Active receptor tyrosine kinases, but not Brachyury, are sufficient to trigger chordoma in zebrafish.

The aberrant activation of developmental processes triggers diverse cancer types. Chordoma is a rare, aggressive tumor arising from transformed notochord remnants. Several potentially oncogenic factors have been found to be deregulated in chordoma, yet causation remains uncertain. In particular, sustained expression of TBXT - encoding the notochord regulator protein brachyury - is hypothesized as a key driver of chordoma, yet experimental evidence is absent. Here, we employ a zebrafish chordoma model to identify the notochord-transforming potential of implicated genes in vivo We find that Brachyury, including a form with augmented transcriptional activity, is insufficient to initiate notochord hyperplasia. In contrast, the chordoma-implicated receptor tyrosine kinases (RTKs) EGFR and Kdr/VEGFR2 are sufficient to transform notochord cells. Aberrant activation of RTK/Ras signaling attenuates processes required for notochord differentiation, including the unfolded protein response and endoplasmic reticulum stress pathways. Our results provide the first in vivo evidence against a tumor-initiating potential of Brachyury in the notochord, and imply activated RTK signaling as a possible initiating event in chordoma. Furthermore, our work points at modulating endoplasmic reticulum and protein stress pathways as possible therapeutic avenues against chordoma.

Aberrant Expression of Extracellular Signal-Regulated Kinase and 15-Hydroxyprostaglandin Dehydrogenase Indicates Radiation Resistance and Poor Prognosis for Patients with Clival Chordomas.

OBJECTIVE: To explore molecular markers of radiosensitivity and prognostic factors in patients with clival chordomas. METHODS: Retrospective review was performed of 35 patients. Mean follow-up interval was 66.37 months (range, 29-106 months). Kaplan-Meier method was used for survival analysis. Immunohistochemical staining was used to detect expression levels of extracellular signal-regulated kinase (ERK) and 15-hydroxyprostaglandin dehydrogenase (HPGD). RESULTS: Total resection was achieved in 12 cases, subtotal resection was achieved in 12 cases, and partial resection was achieved in 11 cases. Radiation-sensitive group comprised 17 cases, and radiation-resistant (RR) group comprised 18 cases. Five-year progression-free survival (PFS) rates in total resection and nontotal resection groups were 46.3% and 10.1%, respectively (P = 0.005). Mean H-scores of ERK in radiation-resistant and radiation-sensitive groups were 110.38 and 82.98, respectively (P = 0.043). Mean H-scores of HPGD in radiation-resistant and radiation-sensitive groups were 178.62 and 203.47, respectively (P = 0.031). Mean PFS in low ERK expression group (58.61 months) was significantly longer than mean PFS in high ERK expression group (24.94 months) (P = 0.022). Mean PFS in high HPGD expression group (39.54 months) was significantly longer than mean PFS in low HPGD expression group (9.5 months) (P = 0.013). CONCLUSIONS: Radical resection with protection of important structures is the most effective treatment of clival chordomas. High HPGD expression and low ERK expression were associated with radiation sensitivity and better prognosis. HPGD and ERK can be used as biomarkers to predict prognosis and guide treatment.

Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in chordoma.

Background: Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods: Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results: PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions: Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

Kinase Activity in Recurring Primary Skull Base Chordomas and Chondrosarcomas: Identification of Novel Pathways of Oncogenesis and Potential Drug Targets.

BACKGROUND: Chordomas and chondrosarcomas can occur in the skull base. Currently, 45% of chordomas and 56% of chondrosarcomas recur within 5 years of surgery. The role of adjuvant therapy is highly debated. No pharmacotherapies have been approved by the U.S. Food and Drug Administration for chordomas or chondrosarcomas. High propensity for recurrence and lack of definitive adjuvant therapy necessitate additional basic science research to identify molecular anomalies associated with recurrent disease. METHODS: We pooled tumor lysates from patients based on clinical criteria into 4 groups: primary chordomas, primary chordomas that recurred, primary chondrosarcomas, and primary chondrosarcomas that recurred. We used a peptide labeling method, isobaric tags for relative and absolute quantitation, to uniquely identify each tumor group. Phosphorylated peptides were identified and quantified via mass spectroscopy to determine and predict active kinases. RESULTS: Six groups of phosphorylated peptides were associated with primary tumors that later recurred. Specific kinases associated with primary chordomas that recurred were FES and FER. Specific kinases associated with primary chondrosarcomas that recurred were FES, FER, SRC family kinases, PKC, ROCK, and mitogen-activated protein kinase signaling (JNK, ERK1, p38). CONCLUSIONS: These data provide clinicians with a means to screen skull base chordomas and chondrosarcomas to help identify tumors with a propensity to recur. Many of these kinases can be efficaciously inhibited by Food and Drug Administration-approved drugs that have not yet been used in clinical trials for treatment of skull base chordomas or chondrosarcomas. Validation of kinases identified in this study may advance treatment options for patients with these tumors.

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

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

Overexpression of Raf-1 and ERK1/2 in sacral chordoma and association with tumor recurrence.

Chordoma is a rare and low-malignant neoplasm which is considered to arise from notochord remnants. Due to its large resistance to chemotherapy and radiotherapy, surgical resection so far is the prior treatment for chordoma. However, the recurrence rate is high even after complete surgical resection. Recently, targeted cancer therapy has been demonstrated to be effective in several other tumors, while the related research on chordoma is rare. Mitogen-activated protein kinase signaling pathway is acknowledged to participate in tumor development, in which Raf-1 and extracellular regulated protein kinase 1/2 (ERK1/2) play vital roles. In this study, we evaluated the expression of Raf-1 and ERK1/2 by immunohistochemical staining in 42 chordoma tissue and 16 distant normal tissue. Moreover, we also investigated the correlations of Raf-1 and ERK1/2 expression with clinical features in sacral chordoma. Expression of Raf-1 and ERK1/2 was both significantly higher in sacral chordoma tissue than distant normal tissue (P = 0.008, P = 0.019). Raf-1 positive expression was related to surrounding muscle invasion (P = 0.032) and chordoma recurrence (P = 0.002), but the results did not indicate any association with patients' age, gender, tumor size and location. ERK1/2 was associated with tumor size (P = 0.044) instead of other clinical factors (P > 0.05). Spearman correlation test showed close relation between ERK1/2 and Raf-1 (P = 0.001, r = 0.518). Kaplan-Meier survival Curve and log-rank test showed that Raf-1 positive expression was associated with shorter continuous disease-free survival time (CDFS) (P = 0.001), while ERK1/2 had no relation to CDFS (P = 0.961). Conclusively, Raf-1 may be an important biomarker in predicting the prognosis of chordoma patients.

Expression of PDGFR-α, EGFR and c-MET in spinal chordoma: a series of 52 patients.

AIM: To investigate the expression of platelet-derived growth factor (PDGF) receptor-A (PDGFRα), epidermal growth factor receptor (EGFR) and c-Met in spinal chordoma. To the authors' knowledge, little is known regarding the prognostic significance of receptor tyrosine kinase in spinal chordoma. MATERIALS AND METHODS: Using immunohistochemical techniques, the authors investigated PDGFR-α, EGFR and c-MET expression in 52 primary and 104 recurrent lesions, and compared these data with clinicopathological parameters. RESULTS: PDGFR-α, EGFR and c-MET were found to be expressed in 75.0%, 83% and 77% of primary, and in 97.0% of recurrent lesions in all investigated receptor tyrosine kinases. Higher PDGFR-α and c-MET expression was found to be correlated with younger patient age. Lesions with a higher expression of PDGFR-α demonstrated significantly higher EGFR scores in both primary and recurrent lesions compared to those with lower PDGFR-α expression. In recurrent lesions, higher c-MET expression was found to be associated with significantly better prognosis than those with lower c-MET expression (p=0.033). Lesions with a higher level of PDGFR-α expression were found to have significantly poorer prognosis than those with lower PDGFR-α expression (p=0.024). Those patients with lower EGFR expression were found to have significantly better prognosis than those with higher EGFR expression (p=0.005). CONCLUSION: In the current study, c-MET expression in patients with spinal chordoma was found to be correlated with a younger patient age and a favorable prognosis. Patients with a higher level of PDGFR-α and EGFR expression were found to have a significantly poorer prognosis than those with lower PDGFR-α and EGFR expression.

Histone deacetylase inhibitors as potential therapeutic approaches for chordoma: an immunohistochemical and functional analysis.

Chordomas are rare malignancies of the axial skeleton. Therapy is mainly restricted to surgery. This study investigates histone deacetylase (HDAC) inhibitors as potential therapeutics for chordomas. Immunohistochemistry (IHC) was performed using the HDAC 1-6 antibodies on 50 chordoma samples (34 primary tumors, 16 recurrences) from 44 patients (27 male, 17 female). Pan-HDAC-inhibitors Vorinostat (SAHA), Panobinostat (LBH-589), and Belinostat (PXD101) were tested for their efficacy in the chordoma cell line MUG-Chor1 via Western blot, cell cycle analysis, caspase 3/7 activity (MUG-Chor1, UCh-1), cleaved caspase-3, and PARP cleavage. p-Values below 0.05 were considered significant. IHC was negative for HDAC1, positive for HDAC2 in most (n = 36; 72%), and for HDACs 3-6 in all specimens available (n = 43; 86%). HDAC6 expression was strongest. SAHA and LBH-589, but not PXD101 caused a significant increase of G2/M phase cells and of cleaved caspase-3 (p = 0.0003, and p = 0.0014 after 72 h, respectively), and a peak of caspase 3/7 activity. PARP cleavage confirmed apoptosis. The presented chordoma series expressed HDACs 2-6 with strongest expression of HDAC6. SAHA and LBH-589 significantly increased apoptosis and changed cell cycle distribution in vitro. HDAC-inhibitors should be further evaluated as therapeutic options for chordoma.

Expression of ezrin, MMP-9, and COX-2 in 50 chordoma specimens: a clinical and immunohistochemical analysis.

STUDY DESIGN: Retrospective study. OBJECTIVE: To investigate the immunohistochemical expression profile of ezrin, matrix metalloproteinase-9 (MMP-9), and cyclooxygenase-2 (COX)-2 in chordomas. SUMMARY OF BACKGROUND DATA: Ezrin, MMP-9, and COX-2 are expressed in different solid tumors, including chordomas. This study investigates the immunohistochemical expression of the aforementioned biomarkers and the clinical outcome in regard to immunohistochemistry, tumor volume, and localization. METHODS: Fifty brachyury-verified chordoma specimens of 34 primary and 16 recurrent tumors of 44 patients were tested for ezrin, MMP-9, and COX-2 as possible therapeutical targets by immunohistochemistry. The clinical evaluation concentrated on tumor location, volume, and age-related data. RESULTS: Ezrin expression was detected in 33 of 34 primary chordomas and in 16 of 16 recurrent cases. The primary chordomas located in the sacrum and the spine demonstrated a significantly higher percentage of positively stained tumor cells (P = 0.034) than the skull-based chordomas. An expression of MMP-9 and COX-2 was observed in 33 of 34 primary chordomas and in 16 of 16 recurrences, and in 13 of 34 primary chordomas and in 11 of 16 recurrences, respectively. Patients' survival was significantly influenced by age (P = 0.01), tumor location (P = 0.029), and tumor volume (P = 0.002). A significant positive correlation between tumor volume and the anatomic distance of the chordoma from the skull was calculated (P = 0.00002). CONCLUSION: En bloc resection with tumor-free margins is seldom feasible, particularly in the sacrum. Intralesional excisions mostly end in early local recurrence; therefore, the demand for further treatment options is frequently posed. The marked trend of the investigated biomarkers of this study may build a starting point for further investigations as molecular targets for future adjuvant therapies in chordomas. Future multicenter studies on larger patients' series are necessary to elucidate these preliminary data and to test new treatment options for patients with chordomas.

Immunohistochemical analysis of receptor tyrosine kinase signal transduction activity in chordoma.

AIMS: Currently, there are no effective chemotherapeutic protocols for chordoma. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumours may respond to kinase inhibitor therapy. However, RTK signalling activity has not been extensively investigated in chordoma. METHODS: A tissue microarray containing 21 cases of chordoma was analysed for expression of a number of proteins involved in signal transduction from RTKs by immunohistochemistry. RESULTS: Platelet-derived growth factor receptor-beta, epidermal growth factor receptor (EGFR), KIT and HER2 were detected in 100%, 67%, 33% and 0% of cases, respectively. Platelet-derived growth factor receptor-beta staining was of moderate-to-strong intensity in 20 of 21 cases. In contrast, KIT immunoreactivity was weak and focal in each of the seven positive cases. Total EGFR staining was variable; weak staining for phosphorylated EGFR was detected in nine cases. Phosphorylated isoforms of p44/42 mitogen-activated protein kinase, Akt and STAT3, indicative of tyrosine kinase activity, were detected in 86%, 76% and 67% of cases, respectively. CONCLUSIONS: Chordomas commonly express RTKs and activated signal transduction molecules. Although there were no statistically significant correlations between the expression of any of the markers studied and disease-free survival or tumour location, the results nonetheless indicate that chordomas may respond to RTK inhibitors or modulators of other downstream signalling molecules.

Overexpressions of nerve growth factor and its tropomyosin-related kinase A receptor on chordoma cells.

STUDY DESIGN: Immunohistochemistry and in situ apoptosis detection assay were performed on chordoma and notochordal cells. OBJECTIVES: To investigate the expression levels of nerve growth factor (NGF) and its 2 receptors, tropomyosin-related kinase A (TrkA) and p75, as well as proliferation potential and apoptosis indexes in chordoma and notochordal cells. SUMMARY OF BACKGROUND DATA: Chordomas arise from primitive notochordal remnants. Why these notochordal remnants undergo malignant transformation to chordoma remains unknown. The binding of NGF to the TrkA receptor promotes cell survival, while its binding to the p75 receptor triggers apoptosis. If there is simultaneous expression of both receptors, the effect of TrkA supersedes and the cells survive. METHODS: We examined 10 surgically obtained sacral chordoma tissue samples to determine the expressions of NGF and TrkA and p75 receptors as well as markers of cellular proliferation and apoptosis. As controls, we used notochordal cells of L4-L5 discs obtained from ten 1-month old rats. We quantified the expressions of NGF and TrkA and p75 receptors as well as markers of cellular proliferation and apoptosis for both groups, respectively. RESULTS: Chordoma and notochordal cells both expressed NGF as well as TrkA and p75 receptors. While the mean percentage of p75 receptor expression was very similar between chordoma and notochordal cells (P = 0.394), the mean percentages of TrkA and NGF expressions were significantly higher in chordoma cells than in notochordal cells (both P = 0.002). The mean proliferation potential index of chordoma cells was significantly higher than in notochordal cells (P < 0.01). Conversely, the mean apoptosis index of chordoma cells was significantly lower compared with that of notochordal cells (P = 0.03). CONCLUSION: The current results suggest that increased expressions of NGF and TrkA receptor in chordoma cells might be a possible mechanism of malignant transformation of notochordal remnants to chordoma by negating apoptotic signal of p75 receptor.

Establishing tumor cell lines from aggressive telomerase-positive chordomas of the skull base. Technical note.

Permanent cell cultures are invaluable tools for understanding the biological characteristics of tumors. In the present study the authors report on the establishment of permanent human cell lines from three cases of aggressive chordomas of the clival region. All of the parental tumors showed telomerase activity. Cultured chordoma cells had a doubling time of 5 to 7 days and grew as a monolayer of cells that retained both the immunophenotype and the p53 status of the parental tumor. In vitro, chordoma cells overexpressed telomerase, supporting the hypothesis that this enzyme is required for the immortalization process.

Expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, cathepsin B, and urokinase plasminogen activator in non-skull base chordoma.

We analyzed the expression of proteases and the clinicopathologic significance in non-skull base chordoma (NSBC). By using immunohistochemical techniques, we studied the expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, cathepsin B (CatB), and urokinase plasminogen activator (uPA) in 29 NSBCs and compared these data with clinicopathologic parameters and the expression of cell differentiation markers. Expression of MMP-1 (P = .092), MMP-2 (P = .041), and CatB (P = .058) was associated with nuclear pleomorphism, a previously described adverse prognostic indicator. Expression of cytokeratin 8 correlated with that of MMP-1 (P = .005), MMP-2 (P = .002), and uPA (P = .032). Patients with higher MMP-2 expression had a poorer prognosis than those with lower MMP-2 expression (P = .013). We believe that NSBCs with nuclear pleomorphism or stronger epithelial character have a higher invasive ability than those without. In addition, high MMP-2 expression was an indicator of an unfavorable clinical outcome in NSBC.

Immunofluorescence localization of prolyl 4-hydroxylase isoenzymes and type I and II collagens in bone tumours: type I enzyme predominates in osteosarcomas and chondrosarcomas, whereas type II enzyme predominates in their benign counterparts.

Prolyl 4-hydroxylase is the key enzyme of synthesis of collagens. Hydroxylation of a sufficient number of proline residues to hydroxyproline is necessary for the stability of triple helices in collagenous proteins, because non-hydroxylated non-triple-helical collagen polypeptide chains are degraded intracellularly. We studied 15 primary chondrosarcomas and osteosarcomas, 17 benign bone tumours and one case of fibrous dysplasia and chordoma using immunofluorescence staining with antibodies against the alpha(I) and alpha(II) subunits of type I and II prolyl 4-hydroxylases, and with antibodies against collagen types I and II. Type I prolyl 4-hydroxylase was found to be the predominant isoenzyme in both types of bone sarcoma, whereas the type II enzyme was more readily expressed by benign tumours. A feature of collagen staining, that was common to both sarcoma types, was that collagen types I and II were mainly found within cancer cells and were rarely present extracellularly. Extracellular collagen staining was more obvious in benign tumours. The results show that expression of prolyl 4-hydroxylase isoenzymes is altered in bone sarcomas as compared with normal bone tissue. Chondrous cells, which normally express mainly the type II isoenzyme, switch their expression pattern to that of type I. The findings provide evidence that type I is the major isoenzyme in malignant bone tumours, and probably in malignant neoplasms in general. The pattern of enzyme expression is considered to be associated with dedifferentiation of cancer cells.

Expression of cathepsin K in chordoma.

Invasive growth of chordoma is accompanied by severe destruction of adjacent bone tissue, a fact that requires high proteolytic activity at the tumor invasion fronts. In this context, cathepsin K is a candidate molecule. It is a protease with high collagenolytic and elastinolytic activity and previously thought to be restricted to osteoclasts and osteoclast-mediated bone resorption. In this study, 44 cases of chordoma of sphenooccipital localization, and 10 embryo-fetal specimens including chorda dorsalis were studied immunohistochemically for their expression of cathepsin K. In 4 additional snap-frozen chordoma cases, the enzyme expression was investigated by reverse transcription polymerase chain reaction and enzyme histochemistry. Ten chondrosarcomas of the skull base served as controls. Various concentrations of cathepsin K mRNA could be seen in all snap-frozen chordoma specimens. The protease was immunohistochemically expressed by the tumor cells. The immunoreactions were accentuated at the tumor invasion fronts. Enzyme histochemistry indicated a strong tumor cell-associated cathepsin K activity in invasive tumor components. In contrast to chordoma, cathepsin K was not significantly expressed in chorda dorsalis and chondrosarcoma of the skull base. In chondrosarcoma, protease expression was limited to osteoclastic cells localized between infiltrative tumor components and regular bone trabeculae. This study shows the significant expression and activity of cathepsin K in chordoma and implicates an important and direct role of this protease in the infiltrative growth of this tumor. This protease expression occurred during neoplastic transformation and did not appear in chorda dorsalis.

Expression and localization of urokinase-type plasminogen activator in human spinal column tumors.

We have sought to determine the production and activity of serine proteases in primary and metastatic spinal tumors and the association of these enzymes with the invasive and metastatic properties of spinal column tumors. Using immunohistochemical techniques, the cellular localization and expression of urokinase-type plasminogen activator (uPA) was assessed, whereas its activity was determined by fibrin zymography, and the amounts of enzyme were measured by an enzyme-linked immunosorbent assay (ELISA) in primary spinal column tumors (chordoma, chondrosarcoma, and giant cell tumor) and metastatic tumors of the spine arising from various malignancies (breast, lung, thyroid, and renal cell carcinomas, and melanomas). Metastatic tumors displayed higher levels of uPA activity than did primary spinal tumors (P<0.001). Immunohistochemical analysis revealed that uPA expression was highest in metastases from lung and breast carcinomas and melanomas, followed by metastatic tumors from thyroid and renal cell carcinomas. Similar results were obtained for uPA activity and enzyme level as determined by fibrin zymography and ELISA, respectively. We conclude that metastatic spinal tumors possess higher levels of uPA expression and activity than the primary spinal tumors, which tend to be less aggressive and only locally invasive malignancies. The results suggest that the plasminogen system may participate in the metastasis of tumors to the spinal column.

Expression and role of matrix metalloproteinases MMP-2 and MMP-9 in human spinal column tumors.

Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immunohistochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.

The expression of Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase and alpha 2,6-linked sialoglycoconjugates in human brain tumors.

CMP-NeuAc: Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase (alpha 2,6-ST) [EC 2.4.99.1] is developmentally regulated, shows a high degree of tissue specificity, and appears to play a role in oncogenic transformation and metastasis. In the present study, we have performed the first detailed analysis of the expression of alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates in human brain tumors. We used a polyclonal, monospecific anti-rat alpha 2,6-ST antibody and the alpha 2,6-linked sialic acid-specific lectin, Sambucus nigra agglutinin (SNA) for histochemical studies, and a human alpha 2,6-ST-specific cDNA probe for Northern analysis. Meningiomas, chordomas and craniopharyngiomas frequently expressed alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates. Among the different meningioma subtypes, meningothelial meningiomas stained more strongly with both anti-alpha 2,6-ST antibody and SNA than the fibroblastic and anaplastic meningiomas. On the other hand, all tumors of glial origin and medulloblastomas were virtually devoid of either alpha 2,6-ST or alpha 2,6-linked sialoglycoconjugate expression. Moreover, very weak to negligible expression of both alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates was observed in brain metastases. In conclusion, alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugate expression is associated with non-neuroectodermal epithelial-like tumors.

Cytogenetic, telomere, and telomerase studies in five surgically managed lumbosacral chordomas.

Lumbosacral chordomas are rare skeletal sarcomas of the spine that originate from the remnant notochord. The understanding of this human cancer is limited to observations of its clinical behavior and its embryonic link. Thus, we performed chromosome and molecular analyses from five surgically harvested chordomas in an effort to document genetic and biochemical abnormalities which might aid in understanding the tumor biology of this understudied neoplasm. Cytogenetic analysis of the five chordomas revealed normal results in four patients and random abnormalities in only one tumor cell in the 100 cells studied from the fifth patient. A repeat telomeric probe (TTAGGG)50 was hybridized to genomic DNA isolated from chordoma cells (and HeLa cells) and digested with HinfI. The tumor DNA was paired with leukocyte DNA from age-matched controls and revealed telomere elongation in four of the four chordoma patients studied with molecular genetic techniques. Conversely, telomere length reduction has been reported during in vitro senescence of human fibroblasts, giant cell tumor of bone, colon cancer, intracranial tumors, childhood leukemia, Wilms tumor, and in HeLa cells. Telomerase activity (telomerase is required to maintain telomere integrity) was also determined by visualizing the extension of radioactive telomeric repeats on DNA sequencing gels. The telomeric fragments were assembled during incubation of the cytoplasmic extract containing telomerase. Telomerase activity was observed in HeLa (positive control and commercially available cell line), giant cell tumor of bone (positive control tumor cells from living patients), and in chordoma cells from one of the two chordoma patients (but to a lesser degree compared with HeLa). As expected, the chordoma patients' fibroblasts exhibited no telomerase activity.