The Anal Canal

362 Pages·2014·30.67 MB·English

Human Papillomavirus Infection of the
Anal Canal
and its Relation to
Anal Disease

This thesis is presented for the degree of Doctor of Philosophy
of the University of Western Australia
September 2012

by

Jenny McCloskey
MBBS, Dip Ven, MPH, FAChSHM, FRCP

School of Medicine and Pharmacology
University of Western Australia

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ABSTRACT/SYNOPSIS

Introduction
Genital warts have been increasing in incidence over the last few decades to being the
most common infection seen in clinics treating sexually transmissible infections prior
to the advent of human papillomavirus quadrivalent vaccination. They have been
thought to be a cosmetic and nuisance problem and only caused by low-risk genotypes
of human papillomavirus. Research has demonstrated that warts are often mixed
infections with both low and high-risk human papillomavirus infection present, and
they are associated with intraepithelial neoplasia, and malignancy. Over the time this
thesis has been written, the importance of anal cancer as an emerging entity has risen,
particularly because even though it is a rare disease, there are increasing disease rates
in both men and women, and it is considered to be in epidemic proportions in some
sub populations (HIV-positive men and women, and HIV-negative men). In addition,
ways of identifying anal cancer precursors have developed and include anal cytology
and anal cytology screening for anal cancer precursors, and a highly specialised clinical
skill called ‘high resolution anoscopy’ to recognise and histologically confirm the
presence of anal precancer. In addition to my surgical skill of scissor excision of
anogenital warts I have become proficient in high resolution anoscopy and
consequently have been able to research the use of this skill along with cytology and
histopathology to identify anal cancer precursor lesions in high-risk individuals. The
HPV vaccines developed commercially to date are designed for HPV naive individuals,
and hence for those with persistent HPV infection there is a need for a therapeutic
vaccine or treatment.

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My research aims were:
1. To assess the prevalence of high-grade anal intraepithelial neoplasia in anogenital
warts.

2. To assess if the prevalence of high-grade anal intraepithelial neoplasia in anogenital
warts had changed over time.

3. To assess ways of diagnosing high-grade anal intraepithelial neoplasia by:
(a) comparing anal cytology to histopathology in patients with anogenital warts
(b) comparing cytology to histology during high resolution anoscopy
(c) comparing different methods of collection of anal cytology during high
resolution anoscopy.

4. To develop a therapeutic strategy for early human papillomavirus infection of the
anogenital tract

Context and collaborations
In 1996 I established a surgical wart database at Royal Perth Hospital. Primarily this
was a tool established for self-audit, and I collected patient demographics, including
age, sex, sexual preference, prior sexually transmitted diseases including genital
herpes, gonorrhoea, chlamydia and HIV status, lifetime partner number and smoking
status. Histology, cytology and high-risk human papillomavirus results were collected.
Additional data on HIV patients including their duration of infection, CD4 cell count
and viral load at the time of surgery, and CD4 nadir were collected. Having noted that
genital warts were being reported as having intraepithelial neoplasia, some with high-
grade intraepithelial neoplasia, I discussed this with Dr Cecily Metcalf in Department of
Anatomical Pathology at Royal Perth Hospital. It became evident they were seeing
more anal intraepithelial neoplasia than other similar pathology services, so it became
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relevant to audit the data to understand the observation we were seeing as clinicians
and pathologists.
In 2010 I established a prospective high resolution anoscopy service at Royal Perth
Hospital, having acquired the skills to perform this highly specialised procedure.
Patient demographics including age, sex, sexual preference, prior sexually transmitted
diseases and HIV status, histology and cytology and high-risk human papillomavirus
results were collected for patients examined. Additional laboratory cytological
information was collected in the PathWest Laboratory Medicine (WA) Royal Perth
Hospital Laboratory. A new research partnership was developed with Dr Vin Williams
in the Western Australian Biomedical Research Institute, (School of Biomedical
Sciences) within the Curtin Health Innovation Research Institute, at Curtin University to
do the cytology research. A patent for treatment of early human papillomavirus
infection of the anogenital tract was lodged initially in Australia, then in Europe, and
the United States of America.

Results
Chapter 1 provides a review of the relevant literature as a background to the
manuscripts submitted for this thesis. Chapter 2 describes the audit of the anogenital
wart database which revealed the frequency of high-grade anal intraepithelial
neoplasia in warts was higher than previously recognised. In this study HIV-positive
men with anal/perianal warts were found to have very high rates (70%) of any grade of
intraepithelial neoplasia (AIN 1-3) with 52% having high-grade intraepithelial
neoplasia. HIV-negative men were found to have 20% high-grade intraepithelial
neoplasia in warts. This is an important observation because warts have previously
been thought to be benign, and the research suggests they may be particularly
important in HIV-positive men, augmenting their risk of developing anal cancer.
Reanalysis of the enlarged database described in Chapter 3 found the initial high
frequency of intraepithelial neoplasia to be maintained over a longer time period thus
strengthening the initial observations by increased power from a cohort approximately
double the original cohort size. Because of auditing the results in the wart and high
resolution anoscopy database, note was taken when microinvasive anal cancer was
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seen for the first time in the sexual health service, and the case series observed are
described in Chapter 4.

Conventional cytology taken at the time of surgery was found to increase the
detection of high-grade intraepithelial neoplasia by about 10% and this research is
presented in Chapter 5. Rather than using any of the established databases in the
Sexual Health Service, the Anatomical Pathology Reports Database was interrogated
for paired histology and cytology samples (taken before the establishment of a high
resolution anoscopy service) and the findings presented in Chapter 6. Anal cytology
was found to be sensitive for the detection of abnormal squamous cells but with low
specificity. Chapter 7 describes a comparative cytology study where conventional
cytology was found to perform better than ThinPrep cytology. In this study the
samples were taken at the time of high resolution anoscopy which was found to be the
gold standard.

Chapter 8 is a comprehensive audit of variables in the wart database and a unique
association between gonorrhoea, hepatitis B and C and high-grade intraepithelial
neoplasia has been observed in HIV-positive men with chlamydia found to be
protective. There are public health ramifications for the association of high-grade anal
intraepithelial neoplasia and gonorrhoea because of the emergence of serious drug
resistance, and the potential from our observations for untreatable gonorrhoea to
contribute to escalation of the anal cancer epidemic in MSM. The risk increased with
multiple infections and there is biological plausibility for the observations.

The Appendix contains two papers I was invited to write. Appendix 1 was written for
an international audience interested in human papillomavirus infection and Appendix
2 was written for a general practitioner audience. The Supplementary material in the
appendix includes a patent of both a treatment device for the cervix, and another
treatment device for the anus for treatment of human papillomavirus. As the HPV
vaccine does not cover all HPV strains, and some individuals are not vaccinated and
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acquire HPV infection, there is still a need for treatment of HPV infection.
Consequently progress with the patent of treatment devices to treat cervical and anal
human papillomavirus is occurring. It has been progressing through the legal system
and the intellectual property has been assigned to me by the Health Department of
Western Australia.

Conclusions
Future directions are discussed in Chapter 9 and indicate further research needs to be
undertaken to understand the clinical observation of high-grade intraepithelial
neoplasia in warts and the association of genital warts with malignancy. The possibility
of low-risk strains contributing to the development of intraepithelial neoplasia in the
early stages of infection needs to be explored. The recognition of high-grade
intraepithelial neoplasia in anal warts has validated the need for surgical removal of
anogenital warts that may have otherwise been left untreated, hence improving
clinical outcomes for patients. Further studies clarifying the natural history of the
various stages of disease are required. The timing of disease intervention including
studies of treatment morbidity including psychological morbidity also need to be
further studied, as does the morbidity of not providing any intervention. The
establishment of a moderate size anal/perianal wart database has established a rare
world resource which has been recognised, and consequently genotyping studies of
the database are being performed collaboratively with colleagues at the Institute
d’Oncologica in Barcelona Spain. This research work will be of assistance in tailoring
the human papillomavirus vaccine strains for men. Commercialisation and clinical trials
of the patented devices are being planned.

Further research is required with larger studies to establish whether conventional
cytology or ThinPrep cytology is best. Additional studies are needed to determine
which swab – Dacron or a cytobrush or another form of swab, should be used to obtain
the best results. Anal cytology can be used to screen for high-grade anal intraepithelial
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neoplasia in larger populations, and screening studies should be promoted in high-risk
groups. Whether histology is in fact the gold standard at high resolution anoscopy
remains to be explored as the detection of high-grade disease depends on the skill of
the operator.

The unique observation of an association between high-risk human papillomavirus
infection, and gonorrhoea in HIV-positive men with high-grade anal intraepithelial
neoplasia and anogenital warts has major public health ramifications for MSM and
their sexual practices. Larger studies are required to confirm this association.

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TABLE OF CONTENTS

ABSTRACT/SYNOPSIS .................................................................... i
TABLE OF CONTENTS ................................................................. vii
Acknowledgements ......................................................................... xi
Statement of Candidate Contribution ............................................. xiii
List of Presentations Relevant to this Thesis ................................ xvii
Oral Presentations ................................................................................................................. xvii
Posters ................................................................................................................................... xvii
List of Figures Chapter 1 ............................................................... xix
List of Tables Chapter 1 ................................................................. xx
List of Abbreviations ...................................................................... xxi
CHAPTER 1 LITERATURE REVIEW ............................................. 1
1. INTRODUCTION TO LITERATURE REVIEW .......................... 3
1.1 OVERVIEW OF HUMAN PAPILLOMAVIRUS INFECTION ...... 4
1.1.1 HPV Biology ............................................................................................................... 4
1.1.2 HPV Replication ......................................................................................................... 6
1.1.3 The intracellular mechanism of the E6 and 7 proteins ............................................. 8
1.2 ANATOMY OF THE ANAL CANAL .......................................... 9
1.3 EPIDEMIOLOGY OF HPV IN THE ANAL CANAL .................. 11
1.3.1 Prevalence of warts ................................................................................................. 11
1.3.2 Impact of HPV Vaccination ...................................................................................... 13
1.3.3 Anal/perianal warts ................................................................................................. 16
1.3.4 Risk factors for anal warts and HPV ........................................................................ 17
1.3.5 HPV prevalence ....................................................................................................... 18
1.3.5.1 Prevalence related to sexual preference and HIV status .................................... 18
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1.3.5.2 Anal HPV in heterosexual men ............................................................................ 19
1.3.5.3 Anal HPV in women ............................................................................................. 20
1.3.5.4 Natural history of HPV in the anal canal .............................................................. 21
1.3.6 Anal HPV genotyping studies ................................................................................... 23
1.3.6.1 Infection with multiple genotypes....................................................................... 27
1.3.7 HPV genotypes types in warts ................................................................................. 28
1.4 ANAL CANCER ...................................................................... 31
1.4.1 Epidemiology of anal cancer .................................................................................... 31
1.4.2 Risk factors for anal cancer ...................................................................................... 37
1.4.2.1 HPV genotyping studies in anal cancer ............................................................... 38
1.4.2.2 Genital warts ....................................................................................................... 40
1.4.2.3 Sexually transmitted infections and other factors .............................................. 41
1.4.2.4 Cigarette smoking ................................................................................................ 42
1.4.2.5 HIV infection and immunosuppression ............................................................... 43
1.4.2.6 Previous genital cancer ........................................................................................ 44
1.5 PARALLELS WITH CERVICAL CANCER .............................. 45
1.6 MICROINVASIVE ANAL CANCER ......................................... 46
1.7 ANAL INTRAEPITHELIAL NEOPLASIA ................................. 47
1.7.1 Histological and cytological definitions of AIN ........................................................ 47
1.7.2 Prevalence of anal intraepithelial neoplasia ........................................................... 49
1.7.3 Natural history of AIN: progression/regression ...................................................... 51
1.7.4 Progression to invasive disease ............................................................................... 54
1.7.5 DIAGNOSIS OF ANAL INTRAEPITHELIAL NEOPLASIA ................................................ 57
1.7.5.1 Anal cytological studies ........................................................................................... 58
1.7.5.1.1 Screening with anal cytology ........................................................................... 62
1.7.5.1.2 Techniques for collection of anal cytology: blind, directly observed, self-
collected 67
1.7.5.1.3 Physician or self-collected ............................................................................... 68
1.7.5.1.4 Blind or anoscope guided? .............................................................................. 70
1.7.5.1.5 Self swabbing studies ...................................................................................... 70
1.7.5.1.6 Which collecting device to use? ...................................................................... 70
1.7.5.1.7 Liquid based or conventional cytology? .......................................................... 71
1.7.5.1.8 Interobservor and intraobservor error in cytology and histology ....................... 73
1.7.5.2 High resolution anoscopy ........................................................................................ 75
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