Cervical cancer is the second most common cause of death from cancer in women worldwide, and the development of new diagnostic, prognostic, and treatment strategies merits special attention. Many efforts have been made to design new drugs and develop immunotherapy and gene therapy strategies to treat cervical cancer. HPV genotyping has potentially valuable applications in triage of low-grade abnormal cervical cytology, assessment of prognosis and followup of cervical intraepithelial neoplasia, and in treatment strategies for invasive cervical cancer. It is known that during the development of cervical cancer associated with HPV infection, a cascade of abnormal events is induced, including disruption of cellular cycle control, alteration of gene expression, and deregulation of microRNA expression. Thus, the identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis, identifying cervical cancer molecular markers, and developing specific targeting strategies against tumor cells. Therefore, in this paper, we discuss the main diagnostic methods, management strategies, and followup of HPV-associated cervical lesions and review clinical trials applying gene therapy strategies against the development of cervical cancer.
Currently, 500,000 new cases of cervical cancer are diagnosed and 280,000 deaths occur each year worldwide, making cervical cancer the second most common malignancy affecting women worldwide [
During the last 50 years, screening programs based on conventional cytology have significantly reduced cervical cancer morbidity and mortality [
Once the disease has spread beyond the confines of a radiotherapy, chemotherapy, or surgical field, no standard treatment is currently available. However, new molecular targeted drugs being tested in clinical trials might inhibit tumor progression and increase apoptosis, resulting in tumor response or stabilization; furthermore, gene therapy strategies could also be employed for the control of HPV-associated precancerous cervical lesions. In this paper, we discuss the role of HPV in cervical cell transformation and the main methods for the detection of high-risk HPVs, with special attention to application of HPV testing in screening, treatment, and followup of precancerous cervical intraepithelial neoplasia. We also examine the major gene therapy strategies against cervical cancer development that have been tested or are being tested in clinical trials.
Currently, over 120 different types of HPV have been identified based on the genotype [
The early region (“E”) of the HPV genomes covers more than 50% of the virus genome and contains the E1, E2, E4, E5, E6, and E7 genes that are carefully expressed at the early and late stages of infection. The early proteins E1 and E2 are involved in viral DNA replication and viral RNA transcription, E4 is involved in cytoskeleton reorganization and E5, E6, and E7 are responsible for cellular transformation, and immortalization [
HPV16
HPV E5 oncoprotein interacts with cellular host proteins, and these interactions are important for HPV’s biological activity in cell transformation and evasion of the host immune response. Recent studies have highlighted the important role of E5 in cell transformation, tumorigenesis, and immune modulation, thus implicating E5 in pivotal steps of carcinogenesis [
An early event in HPV-associated carcinogenesis during HPV DNA integration is a global perturbation of cellular gene expression by the E6 and E7 oncoproteins. The high-risk HPV E6 oncoprotein contains approximately 150 aminoacids and has two zinc finger motives formed by four C-X-X-C motives that are required for E6 functions [
Two new proteins codified in the early gene region have been recently identified, E3 and E8, which are present only in a few papillomavirus types (HPV 1, 11, 16, 31, and 33). A fusion protein, E8
The late region (“L”) of all HPV genomes, comprising almost 40% of the virus genome, is located downstream of the early region and encodes L1 and L2 ORFs for translation of a major (L1) and a minor (L2) capsid protein. Finally, the long control region (LCR), a segment of about 850 bp (
Major functions of HPV proteins.
HPV proteins | Functions | Reference |
---|---|---|
E1 | Involved in productive HPV replication. | [ |
E2 | Regulates transcription from different HPV promoters. | [ |
E3 | Recently identified gene located in early gene region and found only in a few papillomavirus types (HPV1, 11, 16, 31, 33); its function has not been identified. | [ |
E4 | Represent up to 30% of total wart protein produce by HPV-1a and might serve as scaffold, transport, or structural protein. | [ |
E5 | Inactivates with cellular host proteins (MHC I, Bap31); these interactions are important for the biological activity of the protein in cell transformation and evasion of the immune response. |
[ |
E6 | Inactivates the tumor suppressor protein p53, involved in the control of cell proliferation and cell response to genotoxic stress and in DNA damage. | [ |
E7 | Interacts with the pRb tumor suppressor protein. These interactions influence the gene expression involved in progression to S-phase of the cell cycle, such as cyclin A, cyclin D, and cyclin E genes. | [ |
E8 | Recently identified gene located in early gene region and found only in a few papillomavirus types (HPV 1, 11, 16, 31, 33). A fusion protein, E8 |
[ |
L1 |
Major capsid protein. |
[ |
There is a close relationship between high-risk HPV infection and cervical cancer development. However, it is well documented that HPV is a very common sexually transmitted virus, and most infections do not cause cervical intraepithelial neoplasia. Epidemiological studies have reported HPV prevalence between 10% and 40% in women with no cytological abnormalities [
The process by which cervical cancer develops is complex, and a combination of environmental, viral, and host factors together increases the risk of progression. Three main HPV-liked molecular events have been identified as triggers of carcinogenesis: (a) viral DNA integration in the host genome; (b) expression of viral proteins (E1, E2, E3, E4, E5, E6, and E7); (c) interactions between E2, E6/E7, and cellular proteins. In general, HPV epithelial infections occur either through mechanical microabrasions or infection of the area, where basal cells undergo a transformation from a columnar to a squamous epithelium. When basal cells are infected, they migrate to the lumen and express the capsid genes L1 and L2. In subclinical infections of low-grade lesions, the viral genome is then replicated as an episome and encapsidated into the nuclei of cells in the upper layer epithelium. Thus, protected viral particles are able to infect new areas of epithelium or be sexually transmitted. In some cases, the infection progresses to high-grade lesions and cervical carcinoma, a process associated with the integration of the HPV genome into the host genome due in part to the loss of the transcriptional repression exerted by the E2 protein.
Invasive cervical carcinoma is preceded by precursor lesions, which are characterized by disturbances of cellular maturation and stratification, as well as atypical nuclei, which tend to promote cellular immortalization and transformation [
Recommendations for cervical cancer screening released in 2012 by the US Preventive Services Task Force (USPSTF) and separately by a partnership among the American Cancer Society/American Society for Colposcopy and Cervical Pathology/American Society for Clinical Pathology (ACS/ASCCP/ASCP) no longer indicate annual screening with cervical cytology but rather screening at three- to five-year intervals with cytology and optional incorporation of testing for high-risk HPV infection. For women aged 21–29, the guidelines indicate screening with cytology (i.e., the Papanicolaou test) every three years; HPV testing is not recommended for this age group because younger women are more likely to experience transient infections and low-grade lesions that do not progress to precancerous lesions and do not require identification or treatment. Women older than 65 with evidence of adequate negative prior screening and no history of CIN2+ within the last 20 years should not be screened. Screening should not be resumed for any reason, even if a woman reports having a new sexual partner. For women aged 30–65, however, both sets of guidelines indicate screening with a combination of cytology and HPV testing every five years, the “preferred” method, or screening with cervical cytology alone every three years (“acceptable”) [
The US Food and Drug Administration (FDA) currently approves three HPV DNA tests for clinical use: Hybrid Capture 2, manufactured by Qiagen; Cervista HPV HR (Hologic); the cobas HPV test (Roche Molecular Systems). The most widely used test is Hybrid Capture 2 (HC2), a second-generation commercial test designed to detect HPV types classified as high-risk. HC2 is a nucleic acid hybridization assay that utilizes an HPV RNA probe mixture containing probes for 13 high-risk HPVs. Samples containing HPV DNA hybridize with the RNA probe and are captured on a plate coated with antibodies conjugated to alkaline phosphatase; when alkaline phosphatase’s substrate is added, light is emitted and measured via luminometer. The test’s limitations include possible cross-hybridization of low-risk with high-risk HPV types as well as possible false positives in contiguous samples due to chemiluminescent emission of high-load HPV samples [
An established method for the detection of HPV DNA is PCR amplification with primers derived from consensus sequences of either the conserved E1 or L1 open reading frames of the viral genome. The primer sets MY09/11 and GP5+/GP6+ are both able to amplify a wide range of HPV types. The MY09/11 primers are synthesized with several degenerate nucleotides and constitute a mixture of 25 primers, while the GP5+/GP6+ primers have fixed nucleotide sequences but use a lower annealing temperature during PCR to detect more HPV types [
Baleriola et al. [
Another detection method, the HPV oligonucleotide microarray (commercially available from Biomedlab Co., Republic of Korea), was evaluated by An et al. [
The application of HPV DNA testing alone or in combination with cervical cytology in screening, treatment, and followup of cervical cancer has been the focus a great deal of research in the past two decades. A 2006 meta-analysis of split-sample studies in Europe and North America that compared HPV testing with routine cytology found that HPV testing had significantly higher sensitivity than cytology, while cytology had a higher positive predictive value (PPV), in detecting a cervical intraepithelial lesion of grade II or worse (CIN2+), confirmed via subsequent histologic examination [
Several recently published studies have examined long-term outcomes of screening programs that incorporate HPV testing. A large Netherlands-based randomized controlled trial, the Population-Based Screening Study Amsterdam (POBASCAM), determined that women who were screened for HPV DNA (with the GP5+/6+ PCR method) in combination with cytology had fewer cervical intraepithelial neoplasias of grade 3 (CIN3) and fewer cervical carcinomas in a subsequent screen five years later, than those who were screened with cytology alone [
High-risk-HPV testing may be particularly valuable in low-resource settings, where the majority (80%) of cervical cancer cases occur, and where the infrastructure does not exist to implement effective cytology-based screening programs. In Colombia, for example, guidelines issued by the National Cancer Institute recommend screening based on a 1-1-3 cytology strategy (annual conventional cytology until two consecutive negative smears and every three years thereafter); however, coverage rates are low, with one-year coverage of women aged 25–69 ranging from 43.3% in the state of Boyacá to 56.8% in Caldas and three-year year coverage ranging from 66.6% in Magdalena to 81.3% in Caldas [
The technical requirements of most HPV-DNA tests, however, make them impractical for use in low-resource settings. A new HPV test designed specifically for use in developing countries was assessed for clinical accuracy in screening tests in county hospitals in rural China [
Following histologic identification of cervical intraepithelial neoplasia, surgical treatment is recommended for preinvasive lesions of grade CIN2 or CIN3. The 2006 Consensus Guidelines published by American Society for Colposcopy and Cervical Pathology (ASCCP) and its partner organizations utilize a two-tiered system in which histologically diagnosed CIN1 is classified as a low-grade lesion and CIN2/3 are classified as high-grade precursors [
Although the risk of CIN2 progressing to invasive cancer is low, diagnosis of CIN2 has limited reproducibility and validity, and thus CIN2 is used as the treatment threshold for safety reasons [
Postsurgical followup of women treated for CIN 2/3 with ablative or excisional methods is critical, since treatment failure rates are estimated at 5%–15% across all treatment modalities [
If lesions are not identified and treated at the precancerous stages, treatment options are limited for women with metastatic or recurrent cervical cancer. Unfortunately, only up to one third of patients with metastatic and recurrent disease will respond to drug chemotherapy, and these responses are short-lived, on the order of months. Gene therapy strategies targeted to HPV products could represent new treatment options for cervical cancer and also other tumors in which HPV participates as a cancer promoter. Currently, it is well known that HPV E6 and E7 interact with a plethora of cellular proteins, both nuclear and cytoplasmic, and participate in molecular pathways involved in the activation and establishment of the tumor phenotype. Hence, it is feasible to design experimental strategies aimed to block the expression of viral oncoproteins. HPV E6 and E7 oncoproteins are excellent candidates for HPV therapeutic vaccination strategies, and the most of the therapeutic vaccines that have been tested in preclinical and clinical trials focus on interacting with antigen-presenting cells to stimulate cytokine production and T-cell activation. In addition, several efforts are focusing on the development HPV therapeutic vaccines based in attenuated virus and bacterial vectors, HPV peptide and proteins, and specific tumor and dendritic cells, as well as naked plasmid DNA-based vaccines.
Table
Active gene therapy clinical trials for cervical cancer worldwide from 1989 to 2012.
ID trial | Trial title/country | Indication/clinical phase | Status/year approved-initiated | Gene(s) transferred | Vector used/administration route | Gene delivery |
---|---|---|---|---|---|---|
BE-0024 | A randomized, double blind, placebo-controlled, parallel group, multicenter study of the safety and response rate of 3 subcutaneously administered doses of 5 × 107 pfu RO5217790 in patients with high grade cervical intraepithelial neoplasia grade 2 or 3 associated with high risk HPV infection. Belgium | Cervical intraepithelial neoplasia. |
Open |
(i) delE6 |
Vaccinia virus/ND | ND |
CN-0010 | Gendicine intratumoral injection combined with radiotherapy for advanced cervical carcinoma. China | Cervical carcinoma. |
Open |
p53 | Adenovirus/intramuscular | ND |
ES-0010 | A randomized, double blind, placebo-controlled, parallel group, multicenter study of the safety and response rate of 3 subcutaneously administered doses of 5 × 107 pfu RO5217790 in patients with high grade cervical intraepithelial neoplasia grade 2 or 3 associated with high risk HPV infection. Spain | Cervical intraepithelial neoplasia. |
Open |
(i) delE6 |
Vaccinia virus/ND | ND |
FR-0032 | Phase II trial to assess efficacy of TG4001 (MVA-HPV-IL2) in patients with grade 2/3 cervical intra epithelial neoplasia (CIN 2/3) linked to HPV16 infection (protocol TH4001.07). France | CIN 2 and 3. |
Open |
(i) IL-2 |
Vaccinia virus/ND | ND |
MX-0001 | Clinical protocol. A phase II study. Efficacy of the gene therapy of the MVA E2 recombinant virus in the treatment of precancerous lesions (NIC I and NIC II) associated with infection of oncogenic human papillomavirus. Mexico | Cervical cancer. |
Open |
ND | Adenovirus/ND | ND |
UK-0041 | Use of a recombinant vaccinia vaccine (TA-HPV) to treat cervical intraepithelial neoplasia III. UK | Cervical intraepithelial neoplasia III. |
Open |
HPV E6 and E7 oncogenes | Poxvirus/ND | ND |
UK-0042 | Use of a recombinant vaccinia vaccine (TA-HPV) to treat cervical intraepithelial neoplasia III. UK | Cervical intraepithelial neoplasia III. |
Open |
HPV E6 and E7 oncogenes | Poxvirus/ND | ND |
UK-0071 | A phase II, multicenter, double-blind, placebo-controlled, dose finding study of ZYC101a in the treatment of high-grade squamous intraepithelial lesions of the uterine cervix. UK | Anogenital neoplasia III. |
Open |
HPV E6 and E7 oncogenes | Naked plasmid DNA/ND | ND |
UK-0074 | TA-HPV recombinant vaccinia virus expressing the human papillomavirus 16 and 18 E6 and E7 proteins: application to amend currently approved protocol to add a clinical trial involving prime-boost strategy of TA-CIN administered in association with TA-HPV in high grade anogenital intraepithelial neoplasia (AGIN) patients (PB-HPV/01). UK | Cervical cancer. |
Open |
HPV E6 and E7 oncogenes | Vaccinia virus/ND | ND |
US-0592 | A phase 1 study to determine the safety and immunogenicity of vaccination with Listeria monocytogenes expressing human papilloma virus type 16 E7 for the treatment of progressive, recurrent, and advanced squamous cell cancer of the cervix. USA | Cervical cancer. |
Open |
HPV E7 oncogene | Listeria monocytogenes/intravenous |
|
US-0595 | A phase I/II clinical trial of pNGVL4a-Sig/E7 (detox)/HSP70 for the treatment of patients with HPV16+ cervical intraepithelial neoplasia 2/3 (CIN2/3). USA | Cervical cancer. |
Open |
HPV16 E7 oncogene | Naked plasmid DNA/intramuscular |
|
US-0916 | Phase I, open-label, dose escalation study to evaluate the safety, tolerability, and immunogenicity of human papillomavirus (HPV) DNA plasmid (VGX-3100) + electroporation (EP) in adult females with histological diagnosis of grade 2 or 3 cervical intraepithelial neoplasia (CIN). USA | Cervical cancer. |
Open |
(i) HPV16 E6 and E7 oncogenes |
Naked plasmid DNA/intramuscular |
|
US-0928 | A phase I efficacy and safety study of HPV16-specific therapeutic DNA-r vaccinia vaccination in combination with topical imiquimod in patients with HPV16+ high grade cervical dysplasia (CIN3). USA | HPV16+ high-grade cervical dysplasia. |
Open |
(i) HPV16 + HPV18 |
Naked plasmid DNA + Vaccinia virus/intramuscular |
|
US-0958 | A randomized, double blind, placebo-controlled, parallel group, multicenter study of the safety and response rate of 3 subcutaneously administered doses of 5 × 107 pfu R05217790 in patients with high grade cervical intraepithelial neoplasia grade 2 or 3 associated with high risk HPV infection. USA | Cervical intraepithelial neoplasia (CIN). |
Open |
(i) HPV E6 and E7 oncogenes |
Vaccinia virus/intramuscular |
|
US-0984 | A pilot study of pNGVL4a-CRT/E7(detox) for the treatment of patients with HPV16+ cervical intraepithelial neoplasia 2/3 (CIN2/3). USA | Cervical cancer. |
Open |
HPV16 E7 oncogene | Naked plasmid DNA/intramuscular |
|
US-1040 | Phase I, open-label study to evaluate the safety, tolerability, and immunogenicity of a fourth dose of human papillomavirus (HPV) DNA plasmid (VGX-3100) + electroporation (EP) in adult females previously immunized with VGX-3100. USA | Cervical cancer. |
Open |
(i) HPV16 E6 and E7 oncogenes |
Naked plasmid DNA/intramuscular |
|
US-1082 | A phase II evaluation of ADXS11-001 (NSC #752718, IND # 13,712) in the treatment of persistent or recurrent squamous or on-squamous cell carcinoma of the cervix. USA | Cervical cancer. |
Open |
HPV E7 oncogene | Listeria monocytogenes/intravenous |
|
US-1093 | Phase II placebo-controlled study of VGX-3100, (HPV16 E6/E7, HPV18 E6/E7 DNA Vaccine) delivered IM followed by electroporation (Ep) with cellectra-5p for the treatment of biopsy-proven CIN 2/3 or CIN 3 with documented HPV 16 or 18. USA | Cervical cancer. |
Open |
(i) HPV16 E6-E7 fusion protein |
Naked plasmid DNA/intramuscular |
|
Clinical trial information obtained from
ND: no data provided.
Note. The table was created by the authors of this paper with the information obtained from
Although there is currently limited clinical information about gene therapy in cervical cancer patients, then trials discussed could establish proof of concept; therefore, it could be feasible to use gene therapy
On the other hand, although many studies have described the induction of cancer cell death
Although early stage cervical cancers have a good prognosis with a 5-year survival rate greater than 80%, clinical and epidemiological evidence suggests that the natural history of HPV in young women (aged <30 years) may be such that establishment of a high-grade CIN lesion occurs early in the course (within 2 years) of a high-risk HPV infection. The consequences of HPV infection will depend on the infecting HPV type and site of infection, as well as on host factors that regulate viral persistence, regression, and latency. HPV testing and identification of high-risk strains and multiple infections has potential applications in the screening, treatment, and followup of cervical intraepithelial lesion. The identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins is the major challenge for their utilization as molecular biomarkers and may provide useful information in understanding cervical carcinogenesis for development of specific targeting strategies against tumor cells. Many experimental HPV vaccine strategies are being developed and tested in preclinical and clinical trials, in combination with immunotherapy and chemotherapy approaches to control of HPV-associated cervical lesions and invasive cancers. The principal factor for the prevention and treatment of cervical cancer is the education of the society, which requires different strategies in developing and industrialized countries [
In conclusion, educational strategies and organized screening programs to detect HPV must be implemented alongside research and development of new therapeutic vaccines infection in order to reduce rates of HPV infection and cervical cancer. New gene therapy and siRNA-based approaches could represent a major step in reducing morbidity and mortality, but more work is required to achieve clinical efficacy at a level that can challenge current therapy.
None of the authors has any financial conflict of interests related to the submitted paper.
This paper was carried out at the National Institute of Public Health in México, and it received federal financial support from the institute, as well as from the National Council of Science and Technology (CONACYT) with File nos. 46151, SALUD-2008-01-87130, and SALUD-2009-01-111892.