Evaluation of a Commercialized In Situ Hybridization Assay for Detecting Human Papillomavirus DNA in Cervical Intraepithelial Neoplasia and Cervical Carcinoma

Departments of Pathology and of Biostatistics and Applied Mathematics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA; Department of Pathology, Taichung Veterans General Hospital, Taichung, Taiwan; Department of Obstetric/Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

^* To whom correspondence should be addressed. Email: mguo{at}mdanderson.org.

	Abstract

To evaluate a commercialized in situ hybridization (ISH) assays for detecting human papillomavirus (HPV) DNA, we compared the ability of the new ISH probe, INFORM HPV 3 (Ventana Medical Systems, Tucson, AZ) with that of PCR assays to detect HPV DNA in cervical tissue specimens with normal cervix (20 cases), cervical intraepithelial neoplasia (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases), and cervical carcinoma (29 cases). General HPV DNA was detected using consensus primer mediated PCR assays. HPV genotyping was performed using EasyChip HPV blot (King Car Yuan Shan Institute, I-Lan, Taiwan). HPV 16 integration status (E2/E6 ratio) was determined using quantitative real-time PCR. Our findings showed that the ISH and PCR had fair to good agreements in detecting HPV DNA across all CIN categories without significant differences (Kappa coefficient: 0.34-0.63, P = 0.13-1.0). However, ISH detected significantly fewer HPV positive cases in carcinoma than PCR did (Kappa coefficient: 0.2; P = 0.03). Eleven cases with ISH-/PCR+ results had HPV types that can be detected by INFORM HPV 3. Five carcinoma cases with ISH-/PCR+ results showed a significantly higher level of integrated HPV 16 (P = 0.008) than did the ISH+ cases. As a consequence, lower copy numbers of episomal HPV16 in carcinoma might be the cause for the false-negative ISH results. Although the punctate signal pattern of HPV significantly increased with the severity of disease (P trend = 0.01), no significant difference in the HPV 16 integration status was observed between the cases with a punctate signal only and the cases with mixed punctate and diffuse signals (P = 0.4). In conclusion, ISH using INFORM HPV 3 probe seems comparable to PCR for detecting HPV DNA in cervical tissue with CINs. False-negative ISH results appears to be associated with the lower copy numbers of the episomal HPV 16 but not with the capability of INFORM HPV 3 probe to detect specific HPV types. In addition, signal patterns, especially mixed punctate and diffuse pattern of HPV cannot be reliably used to predict viral integration status.