Vol. 6, 3511–3515, September 2000 Clinical Cancer Research 3511 CDKN2A Mutation and Deletion Status in Thin and Thick Primary Melanoma1 Adrian R. Cachia,2,3 James O. Indsto,2 involved in the progression rather than initiation of sporadic Kathryn M. McLaren, Graham J. Mann, and malignant melanoma. Mark J. Arends INTRODUCTION Department of Tissue and Cell Pathology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, The incidence of malignant melanoma is rising in devel- New South Wales 2145, Australia [A. R. C.]; Westmead Institute for oped countries. Investigations of potential tumor suppressor Cancer Research, University of Sydney at Westmead Hospital, genes involved in melanoma formation have shown either hem- Westmead, New South Wales 2145, Australia [J. O. I., G. J. M.]; izygosity or LOH4 for DNA markers within chromosome 9p21 Department of Pathology, University Medical School, Teviot Place, in 34–86% of melanoma cell lines and tumors examined by Edinburgh EH8 9AG, Scotland [K. M. M.]; and Molecular several authors (1–7). Histopathology, University of Cambridge Department of Pathology, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom One gene within this deleted region is CDKN2A, which can [M. J. A.] produce two independent transcripts through the alternate splic- ing of a separate exon 1. One transcript encodes the cdk inhib- itor protein, p16INK4A (p16). The p16 protein binds to and ABSTRACT blocks the catalytic activity of two cyclin D-bound, cdks, cdk4 Human melanoma cell lines and tumor tissue from and cdk6. cdk4 is able to phosphorylate the retinoblastoma familial and sporadic melanomas have frequent, nonrandom (pRb) protein, leading to the release of associated proteins that chromosomal breaks and deletions on chromosome 9p21, a activate genes necessary for progression from the G1 to the region that includes the tumor suppressor gene CDKN2A/ S-phase of the cell cycle. It has been directly demonstrated that p16INK4A. Germ-line mutations within this gene have been melanoma-associated mutant p16 proteins are defective in their observed in some familial melanoma kindreds, but somatic ability to inhibit the catalytic activity of the cyclin D1/cdk4 mutation in sporadic primary melanoma is infrequent. Thir- complex (8–12). Loss of functional p16 protein may permit ty-nine archival, paraffin-embedded, sporadic, primary excessive cdk4 activity and therefore represents loss of a regu- cutaneous malignant melanomas (20 >3-mm-thick and 19 latory constraint on progression through the cell cycle. In addi- <0.75-mm-thick cases) were examined for mutations of the tion, experiments have shown that wild-type p16 can prevent CDKN2A gene using single-strand conformational polymor- cellular transformation by H-ras and myc oncogenes (13). Thus, phism analysis and direct sequencing. No mutations were by these mechanisms, CDKN2A can function as a tumor sup- pressor gene (14–18). detected. Loss of heterozygosity for the 9p21 microsatellite ARF marker D9S942 was detected in 6 of 17 informative thick The mRNA for p14 is generated by transcriptional splicing of exons 2 and 3 shared by CDKN2A with an alternative These .(0.006 ؍ lesions (35%) but 0 of 18 thin lesions (P exon 1 (E1␤; Refs. 19–21). The protein p14ARF is translated in results support other studies indicating that intragenic mu- a different reading frame from p16 and acts on a completely tation is an infrequent mechanism of CDKN2A inactivation different pathway, the regulation of the p53 tumor suppressor. in primary melanoma. The finding of loss of heterozygosity Binding of p53 to hdm2 results in its translocation to the for the 9p21 microsatellite D9S942 in thick but not thin cytoplasm and degradation; p14ARF binds to hdm2, sequesters it primary melanoma suggests that deletion or inactivation of in the nucleolus, and thereby enables p53 levels to stabilize and CDKN2A or other tumor suppressor gene(s) at this locus is rise in appropriate conditions (22). We have performed mutational analysis of the CDKN2A gene by SSCP analysis on 39 cases of sporadic primary cuta- neous malignant melanoma. Of the 11 studies to date reporting Received 2/25/00; revised 6/15/00; accepted 6/21/00. somatic CDKN2A mutations in melanoma, only five involved The costs of publication of this article were defrayed in part by the primary sporadic melanomas and in all of these the lesions payment of page charges. This article must therefore be hereby marked examined were either Ͼ0.75 mm in Breslow thickness (23–25) advertisement in accordance with 18 U.S.C. Section 1734 solely to or of unspecified thickness (26, 27). Breslow thickness is the indicate this fact. 1 A. R. C. was supported by a grant from the United Kingdom Cancer most powerful histopathological predictor of melanoma pro- Research Council. M. J. A. is supported by grants from the Scottish gression currently known (28). None of the studies in which Hospitals Endowment Research Trust, Association for International CDKN2A mutations were detected in primary melanoma exam- Cancer Research, and the Cancer Research Campaign. J. O. was sup- ined tumors Ͻ0.75 mm thick (23, 24). Most have been on ported by the New South Wales Cancer Council and the Melanoma Foundation of the University of Sydney. 2 The first two authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Tissue and Cell Pathology, ICPMR, Westmead Hospital, Westmead 4 The abbreviations used are: LOH, loss of heterozygosity; cdk, cyclin- NSW 2145, Australia. dependent kinase; SSCP, single-strand conformational polymorphism. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. 3512 CDKN2A Mutation and 9p21 Deletion in Melanoma Table 1 Characteristics of primary melanomas analyzed Breslow thickness Clark level Mean mitotic Tumor-infiltrating Age Male Nodular mean and count lymphocytes, Melanomas n (yr) (%) (%) (range) (mm) II III IV V (/mm2) positive (%) Thin (Ͻ0.75 mm) 19 47.2 35 0 0.7 (0.50–0.74) 6 13 0.1 75 Thick (Ͼ3 mm) 20 61.6 60 55 5.5 (3.0–11.0) 15 5 7.3 75 metastatic tumors (3, 17, 29–32). To contribute information on the relative role of CDKN2A at different stages of primary melanoma tumorigenesis, we stratified our cases into two groups according to Breslow thickness and Clark level. LOH at the CDKN2A locus was assessed to determine whether there were different rates of deletion in thin versus thick melanomas and whether these rates were in line with those reported previ- ously for sporadic primary melanoma. MATERIALS AND METHODS Melanoma Cases. Twenty sporadic malignant melano- mas of Breslow thickness 3 mm/Clark’s level IV or thicker and 19 sporadic melanomas of Breslow thickness 0.75 mm/Clark’s level III or thinner were retrieved from the Scottish Melanoma Group archives over a 3-year period from 1993 to 1995. The main clinical and histopathological features of the two groups are summarized in Table 1. All cases had been routinely pro- cessed and paraffin embedded. Two 10-␮m sections of tumor and normal skin for control were cut from the paraffin blocks in Fig. 1 Melanoma microdissection. A, H&E-stained section of a thick each case. Using a fresh scalpel, tumor tissue in each 10-␮m melanoma. B, H&E-stained, coverslipped tissue remaining after tumor has been microdissected away from a paraffin section contiguous to the section was carefully dissected from normal tissue with the aid section in the left panel. Contamination with normal stromal cells has of a dissecting microscope. Comparison to directly adjacent been minimized by retrieving only tumor tissue from within the edge of H&E-stained sections was used to minimize cross contamina- the neoplasm, leaving the host-tumor interface behind. tion of tumor with normal tissue, which may mask mutation detection (example dissection in Fig. 1). It is estimated after light microscopic examination of the poststained, dissected slides that contamination of dissected tumor tissue by normal 1 min, annealing for 1 min, and extension at 72°C for 2 min, stromal cells was Ͻ25% for thin lesions and Ͻ10% for thick with further extension for 10 min at the termination of the lesions. reaction. Primer sequences were manually and computer- DNA Preparation and PCR. The sections were depar- matched against the CDKN2A gene sequence published by affinized by sequential exposure to 600 ␮l each of xylene/100% Okamoto et al. (33), which includes corrections of sequence ethanol/100% ethanol in 1.5-ml Eppendorf tubes with 10 min published previously by Kamb et al. (2) and Serrano et al. (14). microcentrifugation at 13,000 rpm between each stage. The Primers used were: CDKN2A exon 1 sense, 5Ј-GGGAGCAG- tissue was then incubated overnight at 55°C in 400 ␮l of lysis CATGGAGCCG- 3Ј (13); CDKN2A exon 1 antisense, 5Ј- buffer [10 mM Tris (pH 8.3), 50 mM KCl, 0.45% Tween 20, and AGTCGCCCGCCATCCCCT-3Ј (13), annealing temperature ϭ Ј 2.5 mM MgCl2] with 0.5 mg of proteinase K (Boehringer Mann- (Tann 63°C); CDKN2A exon 2a sense, 5 -CCTGGCTCT- heim, Basel, Switzerland), followed by boiling for 30 min to GACCATTCTGT- 3Ј (novel, intronic); CDKN2A exon 2a anti- ␮ Ј Ј ϭ destroy proteinase K activity. A 10–30- l DNA aliquot was sense, 5 -GGGCAGCGTCGTGCACGGGT-3 (novel; Tann then subjected to PCR in 10 ␮l of PCR buffer (Life Technolo- 54°C); CDKN2A exon 2b sense, 5Ј-AACTGCGCCGAC- gies, Inc., Gaithersburg, MD), 25 pmol of each primer, 50–75 CCCGCCACTCTCA-3Ј (novel); CDKN2A exon 2b antisense, Ј Ј ϭ nmol of MgCl2 and 3 units of Taq DNA polymerase to a final 5 -CCAGCTCCTCAGCCAGGTCCACG-3 (novel; Tann volume of 100 ␮l. Because no disease-related mutations have 62°C); CDKN2A exon 2c sense, 5Ј-CGATGCCTGGGGC- been recorded in the 11-bp coding region of exon 3, this very CGTCTGC- 3Ј (13); CDKN2A exon 2c antisense, 5Ј-GTA- Ј ϭ short sequence was not examined.