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Overview ideogram of CGH data of all chordoma cell lines. Every cell line corresponds to one color. Lines on the left side are losses, and on the right side are gains of chromosomal material. Arrows mark the CDK6 region on 7q and p16 region on 9p.

Detailed CGH karyograms of the chordoma cell lines as shown in the ideogram in Supplementary Figure 1 (according to ISCN: International System for Human Cytogentic Nomenclature).

Results of the single tandem repeats analysis of the cell lines U-CH1, U-CH2, UCH3, UCH6, UCH7, U-CH10, U-CH11 and UCH12. The results of the sex typing marker AMEL and the detected alleles for the markers D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045 are given.

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

Unsupervised cluster analysis of mRNA expression data of all chordoma cell lines. The T-gene (i.e., brachyury) is overexpressed (red). Cluster Analysis of chordoma cell lines with chondrocyte, liposarcoma and sarcoma expression data from GEO and own data from a cell line with cartilaginous differentiation and from tissue of a vertebral disc.

Effect of Abemaciclib (LY2835219) on the growth of chordoma lines U-CH1, U-CH6, CAL51 (negative control), and MCF7 (positive control). Cells were grown in 96-well-plates were incubated for 3 days at 37{degree sign}C with increasing concentrations of Abemaciclib (3, 6, 10, 30, 60, 100, 300, 600, 1000 nM) in the medium or left untreated for control. The amount of viable cells was determined by using the MTS cell viability test (GraphPad Prism software, Version 6).

Western blot analyses of 8 chordoma cell lines showing expression of brachyury at 47 kDa.

Clinical data of chordoma patients. Every sample was positive for brachyury. Asterisks (*) mark the parental chordomas of the established chordoma cell lines.

Proliferation assay using Alamar Blue assay for U-CH2, 3, 6 and 10. Increasing concentrations of Palbociclib lead to an increase of growth inhibition of chordoma cells.

Inversion microscopy views of the published chordoma cell lines U-CH1, U-CH2, U-CH 3, 6, 7, 10, 11, and 12. The cells showing the typical physaliphorous cytology; bars=100µm.

Supplementary Figure Legend

Immunocytology of U-CH1 control vs inhibited cells with 1000 nM Palbociclib for 3 days. Ki-67 decreases from 20% down to

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

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

List of up and down regulated genes on expression level with a p-value of 10-4 a FC of 10.

FISH results regarding the CDKN2A status in chordoma cell lines and the corresponding parental tumor.

Western blot analysis for cleaved PARP and cleaved caspase 3 of all chordoma cell lines as compared to HEK-293 cells (+/- stimulation with TNFα and Bortezomib for 18h). No corresponding bands are apparent in the cell lines after inhibition with 1000 nM Palbociclib for 3 days. U-CH 1-6 (A) and U-CH 7-12 (A).

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.

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 WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

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/G2fractions. SK-UT-1 did not respond to palbociclib inhibition. To compare thesein vitrofindings with patient tissue samples, a p16, CDK4, CDK6, and p-Rb immunohistochemical staining assay of a large LMS cohort (n=99patients 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.

Chordomas are rare tumours of the bone arising along the spine from clivus to sacrum. We compared three chordoma cell lines of the clivus region including the newly established clivus chordoma cell line, U-CH14, with nine chordoma cell lines originating from sacral primaries by morphology, on genomic and expression levels and with patient samples from our chordoma tissue bank. Clinically, chordomas of the clivus were generally smaller in size at presentation and patients with sacral chordomas had more metastases and more often recurrent disease. All chordoma cell lines had a typical physaliphorous morphology and expressed brachyury, S100-protein and cytokeratin. By expression analyses we detected differentially expressed genes in the clivus derived cell lines as compared to the sacral cell lines. Among these were HOXA7, HOXA9, and HOXA10 known to be important for the development of the anterior-posterior body axis. These results were confirmed by qPCR. Immunohistologically, clivus chordomas had no or very low levels of HOXA10 protein while sacral chordomas showed a strong nuclear positivity in all samples analysed. This differential expression of HOX genes in chordomas of the clivus and sacrum suggests an oncofetal mechanism in gene regulation linked to the anatomic site.

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). 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

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. Cancer Res; 75(18); 3823–31. ©2015 AACR.

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

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. 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. Significant differences were found in four of 20 texture parameters with regard to the different MRI sequences (p

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.

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.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.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%.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.

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.

Die aneurysmatische Knochenzyste ist eine seltene tumorahnliche Erkrankung, die jeden Skelettabschnitt involvieren kann und sich uberwiegend im Kindes- oder Jugendlichenalter manifestiert. Differentialdiagnostische Schwierigkeiten bei der Abgrenzung gegenuber bestimmten benignen und malignen primaren Knochentumoren erfordern neben dem Einsatz differenzierter bildgebender Verfahren haufig die histopathologische Beurteilung durch ein Knochentumorregister. Bedingt durch eine sehr heterogene biologische Aktivitat und den Umstand, das die Erkrankung uberwiegend das wachsende Skelett betrifft, mus bei der chirurgischen Therapie sowohl hinsichtlich der Resektion als auch der Defektrekonstruktion auf ein breites Spektrum an Behandlungsmasnahmen zuruckgegriffen werden konnen. In einer retrospektiven Studie wurden die Behandlungsergebnisse bei 41 operierten Tumoren, davon 31 aktive und jeweils 5 latente bzw. aggressive Lasionen, untersucht. Wahrend die therapieassoziierte Morbiditat auch bei ausgedehnteren Destruktionen sowie aufwendigeren Rekonstruktionen vergleichsweise gering war, lag die Rezidivrate bei 19,5%. Die Analyse der Lokalrezidive zeigte, das– unter Berucksichtigung der biologischen Aktivitat des Primartumors – jeweils ein inadaquates Resektionsverfahren zu Grunde lag. Der Umstand, das das Lokalrezidivrisiko sowohl mit der Aggressivitat der Primarlasion als auch mit der chirurgischen Radikalitat korreliert, macht ein praoperatives Staging und eine stadiengerechte Therapie erforderlich.

Im Rahmen einer prospektiven Studie an 168 Patienten mit Weichteil- oder Knochentumoren, darunter 71 Sarkomen, wurde unter farbdopplersonographischer Kontrolle eine Vollschnittbiopsie mit dem Hochgeschwindigkeitssystem Autovac® gewonnen. Das Ultraschallmonitoring erlaubte eine gezielte Punktion auch von kleinen oder tiefer gelegenen Lasionen und ermoglichte im Falle des Vorliegens von Nekrosen das Aufsuchen vitaler Tumoranteile. Eine adaquate histologische Beurteilung einschlieslich Tumortypisierung und Grading war bei der grosen Mehrzahl der Weichteilsarkome, Weichteilmetastasen, malignen Lymphome, Plasmocytome und osteolytischen Skelettmetastasen moglich. Benigne Weichteil- und Knochentumoren waren dagegen nur zu 66 % diagnostizierbar. In der Gruppe der ossaren Sarkome war trotz histologischem Malignitatsnachweis eine exakte Tumortypisierung aufgrund zu geringer Materialmenge haufig nicht moglich. Mit einer Genauigkeit von 97 % fur das Kriterium Malignitat und von 94 % fur die Diagnose Weichteilsarkom liefert die Methode Ergebnisse, die denen der Incisionsbiopsie, dem Standardverfahren bei Weichteil- und Knochentumoren unklarer Dignitat, vergleichbar sind. In Anbetracht der bekannten Nachteile und onkologischen Risiken des offenen Verfahrens stellt die Nadelbiopsie – bei Einhaltung der fur die Incisionsbiopsie geltenden Kautelen – fur Weichteiltumoren und osteolytische Skelettlasionen die Methode der Wahl dar.

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.

In April 2010, a 19-year-old soccer player presented with a 4-week history of pain after a tap against his right knee. Medical and family histories were unremarkable and no abnormalities were found on physical examination. A radiograph of the right knee showed osteolytic lesions in the distal femur and the head of the tibia. On magnetic resonance imaging, the epiphyseal lesions were visualized as cystic and nonsclerotic and extended to the articular surface of the distal femur and proximal tibia (Figure 1). Soft tissue extension was suspected from the tibia. Laboratory test results showed normal blood cell count, renal function, and concentration of C-reactive protein. Aneurysmatic bone cysts were suspected and a biopsy was performed. Histopathological examination detected spindle cell infiltrates with hemorrhages, fragmented trabecular bone, and numerous osteoclastic giant cells proliferating at a rate of 10% suggesting an aggressive giant cell lesion. Since it was still not determined whether the lesions were malignant, flourodeoxyglucose F 18positron emission tomography/computed tomographywasperformedand foundadditional lesions in the left distal femur and the leftmandible. Biopsyof themandible showed the same type of tissue as seen in the right femur and tibia. Quiz at jamasurgery.com A B