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J. Dopierala, S. E. Coupland, B. Damato, J. Sibbring; Heterogeneity of Genetic Abnormalities in Uveal Melanoma Assessed by Multiplex Ligation-Dependent Probe Amplification (MLPA). Invest. Ophthalmol. Vis. Sci. 2009;50(13):5776.
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© ARVO (1962-2015); The Authors (2016-present)
To study the distribution of genetic abnormalities in primary uveal melanoma by Multiplex Ligation-Dependent Probe Amplification (MLPA) in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues and to compare the results with MLPA data obtained from corresponding fresh material tested in routine diagnostic practice.
The MLPA P027 kit (MRC-Holland) tests 31 loci located on chromosomes 1, 3, 6 and 8. Sections of the whole tumor and 2-6 distinct, microdissected areas were taken from 13 FFPE uveal melanomas. DNA was extracted & tested by MLPA in triplicate. 5 normal FFPE choroid tissues were controls. MLPA data were analysed using an Excel spreadsheet (NGRL, Manchester). The MLPA data obtained from distinct areas from each FFPE block were compared to a whole tumor section, to each other, & to MLPA data obtained from corresponding fresh material. A uveal melanoma was considered "heterogeneous" for a particular locus if the absolute value of the difference of any combination of normalized, equalized probe peak heights was ≥0.2 between different areas of a tumor.
MLPA detected genetic abnormalities in all uveal melanomas. Monosomy 3 was detected in 8/13 tumors in at least 1 microdissected area. Gains of MYC & DDEF1 were present in all samples. In 4/13 uveal melanomas, the genetic abnormalities detected in the microdissected areas were the same as those seen in whole tumour sections. In only 2 of these 4 FFPE tumours concordant MLPA results were seen when compared to data obtained from fresh tissue. The remaining 9 FFPE uveal melanomas showed differences in between 1-30 loci when comparing MLPA results from whole tumour sections and microdissected areas. Differential distribution of gene loss or gain located on chromosome 1 were the most common changes, and present in 8 tumors. Interestingly, one tumor displayed monosomy 3 in 3 microdissected areas and in others, polysomy 3. The MLPA on the corresponding fresh material showed the latter alterations only. Another uveal melanoma showed polysomy 3 in 3 microdissected areas and disomy 3 in one region; the MLPA performed on the corresponding fresh material showed 2 copies of chromosome 3 only.
MLPA analysis of uveal melanoma demonstrated a high frequency of heterogeneity of aberrations of chromosomes 1, 3, 6 and 8. There were geographic differences in at least one locus in 9/13 tumours using microdissection. Our results suggest that when examining chromosomal changes in uveal melanoma, one random sample may not be representative of the whole tumor.
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