[CANCER RESEARCH 55, 2053-2055, May 15, 1995] Advances in Brief No CDKN2 Mutations in Neuroblastomas1 Christian P. Beltinger, Peter S. White, Erik P. Sulman, John M. Maris, and Garrett M. Brodeur2 Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-9786 Abstract Mutations of CDKN2 have been found recently in melanoma and many other tumor types. Neuroblastoma shares with melanoma a neuroectoder- mal origin and a high incidence of deletions of the short arm of chromo some 1. Therefore, we analyzed 18 primary neuroblastomas and 9 tumor- derived cell lines for mutations in CDKN2. We used PCR-single-strand conformation polymorphism to examine exons 1 and 2 of the CDKN2 gene for mutations, but none were detected. Furthermore, no homozygous deletions were detected and there was no loss of heterozygosity at the closely linked II \\ locus. We conclude that disruption of the CDKN2 gene is not required for malignant transformation of human neuroblas tomas. Introduction The CDKN2 (MTSÃŒ) gene encodes piò, a protein which inhibits the CDK43/cyclin D complex. Inhibition by pl6 decreases the phospho- rylation state of the retinoblastoma gene product (pRb), and this cell cycle checkpoint limits the G,-S transition and ultimately inhibits cell growth (1-3). The CDKN2 gene maps to 9p21, a site of frequent deletion in melanoma and other tumor types. Interestingly, mutations and ho mozygous deletions of CDKN2 were found initially in cell lines from many different tumor types (4, 5). However, the overall incidence of mutations or homozygous deletions is generally less in primary tu mors compared to cell lines (6). In primary tumors, homozygous deletions and intragenic mutations are frequently seen in pancreatic adenocarcinoma, glioblastoma, anaplastic astrocytoma (but not low- grade astrocytoma), esophageal squamous cell carcinoma, non-small cell lung cancer, bladder carcinoma, acute lymphocytic leukemia, and non-Hodgkin's lymphoma (4, 7-12). Intragenic alterations of CDKN2 have been detected rarely, if at all, in primary breast cancer (12), primary renal cancer (11), and primary sporadic melanoma (13), although cell lines derived from these tumors often contain homozy gous deletions (5). Although cell lines of osteosarcoma, ovarian tumor, and mesothelioma frequently show homozygous deletions of CDKN2, no data concerning primary tumors have been published yet for these cancers (4). Homozygous deletions of CDKN2 occur in 60% of melanoma cell lines (4, 5), and disease-specific germ line mutations are found in many 9p21-linked melanoma families (14, 15). Melanomas frequently contain deletions of the short arm of chromosome 1 (16), a region that also shows genetic linkage in several familial melanomas (15). Inter estingly, deletion of the short arm of chromosome 1 is the most common genetic abnormality in human neuroblastomas (17, 18). Furthermore, melanocytes and neuroblasts are both derived from the Received 3/9/95; accepted 4/5/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grant CA 39771 (G. M. B.). 2 To whom requests for reprints should be addressed, at Division of Oncology, Room 9(KX). Children's Hospital of Philadelphia, 324 South 34th Street. Philadelphia, PA 19104-9786. 3 The abbreviations used are: CDK4, cyclin-dependent kinasc 4; LOH, loss of het erozygosity; SSCP, single-strand conformation polymorphism. neural crest. Because of these similarities, as well as the frequent involvement of CDKN2 mutations in many other cancers, we exam ined the CDKN2 gene in a series of neuroblastoma cell lines and primary tumors. However, no homozygous deletions or mutations were detected in the coding region, and no LOH was found at the adjacent IFNA locus, suggesting that CDK.N2 is not involved in the pathogenesis of neuroblastomas. Materials and Methods Tumors and Cell Lines. Nine neuroblastoma cell lines were analyzed, six of which had amplification of the MYCN oncogene as well as Ip deletions or rearrangements (NGP, NLF, NAB. SMS-KAN, SMS-KCN, and LA-N-5). Three cell lines had neither MYCN amplification nor abnormalities of Ip (NBL-S, SK-NSH, and LHN). Eighteen primary tumor samples were selected to represent the distribution of stage and MYCN copy number typically found in neuroblastoma. One of six selected stage I-II tumors and 4 of 10 selected stage 1II-IV tumors had MYCN amplification. Two tumors were stage IV-S and not amplified. For LOH studies, constitutional DNA corresponding to the neuroblastoma samples was extracted and processed as described previously (17). DNA samples from unrelated probands of 31 Centre d'étudedu poly morphisme humain pedigrees were provided by H. Donis-Keller. Constitu tional DNA samples of eight patients with germ line CDKN2 substitutions (one substitution in exon 1 and seven substitutions in exon 2) were a gift from N. Dracopoli (15). PCR-SSCP and DNA Sequencing. PCR of CDKN2 was performed as described previously (15). One primer pair covered exon 1. Three primer pairs spanning overlapping regions designated as A, B, and C covered exon 2. SSCP was performed as described (15), with the following modifications. Samples were electrophoresed at 25°Con a 4.5% polyacrylamide gel in IX TBE (90 mm Tris-borate, 2 IHMEDTA) at 25 W for 4 h. Duplicates were run at 25°Con a 0.5X MDE gel (AT Biochem, Malvern. PA) with 5% glycerol in 0.6X TBE at 6 W for 12 h. Variant bands were cut from the gel and used directly as a template for a second PCR reaction under the same conditions as described above. The PCR product was then sequenced in both directions with the same primers using methods described previously (19). LOH Analysis. A microsatellite repeat within IFNA, which also maps to 9p21 and is adjacent to CDKN2, was PCR amplified from 17 paired normal and tumor DNA samples using primers derived from the Genome Data Base. One primer was end labeled with y-'2P using T4 polynucleotide kinase. PCR was performed in 20-/il volumes containing 2 ¡u10X PCR buffer II (Perkin Elmer/Cetus, Branchburg, NJ), 0.4 mM each primer, 0.1 mM each dNTP, 0.2 units AmpliTaq DNA polymerase (Perkin Elmer/Cetus), 1.5 mM MgCK, and 20 ng DNA. Reactions were amplified for one cycle at 95°C(3 min); for 16 cycles at 95°C(45 s) with the annealing/extension temperature starting at 70°C and decreasing by 0.7°Ceach cycle (1 min); 25 cycles at 95°C(45 s), 55°C(30 s), and 72°C(1 min); and for one cycle at 72°C(5 min). LOH analysis was performed as described (16). Results To first confirm the sensitivity of our PCR-SSCP method, we examined exons 1 and 2 of CDKN2 in the constitutional DNA of eight melanoma patients with known germ line CDKN2 substitutions. In each case, a variant band was clearly detectable by PCR-SSCP. Fig. 1 shows the three germ line samples with substitutions in exon 2 (region C) used as controls (Lanes 6-9). While not all mutations were seen on the polyacrylamide gels without glycerol, glycerol-supple- mented MDE gels detected all substitutions. 2053 on March 7, 2021. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from