Clinical Orthopaedics and Related Research ®

A Publication of The Association of Bone and Joint Surgeons ®

Symposium: Molecular Genetics in Sarcoma 18 articles

Articles

S100A6 Expression and Function in Human Osteosarcoma

Xiaoji Luo MD, PhD, Katie A. Sharff BA, Jin Chen MD, Tong-Chuan He MD, PhD, Hue H. Luu MD There is a critical need to identify markers that can accurately identify existing or predict future metastatic disease in patients with osteosarcoma since the majority of patients present with undetectable micrometastatic disease. We previously reported S100A6 is overexpressed in human osteosarcoma and increased expression of S100A6 by immunohistochemistry correlated with decreased clinical metastasis. We have established 11 primary cultures from biopsies of patients with osteosarcoma and ten of the 11 primary cultures have increased expression of S100A6 relative to normal human osteoblasts. To further explore possible mechanisms for metastasis suppression previously reported, we used in this report siRNA-mediated knockdown of S100A6 in four commonly used human osteosarcoma lines, then examined their cell adhesion, migration, and invasion properties. Knockdown of S100A6 expression inhibited cell adhesion and promoted cell migration and invasion in these lines. Conversely, S100A6 overexpression enhanced cell adhesion and inhibited cell invasion. Our data demonstrate S100A6 is commonly overexpressed in human osteosarcoma. S100A6 may inhibit osteosarcoma metastasis by promoting cell adhesion and inhibiting cell motility and invasion. Thus, S100A6 may be considered a potential marker for human osteosarcoma with prognostic value for identifying patients without metastases.

The Classic: Integration of Deoxyribonucleic Acid Specific for Rous Sarcoma Virus after Infection of Permissive and Nonpermissive Hosts

Harold E. Varmus, Peter K. Vogt, J. Michael Bishop A relatively simple but stringent technique was developed to detect the integration of virus-specific DNA into the genomes of higher organisms. In both permissive (duck) and nonpermissive (mammalian) cells which normally contain no nucleotide sequences specific for Rous sarcoma virus, transformation by the virus results in the appearance of DNA specific for Rous sarcoma virus covalently integrated into strands of host-cell DNA containing reiterated sequences. Early after infection of mouse or duck cells by Rous sarcoma virus, unintegrated DNA specific for the virus can be demonstrated.

Dendritic Cell-Ewing’s Sarcoma Cell Hybrids Enhance Antitumor Immunity

Wei Guo MD, PhD, Yi Guo MD, Shun Tang MD, Huayi Qu MD, Hui Zhao MD Given the effective immunotherapy of DC-based vaccine in other cancers, we hypothesized DC-based vaccines would induce effective immune responses against Ewing’s sarcoma. To verify this hypothesis and develop the most effective dendritic cell vaccine against Ewing’s sarcoma, we evaluated the antitumor efficacy of dendritic cell-Ewing’s sarcoma hybrids and dendritic cells pulsed with other antigen-loading methods, including cell lysates and the characteristic EWS-FLI1 gene of Ewing’s sarcoma, using an A673 cell line as a model. The hybrids were generated by electrofusion with fusion efficiency and viability determined by flow cytometry and fluorescent microscopy analyses. By interferon-γ secretion assay, the capacity of hybrids to stimulate cytotoxic T-lymphocytes (CTLs) is higher than that of other antigen-loading methods showing stronger tumor antigen-specific CTL cytotoxicity to A673. By in vivo experiment in SCID mice, all dendritic cell-based strategies induced specific immune responses to Ewing’s sarcoma after mice-human immune system reconstitution by inoculating human peripheral blood mononuclear cells into the peritoneal cavity of SCID mice. However, the hybrids most inhibited the subcutaneous tumor growth in SCID mice compared with dendritic cells pulsed with other loading methods. The data suggest A673 cells respond to dendritic cell-based immunotherapy.

Dominant Negative LRP5 Decreases Tumorigenicity and Metastasis of Osteosarcoma in an Animal Model

Yi Guo MD, PhD, Elyssa M. Rubin MD, Jun Xie BA, Xiaolin Zi MD, PhD, Bang H. Hoang MD Osteosarcoma (OS) is a primary malignant bone tumor with a high propensity for local recurrence and distant metastasis. We previously showed a secreted, dominant-negative LRP5 receptor (DNLRP5) suppressed in vitro migration and invasion of the OS cell line SaOS-2. Therefore, we hypothesized DNLRP5 also has in vivo antitumor activity against OS. We used the 143B cell line as a model to study the effect of DNLRP5 by stable transfection. Inhibition of Wnt signaling by DNLRP5 was verified by a reduction in TOPFLASH luciferase activity. In soft agar, DNLRP5-transfected 143B cells formed fewer and smaller colonies than control transfected cells. DNLRP5 transfection reduced in vivo tumor growth of 143B cells in nude mice. DNLRP5 also decreased in vitro cellular motility in a scratch wound assay. In a spontaneous pulmonary metastasis model, DNLRP5 reduced both the size and number of lung metastatic nodules. The reduction in cellular invasiveness by DNLRP5 was associated with decreased expression of matrix metalloproteinase-2, N-cadherin, and Snail. Our data suggest canonical Wnt/LRP5 signaling reflects an important underlying mechanism of OS progression. Therefore, strategies to suppress LRP5-mediated signaling in OS cells may lead to a reduction in local or systemic disease burden.

Neuronal Differentiation of Synovial Sarcoma and Its Therapeutic Application

Tatsuya Ishibe MD, PhD, Tomitaka Nakayama MD, PhD, Tomoki Aoyama MD, PhD, Takashi Nakamura MD, PhD, Junya Toguchida MD, PhD Synovial sarcoma is a rare sarcoma of unknown histologic origin. We previously reported the gene expression profile of synovial sarcoma was closely related to that of malignant peripheral nerve sheath tumors, and the fibroblast growth factor (FGF) signal was one of the main growth signals in synovial sarcoma. Here we further demonstrate the neural origin of synovial sarcoma using primary tumors and cell lines. The expression of neural tissue-related genes was confirmed in synovial sarcoma tumor tissues, but the expression of some genes was absent in synovial sarcoma cell lines. Treatment of synovial sarcoma cell lines with BMP4 or FGF2 enhanced or restored the expression of neural tissue-related genes and induced a neuron-like morphology with positive Tuj-1 expression. Treatment with all-trans-retinoic acid also induced the expression of neural tissue-related genes in association with growth inhibition, which was not observed in other cell lines except a malignant peripheral nerve sheath tumor cell line. A growth-inhibitory effect of all-trans-retinoic acid was also observed for xenografted tumors in athymic mice. The simultaneous treatment with FGF signal inhibitors enhanced the growth-inhibitory effect of all-trans-retinoic acid, suggesting the combination of growth signaling inhibition and differentiation induction could be a potential molecular target for treating synovial sarcoma.

Gene Translocations in Musculoskeletal Neoplasms

Balaji Krishnan MS, Gaurav Khanna MD, Denis Clohisy MD Establishing the best diagnosis for musculoskeletal neoplasms requires a multidisciplinary approach using clinical, radiographic, and histologic analyses. Despite this rigorous approach, establishing accurate diagnoses and prognoses remains challenging. Improved diagnostic methods are expected as unique molecular signals for specific bone and soft tissue cancers are identified. We performed a systematic review of the best available evidence to explore three major applications of molecular genetics that will best benefit clinical management of musculoskeletal neoplasms: diagnostic, prognostic, and therapeutic applications. The specific questions addressed in this systematic review are: (1) What sets of histopathologic sarcoma subtypes will benefit from molecular evaluation and diagnosis? (2) What molecular methods are best applied to histopathologic sarcomas to distinguish between major subtypes? (3) How do the molecular patterns discovered on genetic diagnosis affect prognosis of certain sarcomas? (4) Which sarcoma translocations can benefit from an improved response and outcome using existing and forthcoming pharmacogenetic approaches targeting molecular events? This review summarizes recent advances in molecular genetics that are available and will soon be available to clinicians to better predict outcomes and subsequently help make future treatment decisions.,[object Object]

Synovial Sarcoma: From Genetics to Genetic-based Animal Modeling

Malay Haldar PhD, R. Lor Randall MD, Mario R. Capecchi PhD Synovial sarcomas are highly aggressive mesenchymal cancers that show modest response to conventional cytotoxic chemotherapy, suggesting a definite need for improved biotargeted agents. Progress has been hampered by the lack of insight into pathogenesis of this deadly disease. The presence of a specific diagnostic t(X;18) translocation leading to expression of the unique SYT-SSX fusion protein in effectively all cases of synovial sarcoma suggests a role in the etiology. Other nonspecific anomalies such as overexpression of Bcl-2, HER-2/neu, and EGFR have been reported, but their role in the pathogenesis remains unclear. Using gene targeting, we recently generated mice conditionally expressing the human SYT-SSX2 fusion gene from mouse endogenous ROSA26 promoter in chosen tissue types in the presence of Cre recombinase. These mice develop synovial sarcoma when SYT-SSX2 is expressed within myoblasts, thereby identifying a source of this enigmatic tumor and establishing a mouse model of this disease that recapitulates the clinical, histologic, immunohistochemical, and transcriptional profile of human synovial sarcomas. We review the genetics of synovial sarcoma and discuss the usefulness of genetics-based mouse models as a valuable research tool in the hunt for key molecular determinants of this lethal disease as well as a preclinical platform for designing and evaluating novel treatment strategies.

Specific Tyrosine Kinase Inhibitors Regulate Human Osteosarcoma Cells In vitro

Patrick J. Messerschmitt MD, Ashley N. Rettew BS, Robert E. Brookover BS, Ryan M. Garcia MD, Patrick J. Getty MD, Edward M. Greenfield PhD [object Object]

Osteosarcoma Development and Stem Cell Differentiation

Ni Tang MD, PhD, Wen-Xin Song MD, Jinyong Luo MD, Rex C. Haydon MD, PhD, Tong-Chuan He MD, PhD Osteosarcoma is the most common nonhematologic malignancy of bone in children and adults. The peak incidence occurs in the second decade of life, with a smaller peak after age 50. Osteosarcoma typically arises around the growth plate of long bones. Most osteosarcoma tumors are of high grade and tend to develop pulmonary metastases. Despite clinical improvements, patients with metastatic or recurrent diseases have a poor prognosis. Here, we reviewed the current understanding of human osteosarcoma, with an emphasis on potential links between defective osteogenic differentiation and bone tumorigenesis. Existing data indicate osteosarcoma tumors display a broad range of genetic and molecular alterations, including the gains, losses, or arrangements of chromosomal regions, inactivation of tumor suppressor genes, and the deregulation of major signaling pathways. However, except for p53 and/or RB mutations, most alterations are not constantly detected in the majority of osteosarcoma tumors. With a rapid expansion of our knowledge about stem cell biology, emerging evidence suggests osteosarcoma should be regarded as a differentiation disease caused by genetic and epigenetic changes that interrupt osteoblast differentiation from mesenchymal stem cells. Understanding the molecular pathogenesis of human osteosarcoma could ultimately lead to the development of diagnostic and prognostic markers, as well as targeted therapeutics for osteosarcoma patients.

Expression of Macrophage Migration Inhibitory Factor Relates to Survival in High-grade Osteosarcoma

Ilkyu Han MD, PhD, Mi Ra Lee BS, Kwang Woo Nam MD, Joo Han Oh MD, PhD, Kyung Chul Moon MD, PhD, Han-Soo Kim MD, PhD Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is implicated in many aspects of tumor progression, including cell proliferation, invasion, and angiogenesis. We asked if MIF expression predicts survival and if it is associated with angiogenesis and cell invasion in osteosarcoma. We performed immunohistochemistry for MIF expression in prechemotherapy biopsy specimens of 58 patients with osteosarcoma. To investigate the role of MIF in angiogenesis, microvessel density was measured and compared with MIF expression. We also treated osteosarcoma cell lines (U2-OS and MG63) with MIF and measured vascular endothelial growth factor, a potent proangiogenic factor, by enzyme-linked immunosorbent assay. To study the role of MIF in cell invasion, Boyden chamber assay was performed after knockdown of MIF by short interfering RNA. MIF independently predicted overall survival and metastasis-free survival. MIF expression correlated with microvessel density and induced a dose-dependent increase in vascular endothelial growth factor. Knockdown of MIF by short interfering RNA resulted in decreased cell invasion. These results suggest MIF could serve as a prognostic marker and a potential therapeutic target for osteosarcoma.,[object Object]

PATCHED-ONE or SMOOTHENED Gene Mutations Are Infrequent in Chondrosarcoma

Taiqiang Yan MD, Mark Angelini MD, FRCSC, Benjamin A. Alman MD, FRCSC, Irene L. Andrulis PhD, Jay S. Wunder MD, MSc, FRCS(C)

Global Protein-expression Analysis of Bone and Soft Tissue Sarcomas

Akira Kawai MD, PhD, Tadashi Kondo MD, PhD, Yoshiyuki Suehara MD, PhD, Kazutaka Kikuta MD, Setsuo Hirohashi MD, PhD Analysis of global protein expression, an approach known as expression proteomics, can offer important clues for understanding tumor biology that cannot be obtained by other approaches (e.g., genome or transcriptome analysis). Using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry, we performed global protein expression studies of bone and soft tissue sarcomas to develop novel diagnostic and therapeutic biomarkers and allow molecular classification of the tumors. Among 1500 protein variants identified in the two-dimensional gel, 67 proteins correctly distinguished the eight subtypes of 99 histologically classified soft tissue sarcomas. Hierarchical clustering demonstrated leiomyosarcoma and MFH shared a similar protein expression profile, and clear cell sarcoma, synovial sarcoma, and MPNST could be grouped according to their protein expression patterns. Pleomorphic leiomyosarcoma and MFH showed similar tropomyosin isoform expression patterns. Patients with gastrointestinal stromal tumors expressing pfetin protein had better survival than those whose tumors lacked it. We identified 10 protein spots associated with the chemosensitivity of osteosarcoma to preoperative chemotherapy. These 10 spots could be new diagnostic and prognostic markers for osteosarcoma and new therapeutic targets for the disease. Proteomic analysis using 2D-DIGE provides novel information on the biology of bone and soft tissue sarcomas that could be used to diagnosis and treat these tumors.,[object Object]

Hypoxia Markers in Human Osteosarcoma: An Exploratory Study

Hiroo Mizobuchi MD, José Manuel García-Castellano MD, PhD, Shaji Philip BA, John H. Healey MD, Richard Gorlick MD Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1α) and vascular endothelial growth factor (VEGF). The roles of these markers have been studied previously in several cancer lines. We ascertained the frequency of HIF-1α expression, VEGF expression, the degree of neovascularization, and cell proliferation in osteosarcoma samples. Samples from osteosarcoma patients were assessed for HIF-1α and VEGF protein expression using immunohistochemistry, neovascularization using antibodies for Factor VIII, and cell proliferation using the Ki-67 labeling index. Associations between these parameters and clinical features were examined. HIF-1α staining was positive in 35% of patients and metastases were present in 61% of these HIF-1α-positive patients. VEGF protein expression was detected in 69% of patients, 92% of whom were female. We observed an insignificant trend for a higher frequency of VEGF expression in the high-grade as compared to low-grade osteosarcoma. We observed no association between vascular density and proliferation index and any clinical parameters. We found an association between HIF-1α expression and metastatic disease and between VEGF expression and female gender.

Histogenetic Characterization of Giant Cell Tumor of Bone

Manuela Salerno MSc, Sofia Avnet PhD, Marco Alberghini MD, Armando Giunti MD, Nicola Baldini MD [object Object]

Polymorphisms and Methylation of the Reduced Folate Carrier in Osteosarcoma

Rui Yang MD, Jing Qin PhD, Bang H. Hoang MD, John H. Healey MD, Richard Gorlick MD High-dose methotrexate is a standard component in the treatment of osteogenic sarcoma. Impaired methotrexate uptake associated with decreased reduced folate carrier expression is a common mechanism of methotrexate resistance in osteogenic sarcoma samples. We investigated whether promoter methylation and polymorphisms in the 3′ untranslated region are involved in regulating reduced folate carrier expression. In a cohort of 66 osteogenic sarcoma specimens, quantitative methylation-specific polymerase chain reaction and single-strand conformation polymorphism were performed. We found detectable levels of promoter methylation in 84.3% of samples. When related to the reduced folate carrier mRNA levels, a trend was observed that reduced folate carrier expression is lower in samples (median, 0.7) with greater than 10% DNA methylation as compared with those (median, 2.3) with less than 10% DNA methylation. The heterozygous polymorphisms of 2582 T/G and 2617C/T in the 3′ untranslated region showed reduced folate carrier expression (median, 0.9) as compared with the wild-type 2582T and 2617C (median, 4.2). The data suggest promoter methylation and polymorphisms in the 3′ untranslated region of the reduced folate carrier may be involved in its transcriptional regulation in osteogenic sarcoma. Further study is required to confirm this finding.

Fas Death Pathway in Sarcomas Correlates with Epidermal Growth Factor Transcription

David E. Joyner PhD, Albert J. Aboulafia MD, Timothy A. Damron MD, R. Lor Randall MD, FACS Modulation of apoptosis may influence sarcoma pathogenesis and/or aggressiveness. The Fas death pathway, mediated by FasL or TGFβ, is one of two apoptotic pathways. Recent studies report that EGF can modulate TGFβ and/or FasL expression/activity; thus, EGF has the potential to influence activation of the Fas pathway. EGF is not always detectable in mesenchymal tumors; therefore, we hypothesized EGF would define which Fas ligand predominates. We assayed 57 surgically removed human sarcomas for 10 genes involved in the Fas pathway. Skeletal muscle biopsies from eight patients served as controls. Sample transcripts were detected by real-time RT-PCR. We attempted to identify relevant predictor variables. The 57 sarcomas were segregated into two categories defined by EGF mRNA content: (1) 23 tumors with EGF concentrations that approximated muscle EGF transcript levels (high-EGF tumors); and (2) 34 tumors that either lacked EGF mRNA, or whose mRNA levels were very low and frequently undetected by PCR (low-EGF tumors). TGFβ1 expression best predicted Fas transcript concentrations in the 34 low-EGF sarcomas, while FasL predicted Fas mRNA levels in the remaining 23 high-EGF sarcomas. The results suggest ligand activity in the Fas death pathway correlates with EGF transcription in sarcomas.

Metastatic Osteosarcoma Gene Expression Differs In Vitro and In Vivo

Jennifer W. Lisle MD, Joseph Y. Choi MD, PhD, Jason A. Horton BS, Matthew J. Allen VetMB, PhD, Timothy A. Damron MD An understanding of differential gene expression in highly metastatic osteosarcoma could provide gene targets for treatment of metastases. We compared gene expression profiles of high- (LM7) and low- (LM2) metastatic SaOS2-derived cell lines in an in vitro tissue culture model and examined several differentially regulated genes in vivo in a murine orthotopic xenograft model. We hypothesized an orthotopic inoculation of LM2 and LM7 cells would establish a primary lesion and the gene expression profile of cells grafted in this fashion would resemble the gene expression profile observed in an in vitro model. Thirty-five days after inoculation, animals were euthanized and both tibiae were harvested and rapidly frozen in liquid nitrogen. Human-specific GAPDH mRNA was present in two of four tibias inoculated with LM2 cells and three of four tibias inoculated with LM7 cells. Tibiae displaying the presence of human cells were assayed by semiquantitative reverse transcriptase polymerase chain reaction. We observed poor correspondence of in vitro to in vivo gene expression for either cell line. Accordingly, in vitro osteosarcoma gene expression data must be interpreted with caution until confirmed in vivo. Our orthotopic injection model allowed in vivo study of differential gene expression between these two cell lines but did not show radiographic evidence of an established primary lesion.