Presentations

[PS 7-6] Developing a Lateral Flow Strip Test for E6 Oncoprotein to Identify Cervical Intraepithelial Neoplasia (CIN) at Increased Risk of Progression to Cervical Cancer.

R. B. Peck, B. H. Weigl, J. Schweizer, C. Somoza, J. Silver, L. Liu, P. S. Lu, J. W. Sellors. Arbor Vita Corporation, Sunnyvale, U.S.A.; PATH, Seattle, U.S.A.

Arbor Vita Corporation (AVC) and PATH are collaborating to develop an affordable, rapid, point-of-care strip test to identify women with CIN and expressing HPV-E6 oncoprotein. Expression of E6 is necessary for cellular transformation to cervical cancer and a potentially useful biomarker for screening or triage of women at risk of developing malignant cervical lesions. The strip test is based on AVC's proprietary technology, which takes advantage of PDZ binding to all of the high-risk—but not the low-risk—HPV-E6 oncoproteins. The test will utilize a provider-collected cervical swab and provide results within 30 minutes.The test, which is in the research and development phase, is an immunochromatographic strip with recombinant PDZ immobilized on the strip and novel antibodies to E6 for detection. A buffer has been developed for assay running, which also releases the sample from the swab, lyses the cells, and extracts E6. Two versions of the strip test are being evaluated. One is a qualitative test utilizing antibody conjugated colloidal gold as the visual detector; the other is a quantitative test utilizing antibody conjugated to super paramagnetic particles and read on a simple magnetic strip reader.The visual and magnetic reader-based test formats have been evaluated with recombinant E6 of HPV type 16 and exhibit sensitivity below 200 pg. Based on the number of cells collected by a cervical swab and the level of expression of E6, it is estimated that the test will need to detect down to approximately 50pg of E6. Ongoing work focuses on sensitivity improvement of both test formats and to evaluate test performance with additional HPV types, cell lines, and clinical samples. Data will be presented on the assay formats and level of sensitivity achieved for E6 from oncogenic types of HPV.

Date: Monday, September 04, 2006

[PS 9-6] High-Risk HPV-E6 Activates the JNK Pathway by Interacting with PDZ-Protein MAGI1

C. Somoza, C. P. Bagowski, L. Peysakhovich, S. Mahoney, R. Khanna, C. Lynch, J. Silver, P. S. Lu, J. Schweizer. Arbor Vita Corporation, Sunnyvale, U.S.A.; Harvard University, Boston, U.S.A.; Leiden University, Institute Of Biology, Leiden, Netherlands

Human papillomavirus (HPV) causes > 99% of cervical cancers. High-risk HPV oncoproteins E6 and E7 are necessary for cervical epithelial cell transformation and progression to cancer. All oncogenic E6 proteins contain C-terminal PDZ ligands (PL), which are absent in low-risk HPV-E6. We previously reported that (i) MAGI1-PDZ1 is the only PDZ that binds all high-risk HPV-E6 proteins, that (ii) expression of HPV16-E6 leads to downregulation of MAGI1 in a PL-dependent manner, and that (iii) cervical cancer cell lines and cervical carcinoma biopsies have substantially reduced levels of MAGI1. We have investigated the molecular pathways regulated by the E6 / MAGI1 interaction and we found that HPV positive cervical cancer cell lines have elevated basal Jun-N-terminal kinase (JNK) activity. JNK is an activator of the transcription factor AP-1 that regulates E6 and E7 expression during cervical carcinogenesis. Recently, it was shown that elevated JNK activity plays a role in the pathogenesis of several human cancers and that JNK activation is required for tumor cell growth. We now show that HPV16-E6, but not a E6?PL mutant, induces JNK activation in human epithelial cells. In the Xenopus oocyte, co-injection of a peptide representing the 20 C-terminal amino acids of HPV16-E6 blocks the HPV16- and HPV18-E6-dependent JNK activation in a dose-dependent manner. We tested whether E6 mediated MAGI1 dowregulation is linked to JNK activation and show that elimination of MAGI1 by RNAi leads to JNK activation, suggesting that high-risk E6 proteins activate JNK by downregulating MAGI1. Finally, we find that the E6-PL mediated downregulation of MAGI1 is reversible, as inhibition of E6 expression in cervical cancer cells via siRNA restores MAGI1 protein expression. We conclude that high-risk HPV-E6 mediates cervical carcinogenesis in part by activating the oncogenic JNK pathway, and that JNK and MAGI1 are potential new targets for the treatment of cervical cancer.

Date: Monday, September 04, 2006

[P-530] Detection of High RiskHPV-E6 Oncoprotein in Low and High-Grade CervicaL Intraepithelial Neoplasia (CIN): Emerging Evidence for Predictive Validity

J. Schweizer, C. Somoza, C. Mahoney, J. Silver, R. Peck, B. Weigl, Y.L. Qiao, J. Sellors, P. S. Lu. Arbor Vita Corporation, Sunnyvale, U.S.A.; Cancer Institute, Chinese Academy Of Sciences, Beijing, China; PATH, Seattle, U.S.A.

Infections with high risk HPV types result in approximately 230,000 deaths from cervical cancer per year, over 80% in developing countries. Accumulating evidence suggests that expression of the high risk HPV-E6 oncoprotein is necessary for cervical cancer progression. Previously, we had demonstrated that all high-risk, but not the low-risk, HPV-E6 oncoproteins bind specifically to a cellular PDZ domain with high affinity. This property is the basis of a diagnostic assay in which high-risk E6 oncoprotein is captured via a PDZ domain and detected via anti-E6 monoclonal antibodies. To elucidate the correlation of E6 oncoprotein expression with the severity of the clinical phenotype, we used cervical swab specimens collected from women screened, diagnosed, and managed in rural settings in China, and we determined the quantity of E6 oncoprotein by Western blot. All specimen were derived from women with histologically-confirmed CIN and cancer in cross-sectional surveys and in a prospective follow up study of women with CIN1. Samples from 18 women who were positive for HPV16-E6 DNA by PCR were analyzed. We detected HPV16-E6 oncoprotein in cervical specimens from 6 out of 7 women with CIN3, in 1 out of 2 with CIN2, but only in 1 out of 9 women with CIN1. Interestingly, the CIN1 specimen that showed expression of HPV16-E6 was from the only woman among the 9 who progressed to CIN3 during the follow-up study. These preliminary findings suggest, that detection of HPV16-E6 oncoprotein in CIN1 lesions may indicate an increased risk for progression. Further studies to elucidate the predictive validity of E6 oncoprotein as a diagnostic marker are currently underway using commercially viable assay formats.

Date: Wednesday, September 06, 2006