Immunocytochemistry of p16^INK4a in liquid-based cervicovaginal specimens with modified Papanicolaou counterstaining

Abstract

Aim: To evaluate the feasibility and value of a modified Papanicolaou counterstain for p16^INK4a immunostaining in liquid-based cervicovaginal samples.

Methods: Immunocytochemical analyses were carried out with p16^INK4a and modified Papanicolaou counterstain on 81 liquid-based samples, including 23 of within normal limits (WNL), 6 of low-grade squamous intraepithelial lesion (LSIL), 20 of high-grade squamous intraepithelial lesion (HSIL), 16 of atypical squamous cells of undetermined significance (ASC-US) and 16 of atypical squamous cells, high-grade lesion cannot be excluded (ASC-H). Results were compared with histological or cytological follow-up. For comparison, samples from 29 more cases (10 of LSIL, 10 of ASC-H and 9 of HSIL) were immunostained with p16^INK4a and conventionally counterstained with haematoxylin. The intensity of immunostaining in cases of squamous intraepithelial lesion (SIL) was assessed using a 0–3 scoring system. Interobserver agreement was calculated by κ statistics.

Results: Expression of p16^INK4a was detected in 3 of 23 cases of WNL, 4 of 6 cases of LSIL, all cases of HSIL, 5 of 16 cases of ASC-US and 13 of 16 cases of ASC-H. Excluding two cases with no residual dysplastic cells in the immunocytochemistry, all cases of cervical intraepithelial neoplasia (CIN)2 or CIN3 at follow-up expressed p16^INK4a and none of the p16^INK4a-negative cases showed a high-grade lesion at follow-up. No evident differences in pattern or intensity of p16^INK4a expression were observed between the specimens of the study and control groups. Interobserver agreement was significantly better in the study group than in the group with conventional immunostaining (combined κ 0.773 v 0.549; p<0.05), and still better, albeit statistically not significant, than with conventional immunostaining and cervical smear test together (combined κ 0.773 v 0.642).

Conclusion: Immunocytochemistry with p16^INK4a and modified Papanicolaou counterstain may add to the cervicovaginal cytology the full potentiality of p16^INK4a without the need of a further slide and the risk of loss of dysplastic cells, yet maintaining the typical morphological features of the smear test.