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30 Current and Future Vaccines to Prevent HPV-associated Cancers
Symposia
european journal of cancer 48, suppl. 5 (2012) S5–S12
more that 70% of patients can now be successfully treated. The future is even brighter with many compounds in the pipeline against both virus and host targets, raising hopes for an all oral interferon-free regimen with a high cure rate. 28 Epstein–Barr Virus − Pathogenesis and Immunobiology A. Rickinson1 . 1 Institute of Cancer Studies, University Edgbaston, Birmingham, United Kingdom Epstein–Barr virus (EBV), a B-lymphotropic herpesvirus widespread in human populations, is aetiologically linked to a range of malignancies. These include (i) three tumours of B cell origin, post-transplant lymphoproliferative disease (PTLD), endemic Burkitt lymphoma (BL) and a subset of Hodgkin lymphomas, (ii) a specific subset of T or NK cell lymphomas, often of nasal type, and (iii) nasopharyngeal carcinoma (NPC) and a minor subset of gastric carcinomas with similar histology. Each of these tumours carry episomal EBV genomes in every malignant cell but display distinct patterns of virus latent gene expression. These patterns not only reflect the different contributions EBV makes to disease pathogenesis in different tumour contexts, but also have implications for immune-based therapies by restricting the number of viral antigens that are available for tumour targeting. To illustrate the versatility of EBV as a tumour virus, recent studies of B cell infections in vitro have shed new light on how virus-driven growth transformation, with full latent gene expression, can contribute to PTLD pathogenesis. By contrast, two more-restricted forms of latency are found in endemic BL, both of which act not to promote growth but to protect tumour cells from the pro-apoptotic effects of c-myc gene deregulation. While the c-myc translocation is a characteristic of both endemic (EBV-positive) and sporadic (largely EBV-negative) BL, recent work is identifying additional cell gene mutations whose patterns differ between the endemic and sporadic diseases, again reflecting their subtly different pathogenetic routes. Much still remains to be learned about the biology of EBV infection in vivo, its persistence within the B cell system, its occasional transmission to T and NK cells, and its varied contributions to human lymphomagenesis. 29 Proffered Paper: Detection of Hypermutation in Human Papillomavirus DNA Sequences From Cervical Samples V. Correa Vieira1 , J.D. Siqueira1 , A.R.I. Meyrelles2 , G.L. Almeida2 , H.N. Seuanez1 , E.S. Machado2 , E.A. Soares1 , M.A. Soares1 . 1 Instituto ˆ Nacional de Cancer Jose´ Alencar Gomes da Silva − INCA, Programa de ´ Genetica, Rio de Janeiro − RJ, Brazil, 2 Universidade Federal do Rio de ´ Janeiro, Hospital Universitario Clementino Fraga Filho, Rio de Janeiro − RJ, Brazil Persistent infection with high-risk human papillomavirus (HPV) is the main risk factor for development of cervical neoplasia. However, the factors leading to viral clearance or persistence are poorly understood. It has been shown that APOBEC3 proteins have antiviral effect against several viruses, such as HIV, inducing extensive G-to-A hypermutation into the viral genome. However, its role in the course of HPV infection and cancer development is unknown. Recently, evidence was shown for editing of HPV-16 DNA by APOBEC3 proteins in precancerous lesions. The aim of this study was to verify the presence of G-to-A mutations introduced by APOBEC3 into HPV sequences and to investigate its correlation with HPV-associated cervical lesions. DNA was extracted from genital scrapes using a commercial kit (QIAamp DNA Mini Kit® ). To detect hypermutation, a 370 bp fragment of the HPV LCR region was amplified by 3D-PCR, which enriches the amplification of ATrich sequences. PCR products were cloned, and at least ten clones from each sample were analyzed by colony PCR. Products were sequenced and hypermutation analysis was performed using Hypermut 2.0. A total of 68 viral sequences recovered from 5 patients were analyzed. At the time of sample collection, patients had normal cytology (n = 2), LSIL (n = 1) or HSIL (n = 2), and all were infected by HPV-16. Among the sequences analyzed, four drastically hypermutated sequences were found (p < 0.05), in addition to several sequences with elevated levels of G-to-A changes. Hypermutated sequences had a total of 65 G-to-A changes versus 4 non G-to-A changes. The preferential dinucleotide context of changes was GG (58%), suggesting a predominant activity of specific APOBEC3 proteins. All altered sequences belonged to a single patient, who had normal cytology at time of sample collection and progressed to LSIL within 6 months of follow-up. These results corroborate a previous report showing evidence for APOBEC3 editing of HPV genome in vivo. However, this is the first time that HPV hypermutation is found in cervical samples in the absence of cytological abnormalities, suggesting an early role of APOBEC3 proteins in the natural course of HPV cervical infection, despite clinical evolution to intraepithelial lesion. Considering that targeting APOBEC enzymes represents a potential antiviral strategy, further studies should be conducted to elucidate the role of hypermutation in the progression of HPV-associated cervical lesions.
S7
30 Current and Future Vaccines to Prevent HPV-associated Cancers D.R. Lowy1 , P.M. Day1 , R.C. Kines1 , C.D. Thompson1 , J.T. Schiller1 . 1 National Cancer Institute, Center for Cancer Research, Bethesda, USA Background and Rationale: HPV infection causes virtually all cases of cervical cancer, and a proportion of other non-cervical malignancies, including vulvar, vaginal, penile, anal, and oropharyngeal cancer. In many countries that lack widespread effective cervical cancer screening programs, the incidence of cervical cancer is high and greatly surpasses the incidence of non-cervical cancers. The incidence of cervical cancer is substantially lower in countries with effective screening programs. In some of those countries, the incidence of HPV-associated non-cervical cancers may be similar that of cervical cancer, largely because of recent increases in HPV-associated oropharyngeal cancer. These epidemiologic differences imply that the main public health goals of HPV vaccination may vary with the setting. In high incidence cervical cancer regions, reduction of this cancer is the primary goal. Given the long interval between incident infection and cervical cancer, vaccine-induced reductions in cervical cancer incidence and mortality will take many years. Therefore, in regions that lack widespread effective cervical cancer screening programs, serious consideration should be given to their implementation for adult women, in addition to vaccination of adolescent girls, as screening can have an impact on cervical cancer much sooner than vaccination. In regions with effective cervical cancer screening programs, cervical cancer reduction is a major goal of vaccination, but the role of the vaccine in reducing the incidence of HPVassociated non-cervical cancers may also be important. In addition, it might eventually be possible to safely modify screening guidelines in vaccinated populations, resulting in lower cost without reduced effectiveness. The current vaccines: Persistent infection by HPV16 and HPV18 accounts for about 70% of cervical cancer, and for more than 90% of the HPV-associated non-cervical cancers. Infection by another subset of human papillomaviruses (HPV), especially HPV6 and 11, causes most cases of most cases of genital warts and recurrent respiratory papillomatosis. The importance of HPV as human pathogen stimulated development of prophylactic HPV vaccines, based on the observation that the L1 main structural protein of the HPV virion can self-assemble into empty virus-like particles (VLPs) which contain the conformationally-dependent neutralization epitopes of L1 and can induce high levels of neutralizing antibodies. There are two FDA/EMEA-approved commercial versions of the VLP vaccine. Merck’s is a quadrivalent vaccine composed of VLPs from HPV6, 11, 16, and 18, while GlaxoSmithKline’s is a bivalent vaccine composed of VLPs from HPV16 and 18. Both vaccines are administered in three doses, given over 6 months. Efficacy trials in young adults have shown that, for fully vaccinated subjects, both vaccines induce almost complete protection against incident persistent anogenital infection and the associated lesions attributable to the HPV types targeted by the vaccine. Immunogenicity and protection induced by the vaccines are so strong that a two dose regimen is being used in some countries outside the United States, such as parts of Canada and Mexico. Experimental studies indicate that the neutralizing antibodies induced by the VLP vaccine are the main mechanism by which it protects against infection and disease. Potential utility of second generation vaccines: The limitations of the current HPV vaccines provide a rationale for development of candidate second generation vaccines. Low cost second generation vaccines that could induce long-term protective immunity with fewer doses would be especially attractive. Vaccines with activity against a broader range of HPV types that cause cervical cancer would increase their effectiveness. Their development might make it feasible to alter cervical cancer screening to an even greater degree than the current vaccines.
Sunday 8 July 2012
10:15−12:00
Symposium
Ageing and Cancer 31 Telomeres in Cancer and Ageing M.A. Blasco1 . 1 CNIO − Spanish National Cancer Research Centre, Molecular Oncology Programme, Madrid, Spain Telomeres protect the ends of chromosomes against DNA repair and degradation activities. Telomere length and integrity of telomere binding proteins are both important factors in telomere protection. Additionally, telomeres are transcribed and the telomeric RNA remains associated to telomeric chromatin. Our group has demonstrated that telomere shortening below critically short telomere lengths suffices to trigger organismal ageing and that if we stop this process of telomere shortening through reactivation of the telomerase enzyme, we are then able of both delaying ageing and increasing longevity.
european journal of cancer 48, suppl. 5 (2012) S5–S12
more that 70% of patients can now be successfully treated. The future is even brighter with many compounds in the pipeline against both virus and host targets, raising hopes for an all oral interferon-free regimen with a high cure rate. 28 Epstein–Barr Virus − Pathogenesis and Immunobiology A. Rickinson1 . 1 Institute of Cancer Studies, University Edgbaston, Birmingham, United Kingdom Epstein–Barr virus (EBV), a B-lymphotropic herpesvirus widespread in human populations, is aetiologically linked to a range of malignancies. These include (i) three tumours of B cell origin, post-transplant lymphoproliferative disease (PTLD), endemic Burkitt lymphoma (BL) and a subset of Hodgkin lymphomas, (ii) a specific subset of T or NK cell lymphomas, often of nasal type, and (iii) nasopharyngeal carcinoma (NPC) and a minor subset of gastric carcinomas with similar histology. Each of these tumours carry episomal EBV genomes in every malignant cell but display distinct patterns of virus latent gene expression. These patterns not only reflect the different contributions EBV makes to disease pathogenesis in different tumour contexts, but also have implications for immune-based therapies by restricting the number of viral antigens that are available for tumour targeting. To illustrate the versatility of EBV as a tumour virus, recent studies of B cell infections in vitro have shed new light on how virus-driven growth transformation, with full latent gene expression, can contribute to PTLD pathogenesis. By contrast, two more-restricted forms of latency are found in endemic BL, both of which act not to promote growth but to protect tumour cells from the pro-apoptotic effects of c-myc gene deregulation. While the c-myc translocation is a characteristic of both endemic (EBV-positive) and sporadic (largely EBV-negative) BL, recent work is identifying additional cell gene mutations whose patterns differ between the endemic and sporadic diseases, again reflecting their subtly different pathogenetic routes. Much still remains to be learned about the biology of EBV infection in vivo, its persistence within the B cell system, its occasional transmission to T and NK cells, and its varied contributions to human lymphomagenesis. 29 Proffered Paper: Detection of Hypermutation in Human Papillomavirus DNA Sequences From Cervical Samples V. Correa Vieira1 , J.D. Siqueira1 , A.R.I. Meyrelles2 , G.L. Almeida2 , H.N. Seuanez1 , E.S. Machado2 , E.A. Soares1 , M.A. Soares1 . 1 Instituto ˆ Nacional de Cancer Jose´ Alencar Gomes da Silva − INCA, Programa de ´ Genetica, Rio de Janeiro − RJ, Brazil, 2 Universidade Federal do Rio de ´ Janeiro, Hospital Universitario Clementino Fraga Filho, Rio de Janeiro − RJ, Brazil Persistent infection with high-risk human papillomavirus (HPV) is the main risk factor for development of cervical neoplasia. However, the factors leading to viral clearance or persistence are poorly understood. It has been shown that APOBEC3 proteins have antiviral effect against several viruses, such as HIV, inducing extensive G-to-A hypermutation into the viral genome. However, its role in the course of HPV infection and cancer development is unknown. Recently, evidence was shown for editing of HPV-16 DNA by APOBEC3 proteins in precancerous lesions. The aim of this study was to verify the presence of G-to-A mutations introduced by APOBEC3 into HPV sequences and to investigate its correlation with HPV-associated cervical lesions. DNA was extracted from genital scrapes using a commercial kit (QIAamp DNA Mini Kit® ). To detect hypermutation, a 370 bp fragment of the HPV LCR region was amplified by 3D-PCR, which enriches the amplification of ATrich sequences. PCR products were cloned, and at least ten clones from each sample were analyzed by colony PCR. Products were sequenced and hypermutation analysis was performed using Hypermut 2.0. A total of 68 viral sequences recovered from 5 patients were analyzed. At the time of sample collection, patients had normal cytology (n = 2), LSIL (n = 1) or HSIL (n = 2), and all were infected by HPV-16. Among the sequences analyzed, four drastically hypermutated sequences were found (p < 0.05), in addition to several sequences with elevated levels of G-to-A changes. Hypermutated sequences had a total of 65 G-to-A changes versus 4 non G-to-A changes. The preferential dinucleotide context of changes was GG (58%), suggesting a predominant activity of specific APOBEC3 proteins. All altered sequences belonged to a single patient, who had normal cytology at time of sample collection and progressed to LSIL within 6 months of follow-up. These results corroborate a previous report showing evidence for APOBEC3 editing of HPV genome in vivo. However, this is the first time that HPV hypermutation is found in cervical samples in the absence of cytological abnormalities, suggesting an early role of APOBEC3 proteins in the natural course of HPV cervical infection, despite clinical evolution to intraepithelial lesion. Considering that targeting APOBEC enzymes represents a potential antiviral strategy, further studies should be conducted to elucidate the role of hypermutation in the progression of HPV-associated cervical lesions.
S7
30 Current and Future Vaccines to Prevent HPV-associated Cancers D.R. Lowy1 , P.M. Day1 , R.C. Kines1 , C.D. Thompson1 , J.T. Schiller1 . 1 National Cancer Institute, Center for Cancer Research, Bethesda, USA Background and Rationale: HPV infection causes virtually all cases of cervical cancer, and a proportion of other non-cervical malignancies, including vulvar, vaginal, penile, anal, and oropharyngeal cancer. In many countries that lack widespread effective cervical cancer screening programs, the incidence of cervical cancer is high and greatly surpasses the incidence of non-cervical cancers. The incidence of cervical cancer is substantially lower in countries with effective screening programs. In some of those countries, the incidence of HPV-associated non-cervical cancers may be similar that of cervical cancer, largely because of recent increases in HPV-associated oropharyngeal cancer. These epidemiologic differences imply that the main public health goals of HPV vaccination may vary with the setting. In high incidence cervical cancer regions, reduction of this cancer is the primary goal. Given the long interval between incident infection and cervical cancer, vaccine-induced reductions in cervical cancer incidence and mortality will take many years. Therefore, in regions that lack widespread effective cervical cancer screening programs, serious consideration should be given to their implementation for adult women, in addition to vaccination of adolescent girls, as screening can have an impact on cervical cancer much sooner than vaccination. In regions with effective cervical cancer screening programs, cervical cancer reduction is a major goal of vaccination, but the role of the vaccine in reducing the incidence of HPVassociated non-cervical cancers may also be important. In addition, it might eventually be possible to safely modify screening guidelines in vaccinated populations, resulting in lower cost without reduced effectiveness. The current vaccines: Persistent infection by HPV16 and HPV18 accounts for about 70% of cervical cancer, and for more than 90% of the HPV-associated non-cervical cancers. Infection by another subset of human papillomaviruses (HPV), especially HPV6 and 11, causes most cases of most cases of genital warts and recurrent respiratory papillomatosis. The importance of HPV as human pathogen stimulated development of prophylactic HPV vaccines, based on the observation that the L1 main structural protein of the HPV virion can self-assemble into empty virus-like particles (VLPs) which contain the conformationally-dependent neutralization epitopes of L1 and can induce high levels of neutralizing antibodies. There are two FDA/EMEA-approved commercial versions of the VLP vaccine. Merck’s is a quadrivalent vaccine composed of VLPs from HPV6, 11, 16, and 18, while GlaxoSmithKline’s is a bivalent vaccine composed of VLPs from HPV16 and 18. Both vaccines are administered in three doses, given over 6 months. Efficacy trials in young adults have shown that, for fully vaccinated subjects, both vaccines induce almost complete protection against incident persistent anogenital infection and the associated lesions attributable to the HPV types targeted by the vaccine. Immunogenicity and protection induced by the vaccines are so strong that a two dose regimen is being used in some countries outside the United States, such as parts of Canada and Mexico. Experimental studies indicate that the neutralizing antibodies induced by the VLP vaccine are the main mechanism by which it protects against infection and disease. Potential utility of second generation vaccines: The limitations of the current HPV vaccines provide a rationale for development of candidate second generation vaccines. Low cost second generation vaccines that could induce long-term protective immunity with fewer doses would be especially attractive. Vaccines with activity against a broader range of HPV types that cause cervical cancer would increase their effectiveness. Their development might make it feasible to alter cervical cancer screening to an even greater degree than the current vaccines.
Sunday 8 July 2012
10:15−12:00
Symposium
Ageing and Cancer 31 Telomeres in Cancer and Ageing M.A. Blasco1 . 1 CNIO − Spanish National Cancer Research Centre, Molecular Oncology Programme, Madrid, Spain Telomeres protect the ends of chromosomes against DNA repair and degradation activities. Telomere length and integrity of telomere binding proteins are both important factors in telomere protection. Additionally, telomeres are transcribed and the telomeric RNA remains associated to telomeric chromatin. Our group has demonstrated that telomere shortening below critically short telomere lengths suffices to trigger organismal ageing and that if we stop this process of telomere shortening through reactivation of the telomerase enzyme, we are then able of both delaying ageing and increasing longevity.