Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells

The Role of RUNX2 in Osteosarcoma Oncogenesis

Osteosarcoma is an aggressive but ill-understood cancer of bone that predominantly affects adolescents. Its rarity and biological heterogeneity have limited studies of its molecular basis. In recent years, an important role has emerged for the RUNX2 “platform protein” in osteosarcoma oncogenesis. RUNX proteins are DNA-binding transcription factors that regulate the expression of multiple genes involved in cellular differentiation and cell-cycle progression. RUNX2 is genetically essential for developing bone and osteoblast maturation. Studies of osteosarcoma tumours have revealed that the RUNX2 DNA copy number together with RNA and protein levels are highly elevated in osteosarcoma tumors. The protein is also important for metastatic bone disease of prostate and breast cancers, while RUNX2 may have both tumor suppressive and oncogenic roles in bone morphogenesis. This paper provides a synopsis of the current understanding of the functions of RUNX2 and its potential role in osteosarcoma and suggests directions for future study

In Vitro Analysis of Integrated Global High-Resolution DNA Methylation Profiling with Genomic Imbalance and Gene Expression in Osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks

Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma

BACKGROUND: Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets. METHODS: We used a genome-wide screening method – array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study. RESULTS: Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (PRDM16), 6p21.1 (CDC5L, HSPCB, NFKBIE), 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 (PMP22 MYCD, SOX1,ELAC27). We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). CONCLUSIONS: This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones

Establishment of a new human osteosarcoma cell line, UTOS-1: cytogenetic characterization by array comparative genomic hybridization

The cytogenetic characteristics of osteosarcoma (OS) remain controversial. The establishment of a new human OS cell line may improve the characterization. We report the establishment of a new human osteosarcoma cell line, UTOS-1, from a typical osteoblastic OS of an 18-year-old man. Cultured UTOS-1 cells are spindle-shaped, and have been maintained in vitro for over 50 passages in more than 2 years. Xenografted UTOS-1 cells exhibit features typical of OS, such as production of osteoid or immature bone matrix, and proliferation potency in vivo. UTOS-1 also exhibit morphological and immunohistochemical characteristics typical of osteoblastic OS. Chromosomal analysis by G-band show 73~85 chromosomes with complicated translocations. Array CGH show frequent gains at locus DAB2 at chromosome 5q13, CCND2 at 12p13, MDM2 at 12q14.3-q15, FLI and TOP3A at 17p11.2-p12 and OCRL1 at Xq25, and show frequent losses at HTR1B at 6q13, D6S268 at 6q16.3-q21, SHGC17327 at 18ptel, and STK6 at 20q13.2-q13.3. The UTOS-1 cell line may prove useful for biologic and molecular pathogenetic investigations of human OS

Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

Background: Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Methods: Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts.Results: RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. Conclusion: These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma. © 2010 Sadikovic et al; licensee BioMed Central Ltd

Chromosome 9p21 gene copy number and prognostic significance of p16 in ESFT

Chromosome 9p21 gene copy number in Ewing's sarcoma family of tumour (ESFT) cell lines and primary ESFT has been evaluated using Multiplex Ligation-dependent probe amplification, and the clinical significance of CDKN2A loss and p16/p14ARF expression investigated. Homozygous deletion of CDKN2A was identified in 4/9 (44%) of ESFT cell lines and 4/42 (10%) primary ESFT; loss of one copy of CDKN2A was identified in a further 2/9 (22%) cell lines and 2/42 (5%) tumours. CDKN2B was co-deleted in three (33%) cell lines and two (5%) tumours. Co-deletion of the MTAP gene was observed in 1/9 (11%) cell lines and 3/42 (7%) tumours. No correlation was observed between CDKN2A deletion and clinical parameters. However, co-expression of high levels of p16/p14ARF mRNA predicted a poor event-free survival (P=0.046, log-rank test). High levels of p16/p14ARF mRNA did not correlate with high expression of p16 protein. Furthermore, p16 protein expression did not predict event-free or overall survival. Methylation is not a common mechanism of p16 gene silencing in ESFT. These studies demonstrate that loss (homozygous deletion or single copy) of CDKN2A was not prognostically significant in primary ESFT. However, high levels of p16/p14ARF mRNA expression were predictive of a poor event-free survival and should be investigated further

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors

<p>Abstract</p> <p>Background</p> <p>Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to <it>EWS/FLI1 </it>gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas.</p> <p>Methods</p> <p>Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5–192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest.</p> <p>Results</p> <p>Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the <it>FLI1 </it>and <it>EWSR1 </it>loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between <it>EWSR1 </it>and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, <it>HDGF</it>, confirmed that its expression was higher than in control samples. However, no association between <it>HDGF </it>expression and patient survival was observed.</p> <p>Conclusion</p> <p>We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.</p

Guidelines for histopathological specimen examination and diagnostic reporting of primary bone tumours

This review is intended to provide histopathologists with guidelines for clinical assessment, specimen handling and diagnostic reporting of benign and malignant primary bone tumours. Information from radiology, surgical, oncology and other clinical colleagues involved in the diagnosis and treatment of primary bone tumours should be properly assessed before undertaking a structured approach to specimen handling and histological reporting. This ensures that the information needed for planning appropriate treatment of these complex tumours is provided. Consistency in diagnostic evaluation with respect to both terminology and report content facilitates liaison at multidisciplinary bone tumour meetings and collaboration between cancer units and networks, as well as providing a common database for audit of the clinical, radiological and pathological aspects of bone tumours