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Low Frequency of p16/CDKN2A Methylation in Sporadic Melanoma: Comparative Approaches for Methylation Analysis of Primary Tumors¹

Department of Biochemistry and Molecular Biology [M. L. G., C. M. B., E. H. Y., J. M. G., J. F. F., G. J. W., P. A. J., J. W. F.], Institute for Genetic Medicine [E. H. Y., J. M. G., J. F. F., G. J. W., J. W. F.], University of Southern California, School of Medicine, Los Angeles, California 90033, and Queensland Institute of Medical Research, Brisbane 4029, Australia [G. J. W., N. K. H.]

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

¹ This work was supported in part by Grant R01CA66021 (to J. W. F.) and USPHS Grant R35CA49758 (to P. A. J.) from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, Institute for Genetic Medicine, University of Southern California, 2250 Alcazar Street, IGM240, Los Angeles, CA 90033.

Received 8/ 1/97. Accepted 10/ 3/97.

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