Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention

Luisa Barzon; Colomba Giorgi; Franco Buonaguro; Giorgio Palù

doi:10.1186/1750-9378-3-14

Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention

Barzon, Luisa; Giorgi, Colomba; Buonaguro, Franco; Palù, Giorgio 2008-12-16 00:00:00 Objective: To provide guidelines for health-care providers on strategies for cervical cancer prevention based on HPV testing and anti-HPV vaccination. Outcomes: Overall efficacy of different preventive strategies, assessing reduction in the incidence of invasive cervical cancer and precancerous lesions. Evidence: Medline and the Cochrane Database were searched for articles in English on subjects related to HPVs, HPV diagnosis, HPV anogenital lesions, cervical cancer, HPV testing, and HPV vaccines, in order to elaborate an up-dated document. Relevant Italian Government publications and position papers from appropriate health and family planning organizations were also reviewed. Values: The quality of the evidence and ranking of recommendations for practice were rated using criteria defined by SIV, which were adapted from the Canadian Task Force on Preventive Health Care. with SIV members, and reviewed and approved by SIV Sci- Introduction This report summarizes recommendations on cervical entific Committee, during the annual SIV Conference of cancer screening and prevention strategies proposed by 2008, following a specific Round table on the subject. experts of the Italian Society for Virology (SIV, Società Ital- These recommendations recognize the diagnostic value of iana di Virologia) on the basis of systematic review of evi- HPV testing and genotyping in the management of dence from the scientific literature and current guidelines women with abnormal PAP test and in screening pro- produced by International Societies, in view of an Euro- grams for prevention of cervical carcinoma and recom- pean consensus strategy for cervical cancer screening, mend HPV vaccination for primary prevention of cervical human papillomavirus (HPV) testing, and anti-HPV vac- cancer. The searching strategies, the study selection criteria cination programs. The guidelines were discussed with and other methodologies adopted for the literature review experts in the field (see acknowledgment section) and can be asked to the correspondig author. Page 1 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 the field; therefore the definition of the oncogenicity of Human papillomavirus and cervical cancer The demonstration that human papillomavirus (HPV) some HPVs could change with time, with obvious impli- infection has a pathogenetic role on cervical cancer devel- cations on the definition of the diagnostic standards for opment and the understanding of the epidemiology of HPV infection, as outlined below. HPV infection and its relationship with the natural history of precancerous lesions and cervical cancer have been cru- High-risk HPV DNA is virtually detectable in all cervical cial in the development of diagnostic and vaccination cancers [15]. Among high-risk or carcinogenic HPVs (cur- strategies for prevention of one of the most common and rently, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, lethal cancers, especially in developing countries. 59) and probably carcinogenic HPVs (HPV types 26, 53, 66, 68, 73, 82) [4,5], HPV-16 is responsible for 60% of Epidemiology of cervical cancer and HPV infection cervical cancer, HPV-18 for 10%, HPV-45 and HPV-31 for Epidemiological data on HPV infection and associated 4% each, whereas HPV-33, HPV-52, and HPV-58 alto- disease have been reviewed in a recently published IARC gether account for 2% of cervical cancer [12,16]. In high- Monograph [1]. Cervical cancer is the second most com- grade squamous intraepithelial lesions (HSIL), the most mon and lethal cancer in women worldwide, with esti- common HPV types identified are similar to those in cer- mated 493,000 new cases and 274,000 deaths in 2002 [2]. vical cancer, except for the under-representation of HPV- More than 80% of cervical cancers and related deaths 18 and HPV-45 [16]. In low-grade squamous intraepithe- occur in developing countries, where cervical cancer is the lial lesions (LSIL), high-risk HPV DNA can be detected in most common cancer, accounting for 15% of female can- 30 to 100% of cases in different studies [17]. Again, HPV- cers, while in developed countries it accounts for only 16 is the most common type, being detected in 26% of 3.6% of new cancers [2]. Cervical cancer has a relatively cases, followed by HPV-31 (12%), HPV-51 (11%), HPV- low incidence in Europe (less than 15 per 100,000), 53 and HPV-56 (10%), and many other HPV types [17]. whereas its incidence is generally over 30 per 100,000 in developing countries [3,4]. The incidence of invasive cer- A recent meta-analysis investigated the worldwide preva- vical cancer has a peak at about 20–25 years after the peak lence and genotype distribution of HPV DNA in cervical age for HPV infection prevalence, and the incidence of cer- samples from women with normal cytology [18]. This vical intraepithelial neoplasia (CIN) peaks in between [1]. meta-analysis estimated the overall HPV prevalence in According to the AIRTum (Associazione Italiana dei Reg- women with normal cytology was 10.4% (95% confi- istri Tumori) database, in Italy, during 1998–2002, cervi- dence interval [CI], 10.2–10.7%). The prevalence was rel- cal cancer represented 1.6% of all newly diagnosed atively high in Africa, Central America and Mexico (about cancers among females, with an average incidence of 9.8 20 to 30%) and lower in northern America, Europe, and cases per 100,000 females per year, and represented 0.6% Asia (about 8 to 11%). In all world regions, HPV preva- of all cancer deaths among females. It has been estimated lence was highest in women younger than 35 years of age, that every year 3,418 new cervical carcinomas are diag- decreasing in women of older age [18]. In Africa, the nosed in Italy; as regards mortality, in 2002 there were Americas, and Europe, a second peak of HPV prevalence 370 deaths due to cervical cancer, and 1,756 deaths due to was observed in women of older age [18]. Overall, the cancer of the uterus not otherwise specified [5]. Survival most common HPV types were HPV-16, HPV-18, HPV-31, rates in patients with cervical cancer vary in different HPV-58, and HPV-52, while the most common types in countries, ranging from 73% at 5 years in US [6] and 63% southern Europe were HPV-16, HPV-66, HPV-45, HPV- in Europe [7] to 30.5% in sub-Saharan Africa [8]. In Italy, 31, and HPV-42 [18]. at 5 years from diagnosis of cervical cancer, survival rates range between 78% in Ferrara and 40% in Naples, with an In the Italian population, the prevalence of high-risk HPV average national survival rate of 65% [5]. in women aged 25–70 years has been reported to be about 9%, and HPV-16 was the most common genotype, being Epidemiological and experimental evidence demonstrate detected in 30–60% of HPV-positive women, followed by that persistent infection with a carcinogenic HPV geno- HPV-66, HPV-45, HPV-31, and HPV-53 [19,20]. HPV gen- type is a necessary cause of cervical cancer and its precan- otyping by restriction fragment length polymorphism cerous lesions, as suggested by zur Hausen in '70 [9]. Over analysis and direct sequencing showed that, in HSIL, 120 different HPV genotypes have been identified so far HPV-16 and HPV-31 were the most commonly detected and at least 40 of these HPV types infect the anogenital types, followed by HPV-67, HP-87, HPV-61, and HPV-58, mucosa [10]. Genital HPVs are designated as high-risk or while HPV-16, HPV-31, HPV-66, HPV-53, HPV-52, and low-risk types, based on their association with cervical can- HPV-56 were the most frequent types in LSIL [21]. By con- cer [11-14]. It should be recognized that the distinction trast, low-risk HPV-6 and HPV-11 were responsible for between high-risk and low-risk HPV genotypes is contin- about 90% of genital warts [22], whereas the other low- uously under revision on the basis of new knowledge in risk HPV types were less frequently associated with ano- Page 2 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 genital lesions. Data at recruitment from the New Tech- sion [33]. Following entry into the suprabasal layers, nologies for Cervical Cancer Screening (NTCC) expression of the late viral genes E4, L1, and L2 is initi- randomized trial, which is ongoing in Italy and includes ated; the circular viral genome is then replicated and struc- women attending routine cervical screening programs, tural proteins form. In the upper layers of the epidermis or showed the rates of high-risk HPV positivity were 13.1% mucosa, complete viral particles are assembled and for women aged 25–34 and 5.8% for women aged 35–60, released [33]. The HPV E1 and E2 proteins are involved in without a second peak of HPV prevalence [23]. In younger replication and segregation of episomal viral genome women aged 18 to 24, the prevalence of high-risk HPV [34]; the E2 protein also acts as a transcriptional activator DNA detection is 17.4% and HPV-16 was the most fre- of viral promoters through its ability to bind to specific quent type, followed by HPV-53, HPV-84, HPV-42, HPV- sequences within the regulatory sequences of HPV 62, HPV-66, and HPV-89 [24]. A higher prevalence has genome, known as long control region (LCR) [35]. How- been reported in a Danish population-based study [25], ever, in some cases, E2 may also inhibit viral gene tran- which showed that about 40% and 45% of women aged scription: in genital tract-associated high-risk HPV, E2 15–19 and 20–24 years, respectively, were infected with protein has been demonstrated to suppress E6 and E7 high-risk HPV types. HPV-16 was the most common HPV expression probably through steric interference with the type, followed by HPV-31, HPV-52, and HPV-51 [25]. binding of positively acting cellular transcription factors Like in the Italian population, the prevalence of infection in the LCR [36]. HPV E4 function is probably to aid the markedly decreased with age, without significant second virus in its egress from the cell [37]. E5 seems to be impor- prevalence peak [25]. The EDITH (Etude de la Distribu- tant in the early course of infection. It probably stimulates tion des Types d'HPV en France) study investigated the cell growth by forming a complex with the epidermal prevalence and distribution of HPV types in France. The growth factor receptor or a subunit of the vacuolar results of this study showed that HPV DNA could be ATPase, thus inhibiting the acidification of endosomes detected in 98% of LSIL smears, in 98% of high-grade cer- and growth factor receptor recycling [38,39]. However, vical intraepithelial neoplasia (CIN2/3), and in 97% of since the E5 gene is not expressed in most HPV-positive invasive cancers [26-28]. In LSIL, the most prevalent gen- cancers, E5 does not seem to be obligatory in late events otypes were HPV-66, HPV-16, HPV-53, HPV-51 and HPV- of HPV-mediated carcinogenesis [33]. A more significant 52 [26]; in CIN2/3, HPV-16 was by far the most prevalent role for malignant transformation can be assigned to the genotype (62%), followed by HPV-31, HPV-33, HPV-52, E6 and E7 genes and their respective proteins. They are HPV-51, and HPV-58 [27], while the most prevalent gen- consistently expressed in malignant tissue, and inhibiting otypes in invasive cervical cancer were HPV-16 (73%) and their expression blocks the malignant phenotype of cervi- HPV-18 (19%), followed by HPV-31 (7%), HPV-33, HPV- cal cancer cells [40]. They are independently able to 68, HPV-45, HPV-52, and HPV-58 (4.1-2.3%) [28]. While immortalize various human cell types in tissue culture, HPV-16 was the most prevalent type in both squamous and show synergistic activity when they are expressed cell carcinoma (74%) and adenocarcinoma (64%), HPV- together. Interestingly, in high risk HPVs, E6 and E7 pro- 18 was more prevalent in adenocarcinoma (37%) com- tein are translated together from a polycistronic mRNA pared to squamous cell carcinoma (16%) [28]. [33]. Besides cervical cancer, HPV infection is associated with The E7 protein encoded by high-risk HPV is a small mul- over 70% of cases of anal carcinoma and squamous tifunctional nuclear protein that binds zinc, is phosphor- oropharyngeal carcinoma, with 50% of vulvar, vaginal, ylated by casein kinase II, and shares structural similarities and penile cancer, and, in immunocompromised with oncoproteins of other DNA tumor viruses [41]. The patients, with about 90% of cutaneous squamous cell can- most significant cellular targets of E7 are the retinoblast- cer [12,29,30]. In Italy, high-risk HPV DNA, mainly HPV- oma (Rb) tumor suppressor protein and its functionally 16, was detected in 46% of cases of penile carcinoma [31]. related proteins, p107 and p130, especially in their hypo- phosphorylated active forms, which are able to inhibit cell Pathogenesis cycle progression by binding and repressing the activity of Several basic experimental studies have clarified the E2F cellular transcription factors [42]. Binding with high- mechanisms of HPV oncogenesis [29,32]. In its life cycle, risk E7 results in pRb proteasomal degradation, but this HPV requires infection of epidermal or mucosal epithelial event is not sufficient to account for E7 transforming func- cells of the basal layer, that are still able to proliferate, tions [43]. Indeed, E7 has additional cellular targets, such kip1 most probably through wounds or abrasions. In basal as the cyclin-dependent kinase inhibitors p27 and cip1 layer cells, viral gene expression is largely suppressed, p21 , which are inactivated [44-46], and causes although the limited expression of the early viral genes E1, genomic instability leading to chromosomal abnormali- E2, E5, and especially E6 and E7, results in enhanced pro- ties and aneuoploidy [47]. liferation of the infected cells and in their lateral expan- Page 3 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 The first transforming activity identified for the high-risk who have had 3 normal test results in a row may get HPV E6 proteins was the ability to mediate p53 degrada- screened every 2 to 3 years with cervical cytology alone, or tion through the ubiquitin proteolysis pathway [48], a every 3 years with cytology test plus high-risk HPV DNA property not possessed by the low-risk HPV E6 proteins. testing (HPV test). Women aged ≥ 70 years who have had E6 first associates with the cellular ubiquitin ligase protein 3 or more normal Pap tests and no abnormal Pap tests in termed E6-associated protein (E6AP), then the E6-E6AP the last 10 years and women who have had total hysterec- complex binds to p53, stimulating its ubiquitination and tomy may choose to stop cervical cancer screening. Special degradation [49]. In targeting p53, the high-risk HPV E6 recommendations are given to at-risk categories (i.e., his- proteins inhibit DNA damage and oncogene-mediated tory of cervical cancer, in utero exposure to diethyl- cell death signals. E6 from high-risk HPV also interacts stilbestrol, immunosuppression). Similar guidelines have with several other cellular proteins involved in DNA rep- been proposed by the American College of Obstetricians lication, signal transduction (NFX1), activation of telom- and Gynecologists in 2003 http://www.acog.org, by the erase (Myc), cell proliferation (PDZ motif-containing U.S. Preventive Services Task Force in 2003 http:// proteins), and inhibition of apoptosis (Bak, CBP/p300) www.preventiveservices.ahrq.gov, and, more recently, in [33]. 2006, by the American Society for Colposcopy and Cervi- cal Pathology [55,56]. Invasive cervical cancers and precancerous lesions associ- ated with high-risk HPV infection are characterized by In European Union member states, screening programs integration of HPV DNA in cellular chromosomes. Viral for cervical cancer prevention are organized according to DNA integration is associated with deletion of large seg- the European Council [57] and the European Guidelines ments of viral genome, but with the presence of intact E6 [58], which recommend to set up cancer screening as a and E7, and with transcription of sequence downstream population-based public health programme, with identi- from the integrated LCR [50]. Disruption of the viral E1 fication and personal invitation of each woman in the eli- and E2 genes, as well as of downstream viral sequences, gible target population, and to discourage opportunistic may permit higher levels of E6 and E7 transcription, screening. According to these guidelines, cervical cytology whose RNA are stabilized following fusion with down- is the currently recommended standard test for cervical stream cellular sequences [51]. Persistent expression of E6 cancer screening, which should start in the age range 20– and E7 appears to be necessary to maintain the malignant 30 years. Screening is recommended to be continued at 3– phenotype and to favour the occurrence of cellular genetic 5-year intervals until the age of 60–65. In older women, and epigenetic events, which are important for cancer pro- who have had three or more consecutive previous recent gression [52]. normal cytology results, stopping screening is considered appropriate, while special attention has to be paid to those who have never attended screening. While waiting Screening for cervical cancer and diagnosis of HPV infection for the results of ongoing longitudinal trials before recom- Screening programs for cervical cancer prevention derive mending HPV screening, HPV DNA testing is proposed in: from knowledge on the natural history of cervical cancer 1) primary screening for oncogenic HPV types alone or in and its precancerous lesions and the role of HPV infection combination with cytology; 2) triage of women with in cancer development. These programs, which vary equivocal cytological results; 3) follow-up of women widely by country, include screening tests for early detec- treated for CIN to predict success or failure of treatment. tion of cancer, as well as management strategies for treat- Referral for colposcopy is recommended for women with ment and post-treatment follow-up. Several international a high-grade cytological lesion (ASC-H and HSIL or and national scientific societies have drawn guidelines on higher), a repeated low-grade lesion, or with an equivocal cervical cancer screening and prevention, which are sum- cytology result and a positive HPV test. Management marized in Table 1. All these guidelines emphasize the options in case of atypical squamous cells of undeter- utility to incorporate HPV testing as an adjunct to cervical mined significance (ASC-US) include reflex HPV DNA cytology in cervical cancer screening programs in women testing (preferred option when HPV testing is available), aged ≥ 30 years and for management of women with an repeating the Pap smear after 6 to 12 months, or referral abnormal Pap test. for colposcopy and cervical biopsy (when poor follow-up compliance is suspected or when explicit risk factors are Guidelines of the American Cancer Society [53,54] recom- present). Repeat cytology or referral for colposcopy (pre- mend that screening should begin approximately 3 years ferred option) are considered acceptable options for ini- after a woman starts having vaginal intercourse, but no tial management of LSIL, while HPV testing is considered later than age 21 years. Screening should be done every not sufficiently selective. The guidelines of the Italian year with conventional Pap tests or every 2 years using liq- Ministry of Health for the cervical cancer screening suggest uid-based cytology testing. At or after age 30 years, women performing a Pap test every three years in women aged Page 4 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Table 1: European and US guidelines on cervical cancer screening and prevention. European ACS (American ACOG (American ASCCP (American US Preventive guidelines for Cancer Society); College of Society for Service Task Force; quality assurance in 2007 [53,54] Obstetricians & Colposcopy & 2003 http:// cervical cancer Gynecologists); Cervical www.preventiveservic screening; 2007 [58] 2003 http:// Pathology); 2006 es.ahrq.gov www.acog.org [55,56] Initiation of screening 20–30 yr about 3 yr after the about 3 yr after the NR within 3 yr of onset of with Pap cytology onset of sexual onset of sexual sexual activity or age activity but no later activity but no later 21 yr, whichever than age 21 yr than age 21 yr comes first Use of HPV testing in Not recommended With cytology in With cytology in Insufficient evidence screening programs while waiting for the women ≥ 30 yr women ≥ 30 yr results of randomized controlled trials. Screening intervals - conventional Pap 3–5 yr Annually; every 2–3 yr Annually; every 2–3 yr NR At least every 3 yr test for women aged ≥ 30 for women aged ≥ 30 yr with 3 consecutive yr with 3 consecutive negative cytology negative cytology tests. tests. - if HPV testing used NR Every 3 yr if HPV Every 3 yr if HPV NR Insufficient evidence negative and cytology negative and cytology negative. negative. Discontinuation of 60–65 yr with ≥ 3 Women aged ≥ 70 yr Inconclusive evidence NR Women aged ≥ 65 yr screening recent consecutive with ≥ 3 recent to establish upper age with negative tests, negative tests. consecutive negative limit. who are not tests and no abnormal otherwise at high risk tests in prior 10 yr. for cervical cancer. Management of ASC-US: reflex HPV NR NR ASC-US: HPV testing, NR abnormal cervical testing; or repeat cytology, or cancer screening test LSIL: repeat cytology colposcopy in women - ASC-US or colposcopy; ≥ 20 yr; - ASC-H ASC-H: colposcopy; ASC-H: colposcopy - LSIL HSIL: colposcopy and LSIL: colposcopy - HSIL biopsy. HSIL: immediate loop electrosurgical excision or colposcopy with endocervical assessment. NR: not reported. 25–64, as first level of screening, followed by specific rec- The efficacy of conventional cytological screening for cer- ommendations for women with abnormal cytology [59]. vical cancer has never been investigated in randomised In particular, referral for colposcopy is recommended in clinical trials, but evidence of its effectiveness derives from the case of ASC-H and HSIL or higher at Pap test, while the observational studies. Diagnostic performance of cervical following management options are acceptable in the case cytology has been evaluated by several studies in the liter- of ASC-US: triage with reflex HPV DNA test (and referral ature, whose results have been reviewed in recent meta- to colposcopy if HPV DNA test is positive), immediate analyses. These studies have demonstrated that a single referral to colposcopy, or repeat Pap cytology after 6 cytology test has a sensitivity of about 50% for identifying months. Referral to colposcopy is the preferred manage- women with high-grade precancerous lesions or invasive ment strategy for LSIL, although triage with HPV testing is cervical carcinoma (i.e., cervical intraepithelial neoplasia also acceptable [59]. type 2 or higher, CIN2+) [60-63]. In particular, a recent meta-analysis on 8 cervical cancer screening studies done Cytology testing in North American and European countries with estab- Screening programs based on cytology testing led to the lished cytology-based screening activities, including over reduction of the incidence of cervical cancer and cancer- 60,000 women who were tested for both HPV DNA and related mortality of about 70–80% in many industrialized cytology, found that the overall relative sensitivity of countries [1]. cytology for CIN2+ was 53.0% (95% CI, 48.6%–57.4%), Page 5 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 by using ASC-US as cut-off for a positive result [62]. More- PCR system, the Roche Amplicor system, the PGMY over, sensitivity of cytology varied considerably among primer set, and the SPF primer set, to amplify a broad studies, ranging from 18.6% to 76.7% [62]. In fact, cytol- spectrum of HPV types. These primers target the L1 gene ogy testing has been shown to have poor inter-laboratory in a conserved region amongst HPVs. Subsequent to and inter-operator reproducibility [64]. The meta-analysis amplification of HPV DNA, detection of high-risk HPV also showed the sensitivity of cytology testing increases types is performed by hybridization with probes specific with patient's age and is significantly higher in women for high-risk HPVs. HPV genotyping is performed by type- over the age of 50 than in younger women (79.3% vs. specific PCR or by amplification of HPV DNA by using 59.6%, respectively). The overall specificity of cytology for consensus or general primers, followed by sequencing, CIN2+ is very high (96.3%; 95% CI, 96.1–96.5%), and restriction fragment length polymorphism analysis, or this is an important requisite for a screening test, and is reverse hybridization of the amplicon to multiple oligo- even higher in women aged ≥ 35 years than in younger nucleotide probes corresponding to high-risk and low- women (97.1% vs. 95.9%, respectively) [62]. More recent risk HPVs. Reverse hybridization assays, such as the linear meta-analyses involving more European/North American array by Roche Diagnostics, the line probe assay by Inno- studies yielded a pooled sensitivity of cytology at cut-off genetics NV (Belgium), microchips by Biomedlab Co. ASCUS+ for finding CIN2+ of 70% (95% CI: 60–83%). (Korea) and by other companies, Multiplexed Luminex After omission of three German studies, which reported Assay (Multimetrix, Germany), etc., are able to identify very low sensitivity data, the pooled sensitivity increased co-infections by different HPV types, at variance with to 78% (95% CI: 69–87%) [63,65]. sequencing-based assays, which have high accuracy in the definition of HPV types but poor sensitivity in co-infec- Liquid-based cytology has been more recently introduced tions. and is now widely used for primary screening of cervical cancer. It has the advantage of allowing automation, faster Applications of HPV testing in cervical cancer reading times, and to be used for adjunctive testing, screening programs including HPV testing. It has been suggested to be more Using HPV DNA test for triage of abnormal Pap test sensitive than conventional cytology, but this feature has HPV testing has been introduced in programs for cervical cancer prevention after numerous studies had demon- been rebutted by recent randomized studies, which com- pared the accuracy of liquid-based cytology with conven- strated that detection of high-risk HPVs has a higher sen- tional cytology for primary screening of cervical sitivity than cytology testing in predicting high-grade carcinoma [66,67]. In these studies, liquid-based cytology cervical precancerous lesions or invasive cervical carci- showed no significant difference in sensitivity to conven- noma. A meta-analysis on the use of HPV testing to man- tional cytology for detection of CIN2+, besides a lower age women with ASC-US showed that HPV testing has a positive predictive value (PPV) due to a higher number of greater accuracy than repeat cervical cytology [61]. In fact, positive results. It showed however the advantage of a the overall sensitivity and specificity of HPV testing were reduction of unsatisfactory smears [67]. 84.4% and 72.9%, respectively, vs. 57.6% and 81.8% of Pap-test, by using ASC-US as cut-off for a positive result; HPV test by using LSIL as cut-off, Pap test sensitivity was reduced to Currently used molecular tests for the detection of nucleic 45.7%, whereas specificity increased to 89.1% [61]. acids of high-risk HPV in clinical specimens are based on Among the studies included in the meta-analysis, there signal-amplification and hybridization methods, such as was the large ALTS (ASCUS Low SIL Triage Study), a con- the Hybrid Capture II assay (HC2, Diagene Corp., Gaiths- trolled clinical trial, which enrolled 3,488 women, rand- burg, Maryland, USA), or on target amplification, like the omized into three intervention groups: immediate polymerase chain reaction (PCR)-based Amplicor HPV colposcopy, repeated liquid-based cytology, or HPV test- test (Roche Molecular Diagnostics, Switzerland) or other ing. This trial, as confirmed also by other similar studies, PCR-based assays. The HC2 test, to date, is the only com- demonstrated that HPV testing for ASC-US triage had a mercially available HPV DNA detection test that is significantly higher sensitivity and similar specificity for approved by the FDA for cervical cancer screening in com- CIN3+ than repeat cytology [68,69]. Based on these bination with cytology after the age of 30 years. The HC2 results, both the American Society for Colposcopy and test is a nucleic acid hybridization assay with signal ampli- Cervical Pathology (ASCCP) [55,56] and the European fication for the qualitative detection of HPV DNA of 13 Guidelines [58] currently recommend HPV testing using high-risk types in cervical specimens. The HC2 assay can- validated HPV assays as an acceptable method for manag- not identify the specific HPV type, since detection is per- ing women over the age of 20 years with ASC-US. Accord- formed with a combined probe mix, and provides a semi- ing to ASCCP guidelines, women with ASC-US who are quantitative estimate of HPV DNA load. PCR-based assays HPV DNA negative can be followed up with repeat cyto- use consensus or general primers, such as the GP5+/6+ logical testing at 12 months, whereas those who are HPV Page 6 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 DNA positive should be referred for colposcopic evalua- HPV testing at 6–12 months for post treatment manage- tion. If CIN is not identified, repeat HPV testing at 12 ment of CIN2,3 is recommended by current guidelines of months is recommended. However, women with ASC-US the American Society for Colposcopy and Cervical Pathol- who are HPV DNA negative have a very low probability of ogy [56], whereas European Guidelines, while waiting for having CIN2+, lower than the general population with further data from clinical trials, recommend double test- negative cytology and unknown HPV status, who is actu- ing with cytology and an HPV test at 6 months post treat- ally referred to three years. So, a more conservative man- ment [58]. agement strategy than ASCCP recommendations could be appropriate. Using HPV DNA test for cervical cancer screening The potential role of HPV testing as a stand-alone test or HPV testing has not been demonstrated to be a useful in conjunction with cytology in cervical cancer screening management option for women with a cytological result has been investigated in several studies [reviewed in of LSIL. In fact, a meta-analysis of published studies dem- [62,65,77]]. Unfortunately, these studies were performed onstrated HPV triage of LSIL had no significantly higher with different techniques and sometimes gave conflicting sensitivity but lower specificity than repeat cytology in the results. detection of high-grade CIN [70]. In particular, the ALTS study found that over 80% of women with LSIL were A meta-analysis including 25 non-randomized clinical tri- HPV-positive, whereas the prevalence of CIN2+ was con- als, which compared the performance of cytology vs. HPV siderably lower [71]. Nevertheless, reflex HPV testing may test in screening programs for prediction of CIN2+ [63], be cost effective in older women with LSIL, due to consid- showed the overall sensitivity of HPV test was 90%, when erably lower prevalence of HPV infection. In this regard, the HC2 assay was used, and 80.9%, when PCR with con- data from the randomized Italian study New Technolo- sensus primers was employed; overall specificity was gies for Cervical Cancer Screening (NTCC), which com- higher in PCR-based tests than in HC2, being 94.7% and pared accuracy of cytology testing vs. HPV DNA testing, 86.5%, respectively. The accuracy of cytology testing was specificity of HPV testing for triage of LSIL was higher in lower than HPV testing: in fact, with ASC-US as a cut-off, women aged over 35 years than in younger women, since sensitivity was 72.7% and specificity 91.9%. In women the proportion of HPV positive women was much lower aged over 30 yr, the overall sensitivity of the HC2 assay in women aged 35–60 years than in those aged 25–34 was 94.8% and specificity 86% [63]. years (42% vs. 72%) [72]. So, HPV triage could be appro- priate also for LSIL in women over 35 years, in order to Likewise, the meta-analysis by Cuzick et al. [62], which reduce colposcopy burden and to increase the positive included only studies from European and North Ameri- predictive value of the colposcopy referral. can countries, where screening programs are well imple- mented, showed the overall sensitivity and specificity of Using HPV DNA test for follow-up after treatment of HPV testing were 96.1% and 90.7%, respectively, whereas cervical dysplasia the overall sensitivity and specificity of cytology testing Several studies demonstrated that HPV testing, performed were 53.0% and 96.3%, respectively. Moreover, at vari- at 4–6 months intervals following ablative therapy for ance with cytology testing, sensitivity of the HPV test did high-grade lesions (CIN2–3), has a higher sensitivity and not vary among the different Centres and was very high specificity than cytology in detecting residual disease or both in young women and in those over 50 years of age. recurrence [73-75]. In a meta-analysis of 11 studies evalu- Among the studies included in this meta-analysis, there ating HPV DNA testing in monitoring women after treat- was the HART (HPV in Addition to Routine Testing) trial, ment of CIN3, the negative predictive value of HPV testing a multicentric screening protocol which enrolled over for recurrent/residual disease was 98%, whereas the nega- 10,000 women aged 30–60 years attending for routine tive predictive value of cytology was 93% [76]. Overall, screening. In this study, women with borderline cytology the sensitivity of HPV testing for identifying recurrent/per- or negative cytology but positive HPV test were rand- sistent CIN reaches 90% by 6 months after treatment and omized to immediate colposcopy or follow-up with remains high for at least 24 months, at variance with cyto- repeat HPV test, cytology testing, or colposcopy at 12 logical follow-up, which has a sensitivity of approxi- months [78]. The HART study confirmed HPV testing is mately 70%. These findings were confirmed in an updated more sensitive than borderline or worse cytology (97.1%, meta-analysis, which showed HPV testing has a signifi- 95% CI 91.2–99.1%, vs. 76.6%, 95% CI 65.1–85.1%) but cantly higher sensitivity (ratio: 1.27; 95% CI: 1.06–1.51) less specific (93.3%, 95% CI 92.7–93.9%, vs. 95.8%, 95% and not-significantly lower specificity (ratio: 0.94; 95% CI 95.4–96.2%) for detecting CIN2+ [78]. Comparison of CI: 0.87–1.01) than follow-up cytology in the prediction management strategies for borderline cytology and for of residual/recurrent CIN [65]. positive HPV test with negative cytology results at baseline showed that surveillance at 12 months was as effective as Page 7 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 immediate colposcopy [78]. Interestingly, no CIN2+ were more restricted referral criteria (i.e., cut-off of 2 pg/mL and detected in women with a positive HPV test at baseline positive results at repeat HPV testing) was predicted to but negative at follow-up nor in those with an initial neg- increase PPV to that obtained with conventional cytology ative HPV test and borderline or mild cytology [78]. Based [81]. on these results, it was suggested that HPV test could be used as a screening test in women over 30 years of age Results from the second recruitment phase of the NTCC with cytology triage of HPV-positive results. In case of nor- study showed that, in women aged 35–60 years, HPV test- mal or borderline cytology, repeat HPV testing after 12 ing alone with a cut-off of 2 pg/mL achieved a substantial months was indicated [78]. gain in sensitivity over conventional cytology with only a small reduction in PPV (relative sensitivity, 1.81; 95% CI, Randomized longitudinal studies [79], which have been 1.20–2.72; relative PPV, 0.99; 95% CI, 0.67–1.46) [23], conducted more recently, were not included in the meta- confirming the results of the first phase of the study [80]. analyses. One of these studies is the NTCC study, an ongo- In women aged 25–34 years, direct referral to colposcopy ing clinical trial in Italy nested in the routine activity of after a positive HPV test led to detection of a significantly organized screening programmes, which enrolled approx- greater number of CIN2+ and CIN3+ lesions than in the imately 95000 women, who were randomly assigned to first phase of the study [23]. The increased sensitivity of conventional cytology or to an experimental arm that fol- HPV testing for CIN2+ in the second phase of the study as lowed two phases depending on the period of recruitment compared with the first was suggested to be related to a [23]. During the phase 1 of recruitment, women were ran- high rate of high-grade lesions in young women, which domly assigned to the conventional arm for screening by spontaneously regress [23]. So, in order to avoid over- conventional cytology or to the experimental arm, which treatment, it was suggested that HPV-positive women used both HPV DNA testing with HC2 and liquid-based aged 25–34 years should be referred to colposcopy only if cytology. HPV-positive women were managed differently cytology is also abnormal or if infection persists after 1 according to age. Among women aged 35–60 years, those year [23]. In older women, high-grade lesions are less who had a cytological abnormality or were HPV-positive likely to regress [78,80], therefore a more aggressive man- were referred to colposcopy. Among women aged 25–34 agement strategy for HPV-positive results appears to be years, all women with abnormal cytology were referred for justified. On the other hand, referral to colposcopy of colposcopy but those who were HPV-positive with nor- women with abnormal cytological findings (ASC-US+) mal cytology were retested after 1 year and referred for col- but a negative HPV test may led to a high-rate of false pos- poscopy only if HPV positivity persisted or if cytology itive CIN2+ results and, as a consequence, to overtreat- became ASC-US+. During the phase 2 of recruitment, ment [82]. women were randomly assigned to conventional cytology with referral for colposcopy if cytology indicated ASC-US+ Persistence of the additional lesions detected at baseline or to HPV DNA testing alone with referral for colposcopy by HPV testing in older women was also suggested by the if the test was positive [23]. Results at recruitment showed recently published results from two longitudinal rand- that the screening method in the experimental arm was omized controlled trials, that included only women at more sensitive than conventional cytology in women least 30 [83] or 32 [84] years of age. One of the trials is the aged 35–60 years [80]. In particular, referral based on Population Based Screening Study Amsterdam (POBAS- HPV testing with HC2 at a high cut-off value of 2 pg/mL CAM), which aims to assess whether primary HPV DNA showed a higher sensitivity than conventional cytology testing is more effective than cytological testing in the set- for detection of CIN2+ (relative sensitivity, 1.41%; 95% ting of a regular screening programme. This study enrolled CI, 0.98–2.01), but a lower positive predictive value (PPV; women aged 30–56 years and attending the regular cervi- relative PPV, 0.75; 95% CI, 0.45–1.27). The use of the 2 cal cancer screening programme in the Netherlands. pg/mL cut-off led to a higher PPV than the 1 pg/mL cut-off Women were randomly assigned to the intervention [80]. Adding liquid-based cytology improved sensitivity group (screening with HPV DNA testing combined with only marginally but increased false positives [80]. In Pap test) or control group (Pap test only, HPV DNA test women younger than 35 years, HPV testing alone as pri- results blinded). After 5 years, combined cytological and mary test, with triage of HPV-positive women by cytology, HPV DNA testing were done in both groups and follow- appeared to be the best approach, since the use of liquid- up data for ≥ 6.5 years were available for 17,155 women. based cytology did not increased the sensitivity for CIN2+ Management strategies were more conservative than in detection and had a significantly lower PPV as compared the NTCC study. In fact, women with HSIL or worse were with conventional cytology. Moreover, addition of liquid- immediately referred to colposcopy, irrespective of the based cytology as a primary screening test to HPV testing HPV result, whereas repeat testing after 6 and 18 months had a negligible effect on sensitivity but strongly reduced was advised to women with normal cytology but positive PPV compared with HPV testing only [81]. The use of HPV test and to those with ASC-US, ASC-H, or LSIL. Page 8 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Women with repeat positive cytology and HPV test results cohort study which enrolled over 20,000 women in Port- were referred to colposcopy [83]. In this study, detection land who underwent simultaneous screening with a Pap of HPV DNA was done by GP5+/6+ PCR followed by test and HPV testing with HC2, the 5-year cumulative risk enzyme immunoassay detection of 14 high-risk HPV of CIN3, was 4.4% for women who were HPV-positive at types with a cocktail of oligonucleotide probes. The other baseline, but only 0.24% among women with a negative trial was conducted in Sweden and enrolled 12,527 HPV test and 0.16% when both the HPV test and Pap women aged 32 to 38 years, who, like in the POBASCAM smear were negative [85]. study, were randomized to have an HPV test plus Pap test or a Pap test alone [84]. In this study, HPV test was based A direct comparison between Pap test and HPV test (by on GP5+/6+ PCR followed by reverse dot blot hybridiza- using the HC2 assay) as stand alone tests for cervical can- tion to identify 14 high-risk HPVs. Women with a positive cer screening was evaluated in a recent randomized trial in HPV test and a normal Pap test were offered a second HPV Canada, involving 10,154 women aged 30–69 years [86]. test at least 1 year later, and those who were found to be Both tests were performed on all women in a randomly infected with the same high-risk HPV type were offered assigned order but, to the aim of the study, only one first colposcopy with cervical biopsy. To avoid ascertainment test was considered for statistical evaluation. Women with bias, a similar number of Pap smears and colposcopy with abnormal Pap test results or a positive HPV test at 1 pg/mL biopsies were performed in randomly selected women in cut-off underwent colposcopy and biopsy, as a random the control group. All women were followed for a mean of sample of women with negative tests. Results at baseline 4.1 years with annual Pap smears and HPV tests, with col- testing showed sensitivity of HPV testing for CIN2 or poscopy in cases of persistent high-risk HPV infection CIN3 was 94.6% (95% CI, 84.2–100%), significantly [84]. Both studies showed that a significantly higher higher than sensitivity of Pap test, which was 55.4% (95% number of CIN2+ or CIN3+ lesions were detected at base- CI, 33.6–77.2%). HPV test specificity was 94.1% (95% CI, line in the intervention group than in the control group, 93.4–94.8%) and 96.8% for Pap testing (95% CI, 96.3– whereas the number of lesions detected in the subsequent 97.3%) [86]. follow-up visit, when both HPV test and cytology were done, was significantly lower in the intervention group Recommendations on the use of HPV testing in cervical than in the control group [83,84], thus indicating that cancer screening programs (grading of recommenda- implementation of HPV DNA testing in cervical cancer tions is reported in Table 2) screening leads to earlier detection of high-grade lesions, thus allowing longer screening intervals. In the Dutch � HPV testing is recommended for ASC-US triage since study, the 5-year cumulative risk of CIN3+ was 0.1% HPV test is more sensitive than repeat cytology for detec- (95% CI, 0.1–0.2%) after a combined negative HPV DNA tion of CIN2+ (IA). and cytological result at baseline and 0.8% (95% CI, 0.6– 1.0%) after a negative cytology at baseline, but without � HPV testing is, in general, not a management option in HPV testing [83]. After a negative HPV test at baseline, the case of LSIL (ID), even if it may be cost effective in older 5-year cumulative risk of CIN3+ was estimated as 0.2% women with LSIL (IB). Local assess is needed to identify a (95% CI, 0.1–0.3%) [83]. Likewise, in a longitudinal good triage test for women with LSIL (research need). Table 2: Key to evidence statements and grading recommendations defined by SIV.* Strength of Recommendation A. Good evidence for efficacy and substantial clinical benefit support recommendation for use. B. Moderate evidence for efficacy or only limited clinical benefit supports recommendation for use. C. Evidence for efficacy is conflicting and does not allow supporting a recommendation for or against use, but recommendations may be made on other grounds. D. Moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use. E. Good evidence for lack of efficacy or for adverse outcome supports a recommendation against use. I. There is insufficient evidence (in quality and quantity) to make a recommendation; however, other factors may influence decision-making. Quality of Evidence I. Evidence from at least 1 randomized, controlled trial. II. Evidence from at least 1 clinical trial without randomization, from cohort or case-controlled analytic studies (preferably from more than 1 centre) or from multiple time-series studies or dramatic results from uncontrolled experiments. III. Evidence from opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert committees. *: Quality of evidence and grading of recommendations were defined and approved by SIV; they were adapted from The Evaluation of Evidence and the Classification of Recommendations criteria described in The Canadian Task Force on Preventive Health Care. Page 9 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 � HPV testing, alone or in combination with cytology, is New molecular tests for management of women recommended for follow-up after treatment of high-grade with HPV infection Due to the relatively low specificity of HPV-DNA testing cervical lesions, since it is more accurate than repeat cytol- ogy in diagnosing residual disease or relapse (IA). and the high rate of false negative results at Pap test, new molecular tests have been proposed in order to identify � HPV testing, both based on hybridization (HC2) and women with a higher risk of progression to invasive can- PCR amplification with detection of high-risk HPVs, has cer. These tests include (i) detection of type-specific HPV been widely used in women aged >30 years in addition to persistence; (ii) detection of high-risk HPV E6/E7 mRNAs; Pap cytology because of the high NPV of a combined neg- (iii) HPV genotyping to detect HPV-16 and HPV-18, ative test (I). Women with normal Pap test result and neg- which have been associated with a higher risk of CIN2,3 ative HPV test have a very low risk to develop cervical or invasive cancer; (iv) measurement of high-risk HPV cancer and should not perform the subsequent follow-up load; (v) demonstration of high-risk HPV DNA integra- visit earlier than 5 years (IA). However, combination of tion; (vi) HPV subtyping by E6, L1, and LCR sequencing; HPV testing and Pap cytology is not a cost-effective strat- (vii) analysis of p16-INK4A expression. The implementa- egy (I), so, strategies based on HPV testing as a stand alone tion of these new molecular tests in cervical cancer screen- test should be preferred in women aged >30 years (IA). ing could be useful to identify the subpopulation of high- risk HPV-positive women who deserve a closer follow-up � Follow-up with HPV testing or repeat cytology at 12 and appropriate treatment. months is recommended for cytology-negative, HPV-pos- itive women aged ≥ 30 years (IA). Women who are persist- Demonstration of HPV persistence ently HPV-positive on repeat testing should undergo In women younger than 25 years of age, 20% of high-risk colposcopy, whereas those who are negative on both tests HPV infections persist, whereas the risk of persistent infec- may be rescreened in 5 years (IA). tion is over 50% in women older than 55 years [18]. Only a few women with HPV infection will develop cancer. � Using the HPV test in screening programs leads to earlier Thus, a single positive HPV test in the absence of clinically detection of CIN2+ and CIN3+ and reduction of the significant lesions does not justify treatment [87]. Several number of high-grade lesions detected at subsequent fol- longitudinal studies demonstrated that persistence of low-up in women aged ≥ 30 years (I), thus allowing high-risk HPV is necessary for cancer initiation and pro- longer screening intervals. The extent of long-term protec- gression and that the majority of high-risk HPV infections tion against high-grade lesions after a negative HPV DNA and associated low-grade lesions spontaneously regress test has been estimated to be longer than 5 years, but fur- within 6–18 months [1,88-93]. Screening strategies based ther research is need to establish the most appropriate on repeat HPV testing at 12 months following detection of screening interval (research need). a high-risk HPV could be useful to identify women at risk of cancer, who should undergo colposcopy, and to avoid � HPV testing as a stand alone test is more effective than overtreatment of lesions which will regress [94,95]. cytology alone in identification of CIN2+ lesions in women aged ≥ 35 years (I) and might be used as a primary From a practical point of view, persistence can be defined screening test in this age category. First follow-up results as the detection of the same HPV type (or, with a higher from ongoing controlled randomized trials, which com- degree of certainty, the same intratypic variant) two or pare longitudinal performance of Pap test vs. HPV testing more times over a certain period. There is no consensus as in screening programs, confirm the higher efficacy of HPV to the length of time that implies persistence, but at least screening that Pap screening (I). Local assessment is 6 months to 1 year is the time frame that is usually chosen needed before screening policies based on primary HPV [1]. A prospective study of type-specific HPV natural his- testing can be recommended (research need). tory, with a median follow-up of 5.1 years, showed partic- ularly pronounced persistence of HPV-16 and markedly � In women aged < 35 years, HPV testing with immediate increased risk of CIN3+ occurrence when HPV-16 per- referral to colposcopy leads to over diagnosis of CIN2+ as sisted, compared with any other HPV type [96]. Evalua- compared with more conservative management strategies tion of type-specific HPV persistence was used as a (I). Thus, in women younger than 35 years of age with a management strategy for women with a positive HPV test positive HPV test, cytology triage or repeat HPV testing at but normal Pap test in a controlled randomized trial in 12 months could be appropriate management strategies Sweden, as above reported [84]. to avoid over diagnosis and over treatment of CIN2+, but this hypothesis needs confirmation in longitudinal stud- Analysis of high-risk HPV E6/E7 mRNA ies (research need). Cervical cancer is characterized by overexpression of E6/ E7 mRNA of high-risk HPVs. A multiplex nucleic acid Page 10 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 sequence based amplification system (NASBA) assay was ally, a positive relationship between viral load and sever- developed for the identification of E6/E7 mRNA from ity of cervical lesions has been reported [107-109]. HPV types 16, 18, 31, 33, and 45 (PreTect™ HPV Proofer kit, NorChip AS, Norway; now also distributed as Nucli- HPV typing and subtyping SENS EasyQ HPV by bioMérieux SA, Marcy l'Etoile, Women with HPV-16 and HPV-18 infection have a higher France). Analysis of E6/E7 mRNA expression with the Pre- probability to develop cancer than women with infection Tect™ HPV Proofer kit in 204 histologically confirmed by other high-risk HPV types [110-113]. Some studies invasive cervical squamous cell carcinomas from Norwe- demonstrated that detection of HPV-16 and HPV-18 types gian women demonstrated the presence of oncogenic represents a risk factor for CIN2–3 [111,112], but also transcripts of HPV-16, HPV-18, HPV-31, HPV-33, and identification of other high-risk HPV types could be useful HPV-45 in 97% of HPV-positive cases, thus confirming to select at risk patients [114,115]. Investigation of intra- the expression of oncogenic E6/E7 in most invasive can- typic variants of HPV, which are defined by a difference in cers and the involvement of the above HPV types in most the L1 gene sequence less than 2% of the reference HPV cases [97]. A cross-sectional study was performed in 4,136 prototype, have been proven to be useful in epidemiolog- women >30 years of age to compare the performance of ical studies. In multiethnic populations, Asiatic-American HPV E6/E7 mRNA detection with detection of HPV-DNA and other non-European variants of HPV-16 and HPV-18 by Gp5+/6+ consensus PCR [98]. No significant differ- are associated with a higher risk of persistent infection and ence in sensitivity and specificity was observed between development of cervical precancerous lesions and inva- the two tests for detection of HPV in high-grade squamous sive cancer [116-118]. intraepithelial lesion (HSIL). On the contrary, only a small proportion of the HPV DNA-positive women with a Investigation of high-risk HPV E6 e E7 variants also gave normal, ASC-US, or LSIL diagnosis had a detectable E6/E7 interesting results. The T350G sequence variant of the mRNA expression [98]. A comparative study between HPV16 E6 gene is associated with increased viral persist- HPV testing with Gp5+/6+ consensus PCR and high-risk ence and oncogenicity [119,120]. The aminoacid change HPV E6/E7 mRNA testing with the PreTect™ HPV Proofer caused by this mutation could affect E6 degradation activ- kit showed the latest test had equal sensitivity and signifi- ity on p53, besides changing its immunogenicity. Moreo- cantly higher specificity for CIN2+ than PCR [99]. Moreo- ver, the L83V mutation of HPV16 E6 increases activation ver, E6/E7 mRNA-positive women had a significantly of the MAPK pathway and cooperates with Notch 1 in higher risk to have a CIN2+ within 2 years than negative tumorigenesis [121]. HPV-16 sequence variants may also women [99]. Interestingly, quantitative analysis of E6/E7 be associated with different oncogenic potential, even in mRNA in cervical cancers, but not HPV DNA load, corre- the absence of virus integration, due to derepression of the lated with patient survival [100]. The APTIMA HPV Assay, E6 and E7 promoter [122,123]. Thus, typing, subtyping, developed by Gen-Probe Incorporation (San Diego, CA), and sequencing of HPV oncogenes and the upstream reg- employs transcription-mediated amplification and target ulatory region could be useful for epidemiological pur- capture to detect HPV E6/E7 mRNA from 14 high-risk poses but also as prognostic markers. HPV types, but it does not differentiate among the 14 high-risk types. Preliminary results obtained with this Number of HPV types included in HPV tests assay suggest it has higher sensitivity and specificity than Demonstration that some new HPV types, defined as intermediate-risk, such as HPV-26, 53, 66, 73, and 82, HPV testing for identification of CIN2+ lesions [101]. may be associated with the risk of cervical cancer has sug- Measurement of viral load gested to extend the number of HPV types included in As above reported, higher cut-off values improve the spe- HPV DNA tests. To assess the clinical impact of such an cificity of the HC2 test [80-82] and correlate with an extended test, the results from two randomized trials increased risk of HPV persistence and development of which used HPV test for cervical cancer screening and high-grade cervical lesions [78]. A more accurate quantifi- ASC-US triage, respectively, were analyzed [124]. In the cation of viral load can be achieved by using real-time cancer screening protocol, addition of other high-risk PCR to detect specific HPV types. This method allowed HPV types, besides the 12 most common, did not amelio- demonstrating that high HPV-16 DNA load is associated rate test sensitivity. Likewise, when HPV testing was used with CIN3 and invasive cervical carcinoma or with an for ASC-US triage, test sensitivity was the greatest with 17 increased risk to develop CIN2–3 during follow-up genotypes, but specificity was very low [124]. Thus, at the [92,102-106]. Thus, measurement of HPV-16 load by moment, increasing the number of high-risk HPV types, real-time quantitative PCR has been proposed as a marker besides those already present in the FDA-approved HC2 of risk for CIN progression. Contrasting results have been test, does not seem to be advantageous neither for screen- obtained with other high-risk HPV types, although, gener- ing nor for triage of an abnormal Pap smear. On the other hand, reduction of the number of high-risk types included Page 11 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 in the HPV test to those most common in invasive cervical some individuals with documented HPV infection [134]. cancer (e.g., HPV-16, HPV-31, HPV-33, etc.) could On the other hand, high anti-HPV antibody titre is more improve HPV test specificity [125]. common in women with persistent infection than in those who clear infection [134-136]. Moreover, seroposi- Analysis of HPV DNA integration tivity for different high-risk HPV types is associated with Integration of HPV-16 and other high-risk HPVs into the high-grade CIN [137]. Unfortunately, no standardized genome of infected cells is an important step in tumori- and validated HPV serological assays are available genesis, since it promotes expression of E6/E7 oncogenes. [138,139]. Clinical studies demonstrated HPV DNA integration is associated with a higher risk of treatment failure and with Cell-mediated immune response is important for HPV a shorter disease-free interval than cases without integra- clearance, as suggested by the high incidence of persistent tion [126,127]. Thus, assessment of HPV genome integra- HPV infection in AIDS or transplanted patients. Persistent tion, by Southern-blot or quantitative real-time PCR infection and cancer progression is also associated with analysis of E2/E6 ratio, could represent a good prognostic abnormal response of CD4+ T cells [140] and CD8+ T marker. cells [141]. Analysis of p16-INK4A expression Evidence synthesis on the use of new tests for diagnosis of HPV In high-risk HPV infection, E7 binds to and degrades RB infection which results in substantial up regulation of p16-INK4A � Most high-risk HPV infections recover spontaneously synthesis. In spite of the high level of p16-INK4A synthe- within 6–12 months (I); persistent high-risk HPV infec- sis, this protein remains functionally inactive, as E7 tion and high-risk HPV E6/E7 expression are necessary induces cyclin A and cyclin E expression, thereby func- events for cervical cancer development and represent risk tionally bypassing its interference with the cell cycle. Anti- factors for cervical cancer (I). bodies that are directed against p16-INK4A allow selective staining of high-risk HPV-infected histological sections or � HPV typing and subtyping provides epidemiological of cytological smears, but not other cervical epithelia, sug- data on HPV distribution and diffusion (II). gesting that detection of this marker could provide diag- nostic support to distinguish true CIN/dysplasia from � In epidemiological surveys, type-specific HPV detection immature metaplasia or other non-neoplastic changes of could contribute to the evaluation of anti-HPV vaccine the cervix [128-131] and to improve interpretation of his- efficacy (III). tology [132]. Sensitivity and specificity for CIN2+ of p16- INK4A overexpression testing by immunostaining in cer- � High HPV DNA load is a risk factor for cervical cancer vical cell samples from HPV-DNA-positive women were (II). estimated in a nested sub-study of the NTCC trial [133]. This study demonstrated that HPV testing with p16- � HPV testing with p16-INK4A triage improves test specif- INK4A triage improves test specificity for CIN2+. In fact, icity for CIN2+ (II). p16-INK4A triage allows maintaining all the gain in sen- sitivity obtained by HPV testing alone with respect to con- � Detection of anti-HPV antibodies demonstrates previ- ventional cytology, but with referral to colposcopy similar ous HPV exposure, but this test has low sensitivity (II); a to that of conventional cytology [133]. high antibody titre might indicate persistent infection (II). Analysis of immune response against HPV � Patients with invasive cervical carcinoma have a defi- Both antibody-mediated and cell-mediated immune cient HPV-specific cell-mediated immune response (II). responses are essential for clearance of HPV and HPV- related cervical lesions. The immune response against Recommendations on the use of new tests for diagnosis of HPV HPV (and HPV virus-like particles, VLPs) is mainly directed infection against type-specific immunodominant conformational � In order to demonstrate HPV persistence, repeat HPV epitopes, although cross-reactivity has been documented, testing should be done at intervals of at least 12 months such as between HPV-6 and HPV-11, HPV-31 and HPV- (IA). 33, HPV-18 and HPV-45. Serum levels of anti-HPV anti- bodies are stable, even after virus clearance, so HPV sero- � HPV typing and subtyping in women with a normal or logical assays simply demonstrate previous exposure to dubious Pap test is useful since it allows to document per- the virus. Sensitivity of HPV serology is low (50–60%) sistent infection by the same oncogenic HPV type (II) and while specificity is high (about 90%). The low sensitivity to demonstrate the presence of HPV-16 and HPV-18 DNA of the test is in part explained by lack of seroconversion in which are markers of increased risk for cervical cancer (II). Page 12 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Women with persistent high-risk HPV infection or with which is administered i.m. in three doses (time 0, 1, and the presence of HPV-16 and HPV-18 DNA should be 6 months), and a quadrivalent alum adjuvanted L1 VLP vaccine against HPV-16, HPV-18, HPV-6, and HPV-11 referred to colposcopy (B). (produced by Merck & Co., Inc.; distributed in Europe by � Measurement of HPV-16 and other high-risk HPV DNA Sanofi Pasteur MSD as Gardasil and by Merck Sharp & load could be employed for triage of women with dubious Dohme as Silgard ), which is administered i.m. in three Pap test or with a positive HPV test (B), but further doses (time 0, 2, and 6 months). The bivalent vaccine is research is needed to identify appropriate management advised for reduction of precancerous cervical lesions and strategies for women with high HPV load (research need). cancer incidence. The quadrivalent vaccine, besides cervi- cal lesions, is advised for reduction of vulvar precancerous � Detection of high-risk HPV E6/E7 mRNA could be lesions and genital condylomas. employed for triage of women with dubious Pap test or with a positive HPV DNA test (IIB). Due to its high sensi- The USA and several other Countries are targeting 11–12- tivity and specificity (II) HPV E6/E7 mRNA testing should year-old girls for vaccination. In particular, the Italian be investigated as a stand alone test in primary screening HPV vaccination program is universally offered free-of- for cervical cancer (research need). charge to all 12-year old girls, and advised to all adoles- cent women before sexual debut. The choice of a target � HPV typing and subtyping is useful for epidemiological population of young girls is based on the results of immu- investigation of HPV distribution and diffusion (B). nogenicity and safety studies, which were conducted in girls and boys aged 9–15 years (quadrivalent vaccine) and � Detection of p16-INK4A expression could be used as aged 10–14 years (bivalent vaccine). These studies dem- triage of HPV positive cases (B) and abnormal Pap smears onstrated that both vaccines are safe and immunogenic, (B) and to confirm histological analysis of cervical biop- and induce antibody titre which is more than 50 times the sies (B). titres induced by natural infection [145,146,150-156]. � Further research is needed to develop standardized Efficacy of HPV VLP vaccines Evaluation of vaccine efficacy in phase II-III clinical trials methods to measure anti-HPV immune response (research need). was based on the demonstration of reduction of the inci- dence of persistent HPV infection (two positive detections � In epidemiological surveys, evaluation of antibody- at 4–6 months intervals) and on reduction of the inci- mediated and cell-mediated anti-HPV immune response dence of precancerous lesions (CIN2 and CIN3) caused can be used to assess immune response to anti-HPV vacci- by HPV vaccine types. International standards for treat- nation and to monitor duration of vaccine protection (B). ment of CIN2/3 are ablative therapy. Moreover, in pro- spective studies, it is not ethical to allow a woman to Prophylactic HPV virus-like particle vaccines develop invasive disease in order to demonstrate efficacy Two vaccines targeting HPV-16 and HPV-18, the types of prevention strategies. In this regard, WHO and other that are responsible for 70% of invasive cervical cancers international agencies indicated CIN2/3 as clinical end- [13,16], have been recently developed and experimented point surrogate to demonstrate efficacy of cancer preven- for immunogenicity, safety, and efficacy in randomized tion. clinical trials. These vaccines are based on the self-assem- bly of recombinant L1 protein into VLPs, that are non- Randomised clinical trials demonstrated that three doses infectious capsids that contain no genetic material. They vaccination is effective for the prevention of persistent were first developed and evaluated as monovalent vac- infection and for precancerous lesions caused by HPV cines targeting HPV-16 [142,143], thereafter as a bivalent genotypes included in the vaccine. Results on efficacy vaccine targeting HPV-16 and HPV-18 [144-146] and a reported in published studies are mainly two types: (i) per quadrivalent vaccine against low-risk HPV-6 and HPV-11, protocol analysis, which demonstrates the pure efficacy of besides HPV-16 and HPV-18 [147-149]. Intramuscular the vaccine, obtained by analysis, in the experimentation injection of the vaccine induces high titres of neutralising context, of clinical data of women who did not violate the antibody in almost all subjects within a month from com- protocol (woman who received three doses of vaccine or pletion of the vaccination protocol [145,146,148-153]. placebo as expected), who resulted negative for all HPV genotypes included in the vaccine both at enrolment or Two HPV VLP vaccines have been developed and are avail- during the administration of the vaccine. (ii) modified able for primary vaccination in the European Union: an intention to treat analysis, where evaluation of efficacy is AS04 adjuvanted L1 VLP vaccine against HPV-16 and more similar to the clinical setting in the general popula- HPV-18 (Cervarix , produced by GlaxoSmithKline), tion, since it includes all enrolled women, as long as they Page 13 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 receive at least the first dose of vaccine or placebo and vaginal and vulvar intraepithelial neoplasia related to who resulted negative for all HPV genotypes of the vaccine HPV-6/11/16/18 and 100% efficacy (95% CI 94–100%) merely at the time of administration of first vaccine dose. for prevention of CIN1–3 or in situ adenocarcinoma asso- ciated with vaccine HPV genotypes [149]. Modified inten- Efficacy of the quadrivalent HPV VLP vaccine tion-to-treat analysis (also defined unrestricted susceptible A phase II randomised trial was carried out on 552 population) showed 95% efficacy (95% CI 87–99%) women (277 randomised in the vaccine group and 275 in regarding precancerous genital lesions, vulvar or vaginal, the placebo group). Per protocol efficacy analysis demon- or condylomas and 98% efficacy (95% CI 92–100%) for strated that Gardasil reduced by 90% (95% CI, 71–97%) CIN1+ or in situ adenocarcinoma caused by vaccine HPV the incidence of persistent infection and genital lesion types. The intention-to-treat analysis showed a 73% efficacy associated with the HPV genotypes included in the vac- (95% CI, 58–83%) in preventing external anogenital or cine [147]. A subset of subjects participated to a subse- vaginal lesions of any degree and a 55% efficacy (95% CI, quent follow-up of this study. Following five years from 40–66%) in preventing incident and prevalent cervical enrolment, efficacy on combined incidence of persistent lesions of any grade associated with vaccine HPV geno- infection of HPV-6/11/16/18-related disease was 96% types, at 3 years follow up. In both FUTURE I and II stud- (95% CI 83.8–99.5%) [143]. Modified intention to treat ies, vaccination did not appear to significantly alter the analysis demonstrated that vaccination reduced incident course of infection or lesions due to HPV which were infections and cervical lesions by 94% (95% CI, 83– already present before the first dose of vaccine. 98.3%) [152]. - Per-protocol efficacy analysis of the results achieved by the Phase III efficacy studies of the quadrivalent vaccine combination of three randomized clinical trials of vacci- Gardasil were conducted in the context of the two proto- nation (n = 7,811) versus placebo (n = 7,785) showed cols Females United to Unilaterally Reduce Endo/Ectocer- that vaccination, after an average follow-up period of 3 vical Disease (FUTURE) I and FUTURE II: years, has an efficacy of 100% (95% CI 72–100%) in pre- venting vulvar and vaginal lesions grade 2 and 3 associ- - FUTURE II trial. Per protocol analysis in over 10,000 ated with HPV-16 and HPV-18. Modified intention-to-treat women (5,305 randomised in the vaccination group and analysis demonstrated an efficacy of 97% (95% CI 79– 5,260 randomised in the placebo group) aged 15–26 100%) and intention-to-treat analysis, on a total of 18,174 years demonstrated that, a after a mean 3 year follow-up randomised women, showed an efficacy of the vaccine at period, vaccine efficacy was 98% (95% CI, 86–100%) for three years of 71% (95% CI, 37–88) in the prevention of prevention of CIN2,3 and in situ adenocarcimoma associ- incident/prevalent HPV-associated lesions [153]. ated with HPV-16 and HPV-18 [148]. It is to notice that the unique woman, included in the vaccine group, who - Per-protocol efficacy analysis of the results achieved by the developed a HPV-16 or HPV-18 associated lesion, had a combination of four randomized clinical trials of vaccina- positive CIN3 for HPV-52 at the initial control and in all tion with the quadrivalent vaccine (n = 9,087) and its the following 5 samplings during follow-up, only one HPV-16 L1 component (n = 1,204) vs. placebo (n = these samples resulted to be HPV-16 positive. Modified 10,292) showed that, after a follow-up of 3 years, in HPV- intention to treat analysis (definedalso unrestricted suscepti- 16 and HPV-18 negative women, vaccine efficacy was ble population) in randomised women (95% of the study 99% (95% CI 93–100) in preventing development of population), who were negative for the vaccine types at CIN2/3, in situ adenocarcinoma or invasive cervical cancer the first dose administration, and who did not strictly associated with HPV-16 and HPV-18. Efficacy was 98%, adhere to the study protocol, demonstrated vaccine effi- (95% CI 93–100) in the population of modified intention- cacy was 95% (95% CI, 85–99%). Intention to treat analy- to-treat analysis. The modified intention-to-treat analysis dem- sis including all randomised subjects, without any pre- onstrated an impact of the vaccine of 44% (95% CI, 31– vaccine screening, was used to predict efficacy of vaccina- 55) in reducing the incidence of cervical lesions associated tion on incident/prevalent HPV infections and related with HPV-16 and HPV-18, in the case of HPV type vaccine lesions in the general population, who demonstrated an diseases already prevalent/incident in the population impact of 44% (95% CI 26–58%) at 3 years follow-up. [157]. - FUTURE I trial. Per protocol analysis on over 5,000 Efficacy of the AS04 adjuvanted bivalent VLP vaccine women (2,261 randomised in the vaccination group and Efficacy the AS04 adjuvanted bivalent VLP vaccine has 2,279 randomised in the placebo group) aged between 16 been assessed in phase II-III studies. A pilot randomized and 24 years showed that after an average period of 3 years phase II study on safety, immunogenicity, and efficacy of since vaccine administration, the vaccine had 100% effi- the vaccine for prevention of incident and persistent HPV- cacy (95% CI 94–100%) for prevention of condylomas, 16 and HPV-18 infection enrolled and randomized 1,113 Page 14 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 women aged 15–25 years to receive either the vaccine or Cross-protection against oncogenic HPVs placebo [144]. Study participants were initially seronega- Two clinical trials have evaluated the efficacy of Cervarix™ tive for HPV-16 and HPV-18 by ELISA and negative for on other oncogenic types [144-146]. They demonstrated HPV-16 and HPV-18 DNA by PCR and had negative vaccination led to sustained cross-protection against inci- results at cervical cytology. In the per protocol analysis, vac- dent infection with HPV-45 and -31 for the entire follow- cine efficacy was 91.6% (95% CI, 64.5%–98.0%) against up period up to 5.5 years and cross-protection against six- incident infection and 100% against persistent infection month persistent infection due to HPV-45, -31 and -52 (95% CI, 47%–100%) with HPV-16/18 at 18 months [160]. Furthermore, broad protection was observed post-vaccination. In the intention-to-treat analyses, vaccine against 12-month persistent infections (defined as detec- efficacy was 95.1% (95% CI, 63.5%–99.3%) against per- tion of the same HPV type in all available cytology sam- sistent cervical infection with HPV-16/18 and 92.9% ples collected over any 12-month period) with 12 (95% CI, 70%–98.3%) against cytological abnormalities combined oncogenic HPV types, not including HPV-16 associated with HPV-16/18 infection. In a subsequent and -18 (vaccine efficacy 27.1%; 97.9% CI: 0.5–46.8) report from the same study, after a follow-up of 4–5 years, [160]. The impact of Gardasil HPV quadrivalent vaccine per protocol analysis on the results obtained from a group on the rates of infection and disease associated with 10 of 732 women (367 in the vaccination group and 365 in HPV types not included in the vaccine was estimated in a the placebo group) who received all three doses of vac- population of women that was, pre-vaccination, seroneg- cine, demonstrated vaccine efficacy against incident infec- ative and PCR-negative to all HPV types for which testing tion was 96.9% (95% CI, 81.3%–99.9%) and 100% was available and had a normal Pap test [161]. Within (33.6%–100%) against persistent infection for 12 months approximately 3 years of follow-up, combined efficacy for [145]. Efficacy against CIN lesions associated with CIN2/3 or adenocarcinoma in situ caused by all 10 non- HPV16/18 infection was 100% (95% CI, 42.4%–100%), vaccine HPV types was 38% (95% CI:6–60). The final since 8 cases of CIN1/2 occurred in the placebo group and end-of study data with approximately 3.6 years follow-up none in the vaccination group. Interim results at a mean showed similar high vaccine efficacy [161]. follow-up of 14.8 months (SD 4.9) from a large, interna- tional phase III study have been published [149]. In this Side effects of prophylactic HPV VLP vaccines Data on safety of the quadrivalent HPV VLP vaccine trial, 18525 women aged 15–25 years from North Ameri- can, South American, European, Asian, and Australian reported a higher rate of side effects in the vaccination countries were randomly assigned to receive either group than in the placebo group. Side effects included HPV16/18 vaccine (n = 9,258) or hepatitis A vaccine (n = local reaction at the site of injection (erythema, pain, 9,267) at 0, 1, and 6 months. Two cases of CIN2+ associ- edema) (altogether, 86.8% in the vaccine group vs. 77.4% ated with HPV16 or HPV18 DNA were seen in the HPV16/ in the placebo group, respectively) and systemic side 18 vaccine group; 21 were recorded in the control group. effects, such as fever (13.5% vs. 10.2%, respectively) Based on these data, vaccine efficacy against CIN2+ con- [149]. Also data on safety of the bivalent vaccine reported taining HPV16/18 DNA was estimated to be 90.4% a higher rate of injection site symptoms and systemic side (97.9% CI, 53.4–99.3), but, a post hoc analysis demon- effects (fatigue, headache, myalgia) in the vaccine group strated that the HPV16 or HPV18 DNA in the 2 CIN2+ than in the control group [146]. No increased incidence of cases in the vaccination group and in 1 case of the control spontaneous abortion, late fetal deaths, or congenital group were already present at study entry. Efficacy against anomalies was observed in pregnancies occurring in 12-month persistent HPV-16/18 infection was 75.9% women receiving either type of vaccines as compared with (97.9% CI, 47.7–90.2). controls [146,148]. Efficacy of HPV VLP vaccination in HPV-positive women Recommendations on the use of prophylactic HPV vaccines In women positive for HPV DNA, HPV VLP vaccination � HPV vaccination with the quadrivalent vaccine is safe does not accelerate clearance of the virus nor prevents and efficacious for the prevention of incident high-grade incident CIN2+ caused by HPV vaccine types. This finding precancerous lesions, in situ adenocarcinoma, invasive has been demonstrated in a randomized phase III trial in cervical cancer, high-grade vulvar and vaginal lesions, and 2189 women aged 18–25 years, who received the AS04 genital warts correlated with HPV-16, HPV -18, HPV -6, adjuvanted bivalent VLP vaccine [158] and in two rand- and HPV -11 (I). omized trials of the quadrivalent HPV vaccine enrolling 17,622 women without consideration of HPV status � HPV vaccination with the AS04 adjuvanted bivalent vac- [159]. cine is safe and efficacious for the prevention of incident and persistent HPV-16 e HPV-18 infection and CIN1+ cor- related with HPV vaccine types (I) Page 15 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 � Long-term efficacy (> 5 years) of HPV vaccination is cur- � HPV typing and subtyping can be used for epidemiolog- rently under investigation (research need). ical evaluation of HPV distribution and diffusion (B), for identification of persistent infection (B), for identification � Efficacy of HPV vaccination has been demonstrated in of HPV-16 and HPV-18, which are associated with a women aged 15–26 years, who had not been previously higher risk of precancerous lesions (B) or invasive cervical exposed to HPV vaccine types (I); results on efficacy in cancer (B), and for monitoring the efficacy of vaccination women older than 26 years are not available yet (research (A). But, there is insufficient evidence to define the best need). protocol to manage women with positive tests (I). � Results on immunogenicity and safety (but not efficacy) � Detection of high-risk HPV E6/E7 mRNA may be useful justify vaccination in girls aged 9–14 years (II). to identify women at risk for cervical cancer and with worse prognosis (IIB). Further research is needed to define � Efficacy of HPV vaccination in males is currently under the best protocol to manage women with a positive test investigation (research need). and to evaluate test efficacy in primary screening pro- grams. � HPV vaccination in males is not considered to be cost- effective (III). � Measurement of HPV-16 DNA load in women with nor- mal cytology may be useful to identify women at risk for � HPV vaccination should be done before exposure to cervical cancer (IIB). But, there is insufficient evidence to genital HPV, since it lacks efficacy in women who have define the best protocol to manage women with positive already been infected (IA), and it does not enhance virus tests (I). clearance (IA). � In epidemiological surveys, HPV serological testing and � Screening for cervical cancer should be recommended measurement of anti-HPV cellular immune response both in vaccinated and non-vaccinated women according could be useful to monitor the duration of vaccine protec- to current guidelines (II). Screening protocols could be tion (B). modified only after appropriate clinical trials (research need). Who should be vaccinated and how should vaccine efficacy be evaluated? � Further research is needed to assess the duration of vac- � Quadrivalent HPV VLP vaccine is safe and effective in cine protection, variations in the prevalence of HPV types the prevention of cervical and vulvar precancerous in vaccinated women and in the general population, and lesions, in situ adenocarcinoma, invasive cervical cancer surveillance of side-effects of vaccination (research need). and genital warts associated with vaccine HPV types in women aged 15–26 yrs (I). Summary of recommendations and guidelines When should HPV testing be used in cervical-cancer � HPV vaccination with the AS04 adjuvanted bivalent vac- screening programs? cine is safe and efficacious for the prevention of incident � in ASCUS triage (IA). and persistent HPV-16 e HPV-18 infection and CIN1+ cor- related with HPV vaccine types in women aged 15–26 yrs � in the follow-up after treatment of CIN2/3 (IA). (I) � as a screening test for cervical cancer, as an alternative to � HPV vaccine should be administered before genital HPV cytology, in women older than 30 years, with cytology or exposure, since the benefit of vaccination declines in repeat HPV testing as triage tests (IA). women already positive for HPV DNA (IA). Efficacy in cervical cancer screening has been proven for � In epidemiological surveys, HPV serological testing and both PCR-based and signal amplification-based HPV tests HPV genotyping could be useful to monitor vaccine effi- (I). cacy (research need). What is the role of new HPV tests in the diagnosis of HPV � Cervical cancer screening is recommended both in vac- infection? cinated and non-vaccinated women, in agreement with � Evaluation of HPV persistence may be useful to identify current guidelines for cervical cancer prevention (III). patients at risk for cervical cancer, who should be referred for colposcopy (A). � Further research is needed to assess the duration of vac- cine protection, variations in the prevalence of HPV types Page 16 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 9. zur Hausen H, Meinhof W, Scheiber W, Bornkamm GW: Attempts in vaccinated women and in the general population, and to detect virus-specific DNA sequences in human tumors: I. surveillance on side-effects of vaccination (research need). Nucleic acid hybridization with complementary RNA of human wart virus. Int J Cancer 1974, 13:650-656. 10. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Competing interests Classification of papillomaviruses. Virology 2004, 324:17-27. The authors declare that they have no competing interests. 11. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ, International Agency for Research on Can- cer Multicenter Cervical Cancer Study Group: Epidemiologic clas- Authors' contributions sification of human papillomavirus types associated with LB drafted the manuscript. CG, FMB, and GP contributed cervical cancer. N Engl J Med 2003, 348:518-527. 12. Munoz N, Castellsague X, de Gonzalez AB, Gissmann L: HPV in the to the preparation of guidelines. All authors read and etiology of human cancer. Vaccine 2006, 24(Suppl 3):S1-S10. approved the final manuscript. 13. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL: HPV type dis- tribution in women with and without cervical neoplastic dis- eases. Vaccine 2006, 24(Suppl 3):S26-34. Acknowledgements 14. Trottier H, Franco EL: The epidemiology of genital human pap- These guidelines were reviewed by SIV Board Members and approved by illomavirus infection. Vaccine 2006, 24(Suppl 1):S1-S15. the Executive and Council of SIV. We thank the members of SIV who 15. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N: Human papillomavirus reviewed the draft version of this guideline when it was posted on the SIV is a necessary cause of invasive cervical cancer worldwide. J website and who sent a great number of comments, most of which were Pathol 1999, 189:12-19. incorporated into the final version of the manuscript. Moreover, we thank 16. Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, Clifford for their helpful suggestions: K. Soldan, Health Protection Agency Centre GM: Human papillomavirus type distribution in invasive cer- for Infections, London, UK; L. Banks, International Centre for Genetic Engi- vical cancer and high-grade cervical lesions: A meta-analysis update. Int J Cancer 2007, 121:621-632. neering and Biotechnology, Trieste, Italy; R. Kirnbauer, Viral Oncology, 17. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, Pimenta JM: Medical University Vienna, Austria; S. Pecorelli, Department of Obstetrics Human papillomavirus genotype distribution in low-grade and Gynaecology, University of Brescia, Brescia, Italy; M.G. Pompa, Ministry cervical lesions: comparison by geographic region and with of Health, Directorate General of Health Prevention, Communicable Dis- cervical cancer. Cancer Epidemiol Biomarkers Prev 2005, 14:1157-1164. eases Unit, Rome, Italy; F. Carozzi, Centre for Cancer Study and Preven- 18. de Sanjosé S, Diaz M, Castellsagué X, Clifford G, Bruni L, Muñoz N, tion, Florence, Italy; C.F. 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Publisher: Springer Journals
Copyright: Copyright © 2008 by Barzon et al; licensee BioMed Central Ltd.
Subject: Biomedicine; Cancer Research; Infectious Diseases; Oncology
eISSN: 1750-9378
DOI: 10.1186/1750-9378-3-14
pmid: 19087272
Publisher site: See Article on Publisher Site

Abstract

Objective: To provide guidelines for health-care providers on strategies for cervical cancer prevention based on HPV testing and anti-HPV vaccination. Outcomes: Overall efficacy of different preventive strategies, assessing reduction in the incidence of invasive cervical cancer and precancerous lesions. Evidence: Medline and the Cochrane Database were searched for articles in English on subjects related to HPVs, HPV diagnosis, HPV anogenital lesions, cervical cancer, HPV testing, and HPV vaccines, in order to elaborate an up-dated document. Relevant Italian Government publications and position papers from appropriate health and family planning organizations were also reviewed. Values: The quality of the evidence and ranking of recommendations for practice were rated using criteria defined by SIV, which were adapted from the Canadian Task Force on Preventive Health Care. with SIV members, and reviewed and approved by SIV Sci- Introduction This report summarizes recommendations on cervical entific Committee, during the annual SIV Conference of cancer screening and prevention strategies proposed by 2008, following a specific Round table on the subject. experts of the Italian Society for Virology (SIV, Società Ital- These recommendations recognize the diagnostic value of iana di Virologia) on the basis of systematic review of evi- HPV testing and genotyping in the management of dence from the scientific literature and current guidelines women with abnormal PAP test and in screening pro- produced by International Societies, in view of an Euro- grams for prevention of cervical carcinoma and recom- pean consensus strategy for cervical cancer screening, mend HPV vaccination for primary prevention of cervical human papillomavirus (HPV) testing, and anti-HPV vac- cancer. The searching strategies, the study selection criteria cination programs. The guidelines were discussed with and other methodologies adopted for the literature review experts in the field (see acknowledgment section) and can be asked to the correspondig author. Page 1 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 the field; therefore the definition of the oncogenicity of Human papillomavirus and cervical cancer The demonstration that human papillomavirus (HPV) some HPVs could change with time, with obvious impli- infection has a pathogenetic role on cervical cancer devel- cations on the definition of the diagnostic standards for opment and the understanding of the epidemiology of HPV infection, as outlined below. HPV infection and its relationship with the natural history of precancerous lesions and cervical cancer have been cru- High-risk HPV DNA is virtually detectable in all cervical cial in the development of diagnostic and vaccination cancers [15]. Among high-risk or carcinogenic HPVs (cur- strategies for prevention of one of the most common and rently, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, lethal cancers, especially in developing countries. 59) and probably carcinogenic HPVs (HPV types 26, 53, 66, 68, 73, 82) [4,5], HPV-16 is responsible for 60% of Epidemiology of cervical cancer and HPV infection cervical cancer, HPV-18 for 10%, HPV-45 and HPV-31 for Epidemiological data on HPV infection and associated 4% each, whereas HPV-33, HPV-52, and HPV-58 alto- disease have been reviewed in a recently published IARC gether account for 2% of cervical cancer [12,16]. In high- Monograph [1]. Cervical cancer is the second most com- grade squamous intraepithelial lesions (HSIL), the most mon and lethal cancer in women worldwide, with esti- common HPV types identified are similar to those in cer- mated 493,000 new cases and 274,000 deaths in 2002 [2]. vical cancer, except for the under-representation of HPV- More than 80% of cervical cancers and related deaths 18 and HPV-45 [16]. In low-grade squamous intraepithe- occur in developing countries, where cervical cancer is the lial lesions (LSIL), high-risk HPV DNA can be detected in most common cancer, accounting for 15% of female can- 30 to 100% of cases in different studies [17]. Again, HPV- cers, while in developed countries it accounts for only 16 is the most common type, being detected in 26% of 3.6% of new cancers [2]. Cervical cancer has a relatively cases, followed by HPV-31 (12%), HPV-51 (11%), HPV- low incidence in Europe (less than 15 per 100,000), 53 and HPV-56 (10%), and many other HPV types [17]. whereas its incidence is generally over 30 per 100,000 in developing countries [3,4]. The incidence of invasive cer- A recent meta-analysis investigated the worldwide preva- vical cancer has a peak at about 20–25 years after the peak lence and genotype distribution of HPV DNA in cervical age for HPV infection prevalence, and the incidence of cer- samples from women with normal cytology [18]. This vical intraepithelial neoplasia (CIN) peaks in between [1]. meta-analysis estimated the overall HPV prevalence in According to the AIRTum (Associazione Italiana dei Reg- women with normal cytology was 10.4% (95% confi- istri Tumori) database, in Italy, during 1998–2002, cervi- dence interval [CI], 10.2–10.7%). The prevalence was rel- cal cancer represented 1.6% of all newly diagnosed atively high in Africa, Central America and Mexico (about cancers among females, with an average incidence of 9.8 20 to 30%) and lower in northern America, Europe, and cases per 100,000 females per year, and represented 0.6% Asia (about 8 to 11%). In all world regions, HPV preva- of all cancer deaths among females. It has been estimated lence was highest in women younger than 35 years of age, that every year 3,418 new cervical carcinomas are diag- decreasing in women of older age [18]. In Africa, the nosed in Italy; as regards mortality, in 2002 there were Americas, and Europe, a second peak of HPV prevalence 370 deaths due to cervical cancer, and 1,756 deaths due to was observed in women of older age [18]. Overall, the cancer of the uterus not otherwise specified [5]. Survival most common HPV types were HPV-16, HPV-18, HPV-31, rates in patients with cervical cancer vary in different HPV-58, and HPV-52, while the most common types in countries, ranging from 73% at 5 years in US [6] and 63% southern Europe were HPV-16, HPV-66, HPV-45, HPV- in Europe [7] to 30.5% in sub-Saharan Africa [8]. In Italy, 31, and HPV-42 [18]. at 5 years from diagnosis of cervical cancer, survival rates range between 78% in Ferrara and 40% in Naples, with an In the Italian population, the prevalence of high-risk HPV average national survival rate of 65% [5]. in women aged 25–70 years has been reported to be about 9%, and HPV-16 was the most common genotype, being Epidemiological and experimental evidence demonstrate detected in 30–60% of HPV-positive women, followed by that persistent infection with a carcinogenic HPV geno- HPV-66, HPV-45, HPV-31, and HPV-53 [19,20]. HPV gen- type is a necessary cause of cervical cancer and its precan- otyping by restriction fragment length polymorphism cerous lesions, as suggested by zur Hausen in '70 [9]. Over analysis and direct sequencing showed that, in HSIL, 120 different HPV genotypes have been identified so far HPV-16 and HPV-31 were the most commonly detected and at least 40 of these HPV types infect the anogenital types, followed by HPV-67, HP-87, HPV-61, and HPV-58, mucosa [10]. Genital HPVs are designated as high-risk or while HPV-16, HPV-31, HPV-66, HPV-53, HPV-52, and low-risk types, based on their association with cervical can- HPV-56 were the most frequent types in LSIL [21]. By con- cer [11-14]. It should be recognized that the distinction trast, low-risk HPV-6 and HPV-11 were responsible for between high-risk and low-risk HPV genotypes is contin- about 90% of genital warts [22], whereas the other low- uously under revision on the basis of new knowledge in risk HPV types were less frequently associated with ano- Page 2 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 genital lesions. Data at recruitment from the New Tech- sion [33]. Following entry into the suprabasal layers, nologies for Cervical Cancer Screening (NTCC) expression of the late viral genes E4, L1, and L2 is initi- randomized trial, which is ongoing in Italy and includes ated; the circular viral genome is then replicated and struc- women attending routine cervical screening programs, tural proteins form. In the upper layers of the epidermis or showed the rates of high-risk HPV positivity were 13.1% mucosa, complete viral particles are assembled and for women aged 25–34 and 5.8% for women aged 35–60, released [33]. The HPV E1 and E2 proteins are involved in without a second peak of HPV prevalence [23]. In younger replication and segregation of episomal viral genome women aged 18 to 24, the prevalence of high-risk HPV [34]; the E2 protein also acts as a transcriptional activator DNA detection is 17.4% and HPV-16 was the most fre- of viral promoters through its ability to bind to specific quent type, followed by HPV-53, HPV-84, HPV-42, HPV- sequences within the regulatory sequences of HPV 62, HPV-66, and HPV-89 [24]. A higher prevalence has genome, known as long control region (LCR) [35]. How- been reported in a Danish population-based study [25], ever, in some cases, E2 may also inhibit viral gene tran- which showed that about 40% and 45% of women aged scription: in genital tract-associated high-risk HPV, E2 15–19 and 20–24 years, respectively, were infected with protein has been demonstrated to suppress E6 and E7 high-risk HPV types. HPV-16 was the most common HPV expression probably through steric interference with the type, followed by HPV-31, HPV-52, and HPV-51 [25]. binding of positively acting cellular transcription factors Like in the Italian population, the prevalence of infection in the LCR [36]. HPV E4 function is probably to aid the markedly decreased with age, without significant second virus in its egress from the cell [37]. E5 seems to be impor- prevalence peak [25]. The EDITH (Etude de la Distribu- tant in the early course of infection. It probably stimulates tion des Types d'HPV en France) study investigated the cell growth by forming a complex with the epidermal prevalence and distribution of HPV types in France. The growth factor receptor or a subunit of the vacuolar results of this study showed that HPV DNA could be ATPase, thus inhibiting the acidification of endosomes detected in 98% of LSIL smears, in 98% of high-grade cer- and growth factor receptor recycling [38,39]. However, vical intraepithelial neoplasia (CIN2/3), and in 97% of since the E5 gene is not expressed in most HPV-positive invasive cancers [26-28]. In LSIL, the most prevalent gen- cancers, E5 does not seem to be obligatory in late events otypes were HPV-66, HPV-16, HPV-53, HPV-51 and HPV- of HPV-mediated carcinogenesis [33]. A more significant 52 [26]; in CIN2/3, HPV-16 was by far the most prevalent role for malignant transformation can be assigned to the genotype (62%), followed by HPV-31, HPV-33, HPV-52, E6 and E7 genes and their respective proteins. They are HPV-51, and HPV-58 [27], while the most prevalent gen- consistently expressed in malignant tissue, and inhibiting otypes in invasive cervical cancer were HPV-16 (73%) and their expression blocks the malignant phenotype of cervi- HPV-18 (19%), followed by HPV-31 (7%), HPV-33, HPV- cal cancer cells [40]. They are independently able to 68, HPV-45, HPV-52, and HPV-58 (4.1-2.3%) [28]. While immortalize various human cell types in tissue culture, HPV-16 was the most prevalent type in both squamous and show synergistic activity when they are expressed cell carcinoma (74%) and adenocarcinoma (64%), HPV- together. Interestingly, in high risk HPVs, E6 and E7 pro- 18 was more prevalent in adenocarcinoma (37%) com- tein are translated together from a polycistronic mRNA pared to squamous cell carcinoma (16%) [28]. [33]. Besides cervical cancer, HPV infection is associated with The E7 protein encoded by high-risk HPV is a small mul- over 70% of cases of anal carcinoma and squamous tifunctional nuclear protein that binds zinc, is phosphor- oropharyngeal carcinoma, with 50% of vulvar, vaginal, ylated by casein kinase II, and shares structural similarities and penile cancer, and, in immunocompromised with oncoproteins of other DNA tumor viruses [41]. The patients, with about 90% of cutaneous squamous cell can- most significant cellular targets of E7 are the retinoblast- cer [12,29,30]. In Italy, high-risk HPV DNA, mainly HPV- oma (Rb) tumor suppressor protein and its functionally 16, was detected in 46% of cases of penile carcinoma [31]. related proteins, p107 and p130, especially in their hypo- phosphorylated active forms, which are able to inhibit cell Pathogenesis cycle progression by binding and repressing the activity of Several basic experimental studies have clarified the E2F cellular transcription factors [42]. Binding with high- mechanisms of HPV oncogenesis [29,32]. In its life cycle, risk E7 results in pRb proteasomal degradation, but this HPV requires infection of epidermal or mucosal epithelial event is not sufficient to account for E7 transforming func- cells of the basal layer, that are still able to proliferate, tions [43]. Indeed, E7 has additional cellular targets, such kip1 most probably through wounds or abrasions. In basal as the cyclin-dependent kinase inhibitors p27 and cip1 layer cells, viral gene expression is largely suppressed, p21 , which are inactivated [44-46], and causes although the limited expression of the early viral genes E1, genomic instability leading to chromosomal abnormali- E2, E5, and especially E6 and E7, results in enhanced pro- ties and aneuoploidy [47]. liferation of the infected cells and in their lateral expan- Page 3 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 The first transforming activity identified for the high-risk who have had 3 normal test results in a row may get HPV E6 proteins was the ability to mediate p53 degrada- screened every 2 to 3 years with cervical cytology alone, or tion through the ubiquitin proteolysis pathway [48], a every 3 years with cytology test plus high-risk HPV DNA property not possessed by the low-risk HPV E6 proteins. testing (HPV test). Women aged ≥ 70 years who have had E6 first associates with the cellular ubiquitin ligase protein 3 or more normal Pap tests and no abnormal Pap tests in termed E6-associated protein (E6AP), then the E6-E6AP the last 10 years and women who have had total hysterec- complex binds to p53, stimulating its ubiquitination and tomy may choose to stop cervical cancer screening. Special degradation [49]. In targeting p53, the high-risk HPV E6 recommendations are given to at-risk categories (i.e., his- proteins inhibit DNA damage and oncogene-mediated tory of cervical cancer, in utero exposure to diethyl- cell death signals. E6 from high-risk HPV also interacts stilbestrol, immunosuppression). Similar guidelines have with several other cellular proteins involved in DNA rep- been proposed by the American College of Obstetricians lication, signal transduction (NFX1), activation of telom- and Gynecologists in 2003 http://www.acog.org, by the erase (Myc), cell proliferation (PDZ motif-containing U.S. Preventive Services Task Force in 2003 http:// proteins), and inhibition of apoptosis (Bak, CBP/p300) www.preventiveservices.ahrq.gov, and, more recently, in [33]. 2006, by the American Society for Colposcopy and Cervi- cal Pathology [55,56]. Invasive cervical cancers and precancerous lesions associ- ated with high-risk HPV infection are characterized by In European Union member states, screening programs integration of HPV DNA in cellular chromosomes. Viral for cervical cancer prevention are organized according to DNA integration is associated with deletion of large seg- the European Council [57] and the European Guidelines ments of viral genome, but with the presence of intact E6 [58], which recommend to set up cancer screening as a and E7, and with transcription of sequence downstream population-based public health programme, with identi- from the integrated LCR [50]. Disruption of the viral E1 fication and personal invitation of each woman in the eli- and E2 genes, as well as of downstream viral sequences, gible target population, and to discourage opportunistic may permit higher levels of E6 and E7 transcription, screening. According to these guidelines, cervical cytology whose RNA are stabilized following fusion with down- is the currently recommended standard test for cervical stream cellular sequences [51]. Persistent expression of E6 cancer screening, which should start in the age range 20– and E7 appears to be necessary to maintain the malignant 30 years. Screening is recommended to be continued at 3– phenotype and to favour the occurrence of cellular genetic 5-year intervals until the age of 60–65. In older women, and epigenetic events, which are important for cancer pro- who have had three or more consecutive previous recent gression [52]. normal cytology results, stopping screening is considered appropriate, while special attention has to be paid to those who have never attended screening. While waiting Screening for cervical cancer and diagnosis of HPV infection for the results of ongoing longitudinal trials before recom- Screening programs for cervical cancer prevention derive mending HPV screening, HPV DNA testing is proposed in: from knowledge on the natural history of cervical cancer 1) primary screening for oncogenic HPV types alone or in and its precancerous lesions and the role of HPV infection combination with cytology; 2) triage of women with in cancer development. These programs, which vary equivocal cytological results; 3) follow-up of women widely by country, include screening tests for early detec- treated for CIN to predict success or failure of treatment. tion of cancer, as well as management strategies for treat- Referral for colposcopy is recommended for women with ment and post-treatment follow-up. Several international a high-grade cytological lesion (ASC-H and HSIL or and national scientific societies have drawn guidelines on higher), a repeated low-grade lesion, or with an equivocal cervical cancer screening and prevention, which are sum- cytology result and a positive HPV test. Management marized in Table 1. All these guidelines emphasize the options in case of atypical squamous cells of undeter- utility to incorporate HPV testing as an adjunct to cervical mined significance (ASC-US) include reflex HPV DNA cytology in cervical cancer screening programs in women testing (preferred option when HPV testing is available), aged ≥ 30 years and for management of women with an repeating the Pap smear after 6 to 12 months, or referral abnormal Pap test. for colposcopy and cervical biopsy (when poor follow-up compliance is suspected or when explicit risk factors are Guidelines of the American Cancer Society [53,54] recom- present). Repeat cytology or referral for colposcopy (pre- mend that screening should begin approximately 3 years ferred option) are considered acceptable options for ini- after a woman starts having vaginal intercourse, but no tial management of LSIL, while HPV testing is considered later than age 21 years. Screening should be done every not sufficiently selective. The guidelines of the Italian year with conventional Pap tests or every 2 years using liq- Ministry of Health for the cervical cancer screening suggest uid-based cytology testing. At or after age 30 years, women performing a Pap test every three years in women aged Page 4 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Table 1: European and US guidelines on cervical cancer screening and prevention. European ACS (American ACOG (American ASCCP (American US Preventive guidelines for Cancer Society); College of Society for Service Task Force; quality assurance in 2007 [53,54] Obstetricians & Colposcopy & 2003 http:// cervical cancer Gynecologists); Cervical www.preventiveservic screening; 2007 [58] 2003 http:// Pathology); 2006 es.ahrq.gov www.acog.org [55,56] Initiation of screening 20–30 yr about 3 yr after the about 3 yr after the NR within 3 yr of onset of with Pap cytology onset of sexual onset of sexual sexual activity or age activity but no later activity but no later 21 yr, whichever than age 21 yr than age 21 yr comes first Use of HPV testing in Not recommended With cytology in With cytology in Insufficient evidence screening programs while waiting for the women ≥ 30 yr women ≥ 30 yr results of randomized controlled trials. Screening intervals - conventional Pap 3–5 yr Annually; every 2–3 yr Annually; every 2–3 yr NR At least every 3 yr test for women aged ≥ 30 for women aged ≥ 30 yr with 3 consecutive yr with 3 consecutive negative cytology negative cytology tests. tests. - if HPV testing used NR Every 3 yr if HPV Every 3 yr if HPV NR Insufficient evidence negative and cytology negative and cytology negative. negative. Discontinuation of 60–65 yr with ≥ 3 Women aged ≥ 70 yr Inconclusive evidence NR Women aged ≥ 65 yr screening recent consecutive with ≥ 3 recent to establish upper age with negative tests, negative tests. consecutive negative limit. who are not tests and no abnormal otherwise at high risk tests in prior 10 yr. for cervical cancer. Management of ASC-US: reflex HPV NR NR ASC-US: HPV testing, NR abnormal cervical testing; or repeat cytology, or cancer screening test LSIL: repeat cytology colposcopy in women - ASC-US or colposcopy; ≥ 20 yr; - ASC-H ASC-H: colposcopy; ASC-H: colposcopy - LSIL HSIL: colposcopy and LSIL: colposcopy - HSIL biopsy. HSIL: immediate loop electrosurgical excision or colposcopy with endocervical assessment. NR: not reported. 25–64, as first level of screening, followed by specific rec- The efficacy of conventional cytological screening for cer- ommendations for women with abnormal cytology [59]. vical cancer has never been investigated in randomised In particular, referral for colposcopy is recommended in clinical trials, but evidence of its effectiveness derives from the case of ASC-H and HSIL or higher at Pap test, while the observational studies. Diagnostic performance of cervical following management options are acceptable in the case cytology has been evaluated by several studies in the liter- of ASC-US: triage with reflex HPV DNA test (and referral ature, whose results have been reviewed in recent meta- to colposcopy if HPV DNA test is positive), immediate analyses. These studies have demonstrated that a single referral to colposcopy, or repeat Pap cytology after 6 cytology test has a sensitivity of about 50% for identifying months. Referral to colposcopy is the preferred manage- women with high-grade precancerous lesions or invasive ment strategy for LSIL, although triage with HPV testing is cervical carcinoma (i.e., cervical intraepithelial neoplasia also acceptable [59]. type 2 or higher, CIN2+) [60-63]. In particular, a recent meta-analysis on 8 cervical cancer screening studies done Cytology testing in North American and European countries with estab- Screening programs based on cytology testing led to the lished cytology-based screening activities, including over reduction of the incidence of cervical cancer and cancer- 60,000 women who were tested for both HPV DNA and related mortality of about 70–80% in many industrialized cytology, found that the overall relative sensitivity of countries [1]. cytology for CIN2+ was 53.0% (95% CI, 48.6%–57.4%), Page 5 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 by using ASC-US as cut-off for a positive result [62]. More- PCR system, the Roche Amplicor system, the PGMY over, sensitivity of cytology varied considerably among primer set, and the SPF primer set, to amplify a broad studies, ranging from 18.6% to 76.7% [62]. In fact, cytol- spectrum of HPV types. These primers target the L1 gene ogy testing has been shown to have poor inter-laboratory in a conserved region amongst HPVs. Subsequent to and inter-operator reproducibility [64]. The meta-analysis amplification of HPV DNA, detection of high-risk HPV also showed the sensitivity of cytology testing increases types is performed by hybridization with probes specific with patient's age and is significantly higher in women for high-risk HPVs. HPV genotyping is performed by type- over the age of 50 than in younger women (79.3% vs. specific PCR or by amplification of HPV DNA by using 59.6%, respectively). The overall specificity of cytology for consensus or general primers, followed by sequencing, CIN2+ is very high (96.3%; 95% CI, 96.1–96.5%), and restriction fragment length polymorphism analysis, or this is an important requisite for a screening test, and is reverse hybridization of the amplicon to multiple oligo- even higher in women aged ≥ 35 years than in younger nucleotide probes corresponding to high-risk and low- women (97.1% vs. 95.9%, respectively) [62]. More recent risk HPVs. Reverse hybridization assays, such as the linear meta-analyses involving more European/North American array by Roche Diagnostics, the line probe assay by Inno- studies yielded a pooled sensitivity of cytology at cut-off genetics NV (Belgium), microchips by Biomedlab Co. ASCUS+ for finding CIN2+ of 70% (95% CI: 60–83%). (Korea) and by other companies, Multiplexed Luminex After omission of three German studies, which reported Assay (Multimetrix, Germany), etc., are able to identify very low sensitivity data, the pooled sensitivity increased co-infections by different HPV types, at variance with to 78% (95% CI: 69–87%) [63,65]. sequencing-based assays, which have high accuracy in the definition of HPV types but poor sensitivity in co-infec- Liquid-based cytology has been more recently introduced tions. and is now widely used for primary screening of cervical cancer. It has the advantage of allowing automation, faster Applications of HPV testing in cervical cancer reading times, and to be used for adjunctive testing, screening programs including HPV testing. It has been suggested to be more Using HPV DNA test for triage of abnormal Pap test sensitive than conventional cytology, but this feature has HPV testing has been introduced in programs for cervical cancer prevention after numerous studies had demon- been rebutted by recent randomized studies, which com- pared the accuracy of liquid-based cytology with conven- strated that detection of high-risk HPVs has a higher sen- tional cytology for primary screening of cervical sitivity than cytology testing in predicting high-grade carcinoma [66,67]. In these studies, liquid-based cytology cervical precancerous lesions or invasive cervical carci- showed no significant difference in sensitivity to conven- noma. A meta-analysis on the use of HPV testing to man- tional cytology for detection of CIN2+, besides a lower age women with ASC-US showed that HPV testing has a positive predictive value (PPV) due to a higher number of greater accuracy than repeat cervical cytology [61]. In fact, positive results. It showed however the advantage of a the overall sensitivity and specificity of HPV testing were reduction of unsatisfactory smears [67]. 84.4% and 72.9%, respectively, vs. 57.6% and 81.8% of Pap-test, by using ASC-US as cut-off for a positive result; HPV test by using LSIL as cut-off, Pap test sensitivity was reduced to Currently used molecular tests for the detection of nucleic 45.7%, whereas specificity increased to 89.1% [61]. acids of high-risk HPV in clinical specimens are based on Among the studies included in the meta-analysis, there signal-amplification and hybridization methods, such as was the large ALTS (ASCUS Low SIL Triage Study), a con- the Hybrid Capture II assay (HC2, Diagene Corp., Gaiths- trolled clinical trial, which enrolled 3,488 women, rand- burg, Maryland, USA), or on target amplification, like the omized into three intervention groups: immediate polymerase chain reaction (PCR)-based Amplicor HPV colposcopy, repeated liquid-based cytology, or HPV test- test (Roche Molecular Diagnostics, Switzerland) or other ing. This trial, as confirmed also by other similar studies, PCR-based assays. The HC2 test, to date, is the only com- demonstrated that HPV testing for ASC-US triage had a mercially available HPV DNA detection test that is significantly higher sensitivity and similar specificity for approved by the FDA for cervical cancer screening in com- CIN3+ than repeat cytology [68,69]. Based on these bination with cytology after the age of 30 years. The HC2 results, both the American Society for Colposcopy and test is a nucleic acid hybridization assay with signal ampli- Cervical Pathology (ASCCP) [55,56] and the European fication for the qualitative detection of HPV DNA of 13 Guidelines [58] currently recommend HPV testing using high-risk types in cervical specimens. The HC2 assay can- validated HPV assays as an acceptable method for manag- not identify the specific HPV type, since detection is per- ing women over the age of 20 years with ASC-US. Accord- formed with a combined probe mix, and provides a semi- ing to ASCCP guidelines, women with ASC-US who are quantitative estimate of HPV DNA load. PCR-based assays HPV DNA negative can be followed up with repeat cyto- use consensus or general primers, such as the GP5+/6+ logical testing at 12 months, whereas those who are HPV Page 6 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 DNA positive should be referred for colposcopic evalua- HPV testing at 6–12 months for post treatment manage- tion. If CIN is not identified, repeat HPV testing at 12 ment of CIN2,3 is recommended by current guidelines of months is recommended. However, women with ASC-US the American Society for Colposcopy and Cervical Pathol- who are HPV DNA negative have a very low probability of ogy [56], whereas European Guidelines, while waiting for having CIN2+, lower than the general population with further data from clinical trials, recommend double test- negative cytology and unknown HPV status, who is actu- ing with cytology and an HPV test at 6 months post treat- ally referred to three years. So, a more conservative man- ment [58]. agement strategy than ASCCP recommendations could be appropriate. Using HPV DNA test for cervical cancer screening The potential role of HPV testing as a stand-alone test or HPV testing has not been demonstrated to be a useful in conjunction with cytology in cervical cancer screening management option for women with a cytological result has been investigated in several studies [reviewed in of LSIL. In fact, a meta-analysis of published studies dem- [62,65,77]]. Unfortunately, these studies were performed onstrated HPV triage of LSIL had no significantly higher with different techniques and sometimes gave conflicting sensitivity but lower specificity than repeat cytology in the results. detection of high-grade CIN [70]. In particular, the ALTS study found that over 80% of women with LSIL were A meta-analysis including 25 non-randomized clinical tri- HPV-positive, whereas the prevalence of CIN2+ was con- als, which compared the performance of cytology vs. HPV siderably lower [71]. Nevertheless, reflex HPV testing may test in screening programs for prediction of CIN2+ [63], be cost effective in older women with LSIL, due to consid- showed the overall sensitivity of HPV test was 90%, when erably lower prevalence of HPV infection. In this regard, the HC2 assay was used, and 80.9%, when PCR with con- data from the randomized Italian study New Technolo- sensus primers was employed; overall specificity was gies for Cervical Cancer Screening (NTCC), which com- higher in PCR-based tests than in HC2, being 94.7% and pared accuracy of cytology testing vs. HPV DNA testing, 86.5%, respectively. The accuracy of cytology testing was specificity of HPV testing for triage of LSIL was higher in lower than HPV testing: in fact, with ASC-US as a cut-off, women aged over 35 years than in younger women, since sensitivity was 72.7% and specificity 91.9%. In women the proportion of HPV positive women was much lower aged over 30 yr, the overall sensitivity of the HC2 assay in women aged 35–60 years than in those aged 25–34 was 94.8% and specificity 86% [63]. years (42% vs. 72%) [72]. So, HPV triage could be appro- priate also for LSIL in women over 35 years, in order to Likewise, the meta-analysis by Cuzick et al. [62], which reduce colposcopy burden and to increase the positive included only studies from European and North Ameri- predictive value of the colposcopy referral. can countries, where screening programs are well imple- mented, showed the overall sensitivity and specificity of Using HPV DNA test for follow-up after treatment of HPV testing were 96.1% and 90.7%, respectively, whereas cervical dysplasia the overall sensitivity and specificity of cytology testing Several studies demonstrated that HPV testing, performed were 53.0% and 96.3%, respectively. Moreover, at vari- at 4–6 months intervals following ablative therapy for ance with cytology testing, sensitivity of the HPV test did high-grade lesions (CIN2–3), has a higher sensitivity and not vary among the different Centres and was very high specificity than cytology in detecting residual disease or both in young women and in those over 50 years of age. recurrence [73-75]. In a meta-analysis of 11 studies evalu- Among the studies included in this meta-analysis, there ating HPV DNA testing in monitoring women after treat- was the HART (HPV in Addition to Routine Testing) trial, ment of CIN3, the negative predictive value of HPV testing a multicentric screening protocol which enrolled over for recurrent/residual disease was 98%, whereas the nega- 10,000 women aged 30–60 years attending for routine tive predictive value of cytology was 93% [76]. Overall, screening. In this study, women with borderline cytology the sensitivity of HPV testing for identifying recurrent/per- or negative cytology but positive HPV test were rand- sistent CIN reaches 90% by 6 months after treatment and omized to immediate colposcopy or follow-up with remains high for at least 24 months, at variance with cyto- repeat HPV test, cytology testing, or colposcopy at 12 logical follow-up, which has a sensitivity of approxi- months [78]. The HART study confirmed HPV testing is mately 70%. These findings were confirmed in an updated more sensitive than borderline or worse cytology (97.1%, meta-analysis, which showed HPV testing has a signifi- 95% CI 91.2–99.1%, vs. 76.6%, 95% CI 65.1–85.1%) but cantly higher sensitivity (ratio: 1.27; 95% CI: 1.06–1.51) less specific (93.3%, 95% CI 92.7–93.9%, vs. 95.8%, 95% and not-significantly lower specificity (ratio: 0.94; 95% CI 95.4–96.2%) for detecting CIN2+ [78]. Comparison of CI: 0.87–1.01) than follow-up cytology in the prediction management strategies for borderline cytology and for of residual/recurrent CIN [65]. positive HPV test with negative cytology results at baseline showed that surveillance at 12 months was as effective as Page 7 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 immediate colposcopy [78]. Interestingly, no CIN2+ were more restricted referral criteria (i.e., cut-off of 2 pg/mL and detected in women with a positive HPV test at baseline positive results at repeat HPV testing) was predicted to but negative at follow-up nor in those with an initial neg- increase PPV to that obtained with conventional cytology ative HPV test and borderline or mild cytology [78]. Based [81]. on these results, it was suggested that HPV test could be used as a screening test in women over 30 years of age Results from the second recruitment phase of the NTCC with cytology triage of HPV-positive results. In case of nor- study showed that, in women aged 35–60 years, HPV test- mal or borderline cytology, repeat HPV testing after 12 ing alone with a cut-off of 2 pg/mL achieved a substantial months was indicated [78]. gain in sensitivity over conventional cytology with only a small reduction in PPV (relative sensitivity, 1.81; 95% CI, Randomized longitudinal studies [79], which have been 1.20–2.72; relative PPV, 0.99; 95% CI, 0.67–1.46) [23], conducted more recently, were not included in the meta- confirming the results of the first phase of the study [80]. analyses. One of these studies is the NTCC study, an ongo- In women aged 25–34 years, direct referral to colposcopy ing clinical trial in Italy nested in the routine activity of after a positive HPV test led to detection of a significantly organized screening programmes, which enrolled approx- greater number of CIN2+ and CIN3+ lesions than in the imately 95000 women, who were randomly assigned to first phase of the study [23]. The increased sensitivity of conventional cytology or to an experimental arm that fol- HPV testing for CIN2+ in the second phase of the study as lowed two phases depending on the period of recruitment compared with the first was suggested to be related to a [23]. During the phase 1 of recruitment, women were ran- high rate of high-grade lesions in young women, which domly assigned to the conventional arm for screening by spontaneously regress [23]. So, in order to avoid over- conventional cytology or to the experimental arm, which treatment, it was suggested that HPV-positive women used both HPV DNA testing with HC2 and liquid-based aged 25–34 years should be referred to colposcopy only if cytology. HPV-positive women were managed differently cytology is also abnormal or if infection persists after 1 according to age. Among women aged 35–60 years, those year [23]. In older women, high-grade lesions are less who had a cytological abnormality or were HPV-positive likely to regress [78,80], therefore a more aggressive man- were referred to colposcopy. Among women aged 25–34 agement strategy for HPV-positive results appears to be years, all women with abnormal cytology were referred for justified. On the other hand, referral to colposcopy of colposcopy but those who were HPV-positive with nor- women with abnormal cytological findings (ASC-US+) mal cytology were retested after 1 year and referred for col- but a negative HPV test may led to a high-rate of false pos- poscopy only if HPV positivity persisted or if cytology itive CIN2+ results and, as a consequence, to overtreat- became ASC-US+. During the phase 2 of recruitment, ment [82]. women were randomly assigned to conventional cytology with referral for colposcopy if cytology indicated ASC-US+ Persistence of the additional lesions detected at baseline or to HPV DNA testing alone with referral for colposcopy by HPV testing in older women was also suggested by the if the test was positive [23]. Results at recruitment showed recently published results from two longitudinal rand- that the screening method in the experimental arm was omized controlled trials, that included only women at more sensitive than conventional cytology in women least 30 [83] or 32 [84] years of age. One of the trials is the aged 35–60 years [80]. In particular, referral based on Population Based Screening Study Amsterdam (POBAS- HPV testing with HC2 at a high cut-off value of 2 pg/mL CAM), which aims to assess whether primary HPV DNA showed a higher sensitivity than conventional cytology testing is more effective than cytological testing in the set- for detection of CIN2+ (relative sensitivity, 1.41%; 95% ting of a regular screening programme. This study enrolled CI, 0.98–2.01), but a lower positive predictive value (PPV; women aged 30–56 years and attending the regular cervi- relative PPV, 0.75; 95% CI, 0.45–1.27). The use of the 2 cal cancer screening programme in the Netherlands. pg/mL cut-off led to a higher PPV than the 1 pg/mL cut-off Women were randomly assigned to the intervention [80]. Adding liquid-based cytology improved sensitivity group (screening with HPV DNA testing combined with only marginally but increased false positives [80]. In Pap test) or control group (Pap test only, HPV DNA test women younger than 35 years, HPV testing alone as pri- results blinded). After 5 years, combined cytological and mary test, with triage of HPV-positive women by cytology, HPV DNA testing were done in both groups and follow- appeared to be the best approach, since the use of liquid- up data for ≥ 6.5 years were available for 17,155 women. based cytology did not increased the sensitivity for CIN2+ Management strategies were more conservative than in detection and had a significantly lower PPV as compared the NTCC study. In fact, women with HSIL or worse were with conventional cytology. Moreover, addition of liquid- immediately referred to colposcopy, irrespective of the based cytology as a primary screening test to HPV testing HPV result, whereas repeat testing after 6 and 18 months had a negligible effect on sensitivity but strongly reduced was advised to women with normal cytology but positive PPV compared with HPV testing only [81]. The use of HPV test and to those with ASC-US, ASC-H, or LSIL. Page 8 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Women with repeat positive cytology and HPV test results cohort study which enrolled over 20,000 women in Port- were referred to colposcopy [83]. In this study, detection land who underwent simultaneous screening with a Pap of HPV DNA was done by GP5+/6+ PCR followed by test and HPV testing with HC2, the 5-year cumulative risk enzyme immunoassay detection of 14 high-risk HPV of CIN3, was 4.4% for women who were HPV-positive at types with a cocktail of oligonucleotide probes. The other baseline, but only 0.24% among women with a negative trial was conducted in Sweden and enrolled 12,527 HPV test and 0.16% when both the HPV test and Pap women aged 32 to 38 years, who, like in the POBASCAM smear were negative [85]. study, were randomized to have an HPV test plus Pap test or a Pap test alone [84]. In this study, HPV test was based A direct comparison between Pap test and HPV test (by on GP5+/6+ PCR followed by reverse dot blot hybridiza- using the HC2 assay) as stand alone tests for cervical can- tion to identify 14 high-risk HPVs. Women with a positive cer screening was evaluated in a recent randomized trial in HPV test and a normal Pap test were offered a second HPV Canada, involving 10,154 women aged 30–69 years [86]. test at least 1 year later, and those who were found to be Both tests were performed on all women in a randomly infected with the same high-risk HPV type were offered assigned order but, to the aim of the study, only one first colposcopy with cervical biopsy. To avoid ascertainment test was considered for statistical evaluation. Women with bias, a similar number of Pap smears and colposcopy with abnormal Pap test results or a positive HPV test at 1 pg/mL biopsies were performed in randomly selected women in cut-off underwent colposcopy and biopsy, as a random the control group. All women were followed for a mean of sample of women with negative tests. Results at baseline 4.1 years with annual Pap smears and HPV tests, with col- testing showed sensitivity of HPV testing for CIN2 or poscopy in cases of persistent high-risk HPV infection CIN3 was 94.6% (95% CI, 84.2–100%), significantly [84]. Both studies showed that a significantly higher higher than sensitivity of Pap test, which was 55.4% (95% number of CIN2+ or CIN3+ lesions were detected at base- CI, 33.6–77.2%). HPV test specificity was 94.1% (95% CI, line in the intervention group than in the control group, 93.4–94.8%) and 96.8% for Pap testing (95% CI, 96.3– whereas the number of lesions detected in the subsequent 97.3%) [86]. follow-up visit, when both HPV test and cytology were done, was significantly lower in the intervention group Recommendations on the use of HPV testing in cervical than in the control group [83,84], thus indicating that cancer screening programs (grading of recommenda- implementation of HPV DNA testing in cervical cancer tions is reported in Table 2) screening leads to earlier detection of high-grade lesions, thus allowing longer screening intervals. In the Dutch � HPV testing is recommended for ASC-US triage since study, the 5-year cumulative risk of CIN3+ was 0.1% HPV test is more sensitive than repeat cytology for detec- (95% CI, 0.1–0.2%) after a combined negative HPV DNA tion of CIN2+ (IA). and cytological result at baseline and 0.8% (95% CI, 0.6– 1.0%) after a negative cytology at baseline, but without � HPV testing is, in general, not a management option in HPV testing [83]. After a negative HPV test at baseline, the case of LSIL (ID), even if it may be cost effective in older 5-year cumulative risk of CIN3+ was estimated as 0.2% women with LSIL (IB). Local assess is needed to identify a (95% CI, 0.1–0.3%) [83]. Likewise, in a longitudinal good triage test for women with LSIL (research need). Table 2: Key to evidence statements and grading recommendations defined by SIV.* Strength of Recommendation A. Good evidence for efficacy and substantial clinical benefit support recommendation for use. B. Moderate evidence for efficacy or only limited clinical benefit supports recommendation for use. C. Evidence for efficacy is conflicting and does not allow supporting a recommendation for or against use, but recommendations may be made on other grounds. D. Moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use. E. Good evidence for lack of efficacy or for adverse outcome supports a recommendation against use. I. There is insufficient evidence (in quality and quantity) to make a recommendation; however, other factors may influence decision-making. Quality of Evidence I. Evidence from at least 1 randomized, controlled trial. II. Evidence from at least 1 clinical trial without randomization, from cohort or case-controlled analytic studies (preferably from more than 1 centre) or from multiple time-series studies or dramatic results from uncontrolled experiments. III. Evidence from opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert committees. *: Quality of evidence and grading of recommendations were defined and approved by SIV; they were adapted from The Evaluation of Evidence and the Classification of Recommendations criteria described in The Canadian Task Force on Preventive Health Care. Page 9 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 � HPV testing, alone or in combination with cytology, is New molecular tests for management of women recommended for follow-up after treatment of high-grade with HPV infection Due to the relatively low specificity of HPV-DNA testing cervical lesions, since it is more accurate than repeat cytol- ogy in diagnosing residual disease or relapse (IA). and the high rate of false negative results at Pap test, new molecular tests have been proposed in order to identify � HPV testing, both based on hybridization (HC2) and women with a higher risk of progression to invasive can- PCR amplification with detection of high-risk HPVs, has cer. These tests include (i) detection of type-specific HPV been widely used in women aged >30 years in addition to persistence; (ii) detection of high-risk HPV E6/E7 mRNAs; Pap cytology because of the high NPV of a combined neg- (iii) HPV genotyping to detect HPV-16 and HPV-18, ative test (I). Women with normal Pap test result and neg- which have been associated with a higher risk of CIN2,3 ative HPV test have a very low risk to develop cervical or invasive cancer; (iv) measurement of high-risk HPV cancer and should not perform the subsequent follow-up load; (v) demonstration of high-risk HPV DNA integra- visit earlier than 5 years (IA). However, combination of tion; (vi) HPV subtyping by E6, L1, and LCR sequencing; HPV testing and Pap cytology is not a cost-effective strat- (vii) analysis of p16-INK4A expression. The implementa- egy (I), so, strategies based on HPV testing as a stand alone tion of these new molecular tests in cervical cancer screen- test should be preferred in women aged >30 years (IA). ing could be useful to identify the subpopulation of high- risk HPV-positive women who deserve a closer follow-up � Follow-up with HPV testing or repeat cytology at 12 and appropriate treatment. months is recommended for cytology-negative, HPV-pos- itive women aged ≥ 30 years (IA). Women who are persist- Demonstration of HPV persistence ently HPV-positive on repeat testing should undergo In women younger than 25 years of age, 20% of high-risk colposcopy, whereas those who are negative on both tests HPV infections persist, whereas the risk of persistent infec- may be rescreened in 5 years (IA). tion is over 50% in women older than 55 years [18]. Only a few women with HPV infection will develop cancer. � Using the HPV test in screening programs leads to earlier Thus, a single positive HPV test in the absence of clinically detection of CIN2+ and CIN3+ and reduction of the significant lesions does not justify treatment [87]. Several number of high-grade lesions detected at subsequent fol- longitudinal studies demonstrated that persistence of low-up in women aged ≥ 30 years (I), thus allowing high-risk HPV is necessary for cancer initiation and pro- longer screening intervals. The extent of long-term protec- gression and that the majority of high-risk HPV infections tion against high-grade lesions after a negative HPV DNA and associated low-grade lesions spontaneously regress test has been estimated to be longer than 5 years, but fur- within 6–18 months [1,88-93]. Screening strategies based ther research is need to establish the most appropriate on repeat HPV testing at 12 months following detection of screening interval (research need). a high-risk HPV could be useful to identify women at risk of cancer, who should undergo colposcopy, and to avoid � HPV testing as a stand alone test is more effective than overtreatment of lesions which will regress [94,95]. cytology alone in identification of CIN2+ lesions in women aged ≥ 35 years (I) and might be used as a primary From a practical point of view, persistence can be defined screening test in this age category. First follow-up results as the detection of the same HPV type (or, with a higher from ongoing controlled randomized trials, which com- degree of certainty, the same intratypic variant) two or pare longitudinal performance of Pap test vs. HPV testing more times over a certain period. There is no consensus as in screening programs, confirm the higher efficacy of HPV to the length of time that implies persistence, but at least screening that Pap screening (I). Local assessment is 6 months to 1 year is the time frame that is usually chosen needed before screening policies based on primary HPV [1]. A prospective study of type-specific HPV natural his- testing can be recommended (research need). tory, with a median follow-up of 5.1 years, showed partic- ularly pronounced persistence of HPV-16 and markedly � In women aged < 35 years, HPV testing with immediate increased risk of CIN3+ occurrence when HPV-16 per- referral to colposcopy leads to over diagnosis of CIN2+ as sisted, compared with any other HPV type [96]. Evalua- compared with more conservative management strategies tion of type-specific HPV persistence was used as a (I). Thus, in women younger than 35 years of age with a management strategy for women with a positive HPV test positive HPV test, cytology triage or repeat HPV testing at but normal Pap test in a controlled randomized trial in 12 months could be appropriate management strategies Sweden, as above reported [84]. to avoid over diagnosis and over treatment of CIN2+, but this hypothesis needs confirmation in longitudinal stud- Analysis of high-risk HPV E6/E7 mRNA ies (research need). Cervical cancer is characterized by overexpression of E6/ E7 mRNA of high-risk HPVs. A multiplex nucleic acid Page 10 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 sequence based amplification system (NASBA) assay was ally, a positive relationship between viral load and sever- developed for the identification of E6/E7 mRNA from ity of cervical lesions has been reported [107-109]. HPV types 16, 18, 31, 33, and 45 (PreTect™ HPV Proofer kit, NorChip AS, Norway; now also distributed as Nucli- HPV typing and subtyping SENS EasyQ HPV by bioMérieux SA, Marcy l'Etoile, Women with HPV-16 and HPV-18 infection have a higher France). Analysis of E6/E7 mRNA expression with the Pre- probability to develop cancer than women with infection Tect™ HPV Proofer kit in 204 histologically confirmed by other high-risk HPV types [110-113]. Some studies invasive cervical squamous cell carcinomas from Norwe- demonstrated that detection of HPV-16 and HPV-18 types gian women demonstrated the presence of oncogenic represents a risk factor for CIN2–3 [111,112], but also transcripts of HPV-16, HPV-18, HPV-31, HPV-33, and identification of other high-risk HPV types could be useful HPV-45 in 97% of HPV-positive cases, thus confirming to select at risk patients [114,115]. Investigation of intra- the expression of oncogenic E6/E7 in most invasive can- typic variants of HPV, which are defined by a difference in cers and the involvement of the above HPV types in most the L1 gene sequence less than 2% of the reference HPV cases [97]. A cross-sectional study was performed in 4,136 prototype, have been proven to be useful in epidemiolog- women >30 years of age to compare the performance of ical studies. In multiethnic populations, Asiatic-American HPV E6/E7 mRNA detection with detection of HPV-DNA and other non-European variants of HPV-16 and HPV-18 by Gp5+/6+ consensus PCR [98]. No significant differ- are associated with a higher risk of persistent infection and ence in sensitivity and specificity was observed between development of cervical precancerous lesions and inva- the two tests for detection of HPV in high-grade squamous sive cancer [116-118]. intraepithelial lesion (HSIL). On the contrary, only a small proportion of the HPV DNA-positive women with a Investigation of high-risk HPV E6 e E7 variants also gave normal, ASC-US, or LSIL diagnosis had a detectable E6/E7 interesting results. The T350G sequence variant of the mRNA expression [98]. A comparative study between HPV16 E6 gene is associated with increased viral persist- HPV testing with Gp5+/6+ consensus PCR and high-risk ence and oncogenicity [119,120]. The aminoacid change HPV E6/E7 mRNA testing with the PreTect™ HPV Proofer caused by this mutation could affect E6 degradation activ- kit showed the latest test had equal sensitivity and signifi- ity on p53, besides changing its immunogenicity. Moreo- cantly higher specificity for CIN2+ than PCR [99]. Moreo- ver, the L83V mutation of HPV16 E6 increases activation ver, E6/E7 mRNA-positive women had a significantly of the MAPK pathway and cooperates with Notch 1 in higher risk to have a CIN2+ within 2 years than negative tumorigenesis [121]. HPV-16 sequence variants may also women [99]. Interestingly, quantitative analysis of E6/E7 be associated with different oncogenic potential, even in mRNA in cervical cancers, but not HPV DNA load, corre- the absence of virus integration, due to derepression of the lated with patient survival [100]. The APTIMA HPV Assay, E6 and E7 promoter [122,123]. Thus, typing, subtyping, developed by Gen-Probe Incorporation (San Diego, CA), and sequencing of HPV oncogenes and the upstream reg- employs transcription-mediated amplification and target ulatory region could be useful for epidemiological pur- capture to detect HPV E6/E7 mRNA from 14 high-risk poses but also as prognostic markers. HPV types, but it does not differentiate among the 14 high-risk types. Preliminary results obtained with this Number of HPV types included in HPV tests assay suggest it has higher sensitivity and specificity than Demonstration that some new HPV types, defined as intermediate-risk, such as HPV-26, 53, 66, 73, and 82, HPV testing for identification of CIN2+ lesions [101]. may be associated with the risk of cervical cancer has sug- Measurement of viral load gested to extend the number of HPV types included in As above reported, higher cut-off values improve the spe- HPV DNA tests. To assess the clinical impact of such an cificity of the HC2 test [80-82] and correlate with an extended test, the results from two randomized trials increased risk of HPV persistence and development of which used HPV test for cervical cancer screening and high-grade cervical lesions [78]. A more accurate quantifi- ASC-US triage, respectively, were analyzed [124]. In the cation of viral load can be achieved by using real-time cancer screening protocol, addition of other high-risk PCR to detect specific HPV types. This method allowed HPV types, besides the 12 most common, did not amelio- demonstrating that high HPV-16 DNA load is associated rate test sensitivity. Likewise, when HPV testing was used with CIN3 and invasive cervical carcinoma or with an for ASC-US triage, test sensitivity was the greatest with 17 increased risk to develop CIN2–3 during follow-up genotypes, but specificity was very low [124]. Thus, at the [92,102-106]. Thus, measurement of HPV-16 load by moment, increasing the number of high-risk HPV types, real-time quantitative PCR has been proposed as a marker besides those already present in the FDA-approved HC2 of risk for CIN progression. Contrasting results have been test, does not seem to be advantageous neither for screen- obtained with other high-risk HPV types, although, gener- ing nor for triage of an abnormal Pap smear. On the other hand, reduction of the number of high-risk types included Page 11 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 in the HPV test to those most common in invasive cervical some individuals with documented HPV infection [134]. cancer (e.g., HPV-16, HPV-31, HPV-33, etc.) could On the other hand, high anti-HPV antibody titre is more improve HPV test specificity [125]. common in women with persistent infection than in those who clear infection [134-136]. Moreover, seroposi- Analysis of HPV DNA integration tivity for different high-risk HPV types is associated with Integration of HPV-16 and other high-risk HPVs into the high-grade CIN [137]. Unfortunately, no standardized genome of infected cells is an important step in tumori- and validated HPV serological assays are available genesis, since it promotes expression of E6/E7 oncogenes. [138,139]. Clinical studies demonstrated HPV DNA integration is associated with a higher risk of treatment failure and with Cell-mediated immune response is important for HPV a shorter disease-free interval than cases without integra- clearance, as suggested by the high incidence of persistent tion [126,127]. Thus, assessment of HPV genome integra- HPV infection in AIDS or transplanted patients. Persistent tion, by Southern-blot or quantitative real-time PCR infection and cancer progression is also associated with analysis of E2/E6 ratio, could represent a good prognostic abnormal response of CD4+ T cells [140] and CD8+ T marker. cells [141]. Analysis of p16-INK4A expression Evidence synthesis on the use of new tests for diagnosis of HPV In high-risk HPV infection, E7 binds to and degrades RB infection which results in substantial up regulation of p16-INK4A � Most high-risk HPV infections recover spontaneously synthesis. In spite of the high level of p16-INK4A synthe- within 6–12 months (I); persistent high-risk HPV infec- sis, this protein remains functionally inactive, as E7 tion and high-risk HPV E6/E7 expression are necessary induces cyclin A and cyclin E expression, thereby func- events for cervical cancer development and represent risk tionally bypassing its interference with the cell cycle. Anti- factors for cervical cancer (I). bodies that are directed against p16-INK4A allow selective staining of high-risk HPV-infected histological sections or � HPV typing and subtyping provides epidemiological of cytological smears, but not other cervical epithelia, sug- data on HPV distribution and diffusion (II). gesting that detection of this marker could provide diag- nostic support to distinguish true CIN/dysplasia from � In epidemiological surveys, type-specific HPV detection immature metaplasia or other non-neoplastic changes of could contribute to the evaluation of anti-HPV vaccine the cervix [128-131] and to improve interpretation of his- efficacy (III). tology [132]. Sensitivity and specificity for CIN2+ of p16- INK4A overexpression testing by immunostaining in cer- � High HPV DNA load is a risk factor for cervical cancer vical cell samples from HPV-DNA-positive women were (II). estimated in a nested sub-study of the NTCC trial [133]. This study demonstrated that HPV testing with p16- � HPV testing with p16-INK4A triage improves test specif- INK4A triage improves test specificity for CIN2+. In fact, icity for CIN2+ (II). p16-INK4A triage allows maintaining all the gain in sen- sitivity obtained by HPV testing alone with respect to con- � Detection of anti-HPV antibodies demonstrates previ- ventional cytology, but with referral to colposcopy similar ous HPV exposure, but this test has low sensitivity (II); a to that of conventional cytology [133]. high antibody titre might indicate persistent infection (II). Analysis of immune response against HPV � Patients with invasive cervical carcinoma have a defi- Both antibody-mediated and cell-mediated immune cient HPV-specific cell-mediated immune response (II). responses are essential for clearance of HPV and HPV- related cervical lesions. The immune response against Recommendations on the use of new tests for diagnosis of HPV HPV (and HPV virus-like particles, VLPs) is mainly directed infection against type-specific immunodominant conformational � In order to demonstrate HPV persistence, repeat HPV epitopes, although cross-reactivity has been documented, testing should be done at intervals of at least 12 months such as between HPV-6 and HPV-11, HPV-31 and HPV- (IA). 33, HPV-18 and HPV-45. Serum levels of anti-HPV anti- bodies are stable, even after virus clearance, so HPV sero- � HPV typing and subtyping in women with a normal or logical assays simply demonstrate previous exposure to dubious Pap test is useful since it allows to document per- the virus. Sensitivity of HPV serology is low (50–60%) sistent infection by the same oncogenic HPV type (II) and while specificity is high (about 90%). The low sensitivity to demonstrate the presence of HPV-16 and HPV-18 DNA of the test is in part explained by lack of seroconversion in which are markers of increased risk for cervical cancer (II). Page 12 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 Women with persistent high-risk HPV infection or with which is administered i.m. in three doses (time 0, 1, and the presence of HPV-16 and HPV-18 DNA should be 6 months), and a quadrivalent alum adjuvanted L1 VLP vaccine against HPV-16, HPV-18, HPV-6, and HPV-11 referred to colposcopy (B). (produced by Merck & Co., Inc.; distributed in Europe by � Measurement of HPV-16 and other high-risk HPV DNA Sanofi Pasteur MSD as Gardasil and by Merck Sharp & load could be employed for triage of women with dubious Dohme as Silgard ), which is administered i.m. in three Pap test or with a positive HPV test (B), but further doses (time 0, 2, and 6 months). The bivalent vaccine is research is needed to identify appropriate management advised for reduction of precancerous cervical lesions and strategies for women with high HPV load (research need). cancer incidence. The quadrivalent vaccine, besides cervi- cal lesions, is advised for reduction of vulvar precancerous � Detection of high-risk HPV E6/E7 mRNA could be lesions and genital condylomas. employed for triage of women with dubious Pap test or with a positive HPV DNA test (IIB). Due to its high sensi- The USA and several other Countries are targeting 11–12- tivity and specificity (II) HPV E6/E7 mRNA testing should year-old girls for vaccination. In particular, the Italian be investigated as a stand alone test in primary screening HPV vaccination program is universally offered free-of- for cervical cancer (research need). charge to all 12-year old girls, and advised to all adoles- cent women before sexual debut. The choice of a target � HPV typing and subtyping is useful for epidemiological population of young girls is based on the results of immu- investigation of HPV distribution and diffusion (B). nogenicity and safety studies, which were conducted in girls and boys aged 9–15 years (quadrivalent vaccine) and � Detection of p16-INK4A expression could be used as aged 10–14 years (bivalent vaccine). These studies dem- triage of HPV positive cases (B) and abnormal Pap smears onstrated that both vaccines are safe and immunogenic, (B) and to confirm histological analysis of cervical biop- and induce antibody titre which is more than 50 times the sies (B). titres induced by natural infection [145,146,150-156]. � Further research is needed to develop standardized Efficacy of HPV VLP vaccines Evaluation of vaccine efficacy in phase II-III clinical trials methods to measure anti-HPV immune response (research need). was based on the demonstration of reduction of the inci- dence of persistent HPV infection (two positive detections � In epidemiological surveys, evaluation of antibody- at 4–6 months intervals) and on reduction of the inci- mediated and cell-mediated anti-HPV immune response dence of precancerous lesions (CIN2 and CIN3) caused can be used to assess immune response to anti-HPV vacci- by HPV vaccine types. International standards for treat- nation and to monitor duration of vaccine protection (B). ment of CIN2/3 are ablative therapy. Moreover, in pro- spective studies, it is not ethical to allow a woman to Prophylactic HPV virus-like particle vaccines develop invasive disease in order to demonstrate efficacy Two vaccines targeting HPV-16 and HPV-18, the types of prevention strategies. In this regard, WHO and other that are responsible for 70% of invasive cervical cancers international agencies indicated CIN2/3 as clinical end- [13,16], have been recently developed and experimented point surrogate to demonstrate efficacy of cancer preven- for immunogenicity, safety, and efficacy in randomized tion. clinical trials. These vaccines are based on the self-assem- bly of recombinant L1 protein into VLPs, that are non- Randomised clinical trials demonstrated that three doses infectious capsids that contain no genetic material. They vaccination is effective for the prevention of persistent were first developed and evaluated as monovalent vac- infection and for precancerous lesions caused by HPV cines targeting HPV-16 [142,143], thereafter as a bivalent genotypes included in the vaccine. Results on efficacy vaccine targeting HPV-16 and HPV-18 [144-146] and a reported in published studies are mainly two types: (i) per quadrivalent vaccine against low-risk HPV-6 and HPV-11, protocol analysis, which demonstrates the pure efficacy of besides HPV-16 and HPV-18 [147-149]. Intramuscular the vaccine, obtained by analysis, in the experimentation injection of the vaccine induces high titres of neutralising context, of clinical data of women who did not violate the antibody in almost all subjects within a month from com- protocol (woman who received three doses of vaccine or pletion of the vaccination protocol [145,146,148-153]. placebo as expected), who resulted negative for all HPV genotypes included in the vaccine both at enrolment or Two HPV VLP vaccines have been developed and are avail- during the administration of the vaccine. (ii) modified able for primary vaccination in the European Union: an intention to treat analysis, where evaluation of efficacy is AS04 adjuvanted L1 VLP vaccine against HPV-16 and more similar to the clinical setting in the general popula- HPV-18 (Cervarix , produced by GlaxoSmithKline), tion, since it includes all enrolled women, as long as they Page 13 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 receive at least the first dose of vaccine or placebo and vaginal and vulvar intraepithelial neoplasia related to who resulted negative for all HPV genotypes of the vaccine HPV-6/11/16/18 and 100% efficacy (95% CI 94–100%) merely at the time of administration of first vaccine dose. for prevention of CIN1–3 or in situ adenocarcinoma asso- ciated with vaccine HPV genotypes [149]. Modified inten- Efficacy of the quadrivalent HPV VLP vaccine tion-to-treat analysis (also defined unrestricted susceptible A phase II randomised trial was carried out on 552 population) showed 95% efficacy (95% CI 87–99%) women (277 randomised in the vaccine group and 275 in regarding precancerous genital lesions, vulvar or vaginal, the placebo group). Per protocol efficacy analysis demon- or condylomas and 98% efficacy (95% CI 92–100%) for strated that Gardasil reduced by 90% (95% CI, 71–97%) CIN1+ or in situ adenocarcinoma caused by vaccine HPV the incidence of persistent infection and genital lesion types. The intention-to-treat analysis showed a 73% efficacy associated with the HPV genotypes included in the vac- (95% CI, 58–83%) in preventing external anogenital or cine [147]. A subset of subjects participated to a subse- vaginal lesions of any degree and a 55% efficacy (95% CI, quent follow-up of this study. Following five years from 40–66%) in preventing incident and prevalent cervical enrolment, efficacy on combined incidence of persistent lesions of any grade associated with vaccine HPV geno- infection of HPV-6/11/16/18-related disease was 96% types, at 3 years follow up. In both FUTURE I and II stud- (95% CI 83.8–99.5%) [143]. Modified intention to treat ies, vaccination did not appear to significantly alter the analysis demonstrated that vaccination reduced incident course of infection or lesions due to HPV which were infections and cervical lesions by 94% (95% CI, 83– already present before the first dose of vaccine. 98.3%) [152]. - Per-protocol efficacy analysis of the results achieved by the Phase III efficacy studies of the quadrivalent vaccine combination of three randomized clinical trials of vacci- Gardasil were conducted in the context of the two proto- nation (n = 7,811) versus placebo (n = 7,785) showed cols Females United to Unilaterally Reduce Endo/Ectocer- that vaccination, after an average follow-up period of 3 vical Disease (FUTURE) I and FUTURE II: years, has an efficacy of 100% (95% CI 72–100%) in pre- venting vulvar and vaginal lesions grade 2 and 3 associ- - FUTURE II trial. Per protocol analysis in over 10,000 ated with HPV-16 and HPV-18. Modified intention-to-treat women (5,305 randomised in the vaccination group and analysis demonstrated an efficacy of 97% (95% CI 79– 5,260 randomised in the placebo group) aged 15–26 100%) and intention-to-treat analysis, on a total of 18,174 years demonstrated that, a after a mean 3 year follow-up randomised women, showed an efficacy of the vaccine at period, vaccine efficacy was 98% (95% CI, 86–100%) for three years of 71% (95% CI, 37–88) in the prevention of prevention of CIN2,3 and in situ adenocarcimoma associ- incident/prevalent HPV-associated lesions [153]. ated with HPV-16 and HPV-18 [148]. It is to notice that the unique woman, included in the vaccine group, who - Per-protocol efficacy analysis of the results achieved by the developed a HPV-16 or HPV-18 associated lesion, had a combination of four randomized clinical trials of vaccina- positive CIN3 for HPV-52 at the initial control and in all tion with the quadrivalent vaccine (n = 9,087) and its the following 5 samplings during follow-up, only one HPV-16 L1 component (n = 1,204) vs. placebo (n = these samples resulted to be HPV-16 positive. Modified 10,292) showed that, after a follow-up of 3 years, in HPV- intention to treat analysis (definedalso unrestricted suscepti- 16 and HPV-18 negative women, vaccine efficacy was ble population) in randomised women (95% of the study 99% (95% CI 93–100) in preventing development of population), who were negative for the vaccine types at CIN2/3, in situ adenocarcinoma or invasive cervical cancer the first dose administration, and who did not strictly associated with HPV-16 and HPV-18. Efficacy was 98%, adhere to the study protocol, demonstrated vaccine effi- (95% CI 93–100) in the population of modified intention- cacy was 95% (95% CI, 85–99%). Intention to treat analy- to-treat analysis. The modified intention-to-treat analysis dem- sis including all randomised subjects, without any pre- onstrated an impact of the vaccine of 44% (95% CI, 31– vaccine screening, was used to predict efficacy of vaccina- 55) in reducing the incidence of cervical lesions associated tion on incident/prevalent HPV infections and related with HPV-16 and HPV-18, in the case of HPV type vaccine lesions in the general population, who demonstrated an diseases already prevalent/incident in the population impact of 44% (95% CI 26–58%) at 3 years follow-up. [157]. - FUTURE I trial. Per protocol analysis on over 5,000 Efficacy of the AS04 adjuvanted bivalent VLP vaccine women (2,261 randomised in the vaccination group and Efficacy the AS04 adjuvanted bivalent VLP vaccine has 2,279 randomised in the placebo group) aged between 16 been assessed in phase II-III studies. A pilot randomized and 24 years showed that after an average period of 3 years phase II study on safety, immunogenicity, and efficacy of since vaccine administration, the vaccine had 100% effi- the vaccine for prevention of incident and persistent HPV- cacy (95% CI 94–100%) for prevention of condylomas, 16 and HPV-18 infection enrolled and randomized 1,113 Page 14 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 women aged 15–25 years to receive either the vaccine or Cross-protection against oncogenic HPVs placebo [144]. Study participants were initially seronega- Two clinical trials have evaluated the efficacy of Cervarix™ tive for HPV-16 and HPV-18 by ELISA and negative for on other oncogenic types [144-146]. They demonstrated HPV-16 and HPV-18 DNA by PCR and had negative vaccination led to sustained cross-protection against inci- results at cervical cytology. In the per protocol analysis, vac- dent infection with HPV-45 and -31 for the entire follow- cine efficacy was 91.6% (95% CI, 64.5%–98.0%) against up period up to 5.5 years and cross-protection against six- incident infection and 100% against persistent infection month persistent infection due to HPV-45, -31 and -52 (95% CI, 47%–100%) with HPV-16/18 at 18 months [160]. Furthermore, broad protection was observed post-vaccination. In the intention-to-treat analyses, vaccine against 12-month persistent infections (defined as detec- efficacy was 95.1% (95% CI, 63.5%–99.3%) against per- tion of the same HPV type in all available cytology sam- sistent cervical infection with HPV-16/18 and 92.9% ples collected over any 12-month period) with 12 (95% CI, 70%–98.3%) against cytological abnormalities combined oncogenic HPV types, not including HPV-16 associated with HPV-16/18 infection. In a subsequent and -18 (vaccine efficacy 27.1%; 97.9% CI: 0.5–46.8) report from the same study, after a follow-up of 4–5 years, [160]. The impact of Gardasil HPV quadrivalent vaccine per protocol analysis on the results obtained from a group on the rates of infection and disease associated with 10 of 732 women (367 in the vaccination group and 365 in HPV types not included in the vaccine was estimated in a the placebo group) who received all three doses of vac- population of women that was, pre-vaccination, seroneg- cine, demonstrated vaccine efficacy against incident infec- ative and PCR-negative to all HPV types for which testing tion was 96.9% (95% CI, 81.3%–99.9%) and 100% was available and had a normal Pap test [161]. Within (33.6%–100%) against persistent infection for 12 months approximately 3 years of follow-up, combined efficacy for [145]. Efficacy against CIN lesions associated with CIN2/3 or adenocarcinoma in situ caused by all 10 non- HPV16/18 infection was 100% (95% CI, 42.4%–100%), vaccine HPV types was 38% (95% CI:6–60). The final since 8 cases of CIN1/2 occurred in the placebo group and end-of study data with approximately 3.6 years follow-up none in the vaccination group. Interim results at a mean showed similar high vaccine efficacy [161]. follow-up of 14.8 months (SD 4.9) from a large, interna- tional phase III study have been published [149]. In this Side effects of prophylactic HPV VLP vaccines Data on safety of the quadrivalent HPV VLP vaccine trial, 18525 women aged 15–25 years from North Ameri- can, South American, European, Asian, and Australian reported a higher rate of side effects in the vaccination countries were randomly assigned to receive either group than in the placebo group. Side effects included HPV16/18 vaccine (n = 9,258) or hepatitis A vaccine (n = local reaction at the site of injection (erythema, pain, 9,267) at 0, 1, and 6 months. Two cases of CIN2+ associ- edema) (altogether, 86.8% in the vaccine group vs. 77.4% ated with HPV16 or HPV18 DNA were seen in the HPV16/ in the placebo group, respectively) and systemic side 18 vaccine group; 21 were recorded in the control group. effects, such as fever (13.5% vs. 10.2%, respectively) Based on these data, vaccine efficacy against CIN2+ con- [149]. Also data on safety of the bivalent vaccine reported taining HPV16/18 DNA was estimated to be 90.4% a higher rate of injection site symptoms and systemic side (97.9% CI, 53.4–99.3), but, a post hoc analysis demon- effects (fatigue, headache, myalgia) in the vaccine group strated that the HPV16 or HPV18 DNA in the 2 CIN2+ than in the control group [146]. No increased incidence of cases in the vaccination group and in 1 case of the control spontaneous abortion, late fetal deaths, or congenital group were already present at study entry. Efficacy against anomalies was observed in pregnancies occurring in 12-month persistent HPV-16/18 infection was 75.9% women receiving either type of vaccines as compared with (97.9% CI, 47.7–90.2). controls [146,148]. Efficacy of HPV VLP vaccination in HPV-positive women Recommendations on the use of prophylactic HPV vaccines In women positive for HPV DNA, HPV VLP vaccination � HPV vaccination with the quadrivalent vaccine is safe does not accelerate clearance of the virus nor prevents and efficacious for the prevention of incident high-grade incident CIN2+ caused by HPV vaccine types. This finding precancerous lesions, in situ adenocarcinoma, invasive has been demonstrated in a randomized phase III trial in cervical cancer, high-grade vulvar and vaginal lesions, and 2189 women aged 18–25 years, who received the AS04 genital warts correlated with HPV-16, HPV -18, HPV -6, adjuvanted bivalent VLP vaccine [158] and in two rand- and HPV -11 (I). omized trials of the quadrivalent HPV vaccine enrolling 17,622 women without consideration of HPV status � HPV vaccination with the AS04 adjuvanted bivalent vac- [159]. cine is safe and efficacious for the prevention of incident and persistent HPV-16 e HPV-18 infection and CIN1+ cor- related with HPV vaccine types (I) Page 15 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 � Long-term efficacy (> 5 years) of HPV vaccination is cur- � HPV typing and subtyping can be used for epidemiolog- rently under investigation (research need). ical evaluation of HPV distribution and diffusion (B), for identification of persistent infection (B), for identification � Efficacy of HPV vaccination has been demonstrated in of HPV-16 and HPV-18, which are associated with a women aged 15–26 years, who had not been previously higher risk of precancerous lesions (B) or invasive cervical exposed to HPV vaccine types (I); results on efficacy in cancer (B), and for monitoring the efficacy of vaccination women older than 26 years are not available yet (research (A). But, there is insufficient evidence to define the best need). protocol to manage women with positive tests (I). � Results on immunogenicity and safety (but not efficacy) � Detection of high-risk HPV E6/E7 mRNA may be useful justify vaccination in girls aged 9–14 years (II). to identify women at risk for cervical cancer and with worse prognosis (IIB). Further research is needed to define � Efficacy of HPV vaccination in males is currently under the best protocol to manage women with a positive test investigation (research need). and to evaluate test efficacy in primary screening pro- grams. � HPV vaccination in males is not considered to be cost- effective (III). � Measurement of HPV-16 DNA load in women with nor- mal cytology may be useful to identify women at risk for � HPV vaccination should be done before exposure to cervical cancer (IIB). But, there is insufficient evidence to genital HPV, since it lacks efficacy in women who have define the best protocol to manage women with positive already been infected (IA), and it does not enhance virus tests (I). clearance (IA). � In epidemiological surveys, HPV serological testing and � Screening for cervical cancer should be recommended measurement of anti-HPV cellular immune response both in vaccinated and non-vaccinated women according could be useful to monitor the duration of vaccine protec- to current guidelines (II). Screening protocols could be tion (B). modified only after appropriate clinical trials (research need). Who should be vaccinated and how should vaccine efficacy be evaluated? � Further research is needed to assess the duration of vac- � Quadrivalent HPV VLP vaccine is safe and effective in cine protection, variations in the prevalence of HPV types the prevention of cervical and vulvar precancerous in vaccinated women and in the general population, and lesions, in situ adenocarcinoma, invasive cervical cancer surveillance of side-effects of vaccination (research need). and genital warts associated with vaccine HPV types in women aged 15–26 yrs (I). Summary of recommendations and guidelines When should HPV testing be used in cervical-cancer � HPV vaccination with the AS04 adjuvanted bivalent vac- screening programs? cine is safe and efficacious for the prevention of incident � in ASCUS triage (IA). and persistent HPV-16 e HPV-18 infection and CIN1+ cor- related with HPV vaccine types in women aged 15–26 yrs � in the follow-up after treatment of CIN2/3 (IA). (I) � as a screening test for cervical cancer, as an alternative to � HPV vaccine should be administered before genital HPV cytology, in women older than 30 years, with cytology or exposure, since the benefit of vaccination declines in repeat HPV testing as triage tests (IA). women already positive for HPV DNA (IA). Efficacy in cervical cancer screening has been proven for � In epidemiological surveys, HPV serological testing and both PCR-based and signal amplification-based HPV tests HPV genotyping could be useful to monitor vaccine effi- (I). cacy (research need). What is the role of new HPV tests in the diagnosis of HPV � Cervical cancer screening is recommended both in vac- infection? cinated and non-vaccinated women, in agreement with � Evaluation of HPV persistence may be useful to identify current guidelines for cervical cancer prevention (III). patients at risk for cervical cancer, who should be referred for colposcopy (A). � Further research is needed to assess the duration of vac- cine protection, variations in the prevalence of HPV types Page 16 of 22 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:14 http://www.infectagentscancer.com/content/3/1/14 9. zur Hausen H, Meinhof W, Scheiber W, Bornkamm GW: Attempts in vaccinated women and in the general population, and to detect virus-specific DNA sequences in human tumors: I. surveillance on side-effects of vaccination (research need). Nucleic acid hybridization with complementary RNA of human wart virus. Int J Cancer 1974, 13:650-656. 10. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Competing interests Classification of papillomaviruses. Virology 2004, 324:17-27. The authors declare that they have no competing interests. 11. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ, International Agency for Research on Can- cer Multicenter Cervical Cancer Study Group: Epidemiologic clas- Authors' contributions sification of human papillomavirus types associated with LB drafted the manuscript. CG, FMB, and GP contributed cervical cancer. N Engl J Med 2003, 348:518-527. 12. Munoz N, Castellsague X, de Gonzalez AB, Gissmann L: HPV in the to the preparation of guidelines. All authors read and etiology of human cancer. Vaccine 2006, 24(Suppl 3):S1-S10. approved the final manuscript. 13. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL: HPV type dis- tribution in women with and without cervical neoplastic dis- eases. Vaccine 2006, 24(Suppl 3):S26-34. Acknowledgements 14. Trottier H, Franco EL: The epidemiology of genital human pap- These guidelines were reviewed by SIV Board Members and approved by illomavirus infection. Vaccine 2006, 24(Suppl 1):S1-S15. the Executive and Council of SIV. We thank the members of SIV who 15. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N: Human papillomavirus reviewed the draft version of this guideline when it was posted on the SIV is a necessary cause of invasive cervical cancer worldwide. J website and who sent a great number of comments, most of which were Pathol 1999, 189:12-19. incorporated into the final version of the manuscript. Moreover, we thank 16. Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, Clifford for their helpful suggestions: K. Soldan, Health Protection Agency Centre GM: Human papillomavirus type distribution in invasive cer- for Infections, London, UK; L. Banks, International Centre for Genetic Engi- vical cancer and high-grade cervical lesions: A meta-analysis update. Int J Cancer 2007, 121:621-632. neering and Biotechnology, Trieste, Italy; R. Kirnbauer, Viral Oncology, 17. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, Pimenta JM: Medical University Vienna, Austria; S. Pecorelli, Department of Obstetrics Human papillomavirus genotype distribution in low-grade and Gynaecology, University of Brescia, Brescia, Italy; M.G. Pompa, Ministry cervical lesions: comparison by geographic region and with of Health, Directorate General of Health Prevention, Communicable Dis- cervical cancer. Cancer Epidemiol Biomarkers Prev 2005, 14:1157-1164. eases Unit, Rome, Italy; F. Carozzi, Centre for Cancer Study and Preven- 18. de Sanjosé S, Diaz M, Castellsagué X, Clifford G, Bruni L, Muñoz N, tion, Florence, Italy; C.F. Perno, Department of Experimental Medicine, Bosch FX: Worldwide prevalence and genotype distribution University of Tor Vergata Rome, Italy; M.L. Tornesello, Molecular Biology of cervical human papillomavirus DNA in women with nor- and Viral Oncology and AIDS Reference Centre, National Cancer Institute, mal cytology: a meta-analysis. Lancet Infect Dis 2007, 7:453-459. 19. Ronco G, Ghisetti V, Segnan N, Snijders PJ, Gillio-Tos A, Meijer CJ, Fond. Pascale, Naples, Italy; A. Vecchione, Department of Gynaecology, Merletti F, Franceschi S: Prevalence of human papillomavirus Perinatology and Child Health, University of Rome La Sapienza, Rome, Italy; infection in women in Turin, Italy. Eur J Cancer 2005, M. Ciofi degli Atti, Bambino Gesù Children's Hospital, Rome, Italy; A. 41:297-305. Venuti, Laboratory of Virology, Regina Elena Institute for Cancer Research, 20. Tornesello ML, Duraturo ML, Botti G, Greggi S, Piccoli R, De Palo G, Rome, Italy, F. 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Jenkins D: A review of cross-protection against oncogenic HPV by an HPV-16/18 AS04-adjuvanted cervical cancer vac- cine: Importance of virological and clinical endpoints and implications for mass vaccination in cervical cancer preven- tion. Gynecol Oncol 2008, 110:S18-S25. 161. Barr E, Sings HL: Prophylactic HPV vaccines: New interven- tions for cancer control. Vaccine 2008, 26:6244-6257. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention

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Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention

Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention

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