List of experimental genes and control genes assayed.

<p>Twenty one probes for 17 genes and three controls were assayed for expression in the nanoString code set. Probes for each of the two main promoter regions of <i>RUNX2</i> were constructed.</p

Digital Expression Profiling Identifies <i>RUNX2</i>, <i>CDC5L</i>, <i>MDM2</i>, <i>RECQL4</i>, and <i>CDK4</i> as Potential Predictive Biomarkers for Neo-Adjuvant Chemotherapy Response in Paediatric Osteosarcoma

<div><p>Osteosarcoma is the most common malignancy of bone, and occurs most frequently in children and adolescents. Currently, the most reliable technique for determining a patients’ prognosis is measurement of histopathologic tumor necrosis following pre-operative neo-adjuvant chemotherapy. Unfavourable prognosis is indicated by less than 90% estimated necrosis of the tumor. Neither genetic testing nor molecular biomarkers for diagnosis and prognosis have been described for osteosarcomas. We used the novel nanoString mRNA digital expression analysis system to analyse gene expression in 32 patients with sporadic paediatric osteosarcoma. This system used specific molecular barcodes to quantify expression of a set of 17 genes associated with osteosarcoma tumorigenesis. Five genes, from this panel, which encoded the bone differentiation regulator <i>RUNX2</i>, the cell cycle regulator <i>CDC5L</i>, the <i>TP53</i> transcriptional inactivator <i>MDM2</i>, the DNA helicase <i>RECQL4</i>, and the cyclin-dependent kinase gene <i>CDK4</i>, were differentially expressed in tumors that responded poorly to neo-adjuvant chemotherapy. Analysis of the signalling relationships of these genes, as well as other expression markers of osteosarcoma, indicated that gene networks linked to RB1, TP53, PI3K, PTEN/Akt, myc and RECQL4 are associated with osteosarcoma. The discovery of these networks provides a basis for further experimental studies of role of the five genes (<i>RUNX2</i>, <i>CDC5L</i>, <i>MDM2</i>, <i>RECQL4</i>, and <i>CDK4</i>) in differential response to chemotherapy.</p></div

Cluster map constructed using Cluster 3.0, showing the differential expression of the 17 gene set in osteosarcoma biopsy and resection cases in the cohort.

<p>Patients with >90% tumor necrosis in response to chemotherapy (good response) are shown in blue and those with <90% (poor response) are shown in red. Sample numbers 1–36 were analyzed in triplicates whereas samples 41–116 were analysed in duplicates. Detailed data manipulation for the cohort is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095843#pone.0095843.s004" target="_blank">Table S2B</a>.</p

Mean expression of the most significantly discriminating five genes in the osteosarcoma cohort when poor response (<90% tumor necrosis in response to chemotherapy) was compared to good response (>90% tumor necrosis).

<p>Unpaired Student’s t-test was applied to derive the genes that were differentially expressed in the two groups.</p

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-1

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>ice (panel B) using immunohistochemical staining with 5-mc-Ab. To the right a 20X enlargement of representative U2OS histology is shown. More 5-mc-Ab staining is evident in the nuclei from control sections (panel A-right side) in comparison to nuclear staining in the sections derived from decitabine treated mice (panel B right side). These data are consistent with a reduction in 5-methylcytidine nuclear content in U2OS xenografts derived from mice treated with decitabine. Enlargements of darkly stained host murine kidney cells correspond to heavily methylated differentiated renal tissue (denoted 2 in panels A and B). In panel C the staining intensity from control (dark columns) and decitabine treated (light columns) is quantitated using Aperio scanning image analysis of sections. The graph to the right of panel C confirms that the 5-mc-Ab staining in control untreated (dark columns) U2OS xenograft sections is more intense than the decitabine treated (light columns) U2OS xenografts. This decrease in staining intensity was significant (p < 0.05). In contrast host kidney cells from both control and treated mice do not exhibit any significant difference in staining intensities

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-3

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>from control (no treatment) as base lines and for a reference gene by applying the ΔΔCt method. Each column is the Mean of three replicas and error bars indicate standard deviation from the Mean. The data is expressed as fold change relative to control (no-treatment). Xeno-4, Xeno-5, and Xeno-6 = decitabine treated xenografts. NHOst = normal human osteoblasts

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-4

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>are indicated on the x-axis. The location of CpG positions relative to the gene start site and to each other is shown in Figure 5. The results of 5 CpG positions are shown to represent the methylation percentage in the four genes across the samples. There was a significant decrease (p < 0.001) in methylation quantity for each CpG position after decitabine treatment both and for the four genes in all sample but not in the NHOst (normal human osteoblasts). p-values were calculated by comparing the percentage of methylation for each individual CpG position in control cells with the same CpG position in the treated cells using student t-test and they all resulted in p < 0.001

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-0

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>e y-axis indicates cell number in millions and the x-axis indicates time points in days. The results are expressed as cell counts at each corresponding time point. Each data point is the Mean of cell counts from 2 experiments (5 passages apart) each consist of 2 independent cultures and the error bars indicate the standard deviation. The findings indicate a slight increase of U2OS cells' doubling time and a decrease of 18% (p = 0.045) in the viability of treated cells compared to untreated control , Cell death in U2OS cells caused by decitabine treatment at 1 μM concentration (light column) compared to no-treatment (dark column). The results are expressed as percentage of cell death (fraction of cells with positive PI stain). The y-axis indicates the percentage of cells with PI staining (dead cells). Each column is the Mean of 3 experiments with error bars indicating standard deviation. The asterisk indicates significant increase in cell death (p < 0.05) as a result of decitabine treatment

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-6

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>e y-axis indicates cell number in millions and the x-axis indicates time points in days. The results are expressed as cell counts at each corresponding time point. Each data point is the Mean of cell counts from 2 experiments (5 passages apart) each consist of 2 independent cultures and the error bars indicate the standard deviation. The findings indicate a slight increase of U2OS cells' doubling time and a decrease of 18% (p = 0.045) in the viability of treated cells compared to untreated control , Cell death in U2OS cells caused by decitabine treatment at 1 μM concentration (light column) compared to no-treatment (dark column). The results are expressed as percentage of cell death (fraction of cells with positive PI stain). The y-axis indicates the percentage of cells with PI staining (dead cells). Each column is the Mean of 3 experiments with error bars indicating standard deviation. The asterisk indicates significant increase in cell death (p < 0.05) as a result of decitabine treatment

Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-2

<p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>n is the Mean of tumor volumes measured in 12 xenograft tumors. There was a significant decrease (p < 0.05) in tumor volumes as a result of decitabine treatment. , Osteoid assessment . Each column is the Mean of osteoid evaluation of 9 sections. There was significant increase (p < 0.0001) in osteoid formation as a result of decitabine treatment. , Representation images of H&E sections. Control xenograft tumors (left) show solid sheets of poorly differentiated cells with minimal osteoid (image magnification × 100). Decitabine treated tumors shows less dense cell population and increased areas of osteoid seen as light pink and lacy matrix with the nuclei of osteoblasts sitting closer to the produced matrix (image magnification × 100). The arrows in the enlargement show the osteoid matrix surrounding osteoblasts (defined as eosinophilic osteoid-like material). And , Mitotic count as identified in the same sections used to asses for osteoid evaluation. Each column is the Mean mitotic count in 9 sections (a minimum of 1100 nuclei were scanned per section). Decitabine treatment resulted in a significantly lower mitotic count (p < 0.0001). The arrows in the enlargement image in (left) indicate mitotic nuclei. , Results for apoptosis analysis by TUNEL assay. Each column is the Mean count of TUNEL positive nuclei seen in 9 images representing 9 sections (≥1000 nuclei were scanned per section). There was a significant increase (p < 0.05) of apoptotic cells as a result of decitabine treatment. , Representation images from TUNEL assay of control tumors (left) and decitabine treated tumors [50] (image magnification × 200). The arrows in the enlargement image show the TUNEL-positive nuclei (apoptotic nuclei). Error bars indicate standard deviation from the Mean values and p-values are based on comparison between control and decitabine treated tumors using student t-test