, affect the balance between mutant and WT cells. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: Osteosarcoma, the most common primary tumor of bone, generally harbors complex structural rearrangements. In this study, we identified frequent mono- and bi-allelic deletions of the two long non-coding RNAs TUSC7 and LINC00901, both located on the long arm of chromosome 3 (3q13.31). Their precise biological function remain largely unknown, although it was suggested that TUSC7 may participate in micro-RNA trapping and thereby could be involved in tumor suppressor gene regulation. Moreover, the presence of a TP53 responsive element upstream of their coding region suggests an involvement in the convoluted TP53 signaling pathway. Methods: Whole genome sequencing data (n=104) and Affymetrix Cytoscan arrays (n=52) were used to derive copy-number profiles of each tumor and interrogated for LINC00901 and TUSC7 deletions. These findings were validated in an independent set of 102 formalin-fixed and paraffin-embedded tissue samples using RNA in situ hybridization techniques. Results: Mono- and bi-allelic deletions were detected in 68 (43.5%) sequenced and array-profiled osteosarcomas. Of these, 34 tumors (50%) acquired deletions in both genes whilst additional focal deletions of TUSC7 and LINC00901 were acquired in 11 (16%) and 19 (28%), respectively. The four remaining cases (6%) revealed copy number losses within the intergenic region between TUSC7 and LINC00901. In addition, reduced amounts or complete losses of hybridisation signals were detected in similar proportions in the independent set of FFPE samples. Conclusions: We identified recurrent 3q13.31 deletions in 68/156 (43,5%) human osteosarcomas which seems remarkable in a tumor well known for its high amount of genomic complexity and intertumoral heterogeneity. TUSC7 and LINC00901 might act as downstream effectors of the TP53 pathway and could be functionally equivalent to TP53 inactivation in case of copy number loss. To further elucidate the consequences of 3q13.31 aberrations we aim to correlate our findings with transcriptome data which is currently ongoing. Citation Format: Baptiste Ameline, Michal Kovac, Karim H. Saba, Maxim Barenboim, Michaela Nathrath, Karolin H. Nord, Daniel Baumhoer. Recurrent deletions of 3q13.31 in human osteosarcoma commonly affect TUSC7 and LINC00901 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3654.

International audience; Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

International audience; Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.

International audience; We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.