Bone Tumor In Children Research Articles

Ewing Sarcoma

The 2 most common types of primary bone tumors in children and adolescents are osteosarcoma and Ewing sarcoma (ES). Common sites of presentation for ES include the long bones, pelvis, chest wall, and spine. Definitive diagnosis of ES is made via open or core needle biopsy. The standard treatment regimen of ES includes neoadjuvant chemotherapy, followed by local primary tumor control and then consolidative chemotherapy. In addition, ES is sensitive to radiation, and thus, radiotherapy is often implemented. Overall, the ES survival rates range from 70% to as low as 15% to 30% based on the stage.

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1‐ETS fusion oncogenes, most often EWSR1‐FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F‐dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate‐independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho‐dependent cell migration, and impaired caspase‐3‐mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5‐benzenetetraamine tetrahydrochloride (Y15) enhanced caspase‐mediated apoptosis and EwS cell migration, independent from the respective EWSR1‐ETS fusion type, mimicking an anoikis‐like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

Segmentation of osteosarcoma tumor using diffusion weighted MRI: a comparative study using nine segmentation algorithms

Osteosarcoma is a primary malignant bone tumor in children and adolescents with significant morbidity and poor prognosis. Diffusion weighted imaging (DWI) plays a crucial role in diagnosis and prognosis of this malignant disease by capturing cellular changes in tumor tissue early in the course of treatment without any contrast injection. Segmentation of tumor in DWI is challenging due to low signal-to-noise ratio, partial-volume effects, intensity inhomogeneities and irregular shape of osteosarcoma. The purpose of this study was to segment osteosarcoma solely utilizing DWI and identify effective and robust technique(s) for tumor segmentation. DWI dataset of fifty-five (N = 55; male:female = 41:14; Age = 17.8 ± 7.4 years) patients with osteosarcoma was acquired before treatment. Total nine automated and semi-automated segmentation algorithms based on (1) Otsu thresholding (OT), (2) Otsu threshold-based region growing (OT-RG), (3) Active contour (AC), (4) Simple linear iterative clustering Superpixels (SLIC-S), (5) Fuzzy c-means clustering (FCM), (6) Graph cut (GC), (7) Logistic regression (LR) (8) Linear support vector machines (L-SVM) and (9) Deep feed-forward neural network (DNN) were implemented. Segmentation accuracy was estimated by Dice coefficient (DC), Jaccard Index (JI), precision (P) and recall (R) using manually demarcated ground-truth tumor mask by a radiologist. Evaluated apparent diffusion coefficient (ADC) in segmented tumor mask and ground-truth tumor mask was compared using paired t test for statistical significance (p < 0.05) and Pearson correlation coefficient (PCC). Automated SLIC-S and FCM showed quantitatively and qualitatively superior segmentation with DC: ~ 79–82%; JI: ~ 67–71%; P: ~ 81–83%; R: ~ 80–86% and PCC = 0.89, 0.88 among all methods. Among semi-automated methods, AC was quantitatively more accurate (DC: ~ 77%; JI: ~ 65%; P: ~ 72%; R: ~ 88%; PCC = 0.85) than OT-RG and GC (DC: ~ 74–75%; JI: ~ 60–61%; P: ~ 67–72%; R: ~ 84–89%; PCC = 0.78, 0.73). Among machine learning algorithms, DNN showed the highest accuracy (DC: ~ 73%; JI: ~ 62%; P: ~ 77%; R: ~ 86%; PCC = 0.79) than LR and L-SVM (DC: ~ 70–71%; JI: ~ 58–63%; P: ~ 73%; R: ~ 74–85%; PCC = 0.69, 0.71). Execution times were instantaneous for SLIC-S, FCM and machine learning methods, while OT-RG, AC and GC took comparable ~ 1–6 s/slice image. Automated SLIC-S, FCM and semi-automated AC methods produced promising tumor segmentation results using DWI of the osteosarcoma dataset.

Prospects for the involvement of cancer stem cells in the pathogenesis of osteosarcoma.

Osteosarcoma (OS) is one of the most common bone tumors in children and adolescents that cause a high rate of mortality in this age group and tends to be metastatic, in spite of chemotherapy and surgery. The main reason for this can be returned to a small group of malignant cells called cancer stem cells (CSCs). OS-CSCs play a key role in the resistance to treatment and relapse and metastasis through self-renewal and differentiation abilities. In this review, we intend to go through the different aspects of this malignant disease, including the cancer stem cell-phenotype, methods for isolating CSCs, signaling pathways, and molecular markers in this disease, and drugs showing resistance in treatment efforts of OS.

Slow skeletal muscle troponin T, titin and myosin light chain 3 are candidate prognostic biomarkers for Ewing's sarcoma.

Ewing's sarcoma (ES) is a common malignant bone tumor in children and adolescents. Although great efforts have been made to understand the pathogenesis and development of ES, the underlying molecular mechanism remains unclear. The present study aimed to identify new key genes as potential biomarkers for the diagnosis, targeted therapy or prognosis of ES. mRNA expression profile chip data sets GSE17674, GSE17679 and GSE45544 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened using the R software limma package, and functional and pathway enrichment analyses were performed using the enrichplot package and GSEA software. The NetworkAnalyst online tool, as well as Cytoscape and its plug-ins cytoHubba and NetworkAnalyzer, were used to construct a protein-protein interaction network (PPI) and conduct module analysis to screen key (hub) genes. LABSO COX regression and overall survival (OS) analysis of the Hub genes were performed. A total of 211 DEGs were obtained by integrating and analyzing the three data sets. The functions and pathways of the DEGs were mainly associated with the regulation of small-molecule metabolic processes, cofactor-binding, amino acid, proteasome and ribosome biosynthesis in eukaryotes, as well as the Rac1, cell cycle and P53 signaling pathways. A total of one important module and 20 hub genes were screened from the PPI network using the Maximum Correlation Criteria algorithm of cytoHubba. LASSO COX regression results revealed that titin (TTN), fast skeletal muscle troponin T, skeletal muscle actin α-actin, nebulin, troponin C type 2 (fast), myosin light-chain 3 (MYL3), slow skeletal muscle troponin T (TNNT1), myosin-binding protein C1 slow-type, tropomyosin 3 and myosin heavy-chain 7 were associated with prognosis in patients with ES. The Kaplan-Meier curves demonstrated that high mRNA expression levels of TNNT1 (P<0.001), TTN (P=0.049), titin-cap (P=0.04), tropomodulin 1 (P=0.011), troponin I2 fast skeletal type (P=0.021) and MYL3 (P=0.017) were associated with poor OS in patients with ES. In conclusion, the DEGs identified in the present study may be key genes in the pathogenesis of ES, three of which, namely TNNT1, TTN and MYL3, may be potential prognostic biomarkers for ES.

Lung metastases at the initial diagnosis of high-grade osteosarcoma: prevalence, risk factors and prognostic factors. A large population-based cohort study.

Osteosarcoma is the most prevalent malignant bone tumor in children and adolescents. Lung metastases are associated with poor prognosis. The aim here was to explore the prevalence of and risk and prognostic factors for lung metastases in high-grade osteosarcoma patients. Retrospective cohort study based on the Surveillance, Epidemiology and End Results (SEER) database in the United States. Data on 1,408 high-grade osteosarcoma patients registered in the SEER database between 2010 and 2015 were extracted. From these, all patients with high-grade osteosarcoma and initial lung metastasis were selected for analysis on risk and prognostic factors for lung metastases. Overall survival was estimated. There were 238 patients (16.90%) with lung metastases at diagnosis. Axial location, tumor size > 10 cm (odds ratio, OR 3.19; 95% confidence interval, CI: 1.58-6.45), higher N stage (OR 4.84; 95% CI: 1.94-12.13) and presence of bone metastases (OR 8.73; 95% CI: 4.37-17.48) or brain metastases (OR 25.63; 95% CI: 1.55-422.86) were significantly associated with lung metastases. Younger age and surgical treatment (hazard ratio, HR 0.46; 95% CI: 0.30-0.71) favored survival. Median survival was prolonged through primary tumor surgery. The factors revealed here may guide lung metastasis screening and prophylactic treatment for osteosarcoma patients. A primary tumor in an axial location, greater primary tumor size, higher lymph node stage and presence of bone or brain metastases were significantly correlated with lung metastases. The elderly group (≥ 60 years) showed significant correlation with poor overall survival. For improved survival among high-grade osteosarcoma patients with lung metastases, aggressive surgery on the primary tumor site should be encouraged.

Apiosporamide, A 4-hydroxy-2-pyridone Alkaloid, Induces Apoptosis Via PI3K/Akt Signaling Pathway In Osteosarcoma Cells.

BackgroundOsteosarcoma (OS) is a common primary malignant bone tumour in children and young adults. Apiosporamide, a 4-hydroxy-2-pyridone alkaloid from a deep-sea-derived fungus, Arthrinium sp. UJNMF0008, showed anti-proliferative effects toward a panel of human cancer cell lines, and the molecular mechanism in MG63 cells was then investigated in the current work.MethodsCell viability was determined with MTT method. Cell proliferation was detected using colony-formation assay. Screening electron microscope was used for morphology observation. Cell cycle and apoptosis was analysed via flow cytometry. Real-time PCR was conducted to evaluate the mRNA expression related with cell apoptosis. The expression levels of proteins related to capase-mediated apoptotic pathway and PI3K/Akt signalling pathway were detected by Western blotting.ResultsApiosporamide significantly decreased cell viability in cancer cells, and also exhibited excellent anti-proliferative effect. Apiosporamide caused cell cycle arrests at G0/G1 phase in MG63 cells. Moreover, apiosporamide induced apoptosis, activated caspase-3, caspase-8 and caspase-9, and regulated expression of Bax and Bcl-2 in MG63 cells. In addition, apiosporamide also attenuated PI3K/Akt signaling pathway.ConclusionApiosporamide effectively suppressed MG63 cells proliferation by inducing apoptosis through PI3K/Akt and caspase-associated apoptotic pathway.

PTEN loss activates a functional AKT/CXCR4 signaling axis to potentiate tumor growth and lung metastasis in human osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Loss of the tumor suppressor PTEN or activation of chemokine receptor CXCR4 has been demonstrated to associate with OS respectively. However, the signaling mechanism underlying PTEN-mediated antitumor effect remains largely unknown, and the crosstalk between PTEN and CXCR4 in OS has not been investigated. Here, we uncover a PTEN/AKT/CXCR4 pathway nexus in highly tumorigenic and metastatic human 143B OS cells. Loss of PTEN activates AKT/CXCR4 signaling axis and regulates a series of tumor cell behaviors. Notably, ERK is inversely regulated by PTEN and its activation occurs downstream of AKT but upstream of CXCR4, suggesting this kinase to be an important mediator between AKT and CXCR4. In vivo studies show that overexpression of PTEN dramatically attenuates bone destruction, and this inhibition is associated with reduced CXCR4 expression in tumors. CXCR4 inhibitor AMD3100 also markedly suppresses tumor growth in the bone. In addition, PTEN overexpression or AMD3100 substantially inhibits tumor expansion in the lung. Our studies highlight a novel PTEN/AKT/CXCR4 signaling nexus in OS tumor growth and lung metastasis, and provide a strong rationale to consider PTEN restoration or CXCR4 blockade for the treatment of aggressive OS in humans.

Small round cell tumor of cervical region: a case report with unusual site &amp; course

Ewing’s sarcoma belongs to a spectrum of neoplastic diseases known as Ewing’s family of tumors. Ewing sarcoma can arise in either bone or soft tissue. Small round cell tumors comprise a heterogeneous group of neoplasms that predominate in childhood and adolescence, share similar morphological features, consisting of dense cellular proliferation of small round cells with primitive appearance. Ewing sarcoma is the second most common malignant bone tumour in children and young adults, although, rarely, it may be of extraskeletal origin. Extra-osseous tumor with metastatic or recurrent disease has a worse outcome; 5-year overall survival remains about 25 %. A female patient presented in surgical OPD for the first time at the age of 12 years with swelling in right supraclavicular region. The swelling was excised & a provisional diagnosis of small round cell tumor was given & patient was lost to follow up. Swelling recurred at the age of 23 years & was gradually increasing in size, measuring 20 x 20 x 20 cm. Radiologically, lesion was suggestive of sarcoma / lymphoma. Patient was operated & histopathological diagnosis of malignant round cell tumor was given with differential diagnosis of Ewing’s sarcoma, PNET, lymphoma. Patient had third recurrence of swelling at the age of 28 years at same site. On IHC, diagnosis of Soft Tissue Ewing sarcoma was given. Inspite of recurrence & huge size of tumor, patient was otherwise asymptomatic. Ours is a rare case of more than 16 years of survival inspite of recurrence. Immunochemistry is mandatory for diagnosis of Extraskeletal Ewing sarcoma in younger age group patients. Regular follow up of such patients is essential for timely detection & complete treatment of recurrent tumors.

Sodium cantharidate targets STAT3 and abrogates EGFR inhibitor resistance in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Overactive EGFR signaling is frequently seen in osteosarcoma cells, and represents a potential therapeutic target. However, feedback activation of STAT3 after EGFR inhibition is linked to treatment resistance, suggesting that combined EGFR/STAT3 inhibition may be needed to overcome this effect. Cantharidin and its analogues have shown strong anticancer effects, including STAT3 inhibition, in several tumor cells. Therefore, we investigated the effects of sodium cantharidate (SC), either as monotherapy and in combination with the EGFR inhibitor erlotinib, on STAT3 activation and osteosarcoma cell growth. Cell viability, migration, and apoptosis assays were performed in human MG63 and U2OS cells, and MG63 xenografts were generated in nude mice to verify the suppression of tumor growth in vivo. Additionally, western blotting and immunohistochemistry were used to verify the STAT3 and EGFR phosphorylation statuses in xenografts. We found that SC repressed cell viability and migration and induced apoptosis in vitro, while combined SC and erlotinib treatment enhanced osteosarcoma growth suppression by preventing feedback activation of STAT3. These data support further development of cantharidin-based combination therapies for metastatic and recurrent/refractory osteosarcoma.

Tumeurs osseuses bénignes de la hanche

The etiological diagnosis of hip benign bone tumour in children and adults is a clinical and radiological challenge. We discuss the pseudotumours common in the hip, then propose a diagnostic approach based on the patient's age, clinical presentation, anatomical site and radiological semiology.

AUF1 positively controls angiogenesis through mRNA stabilization-dependent up-regulation of HIF-1α and VEGF-A in human osteosarcoma.

Osteosarcoma is the most common malignant bone tumor in children, adolescents, and young adults. This pleiomorphic tumor depends on new blood vessel development, also known as angiogenesis, for tumor growth and metastasis. Therefore, it’s of utmost importance to identify the key genes and pathways that regulate this pro-metastatic process in order to develop more efficient therapies. Here, we have shown that the RNA-binding protein AUF1 positively regulates the expression of the pro-angiogenic factor VEGF-A and its positive regulator HIF-1alpha through direct binding and stabilization of their mRNAs. This effect is mediated through the seeding sequence of the AUF1 protein in the VEGF-A and HIF-1alpha 3’UTR sequences. As a consequence, the expression of the 3 genes was highly correlative in various osteosarcoma cell lines, and AUF1 enhanced the pro-angiogenic capabilities of osteosarcoma cells both in vitro and in vivo. Indeed, while inhibition of AUF1 using specific siRNA suppressed the pro-angiogenic effects of osteosarcoma cells, ectopic expression of AUF1 enhanced the pro-angiogenic effect in a VEGF-A-dependent manner. Therefore, in the era of targeted therapy, anti-angiogenic therapies targeting AUF1 could provide effective methods for treating osteosarcoma.

Tangeretin-Assisted Platinum Nanoparticles Enhance the Apoptotic Properties of Doxorubicin: Combination Therapy for Osteosarcoma Treatment.

Osteosarcoma (OS) is the most common type of cancer and the most frequent malignant bone tumor in childhood and adolescence. Nanomedicine has become an indispensable field in biomedical and clinical research, with nanoparticles (NPs) promising to increase the therapeutic efficacy of anticancer drugs. Doxorubicin (DOX) is a commonly used chemotherapeutic drug against OS; however, it causes severe side effects that restrict its clinical applications. Here, we investigated whether combining platinum NPs (PtNPs) and DOX could increase their anticancer activity in human bone OS epithelial cells (U2OS). PtNPs with nontoxic, effective, thermally stable, and thermoplasmonic properties were synthesized and characterized using tangeretin. We examined the combined effects of PtNPs and DOX on cell viability, proliferation, and morphology, reactive oxygen species (ROS) generation, lipid peroxidation, nitric oxide, protein carbonyl content, antioxidants, mitochondrial membrane potential (MMP), adenosine tri phosphate (ATP) level, apoptotic and antiapoptotic gene expression, oxidative stress-induced DNA damage, and DNA repair genes. PtNPs and DOX significantly inhibited U2OS viability and proliferation in a dose-dependent manner, increasing lactate dehydrogenase leakage, ROS generation, and malondialdehyde, nitric oxide, and carbonylated protein levels. Mitochondrial dysfunction was confirmed by reduced MMP, decreased ATP levels, and upregulated apoptotic/downregulated antiapoptotic gene expression. Oxidative stress was a major cause of cytotoxicity and genotoxicity, confirmed by decreased levels of various antioxidants. Furthermore, PtNPs and DOX increased 8-oxo-dG and 8-oxo-G levels and induced DNA damage and repair gene expression. Combination of cisplatin and DOX potentially induce apoptosis comparable to PtNPs and DOX. To the best of our knowledge, this is the first report to describe the combined effects of PtNPs and DOX in OS.

Chemotherapeutic Targets in Osteosarcoma: Insights from Synchrotron-MicroFTIR and Quasi-Elastic Neutron Scattering

This study aimed at the development of improved drugs against human osteosarcoma, which is the most common primary bone tumor in children and teenagers with a low prognosis. New insights into the impact of an unconventional Pd(II) anticancer agent on human osteosarcoma cells were obtained by synchrotron radiation-Fourier transform infrared microspectroscopy and quasi-elastic neutron scattering (QENS) experiments from its effect on the cellular metabolism to its influence on intracellular water, which can be regarded as a potential secondary pharmacological target. Specific infrared biomarkers of drug action were identified, enabling a molecular-level description of variations in cellular biochemistry upon drug exposure. The main changes were detected in the protein and lipid cellular components, namely, in the ratio of unsaturated-to-saturated fatty acids. QENS revealed reduced water mobility within the cytoplasm for drug-treated cells, coupled to a disruption of the hydration layers of biomolecules. Additionally, the chemical and dynamical profiles of osteosarcoma cells were compared to those of metastatic breast cancer cells, revealing distinct dissimilarities that may influence drug activity.

Abstract 3231: Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells. Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues. Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response. Citation Format: Koji Demiya, Hiroshi Tazawa, Yusuke Mochizuki, Miho Kure, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma [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 3231.

Abstract 68: Comparative transcriptome analysis of primary tumor and matched tumor thrombus reveals that coagulation cascades activation contributes to the venous metastases of osteosarcoma

Abstract Comparative transcriptome analysis of primary tumor and matched tumor thrombus reveals that coagulation cascades activation contributes to the venous metastases of osteosarcoma Osteosarcoma is the most frequent malignant primary tumor of bone in children and adolescents with an approximately 5-year survival about 50-60%. Lung metastasis remains as the most challenging condition during treatment and follow-up, which has been regarded as the main reason for bottle neck of prognosis. Tumor thrombus is occasionally diagnosed in patients with malignant tumor. It usually exists during tumor angiogenesis associated with proliferation and metastasis. The presentation shows aggressive features and advanced stage of the disease. Tumor thrombus is associated with highly metastasis potential. Based on the evolution theory of tumor metastasis, tumor thrombus can be the intermediate phase between local invasion and distal metastasis. In order to investigate the underlying mechanism and identify candidate metastasis driver genes, we collected a set of high quality paired tumor tissue, both the primary site and corresponding tumor thrombus that were treated by thrombectomy. In this study, we present a large-scale transcriptome analysis, by RNA sequencing, of 4 patients diagnosed with large pelvic osteosarcoma with tumor thrombus. The dysregulated genes between primary site and thrombus suggested that the complement and coagulation cascades, focal adhesion and extracellular matrix receptor was correlated with the formation of tumor thrombus. Tissue factor was identified as a major marker in coagulation pathway activation. We also identified NOTCH2NL-NBPF1 fusion genes in all four patients. All of these findings broaden our knowledge of osteosarcoma thrombus and may also contribute to the novel mechanism of metastasis in osteosarcoma. Citation Format: Tao Ji, Yifei Wang, Yi Guo, Dasen Li, Haijie Liang, Tingting Ren, Wei Guo. Comparative transcriptome analysis of primary tumor and matched tumor thrombus reveals that coagulation cascades activation contributes to the venous metastases of osteosarcoma [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 68.

Abstract 4494: HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction

Abstract Ewing sarcoma (EWS) is the second most common bone tumor in children and adolescents. The primary oncogenic event in ~85% of EWS is a chromosomal translocation that generates a fusion gene encoding the aberrant transcription factor EWS-FLI1. A third of EWS-FLI1 driven tumors harbor translocations that retain exon 8 of the EWSR1 gene, and in these tumors, skipping of this exon is vital to express an in-frame EWS-FLI1 mRNA. We have shown previously that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and facilitates the exclusion of this exon from distinct EWS-FLI1 pre-mRNAs. Guanine-rich nucleic acids may fold into stable tertiary structures, such as RNA guanine quadruplexes (rG4s). Our current studies are focused on the tertiary RNA structure of the G-rich regions within EWSR1 exon 8 and its role in HNRNPH1-dependent RNA processing. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of wild-type EWSR1. We demonstrate that guanine-rich sequences within EWSR1 exon 8 can fold into RNA G-quadruplex structures and that this structure favors the recruitment of HNRNPH1. Bioinformatic analysis of transcriptome-wide profiles for RNA G-quadruplexes and RNA targets of HNRNPH1 revealed sequences containing two-quartet G-quadruplex configurations are central for HNRNPH1 binding to exonic elements. Critically, we also demonstrate that the glycine-tyrosine-arginine-rich domain of HNRNPH1 is the minimal domain required for G-quadruplex recognition. Small molecules that can displace HNRNPH1 binding may provide a therapeutic vulnerability in a subset of Ewing sarcoma. To evaluate if this protein-RNA interaction is amenable to small molecules, we performed displacement assays with HNRNPH1 protein and an EWSR1 exon 8 RNA oligomer at varying concentrations of the pan-quadruplex binding molecule, pyridostatin (PDS). PDS disrupts the HNRNPH1-RNA complex with an IC50 of 7 micromolar. Furthermore, treatment with PDS selectivity inhibits the growth of EWS cells harboring EWSR1 exon 8 fusions, decreases EWS-FLI1 activity in cell-based reporter assays, and restores mRNA expression of EWS-FLI1 deregulated transcriptional targets. Our findings illustrate that splicing modulation of EWS-FLI1 pre-mRNA is a promising strategy for future therapeutics against this fusion oncogene expressed in a third of Ewing sarcoma. Citation Format: Carla Neckles, Robert Boer, Nicholas Aboreden, Robert L. Walker, Bong-Hyun Kim, Suntae Kim, John S. Schneekloth, Natasha J. Caplen. HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction [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 4494.

Abstract 3115: High-capacity adenoviral vectors with controlled expression of interleukin 12 as a new strategy against pediatric osteosarcoma

Abstract Osteosarcoma (OS) is the most frequent and aggressive primary bone tumor in children and young adults, with a high propensity to metastasize to the lungs. Despite therapeutic efforts, the survival rate for metastatic or relapsed patients in under 30%. For this reason, we need new strategies to halt the progression of primary bone tumor and impede the metastatic stage. Interleukin-12 (IL12) is a cytokine implicated in a variety of immune activities including the stimulation of T and natural killer (NK) cells and the production of high levels of IFNγ. In addition, this process triggers an anti-angiogenic response leading to T reg inhibition and tumor death. This cytokine has shown significant antitumor activity in several animal models and clinical trials. The aim of this work was to evaluate the antisarcoma effect of a high-capacity adenovirus vector carrying the mouse IL12 (mIL12). This vector harbours a mifepristone (RU486)-inducible system for controlled expression of IL12 (HCA-EFZP-IL12) in order to improve the treatment of OS and reduced the systemic toxicity associated with IL12. The murine K7M2 OS cell line was injected intratibially in Balbc mice to induce bone tumor. HCA-EFZP-IL12 was administered intratumorally (two doses 2 days apart) in the orthotopic model with mifepristone induction during two weeks. Blood samples were collected 10 h after the first RU486 induction; mIL12 protein level was measured by ELISA, and tumor volumes were measured using a calliper. At the end of the experiment we collected tibias, lungs and livers for immunohistochemical studies. Our results showed a reduction of local tumor growth and also a significant extended overall survival. Importantly, the treatment led to long-term survivors free of disease. We performed a re-challenge experiment to assess the generation of antitumor memory and we are currently waiting for those results. Immunohistochemical analyses showed an increase in CD3, mainly CD4 and CD8 and a decrease of T-reg cells. Moreover, splenocytes from treated mice produced significant higher amounts of IFNγ than those of control mice. In summary, the role of the microenvironment and the potent antitumor immune response mediated by controlled expression of IL12 by an HC-Ad equipped with a drug-inducible system can be a real therapeutic option for primary pediatrics OS. Citation Format: Marta Zalacain, Virginia Laspieda, Naiara Martinez-Vélez, Lucía Marrodán, María Buñuales, Marisol Gonzalez-Huarriz, Maider Varela, Montserrat Puigdelloses, Manuela Gonzalez-Aparicio, Marta María Alonso, Rubén Hernandez, Ana Patiño-García. High-capacity adenoviral vectors with controlled expression of interleukin 12 as a new strategy against pediatric osteosarcoma [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 3115.

Abstract 3655: Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Neo-adjuvant chemotherapy combined with wide resection increased the 5-year survival rate to 60%-70%. However, treatment and outcome of OS have not changed in several decades. OS is histologically defined by the presence of malignant osteoid, suggesting it could be derived from anywhere in the osteogenic lineage between mesenchymal stem cells (MSC) and mature osteoblasts, but the cell of origin of OS is still undefined. Tracing the cells from which most cancers formed are characterized by its multipotent and/or unipotent stem cell nature. Studies on the cellular origin of tumors from phenotypic lineage-tracking and its derived progeny can reveal tissue-specific tumor-initiating cells. Investigating cell of origin of OS and osteogenic differentiation using developmentally relevant model using MSC to OB differentiation is promising. Here we developed a high-dimensional analytic pipeline using single-cell mass cytometry (CyTOF) and RNA sequencing to discover the unrecognized progenitor-osteoblast populations in the osteogenic lineage. For the data analysis, the population clustering using Flowsom and ConsensusClusterPlus on arcsinh transformed FCS data combined with t-SNE plot visualization shows that combination of CD49f, CD95, CD56, CD117 expression enables the identification and future isolation of the progenitor populations. Also, when comparing OS with the populations from MSC to OB, there are progenitor -osteoblast / mature osteoblast-like populations in tumor cells, but no MSC-like component is found. The expression level of CD56 and CD117 are significantly higher in OS compare to MSC and OB, which warrants future investigation of new therapeutic strategy. Isolation of the progenitor-osteoblast cells, gene expression comparison between OS and these progenitors, validation of their differentiation potential and tumor-initiating capacity is still underway. In this study, we developed a CyTOF based pipeline to discover the progenitor populations in the osteogenic lineage. By comparison of the OS and these progenitor cells, we may be able to unveil the cell of origin and new targeted treatment strategy of OS. Citation Format: Yifei Wang, Sankaranarayanan Kannan, Zhongting Zhang, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis [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 3655.

Abstract 4292: Molecular mechanism of APIO-EE-7 suppresses cell metastasis by targeting Aurora B in osteosarcoma

Abstract Osteosarcoma is one of the most prevalent malignant bone tumors in children and adolescents and has the characteristics of early metastasis, rapid progression and poor prognosis. Although with the advancement in techniques and treatment increasing the survival rates up to 60%, the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma. Identifying the molecular mechanism and developing therapeutic agents for metastasis is urgent. Aurora B kinase play a key role in mitosis and have been reported to be associated with cancer metastasis. However, the role of Aurora kinase in osteosarcoma is still unclear. In this study, we identified a novel compound, referred to as APIO-EE-7, which inhibits growth and colony formation and induces apoptosis in osteosarcoma. Using computer modeling, we found that APIO-EE-7 can interact with Aurora B at ATP-binding pocket. Treatment with APIO-EE-7 can increase EMT marker expression level of E-cadherin and decrease N-cadherin, vimentin and survivin expression. Importantly, APIO-EE-7 dramatically attenuated tumor incidence and volume in osteosarcoma metastasis mouse model. Our findings suggest that APIO-EE-7 is a potential therapeutic agent against osteosarcoma metastasis by targeting Aurora B, which provides hope for rapid clinic translation. Citation Format: Guoguo Jin, Jitian Li, Zhiping Guo, Junyan Teng, Xiaoling Li, Zhenjiang Zhao. Molecular mechanism of APIO-EE-7 suppresses cell metastasis by targeting Aurora B in osteosarcoma [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 4292.