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The P16 (Cdkn2a/Ink4a) Tumor-Suppressor Gene in Head

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The p16 (CDKN2a/INK4a) Tumor-Suppressor Gene in Head and Neck Squamous Cell Carcinoma: A Promoter Methylation and Protein Expression Study in 100 Cases Lingbao Ai, M.D., Krystal K. Stephenson, Wenhua Ling, M.D., Chunlai Zuo, M.D., Perkins Mukunyadzi, M.D., James Y. Suen, M.D., Ehab Hanna, M.D., Chun-Yang Fan, M.D., Ph.D. Departments of Pathology (LA, KKS, CZ, PM, CYF) and Otolaryngology-Head and Neck Surgery (CYF, JYS, EH), University of Arkansas for Medical Sciences; and School of Public Health (LA, WL), Sun-Yat Sen University, Guangzhou, China apparent loss of p16 protein expression appears to The p16 (CDKN2a/INK4a) gene is an important be an independent prognostic factor, although loss tumor-suppressor gene, involved in the p16/cyclin- of p16 protein may be used to predict overall pa- dependent kinase/retinoblastoma gene pathway of tient survival in early-stage head and neck squa- cell cycle control. The p16 protein is considered to mous cell carcinoma. be a negative regulator of the pathway. The gene encodes an inhibitor of cyclin-dependent kinases 4 KEY WORDS: Gene inactivation, Head and and 6, which regulate the phosphorylation of reti- neck squamous cell carcinoma, p16, Promoter noblastoma gene and the G1 to S phase transition of hypermethylation. the cell cycle. In the present study, p16 gene pro- Mod Pathol 2003;16(9):944–950 moter hypermethylation patterns and p16 protein expression were analyzed in 100 consecutive un- The development of head and neck squamous cell treated cases of primary head and neck squamous carcinoma is believed to be a multistep process, in cell carcinoma by methylation-specific PCR and im- which genetic and epigenetic events accumulate as munohistochemical staining. The p16 promoter hy- a result of chronic exposure to environmental car- permethylation and apparent loss of p16 protein cinogens, resulting in loss of cell cycle control, se- expression were detected in 27% and 74% of head lected cell growth, and finally the development of and neck squamous cell carcinoma, respectively. By cancer. ␹2 test, history of alcohol or tobacco use was signif- On chromosome 9p21, the INK4a locus encodes icantly correlated with the loss of p16 protein ex- both p16 and p14ARF proteins by alternative splicing -and .05, respectively). When pa 005. ؍ pression (P (1, 2), in which exon 1␣ is used by p16 and exon 1␤ tient follow-up data were correlated with various is used by p14ARF. Although the role of p14ARF as a clinical and molecular parameters, tumor size and true tumor suppressor has yet to be further charac- nodal and clinical stage were the strongest prognos- terized, p16 fulfills all the criteria for a bona fide tic predictors for disease-free survival (tumor recur- tumor-suppressor gene, even judged with the rence) and for cause-specific and overall survival in strictest measures (3). The human p16 protein con- patients with head and neck squamous cell carci- tains 156 amino acids and was first discovered in a noma. Neither p16 promoter hypermethylation nor yeast two-hybrid system to detect proteins that in- teract with human cyclin-dependent kinase 4 (4). The tumor suppressor function of p16 is attributed Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. to its ability to inhibit the catalytic activity of the VOL. 16, NO. 9, P. 944, 2003 Printed in the U.S.A. cyclin-dependent kinase 4–6/cyclin D complex Date of acceptance: May 13, 2003. This work was supported by research funds from the Departments of that is required for phosphorylation of retinoblas- Pathology and Otolaryngology, University of Arkansas for Medical Sci- toma protein (5, 6). ences and Biomedical Research Foundation, John L. McClellan Memorial Veteran’s Hospital, Little Rock, Arkansas. The E2F family of transcription factors is essen- Presented at the 92nd Annual Meeting of the United States and Canadian tial for cell cycle progression, and their transcrip- Academy of Pathology, Washington, D.C., March 22–28, 2003. Address reprint requests to: Chun-Yang Fan, M.D., Ph.D., Department of tional activity is inhibited through the specific Pathology (113/LR), University of Arkansas for Medical Sciences and binding to retinoblastoma protein b (7). This reti- Central Arkansas Veterans Healthcare System, 4300 W. 7th Street, Little Rock, AR 72205; fax: 501-257-6430; e-mail: [email protected]. noblastoma protein/E2F complex will release E2F DOI: 10.1097/01.MP.0000085760.74313.DD transcriptional factors upon phosphorylation of ret- 944 inoblastoma protein by cyclin dependent kinase ity, pharynx, and larynx); (3) no prior history of 4–6/cyclin D complex and promote cell cycle pro- head and neck squamous cell carcinoma; and (4) gression past the G1/S checkpoint (3, 7). By binding no prior history of chemotherapy or radiation ther- to cyclin-dependent kinases 4 and 6 and inhibiting apy. The histology of each case was reviewed, and retinoblastoma protein phosphorylation, p16 pre- representative tissue sections containing invasive vents the formation of cyclin-dependent kinases squamous cell carcinoma were selected for immu- 4–6/cyclin D stimulatory complex and promotes nohistochemical staining and extraction of DNA for the formation of retinoblastoma protein/E2F re- promoter methylation analysis. Clinical follow-up pressive complex, with the end result of blockage of was available for all patients until May 2002. Perti- cell cycle progression at the G1/S checkpoint (3). nent patient information was retrieved from the In head and neck squamous cell carcinoma, a Computerized Patient Record System of the De- number of studies have identified three major partment of Veterans Affairs. mechanisms by which p16 gene is inactivated, and these are homozygous deletions, inactivating mu- Immunohistochemical Staining for the p16 tation, and aberrant promoter methylation. In the Protein review by Rocco and Sidransky (3), the p16 inacti- Immunohistochemical staining was performed Յ vation was seen in 68% of primary human tumors as described elsewhere (15), except that the primary and was caused by homozygous deletions in 27%, antibody was incubated at the dilution of 1:50 for by inactivating mutations in 11%, and by promoter 1.5 hours at room temperature, using a monoclonal hypermethylation in 30% of the cases respectively. antibody against the p16 protein (Ab-4, clone 16P04 It has been established that loss of p16 function or JC2; NeoMarkers, Fremont, CA). The p16 immu- occurs early in cancer progression (8, 9), and some nohistochemical staining results were interpreted studies even indicate that p16 loss is the earliest semiquantitatively as follows: negative, Ͻ10% pos- detectable genetic abnormality in head and neck itive cells (Ϫ); mostly negative, 10 to 20% positive squamous cell carcinoma (10, 11). cells (Ϯ); weakly positive, 30 to 40% positive cells Consistent with the results of genetic and epige- (ϩ); moderately positive, 50 to 70% positive cells netic analyses in head and neck squamous cell car- (ϩϩ); and strongly positive, 80 to 100% positive cinoma, the p16 gene expression is frequently lost cells (ϩϩϩ). The above percentages were deter- at the protein levels. The frequency of absent p16 mined by visual estimation of the positively stained protein expression by immunohistochemical stain- area as compared with all areas containing tumor ing method in head and neck squamous cell carci- cells. noma ranges from 55 to 90% in three studies in- Two pathologists (CLZ and CYF) evaluated the volving 332 cases, with an average frequency of 74% slides independently without prior knowledge of (12–14). the methylation-specific PCR results. Any discrep- In the present study, we analyze promoter meth- ancy in the immunohistochemical staining inter- ylation and protein expression patterns of the p16 pretation was resolved by reexamination of the gene in 100 patients with primary head and neck cases by both pathologists. One head and neck squamous cell carcinomas that have not been pre- squamous cell carcinoma case that showed un- viously treated by radiation and chemotherapy. The equivocal positive reaction in both nucleus and epigenetic alteration and protein expression data cytoplasm was used as the positive control and was are then correlated with various clinical parameters included every time when immunohistochemical in an attempt to determine the prognostic signifi- staining was performed. cance of the p16 gene in head and neck squamous cell carcinoma. Sample Collection and DNA Extraction DNA samples were collected using the EX-WAX DNA Extraction Kit (Intergen Co., New York, NY) MATERIALS AND METHODS from five deparaffinized, 5-␮m-thick tissue sections A total of 100 consecutive untreated cases of head from each tissue block. Human placental DNA and neck squamous cell carcinoma were gathered (Sigma) was used as a negative control, and CpGe- from the paraffin block archives in the Department nome universal methylated human DNA (Intergen of Pathology at John L. McClellan Memorial Veter- Co.) served as a positive control, in which all CpG sites ans’ Hospital, spanning a period from 1993 to 1998. within the genomic DNA are chemically methylated. Criteria for tissue selection were as follows: (1) pri- mary surgical resection with curative intent or first- Bisulfite Modification of DNA for Methylation- time biopsy for adjuvant therapy; (2) an established Specific PCR diagnosis of squamous cell carcinoma by histomor- In DNA samples from head and neck squamous phology of upper aerodigestive tract (lip, oral cav- cell carcinoma cases, negative and positive controls P16 (CDKN2a/INK4a) Tumor-Suppressor Gene (L. Ai et al.) 945 were subjected to bisulfite modification before MSP chemo/radiation therapies. All P values were two- using CpGenome DNA modification Kit (Intergen sided. SPSS software (Version 11.0, SPSS Inc., Chi- Co.). cago, IL) was used for all statistical computation. PCR Amplification and Primers RESULTS Amplification of the promoter region of the p16 gene was carried out in a Touchgene Gradient Of 100 patients, 91 underwent curative surgical Thermal Cycler (Techne Inc., Princeton, NJ) in a resection, and 9, chemoradiation therapies as pri- 50-␮L PCR reaction mixture containing 2 ␮Lof mary treatment for their tumors.
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  • DNA Methylation and the Mechanisms of CDKN2A Inactivation in Transitional Cell Carcinoma of the Urinary Bladder Andrea R

    DNA Methylation and the Mechanisms of CDKN2A Inactivation in Transitional Cell Carcinoma of the Urinary Bladder Andrea R

    0023-6837/00/8010-1513$03.00/0 LABORATORY INVESTIGATION Vol. 80, No. 10, p. 1513, 2000 Copyright © 2000 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. DNA Methylation and the Mechanisms of CDKN2A Inactivation in Transitional Cell Carcinoma of the Urinary Bladder Andrea R. Florl, Knut H. Franke, Dieter Niederacher, Claus-Dieter Gerharz, Hans-Helge Seifert, and Wolfgang A. Schulz Urologische Klinik (ARF, KHF, H-HS, WAS), Frauenklinik (DN), and Institut für Pathologie (C-DG), Heinrich Heine Universität, Düsseldorf, Germany SUMMARY: Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator proteins are frequently found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivation of this locus. Multiplex quantitative PCR analysis for five microsatellites around the locus showed that 34 tumors (39%) had loss of heterozygosity (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking microsatellites, as detected by quantitative PCR. Analysis by restriction enzyme PCR and methylation-specific PCR showed that only three specimens, each with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1␣ CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA as reflected in the methylation status of LINE L1 sequences. The extent of DNA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectively, on average, versus 11%, p Ͻ 0.0015).