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J.-C. A. Marshall, S. Di Cesare, S. Maloney, B. F. Fernandes, E. Antecka, M. N. Burnier, Jr.; Comparative Genomic Hybridization Array of Paraffin Embedded Tissue From an Animal Model of Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4756.
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© ARVO (1962-2015); The Authors (2016-present)
Several different genetic abnormalities have been characterized in uveal melanoma patients, with monosomy of chromosome 3 being the best known. A new generation of genetic tools, including array comparative genomic hybridization (aCGH), now allow for high-resolution (>35 base pair) detection of chromosomal amplifications and deletions. Current treatment for uveal melanoma has reduced the percentage of patients who undergo enucleation, and therefore reduced the fresh tissue available for analysis. The aim of this study was to establish a method for using archival paraffin embedded material for aCGH analysis.
Formalin-fixed, paraffin-embedded intraocular tumour specimens from a previously published immunosuppressed rabbit model using human uveal melanoma cells were utilized for this study. 10 micron thick serial sections were made for each of six different eyes with large intraocular tumors, and five different DNA extraction techniques were used for each section. DNA yield was measured by UV spectrophotometer and degradation checked using agarose gel electrophoresis. Those samples with a minimum of 200ng of non-degraded DNA were directly labelled. Those samples with less than 200ng of DNA were amplified using two rounds of ligation mediated PCR prior to labelling.
The DNA samples that were the least degraded after purification from paraffin and had the highest quantity of DNA were those isolated using a Gentra PureGene tissue kit. Direct labelling of these samples gave the best signal to noise ratio along with highest signal intensity and reproducibility. Several different regions of amplification and deletion were seen throughout the chromosome for all samples. A large area of amplification of the q arm of chromosome 7 was seen in all samples. The area of amplification included the genes Wnt2, and C-Met. A small area of deletion in chromosome 3, near q12.2 was also observed.
To the best of our knowledge this is the first time that genomic DNA has been extracted from paraffin embedded tissue of uveal melanoma and used for aCGH analysis. We identified several genomic amplifications and deletions including an area of amplification of Wnt2, which is involved in beta-catenin regulation and C-Met which plays a role in tumour cell homing to the liver in patients. The majority of archival tissue from patients with follow up data that is available is paraffin embedded, therefore this method has broad implications to evaluate ocular and systemic disease in uveal melanoma patients.
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