P16INK4A as an adjunct test in liquid-based cytology.

OBJECTIVE: To assess the utility of P16INK4A as an adjunct test in liquid-based cytology in cases with equivocal morphologic changes of high grade squamous intraepithelial lesion (HSIL). STUDY DESIGN: P16INK4A immunoreactivity was investigated in residual ThinPrep material (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) from 30 cases with equivocal diagnoses of HSIL that had corresponding follow-up biopsies. Two control ThinPrep cases were included: 1 HSIL with biopsy-confirmed cervical intraepithelial neoplasia (CIN) 3 and a negative specimen with a corresponding biopsy of squamous metaplasia. The expression of P16INK4A in ThinPrep specimens and corresponding biopsies was scored as previously described. A ThinPrep case was scored positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as having negative, sporadic, focal or diffuse staining. RESULTS: The P16INK4A antibody assay was positive in 19 of 30 ThinPrep cases (63.3%). Seventeen of the 19 (89.4%) biopsies corresponding to the positively stained ThinPreps also were positive, with a score of at least focal positivity in the dysplastic regions (2 CIN 1, 4 CIN 2, 11 CIN 3; 2 lesions lost in the tissue recut). The assay was negative in 11 ThinPreps (36.6%) and 10 biopsies (33.3%) with tissue confirmation of chronic cervicitis (5), squamous metaplasia (2), CIN 1 (3) and 1 lesion lost in the tissue recut. Seventeen of 18 (94.4%) ThinPreps confirmed as high grade lesions upon biopsy showed P16INK4A positivity. The control HSIL case with a CIN 3 biopsy was diffusely positive for P16INK4A, and the control negative case with biopsy diagnosis of squamous metaplasia was negative. Nondysplastic squamous and metaplastic epithelium in 7 biopsies and nondysplastic squamous or metaplastic cells in ThinPrep cases were negative. Sporadic staining of bacteria, inflammatory cells and endocervical cells was noted. CONCLUSION: ThinPrep cases in the equivocal cytologic category with the corresponding tissue biopsy assayed for P16INK4A expression showed that there was utility for this type of testing. A larger series comparing corresponding ThinPrep and tissue biopsies will be undertaken. The role of HPV infection in these cases will also be explored.

Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens.

OBJECTIVE: To develop a procedure for the immunocytochemical detection of P16INK4A in ThinPrep specimens. STUDY DESIGN: 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). RESULTS: 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. CONCLUSION: 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.