Viral infections contribute as a cause of 15–20% of all human cancers. Infection by oncogenic viruses can promote different stages of carcinogenesis. Among many types of HPV, around 15 are linked to cancer. In spite of effective screening methods, cervical cancer continues to be a major public health problem. There are wide differences in cervical cancer incidence and mortality by geographic region. In addition, the age-specific HPV prevalence varies widely across different populations and showed two peaks of HPV positivity in younger and older women. There have been many studies worldwide on the epidemiology of HPV infection and oncogenic properties due to different HPV genotypes. However, there are still many countries where the population-based prevalence has not yet been identified. Moreover, cervical cancer screening strategies are different between countries. Organized cervical screening programs are potentially more effective than opportunistic screening programs. Nevertheless, screening programs have consistently been associated with a reduction in cervical cancer incidence and mortality. Developed countries have achieved such reduced incidence and mortality from cervical cancer over the past 40 years. This is largely due to the implementation of organized cytological screening and vaccination programs. HPV vaccines are very effective at preventing infection and diseases related to the vaccine-specific genotypes in women with no evidence of past or current HPV infection. In spite of the successful implementation of the HPV vaccination program in many countries all over the world, problems related to HPV prevention and treatment of the related diseases will continue to persist in developing and underdeveloped countries. According to the World Health Organization's (WHO) statistics, common cancers are one of the most prevalent causes of mortality worldwide with 8.2 million deaths in 2012, and this trend has not changed in recent years. Viral infections contribute to 15–20% of all human cancers, whereby several viruses play considerable roles in the multistage development of malignant cancers. Over the past two decades, it has become obvious that several viruses play an important role in the development of human cancers. Around 15% to 20% of cancer cases are associated with viral infections. Oncogenic viruses can facilitate various stages of carcinogenesis .HPV is a double-stranded DNA virus belonging to the Papovaviridae family. Almost 200 HPV types have been identified with more than 40 types colonizing the genital tract. All HPV infection types are divided into two groups based on their carcinogenic properties; these are high risk and low risk. High-risk types include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68, and 59. Others are classified as potential high-risk . Currently, it is well known and proven that HPV16 and 18 are the most virulent high-risk genotypes, causing about 70% of all invasive cervical cancer in the world [At the present time, we have a relatively clear picture of HPV infection's natural history, oncogenic properties, screening, and prevention algorithms. However, HPV infection rates continue to persist, especially in developing countries, where cervical cancer incidence and prevalence are still high. This is due to different reasons, which include low socioeconomic status, lack of population awareness, and inadequately implemented screening and vaccination programs. Thus, it is necessary to continue this discussion and to refocus attention of specialists and population worldwide to HPV infection and related diseases. The aim of this review article is to summarize updated information regarding the aforementioned aspects of HPV infection and related cancers, including also discussions about the HPV genome and molecular events leading to cancer development following an HPV infection. Enhanced knowledge of HPV status and cancer progression events contributes to the improvement of the future management of patients with cervical lesions; this in turn can help mitigate cervical cancer progression among HPV-infected women.Papillomavirus genome is comprised of a small double-stranded and highly conserved DNA with an approximate size of 8000 base pairs and consists of three regions. The molecular biology of this small DNA molecule is complex. There are six early proteins, three regulatory proteins  and three oncoproteins  encoded in 4000 base pairs (bp) that participate in viral replication and transformation of cell. Another 3000 bp region of DNA molecule encodes two structural proteins L1 and L2 that compose the capsid of virus. The viral DNA replication and transcriptional regulatory elements are controlled by a long control region (LCR) that is encoded in a 1000 bp region [Upon the viral evolution, accumulation of numerous lineage-defining genetic variations in these regions can lead to speciation into separate HPV types. Sequence variations such as single-nucleotide polymorphisms or genetic mutations within L1, LCR, E6, and E7 regions of HPV can determine families, relatedness, and phylogeny of the HPV types. HPV type can be defined as an entity based on the more than 10% difference in the DNA sequence of the L1 gene between two genomes. However, the difference between 2% and 10% determines the HPV subtypes. In addition, the variants are entities that define less than 2% of dissimilarities between HPV genomes. According to recent studies, there are 60 out of 160 HPV types associated with mucosal epithelia and categorized as Alphapapillomavirus genus  .According to Papillomavirus Nomenclature Committee, each HPV type can be differentiated into phylogenetic lineages in terms of geographic distribution, pathogenicity, regulation of transcription, and immunological response . The alpThe vast majority of HPV infections are transitory and become undetectable in 12–24 months –14. HoweNatural history of CIN lesions is different depending on its grade. CIN1 is a low-grade squamous intraepithelial lesion (LSIL). According to statistical data, over 70–80% of CIN1 lesions spontaneously regress without treatment or become undetectable , 16. ThuCIN2 and CIN3 are considered high-grade dysplasia or high-grade squamous intraepithelial lesion (HSIL); however, they are different whereby CIN2 less commonly progresses to cancer. CIN2 develops in two different ways; the annual regression rate of CIN2 in adult women is estimated to range from 15 to 23%, with up to 55% regressing by 4–6 years , 17, wheThe link between high-risk HPV types and cervical cancer development contributed to the introduction of novel screening programs. For example, testing for the presence of high-risk HPV is recommended as a screening tool by the WHO and the European Guidelines for Quality Assurance for Cervical Cancer Screening , 22. HPVCervical cancerogenesis can be defined as the complex mechanism of uncontrolled cellular division that can involve HPV gene integration together with other cellular changes and epigenetic factors. As the HPV infection occurs, the DNA can undergo mutations under the cellular and other environmental conditions leading to viral DNA integration and operation with the host DNA synthesis machinery. As a result, virus can escape cellular and immune defense mechanisms while promoting cell proliferation and inhibiting cellular apoptotic mechanisms.Oncogenic potential of HPV16 depends on the regulation of viral transcriptional factors. At the initiation of viral infection, the HPV16 genome can be presented as unintegrated small DNA molecule also called episome and results in benign and precancerous lesions of the cervix. However, HPV16 can integrate its genome into the host genome, which in turn can lead to the development of cervical carcinoma and cervical intraepithelial neoplasia grade III . Viral gThere are many types of HPV, which are found to be associated with cancerous diseases—16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 types . In HPV1This binding leads to the degradation of p53 and thus leads to cell proliferation. E7, on the other hand, binds pRb causing its inactivation and degradation. Both the low-risk and high-risk E7 protein has been shown to target the pRB family members including p107 (RBL1) and p130 (RBL2) for degradation . pRb dowmiRNA plays an important role in the posttranscriptional control of the expression of host genes. Recent studies proposed that HPV E6, E7, and E5 oncoproteins regulate the host miRNA profile. In HPV-associated cervical cancer cells, a number of miRNAs such as miR-21, miR-143, and miR-9 are overexpressed, thus targeting CCL20 (chemokine (C-C) motif ligand) and promoting migration of HPV16-positive cancerous cells. However, overexpression of some miRNAs such as miR-203 inhibits HPV amplification. Thus, in the HPV-infected cancer cells, miR-203 is suppressed by HPV E7 gene overexpression, leading to the induction of viral replication. Deregulation of miRNA expression can occur mostly due to epigenetic methylation of miRNA promoters .E6 belonging to tumorigenic HPV types harbors a PDZ binding motif (PBM) at the C terminus which facilitates the binding of E6 to a number of proteins containing the PDZ site. The binding of E6 to these proteins leads to inactivation and degradation. Examples of such proteins include potential tumor suppressors such as Dlg , MAGI-1 The epigenetic control of viral and host gene expression plays an important role in carcinogenesis by involving changes in DNA methylation, modifications of histones, and noncoding RNA profile. Cervical carcinogenesis is strongly associated with persistent HPV infection that can further affect both the host genome and the viral genome methylation process .E6 and E7 have been shown to bind DNA methyltransferases (DNMT) which impairs their activity leading to hypermethylation of CpG islands which can eventually lead to possible silencing of host tumor suppressors , 39. SomIt has been suggested that increased methylation of CpG dinucleotides within E2 binding site (E2BS) on the host genome can modify interaction of different factors and result in abnormal cell differentiation with further disease progression . As a reCervical cancer is the leading genital cancer among women worldwide, with almost half a million new cases per year  . In 2015The mortality from cervical cancer varies in different geographic regions. The age-standardized incidence rate for cervical cancer is much lower in developed countries at 5.0 per 100,000 compared to developing countries at 8.0 per 100,000 . SimilarLimited statistical data are available on cervical cancer in Central Asia . From thCervical cancer has a bimodal age distribution with the majority of cases occurring among women in their 30s and 40s, the age at which women are often raising families and ensuring the financial viability of their families and communities. In addition to the risk of death, cervical cancer is associated with increased morbidity, including bleeding, pain, and kidney failure, which are difficult to treat, especially in communities with poor access to healthcare .HPV infections are widespread all over the world; however, prevalence and type distribution are heterogeneous . The ageThere have been many studies worldwide on the epidemiology of HPV infection and oncogenic properties due to different HPV genotypes . One of According to the Extended Middle East and North Africa (EMENA) study, in the Middle East, the incidence of HPV shows lower rates compared to the rest of the world . For insVery limited data are available on HPV prevalence, incidence, and genotype-specific dissemination in Central Asia and Eastern Europe. For example, according to the report of HPV Information Centre (2017), no data on the epidemiology of HPV infection are available in Kazakhstan , and only a few articles on the epidemiology of HPV infection in Kazakhstan were published in international peer-reviewed journals and several articles in local medical journals . The autThe prevalence of HPV infection among Africans is higher than in the European population with 26.3% in Nigeria, 47.9% in Guinea, 41% in South Africa, and 38.8–42.3% in Kenya , 55. PosOther studies highlighted that some special populations have a higher risk of acquiring HPV infection. A study investigated the prevalence of HPV infection among the adolescent population in Uganda has shown significantly high distribution of high-risk HPV types , which is 51.4% . The reaWith the development of highly sensitive HPV DNA testing, studies have confirmed that most cervical cancer specimens have detectable HPV DNA, and greater than 90% contain DNA for HPV16, 18, 31, 33, 39, 45, 52, or 58 . It shouSome investigators have identified regional differences in the prevalence of squamous cell carcinoma linked to HPV infection. In a meta-analysis of 85 studies, which included 10,058 women with cervical cancer, HPV16 prevalence predominated in squamous cell carcinoma, ranging from 46% in Asia to 63% in North America. The second most prevalent type was HPV18, found in 10–14% of squamous cell carcinoma specimens. The frequency of adenocarcinoma among all invasive cervical cancers also remains significant. It ranges from 4% in Africa to 32% in North America. As expected, high-risk HPV type 18 was found to be dominant in adenocarcinoma cases with a prevalence that ranges from 37% to 41%. The next most common HPV types are type 16 and type 45, which were found in 26–36% and 5–7% of samples, respectively . AccordiSeveral international studies investigated the prevalence of HPV types in invasive cervical cancer specimens. One of those studies explored the most prevalent types in 1918 women with cervical cancer. For that purpose, cervical cancer cells were directly tested for HPV types and the researchers found the following HPV types to be the most prevalent: HPV16, 18, 45, 31, 33, 52, 58, and 35 . SimilarHaving a high incidence and mortality from cervical cancer makes the screening program very important. Enhancing public awareness of underlying causal factors is a high priority for developing an appropriate cancer control and prevention program.It is well known that cervical cancer screening can reduce cervical cancer incidence and mortality . CervicaHPV vaccine was introduced later. Developed countries have accomplished reduction of cervical cancer incidence and mortality during the last 40 years due to the introduction of cytological smear screening . For insAt the same time, cervical cancer prevalence remains at a high level in developing countries of Central and South-East Asia, Africa, and Eastern Europe, where cervical cancer screening programs are not properly implemented due to a variety of reasons . Population coverage by screening program in developing countries ranges between 6 and 8% . In receThere is also discrepancy in the frequency of the screening tests among countries and age groups . In deveIn most of the developing countries of Africa, Central Asia, South-East Asia, Eastern Europe, screening is scheduled every 5 years or even rarer , 76. HowAlthough the recommended screening modalities for cervical cancer have contributed to a reduction in cervical cancer incidence and mortality due to cervical cancer, the benefits of cervical cancer screening are yet to be fully realized in countries with poorly organized screening programs for women at risk. The updated WHO recommendations for cervical cancer screening and prevention are summarized in It is also noteworthy that even in countries with organized screening services, these benefits are not maximized in underserved, uninsured, and underrepresented populations due to factors such as cost, access problems, anxiety, discomfort with the screening procedure, and fear of cancer or poor health literacy, all of which contribute to poor outcomes for cervical cancer .Incorporation of HPV testing into cervical cancer screening strategies has the potential to allow both increased disease detection and increased length of screening intervals .Statistical data from the recent years show that utilization of HPV vaccines is very effective for preventing infection and disease related to the specific HPV genotypes . VaccinaThere are three commercially prophylactic vaccines available; these are Cervarix , Gardasil , and Gardasil 9 . They are noninfectious subunit vaccines containing viral-like particles (VLP) derived from the assembly of the recombinant expression of L1 major capsid protein of the HPV in yeast (Gardasil) and in insect cells (Cervarix). Administration of the vaccine is carried out by intramuscular injection with three doses of prime/boost series over a 6-month period. Early analysis shows that even a single dose can reduce infection and is effective in preventing the persistent incidence of infection and premalignant neoplasia . The excAt the present time, we have an abundance of evidence from multiple countries, with a different level of HPV vaccination coverage and implementation strategies that show the vaccines are effective . In deveTaking into consideration the present efforts to increase HPV vaccinations for primary cervical cancer prevention, early detection of precancerous cervical lesions through screening remains to be very important in order to timely diagnose and reduce cervical cancer incidence and mortality. This is especially true in low-income regions where HPV vaccination has not yet implemented and supported at the governmental level . DevelopHPV vaccination for the prevention of high-risk HPV types is expected to reduce cervical cancer burden . SupportThere are also apparent limitations and the public health challenges in attempting to implement HPV vaccination programs. These limitations and challenges include the vaccine's type specificity, required to be given prior to exposure, the three-dose schedule, ethical issues in targeting age group of early adolescence, and potential communication challenges around HPV being a sexually transmitted infection . TherefoIn developing countries, reduction of cervical cancer incidence and mortality could be achieved only with governmental guidance by the implementation of sustainable and effective screening and vaccination programs.Cervical cancer is associated with considerable morbidity and mortality all over the world. It is well known that one of the main causative agents for cervical cancer is high-risk HPV strains, and this type of malignancy is preventable. High incidence of cervical cancer with considerable mortality is an evidence of HPV infection abundance with the absence of the HPV screening and low public awareness of the problem. Substantial incidence and mortality from cervical cancer make the screening program very important. Enhancing public awareness of underlying causal factors is a priority that should be emphasized for prevention programs. Incorporation of HPV testing into screening strategies has a high potential to decrease morbidity and mortality from cervical cancer. The knowledge of HPV prevalence and type distribution could contribute to the successful vaccination program implementation. The educational health promotion projects for the population should be provided to reinforce the knowledge and conversance of this public health problem. From the review given here, it is clear that the HPV screening along with the vaccination program should be implemented and supported at a governmental level in developing countries with high incidence and mortality of cervical cancer.


The pathogenesis of lung cancer is triggered by a combination of genetic and environmental factors, being the tobacco smoke the most important risk factor. Nevertheless, the incidence of lung cancer in non-smokers is gradually increasing, which demands the search for different other etiological factors such as occupational exposure, previous lung disease, diet among others. In the early 80’s a theory linked specific types of human papillomavirus (HPV) to lung cancer due to morphological similarities of a subset of bronchial squamous cell carcinomas with other HPV-induced cancers. Since then, several studies revealed variable rates of HPV DNA detection. The current study aimed to provide accurate information on the prevalence of HPV DNA in lung cancer.Biopsies were collected from 77 newly diagnosed non-small cell lung cancer (NSCLC) patients treated at the Thoracic Oncology Department at Barretos Cancer Hospital. The samples were formalin fixed and paraffin embedded (FFPE), histologic analysis was performed by an experienced pathologist. DNA was extracted from FFPE material using a commercial extraction kit and HPV DNA detection was evaluated by multiplex PCR and HPV16 specific real-time PCR.HPV was not identified in any of the samples analysed (69).Our data demonstrated a lack of HPV DNA in a series of NSCL cancers. Lung cancer is the major cause of cancer-related death worldwide. The International Agency for Research on Cancer (IARC) estimated 1.8 million new cases for 2016 . HoweverSyrjanen 2012)  performe12  perfoHPV infection in lung tissue was evaluated by another meta-analysis using the search terms “lung cancer”, “human papillomavirus”, “HPV” and their combinations and, antagonistically, the results suggested that HPV infection in lung tissue have a strong association with lung cancer development . AnotherAdditionally, a Chinese a study developed to identify the association between HPV positive rate and smoking in lung cancer (LC) patients observed that HPV infections are associated with smoking in LC patients and the association may relate to different regions . Lung caDue to discrepancies among the results across the world and previous work from our country, we aimed to provide detailed additional data on the HPV burden in lung cancer by evaluating the prevalence of HPV DNA in samples of non-small cell lung cancer (NSCLC) using a specific method for this. Furthermore, p16 expression was accessed by immunohistochemistry (IHC) to further address if it’s immunoexpression could be considered as a surrogate marker of HPV activity in NSCLC specimens.Samples were achieved from a prospective analysis of patients submitted to the bronchoscopy admitted at Barretos Cancer Hospital between 2013 and 2015. All biopsies were performed for diagnosis and all the patients (77) were naïve of treatment. Patients with NSCLC diagnosis that agreed to participate were included.Written informed consent was provided by all patients and the study was approved by Barretos Cancer Hospital Ethic Committee (number 920.225).All 77 patients were subjected to a detailed trained interviewer assisted interview for assessment of demographic, NSCLC risk factors and previous history of HPV infection variables.Tumor samples were obtained by endobronchial and transbronchial biopsies. Specimens were routinely processed for histological diagnosis, HPV testing and for immunohistochemical analysis of p16. Pathologic stages were determined according to the American Joint Committee on Cancer (AJCC) , and hisFormalin-fixed, paraffin-embedded (FFPE) tumor tissue sections, previously evaluated and delimited by an expert pathologist (CSN), were deparaffinized, and using QIAamp DNA Micro kit  DNA was extracted according to the manufacturer’s recommendations. Briefly, 4–6 10 μm FFPE sections were submitted to deparaffinization with 100% xylene, followed by washes of ethanol  and incubation with DNA extraction buffers overnight.All 77 DNA samples were quantified by NanoDrop 2000 (ThermoScientific) and subjected to β-globin gene amplification using PCO3 (ACACAACTGTGTTCACTAGC 5′-3 ‘) and PCO4 (CAACTTCATCCACGTTCACC 5’-3′) primers. The amplification of a short fragment of 110 pair base of human β-globin gene indicates the integrity of DNA for subsequent analysis. Only β-globin positive samples (62) were used for HPV analysis.HPV DNA in lung tumors was independently assessed using two different techniques based on real-time PCR. The first one consisted of TaqMan singleplex based type-specific real-time PCR targeting the E7 region of HPV 16 type using specific TaqMan probe (5′- 6FAMCAAGCAGAACCGGACAG-MGBNFQ-3′) and primers (forward 5′-GATGAAATAGATGGTCCAGC-3′ and reverse 5′- GCTTTGTACGCACAACCGAAGC-3′) . Among 7Additionally, 29 samples were randomly chosen for testing using a type-specific PCR bead-based multiplex genotyping assay that combine multiplex PCR and bead-based Luminex technology  that is able to identify 21 HPV types , 29, as Sections of 4 μm containing representative tumour areas were used for IHC, which was performed using Ventana Benchmark ULTRA automated system . Antigen retrieval was performed using cell conditioning 1 buffer (CC1) at 95 °C for 64 min. For detection of immune reaction we used the ultraView Universal DAB Detection Kit polymer amplification system  according to manufacturer’s instructions. Mouse monoclonal anti-human antibody against p16INK4A protein, Clone E6H4™, ready for use  was used as primary antibody. Samples with strong and diffuse nuclear and cytoplasmic staining in more than 70% of the cells were considered positive , 26. AllFrequencies were obtained using the IBM® SPSS® Statistics 21.0 software for Windows .n = 47, 75.8%), married , and 54 (87.1%) have lived in rural area. As a consequence, 21 (33.9%) patients were farmer. Further characteristics of the patients included in this study are summarized in Table Among 77 samples included in this study, 15 were excluded due to the lack of β-Globin amplification and 62 were suitable for further analysis: 41 (66.1%) squamous cell carcinomas (SCC) and 21 (33.9%) adenocarcinomas. The SCC staging was as follow: 2 (3.4%) stage I, 4 (6.8%) stage II, 26 (44.1%) stage III and 27 (45.8%) stage IV. Additionally, 27 tissue samples (51.9%) were classified as well or moderately differentiated, and 25 (48.1%) as poorly differentiated. Most patients were male  cases. The analysis of positive cases for the expression of p16 (10/70) in relation to socio-demographic information, lifestyle and clinical-pathological findings showed no statistically significant correlation (Table The main goal of this study was to evaluate the presence of HPV DNA in NSCLC. Many contradictory results have been found among the reports published about this issue; the majority of discordances may likely be attributable to the differences in methodology used to identify HPV and the fact that many studies have no control regarding the quality of the samples preservation retrospectively selected for analysis. After all, it is not a surprise that an assertive attention about the real meaning of HPV in lung cancer is currently questionable. We did not find any NSCLC positive sample for HPV DNA among the samples we evaluated, despite the use of a very sensitive methodology we used to identify HPV, and the stringent conditions to preserve the samples prospectively collected, including rigorous control of quality in each step of the study. This final result is in agreement with some results obtained worldwide from different studies groups , 35. YwaOn the other hand, some studies have reported correlation between HPV and lung cancer . SyrjäneStill in an attempt to explore the relationship of HPV with NSCLC, IHC was performed in NSCLC paraffin samples to evaluate the expression of p16, a protein considered an indirect marker for HPV infection, since in SCC and adenocarcinoma of uterine cervix and, in a fraction of oropharyngeal squamous cell carcinomas, the overexpression of p16 is strongly related to HPV infection . HoweverOur data shows lack of HPV infection in a series of NSCL cancers. The few p16-positive cases are therefore unrelated to HPV. Several reports evaluating the prevalence of HPV DNA are increasing and new evidences with the HPV infection and NSCL coexistence are emerging –43. Howe


ASCL1, LHX8, and ST6GALNAC5, as markers of advanced cervical disease, in relation to type-specific HPV infection. Overall, HPV prevalence was 56% and positivity increased with severity of cervical disease: from 28.0% in cervical intraepithelial neoplasia (CIN) grade 1 or less (≤CIN1) to 100% in invasive cervical cancer (ICC). HPV16 was the most prevalent type, accounting for 9.9% of HPV-positive ≤CIN1, 14.3% of CIN2, 31.7% of CIN3, and 45.5% of ICC. HPV16 was significantly more associated with ICC and CIN3 than with ≤CIN1  and 4.37 (95% CI 1.81–10.58), respectively), as opposed to non-16 high-risk HPV types. Methylation levels of ASCL1, LHX8, and ST6GALNAC5 in cervical scrapes of women with CIN3 or worse (CIN3+) associated with HPV16 were significantly higher compared with methylation levels in cervical scrapes of women with CIN3+ associated with non-16 high-risk HPV types . When CIN3 and ICC were analysed separately, the same trend was observed, but the differences were not significant. Our results confirm the key role that HPV16 plays in uterine cervix carcinogenesis, and suggest that the evaluation of host cell gene methylation levels may monitor the progression of cervical neoplasms also in WLHIV.Data on human papillomavirus (HPV) type-specific cervical cancer risk in women living with human immunodeficiency virus (WLHIV) are needed to understand HPV–HIV interaction and to inform prevention programs for this population. We assessed high-risk HPV type-specific prevalence in cervical samples from 463 WLHIV from South Africa with different underlying, histologically confirmed stages of cervical disease. Secondly, we investigated DNA hypermethylation of host cell genes  Human papillomavirus (HPV) is one of the most common sexually transmitted infections, with a worldwide prevalence of approximately 10% in women ,3,4. Of It has been suggested that the oncogenic potential of high-risk genital HPV types may be influenced by a concomitant infection with the human immunodeficiency virus (HIV) ,9,10. HIManipulation of the host cell DNA methylation machinery is known to be an important mechanism by which HPV influences cellular and viral gene expression and is likely one of the mechanisms by which HPV evades antiviral immunity . DNA metASCL1, LHX8, and ST6GALNAC5. These genes were identified in a recent genome-wide methylation profiling study on HPV-positive self-collected cervico-vaginal specimens [In the present report, we describe HPV type-specific prevalence in cervical samples from WLHIV from South Africa with different underlying, histologically confirmed stages of cervical disease, and explore a possible association with hypermethylation of host cell genes pecimens . While tpecimens . To furtAs shown in HPV prevalence within disease categories is shown in HPV was detected in 28.0% of ≤CIN1 and most of these lesions had a single-type infection (77.5%). Almost all HPV types tested were represented in this group. HPV52 was the most prevalent type, accounting for 16.9% of HPV-positive ≤CIN1. HPV18 and HPV45 accounted for another 11.3% and 14.1%, respectively.HPV was detected in 77.8% of CIN2 lesions and almost half of these had multiple types (48.8%). HPV35 was the most prevalent type, accounting for 21.4% of HPV-positive CIN2 lesions. HPV18 and HPV45 accounted for another 19.0% and 14.3%, respectively.MH 4.37; 95% CI 1.81–10.58); this was also true when only single infections were taken into account.HPV was detected in 92.5% of CIN3 lesions and most of these lesions had a single-type infection (67.5%). HPV16 was the most prevalent type, accounting for 31.7% of HPV-positive CIN3 lesions. HPV18 and HPV45 were each present in 13.6% of HPV-positive CIN3 lesions. HPV16, -33, -35, -51, -58, and -66 were found more frequently in CIN3 than in ≤CIN1 , but for HPV66 this was not observed when only single infections were considered. When adjusted for age, women with CIN3 were significantly more likely to carry HPV16 than women with ≤CIN1 ; this was also true when only single infections were taken into account.All ICC tested positive for HPV. The majority were associated with a single HPV infection (86.4%), but three ICC were positive for two HPV types: one contained HPV31 and HPV52, one contained HPV52 and HPV66, and one contained HPV35 and HPV66. HPV16 was the most prevalent type in ICC, accounting for 45.5% of ICC, followed by HPV18 and HPV45, each accounting for 13.6% of ICC. HPV16, -18, and -66 were found more frequently in ICC than in ≤CIN1 , but HPV66 was only found in ICC with multiple infections, and therefore lost its importance when only single infections were considered. HPV16 and -18, but also HPV31 and -45, were more frequently observed in ICC than in CIN3 . When adjusted for age, women with ICC were significantly more likely to carry HPV16 compared with women with ≤CIN1  of ASCL1, LHX8, and ST6GALNAC5 in cervical scrapes of women with HPV16 versus non-16 HPV, HPV18 versus non-18 HPV, and HPV45 versus non-45 HPV infections, stratified for CIN3 and ICC (n = 3) over non-18 HPV-positive ICC were observed, and after the exclusion of HPV16-positive cases, this difference reached significance for LHX8 and ST6GALNAC5 . This was not observed for HPV18-positive CIN3 lesions. For HPV45, methylation levels in HPV45-positive lesions were comparable to methylation levels in non-45 HPV-positive lesions, also after the exclusion of HPV16-related cases. From 197 HPV-positive women, methylation results from a cervical scrape were available.  and ICC A and str and ICC B. A trenCountry-based HPV type-specific prevalence ratios across cervical disease categories in WLHIV are needed to make appropriate recommendations on screening and vaccination policies for this high-risk population. In line with previous reports on HPV type-specific prevalence in WLHIV, HPV16 was the most prevalent high-risk type in our study group of WLHIV in South Africa . A steadASCL1, LHX8, and ST6GALNAC5, increase with the severity of the underlying cervical disease and are particularly high in cervical scrapes of women with cervical cancer [This preferential cancer risk of HPV16 in WLHIV, which has also been described for HIV-uninfected women , might bl cancer ,33,34,35So far, a limited number of studies on host cell DNA methylation have included WLHIV ,37,38,39ASCL1, LHX8, and ST6GALNAC5. However, methylation-dependent expression regulation and the functional role of the genes ASCL1, LHX8, and ST6GALNAC5 in cervical carcinogenesis remain to be established. Methylation of ASCL1, LHX8, and ST6GALNAC5, as found in cervical cancer, has also been detected in oral cancer (ASCL1) [ASCL1 and ST6GALNAC5) [LHX8) [ASCL1 and ST6GALNAC5 have been described as protumorigenic in other cancer types [It may be speculated that DNMT inhibitors could be effective in the treatment of HPV-induced lesions by reversing the expression of methylation targets such as  (ASCL1) , colorecGALNAC5) ,45, and ) [LHX8) , suggester types ,48,49,50Some limitations to this study may apply. First, the sample size may have been too small to demonstrate a preferential cancer risk or a specific influence on methylation levels of rare high-risk HPV types. Second, the long-term type-specific cancer risk could not be calculated, as only cross-sectional results were available. Further (prospective) research on the influence of HPV type on host cell methylation levels and the associated cervical cancer risk is needed and should include both HIV-infected and HIV-uninfected women. These studies are in progress.Our data confirm that although HPV16 is relatively under-represented in WLHIV with cervical cancer, it remains the most important risk factor for ICC. At the same time, the increased prevalence of non-16 high-risk HPV types, most notably HPV45, in cervical carcinomas and CIN3 in WLHIV compared with the general population suggests that specific cervical cancer prevention methods, targeting not only HPV16 but also other high-risk HPV types, are required for this population. Our finding that, in WLHIV, HPV16-associated CIN3 lesions and cervical cancer have increased methylation levels compared with lesions associated with other HPV types requires further exploration and advocates further research into screening algorithms that combine HPV genotyping with methylation analysis.Between February 2013 and March 2015, women aged 18 years and above who had not been treated for cervical cancer or precancer in the preceding two years were recruited from a gynaecological outpatient clinic in Steve Biko Academic Hospital and Tshwane District Hospital, Pretoria, South Africa, as part of a study comparing different cervical screening strategies. The the Faculty of Health Sciences Research Ethics Committee of the University of Pretoria approved the study . Written informed consent was obtained from all participants.®  and, after the preparation of a conventional slide, the cells were stored in Thinprep PreservCyt® . Colposcopy was then performed in all participants and two mandatory biopsies were taken. Women included in the referral population were seen at the gynaecologic outpatient department for evaluation of an abnormal Pap smear , n = 104) or a histologically proven cervical carcinoma (n = 18). Prior to gynaecological examination, a cervical specimen was collected by either the physician using a Cervix brush (n = 67), or by the participant herself using a Delphi Screener , and the cellular material was stored in Thinprep PreservCyt solution.In the present report, HIV-seropositive women from two study groups were included see : 355 womClinical data including HIV status, most recent CD4+ count, and the use of antiretroviral treatment (ART) were recorded. All histology specimens were classified as either no dysplasia, CIN1, CIN2, CIN3, or invasive cervical cancer (ICC) according to international criteria . Women wMolecular analyses were performed at the Department of Pathology of Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. Nucleic acids were isolated from physician-taken and self-collected cervical specimens using the Nucleo-Spin 96 Tissue kit  and a Microlab STAR robotic system  according to the manufacturer’s instructions. DNA isolates were tested for the presence of high-risk HPV DNA using the clinically validated GP5+/6+ PCR enzyme immunoassay (EIA) as described previously . SeparatASCL1, LHX8, and ST6GALNAC5 was performed using 50 ng of bisulphite-converted DNA, as described previously [Ct ratios >30 were excluded from the analysis. The comparative Ct method (2Ct−∆∆ × 100), which normalises methylation values of all targets to the reference gene and the calibrator, was used to obtain ∆∆Ct ratios [For methylation analysis, DNA isolated from cervical scrapes was used. Multiplex quantitative methylation-specific PCR (qMSP) for eviously . Sample t ratios .MH) and 95% confidence intervals (95% CI) were calculated to adjust for age. Only ORMH values of 1.0 and higher are reported. Data were adjusted for 10-year age categories . Breslow–Day’s test of homogeneity was used to determine the presence of an association between ORMH and age. Analyses were performed separately for women with any HPV infection (single and multiple infections combined) and for women with single infections only.Overall HPV prevalence was assessed within disease categories based on histology , CIN2, CIN3, and ICC). Type-specific positivity is reported as the proportion of HPV-positive cases in which the particular HPV type was detected. Differences in type-specific prevalence for women with CIN3 compared with women without evidence of high-grade cervical disease (CIN3:≤CIN1), and women with ICC compared with women with ≤CIN1 (ICC:≤CIN1), were examined using prevalence ratios. The same was done to compare type-specific prevalence in women with ICC to type-specific prevalence in women with CIN3 (ICC:CIN3). The Mantel–Haenszel common odds ratios  in women with and without CIN3 or worse  using Mann–Whitney U tests.


HPV related OPSCC has a distinct behavior and improved outcome. As immunotherapy has recently evolved into a new standard for advanced (SCCHN), trials for high-risk SCCHN have trended to encompass both HPV related and unrelated diseases. In this invited editorial, we question the wisdom of this approach and argue for the design of trials focused specifically on HPV related locally advanced oropharyngeal squamous cell carcinoma as a unique disease entity. Since the MACH meta-analysis and early phase III trials, the treatment of locally advanced SCCHN have relied on chemoradiotherapy . For morAs immunotherapy has recently evolved into a new standard for advanced HPV related and unrelated squamous cell carcinoma of the head and neck (SCCHN) , trials Even though the rationale for these approaches we believe is valid, the wisdom of encompassing both HPV related and unrelated disease is questionable. Assuming a similar outcome for diseases with different etiologies, presentations, pathologic and radiologic characteristics, degree of co-morbidities, clinical staging, and pattern of distant metastases we believe deserves a closer examination , 9. NoteECOG ACRIN EA3161 is a recently activated phase II/III trial examining the question of whether a maintenance approach with a single agent ICPI following definitive therapy would alter the progression-free survival (PFS) or overall survival (OS) for high-risk HPV related disease. As this study focuses on HPV related disease, it also avoids intensification of concurrent therapy and relies instead on abrogating the risk of distant metastases through a maintenance approach. We believe therefore EA3161 it uniquely positioned to answer an important question for a group of healthier patients with better prognosis yet equivalent risk of distant metastases.It is likely that adding ICPI to the backbone standard concurrent regimens will be proven effective for locally advanced SCCHN soon. The questions of whether intensification of concurrent therapy for the HPV related group is the correct approach and the degree to which this group will benefit from a maintenance ICPI, we believe will continue to linger for some time to come. EA3161 is poised to answer these questions.


Cervical cancer is a largely preventable disease mediated by persistent infection with high-risk Human Papillomaviruses (Hr-HPV). There are now three approved vaccines against the most common HPV genotypes. In Ghana, mortality due to cervical cancer is on the rise, due to the absence of an organized and effective cervical cancer prevention and control program. Data on circulating HPV genotypes is important for studying the likely impact of mass introduction of HPV vaccination of the female population before sexual debut. High HPV prevalence has been reported in Female Sex Workers (FSWs), who constitute an important active group for maintenance of HPV in the population. This study was conducted to determine the size of HPV prevalence in this group and to provide information for future assessment of the impact of vaccine introduction in the country. We conducted a cross-sectional study where the snowballing technique was used to identify and select FSW's ≥18 years, operating within suburbs of Greater Accra Region (GAR). A risk factor assessment interview was conducted and cervical swabs were collected for HPV-DNA detection and genotyping by Nested Multiplex PCR. Hundred participants, age ranging from 18 to 45 years, median 24 years, were studied. The prevalence of Cervical HPV was 26%. Eleven genotypes were detected comprising 9 high-risk in order of decreasing prevalence HPV-16 (8%), HPV-35 (5%), HPV-33/39/-68 (3%), HPV-52/51/59 (2%) and HPV-18 (1%) and 2 Low-risk types, HPV-42(3%), and HPV-43 (1%). Three women had HPV types that could not be genotyped by our method. Oral contraceptives use was associated with a reduced chance of HPV infection . This study found a high HPV prevalence among FSWs in the GAR. A high number of Hr-HPV genotypes seen are vaccine preventable, providing additional compelling argument for implementing a national cervical cancer prevention plan including vaccination. Globally, cervical cancer remains a primary cause of morbidity and mortality, with estimated 569,847 new cases and 311,365 attributable deaths in 2018 [Infection of the cervix by a high-risk Human Papillomavirus (HPV), a common sexually transmitted infection, is necessary for the development of cervical cancer Many studies have found a direct association of HPV infection with sexual behaviour and have indicated that a high number of lifetime partners may lead to a higher transmission of HPV leading to higher cervical cancer rates –8.Female sex workers (FSWs) are a group of females who provide sexual services for economic remuneration. Due to exposure to multiple sexual partners in their occupation, they are prone to various sexually transmitted infections, including HPV. Sexual contact with FSWs plays an important role in HPV transmission and might be a major contributor to the prevalence of HPV and cervical cancer among women in the general population. Also, through the transmission of the virus to their male clients, they increase the risk of penile cancers among these men . FurtherIn Ghana, mortality due to cervical cancer is on the rise, most likely due to the lack of an organized prevention and control program. Information on circulating HPV genotypes is crucial for determining the impact of cervical cancer control programmes, including HPV immunization. The high rates of HPV reported among FSWs make them a priority group for study as we seek to characterize the prevalent HPV genotypes in the country in order to predict the likely impact of the current vaccines in reducing the incidence of cervical cancer after introduction of mass female vaccination in the country. The study also looked at other factors that may increase the risk of HPV, contribute to persistence of infection, and/or promote progression of HPV-induced changes in the cervical epithelium. Here, we report the first study of cervical HPV and its associated risk factors among FSW's in Ghana.This was a cross-sectional study undertaken between February and July 2016 in Greater Accra, one of the ten administrative regions of Ghana  with a predominantly urban population. A risk factor assessment interview was conducted for 100 out of the 109 FSWs who were reached through snowballing, to elicit data on their basic demographics, sexual activities and behaviours , reproductive history, menarche, sexually transmitted disease (STD) history, screening history, and smoking habit (past and present). Only FSWs of age 18 years and above and had been a sex worker for at least 6 months were included in the study. The Ethics and Protocol Review Committee of School of Biomedical and Allied Health Sciences, University of Ghana, approved this study (SBAHS/10161447/AA/MLS/2015-2016). Participants were fully informed about the purpose, procedures, risks, and benefits of participating in this study and Informed consent was obtained from all subjects.Following the interviews, a Gynaecologist collected exfoliated cells from the cervix into tubes containing DNAgard™  for HPV-DNA detection and genotyping. Samples were collected by single use, disposable equipment.μl contained 10X PCR buffer, 2.5mM MgCl2 200μM of each of the four deoxyribonucleoside triphosphates (dNTP's), 15pmols of each E6/E7 consensus primers, and 1.25 units of Taq polymerase enzyme. Five microliters (5μl) of DNA extracts was used as a template for the amplification reactions using a thermal cycler . The cycling parameters for the first round PCR with E63F/E75B/E76B consensus primers were as follows: 94°C for four minutes, followed by 40 cycles of 94°C for one minute, 40°C for two minutes, 72°C for two minutes, and a single final elongation step of 72°C for 10 minutes. In the second round PCR, Primers for the identification of high-risk genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 and low-risk genotypes 6/11, 42, 43, and 44 were used. The primers were used in four cocktails, each containing four to five different primer pairs. Two microliters of first round PCR product, 15 pmols of forward, and reverse primers for genotyping were used. The other parameters remained the same as used in the first round PCR. However, the cycling parameters were as follows: 94°C for four minutes followed by 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds, and a single final elongation step of 72°C for four minutes [HPV detection and typing were carried out by Nested multiplex PCR. . A singlμg/ml ethidium bromide. Ten microliters of each sample was added to 2μl of orange G (10X) gel loading dye for the electrophoresis. Hundred base pair DNA molecular weight marker  was run alongside the PCR products. The gel was prepared and electrophoresed in 1X TAE buffer using an electrophoresis tank at 80 volts for one hour and the gel photographed over an Ultraviolet (UV) transilluminator [The amplicons were resolved on 2% agarose gel stained with 0.5orange G X gel loaThe data obtained through the questionnaire was checked for accuracy and entered into the computer using Microsoft Excel (2016) Programme and was analysed using SPSS version 20 . Exploratory analysis was first carried out to obtain descriptive statistics. Charts and tables were used to summarize data and display figures where appropriate. The number and proportion of HPV DNA positives and type-specific HPV infection were calculated. To assess the association between HPV DNA positivity and sociodemographic and sexual behavioural factors, odds ratios (ORs) and 95% CIs were calculated using logistic regression. In all statistical considerations a p-value <0.05 was considered statistically significance.A total of 100 FSW, ages ranging from 18 to 45 years with a median age of 24 years, were interviewed. More than half (58%) of them were below 25 years. The level of education among the women was generally low, with the vast majority, 74% (74/100), having at most basic education while 6% (6/100) reported to be illiterates. The median length of sex work was 2 years with a range of 0.5-26 years. About a half (53%) of the sex workers reported engaging in other economic activities (mostly petty trading) while the rest (47%) were full time sex workers. Seventy-seven percent of the subjects who remember their age at sexual debut recalled it to be ≥16 years and 59% of them had at least a child. Condom use always was reported by 82% (82/100) of the study participants whereas oral contraceptive use was reported by 20%. Two (2%) subjects reported to be HIV positive while 14/100 (14%) had had gonorrhoea, 4/100 (4%) syphilis, 3/100 (3%) genital warts, and 2/100 (2%) herpes before. A third (33%) of them had ever smoked cigarette whist 17% are active smokers. Only 9/100 (9%) had ever undergone cervical cancer screening.The overall HPV prevalence (any HPV) was 26% (26/100). Eleven genotypes were detected comprising 9 high-risk types and 2 low-risk types. The high-risk HPV types in order of decreasing prevalence were HPV-16 , HPV-35 , HPV-33/39/68 , and HPV-51/52/59  HPV-18 . HPV 42,  and HPV-43  were the low-risk HPV types found. In addition 3% (3/100) had HPV that could not be typed by our method . There wTo identify putative risk factors for HPV infection, we performed univariate regression analysis and the results are presented in Studies have indicated that a high number of lifetime partners may lead to a higher transmission of HPV , 7. The A high number of HPV positive FSWs (17/26), though not statistically significant, were below 25 years. This is consistent with reports in a global review of HPV prevalence among female sex workers . This obThe high-risk HPV genotypes detected in decreasing order of prevalence were 16 (8%), 35 (5%), 33/39/68 (3%), 51/52/59 (2%), and 18 (1%). These genotypes are similar to what is seen in studies in Ghanaian women with and without cervical cancer and elsewhere in Africa but with varying individual genotype prevalence –25. AbouOral contraceptives use significantly influenced HPV infection (P=0.002), with about 19% decreased chance of HPV infection in oral contraceptive users compared to nonusers . However, a systematic review of 19 epidemiological studies of the risk of genital HPV infection and oral contraceptive use concluded that there was no evidence for a strong positive or negative association between HPV positivity and ever use or long duration use of oral contraceptives . There wThis is the first report of cervical HPV and associated risk factors among FSW's in Ghana. Our study also has some limitations: this study was done on a small scale; therefore a larger study with a higher statistical power is needed to determine the extent of HPV infection in this population. Also, the convenience sampling method employed means the prevalence of HPV cannot be generalised to the whole female sex worker population in the Greater Accra.This study found a high HPV prevalence among a cohort of female sex workers in the Greater Accra Region. A high number of the high-risk HPV seen in this population are vaccine preventable, providing additional compelling argument for implementing a national cervical cancer prevention plan including vaccination.


HPV is well known as a potential cause of cervical cancer. Less well known is its link to temporal subfertility that is caused by binding of infectious virions to the spermatozoa’s head which induces sperm-DNA damage and causes a reduction in clinical pregnancy rates in women receiving HPV positive semen.This impact on the global fertility burden remains greatly underestimated and underexplored. This risk of reduced fertility due to infectious HPV in sperm is especially important when donor sperm insemination is considered, since testing for the presence of HPV virions before use seems warranted.We tested 514 donor sperm samples from 3 different sperm banks for 18 different HPV types.Overall 3.9% (20/514) of tested donor sperm was positive for HPV, with different prevalence among the 3 different sperm banks . Also the HPV virion per spermatozoon ratio in donor samples was similar across the different sperm banks .When HPV positive donor sperm was used, no clinical pregnancies resulted, whereas when HPV negative donor sperm was used the clinical pregnancy rate was 14.6%.From both a cost/benefit and a safety point of view we recommend that donor sperm should always be tested for HPV before using it for insemination. Although infection with human papillomavirus (HPV) is very common worldwide, until recently there was only scarce evidence linking HPV infection to reduced fertility outcomes .One of the major reasons why it took so long to realize infectious HPV virions impair fertility is because formerly we often failed to see the different impact of infection and disease, HPV viruses and HPV virions . On one Because of the absence of dividing cells in semen, the measured HPV DNA in sperm samples always originates from virions that can only be produced in non-dividing desquamating cells. Despite the fact that only 1-10 virions are sufficient to infect a basal cell and establish infection , only a It has been reported before that HPV virions can physically bind to the spermatozoon’s head  and induIn order to formulate a recommendation regarding HPV donor sperm testing for sperm banks, we assessed the impact of the HPV status on success rates of donor sperm by analyzing donor sperm that was used for inseminations originating from 3 different donor banks.We measured HPV DNA in donor sperm that was destined for IUI as previously described . BrieflyIf in one of the sperm fractions HPV DNA was detected, the sperm sample was considered HPV positive. Donor sperm originated from one of three donor banks . The study was approved by both the Institutional Review Board of the University hospital of Antwerp and the University of Antwerp .DNA extraction on donor sperm was performed on the ABBOTT M200sp as previously described . Each DNThe number of HPV copies (virions) per ml sperm was divided by the number of spermatozoa per ml sperm to calculate the virions to spermatozoon ratio (HPV copies/spermatozoon). When different HPV types were present in one sperm sample, the number of HPV copies per ml of sperm for each individual HPV type was added and divided by the number of spermatozoa per ml as previously described .Statistical analysis was performed with MedCalc®  .From the 365 donor sperm samples tested from bank A, 13 tested positive for one or two HPV types (3.6%). From bank B, 4 donor sperm samples tested HPV positive out of 131 (3.1%) and from bank C, 3 samples tested positive out of 18 16.7%; .From the 514 donor sperm samples tested, 20 (3.9%) tested positive for at least one HPV type and 2 (0.4%) tested positive for two HPV types . The majThe median HPV virion per spermatozoon ratio in HPV positive donor sperm was 0.03 (95% CI 0.01 to 1.07 HPV virions/spermatozoon). There was no difference between the median HPV virion per spermatozoon ratio of HPV positive donor sperm from the 3 different sperm banks . We prevAlthough most donor samples (14/20) had a HPV virion per spermatozoon ratio below this cut-off ratio of 0.66 HPV virions per spermatozoon, still no clinical pregnancies were achieved with HPV positive donor sperm. Whereas insemination with HPV negative donor sperm led to clinical pregnancies in 14.6% per IUI cycle.Our data on HPV typing of 514 semen samples from sperm donors originating from 3 different sperm banks clearly shows that HPV positive donor sperm was found in all three sperm banks. In total 20 donor sperm samples tested positive for HPV (3.9%), comparable to the 4.8% HPV prevalence previously found in the sperm bank of the University Hospital of Antwerp . The varNone of the inseminations from the HPV positive donors in the current study resulted in a pregnancy , as compAccording to the results report generated from European registers by ESHRE for the year 2012  a total In our opinion, donor sperm should be systematically screened for HPV, not only from a cost/benefit point of view, but also for safety reasons. Indeed since no dividing cells are present in sperm, HPV DNA detected in sperm always originates from infectious HPV virions, potentially resulting in transmission and potential infection of the inseminated women. As a consequence, most women (81%)  will devBased on these data we strongly recommend from both a cost/benefit and a safety point of view that donor sperm should always be tested for HPV prior to insemination.


The aim of this study was to investigate the short-term efficacy and safety of Poly-gamma-glutamic acid (γ-PGA) and the immunologic changes in patients with CIN 1.Participants were randomly assigned to one of two groups and orally treated with placebo or 1,500 mg of γ-PGA for 4 weeks. The primary endpoint of the study was histologic regression rate of CIN 1 at 12 weeks between γ-PGA and control groups. The secondary endpoints were HPV clearance and change in immune responses.From April 2013 to December 2015, 195 patients participated in the study. In the intention-to-treat analysis, 42 (42.4%) of the women who received γ-PGA experienced histologic remission versus 26 (27.1%) in the control group, with a statistically significant difference (p = 0.018). In the γ-PGA group, HPV clearance was found in 37 (43.5%) of 85 patients infected with high-risk HPV, showing a significant difference compared to the control group, in which 20 (26.7%) of 75 patients exhibited HPV clearance (p = 0.026). However, there was no significant difference between the two groups in the change of NK cell activity, major histocompatibility complex (MHC) class II CD8 count, and CD56 count.γ-PGA showed a short-term therapeutic effect on CIN 1 and high-risk HPV infection. It is a non-invasive, promising oral medication for women with these conditions.NCT01826045.Clinical Trials  Cervical intraepithelial neoplasia (CIN) can be a precursor to invasive cervical cancer and is the most common significant gynecologic disease in women of reproductive age . In geneγ-PGA is a non-toxic, water-soluble, and biodegradable substance known as an immune modulator. In vivo tumor regression activity of high-molecular weight γ-PGA from Bcaillus subtilis (chungkookjang) was observed with high levels of natural killer (NK) cell-mediated cytotoxicity and interferon (IFN)-gamma secretion in mice , 10. γ-PThe study was carried out according to the principles expressed in the Declaration of Helsinki and Korean good clinical practice (KGCP). The study received institutional review board (IRB) of Korea university guro hospital (2013GR0058) and was registered as clinicaltrials.gov study NCT01826045. Each subject gave written consent for their participation. The Protocol for this Trial and supporting CONSORT checklist are available as supporting information .From April 2013 to December 2015, women were recruited from six medical centers in Korea. Women were eligible if they met the following inclusion criteria: aged between 20 and 49 years and histologically confirmed with CIN 1 by colposcopic punch biopsy. Women with other malignancies or those who were immunosuppressed or pregnant were excluded from the study. All subjects were required to use effective contraception during the study.Based on published literature, we assumed that 31% of CIN 1 cases would resolve spontaneously, and a clinically significant difference was estimated at 20% –17. A saSubjects were enrolled and randomized to either placebo or γ-PGA group. Randomization was performed using a computer-generated assignment scheme designed and performed in a masked manner by the data coordinating center. Participants were assigned to a group by block randomization with an appropriate block size. After randomization, subjects received oral administration of 100 mL of a syrup containing 1000 mg γ-PGA or placebo once daily for 4 weeks. The dose was based on Phase 1 clinical trial results: immunohistochemical (IHC) staining showed that the γ-PGA group had a higher level of NK cell activity than the placebo group, and the low dose group (1.5 g/day) showed a higher level than the high dose group (3.0 g/day) at 3 and 5 days after administration . At the TM II HPV28 detection at the Department of Pathology, Cheil General Hospital. All specimens were tested with HC2 and Anyplex II HPV detection kits according to the manufacturer’s protocol.The primary outcome was disease regression at three months after diagnosis. Secondary outcomes were high-risk HPV status, viral load of HPV, immune response, and safety data. HPV testing was performed at the 12-week visit using the Digene Hybrid Capture (HC) 2 High-Risk HPV DNA Test and Anyplex5) from participants were cultured in the presence or absence of γ-PGA (0.1mg/mL) for 8 hours and then stimulated with K562 tumor cells (2 x105) for an additional 7 hours in the presence of monensin or brefeldin A, which detect surface CD107a and intracellular interferon-γ (IFN-γ), respectively. Cells were stained with CD56 , CD3, IFN-γ, CD107a , CD14, and CD19  for flow cytometry, and CD56dim, CD3-, CD14-, CD19- cells were gated as previously described [Immune status was evaluated at each visit (every 4 weeks) using an NK cell activity assay and measurement of the MHC class II CD8 and CD56 populations. Laboratory protocol for assay of the cytotoxic activity of NK cells was adapted from a previous phase I study . Peripheescribed .To measure phenotypic changes in peripheral blood mononuclear cells (PBMNCs), the cells were separated by a density gradient method using a boiling solution of Ficoll-Hypaque. Peripheral blood samples were superimposed on Histopaque  solution using SEPMATE tubes  and were centrifuged at 1200 × g for 10 minutes. The upper layer was removed and washed with PBS + 2% to separate mononuclear cells. PBMNCs were treated with FACS buffer  containing fluorescent-labeled antibodies  and left on ice for 30 minutes to confirm the change in peripheral blood mononuclear cell immunity phenotype using a BD truCountTM method . Fluorescent-stained cells were washed twice with FACS buffer and fixed with phosphate buffer containing 2% paraformaldehyde. Cell fluorescence was measured and analyzed using a Gallios flow cytometer .The primary efficacy variable was based on histologic analysis of biopsy-derived cervical tissue for the presence or absence of CIN 1. Histologic resolution was defined as an outcome of “normal.” In general, continuous variables were summarized by descriptive statistics, including number, mean, median, standard deviation, minimum, and maximum. Categorical variables were summarized by number and percentage. All percentages were calculated using non-missing data as the denominator. All statistical tests were 2-sided with a 0.05 level of significance. The demographic and baseline diagnostic data between placebo and γ-PGA groups were calculated. The regression rate, which is the primary endpoint, was analyzed with a one-sided test at a significance level of 5%. Type-specific HPV infection, viral load of HPV, NK cell activity, CD8 count, and CD56 count were measured at 12 weeks of follow-up and compared with the baseline values. A generalized estimating equation or generalized linear mixed model was used to identify differences and time and intergroup interactions. Analyses of general characteristics and histopathologic response by treatment group were completed using intention-to-treat (ITT) methodology and by imputing histologic biopsy results for women with missing 12-week measurements. Imputed values were found using worst-rank score .Patients with missing data were excluded from statistical analysis of HPV clearance and immunologic response. All statistical analyses were performed using PC SAS 6.12 .From April 2013 to December 2015, 195 subjects meeting inclusion criteria were enrolled from six medical centers in Korea . Forty-sThe general characteristics of the two groups are described in The primary endpoint of this study was histopathologic response in women with CIN 1 . In the The type-specific clearance and viral load of HPV are described in NK cell activity and MHC class II CD8 and CD56 counts were performed at baseline and at 4 weeks, 8 weeks, and 12 weeks to check immune responses. In the γ-PGA group, NK cell activity increased at 4 and 8 weeks, but there was no statistically significant difference between the two groups . There wAccording to the safety evaluation, 76 (38.9%) subjects had 157 adverse events: 34 patients (35.4%) in the placebo group and 42 (42.4%) in the γ-PGA group. The most common adverse effects in the placebo group were headache, cough, and dyspepsia, whereas those in the γ-PGA group were premenstrual pain, headache, and cough. There was no significant difference in the incidence of adverse effects between the two groups. Adverse events related to the drug were very infrequent in both the placebo group  and the γ-PGA group , and no severe adverse events were reported.Our data suggest that γ-PGA is efficacious in promoting histologic regression and clearance of high-risk HPV in patients with CIN 1 in the short term. To our knowledge, γ-PGA is the only oral medication proven to be efficacious for regression of CIN I and clearance of high-risk HPV in a randomized controlled trial (RCT). γγ-PGA is extracted from the traditional Korean food Chungkukjang and it exhibits excellent tolerability and safety.In this study, the histologic regression rate was significantly higher in the γ-PGA group than in the placebo group (42.4% versus 27.1%). The spontaneous regression rate of the placebo group was consistent with previous studies that reported 28% to 31% at 3 months after diagnosis , 16. In Unfortunately, PGA did not reduce progression of CIN 1 in this study. However, CIN1 remission is also meaningful. In several studies investigating treatment preferences, most patients desire more active treatment rather than only follow-up visits; repeated examinations and follow-up visits were associated with anxiety and discomfort, which negatively affected their daily lives –31. AccoIn this study, γ-PGA was associated with a significantly higher clearance rate of high-risk HPV infection compared with placebo (difference of 17%). However, there was no significant difference in the type-specific HPV clearance rate except for a borderline significant difference in HPV 53 . Similar results were reported in a previous study, suggesting that the HPV therapeutic vaccine cleared HPV infections and CIN lesions. Four of nine patients vaccinated with GX-188E cleared their HPV infection in 12 weeks, and 6 patients cleared infections in 20 weeks . AlthougNK cell activity and CD8 and CD56 cell counts were measured at 4 and 8 weeks after treatment to evaluate the mechanism of γ-PGA action. Kim et al. reported that NK cells and IFN-gamma play important roles in the anticancer activity of γ-PGA . Unlike Many studies investigating treatments for CIN and HPV infection have been reported. Several therapeutic vaccines targeting high risk HPV have been tested in clinical trials and recent phase IIb trials have shown regression of high-grade CIN after vaccine treatment . StudiesBacillus subtilis) is a biopolymer used in a wide range of applications, including cosmetics, bone care, and nanoparticles for drug delivery. Recent studies have shown that it can be used as an immune-stimulating agent, especially the high molecular weight form [γ-PGA extracted from chungkukjang , and the difference between the two groups was slightly lower than 20% (15%). However, 45.8% (42 patients) of the experimental group and 30.2% (26 patients) of the control group were regressed to normal of 178 patients (92 patients in the PGA group and 86 patients in the placebo group), except those who did not undergo biopsy at the 12-week visit. The difference in the regression rate between the two groups was statistically significant at 15.5% (p = 0.025). Therefore, the assumptions of the sample size computation of this study may be considered partially realized.γ-PGA showed a short-term therapeutic effect in CIN 1 and high-risk HPV infection. It is a promising new oral medication for women with CIN 1 and women with high-risk HPV infection. However, no significant effect was observed on specific HPV types, such as 16 and 18, and the immunologic response did not change with γ-PGA treatment. Therefore, further studies of the mechanism of γ-PGA activity are needed.S1 Text(DOCX)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Highly efficient single-stack hybrid cool white organic light-emitting diodes (OLEDs) having blue-yellow-blue multiple emitting layers (EMLs) are designed and constructed by utilizing blue thermally activated delayed fluorescent (TADF) and yellow phosphorescent emitters. The out-coupling efficiencies of yellow and blue emissions are maximized by tuning the ITO and total device thickness that satisfies both of antinode positions for yellow and blue emissions in a limited multiple EML thickness. To obtain a cool white emission, the exciton formation ratio in the blue-yellow-blue multiple EML system is controlled by manipulating the recombination zone through charge conductivity variation of host medium in the blue TADF EML. The resulting device exhibits cool white emission with very high maximum external quantum efficiency of 23.1% and CIE color coordinates of . We anticipate that the studied approach will raise the viability of single-stack hybrid cool white OLEDs for high performance display applications. Recently, the importance of WOLED is increasing for large size OLED TV displays. OLED TV is successfully commercialized by adopting the OLED panel comprising WOLED with fluorescent blue and phosphorescent yellow green units in two-tandem configuration and red, green and blue color filters5. Indeed, in the cool WOLEDs (CWOLEDs), hybrid system utilizing fluorescent blue and phosphorescent green and red or yellow emitters are used to secure sufficient device lifetime because highly stable blue phosphorescent emitter has not been developed so far7. Generally, hybrid CWOLED structures are especially divided into single-stack and tandem configurations12. Among them, only three-stack tandem structure is adopted for display applications due to its high efficiency and long device life. In this tandem structure, two blue emitting layers (EML) are used to compensate low intensity of blue fluorescence, and thereby highly efficient cool white emission and long device life can be achieved. However, such a complicate structure not only increases manufacturing cost and driving voltage but also degrades viewing angle characteristics. On the other hand, single-stack hybrid WOLED has more simple structure compared to that of tandem structure. Hence, the device manufacturing cost can be reduced and it also provides much better viewing angle characteristics. However, low efficiency is the most challenging issue in single-stack hybrid WOLEDs.White organic light-emitting diode (WOLED) is a core technology for large-area active matrix OLED (AMOLED) displays and a promising candidate for next generation lighting applications14.In the single-stack hybrid CWOLED, 100% internal quantum efficiency (IQE) can be achieved if all blue triplet excitons are converted to yellow triplet excitons via Dexter energy transfer as shown in Fig. In this work, we investigate a device design method to achieve high efficiency and cool white emission from single-stack hybrid CWOLED. The blue-yellow-blue multiple EML structure is employed to harvest all electrically generated excitons and to balance the intensities of blue and yellow emissions. To maximize out-coupling efficiencies of yellow and blue emissions, the ITO and total device thickness that satisfy both of antinode positions for yellow and blue emission in the limited multiple EML thickness are considered. Also, the exciton formation ratio in the blue-yellow-blue multiple EML system is tuned by controlling recombination zone via charge conductivity variation of host materials in the blue TADF EML. By utilizing the optimum device thickness and the multiple EML system, very high external quantum efficiency (EQE) of 23.1% and cool white emission with CIE color coordinate of  are achieved in single-stack hybrid CWOLED.2(acac) is selected because of its pure yellow emission with almost 100% photoluminescence quantum yield (PLQY)16. As a high-efficiency blue TADF emitter, DMAC-DPS is the most famous and widely used material with very high quantum yield, but it has a sky-blue emission color due to broad emission spectrum resulting from rotational motion of phenyl units. Therefore, this emitter cannot be efficient for cool white emission in combination with the yellow emitter as shown in Fig. 17. On the other hand, DMAC-DMT increases the rigidity of molecular structure compared with DMAC-DPS by interlocking the phenyl units, resulting in higher PLQY and deep blue color emission due to suppressed rotational motion of phenyl units17. Indeed, DMAC-DMT can make cool white emission in combination with the yellow emitter. Hence, we selected DMAC-DMT as a blue emitter for further evaluation.CIE Color coordinates of blue and yellow emitters are very important parameter for making cool white emission. Thus, we carefully selected blue TADF emitter and yellow phosphorescent emitter. Herein, for the yellow phosphorescent emitter, Ir(tptpy)19. Hence, it is very important in designing OLED structure to determine the device thickness (distance between transparent and reflective electrode) and the EML position by considering antinode condition of the EML emission spectrum. In particular, WOLED requires precise device engineering for high efficiency because two or more EMLs with different emission wavelengths are placed within the same device thickness. The antinode positions of the EMLs with different emission wavelengths are placed at different location for same device thickness condition. Unlike tandem WOLEDs, the difference in antinode position of the EML with respect to the emission wavelength makes the device design of single-stack WOLED more difficult. In the case of tandem structure with two EMLs (yellow and blue emission), blue EML and yellow EML can be easily placed in the respective antinode position because charge generation layer (CGL) generates two spatially separated recombination zones as shown in Fig. To maximize the out-coupling efficiency of OLEDs, EMLs should be located at the antinode positions, wherein the electromagnetic waves generated from the emitter in the EML are constructively interfered2(acac)) and blue (DMAC-DMT) radiance as a function of the distance from EML to ITO and to reflective cathode (Al). Wherein, the thickness of yellow and blue EMLs is 2.5 and 25 nm, respectively. The distance from EML to Al (DEML-Al) greatly affects the change in radiance, and the DEML-Al at the antinode position is independent on the ITO thickness variation. However, the distance from EML to ITO (DEML-ITO) weakly affects the change in radiance, and the DEML-ITO at the antinode position is dependent on the ITO thickness variation. Therefore, it is necessary to find the accurate thickness of ITO that matches the DEML-ITO at the blue and yellow antinode positions. Figure EML-Al of yellow and blue devices were fixed to 30 and 47.5 nm, respectively, because thin yellow EML should be located in the blue EML to form blue-yellow-blue multi EML system. In the first antinode condition, the thickness of ITO that makes an equivalent DEML-ITO for both yellow and blue devices was not found. On the other hand, in the second antinode position, 150 nm thick ITO demonstrated a similar DEML-ITO (~200 nm) for blue and yellow devices. However, large thickness in both ITO and HTL may induce large ratios of waveguide modes and reduce light extraction. Hence, optical evaluation of devices with thin (70 nm) and thick (150 nm) ITO and different antinode conditions were performed. In the case of thin ITO (70 nm) and 1st antinode condition, inappropriate emission pattern was observed due to strong emission from the yellow emitter. As a result, spectral ratio between blue and yellow was closer to the warm white emission . In contrast, device with 150 nm thick ITO and 2nd antinode condition showed cool white color emission . Therefore, by considering the thickness condition shown in Fig. Fig. EML-Al for yellow EML that located between the blue EML, the recombination zone of blue TADF-OLED should be located near the HTL side. Thus, we selected DBFPO and DPEPO as host materials because their electron-transporting characteristics help to form the recombination near the HTL side, and also their higher triplet energy compared to that of DMAC-DMT prevents back energy transfer from the triplet of DMAC-DMT to the triplet of host. Prior to the evaluation of blue TADF-OLEDs, we optimized the doping concentration of DMAC-DMT in OLEDs. The device with 40% doped TADF emitter shows relatively higher luminance and EQE as well as good charge balance than those of other doping concentrations , indicating that the electron conductivity of host materials is higher than the DMAC-DMT and additionally, DMAC-DMT is indeed acting as a deep electron trap, which leads to reduction of electron conductivity in the EMLs (host:DMAC-DMT). Furthermore, EOD with DBFPO has higher electron current density than that of DPEPO contained device despite of the same lowest unoccupied molecular orbital (LUMO) level of DBFPO and DPEPO, which signifies higher electron conductivity of DBFPO compared to that of DPEPO. It is important to note that, due to the higher electron conductivity of DBFPO and lower hole conductivity of DMAC-DMT in the EML of DBFPO based blue device, the recombination zone has formed close to the HTL interface than that of DPEPO device. Hence, this result provides the evidence of slightly red-shifted EL spectrum  films, hole only device (HOD) and electron only device (EOD) were fabricated with the device structure shown in inset of Fig. rum Fig.  and highrum Fig.  in DBFPO22. As can be seen, the transient PL decay characteristics of host:DMAC-DMT(40%) films are clearly distinguished by prompt and delayed components. The prompt and strong delayed components are originated from the fluorescence and TADF of DMAC-DMT, respectively. The exciton lifetime of the delayed component (τd) of DBFPO:DMAC-DMT(40%) film (2.45 μs) is shorter than that of DPEPO:DMAC-DMT(40%) (3.88 μs) film. A shorter delayed exciton lifetime is beneficial for reducing triplet-triplet and triplet-polaron annihilation in the EML of OLED, which leads to suppression of efficiency roll-off characteristic25. This result indicates the main reason behind the lower efficiency roll-off characteristic of DBFPO device compared to that of DPEPO device  film exhibits not only higher rate constant for RISC (kRISC) of about 1.6 × 106 s−1 but also high PLQY of 91% compared to that of DPEPO:DMAC-DMT(40%) (1.0 × 106 s−1 and 78%), which results in high EQE for DBFPO contained blue device than the DPEPO based blue device.To get the deep insight of OLED performances, photophysical properties of host:DMAC-DMT(40%) films are compared by measuring PLQY and transient PL, and their rate constants are also calculated using previously reported method as displayed in Table ice Fig. . Moreove2:(6%) Ir(tptpy)2(acac) (2.5 nm)/Host:(40%) DMAC-DMT (22.5 - x nm)/Host (5 nm)/TPBI (25 nm)/LiF (1.5 nm)/Al (100 nm). Herein, the thin yellow EML position has varied to adjust the intensity ratio of yellow and blue emission. As shown in Table By considering the position of recombination zone and high EQE of DBFPO based blue device, it may seem that DBFPO is more suitable for blue host material in high performance WOLED device. More interestingly, the use of DBFPO as a host also allows us to place thin yellow EML close to the HTL side; thereby the distance between yellow EML and Al for the antinode position of yellow emission can be obtained. On the other hand, DPEPO host has an advantage in color coordinate characteristics. To investigate the effect of host materials in blue-yellow-blue multiple EML system and to achieve cool white emission, single-stack hybrid WOLED Fig.  was fabr2 are shown in Fig. On the other hand, due to relatively lower electron conductivity of DPEPO and higher hole mobility of DMAC-DMT in DPEPO medium, the recombination zone can be broadly formed over the whole blue-yellow-blue EMLs of DPEPO comprised WOLED as depicted in the inset of Fig. In conclusion, single-stack CWOLED with blue-yellow-blue hybrid multiple EML structure was developed by utilizing blue TADF and yellow phosphorescent emitters. The device structure was optimized to achieve balanced and highly efficient blue and yellow emission by using constructive interference condition of the blue and yellow emission as well as exciton dynamics in the multiple EML system. Both of constructive interference condition for yellow and blue emissions are satisfied even in the single-stack OLED using 150 nm thick ITO and second antinode condition, which results in high out-coupling efficiency and very high EQE of 23.1%. The well-balanced intensities of blue and yellow emissions are attained in the multiple EML system by controlling recombination zone and utilizing host materials with different electron conductivity, thereby cool white emission  with high color stability depending on luminance are realized. These results demonstrated that even single-stack hybrid CWOLEDs can achieve high efficiency comparable to those of tandem hybrid CWOLEDs. This work provides an approach to obtain highly efficient cool white emission from single-stack hybrid WOLED and increases the feasibility of single-stack hybrid WOLED for large area and high-resolution display applications. Additionally, we anticipate that our approach can be realized for RGB three-component WOLED either by doping ultra-low concentration of red phosphorescent emitter in yellow EML or by inserting separate ultra-thin red EML.28. The other materials such as 1,4,5,8,9,11-hexaazatriphenylene-hexacarbonitrile (HATCN), 1,1-bis[(di-4-tolylamino) phenyl] cyclo-hexane (TAPC), Bis(2-(2-hydroxyphenyl)pyridinato)beryllium (Bepp2), Acetylacetonatobis(4-(4-tert-butylphenyl)-thienopyridinato-C2,N)iridium (Ir(tptpy)2(acac)), 2,2′,2″--tris(1-phenyl-1-H-benzimidazole) (TPBi), lithium fluoride (LiF), and aluminum (Al) were purchased from EM Index, Jilin OLED Material Tech Co., Ltd., and Luminescence Technology Corp., respectively. The DCDPA was used as an exciton-blocking layer. HATCN and TAPC were incorporated as hole-injecting and hole-transporting layer, respectively. DBFPO and DPEPO were used as blue TADF host and DMAC-DMT was used as blue TADF emitter. For the yellow EML, Ir(tptpy)2(acac) and Bepp2 were used as yellow emitter and yellow host, respectively. TPBi was used as electron-transporting layer.3,5-Di(9H-carbazol-9-yl)-N,N-diphenylaniline (DCDPA), bis[2-(diphenylphosphino) phenyl] ether oxide (DPEPO), 2,8-bis(diphenylphosphine oxide) dibenzofuran (DBFPO), and 2,7-bis-yl)-9,9-dimethyl-9H-thioxanthene 10,10-dioxide (DMAC-DMT) were synthesized according to the previously reported methodsThe extensive optical simulations of yellow and blue OLEDs were performed using semiconducting emissive thin film optics simulator (SETFOS 4.1). The n and k values of studied organic materials were obtained from the spectroscopic ellipsometry measurements.2 were sequentially cleaned by using ultrasonic cleaner in acetone, isopropyl alcohol, and deionized water for 10 minutes each. All the organic and inorganic layers were evaporated on the pre-cleaned ITO glass substrates in thermal evaporator under high vacuum pressure of 10−7 Torr. After the deposition process, complete device was encapsulated by using desiccant attached glass cover and UV-cured resin under nitrogen atmosphere.The ITO glass substrates with active area of 4.0 mm2 atmosphere at room temperature. The exciton lifetimes of prompt and delayed components of the PL decay curves were calculated using single and triple-exponential decaying functions22.The current density-voltage-luminance (J-V-L) curves, Commission Internationale de l’Eclairage (CIE) 1931 color coordinate, and electroluminescence (EL) spectra were obtained by using a spectroradiometer . Photoluminescence (PL) spectra of 50 nm-thick EML films (DBFPO:DMAC-DMT(40%) and DPEPO:DMAC-DMT(40%)) were measured using fluorescence spectrophotometer (Hitachi F-7000) at room temperature. The photoluminescence quantum yields (PLQY) of the EML films were obtained from fluorescence spectrometer (JASCO FP-8500) installed with an integrated sphere. The transient PL decay curves of the EML films were recorded from Quantaurus-Tau fluorescence lifetime measurement system  under NSupplementary Information