E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

Purpose: Persistent infections by high-risk human papillomavirus (HPV) types are the main etiologic factor for cervical cancer. The objective of this study was to evaluate whether high-risk E7 oncoprotein is adequate as a marker for the detection of cervical cancer. Experimental Design: HPV typing was done in biopsies from 58 cervical carcinoma and 22 normal cervical squamous epithelia. The HPV-16 E7, HPV-18 E7, and HPV-45 E7 oncoprotein levels were monitored by immunohistochemistry and compared with those of p16INK4a and Ki67. Results: Fifty-five (94.8%) tumors were high-risk HPV-DNA^positive (46 HPV-16, 2 HPV-16 and HPV-18, 4 HPV-18, 1 HPV-33, and 2 HPV-45). HPV-DNA could not be detected in three tumors (5.2%). High HPV E7 oncoprotein levels were shown in 57 cervical cancers (98.3%), without correlation between expression levels and tumor stages. Conclusion: This is the first study which systematically analyzes the levels of the major HPV oncoproteins in cervical carcinomas demonstrating that the high-risk HPV E7 proteins are regularly expressed in these cancers. This suggests that high-risk E7 oncoproteins are necessary for cervical cancers and apparently essential as tumor marker.

Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6-to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears. P ersistent infections by human papillomaviruses (HPVs) are the main etiologic factor for cervical cancer (41). Approximately 85% of cervical cancers are squamous cell carcinomas (SCCs) which arise from dividing keratinocytes in the squamous epithelium of the ectocervix and 15% are adenocarcinomas (ACs) which arise from glandular cells located in the endocervix (3, 25). About 40 HPV genotypes that can infect epithelial squamous and glandular cells in the cervical mucosa have been described. On the basis of epidemiological and biochemical data, only a subgroup of HPV types, referred to as high-risk HPVs, is associated with intraepithelial lesions that have a high potential for progression to invasive carcinoma. Infections by high-risk HPV genotypes have been detected in virtually all cervical cancers (37). At least 15 highrisk HPV types have been associated with these cancers. HPV type 16 (HPV16) and HPV18 are the most prevalent genotypes worldwide in SCCs as well as in ACs (23).A persistent infection with oncogenic HPVs is necessary for the development of cervical precancer and cancer (3, 37, 40, 41). Initial events of cervical carcinogenesis after viral infection by highrisk HPV types are specific changes that overcome the transcriptional control of viral gene expression in the infected keratinocytes (40). Inactivation of these cellular control functions permits deregulated transcription of the early viral genes E6 and E7. This event triggers reprogramming of cell proliferation, apoptosis, differentiation, metabolism, epigenetic reorganization, and genomic instability (21). These changes can support the integration ...