Bibbo Marluce, Klump William J, DeCecco Jennifer, Kovatich Albert J
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Acta Cytol. 2002 Jan-Feb;46(1):25-9. doi: 10.1159/000326711.
To develop a procedure for the immunocytochemical detection of P16INK4A in ThinPrep specimens.
Archived ThinPrep, liquid-based cervical/endocervical cytology specimens (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) diagnosed as LSIL, HSIL and WNL were resampled and fixed in 95% ethanol for at least three days. Rehydration and endogenous peroxidase blocking of both ThinPreps and formalin-fixed, paraffin-embedded tissues were accomplished on a Leica Autostainer (Leica, Deerfield, Illinois, U.S.A.). Microwave antigen retrieval with CitraPlus (Biogenex, San Ramon, California, U.S.A.) was performed using a Panasonic microwave oven (Matsushita Cooking Appliances, Franklin Park, Illinois, U.S.A.) on the high setting twice for five minutes each. After cooling for 20 minutes and undergoing a buffer rinse, the slides were placed in a Dako autostainer (Dako-USA, Carpinteria, California, U.S.A.). The P16INK4A primary antibody, clone E6H4 (MTM Laboratories, Heidelberg, Germany) was diluted 1:200 in antibody diluent buffer. Detection was accomplished with a mouse non-avidin-biotin EnVision+ polymer (Dako). The expression of P16INK4A in ThinPreps and corresponding biopsies were scored by two pathologists. A ThinPrep case was scored as positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as exhibiting negative, sporadic, focal or diffuse staining, as described by Klaes et al, Overexpression of P16INK4A as specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri (Int J Cancer 2001;92:276-284).
The P16INK4A antibody assay was positive in 14 of 19 (73.68%) LSIL ThinPrep cases and in 25 of 26 (96.15%) HSIL ThinPrep cases. Thirty-eight of the 39 (97.44%) biopsies corresponding to the positively stained ThinPreps also were positive, with a staining score of at least focal positivity in the dysplastic regions. The P16INK4A antibody assay was negative in 5 of 19 (26.32%) LSIL ThinPrep cases and negative in 1 of 26 (3.85%) HSIL ThinPrep cases. The six biopsies corresponding to the negative ThinPreps were similarly negative. The two cytologic specimens diagnosed as WNL were negative for P16INK4A, as were two tissue control cases with benign diagnoses. Nondysplastic squamous epithelium, identified in 17 biopsy cases, did not stain, nor did nondysplastic squamous cells identified in ThinPrep cases. Sporadic staining of bacteria, inflammatory cells and occasional endocervical glandular cells was identified.
P16INK4A expression in ThinPrep specimens correlates with tissue expression of P16INK4A, as implemented in the above protocol. P16INK4A may thus serve as a surrogate marker in gynecologic cytology for high-risk HPV infection and for the development of cervical neoplasia.
建立一种在ThinPrep标本中免疫细胞化学检测P16INK4A的方法。
对存档的ThinPrep液基宫颈/宫颈管细胞学标本（Cytyc公司，美国马萨诸塞州博克斯伯勒）进行重新采样，这些标本诊断为低度鳞状上皮内病变（LSIL）、高度鳞状上皮内病变（HSIL）和未见上皮内病变或恶性病变（WNL），并在95%乙醇中固定至少三天。ThinPrep标本和福尔马林固定、石蜡包埋组织的水化及内源性过氧化物酶阻断在徕卡自动染色仪（徕卡，美国伊利诺伊州迪尔菲尔德）上完成。使用松下微波炉（松下烹饪电器，美国伊利诺伊州富兰克林公园）在高功率设置下，用CitraPlus（Biogenex公司，美国加利福尼亚州圣拉蒙）进行微波抗原修复，每次五分钟，共两次。冷却20分钟并经过缓冲液冲洗后，将玻片放入达科自动染色仪（达科美国公司，美国加利福尼亚州卡平特里亚）。P16INK4A一抗，克隆E6H4（MTM实验室，德国海德堡）在抗体稀释缓冲液中按1:200稀释。用小鼠非抗生物素蛋白-生物素EnVision+聚合物（达科）进行检测。两名病理学家对ThinPrep标本和相应活检组织中P16INK4A的表达进行评分。如果一个ThinPrep病例含有超过10个具有核和细胞质免疫细胞化学染色的异常细胞，则评分为阳性。相应的活检组织按Klaes等人所述的阴性、散在性、局灶性或弥漫性染色进行评分，P16INK4A过表达作为子宫颈发育异常和肿瘤上皮细胞的特异性标志物（《国际癌症杂志》2001年；92:276 - 284）。
在19例LSIL ThinPrep病例中的14例（73.68%）以及26例HSIL ThinPrep病例中的25例（96.15%），P16INK4A抗体检测呈阳性。与染色阳性的ThinPrep标本对应的39例活检组织中的38例（97.44%）也呈阳性，在发育异常区域的染色评分至少为局灶性阳性。在19例LSIL ThinPrep病例中的5例（26.32%）以及26例HSIL ThinPrep病例中的1例（3.85%），P16INK4A抗体检测呈阴性。与阴性ThinPrep标本对应的6例活检组织同样呈阴性。诊断为WNL的2例细胞学标本以及2例诊断为良性的组织对照病例，P16INK4A均为阴性。在17例活检病例中识别出的非发育异常鳞状上皮未染色，在ThinPrep病例中识别出的非发育异常鳞状细胞也未染色。发现细菌、炎性细胞和偶尔的宫颈管腺细胞有散在染色。
按照上述方案，ThinPrep标本中P16INK4A的表达与P16INK4A的组织表达相关。因此，P16INK4A可作为妇科细胞学中高危人乳头瘤病毒（HPV）感染和子宫颈肿瘤发生的替代标志物。
