Chordoma Development Research Articles

Brachyury as a Diagnostic Biomarker and Therapeutic Molecular Target in Chordomas: A Systematic Review, Meta-Analysis, and Bioinformatics Analysis

Background: Brachyury has recently garnered attention as both a biomarker and a causative factor in chordoma development, making it a promising therapeutic target. Objectives: Herein, we report a systematic review and meta-analysis to evaluate brachyury as a diagnostic biomarker and therapeutic molecular target in chordomas. Methods: Scopus, PubMed/Medline, Cochrane Library databases, and Web of Science were searched up to July 18, 2024, without any restrictions. The Comprehensive Meta-Analysis version 3.0 software was used to calculate the event rate (ER) with a 95% confidence interval (CI). We utilized STRING to explore functional interactions among the studied genes. Results: Twenty-five studies related to the expression of brachyury and five related to the treatment vaccine of brachyury were included in this category. The ER of brachyury expression among chordoma patients was 92.2% (95% CI: 87.3% - 95.3%), with I² = 74.65%. Among the five studies related to treatment vaccines, there were three studies with Phase 1, one with Phase 2, and a case report. Conclusions: The high expression suggests that brachyury serves as a reliable diagnostic marker for chordomas. The findings indicate potential therapeutic implications for targeting brachyury in chordoma treatment.

EXPRESSION PATTERN OF THE MRPS18 FAMILY PROTEINS IN CHORDOMA

Chordomas are rare tumors of the axial skeleton that arise from the notochord; they are observed usually at the base of the skull and rarer along the spine. Chordomas are resistant to conventional chemotherapy, that is why it is important, to find the new diagnostic and/or prognostic markers. We have shown recently, that genes of the mitochondrial ribosomal protein S18 (MRPS18) family are differentially expressed in gliomas. The MRPS18-2 oncoprotein is expressed at higher levels in cancerous cells compared to their normal counterpart. Aim. We wanted to investigate the peculiarities of the expression pattern of the MRPS18 family genes in chordoma to better understand their role in cancerogenesis, using an immunohistochemical analysis on chordoma clinical samples. Methods. 15 specimens of chordoma of the base of the skull and 5 samples of the spine localization were studied retrospectively. The specific antibodies against MRPS18-1, MRPS18-2, MRPS18-3, and RB was used for an immunohistochemical analysis. All cases were stained in parallel with appropriate negative control. Results. We found that MRPS18 family proteins are differentially expressed in chordoma tissues. MRPS18-1 showed the strongest signal in all tumor samples. The lowest level of the signal intensity was recorded for the MRPS18-3 protein. In several samples the RB signal was detected in cytoplasm of tumor cells. Importantly, in these samples the strong staining for MRPS18-2 was observed as well. The fine mechanism of sequestering of RB in cytoplasm of cancer cells should be further elucidated. Conclusion, These preliminary results should lead to a larger work on a role of RB-MRPS18 family proteins interaction for chordoma development.

Abstract LB194: Methylation subtypes of skull base chordoma

Abstract Background: Chordoma is a rare bone tumor with a high recurrence partially due to incomplete tumor resection. Recent studies suggest that chordoma is a heterogeneous disease that may comprise of molecular subtypes manifesting distinct clinical outcomes. Molecular classification may improve our knowledge of chordoma biology and patient stratification. However, due to its rareness, tumor profiling data is limited. The aim of this study was to identify molecular subtypes of chordoma that may improve clinical management using methylation profiling data. Methods: Genomic DNA was extracted from fresh frozen tissues in 67 patients with skull-base chordoma from Beijing Tiantian Hospital, China. DNA was then profiled using an Infinium Methylation 850K BeadChip array. CpG sites involving low-quality data (CpG detection with p > 0.01), those on the X and Y chromosomes, and the sites overlapping with single-nucleotide polymorphisms were removed. RNA sequencing (RNA-Seq) data was available for 51 out of 67 tumor samples. Results: We conducted unsupervised clustering analysis using the top 8000 most variable methylation probes and identified two distinct clusters (Cluster 1 n = 40, Cluster 2 n = 27). Thirty-three significant differentially methylated regions (DMRs, p-adj < 0.05) were identified between the two clusters using Bumphunter. Gene set enrichment analysis (GSEA) Hallmark analysis identified four enriched pathways of interest: IL6 JAK STAT3 Signaling, Mitotic Spindle, TGF Beta Signaling, and Apical Junction among genes included in these DMRs (p-adj < 0.05). We used RNA-Seq data to identify significant correlations between methylation probes and gene expression associated with DMR genes (n=30 genes; n=150 probes). We found probes in four genes of interest (HOXA3, MBP, NEDD4L, and TEX14) showed consistent negative correlations with gene expression levels. HOXA3 encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation in pathways that are highly relevant to chordoma development. Previous research has cited HOX complex inhibition in highly therapy resistant chordoma relapses. NEDD4L is associated with regulation of the epithelial NA+ channels and plays an important role in regulating tumor cell function through ubiquitinating substrates. MBP is involved in nervous system development, while the role of TEX14 in cancer is less understood. Conclusion: Our results confirmed findings from previous studies that chordoma is molecularly heterogeneous. In addition to immune pathways, we showed that the two subtypes also demonstrated different epigenetic regulation in mitosis and cell adhesion related genes/pathways. Next, we will expand the analysis by including more samples to increase statistical power and to associate our epigenetic findings with patient outcomes. Our goal is to build a reliable classifier to facilitate clinical management. We will integrate whole genome/exome sequencing data to identify potential epigenetic drivers. Citation Format: Gulzar N. Daya, Yujia Xiong, Mingxuan Li, Hela Koka, Xiaoyu Wang, DiFei Wang, Shilpa Katta, Jiwei Bai, Xiaohong R. Yang. Methylation subtypes of skull base chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB194.

KIF15 promotes the development and progression of chordoma via activating PI3K-AKT signalling pathway

AimsDespite its implication in various human cancers, the expression and functional significance of Kinesin family member 15 (KIF15) in chordomas remain unexplored. Main methodsThe evaluation of KIF15 protein levels was conducted through immunohistochemistry (IHC) staining and Western blot analysis. Cell proliferation was quantified using MTT and CCK8 assays, whereas cell migration was examined using wound healing and Transwell assays. Furthermore, flow cytometric analysis was utilized to assess cell apoptosis and the cell cycle. Additionally, in vivo experiments were performed using a mouse xenograft model. Key findingsOur study revealed significantly higher expression of KIF15 in stage III chordoma tissues compared to stage II tissues. Knockdown of KIF15 led to notable inhibition of cell proliferation and migration, along with enhanced apoptosis and cell cycle arrest. In vivo studies further confirmed the inhibitory effects of KIF15 knockdown on chordoma tumour growth. In terms of mechanism, we identified the involvement of the PI3K-AKT signalling pathway mediated by KIF15 in chordomas. Notably, the anti-tumour effects of KIF15 deficiency on chordomas were partially reversed by the addition of an AKT activator. SignificanceKIF15 promotes chordoma development and progression through the activation of the PI3K-AKT signalling pathway. Thus, targeting KIF15 might be a promising therapeutic strategy for treating chordomas.

Transcriptional Profiling Supports the Notochordal Origin of Chordoma and Its Dependence on a TGFB1-TBXT Network

Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-β)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-β. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-β as a prime druggable candidate.

Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T-ARMS-PCR assay.

BackgroundChordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T‐box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells.MethodsHerein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra‐primer amplification refractory mutation system‐polymerase chain reaction (T‐ARMS‐PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T‐ARMS‐PCR. The results were subsequently validated by Sanger sequencing.ResultsThe genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p‐value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p‐value <0.05). DNA sequencing verified the T‐ARMS‐PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost‐effective T‐ARMS‐PCR assay.ConclusionsOur results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T‐ARMS‐PCR assay to determine the genotype of rs2305089.

LncRNA NONHSAT114552 Sponges miR-320d to Promote Proliferation and Invasion of Chordoma Through Upregulating NRP1.

Chordoma is a relatively rare malignant bone tumor with high local recurrence. To date, the mechanism remains unclear. lncRNAs play a pivotal role in tumorigenesis by acting as competitive endogenous RNAs of microRNAs. However, the biological role of lncRNA is still unclear in chordoma. In this research, our aim is to investigate the roles and regulation mechanisms of lncRNA NONHSAT114552 in chordoma development. The expression level of NONHSAT114552 and miR-320d in chordoma tissues was determined by qRT-PCR. Meantime, the correlation between NONHSAT114552 and clinical prognosis was also studied. Bioinformatics analysis and luciferase reporter assays were used to verify the relationship between NONHSAT114552 and miR-320d, and between miR-320d and Neuropilin 1 (NRP1). In addition, effects of NONHSAT114552 on chordoma cells (U-CH1 and U-CH2) proliferation and invasion and its regulation on miR-320d were also evaluated. Furthermore, the influences of NONHSAT114552/miR-320d/NRP1 axis on chordoma tumorigenesis were investigated in vivo. NONHSAT114552 was overexpressed while miR-320d was down-regulated in chordoma tissue compared to fetal nucleus pulposus. Kaplan-Meier survival analysis showed that NONHSAT114552 overexpression was associated with patients’ poor prognosis. Knockdown of NONHSAT114552 significantly suppressed chordoma cell proliferation and invasion. In vitro studies confirmed that NONHSAT114552 acted as ceRNA to regulate NRP1 by directly sponging miR-320d, thus facilitating chordoma cell proliferation and invasion. In vivo study demonstrated that NONHSAT114552 moderated chordoma growth by sponging miR-320d to regulating NRP1. Our findings indicate that lncRNA NONHSAT114552 exhibits a critical role in the tumorigenesis and development of chordoma and it may become one potential prognostic marker and therapeutic target for this disease. .

CMTM3 suppresses chordoma progress through EGFR/STAT3 regulated EMT and TP53 signaling pathway

BackgroundChordomas are rare, slow-growing and locally aggressive bone sarcomas. At present, chordomas are difficult to manage due to their high recurrence rate, metastasis tendency and poor prognosis. The underlying mechanisms of chordoma tumorigenesis and progression urgently need to be explored to find the effective therapeutic targets. Our previous data demonstrates that EGFR plays important roles in chordoma development and CKLF-like MARVEL transmembrane domain containing (CMTM)3 suppresses gastric cancer metastasis by inhibiting the EGFR/STAT3/EMT signaling pathway. However, the roles and mechanism of CMTM3 in chordomas remain unknown.MethodsPrimary chordoma tissues and the paired adjacent non-tumor tissues were collected to examine the expression of CMTM3 by western blot. The expression of CMTM3 in chordoma cell lines was tested by Real-time PCR and western blot. CCK-8 and colony forming unit assay were performed to delineate the roles of CMTM3 in cell proliferation. Wound healing and Transwell assays were performed to assess cell migration and invasion abilities. A xenograft model in NSG mice was used to elucidate the function of CMTM3 in vivo. Signaling pathways were analyzed by western blot and IHC. RNA-seq was performed to further explore the mechanism regulated by CMTM3 in chordoma cells.ResultsCMTM3 expression was downregulated in chordoma tissues compared with paired normal tissues. CMTM3 suppressed proliferation, migration and invasion of chordoma cells in vitro and inhibited tumor growth in vivo. CMTM3 accelerated EGFR degradation, suppressed EGFR/STAT3/EMT signaling pathway, upregulated TP53 expression and enriched the TP53 signaling pathway in chordoma cells.ConclusionsCMTM3 inhibited tumorigenesis and development of chordomas through activating the TP53 signaling pathway and suppressing the EGFR/STAT3 signaling pathway, which suppressed EMT progression. CMTM3 might be a potential therapeutic target for chordomas.

Knockdown of TMED3 inhibits cell viability and migration and increases apoptosis in human chordoma cells.

Chordoma is a rare low-grade tumor of the axial skeleton. Over previous decades, a range of targeted drugs have been used for treating chordoma, with more specific and effective therapies under investigation. Transmembrane Emp24 protein transport domain containing 3 (TMED3) is a novel gene reported to be a regulator of oncogenesis, cancer development and metastasis; however, its role in chordoma remains unclear. In the present study, the expression of TMED3 was investigated in chordoma cells, and the effect of TMED3 knockdown on chordoma development was examined in vitro and in vivo, followed by exploration of differentially expressed proteins in TMED3-silenced chordoma cells via an apoptosis antibody array. Reverse transcription-quantitative PCR and western blot assays were performed to determine the expression levels. It was revealed that TMED3 was highly expressed in chordoma, and that knockdown of TMED3 inhibited cell viability and migration, and enhanced the apoptosis of chordoma cells. Additionally, knockdown of TMED3 inhibited the expression of Bcl-2, heat shock protein 27, insulin-like growth factor (IGF)-I, IGF-II, IGF binding protein-2, Livin, Akt, CDK6 and cyclin D1 proteins, whereas MAPK9 was upregulated. Furthermore, a xenograft nude mice model demonstrated that TMED3 expression promoted tumor growth. Collectively, the present findings suggested that knockdown of TMED3 inhibited cell viability and migration, and enhanced apoptosis in chordoma cells, and that TMED3 may be a novel target for chordoma therapy.

Transformed notochordal cells trigger chronic wounds in zebrafish, destabilizing the vertebral column and bone homeostasis.

ABSTRACTNotochordal cells play a pivotal role in vertebral column patterning, contributing to the formation of the inner architecture of intervertebral discs (IVDs). Their disappearance during development has been associated with reduced repair capacity and IVD degeneration. Notochord cells can give rise to chordomas, a highly invasive bone cancer associated with late diagnosis. Understanding the impact of neoplastic cells during development and on the surrounding vertebral column could open avenues for earlier intervention and therapeutics. We investigated the impact of transformed notochord cells in the zebrafish skeleton using a line expressing RAS in the notochord under the control of the kita promoter, with the advantage of adulthood endurance. Transformed cells caused damage in the notochord and destabilised the sheath layer, triggering a wound repair mechanism, with enrolment of sheath cells (col9a2+) and expression of wt1b, similar to induced notochord wounds. Moreover, increased recruitment of neutrophils and macrophages, displaying abnormal behaviour in proximity to the notochord sheath and transformed cells, supported parallels between chordomas, wound and inflammation. Cancerous notochordal cells interfere with differentiation of sheath cells to form chordacentra domains, leading to fusions and vertebral clefts during development. Adults displayed IVD irregularities reminiscent of degeneration, including reduced bone mineral density and increased osteoclast activity, along with disorganised osteoblasts and collagen, indicating impaired bone homeostasis. By depleting inflammatory cells, we abrogated chordoma development and rescued the skeletal features of the vertebral column. Therefore, we showed that transformed notochord cells alter the skeleton during life, causing a wound-like phenotype and activating chronic wound response, suggesting parallels between chordoma, wound, IVD degeneration and inflammation, highlighting inflammation as a promising target for future therapeutics.This article has an associated First Person interview with the first author of the paper.

GSK-3β Regulates the Expression of P21 to Promote the Progression of Chordoma.

PurposeChordoma is a rare malignant bone tumor transformed from the remnants of notochord. It is characterized as highly aggressive and locally invasive, difficult to be completely removed by surgery, and has a poor clinical prognosis. Glycogen synthase kinase 3 beta (GSK-3β) is involved in many cellular processes. GSK-3β overexpression has been shown to promote the development of many cancers, according to previous studies. However, the role of GSK-3β in chordoma remains unclear.MethodsImmunohistochemistry (IHC) and Western blotting (WB) were performed on clinical specimens to measure GSK-3β expression in chordoma, and immunofluorescence and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to examine the expression of GSK-3β and P21 in cell lines. Cell proliferation was detected by the CCK-8 assay and colony formation analysis, cell migration and invasion checked by Transwell experiments, and cell apoptosis was determined by Annexin V/propidium iodide staining. P21 was predicted as a downstream target gene of GSK-3β using STRING and UNIHI databases. Moreover, we used immunoprecipitation to confirm that GSK-3β and P21 interacted with each other. The double luciferase reporter gene assay showed that GSK-3β could regulate the promoter activity of P21. Finally, the role of the GSK-3β -P21 pathway in chordoma tumorigenesis was analyzed in vivo in nude mice.ResultsOur study showed that GSK-3β was significantly higher in chordoma tissues than in paracancer tissues, and siRNA knockdown of GSK-3β inhibited chordoma cell proliferation and promoted cell apoptosis. Additionally, our research found that GSK-3β bound and downregulated the expression of the P21 gene, and the expression of silencing P21 partially reversed the inhibitory effect of knockdown GSK-3β on chordoma. Furthermore, xenografts showed that knockdown GSK-3β inhibited the formation of chordomas in vivo.ConclusionOur results indicated that the GSK-3β-P21 axis may be an important signaling pathway for the occurrence and development of chordoma, providing a new therapeutic target for the clinical treatment of this disorder.

Gene expression profile and bioinformatics analysis revealed key molecular characteristics of chordoma-before and after TNF- a treatment.

Background:Chordoma is a rare malignant tumor with limited treatment. Recent studies have shown that the proliferation and invasion ability of chordoma after Tumor necrosis factor alpha (TNF-α) treatment is enhanced, which may activate the gene pathway involved in the development of chordoma. This study tends to identify differentially expressed genes (DEGs) before and after treatment of TNF-α in chordoma cell line, providing a new target for future molecular therapy of chordoma.Methods:The gene expression profile of GSE101867 was downloaded from the Gene Expression Omnibus database, and the differentially expressed genes were obtained using GEO2R. Based on the CLUEGO plugin in Cytoscape, DEGs functionality and enrichment analysis. A protein-protein interaction (PPI) network was constructed using Cytoscape based on data collected from the STRING online dataset. The Hub genes are selected from the CytoHubba, the first 20 genes that coexist with the KEGG tumor-related pathway.Results:A total of 560 genes, including 304 up-regulated genes and 256 down-regulated genes, were selected as DEGs. Obviously, GO analysis shows that up-regulated and down-regulated DEGs are mainly enriched in biological processes such as synaptic tissue, cell adhesion, extracellular matrix organization and skeletal system development. DEGs are mainly enriched in tumor-associated pathways such as Pi3k-akt Signal path, Rap1 signal path. Three key genes were identified: PDGFRB, KDR, FGF2. All of these genes are involved in the tumor-associated pathways described previously.Conclusion:This study is helpful in understanding the molecular characteristics of chordoma development. Hub genes PDGFRB, KDR, FGF2 and pi3k-akt signaling pathway, Rap1 signaling pathway will become a new target for the future treatment of chordoma.

MiR-1290 inhibits chordoma cell proliferation and invasion by targeting Robo1.

BackgroundChordoma is a low-grade aggressive bone tumor with a high local recurrence. MicroRNAs (miRNAs) have been reported to play crucial roles in the development of chordoma. Our previous study has shown miR-1290 is associated with muscle invasion and the prognosis of chordoma. However, the underlying mechanism of miR-1290 in chordoma remains unclear. In this study, we aimed to explore the function of miR-1290 in the biological behaviors of chordoma.MethodsSixteen sacral chordoma samples and 10 fetal nucleus pulposus specimens were collected for the detection of miR-1290 and Robo1 at the First Affiliated Hospital of Soochow University. Bioinformatic analysis and a luciferase reporter assay was used to verify the interaction between miR-1290 and the target gene robo1 in chordoma. Effects of miR-1290 expression on chordoma cell proliferation and invasion were explored by clone formation and Transwell assay in vitro. The underlying mechanisms of miR-1290 and Robo1 in chordoma cell proliferation and invasion were also explored in the U-CH1 cell line.ResultsIn vitro functional analysis, including clone formation, and Transwell assays indicated overexpression of miR-1290 significantly suppressed chordoma cell proliferation and invasion. Bioinformatic analysis revealed Robo1 as a potential target of miR-1290, and luciferase reporter assays demonstrated the association between miR-1290 and the Robo1 gene in U-CH1 cells. Robo1 was further confirmed to be up-regulated in chordoma tissues by immunohistochemistry (IHC), which is negatively correlated with miR-1290 expression in chordoma tissue. Additionally, we found down-regulation of miR-1290 could induce the expression of Robo1 in chordoma cells, while the elevation of miR-1290 expression could inhibit Robo1 expression in chordoma cells.ConclusionsmiR-1290 inhibits chordoma cell proliferation and invasion by negatively regulating the Robo1 gene.

MiR-1290 inhibits chordoma cell proliferation and invasion by targeting Robo1

Background: Chordoma is a low-grade aggressive bone tumor with a high local recurrence. MicroRNAs (miRNAs) have been reported to play crucial roles in the development of chordoma. Our previous study has shown miR-1290 is associated with muscle invasion and the prognosis of chordoma. However, the underlying mechanism of miR-1290 in chordoma remains unclear. In this study, we aimed to explore the function of miR-1290 in the biological behaviors of chordoma. Methods: Sixteen sacral chordoma samples and 10 fetal nucleus pulposus specimens were collected for the detection of miR-1290 and Robo1 at the First Affiliated Hospital of Soochow University. Bioinformatic analysis and a luciferase reporter assay was used to verify the interaction between miR-1290 and the target gene robo1 in chordoma. Effects of miR-1290 expression on chordoma cell proliferation and invasion were explored by clone formation and Transwell assay in vitro. The underlying mechanisms of miR-1290 and Robo1 in chordoma cell proliferation and invasion were also explored in the U-CH1 cell line. Results: In vitro functional analysis, including clone formation, and Transwell assays indicated overexpression of miR-1290 significantly suppressed chordoma cell proliferation and invasion. Bioinformatic analysis revealed Robo1 as a potential target of miR-1290, and luciferase reporter assays demonstrated the association between miR-1290 and the Robo1 gene in U-CH1 cells. Robo1 was further confirmed to be up-regulated in chordoma tissues by immunohistochemistry (IHC), which is negatively correlated with miR-1290 expression in chordoma tissue. Additionally, we found down-regulation of miR-1290 could induce the expression of Robo1 in chordoma cells, while the elevation of miR-1290 expression could inhibit Robo1 expression in chordoma cells. Conclusions: miR-1290 inhibits chordoma cell proliferation and invasion by negatively regulating the Robo1 gene.

Comprehensive analysis of mRNA-lncRNA co-expression profile revealing crucial role of imprinted gene cluster DLK1-MEG3 in chordoma.

Chordoma is a rare bone tumor with high recurrence rate, but the mechanism of its development is unclear. Long non-coding RNAs(lncRNAs) are recently revealed to be regulators in a variety of biological processed by targeting on mRNA transcription. Their expression profile and function in chordoma have not been investigated yet. In this study, we firstly performed the comprehensive analysis of the lncRNA and coding genes expression analysis with three chordoma samples and three fetal nucleus pulposus tissues. lncRNA and gene microarrays were used to determine the differentially expressed lncRNAs and protein coding genes. 2786 lncRNAs and 3286 coding genes were significantly up-regulated in chordoma, while 2042 lncRNAs and 1006 coding genes were down-regulated. Pearson correlation analysis was conducted to correlate differentially expressed lncRNAs with protein coding genes, indicating a comprehensive lncRNA-coding gene co-expression network in chordoma. Cis-correlation analysis showed that various transcripts of MEG3 and MEG8 were paired with the most differentially expressed gene DLK1. As located in the same locus, we further analyzed the miRNA clusters in this region, and identified that 61.22% of these miRNAs were significantly down-regulated, implying the silence of the imprinted gene cluster DLK1-MEG3. Overexpression of MEG3 suppressed the proliferation of chordoma cells. Our study pointed out the potential role of lncRNAs in chordoma, presented the lncRNA-coding genes co-expression profile, and revealed that imprinted gene cluster DLK1-MEG3 contributes to the pathogenesis of chordoma development.

MicroRNA profiling and bioinformatics analyses reveal the potential roles of microRNAs in chordoma.

Chordoma is a rare aggressive bone tumor arising from remnants of the notochord, and patients with chordoma have a poor prognosis. However, the unique expression profiles of microRNAs (miRNAs/miRs) and their downstream signaling pathways in chordoma remain incompletely characterized. The aim of the present study was to delineate the global miRNA expression profile and associated signaling networks in chordoma. miRNA profiling was performed on chordoma and fetal notochord tissues. Differentially expressed miRNAs in chordoma were analyzed using microarrays with hierarchical clustering analysis. The target genes of the differentially expressed miRNAs were predicted, and Gene Ontology (GO) and pathway analyses were performed for the intersecting genes. A total of 42 miRNAs were significantly dysregulated in chordoma compared with that in fetal nucleus pulposus tissues. The expression of hsa-miR-21-3p, hsa-miR-150-5p, hsa-miR-1290 and hsa-miR-623 were validated using the reverse transcription-quantitative polymerase chain reaction. On the basis of the intersection predicted by three databases (Targetscan, microRNA.org and PITA), 10,292 potential miRNA targets were identified. Bioinformatic analyses suggested that these dysregulated miRNAs and their predicted targets were functions of signaling pathways in cancer, the mitogen-activated protein kinase signaling pathway, regulation of actin cytoskeleton, focal adhesion and endocytosis. In particular, human (hsa-)miR-185-5p was identified as a crucial miRNA in chordoma development via the Wnt signaling pathway. The results of the present study provide a comprehensive expression and functional profile of differentially expressed miRNAs associated with chordoma. This profile may serve as a potential tool for biomarker and therapeutic target identification in patients with chordoma.

Immunophenotypic features of dedifferentiated skull base chordoma: An insight into the intratumoural heterogeneity.

Chordomas are rare and low-grade malignant solid tumours, despite their histologically benign appearance, that arise in the bone from embryonic notochordal vestiges of the axial skeleton, a mesoderm-derived structure that is involved in the process of neurulation and embryonic development. Chordomas occurring in the skull base tend to arise in the basiocciput along the clivus. Three major morphological variants have been described (classical, chondroid, and atypical/dedifferentiated). The pathogenesis and molecular mechanisms involved in chordoma development remain uncertain. From a pathological standpoint, the microenvironment of a chordoma is heterogeneous, showing a dual epithelial-mesenchymal differentiation. These tumours are characterised by slow modality of biologic growth, local recurrence, low incidence of metastasis rates, and cancer stem cell (CSC) phenotype. The main molecular findings are connected with brachyury immunoexpression and activation of the downstream Akt and mTOR signalling pathways. The differentiation between typical and atypical chordomas is relevant because the tumoural microenvironment and prognosis are partially different. This review provides an insight into the recent and relevant concepts and histochemical markers expressed in chordomas, with special emphasis on dedifferentiated chordomas and their prognostic implications.

Notochord isolation using laser capture microdissection

BackgroundChordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. MethodsExamination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. ResultsThis study illustrates notochord of embryonic spines in three different stages of gestation (9–11–13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. ConclusionLCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Genomic and transcriptomic characterization of skull base chordoma.

Skull base chordoma is a primary rare malignant bone-origin tumor showing relatively slow growth pattern and locally destructive lesions, which can only be characterized by histologic components. There is no available prognostic or therapeutic biomarker to predict clinical outcome or treatment response and the molecular mechanisms underlying chordoma development still remain unexplored. Therefore, we sought out to identify novel somatic variations that are associated with chordoma progression and potentially employed as therapeutic targets. Thirteen skull base chordomas were subjected for whole-exome and/or whole-transcriptome sequencing. In process, we have identified chromosomal aberration in 1p, 7, 10, 13 and 17q, high frequency of functional germline SNP of the T gene, rs2305089 (P = 0.0038) and several recurrent alterations including MUC4, NBPF1, NPIPB15 mutations and novel gene fusion of SAMD5-SASH1 for the first time in skull base chordoma.

Spinal column chordoma: prognostic significance of clinical variables and T (brachyury) gene SNP rs2305089 for local recurrence and overall survival.

Chordomas are rare, locally aggressive bony tumors associated with poor outcomes. Recently, the single nucleotide polymorphism (SNP) rs2305089 in the T (brachyury) gene was strongly associated with sporadic chordoma development, but its clinical utility is undetermined. In 333 patients with spinal chordomas, we identified prognostic factors for local recurrence-free survival (LRFS) and overall survival and assessed the prognostic significance of the rs2305089 SNP. The median LRFS was 5.2 years from the time of surgery (95% CI: 3.8-6.0); greater tumor volume (≥100cm3) (hazard ratio [HR] = 1.99, 95% CI: 1.26-3.15, P = .003) and Enneking inappropriate resections (HR = 2.35, 95% CI: 1.37-4.03, P = .002) were independent predictors of LRFS. The median overall survival was 7.0 years (95% CI: 5.8-8.4), and was associated with older age at surgery (HR = 1.11 per 5-year increase, 95% CI: 1.02-1.21, P = .012) and previous surgical resection (HR = 1.73, 95% CI: 1.03-2.89, P = .038). One hundred two of 109 patients (93.6%) with available pathologic specimens harbored the A variant at rs2305089; these patients had significantly improved survival compared with those lacking the variant (P = .001), but there was no association between SNP status and LRFS (P = .876). The ability to achieve a wide en bloc resection at the time of the primary surgery is a critical preoperative consideration, as subtotal resections likely complicate later management. This is the first time the rs2305089 SNP has been implicated in the prognosis of individuals with chordoma, suggesting that screening all patients may be instructive for risk stratification.