The HPV prevalence and genotype distribution are important for the estimation of the impact of HPV-based cervical cancer screening and HPV vaccination on the incidence of diseases etiologically linked to HPVs. The HPV genotype distribution varies across different geographical regions. Therefore, we investigated the type-specific HPV prevalence in Czech women and men with anogenital diseases.We analyzed 157 squamous cell carcinoma samples, 695 precancerous lesion samples and 64 cervical, vulvar and anal condylomata acuminate samples. HPV detection and typing were performed by PCR with GP5+/6+ primers, reverse line blot assay and sequencing.The presence of HPV types related to the vaccinal ones  were detected in 16% of carcinoma samples and 18% of precancerous lesion samples. HPV 16 and/or 18 were present in 76% of cervical cancer samples, 33% of CIN1, 43% CIN2 and 71% of CIN3 samples. HPV types 6 and/or 11 were detected in 84% samples of condylomata acuminate samples.Thirty different HPV genotypes were detected in our study, HPV 16 being the most prevalent type both in precancerous lesions (45%) and squamous cell carcinomas (59%). In benign lesions, HPV 6 (72%) was the most common type. Altogether, 61% of carcinoma samples and 43% of precancerous lesion samples contained HPV 16 and/or 18. The prevalence of vaccinal and related HPV types in patients with HPV-associated diseases in the Czech Republic is very high. We may assume that the implementation of routine vaccination against HPV would greatly reduce the burden of HPV-associated diseases in the Czech Republic. Human papillomaviruses (HPVs) have been established as etiological agents of invasive cervical cancer (CC) In spite of the high burden of cervical cancer in Central and Eastern Europe Squamous cell cervical carcinoma (SCC) samples as well as precancerous lesion samples from different anatomical locations were selected from the biobank of the National Reference Laboratory for Papillomaviruses in Prague. These samples were collected between 1993 and 2005, stored at −20°C and analyzed in previous studies.Cervical scrape and biopsy specimens were obtained from women visiting hospital gynecology departments and selected centers of gynecologic-oncology prevention in the Czech Republic nd Faculty of Medicine, Charles University, Prague for squamous cell vulvar carcinoma (VC), vulvar intraepithelial neoplasia (VIN) and vulvar condylomata acuminata (VCA) were also included in the study. The patientś characteristics and histological data were published before Samples from patients surgically treated in the Department of Obstetrics and Gynecology of the 2Twenty-two samples from patients with squamous cell carcinoma of the anus (AC)  and 10 samples of anal condylomata acuminata (ACA) samples , were analyzed. Details on the population, sample preparation and pathological classification were published before Overall, 157 cancer samples from multiple locations, 695 pre-malignant neoplasia samples, and 64 condylomata acuminata samples were included in this study.No informed consent was needed from the patient by the course of law in the Czech Republicbefore 2000. All patients enrolled after the year 2000 signed an informed consent form and the study was approved by the institutional ethics committee PCR and reverse line blot hybridization (RLB) were used for the detection and genotyping of the HPV DNA in samples Detected HPV types were classified into low-risk (LR) , high-risk (HR)  and probably high-risk (pHR)  types of the genus Alpha that contains the mucosal types of HPV To confirm the presence and integrity of the human DNA, beta-globin PCR analysis by PC03/04 primer set The laboratory is accredited according to ČSN EN ISO 15 189 and participates regularly in external control of quality programs organized by INSTAND (Germany) and Mendel Center for Biomedical Sciences (Cyprus). Furthermore, the laboratory participated twice in WHO HPV LabNet Proficiency Study of HPV DNA Typing organized by the WHO HPV Global Reference Laboratory http://www.ncbi.nlm.nih.gov/BLAST/).To determine the type of HPV in the specimens positive by RLB only on the agarose gel but not by RLB hybridization, the remaining aliquots of PCR amplicons were used for nucleotide sequencing. The 150 bp products were cut out of the 2% NuSieve GTG agarose gel , purified using the MinElute™Gel Extraction Kit  and directly sequenced using the BigDye® Terminator v1.1 Cycle Sequencing Kit . The sequence analysis was performed on the ABI PRISM 310 genetic analyzer (Applied Biosystems) and the sequences were analyzed by Chromas software and evaluated by BLAST software  test. For contingency tables, the standard chi-square test and the Fisher exact test were used. The prevalence ratios in SCC in comparison to CIN2, 3 and CIN1 with 95% confidence intervals (CI) were determined using GraphPad InStat (version 3.00) . All tests were two sided and the significance level was p = 0.05. For assessing the possible impact of HPV vaccines on the prevention of HPV-associated cancer, we estimated the number of cervical cancer cases attributed to 8 HPV types most prevalent in the Czech Republic. We used the numbers of incident cases of cervical, vulvar and anal cancers in the Czech Republic published in 2010 Altogether, 157 carcinoma samples were available for HPV DNA testing. Patients with SCC were significantly younger (P<0.001) than those with other types of carcinomas see . One hunOverall, we detected 9 HR  2 pHR  and 1 LR (HPV 42) HPV types in different types of carcinomas, of which 11 different HPV genotypes were found in SCC samples while the spectrum of HR HPV types in other types of carcinomas was much narrower. Only HR HPV types 16, 33 and 45 were found as a single infection. HPV 16 was the most prevalent type in cervical 73% (63/86), vulvar 25% (12/49) and anal 82% (18/22) carcinomas, followed by HPV 33, 45, 18, and 31 in descending order.HPV vaccinal types (HPV 16 and/or 18) were detected as a single infection in 50% (79/157) of tumors and as a coinfection with other HR HPV types in additional 10% (16/157) of samples. Altogether, 61% (95/157) of analyzed malignant tumors contained one or both vaccinal types. The presence HPV types either closely or distantly related to the vaccinal ones, i.e. HPV 31 and 45 and HPV 33, 52, and 58, respectively, was detected in additional 5% (8/157) and 11% (11/157) of carcinoma specimens, respectively. Sixty-five (76%) of 86 cervical cancer samples contained HPV 16 and/or 18 as a single or multiple infection. The presence of HPV types either closely or distantly related to the vaccinal ones, i.e. HPV 31 and 45 and HPV 33, 52, and 58, was detected in additional 8% (7/86) and 8% (7/86) of SCC samples, respectively.A total of 695 precancerous lesion samples were available for our analyses: 338 from CIN1 cases, 111 from CIN2 and 200 from CIN3 cases and 46 from VIN cases. Median age of women with cervical lesions was substantially and statistically significantly lower (P<0.0001) compared to that of patients with VIN (see study design). Overall, the prevalence of HPV DNA was 76% (528/695). HPV infection was detected in 62% (209/338) of CIN1 samples, 77% (85/111) of CIN2 and 94% (188/200) of CIN3 samples and 100% (46/46) of VIN samples . Among tIn comparison to carcinomas, precancerous lesions contained a larger variety of HPV types. As shown in More than a half of samples contained a single HPV type (56%). Multiple infection was found most commonly in cervical precancerous lesion samples: in 20% (66/338) of CIN1, 21% (23/111) of CIN2 and 22% (44/200) of CIN3 samples. Most multiple infections were coinfections with two or three HPV types. Coinfection with four HPV genotypes was only found in six cervical lesion samples.The vaccinal types HPV 16 and/or 18 were present in 34% (239/695) of precancerous lesion samples as a single infection, in 1% (8/695) as a multiple infection (combined HPV16/18 infection), and in 13% (87/695) in combination with other HPV types. The presence of the types either closely or distantly related types to the vaccinal ones: HPV 31 and 45 and HPV 33, 52, and 58, respectively, was detected in 9% (59/695) and 11% (79/695) of samples, respectively.Altogether, 33% (110/338), 43% (48/111) and 71% (141/200) of CIN1, CIN2 and CIN3 samples, respectively, were positive for HPV 16 and/or 18. Closely related types HPV 31 and 45 were present in 9% (31/338),11% (12/111) and 7% (14/200) of samples, respectively, and distantly related types HPV 33, 52, and 58 in 9% (30/338), 14% (16/) and 13% (25/100) of samples, respectively. LR HPV types were detected in 9% of precancerous lesion samples. HPV 6 and/or 11 were present as either a single or multiple infection in 3% of all precancerous lesion samples, more precisely in 5% (18/338) of CIN1 samples, 1% (1/111) of CIN2 and 1% (1/200) of CIN 3 samples and 9% (4/46) of VIN samples.HR HPV types as a single infection were only detected in 4% (2/54) of VCA samples and as a multiple infection together with LR types in additional 15% (8/54) of VCA samples, while ACA samples did not contain any HR HPV type, with a single sample being positive for two LR types. In VCA samples, 11 different HPV types were detected: 4 were HR , 2 pHR  and 5 LR  HPV types .Altogether, vaccinal HPV types 6 and/or 11 were present in 84% (54/64) of condyloma acuminatum samples from different anatomical locations, in 87% (47/54) of VCA samples, and 70% (7/10) of ACA samples.The overall prevalence of HPV was higher in SCC (95%) in comparison to CIN1 (62%) , CIN2 (77%)  but not in CIN3 (94%)  cases. Similar results were obtained for any HR HPV type detected and for seven HR HPV types most prevalent in SCC samples . For eacIn the Czech Republic 1300 cervical, vulvar and anal incident cancer cases occur, more precisely 990 cervical cancer cases, 189 vulvar cancer cases, and 121 anal cancer cases In this study we provide the largest summary data on the type-specific HPV type specific prevalence in the population of Czech women and men with diseases of the anogenital tract associated with HPV infection. Importantly, the prevalence rates of the vaccinal HPV types as well as those of HPV types which have shown a partial cross protection in clinical trials In this study we analyzed 157 squamous cell carcinoma samples, 695 precancerous lesion samples and 64 condylomata acuminata samples from different anatomical locations. A very sensitive method was used, which is based on the amplification of a short DNA fragment of HPV L1 ORF and allows detection of multiple HPV infections The data on HPV prevalence in precancerous cervical lesions and invasive cervical cancer cases in the Czech Republic were evaluated previously on another set of specimens The published data on the type-specific HPV prevalence in patients with HPV-associated diseases in the Central and East European countries are scarce and most studies have analyzed only very small numbers of specimens. While more studies from Central and East Europe on HPV prevalence in CC were published and included in the meta-analyses The type-specific HPV prevalence in CIN2+ samples found in our study, is the same as the data reported for Europe (88%), with the exception of HPV 16 and/or 18 (61 vs. 52%) In comparison to the summary data for Europe as published by Clifford et al. The present study has shown a significantly higher prevalence of any HPV type, as well as of HR HPV type among SCC cases in comparison to CIN1 (p<0.0001 for both)and CIN2 cases  . The preA meta-analysis of HPV prevalence studies in precancerous lesions and vulvar and anal carcinomas The overall prevalence of HPV as well as HPV type distribution in VC is in our study comparable to other studies. Nowadays it is widely accepted that about 40% of VC cases can be etiologically linked to HPV.Importantly, focused on the prevalence of both vaccinal and cross-reactive HPV types, this study revealed that altogether 43% of precancerous lesions of the cervix and vulva  are caused by HPV 16 and/or 18 and additional 20% by HPV types related to the vaccinal ones . Therefore, a substantial number of precancerous lesions can be considered preventable by prophylactic vaccination in the Czech Republic. The vaccinal LR HPV types HPV 6 and/or 11 were detected in 5% of CIN1, as few as 1% of CIN2 and 0.5% CIN3 and 9% of VIN cases.The overall prevalence of HPV 16 and/or 18 among the analyzed cancer cases was 61% and that of the closely or distantly related types was 12%. The lowest prevalence of HPV 16/18 was observed in VC cases. Based on our data, the development of vulvar cancer can be prevented in about half of cases, thus reducing the need for mutilating surgery that dramatically reduces quality of life for patients.The rate of SCC cases attributable to HPV 16/18 infection in the Czech population is 76%. Even higher is the involvement of HPV 16/18 in AC cases (82%). In view of cross-protective effect of the available vaccines, we can expect the potential benefit from vaccination against HPV in preventing SCC to be as high as 92% for the Czech population.Finally, specimens with the histologically confirmed presence of condylomata acuminata were analyzed. Even though only a limited number of samples were available, the information is very important for the planning of the preventive strategies. We have shown that 89% of these lesions are infected by LR HPV types, with vaccinal types HPV 6/11 being present in 84% of them. These data should be taken into account when considering population-based prophylactic vaccination against HPV.In conclusion, our study reports on the type-specific prevalence of HPV in benign, premalignant and malignant lesions of the anogenital tract in women and man. The prevalence and spectrum of HPV types detected in the Czech Republic are comparable to the data reported for European countries. The observed differences can be mostly attributed to the variation in the methods used for HPV detection. The proportion of patients infected with vaccinal and closely or distantly related HPV types is much higher than originally proposed. Approximately 952 of 1300 incident cancer cases  and 921 of 990 CC cases can be attributed to these HPV types in the Czech Republic. Furthermore, it has been shown that the implementation of routine vaccination not only resulted in decrease in incidence of atypical cervical cytology and precancerous cervical lesions but also in the reduced need for colposcopy and invasive treatment procedures


Background. Oral lichen planus (OLP) is a common chronic inflammatory immune-mediated disease, with an etiopathogenesis associated with cell-mediated immunological dysfunction. Viral infection has been hypothesized as a predisposing factor in the pathogenesis of this disease. Viruses may alter host cell function by inducing the abnormal expression of cellular proteins leading to disease development. However, reports on the relationship between human papillomavirus (HPV) and OLP are inconclusive. Objective. To explore the association between HPV and OLP in Thai patients. Materials and Methods. DNA was extracted from thirty-seven fresh-frozen tissue biopsy specimens from OLP lesions, and polymerase chain reaction assay for the L1 and E1 genes covering 32 types of high- and low-risk HPV was performed. Results. HPV DNA was detected in one tissue biopsy from an atrophic-type OLP lesion. All control samples were negative. Genomic sequencing of the E1 gene PCR product demonstrated that the HPV-type 16 found in the lesion is closely related to the East Asian type. Conclusion. Our data indicate a low prevalence of HPV infection in OLP lesions in Thai patients. Oral lichen planus (OLP) is a chronic inflammatory disease in which the immunopathogenesis involves cell-mediated immune dysregulation . OLP aff+T cells trigger apoptosis in oral epithelial cells [+T cells is similar to what occurs during a viral infection where a virus can act as a cytoplasmic antigen or induce the expression of host cell proteins, resulting in an altered host cell protein profile [OLP is classified as a potentially malignant disorder (PMD) of the oral mucosa with a transforming rate of 0–6.25% , 4. Histal cells . Exogeno profile . TherefoHuman papillomaviruses (HPVs) are epitheliotropic DNA viruses with more than 150 genotypes. Clinically, HPV infection is usually characterized by hyperplastic, papillomatous, or verrucous lesions in the stratified epithelium. HPV classification has been based on the degree of HPV DNA homology. HPV has been detected in various types of oral lesions, ranging from benign to malignant , 9. HPV A causal role of HPV has been reported for OLP and OSCC, but there are wide variations in disease prevalence with regard to different geographic populations , 18. FurMost HPV infections are asymptomatic; however, lesions can develop any time after infection. Currently, there is no clear understanding of HPV latency, reactivation, or subclinical infection without apparent disease . The couThe aims of this study were to investigate the presence of HPV DNA in OLP lesions of Thai patients using PCR and to characterize the virus type in those lesions by nucleotide sequencing. Participants with a clinical diagnosis of OLP who visited the Department of Oral Medicine, Faculty of Dentistry, Chulalongkorn University from November 2008 to August 2010 were recruited into the study. Prior to enrollment, the patients signed informed consent forms approved by the Ethics Committee of the Faculty of Dentistry. None of the participants received any medication for their OLP before specimens were obtained. Specimens were collected from tissue biopsies for histopathological examination. Thirty-seven cases that were confirmed as OLP positive based on histopathological evaluation were selected for DNA extraction. Each specimen was transferred to a 1.5 mL sterile microcentrifuge tube containing 0.5 mL of virus transport media. Matching controls from each subject were obtained by scraping healthy normal mucosa into a 1.5 mL sterile microcentrifuge tube containing virus transport media. All samples were kept on ice, immediately taken to the laboratory, and stored at −80°C until further analysis.μL.DNA extraction and purification from both tissue biopsies and negative control cells were performed using the Qiamp DNA mini kit  per the manufacturer's instructions. DNA extraction from control sites was performed by standard organic extraction (phenol-chloroform) and alcohol precipitation. Purified DNA was re-suspended in deionized water to a final volume of 30 μL of product from the first round as template and HPV-E1F2_1383 (nt 1315–1335) sequences; 5′-GCGAAGACAGCGGNTATGGC-3′ and HPV-E1R1_2119 as primers. These primers yield a PCR product of 785 bp. The use of these consensus primers allowed the amplification of a broad spectrum of HPV-type DNA. The MY09/MY11 and GP5+/GP6+ primer sets are the most widely used in detecting the HPV L1 gene region. The house keeping β-globin gene was used as an internal control. The PCR products were subjected to 2% agarose gel  electrophoresis, and appropriately sized bands were purified using the HiYield Gel/PCR DNA Fragments Extraction kit (Bioscience). The purified DNA fragments were sequenced by FirstBASE Laboratories SDNBHD  and were analyzed using the BLAST analysis tool in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). We assayed for HPV DNA using primers to both the E1 and L1 regions. The primer design, amplification, and sequencing were performed . BrieflyThe sequences were aligned with the BioEdit program (version 7.0.4.1). The phylogenetic trees were constructed using the MEGA4 program with neighbor-joining analysis, and Bootstrapping applied with 1,000 replicates was used to support tree topologies. The study comprised 37 participants, ranging from 19–78 years old, with a median and mean age of 48 and 49 (±12.58) years, respectively. The male to female ratio was 12 : 25. The 37 specimens were obtained from 12 hyperplastic-, 23 atrophic-, and 2 ulcerative-type OLP lesions. All subjects had lesions on both the buccal mucosa and gingiva at the time of biopsy. All specimens from the tissue biopsies had a confirmed diagnosis of OLP . HPV DNAOLP affects 1.27% of the global population with a prevalence varying according to geographic locations [The present study supports the finding of a previous report on HPV prevalence in OLP in Thai patients. A prior study in Thai patients did not find HPV in any of the sixteen OLP lesions examined , while iA systematic review shows a statistically significant twofold difference (11% versus 23%) between HPV associated with OLP lesions and HPV found in normal tissue . Among sin vitro study of HPV is difficult since HPV cannot be grown using conventional cell culture methods, requiring other techniques. The choice of analysis technique used is also important. Kellokoski et al. [2 copies μL−1 and also allows an accurate identification since it can amplify long nucleotide sequences [The i et al.  reportedi et al. . PCR ampi et al. , 28. Theequences . in situ hybridization on paraffin embedded specimens did not find any relationship between HPV and OLP [Efforts in exploring the correlation between HPV and OLP have mainly focused on epidemiological studies of different populations. It is noteworthy that there have been profound variations in the results found among geographically different populations . Studies and OLP , 30. In  and OLP , 23–26.  and OLP , 31, 32. and OLP , 24, 33. and OLP  reportedThe DNA sequence of the HPV found in the present study was identified as the high-risk-type 16. The lesion positive for HPV DNA was of the atrophic type. Mattila et al.  reportedA predisposition of the oropharyngeal mucosa to malignant transformation by HPV was first suggested when HPV16 was detected in tumors of the tongue, tonsil, and pharynx but not in control tissues . FurtherOLP is an inflammatory immune-mediated disease associated with cell-mediated immunological dysfunction. Infectious agents have been proposed as one of the causes of OLP. Our data showed a low prevalence of HPV infection in OLP lesions. We conclude that HPV may not play a significant role in Thai patients with OLP. Genomic sequencing of the E1 gene demonstrated that the HPV-type 16 from the sole positive lesion was closely related to the East Asian type. Although it is likely that additional factors affect malignant transformation, identifying these will require the long-term followup of HPV-positive OLP patients.


Risk factors for lung cancer, such as cigarette smoking, environmental pollution, asbestos, and genetic determinants, are well-known, whereas involvement of the human papillomavirus (HPV) is still unclear.We examined a series of 100 lung cancer patients from Italy and the UK for the presence of HPV DNA in both lung tumor specimens and adjacent non-tumoral specimens from the same patients. Thirty-five of the most clinically relevant HPV types were assayed using PCR amplification of the highly conserved L1 region of the viral genome followed by hybridization with specific probes.No HPV was detected in tumor specimens nor in normal lung tissue of any patient.These data indicate that, in this Western series, HPV is not associated with the risk of lung cancer. Our findings will help refine estimates of lung cancer risk in patients affected by a common viral infection involved in other types of human cancer. Human papillomavirus (HPV) is the causal agent of cervical carcinoma and the high-risk HPV types 16 and 18 are consistently detected in about 70% of cervical carcinomas in the world , for lunA recent meta-analysis highlighted the wide variability in HPV prevalence in primary lung cancer specimens, ranging from 0% to 78.3% worldwide, and noted higher frequencies of HPV in Asian than in European countries . A recenPatients were recruited from three hospitals: Istituto Nazionale Tumori, Istituto Europeo di Oncologia  and Royal Brompton Hospital . Study protocols for recruitment were approved by the ethics committees of the three hospitals. Each subject gave informed consent to the use of their biological samples for research purposes. All patients underwent lung lobectomy and the excised specimens were pathologically examined and stored frozen. In addition, from each patient, a small section of normal lung parenchyma distant from the macroscopic lung cancer tissue was removed at surgery and stored frozen. All the tumors were clinically staged, with a prevalence of stage I patients (54%); 19% of patients were non-smokers Tables . Within Genomic DNA was extracted from both tumoral and adjacent non-tumoral lung tissue using the DNeasy Blood & Tissue Kit ; it was quantified using Picogreen dsDNA Quantitation Kit .For the detection of HPV infection, we used the Clinical Array Technology (CLART) HPV 2 kit , which combines highly specific and highly sensitive PCR with the technology of low-density arrays. The method is based on the PCR amplification of a 450-bp fragment within the highly conserved L1 region of the viral genome followed by hybridization with specific probes for each HPV type. This method allows detection of minimal quantities of viral DNA of up to 35 of the most clinically relevant HPV types, including 20 types considered high risk  and 15 types classified as low risk  for cervical cancer.The whole procedure was performed in two physically separated areas: the pre-PCR area, where samples were prepared and DNA was extracted, and the post-PCR area, where products were amplified and then visualized, and strict procedures were developed to avoid specimen contamination. For each HPV test, a pair of primers permitting the amplification of a fragment of the human CFTR gene was used as a genomic DNA control; this was essential for confirming a negative result, since it indicated the presence of DNA from the patient even if HPV was not found. Also, a pair of primers for the amplification of a modified plasmid was used as a PCR control; this was essential to distinguish between an inhibited amplification reaction and a sample that contained no DNA.We have used previously analyzed cervical cytobrush specimens that were selected as either negative or positive controls. Negative controls derived from patients with negative histological and cytological findings, and resulted HPV negative; positive controls derived from patients with positive histological and cytological findings and found positive for either HPV-6 or HPV-16 genotype (Figure All samples were successfully amplified using the CFTR gene as a positive control for DNA quality and PCR reaction Figure .Among the lung tumor specimens tested for HPV, five were initially found weakly and doubtfully positive for HPV44 DNA, but repetition of the test with a new assay kit lot indicated that they were negative. Therefore, all the tumor samples were deemed free of HPV infection Table . Among tOur findings of no HPV DNA in lung cancer specimens in the present Italian-British series is in agreement with Koshiol et al.’s previous study on an independent Italian series . The facAltogether, our findings strongly indicate the absence of a pathogenic role of HPV in lung cancer in a Western population.The authors declare no conflict of interest.AG wrote the paper and discussed results; SN collected samples, and isolated DNA; FT and CL carried out HPV genotype detection; SF designed the study and analyzed results; UP recruited patients and collected clinical data; TAD supervised all experiments performed as principal investigator. All participants contributed commentary on and corrected the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/512/prepub


Background. There are limited data on high-risk human papillomavirus (hr-HPV) genotypes among HIV-positive women in Africa, and little is known about their relationship with cervical cytology in these populations. Methods. We conducted a cross-sectional study among 194 HIV-positive women  to evaluate HPV genotypes among HIV-positive women with normal and abnormal cytology. Cervical samples were genotyped for HPV types, and slides were evaluated for atypical squamous cell changes according to the Bethesda classification system. Results. Prevalence of high grade squamous intraepithelial dysplasia (HSIL) was 9%. Overall, more than half (56%) of women were infected with an hr-HPV type; 94% of women with HSIL (n = 16), 90% of women with LSIL (n = 35), and 42% of women within normal limits (WNL) (n = 58) tested positive for hr-HPV. Overall, the most prevalent hr-HPV subtypes were HPV16 (26%) and HPV52 (30%). Regional differences in the prevalence of HPV18 and HPV35 were found. Conclusion. Regional differences in HPV genotypes among African women warrant the need to consider different monitoring programmes for cervical preneoplasia. HPV-based screening tests for cervical preneoplasia would be highly inefficient unless coupled with cytology screening of the HPV-positive sample, especially in HIV-positive women. Cervical carcinoma is the second most prevalent cancer in the world, and the most common female cancer in sub-Saharan Africa. Infection with high-risk human papillomavirus (hr-HPV) is the main risk factor for the development of cervical carcinoma. HIV-positive women are reported to be almost twice as likely to be concurrently infected with HPV than HIV-negative women. Recently introduced HPV vaccines against genotypes 16 and 18 are designed to prevent about 70% of cervical cancers. The basis for selecting these genotypes was their prevalence in Europe and North America . In thisThere are limited data on hr-HPV genotypes among HIV-positive women in Africa, and little is known about their relationship with cervical cytology in these populations. We carried out a study to determine the prevalence of HPV genotypes among HIV-positive women in Africa with normal and abnormal cervical cytology scores. Cervical samples were collected from women attending the HIV treatment clinic of Sekou-Toure regional hospital and surrounding hospitals in Mwanza, Tanzania between February and March 2010, as part of a randomized controlled trial. Eligible subjects were nonpregnant, HIV-positive females, over 18 years of age who had used antiretroviral therapy (ART) for at least 6 months. Subjects were excluded if pregnant, breastfeeding, intolerant to lactose or fermented milk. In total 168 Tanzanian women were included in the study, of whom 143 women had cervical swabs adequate for HPV testing at baseline. The Medical Research Coordinating Committee of the National Institute for Medical Research, Tanzania, approved the study design and protocol. Participants were informed of the purpose of the study and gave their signed or thumb-printed informed consent before participation  [Cervical samples collected in 2008 from HIV-positive women attending the Ndlovu Medical Centre in Elandsdoorn in Moutse District, South Africa, were also included in this analysis. In total, 51 women testing positive for HIV were included. Information was provided to the study participants and oral consent was obtained by the doctors for a cervical sample to be examined. Pregnant women and those aged ≤16 years were excluded. None of the HIV-positive women were receiving ART at the time of sample collection. Cervical samples from all participants were collected by a clinician using a standard sampling brush, and placed into a vial with coagulant fixative Boonfix, which consists of ethyl alcohol, low molecular weight PEG, and acetic acid. This substance has no detrimental effects on DNA preservation or DNA isolation . The fixA ThinPrep Cytology slide was made of the Boonfixed samples, and stained with the Papanicolaou method. The Cytology ThinPrep slides were classified according to the Bethesda classification system as WNL , ASCUS or LSIL , or HSIL . Of the cases with an abnormal cytology score , the residue of the cervical sample was used to prepare histologic paraffin slides , 8. BiopThe purification of DNA from the Boonfix solution was performed in the BioRobot M48, Qiagen (Germany), which uses the MagAttract DNA Mini M48 Kit. This magnetic-particle technology provides high-quality DNA. Samples were analyzed using a sensitive SPF 10 PCR-reverse hybridization line probe assay . This system is based on PCR of part of the L1 region of the HPV genome, which is amplified using SPF10 primers . DetectiType-specific HPV prevalence was compared between women with HSIL, LSIL, and WNL. The chi-square test was used to test for significant associations between country and HPV type. Analyses were conducted using SPSS and Mathematica. One hundred three (72%) Tanzanian women and 35 (69%) South African women had a normal cytology score (WNL) . Nine  women tested positive for hr-HPV ; 80 , and the prevalence of hr-HPV 35 was 24% in South Africa and 8% in Tanzania   and hr-HPV 52 (30%), and the prevalence of hr-HPV genotypes varied by country. Among Tanzanian women testing positive for any hr-HPV, the most common genotype was hr-HPV 52 (35%), followed by hr-HPV 16 (23%) and hr-HPV 66 (20%). In contrast, among South African women testing positive for any hr-HPV, the most common genotype was hr-HPV 16 (34%), followed by hr-HPV 35 (24%), hr-HPV 18 (21%), hr-HPV 33 (21%), and hr-HPV 66 (21%). Genotypes 18 and 35 were significantly more common in South Africa than Tanzania; the prevalence of genotype 18 was 21% in South Africa compared to 4% in Tanzania ( = 0.03) . The mos = 0.03) .P < 0.01). Hr-HPV 18 was found in only 11.8% of women with HSIL .All women with abnormal cytology  had detectable HPV . Ninety-ith HSIL  and the  This study found that hr-HPV genotypes not included in the currently licensed HPV vaccines are highly prevalent in HIV-positive women from Tanzania and South Africa. As expected, among women with hr-HPV the prevalence of hr-HPV 16 was high (26%).  Despite the high rate of abnormal cytology among women in Tanzania, hr-HPV 18 was uncommon in women with abnormal cytology (4%). In contrast, hr-HPV 18 was significantly more common among South African women with abnormal cytology 21%). We also found significant regional differences in the prevalence of hr-HPV 35; the prevalence of hr-HPV 35 was 8% among Tanzanian women compared to 24% for South African women. In rural Mozambique, Castellsagué et al. also found a high prevalence of hr-HPV 35 , compar1%. We alThe hr-HPV 52 prevalence found in this study (35% of Tanzanian women and 17% of South African women with any HPV) is vastly different from that in women in The Netherlands (5.5%) . A studyThe prevalence of hr-HPV 45 was low in our cohort , as alsoThe strengths of our study include using a standardized HPV type-specific PCR protocol and cytology diagnosis across the two sites. There were differences between the women studied that may have contributed to the different prevalence estimates of hr-HPV types. Tanzanian women were all using ART. In contrast, the women in South Africa were recently diagnosed with HIV and not yet eligible for ART. In HIV positive women there is less HPV clearance due to a lack of an appropriate immune response. The impact of ART in HPV infection and cervical cancer still needs to be defined . A limitFor an HPV vaccine to effectively reduce incidence of cervical cancer, it will need to protect against the major hr-HPV infection types progressing to cervical cancer. Because infection types may vary across settings and ethnicities, the impact a vaccine has in preventing cancer in particular settings needs to be considered. In this study, hr-HPV16 was found in 29% of women diagnosed with HSIL; however, hr-HPV 18 was only found in 11%.Certainly the high prevalence of hr-HPV found here and reported by others,  emphasizIn summary, the present study showed some differences in the prevalence of HPV genotypes among HIV-positive women in two African countries, and an overall high HPV prevalence. The differences found in hr-HPV genotypes warrant the need to consider different monitoring programmes for cervical preneoplasia, especially in HIV-positive women.


Eleven novel human papillomavirus (HPV) types were isolated and characterized from healthy individuals in China. HPV163 belongs to the γ-1 species, HPV 164 and HPV 168 fit in the γ-8 species, HPV 165 and KC5 belongs to the γ-12 species, HPV 168 is closely allied with the γ-4 species, HPV 169 is closely related to the γ-11 species, and HPV 170 is related to the γ-12 species. In addition, HPV 161, HPV 162, and HPV 166 may form a new HPV species of the γ-PV genus. The prevalence of these HPV types in the normal population is low.  A typical novel PV type is identified and defined by cloning full-length PV genome whose L1 gene nucleotides sequences is at least 10% dissimilar from any other known PV type. The classification of PV types is based on construction of a phylogenetic tree using the L1 genes , and six HPVs  have the LxCxE motif for binding to the pRB protein, but the remainder of these novel HPV types lack this motif. The ATP binding site of the ATP-dependent helicase (GPPDTGKS) is conserved in the carboxy-terminal region of E1 in HPV 161, HPV 162, HPV 163, HPV 165, HPV 166, HPV 168, and KC5. The ATP binding site is modified slightly in HPV 164 and HPV 167 as “GPPDSGKS”, in HPV 169 as “GVPDSGKS”, and in HPV 170 as “GPPNTGKS” (the amino acid in bold indicate mutation site). The length of the upstream regulatory region (URR) is from 447 bp (HPV 165) to 581 bp (HPV 170) (6GGT) were identified (HPV 170) , similarentified . http://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?cmdresult=main&id=347), HPV 161 and HPV 162 are both most related (67%) to HPV 135, HPV 163 share 73% nucleotide sequence identity with the whole genome of HPV 95, the sequence of HPV 164 is 78% identical to HPV 112, HPV 165 is 70% identical to HPV 148, HPV 166 is 66% identical to HPV 130, HPV 167 is 76% identical to HPV 112, HPV 168 is 67% identical to HPV 60, HPV 169 is 74% identical to HPV 126, HPV 170 is 76% identical to HPV 123, and the KC5 is most related (72%) to HPV 148. L1 nucleotide sequence similarities among these novel HPV types and closest known HPVs are listed in As determined using the PASC global alignment analysis searching for the best matches within the papillomavirus family . The tree is rooted with selected Mu and Nu species. The analysis involved 68 nucleotide sequences. All positions containing gaps and missing data were eliminated.(TIF)Click here for additional data file.Figure S2ML tree constructed based on the E1-E2 ORFs. Numbers on branches are bootstrap support values to the clusters to their right . The tree is rooted with selected Mu and Nu species. The analysis involved 68 nucleotide sequences. All positions containing gaps and missing data were eliminated.(TIF)Click here for additional data file.Figure S3ML tree constructed based on the L1 ORFs. Numbers on branches are bootstrap support values to the clusters to their right . The tree is rooted with selected Mu and Nu species. The analysis involved 68 nucleotide sequences. All positions containing gaps and missing data were eliminated.(TIF)Click here for additional data file.Figure S4Bayesian tree constructed based on the L1 ORFs. Bayesian posterior probabilities . The tree is rooted with selected Mu and Nu species. The analysis involved 68 nucleotide sequences. All positions containing gaps and missing data were eliminated.(TIF)Click here for additional data file.Table S1Positions* of Zinc-binding domains of E6 and E7 ORFs, LxCxE motif of E7 ORF, ATP binding site of the ATP-dependent helicase (GPPDTGKS), and E2-binding site motifs.(DOCX)Click here for additional data file.Table S2Primers and clone information about the 11 novel HPV types.(DOCX)Click here for additional data file.


INK4a, a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS.The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16INK4a as well as markers of EMT.In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16INK4a. High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16INK4a and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib.We found overexpression of p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.Our results show that overexpression of p16 Cervical and head and neck cancers display overexpression of the protein upon oncogenic infection with high-risk genotypes of human papillomavirus (HPV). Viral oncoprotein E7 interacts with p16INK4a target RB, leading to its dissociation from the transcription factor E2F which then initiates transition from G to S phase of the cell cycleINK4a protein expression, which is regarded as a surrogate marker for high-risk HPV infection in these tumours, is understood as an “unsuccessful attempt” to stop cell proliferation. However, it is still unclear whether high-risk HPV infection also promotes urothelial tumourigenesisINK4a in UCIS is associated with HPV infection and, if this is not the case, to reveal the mechanism of p16INK4a upregulation in these lesions.Urothelial bladder cancer is the 7th, 2012).All tissue samples were collected for histologic examination and diagnostic purposes and were thoroughly anonymized for the use in this study. Thus no informed consent was needed. This study was approved by the University of Ulm ethics committee . P16INK4a was regarded as positive if there was a diffusely distributed, strong cytoplasmic and nuclear staining signal.Immunohistochemistry was done according to standard protocols as previously described Whole genomic DNA was extracted from paraffin slides using the automated Maxwell® 16 FFPE Plus LEV DNA Purification Kit . Amplification of HPV DNA with biotinylated primers and hybridization of the PCR products to strips precoated with specific oligonucleotide probes was performed using the HPV typing kit from AID diagnostics  according to the manufacturer's protocol. The kit detects HPV genotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58 and 59.INK4a-positive UCIS. 5 µm-thick slides were cut off paraffin blocks and pretreated using standard procedures. Slides were incubated with the Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen DNA probe  and mounted in DAPI/Antifade-Solution . The CDKN2A locus encodes for p16INK4a, while centromere 9 (CEP 9) was used as an internal positive control. Gene copy numbers and centromere 9 signals in each 100 nuclei of 5 UCIS lesions and of the underlying stroma were counted out and each nucleus was assigned to one of the following groups according to the number of visible CDKN2A SpectrumOrange DNA probe signals: 1 orange/2 green (1O2G), 2 orange/2 green (2O2G) or 3 or more orange/2 green (3+O/2G). The 3+O/2G pattern would therefore indicate gene amplification of the CDKN2A locus encoding for p16INK4a.FISH analysis was performed on five representative cases out of the 25 p16KRAS mutation status was tested with KRAS strip assays  as previously described . KRAS muescribed [17]. Traescribed . Quantif testing [17].PcDNA3-KRAS-wild type/pcDNA3-KRAS-G12D (GGT/GAT transition) mammalian expression vectors were kind gifts of Dr. Patrizio Castagnola, National Cancer Research Center, Genova, Italy, and have been previously publishedIF staining was performed according to standard methods as previously described by our work groupThe study contained 27 UCIS samples from 19 patients who had a mean age of 74 years  and 5/5 CIN lesions (100%)  , Table 1ithelium . There w0/5, 0%; .High-risk human papillomavirus (HPV) DNA (genotype 16) could be detected in 4/5 CIN lesions 80%; . For UCICDKN2A gene locus encoding for p16INK4a(orange) and centromere 9  per nucleus in 5 representative samples out of the 25 p16INK4a-positive UCIS specimens revealed two orange and two green signals (2O2G pattern) in 393/500 UCIS nuclei . In the underlying stroma, 454/500 nuclei (90.8%) showed a 2O2G and 46/500 (9.2%) showed a 1O2G pattern. We did not detect three or more orange signals (3+O2G pattern) in any of the examined nuclei. These results show that there was no amplification, but rather loss of the CDKN2A gene locus encoding for p16INK4a in neoplastic urothelium.FlSH analysis highlighting the copy numbers of the i 78.6%; , Table 2INK4a and weak nuclear staining for pERK1/2, but strong membraneous positivity for E-cadherin and Beta-catenin in healthy urothelium . This shows that transfection-induced activation of the RAS/MAPK signaling pathway is sufficient to cause EMT in vitro, and that this effect can be inhibited by Sorafenib.Western immunoblotting for E-cadherin and Beta-catenin in nchanged . Secondlin vivo.Immunohistochemistry revealed a positive staining pattern for uPA in the cytoplasm of apical tumour cells . Here, we show for the first time that this overexpression of p16INK4a in UCIS is neither associated with oncogenic HPV infection nor p16INK4a gene amplification. Furthermore, we point out a mechanism for p16INK4a upregulation in UCIS.The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. In cattle, infection with bovine papillomavirus (BPV) is a central risk factor for development of urothelial cancerINK4a staining in 100% of examined UCIS, we found nuclear and cytoplasmic positivity for p16INK4a in 92.6% of UCIS. This might be due to the higher sample number of isolated UCIS in our group; however, these findings are comparable to figures from literatureINK4a, which is also consistent with data from the literatureINK4a is regarded as a surrogate marker for HPV infection in these lesions, this is comparable to findings in literature, where a positive predictive value (PPV) of p16INK4a for high-risk HPV infection between 59 and 91% has been described In slight contrast to the findings by Yin et al.INK4a immunostaining. Contrary to our study, that study did not focus on flat urothelial lesions/urothelial carcinoma in situ, and accordingly, the same group and others had previously found evidence for an etiologic role of human papillomavirus in papillary and low-grade urothelial lesionsINK4a expression and inverted papillomas of the urinary bladderSurprisingly, we did not detect HPV DNA in any of the examined UCIS samples. This finding is in contrast to results from Shigehara et al, who found HPV DNA in 18 out of 117 (15%) bladder carcinomas in 2011INK4a, and upregulation of p16INK4a in tumor cells independent of HPV infection has also been described for other tumour entitiesINK4a expression did not correlate with the expression of HPV16 E7 oncoproteinINK4a as a surrogate marker for HPV infection or HPV-associated tumorigenesis in urothelial carcinoma. However, the possibility remains that other viral infections contribute to urothelial carcinogenesis. For human polyomavirus (HPyV), however, such an association between infection status and bladder cancer risk could not be shown in a previous studyOur own results are consistent with data for invasive urothelial carcinoma that is widely regarded to be more closely related to UCIS than papillary lesions in terms of molecular tumourigenesisCDKN2A gene locus encoding for p16INK4a in most of examined UCIS nuclei. There was no amplification, but loss of one gene copy in about one fifth of nuclei in the cancerous lesion. This finding is consistent with data from the literature, where multiple studies described loss of CDKN2A in flat urothelial lesions and urothelial carcinomaINK4a is not related to gene amplification in UCIS.FISH analysis showed a regular number of the INK4a in UCIS was accompanied by enhanced kinase phosphorylation as shown by immunohistochemistry. Since overexpression of p16INK4a upon activation RAS/MAPK signaling has been describedin vitro enhanced phosphorylation of ERK and led to upregulation of p16INK4a. However, contrary to the results from immunohistochemistry, we found that treatment with TNFα to simulate an inflammatory setting enhanced kinase phosphorylation and led to upregulation of p16INK4a. Since this in vitro effect has been previously describedin vivo are lower than they are in cell culture. Furthermore, in vitro models lack anti-inflammatory molecules (such as CXCL9–11 and TNFSF14) that have been shown to be upregulated in bladder samples from patients with ulcerative interstitial cystitis and might have influence on apoptotic signaling cascades and p16INK4a expression in vivoStrong expression of p16The observed changes in E-cadherin and Beta-catenin expression and distribution are consistent with epithelial-mesenchymal transition (EMT), a process in which epithelial cells lose cohesiveness and gain a mesenchymal phenotype as a prerequisite for subsequent invasionin vitro. Sorafenib has been previously shown to effectively inhibit EMT and is thus an interesting candidate for pharmacologic prevention of invasivenessINK4a, is controlled by the ETS1/2 transcription factor family downstream the RAS/MAPK signaling pathwayBesides EMT induction, RAS signaling led to overexpression of Urokinase plasminogen activator (uPA), and both effects could be suppressed by application of the multi-kinase inhibitor Sorafenib INK4a in UCIS is independent of HPV infection and p16INK4a gene amplification, but follows enhanced RAS/MAPK signaling accompanied by upregulation of uPA and epithelial-mesenchymal transition. In our model, which is schematically depicted in INK4a would therefore be a byproduct of this process and a candidate marker for the EMT which is occurring in the tumour cells. Since in our in vitro model, uPA expression as well as EMT are both sensitive to kinase inhibition, our results indicate a potential therapeutic use of Sorafenib to prevent UCIS invasiveness and progression.Taken together, we conclude that overexpression of p16Figure S1A, Negative immunostaining for p16INK4a in erosive urocystitis. B, Negative immunostaining for pAKT in urothelium (above left) and UCIS (below left) compared to moderate staining intensity for pERK1/2 in urothelium (above right) and strong staining intensity in UCIS (below right).(TIF)Click here for additional data file.Figure S2Authentication of RT112 urothelial carcinoma cells by short tandem repeat (STR) profiling.(TIF)Click here for additional data file.Figure S3KRAS strip assay testing of RT112 cells upon transfection with wild-type KRAS (1), KRAS G12D (2) and testing of untransfected cells (3). The band in (2) indicates successful insertion of KRAS G12D mutation.(TIF)Click here for additional data file.Figure S4Full-length western blots from the .(TIF)Click here for additional data file.Table S1List of antibodies and concentrations as used in the study.(DOCX)Click here for additional data file.


This is a report on some of the research activities currently ongoing in Italy as outlined at the “Viruses and solid tumors” meeting jointly organized by the Oncology Sections of IRCCS Policlinico “San Matteo” (Pavia) and IRCCS National Cancer Institute (Aviano), held in Pavia, Italy, on October 2011. Experts from the various disciplines involved in the study of the complex relationships between solid tumors and viruses met to discuss recent developments in the field and to report their personal contributions to the specified topics. Secondary end point was to establish a multidisciplinary work group specifically devoted to solid tumors and infectious agents, aimed to identify areas of common interest, promoting and establishing collaborative projects and programs, and to coordinate clinical and research activities. The group, which will be named IVOG , will operate under the patronage of the various scientific societies of interest. SRC) and tumor suppressor genes , and the first vaccination achievements  . Then it was the turn to address the intimate mechanisms of direct and indirect virus oncogenesis, illustrating the key steps and complex regulatory circuits that contribute to the cancer development . At the end, Antonino Carbone  reviewed the molecular techniques for pathologic and virologic investigations, including DNA/RNA detection techniques and conventional immunohistochemistry for EBV, KSHV, MCPyV and HPV.The first introductory lecture dealt with the fundamental milestones of the long lasting and successful research on viruses and cancer, such as the discovery of transforming non-filterable agents that cause cancer, the identification of the first human tumor virus (EBV), the discoveries of proto-oncogenes  who presented an interesting study on HLA-A and -B high-resolution profile in EBV-associated undifferentiated nasopharyngeal carcinoma (UNPC) in Italy, a non endemic area for this disorder ; it was The estimated total number of cancer cases attributable to infection in the year 2002 was 1.9 million, or 17.8% of the global cancer burden . ResultsIn recent studies the involvement of HPV infection in head and neck cancers was investigated. Data indicated that the fraction of oropharynx, tonsils and base of tongue cancers attributed to HPV was 50% in developed countries and 5% in developing countries . The natural history of cervical HPV infection is well studied, whereas the extension of cervical findings to HPV carcinogenesis in the tonsil needs more investigations. Cervical HPV infection is mainly transmitted through sexual intercourse, and is very common and rising in all world population. On the other hand, information on the prevalence and the age at first acquisition of HPV infection to the oropharynx is limited in quantity and quality. HPV-16 was found in 50% of cervical carcinoma and in 90% of HPV-positive carcinomas of the oropharynx. The precancerous HPV-associated lesions that precede cervical carcinoma (CIN1-3) are well defined and widely employed in screening programs. In contrast, precancerous lesions in the oropharynx are poorly understood and not consistently classified. Contrary to cervical carcinoma, very strong risk factors, other than HPV, exist for oropharyngeal carcinoma . In conclusion, Franceschi presented the future objectives emerged from the FP-7 funded IARC Study of human Papillomavirus and precancerous Lesions In the Tonsil (SPLIT study): i) prevalence of HPV infection in cancer-free tonsils and age at acquisition; ii) detection of precancerous lesions in the tonsil using histological and cytological examinations; iii) differences between HPV-related precancerous lesions in the oropharynx and those caused by other risk factors ; iiii) fraction of oropharyngeal carcinomas truly attributable to HPV, in different countries.HPV 16, and secondarily HPV 18, are by far the most relevant human Papillomavirus types involved in oropharyngeal, laryngeal and oral cavity cancer, being observed in a relevant fraction of HN cancers, particularly in oropharyngeal tract. A role cannot be excluded for HPV types 6 and 11 too, since a minority of cancers (less than 5-10%) can be demonstrated to harbor sequences from these types. Several studies reported that detection of HPV DNA is associated with poorly differentiated cancer grade, positive lymph nodes, and late-stage disease, which traditionally indicate poor prognosis. Despite these observations, the outcome of HPV-positive tumors, progression-free survival (PFS) and overall survival (OS), appears to be significantly better than HPV negative tumors . The morOverexpression of p16, a cell cycle regulator, in HPV-positive oropharyngeal squamous cell carcinoma results from degradation of pRb by the viral oncoprotein E7; for this reason, p16 positivity at immunohistochemistry has been proposed as a surrogate marker for high-risk HPV infection. However, although some studies indicated a good correspondence between p16 and HPV status , a recent study from the Licitra group in Milan [10-LIPA, detecting 15 high-risk and 10 low-risk HPV types) on a consecutive series of 1323 women undergoing colposcopy, 69% of whom had a cervical biopsy, and correlated CIN severity with the type and number of HPV [per se was associated with high-grade lesions, independently of HPV-16, stressing the need to study the role of this phenomenon in cervical precancerous lesions.The WHO/ICO 2010 summary report on HPV and Cervical Cancer (HPV Information Centre) indicated 2880 new cervical cancer cases/year in Italy, with a crude incidence rate of about 9.4 per 100,000 women/year, and with a prevalence of HPV-16 and/or HPV-18 among women with high grade cervical lesions  of 55.7%, and with cervical cancer of 72.1%. The crude incidence rate observed in Italy  indicated a slightly lower incidence in comparison to other Southern Europe countries (11.1%), but significantly lower compared to the global overall incidence (15.8%). Arsenio Spinillo  presented studies on multiple HPV strain infection in cervical specimens. HPV DNA was investigated by means of a robust and sensitive PCR method for the simultaneous detection of multiple HPV types , described the viral and cellular oncogenic pathways involved in the development of HCC, focusing on the importance of surveillance that might allow an early diagnosis of HCC in patients with defined risks and consequently reduced mortality. Antiviral treatment can reduce, but not eliminate, the risk of HCC in HBV/HCV infections. A 17 year prospective cohort study coordinated in Pavia [HCC is the 5in Pavia  has demoIn the other contribution Camillo Porta  illustrated the molecular pathogenesis of HCC by showing the most relevant pathways, focusing on angiogenesis and the mechanisms of deregulation of proliferation and survival. Several molecularly target therapies are now available, capable of interfering with these pathways at various levels, including sunitinib, bevacizumab, erlotinib, gefitinib and cetuximab, and promising new drugs such as everolimus, and the Met inhibitor ARQ-187. Presently sorafenib, an oral inhibitor of the serine/threonine kinase Raf-1 and of several tyrosine kinases involved in angiogenesis (VEGFR2-3), is the only approved drug. A multicenter study , with a As previously discussed for cervical cancer, miRNAs may be involved in hepatocellular carcinoma as well. The group of Palù evaluated miRNAs expression in relation with the degree of fibrosis, in 40 chronic hepatitis/cirrhosis patients. Results showed that some miRNAs were up-regulated, whereas other miRNAs appeared down-regulated. Relevant miRNAs had several targets that were involved in some biological pathways that promote collagene synthesis, cell proliferation, migration, and epithelial to mesenchimal transition, suggesting a role in viral hepatitis/cirrhosis progression. In a second set of experiments, miRNAs expression was evaluated in 18 HCC patients. Results showed that some of the miRNAs deregulated in advanced grade fibrosis were deregulated in HCC too, suggesting a link between fibrosis and cancer. Future studies will address the role of miRNAs as prognostic indicators or diagnostic markers for HCC.The first speaker, Mauro Tognon , focused the attention on personal data on polyomaviruses, brain tumors and Merkel cell carcinoma. SV40, a potent transforming polyomavirus, was introduced in the human population about 50 years ago by contaminated polio vaccines, since then its role in the pathogenesis of human brain tumors, mesothelioma and colon cancer has been periodically claimed. SV40 Tag-coding sequences were found in various histotypes of human brain tumors, including 83% of choroid plexus papillomas, 73% of ependymomas, 47% of astrocytomas and 33% of glioblastomas ,14. ExprMerkel cell carcinoma is a rare aggressive human skin cancer, with a high mortality rate and an increasing incidence, which is found prevalently in immunocompromised hosts, such as transplant recipients, HIV patients, and subjects with chronic lymphocytic leukemia. Recently, sequences from a new polyomavirus, named Merkel polyomavirus (MCPyV), were found integrated in the genome of a high proportion of Merkel cell carcinoma primary samples. Clonal integration and the deletions and/or mutations observed in the T antigen gene have suggested a direct oncogenic role for MCPyV ,16. TognThe session proceeded with a contribution on mesothelioma and gastrointestinal cancers. Manola Comar  discussed the SV40 role in the pathogenesis of mesothelioma, as a possible cofactor in the cancerogenetic process. Northeastern Italy may be considered an hyperendemic area for mesothelioma, in association with a massive occupational exposure to asbestos. In a first retrospective Italian study aimed to investigate the presence of SV40 in malignant and normal tissues from mesothelioma patients of this area, Comar and collaborators  detectedBecause of localization, providing exposure to external environment, colon cancer may be a target for viral oncogenesis, and this is area of active investigation. Comar presented two studies on polyomaviruses and colon cancer. In the first case–control study , DNA froEBV has an important role in the pathogenesis of some solid tumors too, namely EBV-related nasopharingeal carcinoma and a variant of gastric cancer, comprising approximately 15% of all gastric cancers. In his talk on EBV-related solid cancers and relative immunotherapy approaches, Fausto Baldanti  reported an interesting case of EBV-Smooth Muscle Tumor (SMT) in the context of kidney transplantation successfully treated by minimizing immunosuppression , providiIn an Italian retrospective study  on post-in vitro. Quadrivalent HPV  and bivalent HPV  L1 VLP vaccines are available, and their efficacy was demonstrated in phase III trials. Results from a phase II randomised placebo controlled trials of a quadrivalent HPV [Franco Buonaguro  introduced the section summarizing features of HPV virus replication, transmission, and immune response. Then the speaker focused the attention on HPV vaccines. Target of preventive vaccines is neutralization of virus before infection of basal keratinocytes. Actual vaccines are based on recombinant L1 protein, a major capsid protein, which is characterized by self assembly to form virus-like particles lent HPV  and bivalent HPV  demonstrlent HPV  HPV-16, lent HPV . The resex vivo.As regards new HPV vaccines technologies, research is focused on the production of capsomers and in low-cost synthesis. Vaccine production in other systems, such as in plants will permit safer and cheaper synthesis of viral molecules for the developing world. The author is actively pursuing vaccine development in tobacco plants. Therapeutic vaccines are also under development, specifically based on several long peptides derived from proteins E6 and E7. While these strategies have proven to be successful in animal models of HPV neoplasia (mice and rabbits), ways to improve the T helper 1 response are being investigated, including the use of dendritic cells exposed to viral peptides The role of antiviral therapy in HCC prevention was introduced by Raffaele Bruno  who presented a collaborative retrospective study of the Italian Association for the Study of the Liver (AISL)  on the iin vivo T-cell expansion, lymphodepletion was employed before high-dose EBV-specific CTLs in eleven EBV-related NPC, but results were comparable [Cell-based immunotherapy against virus-mediated cancers was discussed by Patrizia Comoli . The investigator illustrated the last 15 years experience on cell therapy for EBV-related PTLD in immunocompromised patients . Cell thmparable . These pContributions of the last section were focused on HIV-associated solid tumors. WHO estimates 33.3 million of adults and children infected with HIV in the world, about 820,000 in Europe. In the HAART era, HIV-infected patients survive to older ages, with prolonged exposure to cancer risk factors and, nowadays, non-AIDS Defining Cancers (NADC) represent a much larger fraction of the overall cancer burden. NADC are constituted mainly by Hodgkin’s lymphoma and some solid tumors  associated with alcohol and smoke exposure, immunedepression, and viral infection . Emanuela Vaccher  analyzed data of a recent record-linkage study between the Italian AIDS registry and the Italian cancer registries ,40. ThisAttention was then focused on HCC and HIV. HCC is an increasing cause of mortality in HIV-infected patients in the HAART era; the risk of HCC is increased in HIV patients (SIR = 3-19). Furthermore, HIV is able to accelerate the evolution process of fibrosis/cirrhosis. In the Italian cohort, 1 year survival of 28% in HIV-positive patients versus a survival of 76.2% in HIV-negative patients has been reported. Antonello Malfitano , presented some contributions that explain the main features, clinical presentation and outcome of HCC in HIV-infected patients. The first study presented  was baseLast contribution was focused on AIDS-Kaposi’s sarcoma (AIDS-KS). AIDS-KS is the most aggressive form of KS, and Kaposi’s sarcoma herpesvirus  is the infectious agent associated with this neoplasia. AIDS-KS most frequently manifests in a multifocal mucocutaneous distribution involving the face, trunk, genitalia, and/or lower extremities. Visceral sites most commonly include the lung and gastrointestinal tract. KS predictive factors are immune system impairment, current CD4+ cell count, current viral load, and absence of combination antiretroviral therapy (cART) . KS riskCo-administration of protease inhibitors with anticancer drugs in the management of HIV-related malignancies can cause potential drug-drug interactions. A study  conducteAlthough KS treatment is based on HAART, in rare cases individuals may experience KS flare during immune reconstitution inflammatory syndrome (IRIS), a heterogeneous and sometimes fatal disorder of immune perturbation after initiation of highly active antiretroviral therapy. Such a flare does not necessarily indicate a poor prognosis. Maserati reported a study  based onThe long standing interest and expertise on viral oncology of the several Italian groups participating at the Pavia meeting prompted the organizers to propose a multidisciplinary scientific association with the primary aim to potentiate collaboration throughout the country on basic research, epidemiology, translational and clinical studies on viruses and solid cancer. At the end of the meeting, speakers held a round table discussing these points and expressed intent to participate as investigators at the proposed association.The authors declare that they have no competing interests.VP and MR wrote the manuscript. All authors read and approved the final manuscript.


Cancer of the cervix uteri is the second most common cancer among women worldwide. The association of human papillomavirus (HPV) infection with cervical carcinogenesis is well documented. This is a pilot study aiming to studying the prevalence and the pattern of Human Papilloma Virus Type 16 (HPV16) by immunostaining in the tissues of cervical carcinomas of Ethiopian women.20 specimens of uterine cervical carcinomas were studied histopathologically and immunohistochemically for HPV16. Histologically the specimens were classified as: Ten cases were Non Keratinized Squamous cell carcinoma (NKSCC), six cases were Keratinized Squamous Cell Carcinoma (KSCC) and four cases were Adenocarcinoma (ADC). Immunohistochemistry study showed positivity in eleven cases (55%); seven cases (35%) were non-keratinized squamous cell carcinoma; three cases (15%) were keratinized squamous cell carcinoma and one case (5%) belonged to the adenocarcinomas.This study reveals a significant detection of HPV in Ethiopian women by the use of advanced techniques such as Immunohistochemistry (IHC). The data of this study suggested that the marked expression of the HPV 16 was in the less differentiated uterine cervix carcinomas.  Cervical cancer is an important public health problem for adult women in developing countries in South and Central America, sub-Saharan Africa, and south and south-east Asia ,2. The IRetrospective analysis of the causes of postmenopausal bleeding in Addis Ababa, Ethiopia; showed that cervical carcinoma accounted for 84.8% of all female genital tract malignancies . CervicaThe vast majority of cervical cancer cases are caused by infection with certain subtypes of human papilloma virus (HPV), a sexually transmitted virus that infects cells and may result in precancerous lesions and invasive cancer ,9. The hThe present study is a retrospective pilot study aiming to determining the prevalence of Human Papilloma Virus Type 16 (HPV16) associated histological types of cervical carcinomas in Ethiopian women. This pilot feasibility study was designed prior to a larger immunohistochemistry studies related to cancer cervix and to improve the efficacy and quality of immunostaining procedures in the Department of Medical Laboratory Sciences and Pathology; College of Public Health and Medical Sciences, Jimma University. The study included 20 punch biopsy specimens of uterine cervical carcinomas retrieved randomly from the archives of the Department of Medical Laboratory Sciences and Pathology; Collage of Public Health and Medical Sciences; Jimma University. All patients were presented clinically with irregular vaginal bleeding or post coital bleeding. The retrieved punch biopsies were sectioned and stained by hematoxylin and eosin stain for histopathological classification of cases according to the (IARC/WHO); World Health Organization (WHO) classification of cervical tumors . Immunohistochemistry was performed on the 20 cervical carcinomas specimens. Four micrometer sections of formalin-fixed, paraffin-embedded tissues were cut and placed on coated slides. The sections were dewaxed in xylene, rehydrated in graded alcohol, and rinsed in water. For antigen retrieval, the sections were immersed in 0.01M of citrate buffer, pH 6.0, in a high pressure cooker for 10 minutes, the tissue sections were cooled under tap water for 10 minutes. A peroxidase block reagent was applied and it was incubated for 5-10 minutes at room temperature. Next, a block reagent was added, after draining out the slides, and incubated for 15 minutes at room temperature. The monoclonal antibody  was added for one hour on the specimen followed by the secondary antibody  that was incubated for 30 minutes, followed by rinsing with Tris buffer saline (pH 7.4 to 7.6) for 10 minutes. The slides were drained and blotted around the sections, to which an appropriate volume of substrate  solution was added, and they were incubated for 40-50 minutes at room temperature followed by rinsing in Tris-buffer saline. Finally, the sections were counterstained in a Mayer"s hematoxylin bath for 5 minutes and then rinsed with tap water. Known positive control sections were included in each run to ensure proper immunostaining whereas the negative control consisted of the same section where the primary antibody was omitted. The pilot study included 20 cases of cancer cervix; the patient ages ranged from 32 years to 56 years, the clinical presentations were mostly vaginal bleeding for more than one month. Histologically the specimens were classified as: Ten cases (50%) were Non Keratinized Squamous cell carcinoma (NKSCC), six cases (30%) were Keratinized Squamous Cell Carcinoma (KSCC) and four cases (20%) were Adenocarcinoma (ADC).Immunohistochemistry study showed positivity in eleven cases (55%); the staining reaction was seen granular, cytoplasmic and also perinuclear and nuclear. The immunostaining positive reactions were distributed as; seven cases (35%) non-keratinized squamous cell carcinoma; three cases (15%) were keratinized squamous cell carcinoma and one case (5%) belonged to the adenocarcinomas. In the sheets of tumor cells, HPV was positive in the less mature cells ; meanwhiThe association of Human Papillomavirus (HPV) infection with cervical carcinogenesis is well documented . Nearly Studies have shown that >99% of invasive cervical cancers contain high-risk HPV-DNA and HPV-16 is the most prevalent type with an incidence of ~53% . In the A prevalence of 15.9% was revealed by study done to determine the prevalence of HPV infection among women attending outpatients' clinics in Attat Rural Hospital Southern Ethiopia . A retroAn Indian study reported that out of thirty samples, 15 expressed positive and 15 negative for HPV marker . The preSquamous cell carcinoma is the predominant type of cancer of the uterine cervix, and HPV 16 is the most common type of HPV DNA in these tumors . InfectiIt is known that during HPV infection, cells in the basal layer are infected first and the production of viral progeny takes place in differentiated cells in intermediate layers . This fiAdenocarcinoma, adenosquamous carcinoma, and small-cell carcinoma of the uterine cervix are reported to be low in incidence but clinically important. They usually exhibit a more aggressive biologic behavior and have a poorer prognosis than squamous cell carcinomas at similar stages . Unlike This study reveals a significant detection of HPV in Ethiopian women by the use of advanced techniques such as Immunohistochemistry (IHC). The data of this study suggested that the marked expression of the HPV 16 was in the less differentiated uterine cervix carcinomas. The authors declare no competing interests. Dr MR and Dr AB equally contributed to the study, both are pathologists. Dr. AB supplied the specimens and the data of cases as well as the histopathological interpretation of slides and Dr. MR performed the immunohistochemistry study of the specimens. All authors have read and approved the final version of the manuscript.


We report two cases in which we were able to diagnose bronchopleural fistula through retrograde methylene blue instillation during bronchoscopy. In the first case, methylene blue was injected through an abdominal drain, followed by air instillation and detected in the left bronchial tree, demonstrating the presence of a fistula in the lingula's bronchus. In the second case, methylene blue was injected into a pleural drain, through a breach on a surgical suture and detected in the right bronchial tree, demonstrating the presence of a fistula in the right inferior bronchus. The retrograde instillation of methylene blue, through a drain in the abdomen or the thoracic wall, is a safe, cheap, and practical method that allows the bronchoscopist to identify the presence of a fistula and, more importantly, to identify the exact point on the bronchial tree where a fistula is located. This provides the possibility of sealing the fistula with a variety of devices. It is our opinion that this procedure should be considered a primary method of diagnosis when a bronchopleural fistula is suspected and a drain on the thoracic or abdominal wall is positioned such that effusions are able to drain. Bronchopleural fistulas (BPFs) are communications between the pleural space and the bronchial tree.They are an adverse complication of several pulmonary conditions, particularly in the postoperative period after lung surgery or other invasive lung procedures  . BPFs reinstillation of Xe 133 into the segmental bronchus leading to the fistula ,81mKr and 99mTc diethylene triamine pentaacetic acid ventilation scintigraphy [instillation of other gasses such as tigraphy ,2O [introduction of small metallic probes through the working channel of the bronchoscope, and changes in gas concentration in the pneumonectomy cavity after inhaling different concentrations of oxygen and N2O , 12,instillation of Indocianine green during bronchoscopy ,antherograd instillation of methylene blue through the drain and its detection in the chest tube ,retrograde instillation of methylene blue .Bronchoscopy can be integrated withMethylene blue is an organic compound consisting of dark green crystals or crystalline powder, having a bronze-like luster. Solutions in water or alcohol have a deep blue color. Methylene blue is used as a bacteriologic stain and as an indicator. It inhibits guanylate cyclase and has been used to treat cyanide poisoning and to lower levels of methemoglobin . It is aWe report two cases of BPF diagnosis achieved through retrograde instillation of methylene blue during bronchoscopy.G. B. male, 34 years old. 11 years ago the patient underwent a posttraumatic splenectomy. This was the result of 40 days experience of nausea and vomiting (food and biliary acids) for which he went to see his GP. The patient undergoes a thorax-abdomen CT scan (with contrast enhancement), which shows herniation of colon, peritoneum, stomach, and vessels trough the diaphragm. Admitted to General Surgery division, he undergoes an election hernioplasty procedure, complicated by haemoperitoneum. An urgent laparotomy is performed to stop the bleeding. In the postoperative period, amylase is detected from one of the abdominal stumps. After some days, the patient develops pneumonia with fever and intense cough. He performs a bronchoscopy, which shows biliary fluid in the right and left bronchial tree. Suspecting an empyema, Mr. G. B. undergoes a new CT scan which shows the presence of a loculated effusion. It extends from the retro-pancreatic subphrenic space to the base of the left lung, through a breach located on the surgical suture. Furthermore, cough is noted during the abdominal drains' washings. A new FBS is performed. During the procedure, methylene blue 5 cc is injected through the abdominal drain, followed by air instillation (Retrograde methylene Blue Injection). Methylene blue appears then in the left bronchial tree, demonstrating the presence of a fistula located in the lingula's bronchus associated with a pleural-peritoneal fistula. The pleural-peritoneal fistula could have played a role in the development of a BPF. The pneumonia is successfully treated with antibiotics. Considering the improvement of clinical conditions in the days following the procedure, the patient is discharged and followed up (conservative treatment). He is now well and has no symptoms.R. R. male, 77 years old, history of pulmonary TB and diabetes mellitus. On 2008 he undergoes right inferior lobectomy for a lung squamosus carcinoma (T1N1 M0-IA). On April 2009 a chest CT scan (with contrast enhancement) shows the presence of free air and “low density deposits of uncertain attribution” in the pleural space, leading to a suspected BPF. He is admitted to our Pneumology department for further investigation and a thoracic drain is positioned. During an FBS session, 2 cc of concentrated methylene blue are instilled through the work channel of the bronchoscope, in the right bronchial tree, close to the surgical suture. The aim is to show the presence of blue colorant in the boulau connected to the drain in the thoracic wall, however, the test fails. Considering the relative good health of the patient, he is discharged. Between September 2011 and November 2011, the patient begins to complain of a chronic cough; he is subsequently admitted to our Pneumology Department and undergoes two FBSs with Anterograd methylene blue injection (5 cc); however, the test fails to provide diagnosis as no methylene blue is detected in the pleural drain. On December 2011 there is still evidence of air and pleural effusion on chest radiography, so a new pleural drain is positioned. Suspecting an esophagus-pleural fistula, the patient undergoes a barium esophagram, but no fistula is detected. The only way for air to reach the pleural cavity is in our opinion through a BPF. In order to confirm our suspicions, we propose to Mr. RR a Bronchoscopy with Retrograde methylene blue Injection. During this final FBS session, a solution of 5 cc of methylene blue mixed in 50 cc of saline solution (NaCl 0.9%) is injected into the pleural drain. The colorant finally appears in the right bronchial tree, demonstrating the presence of a BPF, located in the right inferior bronchus, through a breach on the surgical suture. After the diagnosis of BPF, an FBS with rigid bronchoscope is then performed and histo-acryl glue (1 cc -BluGran) is used to close the fistula. One week later, the chest drain is removed and the patient is discharged.133, 81mKr, or 99mTc diethylene triamine pentaacetic acid can be a useful method of BPF diagnosis; however, there is difficulty in detecting the exact point where BPF is located. In contrast, a combination of FBS and instillation of a liquid colorant is not only able to accurately diagnose a BPF, but also to indicate the exact point of the BPF location, allowing posttreatment success to be easily determined. An anterograde instillation of methylene blue can help the physician with the diagnosis of BPF but is unable to determine the exact location of a BPF. In contrast, the retrograde instillation of methylene blue, through the drain in the abdomen or the thoracic wall, allows the bronchoscopist to identify the exact point on the bronchial tree where the BPF is located. Once the leak is located, it can be sealed with several devices, like histo-acril glue, valves, stents, and other devices aiming to stop air and liquid leaks . They have been reported after infections, gastroesophageal reflux disease with Barrett esophagus, malignancy, thoracic trauma, ARDS, necrotizing lung disease associated with radiation, and/or chemotherapy and could also be idiopathic. In lung cancer patients, BPFs are associated with an advanced stage, presence of residual tumor on the stump after surgery, and intrathoracic use of chemotherapy . It is eeaks see .Retrograde methylene blue instillation during bronchoscopy is a useful and practical method used to accurately diagnose bronchopleural fistulas. This methodology is able to determine the exact point where the fistula is located, allowing the bronchoscopist to treat it properly. It has been shown to be more accurate than chest imaging and antherograd methylene blue instillation, because it is able to diagnose small Bronchopleural fistulas. With this cheap and practical method, we were able to achieve a diagnosis that several colleagues, using other methods, were not able to reach. It is our opinion that this procedure should be considered a primary method of diagnosis when a bronchopleural fistula is suspected and a drain on the thoracic or abdominal wall is positioned such that effusions are able to drain.


Human papillomavirus (HPV) infection has traditionally been regarded as a sexually transmitted disease (STD), but recent evidence implicates that an infected mother can transmit HPV to her newborn during pregnancy, at delivery, perinatal period or later. Given the lack of any studies on HPV-specific immune responses in children, we conducted HPV16-specific cell-mediated immune (CMI) monitoring of the mother-child pairs with known oral and genital HPV follow-up (FU) data since the delivery. In the Finnish Family HPV Study, 10 out of 331 mothers developed incident cervical intraepithelial neoplasia (CIN) during their 14-year FU. Our hypothesis according to the common dogma is that there is no HPV16 specific immune response in offspring of the CIN mother as she/he has not started the sexual life yet.We used overlapping 30–35 mer peptides covering the entire HPV16 E2, E6 and E7 protein sequences. Assays for lymphocyte proliferation capacity, cytokine production and HPV16-specific Foxp3 + CD25 + CD4+ regulatory T-cells were performed.HPV16-specific proliferative T-cell responses were broader in children than in their mothers. Nine of 10 children had responses against both E2 peptide pools compared to only 4 of the 10 mothers. Six of the 10 children and only 2 mothers displayed reactivity to E6 and/or E7. The cytokine levels of IL-2 (p = 0.023) and IL-5 (p = 0.028) induced by all peptide pools, were also higher among children than their mothers. The children of the mothers with incident CIN3 had significantly higher IFN-γ (p = 0.032) and TNF-α (p = 0.008) levels than other children.Our study is the first to show that also children could have HPV-specific immunity. These data indicate that the children have circulating HPV16-specific memory T-cells which might have been induced by previous HPV16 exposure or ongoing HPV 16 infection. Persistent high-risk (HR) human papillomavirus (HPV) infection is the most important risk factor for cervical cancer (CC). Of the HR-HPV genotypes, HPV16 is the most prevalent, present in 50% of all CC cases worldwide . It has HPV-induced immunity is crucial for clearance of the infection. However, HPV is known to have several mechanisms to evade the host immune system, as recently discussed -12. HPV Until now, most research on HPV immunity has been focused on cervical HPV infections in women. HPV immunity is based on circulating HPV-specific CD4+ T helper (Th) and CD8+ cytotoxic T lymphocytes (CTLs) of the adaptive, cell mediated immune (CMI) system . These cHPV infection has been traditionally considered as a sexually transmitted disease (STD). However, recent meta-analysis confirmed that newborns of the mothers with genital HPV infection had 33% higher risk to acquire HPV infection than those of HPV-negative mothers ,22. VertThe Finnish Family HPV study was designed in 1998 to elucidate the dynamics of HPV transmission between the family members ,31. In tThe Finnish HPV Family Study is a longitudinal cohort study conducted at the Department of Oral Pathology, Institute of Dentistry, University of Turku and Department of Obstetrics and Gynecology, Turku University Central Hospital. The study plan was approved by the Research Ethics Committee of Turku University Hospital (#3/1998 and 45/180/2010). Originally, 329 pregnant women at their 3rd trimester pregnancy and all their newborns  were enrolled in the study between 1998 and 2001, as described previously ,24,32,33The authors acknowledge the concept of the Minimal Information About T cell Assays (MIATA) framework, which was recently published ,35. Ther6 PBMCs were used and the remaining cells were frozen in 80% Fetal Bovine Serum  and 20% DMSO   using Nalgene™ Cryo 1°C Freezing container, which ensures a −1°C/min rate of cooling, at −70°C, and after 1–2 days stored the cryopreserved PBMCs in liquid nitrogen until further use. The median time from sample collection until beginning of LST or cryopreservation was 3 hours. From 9 mL of blood collected into clotting tube the serum was isolated by centrifugation for 7 minutes at 1000 g. Obtained autologous serum was used for short-term T-cell proliferation assay as described previously -Thymidine  per well was added. After 18 hours of incubation, the cells were harvested into Unifilter plates (PerkinElmer) using the FilterMateTM Cell Harvester (PerkinElmer). Subsequently, the filter plates were dried and counted on the 1450 MicroBeta + counter (PerkinElmer). The cut-off value for counts per minute (CPM) values was determined by the average plus 3 × SD of the eight medium-only control wells. Stimulation index (SI) was calculated as the average of tested eight wells divided by the average of the medium-only control wells. The proliferative response was defined positive if the CPM values of at least six of the eight wells were above the cut-off value and if the SI was ≥3 [The freshly isolated PBMCs with a density of 1.5 × 10I was ≥3 ,14.The supernatants collected from LST at day 6 were analyzed using Cytometric Bead Array (CBA) human enhanced sensitivity flex set system  according to the manufacturer’s instructions. In this array, the levels of IFN-γ, TNF-α, IL-2, IL-5, IL-10, and IL-17A were determined. The detection limits for the cytokines were based on standard curves complying with the limit of 274 fg/mL described by the manufacturer. The positive antigen-induced cytokine production was defined as a cytokine concentration > 2× the concentration of the medium-only control . Cytokin6 cells/well). The cells were cultured in IMDM containing 10% Human AB serum  and the indicated peptide pools  software package . Frequency tables were analysed using the χ2-test, with the likelihood ratio or Fisher’s exact test for categorical variables. Differences in the means of continuous variables were analysed using non-parametric  tests for two- and multiple independent samples, respectively. Paired-samples test (Wilcoxon) was used to analyse the response levels in mother-child pairs. All statistical tests were two-sided and declared significant at p-value ≤0.05.The laboratory of the Oral Pathology at the Institute of Dentistry, Faculty of Medicine, University of Turku, Turku, Finland, is a research laboratory where the T-cell assays are performed according to SOPs, including the predefined criteria for positive responses. All the methods needed for the CMI studies were transferred from the laboratory of van der Burg under his and Dr. Welter’s close guidance and quality control.Additional file PBMCs of all ten mothers and children showed proliferative response against MRM Figure . The MRMOne of the mothers had no response to any of the peptide pools (ID10A). She had a persistent HPV16 infection detectable for at least 72 months and eventually developed a CIN1 lesion with an HPV16/66 double infection. She had HPV16 antibodies detectable only once; one year after study entry, despite persisting HPV16. In addition, she showed the lowest proliferative T-cell response to the common recall antigens in the MRM , E2.2 , E6.2 , E6.3 , E6.4 , E7.2  and MRM . The control group of mothers showed slightly more E6 and E7 responses than the study group and the control group of children showed fewer responses to peptide pools E6 and E7 than the study group (data not shown). One mother showed positive LST responses only for E2.2 peptide pool when two others showed positive responses for both E2.1 and E2.2 peptide pools. In addition to responses for peptide pools of E2, the one mother showed responses also for peptide pools E6.2 and E6.4 and the other mother showed responses also for E6.3, E6.4 and E7.2 peptide pools. Control group of children showed responses only for peptide pools E2.1 , E2.2 , E6.4  and MRM . All responded for peptide pools E2.1 and E2.2 and only one child also for peptide pool E6.4 (data not shown).Figures HPV16 E2-specific IFN-γ producing T-cells to both E2.1 and E2.2 peptides were detected in 7 mothers . HPV16 induced antigen-specific TNF-α secretion was detected in six mothers and in five children  being usually higher than those of IL-17A (up to 75.26 pg/mL) for the individual peptide pools Figure . The motAmong control group of mothers, the highest cytokine levels were with IFN-γ, IL-17 and IL-10 followed by IL-2, IL-5 and TNF-α. The levels of all cytokines are presented in the legend of the Figure To summarize, PBMC cultures displaying HPV16-specific proliferative responses produced slightly higher levels of inflammatory cytokines than PBMC cultures not responding by proliferation. Especially Th1 –, Th2 –, and Th17-type cytokines were identified in response to HPV16 peptides Figure . In the The frequency of CD4 + CD25 + Foxp3+ cells after 7-day stimulation was analyzed  all these children harbored a T-cell response against HPV16 peptides, ii) the responses of the children were broader than those of their mothers (p = 0.0146).In the present study, the children mounted concomitant T-cell responses to HPV16 E2, E6 and/or E7. Previously we reported that there are highly conserved domains in HPV E2 protein sequence among different HPV genotypes, which could lead to cross-reactivity between the E2 peptides of different types of HPV ,14. HoweInterestingly, rarely an HPV16 E6-specific and no HPV16 E7-specific proliferative T-cell responses were found in the mothers, despite the facts that all had developed an incident CIN as a consequence of a persistent genital HPV16 infection and that the HPV16 E6 and E7 proteins should be expressed in all HPV16–induced CIN3 and CC lesions . Five chThe children’s oral sample tested HPV16-positive in two cases, one was HPV18 positive and one HPV6 positive while two were HPV DNA-negative. Thus, there was no straightforward correlation between the detection of HPV in the children and their HPV-specific immune response, except for the fact that five of the mothers of these six children tested HPV16 and one HPV18 DNA-positive in their cervical samples, and four were seropositive for HPV16 or multiple types.T helper type 1 cytokine IFN-γ and T helper type 2 cytokine IL-5 production is the characteristic for HPV16-specific immune response among healthy subjects but this is lost in patients with CC . In the Many studies have described HPV16–specific CMI reactivity among women with HPV-induced CC or CIN -38. In tTo conclude, our data support the view that HPV infection can be transmitted by non-sexual routes. The overall higher HPV16-specific responses of the children who have not started their sexual life than that of their mothers with CIN suggest that an early transmission may also lead to protective immunity. The exact mechanism remains yet unknown and further studies are urgently needed.CBA: Cytometric bead array; CC: Cervical cancer; CIN: Cervical intraepithelial neoplasia; CMI: Cell-mediated immune; CPM: Counts per minute; CTLs: Cytotoxic T lymphocytes; E: Early protein; Foxp3: Forkhead box P3; FU: Follow up; HPLC: High-performance liquid chromatography; HSIL: High-grade squamous intraepithelial neoplasia; HPV: Human papillomavirus; HR: High-risk; LC: Langerhans cells; LSIL: Low-grade squamous intraepithelial neoplasia; LST: Lymphocyte stimulation test; MRM: Memory response mix; NCIN: Non-cervical intraepithelial neoplasia; NSIL: Non-squamous intraepithelial lesion; SI: Stimulation index; SPPS: Solid phase peptide synthesis; STD: Sexually transmitted disease.The authors declare that they have no competing interest.This study has been supported by the Academy of Finland , Finnish Cancer Foundation, Sohlberg Foundation, Finnish Dental Society, and the Government Special Foundation (EVO) to Turku University Hospital. The funders had no role in the study design and data analysis, decision to publish, or preparation of the manuscript.HMK and AEP performed the research, provided essential reagents and tools. SMS and SEG designed the cohort and the current study. HMK, AEP, SMS, MJPW, SHVDB, and KJS analyzed data and HMK, AEP, SHVDB, MJPW, SMS and KJS contributed in writing the manuscript. All authors read and approved the final manuscript.Characteristics of the mothers with CIN and their children. The table summarizes the mothers’ medical history of cervical lesions, oral and genital HPV status and HPV-specific serology, as well as the oral HPV DNA status and HPV serology of their children, followed since birth until present. The red color represents HPV16 positive DNA sample, the purple HPV16 seropositive sample, the light green color cervical disease of grade LSIL/NCIN/ASCUS, the yellow color CIN2, the blue color represents CIN3 and the light grey negative sample. A = mother, B = child, baseline = time of the birth, 3d = age of 3 days, 1 mo = age of one month etc.Click here for file