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HUMAN PAPILLOMAVIRUS GENOME STATUS IN CERVICAL SAMPLES
LUIGI MARONGIU Submitted to the University College London for the degree of Doctor of Philosophy
14 February 2014
Delux version University College London Department of Infection and Immunity Cruciform Building – Gower street WC1E 6BT London (UK)
Abstract
Infection with oncogenic Papillomavirus genotypes is considered a major risk factor for the development of cervical cancer; nonetheless only a small proportion of the infected patients actually develop malignancies. The specific identification of high grade lesions is pivotal to increase the effectiveness of the cervical cancer screening. In the present study promising candidate molecular biomarkers based on the Human Papillomavirus (HPV) genomes were assessed in a cross-sectional cohort formed by samples singly infected with the most prevalent oncogenic genotypes 16, 18, 31 and 45. The candidate markers under investigation were the DNA viral load (VL), viral CpG methylation and viral integration. For the HPV16 samples, sequence variation within the regulatory region was also assessed. The viral integration was evaluated in a smaller longitudinal set. The results obtained showed that the DNA VL was lowest in subclinical lesions. The viral methylation was highest in severe dysplastic samples and differences in methylation profiles were observed between HPV species. The viral integration displayed a significant depletion of HPV16 episomal forms in cancerous lesions and the presence of viral integrants in all cytology grades. The analysis of the HPV16 variants identified eight novel polymorphisms and mutation profiles specifically recovered in high cervical disease grades. It was concluded that the viral genome modifications allowed the prediction of the cervical lesions. The combination of the molecular markers might allow a higher clinical specificity in the identification of cervical cancer precursor lesions.
Declaration I, Luigi Marongiu, confirm that the work presented in this dissertation is my own. Where information has been derived from other sources, I confirm that this has been indicated.
Oral presentations based on data derived from the study 2011 Eurogin congress: ‘HPV genome methylation: relationship with cervical disease progression’. 2010 Eurogin congress: ‘HPV DNA viral load and methylation status in liquid-based cytology samples stratified by disease stage’.
Acknowledgements I am indebted to Prof. Vincent Emery, formerly at the University College London, for the suggestions given to the present work and for leading me to the final step of this endevour. I would also like to thank Lauren Collins and Ben Chain, Division of Infection of Immunity of the University College of London, for their administrative support. I would like to acknowledge Prof. Kitchener and Dr. Kate Soldan for kindly providing the cancerous specimens. A special greeting goes to Prof. Clementina Cocuzza for her kindness and for providing the longitudinal samples and the related data. I also would like to thank Ms Paola Margarito, Biblioteca Nazionale Marciana of the University of Venice (Italy) for providing the paper of Rigoni-stern (1842). A great thank goes to Rupesh Vyas for his help during the methylation analysis. I am particularly grateful to Dr. Saranya Sridhar, Imperial College London, for the attention he dedicated to the drafting of the present manuscript. The present project would not have been realized without financial assistance from my parents and the emotional support of my beloved wife.
Et Sisyphum vidi, a terribili dolore oppressum
Acronyms °C
Degrees Celsius
AA
Asian-American HPV16 variant
ADC
Adenocarcinoma
Af
African HPV16 variant
ALTS
ASCUS-LSIL Triage Study
ARTISTIC
A randomised trial in screening to improve cytology
ASC
Atypical squamous cells
ASC-H
Atypical squamous cells not excluding HSIL
ASC-US
Atypical squamous cells of undetermined significance
ATM
Ataxia-telangiectasia mutated
AUC
Area under the curve
B
Borderline cytology
BLAST
Basic local alignment search tool
bp
Basepair
BSA
Bovine serum albumin
c/µl
viral copies per microlitre
c/c
viral copies per cell
c/µl
Copies per microlitre
CDC-ACIP
Centre for Disease Control Advisory Committee for Immunization Practices
CFS
Common fragile sites
CI
Confidence interval
CIN
Cervical intraepithelial neoplasia
CISOE
Composition, inflamation, squamous epithelium, other, epithelium
CpG
CG dinucleotide
Cq
Quantitative cycle
CV
Coefficient of variation
DEPC
Diethylpyrocarbonate
DIPS
Detection of integrated papillomavirus sequences
DNA
Deoxyribonucleic acid
DNMT
DNA methyl-transferases
dNTP
Deoxyribonucleic tri-phosphate nucleotide
dpi
Dots per inch
E
European HPV16 variant
E2BS
E2 binding site
E6AP
E6 associated protein
EA
East-Asian HPV16 variant
EBV
Epstein-Barr virus
ECM
Extra-cellular matrix
EDTA
Ethylenediaminetetraacetic acid
fg/µl
Femtogram per microlitre
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
HDAC
Histone deacetylases
HIV
Human immunodeficiency virus
HPA
Health Protection Agency
HPV
Human Papillomavirus
HSIL
High grade squamous intraepithelial lesions
HSPG
Heparan sulphate proteo-glycan
HSV
Herpes Simplex virus
IARC
International Agency for Research on Cancer
ICBP90
Inverted CCAAT box binding protein
ICTV
International Committee on Taxonomy of Viruses
IFN
Interferon
IL-10
Interleukin 10
IQR
Interquartile range
ISH
In situ hybridization
Kb
Kilobases
LB
Luria-Bertani medium
LBC
Liquid-based cytology
LCR
Long control region
LN5
Laminin 5
LOD
Limit of detection
LSIL
Low grade squamous intraepithelial lesions
m
Mild dyskaryosis cytology
mA
milliampere
MALDI-TOF Matrix-assisted laser desorption ionization time of flight MAR
Matrix attachment region
MeCP2
Methyl-CpG-binding protein 2
MGMT
O6-methylguanidine-DNA methyltransferase
min
Minutes
MIQE
Minimum information for publication of quantitative digital PCR experiments
MISCAN
Microsimulation screening analysis
ml
Millilitre
MND
Methyl binding domain
Mo
Moderate dyskaryosis cytology
MSP
Methylation specific PCR
MSRA
Methylation sensitive restriction analysis
MW
Molecular weight
n
Number of samples
N
Normal cytology
NASBA
Nucleic acid sequence based amplification
NCBI
National Center for Biotechnology Information
ND10
Nuclear domain 10
NHSCSP
National Health Service Cervical Screening Programme
NJ
Neighbour joining algorithm
nmol
Nanomolar
NPV
Negative predictive value
NTC
Non template control
OR
Odds ratio
ORF
Open reading frame
ori
Origin of replication
PBS
Phosphate-buffered saline
PCR
Polymerase chain reaction
PCR-RFLP
PCR restriction lenght polymorphism
PE
Early promoter
PL
Late promoter
PPV
Positive predictive value
qPCR
Quantitative PCR
RNA
Ribonucleic acid
ROC
Receiver operating characteristics
RPA
Replication protein A
rpm
Rotations per minute
RT-qPCR
Reverse-transcription quantitave PCR
S
Severe dyskaryosis cytology
s
seconds
SCC
Squamous cell carcinoma
SD
Standard deviation
SDS
Sodium dodecyl sulfate
SMRT
Single molecule real time sequencing
SNP
Single nucleotide polymorphisms
SNR
Non-coding region
SNR
Short non coding region
Sp1
Specificity protein 1
SV40
Simian vacuolating virus 40
TAE
Tris acetate EDTA
TBP
TATA binding protein
TE
Tris EDTA
TEF-1
Transcriptional enhancer factor 1
TERT
Telomerase catalytic subunit
TMB
Tetramethylbenzidine
TNF
Tumor necrosis factor
tPA
Tissue plasminogen activator
U
Units
UK
United Kingdom
URR
Upstream regulatory region
V
Volts
ver.
Version
VL
Viral load
VLP
Virus like particle
VRD
Virus reference departement
w/v
Weight/volume solution
WHO
World health organization
X-gal
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
XRCC1
X-ray cross complementing protein 1
YY1
Yin-yang protein 1
µg/ml
Micrograms per millilitre
µl
Microlitre
µM
Micromolar
Table of contents
Abstract..................................................................................................................... i Declaration................................................................................................................ ii Oral presentations based on data derived from the study............................................. iii Acknowledgements.................................................................................................... iv Acronyms.................................................................................................................. v Table of Contents...................................................................................................... ix List of figures............................................................................................................ xvi List of tables.............................................................................................................. xxi
Introduction.............................................................................................................. 1 Cervical cancer................................................................................................ 1 Epidemiology....................................................................................... 1 Aetiology............................................................................................. 3 Grading of the cervical lesions............................................................... 7 Progression of the cervical lesions.......................................................... 10 Human Papillomavirus..................................................................................... 11 Virion structure..................................................................................... 11 Classification and phylogeny................................................................. 13 Life cycle.............................................................................................. 18 Transmission, attachment and entry........................................... 18 Replication................................................................................ 19 Transcription............................................................................. 21 Extension of the S phase............................................................ 22 Viral latency............................................................................... 25 Encapsidation and release.......................................................... 26
Viral transformation.............................................................................. 26 HPV and cervical cancer............................................................. 26 Viral integrants and cervical cancer............................................. 28 Viral episomes and cervical cancer.............................................. 31 Immunity and vaccination..................................................................... 32 Immunity.................................................................................. 32 Vaccination................................................................................ 33 Screening of cervical cancer............................................................................. 36 Overview of the cervical cancer screening protocol................................ 36 Colposcopy.......................................................................................... 37 Cytological analysis.............................................................................. 37 Molecular testing.................................................................................. 38 HPV DNA testing....................................................................... 41 Genotyping............................................................................... 44 DNA viral load........................................................................... 46 RNA viral load............................................................................ 49 Viral variants.............................................................................. 50 Methylation profiles.................................................................. 51 Viral integration........................................................................ 54 Immunological testing......................................................................... 57 Serology................................................................................... 57 Immuno-histochemistry............................................................. 58 Performances of the screening assays.................................................... 59 Improving the effectiveness of the cervical cancer screening.............................. 61 Aim of the present work.................................................................................. 63
Materials and methods............................................................................................... 65 Study design................................................................................................... 65 Cross-sectional study............................................................................ 65
Longitudinal study.............................................................................. 69 Plasmids and cells.......................................................................................... 72 Plasmids............................................................................................. 72 Reference sequences and cell lines....................................................... 73 DNA extraction.............................................................................................. 73 Total extraction with commercial kits................................................... 73 Selective extraction............................................................................. 74 General laboratory techniques........................................................................ 74 Prevention of contamination................................................................ 74 Gel electrophoresis.............................................................................. 75 Cloning.............................................................................................. 75 Sequencing........................................................................................ 76 DNA viral load............................................................................................... 76 Experimental protocol......................................................................... 76 Reproducibility and specificity.............................................................. 79 CpG methylation........................................................................................... 79 Pyrosequencing approach................................................................... 79 HPV16..................................................................................... 79 HPV18..................................................................................... 82 Accuracy.................................................................................. 84 Cloning approach............................................................................... 85 Identification of a suitable enzyme............................................ 85 HPV18, HPV31 and HPV45....................................................... 86 Assessment of the chemical stability of the CpG moiety.................................................................. 87 Specificity analysis.................................................................... 88 Late promoter methylation profile............................................. 88 CpG mapping..................................................................................... 88 Analysis of the data............................................................................. 88
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Viral integration.............................................................................................. 90 Variants analysis............................................................................................. 93 Southern blot................................................................................................. 94 Statistical analysis........................................................................................... 95
HPV DNA viral load as a discriminator of cervical disease............................................. 97 Introduction................................................................................................... 97 Analysis of the HPV DNA VL in the cross-sectional samples............................... 98 Validation of the experimental protocol................................................ 98 Distribution of HPV DNA VL in clinical samples...................................... 100 Analysis of the stratification of the viral load by cytology cluster......................................................................... 102 Receiver operating curve analysis to discriminate cytology grades......................................................................... 104 Genotype specific cut-off of the HPV DNA VL.............................. 104 Comparison between HPV DNA VL and genotyping in the dscrimination of cytology grades........................... 107 Conclusions.................................................................................................... 109
HPV methylation as a predictor of cervical lesions........................................................ 113 Introduction.................................................................................................... 113 Analysis of viral methylation in clinical samples and cell lines............................. 114 Validation of the HPV16 pyrosequencing protocol.................................. 114 Characterization of the viral methylome in HPV16-containing cell lines........................................................ 115 Methylation analysis in HPV16 samples................................................. 118 Validation of the HPV18 pyrosequencing protocol.................................. 121 Methylation profiles of the HeLa cell line................................................ 123
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HPV18 methylation in clinical samples obtained by pyrosequencing.................................................................... 124 HPV18 methylation in clinical samples obtained by cloning................... 127 HPV31 methylation in clinical samples................................................... 131 HPV45 methylation in clinical samples................................................... 131 Optimal methylation cut-off to discriminate cytology grades.................. 134 Genotype specific 3’-L1 and LCR overall methylation................... 134 Comparison between viral methylation, viral load and genotyping to discriminate cytology grades.............. 140 Association between HPV DNA viral load and methylation...................... 142 Conclusions.................................................................................................... 144
Association between viral genomic physical status and cervical lesions.......................... 151 Introduction.................................................................................................... 151 Analysis of the HPV genomic physical status in the clinical samples.................... 153 Validation of the experimental protocol................................................. 153 Establishment of the episomal thresholds.............................................. 155 Decrease of the E2-5’/E6 ratio in cancerous samples corresponded to depletion of episomal forms in these lesions............................... 157 Viral integration analysis of the longitudinal samples.............................. 161 Identification of optimal methylation cut-off to dicriminate cytology grades................................................... 161 Association between HPV DNA viral load and physical status.................. 163 Association between HPV methylation and physical status...................... 165 Conclusions.................................................................................................... 166
Discussion..................................................................................................................171 Discrimination of the subclinical lesions from higher grades.............................. 172
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Discrimination of the severe dysplastic lesions based on the viral methylation............................................................................. 175 Discrimination of cancerous lesions based on the depletion of viral episomes................................................................................... 179 Strengths of the study..................................................................................... 181 Limitations of the study................................................................................... 182 Conclusions.................................................................................................... 184
Appendix 1 – Additional work carried out in the present study..................................... 187 Foreword....................................................................................................
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Methylation around the late promoter..............................................................188 Introduction......................................................................................... 188 Results................................................................................................. 189 Discussion............................................................................................ 190 Southern blot as confirmatory assay of integration............................................ 191 Introduction........................................................................................ 191 Results................................................................................................. 192 Discussion............................................................................................ 193 Hirt extraction to separate viral episomes from the bulk of the chromosomal DNA......................................................................... 194 Introduction......................................................................................... 194 Results................................................................................................. 196 Discussion............................................................................................ 196 LCR and E6 polymorphisms in the cervical disease............................................ 197 Introduction......................................................................................... 197 Results................................................................................................. 198 Nucleic acid substitutions........................................................... 198 Amino acid substitutions............................................................ 203 Discussion........................................................................................... 204
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Appendix 2 – Scientific publication derived from the present study............................. 209 A. Human Papillomavirus 16, 18, 31 and 45 viral load, integration and methylation status stratified by cervical disease stage............................ 209 Abstract.............................................................................................. 211 Background........................................................................................ 213 Methods............................................................................................. 216 Results and discussion.......................................................................... 219 Conclusions......................................................................................... 228 B. Human Papillomavirus type 16 long control region and E6 variants stratified by dervical disease stage...................................................................... 233 Abstract.............................................................................................. 234 Background........................................................................................ 235 Methods............................................................................................. 237 Results and discussion.......................................................................... 239
Appendix 3 – Sequences derived from the present study............................................ 244 References................................................................................................................ 271
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List of figures
Figure 1-1. Diagram of the human cervix.................................................................... 2 Figure 1-2. World prevalence of HPV genotypes.......................................................... 8 Figure 1-3. Classification of cytological and histological lesions.....................................10 Figure 1-4. Structure of the HPV16 genome................................................................ 11 Figure 1-5. HPV16 transcription map.......................................................................... 13 Figure 1-6. Structure of the LCR of the genital HPVs.................................................... 14 Figure 1-7. HPV virion structure.................................................................................. 15 Figure 1-8. Phylogenetic tree of the Papillomaviridae family......................................... 16 Figure 1-9. Geographical distribution of the HPV16 lineages........................................ 17 Figure 1-10. HPV life cycle.......................................................................................... 19 Figure 1-11. Replication in HPV................................................................................... 20 Figure 1-12. Functions of the HR HPV oncoproteins..................................................... 23 Figure 1-13. Progression of the HR HPV-associated lesions........................................... 27 Figure 1-14. Model of carcinogenesis in HR HPV.......................................................... 29 Figure 1-15. Model of replication-induced chromosomal instability.............................. 30 Figure 1-16. Diagnosis of cervical disease.................................................................... 37 Figure 1-17. Localization of genotypes and lesions within a cervical sample................. 41 Figure 1-18. Algorithm of the cervical cancer screening in UK...................................... 43 Figure 1-19. Proposed algorithm for the cervical cancer screening............................... 44 T
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Figure 1-20. Impact of sampling on the outcome of the assays.................................... 48 Figure 1-21. Methylome of HPV16 in established cell lines........................................... 53 Figure 1-22. Methylome of HPV18 in HeLa cell line...................................................... 54 Figure 1-23. Clinical sensitivity and specificity of molecular tests.................................. 61
Figure 2-1. Prevalence of single and multiple infections in the cross-sectional study...... 67 Figure 2-2. Age distribution for different populations................................................... 69 Figure 2-3. Dynamics of pooled DNA VL by sampling time.......................................... 71 Figure 2-4. Localization of the amplicons used for the HPV16 CpG pyrosequencing analysis............................................................... 82 Figure 2-5. Localization of the amplicons used for the HPV18 CpG pyrosequencing analysis............................................................... 84 Figure 2-6. Comparison between DNA polymerases.................................................... 86 Figure 2-7. Map illustrating the position of the late promoter....................................... 89 Figure 2-8. Localization of the CpG sites studied.......................................................... 90 Figure 2-9. Clustering method for the methylation analysis.......................................... 91
Figure 3-1. Comparison of the standard curves........................................................... 100 Figure 3-2. DNA VL of the positive controls................................................................. 102 Figure 3-3. Specificity of the qPCRs used in the present study...................................... 103 Figure 3-4. Limits of detection of the installed qPCRs................................................... 104 Figure 3-5. Distribution of HPV16 and HPV31 DNA VL................................................. 105 Figure 3-6. Distribution of HPV18 and HPV45 DNA VL................................................. 106
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Figure 3-7. Identification of optimal cut-off point for the DNA VL................................. 108
Figure 4-1. Methylomes for CaSki and SiHa................................................................. 116 Figure 4-2. Correlation between the methylation profiles obtained by pyrosequencing and cloning strategy............................................................... 117 Figure 4-3. Methylation of HPV16 clinical samples....................................................... 119 Figure 4-4. Stratification of the methylation values in HPV16........................................ 120 Figure 4-5. Stratification of the methylation in the HPV16 LCR..................................... 122 Figure 4-6. Methylation profile for HeLa CCL2 obtained by pyrosequencing.................. 123 Figure 4-7. Methylation of HeLa CCL2 obtained by cloning.......................................... 124 Figure 4-8. Methylomes for the HPV18 clinical samples obtained by pyrosequencing.......................................................................................... 125 Figure 4-9. Methylation profiles of HeLa obtained by cloning analysis........................... 127 Figure 4-10. Methylation of HPV18 clinical samples obtained by cloning....................... 128 Figure 4-11. Stratification of the methylation values in HPV18...................................... 130 Figure 4-12. Methylation of HPV31 clinical samples obtained by cloning....................... 132 Figure 4-13. Methylation of HPV45 clinical samples obtained by cloning....................... 133 Figure 4-14. Optimal cut-off for the HPV16 3’-L1 methylation...................................... 135 Figure 4-15. Optimal cut-off for the HPV16 LCR overall methylation............................. 136 Figure 4-16. Optimal cut-off for the HPV18 3’-L1 methylation...................................... 137 Figure 4-17. Optimal cut-off for the HPV18 LCR overall methylation............................. 138 Figure 4-18. Optimal cut-off for the HPV31 3’-L1 methylation...................................... 139 Figure 4-19. Optimal cut-off for the HPV45 3’-L1 methylation...................................... 141
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Figure 4-20. Stratification of HPV31 DNA VL according to the methylation cut-off.................................................................................... 143
Figure 5-1. Alignment of PCR primer sets.................................................................... 152 Figure 5-2. Empirical assessment of the lower episomal E2/E6 ratio...............................156 Figure 5-3. Comparison between tests........................................................................ 157 Figure 5-4. E2/E6 ratios obtained on the HPV16 clinical samples................................... 158 Figure 5-5. Stratification of the clinical samples according to the viral physical status.................................................................................... 159 Figure 5-6. Concordance between the two E2 fragments............................................. 160 Figure 5-7. HPV16 ROC analysis of viral integration...................................................... 163 Figure 5-8. Stratification of DNA VL by viral physical status........................................... 164 Figure 5-9. Methylation of clinical samples bearing mostly integrative viral forms.......... 166
Figure 6-1. Association between viral genomic status and cytology grade..................... 172 Figure 6-2. Proposed cervical screening algorithm....................................................... 185
Figure A-1. Methylation profile around the late HPV16 promoter.................................. 189 Figure A-2. Incorporation of digoxigenin..................................................................... 193 Figure A-3. Detection of viral DNA by Southern blot.................................................... 194 Figure A-4. Dendrogram of the HPV16 variants............................................................ 199 Figure A-5. Location of the identified SNPs within the LCR........................................... 203 Figure A-6. Location of the identified mutations within the E6 ORF............................... 204
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Figure A-7. Viral load.................................................................................................. 220 Figure A-8. Integration............................................................................................... 222 Figure A-9. Methylation.............................................................................................. 224 Figure A-10. Sensitivity and specificity plot.................................................................. 226 Figure A-11. (Supplementary) Impact of varying integration thresholds........................ 230 Figure A-12. Phylogenic distribution of LCR-E6 variants................................................ 240 Figure A-13. Site-specific intra-type sequence diversity................................................. 241
List of tables
Table 1-1. HPV types as defined by the IARC classification............................................ 6 Table 1-2. The five most prevalent HPV genotypes in worldwide assessments............... 9 Table 1-3. Molecular-based HPV assays....................................................................... 39
Table 2-1. Overall cytology fluctuation in the longitudinal study set............................. 70 Table 2-2. Details of the cell lines used in the present study......................................... 73 Table 2-3. Primers used for the DNA VL analysis.......................................................... 77 Table 2-4. Concentration of the oligonucleotides used for the DNA VL assessment................................................................................... 78 Table 2-5. Primers used for the HPV16 CpG methylation analysis................................. 81 Table 2-6. Primers used for chemical status analysis of HPV18 by pyrosequencing......................................................................................... 83 Table 2-7. Primers used for the CpG methylation analysis by cloning strategy......................................................................................... 87 Table 2-8. Primers used for viral integration analysis.................................................... 92 Table 2-9. Primers used for viral variants analysis......................................................... 93
Table 3-1. Performances of the qPCR used to assess the DNA VL.................................. 99 Table 3-2. Variability over different runs of the qPCR used to essay the DNA VL............ 101
Table 3-3. Discrimination of grades based on genotyping and DNA VL......................... 109
Table 4-1. Discrimination of severe dyskaryotic samples from lower cytology grades based on genotyping, viral load and methylation............ 142
Table 5-1. Performances of the qPCR used for viral integration analysis......................... 154 Table 5-2. Specificity of the qPCR used for the assessment of the viral integration......................................................................................... 155 Table 5-3. Analysis of the viral physical status in the longitudinal cohort....................... 162
Table 6-1. Nucleic acid variation profiles in HPV16...................................................... 175 Table 6-2. Prevalence of the variation profiles in the HPV16 samples............................. 176
Table A-1. Nucleic acid variation profiles in HPV16...................................................... 201 Table A-2. Prevalence of the variation profiles in the HPV16 samples............................. 201 Table A-3. (Supplementary) PCR primers and probe sequences.................................... 231
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Introduction
Cervical cancer Epidemiology More than 2 billion women worldwide are at risk of developing cancer of the cervix (figure 1-1) due to infection with oncogenic Human Papillomaviruses (HPV) (WHO Information Centre on HPV and cervical cancer, 2010b). The global annual incidence of cervical cancer is over 520,000 while 275,000 women die each year from the disease. Such figures are expected to increase in the next decade unless proper implementation of vaccine programme and early identification of patients at risk is achieved (Arbyn et al., 2012; Patel et al., 2012). In Europe, cervical infection with HPV has a prevalence of 9.7% and cervical cancer represents the fifth most common type of cancer for the European women, with more than 54,000 new cases of cervical cancer and 25,000 deaths per year (WHO Information Centre on HPV and cervical cancer, 2010a). In particular, for women aged 15-44 years, cervical cancer represents the second most common form of neoplasia after breast cancer. The seminal work carried out by Rigoni-Stern in the 19th century has shown an increased ratio of uterine cancers in married women with respect to nuns (Rigoni-Stern, 1842) and further assessments have established that the probability of development of uterine and cervical cancers was associated to sexual activity (Smith et al., 1980; Taylor et al., 1959; Wynder et al., 1954). In particular the most important risk factors for the cervical cancer were identified in the age of the onset of coitus, the number of sexual partners, parity and even circumcision (Lombard and Potter, 1950; Rotkin, 1967a, b). Several studies have established an increased risk of cervical and uterine cancers in women from the lowest ¨
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FIGURE 1-1. Diagram of the human cervix. The position of the transformation zone, where the stratified squamous epithelial cells of the ectocervix (vagina) are substituted by columnar epithelial cells, is indicated with an arrow. (Hafez, 1982, with modifications). social classes; ethnicity, which also display differences in the risk of development of these malignancies, might be a surrogate factor for the overall income or a pointer to a varied lifestyle associated with the race groups (Akers et al., 2007; Downs et al., 2008; Faggiano et al., 1997; Kennaway, 1948; Rotkin, 1973). Despite the fact that national screening and vaccination programmes are implemented in many nations (allowing the early identification of, and protection from, the cervical cancer and thus drastically reducing the morbidity and mortality associated with this disease), the coverage is not absolute. Even in a high income country such as the United Kingdom (UK) socio-economical and ethnic factors affect the attendance of the eligible women to the screening and vaccination programmes (Bang et al., 2012; Kumar and Whynes, 2011; ´
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Moser et al., 2009). Thus cervical cancer remains a major worldwide Public Health concern that requires further investigation.
Aetiology Experiments on mice had shown that application of smegma (an amalgam of exfoliated cells and skin oil) by speculum or injection induced cancer; these results, coupled by epidemiological data, have led to the theory of a carcinogenic agent carried by the smegma during the coitus, and one of the first candidate markers was Mycobacterium smegmatis, part of the normal flora of the male genitalia (Alexander, 1973; Dennis et al., 1956; Plaut and Kohn-Speyer, 1947). Subsequent studies suggested that infection with Treponema pallidum might be involved in the cervical cancer development, although others suggested that such connection might only point to more promiscuous sexual behaviours rather than indicate a causal relation (Harding, 1942; Wynder et al., 1954). A significant increase in antibodies against HSV type 2 (genital) was identified in samples derived from cervical cancer lesions, suggesting a role as carcinogenic agent (Nahmias et al., 1970; Rawls et al., 1968; Royston and Aurelian, 1970). Anyway, the attempt to identify HSV DNA by mean of hybridization assays could not recover HSV from cervical carcinoma tissues (Delap et al., 1976; zur Hausen et al., 1974). Conversely DNA hybridization assays definitely proved the consistent presence of HPV DNA in cervical cancer lesions (Boshart et al., 1984; Gissmann et al., 1984; Gissmann et al., 1983). HPVs are acknowledged to cause ‘papillomata’, localized hyperplasic lesions of the epithelium characterized by the presence of protuberances known as papillae (from the Latin for ‘nipple’) (Routh et al., 1997). Papillomata have been described since the time of the ancient Greeks and have been recognized for centuries to affect not only humans but several species of animals (Lancaster and Olson, 1982). The viral aetiology of the papillo-
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mata was proved at the beginning of the 20th century when it was shown the infectivity of cell-free filtrates of warts (Ciuffo, 1907). The first animal Papillomavirus was isolated in the 1930s (Shope and Hurst, 1933) and the first identification of viral particles in wart lesions by electron microscopy was achieved in the 1940s (Strauss et al., 1949). Papillomata are generally referred to as warts or verrucae (from the Latin for ‘height’); specifically, the term condyloma (from the Greek for ‘knob’) is reserved to designate the genital warts (Cardoso and Calonje, 2011). Condylomas manifest either in the external or internal genitalia; in the latter case the transformation zone of the cervix is preferentially affected (Doorbar et al., 2012; Meisels et al., 1985; Paavonen, 1985; Roy et al., 1981; zur Hausen, 1977). The recognition of condylomas as venereal diseases was established after the second world war (Barrett et al., 1954). Nowadays HPVs are recognized as the most common sexually transmitted viruses (Crosbie et al., 2013; Weinstock et al., 2004). Currently HPV shows a worldwide prevalence in the female population of 11.4% corresponding to almost 300 million women infected with a genital HPV at any given time. The association between cervical cancer and HPV infection was recognized in the second half of the 20th century (zur Hausen, 2009). Epidemiological evidence pointed to a correlation between cervical cancer and HPV infection: both diseases were more common in sexually promiscuous subjects than in women with few or none sexual partners and both shared sexually-related epidemiological factors, such as history of other venereal diseases and parity (Larsen et al., 1988; Wright and Richart, 1990). A worldwide study recovered HPV genotypes in 93% of the analysed cancer lesions (Bosch et al., 1995) but further re-evaluation identified false-negative samples, thus increasing the prevalence of HPV DNA in cervical cancer to 99.7% (Walboomers et al., 1999). These findings corroborated the involvement of HPV in the development of this malignancy ruling out other microbiological aetiologies (Herrington, 1999; Walboomers and Meijer, 1997).
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Clinical and experimental data ascertained that papillomata could progress into malignancies (Rous and Beard, 1935; zur Hausen, 1977) and tissue cultures transfected with DNA derived from Papillomavirus particles showed morphological alterations demonstrating the transforming capability of this virus (Lancaster and Meinke, 1975). Grafts of warts and condylomas transplanted on the healthy skin of human volunteers developed into wart lesions (Goldschmidt and Kligman, 1958; Serra, 1924). Anti-sera from patients affected by genital warts reacted with common warts, suggesting a common etiological agent for the two types of lesion (Ogilvie, 1970). It was therefore believed that the different types of papillomata were due to a single transmissible agent; in the 1970s this conjecture has been subsequently revised (Koutsky et al., 1988; zur Hausen, 1977). Epidemiological data, hybridization patterns and serological outcomes revealed the presence of several Papillomavirus genotypes (Gissmann and zur Hausen, 1976). Several studies have shown that certain HPV genotypes could be recovered more frequently in cancerous lesions whereas others were more prevalent in benign injuries (Crum et al., 1984; de Villiers, 1992). In particular, the 6th and 11th genotypes to be characterized (HPV6 and HPV11) have been preferentially recovered from condylomas of the external genitalia (Brown et al., 1993); conversely the 16th and 18th (HPV16 and HPV18) were the most prevalent in cervical cancer samples (Boshart et al., 1984; Durst et al., 1983). Nonetheless genotypes more associated with cervical cancer lesions could be recovered in condylomas (Brown et al., 1999; Sturegard et al., 2013). The risk of developing cervical cancer associated to infection with HPV varied with the genotype, being maximal for the genotype 16 (HPV16, odds ratio = 434.5) (Munoz et al., 2003). Due to the different association with the cervical lesion grades, the HPV genotypes could be subdivided in low risk (LR) and high risk (HR, or oncogenic) for the development of cervical cancer (table 1-1) (Arbyn et al., 2012; Bouvard et al., 2009; Chan et al., 2012).
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Table 1-1 – HPV types as defined by the IARC classification¹. Strains Correlation
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18, 31, 33, 35, 39, 45, 51, Sufficient evidence 52, 56, 58, 59
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26, 30, 34, 53, 66, 67, 69, Limited evidence or phylogenetic analogy 70, 73, 82, 85, 97
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6, 11, 40, 42, 43, 54, 61, Low risk alpha-papillomaviruses 70, 72, 81, CP6108
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Several
Strong epidemiological and molecular evidence
Limited evidence in humans, strong mechanistic correlation
Beta and gamma papillomaviruses
¹ Derived from: Bouvard et al., 2009; Munoz et al., 2003.
The main characteristic of the HR HPV genotypes is the capacity to progress to cervical cancer in the presence of persistency, whereas the LR-HPV are very unlikely to be associated with these lesions in long lasting infections (Schiffman et al., 2005). In addition oncogenic types have been generally found to persist longer than the LR strains (Brown et al., 2005; Franco et al., 1999; Giuliano et al., 2002a). Even among the high risk genotypes, HPV16 has been shown to be the most persistent and progressive (Burk et al., 2009; Carcopino et al., 2011) and precursor lesions associated with this genotype develop at an earlier age than those related to other strains (Wentzensen et al., 2013). HPV18 has a particularly low recovery frequency in low grade lesions, suggesting that its rate of progression towards precursors and cancer might be even faster than that of HPV16 (Cox, 1995). Despite the strong association between persistent infection with HR-HPV infections and cervical cancer, only a small percentage of infected women develop cancer lesions, therefore infection with HR-HPV is considered a necessary but not sufficient cause for neoplasia (Giuliano et al., 2002b; Hildesheim et al., 2001; Munoz et al., 2002; Schiffman and Castle, 2003). Other co-risk factors are required, such as smoking, number of sexual partners, ä
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concomitant infections and prolonged contraceptive use. For instance, co-infection with Human Immunodeficiency virus (HIV) is also an important factor for persistence and progression of HPV infections, possibly due to the impairment of the immune system in the early stages of the Papillomavirus natural history (Palefsky, 2006). Genetic polymorphisms can increase the risk of cancer development: for instance, in presence of HPV infection, three XRCC1 variants have been associated with further increased risk of cervical cancer in comparison to the wild type (Huang et al., 2007); and women with systemic lupus erythematosus have been shown to be at higher risk of developing precancerous lesions than healthy subjects (Nath et al., 2007).
Grading of the cervical lesions Condylomas show two main morphologies: esophytic (also known as ‘acumimatum’ or spiked) and endophytic (otherwise named ‘planum’, that is flat), the latter has been described only in the late 1970s (Meisels et al., 1985; Paavonen, 1985). Esophytic lesions are characterized by vascularization that determines the projection of the affected tissue outward with respect to the rest of the unaffected mass, whereas flat lesions grow inward sometimes involving the glandular cells (Cox, 1995). Condylomas are associated prevalently with LR HPV genotypes and are distinguished by the presence of koilocytes, which bear the productive phase of the viral replicative cycle, and have a high probability of spontaneous regression (Liao and Manetta, 1993; Richart and Wright, 1991). Conversely atypical flat condylomas, known as cervical intraepithelial neoplasia (CIN) lesions, are characterized by a higher proportion of dyskeratocytes (from the Greek ‘dyskaryon’, nuclear condition) and are more frequently associated with HR HPV genotypes (Fu et al., 1988; Fu et al., 1981). The relative frequencies of the HPV genotypes in cervical samples vary with respect to the individual studies, geographical regions (figure 1-2) and
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FIGURE 1-2. World prevalence of HPV genotypes. Percentage of HPV genotypes in patient resulting positive to HPV DNA screening (n=8977). The patients were stratified by world geographic region in Europe (n=2058), America (North, Central and South, n=3564), Oceania (n=170), Africa (n=544) and Asia (n=2641). (de Sanjose et al., 2010, with modifications). cytology grade, but the most common genotypes in normal cytology samples can be identified as 16, 18, 31, and 58 (Bruni et al., 2010; Clifford et al., 2005; de Sanjose et al., 2007; Jacobs et al., 2000); and as 16, 18, 31, 33 and 45 in cervical cancer (Bosch et al., 1995; Clifford et al., 2003; de Sanjose et al., 2010; Li et al., 2011) (table 1-2). Only HPV18 and HPV45 belong to the species alpha-7, whereas the others are included in the species alpha-9 (Bernard et al., 2010). According to the presence of cellular and nuclear abnormalities the CIN lesions can be subdivided in grade 1, 2 or 3 (figure 1-3) (Apgar et al., 2003). Such lesions can also be defined as dysplasia (from the Greek ‘dys-plasis’, growth disorder); CIN1 are also defined as mildly dysplastic cervical lesions and are accepted to represent productive Papillomavirus infections (Doorbar, 2007; Evans et al., 1986). However flat condyloma, koilocytotic atypia and CIN1 are difficult to discriminate one another (Liao and Manetta, 1993) and are
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Table 1-2 – The five most prevalent HPV genotypes in worldwide assessments. Clifford de Sanjose Bruni Anderson et al., 2005 et al., 2007 et al., 2010 et al., 2013 (n=15,613) (n=15,764) (n=215,568) (n=72) Normal cytology 16 (19.7%) 16 (2.5%) 16 (3.2%) 16 (56.9%) 42 (9.4%) 18 (0.9%) 18 (1.4%) 31 (18.1%) 58 (7.6%) 31 (0.7%) 52 (0.9%) 39 (9.7%) 31 (7.5%) 58 (0.6%) 31 (0.8%) 52 (9.7%) 18 (7.2%) 52 (0.6%) 58 (0.7%) 51 (6.9%) Bosch Clifford de Sanjose Li et al., 1995 et al., 2003 et al., 2010 et al., 2011 (n=902) (n=141,275)¹ (n=8,977) (n=19,184) Cervical cancer 16 (49.9%) 16 (16.3%) 16 (61%) 16 (59.3%) 18 (13.7%) 18 (4.4%) 18 (10%) 18 (13.2%) 45 (8.4%) 45 (3.9%) 45 (6%) 58 (5.1%) 31 (5.3%) 31 (3.8%) 31 (4%) 33 (4.9%) 33 (2.8%) 33 (3.7%) 33 (4%) 45 (4.4%) ¹ Accounts also for adenocarcinomas.
grouped together to describe the low grade squamous intraepithelial lesions (LSIL) (Apgar et al., 2003). CIN2 and CIN3, on the other hand, share features and a natural history that can differentiate them from the CIN1 lesions and are grouped together in the high grade squamous intraepithelial lesions (HSIL) (Llewellyn, 2000). The lesion scoring can be based on either the British/European or the American/Bethesda systems (Apgar et al., 2003; Evans et al., 1986). The former, introduced by the British Society for Clinical Cytology, subdivides the nuclear aberrations as borderline and dyskaryosis of mild, moderate and severe grade. More recently a modification of the classification has been proposed but not yet universally accepted (Denton et al., 2008). The Bethesda system uses the dichotomization of the dysplastic lesions in LSIL and HSIL and classifies of lower cervical diseases as Atypical Squamous Cells (ASC) that can be of undetermined significance (ASC-US) or that cannot exclude HSIL (ASC-H).
FIGURE 1-3. Classification of cytological and histological lesions. The diagram is arranged to represent the relationship between the different classifications. The European (British) system is highlighted. AGC = atypical glandular cells; ASC-H = atypical squamous cells cannot exclude HSIL; ASC-US = atypical squamous cells of undetermined significance; LSIL = low grade squamous intraepithelial lesion; HSIL = high grade squamous intraepithelial lesion; SCC = squamous cell carcinoma; ADC = adenocarcinoma. (Bulkmans et al., 2004, with modifications).
Cervical cancer can be subdivided according to the cell type precursors: squamous cell carcinoma (SCC) and adenocarcinoma (ADC) derive from epithelial and glandular cells, respectively (Bulkmans et al., 2004).
Progression of the cervical lesions In a 48 month follow up study, the percentage of women who developed cervical lesions after infection at baseline was 37.9% for CIN1, 6.3% for CIN2, 2.8% for CIN3 and 0.1% for ADC (Rodriguez et al., 2012). Different rates of progression have been associated with the disease grade: CIN1 has a 20% chance of progression to CIN2, which in turn progresses to CIN3 with a rate of 30%, and 40% of CIN3 lesions progress to SCC (Schiffman et al., 2011). Persistent infections with HR-HPV are therefore strongly correlated with the cervical disease progression (Chen et al., 2011). The odds ratio for progression to CIN3 at 4 years in patient infected with HPV16 is 347 as opposed to 10 for cigarette smoking and development of lung cancer (Bosch et al., 2002).
Human Papillomavirus Virion structure The genomes of the Papillomaviruses consist of covalently closed circular double stranded DNA molecules combined with cellular histones to form mini-chromosomes (figure 1-4A) (You, 2010). The nucleosomes are thought to freely slide along the genome (Stunkel and
FIGURE 1-4. Structure of the HPV16 genome. A: nucleosomal organization of the viral genome extracted from virions in mild conditions. B: organization of the viral genome due to the adhesion of the matrix attachment region on the nuclear matrix. C: organization of the Human Papillomavirus episomal genome exemplified by HPV16. Origin of replication, promoters, poly-adenilation sites, splicing donor, acceptors and other features are reported in the legend; SNR = short non-coding region. (A: Favre et al., 1977; B: Tan et al., 1998). !
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Bernard, 1999). Nevertheless some nucleosomes have been shown to maintain specific positions; in particular, one is overlapping the viral enhancer (Ne) and another lays over the viral early promoter and the origin of replication (Np). These nucleosomes are expected to repress viral transcription and replication by concealing the cognate sequences for the transcriptional activators Sp1 and TATA binding protein (TBP) as well as the viral origin of replication. Activation of the late phases of the viral biochemistry requires the re-localization of such viral nucleosomes (del Mar Pena and Laimins, 2001). Papillomavirus genomes contain AT-rich sequences known as matrix attachment regions (MARs) that are assumed to tether the genome to the nuclear scaffold and possibly partitions it into functional loops (figure 1-4B) (McBride, 2008; Tan et al., 1998). The genome can be subdivided into two main transcriptional units: early (E) and late (L), each of them containing a major promoter (indicated as PE or PL, respectively) and a polyadenylation site (figure 1-4C) (Zheng and Baker, 2006). Nonetheless, several promoters have been characterized and have been shown to have multiple starting points; the Papillomavirus transcripts undergo splicing, are polycistronic and supposedly translated by a leaky scanning mechanism (figure 1-5) (Kim and Taylor, 2003; Stacey et al., 2000; Wang et al., 2011). Two ORFs have been identified in the late region, encoding for the capsid proteins L1 and L2, whereas a number of ORFs with regulatory function have been identified in the early region: E1, E2, E4, E5, E6 and E7 (Danos et al., 1982). The Papillomaviruses gene expression is controlled by the long control region (LCR), or upstream regulatory region (URR), which contains the cognate sites for several transcriptional factors and includes an enhancer (figure 1-6) (Bernard, 2000). The LCR region also allocates the origin of replication (ori); this is characterized by the presence of the cognate sequences for the viral polypeptides involved in the viral replication (E1 and E2) (McBride, 2008). The portion of DNA between the E and L regions contains the early poly-adenylation site and the PL and has been named short non-coding region (SNR) (Maki et al., 1996). ,
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FIGURE 1-5. HPV16 transcription map. Linear representation of the HPV16 genome showing the different ORFs; the early (P97) and late (P670) promoters and the early (AE) and late (AL) poly-adenylation sites are illustrated (upper panel). The potential transcripts of HPV16 are depicted (lower panel); for each mRNA the exons (bars), the introns (thin lines) are reported. (Kajitani et al., 2012).
Cryoelectron microscopy has demonstrated that HPV capsids consists of a non-enveloped particles composed of 60 hexavalent (hexons) and 12 pentavalent (pentons) capsomeres, arranged with a right-handed T=7 morphology (figure 1-7A) (Xu et al., 2006). Each capsomere is formed by homopentamers of the major capsid protein L1; variable amounts of the minor capsid protein L2 are suggested to be embedded within the capsomeres (figure 1-7B) (Buck et al., 2008). Expression of L1 proteins by both eukaryotic or prokaryotic systems has resulted in the production of self-assembled empty capsids, known as virus-like particles (VLP), even in absence of L2 (Pereira et al., 2009; Xu et al., 2006).
Classification and phylogeny In 2001 the Papillomaviruses had been classified into a specific family, the Papillomaviridae (Fauquet and Mayo, 2001). The members share the features of a naked icosahedral
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FIGURE 1-6. Structure of the LCR of the genital HPVs. The LCR is delimited between the L1 and E6 ORFs. The localizations of the enhancer, the matrix attachment regions (MARs), the origin of replication ( ), the specifically positioned nucleosomes (grey boxes) and the cognate sequence for transcriptional and replication factors, including CDP ( ), are indicated. The E2 binding sire (E2) are numbered. HPV strains and species are reported. (O’Connor et al., 1995, with modifications). P
capsid of about 55 nm in diameter, containing a circular double-stranded DNA genome of roughly 8 Kb in length in which all open reading frames (ORF) are orientated in the same direction on the same strand. The members of the Papillomaviridae family show a stringent species-specificity and can be recovered all the amniotes (Bennett et al., 2010; Campo, 2002; Herbst et al., 2009; Lange et al., 2011). The evolutionary rate of the Papillomaviruses is low, with a mutation fixation rate estimated in the order of 10–7-10–8 base substitutions per site per year, as opposed to a rate of 10–9 base substitutions per site per year for the human -globin and between 10–3 and 10–5 base substitutions per site per year for Human Immunodeficiency virus (Gojobori et al., 1990; Halpern, 2000).
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FIGURE 1-7. HPV virion structure. A: surface-shaded representations of the HPV capsid showing the pentavalent (5) and hexavalent (6) capsomeres. B: close up of the pentavalent (left) and hexavalent (right) capsomeres (upper panel) and their cross-sections (lower panel); the possible position of L2 is indicated (arrow). (A: Belnap et al., 1996, with modifications; B Trus et al., 1997).
The classification of Papillomaviruses is based on sequence homology of the major capsid protein L1 ORF, the most conserved region within the genome (de Villiers et al., 2004). Based on the L1 sequence, the Papillomaviridae family is currently arranged in 29 genera, identified with Greek letters, further subdivided in species, genotypes and subtypes (figure 1-8) (Bernard et al., 2010). Nucleotide L1 identity between clades is defined as follows: in genera is 98 percent. The genital HR HPVs belong to the Alpha genus (Bernard et al., 2006). The International Committee on Taxonomy of Viruses does not recognize ‘type’ as a taxonomic unit, preferring the term ‘strain’, and applies the norm of naming the species after a prototype virus (Bernard, 2005). Thus, for instance, HPV16 is commonly named type 16 (alpha-9) but it should be referred to as strain 16 (HPV16). Nevertheless ‘type’, used as a synonym of ‘genotype’, is historically accepted as a label. HPV species correlates better than strains to pathological outcomes; consequently the former is a more clinically defining term.
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FIGURE 1-8. Phylogenetic tree of the Papillomaviridae family. Maximum likelihood tree of the known Papillomaviruses based on the E1-E2-L1 sequence, showing the main lineages (in red, green, blue, and yellow) and the not classified clade (black) together with the hosts (silhouettes). Species HPV16 (alpha-9) and HPV18 (alpha-7) are highlighted; high risk strains are indicated with a dot. (Bravo et al., 2010, with modifications). The HPV16 genome can be differentiated into six geographically defined phylogenic subtypes: European (E), African (Af) 1 and 2, East-Asian (EA), Asian-American (AA) and NorthAmerican (NA); the E lineage can be further subdivided according to a T350G single nucleotide polymorphism (SNP) (figure 1-9) (Cornet et al., 2013a; Yamada et al., 1997).
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FIGURE 1-9. Geographical distribution of the HPV16 lineages. The prevalence of the different geographical variants is depicted. The European lineage is further subdivided according to the T350G polymorphism. AA = Asian-American; Af = African; As = Asian; E = European; NA = North America. (Yamada et al., 1997).
More recent analysis allowed a further separation of the AA and Af lineages rising to nine the number of HPV16 geographic lineages (Cornet et al., 2012). The HPV16 prototype has been originally isolated from a German patient belonging to the E-T350 lineage (Durst et al., 1983). The regulatory region sequences from 57 HPV18 isolates allowed the identification of four main geographical clusters distinct from the HPV18 prototype (Boshart et al., 1984): European, African, East Asian and American Indian (Ong et al., 1993). The HPV31 strain could be subdivided in the A, B and C lineages (Ferenczi et al., 2013) whereas HPV45 has been dichotomized in the A and B clades (Chen et al., 2009).
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Life cycle Transmission, attachment and entry The transmission route of the Papillomaviruses is through direct contact; genital HPVs can be communicated by sexual contact (Forcier and Musacchio, 2010) and the identification of virgins infected with genital HPV has indicated the possibility of exposure by nonpenetrative intercourse (Winer et al., 2003). Horizontal transmission of Papillomaviruses by fomites and blood transfusion has been suggested (Bodaghi et al., 2005; Lacour and Trimble, 2012; Pao et al., 1993; Roden et al., 1997); vertical transmission of HR HPV genotypes, possibly by exposure of the newborn to an infected uterus, has been also reported (Cason et al., 1995; Kaye et al., 1996; Pakarian et al., 1994). Papillomaviruses are thought to begin their replicative cycle by gaining access to the basal layer cell through abrasions that expose the innermost layers of the epithelium; here they bind to sulphated polymers, in particular heparan sulphate proteoglycans, present on both the basal layer of the epithelium and on the surface of epithelial cells (figure 1-10) (Doorbar et al., 2012; Sapp and Bienkowska-Haba, 2009; Sapp and Day, 2009). The internalization of Papillomaviruses is a peculiarly slow process lasting for several hours; the genome is believed to gain access to the nucleus after the dissolution of the nuclear membrane during mitosis (Schiller et al., 2010). As in the case of Herpes Simplex virus, Adenovirus and SV40, the ignition of the early stages of the HPV biochemistry is believed to be carried out by cellular proteins present in the nuclear domains 10 (ND10), nuclear bodies associated with transcription and replication activities (Ching et al., 2005; Ishov and Maul, 1996; Ishov et al., 1997; Schiller et al., 2010). Significantly, early promoter transcription and replication independent from the viral proteins have been described, suggesting that viral proteins are not required to activate the viral replicative cycle (Kim et al., 2005; Romanczuk et al., 1990).
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FIGURE 1-10. HPV life cycle. The virus binds to the heparan sulphate proteoglycans present on the extracellular matrix of the basement membrane. The access is obtained through abrasions. The complex virus/ receptor is transported to the cell’s surface, where internalization takes place. The virus replicates in the stem cells located in the basal layer of the epithelium, maintaining a reservoir. The differentiation pathway of the transiently replicating cells migrating towards the upper layers of the epithelium triggers the late phases of the viral biochemistry, with subsequent generation of proliferative lesions. The viral progeny is assembled in the uppermost layers of the epithelium, and released together with the desquamating keratinocytes. In a minority of infected cells the virus can integrate, a suggested risk factor for the development of unproductive infections and cervical cancer. (Forcier and Musacchio, 2010).
Replication The viral replication requires both E1 and E2 as factors in trans and the ori sequence as a cis element (figure 1-11A) (Kajitani et al., 2012). E1 is formed by about 600 residues and is the only Papillomavirus protein with an enzymatic activity. E1 is subdivided in an N-terminal DNA binding domain and a C-terminal domain with 3
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separated by a variable hinge domain. E1 also interacts with the different proteins of the replication machinery and it is loaded at the ori site by E2 (McBride, 2008).
FIGURE 1-11. Replication in HPV. A: alignment of DNA sequences of the ori region of the HPV strains 16, 18, 31, and 45; the E2BS, E1BS and the A/T rich stretches are reported ( ). Sequences upstream and downstream of the position zero are visualised with different colours. B: the replication in Papillomavirus proceeds bidirectionally through a theta structure (i-iii); failure in termination might allow one replication fork to establish a rolling circle (iv-v). (B: Kusumoto-Matsuo et al., 2011).
E2 is a protein of about 400 amino acids, subdivided in two main domains. The N-terminal end has a transactivation function, whereas the C-terminal domain allows the dimerization with both E2 and E1. The C-terminal domain also recognizes the viral DNA palindrome consensus 5 -ACCG–N4–CGGT-3 , known as E2 binding site (E2BS, where the underlined letters indicate a consensus with a certain degree of variability between genotypes) (Rogers et al., 2011). E2 is also recognized to bind to a plethora of regulators which influence the cellular differentiation, apoptosis and cell cycle (Muller and Demeret, 2012).
Papillomaviruses are believed to display different rates of replication that mark the main phases of the infection (Doorbar et al., 2012). In the early stages of the viral infection the viral genome replicates, in a phase named ‘initial amplification’ or ‘establishment replication’, to about 102 copies per cell, ensuring the subdivision of the viral genomes in the daughter cells during mitosis (Bodily and Laimins, 2011). After this primary expansion, the viral genome is replicated at a steady state so that the number of genomes per cell is kept constant (‘maintenance replication’) and the virus can perpetuate in reservoir stem cells within the basal layer of the epithelium. The migration of the differentiating keratinocytes towards the upper layers of the epithelium is coupled with the activation of the late promoter and the ignition of a fast rate of viral duplication, known as ‘vegetative replication’; in this phase the viral copy number expands in the order of thousands of copies per cell (Hoffmann et al., 2006; Pittayakhajonwut and Angeletti, 2010). The replication proceeds bidirectionally through a theta structure and is carried out by the cellular replication machinery with E1 exploiting a helicase function (figure 1-11B) (Chow and Broker, 1994; Melendy et al., 1995). Rolling circle replication of the Papillomavirus genome has been induced in vitro and has been observed in HPV-derived cell lines, fostering the possibility that this modality of replication is associated with the vegetative phase (Geimanen et al., 2011). Alternatively, it has been proposed that the rolling circle might arise upon failure in the termination step of the Papillomavirus replication (Kusumoto-Matsuo et al., 2011). The replication of HPV has been related to the induction of the DNA damage repair system, suggesting the presence of abnormal replicative intermediates that are likely to be involved in the viral biochemistry and that can generate chromosomal aberrations (Gillespie et al., 2012; Olive, 2011).
Transcription Viral transcription is carried out by the cellular RNA polymerase II and it is modulated by E2 in concert with other cellular transcriptional factors (Thierry, 2009). E2 binds with the
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highest affinity to the E2BS#1 (figure 1-6) thus at low concentrations of E2 this position is preferentially occupied and transcription promoted. At higher amounts, E2 binds to E2BS#3 and #4 displacing the transcription factors Sp1 and TBP, repressing the early promoter and activating both replication and late transcription (Kajitani et al., 2012). The engagement of the PL is coupled with replication but it is disjoint from the E2 modulation and is accompanied by chromosomal and nucleosomal rearrangements of the LCR (del Mar Pena and Laimins, 2001; Kajitani et al., 2012). The coordinated stimulation of both late transcription and vegetative replication in the upper layers of the epithelium assures the timely coexistence of viral genomes and capsid proteins to produce viral progeny in the desquamating cells (Pittayakhajonwut and Angeletti, 2010).
Extension of the S phase The epithelial stem cells generates a replicating progeny known as transit amplifying keratinocytes that, during their migration toward the upper layer of the epithelium, differentiate in mature keratinocytes and withdraw the cell cycle (Yan and Owens, 2008). Since both the epithelial stem cells and the transit amplifying keratinocytes are proliferating, the virus is passively replicated during the mitosis (Bodily and Laimins, 2011; McLaughlin-Drubin et al., 2012; Moody and Laimins, 2010). On the other hand Papillomaviruses evolved specific proteins to counteract the withdrawal from the cell cycle of the keratinocytes. The viral antagonists of the G0 state are encoded in the early region and include E7, E6 and E5. These proteins are not enzymes and absolve their functions interacting with several modulators of the cellular biochemistry (figure 1-12). The resulting aberrant cellular proliferation allows the classification of these polypeptides as oncoproteins. The early promoter is regulated to permit the expression of the appropriate amount of viral proteins to sustain the S phase without inducing senescence (Van Tine et al., 2004). Cells transfected with E6/E7-enconding plasmids have been exhibited to become resistant to final differentiation (Sherman and Schlegel, 1996). Another important outcome of the E6/ ¥
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FIGURE 1-12. Functions of the HR HPV oncoproteins. Diagram illustrating the binding partners of the HPV16 oncoproteins E7 (A), E6 (B) and E5 (C). The affected cellular pathways are depicted. (A,B: Moody and Laimins, 2010; C: Venuti et al., 2011). ±
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E7 expression is the induction of chromosomal instability (Duensing and Munger, 2002) and the increased rate of integration of foreign DNA (Kessis et al., 1996). In addition, these oncoproteins have been suggested to alter the cellular expression of tumor suppressive micro RNAs, to stabilize the viral transcripts and to influence the cell cycle (Zheng and Wang, 2011). The viral protein E7 (about 100 amino acids) has been demonstrated to interact with the members of the retinoblastoma family of proteins (pRb, p107, and p130) (Bodily and Laimins, 2011). The disruption of these factors leads to the stabilization of the transcriptional factor E2F, which in turn enhances the expression of genes involved in the S phase. E7 binds to other cellular proteins, in particular with several histone deacetylases (entangled in the activation of several genes, including E2F) and p21 (a repressor of the cell cycle). The unscheduled reactivation of the S phase brings into action p53, an effector of the DNA repair pathway that can trigger apoptosis (Lim et al., 2007). E6 (150 residues in the HPV16 full length form) associates with a wide range of cellular proteins, which can be classified in specific functional groups: DNA replication and repair, cell cycle control, cell adhesion, signal transduction, apoptosis, and immune recognition (Howie et al., 2009). The best characterized activity of E6 is the ubiquitination of the oncosuppressor p53, through the interaction with an E3 ubiquitin ligase known as E6 associated protein (E6AP) (Huibregtse et al., 1991). Interestingly, the most HR HPV genotypes have been shown not to encode the most efficient E6 proteins in inducing p53 degradation, suggesting that the Papillomavirus-associated oncogenicity might rely on other pathways (Mesplede et al., 2012). The E6 ORF generates both the full length form of the protein as well as smaller splicing variants indicated as E6* that are supposed to counteract the activity of the full length form to further modulate the activity of the viral oncogene (Pim et al., 1997).
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E5 (roughly 83 amino acids) is transcribed from both early and late transcripts, albeit at very low levels (Venuti et al., 2011). Its activity is supposed to enhance the functions of E6 and E7 through association with effectors of the cell cycle, apoptosis and cell-cell interactions. Anyhow, the E5 ORF is found disrupted in a substantial proportion of cervical cancers samples, henceforth its involvement in carcinogenesis is less recognized (Chang et al., 2001).
Viral latency Eukaryotic gene expression is regulated by epigenetic pathways which include the addition of methyl moieties to cytosines within the CG dinucleotide (CpG) and several chemical histone modifications; these alterations dynamically influence one another in what has been named ‘epigenetic cross-talk’ (Dupont et al., 2009; Kondo, 2009). Methylation of the eukaryotic genomes is performed by members of the DNA methyltransferases (DNMT) family: specifically, DNMT3 is responsible for the establishment of methylation in naïve DNA, a process known as de novo methylation; the maintenance of the methylation profiles is carried out by DNMT1 (Brenner and Fuks, 2006). Epigenetic modifications have also been shown to modulate Papillomavirus expression: methylation at the CpG sites within the LCR results in decreased transcription (Hublarova et al., 2009). It has been shown that HPV16 is moderately methylated in undifferentiated cells, whereas upon induction of differentiation the methylation is lost (Kalantari et al., 2008; Vinokurova and von Knebel Doeberitz, 2011). It has been proposed that methylation might be involved in the establishment of a latency state (Badal et al., 2003; Kalantari et al., 2004; Kim et al., 2003). Other viruses, for instance Epstein-Barr virus and Hepatitis B virus, have been shown to use epigenetic regulation to establish latency (Poreba et al., 2011). It has been suggested that the modulation of the E2 function could be exploited by methylation of the E2BS, since this modification inhibits the association of E2 and therefore enhances transcription or replication (Kim et al., 2003; Thain et al., 1996). É
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Encapsidation and release Viral DNA, L1 and L2 assemble together in the nucleus; L2 directs the co-localization of viral genome complexed with E2 and pentamers of L1 at the ND10, thus promoting virion assemble (Florin et al., 2002). The Papillomaviruses can spread in the environment inside the defoliated squamous keratinocytes, which provide protection to the viral progeny (Bodily and Laimins, 2011). Papillomaviruses do not induce cell lysis and do not bud at the cell surface.
Viral transformation HPV and cervical cancer Papillomavirus infections of the cervix are usually self-limited, lasting for an average of 4-20 months (Tota et al., 2011), but about one fifth of the infections can persist for several years and progress towards cancer (Stanley, 2010), which represent a dead-end interaction for the virus because there is not production of viral progeny (figure 1-13) (McLaughlin-Drubin et al., 2012). Infection with HPV has been demonstrated to precede the development of the cervical lesions (Kjaer et al., 2002). Lesions of low grade cytology are diagnosed mostly in women aged 20-30 years whereas cervical cancer diagnosis is most common in patients 50-60 years old, suggesting a progressive evolution of the malignant phenotype (Schiffman et al., 2007; Ting et al., 2010; zur Hausen, 1996). Furthermore, the benign lesions have been shown to be polyclonal, whereas high grade tissues were formed by monoclonal cells, implying that the high grade lesions were derived from a single transformed cellular ancestor (Park et al., 1996; Vinokurova et al., 2005). These observations advocated that high grade lesions could effectively represent precursors of cancerous lesions. In precursor tissues, the expression of the viral early transcripts is extended throughout the thickness of the epithelium, whereas in productive infections these are limited to the Õ
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FIGURE 1-13. Progression of the HR HPV-associated lesions. Viral infection of the cells within the epithelium basal layer determines a series of morphological changes in the affected tissue which include koilocytosis and dyskeratocytosis, the latter being most prevalently identified in cervical intraepithelial neoplasia (CIN). CIN of low grade (CIN1) are usually self resolved but those of high grade (CIN3) are likely to progress toward cancer, with integration being considered a major factor of risk for lesion evolvement. CIN1 are a consequence of the productive phase of the viral infection, therefore high amounts of viral genome could be recovered in such lesions; conversely, the high grade CIN and cancer represents unproductive infections: the viral DNA is scarce and no viral progeny is present. A combination of not clearly defined host, environmental and viral factors are believed to be involved in determining the natural history of the Papillomavirus infection. The progression toward cervical cancer is supposed to span several decades. (Cox, 1995).
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inner layers (Doorbar et al., 2012). In addition, cancerous lesions have been characterized by the presence of viral integrants that constitutively transcribe E6/E7-containing mRNAs (Ho et al., 2011). On the contrary neither late transcripts, encoding for L1 and L2, nor viral particles could be recovered from precursor lesions. These data, coupled with the proliferative and destabilizing activities of E6 and E7, led to a model of carcinogenesis based on the uncontrolled expression of the viral oncoproteins due to the disruption of the modulator function of E2 (Yugawa and Kiyono, 2009; zur Hausen, 1994). Compatible with this consensus, ectopic expression of E2 has been shown to repress cellular proliferation and to inhibit early transcription (Goodwin and DiMaio, 2000). In addition, it has been proposed that E2 could inhibit the E7 activities by direct association (Gammoh et al., 2009). Thus the abolishment of the E2 function could release the activity of the viral oncoproteins.
Viral integrants and cervical cancer Viral integration determines a fundamental switch in the natural history of the Papillomavirus infection: since viral integrants do not generate viral progeny, they are the hallmark of the non productive Papillomavirus infections (Brown and Trimble, 2012). An analysis of integration sites from clinical samples and cell lines has shown that integration involves both viral and cellular sequence rearrangements, with no apparent specificity (Schmitz et al., 2012; Wentzensen et al., 2004). A loss in viral sequences, in particular within the E2 ORF, is typical. Common fragile sites and transcriptionally active regions have been reported to be preferentially targeted for integration, possibly because they have a more accessible chromatin structure. The role of insertional mutagenesis is unclear, given the heterogeneity of the targeted sites; however integration has been suggested to be paired with a chromatin remodelling that might expose the binding sites of transcriptional factors, thus enhancing viral transcription (Bechtold et al., 2003; Lazo, 1987). The identification of short stretches of homologies between cellular and viral sequences at the integrant junctions has pointed to a major role for DNA repair pathways in the integration mechanism (Dall et al., 2008).
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These findings fit with the model of carcinogenesis based on E6/E7 over-expression (figure 1-14) (Korzeniewski et al., 2011; von Knebel Doeberitz, 2002; zur Hausen, 1996). Integration is considered a by-product of the viral biochemistry and is assumed to be a rare event with no sequence specificity. The corollary of this suggestion is that the virus randomly integrates in the cellular chromosomes without preference for a target sequence within the viral genome; however clones with disrupted E2 ORFs are eventually selected because they over-express the early transcripts. The unleashed transcription of the oncoproteins might both improve the cellular proliferation and determine the accumulation of further genetic alterations that can lead, step wisely, to cancer.
FIGURE 1-14. Model of carcinogenesis in HR HPVs. A: in productive infections, E2 inhibits the functions of the oncoproteins E6 and E7, releasing the hindrance of the differentiation commitment. The differentiation pathway is also coupled with the replication of the virus, which requires the E1 polypeptide. B: in unproductive infections, the viral integration determines the disruption of the E2 ORF with a twofold outcome. Firstly, the viral replication is abolished; secondly, the oncoproteins are expressed without regulation. E7 stabilizes the transcriptional factor E2F through binding to pRb with subsequent deregulation of the cell cycle. E6 induces the disposal of the p53 regulatory factor, preventing the activation of the DNA repair and the apoptosis pathways, generating chromosomal instability. The accumulation of chromosomal aberrations is believed to induce cancer formation (Desaintes and Demeret, 1996, with modifications). ù
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In addition, integrants can undergo replication under the control of ectopic E1/E2 proteins (Kadaja et al., 2009; Peter et al., 2010). Once activated, the replication could extend through the flanking regions, resulting in disproportionate replication processes, also known as onion skin replication. This aberrant replication could culminate in the generation of linear DNA fragments that can be recognized by the DNA repair machinery, resulting in chromosomal rearrangements (figure 1-15). Remarkably, origins of replication independent from E1/E2 were described in Papillomavirus (figure 1-8), propounding that silent integrants might act as points of genetic instability during the cellular replication (Hoffmann et al., 2006; Pittayakhajonwut and Angeletti, 2010). On the other hand, low grade lesions might contain silent integrants that are inhibited by the viral episomes (Dall et al., 2008; Pett and Coleman, 2007). The elimination of episomes could represent the trigger for the over-expression of the viral oncogenes and the onion skin replication.
FIGURE 1-15. Model of replication-induced chromosomal instability. The origin of replication of the virus can be fired more than once before the termination of the replicative process, inducing onion skin replication. The viral replication requires E1 and E2, that can be provided by episomes of the same or other viral types. (Kadaja et al., 2007).
The demonstration that de novo methylation targets foreign DNA after integration in the host chromosomes has led to the hypothesis that hypermethylation could represent a cellular mechanism of defence to integration by assuring the transcriptional and replicative repression of the integrants (Doerfler et al., 1995; Yoder et al., 1997). A high level of CpG methylation in the L1 ORF has been observed in HPV-related cancerous lesions (Kalantari et al., 2009; Sun et al., 2011; Turan et al., 2006). The methylation profile of the 3 -L1/ LCR/5 -E6 region has been assessed in W12 cells, which contains predominantly episomal forms of HPV16 (Kalantari et al., 2008). Episomal genomes were moderately methylated in undifferentiated cells and virtually un-methylated in differentiated cells; viral integrants instead did not change their profile upon differentiation.
Viral episomes and cervical cancer Despite this body of evidence linking the integrants to cervical cancer, an elevated proportion of high grade and cervical cancer lesions have been described to contain episomal forms of the virus, suggesting that integration might not be the only determinant for the HPV-associated cancerous phenotype (Chen et al., 1994; Das et al., 2010; Gallo et al., 2003). For instance, it has been observed that deletion of the E2BS induces a lower promoter activation than mutations of the cognate site for the transcriptional repressor YY1 (May et al., 1994). Luciferase experiments with constructs containing LCR have demonstrated an about three fold increased activity in the AA and NA lineages in comparison to the European reference sequence (Kammer et al., 2000). This increase is possibly due to a A7729C transition affecting the cognate site for the transcriptional factor TEF-1 (Veress et al., 1999). Similarly, the reduced and increased replicative phenotypes of the African and American variants, respectively, in comparison to the European lineage have been suggested to be linked to SNPs within the Sp-1 and AP1 binding sites, correspondingly (Hubert, 2005).
Furthermore, significantly higher amounts of early transcripts have been detected in cervical cancer samples bearing fully episomal forms of HPV as opposed to those with entirely integrated virus populations (Das Ghosh et al., 2012). In addition, cells bearing episomal forms of the virus with increased early expression has been reported to become more preponderant than clones containing integrative forms, as evidenced in an in vitro system based on W12 cells (Gray et al., 2010). These transcriptionally active episomal viral genomes were characterized by altered chromatin structure and fluctuating expression levels, suggesting epigenetic regulation of the viral transcription and dynamic adaptation of the viral biology to the cellular environment. These data have evinced that sequence mutations within the cognate binding site of transcriptional regulators and epigenetic modulation might be related to cancer development in samples bearing episomal forms of the virus (Dong et al., 1994).
Immunity and vaccination Immunity Viral regression and recurrence are believed to be influenced by the immune response (Palefsky and Holly, 2003; Schneider, 1994). The humoral response to natural Papillomavirus infection is characterized by low antibody titres, sometimes undetectable in some subjects (Carter et al., 2000). It has been proposed that Papillomaviruses minimize the activation of the immune system by not causing cell lysis, by not having a recognized viremic phase, and by limiting the production of the highly immunogenic capsid antigens in the upper layers of the epithelium and in the final stages of productive infections (Bodily and Laimins, 2011). This evasion, coupled with the activities of the early viral proteins, could explain the reduced immune response observed in Papillomavirus infections.
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Nonetheless animal models demonstrated that neutralizing antibodies directed against L1 protect from incident infections even in low titers (Ghim et al., 2000). Natural seroconversion to Papillomavirus takes usually more than six months to become detectable (Carter et al., 2000). After clearance, the antibody levels drop very quickly, although about 50 percent of seropositive and HPV DNA negative individuals retain good levels of antibody for several years. Cell-mediated immunity has been indicated to play a pivotal role in the viral clearance by targeting the early viral proteins (Stanley, 2012). T-helper and T-killer cells are induced in HPV infection (de Jong et al., 2002; Welters et al., 2003) and the cellular response has been suggested to mount during the infection but then to regress after viral clearance, resembling the humoral response (de Gruijl et al., 1996). Lack of cellular immunity has been instead described in women with cervical cancer, strengthening its importance in the control of the HPV infection (de Jong et al., 2004). Significantly, cellular immunosuppression, as observed in patients co-infected with HIV, is an important co-factor for HPV infection persistence and cervical cancer development (Serraino et al., 2002; Sun et al., 1997). It has recently been suggested that complete viral clearance is probably never accomplished: Papillomaviruses might instead enter a stage of latency that can hide the virus from the immune system and maintain a reservoir of viral genomes in few cells within the basal layer of the epithelium that can be subsequently reactivated (Maglennon and Doorbar, 2012).
Vaccination Vaccination provides protection by inducing neutralizing antibodies and has several advantages over natural infection (Schiller and Lowy, 2012). The intramuscular immunization with L1 VLP induces the production of IgGs that can be recovered in the cervical mucus. In addition the VLPs retain the native conformation and the repetitive highly immunogenic structure of the virions. Two prophylactic vaccines based on L1 VLPs have been devel)
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oped and commercially exploited (Schiller and Lowy, 2012). One is the bivalent Cervarix (GlaxoSmithKline), targeting strains 16 and 18 and produced in the baculovirus system. The other is the quadrivalent Gardasil (Merck), targeting strains 6, 11, 16, and 18 and developed in Saccharomyces cerevisiae cells. Both vaccines are administrated parenterally in three doses and contain aluminium salt adjuvant to delay the release into the body and stimulate the monocytes. The formulation of Cervarix also include monophosphoryl lipid A as an immune stimulator (Giannini et al., 2006). The L1 VLPs present in these vaccines are distinctly immunogenic and generate higher titres than those obtained by natural infection (Harper et al., 2006). The neutralizing antibody produced are protective against incidence infections (Frazer, 2010). More than 99% of vaccinated women have seroconverted to the targeted genotypes after 17 months (Koutsky et al., 2002). Cervarix has been shown to protect from incident infections over eight years after the first administration (Roteli-Martins et al., 2012); for Gardasil the protection from incident infection and absence of high grade cervical diseases has been described in studies performed 3.5 years after vaccination (Mao et al., 2006). Mathematical models have estimated that vaccine-induced immunity could last for 10-30 years (Fraser et al., 2007). The Centres for Disease Control Advisory Committee for Immunization Practices has recommended the administration of these vaccines to girls aged 11-12, that is prior to their sexual debut; vaccination of boys is also possible, but is not considered cost effective (Bonanni et al., 2011). To date, these programmes have been implemented in 33 out of 192 countries around the world (Arbyn et al., 2012). In Europe the nationwide HPV immunization programmes have been adopted since 2007; in United Kingdom the vaccination campaign has begun in September 2008 with the introduction of Cervarix, replaced in September 2012 by Gardasil. This programme is based on the routine vaccination of girls aged 12-13 years as part of the juvenile vaccination platform, and to girls up to 18 years old for the catch-up program. Mathematical simulations based on 80% population coverage 5
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significantly decreased from 60.0% in the normal cytology specimens to 43.5% in severe dysplastic samples and reached 0% in the cancerous lesions (p=0.013).
Viral integration analysis of the longitudinal samples Seventeen longitudinal samples identified by qPCR as bearing HPV16 genomes were analysed for the physical status of the viral genome. Only eight patients (47.1%) had data spanning at least six months and three patients had both a samples at baseline and at study end. The physical analysis was performed averaging the results of two different runs; the correlation between E6 viral copy number obtained by the two tests returned rho=0.985 (p
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