Cunningham Larry L, Pagano Giulia M, Li Mengtao, Tandon Rahul, Holm Stephen W, White Dean K, Lele Subodh M
Oral and Maxillofacial Surgery, University of Kentucky College of Dentistry, Lexington, KY 40536-0297, USA.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006 Jul;102(1):77-81. doi: 10.1016/j.tripleo.2005.11.028. Epub 2006 Apr 24.
Human papillomavirus (HPV) infection has been implicated in the development of high-grade squamous dysplasia and carcinoma of the oral cavity in the absence of other known risk factors such as smoking. HPV-induced oral dysplasia or carcinoma may be a unique tumor entity in terms of biologic behavior and treatment decisions. In detecting such cases, most reported studies have used techniques that are less sensitive than DNA amplification. Recent reports have suggested that overexpression of the p16INK4 protein is a surrogate marker of HPV-induced high-grade dysplasia or carcinoma. However, the correlation between expression of p16INK4 and the presence of HPV DNA as determined by polymerase chain reaction (PCR) amplification has not been previously reported. The purpose of this research was to determine if immunohistochemistry for p16 would serve as a marker of HPV-associated high-grade oral squamous dysplasia.
Archival formalin-fixed, paraffin-embedded tissue sections from 41 cases of high-grade oral squamous dysplasia were randomly selected. Expression of p16INK4 protein was assessed by immunohistochemical analysis (16P04 Neomarkers, Fremont, CA). Strong and diffuse nuclear staining restricted to the dysplastic region in the epithelium was scored as positive for protein expression, whereas focal or weak nuclear or cytoplasmic staining was scored as negative. The presence of HPV was determined by microdissection, DNA extraction, and PCR DNA amplification using elongated primers that align with corresponding sequences of the L1 region of 23 mucosotropic HPV genotypes. The HPV type was determined by direct sequencing of the PCR product. Normal squamous epithelium was used as an internal negative control, and cases of severe cervical high-grade squamous dysplasia were used as a positive control for immunohistochemical staining and PCR.
The results of immunohistochemical analysis for overexpression of p16INK4 were positive in 6 of the 41 tissue sections. The results of PCR DNA amplification were also positive for these 6 sections. HPV-16 was identified in 5 of the positive cases; in the other case, the viral strain could not be determined.
Immunohistochemical detection of p16INK4 is a technically simple and potentially reliable assay for diagnosing cases of HPV-induced oral high-grade squamous dysplasia. Detecting such lesions may influence future therapeutic decisions.
在不存在吸烟等其他已知风险因素的情况下，人乳头瘤病毒（HPV）感染与口腔高级别鳞状发育异常及癌的发生有关。就生物学行为和治疗决策而言，HPV诱导的口腔发育异常或癌可能是一种独特的肿瘤实体。在检测此类病例时，大多数已报道的研究使用的技术不如DNA扩增敏感。最近的报告表明，p16INK4蛋白的过表达是HPV诱导的高级别发育异常或癌的替代标志物。然而，此前尚未报道过p16INK4的表达与通过聚合酶链反应（PCR）扩增测定的HPV DNA的存在之间的相关性。本研究的目的是确定p16免疫组织化学是否可作为HPV相关的口腔高级别鳞状发育异常的标志物。
从41例口腔高级别鳞状发育异常病例中随机选取存档的福尔马林固定、石蜡包埋组织切片。通过免疫组织化学分析（16P04 Neomarkers，弗里蒙特，加利福尼亚州）评估p16INK4蛋白的表达。上皮发育异常区域内局限的强而弥漫的核染色被评为蛋白表达阳性，而局灶性或弱核或细胞质染色被评为阴性。通过显微切割、DNA提取以及使用与23种亲黏膜HPV基因型L1区域相应序列对齐的延长引物进行PCR DNA扩增来确定HPV的存在。通过对PCR产物进行直接测序来确定HPV类型。正常鳞状上皮用作内部阴性对照，严重宫颈高级别鳞状发育异常病例用作免疫组织化学染色和PCR的阳性对照。
41个组织切片中有6个p16INK4过表达的免疫组织化学分析结果为阳性。这6个切片的PCR DNA扩增结果也为阳性。5例阳性病例中鉴定出HPV - 16；另一例无法确定病毒株。
p16INK4的免疫组织化学检测是诊断HPV诱导的口腔高级别鳞状发育异常病例的一项技术简单且可能可靠的检测方法。检测此类病变可能会影响未来的治疗决策。
