Advances in Brief Overexpression of the Cyclin-dependent Kinase Inhibitor p16 Is Associated with Tumor Recurrence in Human Prostate Cancer 1 Cheryl T. Lee, Paola Capodieci, Iman Osman, Melissa Fazzari, Joseph Ferrara, Howard I. Scher, and Carlos Cordon-Cardo 2 Urology Service, Department of Surgery [C. T. L.]; Department of Pathology [P. C., C. C-C.]; Genitourinary Service, Department of Medicine [I. O., H. I. S.]; and Department of Biostatistics and Epidemiology [M. F., J. F.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Abstract The INK4A gene maps to the 9p21 region and was initially described [M. Serrano et al., Nature (Lond.), 366: 704 –707, 1993; A. Kamb et al., Science (Washington DC), 264: 436 – 440, 1994] as encoding a 148-amino-acid protein termed p16. The p16 protein associates exclusively with Cdk4 and Cdk6, inhibiting their complexation with D-type cyclins and the consequent phosphorylation of pRb. This contributes to cell cycle arrest. The purpose of the present study was to evaluate patterns of p16 expression in a well- characterized cohort of prostatic adenocarcinomas while exploring potential associations between alterations of p16 and clinicopathological variables. Normal and malignant tissues from 88 patients with prostate carcinoma were ex- amined. In situ hybridization and immunohistochemistry assays were used to determine the status of the INK4A exon 1 transcripts and levels of p16 protein, respectively. Asso- ciations between altered patterns of expression and clinico- pathological variables, including pretreatment prostate- specific antigen (PSA) level, Gleason grade, pathological stage, and hormonal status, were evaluated using the Man- tel-Haenszel  2 test. Biochemical (PSA) relapse after surgery was evaluated using the Kaplan-Meier method and the log- rank test. Levels of p16 expression and INK4A exon 1 transcripts in normal prostate and benign hyperplastic tis- sues were undetectable. However, p16 nuclear overexpres- sion was observed in 38 (43%) prostate carcinomas, whereas the remaining 50 (57%) cases showed undetectable p16 levels. Overexpression of p16 protein was found to correlate with increased INK4A exon 1 transcripts. Moreover, p16 overexpression was associated with a higher pretreatment PSA level (P  0.018), the use of neoadjuvant androgen ablation (P  0.001), and a sooner time to PSA relapse after radical prostatectomy (P  0.002). These data suggest that p16 overexpression is associated with tumor recurrence and a poor clinical course in patients with prostate cancer. Introduction The INK4A gene maps to the short arm of chromosome 9 (9p21) and was initially described as encoding a protein of M r 15,845, termed p16 (1, 2). The p16 protein forms binary com- plexes exclusively with Cdk4 and Cdk6, inhibiting their kinase activity and subsequent pRb phosphorylation during the G 1 phase of the cell cycle (1, 3). Additional complexity results from the presence of a second INK4A product termed p19 ARF (4-6). The p19 ARF protein has recently been shown to interact with mdm2 and to block mdm2-induced p53 degradation and trans- activational silencing (7, 8). The two products, p16 and p19 ARF , share exons 2 and 3 of the INK4A gene, but have distinct promoters and exon 1 units, exon 1 (p16) and exon 1 (p19 ARF ). The INK4A gene is mutated in a wide variety of tumor cell lines and certain primary tumors (2, 9 –14). In addition, methylation of the 5' CpG island of the exon 1 promoter region is a frequent mechanism of p16 inactivation in primary tumors (15, 16). In prostate cancer, the role of INK4A has not been well elucidated, though analyses using microsatellite markers in the vicinity of the INK4A gene have revealed loss of heterozygosity in a subset of primary and metastatic prostate tumors (17). Unlike other primary tumors, INK4A inactivation, through either deletions, mutations, or promoter methylation, seems to be an infrequent event in prostate cancer (17–22). The present study uses immunohistochemical and in situ hybridization assays to examine patterns of p16 expression in a well-characterized cohort of prostate cancer patients treated with radical retropubic prostatectomy. Associations between altered p16 phenotypes and clinicopathological variables were also studied to further define their potential implications in prostate cancer. Materials and Methods Patient Characteristics and Tissues. A cohort of pa- tients with prostatic adenocarcinoma undergoing radical prosta- tectomy at the Memorial Sloan-Kettering Cancer Center (MSKCC) from 1990 –1991 was retrospectively evaluated. A total of 88 patients had adequate clinical follow-up and available pathological materials. The median age at the time of surgery was 65 years (range, 46 –74 years). The median follow-up time was 64.5 months (range, 10 –94 months). Formalin-fixed, paraffin-embedded prostate tissues were obtained from the De- Received 12/16/98; revised 1/18/99; accepted 1/25/99. 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. 1 Supported in part by National Cancer Institute Grant CA-DK-47650 (C. C-C.) and the PepsiCo Foundation (H. I. S.). C. L. is a Urological Oncology Fellow recipient of a Fellowship from National Cancer Insti- tute Training Program CA09501. 2 To whom requests for reprints should be addressed, at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave- nue, New York, NY 10021. Phone: (212) 639-7746; Fax: (212) 794- 3186; E-mail: cordon-c@mskcc.org. 977 Vol. 5, 977–983, May 1999 Clinical Cancer Research Research. on January 4, 2022. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from