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Human papillomaviruses (HPVs), which are small, double-stranded, circular DNA viruses infecting human epithelial cells, are associated with various benign and malignant lesions of mucosa and skin. Intensive research on the oncogenic potential of HPVs started in the 1970s and spread across Europe, including Croatia, and worldwide. Nowadays, the causative role of a subset of oncogenic or high-risk (HR) HPV types, led by HPV-16 and HPV-18, of different anogenital and head and neck cancers is well accepted. Two major viral oncoproteins, E6 and E7, are directly involved in the development of HPV-related malignancies by targeting synergistically various cellular pathways involved in the regulation of cell cycle control, apoptosis, and cell polarity control networks as well as host immune response. This review is aimed at describing the key elements in HPV-related carcinogenesis and the advances in cancer prevention with reference to past and on-going research in Croatia.

Prognostic, diagnostic or predictive biomarkers are urgently needed for assessment of chronic graft-versus-host disease (cGvHD), a major risk for patients undergoing allogeneic hematopoietic stem cell transplantation. The main goal of this review generated within the COST Action EUROGRAFT “Integrated European Network on Chronic Graft Versus Host Disease” was to identify potential novel biomarkers for cGvHD besides the widely accepted molecular and cellular biomarkers. Thus, the focus was on cellular biomarkers, alloantibodies, glycomics, endothelial derived particles, extracellular vesicles, microbiome, epigenetic and neurologic changes in cGvHD patients. Both host-reactive antibodies in general, and particularly alloantibodies have been associated with cGvHD and require further consideration. Glycans attached to IgG modulate its activity and represent a promising predictive and/or stratification biomarker for cGVHD. Furthermore, epigenetic changes such as microRNAs and DNA methylation represent potential biomarkers for monitoring cGvHD patients and novel targets for developing new treatment approaches. Finally, the microbiome likely affects the pathophysiology of cGvHD; bacterial strains as well as microbial metabolites could display potential biomarkers for dysbiosis and risk for the development of cGvHD. In summary, although there are no validated biomarkers currently available for clinical use to better inform on the diagnosis, prognosis or prediction of outcome for cGvHD, many novel sources of potential markers have shown promise and warrant further investigation using well characterized, multi-center patient cohorts.

There is a strong need to find new, good biomarkers of head and neck squamous cell carcinoma (HNSCC) because of the bad prognoses and high mortality rates. The aim of this study was to identify the potential biomarkers in HNSCC that have differences in their DNA methylome and potentially premalignant oral lesions, in comparison to healthy oral mucosa. In this study, 32 oral samples were tested: nine healthy oral mucosae, 13 HNSCC, and 10 oral lesions for DNA methylation by the Infinium MethylationEPIC BeadChip. Our findings showed that a panel of genes significantly hypermethylated in their promoters or specific sites in HNSCC samples in comparison to healthy oral samples, which are mainly oncogenes, receptor, and transcription factor genes, or genes included in cell cycle, transformation, apoptosis, and autophagy. A group of hypomethylated genes in HNSCC, in comparison to healthy oral mucosa, are mainly involved in the host immune response and transcriptional regulation. The results also showed significant differences in gene methylation between HNSCC and potentially premalignant oral lesions, as well as differently methylated genes that discriminate between oral lesions and healthy mucosa. The given methylation panels point to novel potential biomarkers for early diagnostics of HNSCC, as well as potentially premalignant oral lesions.

Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV− OPSCC, 62 to HPV− OSCC, and 31 shared with both. The results for 9 miRNAs (miR-9, -21, -29a, -100, -106b, -143 and -145) were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV− HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA.

Aim: To show the frequency and characteristics of cutaneous manifestations, with emphasis on vitiligo and alopecia areata in patients with chronic graft-versus-host disease (cGVHD). Methods: Patients with cGVHD were prospectively enrolled in the observational study protocol and evaluated by a multidisciplinary team of specialists. Dermatologic evaluation was focused on clinical spectrum of skin and adnexal involvement in each patient, determining the cutaneous GVHD score according to NIH Consensus criteria. Other objective clinical parameters including the existence of vitiligo, alopecia, xerosis, nail changes and dyspigmentation were assessed in each patient. Results: Among fifty cGVHD patients, 28 (56%) had skin involvement, and twenty seven of them (96%) had hypo and/or hyperpigmentations. In 11 patients (39%) the cutaneous NIH cGVHD score was mild, in 22% moderate and in 39% severe. In the whole cohort, nail changes were observed in 15 (30%) patients, while 10 (20%) were diagnosed with vitiligo or alopecia areata. In the univariate analysis patients with vitiligo/alopecia areata received more lines of prior systemic immunosuppressive therapy (P=0.043) ; they had lower Karnofsky performance status (P=0.028), and had higher number of B- cells (P=0.005), platelet count (P=0.022), total protein (P=0.024). Vitiligo and alopecia areata were associated with higher NIH skin score (P=0.001), were receiving higher intensity of immunosuppressive treatment (P=0.020) and TBI conditioning (P=0.040). In a multivariate regression model patients with higher NIH skin scoring were 3.67 times more likely to have alopecia and/or vitiligo (OR=3.67 ; 95% CI=1.26-10.73), controlled for all other factors in the model (age at study entry, number of B-cells, platelet count and global NIH score). Conclusion: These data indicate that vitiligo and alopecia areata occur more frequently in cGVHD than reported previously.

Human papillomavirus (HPV) E6 and E7 oncoproteins are critical for development and maintenance of the malignant phenotype in HPV-induced cancers. These two viral oncoproteins interfere with a plethora of cellular pathways, including the regulation of cell cycle and the control of apoptosis, which are critical in maintaining normal cellular functions. E6 and E7 bind directly with certain components of the Ubiquitin Proteasome System (UPS), enabling them to manipulate a number of important cellular pathways. These activities are the means by which HPV establishes an environment supporting the normal viral life cycle, however in some instances they can also lead to the development of malignancy. In this review, we have discussed how E6 and E7 oncoproteins from alpha and beta HPV types interact with the components of the UPS, and how this interplay contributes to the development of cancer.

The main etiological factor of precancerous lesion and invasive cervical cancer are oncogenic human papillomaviruses types (HPVs). The objective of this study was to establish the distribution of the most common HPVs in different cervical lesions and cancer prior to the implementation of organized population- based cervical screening and HPV vaccination in Croatia. In this study, 4, 432 cervical specimens, collected through a 16-year period, were tested for the presence of HPV-DNA by polymerase chain reaction (PCR) with three sets of broad-spectrum primers and type-specific primers for most common low-risk (LR) types (HPV-6, 11) and the most common high-risk (HR) types (HPV-16, 18, 31, 33, 45, 52, 58). Additional 35 archival formalin-fixed, paraffin embedded tissue of cervical cancer specimens were analyzed using LiPA25 assay. The highest age-specific HPV-prevalence was in the group 18–24 years, which decreased continuously with age (P

The human papillomavirus (HPV) may be associated with various oral, genital, and cutaneous conditions, both benign and malignant. The association between sexually transmitted α-HPV types is the strongest with cervical cancer because almost all such malignancies contain viral DNA, notably HPV types 16 and 18. The contribution of cancer causing HPV types in other anogenital, oral, and oropharyngeal malignancies, plus benign disorders, is lower and with a less significant public health concern. Cervical cytologic screening is a well-established preventive measure that allows early detection and successful treatment of precancerous cervical lesions. In cases of all other HPV-associated disorders, early detection of a precancerous lesion is either difficult or almost impossible. HPV vaccination remains the only preventive measure against most HPV-related diseases.

OBJECTIVE Our aim was to compare HPV and p16INK4a (p16) expression and their influence on survival and prognosis in oral cavity squamous cell cancer (OCSCC), between non-smokers and non-drinkers (NSND) and smokers and drinkers (SD). SUBJECTS AND METHODS Patients with OCSCC treated with surgery from 2000 to 2010 were included in the study. Patients who did not smoke at all or smoked less than 10 pack per years and did not drink alcohol on a daily basis were considered the NSND group. An equal number of SD were the control group. HPV presence was determined from paraffin-embedded blocks investigated by PCR analysis. p16 expression was evaluated with immunohistochemistry. RESULTS The NSND group were mostly younger or older female patients with tongue or gingival cancers. p16 expression was significantly more frequent in NSND patients (27% vs 10%). Patients with stronger p16 expression had significantly worse survival, especially for tongue cancers (P = 0.026). In Cox multivariate analysis, both HPV and p16 expression carried a negative prognosis for NSND patients (P = 0.0351 and P = 0.0260). CONCLUSIONS NSND are a specific population of OCSCC patients. In contrast to oropharyngeal cancer, HPV and p16 expression in OCSCC are negative predictive factors, especially in NSND patients.

The burden of cervical cancer is very high in low-resource countries, while it is lower in several high-income countries. Secondary prevention based on cervical screening has been very successful in countries where the resources exist to ensure high-quality and good coverage of the population at risk. In many developed countries that have effective organized screening programs, cervical cancer incidence and mortality rates have been reduced substantially. However, the cervical screening is generally inefficient and unworkable in the many regions of the world where the appropriate infrastructure is missing. Primary prevention by prophylactic vaccination against the major cause of cervical cancer, the carcinogenic human papillomavirus types 16 and 18, is now available worldwide and offers additional protection from cervical cancer. This review focuses on basic knowledge about cervical cancer and challenges in the new era of cervical cancer prevention.

Objectives Infection with oncogenic human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. In many cases of cervical cancer and all cervical cancer derived cell lines oncogenic HPV DNA is found to be integrated, indicating the importance of integration in disease development. In this study, 176 HPV 16 positive precancerous cervical lesions were analyzed for the physical state of viral genome to determine the sites of integration into a host cell DNA and to evaluate the incidence of the integration in different stages of cervical lesions. Methods The detection of integrated papillomavirus sequences (DIPS) method in combination with the amplification by polymerase chain reaction (PCR) of E1/E2 region was used to identify the physical state of HPV 16 genome. The site of integration within a host cell genome was determined by sequencing of unusual sized DIPS amplicons. Results The combined results of DIPS and E1/E2 PCR revealed the integration of HPV 16 DNA in 7.4% samples. The integration was found only in high grade cervical lesions indicating that it is a late event in disease progression. Sequencing of 11 DIPS amplicons revealed HPV DNA from 6 samples (54.5%) to be integrated in cellular genes ( VMP1 , PVRL1 , CHERP , CEACAM5 , AHR , MRF-2 ) and also 6 (54.5%) within the common fragile sites (CFS). Conclusions Although, the HPV integration is known to be a random event, this study indicates that HPV 16 integrates more than by chance within or close to CFSs. As most of the genes affected by HPV 16 integration can be linked with some aspects of tumor formation, this indicates that the site of HPV DNA integration might play a role in the rate and the nature of tumor development.

Change in the host and/or human papillomavirus (HPV) DNA methylation profile is probably one of the main factors responsible for the malignant progression of cervical lesions to cancer. To investigate those changes we studied 173 cervical samples with different grades of cervical lesion, from normal to cervical cancer. The methylation status of nine cellular gene promoters, CCNA1, CDH1, C13ORF18, DAPK1, HIC1, RARβ2, hTERT1, hTERT2 and TWIST1 was investigated by Methylation Specific Polymerase Chain Reaction (MSP). The methylation of HPV18 L1-gene was also investigated by MSP, while the methylated cytosines within four regions, L1, 5’LCR, enhancer, and promoter of the HPV16 genome covering 19 CpG sites were evaluated by bisulfite sequencing. Statistically significant methylation biomarkers distinguishing between cervical precursor lesions from normal cervix were primarily C13ORF18 and secondly CCNA1, and those distinguishing between cervical cancer from normal or cervical precursor lesions were CCNA1, C13ORF18, hTERT1, hTERT2 and TWIST1. In addition, the methylation analysis of individual CpG sites of the HPV16 genome in different sample groups, notably 7455 and 7694 sites, proved to be more important than the overall methylation frequency. The majority of HPV18 positive samples contained both methylated and unmethylated L1 gene, and samples with L1-gene methylated forms alone had better prognosis when correlated with the host cell gene promoters’ methylation profiles. In conclusion, both cellular and viral methylation biomarkers should be used for monitoring cervical lesion progression to prevent invasive cervical cancer.

Background: Chronic graft-versus-host disease (cGVHD) is a disorder that affects many organ systems in highly variable fashion occurring in approximately 50% of patients following allogeneic hematopoietic stem cell transplantation (alloHSCT). It is the major cause of non-relapse morbidity and mortality after alloHSCT in individuals otherwise cured of their hematologic diseases, inducing poor quality of life, impaired functional status, inability to work, and need for ongoing chronic care, which has also important impact to health-related costs. cGVHD Consensus Conference held in 2005 at the National Institutes of Health (NIH), USA, produced recommendations regarding cGVHD diagnosis, staging, histopathology, response criteria, biomarkers, ancillary and supportive care, and design of clinical trials. In 2014, second cGVHD NIH Consensus Conference updated these recommendations, published during 2015 as 6 papers in Biology of Blood and Marrow Transplantation (BBMT) journal. Although practitioners are generally familiar with the NIH recommendations, many barriers prevent their greater uptake in clinical practice. In order to overcome these challenges, in 2013 multidisciplinary clinic infrastructure was organized at the University Hospital Center (UHC) Zagreb, Croatia, in collaboration with the NIH leading scientists, using established cGVHD-related grading scales and measurements. Methods: Division of Hematology, UHC Zagreb, Croatia, has experience with alloHSCT since 1983, and 827 patients received alloHSCT until the end of 2014. Since the establishment of multidisciplinary cGVHD team in 2013, patients were enrolled into the Unity through Knowledge Fund (UKF) study protocol (funded by World Bank and Croatian Ministry of Science, Education and Sports) and examined by multiple subspecialists, firstly seen by hematologist, with detailed history and physical exam. Standard cGVHD scoring forms are filled according to NIH Consensus recommendations, and extensive laboratory analyses were done. Patients are seen and evaluated by other sub-specialists (Dental, Dermatology, Rehabilitation, Neurology, Ophthalmology, Gynecology, and other) with further workup as needed. Quality of life questionnaires are filled during the visit. All data are collected in a specially developed database and weekly team meetings were established. Blood and small biopsy tissue samples (skin, mouth) are stored for further research. Results: Using multidisciplinary approach since 2013, 46 (6 pediatric) cGVHD patients were assesed, median age was 41 years; range [9-71], 24 were male and 22 were female. The median time from transplant to enrollment was 20 months [2-258], from cGVHD diagnosis to enrollment 7 months [0.03-234] and from transplant to cGVHD diagnosis 10 months [2-128]. Additional 17 post-alloHSCT patients were eveluated, but without confirmation of cGVHD diagnosis. Among cGVHD patients, 31 (67%) of them received transplants from matched related donors, 27 (59%) of them had myeloablative conditioning, and 26 (57%) received peripheral blood stem cells as graft source. Thirty-five (76%) patients had previous acute GVHD, 11 (24%) had de novo cGVHD, 21 (46%) quiescent and 14 (30%) progressive onset; 41 (89%) were classified as classic and 5 (11%) as overlap; 23 (50%) patients had severe, 19 (41%) moderate, and 4 (9%) mild global cGVHD score. The most involved organs were skin (54%), eyes (50%), lungs (48%) and mouth (39%). Due to internationally peer reviewed UKF grant awarded in 2013 doctoral and postdoctoral researcher were hired, and visits of young clinicians to NIH and other cGVHD centers were realized. Several new research subprojects emerged since formation of our cGVHD team and applications to the new project calls were submitted. Also, 2 international cGVHD symposiums were organized in Zagreb, Croatia, in last 2 years stimulating education and networking. Conclusion: Implementation of NIH criteria for standardizationof cGVHD in Croatia showed remarkable results, not just improving quality of medical documentation and management of these long-terms survivors with complex and long-lasting health issues, but also facilitating further international clinical research and collaboration with cGVHD community, with potential positive impact to health-related costs and benefit to society. Disclosures Nemet: Pliva: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria.

The aim of this study was to evaluate the antiviral properties of clinoptilolite, a natural non-toxic zeolite. Herein, a fine powder of micronized zeolite (MZ) was obtained by tribomechanical micronization of natural clinoptilolite. Different viral suspensions were treated with MZ in concentrations ranging from 0.5 to 50 mg/ml. The viral proliferation was evaluated by optical microscope as percentage of cytopathic effect (CPE). Human adenovirus 5, herpes simplex virus type 1 (HSV 1) and human enteroviruses (coxsackievirus B5 and echovirus 7) were used in the antiviral assay. Concentrations of 0.5 and 5 mg/ml of MZ induced a very low antiviral effect or the antiviral was not observed at all, while concentrations of 12, 25 and 50 mg/ml of MZ induced a significant inhibitory effect upon viral proliferation. MZ inhibited the viral proliferation of HSV 1, coxsackievirus B5 and echovirus 7 more efficiently than that of adenovirus 5. The antiviral effect of MZ seems to be non specific and is more likely based on the incorporation of viral particles into pores of MZ aggregates than ion exchange properties of clinoptilolite. Our preliminary results indicate a possibility of therapeutical application of MZ, either locally (skin) against herpesvirus infections or orally in cases of adenovirus or enterovirus infections. Furthermore, MZ could also be used in purification of drinking water from different viruses.

Two hundred eight cervical specimens from two groups of subjects, 165 nonpregnant women and 53 pregnant women with cervical intraepithelial neoplasia (CIN) of grades I to III, were positive by PCR analyses for human papillomaviruses (HPVs), adeno-associated virus type 2 (AAV 2), and human cytomegalovirus (HCMV) in 67, 6, and 4.1% of the cases, respectively. The presence of AAV 2 infection was more frequently associated with pregnancy (17 versus 2.4%) and HPV-positive cervices (odds ratio = 6.358) than HCMV was. Increased HPV infection was strongly associated ( P < 0.001) with a higher CIN grade, but there is no evidence that AAV 2 and HCMV infections have any impact on CIN development.

Objective To determine the role of infections in miscarriages. Chorionic villi from aborted material were subjected to cytogenetic evaluation and analyzed for the presence of Chlamydia trachomatis , Ureaplasma urealyticum , Mycoplasma hominis , human cytomegalovirus (HCMV), adeno-associated virus (AAV), and human papillomaviruses (HPV). Design Retrospective study. Setting University hospital and academic research institution. Main outcome measure(s) Karyotyping and detection of bacterial and viral DNA by means of polymerase chain reaction (PCR) in placenta specimens. Result(s) In 54 (50%) of 108 samples the karyotype was normal, in 38 (35%) samples it was abnormal, and in 16 (15%) samples karyotype was undetermined. No U. urealyticum , M. hominis , HCMV, or AAV-2 DNA was detected, while C. trachomatis DNA was detected in one (1%) and HPV DNA in eight (7%) samples. No significant correlation of HPV-positive findings with karyotype status was established. Conclusion(s) Our findings do not support a role of C. trachomatis , U. urealyticum , M. hominis , HCMV, or AAV infections in miscarriages during the first trimester of pregnancy. However, further investigation should be made to determine a possible involvement of HPVs in the development of genetic abnormalities of the fetus and in miscarriages.

Oral lichen planus (OLP) and oral lichenoid lesions (OLL) are clinically and histologically similar lesions but their treatment planning and prognosis are different. The review of the literature indicates numerous criteria to distinguish these two lesions; however there is a lot of inconsistency. Thus, the aim of this study was to determine the correlation of histopathology and clinical OLP and OLL diagnosis and to clarify which histopathologic criteria could best distinguish these two diagnoses. A retrospective study showed that clinically diagnosed 92 OLPs and 14 OLLs have been confirmed histopathologically in 52.2% and 42.9% of cases, respectively. In addition, histopathology showed statistically significant more eosinophils (P<0.0005), plasma cells (P<0.0005), and granulocytes (P<0.05) in OLL than OLP. To establish histopathological diagnosis of OLP and OLL it should be mandatory to define the type of cells in mononuclear infiltrate, which can be associated more accurately with clinical feature and patient history. Therefore, currently accepted diagnostic criteria for OLP and OLL should be modified and validated on a larger number of patients taking into account particular distinguishing histopathological features.

Human papillomavirus (HPV) genotype 52 is commonly found in Asian cases of cervical cancer but is rare elsewhere. Analysis of 611 isolates collected worldwide revealed a remarkable geographical distribution, with lineage B predominating in Asia (89.0% vs 0%-5.5%; P(corrected) < .001), whereas lineage A predominated in Africa, the Americas, and Europe. We propose that the name "Asian lineage" be used to denote lineage B, to signify this feature. Preliminary analysis suggested a higher disease risk for lineage B, although ethnogeographical confounders could not be excluded. Further studies are warranted to verify whether the reported high attribution of disease to HPV52 in Asia is due to the high prevalence of lineage B.

About 5% of all cancers worldwide can be attributed to human papillomaviruses (HPVs) ; namely, six sites are strongly associated with HPV infections: cervix, penis, vulva, vagina, anus, and oropharynx. Nonmelanoma skin cancers (NMSC), basal cell carcinoma (BCC), and squamous cell carcinoma (SCC) are the most common malignancies in Caucasians. In fact, there is an intense connection between sunlight exposure, fair skin, HPV, and development of NMSC. We have conducted a pilot study that included tissue samples from 26 carcinoma patients, of which there were 13 BCC and 13 SCC. HPV detection and typing was done with DNA amplification and sequencing, respectively. In total, 23.1% of SCC samples (3/13) and 7.7% of BCC samples (1/13) were positive for HPV DNA. The importance of understanding all aspects of NMSC carcinogenesis may be to reveal novel therapeutic options or preventive measures for HPV containing NMSC patients.

To determine the incidence of Ureaplasma urealyticum and Ureaplasma parvum (UP) in symptomatic and asymptomatic women of reproductive age and to estimate antibiotic susceptibility of ureaplasma isolates. This study included 424 ureaplasma positive women of 1,370 tested women who visited gynecological practices during 2010. Cervicovaginal or urethral swab specimens from each patient were obtained for cultivation and molecular typing by RT-PCR. Ureaplasma spp. was identified by cultivation in 424 (34.4 %) cases, of which 79.0 % were from women with symptoms and 21.0 % from women without symptoms. Among ureaplasma positive women, 121 (28.5 %) were pregnant. Genotyping was successful in 244 strains, and the majority of samples were identified as UP (92.6 %). Among genotyped isolates, there were 79.5 % from symptomatic and 20.5 % from asymptomatic women; 29.9 % from pregnant and 70.1 % from non-pregnant women. There was no difference in the incidence of ureaplasma type regarding symptoms. Antibiotic susceptibility of 424 ureaplasma isolates identified by cultivation showed that all strains were susceptible to doxycycline, josamycin, erythromycin, tetracycline, clarithromycin and pristinamycin, but there was lower susceptibility to quinolone antibiotics, i.e., 42.9 and 24.5 % isolates were susceptible to ofloxacin and ciprofloxacin, respectively. This study shows that UP was the most frequent isolated ureaplasma species (92.6 %). Regarding antibiotic susceptibility, quinolones are not the best choice for the treatment of ureaplasma infections, while macrolides and tetracyclines are still effective.

The polymerase chain reaction (PCR) methods enable the detection of large number of human papillomavirus (HPV) genotypes that infect the anogenital tract. In this study, two groups of cervical scrapes with abnormal cytomorphology were analysed. The first group was tested with three sets of consensus primers located within the L1 region of HPV genome, MY09/MY11 (i.e. MY), L1C1/L1C2-1/L1C2-2 (i.e. LC) and pI-1/pI-2 (i.e. pI) primer sets, while the second group of samples, which were all negative with the MY primers, was tested further with the LC primers, as well as with the GP5/GP6 (i.e. GP) primers. The GP primers were used in the nested PCR following amplification with the MY primers (i.e. MY/GP nested PCR). Samples from both groups were also tested with type-specific primers for HPV types 6/11, 16, 18, 31 and 33. In the first study group (N=164) there were 76.2% positive results obtained with at least one set of consensus primers. There were 62.2, 39, 62.2 and 59.1% positive results obtained with the MY, the pI, the LC and the HPV type-specific primer sets, respectively. The best results were obtained when both the MY and the LC primer sets were used, because in combination they detected 75% positive samples compared to 62.2% when used alone. There were 2. 4% samples negative with all consensus primers, but positive with one of the HPV type-specific primers, which increased the overall positivity rate to 78.6%. In the second study group (N=250) there were 8.4, 38.8 and 4% samples positive with the LC primers, the nested MY/GP and the HPV type-specific primer sets, respectively. Thus, the use of the MY/GP nested PCR increased significantly the positivity rate of HPV DNA detection and should be used for samples with a low copy number of HPV DNA. In conclusion, the following diagnostic protocol would be appropriate for detection of cancer-related HPVs: preselection of samples with the MY and the LC primers, additional amplification of the MY- and the LC-negative samples with the MY/GP nested PCR and HPV typing of consensus PCR-positive samples with the HPV type-specific primers.

Immune system provides us protection from infectious pathogens and tumors formation during lifetime. Cervical cancer (CC), and its cause, human papillomavirus (HPV) are both challenges for the immune system. We present here evidence of epigenetic activation of immune system genes in CC. Illumina Infinium Human Methylation 450K BeadChip identified genes, which were all significantly hypomethylated in CC tissue versus normal tissue. The GeneMANIA computer program identified a tight network between those genes. The most strongly correlated genes based on their function are immune effectors' process (AIM2, BST2, BTN3A3, and IL12RB1) and response to virus related genes (AIM2, BST2, and IL12RB1). Thus, activation of those genes through demethylation is probably triggered by HPV oncogenes. In conclusion, the immune system of women who do not develop CC is probably activated earlier through DNA demethylation.

The association between certain human papillomaviruses (HPV) and cervical intraepithelial neoplasia (CIN) is well documented, but still unknown among Croatian women. In 1995, women between the age of 17 and 64 with cytomorphologically abnormal smears (CIN I–IV) were tested for the presence of HPV. Consensus and specific primers were used in the polymerase chain reaction (PCR) to detect the most common types: 6, 11, 16, 18, 31 and 33, as well as the unknown-risk HPV types (HPV X). Out of 379 specimens, 163 (43%) contained one or more HPV types. Coinfection with different HPV types in the same sample was observed in 16 cases. Beside low-risk HPV 6/11 (25.8%) the most frequently observed types were high-risk HPV types 16 (20.2%) and 31 (17.8%). Globally, the HPV positivity rate declines with age. The presence of HPV DNA significantly increased from 35.5 to 61.1% along with the severity of the cervical intraepithelial neoplasia (CIN I– IV). HPV type 6/11 was strongly associated with CIN I (33.8%), HPV type 31 with CIN II (22.9%), and HPV type 16 with CIN III (50%).

The oncogenic human papilloma viruses (HPVs) are associated with precancerous cervical lesions and development of cervical cancer. The DNA methylation signatures of host genome in normal, precancerous and cervical cancer tissue may indicate tissue-specific perturbation in carcinogenesis. The aim of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared to the DNA samples from cervical intraepithelial neoplasia grade 3 (CIN3) and normal cervical scrapes. The Illumina Infinium Human Methylation 450 K BeadChip method identifies genome- wide DNA methylation changes in CpG islands, CpG shores and shelves. Our findings showed an extensive differential methylation signature in cervical cancer compared to the CIN3 or normal cervical tissues. The identified candidate biomarker genes for cervical cancer represent several types of mechanisms in the cellular machinery that are epigenetically deregulated by hypermethylation, such as membrane receptors, intracellular signaling and gene transcription. The results also confirm the extensive hypomethylation in cancer cells of genes in the immune system. These insights into the functional role of DNA methylome alterations in cervical cancer could be clinically applicable in diagnostics and prognostics, and may guide the development of new epigenetic therapies.

As iron ions may participate in the pathogenesis of cancer and viral infections, the aim of this study was to monitor their influence on proliferation, E6 and E7 oncogene expression and reactive oxygen species (ROS) production in two human papilloma virus (HPV) positive cervical carcinoma cell lines (HeLa and SiHa) and one HPV negative vulvar cell line (A431). The anti-anaemic drug, ferric-sorbitol-citric acid complex (FSC) as a source of Fe(III) ions was used. Cells were treated with FSC at the concentrations between 0.001 and 1 mM Fe(III) for different time periods. Fe(III) ions inhibited the viability of HeLa and A431 cells while it had no influence on SiHa cells. Furthermore, Fe(III) treatment showed a time-dependent and a higher stimulatory effect on E6/E7 expression in SiHa cells than in HeLa cells. Fe(III) ion treatment with concentrations lower than 0.1mM showed a time and a concentration dependent intracellular ROS production in all tested cell lines, while the treatment with 1mM concentration decreased ROS production in all tested cell lines. In conclusion, Fe(III) ion treatment apart from having an anti-tumour effect, as we previously described, enhances survival of HPV 16-positive cells and might be associated with HPV oncogenesis.

Summary Background Knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. We aimed to provide novel and comprehensive data about the worldwide genotype distribution in patients with invasive cervical cancer. Methods Paraffin-embedded samples of histologically confirmed cases of invasive cervical cancer were collected from 38 countries in Europe, North America, central South America, Africa, Asia, and Oceania. Inclusion criteria were a pathological confirmation of a primary invasive cervical cancer of epithelial origin in the tissue sample selected for analysis of HPV DNA, and information about the year of diagnosis. HPV detection was done by use of PCR with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay. Sequence analysis was done to characterise HPV-positive samples with unknown HPV types. Data analyses included algorithms of multiple infections to estimate type-specific relative contributions. Findings 22 661 paraffin-embedded samples were obtained from 14 249 women. 10 575 cases of invasive cervical cancer were included in the study, and 8977 (85%) of these were positive for HPV DNA. The most common HPV types were 16, 18, 31, 33, 35, 45, 52, and 58 with a combined worldwide relative contribution of 8196 of 8977 (91%, 95% CI 90–92). HPV types 16 and 18 were detected in 6357 of 8977 of cases (71%, 70–72) of invasive cervical cancer. HPV types 16, 18, and 45 were detected in 443 of 470 cases (94%, 92–96) of cervical adenocarcinomas. Unknown HPV types that were identified with sequence analysis were 26, 30, 61, 67, 69, 82, and 91 in 103 (1%) of 8977 cases of invasive cervical cancer. Women with invasive cervical cancers related to HPV types 16, 18, or 45 presented at a younger mean age than did those with other HPV types (50·0 years [49·6–50·4], 48·2 years [47·3–49·2], 46·8 years [46·6–48·1], and 55·5 years [54·9–56·1], respectively). Interpretation To our knowledge, this study is the largest assessment of HPV genotypes to date. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 should be given priority when the cross-protective effects of current vaccines are assessed, and for formulation of recommendations for the use of second-generation polyvalent HPV vaccines. Our results also suggest that type-specific high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45. Funding Spanish grants from Instituto de Salud Carlos III, Agencia de Gestio d'Ajuts Universitaris i de Recerca, Marato de TV3 Foundation, and unrestricted grants from GlaxoSmithKline Biologicals, Sanofi Pasteur MSD, and Merck.

Following the 2003 Recommendation of the Council of the European Union on cancer screening, equal access to organised cervical cancer screening is supposed to be ensured for all women at risk in all member states. However, the first IARC report on the implementation of the Council Recommendation suggests that a remarkable proportion of women in the new member states are not yet covered with the free Pap tests offered either in organised or opportunistic manners. Cervical cancer incidence and mortality rates in most of these countries are among the highest in Europe. The purpose of this paper is to identify some common challenges and make further proposals in organising and implementing quality-assured cervical cancer screening programmes in these countries. Based on the responses to a corresponding questionnaire, a summary on cervical cancer prevention policies was established for the seven new European Union member states, Czech Republic, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia, and two candidate states, Croatia and Serbia. In most of these countries there are a lot of challenges to overcome before achieving the level of preventive services as seen in Finland and the Netherlands nowadays.

The identification of the etiological factor of many cervical precancerous lesions and cervical cancer, the Human papillomavirus (HPV) is widely used. In this study, we evaluated the consensus and type-specific (TS) Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP), Line Probe Assay (LiPA, Innogenetics) and sequencing to determine the HPV types in cervical specimens. Out of 690 High-grade Squamous Intraepithelial Lesion (HSIL) samples, 86.7% were HPV positive and 13.3% HPV negative by consensus primers (MY09/MY11, L1C1/L1C2-1/L1C2-2 and/or GP5/6) directed PCR. Out of 598 HPV positive samples, 85.3% were typed by TS-PCR being HPV 6/11, 16, 18, 31 and/or 33, while 14.7% remained untyped. The most prevalent HPV type in the study group was HPV 16, identified in 35.5% cases, while HPV 31 was the second most frequent HPV type with a prevalence of 10.5%. They were followed by HPV types 6/11, 33 and 18 with a prevalence of 7.4%, 6.2% and 4.9%, respectively. Multiple HPV infections with two or more HPV types (6/11, 16, 18, 31 and/or 33) were found in 9.4% cases. A subset of 88 samples was further typed by RFLP and LiPA to determine the rare HPV types in HSIL samples. The most frequent low abundant HPV types in single infections in decreasing order were HPV 53, 58, 66, 56 and 52, while HPV 51 was the most frequent low abundant HPV type found in multiple HPV infections. Multiple HPV infections were mostly found by LiPA in 27.3% cases versus 14.8% cases found by RFLP. The perfect agreement between RFLP and LiPA assay pair was observed only for HPV types 16, 18, 34 and 59 (Kappa value of 1). For other HPV types, the inter-assay agreement ranged from very good to no agreement indicating that neither assay is perfect. Sequencing was performed for 33 samples in cases where both RFLP and LiPA were inconclusive. Sequencing was shown to be a very good method in case of single HPV infection but not applicable in case of multiple HPV infections. Both RFLP and LiPA are good assays for epidemiological studies, although RFLP being cumbersome and time-consuming is less applicable than LiPA. Careful consideration has to be made before the implementation of either HPV typing methods in clinical laboratories.

National and international experts in cervical cancer prevention met at the International Workshop on Human Papillomaviruses and Consensus Recommendations for Cervical Cancer Prevention to review the current evidence and assess the potential for improvement in cervical cancer prevention and to develop plans for implementation of cervical cancer prevention programmes in Croatia. Key recommendations were developed and adopted during the course of the meeting. The process of bringing national experts together with internationally recognized experts in an open forum for the development of consensus recommendations could serve as a model for other countries seeking to implement or improve cervical cancer prevention programmes.

The human papillomavirus (HPV) 16 genome has been studied extensively, although no study has focused on the E1 gene that is implicated in viral DNA replication. After analyzing the E1 region of HPV 16 genomes in 429 cervical samples, 11.2% were found to contain a 63 nucleotides duplication in this region. Sequence analysis of the E6 and the E7 regions has shown that all samples containing this duplication were related to E6-G350 variant of the HPV 16 (Chi square test, P = 0.0012). A comparison of cervical lesion severity of the examinees having regular or variant E1 genes has shown that the variant group had a significantly (Fischer's exact test, P = 0.0401) lower percentage of high grade disease cases, suggesting that this particular duplication might reduce the oncogenic potential of HPV 16, and also might clarify the differences of E6-G350 variant oncogenicity observed in European populations. Albeit, a decreased incidence of high grade cervical lesions can be linked to the prevalence of multiple HPV infection, the additional decrease of those cases with the variant E1 gene versus those without (10.5% and 18.6%, respectively) can only be ascribed to the effect of this particular HPV variant. Further research is needed to clarify the biology of these HPV 16 E1 variants.

Anogenital human papillomavirus (HPV) infections are the most frequently diagnosed sexually transmitted diseases (STDs) of viral origin. The HPV types associated with such lesions have been studied extensively during the last several years.1,2 HPV-associated genital pathology represents one of the major problems with STDs, due to the high recurrence rate, difficulty in eradication, and the oncogenic potential of some HPV types.3–5 HPV genital infections are also among the most frequent diagnoses made in the STD Out-patient Clinic of the Department of Dermatology and Venereology of the Zagreb University Medical School. The frequency of HPV genital infections ranged from 125 patients in 1991 to 193 patients in 2000.6,7 Generally, the patients with HPV genital infection visit the STD Clinic regularly, and their compliance rate is mostly satisfactory, as opposed to patients carrying the diagnosis of nongonococcal urethritis. The association between certain HPV types and cervical intraepithelial neoplasia is well documented among Croatian women8–11; however, similar studies are very rare.12 During the last few years, different diagnostic tools have been used for the patients with HPV genital infection, including pathohistology.13–15 The results have not been sufficiently satisfactory in certain cases; ie, the answer was not precise enough as to whether the lesion was HPV induced or not. Because of the small sample size that can only be taken for HPV DNA detection from male genital lesions, we used a very sensitive and specific molecular method, the polymerase chain reaction (PCR) with HPV consensus primers. With type-specific primers, we typed HPV types 6/11, 16, 18, 31, and 33. Restriction fragment length polymorphism (RFLP) was applied on HPV 6/11 PCR products to distinguish further between HPV types 6 and 11. This would permit us to evaluate the value of viral detection by means of PCR in case of male anogenital lesions.

Summary Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of Wistar-Furth rats that had been immunized with a Moloney sarcoma virus (Mo-MuSV)-induced tumour, MFU. Two immunization protocols were designed. In the first, the animals received several injections of irradiated (10000 rad) cells of a tumour cell line established in vitro, MFU-67. The rats received a booster injection 3 days prior to fusion. In the second protocol, immunization was the result of simple tumour growth, and no booster was given. Hybrids were tested by immunofluorescence for the production of immunoglobulins reacting with mouse cells acutely infected with Mo-MuSV. Over 20% of reactive hybrids were observed in the tumour growth protocol, and about 10% in the irradiated cell protocol when the last injection of the series was given 2 weeks before fusion. After 6 months, the proportion fell to 3%. Hybrid lines producing antibody to p30, the major core polypeptide of murine retroviruses, were obtained by cloning. Three of these were selected for closer study and were found to recognize three non-overlapping epitopes on p30. By direct and competitive binding in ELISA tests, the three epitopes were found to have very different distribution patterns among the various strains and isolates of murine retroviruses.