Margaret Stanley

Pathology and epidemiology of HPV infection in females

Human papillomaviruses (HPVs) are a large family of small double-stranded DNA viruses that infect squamous epithelia. It has been established that infection with specific HPV types is a contributing factor to different types of anogenital cancer, including vulval, vaginal, anal, penile, and head and neck cancers. Approximately 4% of all cancers are associated with HPV. HPV infection is the major cause of cervical cancer and genital warts. Genital HPV infections are very common, are sexually transmitted, and have a peak prevalence between ages 18 and 30. Most of these infections clear spontaneously, but in 10-20% of women, these infections remain persistent and are at risk of progression to grade 2/3 cervical intraepithelial neoplasm (CIN) and eventually to invasive cancer of the cervix (ICC). CINs are genetically unstable lesions with a 30-40% risk of progression to ICC. If left untreated, they form a spectrum of increasing cytological atypia, ranging from low-grade CIN1 to high-grade CIN3; the latter are caused almost exclusively by high-risk HPVs, HPV 16 and 18. Infection with HPV requires a microabrasion in the genital epithelium. The oncogenic properties of high-risk HPV reside in the E6 and E7 genes, which if inappropriately expressed in dividing cells deregulate cell division and differentiation. HPV DNA testing has been shown consistently to be superior to cytology in terms of sensitivity and positive predictive value and will become a major tool in cervical cancer screening, at least in the developed countries.

A Randomized, Controlled, Molecular Study of Condylomata Acuminata Clearance during Treatment with Imiquimod

Imiquimod, an immune response modifier, has been demonstrated to be safe and effective in the treatment of external genital and perianal warts caused by human papillomavirus (HPV). To identify the molecular mechanism(s) by which condylomata acuminata clear during topical treatment with imiquimod, wart skin biopsies were taken from patients before treatment, at treatment week 6, and at the end of treatment. Tissues were analyzed for HPV DNA and for mRNA of several cytokines and HPV gene products. Wart clearance was associated with evidence of tissue production of interferon-alpha, -beta, and -gamma and tumor necrosis factor-alpha. Regression of warts was strongly associated with a decrease in HPV DNA and in mRNA expression for both early and late viral proteins. Thus, topical imiquimod treatment of anogenital warts led to significant increases in local production of multiple interferon mRNAs and a significant reduction in virus load as measured by decreases in HPV DNA and mRNA for early HPV proteins.

Immunobiology of HPV and HPV vaccines

Genital human papillomavirus (HPV) infection with both low- and high-risk types is common, but most infections resolve as a result of a cell-mediated immune response. Failure to induce an effective immune response is related to inefficient activation of innate immunity and ineffective priming of the adaptive immune response; this defective immune response facilitates viral persistence, a key feature of high-risk HPV infection. This milieu becomes operationally HPV antigen tolerant, and the hosts defenses become irrevocably compromised. HPV antigen-specific effector cells are poorly recruited to the infected focus and their activity is downregulated; neoplastic HPV containing cervical keratinocytes expressing high levels of E6 and E7 oncoproteins are not killed in this immunosuppressive, tolerant milieu, and progression to high-grade disease and cancer can result. Highly efficacious prophylactic HPV L1 virus-like particle (VLP) vaccines circumvent viral epithelial evasion strategies since they are delivered by intramuscular injection. The stromal dendritic cells of the muscle that encounter the highly immunogenic repeat structure of the VLP then migrate with their cargo to the lymph node, initiating an immune cascade that results in a robust T-cell dependent B-cell response, which generates high levels of L1-specific serum neutralizing antibodies and immune memory.

Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line

The W12 cell line was derived from a low grade cervical lesion, and is unique among HPV16-containing cell lines in carrying its HPV16 genome as a multicopy episome. As such it is thought to be more representative of a premalignant HPV16-induced tumor than the cervical cancers from which other cell lines have been derived. Using the polymerase chain reaction (PCR), we report here the identification and cloning of a number of novel cDNA species, which appear to be characteristic of the W12 cell line. Two species were identified with E6* coding capacity (E6*I and E6*III). The smaller of these (1009 bp) was predicted to encode a novel E6*III polypeptide containing C-terminal amino acids derived from an out of frame region of the E2/E4 ORFs. The larger species (1480 bp) contained, in addition to the E6*I ORF, an intact E7 ORF and probably represents the transcript for E7 expression, as the E7 protein was readily detectable in the W12 cell line. Both species appeared to be transcribed from the p97 promoter which has been shown to be active in other cell lines. A putative E2 repressor cDNA (891 bp), an E1/E4 message (883 bp), and two novel late cDNA species (1757 and 2031 bp) were also detected, allowing the identification of a splice acceptor immediately in front of the L1 open reading frame (nt 5637) and a splice donor at nt 3631. Although the 1757-base species has the capacity to encode a full-length L1 protein, both messages use a splice donor at nt 1301, and are thus not analogous to late species previously identified in HPV11. Of the six cDNAs cloned, only the 1480-bp E7 message has been observed in other HPV16-containing cell lines. The presence of L1 transcripts, and an E2 repressor mRNA, although unexpected, may reflect the different origins of the W12 cell line.

Acquisition of High-Level Chromosomal Instability Is Associated with Integration of Human Papillomavirus Type 16 in Cervical Keratinocytes

Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the “physiologically” elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by “natural” infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3′ deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.

Epithelial Cell Responses to Infection with Human Papillomavirus

SUMMARY Human papillomavirus (HPV) infection of the genital tract is common in young sexually active individuals, the majority of whom clear the infection without overt clinical disease. Most of those who do develop benign lesions eventually mount an effective cell-mediated immune (CMI) response, and the lesions regress. Regression of anogenital warts is accompanied histologically by a CD4+ T cell-dominated Th1 response; animal models support this and provide evidence that the response is modulated by antigen-specific CD4+ T cell-dependent mechanisms. Failure to develop an effective CMI response to clear or control infection results in persistent infection and, in the case of the oncogenic HPVs, an increased probability of progression to high-grade intraepithelial neoplasia and invasive carcinoma. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections. The viral infectious cycle is exclusively intraepithelial: there is no viremia and no virus-induced cytolysis or cell death, and viral replication and release are not associated with inflammation. HPV globally downregulates the innate immune signaling pathways in the infected keratinocyte. Proinflammatory cytokines, particularly the type I interferons, are not released, and the signals for Langerhans cell (LC) activation and migration, together with recruitment of stromal dendritic cells and macrophages, are either not present or inadequate. This immune ignorance results in chronic infections that persist over weeks and months. Progression to high-grade intraepithelial neoplasia with concomitant upregulation of the E6 and E7 oncoproteins is associated with further deregulation of immunologically relevant molecules, particularly chemotactic chemokines and their receptors, on keratinocytes and endothelial cells of the underlying microvasculature, limiting or preventing the ingress of cytotoxic effectors into the lesions. Recent evidence suggests that HPV infection of basal keratinocytes requires epithelial wounding followed by the reepithelization of wound healing. The wound exudate that results provides a mechanistic explanation for the protection offered by serum neutralizing antibody generated by HPV L1 virus-like particle (VLP) vaccines.

HPV: from infection to cancer

Infection with HPV (human papillomavirus) 16 is the cause of 50% or more of cervical cancers in women. HPV16 infection, however, is very common in young sexually active women, but the majority mount an effective immune response and clear infection. Approx. 10% of individuals develop a persistent infection, and it is this cohort who are at risk of cancer progression, with the development of high-grade precursor lesions and eventually invasive carcinoma. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and release is not associated with inflammation. Furthermore, HPV infections disrupt cytokine expression and signalling with the E6 and E7 oncoproteins particularly targeting the type I IFN (interferon) pathway. High doses of IFN can overcome the HPV-mediated abrogation of signalling, and this may be the basis for the therapeutic effects on HPV infections of immune-response modulators such as the imidazoquinolones that induce high levels of type I IFNs by activation of TLR (Toll-like receptor) 7. Using the unique W12 model of cervical carcinogenesis, some of these IFN-related interactions and their relevance in the selection of cells with integrated viral DNA in cancer progression have been investigated. Our data show that episome loss associated with induction of antiviral response genes is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. Exogenous IFN-beta treatment of W12 keratinocytes in which the majority of the population contain episomes results only in the rapid emergence of IFN-resistant cells, loss of episome-containing cells and a selection of cells containing integrated HPV16 in which the expression of the transcriptional repressor E2 is down-regulated, but in which E6 and E7 are up-regulated.

HPV & head and neck cancer: a descriptive update

The incidence of head and neck squamous cell carcinoma (HNSCC) has been gradually increasing over the last three decades. Recent data have now attributed a viral aetiology to a subset of head and neck cancers. Several studies indicate that oral human papillomavirus (HPV) infection is likely to be sexually acquired. The dominance of HPV 16 in HPV+ HNSCC is even greater than that seen in cervical carcinoma of total worldwide cases. Strong evidence suggests that HPV+ status is an important prognostic factor associated with a favourable outcome in head and neck cancers.Approximately 30 to 40% of HNSCC patients with present with early stage I/II disease. These patients are treated with curative intent using single modality treatments either radiation or surgery alone. A non-operative approach is favored for patients in which surgery followed by either radiation alone or radiochemotherapy may lead to severe functional impairment. Cetuximab, a humanized mouse anti-EGFR IgG1 monoclonal antibody, improved locoregional control and overall survival in combination with radiotherapy in locally advanced tumours but at the cost of some increased cardiac morbidity and mortality.Finally, the improved prognosis and treatment responses to chemotherapy and radiotherapy by HPV+ tumours may suggest that HPV status detection is required to better plan and individualize patient treatment regimes.

HPV - immune response to infection and vaccination

HPV infection in the genital tract is common in young sexually active individuals, the majority of whom clear the infection without overt clinical disease. However most of those who develop benign lesions eventually mount an effective cell mediated immune (CMI) response and the lesions regress.Failure to develop effective CMI to clear or control infection results in persistent infection and, in the case of the oncogenic HPVs, an increased probability of progression to CIN3 and invasive carcinoma. The prolonged duration of infection associated with HPV seems to be associated with effective evasion of innate immunity thus delaying the activation of adaptive immunity.Natural infections in animals show that neutralising antibody to the virus coat protein L1 is protective suggesting that this would be an effective prophylactic vaccine strategy. The current prophylactic HPV VLP vaccines are delivered i.m. circumventing the intra-epithelial immune evasion strategies. These vaccines generate high levels of antibody and both serological and B cell memory as evidenced by persistence of antibody and robust recall responses. However there is no immune correlate - no antibody level that correlates with protection. Recent data on how HPV infects basal epithelial cells and how antibody can prevent this provides a mechanistic explanation for the effectiveness of HPV VLP vaccines.

Human Papillomavirus Type 16 E6 Amino Acid 83 Variants Enhance E6-Mediated MAPK Signaling and Differentially Regulate Tumorigenesis by Notch Signaling and Oncogenic Ras

ABSTRACT Oncogenically high-risk human papillomaviruses (HPVs) are causally associated with the progression of major human neoplasia-like cancers of the cervix. Several studies have defined functions of the key E6 and E7 oncoproteins in epithelial cell immortalization. The roles of these oncogenes in the progression of immortalized epithelial cells to invasive tumors are still poorly understood. Here, we establish a novel link between the E6 oncoprotein and activation of mitogen-activated protein kinase (MAPK) signaling and show that this signaling involves Rap1. We find that activated MAPK signaling cooperates with deregulated Notch1 signaling to recreate features of HPV-driven invasive cervical carcinomas. We extend our analysis to evaluate an E6 (amino acid [aa] 83) variant that has been linked to invasive tumors. The variant enhances MAPK signaling and cooperative transformation with deregulated Notch1 signaling. Unlike E6, this variant surprisingly inhibits oncogenic Ras-mediated transformation. Our data reveal that the quantitative differences in activation of MAPK signaling by E6 and its variant correlate with differences in cooperative transformation with other signaling pathways, thus suggesting that thresholds of MAPK activation may define permissive conditions for other signaling pathways in tumorigenesis. Epidemiological studies have suggested the importance of E6 aa 83 variants in invasive carcinomas; our data support a key deterministic role for this variant in human cervical tumorigenesis. These observations, along with our recent data showing that deregulated Notch signaling activates phosphatidylinositol 3-kinase signaling, strengthen the possibility of the existence of Ras-independent mechanisms to recreate signaling through classical Ras effector pathways.