Impact of CDK Inhibitors on TBXT Expression in Chordoma Cell Lines Including the First Stable Cell Line of a High-Grade Chordoma.

Chordomas are very rare malignant neoplasms of the bone occurring almost exclusively along the spine. As the tumours are thought to arise from notochordal remnants, the vast majority of chordomas express the TBXT gene, resulting in detectable nuclear amounts of its gene product brachyury. This T-Box transcription factor is commonly recognised as being essential in chordoma cells, and limiting TBXT expression is thought to be the key factor in controlling this tumour. Although the tumour is rare, distinct molecular differences and vulnerabilities have been described with regard to its location and the progression status of the disease, rendering it mandatory for novel cell lines to reflect all relevant chordoma subtypes. Here, we describe a novel chordoma cell line arising from the pleural effusion of a disseminated, poorly differentiated chordoma. This cell line, U-CH22, represents a highly aggressive terminal chordoma and, therefore, fills a relevant gap within the panel of available cell culture models for this orphan disease. CDK7 and CDK9 inhibition was lately identified as being effective in reducing viability in four chordoma cell lines, most likely due to a reduction in brachyury levels. In this study, we determined the capability of the CDK7 inhibitor THZ1 and the CDK1/2/5/9 inhibitor dinaciclib to reduce TBXT expression at mRNA and protein levels in a broad range of nine cell lines that are models of primary, recurrent, and metastasised chordoma of the clivus and the sacrum.

Histomorphometric Analysis of 38 Giant Cell Tumors of Bone after Recurrence as Compared to Changes Following Denosumab Treatment.

Giant cell tumor of bone (GCTB) is an osteolytic tumor driven by an H3F3A-mutated mononuclear cell with the accumulation of osteoclastic giant cells. We analyzed tissue from 13 patients with recurrence and 25 patients with denosumab therapy, including two cases of malignant transformation. We found a decrease in the total number of cells (p = 0.03), but not in the individual cell populations when comparing primary and recurrence. The patients treated with denosumab showed induction of osteoid formation increasing during therapy. The total number of cells was reduced (p < 0.0001) and the number of H3F3A-mutated tumor cells decreased (p = 0.0001), while the H3F3A wild-type population remained stable. The KI-67 proliferation rate dropped from 10% to 1% and Runx2- and SATB2-positive cells were reduced. The two cases of malignant transformation revealed a loss of the H3F3A-mutated cells, while the KI-67 rate increased. Changes in RUNX2 and SATB2 expression were higher in one sarcoma, while in the other RUNX2 was decreased and SATB2-positive cells were completely lost. We conclude that denosumab has a strong impact on the morphology of GCTB. KI-67, RUNX2 and SATB2 expression differed depending on the benign or malignant course of the tumor under denosumab therapy.

Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p < 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

H3F3A-mutated giant cell tumour of bone without giant cells-clinical presentation, radiology and histology of three cases.

AIMS: Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS: Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS: We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.

Profiling of three H3F3A-mutated and denosumab-treated giant cell tumors of bone points to diverging pathways during progression and malignant transformation.

Giant cell tumor of bone (GCTB) is a locally aggressive lesion of intermediate malignancy. Malignant transformation of GCTB is a rare event. In 2013, the humanized monoclonal antibody against receptor activator of nuclear factor-κb-Ligand (RANKL) denosumab was approved for treatment of advanced GCTB. Since then, several reports have questioned the role of denosumab during occasional malignant transformation of GCTB. We report on three patients with H3F3A-mutated GCTBs, treated with denosumab. The tissue samples were analysed by histomorphology, immunohistochemistry, and in two instances by next generation panel sequencing of samples before and after treatment. One patient had a mutation of ARID2 in the recurrence of the GCTB under treatment with denosumab. One patient developed a pleomorphic sarcoma and one an osteoblastic osteosarcoma during treatment. Sequencing revealed a persisting H3F3A mutation in the osteosarcoma while the pleomorphic sarcoma lost the H3F3A mutation; however, a FGFR1 mutation, both in the recurrence and in the pleomorphic sarcoma persisted. In addition, the pleomorphic sarcoma showed an AKT2 and a NRAS mutation. These data are inconclusive concerning the role denosumab plays in the event of malignant progression/transformation of GCTB and point to diverging pathways of tumor progression of GCTB associated with this treatment.

Characterization of Three Novel H3F3A-mutated Giant Cell Tumor Cell Lines and Targeting of Their Wee1 Pathway.

The giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that is composed of mononuclear stroma cells, scattered macrophages, and multinucleated osteoclast-like giant cells which cause pathologic osteolysis. The stroma cells represent the neoplastic population of the tumor and are characterized by the H3F3A mutation G34W. This point mutation is regarded as the driver mutation of GCTB. We have established three new stable H3F3A mutated GCTB cell lines: U-GCT1, U-GCT2, and U-GCT3M. MK-1775 is a Wee1-kinase inhibitor which has been used for blocking of sarcoma growth. In the cell lines we detected Wee1, Cdk1, Cyclin B1, H3K36me3, and Rrm2 as members of the Wee1 pathway. We analyzed the effect of MK-1775 and gemcitabine, alone and in combination, on the growth of the cell lines. The cell lines showed a significant reduction in cell proliferation when treated with MK-1775 or gemcitabine. The combination of both agents led to a further significant reduction in cell proliferation compared to the single agents. Immunohistochemical analysis of 13 GCTB samples revealed that Wee1 and downstream-relevant members are present in GCTB tissue samples. Overall, our work offers valuable new tools for GCTB studies and presents a description of novel biomarkers and molecular targeting strategies.

Analysis of the CDK4/6 Cell Cycle Pathway in Leiomyosarcomas as a Potential Target for Inhibition by Palbociclib.

Leiomyosarcoma (LMS) is characterized by high genomic complexity, and to date, no specific targeted therapy is available. In a genome-wide approach, we profiled genomic aberrations in a small cohort of eight primary tumours, two relapses, and eight metastases across nine different patients. We identified CDK4 amplification as a recurrent alteration in 5 out of 18 samples (27.8%). It has been previously shown that the LMS cell line SK-LMS-1 has a defect in the p16 pathway and that this cell line can be inhibited by the CDK4 and CDK6 inhibitor palbociclib. For SK-LMS-1 we confirm and for SK-UT-1 we show that both LMS cell lines express CDK4 and that, in addition, strong CDK6 expression is seen in SK-LMS-1, whereas Rb was expressed in SK-LMS-1 but not in SK-UT-1. We confirm that inhibition of SK-LMS-1 with palbociclib led to a strong decrease in protein levels of Phospho-Rb (Ser780), a decreased cell proliferation, and G0/G1-phase arrest with decreased S/G2 fractions. SK-UT-1 did not respond to palbociclib inhibition. To compare these in vitro findings with patient tissue samples, a p16, CDK4, CDK6, and p-Rb immunohistochemical staining assay of a large LMS cohort (n=99 patients with 159 samples) was performed assigning a potential responder phenotype to each patient, which we identified in 29 out of 99 (29.3%) patients. Taken together, these data show that CDK4/6 inhibitors may offer a new option for targeted therapy in a subset of LMS patients.

Diagnostic value of MRI-based 3D texture analysis for tissue characterisation and discrimination of low-grade chondrosarcoma from enchondroma: a pilot study.

OBJECTIVES: To explore the diagnostic value of MRI-based 3D texture analysis to identify texture features that can be used for discrimination of low-grade chondrosarcoma from enchondroma. METHODS: Eleven patients with low-grade chondrosarcoma and 11 patients with enchondroma were retrospectively evaluated. Texture analysis was performed using mint Lesion: Kurtosis, entropy, skewness, mean of positive pixels (MPP) and uniformity of positive pixel distribution (UPP) were obtained in four MRI sequences and correlated with histopathology. The Mann-Whitney U-test and receiver operating characteristic (ROC) analysis were performed to identify most discriminative texture features. Sensitivity, specificity, accuracy and optimal cut-off values were calculated. RESULTS: Significant differences were found in four of 20 texture parameters with regard to the different MRI sequences (p<0.01). The area under the ROC curve values to discriminate chondrosarcoma from enchondroma were 0.876 and 0.826 for kurtosis and skewness in contrast-enhanced T1 (ceT1w), respectively; in non-contrast T1, values were 0.851 and 0.822 for entropy and UPP, respectively. The highest discriminatory power had kurtosis in ceT1w with a cut-off ≥3.15 to identify low-grade chondrosarcoma (82 % sensitivity, 91 % specificity, accuracy 86 %). CONCLUSION: MRI-based 3D texture analysis might be able to discriminate low-grade chondrosarcoma from enchondroma by a variety of texture parameters. KEY POINTS: • MRI texture analysis may assist in differentiating low-grade chondrosarcoma from enchondroma. • Kurtosis in the contrast-enhanced T1w has the highest power of discrimination. • Tools provide insight into tumour characterisation as a non-invasive imaging biomarker.

H3F3A mutation in giant cell tumour of the bone is detected by immunohistochemistry using a monoclonal antibody against the G34W mutated site of the histone H3.3 variant.

AIMS: Giant cell tumour of the bone (GCTB) is a neoplasm predominantly of long bones characterized by the H3F3A mutation G34W. Conventional diagnosis is challenged by the tumour's giant cell-rich morphology, which overlaps with other giant cell-containing lesions of the bone. Recently, a monoclonal antibody specific for the H3F3A mutation has been generated. Our aim was to test this antibody on a cohort of giant cell-containing lesions. METHODS AND RESULTS: We used the antibody for analysis of 22 H3F3A-mutated GCTB, including two patients with recurrences; for comparison we analysed a cohort of 36 H3F3A wild-type giant cell-rich lesions of the bone and soft tissue, containing one brown tumour, six aneurysmal bone cysts (ABC), six chondroblastomas, five non-ossifying-fibromas, two fibrous dysplasias, nine tenosynovial giant cell tumours, one giant cell-rich sarcoma and six osteosarcomas. Furthermore, among the 22 mutated cases, we included one GCTB with two recurrences and lung metastases; the patient was treated with the anti-receptor activator of nuclear factor κB (RANK) ligand denosumab. We show that all 22 H3F3A-mutated GCTB display strong nuclear H3.3 G34W staining in the neoplastic component, while the osteoclastic giant cells are negative. 36 H3F3A wild-type lesions are negative. The GCTB treated with denosumab revealed a reduction in the H3.3 G34W-positive tumour cells and a decrease in osteoclastic giant cells accompanied by matrix and osteoid formation. CONCLUSIONS: We conclude that positive H3.3 G34W staining is a specific and sensitive method for detection of H3F3A-mutated GCTB. Denosumab treatment leads to a pathomorphosis of the lesion characterized by matrix and osteoid producing H3.3 G34W-negative stromal cells.

In chordoma, metastasis, recurrences, Ki-67 index, and a matrix-poor phenotype are associated with patients' shorter overall survival.

PURPOSE: To establish a chordoma tissue cohort (n = 43) and to correlate localization, size, metastasis, residual disease (R-status), recurrences, histological subtype, matrix content, and Ki-67 proliferation index with patients' overall survival (OS). METHODS AND RESULTS: We used routine histopathology supplemented by immunohistochemistry. In our patient cohort (median age 69 years, range 17 to 84 years) the median OS was 8.25 years. 24 chordomas were localized in the sacrum, 6 in lumbar vertebrae, 7 in thoracic and cervical vertebrae, 5 were limited to the clivus, and one was localized in the nasal septum. Ten patients had metastases, with pulmonary, nodal, and hepatic involvement. 23 patients had recurrent disease. 23 chordomas were classified as 'not otherwise specified' (NOS). Besides NOS, we found the following differentiation patterns: renal cell cancer like in six cases, chondroid in four cases, hepatoid differentiation in three cases, and anaplastic morphology in six cases. Ki-67 index of ≥10 %, presence of metastasis, and the low content of extracellular matrix were statistically linked to poor OS (p < 0.05). The matrix-poor phenotype had a higher Ki-67 index (p < 0.05). Furthermore, presence of metastasis was associated with a higher Ki-67 index in the primary lesion, a positive resection margin, and multiple recurrences (p < 0.05 each). CONCLUSION: We propose to include these parameters in the final pathologic report of the resected chordoma.

Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway.

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies.

Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: differential diagnosis and diagnostic algorithm.

Extraskeletal osteosarcoma is a rare neoplasia within the broad differential diagnostic spectrum of calcifying intramuscular lesions. We present a case of a slowly increasing mass within the left vastus lateralis muscle. At first presentation the patient showed a partially calcified well defined mass with a diameter of 5 cm and with no direct contact to the femur. A biopsy from the periphery revealed an ossifying lesion compatible with myositis ossificans. The patient returned 18 months later with the lesion having increased to a diameter of 25 cm. The resection specimen revealed a well delimitated tumor with a central core of partially necrotic neoplastic bone. Besides, histology showed high mitotic areas with pleomorphic spindle cells and regions with cartilaginous differentiation. Immunohistochemistry demonstrated: vimentin+, CD34-, desmin-, actin-, EMA- and pancytokeratin- with focal S100 protein positivity and a Ki-67 index of 20%. Comparative genomic hybridization (CGH) revealed a gain of chromosomal material on 12q; FISH analyses for the CDK4 and MDM2 region showed high level amplifications. Consequently, a high-grade dedifferentiated extraskeletal osteosarcoma was diagnosed. In conclusion, analysis of the MDM2 and CDK4 status is a powerful and discriminating diagnostic tool to distinguish dedifferentiated extraskeletal osteosarcoma from other benign/malignant ossifying lesions in the skeletal muscle.

Molecular profiling of chordoma.

The molecular basis of chordoma is still poorly understood, particularly with respect to differentially expressed genes involved in the primary origin of chordoma. In this study, therefore, we compared the transcriptional expression profile of one sacral chordoma recurrence, two chordoma cell lines (U-CH1 and U-CH2) and one chondrosarcoma cell line (U-CS2) with vertebral disc using a high-density oligonucleotide array. The expression of 65 genes whose mRNA levels differed significantly (p<0.001; ≥6-fold change) between chordoma and control (vertebral disc) was identified. Genes with increased expression in chordoma compared to control and chondrosarcoma were most frequently located on chromosomes 2 (11%), 5 (8%), 1 and 7 (each 6%), whereas interphase cytogenetics of 33 chordomas demonstrated gains of chromosomal material most prevalent on 7q (42%), 12q (21%), 17q (21%), 20q (27%) and 22q (21%). The microarray data were confirmed for selected genes by quantitative polymerase chain reaction analysis. As in other studies, we showed the expression of brachyury. We demonstrate the expression of new potential candidates for chordoma tumorigenesis, such as CD24, ECRG4, RARRES2, IGFBP2, RAP1, HAI2, RAB38, osteopontin, GalNAc-T3, VAMP8 and others. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in chordoma.

Integrated FDG-PET-CT: its role in the assessment of bone and soft tissue tumors.

PURPOSE: The purpose of this study was to evaluate prospectively, whether integrated 2-deoxy-2-[(18)F]fluoro-D: -glucose positron emission tomography-computed tomography (FDG-PET-CT) is more accurate for determination musculoskeletal tumors compared with separate interpretation of CT and FDG-PET, because most of the current clinical data come from patients studied with PET. METHODS: Eighty patients with newly diagnosed musculoskeletal tumors underwent FDG-PET-CT. CT, FDG-PET, and FDG-PET-CT were interpreted separately to determine the performance of each imaging modality. RESULTS: Assuming that equivocal lesions are benign, performance of diagnostic tests was as follows: sensitivity, specificity and accuracy for CT alone was 81, 84, 83%, for PET 71, 82, 76, and for PET-CT 80, 83 and 86%. Assuming that equivocal lesions are malignant, sensitivity, specificity, and accuracy for CT was 61, 100, 70%, for PET 69, 100, 79, and for PET-CT 69, 100 and 79%. CONCLUSIONS: Combined FDG-PET-CT reliably differentiates soft tissue and bone tumors from benign lesions. The value of the information provided by FDG-PET-CT for planning surgical procedures must be evaluated in further studies.

Metachronous and multiple aneurysmal bone cysts: a rare variant of primary aneurysmal bone cysts.

In 1942, Jaffe and Lichtenstein introduced the term aneurysmal bone cyst (ABC). Primary ABC is characterized by the presence of spongy or multi-cameral cystic tissue filled with blood. The process is benign, but it is locally destructive and has a high propensity for recurrence. In this paper, we present the third case of multiple metachronous primary ABCs as a rare variant of ABC. We describe the 10-year history of a 12-year-old boy with metachronous multiple primary ABCs at five different sites (right proximal humerus, right ulna, bilateral distal radius and right lateral clavicle). Furthermore, our patient suffered from vascular malformations, such as aortic isthmus stenosis, hypoplastic thoraco-abdominal aorta and bilateral renal artery stenosis. To date, in contrast to solitary ABC, the multiple lesions have been found more frequently in male individuals. Using interphase cytogenetics, we analyzed three of five of the patient's ABCs and one of these was also analyzed by GTG-banding. No chromosomal abnormalities were found. Significantly, we excluded the missense mutation of codon 201 in guanine nucleotide-binding protein 1 gene consistently found in McCune-Albright syndrome (MAS) and in non-MAS cases of polyostotic fibrous dysplasia of bone with or without secondary ABC.