High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer. The scientific challenge is to determine whether HPVs are causal and not merely passengers or parasites. Studies of HPV-related koilocytes in breast cancer offer an opportunity to address this crucial issue. Koilocytes are epithelial cells characterised by perinuclear haloes surrounding condensed nuclei and are commonly present in cervical intraepithelial neoplasia. Koilocytosis is accepted as pathognomonic (characteristic of a particular disease) of HPV infection. The aim of this investigation is to determine whether putative koilocytes in normal and malignant breast tissues are because of HPV infection.in situ PCR with confirmation of the findings by standard PCR and sequencing of the products, plus immunohistochemistry to identify HPV E6 oncoproteins.Archival formalin-fixed normal and malignant breast specimens were investigated by histology, in situ (DCIS) and invasive ductal carcinomas (IDCs).human papilloma virus-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with ductal carcinoma As koilocytes are known to be the precursors of some HPV-associated cervical cancer, it follows that HPVs may be causally associated with breast cancer. High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer  surrounding condensed nuclei . KoilocyPutative (supposed) HPV-associated koilocytes have been observed in some breast neoplasias . E7-induced degradation of p110RB often results in a reciprocal overexpression of the cyclin-dependent kinase inhibitor p16INK4A, although this is further complicated with increasing cancer stage.The mechanisms by which high-risk HPV infections cause cervical cancer have been studied in detail . In thisIncreasing levels of HPV viral load seem to be associated with an increased risk of developing cervical pre-cancer  is a problem (in situ (DCIS) specimens plus normal breast specimens from cosmetic surgery were used.We investigated the possible presence of koilocytes in normal and malignant breast specimens. Archival formalin-fixed invasive ductal carcinoma (IDCs) and ductal carcinoma in situ PCR together with known positive and negative controls. In addition, we used immunohistochemistry to identify the presence of HPV types 16 and 18 E6 oncoprotein again with both positive and negative controls. We eliminated samples that potentially could give false-positive HPV results, identified by positive in situ PCRs without primers. An unknown number of these eliminated specimens would have been true positives. Accordingly, the data cannot be used to make estimates of prevalence of the presence of these viruses.PCR is by far the most sensitive technique available for the identification of extremely low viral loads, but the technique is associated with problems including both false negatives and false positives, contamination and inconsistent outcomes . For thiin situ PCR conducted on the same specimens are as described in detail in our companion paper  and Koilocytic cytoplasmic vacuolisation: perinuclear haloes surrounding cell nuclei.Koilocytic nuclear change: pyknosis – nuclear material that may be irregular in size, shape and staining properties. The nucleus is frequently acentric. Binucleation may be apparent. It is now known that ‘binucleation’ involves multilobules caused by HPV-influenced activity at the G2 cell cycle checkpoint  commercial antibodies were used. Human papilloma virus E6 is a nuclear, cytoplasmic and membrane stain. Positive  and negative (HPV E6 antibody omitted) controls were used.Putative (supposed) HPV-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with DCIS and IDCs. These data are shown in in situ PCR in the breast lobules and koilocytes of this same specimen. Human papilloma virus oncoprotein E6 is present in the basal layers of breast skin and in the intercellular spaces and cytoplasm of koilocytes, normal breast epithelial cells and malignant cells. There is little E6 nuclear staining. In some specimens stained for HPV E6, there is ‘background’ staining  when stained with the same antibodies (abCam 70)  HPV infections are specifically associated with koilocytosis in cervical and other tissues, (ii) HPVs have been repeatedly identified in breast cancers that have occurred in women from a wide range of populations, (iii) HPV-positive koilocytes are present in normal skin and breast tissues of normal women, (iv) HPV-positive koilocytes are present in many DCIS and some IDC specimens. As HPV-associated koilocytes in the cervix may progress to varying grades of malignancy (Infections of the cervix by HPV types 16 and 18, as identified in these investigations, are successfully inhibited by the new anti-HPV vaccines . It is l


In this paper we develop a Bayesian analysis to estimate the disease prevalence, the sensitivity and specificity of three cervical cancer screening tests  in the presence of a covariate and in the absence of a gold standard. We use Metropolis-Hastings algorithm to obtain the posterior summaries of interest. The estimated prevalence of cervical lesions was 6.4% . The sensitivity of cervical cytology (with a result of ≥ ASC-US) was 53.6%  compared with 52.9%  for visual inspection with acetic acid and 90.3%  for Hybrid Capture II (with result of >1 relative light units). The specificity of cervical cytology was 97.0%  and the specificities for visual inspection with acetic acid and Hybrid Capture II were 93.0%  and 88.7% , respectively. The Bayesian model with covariates suggests that the sensitivity and the specificity of the visual inspection with acetic acid tend to increase as the age of the women increases.The Bayesian method proposed here is an useful alternative to estimate measures of performance of diagnostic tests in the presence of covariates and when a gold standard is not available. An advantage of the method is the fact that the number of parameters to be estimated is not limited by the number of observations, as it happens with several frequentist approaches. However, it is important to point out that the Bayesian analysis requires informative priors in order for the parameters to be identifiable. The method can be easily extended for the analysis of other medical data sets. When the outcomes of the diagnostic test are represented in a continuous scale, a cut-off value should be chosen in order to determine when an individual is classified as positive or negative. Generally, individuals with test outcome larger or at least equal to this fixed cut-off are classified as positive while individuals with test outcomes inferior to this fixed cut-off are classified as negative.The sensitivity  and visual inspection with acetic acid (VIA) in the detection of cervical precursor lesions, using a Bayesian statistical method that allows for the estimation of these measures, although part of the sampled women was not verified by a gold standard. We also consider the presence of covariates in our study. Since Bayesian methods are based on incorporation of historical information and expert opinion into the modelling strategy , these elements could be too subjective, with source for other bias. In other words, inadequate prior information can imply in a biased estimator. However, a careful verification of the prior information and a subsequent analysis of its changes in the outcomes can result in reasonable estimates for the tests performance measures.Se and Sp related to two diagnostic tests in the absence of a gold standard. We also introduce in this section the notation used in the paper. In the following, we introduce the methodology for estimating Se and Sp in the presence of a covariate. The cervical cancer screening data set is described in Section 4. The application of the proposed methodology on the analysis of the data set is presented in Section 5. Concluding remarks are given in Section 6.Thus, the new methodological contribution of the present paper is an extension of the Bayesian method proposed by Joseph et al.  for esti2.South and Central America have some of the highest incidence rates for cervical carcinoma in the world, ranging from 30/100,000 women to 40/100,000 women, or three to four times the incidence in developed countries . In BrazIn assessing the accuracy of cervical cancer screening tests, it is not straightforward to define an ideal gold standard. In many studies, the gold standard for evaluating the accuracy of screening tests in detecting true positive lesions is histopathology. If biopsies are not obtained, colposcopy is accepted as the final diagnosis. However, colposcopy can give many false negative results when used to discriminate between normal and abnormal tissues  ,8.The reference test used in these studies, defined by the results of histology or colposcopy, is thus subject to errors and its estimates for sensitivity and specificity can be biased. Another type of bias is evident when only a part of the sampled individuals will have their real disease status confirmed by the biopsy and the remainders are not included in the calculations of the sensitivity and specificity. This occurs principally when only the women with positive result for one or more diagnostic tests  are submitted to the gold standard and this selection results in an overestimated sensitivity and an underestimated specificity .Despite of the appearance of new methods developed to estimate the sensitivity and specificity of screening tests without a gold standard  or in thMany studies are introduced in the literature in the absence of a gold standard. For instance, Hui and Walter (1980) derived equations that compute estimates and standard errors of sensitivity, specificity and prevalence, without considering a reference test . Joseph 3.k diagnostic tests, let Tm = 1 if the result of test m is positive and Tm = 0 if the result of test m is negative, for m = 1, …, k. Let Sme and Smp be the sensitivity and the specificity of the test m respectively and let g be an observation of a binary latent variable G, introduced in the model aiming to simulate a non-observable gold standard −1, where l = 1, 2, 3, W0i = 1, θ1i = Sei , θ2i = Spi, θ3i = pi , for i = 1, …, n. In this way, we have a vector of parameters given by β = , where βl = , l = 1, 2, 3. Assuming prior independence among the parameters, we consider the prior densities for βlj with normal distribution with fixed hyperparameters alj (means) and blj2 (variances), l = 1, 2, 3, j = 0, 1, …, L. The likelihood function for β is given byLet g is an observation of the latent variable G, given by (2). Combining the prior distributions with L(β), we have the conditional posterior distributions for β given by the vector of unknown parameters is now given bywhere j = 0, 1, …, L and β (β10) is the vector of all parameters except β10 (for example). Observe that we should simulate samples for all parameters considering the Metropolis-Hastings algorithm . In the present study, we considered the data from Campinas, one of the three Brazilian arms of the LAMS study. From February to December 2002, 1,195 women were recruited at a basic health unit and from July to December 2002, 221 women were recruited at the University Hospital . Both services are situated in Campinas, a 969,396 inhabitants city in Brazil’s southeast region. Among these 1.416 women, 809 women were eligible for the study related to the sensitivity and specificity of three cervical cancer screening tests  in the presence of covariates and in the absence of a gold standard and were willing to participate. Women were eligible if they were between 18 and 60 years of age, if they had been submitted at all three diagnostic methods and if they had intact uterus. Patients previously subjected to treatment for condylomas or with history of current abnormal cytology were excluded. Women who presented with confirmed immunossupression, immunodeficiency or HIV infection, who had sexual intercourse or vaginal medication in the last three days were not included. Informed consent was obtained from all participant women. The study protocol was reviewed and approved by the Committee of Ethics in Research of the Medical Science School of the State University of Campinas.Cervical cytology was collected after evaluation and treatment for possible infectious processes. Ayre spatulas and cervical brushes were used for these samplings. The samples were stained according to the Papanicolaou method and evaluated using the Bethesda System . Cytolog5.T1), VIA (T2) and HC II (T3) were estimated by a Bayesian approach proposed by Joseph et al. (1995) [First of all, the sensitivity and specificity of cervical cytology (. (1995) . This meThe prior information about the sensitivity and the specificity of cervical cytology was based on the systematic review of Nanda et al. (2000), who presented sensitivity for atypical squamous cells of undetermined significance (ASC-US) or worse being ranged from 29 percent to 56 percent and specificity from 97 percent from 100 percent . The stuHowever, the choice of informative prior distributions based only in a summary of previous studies can be a complex task, since each study has elements of subjectivity, error-proneness and possible potential for bias. Thus, a panel of experts on cervical cancer was asked to provide their best estimate for the sensitivities and specificities of the tests and the prior distributions that summarise the information provided by the literature review corrected by the experts were derived. The assessment of beta distribution priors for each test parameter considered the method presented by Joseph et al. (1995) , where tThe median age of the 809 women who participated of the study was 34 years. Approximately three quarters of these women lived with a partner (73.0 percent) and one-third had 8 or more years of education (33.3 percent). The majority self-reported to be white (67.2 percent) and 64.3 percent reported not to be a smoker. Half of the women (50.3 percent) reported to have had only one lifetime sexual partner and almost three quarters (72.9 percent) had initiated the sexual life in the teenage. Only 1.5 percent of the women entered in the study with less than one year since her first sexual intercourse and the majority (86.1 percent) reported to have had only one sexual partner during the last 12 months. The percentage of women who are pregnant at the time of the study was around 7.7 percent.Based on the cases for which cervical cytology was available, 758 (93.7 percent) had normal results, 12 (1.5 percent) had low-grade squamous intraepithelial lesion (LSIL), 4 (0.5 percent) had high-grade squamous intraepithelial lesion (HSIL) and 35 (4.3 percent) were ASC-US. The results suggest a low sensitivity for cervical cytology to detect ASC-US or worse (53.6 percent) as well as for VIA (52.9 percent), but indicate a high sensitivity for the HC II (90.3 percent). All screening methods presented relatively high specificities, 97.0 percent for cervical cytology, 93.0 percent for VIA and 88.7 percent for the HC II. As is evident in Another important result from the Bayesian model is related to the estimates for the expected value of true positives for each combination of the three screening methods, as shown in X) as a continuous covariate. The covariate W1 is given by (X − x̄)/10, where x̄ is the sample mean of X. The quotient 10 is only considered for avoiding numerical instability related to large values in exponential functions present in the conditional posterior densities of interest. We also introduced in the model the variable W2, a dichotomous variable that denotes whether or not the woman is actually pregnant (1 if pregnant and zero otherwise). Firstly, we considered 13 the interaction between W1 and W2 in the model. However, all interaction parameters were estimated to be close to zero (ranged from −0.031 to 0.009) and it were excluded from the final model.In a second instance, we introduced in the model the age of the women . The convergence of the Gibbs samples was monitored by standard existing methods [β10 to β70 were assumed with fixed hyperparameters based in estimates obtained in the previous analysis without covariates. For example, we noted that the estimated sensitivity of the cervical cytology was 0.536 (see a10 is thus given by log (0.536/(1 – 0.536)). All the other hyperparameter values were chosen to have noninformative priors. Thus, we used an empirical Bayesian modelling approach [β are interpreted as being the logarithm of the odds ratios (OR). These odds ratios represent an association measure between the variables W1 and W2 and the operating characteristics of the screening tests.From the conditional densities for the parameters in  methods  and the .536 see , and conapproach . For eacβ, interpreted as odds ratios. We observe that the 95 percent credible intervals for the parameters eβ11 to eβ71 included the value 1, suggesting that there is no evidence for the effect of pregnancy in eβ12 and eβ52 Se and Sp measures for all tests. The parameters eβ12 and eβ52 were estimated in 2.033 and 1.615, respectively and its credibility interval does not include the value 1. This result suggests that the sensitivity and the specificity of VIA increases as the age of the women increases. In fact, in the medical literature several authors have described that methods for detection of precursor lesions of cervical cancer have different performances according to the age of women [OR estimated in 0.483 and the respective 95% credible interval do not included the value 1). This effect of age on the prevalence is well-known in the medical literature since the disease is more incident in sexually active women.In of women , 34, 35.6.In this article, we introduced a Bayesian approach based on a Markov chain Monte Carlo (MCMC) algorithm that allows the performance measures estimations of diagnostic tests in the presence of covariates and when a gold standard is not available. An advantage of the proposed methodology is the fact that the number of parameters to be estimated is not limited by the number of observations as it happens when we use the method introduced by Hui and Walter . We usedG (2) that best describe the data simulating a reference test. This latent variable has a Bernoulli distribution with success probability given in function of the performance screening measures and its subjectiveness from the respective prior distributions. Therefore, more accurate results would be given if we incorporate reasonable prior distributions based on prior knowledgement of clinical experts.An important consideration in the use of the proposed model is its dependence on the prior information. In a sensitivity analysis, we noted, for example, that the prevalence of cervical lesions is increased when we used all non-informative prior distributions and other substantial changes in the sensitivity and specificity. In the presented model, the lack of a gold standard is counterbalanced by the introduction of a latent variable Although the proposed model is able to estimate useful performance measures of serial and parallel combinations of the screening tests see  and 5, tThe major shortcoming of the Bayesian estimating method resides in the necessary presumption that the diagnostic tests are statistically and conditionally independent. This presupposition might not be invariably true  and alteIt is also important to point out that the diagnostic tests evaluated in this study have some inherent flaws. The lack of accuracy and reproducibility of cervical cytology is explained by the biological variability, sample quality, subjective interpretation of morphological abnormalities and examiners fatigue derived from repetitive procedures . VIA reqThus, VIA suggested by Belinson et al. (2001) as a screening method is likely to assume a central role in the prevention of cervical cancer in many countries. This simple and inexpensive method does not require complex technical supplies and it allows diagnosis and treatment at a single visit . Coste eThree Indian studies in the late 1990s provided evidence supporting VIA as a viable alternative to cytology as a primary screening test –47. In oThe studies mentioned above yielded valuable information regarding the performances of VIA and cytology. Our Bayesian estimates provided performance values that can be compared with the results obtained in the standard manner, that is, with the use of a gold standard. In our estimates, VIA and cytology had similar sensitivity and specificity, as well as PPV and NPV. These figures contradict the previously published direct estimates that reported a superiority of VIA in detecting cervical lesions ,47. ThesThe performance of HPV test in screening settings has been extensively studied, but to a lesser extent in comparison with VIA and cytology. Denny et al. (2000) published their data on 2,944 women subjected to VIA, cytology and HPV testing. In this study, VIA and HPV (>1 RLU) were similar to cytology in their performance of detecting high-grade lesions, but VIA yielded the largest number of false-positives among the three testing modalities . More reThe shortcomings of the tests  did not hamper the Bayesian estimates but, in contrast, would enhance the search for realistic estimating equations. Thus, the inclusion of covariates (or control variables) should be encouraged in studies designed for the evaluation of estimating methodologies. According to Parmigiani (2002), prediction models used to support the clinical and health policy decision making need to consider the course of the disease over an extended period of time and draw evidence from a broad knowledge base, including epidemiological cohort and case control studies, randomized clinical trials, expert opinions and more . In thesWe can conclude that the estimated performances of VIA, HC II and cytology clearly show that the Bayesian method is a remarkable tool for validating diagnostic tests when a gold standard is available for a very limited number of cases or not available at all.


Human papillomaviruses (HPVs) are sexually transmitted human carcinogens that may play a role in the oncogenesis of penile cancer.INK4A in the pathogenesis of penile cancer.To investigate the role of HPV infection and expression of the tumour suppressor protein p16INK4A and Ki67, we analysed 26 penile squamous cell carcinomas (SCCs) and 20 independent penile lichen sclerosus (LS) lesions from 46 patients.By means of polymerase chain reaction amplification and reverse hybridization line probe assay to detect HPV infection, and immunohistochemical staining for p16INK4A correlated with HPV 16/18 infection in both penile LS and penile SCC. In our penile SCC series the cancer margins were also associated with penile LS in 13 of 26 lesions, and HPV was detected in seven of the 13 SCC cases associated with LS and in six of the 11 SCC lesions not involving LS.HPV DNA was found in 54% of penile SCCs and 33% of penile LS cases in single and multiple infections. High-risk HPV 16 was the predominant HPV type detected. No relationship between Ki67 expression and HPV infection was observed. Strong immunostaining for p16INK4A expression in penile lesions, consistent with an active role for HPV in interfering with the retinoblastoma pathway. High-risk HPV infection could be involved in the tumorigenic process in 50% of penile cancers, and the use of prophylactic HPV vaccines has the potential to prevent these cancers.Our study shows a high prevalence of HPV 16 and p16 Penile cancer accounts for 0·3–0·6% of all cancers in men in the Western world and this incidence is much higher in some African, Asian and South American countries.8Persistent infection with sexually transmitted high-risk HPV is the main cause of cervical cancer,2INK4A.INK4A expression in both types of lesion.HPV contributes to tumorigenesis predominantly through the action of the viral oncoproteins (E6 and E7). The interaction of E7 with the retinoblastoma (Rb) tumour suppressor leads to Rb degradation, E2F activation and overexpression of the cyclin-dependent kinase inhibitor p16Archival paraffin wax-embedded tissue sections from 26 penile SCCs, 20 penile LS cases not associated with SCC and 26 cervical cancers (Barts and The London NHS Trust) were obtained and reviewed by a pathologist (S.B.), with ethical approval from the East London and City Health Authority Research Ethics Committee (P-02-84 and P-03-302). DNA was extracted from paraffin wax-embedded sections using the QIAamp DNA Mini kit . β-Globin polymerase chain reaction (PCR) was performed using primers B1 and B19 to confirm the adequacy of the extracted DNA. Validated samples were tested for the presence of HPV DNA by a broad-spectrum HPV PCR method using SPF10 primers which amplify a 65-bp fragment of the L1 open reading frame.20INK4A  were applied for 1 h and detected using an Envision Kit . p16INK4A staining was assessed by grading the intensity  and percentage of positively stained cells . The combined score total was calculated by adding together the intensity and percentage coverage scores. Ki67 staining was assessed by the percentage of positively stained cells only. Statistical analysis was performed using Fisher’s exact test and SPSS software . P < 0·05 was considered significant.Antigen retrieval using citrate buffer was performed on dewaxed sections prior to blocking with swine serum. Primary antibodies to Ki67  and p16P < 0·05). There was no significant difference in the number of multiple HPV infections between penile LS and SCC. For both penile LS and SCC, HPV-positive cases consisted of 50% single infections and 50% multiple infections. Similar frequencies were observed in our cervical cancer series (58% single infections and 42% multiple infections). High-risk HPV genotypes were detected in all HPV-positive penile lesions and cervical cancers. Only high-risk HPV 16 and 18 were found as single infections in penile cancer. HPV 16, which is strongly associated with malignant mucosal lesions, was the most commonly occurring HPV type in single and multiple HPV infections in penile LS, penile SCC and cervical cancers. HPV 16 was detected in all six HPV-positive penile LS cases, 11 of 14 (79%) HPV-positive penile SCCs and 20 of 22 (91%) HPV-positive cervical cancers. High-risk HPV 18, 39, 45, 52 or 68 infections were detected in the three HPV-positive penile cancers lacking HPV 16 and in some of the multiple infections with HPV 16 and HPV 6, 33, 40, 42, 51 or 56.An analysis of tissue samples from 26 penile carcinomas, 20 penile LS cases not associated with SCC and 26 cervical cancers was performed to determine HPV prevalence and genotype . Two casINK4A in penile LS and SCC by immunohistochemistry. Staining for Ki67 and p16INK4A was observed in both penile LS and SCC (INK4A or HPV infection was observed (INK4A was found in approximately 50% of the penile lesions and this was associated with HPV 16/18 infection in both penile SCC and penile LS (P < 0·05) (We examined the expression of proliferation marker Ki67 and p16 and SCC . No relaobserved . In cont < 0·05) .In our penile SCC series the cancer margins were also associated with penile LS in 13 of 24 cases (54%), with two cases uncharacterized due to insufficient noncancerous material. HPV was detected in seven of the 13 (54%) SCC cases associated with LS and in six of the 11 (55%) SCC cases not involving LS.INK4A expression. Penile LS has been proposed to be a premalignant lesion predisposing for penile cancer development.26We have established the prevalence of HPV DNA in penile LS and SCC and found an association with high-risk HPV 16/18 and p16High-risk HPV 16 was the most prevalent HPV genotype detected in each of our penile series, infecting 100% of HPV-positive penile LS and 79% of HPV-positive penile SCC cases. HPV 16 and HPV 18 were the only HPV genotypes occurring as single infections, suggesting that these genotypes are more likely to contribute to the carcinogenic process. HPV 16 and 18 are the predominant high-risk HPV genotypes associated with cervical cancer.30INK4A expression in penile LS has not previously been reported, and we are aware of only one paper reporting p16INK4A expression in penile cancer.INK4A and the proliferation marker Ki67 has been described in gynaecological pathology.INK4A which occurred in 55% of penile LS and 46% of penile SCC cases was significantly associated with HPV 16/18 infection. This association is in agreement with Ferreux et al. who reported that increased p16INK4A expression correlated with HPV detection and occurred in 29% of penile SCCs,INK4A expression in our series. In the two cervical cancer, three penile SCC and four penile LS cases where p16INK4A was strongly expressed and no HPV was detected, the levels of HPV may be too low for detection or HPV types may be present that are not amplified by the SPF10 system.INK4A expression is frequently inhibited by methylation or mutation in penile SCCINK4A indirectly by HPV or by methylation and mutation therefore appears to be an important step in penile tumorigenesis.p16et al. found HPV in five of nine SCCs without LS association and only one of eight SCCs with LS association,et al. reported HPV 16 infection in two of three penile SCCs that developed in a series of 86 patients with LS.20Our observation that penile SCC was associated with LS in 54% of our cancer cases is consistent with LS being a predisposing factor to the development of penile and vulval SCC.The importance of HPV status in penile cancer progression and patient survival is controversial. High-risk HPV has traditionally been associated with aggressive variants,22INK4A expression. Expression of p16INK4A may therefore be of value as a surrogate marker confirming the diagnosis of HPV-infected premalignant and malignant penile lesions. HPV 16 predominates in penile LS and SCC and it is likely to be an aetiological agent in the development of a significant proportion of penile cancers. These results are important as prophylactic HPV vaccines being developed for prevention of cervical cancer in women could also prevent penile cancers in men.In summary, high-risk HPV infection occurs in both penile LS and SCC and is associated with p16


Oral HPV infections detected six-months apart were compared to those detected bi-weekly, in an HIV-positive cohort, during the intervening months to elucidate systematic biases introduced into natural history studies by sampling interval.Fourteen consecutive oral rinse samples were collected every two weeks for six months from an HIV-positive cohort (n = 112) and evaluated for the presence of 37 HPV types. The cumulative probability of type-specific HPV detection at visits 1 through 14 was determined as a function of infection categorized at visits 1 and 14 as persistent, newly detected, cleared or absent. Transition models were used to evaluate the effect of HPV viral load  on infection persistence.The average point prevalence of oral HPV infection was similar at two-week and six-month sampling intervals , but cumulative prevalence was higher with the former  as was the cumulative prevalence of type-specific infections . Type-specific infections persistent under a six-month sampling interval had a high probability  of detection at 50% or more of the intervening visits and infections that were absent had a high probability  of no interval detection. The odds of detection at any visit significantly increased for each unit increase in HPV viral load at the previous visit.Six-month sampling is appropriate to model factors associated with type-specific oral HPV infection persistence but may misclassify HPV-exposed individuals as unexposed. Oral human papillomavirus (HPV) 16 infection is strongly associated with oropharyngeal squamous cell carcinoma (OPSCC)An important factor for HPV natural history studies is the sampling interval, and six-month sampling intervals are commonly used for cervical studies. We have previously evaluated the feasibility of a six-month sampling interval for oral HPV infectionWe performed a short-term natural history study of oral HPV infections to compare the results of a two-week to a six-month sampling interval to identify potential systematic biases introduced into natural history studies by the frequency of sampling. Oral HPV infections refers to infections of the oral cavity and oropharynx. An HIV-positive cohort, known to be at elevated risk for oral HPV infection and related malignancies, was selected as our study population.This study was conducted according to the principles expressed in the Declaration of Helsinki. This study was approved by the Institutional Review Board of Johns Hopkins Hospital (application number 03-11-24-04). All patients provided written informed consent for the collection of samples and subsequent analysis.A convenience sample of 112 HIV-positive patients from the Johns Hopkins Hospital was recruited during March 2005. Eligibility criteria included known HIV-positive status and willingness to comply with bi-monthly (every two week) visits for six months. Enrollment took into consideration both CD4 count (< or ≥200 cells per µL) and gender to result in a study population with approximately equal proportions. Subjects received monetary compensation ($160) for participation.Oral rinse samples were collected every two weeks for six consecutive months (visits 1–14) using a 30 second Scope™ mouthwash rinse and gargleOral exfoliated cells were separated by centrifugation, washed, and re-suspended with phosphate buffered saline and stored at −80°C.DNA was purified by use of a modified protocol for Puregene DNA Purification HPV viral load for types 16, 18, 31, 33, and 35, chosen because of their association with OPSCC+ cells per microliter of blood (absolute count).HIV viral load was measured by use of the Roche AMPLICOR™ HIV-1 Monitor Test, version 1.5 and reported from <400 to 750,000 copies per mL. CD4 cell counts were measured by use of the Beckton Dickenson BD TriTest CD4 FITC/CD8 PE/CD3 PerCP Reagent and reported as the number of CD4Descriptive statistics were used to report demographic, biological and behavioral characteristics of the study population and to compare subjects who completed the study, defined as having attended visits one to 14 with a maximum of one interval missed visit, to those who were lost to follow-up. HPV infection status was determined at visits one through 14 and evaluated both in terms of individuals and for type-specific infections. In analysis by individual, a subject was considered infected if one or more of the 37 HPV types evaluated were detected at each visit  or detected in any of the samples collected over the course of the study . In analysis by infection, presence of each of the 37 type-specific HPV infections was evaluated for each individual at each visit  or in any of the samples collected over the course of the study .Point and cumulative prevalence of oral HPV ) were calculated for visits one through 14. Average point prevalence represented the weighted average of point prevalence for each visit, using the number of individuals or infections at a given visit as the weight. McNemar's test was used to compare the two-week and six-month cumulative prevalence estimates.The primary goal was to evaluate HPV infection patterns over time, specifically to compare two-week to six-month sampling intervals. These analyses were restricted to the 91 individuals who completed the study. Under the six-month sampling interval, type-specific HPV infections were categorized as “persistent” (+/+) if present at both visit one and 14, “newly detected” (−/+) if absent at visit one and present at visit 14, “cleared” (+/−) if present at visit one and absent at visit 14 or “absent” (−/−) if not detected at either visits one or 14. These categories were distinct from those for the two-week sampling intervals in which infections were categorized as “persistent” if present in two or more consecutive visits, “transient” if detected without persistence, and “absent” if never detected. A persistent infection was considered “cleared” if not detected for at least two consecutive visits. An infection was defined as having “recurred” if it was detected at any visit after clearance.5) HPV copy number  divided by the number of cells in the sample . A logistic transition model was used to evaluate the relationship between the magnitude of the adjusted viral load at one visit and a positive (i.e. non-zero) adjusted viral load at the next visit.To evaluate the relationship between HPV viral load and infection persistence, the adjusted HPV viral load for types 16, 18, 31, 33, and 35 was plotted across visits. The adjusted HPV viral load was the logarithmically converted ratio of  scheduled visits were completed. Almost all ) oral rinse samples were β-globin positive. All controls for specimen collection and processing were appropriately negative for HPV and β-globin.P = 0.52)  . HoweverP = 0.59). However, cumulative prevalence of detection of 37 type-specific infections was significantly higher with a two-week than a six-month sampling interval (9.3% (n = 386 infections) versus 3.8 (n = 158 infections), P<0.0001). The average point prevalence for each of the 37 type-specific infections ranged from 2.1–3.9% at the two-week sampling intervals  in 112 individuals . The aveFor HPV16 infection, the average point prevalence was 5.9%  and the cumulative prevalence was 21.4% . HPV infections with the highest point prevalence (pp) and cumulative prevalence (cp) were HPV39 , HPV52 , HPV35  and HPV16 .The patterns of HPV infection were evaluated for both two-week and six-month sampling intervals among the 91 individuals who completed visits 1 and 14 . Among tAt visit one, 88 HPV infections were detected in 41 of 91 (45%) participants. At the final visit, 69 (78%) were categorized as persistent and 19 (22%) were categorized as cleared , and 56 As would be expected, the number of infections detected with a two-week sampling interval was higher than for a six-month sampling interval . In totaWe then compared the HPV detection results obtained with a two-week to a six-month sampling interval . Almost We evaluated the probability that an infection was detectable for a certain percentage of the two-week visits stratified by their categorization under the six-month sampling interval as persistent, cleared, newly detected or absent . Oral HPP-values range: <0.0001–0.0001). Similar transition analyses focusing on line blot analysis results for the six-month interval sampling (visits 1 and 14) and baseline characteristics  did not show any significant associations (data not shown).We evaluated the relationship between HPV viral load and persistence or clearance of five high-risk HPV types . The pattern of viral load over the course of the study for those individuals with at least one positive line blot assay for a specific high-risk HPV type is shown in Our data indicate a very low probability of interval detection of a type-specific oral HPV infection if the type was not detected at a six-month sampling interval. Furthermore, there was a very high probability that a type-specific infection categorized as persistent at a six-month sampling interval was detectable for the majority of the intervening two-week visits. A six-month sampling interval therefore appears appropriate for natural history studies of oral HPV infection that model factors associated with type-specific persistence.Cervical HPV natural history studies serve as a model for development of analogous studies at other anatomic sites . The clinical algorithm for cervical cancer screening predated natural history studies of cervical HPV infection and predetermined the commonly used six-month sampling interval. Shorter sampling intervals were not examined prior to implementation of large, well-designed landmark natural history studies. The expense, complexity and burden of serial anogenital sampling likely prohibited more frequent sampling. Consequently, cervical infections that would be detected at a shorter interval are generally believed to not be pathologically significant, while persistence of infection at six-month intervals significantly elevates risk for cervical dysplasiaOur initial studies of the natural history of oral HPV infection have focused on a study population with high prevalence, HIV-infected individuals. We acknowledge that the behavior of infections in this population likely differs from what would be observed in an immunocompetent population. For example, transient detection of oral HPV infection was commonly observed with two-week sampling, and the underlying reasons for this observation are unclear. This may be attributable to technical issues, such as variability in factors affecting oral rinse sampling over time, or fluctuation of HPV viral load above and below the lower limit of detection of the Roche line blot, which is known to vary by type. Consistent with such fluctuation around the limit of assay sensitivity is our finding that the majority of cleared infections under the two-week definition subsequently recurred and met criteria for persistence. Although laboratory contamination of the specimen could also be an explanation, we did not observe any evidence of contamination in control samples. Alternatively, in an immunosuppressed population the high frequency of transient, low viral load infection may represent intermittent reactivation of infections due to immunosuppression. Transient detection may also represent exposure in the absence of subsequent establishment of infection. We did not have the interval sexual behavior data which may have allowed us to explore this possibility.The biological significance of transient HPV infections in the oral cavity, oropharynxand cervix is unclear. While the majority of cervical HPV infections are transient infections, persistent infections are significantly more likely to progress to both premalignant and malignant lesionsOur data lend support to the use of a six-month sampling interval for studies designed to evaluate factors associated with type-specific infection persistence. However, misclassification of transient oral HPV infections as absent would result in an overestimation of the median time to clearance of incident oral HPV infections. A further limitation is that HPV viral load was measured only among individuals with at least one positive line blot assay of the corresponding HPV type. Low copy number oral HPV infections below the lower limit of sensitivity of the line blot would not be selected. As these low copy number infections would be expected to have a higher clearance rate than high copy number HPV infection, we may have underestimate the association between viral load and persistence.Oral HPV prevalence in the study population appeared to be a reasonable approximation of the proportion infected over time. However, it is important to note the potential effect of missing data, which may have caused us to underestimate cumulative prevalence. The difference in cumulative prevalence estimates at two-week and six-month sampling intervals indicate that a six-month interval sampling may significantly underestimate the proportion of the study population that has been “exposed” and misclassify individuals as “unexposed”. Therefore, a six-month sampling interval may bias point estimates for factors that increase risk of acquiring an oral HPV infection toward the null.Despite these limitations, this is the first short term natural history study of oral HPV infection and the first to report results on more than two consecutive visits. Based upon this data, a six-month sampling interval is currently being used in our natural history studies of oral HPV infection to evaluate factors affecting type-specific oral HPV persistence.


In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3–6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 ± 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears. © 2000 Cancer Research CampaignThe efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the β-globin gene appeared to be superior for the GTC-based assays. Using competitive β-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 × 10


HIV and HPV can both cause chronic infections and are acquired during sexual contact. HIV infection results in a progressive loss of CD4+ T cells that is associated with an increased prevalence of HPV infections, type-specific persistence and an increase in HPV-associated malignancies. On the one hand this illustrates the important role of HPV-specific CD4+ helper T-cell immunity, on the other it shows the Achilles heel of the HPV-specific immune response. The use of highly active antiretroviral therapy (HAART) results in a rapid reduction of HIV and a reconstitution of systemic CD4+ T-cell levels. The use of HAART thus has the potential to raise immunity to HPV but to the surprise of many, the incidence of HPV-induced diseases has increased rather than declined since the introduction of HAART. Here, the knowledge on how HPV-induced diseases develop in the face of a non-compromised immune system will be used to explain why the effect of HAART on HPV-induced diseases is modest at best. Furthermore, exciting new data in the field of therapeutic vaccines against HPV will be discussed as this may form a more durable and clinically successful therapeutic approach for the treatment of HPV-induced high-grade lesions in HIV-positive subjects on HAART. Human papilloma virus (HPV) is the most commonly sexually transmitted agent worldwide. A high prevalence of HPV has been reported especially among young sexually active individuals. Persistent infection with oncogenic HPV types, in particular HPV16, are causally related to the development of anogenital lesions like cervical intra-epithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN) and anal intraepithelial neoplasia (AIN) as well as their subsequent progression to invasive squamous cell carcinoma . While HThe incidence of cervical as well as anal precursor lesions and cancer is markedly higher in HIV-positive men and women -8 compar+ T-cells and macrophages [Epidemiological studies have shown a strikingly high prevalence of oncogenic HPV types in the general population -15. For rophages . This obrophages -24. In arophages -30. Morerophages . These orophages .Unfortunately, HIV infection results in a progressive loss of CD4+ T cells. When one considers the type of immune response needed to deal with chronic viral infections in general, the frequent detection of HPV-specific CD4+ T-cell reactivity in protected healthy individuals as well as the association between HPV-specific CD4+ T cells and regression of HPV-induced premalignant lesions, it becomes clear why the loss of CD4+ T-cells is associated with an increased prevalence of HPV infections, type-specific persistence and an increase of HPV-associated malignancies . On the HAART consists of an antiretroviral drug regimen which combines inhibitors of the HIV reverse transcriptase and protease. Depending on the HIV disease stage and the level of HIV viral control over time, the use of HAART results in an increase in the number of CD4+ T cells that may reach (near) normal counts within 2-6 years . InitialThe increase in CD4+ T-cell counts coincides with the decrease in several of the AIDS-defining illnesses arising from opportunistic infections and the detection of T-cell responses against agents such as CMV , CandidaThus far, there is no evidence showing that the oncogenic behaviour of HPV is altered by HIV and whilSome previous studies do report a positive effect of HAART on the natural history of HPV-induced pre-malignancies in HIV-infected subjects. A close look at the data shows us that most of the effects noted are among patients with low-grade lesions with response rates of ~35% ,43, whicSo what happens with the HPV-specific immune response in patients on HAART? It is hard to know as there are few studies on the kinetics of HPV-specific immunity during HAART. Fortunately, there is considerable knowledge on HPV-specific immunity in immunocompetent individuals that allows us to understand why HAART cannot simply restore full protective immunity to HPV in HIV-infected subjects.In contrast to the opportunistic pathogens or HIV, HPV is rather a stealthy virus as it causes minimal inflammation, allowing it to persist at detectable levels for 12-18 months in immunocompetent subjects . From a A comparison of immune presentation of opportunistic pathogens and HPV indicates that there is less inflammation and there are lower amounts of antigens available to the immune system with HPV infection. One could compare the presentation of HPV antigens in immunocompetent subjects to that of HIV antigens in patients on HAART, as in both cases the induction of detectable immune responses may take a while. When HPV-induced lesions develop, the production of viral antigens is severely altered due to the loss of a productive infection. Some viral antigens are not produced anymore (e.g. E2) whereas others may increase in time (e.g. E7). Most importantly antigen-presenting cells (APC) that are present in the local region and whose normal role is to ingest and present the viral antigens to T cells, are functionally altered as these APC are exposed to an immunosuppressive environment and become tolerized . As a coIn a large prospective study on the clinical course of low-grade CIN we have studied HPV16-specific immunity in relation to clinical outcome . HPV16-sThe HPV-specific immune response in patients with high-grade CIN lesions is even worse. The accumulated data from a number of different studies on patients with HPV16+ high-grade CIN revealed that HPV16-specific T-cell responses were absent in the circulation of the majority of patients who visit the clinic for treatment of an HPV16+ high-grade lesion. Notably, the quality of the immune response in those patients who did show HPV16-specific reactivity was low in the sense that most of the detected HPV16-specific T-cell responses did not include secretion of pro-inflammatory cytokines such as IFNγ. In the end, more than 75% of all patients with a high-grade lesion failed to develop an HPV16-specific cellular immune response which would remotely resemble that of what was seen in healthy individuals ,59-62. ICurrent literature indicates that HPV-induced lesions are less likely to regress in immunocompetent  or HIV-pIt is not fair to expect that HAART would lead to regression of HPV-induced cancer as this also poses a general problem among immunocompetent patients with cancer. Furthermore, cervical cancer is strongly associated with failure to mount a strong HPV-specific type 1 T-helper and cytotoxic T lymphocyte (CTL) response and the induction of HPV-specific regulatory T cells ,30,70-72Screening and treatment options for CIN and cancer are well established and consequently the incidence of cervical cancer in HIV-positive women has not increased following the implementation of HAART. There is, however, a strong increase in the incidence of anal diseases in both men and women . AlthougIn a recent review of Kreuter et al.  an overvThe clear link between HPV16 and cancers of the cervix, vulva and anal region has prompted the development of two types of vaccines. One type is focused on the prevention of high-risk HPV-infection for which Franceschi and De Vuyst argued that its success to prevent AIN and anal cancer depends on the administration of the vaccine before onset of sexual activity, its protective efficacy in men as well as the willingness to expand vaccine programs to both sexes . The othRecently, a different type of vaccine consisting of overlapping HPV16 E6 and E7 synthetic long peptides (HPV16-SLP), was reported to induce strong and broad CD4+ T-helper and CD8+ CTL responses in >95% of patients with HPV16-induced cervical cancer ,88. The AIN: anal intraepithelial neoplasia; APC: antigen presenting cell; CIN: cervical intra-epithelial neoplasia; CTL: cytotoxic T lymphocyte; HAART: highly active antiretroviral therapy; HIV: Human Immunodeficiency Virus; HPV: Human Papilloma Virus; IFN: interferon; SLP: synthetic long peptides; VIN: vulvar intraepithelial neoplasia.Long peptides of 22-45 amino acid residues that induce and/or enhance antigen specific immune responses). SHvdB is one of the inventors of the patent and reports to serve as a non-paid member of the strategy team and steering committee of ISA Pharmaceuticals, a biotech company which has licensed the patent from the LUMC. SHvdB has not received any payment for speaking, consulting, patents or royalties with respect to the present study.SHvdB in an employee of the Leiden University Medical Center (LUMC), which holds a patent on the use of synthetic long peptides as vaccine (US 7,202,034: JMP has not received any payment for speaking, consulting, patents or royalties with respect to the present study.SHvdB and JMP drafted, read and approved the final manuscript.


E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 104 cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined.To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the  Breast cancer is one of the most prevalent malignancies throughout the world. It is the leading female cancer worldwide and comes second after stomach cancer in Japan , diagnosed during the period between 2000 and 2001, and 82 cases from Kagoshima University Hospital , diagnosed during the period between 2000 and 2006, were examined in this study. In addition, as positive controls, we examined formalin-fixed and paraffin-embedded specimens from six cervical cancer cases diagnosed at Kagoshima University Hospital. Clinicopathological information, including the expression of oestrogen and progesterone receptors, reproductive histories, and family history of breast cancer, was obtained from medical and pathological records. Histological classification for breast cancer was made using the guidelines determined by μm thickness were cut and collected in sterile tubes. To avoid contamination between samples, a cutter blade was changed between each sample cutting. For DNA extraction from paraffin-embedded tissue, each sample was treated with 0.8 ml of lemosol and 0.2 ml of ethanol, and subsequently washed with 1 ml of ethanol. After centrifugation and air-drying, the pellet was resuspended in digestion buffer  containing 200 μg ml−1 of proteinase K  and incubated for 24 h at 56°C. The solution was then heated at 100°C for 10 min, followed by phenol–chloroform extraction and ethanol precipitation of DNA. DNA quality and the absence of PCR inhibitors in samples were tested by PCR for β-globin using PCO3 5′-ACACAACTGTGTTCACTAGC-3′ and PCO4 5′-CAACTTCATCCACGTTCACC-3′ primers under the following PCR conditions: initial denaturation at 95°C for 15 min, 40 cycles with the cycling profile of 95°C for 1 min, 52°C for 1 min, 72°C for 1 min, and final extension for 5 min at 72°C.Sections of 10 L1 gene, were run on a 4% agarose gel and visualized with ethidium bromide staining by electrophoresis. Human papillomavirus typing of the HPV DNA-positive samples was performed using the INNO-LiPA HPV Genotyping v2 test  , and HPV-33-positive cervical cancer specimens were used as positive controls for amplification.E6 ORF amplification were as follows: E6F: 5′-GAGAAACTGCAATGTTTCAGGACC-3′ and E6R: 5′-TGTATAGTTGTTTGCAGCTCTGTGC-3′. The HPV-16 primers for E2 ORF amplification were as follows: E2F: 5′-AACGAAGTATCCTCTCCTGAAATTATTAG-3′ and E2R: 5′-CCAAGGCGACGGCTTTG-3′. The PCR amplification conditions were 2 min at 50°C, 15 min at 95°C, and a two-step cycle of 95°C for 15 s, and 60°C for 60 s for a total of 40 cycles  and 2 × QuantiTect SYBR Green PCR kit . The HPV-16 primers for E2 and E6 genes from 86 to 862 million copies per reaction, served as a standard control. Numbers of the threshold cycle obtained from E2 PCR and E6 PCR were equivalent in each run. Linear plots of the log of copy number vs numbers of threshold cycle were consistently obtained for both genes.Serial dilutions of the full-length HPV-16 genome, containing equivalent amounts of β-globin gene was performed by the 2 × QuantiTect SYBR Green PCR kit (Qiagen) using PC03/PC04 to adjust the differences in the amount of input genomic DNA between samples. A seven-fold dilution series of a human DNA control  was used to generate the standard curve. The amount of β-globin DNA present in each sample was divided by the weight of one genome equivalent  and a factor of 2  to obtain the number of genome equivalents or cell in the sample. Viral loads in each specimen were expressed as the number of HPV copies per 104 cells.Real-time PCR for the neu overexpression was examined, using the Hercep Test kit  following the manufacturer's instructions. Control cell lines provided by the manufacturer (Dako) were used as negative and positive controls. The data on its oevererexpression for 17 HPV-positive breast carcinomas (16 invasive carcinomas and 1 non-invasive carcinoma) and 42 HPV-negative carcinomas (40 invasive carcinomas and 2 non-invasive carcinoma) were obtained from medical records. In the HPV-positive cases, HER2/neu expression was additionally examined in seven cases. Following the Hercep Test criteria, the immunoreaction was scored as follows: 3+, complete and intense membrane staining of >10% tumor cells; 2+, complete but moderate staining of >10% tumor cells; 1+, weak and incomplete staining in >10% tumor cells; and 0, no membrane staining or staining in <10% tumor cells. We defined cases with a score 3+ as HER2/neu overexpression.HER2/P-values presented are two-sided.Fisher's exact test was conducted to test statistical significance using STATA . All the β-globin amplification was carried out to check the quality of DNA in formalin-fixed and paraffin-embedded specimens. All samples were positive for β-globin gene amplification, indicating that DNA was available for molecular analysis.We examined 124 female breast cancer cases, aged from 23 to 90 years with a mean of 55 years. E6 region of HPV genotypes 16, 18, and 33. By using genotype-specific primers, HPV-16 was detected in two new cases. No additional cases of HPV-18 and HPV-33 were detected by genotype-specific approach. Thus, in total, HPV DNA was detected in 26 (21%) breast cancer specimens.First, we examined all samples using novel, broad-spectrum SPF10 primers for HPV detection. Human papillomavirus DNA was positive in 24 out of 124 (19%) breast cancer specimens. As an alternative approach, we examined all specimens using primer sets targeting the neu expression was examined in 23 invasive and 1 non-invasive HPV-positive breast carcinomas. Three (13%) invasive carcinomas had its overexpression (score of 3+) and the non-invasive carcinoma had no HER2/neu overexpression (score=1). On the other hand, in HPV-negative carcinomas, its overexpression was observed in 6 out of 40 (15%) invasive carcinomas, and in 1 out of 2 non-invasive carcinomas. Neither in HPV-positive nor in HPV-negative carcinomas HER2/neu overexpression was associated with any clinicopathological factors, including tumor size, lymph vessel invasion, vascular invasion, and lymph node metastasis (data not shown).HER2/The most prevalent HPV type was HPV-16, which was detected in 24 (92%) out of 26 HPV-positive cases, using the INNO-LiPA HPV genotyping assay and the type-specific approach . Among tE6 and E2 genes per 104 cells, the ratios of E2 to E6, and the viral physical status are compiled in 4 cells was 5.4 in cancers of the breast and 130 480 in cancers of the uterine cervix. In 19 (95%) breast carcinoma cases, E2 DNA was not detected, and in 1 (5%) case, E2 DNA was detected, but the E2/E6 ratio was less than unity. In cervical carcinoma cases, there were two (33%) cases where E2 DNA was not detected. On the other hand, there were four (67%) cases where the E2 DNA was detected, but the E2/E6 ratio was less than unity.All of the HPV-16-positive breast carcinoma specimens were subjected to quantitative real-time PCR analysis to determine the viral load and physical status of HPV-16. The HPV-16 copy numbers of 4 cells, which was higher than that in breast carcinomas (5.4 copies per 104 cells); however, the difference was not statistically significant (P=0.162 by the Mann–Whitney test). The analysis of the E2/E6 ratio in normal epithelium specimens showed that in one (17%) case, E2 DNA was not detected, and in four (66%) cases, E2 DNA was detected but the E2/E6 ratio was less than unity. The remaining one (17%) case had an E2/E6 ratio of approximately one. We also examined 11 normal breast specimens adjacent to HPV-negative breast tumor tissue, but none of them was HPV-positive (P=0.004 by Fisher's exact test).For 11 HPV-16-positive breast carcinoma specimens, we examined the presence of HPV DNA in 1 specimen of morphologically normal breast tissue adjacent to the carcinoma. Human papillomavirus-16 DNA was detected in seven (64%) of these specimens . The geopositive . The difn=26) of breast carcinomas in Japan. Another study conducted in Japan detected HPV DNA in 11.1% of breast carcinomas (P=0.279).In this study, we found HPV DNA in 21% , this assumption does not affect the interpretation of our observations. On the other hand, the lack of amplified HPV-16 E2 genome was considered to indicate the integration of all of the HPV-16 DNA into the host genome. When the E2/E6 ratio was larger than zero and smaller than unity, this condition was considered as a mixture of episomal and integrated forms . It is unlikely, however, that the low viral loads in breast carcinoma specimens were a consequence of formalin fixation and/or heating during paraffin embedding, as a higher viral load was detected in formalin-fixed and paraffin-embedded specimens of cervical cancer obtained from the same institute. Moreover, we performed parallel amplification of a housekeeping gene (β-globin) to obtain an estimate of the amount of amplifiable genomic DNA in each sample, and to help reconcile the inhibitory effects of formalin fixation and paraffin embedding on DNA amplification.In this study, the HPV-16 viral load determined by quantitative real-time PCR in breast carcinoma was with a geometric mean of 5.4 per 10A low copy number of HPV-16 in non-genital carcinomas has also been reported in studies of Chilean lung carcinomas  are known to be involved in breast carcinogenesis  breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered to be integrated into the host genome. However, their extremely low viral loads suggest that it is unlikely that integrated HPV-16 is aetiologically involved in the development of Japanese breast cancer cases examined in this study. As reported by


The origin of adenocarcinomas presenting on the cervix uteri may be doubtful, i.e. whether it is of cervical or endometrial origin, due to the overlapping morphological features. In HPV negative samples, further tests may be needed to ascertain the nature of the tumours. We aimed to explore the use of immunohistochemistry profiles in tissue microarrays in archived samples of adenocarcinoma (ADC) of the cervix from Uganda that tested negative for HPV DNA.Five commercially available antibodies were tested in tissue array sections immunostained utilizing the avidin-biotin (AB) technique. In 26 ADC samples, HPV was detected in 13, p16 in 15 (8 in HPV positive and 7 in HPV negative), CEA in 12, vimentin in 6, ER in 0, and PR in 2. Among the 13/25 HPV negative ADC samples, five were positive for CEA suggesting endocervical origin, and three were vimentin positive .The immunoprofiles of ADC with the antibodies studied are rather nonspecific. By using immunohistochemistry in 13 HPV negative ADC, endocervical tumour origin was suspected in five CEA positive cases while two out of three vimentin positive samples were probably of endometrial origin, suggesting that CEA and vimentin may be valuable in distinguishing HPV negative cervical adenocarcinomas from endometrial adenocarcinomas. Although the role of HPV in cervical adenocarcinoma appears well established -3, the nHPV negative adenocarcinomas in the same site may originate either from cervical or endometrial cells. We postulated that immunohistochemistry could be of use in HPV negative cervical tumours from Uganda.We aimed to explore the utility of immunohistochemistry profiles, i.e. the expression of p16, carcinoembryonic antigen (CEA), vimentin, estrogen receptor alpha (ER) and progesterone receptor (PR), in tissue microarrays in archived samples of ADC that had been tested for HPV DNA, and in particular to assess if it would be possible to define the tissue of origin of HPV negative adenocarcinoma samples. This was based on the use of CEA as a marker of endocervical ADC, vimentin and hormone receptors  and p16 as a proxy marker for HPV infection. p16 is a protein encoded by p16INK4a gene and has been used as an indirect assay for HPV infection . CEA is Cervical carcinoma samples were retrieved from the archives of the Department of Pathology, Makerere University, Kampala, Uganda, and were diagnosed during the period 1968-1990. The study protocol has been approved of Higher Degrees Research & Ethics Committee at Makerere University, Uganda.The cervical carcinoma samples sections selected for HPV testing were digested with proteinase K and the resulting extract was used for PCR. SPF10 PCR was performed using 10 μl of a 1:10 dilution of the DNA extract in a final reaction volume of 50 μl. The amplified PCR products were tested using probe hybridization with a cocktail of conservative probes recognizing at least 54 mucosal HPV genotypes in a microtiter plate format for the detection of HPV DNA. Optical densities (OD450) were read on a microtiter plate reader. HPV DNA positive samples were subsequently analysed by HPV SPF10-LIPA25  , a reverThree punches (gauge 1 mm) of pre-existing paraffin embedded tissues were obtained from each block and then re-embedded in an arrayed master block using the manual tissue microarrayer . The punch specimens were performed in the most representative areas of the tumours, discarding necrosis or artefacts. Four micron paraffin sections were cut from the TMA blocks and deparaffinised through alcohols and xylene before immunostaining.Monoclonal antibodies included: CEA , p16 INK4a, p16 , Vimentin , estrogen receptor alpha, ER , progesterone receptor, PR .Heat Induced Epitope Retrieval (HIER) was done using a pressure cooker as a heating device. Retrieval solutions that were used were: Citrate buffer for Vimentin, PR, ER; EDTA 10% for p16 and Saponin for CEA. Incubation was done at room temperature for 30 min for all the antibodies.The Dako Autostainer universal staining system was used with the EnVision+ Dual Link System-HRP, a two-step IHC staining technique. This system is based on an HRP labelled polymer which is conjugated with secondary antibodies.The final reaction was done with diaminobenzydine and slides were counterstained with hematoxylin. The immunohistochemical stains were interpreted by one of the authors (BL) and the results were reported as positive or negative using the following criteria for each antibody. ER and PR were considered positive when more than 10% of the tumour cells displayed nuclear positive staining. p16 was considered positive/overexpressed if more than 75% of the tumour cells showed cytoplasmic and/or nuclear staining, CEA was considered as positive if more than 10% of the neoplastic cells showed cytoplasmic positive staining, and vimentin was considered positive only when more than 20% tumour cells displayed cytoplasmic staining. The cut-offs for most antibodies were those used at laboratory in Barcelona, while the 75% for p16 was chosen because this cut-off is both sensitive and specific for HPV related cervical adenocarcinoma .The chi-square test was used to determine significant differences between HPV positive and HPV negative adenocarcinomas and expression of, p16, CEA, ER, PR and vimentin.In total 26 samples were successfully analysed for HPV and immunohistochemistry. In these, HPV DNA was detected in 13/26 (50%) of ADC (Table CEA was positive in 12/25 (48%) ADC Table . Seven AHistological subtypes of endocervical and endometrial ADC have some overlapping features, and poorly differentiated tumours or small biopsies are difficult to classify.The differential diagnosis between endocervical and endometrial ADC is a common problem in surgical pathology, that can be approached using different immunohistochemical antibodies, the most accepted panel markers being CEA , vimentin and hormone receptors . HPV DNA detection by molecular techniques has proved valuable . HoweverAs a comparison, 85% of samples were positive in the whole series of the RIS TT study, which used similar HPV testing methods . This coOverexpression of p16 has been used as a surrogate marker of high risk HPV infection . Only 57Thus, endocervical origin seems confirmed in some samples, endometrial cancer were suspected in four others (two vimentin positive and two without conclusive IHC results but were serous ADC) and one could not be further classified due to lack of histological or IHC markers.In view of overlapping and diverge morphologic heterogeneity of both cervical and endometrial adenocarcinomas, the results of this study suggest that the use of a number of markers appear useful in distinguishing the two tumours in HPV negative cases. In a clinical setting, one could use a combination of p16, CEA and vimentin in cervical tumours to determine possible site of origin. Another relevance of our results is in correct classification of cervical tumours for epidemiological research.Our study has a number of strengths: only areas without necrosis were sampled, the use of tissue microarray techniques allowed uniform conditions during immunohistochemical staining, making the results more comparable, and all specimens were tested for HPV using sensitive methods.Our study also has some limitations, such as the small number of samples studied, the probable variation in the fixation of the tissues due to the fact that samples came from different areas of Uganda, leading to loss of antigen epitopes. In particular, estrogen and progesterone receptors are known to be quite labile antigens.In summary, our results showed overlap in expression of p16, CEA and vimentin between HPV positive and HPV negative cervical ADC, and suggest that some samples among the HPV negative ADC, diagnosed as cervical ADC, may be of endometrial origin. In our series amongst 13 HPV negative samples, the endocervical origin seems probable in five CEA positive samples while two vimentin positive samples are suspected to be endometrial cancer. Although the number of samples studied are too few to draw definite conclusions, it suggests that CEA and vimentin may be of value in distinguishing HPV negative cervical tissue from endometrial ADC.ADC: adenocarcinoma; CEA: carcinoembryonic antigen; ER: estrogen receptor; HIER: Heat Induced Epitope Retrieval; HPV: human papillomavirus; IHC: immunohistochemistry; NOS: not otherwise specified; p16: cyclin dependend kinase inhibitor 2A: tumour suppressor protein; PR: progestin receptor; PRC: polymerase chain reaction; SCC: squamous cell carcinoma; TMA: tissue microarrays.The authors declare that they have no competing interests.EW designed the study. MO had responsibility of collection of samples and designing of the database. NG conducted laboratory analysis, while BL contributed to the project with pathological expertise. MO and EW drafted the manuscript, and all authors revised the following versions of the manuscript. All authors read and approved the final manuscript.


Rate of transmission from penis to cervix was lower than that from cervix to penis; 13 different genotypes were sexually transmitted. We examined the transmission of human papillomavirus (HPV) in 25 heterosexual, monogamous couples , followed up over an average of 7.5 months. A total of 53 heterosexual transmission events were observed among 16 couples . Sexual transmission involved 13 different oncogenic and nononcogenic HPV types; 8% were vaccine-covered types transmitted between partners. The overall rate of HPV transmission from the penis to the cervix was 4.9/100 person-months, which was substantially lower than that from the cervix to the penis (17.4/100 person-months). Transmission between the hands and genitals, as well as apparent self-inoculation events (primarily in men), were also observed. Couples who transmitted HPV were more sexually active and used condoms less frequently. These results have implications for HPV prevention and control strategies, including the targeting of prophylactic vaccines. Cervical cancer remains a major source of illness and death among women globally, and infection with oncogenic human papillomaviruses (HPVs) is its principal cause  were collected by using textured paper and a saline-moistened swab  smear was collected, and a swab and cytobrush were used to consecutively sample the ectocervix and endocervix, including the transformation zone. The same methods used for collection of anal, oral, hand, and urine specimens from men were used for collection of specimens from women.DNA was extracted from specimens by using commercial reagents . The PCR used PGMY09/PGMY11 primers to amplify a 450-bp region of the L1 HPV genome  for transmission rates were calculated by assuming a Poisson distribution for the number of events  were enrolled. Six couples left the study, including 2 whose relationships ended. The present analysis focuses on 25 couples with at least 2 visits. Couples were followed up at ≈2-month intervals over an average of 7.5 months.The mean age was 28 years (range 18–59 years) for men and 26 years (range 18–57 years) for women. Participants comprised Caucasians (52%), Asians (8%), Pacific Islanders (6%), and persons of other single- or mixed-race backgrounds (34%). Couples were single/never married (48%), living together as married (32%), separated/divorced/widowed (12%), and married (8%). All participants indicated that the index partner was their only current sexual partner, and all reported vaginal intercourse with penile penetration during the follow-up period.Five men were uncircumcised. At study entry, 1 woman had cervical low-grade squamous intraepithelial lesions, and 2 men had genital warts. Cervical atypia was diagnosed in 2 women during follow-up.Overall, 957 (94%) of 1,015 specimens were adequate based on β-globin detection and were included in the present analyses. At study entry, 7 couples were HPV negative at all anatomic sites, 4 couples had 1 HPV-positive partner , and 14 couples had both partners HPV positive. Of the 14 couples positive for HPV, 3 couples were positive for different HPV types, and 11 were positive for the same HPV type(s) at 1 or more anatomic sites . All 11 All transmission events occurred in couples with 1 or both partners positive for HPV at study entry. A total of 78 transmission events were observed in 16 couples, including 14 male-to-female, 39 female-to-male, 21 male auto-inoculation, and 4 female self-inoculation . OverallMale-to-female transmission was observed in 7 couples. All infections transmitted from male to female partners originated in the penis with or without additional involvement of the scrotum. In particular, the penis shaft was a predominant source of infection either alone or with other genital sites. The cervix and anus were the most frequent targets of transmission from the men. Transmission of oncogenic HPV 16 comprised 1 of the 5 genital-to-cervix events. Male genital to female hand transmission was observed in 3 couples .Female-to-male transmission was observed in 12 couples. Transmission from the cervix and/or urine to the male genitals comprised most female-to-male events. Within penis subsites, the glans was targeted more frequently than the shaft. The anus was frequently an additional source site along with the cervix/urine. In addition, the anus was the sole source of transmission to the male genitals on 4 occasions, of which 3 targeted the partner’s scrotum . The penis and scrotum, respectively, were the most frequent targets of infection from women. There were 4 instances of transmission from the woman’s hands to the man’s genitals, including 1 case in which it was the sole source (couple I). Oncogenic HPV types 16 and 18 were transmitted in only 2 of the 39 female-to-male events.Male self-inoculation was observed in 11 men, including 3 for whom no heterosexual transmission was observed during the entire period of follow-up. Sixteen events involved transmission between different genital sites, 2 involved anal-to-genital transmission, and 3 involved genital-to-hand transmission. Most genital-to-genital events involved transmission between penis subsites. In 3 instances, male self-inoculation immediately preceded transmission of HPV to female partners (couples A and B).Female self-inoculation was observed in 4 females. All involved urine as a source site and 3 of these targeted the hands.Heterosexual transmission of the same HPV genotype to >1 anatomic site (excluding penis subsites) was observed on 13 occasions. Eight of these cases involved transmission from the cervix/urine to the penis and scrotum.Heterosexual transmission of multiple genotypes to the same anatomic site during the same period was observed in 5 instances ; the scrotum was the target site on 3 of these occasions. In 1 case, different genotypes in the cervix and urine were each transmitted to both the penis glans and shaft (couple K).Transmission of some HPV types in preference to others was also observed. In couple L, after cervical infection with HPV 16 and 31 was detected at study entry, only HPV 31 was transmitted to the penis  and scrotum. Alternatively, the female anus may have been the source of infection because HPV 31, not HPV 16, was present in the anus at baseline. In couple D, HPV 16, 59, and 62 were present in the penis shaft at baseline. Subsequently, only HPV 16 was transmitted to the woman’s cervix/urine and anus by the second visit.A number of instances indicated apparent reinfection after viral clearance. In 1 couple (A), the woman’s anus was positive for HPV 39 infection at baseline and at visit 2. By visit 3, HPV 39 had been transmitted to the man’s scrotum, and the woman was negative for HPV 39. HPV 39 had been transmitted (presumably by auto-inoculation) to the penis by visit 4, and the infection remained through visit 5. By visit 6, or 9 months after initial clearance in the woman, HPV 39 had been transmitted to the woman’s anus.Four cases of transmission to the female anus required >4 months of exposure to an infected partner. In 27 incident infections, no source of infection could be ascertained; 17 of these infections were in the male genitals.The rates of HPV transmission by source site are shown in All couples with genital-to-genital transmission reported vaginal intercourse during the period corresponding to the transmission event. Among the 5 couples with penis-to-anus transmission, 4 reported anal intercourse during the corresponding time period.This study demonstrates that HPV is efficiently transmitted between sexual partners and that multiple transmission events can occur within a couple. The rates of genital transmission from women to men were substantially higher than from men to women. Greater rates of female-to-male transmission should imply higher HPV prevalence in men. Studies in men to date, including our own cohort, have reported male genital HPV prevalences at least as high as in women, with most reporting prevalences of at least 20% and up to 73% (Sexual transmission also involved the scrotum, the anus of women, and the hands of both sexes. The oral cavity and semen were not involved in transmission.The anus of women was both a major source and target of heterosexual transmission. We observed consistency between penis-to-female anus transmission and reported anal intercourse during the corresponding period. We previously demonstrated high genotypic concordance between concurrent cervical-anal infections in women, which indicates possible common sources of infection (Transmission through nonpenetrative sexual contact was demonstrated between the female anus and the scrotum, as well as the female hand and male genitals. The male anus was not a major source or a target of HPV transmission. However, 3 of the 4 couples with baseline infection in only 1 partner involved anal infection in men.Male self-transmission frequently involved the scrotum, likely facilitated by passive contact between proximate genital sites. The scrotum may be an important reservoir of infection for penile infections that can subsequently be transmitted to partners. Hands may also serve as reservoirs of infection in both men and women. Auto-inoculation involving the hands may result from casual contact or masturbation.To some extent, our study results suggest that HPV is relatively indiscriminate in its patterns of transmission. We observed the transmission of a given viral genotype to multiple anatomic sites in a partner and concurrent transmission of multiple genotypes to the same site.Other observations suggest that HPV transmission is not entirely arbitrary and may reflect tissue or genotype differences or both. Rates of transmission of oncogenic types to the male genitals from the urine were higher than from the cervix. This may reflect differences in genotypes found in the vagina and vulva compared with those found in the cervix. All transmission events requiring extended periods of exposure involved the female anus target site.A total of 15 genotypes were transmitted, including 13 which were transmitted through heterosexual means. HPV 16 and 18 and nononcogenic HPV 6 and 11, the 4 types included in the current quadrivalent vaccine, comprised <10% of transmitted types. Notably, we observed greater transmission of HPV 16 to the cervix than such transmission by other types, which underscores the possibility of selective transmission of some HPV types.Compared with couples not experiencing HPV transmission, transmitting couples were more sexually active and were more likely to use certain nonbarrier forms of contraception. Few HPV-transmitting couples reported always using condoms during recent sexual activity, compared with over half of nontransmitting couples.A major limitation of our study was the potential for misclassification of HPV transmission events. Variable detection of HPV could be due to natural fluctuation in viral levels or variable sampling of sites could confound the observation of viral transmission. For example, instances of apparent reinfection of sites may alternatively represent possible reactivation of latent infections.Another potential source of misclassification was the lack of a priori knowledge of the time required for HPV to be acquired from an infected partner. Viral transmission could have occurred more frequently than the 2-month visit intervals used in the study, and transmission events could have been missed.Another limitation of the study was the inclusion of couples who had already had sexual contact with one another; initial viral transmission was likely to have occurred before study entry. Indeed, nearly half of the couples had type-specific concordant infections at study entry, indicating previous transmission of HPV, which limited our ability to evaluate incident infections.Because our study relied on self-reported sexual activity, it was subject to recall bias. Furthermore, although all persons reported monogamous relationships, some of the incident infections without a source, most of which involved the male genitals, could have been acquired through sexual activity with another partner. Despite these limitations, the present study included intensive follow-up of a well-characterized cohort, sampling of multiple genital and nongenital sites, and state-of-the-art HPV testing and genotyping methods.The development of comprehensive HPV prevention and control strategies, which incorporate HPV vaccine usage and contraceptive practices, is impeded by lack of information on the risk and routes of sexual transmission between heterosexual partners and potential genotype-specific differences in transmission efficiency. The small size of the cohort and the diversity of genotypes precluded type-specific analysis of transmission.This study contributes to a growing body of knowledge of HPV in men because we directly examined HPV transmission. However, study results are preliminary and need to be verified in larger cohorts. Future HPV transmission studies are critical to address major gaps in our knowledge of the natural history of this virus.HPV transmission events in male-female couples by anatomic site*


At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV.The objective was to assess the clinical and epidemiological correlates of HPV-seroreactivity in a cohort of HIV-negative and HIV-positive women.Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study.No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women . Importantly, this association did not loose its significance when controlled for confounding from age (continuous variable) in multivariate analysis or using Mantel-Haenszel test for age-groups.It is tempting to speculate that HIV-positive current smokers comprise a special high-risk group, with highly impaired immunological response that could prevent eradication of persistent HPV infections and thus contribute to development of CIN3/CC. Human Papillomavirus (HPV) infection, known to be associated with both benign and malignant tumours at different mucosal sites, is widespread among female and male populations ,2. The oVLP-based assays have been recognized as reliable genotype-specific assays ,15,21 anTo improve the understanding of correlation between infection and antibody response in patients with and without cervical lesions, prospective cohort studies using a widespread panel of viral antigens would be needed ,15,26. SWe have recently reported the development of an in-house ELISA test based on five recombinant HPV16 proteins expressed in E. coli: L1 and L2 capsid proteins, E6 and E7 oncoproteins and the non-structural E4 protein, all used in unfolded form . The ideIn the present study we have correlated the antibody response, evaluated with our system, to the baseline epidemiological, clinical and HPV DNA data of women enrolled in a multi-centre cohort study designed to explore pathogenetic mechanisms and prognostic factors of CIN lesions in HIV-positive and HIV-negative women, known as HPV-PathogenISS study .The recorded epidemiological characteristics of the enrolled women have been correlated to their serological response to either all HPV16 antigens, or separately E and L proteins , as compared with 75.9% among all other women , with OR = 0.158 (95%CI 0.036–0.695). This association was not confounded by age, when tested either using Mantel-Haenszel test for age groups  (p = 0.028) or logistic regression analysis for age as continuous variable  (p = 0.020). On the other way round, women who are HIV-negative and not smokers are significantly more likely to mount antibody response .In a previous study we have reported the baseline data of a cohort comprising HIV-positive and HIV-negative women . The preWe have correlated the women's epidemiological characteristics to their serological response to HPV16 antigens, using three possible outcomes; i) seroreactivity to either E or L (= HPV seropositive or not), ii) reactivity to E, and iii) reactivity to L proteins. Only a few of the recorded epidemiological variables proved to be significantly related to serostatus of the women in this cohort. Interestingly current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers  (p = 0.009), while the difference in reactivity to E and E+L antigens was not significant. This is in full agreement with a recent report, where almost identical observations were reported . In thisThe relationship of contraception and HPV infection has been incompletely studied and only in relation with HPV DNA detection ,45,46, bThe relationship between HPV DNA detection and antibody response to different HPV antigens is highly complex. Large sero-epidemiological studies seem indicate that antibody response to viral antigens does not invariably occur during natural HPV infection ,15. SimiInto the HPV-PathogenISS cohort, HIV-positive women were also enrolled . As geneTwo other observations merit some discussion. We noticed that seroresponse to L proteins declined in parallel with the increasing severity of abnormality in the referral PAP smear, from 100% among ASCUS cases to 56.3% among those with HSIL Table . Women were selected for having abnormal PAP test  . The serA careful sampling protocol for HPV testing was followed in all clinics. Both exo- and endocervical specimens were collected and stored at -70°C, until transported to the two virology laboratories (Tor Vergata and ISS) for HPV analysis. HPV genotyping was performed by sequence analysis, as previously described . When thThe patient HIV-serostatus was determined at the entry with ELISA test and confirmed by Western blot, with the informed consent of the patients. The state of HIV disease in sero-positive patients was assessed by CD4+ and CD8+ lymphocytes counting and recording the opportunistic infections or neoplasia. Unfortunately CD4 parameter was not available for all HIV-positive patients. All these patients were being treated with highly active anti-retroviral treatment (HAART). HIV-positive patients in this study were only 12, of whom 9 current smokers.The collection and treatment of sera of the 96 women have been previously described . ControlThe presence in the sera of anti-HPV antibodies was evaluated by an ELISA test based on the recombinant L1, L2, E4, E6 and E7 HPV16 proteins . A cut-oAvidity of the specific anti-HPV IgGs was evaluated and the AI, expressed as a percentage of the absorbance in ELISA with urea wash and the absorbance in ELISA without urea wash × 100 . Because® software package . Frequency tables were analysed using the Chi-square test, with likelihood ratio (LR) or Fisher's exact test being used to assess the correlation between categorical variables. OR and 95% confidence intervals (95%CI) were calculated where appropriate, using the exact method. Differences in the means of continuous variables were analysed using non-parametric tests  or ANOVA . Logistic regression analysis was used to control for the confounding from age to some observed significant univariate predictors of seroreactivity, e.g. HIV/smoking status. Confounding was also controlled by calculating the common odds ratio (OR) for the stratum-specific estimates, using the Mantel-Haenszel test. To evaluate the statistical power of the study (e.g. for age differences), we used STATA/SE 9.2. power analysis option, with the default value (0.05) for alpha, and using the true sample sizes of the women in the two groups, e.g. seropositive and -negative women. In all tests, the values p < 0.05 were regarded statistically significant.Statistical analyses were performed using the SPSSCG conceived the study, participated in his design and coordination and in rafting the manuscript, PDB, FG, SM, MGD participated in acquisition analysis and interpretation of serology data, LA participated in interpretation of data and critically revised the manuscript, MB, SC, LM, AA, MC participated in acquisition of serum samples and epidemiological data, KS participated in design of the study, performed the statistical analysis and participated in drafting the manuscript.