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Connective Tissue Oncology Society |
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| 2001 CTOS Annual Meeting Posters— Biology GENE EXPRESSION
PROFILES OF LOW GRADE AND HIGH GRADE MALIGNANT FIBRO HISTIOCYTOMAS
USING MICROARRAY ANALYSIS OBJECTIVE: Malignant Fibrous Histiocytomas (MFH) are group of malignant mesenchymal tumors that present a dilemma for clinical management. Current staging systems, based on morphological tumor characteristics cannot accurately classify or predict an individual patient’s risk for eventual metastases. We hypothesize that characterization of gene expression patterns of MFH will improve classification of these tumors. To identify clinically meaningful patterns of gene expression in MFH we are taking advantage of a soft tissue sarcoma tumor bank and a high-throughput microarray technology which is capable of profiling gene expression patterns of tens of thousand genes in a single experiment. METHODS: Tumor specimens were obtained from 37 patients with MFH who did not receive preoperative chemotherapy or radiotherapy. Total RNA was extracted with the RNeasy purificaton kit (Qiagen). 5ug of tumor and reference RNA were labeled with Cy3-dCTP and Cy5-dCTP fluorescent tags respectively, using an indirect labeling method and hybridized simultaneously to 19200 cDNA microarray chips. Reproducibility experiments were performed for each tumor/reference pair, in which the tumor and reference sample were re-assessed using the reciprocal fluorescent tag. The standard reference sample (i.e. control) consisted of a pool of RNA from 11 different tumor cell lines. Following hybridization, arrays were scanned using an Axon scanner and spots were quantitated with GenePix 3.0 analysis software. RESULTS: Before using the limited RNA from these specimens, we performed pilot studies to evaluate the feasibility of the technology on a larger scale using cDNA microarrays containing 1700 and 19200 sequence verified human cDNAs. We performed 10 self test experiments in which each pool of control RNA was split, labeled by Cy3 and Cy5 and simultaneously hybridized to the same 1700 cDNA array. The analysis of scatter plots for these experiments showed the consistency of the labeling and hybridization procedures, as evidenced by high correlation coefficients (R2 = 0.92-0.97). We also analyzed 5 MFH cases using 1700 cDNA chips. To allow for comparison of results between tumors each tumor was analyzed using the same control sample. Background subtracted signal intensities between the two fluorescent images were normalized by applying the median of Cy3/Cy5 log ratio as a normalization factor overall and by subarray for each microarray experiment. Comparison of the data across 10 experiments for 5 tumors showed that 12 genes had consistently high ratios and 17 genes had consistently low ratios . We further investigated 7 low grade and 8 high grade MFH tumors by using arrays with 19200 genes. Biostatistical modeling is being used to detect clusters of genes that may distinguish MFH tumors. CONCLUSION: : We have been able to devise a system that may distinguish the variation in gene expression of 19200 genes in low grade and high grade MFH tumors. Statistical approaches will enable us to detect sets of genes that are differentially expressed in high versus low grade tumors. Further evaluation will allow us to determine their potential use in differential diagnosis and early detection of MFH.
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