miR-486-5p expression is regulated by DNA methylation in osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. RESULTS: To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2'-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. CONCLUSIONS: miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Reexpression of LSAMP inhibits tumor growth in a preclinical osteosarcoma model.

BACKGROUND: Osteosarcomas are the most common primary malignant tumors of bone, showing complex chromosomal rearrangements with multiple gains and losses. A frequent deletion within the chromosomal region 3q13.31 has been identified by us and others, and is mainly reported to be present in osteosarcomas. The purpose of the study was to further characterize the frequency and the extent of the deletion in an extended panel of osteosarcoma samples, and the expression level of the affected genes within the region. We have identified LSAMP as the target gene for the deletion, and have studied the functional implications of LSAMP-reexpression. METHODS: LSAMP copy number, expression level and protein level were investigated by quantitative PCR and western blotting in an osteosarcoma panel. The expression of LSAMP was restored in an osteosarcoma cell line, and differences in proliferation rate, tumor formation, gene expression, migration rate, differentiation capabilities, cell cycle distribution and apoptosis were investigated by metabolic dyes, tumor formation in vivo, gene expression profiling, time-lapse photography, differentiation techniques and flow cytometry, respectively. RESULTS: We found reduced copy number of LSAMP in 45/76 osteosarcoma samples, reduced expression level in 25/42 samples and protein expression in 9/42 samples. By restoring the expression of LSAMP in a cell line with a homozygous deletion of the gene, the proliferation rate in vitro was significantly reduced and tumor growth in vivo was significantly delayed. In response to reexpression of LSAMP, mRNA expression profiling revealed consistent upregulation of the genes hairy and enhancer of split 1 (HES1), cancer/testis antigen 2 (CTAG2) and kruppel-like factor 10 (KLF10). CONCLUSIONS: The high frequency and the specificity of the deletion indicate that it is important for the development of osteosarcomas. The deletion targets the tumor suppressor LSAMP, and based on the functional evidence, the tumor suppressor function of LSAMP is most likely exerted by reducing the proliferation rate of the tumor cells, possibly by indirectly upregulating one or more of the genes HES1, CTAG2 or KLF10. To our knowledge, this study describes novel functions of LSAMP, a first step to understanding the functional role of this specific deletion in osteosarcomas.

Correlation of TP53 and MDM2 genotypes with response to therapy in sarcoma.

BACKGROUND: Relatively few sarcomas harbor TP53 (tumor protein p53) mutations, but in many cases, amplification of MDM2 (murine double minute 2) effectively inactivate p53. The p53 pathway activity can also be affected by normal genetic variation. METHODS: The mutation status of TP53 and expression of MDM2, TP53, and their genetic variants SNP309 and R72P (Arg72Pro) were investigated in 125 sarcoma patient samples and 18 sarcoma cell lines. Association of the different genotypes and gene aberrations with chemotherapy response and survival, as well as response to MDM2 antagonists in vitro was evaluated. RESULTS: Twenty-two percent of the tumors had mutant TP53 and 20% MDM2 gene amplification. Patients with wild-type TP53 (TP53(Wt) ) tumors had improved survival (P < .001) and TP53(Wt) was an independent prognostic factor (hazard ratio = 0.41; 95% confidence interval = 0.23-0.74; P = .03). Interestingly, there was a trend toward longer time to progression after chemotherapy for tumors with the apoptosis-prone p53 variant R72 (P = .07), which was strongest with doxorubicin/ifosfamide-based regimens (P = .01). Liposarcomas had low R72 frequency (33% versus 56%), but increased levels of MDM2 and MDM4 (51% and 11%, P < .001). MDM2 overexpression on a TP53(Wt) background predicted better response to MDM2 antagonist Nutlin-3a, irrespective of R72P or SNP309 status. CONCLUSIONS: Improved survival after chemotherapy was found in patients with TP53(Wt) tumors harboring the R72 variant. MDM2 overexpression in TP53(Wt) tumors predicted good response to MDM2 antagonists, irrespective of R72P or SNP309 status. Thus, detailed TP53 and MDM2 genotype analyses prior to systemic therapy are recommended.

Identification of osteosarcoma driver genes by integrative analysis of copy number and gene expression data.

High-grade osteosarcoma is a tumor with a complex genomic profile, occurring primarily in adolescents with a second peak at middle age. The extensive genomic alterations obscure the identification of genes driving tumorigenesis during osteosarcoma development. To identify such driver genes, we integrated DNA copy number profiles (Affymetrix SNP 6.0) of 32 diagnostic biopsies with 84 expression profiles (Illumina Human-6 v2.0) of high-grade osteosarcoma as compared with its putative progenitor cells, i.e., mesenchymal stem cells (n = 12) or osteoblasts (n = 3). In addition, we performed paired analyses between copy number and expression profiles of a subset of 29 patients for which both DNA and mRNA profiles were available. Integrative analyses were performed in Nexus Copy Number software and statistical language R. Paired analyses were performed on all probes detecting significantly differentially expressed genes in corresponding LIMMA analyses. For both nonpaired and paired analyses, copy number aberration frequency was set to >35%. Nonpaired and paired integrative analyses resulted in 45 and 101 genes, respectively, which were present in both analyses using different control sets. Paired analyses detected >90% of all genes found with the corresponding nonpaired analyses. Remarkably, approximately twice as many genes as found in the corresponding nonpaired analyses were detected. Affected genes were intersected with differentially expressed genes in osteosarcoma cell lines, resulting in 31 new osteosarcoma driver genes. Cell division related genes, such as MCM4 and LATS2, were overrepresented and genomic instability was predictive for metastasis-free survival, suggesting that deregulation of the cell cycle is a driver of osteosarcomagenesis.

Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood.

BACKGROUND: DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. RESULTS: Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. CONCLUSIONS: Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.

Preclinical xenograft models of human sarcoma show nonrandom loss of aberrations.

BACKGROUND: Human tumors transplanted into immunodeficient mice (xenografts) are good preclinical models, and it is important to identify possible systematic changes during establishment and passaging in mice. METHODS: High-resolution microarray-based comparative genomic hybridization (array CGH) was used to investigate how well a series of sarcoma xenografts, including 9 patient/xenograft pairs and 8 early versus late xenograft passage pairs, represented the patient tumor from which they originated. RESULTS: In all analyses, the xenografts were more similar to their tumor of origin than other xenografts of the same type. Most changes in aberration patterns were toward a more normal genome complement, and the increased aberrations observed were mostly toward more loss. In general, the changes were scattered over the genome, but some changes were significant in osteosarcomas. These were rather focused and consistent with amplifications frequent in patient samples, involving the genes platelet-derived growth factor receptor A (PDGFRA), cysteine-rich hydrophobic domain 2 (CHIC2), FIP-like 1 (FIP1L1), ligand of numb-protein X1 (LNX1), RAS-like family 11 member B (RASL11B), and sec1 family domain containing 2 (SCFD2), probably a sign of continued tumor progression. Some changes that disappeared may have been involved in host-stroma interactions or chemotherapy resistance, possibly because of the absence of selection in the mouse. CONCLUSIONS: Direct xenografts reflected well the genomic patterns of their tumors of origin. The few significant aberrations that were lost during passaging in immune-defective mice may have been caused by the lack of selection in the new host, whereas aberrations that were gained appeared to be the result of general tumor progression rather than model-specific artifacts.

Genomic alterations reveal potential for higher grade transformation in follicular lymphoma and confirm parallel evolution of tumor cell clones.

Our aim was to examine the genetics of clonal evolution in follicular lymphoma (FL) and to identify genetic alterations associated with disease progression. A total of 100 biopsies from 44 patients diagnosed with t(14;18)-positive FL were examined by array comparative genomic hybridization. In 20 patients the patterns of somatic hypermutations (SHMs) in the variable region of heavy chain gene were additionally analyzed. Gain of chromosome X in male samples was a marker for poor outcome (P < .01). Gains involving chromosome 2, 3q, and 5 were exclusively present in FL biopsies from cases with higher grade transformation and were among the copy number alterations (CNAs) associated with inferior survival. Although we noted a trend for increasing genomic complexity in initial versus late FL samples, the overall frequencies of CNAs in initial and late FL biopsies showed a surprisingly stable pattern through the course of the disease. In 27 of cases the initial samples harbored CNAs that were absent in relapse samples, indicating that tumor cell clones at relapse were not direct descendants of initially dominating clones. The pattern of SHMs confirmed parallel development of tumor cell clones in 14 cases. Our findings support the hypothesis of common progenitor cells in FL.

Global gene expression profiling of human osteosarcomas reveals metastasis-associated chemokine pattern.

Global gene expression analysis was performed on a panel of 23 osteosarcoma samples of primary and metastatic origin using the Applied Biosystems Gene Expression Array System. When comparing the primary tumours with the metastases, we found a significantly increased expression of genes involved in immunological processes, for example coding for cytokines and chemokines, in the metastatic samples. In addition, a comparison of the gene expression in primary samples from patients with or without metastases demonstrated that patients who later developed metastases had high expression of the chemokine (C-X-C motif) receptor 4 (CXCR4), similar to the metastatic samples, suggesting that these signal molecules play an important role in promoting metastasis. Increased knowledge of mechanisms and interactions between specified molecular signalling pathways in osteosarcomas could lead to a more rational strategy for development of targeted therapy.

Targeted genetic and epigenetic profiling of esophageal adenocarcinomas and non-dysplastic Barrett's esophagus.

BACKGROUND: Despite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett's esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses. RESULTS: Overall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21-89%), as well as in a subset of adjacent normal samples (up to 12%). CONCLUSIONS: Our study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.

Molecular characterization of commonly used cell lines for bone tumor research: a trans-European EuroBoNet effort.

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

LSAMP, a novel candidate tumor suppressor gene in human osteosarcomas, identified by array comparative genomic hybridization.

Osteosarcomas are the most common primary malignant tumor of bone, and almost all conventional osteosarcomas are high-grade tumors with complex karyotypes. We have examined DNA copy number changes in 36 osteosarcoma tumors and 20 cell lines using microarray-based comparative genomic hybridization. The most frequent minimal recurrent regions of gain identified in the tumor samples were in 1q21.2-q21.3 (78% of the samples), 1q21.3-q22 (78%), and 8q22.1 (72%). Minimal recurrent regions in 10q22.1-q22.2 (81%), 6q16.1 (67%), 13q14.2 (67%), and 13q21.1 (67%) were most frequently lost. A small region in 3q13.31 (2.1 Mb) containing the gene limbic system-associated membrane protein (LSAMP) was frequently deleted (56%). LSAMP has previously been reported to be a candidate tumor suppressor gene in other cancer types. The deletion was validated using fluorescence in situ hybridization, and the expression level and promoter methylation status of LSAMP were investigated using quantitative real-time reverse transcription PCR and methylation-specific PCR, respectively. LSAMP showed low expression compared to two normal bone samples in 6/15 tumors and 5/9 cell lines with deletion of 3q13.31, and also in 5/14 tumors and 3/11 cell lines with normal copy number or gain. Partial or full methylation of the investigated CpG island was identified in 3/30 tumors and 7/20 cell lines. Statistical analyses revealed that loss of 11p15.4-p15.3 and low expression of LSAMP (both P = 0.011) were significantly associated with poor survival. Our results show that LSAMP is a novel candidate tumor suppressor gene in osteosarcomas.

Array-CGH fine mapping of minor and cryptic HR-CGH detected genomic imbalances in 80 out of 590 patients with abnormal development.

During a 6-year period, 590 patients suspected of having a minor or cryptic genomic imbalance as the cause of mental retardation with dysmorphic signs +/- malformations have been investigated with high-resolution comparative genomic hybridisation (HR-CGH) in our diagnostic laboratory. Thirty-six patients had a small chromosomal aberration detected by routine karyotyping, and 554 patients had a normal G-banded karyotype. In the latter group, a genomic imbalance was detected by HR-CGH in 40 patients (7.2%): 29 deletions, 3 duplications, 4 unbalanced translocations, and 4 occult trisomy mosaicisms. When microarray-based comparative genomic hybridisation (array-CGH) became available, all HR-CGH-positive samples were also investigated by 1 Mb resolution array-CGH for more precise mapping. From the 514 patients with normal HR-CGH findings, a subset of 20 patients with particularly high suspicion of having a chromosomal imbalance was selected for array-CGH. In four of them (20%), an imbalance was detected: three deletions and one duplication. Of note, 73 out of the 80 array-CGH mapped patients had a de novo chromosomal rearrangement (91%). Taken together, this work provides phenotype-genotype information on 80 patients with minor and cryptic chromosomal imbalances.

DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH). RESULTS: Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (LOXL2) was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-alpha (TOP2A), ets variant gene 4 (E1A enhancer binding protein, E1AF) (ETV4) and baculoviral IAP repeat-containing 5 (survivin) (BIRC5) showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for TOP2A, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors. CONCLUSION: Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes TOP2A, ETV4 and BIRC5 are interesting candidate targets for the 17q gain associated with poor survival.

Chromosomal aberrations in head and neck squamous cell carcinomas in Norwegian and Sudanese populations by array comparative genomic hybridization.

We used microarray-based comparative genomic hybridization to explore genome-wide profiles of chromosomal aberrations in 26 samples of head and neck cancers compared to their pair-wise normal controls. The samples were obtained from Sudanese (n=11) and Norwegian (n=15) patients. The findings were correlated with clinicopathological variables. We identified the amplification of 41 common chromosomal regions (harboring 149 candidate genes) and the deletion of 22 (28 candidate genes). Predominant chromosomal alterations that were observed included high-level amplification at 1q21 (harboring the S100A gene family) and 11q22 (including several MMP family members). Regions of copy number increase was also identified at 6p21 (p21), 7p12 (EGFR), 17p13 (p53) and 19p13.2 (p19INK4d), while regions showing deletion included among others 3p25.2 (RAF1) and 9p21 (p15, p16). We found genes from four common biological pathways (MAPK signaling, cytokine-cytokine receptor interaction, ECM-receptor interaction and Jak-STAT signaling) to be predominantly over-represented in areas of gain and loss. The current study provides valuable information on chromosomal aberrations likely to be involved in the pathogenesis of head and neck cancers. An increased copy number of the S100A and MMP gene family members, known to be involved in invasion and metastasis, may play an important role in the development of the tumors. Hierarchical clustering of the chromosomal alterations with clinicopathological parameters showed little correlation, suggesting an occurrence of gains/losses regardless of ethnic differences and clinicopathological status between the patients from the two countries. Our findings indicate the existence of common gene-specific amplifications/deletions in these tumors, regardless of the source of the samples or attributed carcinogenic risk factors.

Array comparative genomic hybridization reveals distinct DNA copy number differences between gastrointestinal stromal tumors and leiomyosarcomas.

Leiomyosarcomas are spindle cell tumors showing smooth muscle differentiation. Until recently, most gastrointestinal stromal tumors (GIST) were also classified as smooth muscle tumors, but now GISTs are recognized as a separate entity, defined as spindle cell and/or epithelioid tumors localized in the gastrointestinal tract. Using microarray-based comparative genomic hybridization (array CGH), we have created a detailed map of DNA copy number changes for 7 GISTs and 12 leiomyosarcomas. Considerable gains and losses of chromosomal segments were observed in both tumor types. The most frequent aberration observed in GISTs was loss of chromosomes 14 and 22, with minimal recurrent regions in 14q11.2-q32.33 (71% of the tumors) and 22q12.2-q13.31 (100%). In leiomyosarcomas, frequent loss of chromosome 10 and 13q was observed, with minimal recurrent regions in 10q21.3 (75%) and 13q14.2-q14.3 (75%). Recurrent high-level amplification of 17p13.1-p11.2 was detected in leiomyosarcomas. Expression profiling using cDNA microarrays revealed four candidate genes in this region with high expression (AURKB, SREBF1, MFAP4, and FLJ10847). Altered expression of AURKB and SREBF1 has been observed previously in other malignancies. Hierarchical clustering of all samples separated GISTs and leiomyosarcomas into two distinct clusters. Statistical analysis identified six chromosomal regions, 1p36.11-p13.1, 9q21.11-9q34.3, 14q11.2-q23.2, 14q31.3-q32.33, 15q24.3-q26.3, and 22q11.21-q13.31, which were significantly different in copy number between GISTs and leiomyosarcomas. Our results show the potential of using array comparative genomic hybridization to classify histologically similar tumors such as GISTs and leiomyosarcomas.

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.

Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH.

BACKGROUND: Amplification of the q21-q23 region on chromosome 1 is frequently found in sarcomas and a variety of other solid tumours. Previous analyses of sarcomas have indicated the presence of at least two separate amplicons within this region, one located in 1q21 and one located near the apolipoprotein A-II (APOA2) gene in 1q23. In this study we have mapped and characterized the amplicon in 1q23 in more detail. RESULTS: We have used fluorescence in situ hybridisation (FISH) and microarray-based comparative genomic hybridisation (array CGH) to map and define the borders of the amplicon in 10 sarcomas. A subregion of approximately 800 kb was identified as the core of the amplicon. The amplification patterns of nine possible candidate target genes located to this subregion were determined by Southern blot analysis. The genes activating transcription factor 6 (ATF6) and dual specificity phosphatase 12 (DUSP12) showed the highest level of amplification, and they were also shown to be over-expressed by quantitative real-time reverse transcription PCR (RT-PCR). In general, the level of expression reflected the level of amplification in the different tumours. DUSP12 was expressed significantly higher than ATF6 in a subset of the tumours. In addition, two genes known to be transcriptionally activated by ATF6, glucose-regulated protein 78 kDa and -94 kDa (GRP78 and GRP94), were shown to be over-expressed in the tumours that showed over-expression of ATF6. CONCLUSION: ATF6 and DUSP12 seem to be the most likely candidate target genes for the 1q23 amplification in sarcomas. Both genes have possible roles in promoting cell growth, which makes them interesting candidate targets.

M-CGH: analysing microarray-based CGH experiments.

BACKGROUND: Microarray-based comparative genomic hybridisation (array CGH) is a technique by which variation in relative copy numbers between two genomes can be analysed by competitive hybridisation to DNA microarrays. This technology has most commonly been used to detect chromosomal amplifications and deletions in cancer. Dedicated tools are needed to analyse the results of such experiments, which include appropriate visualisation, and to take into consideration the physical relation in the genome between the probes on the array. RESULTS: M-CGH is a MATLAB toolbox with a graphical user interface designed specifically for the analysis of array CGH experiments, with multiple approaches to ratio normalization. Specifically, the distributions of three classes of DNA copy numbers (gains, normal and losses) can be estimated using a maximum likelihood method. Amplicon boundaries are computed by either the fuzzy K-nearest neighbour method or a wavelet approach. The program also allows linking each genomic clone with the corresponding genomic information in the Ensembl database http://www.ensembl.org. CONCLUSIONS: M-CGH, which encompasses the basic tools needed for analysing array CGH experiments, is freely available for academics http://www.uio.no/~junbaiw/mcgh, and does not require any other MATLAB toolbox.

Modulation of the osteosarcoma expression phenotype by microRNAs.

BACKGROUND: Osteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed. PRINCIPAL FINDINGS: We identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. SIGNIFICANCE: This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression.

Integrative analysis reveals relationships of genetic and epigenetic alterations in osteosarcoma.

BACKGROUND: Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies. PRINCIPAL FINDINGS: The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2'-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes. CONCLUSIONS: Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology.