[424] p16^INK4a Immunocytochemistry as an Adjunct to Cervical Cytology Potential Reflex Testing on Specially Prepared Cell Blocks from Residual Liquid Based Cytology (LBC) Specimens

GM Varsegi, KL D'Amore, VB Shidham. Medical College of Wisconsin, Milwaukee, WI

Background: p16^INK4a (p16) is a recognized marker of HPV related dysplasia in cervical biopsies. However, its application to cervical cytology specimens has not been evaluated methodically. The main challenge in applying p16 to cytology specimens is commonly associated, non-diagnostic, cytoplasmic immunoreactivity, which envelops and obscures the nuclear immunoreactivity in uncut cells. We standardized a protocol to overcome this and other obstacles. We evaluated the role of p16 immunoreactivity in residual LBC [SurePath] specimens after cytology interpretations.
Design: Out of 120,000 cases, residual cytology specimens were available for prospective evaluation in 133 [50 LSIL, 28 HSIL, 21 ASC-H, 14 ASCUS and 20 negative (WNL)] cases for preparing HistoGel cell blocks. We standardized a method which included a centrifugation step to align randomly dispersed loose cells in LBC specimens along the flat cutting surface. In addition, a visible marker was embedded to monitor the depth while section cutting. This marker also helped serve as a landmark during interpretation of p16 (clone E6H4, mtm laboratories AG) immunostained slides. Only nuclear immunoreactivity in squamous cells was considered positive. Biopsy was considered positive if CIN1 or above (confirmed with p16 in equivocal cases).
Results: Adequately cellular cell block sections showed positivity in 20/28 HSIL, 11/ 21 ASC-H, 13/ 50 LSIL, 0/14 ASCUS and 0/20 WNL. All 7 biopsy positive ASC-H cases were positive for p16 on cytology specimens.

Correlation of follow-up results with p16 immunocytochemistry

Group		T	W/ Bx F/U - P/N*	Sensitivity*	Specificity*	W/ PAP F/U - P/N*	No/ F/U
LSIL	p16P	26% T=50	44%/56% T=27	85%	100%	7%/93% T=14	9
HSIL	p16P	71% T= 28	69%/31% T=16	100%	67%	43%/57% T=7	5

W/, with; Bx, biopsy; F/U, follow-up; p16P, positive for p16; T, total, P, positive, N, negative. Other abbreviations are as per Bethesda Terminology. *Calculated by including only cases with unequivocal biopsy results and cases with adequately cellular specimens for cell blocks. Trouble shooting of false P + N cases showed sampling and biopsy interpretation issues.


Conclusions: 1. p16 on cervical cytology specimens showed excellent correlation with biopsy results. 2. In cases with abnormal but without unequivocal HSIL cytology (such as ASC-H, LSIL, & ASCUS), reflex p16 immunostaining using a properly standardized protocol to prepare cell block sections of cervical cytology specimens is recommended.
Category: Cytopathology

Monday, March 9, 2009 9:30 AM

Poster Session I Stowell-Orbison/Autopsy Award # 69, Monday Morning