[CANCER RESEARCH 63, 8507–8510, December 1, 2003] Fine Mapping of Chromosome 3 in Uveal Melanoma: Identification of a Minimal Region of Deletion on Chromosomal Arm 3p25.1-p25.2 Paola Parrella,1 Vito M. Fazio,1,2 Antonietta P. Gallo,1 David Sidransky,3 and Shannath L. Merbs4 1Istituto di Ricovero e Cura a Carattere Scientifico H. “Casa Sollievo della Sofferenza” Laboratory of Gene Therapy and Oncology, San Giovanni Rotondo (FG), Italy; 2Interdisciplinary Center for Biomedical Research, Laboratory for Molecular Medicine and Biotechnology, Universita` Campus BioMedico, Rome, Italy; and 3Otolaryngology Head and Neck Surgery and 4Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland ABSTRACT rearranged in tumors and are therefore believed to contain TSGs. Two of these regions are located on the centromeric portion of the short To identify minimal common areas of allelic loss on chromosome 3, we arm (12). The 3p12-13 region contains the gene ROBO1/DUTT1 and have mapped both arms of the chromosome in 21 uveal melanomas that the 3p14.2 region spans the FHIT gene. Three other hot spots for did not show monosomy 3 in our previous allelotype study. DNA was isolated from microdissected paraffin sections and amplified by PCR. In rearrangements were reported on 3p21. The first is located on 3p21.1- an initial screening, 14 microsatellite markers on chromosomal arm 3p p21.2 and includes the gene BAP1 that binds the RING finger domain and 13 on chromosomal arm 3q were used. Loss of heterozygosity for at of the BRCA1 gene. The second is the lung cancer TSG region least one marker was found in 9 of 21 tumors (43%) on 3p and 8 of 21 (LUCA) that includes the RASSF1 gene frequently hypermethylated tumors (38%) on 3q. The initial analysis defined two common regions of in cancer. The third is located on 3p21.3 and contains the DLC1 gene allelic loss on 3p, a 7.3-Mb region between markers D3S1263 and D3S3510 found deleted in a number of small cell lung cancer cell lines and spanning 3p25.3-24.3 and a larger region between markers D3S1578 and primary tumors. The telomeric region 3p25-26, containing the VHL D3S1284. The two common regions of allelic loss were further mapped gene, has also been reported to be frequently rearranged (12). with an additional 14 microsatellite markers. A 1.4-Mb minimal region of Although monosomy or isodisomy 3 are very common in uveal allelic loss was identified between microsatellite markers D3S3610 and melanoma, only a few partial deletions of this chromosome have been D3S1554 on 3p25.1-3p25.2. A total of 10 tumors had allelic loss in this region; 2 of these tumors had corresponding putative homozygous dele- described (13, 14). To gain more insight into the patterns of chromo- tions. These homozygous deletions may further narrow the region of some 3 deletion and possible genes involved in the carcinogenesis of interest to 0.1 Mb. This 1.4-Mb minimum region of deletion includes uveal melanoma, we analyzed 21 tumors from our allelotype cohort several genes that might be involved in the carcinogenesis of uveal mela- that did not show monosomy 3, with a total of 41 microsatellite noma as well as other important tumor types. markers (11). We identified a 1.4-Mb shared MRD on chromosome 3p25.1-p25.2 between markers D3S3610 and D3S1554 that contains INTRODUCTION 9 National Center for Biotechnology Information reference sequences and a number of predicted genes. Carcinogenesis occurs as a stepwise accumulation of genetic alter- ations in genes that control cell growth, cell death, and DNA repair (1). One of the critical events that lead to carcinogenesis is the MATERIALS AND METHODS inactivation of one or more TSGs.5 Allelic loss, manifested as LOH of microsatellite markers flanking the TSG, is recognized as hallmark of Specimens and DNA Extraction. We evaluated 21 cases of posterior sporadic tumors, with inactivation of the second allele by gene dele- uveal melanoma treated by enucleation from 1993 to 1998 and archived as tion, point mutations or promoter hypermethylation (1). paraffin blocks by the Eye Pathology Laboratory of the Wilmer Ophthalmo- logical Institute. DNA was extracted from paraffin-embedded tissue as de- Posterior uveal melanoma is a relatively rare tumor, but it is scribed previously (15). Briefly, unstained paraffin sections of each block were associated with a high visual morbidity and can pose a serous treat to manually dissected into tumor and normal tissue samples under a microscope life (2). About 30% of patients will develop distant metastases within to ensure that tumor samples contained at least 80% cancer cells (15). The 10 years of successful management of the local tumor (3). The most normal ocular tissue was used as the source of normal control DNA for each common genetic abnormality in uveal melanoma is the loss of one patient. Normal and tumor tissue was digested with SDS/proteinase K for 24 h copy of chromosome 3. Cytogenetic and comparative genomic hy- at 48°C, and samples were extracted with phenol/chloroform. Pigments, which bridization studies have demonstrated monosomy 3 in ϳ50% of cases coisolated with the DNA after the extraction, tended to inhibit the PCR. (4–8). Complete or partial deletions of chromosome 3 have been Therefore, DNA samples were additionally purified using Geneclean II ac- detected by microsatellite marker analysis (9, 10). In our allelotype of cording to the manufacturer’s instructions (Bio101; Vista, CA). 50 uveal melanomas, ϳ60% of tumors had LOH of all markers on Fine Deletion Mapping. Twenty-one uveal melanomas from the original allelotype cohort (15 tumors with retention of all tested markers on 3p, 4 chromosome 3 consistent with monosomy or isodisomy 3 (11). tumors with LOH of 3p or 3q, and 2 tumors noninformative for markers on 3p Genetic abnormalities of chromosome 3 and, in particular 3p, are or 3q) were tested (11). DNA from normal and tumor tissues were analyzed for also critical to the development of in several other tumor types, LOH by amplification of STS microsatellite markers using PCR and the including small cell lung carcinoma, breast cancer, ovarian cancer, conditions described below. adenocarcinoma of the cervix, renal cell carcinoma, and head and For an initial screening, 27 primer pairs (Research Genetics. Huntsville, neck tumors (12). Several regions on chromosome 3p are frequently AL) were analyzed: 14 on chromosomal arm 3p (D3S1270, D3S1597, D3S1263, D3S1252, D3S1286, D3S3510, D3S1293, D3S1283, D3S1007, Received 5/14/03; revised 8/10/03; accepted 9/16/03. D3S1358, D3S1578, D3S1234, D3S1210, D3S1284) and 13 on chromosomal Grant support: National Eye Institute K08-EY00378 (to S. L. M.), the Research to arm 3q (D3S2318, D3S1251, D3S1603, D3S1546, D3S1310, D3S1292, Prevent Blindness, Inc. (to S. L. M.), the Niuta and Roy Titus Faculty Development D3S1238, D3S1764, D3S1268, D3S3715, D3S1580, D3S2748, D3S1311). The Award (S. L. M.), and Progetto Ministero della Salute Istituto di Ricovero e Cura a microsatellite markers were chosen to span ϳ5-Mb intervals on chromosome Carattere Scientifico Grant RCO202TG08 (to P. P.). ϳ The costs of publication of this article were defrayed in part by the payment of page 3p and 8-Mb intervals on chromosome 3q. The relative positions of the charges. This article must therefore be hereby marked advertisement in accordance with markers were determined using the Human Genome Working Draft Sequence 18 U.S.C. Section 1734 solely to indicate this fact. Browser November 2002 (Ref. 16; Fig. 1).6 Markers D3S1597, D3S1284, Requests for reprints: Shannath L. Merbs. 5 The abbreviations used are: TSG, tumor suppressor gene; LOH, loss of heterozy- gosity; MRD, minimal region of deletion; STS, sequence-tagged site. 6 Internet address: http://www.ucsc.edu. 8507 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. FINE MAPPING OF CHROMOSOME 3 IN UVEAL MELANOMA Fig. 1. Chromosome 3 mapping. Twenty-one uveal melanomas were tested with 27 microsatellite markers (14 for chromosomal arm 3p and 13 for chromosomal arm 3q), , retention of both alleles; f, loss of heterozygosity; NI, noninformative; ND, not determined; the first row indicates tumor sam- ple number; the first column indicates the STS microsatellite marker. Two common regions of al- lelic loss (CRL) were identified on 3p, D3S1263- D3S3510 (CRL1) and D3S1578-D3S1284 (CRL2). Positions of the markers on the physical map were determined using the Human Genome Working Draft Sequence Browser (16). D3S1292 and D3S1268 had been previously used in the allelotype (11). The used to map the 3p14-12 region (Fig. 2B). With the additional mark- common regions of loss on 3p were further mapped with additional 14 ers, 4 more tumors (UM22, UM13, UM14, and UM26) were found to microsatellite markers (D3S1259, D3S3701, D3S656, D3S3610, D3S100, have LOH in the common region of loss on 3p25.3-24.3, and 2 more D3S2385, D3S1585, D3S1554, D3S1479, D3S1360, D3S1076, D3S1295, tumors (UM14 and UM57) were found to have LOH in the common D3S3635, D3S3507). In total, each tumor was tested with 41 markers. The region of loss on 3p14-11. In total, the 3p chromosomal arm was MRD identified on chromosome 3p25.1-p25.2 was defined using only tumors mapped with 28 STS microsatellite markers, and 15 of the 21 uveal with at least two consecutive losses. Using T4 kinase (Life Technologies, Inc., Gaithersburg, MD), 500 ng of one melanomas (71%) harbored LOH of at least one marker. primer from each pair was end labeled with [32P]ATP (20 mCi; Amersham, A 1.4-Mb shared MRD between microsatellite markers D3S3610 Piscataway, NJ) in a total volume of 20 ␮l. PCR reactions were carried out in and D3S1554 on 3p25.1-p25.2 was identified (Figs.