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Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference

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Abstract …

Abstract
The 18th international papillomavirus
conference took place in Barcelona, Spain
in July 2000. The HPV clinical workshop
was jointly organised with the annual
meeting of the Spanish Association of Cervical
Pathology and Colposcopy. The conference
included 615 abstracts describing
ongoing research in epidemiology,
diagnosis/screening, treatment/prognosis,
immunology/human immunodeficiency
virus, vaccine development/trials,
transformation/progression, replication,
transcription/translation, viral protein
functions, and viral and host interactions.
This leader summarises the highlights
presented at the conference (the full text
of the abstracts and lectures can be found
at www.hpv2000.com). Relevant material
in Spanish can be found at www.aepcc.
org


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  • 1. J Clin Pathol 2001;54:163–175 163 Leaders Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference F X Bosch, T Rohan, A Schneider, I Frazer, H Pfister, X Castellsagué, S de Sanjosé, V Moreno, L M Puig-Tintore, P G Smith, N Muñoz, H zur Hausen Servei Abstract www.hpv2000.com and the numbers in paren- d’Epidemiologia i The 18th international papillomavirus thesis in the text below refer to the abstract Registre del Càncer, conference took place in Barcelona, Spain number on the website. Key publications for Institut Català d’Oncologia, Av Gran in July 2000. The HPV clinical workshop further reading are listed at the end of this Via KM 2,7, E.08907 was jointly organised with the annual paper.1–19 L’Hospitalet de meeting of the Spanish Association of Cer- The opening session was given by PG Smith Llobregat, Barcelona, vical Pathology and Colposcopy. The con- (London School of Hygiene and Tropical Spain ference included 615 abstracts describing Medicine) who lectured on vaccination trials in F X Bosch ongoing research in epidemiology, developing countries. Professor Smith stressed X Castellsagué S de Sanjosé diagnosis/screening, treatment/prognosis, that the next decade holds the promise of new V Moreno immunology/human immunodeficiency vaccines against some of the major infectious virus, vaccine development/trials, diseases aVecting mankind. Most of the Department of transformation/progression, replication, research and deployment eVorts are, and will Epidemiology and transcription/translation, viral protein continue to be, directed at vaccines against the Social Medicine, functions, and viral and host interactions. major infectious diseases, such as malaria, Albert Einstein College of Medicine, This leader summarises the highlights tuberculosis, pneumonia, meningitis, and New York NY–10461, presented at the conference (the full text AIDS. However, some will be directed at what USA of the abstracts and lectures can be found have traditionally been called “chronic” dis- T Rohan at www.hpv2000.com). Relevant material eases, an increasing number of which appear to in Spanish can be found at www.aepcc. have an infectious aetiology. Hepatitis B vacci- Department of org. nation should have a profound influence on the Gynaecology, (J Clin Pathol 2001;54:163–175) rates of chronic liver disease and liver cancer. Friedrich Schiller University D–07743, Similarly, the aetiological association between Keywords: papillomavirus; epidemiology; immunology; Jena, Germany biology; screening HPV and cervical cancer has become increas- A Schneider ingly clear and vaccines that prevent or treat these infections are on the near horizon. Centre for The need for HPV vaccines is greatest in The 18th international papillomavirus confer- Immunology and Cancer Research, ence took place in Barcelona, Spain in July areas of high incidence of cervical cancer and University of 2000. The human papillomavirus (HPV) clini- especially in those settings where diagnostic Queensland, 4102 cal workshop was jointly organised with the and treatment services are poor. In most of the Brisbane, Australia annual meeting of the Spanish Association of industrialised countries eVective screening and I Frazer Cervical Pathology and Colposcopy. The treatment programmes for early lesions have conference included 615 abstracts describing the potential to reduce cervical cancer mor- Institute of Virology, University of Koln, ongoing research as follows: epidemiology tality rates to low levels, but even in these D–50935 Koln, (127 abstracts), diagnosis/screening (129 countries it is often the case that those at high- Germany abstracts), treatment/prognosis (39 abstracts), est risk of disease are least likely to enter H Pfister immunology/human immunodeficiency virus screening programmes. In the developing (HIV) (61 abstracts), vaccine development/ countries, the resources for screening pro- Secció de Ginecologia grammes are sparse, as is access to eVective trials (56 abstracts), transformation/progression Oncològica ICGON, Hospital Clínic (53 abstacts), replication (20 abstracts), treatment for any lesions identified. An eYca- Universitari, E–08036 transcription/translation (30 abstracts), viral cious vaccine against HPV infections, espe- Barcelona, Spain protein functions (40 abstracts), and viral and cially one that acted against established infec- L M Puig-Tintore host interactions (60 abstracts). A special work- tions, as well as preventing new ones, could shop devoted to presenting ongoing research in have a rapid and profound impact on one of the continued over developing countries was also organised and major cancers aZicting women globally. Correspondence to: included 138 abstracts. The conference also Before new vaccines are introduced into Dr Bosch featured 80 invited lectures. The highlights pre- widespread use it is essential that their safety x.bosch@ico.scs.es sented at the conference were selected and are and eYcacy should be properly established. Accepted for publication briefly reported below. The full text of the This will require large randomised controlled 6 December 2000 abstracts and lectures can be found at trials, usually double blind, preceded by www.jclinpath.com
  • 2. 164 Bosch, Rohan, Schneider, et al Department of appropriate smaller scale studies (preclinical HPV-16, 182; HPV-18, 231; HPV-45, 148; and Infectious and Tropical and phase I/II trials) to establish immuno- HPV-31, 71.5. The study updated the conclu- Diseases, London genicity and to detect any major adverse sions of the HPV IARC monograph providing School of Hygiene and Tropical Medicine, eVects. Initial trials may be conducted in the strongest evidence to support the identifi- London WC1E 7HT, developed countries, where the candidate vac- cation of HPV types 16, 18, 31, 33, 35, 45, 51, UK cines are most likely to be produced, at least 52, 58, and 59 as human carcinogens. P G Smith initially. But even if they are found to be safe Most of the epidemiology presentations and eYcacious in these settings it cannot be were on genital HPV infections and cervical Unit of Field and assumed that the same eYcacy will hold in neoplasia. One descriptive epidemiological Intervention Studies, other geographical and ecological situations. report of interest was from Lazcano-Ponce et International Agency Further large scale trials will be required in for Research on al (100) who, in studying a population based Cancer, F–69372 developing countries, where the main market sample of 1340 Mexican women, observed Lyon, France for the vaccines is likely to be, in public health two peaks of HPV DNA prevalence, one in N Muñoz terms if not economically. women aged < 25 years, and the other in Special methods need to be developed to women over the age of 65 years, with the low- Deutsche ensure that individuals included in trials, many est prevalence being seen in women aged Krebsforschungs- of whom may be illiterate, can be followed up 35–44 years. In all age groups, high risk HPV zentrum, D–69120 and the endpoints of interest ascertained, often Heidelberg, Germany types predominated. These data are intriguing many years after an individual has entered into because they seem to contradict the previously H zur Hausen the trial. Furthermore, in such situations, widely accepted notion; namely, that HPV major ethical issues need to be dealt with. The prevalence declines with age. They are, level of medical care that must be provided for however, in agreement with data from a recent those in a trial has been the subject of heated JNCI report expanded by an abstract by Her- debate. rero et al (054) from the Costa Rica study, and Conducting trials of new “expensive” vac- with those from some of the studies included cines in developing countries has been consid- in his international prevalence survey reported ered unethical because it is unlikely that those at this meeting. The interpretation of the peak countries will be able to aVord to include a in prevalence in older women is not clear, but vaccine that is shown to be eYcacious in the could represent either a cohort eVect or reac- routine vaccination programme. An issue that tivation of latent infection. Evidence for this must be discussed before a trial starts is the extent to which an eYcacious vaccine will be being a cohort eVect was provided by Peto et al made available after a trial. At the very least (308), who showed a positive correlation those in the “control” group should be oVered between the HPV prevalence by birth cohort the vaccine, if they are still likely to benefit. and the corresponding lifetime risk of dying However, many consider the responsibility of from cervical cancer in the UK. the sponsors goes beyond this to the wider In work that has some bearing on the community. It is common now to incorporate a Bethesda system, Peto et al (179) presented cost–benefit analysis into field trials of new data from the Manchester cohort showing that interventions. the prevalence of cervical intraepithelial neo- Most existing vaccines that are in widespread plasia 3 (CIN3) increased linearly with increas- use in developing countries are directed at ing time since last normal smear (for intervals infections usually acquired in infancy or child- up to six years), suggesting therefore that the hood. However, it seems unlikely that HPV true incidence of new disease can be estimated vaccines will be directed at this age group, at reliably in a screened cohort and that CIN3 least initially. The target group, for preventive cases persist for at least this interval. The vaccines, is likely to comprise girls or young prevalence of other abnormalities, including women shortly before the onset of sexual activ- notably CIN2, was independent of the screen- ity. This age group is more diYcult to recruit ing interval and essentially unrelated to HPV in into trials, especially because in many develop- a previous normal smear, suggesting that these ing countries girls of this age are unlikely to be abnormalities are part of the normal morpho- in school. Those planning HIV vaccine trials logical spectrum of transient HPV infection. If will have similar diYculties and the ground- correct, this would suggest that the inclusion of work already done in preparing for HIV trials CIN2 in the high grade squamous intraepithe- may be of great value for the planning of HPV lial lesion (HSIL) category of the Bethesda vaccine trials. Indeed, a successful HPV classification system might be both a clinical vaccine looks to be closer than an HIV vaccine. and scientific error, a conclusion supported by some recent cohort data from Toronto. Epidemiology HPV serology raised some attention at the The results of combined analyses of the Inter- meeting. Two population based studies by national Agency for Research on Cancer Herrero et al, one in Argentina (165) and one (IARC) based multicentric case control study in Thailand (167), examined correlates of type were presented (Muñoz et al, 053). The studies specific virus-like particles (VLPs). Both stud- included 2288 cases and 2513 controls from ies were similar in design and both showed that seven countries and used advanced polymerase the prevalence of antibodies against VLPs of chain reaction (PCR) technology for HPV HPV types 16, 18, 31, 33, and 58 increased detection and typing. The combined odds ratio with increasing number of sexual partners. (OR) for viral detection was 83.3 (95% They also showed a strong correlation between confidence interval (CI) 54.9 to 105.3) and the the detection of antibodies against a specific ORs for the most relevant viral types were: HPV type and the presence of the same type in www.jclinpath.com
  • 3. Highlights of HPV 2000 165 the cervix, and that there is considerable cross- COFACTORS IN CERVICAL CARCINOGENESIS reactivity. Antibodies were rare in virgins. In a Cofactors continue to be of interest in relation study of anti-VLP-16 responses in young to HPV related cervical neoplasia because not women, Ho et al (058) showed that those with all HPV positive women develop cervical persistent IgG antibodies to HPV-16 VLP were cancer. In this regard, Moreno et al (061), using half as likely to acquire HPV of any type, data from the IARC multicentric case control suggesting that persistent antibody may cross study, showed strong evidence for the role of protect against infection with other HPV types. OCs as a late stage factor in HPV induced car- Thomann et al (157), in the Ludwig-McGill cinogenesis. According to this study, women cohort, showed that IgM seropositivity at who had used OCs for six or more years and enrolment was predictive of the risk of acquisi- were found to be positive for a high risk HPV tion of incident HPV-16 infection, whereas IgG type had a four times greater risk of developing seropositivity was more consistently associated squamous cell cervical cancer than women with HPV DNA who were not exposed to OCs. with prevalent or persistent oncogenic HPV OCs did not modify the chances of being HPV infection. IgM seropositivity had no diagnostic positive among controls. This finding might value with respect to the detection of HPV have clinical implications. Two studies sug- infection or cervical lesions. gested that Chlamydia trachomatis infection In an intriguing report, Lewis et al (145) might increase the risk of invasive squamous described the use of capture enzyme linked cervical cancer. Wallin et al (113) presented immunosorbent assay (ELISA) to study anti- results from a prospective study in Sweden in bodies against HPV types 6/11 and 16 in saliva, which C trachomatis detected in cervical smears oral mucosal transudates, and serum. IgG anti- collected on average 5.6 years before diagnosis bodies to HPV types 6/11 and 16 were detected or termination of follow up was associated with less frequently in the oral samples than in a 17-fold increase in risk of invasive cervical serum, possibly because of suboptimal collec- cancer after adjustment for HPV status, and tion of oral specimens or because antibody Smith et al (071) found a positive association concentrations in the oral specimens are truly between C trachomatis seropositivity and inva- lower. Nevertheless, HPV IgG values in oral sive squamous cervical cancer risk in HPV specimens correlated with seropositivity, and if positive subjects in case control studies in Bra- this approach can be optimised, it is conceiv- zil and the Philippines, with the ORs increasing able that oral samples could be useful in epide- with increasing C trachomatis antibody titres. In miological studies as a non-invasive alternative contrast, van den Brule et al (063) found no to serological specimens. association between genital C trachomatis infec- Some data were presented on infections with tion and risk of subsequent HSIL in HPV multiple HPV types, which is of interest in positive women, but a possible association with relation to vaccine development. For example, the risk of abnormal squamous cells of indeter- in the Ludwig-McGill cohort, Rousseau et al mined significance/low grade squamous in- (137) showed that multiple infections were traepithelial lesion (ASCUS/LSIL), and Mo- relatively common, occurring in about 19% of scicki et al (075) did not show an association women each visit. Furthermore, assuming between C trachomatis (and other sexually independence of types, she observed an excess transmitted diseases) and the risk of HSIL. If, of joint cumulative positivity of types involving indeed, C trachomatis does influence the devel- HPV types 16, 31, and 53, suggesting that opment of cervical neoplasia, the mechanism individual type infections are not independent. by which it does so is not clear, but might To a large extent, risk factors for single and involve the induction of a chronic inflamma- multiple infections were similar, although age tory process. In relation to this, Castle et al and number of sexual partners were more (070) presented evidence from the Costa Rican strongly associated with multiple infections cohort suggesting that cervical inflammation was positively associated with the risk of the than with single infections. In addition, Slavin- progression of oncogenic HPV to HSIL or car- sky et al (146) showed that HIV positive cinoma. They also found that bacterial vagino- women were more likely to be infected with sis was inversely associated with the risk of multiple HPV types than HIV negative women. progression. In terms of other possible cofac- Bosch et al (064) presented data from the tors, Smith et al (082) also found a positive IARC multicentre case control study of HPV association between herpes simplex virus 2 and cervical adenocarcinoma, the largest of (HSV-2) serology and cervical cancer risk such studies to date. He showed a very strong overall, and in HPV positive subjects in the positive association between HPV infection Philippine component but not the Brazilian and risk (OR, 68.7; 95% CI, 36.2 to 130.5) and component of their Brazilo-Philippine study. similarly high ORs for individual HPV types. One phenomenon of interest in relation to The report also showed that risk factors for risk of cervical cancer is the so called “male” adenocarcinoma are largely the same as those factor. Essentially, this is taken to mean that the for squamous cell carcinoma, the exception sexual behaviour of men (for example, the being an apparent inverse association between number of sexual partners that they have had) parity and risk. An observation of interest, con- can influence the risk of cervical cancer in their sistent with the results of squamous cell carci- female sexual partners. In this regard, Bleeker nomas, is the likely interaction between long et al (140) presented data showing that 80% of term use of oral contraceptives (OCs) and cer- the male sexual partners of women with CIN vical adenocarcinoma. had penile lesions, of which a substantial www.jclinpath.com
  • 4. 166 Bosch, Rohan, Schneider, et al proportion were infected with HPV, and they detection methods (usually nested PCR) indi- speculated that penile lesions in sexual partners cates that these lingering viruses replicate at of women with CIN are probably productive very low levels, which helps them to escape and that they might play an important role in from immune detection. However, the detec- influencing the course of cervical lesions in tion of antibodies to capsid L1 protein these women by continuously reinfecting them indicates that productive infection does takes with HPV. Castellsagué et al (060), in a pooled place (Feltkamp et al, 102). Persistent infec- analysis of data from the seven IARC multicen- tions are obviously activated by sunlight expo- tre case control studies of CIN3 and invasive sure, immunosuppression, or hyperprolifera- cancer, showed that the prevalence of penile tion of the epithelium such as in psoriasis. An HPV was generally higher in husbands of cases interesting report on the transient generation of than in husbands of controls. However, penile anti-HPV-5 antibodies after epidermal repair HPV was not associated with the risk of cervi- in burns supports the view of transient cal neoplasia in high or intermediate risk coun- activation of persisting viruses as a result of tries, but was associated with an eightfold epidermal proliferation (Favre et al, 676). A increase in risk in Spain. similar scenario has been proposed for the cervical-vaginal mucosa by Jin et al (197), who HPV AND SKIN CANCER found a strikingly high prevalence of HPV During the past few years, the association of types 61, 72, and 11 related, novel sequences in HPV with skin cancer in the general population lavages of HIV infected women. These previ- apart from the epidermodysplasia verruci- ously rare members of the phylogenetic sub- formis (EV) syndrome has been a subject of group A3 seem to be activated during AIDS intense research. Non-melanoma skin cancer is associated immunodeficiency, but must cer- the most frequent cancer among white indi- tainly have a reservoir in the general popula- viduals worldwide and is particularly wide- tion. The growing awareness of this type of spread among the increasing number of immu- ubiquitous infection will have a strong impact nosuppressed patients. Using newly developed, on future plans for disease prevention and highly sensitive PCRs, a large and still growing treatment. number of new partial sequences could be identified in non-melanoma skin cancers, which all probably represent new PV types. HPV AND CANCERS OF THE ORAL CAVITY AND THE Most of them are related to EV associated ANAL AND PERIANAL REGIONS viruses but, depending on the PCR used, In relation to epidemiological presentations on HPV-4 related types have also been identified HPV and cancer sites other than the cervix, the (Antonsson et al, 116). These days, genital study by Björge et al (148) provided the first HPV types are less frequently detected. The prospective evidence that previous infection by HPV prevalence is highest in squamous cell HPV-16 (as indicated by the presence of IgG carcinomas of immunosuppressed patients (up antibodies in serum samples collected on aver- to 90%) and lowest in basal cell carcinomas of age 10 years before cancer diagnosis) is associ- immunocompetent patients (about 40%, ated with an increased risk of anal or perianal which is close to the prevalence in normal skin cancers. samples) (Rust et al, 497; Wieland et al, 115). There were several presentations on various Viral load determinations revealed only one aspects of HPV and oropharyngeal cancers. An HPV DNA copy/20–1000 cells in the tumour early report by Herrero et al (166) from the (Wieland et al, 115; Stockfleth et al, 125), indi- IARC international case control study of HPV cating that probably not every tumour cell har- and cancer of the oral cavity and pharynx sug- bours an HPV genome. This suggests that the gested that HPV is not a major aetiological fac- contributions of cutaneous HPV types to tor, based on the comparison of cases and con- carcinogenesis are quite diVerent from genital trols with respect to HPV prevalence in PVs in cervical cancer. It underlines the need exfoliated cells. However, the HPV detection for studies of the molecular biology of these rate was three times higher in tumour biopsies viruses so that their activity can be better than in exfoliated cells. In contrast, Mork et al understood. Preliminary experiments to this (065), in a case control study nested within a end were presented that dealt with the Nordic cohort of almost 900 000 subjects who biochemical functions of cutaneous HPV had donated serum samples, showed that proteins, with the ultraviolet light eVect on viral HPV-16 positivity in serum collected on promoter activity, and with the eVects of cuta- average 9.4 years before diagnosis was associ- neous HPV oncoproteins in organotypic cul- ated with a 14-fold increase in the risk of sub- tures (Caldeira et al, 604; Ruhland and de Vil- sequent oropharyngeal cancer (OR, 14.4; 95% liers, 661; Mulder et al, 629, respectively). CI, 3.6 to 58.1). Seropositivity to HPV types 18, 33, and 73 was not associated with altered NATURAL HISTORY OF HPV INFECTION IN THE risk. The reason for the discrepancy between SKIN the findings of these two studies is unclear— The frequent detection of viral DNA identical possible explanations include diVerences in with or related to EV associated HPVs in study design or specimen types, diVerences in healthy skin samples or plucked hairs of the methods of assessing HPV status, or lack of individuals in all age groups supports the good information on confounders in the notion that probably everybody is infected over Nordic study. The preliminary results of the long periods, if not throughout life, with these IARC study are consistent with previous stud- viruses. The general need for highly sensitive ies indicating that HPV might have aetiological www.jclinpath.com
  • 5. Highlights of HPV 2000 167 relevance for tonsillar carcinomas. This is sup- protein sequences were identified (Baines et al, ported to some extent by the results of HPV 126). Specific primers were designed which, in analysis of tumours in the case series reported contrast to the MY 09/11 and the GP5+/6+ by Weissenborn et al (114) and Summersgill et system, allowed the detection of PVs in all al (172), who found HPV DNA in 58% and groups of the HPV Sequence Database. To 63%, respectively, of tonsillar tumours. improve sensitivity, a PCR system was de- signed by Kornegay et al (282), where a CERVICAL CANCER IN DEVELOPING COUNTRIES non-degenerate pool of five upstream and The 18th international conference included for seven downstream primers was designed in the first time a special workshop entitled “HPV conserved regions of the L1 gene, resulting in a in developing countries: screening, diagnosis, 170 bp product. In clinical specimens, the therapy and prevention”. The workshop was short amplification detected up to 10% more chaired by T Wright (University of Columbia) HPV positives compared with the MY 09/11 and R Sankaranarayanan (IARC) and included system. 138 abstracts. The presentations in this workshop dealt LOCALISATION OF HPV DNA WITHIN TISSUE mainly with the prevalence and type distribu- SPECIMENS AND VIRUS ASSOCIATED PROGRESSION tion of genital HPV infections in central MARKERS America and in Africa, and in the quality of A combination of the SPF10 system with in cytological screening for cervical neoplasia in situ hybridisation (Vermeulen et al, 236) developing countries. The former did not allow allowed the detection and localisation of multi- firm conclusions to be drawn concerning ple HPV infections in up to 20% of HPV posi- diVerences in HPV type distribution from tive cervical cancers. In situ hybridisation those reported elsewhere, and the latter results indicated the presence of multiple HPV highlighted the need for improvements in the subclones. quality of screening, which is handicapped by Several studies looked at the potential value the lack of resources in developing countries. of virus associated progression markers, such Clearly, these are important topics given the as the presence of certain viral RNA species or relatively high rates of cervical cancer in these the E4 protein (Sotlar et al, 195), viral load areas, and most presentations highlighted the (Swan et al, 280), and integration (Klaes et al, need for further investigations in these areas. 194; Counio Coste et al, 223; Blennow et al, Visual inspection of the cervix was discussed 231). Lamarcq et al (187) showed that the extensively at the meeting. Ferreccio et al (200) expression of E6/E7 transcripts and vascular described a cervical cancer prevention pro- endothelial growth factor (VEGF) mRNA was gramme in Peru that uses visual inspection of higher in women with dysplastic cervical the cervix with acetic acid and immediate smears than in those with normal smears, sug- treatment—it is anticipated that this will lead to gesting that these markers might be a useful a far higher detection and treatment rate for prognostic and diagnostic tool for cervical dis- high grade lesions than in neighbouring control ease. Because for most of these markers only areas in which Papanicolaou (Pap) based data from cross sectional studies are available programmes operate. It is expected that further at this point, their clinical usefulness is still developments in this important area of field unknown. research and implementation will be stimu- lated by the growing presence of international NEW BIOMARKERS AS A QUALITY CONTROL OF cooperative networks such as the Alliance for THE HISTOLOGICAL OR CYTOLOGICAL DIAGNOSIS Cervical Cancer Prevention (PAHO, PATH, A non-viral marker might be useful for improv- IARC, AVSC Int. JHPIEGO (www.alliance- ing the quality of the histological or cytological cxca.org)), the Challenge/ESO Group (www. evaluation of cervical specimens. Enhanced cancerworld.org), and the International Net- expression of a cyclin dependent kinase inhibi- work for Control of Gynaecological Cancers tor p16ink4a can be used to increase the (www.matweb.hcuge.ch/matweb). specificity and interobserver agreement of the histological assessment of cervical lesions Diagnosis and detection of HPV DNA (Klaes et al, 188). In addition, it was shown that Several papers concerned new PCR based immunocytological staining for p16ink4a can techniques for HPV detection. The accurate identify dyskaryotic exfoliated cells in cervical detection of various HPV types was described smears (Friedrich et al, 129). by van Doorn et al (254 and 255), where a Few studies compared the quality of HPV broad spectrum PCR primer set (SPF10) is detection systems, and there were contradic- used to amplify a fragment of 65 bp from the tory results with respect to sensitivity and type L1 region. Genotyping is done by reverse specificity of the various systems (de Sanjosé et hybridisation on a line probe assay discriminat- al, 220; Perrons et al, 302; Molijn et al, 254). ing between 25 HPV genotypes. Seth et al These discrepancies between diVerent HPV (253) described the application of the same detection systems, which are in wide clinical technique to 12 year old archived smears; 31% use, show that there is an urgent need for of cases were HPV positive, with higher rates in standardisation and quality control—an indis- women born after 1940 compared with those pensable prerequisite if HPV testing is to be born before 1940. The SPF10 system was integrated into clinical practice. This problem more sensitive than the GP5+/6+ system. To has been recognised and led to the establish- facilitate the detection of a wide range of vari- ment of the “first human papillomavirus inter- ous HPV types, 11 blocks of conserved L1 national quality control proficiency panel” at www.jclinpath.com
  • 6. 168 Bosch, Rohan, Schneider, et al this meeting, an activity which should be high grade CIN. HPV screening in addition to supported by the scientific community and by cytology increased eYcacy significantly. industry. In the Dutch screening programme (182), the prevalence of HPV DNA in cytological negatives was 3.4%. Cytology negativity and HPV TESTING AS A TRIAGE METHOD FOR high risk HPV positivity was associated with CYTOLOGY RESULTS OF UNDETERMINED NATURE HPV persistence in 60% and led to abnormal Several studies concerned the value of HPV cytology in 26% of HPV positives. Cytology testing for triaging women with a diagnosis of positivity and high risk HPV positivity was ASCUS or low grade CIN (Solomon et al, 181; associated with HPV persistence in 70% and Ronco et al, 224; McGoogan et al, 256; Ponce led to high grade CIN in 24%. et al, 294). Ponce et al (294) reported an equal In the Seattle cohort (183), Thin-Prep value of HPV testing (hybrid capture II (HC cytology showed a sensitivity of 56% for the II)) and expert cytology in predicting HSIL in detection of CIN2/3, compared with a sensitiv- women with ASCUS/LSIL. Ronco et al (244) ity of 67% by PCR based HPV DNA testing showed that the use of HPV detection to triage and 68% for HC II. Specificity for cytology was women with cytological evidence of ASCUS 93% and for PCR 91%, compared with 79% and LSIL could reduce substantially the for HC II. With cytology, 12.5% of women had number of colposcopies, with little loss in to be referred for colposcopy. Comparable fig- histological detection of CIN2/3. Solomon et al ures were 15% using PCR and 23% with HC (181), reporting from the ALTS trial (a II. Thus, PCR had a higher sensitivity and randomised, multicentre trial comparing three comparable specificity when compared with management strategies for women with cyto- liquid based cytology. logical diagnoses of ASCUS or LSIL— Two German studies compared the perform- immediate colposcopy, conservative manage- ance of routine cytology and HPV testing by ment, or colposcopic triage using HPV DNA GP5+/6+ PCR (Schneider et al, 184) or HC II testing added to repeat thin layer cytology), (Iftner et al, 185). The sensitivity of cytology showed that the negative predictive value of a for the detection of HSIL was 20% (184) and negative HPV DNA test was high in all groups 34% (185), respectively. HPV testing showed a of study subjects, but that the positive predic- sensitivity of 89% and a specificity of 94% in tive value of finding HPV was greatest in older both studies. The positive and negative predic- women (> 30) with ASCUS as a referral diag- tive values for HPV testing using PCR were nosis on cytology. Thus, HPV testing was 35.8% and 99.6%, respectively. clearly superior to cytology at the expense of a referral rate of 56% for colposcopy. In another SELF SAMPLING FOR HPV TESTING: A study including 270 women with a borderline POTENTIALLY IMPORTANT DEVELOPMENT or mildly dyskaryotic smear, no cases of HSIL Several studies reported the value of self would have been missed by HPV testing collected samples for HPV testing by PCR sys- (McGoogan et al, 256). The authors conclude tems (Harper et al, 186; Nobbenhuis et al, 265; that HPV testing is likely to be useful in triag- Mire et al, 275) or HC II (Salmerón et al, 270; ing women with low grade cervical abnormali- Hillemanns et al, 303) compared with physi- ties. cian directed sampling. These studies con- The value of HPV testing in the triaging of cluded that HPV detection rates in self women with ASCUS seemed to be consistently collected vaginal samples were similar to those superior to repeated cytology for the detection in physician collected cervical samples, and of HSIL. HPV testing in the triaging of LSIL that the use of self collected samples might be cases is less consistent because of the high useful in cervical cancer screening in under- prevalence rates of HPV DNA found in some served women and women in developing coun- studies, such as ALTS. However, many authors tries (303 and 265). In a similar vein, noted the diYculties of comparing studies Brinkman et al (274) showed that HPV detec- because of the additional variability introduced tion rates using a PCR based urine test agreed by the cytology methods used (standard v reasonably with those derived from matching expert v liquid based) and the use of diVerent cervical swabs, raising the possibility that such axes of classification of the neoplastic lesions samples might be useful. (CIN, the Bethesda system (TBS), Pap, and others). ACCEPTANCE OF HPV TESTING BY THE POPULATION AND THE MEDICAL COMMUNITY HPV TESTING AS A PRIMARY SCREENING METHOD An important prerequisite for the acceptance The validity of HPV testing in screening of HPV testing by individuals undergoing programmes was investigated in various co- screening is the evaluation of socioeconomic horts (Franco et al, 180; Meijer et al, 182; and psychological factors, counselling of af- Kulasingam et al, 183; Schneider et al, 184; Ift- fected individuals, and information to the pub- ner et al, 185). In the Newfoundland study lic, issues that were considered in various stud- (180), the adjusted sensitivity for cytology for ies (Johnson et al, 228; Phillips et al, 229; detection of HSIL with cut point LSIL was Whynes et al, 230; Flores et al, 296). The clini- 28%, and for HPV testing 68%, at the expense cal workshop included special presentations on of specificity, which was 90.6% for HPV testing the topics of “Counseling persons with HPV versus 99.1% for cytology. A combination of infections” (Steben, 008) and “Medical, social HPV testing and cytology yielded a negative and ethical issues and challenges of HPV predictive value of 100% for the detection of infections: what can we do?” (Alexander, 007). www.jclinpath.com
  • 7. Highlights of HPV 2000 169 HPV IN IMMUNOCOMPROMISED INDIVIDUALS immunodeficient (SCID) mouse model (Bon- The natural history of HPV might have to be nez et al, 339; 340; 341). It was shown that the redefined based upon the information gathered antisense oligonucleotides ORI-1001 comple- in immunosuppressed individuals (Hoesley et mentary to the 5' end of HPV-6 E1 mRNA has al, 154; Jin et al, 197): 76% of HIV positive an inhibitory eVect on the growth of HPV-6 women were found to be HPV positive by induced human condylomata (339). nested PCR, with 65% harbouring multiple In vivo studies in humans: experiences with infections and with one third of isolates indole-3-carbinol (I3C), a compound found in belonging to the phylogenetic subgroup A3. It cruciferous vegetables that detoxifies carcino- is concluded that HPV infection might be gens and alters oestrogen metabolism, were acquired early in life, and that it might be part reported by Bell et al (311) and Da-Zhi Chen et of the commensal viral microflora of the skin al (312). It was shown that I3C and its and the anogenital mucosa, with sporadic sub- metabolite DIM caused apoptotic changes in clinical shedding caused by transient reactiva- cervical cancer cells in vitro. In vivo, HPV tion in specific settings such as immunosup- transgenic mice exposed to oestrogen and fed pression and overt disease being unusual. I3C showed evidence of apoptotic cells in their cervical epithelium (312). HPV ECONOMICS To evaluate the eVectiveness of I3C in HPV 2000 introduced the topic of HPV humans, 27 patients with biopsy confirmed economics to stress the relevance of conduct- CIN2 or 3 were randomised to oral I3C or pla- ing cost–benefit analyses and studies evaluating cebo (311). Eight of 17 patients treated for 12 quality of life. Most of the work presented did weeks had complete regression of lesions. None so in relation to screening and treatment of of the 10 patients in the placebo group experi- preinvasive neoplastic lesions through conven- enced regression. In another study, a fusion tional cytology screening programmes or protein consisting of the heat shock protein through primary HPV testing. Hsp65 and Mycobacterium bovis BCG linked to The addition of HPV testing was favourably the HPV-16 E7 protein was constructed. In a evaluated as an adjuvant to the follow up of randomised double blind placebo controlled HIV positive women (Goldie et al, 267), and as trial, three monthly subcutaneous doses were a single lifetime screening method in low applied to 10 patients with high grade anal resource settings (Goldie et al, 693; Sherlaw- intraepithelial neoplasia (Goldstone et al, Johnson et al, 260; Kulasingam et al, 272). HPV 347a). All patients had a clinical response after testing when combined with a low technology a course of three doses. treatment, such as cryosurgery for HPV Immunomodulatory substances such as imi- positive women, resulted in cost saving (Goldie quimod (Ikenberg et al, 310), leukonorm et al, 693). An ongoing project in Vietnam (Metzner et al, 322), viscum album (Romero showed that five yearly cytology based screen- Montes et al, 327), and a combination of ing programmes were both feasible and cost iscador and interferon (IFN) (Basta et al, 318) beneficial (Chan Hung et al, 694). Quality of were used in various studies for the treatment life issues about HPV testing (self sampling of CIN and anal condylomata in phase 2 trials acceptability, cost acceptability, diagnostic re- and showed promising responses with tolerable lated stress) form a novel research area that side eVects. Medical treatment of LSIL presents several challenges before primary emerged as an area of growing research interest HPV testing can be implemented (Flores et al, and a plausible alternative to reduce surgical 296; Johnson et al, 228; Philips et al, 229). overtreatment. Some exercises based on mathematical models for predicting outcomes of screening pro- HPV DNA AS A PROGNOSTIC FACTOR IN CIN CASES grammes under a variety of assumptions were AFTER TREATMENT also presented (Gallivan et al, 259; Whynes et Studies evaluating the presence of HPV DNA al, 230) and may become an area of increasing after surgical treatment of CIN (Kjellberg et al, interest. 196; Tachezy et al, 242; Nobbenhuis et al, 317; Ramírez Porras et al, 320; Simon et al, 323; Bettinger et al, 193; Elfgren et al, 329) showed Treatment of HPV infections and of HPV that the detection of HPV increases the risk of related cancer recurrent disease. HPV persistence is rare after In vitro studies: various gene therapeutic conisation with free margins, but not after modalities were defined such as HSV thymi- conisation with positive margins (323). A high dine kinase (HSV-TK) (Sethi et al. 326), HSV risk HPV positive test three months after treat- vectors (Kari et al, 345), HPV-16 E2 derivates ment is more predictive for residual or (HoVmann et al, 333), and anti-HPV hairpin recurrent CIN disease than abnormal cytology ribozymes (Enriquez-Aragon et al, 328). In (317). Persistence of HPV infection after treat- addition, growth factor inhibition (Michael et ment was more common in patients less than al, 342) and endothelin receptor antagonists 35 years of age and with a history of (Venuti et al, 313) were used. The in vitro cryotherapy (329). Conisation resulted in a results of these studies look promising and decline of cervical HPV IgA antibodies development to clinical application is a realistic whereas cryotherapy had much less eVect goal. (329). Using HPV-16 E6 and E7 peptides, a In vivo studies in animals: for preclinical cellular immune response was seen in disease studies, HPV-6 or HPV-11 induced human free patients but not in those with disease xenografts were used in the severe combined recurrence (Sarkar et al, 314). www.jclinpath.com
  • 8. 170 Bosch, Rohan, Schneider, et al HPV TESTS AS PROGNOSTIC MARKERS IN INVASIVE capsid specific antibody within four years of CANCER infection, with a median time to seroconversion In patients with invasive cancer (Lizano et al, of over six months (Carter et al, 057). HPV-18 325), viral DNA sequences were detected in seroconversion is similarly slow, whereas sero- the plasma of four of five patients with conversion for HPV-6b is apparently faster, recurrence and all three patients with tumour and occurs at the same time as the acquisition persistence, but in only one patient of 17 with of viral DNA (057). The definition of a positive regression. A prognostic eVect of the presence serological reaction to the capsid remains of HPV antibodies, HPV DNA (Bravo et al, mathematical (Stone et al, 056) because the 338), certain HPV types (Lo Keith et al, 332; identification of a cohort of individuals never Dreier et al, 335), VEGF serum concentrations infected with a particular HPV genotype is not (Bachtiary et al, 336), or marker chromosomes straightforward, and there is also some evi- (Macville et al, 344) was shown in case series, dence of genotype crossreactivity for serologi- but larger prospective series are needed to sub- cal reactions (Combita et al, 412). Hence, the stantiate these preliminary observations. usefulness of serology for epidemiological studies is limited. For virus encoded non- Immunology and vaccines structural proteins, serological tests are not yet Recognition of the association between HPV as well standardised as for PV virions. Antibody infection and cervical cancer in the 1980s to the E7 protein, which is expressed in acute fuelled interest in HPV immunology, and in the infection (Hamsíková et al, 397) and also in development of vaccines to prevent infection. many PV associated tumours, is seen in about However, data were initially slow to emerge, in 50% of subjects with invasive cervical cancer. part because two problems hindered research, However, it is not seen after acute HPV infec- namely: (1) PV could not be grown in the tion with several genotypes (Gaenzler et al, laboratory as a source of antigen for the devel- 398), even in the presence of antibody to the opment of serological tests and for conven- L1 capsid protein. Low amounts of antibody tional killed vaccines; and (2) no PV could be reactive with peptides derived from other open identified that infected an immunologically reading frames (E2 and E4) are seen in some well characterised laboratory animal. patients with HPV infection and invasive Improved systems for expressing genes in cancer (Sehr et al, 399), although sensitivity eukaryotic cells through the 1990s has allowed and specificity for infection appear to be low. a partial solution to the first problem, which is These results are reminiscent of the results exemplified by the production of VLPs and the obtained with L1 protein before the use of cor- recent resolution of a crystal structure for the rectly conformed VLP substrate. capsid of HPV-16 (Chen et al, 359), and has led to a considerable worldwide interest in the development of vaccines to prevent and to treat CELL MEDIATED IMMUNE RESPONSES PV infection. In addition, epithelial raft cul- Several presentations describing the conse- tures (Thomas et al, 535) have recently allowed quences of HIV infection on the natural history the production of authentic virions from of HPV infection confirmed that HPV infec- epithelial cells infected in vitro. The lack of an tion is more likely to persist and less likely to animal model for the study of PV immunology respond to treatment in individuals with has been circumvented by the development of systemic immune suppression (Palefsky, 349; more robust tests for cell mediated immunity Ebrahim et al, 350). These data strongly to defined antigens in humans, including suggest a role for cell mediated immune tetramer and ELISPOT technology, and key responses in the control of HPV infection, as experiments on the immunological conse- would be expected from data for other viruses. quences of PV infection can now be under- The newer assays (such as ELISPOT and taken in the natural host. Such research has tetramer technologies) allow more precise demonstrated the ability of PV to elicit both measurement of virus specific cytotoxic T cells humoral and cellular immunity and, possibly as (CTLs) and to some extent of virus specific T a consequence of preferential uptake by helper responses (Steele et al, 384). Assay dendritic cells (DCs) into the major histompat- reproducibility and sensitivity remain impor- ibility complex (MHC) class I presentation tant issues, and there is only a limited number pathway, PV VLPs can also induce host of defined viral protein epitopes restricted by protective cytotoxic T cell responses. Interest defined MHC class I and II haplotypes. T cell in PV immunology has now extended outside assays, to be suitable for immunoepidemiology, the narrow confines of viral epidemiology, and or for modelling mechanisms of disease abstracts on PV immunology and immuno- control, need to: (1) produce positive results serology, and on vaccine development, ac- that are reproducible from day to day in the counted for nearly a quarter of the material same patient, (2) correlate with the genotype of presented at the workshop. clinical HPV infection, and (3) demonstrate MHC restriction and antigen specificity. Few SEROLOGICAL RESPONSES TO HPV INFECTION of the presentations gave results from human Data presented from prospective studies of studies that met these stringent criteria. infection with genital genotypes of HPV Positive control reagents, including immortal- suggest that serological responses to the viral ised antigen specific T cell lines, using capsid are slow to appear, when compared with telomerase reverse transcriptase (TERT) most viruses. Only 50% of individuals acquir- transfer (Hooijberg et al, 427), promise to help ing HPV DNA of genotype HPV-16 develop with assay standardisation in the future. www.jclinpath.com
  • 9. Highlights of HPV 2000 171 Cytotoxic T cell mediated immune re- immune responses including CTLs. This para- sponses are not easily demonstrable after natu- dox can be explained by noting that PV infec- ral HPV infection. Low frequencies of CTLs tion is non-lytic and superficial, and because specific for the E6 protein of HPV-16 were most virus genes are expressed at low levels associated in one study with the resolution of they might simply be ignored by the immune cervical infection, whereas E7 specific CTLs system. This has been demonstrated in a skin were found both in patients with regression and transplant model using HPV-16 E7 transgenic in patients with persisting infection (Nakagawa skin, where rejection only occurs after the et al, 354). Of potential importance for vaccine administration of powerful proinflammatory development, some E7 specific CTL epitopes signals, and is mediated via MHC class II were shown to be crossreactive between dependent mechanisms (Frazer et al, 378). HPV-16 and HPV-52 (Youde et al, 357). More Alternatively, if PV antigen expression is studies in this area will be needed to determine increased, presentation in skin without inflam- whether host protective crossreactive responses mation may be tolerogenic, because skin asso- exist after natural HPV infection or vaccina- ciated E7 antigen can be cross presented by tion. Virus specific T helper cells are, predict- bone marrow derived DCs and anergise ably, harder to demonstrate than specific CTL specific T cells, at least in the mouse (Doan et responses, given the much lower frequency of al, 356). In addition, and in common with most T helper cells after viral infection. The E7 pro- viruses, PV genes encode mechanisms for tein contains sequences recognised as MHC modulating both the induction of the host class II restricted epitopes by most normal immune response and virus specific eVector subjects (van der Burg et al, 358), as does E2 mechanisms. Initiation of the innate and adap- (de Jong et al, 383; Dillon et al, 408). Patients tive immune responses to PV antigens may be with cervical cancer but not patients with impaired by E7 mediated downregulation of squamous intraepithelial lesions (SIL) respond the host response to IFN (Barnard and to E6 peptides in vitro (Timmins et al, 353), McMillan, 579; Nees et al, 630). E5 and E7 and patients with progressive cervical disease also interfere with the TAP (transporter associ- respond less to E7 peptides (Luxton et al, 400), ated with antigen presentation) antigen trans- although multiple in vitro restimulations are porter mechanism in infected cells (Vambutas required to elicit positive proliferative re- et al, 360), and with MHC class I presentation sponses. Responses to E6 and E7 peptides are of antigen. Local proinflammatory chemokines better in patients who are disease free after (Fife et al, 388) and cytokine B (Bermúdez treatment (Sarkar et al, 314). Cytokine secre- Morales et al, 379; Jacobs et al, 416) production tion by T cells in response to E6 and E7 is altered in PV infected as opposed to proteins is altered in recurrent respiratory pap- uninfected keratinocytes. Additional mutations illomatosis (RRP) (DeVoti et al, 406), suggest- in the antigen processing and presentation ing that failure of resolution after infection pathways are seen in cervical cancers (Garrido, might reflect immune deviation to a more T 370): SCID/human (HU) tumour grafts, facili- helper type 2-like immune response. In vivo tated with steroid, might prove an interesting testing of delayed type hypersensitivity (DTH) model to examine defects of the antigen to antigen is a cheap, simple, and robust tech- presenting machinery in tumours, free of nique for measuring CD4+ T cell responses to stroma (Baines et al, 625). viral antigens worthy of further investigation, and in one study DTH to E7 peptides was found to be more common in regressing than PROPHYLACTIC VACCINES AGAINST PV: PHASE I progressing CIN (Höpfl et al, 351), in general AND II HUMAN TRIALS support of the hypothesis that cell mediated All existing eVective prophylactic vaccines immunity is important for the regression of against viral infection induce neutralising anti- HPV infection. In keeping with this, regressing body, and none is thought to produce sterile as opposed to persistent CIN appears to be immunity. Some epidemiological evidence was associated with increased numbers of activated presented suggesting that the development of T cells locally within the lesion (Petry et al, an antibody response in humans protects 352). Local immune responses to PV antigen against repeat PV infection (Ho et al, 058), in the female genital tract most probably reflect which is reassuring for the development of pro- systemic as well as mucosal immunity and, phylactic vaccines, because to date the data perhaps in consequence, IgA anticapsid anti- supporting such vaccines have been based on body in the genital secretions, although com- studies in animal models. Most work on mon in HPV infection, is not a marker for per- prophylactic vaccine development has focused sisting local infection (Pretet et al, 396). on vaccines that are based on VLPs comprising the PV L1 capsid protein, produced in yeast or PV EVASION IN HOST IMMUNITY insect cells (Schiller, 348). Were such vaccines The lack of demonstrable immunity to non- to be available, one study indicated that they structural PV proteins after acute HPV infec- would be generally acceptable to high risk tion, together with the slow and variable communities (Lazcano et al, 446). Early phase response to the capsid proteins, suggests that clinical trials of vaccines for HPV-11 (Fife et al, there is a problem with the generation of PV 364) and HPV-16 (Harro et al, 362; Poland et specific immunity. In contrast, PV VLPs are al, 363; Balsley et al, 366) were presented by highly immunogenic when delivered as a several groups. HPV-11 VLPs produced neu- vaccine without adjuvant, stimulate DCs (Ru- tralising antibody for up to 18 months. HPV-16 dolf et al, 355; Lenz et al, 395), and induce VLPs are similarly immunogenic even without www.jclinpath.com
  • 10. 172 Bosch, Rohan, Schneider, et al adjuvant. In general, antibodies are PV geno- THERAPEUTIC VACCINES type specific, and immunogenic at relatively Currently, there are no licensed therapeutic low antigen doses, although some vaccines vaccines for humans, and no clear evidence of might have stability problems. However, in the which PV viral protein(s) might produce a beagle dog, VLP delivered intranasally, al- therapeutic immune response to PV infection. though inducing a good mucosal IgA response, Cottontail rabbits undergo regression of cot- failed to protect against virus challenge (Yuan tontail rabbit PV (CRPV) induced warty et al, 369), suggesting that systemic vaccine lesions after the administration of a polynucleo- delivery may have advantages even for mucosal tide vaccine encoding E1, E2, E6, and E7 PV genotypes. Although no polynucleotide (374), and the same proteins fused to ubiquitin are equally successful (Leachman et al, 455). vaccines have been tried in humans, DNA Although data on eYcacy in natural PV infec- encoding canine oral papillomavirus (COPV) tion are limited, a wide array of potential capsid proteins protects dogs against viral vaccine delivery systems have been tested for challenge (Stanley et al, 375) and may clear immunogenicity in transplantable tumour virus from existing infections (Moore et al, models. Immunotherapy with polynucleotide 452): modification of codon usage of the PV vaccines, either unmodified (Velders et al, 382; capsid genes to match that of more conven- Doan et al, 441; Smahel et al, 445; De Marco et tional mammalian genes improves the immu- al, 449), codon modified (Liu et al, 451), shuf- nogenicity of PV DNA vaccines in animals (Liu fled to avoid potentially harmful consequences et al, 451), and may therefore facilitate their use of gene expression (Osen et al, 372), or fused in humans. with ubiquitin to improve antigen presentation Minor HPV variants share epitopes (Pas- (Brandsma et al, 373), has been eVective in the trana et al, 368), but crossreactivity of neutral- prevention or elimination of PV protein ising antibody between major types is limited, expressing transplantable tumours. Similarly, although the requirement for multicomponent vaccines based on cell derived (Navabi et al, vaccines can be overcome with chimaeric VLPs 447) or recombinant (Giannouli et al, 429; displaying two or more types of HPV, at least in O’Neill et al, 438) proteins, derivative peptides animals (Wilson et al, 462). PV neutralisation (Melief et al, 437), recombinant bacteria assays, which until recently have relied on a (Benyacoub et al, 450; Revaz-Fellay et al, 453; labourious in vivo xenograft model, are now Maclean et al, 457), recombinant viruses available for in vitro use, based on reporter (Jifeng et al, 430; Chen et al, 436; Herd et al, 440), and chimaeric virus-like particles (Liu et genes (Yeager et al, 371) or detection of viral E2 al, 386; Street et al, 439; Liu et al, 442; Shi et al, mRNA by reverse transcription PCR (Hall et 448; Revaz-Fellay, 453; Kaufmann et al, 460) al, 426). Such assays should allow the valida- are all protective in at least one animal tumour tion of vaccines for the less common genotypes model system. Responses to such vaccines can based on surrogate markers of eYcacy, without be improved by cytokines (Hung et al, 434), by the need for extensive clinical trials. targeted delivery systems (Wang et al, 435; Several studies (Chackerian et al, 389; Liu et Michel et al, 458), and by new adjuvants such al, 390) examined the use of PV VLPs as deliv- as Hsp (Kadish et al, 365; Randall Chu et al, ery systems for other antigens. VLPs are potent 377; Cheng et al, 433), whereas “mix and immunogens that can induce DTH in humans match” prime boost strategies (Chen et al, 381) (Lian et al, 431), and T cell proliferation and produced improved responses in animal mod- occasional CTL responses in chimps (Pretet et els when compared with single delivery sys- al, 396). Chimaeric VLPs can activate DCs tems. The human trials of these therapeutic (Balmelli et al, 391; Lenz et al, 395) and induce vaccines to date have been more limited. They epitope specific CTL responses with ex vivo have shown safety and some immunogenicity human cells, and in vivo in mice. Adjuvants of E7 based vaccines. A fusion of Hsp with E7 such as CpG DNA enhance VLP induced induced transient proliferative responses to E7 mucosal immunity (Gerber et al, 465), al- peptides in T cells in four of the eight subjects though as mentioned above a role for mucosal immunised. E7 peptides induce some immune immunity in the control of PV infection responses but CIN regression was no more remains to be demonstrated. Although most than might be expected without vaccine (Kast et al, 367), and no regression of cervical cancer VLPs for vaccine use have been produced in was reported with any vaccine (Khleif et al, yeast or insect cells, alternative expression sys- 380), although many trials are ongoing. Ex vivo tems that might better suit developing coun- DCs pulsed with E7 induce E7 specific CTLs tries are being explored, including bacterial (Evans et al, 410; Nonn et al, 415; Hermonat et systems (Escherichia coli, salmonella, BCG), al, 464), but eYcacy data in humans are not which produce immunogenic and host protec- available. tive L1 pentamers, and plant derived VLPs are It will not be feasible to conduct human eY- also under development (Accardi et al, 456). cacy studies of all the available PV therapeutic Although PV capsid based vaccines are cur- delivery systems and antigens, and to date no rently favoured for prophylaxis against PV single immunological assay has shown a corre- infection, vaccines based on multiple non- lation with eYcacy of immunotherapy for PV structural proteins were found in one study to induced disease in humans or in natural animal protect against infection in the rabbit (Han et PV infection. How can we progress from here? al, 374), an observation worth following up for The usefulness of further studies of vaccine combined prophylactic/therapeutic vaccines. eYcacy using transplantable E7 expressing www.jclinpath.com
  • 11. Highlights of HPV 2000 173 tumours in the mouse is limited, and although expressed in HPV transformed cell lines and in the cottontail rabbit and the beagle dog provide high grade squamous intraepithelial lesions, attractive models for testing immunotherapy, indicating that loss of this complex and the natural history of PV infection in these ani- increased phosphorylation of E7 might con- mals does not closely resemble that of HPV tribute to malignant transformation (Tugizov et infection in humans. Tumour xenografts to al, 589). SCID/HU and transgenic skin/tumour models Regarding genetic instability of persistently might more closely resemble natural human infected keratinocytes, it is of interest that E7 PV infection, but are not ideal for establishing uncouples centrosome duplication from the vaccine eYcacy because the immune system of cell division cycle and that E6 allows the accu- these animal models is compromised. It will mulation of centrosome abnormalities (Duens- probably be necessary to validate surrogate ing and Münger, 471). This will increase the markers of vaccine therapeutic eYcacy in likelihood of chromosomal mis-segregation phase I clinical trials, and discard non-valid followed by genomic changes. markers. Phase I trials of immunotherapy for In addition to its role in the epidermal warts/anal intraepithelial neoplasia (AIN)/ growth factor receptor signal transduction CIN/vulvar intraepithelial neoplasia (VIN)/ pathway, HPV-16 E5 has been shown to down- RRP will be preferable for evaluating surrogate regulate the cyclin dependent kinase inhibitor eYcacy markers against outcome because p27 (Pedroza-Saavedra et al, 583). problems with mutated antigen presentation All oncoproteins are involved in the protec- machinery, and with the eVects of previous tion of the infected keratinocyte from the host treatments, complicate interpretation of the immune response. HPV-16 E6 leads to re- data in patients with cancer. True validation of duced expression of E-cadherin, which has the surrogate eYcacy markers will not be pos- been postulated to play a role in the retention sible from such studies unless studies are and localisation of Langerhans cells (Matthews blinded, placebo controlled, and randomised et al, 665). The E7 protein blocks the antiviral because of the subjective nature of the and antigrowth activities of IFN- , possibly by outcomes from such studies. binding to cellular p48 (Barnard and McMil- lan, 579). However, it does not alter the activ- Biology of PV and virus and host ity of IFN- . In cells transformed by E5 or the interactions homologous E8 protein of bovine PV-4, the Contributions to biology improved the basic export of MHC class I molecules to the cell knowledge of this virus family and paved the surface is downregulated, impairing antigen way for epidemiology, diagnosis, and immunol- presentation, and thereby helping the virus to ogy by identifying and cloning HPV types, escape immune recognition (Ashrafi and demonstrating their association with benign Campo, 592). and malignant tumours in humans, and reveal- ing molecular mechanisms of immortalisation REPLICATION AND TRANSCRIPTION and tumour progression. This provided rea- The viral proteins E1 and E2 are the major gents and tools for epidemiology and HPV players in the control of replication and specific, diagnostic screening programmes. It transcription. E7 seems to promote DNA syn- also defined targets for prophylactic and thera- thesis by directly activating the cyclin depend- peutic vaccination. ent kinase CDK2 and enhancing the phospho- rylation of histones and possibly E1 and E2 VIRAL ONCOPROTEINS (He and Fisher, 586). The interaction of E1 Relevant to oncogenicity are the viral proteins and E2 with CBP/p300 has been shown to E6, E7, and to a lesser degree E5. They are stimulate transcription and replication in mitogenic and modulate apoptosis; in addition, HPV-18 and HPV-8 (Lee and Choe, 526; E6 and E7 induce genetic instability. A prime Müller et al, 540). Furthermore, E2 interacts cause for chromosomal instability is E6 medi- with the transcription factor NF-IL6 to syner- ated degradation of the cellular protein p53 by gise in transcriptional activation (Hadaschik et the ubiquitin–proteasome pathway. De- al, 541). A new mechanism for the repression genkolbe et al (639) showed that E6 targets p53 of transcription by E2 has been proposed. The in yet another way by binding to the transcrip- E8–E2C fusion protein of HPV-31 not only tional coactivator CBP/p300 required by p53, represses by competition for DNA binding thereby inactivating this factor. It is interesting with transactivation competent full length E2, to note that E6 of bovine PV-1, which does not but also possesses an active repression domain induce the degradation of p53, can also down- in the E8 part, which mediates repression over regulate CBP/p300, and that this interaction long distances (Zobel et al, 531). appears necessary but not suYcient for cell The very low concentrations of E1 in PV transformation. Regarding the interaction of infected cells always suggested that this replica- E7 with the retinoblastoma protein pRb, the tion initiator is tightly regulated. It has now importance of enhanced pRb degradation in been shown that E1 is degraded by the comparison with the molar E7–pRb interaction ubiquitin–proteasome pathway unless pro- has been stressed (Gonzalez and Münger, tected in a complex with cyclin E–CDK2 587). Phosphorylation of the E7 protein has before the start of DNA synthesis (Malcles et been shown to be crucial for promoting S phase al, 622). E1 independent replication has been entry (Chien et al, 588). Phosphorylation is described for HPV types 16, 18, and 31, start- inhibited by a macrophage inhibitory factor ing from origins outside the long control related protein complex, which is no longer region, and is possibly functional during the www.jclinpath.com
  • 12. 174 Bosch, Rohan, Schneider, et al non-productive state of the viral life cycle or prevented infection (Giroglou et al, 619). The during latency (Kim and Lambert, 528). This N-terminal region of L2 has been shown to is important news regarding antiviral therapeu- bind to cells and to enter the cytoplasm tic approaches targeting the E1 protein. (Kawana et al, 621). This suggests that the The E1–E4 fusion protein has so far been minor capsid protein plays an important role in regarded mainly as a factor in virus maturation virus–host cell interaction. but now also seems to play a role in replication. Organotypic raft cultures of keratinocytes It interacts with the human replication initia- were introduced long ago into PV research but tion protein Mcm7 and thereby inhibits cellu- it is only now possible to produce infectious lar DNA synthesis (Roberts et al, 631). This viral stocks with titres of 1000 and more in should allow the recruitment of host cell vitro (Meyers et al, 632). This will be useful in factors to sites of viral DNA synthesis. future studies of the viral life cycle. New ways to control transcription and repli- cation have been revealed by the analysis of RELEVANCE OF CELLULAR GENES nucleosomes specifically positioned on the viral The importance of cellular genes in tumour enhancer and the E6 promoter, and flanking progression has been undisputed. Using the nuclear matrix attachment regions, which bind array technique, several groups have started to the diVerentiation regulated CCAAT displace- collect data on diVerential gene expression, at ment protein (CDD) factor (Bernard et al, the moment mainly using archival material. 532). In transient transfection assays, these act Other approaches are already more focused. as silencers of transcription by deacetylating The E6 mediated induction of transcription of the nucleosomes. However, after integration of human TERT (hTERT), the catalytic subunit the viral DNA, the matrix attachment regions of the telomerase, is known to be an important change into potent enhancers, which may par- step in immortalisation. This is followed by the tially explain the deregulation of viral gene loss of cellular telomerase inhibitors. Using expression seen after the integration of viral microcell mediated chromosomal transfer, genomes into cellular DNA in the course of Snijders et al (485) and Backsch et al (486) tumour progression. demonstrated telomerase suppression by chro- mosomes 3, 4, and 6, thereby mapping repres- INTRATYPE VARIANTS sors, the loss of which is involved in HPV A newly identified variant of the CRPV diVers mediated immortalisation. Another putative in biological behaviour from the prototype. senescence gene locus has been mapped to the CRPVb induced papillomas regress in the short arm of chromosome 10 (Poignée et al, vicinity of still proliferating CRPVa induced 476). papillomas (Nasseri et al, 674; Nonnenmacher A great success has been achieved in charac- et al, 479). DiVerences in risk for persistence terising the genetic abnormality of patients and malignant conversion were mapped to the with EV, who are susceptible to infection with control and E6–E7 regions of the variant the subgroup of HPV associated with this dis- genomes (diVerences in wart regression espe- ease. Genetic linkage analyses on EV consan- cially to E6) and were strongly dependent on guineous families has recently identified a first the DR–DQ haplotype of the host. susceptibility locus on chromosome 17q25 and Similar important diVerences in oncogenic a second locus has now been mapped to chro- potential seem to exist among variants of mosome 2 p21–24 (Ramoz et al, 628). This is HPVs. The oncogene promoter of so called the first identification of genomic regions Asian–American and North American variants involved in the control of HPV infections. It is of HPV-16 turned out to be about three times of particular interest that the EV susceptibility more active than the European prototype locus on chromosome 17 overlaps with a (Kämmer et al, 548), and African 1 isolates psoriasis susceptibility locus, in view of recently from penile cancers were more active than the published papers on the high prevalence of EV respective prototype (Tornesello et al, 518). associated PVs in skin lesions of patients with Certain E7 variants of HPV-45 have in- psoriasis. creased transforming potential (Geisbill et al, 515). The association of HPV-16 E6 variants Summary and prospects with cervical cancer appear to be linked to CLINICAL human MHC (HLA) class II distribution In terms of prospects for the near future, it (Voglino et al, 650), which may indicate that seems that the most exciting developments will the risk of specific E6 polymorphism is linked relate to the addition of HPV testing to cervical to host immune surveillance, analogous to the cancer screening programmes. In this regard, above data on CRPV. the Dutch trial, which ultimately will involve 44 000 women (Meijer et al, 182), will yield LIFE CYCLE important data in the next few years. Also, sev- Adsorption of the virus to its host cell is the eral cost–benefit analyses of HPV detection first step in the life cycle. So far, 6/ 4 integrin (Sherlaw-Johnson et al, 260; Goldie et al, 267; has been put forward as a potential receptor Kulasingam et al, 183) for screening for cervi- but turned out to be not obligatory for bovine cal neoplasia were presented at the meeting, PV-4. Studies with HPV-11 VLPs pointed to and analyses of this type will be important in glycosaminoglycans as receptors. A convincing evaluating the feasibility of this approach to study with HPV-33 pseudovirions showed that screening. The other important development heparan sulphate proteoglycans are crucial for in relation to screening is the potential applica- HPV binding because heparinase completely bility of HPV testing, performed perhaps on www.jclinpath.com
  • 13. Highlights of HPV 2000 175 self collected samples, to screening for cervical sponsor. The HPV 2000 conference was supported by public grants from the European Commission (QLFK2–1999–30059 neoplasia in underserved women and women and HPCF-1999–00208), the National Cancer Institute of the in developing countries. Relatively little work US (R13CA90191 to the University of South Carolina School of Medicine), the II Pla de Recerca de Catalunya 1997/2000 has been done in this area, although there now (2000 ARCS 00111), and the Spanish Ministry of Education appears to be a steady growth of activity. and Culture (C099–0430). Support was also received from pri- vate sponsors with interest in HPV research and clinical uses. Finally, one other area about which we can The major sponsors were 3M Pharmaceuticals, 3M Farmacéu- expect to learn more in the near future is sero- tica, Digene, Merck Vaccine Division, Smith Kline Beecham, Abbott Cientifica, Cytyc Corporation, and Roche Diagnostics logical testing for HPV. New HPV detection (for full credit description see www.hpv2000.com). The organ- systems have been created that should be used isers of the HPV 2000 conference acknowledge the support and contribution of the Institut Català d’Oncologia for handling the to improve the performance of currently used administration and the staV at the Servei d’Epidemiologia i tests, especially with respect to HPV type spe- Registre del Cancer, SERC and the Seccion de Ginecologia Oncologica (ICGON), Hospital Clinic i Universitari de Barce- cificity. Clinically useful prognostic markers lona. The organisers acknowledge with gratitude the support and non-invasive treatments still have to be received from all sources and wish to express the continuous independence of the organising committee in establishing the developed. programme, inviting the coauthors of this review, and editing the final version of the text. IMMUNOLOGY AND VACCINES 1 ALTS Group. Human papillomavirus testing for triage of Improved assays to measure immune responses women with cytologic evidence of low-grade squamous to PV antigens in humans will allow seroepide- intraepithelial lesions: baseline data from a randomized trial. J Natl Cancer Inst 2000;92:397–402. miological studies to explore the natural 2 Cuzick J, Sasieni P, Davies P, et al. A systematic review of the history of PV infection, and the role of specific role of human papillomavirus testing within a cervical screening program. Health Technol Assess 1999;3:1–196. PVs in diseases in which the link with PV is not 3 Gross GE, Barrasso R, Human papillomavirus infection. In: yet clarified. Studies of the immune responses A clinical atlas. Berlin: Ullftein Hosby; 1997:1–432. associated with the resolution of PV infections 4 Harwood CA, McGregor JM, Proby CM, et al. Human pap- illomavirus and the development of non-melanoma skin in humans will pave the way for immuno- cancer. J Clin Pathol 1999;52:249–53. therapy for PV associated diseases. VLP based 5 Holowaty P, Miller AB, Rohan T, et al. The natural history of dysplasia of the uterine cervix. J Natl Cancer Inst vaccines are likely to play a part in preventing 1999;91:252–8. PV infection and hence cervical cancer in the 6 Kuhn L, Denny L, Pollack A, et al. Human papillomavirus DNA testing for cervical cancer screening in low-resource near future. Studies of immunotherapy for PV settings. J Natl Cancer Inst 2000;92:818–25. infection will assist in evaluating the role of 7 Manos MM, Kinney WK, Hurley LB, et al. Identifying women with cervical neoplasia. Using human papillomavi- antigen specific immunotherapy in the control rus DNA testing for equivocal Papanicolaou results. JAMA of PV associated and other cancers. PV VLPs 1999;281:1605–10. 8 Meijer CJLM, Walboomers JMM. Cervical cytology after have a promising future as candidate delivery 2000: where to go? J Clin Pathol 2000;53:41–3. systems for other antigens, both for prophylaxis 9 Nobbenhuis MAE, Walboomers JMM, Helmerhorst TJM, et al. Relation of human papillomavirus status to cervical and for treatment. lesions and consequences for cervical-cancer screening: a prospective study. Lancet 1999;354:20–5. 10 Ramoz N, Taieb A, Rueda LA, et al. Evidence for a BIOLOGY nonallelic heterogeneity of epidermodysplasia verruci- formis with two susceptibility loci mapped to chromosome Several excellent studies were presented on regions 2p21–p24 and 17q25. J Invest Dermatol 2000;114: novel functions of viral proteins, transcription 1148–53. 11 Rozendaal L, Westerga J, van der Linden JC, et al. PCR control mechanisms, and intratype variants, based high risk HPV testing is superior to neural network which diVer considerably in biological activi- based screening for predicting incident CIN III in women ties. The availability of pseudovirions and the with normal cytology and borderline changes. J Clin Pathol 2000;53:606–11. use of raft cultures continued to be useful for 12 Salmon J, Nonnenmacher M, Caze S, et al. Variation in the unravelling the life cycle of PVs, which will nucleotide sequence of cottontail rabbit papillomavirus a and b subtypes aVects wart regression and malignant trans- allow new approaches for therapeutic interven- formation and level of viral replication in domestic rabbits. tions. Studies of virus–host interactions are J Virol 2000;74:10766–77. 13 SchiVman M, Herrero R, Hildesheim A, et al. HPV DNA beginning to profit from data from the human testing in cervical cancer screening. Results from women in genome project and techniques such as micro- a high-risk province of Costa Rica. JAMA 2000;283:87–93. 14 Stubenrauch F, Hummel M, Iftner T, et al. The E8E2C pro- cell mediated chromosomal transfer or micro- tein, a negative regulator of viral transcription and replica- array analyses. These techniques enhance the tion, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes. J Virol understanding of the regulation of senescence 2000;74:1178–86. and predisposition to PV infection. The role of 15 Tindle RW. Vaccines for human papillomavirus infection and anogenital disease. Texas: R.G. Landes Company (Medical PVs in the aetiology of skin cancer came into Intelligence, Unit 14), 1999. focus by identifying new cutaneous HPV types 16 Vernon SD, Unger ER, Williams D. Comparison of human papillomavirus detection and typing by cycle sequencing, and through preliminary experiments on the line blotting, and hybrid capture. J Clin Microbiol 2000;38: biochemical activities of cutaneous HPV onco- 651–5. 17 Walboomers JMM, Jacobs MV, Manos MM, et al. Human proteins. papillomavirus is a necessary cause of invasive cervical can- cer worldwide. J Pathol 1999;189:12–19. 18 Wells SI, Francis DA, Karpova AY, et al. Papillomavirus E2 The conference was credited for CME purposes by the induces senescence in HPV-positive cells via pRB- and European School of Oncology (ESO), the Federation of p21(CIP)-dependent pathways. EMBO J 2000;19:5762– European Cancer Societies, the American Medical Association 71. (AMA), and by the Spanish CME University System (Consell 19 zur Hausen H, Papillomaviruses causing cancer: evasion Coordinador de la Formacio Medica Continuada). The Union from host-cell control in early events in carcinogenesis. J Internatinonale Contre le Cancer (UICC) acted as a scientific Natl Cancer Inst 2000;92:690–8. www.jclinpath.com