[CANCER RESEARCH 56. 3630-3633, August 15. 19961 Advances in Brief High Frequency of pió (CDKN2/MTS-1/INK4A) Inactivation in Head and Neck Squamous Cell Carcinoma1'2 Andre L. Reed, Joseph Califano, Paul Cairns, William H. Westra, Richard M. Jones, Wayne Koch, Steven Ahrendt, Yolanda Eby, Duane Sewell, Homaira Nawroz, Jiri Bartek, and David Sidransky3 Department of Otolaryngology-Hrad and Neck Surgery, Head and Neck Cancer Research Division ¡A.L R., J. C.. P. C.. R. M. J.. W. K.. Y. E., D. Se.. H. N., D. SiJ, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196: Departments of Pathology ¡W.H. W.} and General Surgery ¡S.A.¡,Jolin\ Hopkins Hospital, Baltimore. Maryland 21287: and Danish Cancer Society, Division for Cancer Biologv, Strandboulevarden 49, opg. Bygn. 4, DK-2100 Copenhagen 0, Denmark, ¡J.B.I Abstract The tumor suppressor genep/6 (CDKN2/MTS-1/INK4A) can be inacti vated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) far pl6 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. (ito staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the ¡ilftlocus to detect homozygous deletion; (ft) sequence analysis ofplo; and !<•) Southern blot analysis to determine the methylation status of the 5' < |i(, island of p 16. Twenty-four of 29 (83% ) head and neck squamous cell carcinoma tumors displayed an absence of pl6 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the pl6 locus, and I displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the pi6 tumor sup pressor gene is a frequent event in squamous cell carcinomas of the head and neck; tin pl6 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methyla tion; and (<•) immunohistochemical analysis for expression of thep/6 gene product is an accurate and relatively simple method for evaluating pl6 gene inactivation. Introduction HNSCCs4 comprise 5% of the 1,040,000 new cancers afflicting Americans annually; however, little is known about the molecular changes associated with oncogenesis (1, 2). Perturbations in cellular proliferation driven by the accumulation of genetic alterations in oncogenes and tumor suppressor genes give rise to neoplasms (3). Tumor suppressor gene inactivation is among the most common genetic mechanisms resulting in malignant transformation (4). For example, mutational inactivation of the p53 tumor suppressor gene is one of the most common genetic changes in human cancer, and it is also found in approximately 45% of primary HNSCCs (5, 6). Evidence for inactivation of additional tumor suppressor genes in Received 5/7/96; accepted 6/28/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this faci. 1 Supported by Lung Spore Grant CA-58184-01 and a Collaborative Research Agree ment with Oncor. Inc. (Gailhersburg. MD). 2 Oncor. Inc. provided research funding for the study described in this article. Under an agreement between Oncor and The Johns Hopkins University. Dr. Sidransky is entitled to a share of sales royalty received by the University from Oncor. Under that agreement, the University and Dr. Sidransky also have received Oncor stock which, under University policy, cannot be traded until 2 years after the first commercial sales of the products related to this research. Dr. Sidransky also serves as a member of the Scientific Advisory Board of OncorMcd. Inc., an Oncor subsidiary, which is commercializing some of Oncor's technology. The terms of this arrangement have been reviewed and approved by the University in accordance with its conflict of interest policies. 1To whom requests for reprints should be addressed, at Head/Neck Surgery. Johns Hopkins University School of Medicine. 818 Ross Research Building. 720 Rutland Avenue. Baltimore, MD 2I205-2I96 4 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; LOH, loss of heterozygosity; IHC. ¡mmunohistochemistry; mAb. monoclonal antibody. HNSCC is supported by frequent and early LOH at the 9p21 locus in these neoplasms (7). Located within this minimal region is the puta tive tumor suppressor gene pió (CDKN2/MTS-I/1NK4A; Refs. 8 and 9). The ¡>I6gene encodes a cell cycle protein which inhibits cyclin- dependent kinases 4 and 6, preventing phosphorylation of Rb protein and causing inhibition of cell cycle progression from G, to S-phase (10-12). Genetic alterations of the p!6 gene lead to its inactivation, resulting in deregulation of cell proliferation and tumorigenesis. Pre vious reports suggest that the incidence of pió gene mutations in HNSCC cell lines (44%) is much greater than that found in primary HNSCC tumors (10%; Refs. 13 and 14)). This would suggest that the gene encoding pI6 may not be the primary target of LOH at 9p21 in HNSCC. implicating the existence of (an) additional tumor suppressor genes at this locus. However, alternative mechanisms of pió inacti vation include homozygous deletion (8, 15) and methylation (16) of the 5' CpG island within the promotor region leading to pió inacti vation. For most tumor types, these alternative mechanisms of pl6 inactivation are much more frequent than point mutation alone (14). To delineate the role of p!6 as a tumor suppressor in the genesis of HNSCC, we examined 29 primary HNSCC tumors for pió gene inactivation using IHC and correlated these findings with genetic analysis of the pió locus. We found that p¡6 is the most commonly inactivated tumor suppressor gene detected thus far in primary HNSCC and that immunohistochemistry is a sensitive method of detecting ¡>I6 inactivation. Materials and Methods Primary Tumor and Margin Samples and DNA Extraction. Twenty- nine randomly selected primary HNSCC tumors were collected from July 1996 through August 1996 following surgical resection with prior consent from Johns Hopkins Hospital patients. These specimens were fresh frozen, then microdissecled on a cryostat to select for greater than 70% neoplastic cells/ tumor. Blood was obtained by venipuncture from patients, and lymphocyte DNA was isolated as described for use as a normal control (7). Histopathology. Fresh-frozen specimens were embedded in OCT (Tissue- Tek; Miles. Elkhart, IN). Tissue sections (5 mm) were cut on a cryostat, and the first two sections of each tumor were mounted on lysine-coated glass slides and stained with H&E. These slides were examined by a pathologist (W. H. W.). and microdissection was performed as needed to obtain greater than 70% neoplastic cells. Twenty-four sections were obtained from each tumor and mounted on 12 lysine-coated slides and stored at -20°C to be used tor IHC staining. Fifty sections. 12-mm thick, were cut from each tumor and placed in SDS/proteinase K at 60°Cfor 4 h. This was followed by phenol- chloroform extraction of DNA and ethanol precipitation as described previ ously (17). IHC. IHC was performed as described previously (18) with the use of a pl6 monoclonal antibody, DCS-50 (12) (Lab Vision. Fremont, CA), which pref erentially recognizes denaturation-resistant epitopes on the carboxy terminus of the pl6 protein. Briefly, 5-mm thick frozen tumor sections were fixed on lysine-coated slides ina 1:1 cold acetone/methanol solution. After blocking, serum was applied for 15 min. and sections were reacted with primary pl6 3630 Research. on December 13, 2021. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from