HPV-associated Neoplasia Research Articles

Abstract LB-268: Loss of HPV16 E2 protein expression and transcriptional modulation of cellular and viral genes correlate with progression to cervical carcinoma

Abstract Integration of the viral DNA in the cellular genome has been suggested to be critical in carcinogenic progression of HPV-associated cervical neoplasia. This event can be accompanied by disruption of the open reading frame (ORF) encoding the E2 repressor, thus leading to transcriptional up-regulation of the E6 and E7 viral oncogenes. At this stage, it is unclear whether disruption of the E2 ORF is mandatory for carcinogenic progression. We measured E2 RNA and protein expression in clinical samples of various grades of HPV16-associated cervical neoplasia and compared it with the status of the viral genome. RNA extracted from paraffin embedded tissues was hybridized to specific probes and quantified by the NanoString technology. Protein expression was appreciated by immunohistochemistry and the status of viral DNA was determined by in situ hybridization, all performed on serial sections of the same samples. E2 protein was found highly expressed in CIN2 lesions where the HPV DNA was highly replicating, while it was decreased in more advanced grade lesions where replication is decreased or lost (CIN3 and SCC). Our data demonstrate that integration of the viral DNA in the cellular genome is always accompanied by drastic reduction of expression of the E2 protein, even when the E2 ORF is not disrupted by integration. The viral genes appear differentially expressed during progression as transcription of the HPV16 E6/E7 oncogenes, only when compared to E2-E4, is significantly higher in high grade lesions than in CIN2. In addition, transcription of 400 cellular genes of the cell cycle and immune-response were also studied in 20 clinical samples that show modulation during progression. We will discuss the establishment of a transcriptional signature for progression in cervical cancer and the role of local immune response in the fate of cervical lesions associated with HPV16. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-268. doi:1538-7445.AM2012-LB-268

HPV-assoziierte Plattenepithelkarzinogenese

About 7-8% of all human cancers are thought to be related to infections with high-risk (HR) human papilloma virus (HPV). Besides cervical cancer, especially squamous cell carcinomas of the anogenital and oropharyngeal regions are associated with HR-HPV. Transmission of HPV is due to sexual activity. Harald zur Hausen was awarded in 2008 with the Nobel price in medicine for the establishment of a causal link between certain HPV infections and cervical cancer. Meanwhile potent prophylactic vaccines are available to prevent infections with HPV-16 and HPV-18, the two most frequently observed HR HPV types worldwide. On molecular grounds a persistent HPV infection is the central risk factor for the development of HPV-associated neoplasias. Continued expression of the viral E6 and E7 oncogenes disrupts cell cycle control mechanisms in infected cells, thereby gaining limitless proliferative capacity and resistance against apoptotic signals. However acquisition of mutations and genomic instability might cause malignant transformation in these cells.

A novel mucosal orthotopic murine model of human papillomavirus‐associated genital cancers

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

Konservative Behandlungsmethoden anogenitaler HPV-Infektionen

Despite some recent hopeful therapeutic developments such as topical green tea derivatives, the therapy of anogenital warts remains a medical problem. Both physician- and self-administered therapeutic approaches are not fully satisfactory. The high recurrence rates associated with the current therapy of anogenital warts together with their increasing incidence and burden of disease further support the use of the prophylactic quadrivalent VLP HPV6,11,16,18 vaccine for control of HPV-associated neoplasias in women and men.

Virostatische und immunmodulatorische Lokaltherapie bei rezidivierenden HPV-assoziierten hochgradigen vulvären intraepithelialen Neoplasien: Eine Pilotstudie

OBJECTIVES: Aim of the study was to test the efficacy of a local virostatic and immunomodulatory therapy for recurrent HPV-associated vulvar intraepithelial neoplasias (VIN) and to evaluate the effect of imiquimod on dendritic cells. METHODS: Three women with recurrent VIN treated previously with surgical excision and laser destruction developed new VIN lesions. They received local treatment with 5 % imiquimod cream for 2 weeks alternating with 1 % cidofovir gel for 2 weeks for a total period of 12 weeks. Expression surface markers on antigen-presenting dendritic cells after treatment with imiquimod were analyzed. RESULTS: All biopsies revealed HPV 16 DNA. At 1 and 5 months after completion of local therapy, all 3 patients had clinical and histological remission without detectable HPV 16 DNA. One patient was disease-free for 5 years before developing a recurrent VIN lesion again. She was treated locally with imiquimod and cidovir for a second time, which resulted in complete remission. Both other patients remained free of recurrence during the whole period. After incubation with imiquimod, dendritic cells revealed an increased expression of surface markers CD80/B7.1, CD 86/B7.2 and CD40, CD54/ICAM-1 and CD83. CONCLUSION: Virostatic and immunomodulatory local therapy is an effective noninvasive treatment alternative for recurrent HPV-associated high grade VIN. The effect of imiquimod might be based on an enhanced presentation of HPV-antigens to lymphocytes with subsequent destruction. This might be the result of an increased expression of co-stimulatory molecules and surface markers on the cell membrane of antigen presenting dendritic cells.

Global cooperation in gynecologic cancer

Global cooperation in gynecologic cancer

O893 K-ras oncogene expression and viral DNA status in HPV-associated human cervical neoplasia

International Journal of Gynecology & ObstetricsVolume 107, Issue S2 p. S348-S348 Free communication (oral) presentations O893 K-ras oncogene expression and viral DNA status in HPV-associated human cervical neoplasia A. Stanescu, A. StanescuSearch for more papers by this authorC. Goia, C. GoiaSearch for more papers by this authorE. Popa, E. PopaSearch for more papers by this authorI. Huica, I. HuicaSearch for more papers by this authorE. Nistor, E. NistorSearch for more papers by this authorG. Anton, G. AntonSearch for more papers by this authorA. Plesa, A. PlesaSearch for more papers by this authorR. Chiriac, R. ChiriacSearch for more papers by this author A. Stanescu, A. StanescuSearch for more papers by this authorC. Goia, C. GoiaSearch for more papers by this authorE. Popa, E. PopaSearch for more papers by this authorI. Huica, I. HuicaSearch for more papers by this authorE. Nistor, E. NistorSearch for more papers by this authorG. Anton, G. AntonSearch for more papers by this authorA. Plesa, A. PlesaSearch for more papers by this authorR. Chiriac, R. ChiriacSearch for more papers by this author First published: 20 November 2009 https://doi.org/10.1016/S0020-7292(09)61266-2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume107, IssueS2Abstracts of XIX FIGO World Congress of Gynecology and ObstetricsOctober 2009Pages S348-S348 RelatedInformation

Delocalization of the Microtubule Motor Dynein from Mitotic Spindles by the Human Papillomavirus E7 Oncoprotein Is Not Sufficient for Induction of Multipolar Mitoses

Dynein is a minus end-directed microtubule motor that transports numerous cargoes throughout the cell. During mitosis, dynein motor activity is necessary for the positioning of spindle microtubules and has also been implicated in inactivating the spindle assembly checkpoint. Mutations in dynein motor and/or accessory proteins are associated with human disease, including cancer, and the delocalization of dynein from mitotic spindles has been correlated with an increased incidence of multipolar spindle formation in some cancer cells that contain supernumerary centrosomes. The high-risk human papillomavirus type 16 (HPV16) E7 oncoprotein induces centrosome overduplication and has been shown to cause multipolar mitotic spindle formation, a diagnostic hallmark of HPV-associated neoplasias. Here, we show that HPV16 E7 expression leads to an increased population of mitotic cells with dynein delocalized from the mitotic spindle. This function maps to sequences of HPV16 E7 that are distinct from the region necessary for centrosome overduplication. However, contrary to previous reports, we provide evidence that dynein delocalization by HPV16 E7 is neither necessary nor sufficient to cause the formation of multipolar mitoses.

Selective induction of apoptosis by leptomycin B in keratinocytes expressing HPV oncogenes

Human papillomavirus (HPV) infection is strongly associated with the development of anogenital neoplasia, particularly cervical cancer. It has been estimated that 99.7% of all cervical carcinomas are attributable to infection with HPV, and types 16 and 18 account for the vast majority of such cases. Both of these 'high risk' HPV types encode the oncoproteins E6 and E7, which exert multiple effects on many proteins involved in cell-cycle regulation, including p53. The nuclear export protein inhibitor leptomycin B (LMB) has been shown to cause the nuclear sequestration of p53 in cervical carcinoma cells. We demonstrate that LMB induces apoptosis selectively at nanomolar concentrations in primary human keratinocytes (PHKs) expressing HPV oncogenes. Both monolayer and organotypic raft cultures of transduced PHKs were highly susceptible to treatment with LMB. By contrast, although LMB stimulated p53 accumulation in normal PHKs, no significant induction of apoptosis was detected on Western blots or immunostained monolayer/raft cells, or following pulsed exposure to the drug. Furthermore, topical application of microM concentrations of LMB to mouse skin was non-toxic. These data suggest that the topical application of LMB to HPV-infected intra-epithelial lesions may represent a specific and effective therapeutic strategy against HPV-associated anogenital neoplasia.

CHAPTER 5 HPV infection and HPV-associated neoplasia in immunocompromised women

Human immunodeficiency virus (HIV)-positive women and women with transplant-associated immunocompromise are at increased risk for cervical intraepithelial neoplasia (CIN) and cervical cancer compared with healthy, immunocompetent women. HPV often manifests as a “field” effect in immunocompromised women who are also at increased risk for vaginal, vulval, and anal intraepithelial neoplasia. Immunocompromised women require careful follow-up with regular cy-tologic screening, and there should be a low threshold for performing colposcopic evaluation in these women. Once detected, CIN should be treated aggressively and the patient followed up closely for recurrence. Although treatment regimens are similar for immunocompromised and healthy women, the former may need multiple treatment modalities. Data on the ability of highly active antiretroviral therapy (HAART) to reduce the incidence of high-grade CIN and on the regression of existing CIN are mixed, some studies showing no benefit and others a modest benefit from HAART. However, the incidence of cervical cancer has not declined since the introduction of HAART, and the use of HAART among HIV-positive women has not changed the suggested approach to cervical cancer screening and treatment. Finally, prophylactic HPV vaccination offers the possibility of reducing the burden of disease among immunocompromised women, particularly if they are vaccinated before the onset of both sexual activity and immunocompromise. However, studies are needed to document safety and immunogenicity, and —given their high rate of prior HPV exposure —the effectiveness of the vaccine in these women.

P16 and Ki67 Immunostaining is a Useful Adjunct in the Assessment of Biopsies for HPV-Associated Anal Intraepithelial Neoplasia

P16 is a tumor suppressor gene product, shown to be overexpressed in most cervical carcinomas and dysplasias associated with high-risk human papilloma virus (HPV) infection. HPV is also associated with anal squamous dysplasias and carcinomas. Significant interobserver and intraobserver variation exists in the interpretation of biopsies for anal intraepithelial neoplasia (AIN). This study was undertaken to assess the potential role of p16 and Ki67 immunohistochemical expression in refining the diagnosis and grading of AIN.One-hundred and four anal biopsies from 74 patients were retrieved from the surgical pathology files of the department. After discrepancies were resolved and concurrence was achieved by at least 2 of 3 reviewing pathologists, the diagnoses were as follows: 37 negative, 12 condylomas without overt dysplasia, 14 AIN I, 25 AIN II, and 16 AIN III. p16 and Ki67 expression was evaluated by ABC immunoperoxidase staining whereas the presence of the high-risk subtypes of HPV virus was determined by in situ hybridization on a subset of the biopsies. Results were reviewed by 2 pathologists and positive and negative staining was correlated with H&E diagnoses. Nuclear and/or nuclear and cytoplasmic staining was considered as positive for p16 when present in >10% of squamous cells. Two patterns of positive p16 staining were observed: (1) "spotty" in which positive cells were scattered throughout the lesion and (2) "band" in which >90% of contiguous cells in the lesion stained positive. A band-like pattern of p16 immunoreactivity was seen in 21.4% AIN I, 80% AIN II, and 87.5% AIN III cases. None of the condylomas and only 1 of the negative cases showed a band of p16 positive staining. Spotty p16 immunoreactivity was observed in 8.1% negative, 8.3% condyloma, 14.3% AIN I, 12.0% AIN II, and 12.5% AIN III cases. More than 50% of nuclei stained positive for Ki67 in 28.6% AIN I, 48.0% AIN II, and 75.0% AIN III cases but in none of the negative or condyloma cases. On the basis of these results, a band-like pattern of p16 staining and Ki67 positivity in >50% of the squamous cell nuclei were strongly associated with high-grade AIN. Conversely, absence of a p16 band of positivity coupled with Ki67 positivity in <50% of nuclei was frequently associated with benign lesions. Band like p16 staining also correlated strongly with the presence of high-risk HPV. Most AIN I lesions stained similar to the nondysplastic cases. A small subset of biopsies studied did not conform to the pattern described above: 4 of 14 (28.6%) AIN I lesions showed a band-like pattern of p16 staining and/or >50% Ki67 positive nuclei. Two of these cases were positive for high-risk HPV DNA. 4 of 25 (16.0%) AIN II lesions comprising 9.8% of the 41 high-grade AINs (AIN II and III) showed spotty p16 positivity and <50% Ki67 positive nuclei. One was positive for high-risk HPV DNA. We conclude that when used together and evaluated in conjunction with H&E stained sections, p16 and Ki67 immunoexpression is a useful adjunct in the diagnosis and grading of AIN.

More men than women make mucosal IgA antibodies to Human papillomavirus type 16 (HPV-16) and HPV-18: a study of oral HPV and oral HPV antibodies in a normal healthy population

BackgroundWe have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV.MethodsThe prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study.ResultsOral HPV infection was highest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09–12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97–6.2, P = 0.04).ConclusionThe increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation.

Biology of HPV in HIV Infection

HIV-positive men and women are at increased risk of anogenital and oral HPV infection. The risks for HPV-associated high-grade intra-epithelial neoplasia (IN) and cancer are also increased. The prevalence of oral, anal, and cervical HPV infection in HIV-positive individuals compared with HIV-negative individuals increases with progressively lower CD4+ levels, as does incident high-grade IN. In contrast to IN, development of cancer is not related to lower CD4+ level. With increasing grades of IN and cancer, the proportion of tissues with copy-number abnormalities (CNA) increases, with one of the most common genetic changes being amplification of chromosome 3q. The presence of CNA is associated with the integration of HPV DNA into the host genome, with loss of HPV E2 and/or E2 rearrangement. This suggests a link between CNA and increased HPV-induced chromosomal instability mediated through de-repressed E6 and E7 expression consequent to loss of functional E2 protein. In addition, epigenetic changes occur with increasing frequency in high-grade IN and cancer, such as hypermethylation leading to down-regulation of potential tumor suppressor genes. Analysis of these data together suggests that immune suppression plays a more prominent role in the earlier stages of HPV-associated disease, up to and including incident high-grade IN. Persistent high-grade IN and development of cancer may be more strongly related to the cumulative effect of HPV-associated genetic instability and the resulting host genetic changes. There are few data to suggest a direct role for HIV in the pathogenesis of HPV-associated neoplasia, but HIV-associated attenuation of HPV-specific immune responses may allow for persistence of high-grade IN and sufficient time for accumulation of genetic changes that are important in progression to cancer.

HPV and cervical cytology

Summary Infection with high-risk human papillomaviruses (HPV) is the major risk factor for the development of cervical neoplasia. It is therefore reasonable to propose that the identification of these viruses may be helpful in the context of cervical screening. High-risk HPV infection is strongly associated with cervical neoplasia, particularly if persistent and present in women over the age of 35 years. Therefore, tests for HPV in the context of cervical cytology only need to identify high-risk HPV types. The strengths of HPV testing are high sensitivity and negative predictive value, indicating that the absence of HPV may be of more clinical value than its presence. However, the clinical application of HPV testing should be evaluated in the context of specific populations. HPV testing may have a role in: (i) primary screening; (ii) the assessment of women with mildly abnormal smears; (iii) post-treatment follow-up; and (iv) quality control of cervical cytology. HPV vaccination, if implemented, is likely to alter the epidemiological characteristics of HPV infection and cervical neoplasia. The effects of vaccination on HPV prevalence, HPV-type distribution and HPV-associated neoplasia will need to be monitored closely.

Antiproliferative and antiviral mechanisms of ursolic acid and dexamethasone in cervical carcinoma cell lines

The chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid. Herein, we investigated the antiproliferative and antiviral effects of ursolic acid and dexamethasone in human papillomavirus (HPV)-associated cervical cancer cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay to measure antiproliferative activity, and also characterized apoptosis by DNA fragmentation, 4'-6-diamidino-2-phenylindole (DAPI) staining, and flow cytometry (FACS) analysis. We investigated apoptosis-related proteins using western blots. After in vitro treatment, we used reverse transcription-polymerase chain reaction for the expression of the HPV E6/E7 gene to observe the antiviral effects. Ursolic acid suppressed the growth of HPV-positive cervical carcinoma cells (HeLa, CaSki, and SiHa) in a dose- and time-dependent manner, but not the HPV-negative cervical cancer cell line (C33A). Ursolic acid-treated HeLa cells showed typical apoptosis characteristics in DNA fragmentation, DAPI staining, and FACS analysis. The expression of Fas protein was induced, and caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP) proteins were cleaved after ursolic acid treatment. HPV-18 E6/E7 gene expression decreased after ursolic acid treatment in HeLa cells, but the levels of p53 and Rb proteins did not change. In contrast, dexamethasone, which has a similar structure, did not inhibit proliferation. Our findings may offer new insight into the mechanism of antiproliferative and antiviral effect of ursolic acid. Also, these results suggest that ursolic acid might be a useful anticancer drug in treatment of HPV-associated cervical neoplasia.

P16 Expression in the Female Genital Tract and Its Value in Diagnosis

p16 is a cyclin-dependent kinase-4 inhibitor that is expressed in a limited range of normal tissues and tumors. In recent years, immunohistochemistry with p16 antibodies has been used as a diagnostic aid in various scenarios in gynecologic pathology. Diffuse (as opposed to focal) positivity with p16 in the cervix can be regarded as a surrogate marker of the presence of high-risk human papillomavirus (HPV). In cervical squamous lesions, p16 is positive in most high-grade cervical intraepithelial neoplasia (CIN) and in some cases of low-grade CIN, usually those associated with high-risk HPV. p16 may be useful to identify small focal high-grade CIN lesions, to distinguish some cases of CIN involving immature metaplastic squamous epithelium from immature metaplastic squamous epithelium not involved by CIN and to distinguish high-grade CIN from benign mimics. Most cervical carcinomas of squamous, glandular, and small cell type are p16-positive. In cervical glandular lesions, p16 is useful, as part of a panel, in the distinction between adenocarcinoma in situ (diffusely positive) and benign mimics, including tuboendometrial metaplasia and endometriosis, which are usually p16-negative or focally positive. p16 may be used, in combination with other markers, to distinguish between a cervical adenocarcinoma (diffuse positivity) and an endometrioid-type endometrial adenocarcinoma (negative or focally positive). Some uterine serous carcinomas are diffusely positive. In the vulva, p16 is positive in HPV-associated vulval intraepithelial neoplasia (VIN) but negative in VIN not associated with HPV. Similarly, HPV-associated invasive squamous carcinomas are p16-positive, whereas the more common non-HPV-associated neoplasms are largely negative or focally positive. In the uterus, p16 positivity is more common and widespread in leiomyosarcomas than leiomyomas, and this may be a useful aid to diagnosis, although problematic uterine smooth muscle neoplasms have not been extensively studied. Metastatic cervical adenocarcinomas in the ovary are usually diffusely p16-positive, and because these may closely mimic a primary ovarian endometrioid or mucinous adenocarcinoma, this may be a valuable diagnostic aid, although p16 expression in primary ovarian adenocarcinomas of these morphologic subtypes has not been widely investigated. Some ovarian serous carcinomas, similar to their uterine counterparts, are p16-positive.

Human Papillomavirus (Hpv) Typing in Relation to ras Oncogene mRNA Expression in HPV-Associated Human Squamous Cervical Neoplasia

Human papillomavirus (HPV) has been identified as the principal etiologic agent for cervical cancer and its precursors. Different HPV types have been associated with different oncogenic potential. The purpose of this study was to evaluate the relationship between specific HPV type infection and expression pattern of the ras family oncogenes in different grades of HPV-associated human cervical neoplasia. HPV typing was performed using polymerase chain reaction (PCR) in 31 HPV-positive human cervical specimens from patients with squamous intraepithelial lesions (SIL) or squamous cervical carcinoma (SCC). The mRNA expression levels of H-, K- and N-ras oncogenes were examined using the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Statistical analyses were performed using SPSS software. Among patients with SCC, H-, K- and N-ras expression levels were higher in HPV 16/18-associated cases compared to HPV 16/18-unassociated samples (p=0.003, p=0.004 and p=0.0001, respectively). The expression levels for H-, K- and N-ras were significantly higher in SCC patients with multiple HPV infection compared with SCC patients with single HPV infection (p=0.009, p=0.01 and p=0.021, respectively). Among patients with SIL, no statistically significant relationship was found between ras expression and HPV status. Our findings indicate the possible role of ras signaling interaction with "high-risk" HPV 16/18 and multiple HPV infection in cervical cancer development.

Therapeutic Vaccines for Cervical Cancer: Dendritic Cell-Based Immunotherapy

Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical dysplasia and cervical cancer. Several lines of evidence suggest that cell-mediated immune responses are important in controlling both HPV infections and HPV-associated neoplasia. Since HPV E6 and E7 oncoproteins are expressed in these lesions and are necessary for the maintenance of the malignant phenotype, these proteins might be potential tumor-specific target antigens for immunotherapy of cervical cancer. The gold standard treatment for locally advanced cervical cancer is primary radiation therapy combined with chemotherapy. A potential drawback of this potentially curative treatment is a profound and long lasting negative effect on the immune system. Treatment-induced immunosuppression combined with tumor-induced subversion of the immune system may therefore impose severe limitations on the efficacy of conventional vaccination strategies in late stage cervical cancer patients. The recognition of dendritic cells (DC) as powerful antigen-presenting cells capable of inducing primary T cell responses in vitro and in vivo, has recently generated widespread interest in DC-based immunotherapy of several human malignancies. Here, we review various therapeutic HPV vaccines being developed and implemented in human clinical trials, with a particular emphasis on the use of autologous DC pulsed with full-length HPV 16 or 18 E7 oncoproteins as a novel strategy to induce HPV E7-specific and tumor-specific T cell responses in cervical cancer patients following conventional treatment.

Molecular Basis for Advances in Cervical Screening

Human papillomaviruses (HPVs) cause cervical lesions, which can, in some instances, progress to high-grade neoplasia and cancer. Around half a million cases of cervical cancer occur each year, with most occurring in developing countries where cervical cancer is a major cause of cancer-related death. The reduction in cervical cancer incidence in developed countries is largely attributed to the introduction of cervical screening. Cervical screening currently depends on the identification by cytology of abnormalities in cells taken from the surface of the cervix. The standard Pap test was developed >50 years ago, and despite modifications, still forms the basis of the test currently in use in most routine screening laboratories. Advances in our understanding of the molecular mechanisms that lead to the development of cervical cancer have been slow to impact on screening, despite the relatively high false-negative rates that can be associated with the conventional Pap smear. Improvements in screening strategies fall into a number of categories. Methods that improve cell presentation and attempt to eliminate artefacts/obscuring debris can be combined with image analysis systems in order to enhance diagnostic accuracy. Such approaches still rely on cytological evaluation and do not incorporate advances in our knowledge of how HPV causes cancer. By contrast, markers of virus infection or cell cycle entry, particularly those that offer some degree of prognostic significance, may be able to highlight abnormal cells more reliably than cytology, and could be combined with cytology to improve the detection rate. Our understanding of the molecular biology of HPV infection and the organization of the HPV life-cycle during cancer progression provides a rational basis for marker selection. The general assumption that persistent active infection by high-risk HPV types is the true precursor of cervical cancer provides the rationale for HPV DNA testing in conjunction with enhanced cytology, while the development of RNA-based approaches should allow active infections to be distinguished from those that are latent. The detection in superficial cells of marker combinations at the level of RNA or protein has the potential to predict disease status more precisely than the detection of markers in isolation. There is also a need for better prognostic markers if the predictive value of screening is to be improved. The potential to control infection by vaccination should reduce the incidence of HPV-associated neoplasia in the population, and this may cause a change in the way that screening is carried out. Nevertheless, the lack of a therapeutic vaccine, and the difficulties associated with eliminating infection by multiple high-risk HPV types, means that some form of screening will still be required as a preventive measure for the control of cervical cancer for the foreseeable future.

Anogenital neoplasia in AIDS.

We review the recent literature on anogenital neoplasms in AIDS, with emphasis on cancers associated with HPV infection. Immune reactivity to HPV as well as novel immunotherapeutic and preventative strategies are discussed. Many AIDS-associated neoplasms are associated with HPV infection. Whether cervical cancer is truly an AIDS-associated neoplasm has recently been questioned, while the association of anal cancer with AIDS in both males and females is more convincing. Recent reports cast doubt on the efficacy of HAART therapy for HPV-induced anogenital neoplasms, despite efficacy in improving disease caused by other infectious agents. We include here new data on humoral and cellular immune responses to HPV. VLP serology has been reported to be associated with outcome of cervical cancer. VLP seropositivity has been reported to be a favorable prognostic sign in women with HPV 16 positive cervical carcinoma. Several investigators have questioned the immunogenicity of the oncogenic HPV type 16 compared with other HPV types. It has recently been found that in HIV-infected patients, lymphoproliferative cellular immune responses (CMI) to HPV 16 peptides are not associated with CD4 counts, whereas responses to recall antigens and mitogens are associated with CD4 counts. CD4 + T cells may not be responsible for protective cellular immune responses to HPV. VLP serology and CMI responses may be the future intermediate surrogate biomarkers for HPV-associated anogenital neoplasia trials. Several new therapeutic vaccine strategies for management of HPV-induced neoplasia are reviewed. Most anogenital neoplasms occurring with increased frequency in patients with HIV/AIDS are associated with HPV infections. Current treatment strategies are not effective in clearing anogenital HPV infection and need improvement. Immunotherapy with novel vaccines will provide both prevention and therapy for these common malignancies.