Understanding of oropharyngeal squamous cell carcinoma has significantly progressed over the last decades, and the concept that this disease can be subdivided into two distinct entities based on human papilloma virus (HPV) status has gained acceptance. To combat the constantly growing epidemic of HPV+ oropharyngeal cancer, further investigation and characterization the unique features of the disease, along with the development and implementation of new, targeted therapies, is crucial. In this review, we summarize the etiology, pathogenesis, diagnosis, treatment, and molecular characteristics of HPV-associated oropharyngeal squamous cell carcinoma. Head and neck squamous cell carcinomas comprise a diverse group of tumors, which are classified into anatomical subsites including oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx. Cancers of different subsites are known to have unique epidemiology, anatomy, clinical behavior, and association with human papilloma virus (HPV) infection , 2. In tIn the late 20th and early twenty-first century, the campaign to reduce smoking decreased rates of tobacco-related cancers, including oral cavity and laryngeal cancers. During this same period, rates of oropharyngeal cancers increased –6. With Over 90% of HPV(+) OPSCC is caused by the high-risk HPV genotype 16, with almost all oral HPV infections thought to be sexually acquired , 18, 19.Intense interest regarding the benefits of primary prevention of HPV infection has followed the introduction of HPV vaccines. The Gardasil four-valent vaccine covers HPV types 6, 11, 16, and 18 and received FDA approval for use in females in 2006 and in males in 2011. Since January 2017, the nine-valent Gardasil vaccine with expanded coverage, adding HPV types 31, 33, 45, 52, and 58, has been the only HPV vaccine available in the US. The CDC currently recommends routine vaccination for both girls and boys at age 11–12, with vaccination recommended for females through age 26 and for males through age 21 .A US-based study that examined the effects of HPV vaccination on the burden of oral HPV16 infections found that between 2011 and 2014, vaccination potentially prevented almost one hundred thousand infections . HoweverINK4a) IHC has been widely adopted because it is cost effective, reliable, examines paraffin-embedded tissue, and has high sensitivity (94%) [The 2018 version of The National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (USA) directs that OPSCCs be tested for HPV by p16 immunohistochemistry (IHC) . p16 (p1ty (94%) , 26. IHCty (94%) . Howeverty (94%) . To morety (94%) . In the ty (94%) . Becausety (94%) . HoweverNext-generation sequencing (NGS) has emerged as an exciting new technology with the potential to identify HPV(+) tumors and provide rich mechanistic and prognostic information distinguishing subsets even within the HPV(+) group. A 2014 study using NGS found that HPV(+) tumors could be further categorized by presence of integrated versus nonintegrated HPV genes and that integration status corresponded with different patterns of DNA methylation and human and viral gene expression profiles in genes with known roles in carcinogenesis . While iHPV-positive OPSCC carries a favorable prognosis compared to HPV-negative tumors. Five-year survival rates for patients with advanced stage HPV(+) OPSCC are 75–80%, versus survival rates of less than 50% among patients with similarly staged HPV(−) tumors . The impInterest in identifying prognostic biomarkers in HPV-associated OPSCC has stemmed from the desire to decrease treatment morbidity while maintaining high cure rates. While a positive p16 by IHC predicts a favorable prognosis regardless of HPV status, recent data has shown that when used together with HPV status, further prognostic stratification is achieved . A 2017 Recent analysis of an HNSCC cohort in The Cancer Genome Atlas (TCGA) identified potential molecular biomarkers that can be used for prognostication , 39. DelDespite the prognostic significance of HPV in HNSCC, HPV status has not altered treatment guidelines. For the first time in 2018, The National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology , separated treatment pathways for p16(+) and p16(−) OPSCCs ; howeverStandard therapy for advanced OPSCC regardless of HPV status as either definitive or post-operative therapy includes chemotherapy and radiation, which is associated with dose-related adverse side effects, from acute toxicities like mucositis and loss of taste to long-term problems including dysphagia, renal dysfunction, hearing loss, xerostomia, osteoradionecrosis, accelerated arteriosclerosis, neck muscle fibrosis, and trismus. These side effects can lead to a cascade of events, including infections, difficulty eating, and increased hospitalizations, that can markedly erode quality of life. Based on analysis of long-term survivors from the RTOG 91–11 clinical trial, there is also the possibility that treatment-associated morbidity may impact 10-year or longer survival . Given tThe distinct tumor biology, higher treatment sensitivity, and better prognosis of HPV(+) OPSCCs has piqued interest in therapies that can minimize side effects, including new treatment approaches and de-escalation of current therapies. A single-arm phase II clinical trial, ECOG 1308, examined if response to induction chemotherapy could select stage III-IV (AJCC 7th edition) HPV(+) OPSCC patients for reduced-dose radiation . This trSeveral clinical trials (see Table 2 for 5–7 days) reinforced the pre-clinical data, showing increased p53 expression, increased apoptosis and decreased expression of HPV genes. In a mouse xenograft model, 5-aza was also found to reduce the metastatic potential of HPV(+) tumors. A larger clinical trial is needed to fully characterize the therapeutic potential and safety of this promising therapy in HPV(+) HNSCC.In addition to de-escalation of standard therapy, new treatment modalities for HPV+ HNSCC are being developed with the hope of decreasing morbidity of current therapies. Early promising results from an ongoing clinical trial published in 2017 from the Yale Cancer Center examined molecular effects of DNA-demethylation using 5-azacytidine (5-azaC) for treatment of HPV(+) HNSCC patients , Table 1One mechanism of immune escape in HNSCCs is mediated by the receptor programmed death − 1 (PD-1) interacting with its ligand, PD-L1, which is expressed in 50–60% of HNSCC and 70% of HPV(+) HNSCCs . PD-L1, In addition to the epidemiological, pathological, and clinical characteristics distinguishing HPV(+) and HPV(−) OPSCCs, TCGA and other efforts have elucidated molecular and epigenetic differences –52. HereAltered DNA-repair pathways, differences in mitogenic signaling pathways, dysregulation of cell cycle control, and changes in the tumor micro-environment of HPV(+) tumors have all been proposed as possible explanations of their enhanced sensitivity to radiation . SMG-1  tumors. Amplification and overexpression of cyclin D1, inactivation of the cyclin-dependent kinase inhibitor p16, and mutations of the tumor suppressor p53 are common defects found in HPV(−) HNSCC, but are lacking in HPV(+) tumors , 61. On In addition to differences distinguishing HPV(+) and HPV(−) head and neck tumors, significant molecular heterogeneity exists within HPV(+) tumors themselves. Gene expression profiling has classified HPV(+) tumors into two subgroups with one (HPV-KRT) having elevated expression of genes involved in keratinization, viral integrations, spliced E6, chr3q amplifications, and PIK3CA mutations, and the other subgroup (HPV-IMU) having more mesenchymal differentiation, full-length E6 activity, chr16q deletions, and a stronger immune response . AlthougFurther proving the heterogeneity of HPV(+) HNSCCs, a 2014 study of HNSCCs from TCGA found that of the 35 HPV(+) tumors, 25 had integration of the viral genome while 10 tumors lacked integration . The canOPSCC with integrated versus nonintegrated HPV have differences in somatic gene methylation, gene expression patterns, mRNA processing, and inter- and intrachromosomal rearrangements . For tumThe mechanisms of HPV-driven OPSCC have not been intensely studied, as many have assumed that HPV carcinogenesis in OPSCC is identical to the accepted mechanism of HPV carcinogenesis in the uterine cervix; however, there are many differences between HPV(+) OPSCC and uterine cervical cancer. HPV(+) OPSCC and cervical cancer diverge in epidemiologic factors, molecular patterns, HPV type, mutational profile, cell-of-origin, treatment response, and clinical behavior Table , suggestThe productive HPV life cycle has been studied and is outlined here . In the Studies of the transformation process initiated by HPV infection have relied heavily on the study of premalignant uterine cervical cells and have led to a canonical model of HPV carcinogenesis. In this model, initial infection, establishment and maintenance are thought to parallel the normal HPV life cycle; however, with persistent infection of basal or stem cells carcinogenesis can be initiated. The model details that as cells progress from early dysplasia (CIN1) to pre-malignant lesions (CIN3), the HPV genome integrates and disrupts the HPV E2 gene, which relieves negative feedback and increases expression of HPV oncoproteins, E6 and E7 , 75–77. TCGA analysis of HPV-associated OPSCC provides some characterization of the role of HPV integration in tumors . GenomicDirect analysis of the HPV carcinogenesis process in the tonsil is not possible due to the absence of a defined pre-malignant lesion. The area infected by HPV and prone to transformation within the tonsil – the tonsillar crypt – lacks tight epithelial junctions and is characterized by incomplete basement membranes making pathological distinction between invasive cancer and intra-epithelial neoplasia impossible , 81. In INK4a, confirming the importance of HPV oncogenes in tumorigenesis [Given the difficulties of studying progression of HPV pre-malignancies in the oropharynx, comparison of molecular characteristics of OPSCC and uterine cervical cancer may shed light on mechanisms of carcinogenesis in these distinct cancers. APOBEC  is a cytidine deaminase whose mutations have been implicated in carcinogenesis. Both OPSCC and uterine cervical cancers have enrichment for APOBEC mutational signatures , 83, 84.igenesis , 84. Intigenesis , 84.For tumors caused by HPV type 16, integration of the HPV genome occurs at similar rates in OPSCC (72%) and cervical cancer (76%) , 84. DesTogether these data raise the intriguing possibility of an alternative mechanism of HPV carcinogenesis uncovered through the study of HPV(+) OPSCC. Instead of HPV integration as a driver for increased oncoprotein expression, a subset of OPSCC may rely on maintenance of unintegrated HPV that in turn requires molecular defects in TRAF3, CYLD or other genes to activate NF-kB and inhibit innate immune responses.Recent studies of OPSCC are increasing our understanding of HPV-associated carcinogenesis, including the possibility of an alternative mechanism reliant on activation of NF-kB, inhibition of interferon and maintenance of non-integrated HPV. In addition, markers to identify HPV(+) OPSCC patients with improved prognosis are emerging. These insights are critical to improving our management of this rising disease and exploring effective new treatments and identification of patients for de-escalated therapy.


Objective: Provide evidence of HPV, C. trachomatis, and HSV infection in the oral cavity from patients with different types of stomatological lesions.Materials and Methods: Oral swabs samples were collected from a total of 318 patients. The infectious agents were analyzed using the PCR technique. HPV genotyping and HSV type were studied using the RFLP method.Results: We studied 137 benign lesions (B), 96 potentially malignant disorders (PMD) and 85 oral squamous cell carcinomas (OSCC). The prevalence of HPV was 34%. The most frequently genotypes detected were 6 low risk and 16 high risk. The prevalence of C. trachomatis was 16% and HSV 3%. Co-infections were detected mostly in benign lesions as following: HPV-C. trachomatis in 4%, C. trachomatis- HSV in 1.8% and HPV-HSV in 0.3%.Conclusion: This report is the first contribution to the identification and genotype characterization of HPV in a scenario little studied in our area, and it also contributes to improving our understanding on sexually transmitted infectious agents and their associations with the oral cavity. Besides, we detect the presence of C. trachomatis and HSV and co-infection with HPV in the oral cavity, which they should be taken into account for diagnostic and treatment purposes. Human Papillomavirus (HPV) is one of the most frequent sexually transmitted infections (STIs) that could play a role in the pathogenesis of head and neck cancer. This infection is a major health problem worldwide that manifests as progressive squamous dysplasia and results from cumulative genetic and epigenetic alterations induced by exposure to carcinogenic agents ,2.Despite the well-established role of HPV in cervical cancer, new lines of evidence suggest that HPV may also be an independent risk factor for oral cancer , and co-Chlamydia trachomatis (C. trachomatis) infection could act as a co-factor which facilitates HPV infection and contributes to the viral persistence, increasing the risk of developing cervical neoplasia [C. trachomatis is a gram-negative obligate intracellular bacterium with a biphasic development that often remains asymptomatic and can infect genital and oral mucosa [C. trachomatis have a higher risk of developing cervical cancer.There is evidence that eoplasia . C. tracl mucosa ,7. In a l mucosa  reportedC. trachomatis could induce chlamydial persistence [On the other hand, Herpes simplex virus (HSV) in conjunction with HPV infection may increase the risk of cervical cancer . HSV is sistence ,10.C. trachomatis and HSV infection in oral cavity samples collected from patients from the central region of Argentina with different types of stomatological lesions.The aim of this work was to provide evidence of HPV, Oral swabs samples from a total of 318 patients over 18 years were clinically evaluated in the Department of Stomatology–Faculty of Dentistry–National University of Córdoba–Argentina. Samples were obtained of oral lesions which were classified by specialized stomatologist and confirmed by histopathological study, according to three types: benign lesions (B), potentially malignant disorders (PMDs) and oral squamous cell carcinoma (OSCC).This study was approved by the Institutional Committee of Ethics of Health ‘Oulton Romagosa’ (RePIS 007) of Córdoba- Argentina, according to the ethical principles stated in the declaration of Helsinki.C.trachomatis was detected using CTP1 and CTP2 primers to cryptic plasmid obtained amplicons size of 201 bp [Viral and bacterial DNA was extracted using the commercial AccuPrepGenomic DNA Extraction Kit-Bioneer, following the manufacturer’s instructions. Then, HPV L1 genomic region (450 bp) was amplified with degenerate primers MY09 and MY11 following the protocol previously described by Manos et al. . C.trachf 201 bp . We usedf 201 bp .The PCR products were visualized by electrophoresis in 1.5% agarose gel using a UV transilluminator. The β-globin gene was used as a DNA preservation marker.2) and Fisher Exact with a significance level 5% (95% CI), Epi info™ CDC software.Results were analyzed using Chi-square . Of these, 137 were classified among benign lesions (B), 96 potentially malignant disorders (PMDs) and 85 oral squamous cell carcinomas (OSCC). Positivity of DNA-HPV was observed in 34.3% (109/318) . It was C. trachomatis was 16.9% (54/318) and for HSV was 3% (10/318)  . HSV 1 anfection .10.1080/C. trachomatis detection by sex, age and type of oral lesion (p > 0.05), although the presence of HSV was significantly associated to sex and type of lesion variables (p = 0.02)  .C. trachomatis (4%), followed by C. trachomatis- HSV (1.8%), finally HPV-HSV co-infection was 0.3%. In most of the co-infections, HPV LR genotypes were detected , the lack of global standardization techniques used for C. trachomatis detection and the diversity of population study groups , female sex workers, etc.). As a result, it is difficult to make a direct comparison between those studies. Thus, our results show 16% and 18% of C. trachomatis DNA positive in oral lesions from women and men, respectively. These frequencies were higher than those in spittle samples among Japanese men (4.2%) [The frequency of C. n (4.2%) , in oraln (4.2%) . In addin (4.2%) .C. trachomatis was detected in 44% of pharyngeal swabs and in 61% of oral wash fluid samples [However, in a Japanese population of female sex workers,  samples .A limitation of this study is that the collected data do not provide us with information concerning the sexual behavior of patients.With respect to HSV, it has been described that direct contact with lesions or with infected oral or genital secretions during asymptomatic shedding can be a transmission way of the virus . Thus, HBased on this information, we consider it important to study HSV and co-infections with other STIs, which is the aim of the present study. Although a high frequency of HSV in the oral cavity would be expected, in this study the frequency observed was low. This could be due to the fact that lesions directly associated with HSV were not studied, since the biosecurity protocol of dentists does not allow it. In case of visible herpetic injuries, the patients received antiviral treatment and when the treatment is finished, a new sample is taken.Our study detected HSV DNA in 3% of the cases, represented primarily by HSV-1 (2%) followed by HVS-2 (1%). Most of these were benign lesions and female patients. These results are in agreement with a study carried out in the USA among individuals with HSV-1 and HSV-2 antibody  in whichC. trachomatis (4%).Globally, reports show that the frequency of HSV positivity varies according to the population and type of sample used. Thus, some studies report that HSV-1 was detected in 36% of the oral leukoplakia samples (PMD), and in 52% of the OSCC samples among Sweden patients . SimilarC. trachomatis and HSV. The current findings confirm that our knowledge on the variety of infectious agents in the oral cavity is only partial.In the literature, studies of sexually transmitted viruses in lesions of the oral cavity have usually been confined to the search for HPV, so little is known about the presence and co-infection with other infectious agents such as In this study, the three studied agents were found in all lesion type regardless of the sex of the patients; therefore, they should be taken into account for diagnostic, treatment purposes, and their epidemiological importance should be considered.This report is the first contribution to the identification and genotype characterization of HPV in a scenario little studied in our area, and it also contributes to improving our understanding on sexually transmitted infectious agents and their associations with the oral cavity.


Penile cancer is a rare tumor in developed countries; since its diagnosis is made usually in a late stage, it presents high morbidity and mortality. It is more prevalent in developing countries in Asia, Africa and South America, particularly in non-circumcised males .More common histology type is penile squamous cell carcinoma - PSCC (95%) related to the infection by the Human Papillomavirus (HPV) . HPV-DNATreatment of PSCC is mainly surgical, with local resection of lesion, partial or total penectomy and occasionally inguinal and eventually pelvic lymphadenectomy. Dissemination is mainly by lymph nodes  and by bloodstream.balanitis xerotica obliterans). Other important risk factors include: high number of sexual partners, genital wart disease of sexually transmitted diseases (STD)  .HPV infection is the most common sexually transmitted disease in the World, and 50% to 80% of sexually active persons have already been infected by that virus at any moment in their life . It was Cervical uterine cancer is also a disease caused by the persistent oncogenic HPV infection. At the uterine canal, the transformation zone, characterized by areas of immature metaplastic squamous cells, is particularly vulnerable to HPV infection. Apparently, virions infect only human cells, in special stratified squamous epithelium. Usually, HPV infection occurs in microcracks at the surface of epithelium, invading basal membrane cells, where the viral genome is introduced in the cell nucleus .HPV infection leads to displasic transformation of basal cells, that may heal spontaneously or evolve to neoplastic precursor cells and developing high grade lesions, resulting in tumors. During carcinogenesis, infection may alter oncotic cytology, also known as uterine cervix Papanicolau exam, that is used as screening method in women. Persistent high risk HPV infection is the key to lesion progression.Papillomaviridae family, small non-enveloped virus with double chain of DNA. More than 200 types of HPV have already been identified and 140 have been submitted to DNA sequencing . Most patients are uncircumcised. HPV infection is usually asymptomatic and may cause two types of lesions: acuminate condyloma , low grade lesions, and tumors, high grade lesions.Infected patients by serotypes 16 and 18 present high levels of specific antibodies, reducing the risk of subsequent infections by the same serotype. This evidence allowed the development of vaccines with viral parts of each subtype.The first HPV vaccine approved by the American Federal Drug Administration (FDA) was marketed in 2006. At present, there are three different vaccines: bivalent (2014), that targeted the infection of serotypes 16 and 18; quadrivalent ; and nonavalent (last version), that protects against infection of subtypes 6, 11, 16, 18, 31, 33, 45, 52 and 58 .The number of women, adolescents and young adults vaccinated against HPV is increasing worldwide, due to the increase of patients infected by HPV (including developed countries), the correlation of HPV and tumor in both sexes showed in recent studies, and the availability and easy of access to vaccination of young adults and adolescents, with direct impact in the prevention of neoplastic diseases of oropharynx, anus and genitals, such as uterine cervix and penis.Therefore, it is recommended vaccination of adolescents (boys and girls) with 11-12 years old, or before the beginning of sexual life (preferably) to prevent direct infection of HPV in both sexes, and to prevent cross-infection, actively preventing neoplastic diseases beyond penile tumors, such as ano-genital  and oropharynx cancers.


META-pipe is a complete service for the analysis of marine metagenomic data. It provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling. The functional annotation is computationally demanding and is therefore currently run on a high-performance computing cluster in Norway. However, additional compute resources are necessary to open the service to all ELIXIR users. We describe our approach for setting up and executing the functional analysis of META-pipe on additional academic and commercial clouds. Our goal is to provide a powerful analysis service that is easy to use and to maintain. Our design therefore uses a distributed architecture where we combine central servers with multiple distributed backends that execute the computationally intensive jobs. We believe our experiences developing and operating META-pipe provides a useful model for others that plan to provide a portal based data analysis service in ELIXIR and other organizations with geographically distributed compute and storage resources. ELIXIR was established to unite European life science resources. It has 21 member states and more than 180 research organizations that each take responsibility for an analysis service, database, software tool, training material, or provide cloud storage and compute resources. For example, one of the deliveries from ELIXIR Norway are marine metagenomics analysis services, whereas ELIXIR Finland provides cloud storage and compute resources. An ELIXIR user from Portugal may therefore use a service maintained in Norway run on resources in Finland. In this paper, we describe our approach for setting up distributed execution of such analyses.1 is a complete workflow for the analysis of marine metagenomic data. It provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling. We provide META-pipe as an Analysis-as-a-Service for Norwegian and Finnish ELIXIR users. However, additional compute resources are necessary to open the service to all ELIXIR users. Users log into the META-pipe web application where they can upload data to analyze, select tool parameters, start analyses, and download analysis results. The functional annotation is computationally demanding and must therefore be run on a high-performance computing (HPC) cluster or a compute cloud. Job execution is handled by the META-pipe backend, such that resource allocation, parallel execution, and fault handling is hidden from the user.META-pipeSigma2 Stallo supercomputer in Tromsø, which is a Norwegian academic HPC; (ii) theCSC cPouta OpenStack based Infrastructure-as-a-Service cloud, Finland; (iii) the CESNET-MetaCloud OpenNebula cloud that supports the open cloud computing interface, Czech Republic; and (iv) the commercial Amazon EMR cloud service. cPouta is anELIXIR compute service. CESNET-MetaCloud is part of theEGI Federated Cloud.META-pipe has a distributed architecture with three central servers and geographically distributed execution managers . We have1. We demonstrate the use of geographically distributed compute resources for life science data analysis.2. We describe the design and implementation of cloud setup tools for our analysis service.3. We describe our experiences developing and operating the META-pipe analysis service.An important design goal for the META-pipe backend is to make the execution managers portable. In addition, we have taken care to make setup and maintenance of the execution managers easy. We achieve these goals since our backend is designed such that all state is maintained at the central servers. We therefore reduce the amount of code that needs to be ported, maintained, and optimized for a new execution environment. The execution managers are stateless, and the jobs are idempotent. This simplifies failure handling. We also use the widely available Apache Spark framework to execute the pipeline analyses. In addition, we have implemented tools that make it easy to set up and administer the execution managers. These pull the META-pipe tools, dependencies, and jobs from the centralized servers. In this paper, we describe these tools and their use. We make the following contributions:We designed our analysis service to be powerful, easy to use, and easy to maintain. We believe our work provides a useful model for others that plan to provide a portal-based data analysis service in ELIXIR and other organizations with geographically distributed compute and storage resources.META-pipe cloud setup design document.The META-pipe is deployed as shown inBastion VM: acts as the gateway machine used for cluster management and gateway to an initiated cluster.Cluster Master VM: acts as Spark Master, NFS Server, and it runs the main META-pipe executable.Cluster Worker VMs: act as Spark Workers, NFS Clients, and the runners of parallelized tasks of META-pipe jobs.A META-pipe execution environment has three types of virtual machines (VMs):NFS-shared storage used by the worker VMs to read and write temporary computation data including intermediate result files. The master VM contains a NFS-server that serves the access to the storage. The worker VMs are NFS-clients that have full read-write access to the shared storage. The NFS server also has the Spark and Scala installations. The NFS storage can be either a Master VM internal volume, or a virtual volume attached to the Master. We typically use the latter, since it makes deployment easier and it allows META-pipe volume caching (as described below). We have, however, not compared the performance of these two approaches.In addition, META-pipe requires the use ofrd party tools used by the pipeline, tool libraries, and the reference databases used by the tools. These dependencies must be downloaded from our artifacts web server. The dependencies are 44 Gigabytes, of which most of the space is used for the reference databases. Each worker VM must have access to the META-pipe dependencies, so they are typically stored locally on each cluster VM. If there are not enough volume resources, NFS storage is used.The META-pipe executable is downloaded from our artifacts server. The executable is a (42 Megabyte) jar file that contains the Spark job, and it is submitted as a Spark job from the Master VM. The executable jar does not contain 3After setting up the Spark, a simple parallelized script should be used to test that Spark and META-pipe is set up correctly. We first test Spark by submitting a parallelized version of prime number counter, wait for all Workers to be done, and then ensure that there were no error messages and that the result is correct.To validate the correctness of META-pipe installation, we use the built-in validation procedure in the META-pipe executable. This procedure will check the state of all tools required and their dependencies that are required for META-pipe execution. It will also check that the tools do not return errors.After initialization, the submitted executable will listen for, and run, new jobs until the spark-submit is stopped. The jobs are submitted to the central META-pipe job server that checks the tag in the job and submits it to a specific META-pipe executable. The executable downloads the input data from the META-pipe storage server to a Spark RDD data structure and launches the META-pipe job using the spark-submit command. When the job is completed, the executor uploads the output datasets to the META-pipe storage server, which are then accessible by the user on META-pipe portal. After the execution of each tool in the pipeline, the intermediate output datasets are also uploaded to the storage server, so that if the job fails, it can be restarted from the last successful pipeline step.an execution manager setup tool that setups the virtual machines, storage, Spark, and META-pipe as described above. It is implemented as a command line tool written in Java, with some parts implemented in Bash, Ansible and Python. The tool’s requirements, usage information, as well as more detailed technical information is in ourdesign document.To set up the META-pipe execution manager on cPouta we createdWe have optimized cluster provision by caching virtual volumes with the META-pipe execution manager and dependencies. To avoid downloading, unpacking and preparing META-pipe files for each new cluster instance, we store these in a virtual volume the first time a cluster is set up. This volume is used as the storage of prepared META-pipe files (a cache), which is used to create volumes for cluster VMs in later cluster provision. This reduces the time to create a cluster from 30 minutes to 10 minutes. In comparison, the execution time for a typical META-pipe job is several hours.OCCI). It is therefore compatible with allEGI Federated Clouds, since they all support OCCI. The tool is a rOCCI Client implemented in Python and ansible that uses X509 VOMS certificates. It implements aTerraform OCCI plugin. Additional details are inhttps://github.com/cduongt/mmg-cluster-setup-CESNET.To set up the META-pipe execution manager on the CESNET-MetaCloud cloud we adapted the cPouta tool to create a tool that uses the Open Cloud Computing Interface  and command line interface (CLI), and CloudFormation.To set up the META-pipe execution manager on Amazon Web Services (AWS), we use the AWS Elastic MapReduce (EMR) console and a custom cluster boot-time script. We use AWS’s EMR managed framework since it natively supports Spark. The cluster setup is therefore simpler than on OpenStack and OpenNebula, but it is not as configurable. For instance, EMR clusters always use the YARN resource allocator and cannot be configured to use Spark’s “standalone” mode instead, which we use on the other platforms. This has not been a big problem in practice, but it constitutes an uncontrollable variable when optimizing the execution for various cloud platforms. A detailed description of the setup is inFor META-pipe, we make AWS EMR clusters on demand. Our clusters use spot instances with VM flavors that provide the best cost-performance for META-pipe (currently we use c4.4xlarge). An EMR cluster boots up with a compatible version of Spark provisioned by Amazon. Our automatically started boot-time script then provisions META-pipe tools and dependencies from an S3 bucket. The whole cluster creation process takes about 10 minutes. Similar to other execution environments, the cluster is idle until we start the META-pipe execution manager on the cluster’s master node. The execution manager continually fetches jobs submitted using the META-pipe web app and submits them to Spark on the cluster. We must terminate the cluster ourselves, either by a script through the AWS API or manually in the EMR console.ELIXIR webinar (November 2016) about the ELIXIR compute platform.Here we describe a use case where the computationally demanding parts of META-pipe are setup to execute on a cloud resource provided for a new user community. The new user group first applies for computational resources from its partner clouds, and then utilizes these resources easily through the interface provided by the META-pipe web application. The tools described here makes it easy for a resource provider to administer the META-pipe job executions, and the use of standardized technologies and protocols ensure compatibility and portability of the META-pipe analysis backend across clouds. Below we describe the execution of analysis jobs from the point of view from META-pipe end users, compute resource providers, and META-pipe service providers. We have also described these use cases in an1. META-pipe web application using their home institution credentials. The login page is the single sign on provided byELIXIR AAI. The datasets are typically up to a few GB in size and they can quickly be uploaded using the browser.The user logs into the2. The user uploads their dataset to be analyzed, and possibly changes some of the analysis parameters. This is done in the META-pipe web app.3. The user tags the cloud to use for the analysis and submits the job for analysis using the web app GUI.4. 2, Artemis3, or METAREP4.Once computations finish, the data is returned to the portal, and the user can download the enriched results for further analysis or visualization using separate tools such as KronaThe new user community, represented for example by their national ELIXIR node, have applied and received compute resources from an academic cloud provider. One of the academic users has a marine metagenomics dataset they want to analyze:The resource provider must setup the META-pipe execution manager that executes the Spark job that implements the analysis. In addition, the resource provider must test and maintain the execution manager. The execution manager setup tools described in the previous section simplifies this task.1. create-env: to set up the environment and META-pipe files caching volume that will be used in create-cluster.2. create-cluster: provision cluster resources, install and configure the execution manager (Spark and NFS), install META-pipe tools, dependencies, and reference databases on the provisioned cluster VMs, and test the setup.The first time the META-pipe backend is set up on a cloud environment, the resource administrator needs to edit a configuration file that defines the virtual cluster to be created. After that the administrator runs the following commands in the tool:To set up a cluster as second time, only step 2 is run. It will use cached META-pipe volumes created previously.3. sw-launch: start Spark and the server that listens for new META-pipe jobs to execute.Then to accept META-pipe jobs from the job server:4. sw-kill: stop all META-pipe related processes on all cluster VMs.To stop accepting new jobs:5. remove-cluster: remove the cluster and keep the environment for future use.6. remove-env: remove the environment, including cached volumes.To free the allocated resources:Step 6 is only done when the resource is not intended to be used for META-pipe jobs anymore. Steps 2–5 may be automated.The META-pipe team providing the service do not need to make any changes to the central services since the new execution manager is authorized using the META-pipe authorization server, and since the end-user specifies the tag for the new execution manager.https://www.csc.fi/web/training/-/metagenomics also described in the report “EGI-Engage D6.15 Demonstrator for ELIXIR workflows implemented in the EGI Federated cloud”). We setup two META-pipe execution environments. The main execution environment was running in the cPouta cloud environment at CSC, and a backup execution environment that was running in EGI Federated Cloud (CESNET-MetaCloud). These META-pipe backends were set up by the course organizers and so the students did not need to do any technical preparations to use the cloud services. Instead, they only needed to define one extra parameter in the web interface to guide their analysis tasks to a specific external META-pipe backend. 42 students participated to the metagenomics course and successfully used these temporary execution environments through the META-pipe web interface without any interference.In April 2017 we used META-pipe in a metagenomics course organized by the Finnish ELIXIR node  manager, such as GalaxyBioContainers provides many tools. Several pipeline managers support containers, including Nextflow9, Toil,Pachyderm, andLuigi10. META-pipe does not use containers, since our artifacts server and the ansible scripts used by the setup tools take care of META-pipe dependencies. In addition, even when using containers, there is a need to set up container orchestration for parallel execution on distributed resources. Systems such asKubernetes andDocker Swarm can be used to orchestrate containers. The META-pipe execution manager use Spark for orchestration.Another approach for portable bioinformatics tool execution is to package the tools as containers. Repositories such asEBI Cloud Portal enables execution of pipelines on cloud resources. Users can sign on using ELIXIR AAI, add their applications, pipelines as virtual machine images, and configure cloud compute and storage resources. We attempt to hide these details to the end users. Commercial solutions include platform such asIllumina BaseSpace, where developers can provide apps for analysis on AWS of data uploaded to BaseSpace. Currently, most of the provided apps are single tools instead of complete pipelines, such as META-pipe.TheAn important limitation of our approach is that we do not handle resource allocation for end users. Instead users must contact a service provider in their country ) to allocate resources and setup an execution environment. This is not something all users know how to do, and it is unnecessarily complicated for small projects. There are four possible solutions. First, for small projects an ELIXIR node may provide the computation resources for all users. Second, an ELIXIR node may provide computation resources for all their users. Third, federated cloud resources can be used through EGI Federated Cloud or the ELIXIR federated cloud testbed. Long term usage of federated approach requires that access guarantees  are arranged. Fourth, the users can allocate and pay for resources from a commercial cloud provider. Such pay-by-use is especially easy for industry users.We do not provide a service for predicting resource usage for META-pipe jobs. However, we believe that we can make good estimates based on the input file size. We are also currently evaluating and optimizing META-pipe job execution. An important part of such optimization is to choose the most cost efficient virtual machine flavors and storage solutions on a cloud.We have described our approach for setting up and executing the functional analysis of META-pipe on academic and commercial clouds. To make our analysis service easy to use and to maintain, we use a distributed architecture where we combine central servers with multiple distributed backends that execute the computationally intensive jobs. A key issue in ELIXIR is to obtain computing resources for META-pipe end users. It is still to be decided how resources will be obtained, managed and allocated to the individual end users. For all solutions, an up-to date and highly automatized tool for launching a META-pipe execution environment is needed. We will continue improving the META-pipe backend and to add support for additional resource providers.No data is needed to use the cloud setup tools.https://gitlab.com/uit-sfb/METApipe-cPouta-cloud-setup.CSC cPouta OpenStack cloud:https://github.com/cduongt/mmg-cluster-setup-CESNET.CESNET-MetaCloud OpenNebula:https://gitlab.com/uit-sfb/metapipe-on-aws.Amazon Web Services EMR:The code for the three setup tools are open source at:http://doi.org/10.5281/zenodo.105380711. All use theMIT license. The above repositories include user guides.Archived code at time of publication for all: This manuscript described META-pipe as a cloud-based workflow for marine metagenomic analysis. The computation-intensive analysis is parallelized via a distributed Spark-compatible infrastructure. The unique feature of META-pipe is its support of heterogeneous cloud environment for job execution, including those available from academic institutes and commercially available AWS. This is the 2nd version of the manuscript. The authors have addressed all remarks raised by the previous reviewers. A few minor remarks are listed below:Cannot evaluate the application without a valid ELIXIR AAI login credential. Should either provide a demo account or a recorded screencast for the review purpose. Use cases->End user section:"(or be added to an EGI based VO)": What's "VO"?"requires that access guarantees ": the first appearance of acronyms  should be spelled out and/or include links. Discussion->Limitations section:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your suggestion to improve the readability of our paper. We have now spelled out the acronyms. Also note that in the "Use cases" section we provide a link (https://elixir-europe.org/documents/elixir-webinar-elixir-compute-platform-roapmap-november-2016) to a seminar where we show the end user interfaces. This article describes the setup of a multi-cloud architecture that can support the execution of META-pipe jobs for marine metagenomic data analysis. And META-pipe provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling.Remarks:The introduction of META-pipe backend architecture and deployment is detailed and clearIt achieves the goal which is to provide a powerful analysis service that is easy to use and to maintain since META-pipe has web app and GUI that the users just set parameters and then they can make analysis of data.In the chapter concerning resource provider (page 6), “The tools described in the previous section simplifies this task“ needs more details like names of the tools since there are some tools referred in the previous section. It would be easy to understand.There is less example to indicate tasks in META-pipe can be easy and fast. It would be better to give some data like running time and volume to state. Giving other projects differed from this as ‘ control group ’and setting characters to make comparisons will be more persuasive.There are some spelling mistakes like ‘manger’.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for your remarks. We have specified that the tools used by the resource provider are the execution manager setup tools. Performance and scalability evaluation of META-pipe on different execution environments is important, but outside the scope of this paper. Here we describe the work done before and after executing pipeline jobs. This approach can be used for other bioinformatics pipelines (implemented as Spark jobs). A performance evaluation would require a detailed explanation of the META-pipe tools. To make this point clearer we have added a line in “cPouta Open Stack setup” about META-pipe job execution time being several hours (compared to 10-30 minutes of setup time). The article describes the setup of a multi-cloud architecture that can support the execution of META-pipe jobs for marine metagenomics data analysis. It is intended for a technically inclined audience and focuses on the different configuration steps required for the setup of a complex computing architecture involving several clouds in several countries. The purpose is to be able to open the META-pipe service to a large number of ELIXIR users.  Several cloud middleware have been configured . The description of the infrastructure is clear and additional links to github repositories or online documents allow the reader to fetch extra technical information. Remarks : In the introduction, the authors indicate that the design goal is to make execution mangers (sic) portable and that the setup and maintenance of execution mangers (sic) is easy. The authors state that these goals are achieved because all state is maintained on central servers. This section is not completely clear and should be rewritten to highlight the advantages of the central servers architecture. Emphasizing the fact that the META-pipe environment will be pushed from a central server to the execution clouds could clarify things. In the chapter concerning the META-pipe executables and dependencies (page 4), it could be interesting to better describe these dependencies. These dependencies are downloaded from the artifact server but no technical information is given about this server . Are the reference databases provided by the artifact server ? The infrastructure is articulated around a central server hosting the configuration of META-pipe. This creates a single point of failure. It could be interesting to describe the actions that are taken in order to have a more resilient system. In the META-pipe job execution paragraph, the input data is downloaded to a data structure . What is this data structure ?  Typos: Page 3 / Col 2 / Line 4, 5, 11 : manager/manger Page 3 / Col 2 / Line 27 : executables instead of executable Page 3 / Col 2 / Line 29 : is set up Page 5 / Col 1 / Line 3 : is set up Page 5 / Col 1 / Line 27 : to set up Page 5 / Col 2 / Line 42 : are set up Page 5 / Col 2 / Line 45 : utilizes Page 6 / Col 1 / Line 13 : its dataset Page 6 / Col 1 / Line 23 : set up Page 6 / Col 1 / Line 28 : set up Page 6 / Col 1 / Line 38 : To set up a cluster for a second time Page 6 / Col 2 / Line 19  : We set up Page 6 / Col 2 / Line 24 : did not need to :  Missing word Page 6 / Col 2 / Line : several pipeline mangers : managers ? Page 7 / Col 1 / Line 19 : ELIXIRI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We improved the description of the centralized server and the execution environments in the introduction.We added additional details about the dependencies and the artifacts server in “META-pipe executable and dependencies”We now specify that the data is downloaded into a Spark RDD.We have fixed the typos. We agree that centralized servers create a single-point-of-failure, but at the same time it simplifies the implementation of the backend. We are not a stage at the project yet where we believe the improved availability justifies prioritizing this issue.Thank you for your remarks. We have made the following changes in our revised version:


Local treatment for cervical intraepithelial neoplasia (CIN) by Loop Electrosurgical Excision Procedure (LEEP) has been correlated with reproductive morbidity, while the cervicovaginal microbiota is also known to affect the risk of preterm delivery. CIN and treatment by LEEP might change the cervical microbiota. The main aim of this study was to describe the cervical microbiota before and after LEEP and assess its associaton with cone depth and HPV persistence. Further, we aimed to compare the microbiota to references with normal cervical cytology.n = 77) and 12 months (n = 72) post LEEP for bacterial culture and PCR, and post LEEP for DNA testing for human papillomavirus (HPV). We compared the cervical microbiota composition before and after treatment and between women planned for LEEP vs references.Between 2005 and 2007, we prospectively identified 89 women planned for LEEP in a Norwegian hospital and recruited 100 references with a normal cervical cytology. Endocervical swabs were collected prior to treatment and at six  contains supplementary material, which is available to authorized users. Local excisional treatment appears to alter the cervical microbiota towards a less diverse microbiota. Women with CIN have a more diverse microbiota than women without CIN.Chlamydia trachomatis, Trichomonas vaginalis and herpes simplex virus-2 have been associated with HPV-related cervical neoplastic lesions and carcinoma [Persistent genital infection with high-risk human papillomavirus (HPV) is causally associated with cervical cancer and its precursors  . A numbeLactobacillus [Lactobacillus with subsequent overgrowth of anaerobes, in the vagina also referred to as bacterial vaginosis (BV). BV has been associated with higher prevalence and persistence of HPV infection and with development of CIN [Lactobacillus spp. is involved in HPV acquisition and persistence, and in the development of CIN and cancer [Health in the female reproductive tract is commonly associated with low microbial diversity and dominance by one or a few species of bacillus . Dysbiost of CIN –6. Recend cancer –11.Lactobacillus in a small non-pregnant population three months after LEEP [Loop Electrosurgical Excision Procedure (LEEP) is the most commonly used treatment technique for CIN. The majority of the women treated are young and have not started or completed their reproductive career. Local excisional treatment has been associated with an increased risk of preterm delivery (PTD) in subsequent pregnancies –14. The ter LEEP .The main aim of this prospective observational study was to compare the cervical microbiota in women with CIN before and after LEEP and to assess whether cone depth and/or HPV persistence affect bacterial composition post-treatment. An additional aim was to compare the cervical microbiota in women with CIN to healthy reference women with normal cervical cytology.We prospectively identified all women referred for investigation of cervical dysplasia at Østfold Hospital Trust, Health Region East, Norway between October 2005 and February 2007. Women planned for their first LEEP for suspected high-grade disease were eligible Fig. . Women oA population of 100 healthy references, defined by the presence of normal cytology, was recruited at a private gynecological practice, and termed the “reference group”  for detection of Chlamydia trachomatis, using the COBAS TaqMan 48 CT test , and for detection of Mycoplasma hominis, Ureaplasma urealyticum and Ureaplasma parvum. DNA was extracted from the swabs using the BioRobot M48  and the Mag Attract DNA bact protocol. Amplification and detection of U. parvum, U. urealyticum and M. hominis were performed using the ABI Prism 7900HT Sequence Detection System  [With sterile speculum examination, serial Liquid Based Cytology (LBC) and five endocervical bacterial swabs were collected prior to treatment and at six and 12 months post-treatment as well as at baseline for the reference population. One swab was transported on Stuart medium for cultivation of anaerobic and aerobic bacteria on blood and lactose agar plates. Growth results were reported as growth or no growth for the following bacteria; ity, CA) , 21.Lactobacillus bacteria were included in a constructed group called“ any non-Lactobacillus”.In every woman all bacterial species found were recorded. All women that had at least one type of one non-Cervical LBC specimens were collected with a Cytobrush Plus  and transferred to PreservCyt medium  in the follow-up samples of the treated population. Nucleic acid extraction and high-risk HPV DNA testing  was performed as previously described . All bioBackground characteristics of sociodemographical data and contraception in the reference group and the LEEP group were compared using chi-square or independent t-tests.Lactobacillus species at each assessment was recorded. The mean number of non-Lactobacillus before and after LEEP was compared with paired t-test (at six or 12 months). The change in mean number of non-Lactobacillus after LEEP was compared between women below 46 years and women 46 years and above with independent t-test. This change in mean number of non-Lactobacillus after six and 12 months follow up was also compared between women without hormonal contraceptive and women with hormonal contraceptive and between women in a relationship (married/cohabiting) and single women. To study if LEEP resulted in a change of different bacterial species in each individual we focused on the women with a change in detection of bacteria after LEEP compared to before LEEP (those who either lost or aquired bacteria after LEEP compared to before LEEP). We compared the number of women who lost a specific species of bacteria that were present before LEEP to the number of women that aquired that particular bacteria species after LEEP. This was performed using paired data from before LEEP and from follow up (at six or 12 months) with McNemar test.The number of different non-Lactobacillus was compared between HPV negative and HPV positive women with independent t-test at six and 12 months follow up. Difference in detection of Lactobacillus and the constructed group any non-Lactobacillus according to HPV DNA positivity after LEEP was analysed with Fisher’s exact test. The mean aquisition or loss of detectable non-Lactobacillus bacterial species at six and 12 months follow up compared to before treatment was analysed in relation to HPV-DNA status after LEEP using independent t-test.HPV DNA status post-treatment for assessment of persistence of HPV was studied. The mean number of non-Lactobacillus) at six and 12 months after treatment was correlated to the cone depth using the Pearson Correlation test.We compared the cone depth between women testing positive or negative for HPV DNA at both six and 12 months with independent t-test. We further assessed whether change in the microbiota , marital status (living together (married/cohabiting) or living alone (single)), smoking  and use of hormonal contraception (yes or no).The mean number of detected bacterial species in the reference group and the LEEP group before treatment was compared using an independent t-test. The differences in individual bacterial species detected and the constructed broader group“ any non-Statistical analyses were performed for the whole population and in a subgroup including only women below the age of 46 .p-value < 0.05 was accepted as statistically significant. Statistical analyses were performed using IBM SPSS software, version 24.0.For all statistical tests a Women planned for treatment were significantly younger than the healthy references, more likely to use contraception, to smoke and less likely to be married Table . No womaUreaplasma parvum was the most frequently detected bacteria in the LEEP group before treatment (46%), followed by Lactobacillus spp. (36%) and Gardnerella vaginalis (32%). Lactobacillus spp. was the most frequently detected bacteria in the reference group (39%) followed by Ureaplasma parvum (16%).The bacteria composition in the LEEP group before and after LEEP as well as in the reference group are described in Table Lactobacillus bacterial species decreased after treatment . No significant difference in the change of non-Lactobacillus species numbers after LEEP was found when comparing women below 46 years to women 46 years and above, women without hormonal contraceptive to women with hormonal contraceptive or women in a relationship (married/cohabiting) to single women (data not shown).The individual bacterial species detected before and after LEEP were largely similar. The number of non-Streptococcus spp. (p = 0.04) and Ureaplasma parvum (p = 0.04) at 6 months follow up. These differences were no longer significant at the 12 months follow up .There was a tendency towards an increase in Lactobacillus in the HPV pos and the HPV neg group at 6 months  and at 12 months  did not differ. No apparent difference was seen when restricting the analysis to women below 46 years of age. No difference in the detection of Lactobacillus or the occurrence of the any non-Lactobacillus group according to HPV DNA positivity after LEEP was found  and at 12 months 24 of 72 (33%) treated women had a positive HPV DNA test. The mean number of non-p = 0.28; and after 12 months HPV pos 0.29 vs HPV neg 0.31 p = 0.95).The mean loss of detectable bacterial species after LEEP was not associated with HPV status post treatment (mean loss after 6 months HPV pos 0.63 vs HPV neg 0.30 p = 0.9) and 12 months . There was no correlation between cone depth and change in the number of non-Lactobacillus at follow up compared to before treatment (data not shown).The cone depth of the 77 treated cases varied between 10 and 25 mm (mean 18.0 mm). The mean cone depth was similar for women testing positive or negative for HPV DNA at six months , Fig. Lactobacillus bacterial specie, the women in the LEEP group had a significantly higher bacterial diversity before treatment as compared to the reference group . Bacteroides spp., Gardnerella vaginalis, Mycoplasma hominis and Ureaplasma parvum were more often detected in the LEEP group before treatment compared to the reference group (Table Lactobacillus spp. (p = 0.76) , p = 0.009 and p = 0.01. For analyses of differences in detection of specific bacterial species see Additional file In order to assess whether the cervical bacteria changes after LEEP were a result of treatment rather than the removal of the disease, we compared the findings post-treatment at six and 12 months in the LEEP group to the healthy references. Women in the LEEP group had more non-Lactobacillus bacterial species six and 12 months after LEEP compared to before treatment and a tendency towards a concomitant increase in Lactobacillus. The results were not significant for individual bacterial species, possibly due to the small sample size. No association between the detection of cervical bacteria and HPV persistence was found. The cone depth had no impact on the cervical microbiota or HPV status.In this study we found a reduction in the number of non-Lactobacillus iners dominated microbiome. Due to the culture and PCR-based bacterial detection techniques used in our study, we were unable to describe the full taxonomy and different Lactobacillus species in the same way as Zhang et al.Our findings are in agreement with, to our knowledge, the only other study published on this topic so far. Zhang et al. recently published their results of comparing the cervical microbiota, studied with next generation sequencing (NGS) technique, before LEEP and at three months follow up in 26 women . They reLactobacillus spp. in association with preinvasive cervical disease states [In our additional analysis, we found a higher number of bacterial species in women with CIN prior to LEEP as opposed to references with a normal cytology, indicating that women with high-grade dysplasia harbour a more diverse cervical microbiota than the healthy female population. Our results are in agreement with earlier studies showing increased vaginal microbiota diversity and reduced levels of e states –9, 11.The pathogenesis of the association between dysbiosis and development of CIN is unknown. It has earlier been suggested that dysbiosis may result in epithelial cell damage that potentially facilitates entry of HPV into the basal epithelial cells as well as creating an environment that promotes the viral life cycle, persistence of infection and ultimately development of dysplasia . Levels M. hominis, Gardnerella vaginalis and Bacteroides [U. Parvum, that also has been associated with PTD [Lactobacillus spp. Furthermore, although the cone depth correlates with the risk of PTD [Having CIN alone has been shown to confer a small increased risk of PTD , 27, 28 teroides . In our with PTD , was alsk of PTD  this hadTo our knowledge this is the second study, and the study with the largest sample size, that monitor changes in cervical microbiota before and after LEEP. This study includes follow up at two serial time points, including also a reference populaton of healthy individuals. However, there are some limitations in study design which have to be considered when interpreting the results: The history of recent sexual activity, the menopausal status and the use of postmenopausal hormone replacement therapy was unknown, and these factors may have an impact on the results. We performed subgroup analyses in women below the age of 46 ; the results were largely consistent with the total population although the sample size was smaller, facing the risk of type II-error, especially when studying specific bacteria species. The highest diversity and instability in microbiota is usually observed at the time of menstruation when oestrogens and progesterones are at their lowest . The exaRegarding the additional analysis, there were considerable differences in the characteristics between the LEEP and reference group, for example age. This may have affected the results as we know that several environmental and hormonal factors may affect the microbiota. Furthermore there was no follow-up in the reference group to compare any changes in microbiota composition over time. When planning similar studies in the future we recommend detailed collection of information about menopausal status, use of hormone replacement therapy, the exact time of menstruation, menstrual hygiene practices, use of vaginal douching, use of hormonal or barrier contraceptive products, sexual behaviour, number of sexual partners, history of recent sexual activity and use of antibiotics/probiotics  since these factors could affect the cervical microbiota.Lactobacillus spp. six and 12 months after treatment, possibly reflecting the impact of the removal of the disease. These results support recent observations at three months follow up by Zhang et al. [We observed a tendency of decreased bacterial diversity and a concomitant increase in g et al. . FurtherThe sample sizes for some of the comparisons were small. Further, larger longitudinal studies with NGS technique in pre-menopausal women are needed to investigate the changes and stability of the microbiota after local excisional treatment. The analysis based on bacterial 16S rRNA used in NGS will permit an in-depth study of the vaginal microbial community structures to a level of detail not possible with standard culture-based microbiological techniques. Sampling should be expanded to include pregnant populations post-LEEP together with markers of inflammation to correlate results to clinical outcomes. Longitudinal studies are also necessary to confirm temporal relationships between cervicovaginal microbiota and HPV infection and development of dysplasia.Additional file 1:Table S1. The cervical microbiota in the LEEP group and the Reference group in women aged <46 years. Table S2. The cervical microbiota six months post treatment in the LEEP group and the cervical microbiota in the Reference group. Table S3. The cervical microbiota 12 months post treatment in the LEEP group and the cervical microbiota in the Reference group. (DOC 88 kb)


About 30% of the cervical cancer cases in Sweden occur in women older than 60. The primary aim was to evaluate the acceptability of repeated self-sampling at home for HPV-testing in elderly women. The prevalence of HPV and HPV related dysplasia as well as the sensitivity of cytology was evaluated.Repeated self-sampling at home for HPV testing was offered 375 women in each of the four age groups 60, 65, 70 and 75 years. Women with two consecutive positive HPV tests were examined with sampling for histology and cytology.A self-sample was provided by 59.5% (893/1500) of the invited women. The overall prevalence of HPV was 4.4%  in the first test, and 2.5% were persistent positive in the second test  collected on average 5.5 months later. Dysplasia, was found in 1.8% (16/893) (95% CI 1.1–3.0) and CIN 2+ in 1.0% (9/893) (95%CI 0.5–2.0) of the women. Of the 16 women with dysplasia in histology, 13 (81.2%) had a normal cytology.Repeated self-sampling at home combined with HPV testing was well accepted among elderly women. A high prevalence of CIN was diagnosed by histology. Cytology showed extremely low sensitivity and should not be recommended for this age group. In Sweden, about 30% of CC cases occur in women over 60 and the mortality rate is about 70% in this age group –3. Cerviommended .It is well known that DNA based HPV tests are more sensitive than cytology in detecting pre-malignant lesions on the cervix –11. ImplIn post-menopausal women, due to hormonal changes, the transformation zone where precursor lesions develop is situated higher up in the cervical canal, and is therefore not accessible for proper examination and sampling . CytologIn elderly women, there is a lack of knowledge concerning the prevalence of HPV and HPV related dysplasia. There are few studies focusing on women who are older than those included in the screening programmes . In a reThe primary aim of the current study, therefore, was to evaluate the acceptability of repeated self-sampling for HPV testing in elderly women. The prevalence of HPV and HPV related dysplasia, as well as the sensitivity of cytology, was also evaluated.This prospective population based longitudinal descriptive study was conducted in Dalarna County, Sweden, between 2014 and 2016, and 1 500 women were invited to perform repeated self-sampling for HPV testing. Women were randomly selected, by using the random generator in SPSS, from the population register, with 375 women in each of the four age groups 60, 65, 70 and 75. Written informed consent was obtained from women who agreed to participate in the study, and self-sampling kits were sent through the postal service. In brief, self-sampling was performed at home  and the Women who were repeatedly positive in the second HPV test were offered examination by colposcopy, sampling for histology, and liquid-based cytology (LBC). One of the authors (RSH) performed the vast majority of the colposcopies, cervical biopsies, abrasions, and conisations for histological diagnosis.All LBC specimens were screened by cytotechnicians, and those considered abnormal were reviewed by a surgical pathologist. For cervical cytology, the Thin Prep Pap Test was used. The cervical smear was collected with a plastic spatula and a cytobrush. LBC specimens were placed in PreserveCyt solution and processed in the Thin Prep 5000-processor  . The terSpecialists in surgical pathology examined the cervical biopsy samples and cones for histologic diagnosis. One senior pathologist re-evaluated all LBCs, cervical biopsies and cones, focusing on glandular atypia and adenocarcinoma. All cytology and histology specimens were examined at the Department of Pathology and Cytology, Falun County Hospital, Falun, Sweden.For statistical analysis, Excel and the Statistical Package for Social Sciences (SPSS) version 22 for Windows were used. The data was analysed using both a per-protocol approach (PP), including only women who complied with the protocol, and also with an intention-to-treat approach (ITT), by also including women who on their own initiative had a clinical second HPV test and follow up. A p-value less than 0.05 was considered statistically significant. For statistical significance testing between age groups, the chi-squared test in SPSS was used. Confidence intervals (CI) of proportions (Fleiss) were calculated using Excel . The stuThe number of women included and excluded at each stage of the study, as well as the number of end diagnoses in cytology and histology, is presented in Overall 39 women  were HPV positive in the first test and 22  in the second test  i.e. 56.Among the women in the PP approach, 68.4% (13/19) with a positive second HPV test had dysplasia in histology. Out of all 893 women, 16 (1.8%) had CIN, 0.8% (7/893)CIN 1 and 1.0% (9/893) had CIN 2+ . In one The positive predictive value (PPV) for any CIN (CIN 1+) was 41.0% (16/39) after the first HPV test and 68.4% (13/19) after the second HPV test in the PP approach, and 68.2% (15/22) in the ITT approach. The PPV for CIN 2 + was 23.1% (9/39) after the first HPV test and 36.8% (7/19) after the second HPV test in the PP approach, and 36.4% % (8/22) with the ITT approach.Self-sampling was well accepted among elderly women, where 59.5% of those invited participated in the study. In a recent publication based on women aged 30–49 years, where Pap-smear was compared with repeated self-sampling for HPV analysis, the participation rate was 47% in the self-sampling arm . There aAn HPV prevalence of 4.4% in this age group (average age 67 years), is similar to the HPV prevalence of 4.1% found when the sample was collected from the cervix (average age 68 years) by a gynaecologist . There aAlthough the women included in this study were from one Swedish county, we believe that data on HPV prevalence and dysplasia are generalizable to most parts of Sweden. An obvious limitation of this study is the lack of verification of possible disease among women testing HPV negative. It is, however known that the vast majority of the high-grade dysplasias are caused by oncogenic HPV and this study aimed at detecting HPV-related dysplasia. In a previous study we have data on HPV and cytology from elderly women that were HPV-negative in their second HPV test and among them the cytology was normal . Loss ofIn line with what has been shown in other studies , 17, we We found a CIN 2+ prevalence of 1.0% in women aged 60–75 years. This is slightly lower than that which was reported in the Swedish screening population aged 23–60 years, with a CIN 2+ prevalence of 1.3–1.4% . The truThe importance of CIN 1 in elderly women has not been studied. Several studies show that there is a high level of regression of CIN 1 in younger women but whether this is also true in the elderly is not known. According to Bekos et al, a patient's age has a considerable influence on the natural history of CIN, independent of CIN grade and HPV high-risk infection .CIN 2+ is the treatment threshold for women of a fertile age,  but in mWang et al. found that Swedish women born between 1919 and 1945 who were unscreened, or screened with abnormal results in their 50s, had a relatively high risk of cervical cancer after age 60, but being screened at age 61–65 was associated with an evident risk decrease up to age 80 . In womeSelf-sampling was well accepted among elderly women, and thus constitutes a potentially appropriate strategy for cervical cancer prevention in this age group if confirmed in larger studies. We also found that among women with two HPV positive tests there was a high prevalence of CIN 2+ diagnosed by histology, which motivates screening to continue at older ages. Cytology showed extremely low sensitivity in elderly women and should therefore not be used in this age group. These data support strong preliminary evidence for a novel screening paradigm in the elderly, which must be more fully evaluated in a screening trial.


This descriptive study assessed the association of specific microbial STI and socio-demographic characteristics and practices with cervical cytomorphologic presentations in regards to pre cervical cancer grades amongst health seeking patients attending the reproductive health clinic of Nakuru County referral hospital, a public hospital under newly devolved health services governance.a total of 142 patients  whose median age ranged between 20-70 years were purposively sampled. A structured questionnaire with closed and open ended entries was administered and STI screening including Pap smear examination for cytomorphological profiling done according to revised 2014 Bethesda classification. Associations were established using chi-square and multivariate logistic regression model to determine prediction of cervical atypia manifestations.a majority of the study participants had only primary education or no education in AGC/AIS (63%) and HSIL (73%) relative to LSIL (49%) and controls (53%) (P=0.017). Koilocyte rates were higher in AGC/AIS (25%), HSIL (52%) and LSIL (77%) compared controls (12.5%) (P<0.0001). ASCUS predominated in HSIL (61%) and LSIL (86%), while almost all AGC/AIS had AGCUS (88%). HR HPV 16/18 infection rates were higher in AGC/AIS (100%), HSIL (80%) and LSIL (83%) relative to controls (10%) (P<0.0001), and was associated with higher risk of having AGC/AIS ; HSIL, ; and LSIL .altogether, pre-cervical cancer in Kenyan women is characterized by koilocytosis and ASCUS probably from the high rates of HPV 16/18 infections. Promoting cancer education and screening for high risk HPV infections and pre-cancerous lesions will improve women's reproductive health. Cervical cancer disease causes reproductive ill health and is among leading global women cancers accounting for at least 68% annual women mortality in the developing world , 2. The Treponema pallidum and HIV1/2 antibodies [Neisseria gonorrhea, Chlamydia trachomatis and HPV antigen detection [®programme mounted on computer CPU (specific for Venus camera imagery) launched on a Leitz compound microscope. Various photomicrograph images were captured in different pre cancer grades.We assessed the association of HR 16/18 HPV and socio-demographic characteristics and practices with cervical cytomorphologic presentations. This was a descriptive study among women patients attending the reproductive health clinic of Nakuru County referral hospital during the period of January 2014 to December 2014. Ethical approval for this study was obtained from the Kenyatta University Ethics Review Committee (KUERC-KU/R/COMM/51/228) and Nakuru County Referral Hospital Research and Ethics Committee (RII/VOL.I/08) before commencement of research. A total of 142 study subjects were consecutively recruited into the study during reproductive health clinic visits. A structured closed and open ended questionnaire was administered. Questionnaire administration was conducted through a semi interview process as a number of subjects were illiterate and could not interpret the questions on the list. Cervical screening by visual cervical inspection (VIA/VILI test) was conducted. Endocervical scrape smear examination for cyto-morphological profiling was conducted using the revised 2014 Bethesda classification  into foutibodies , 10. Endetection , 12, as etection . All PapData management and analysis: data capture was done using Microsoft office Excel software [software . After csoftware . DescripEthics approval and consent to participate: ethical approval for this study was obtained from the Kenyatta University Ethics Review Committee (KUERC-KU/R/COMM/51/228) and Nakuru County Referral Hospital Research and Ethics Committee (RII/VOL.I/08). Written informed consent was obtained from all the study participants prior to administering the questionnaire and collection of blood specimens and endocervical swabs and scrapes specimens.2= 93.952, df, 24; squamous cells; χ2 = 193.573, df, 18 and glandular cells; χ2 = 139.0, df, 3, . Analysis of smear inclusions revealed higher bacteria, fungi and inflammatory cells presence in AGC/AIC study group (<62.5%) relative to the control, LSIL and HSIL study groups collectively (<58%). One parasite  was detected only in the control group. Corresponding with the presence of microbes, inflammatory cells  were present in all the study groups, aThis study group were merged subjects presenting with both pre cervical cancer grade AGC and AGC/AIS since only one participant was found to have pre cancer of AIS. bStatistical comparisons for categorical variables were performed using the chi-square test for proportions. χ2, chi-square statistic; df, degrees of freedom) indicated that the prevalence of HIV1/2 was higher in women presenting with HSIL grade (25.5%) relative to LSIL and control, 11.4% and 12.5%, respectively .The prevalence of cervical atypia from Pap smear test protocol  which de2= 62.681; df, 3; P<0.0001). Subsequent regression analysis to determine whether HIV1/2 and HPV16/18 independently predicted pre cancer grades, . bHR HPV16/18 . Pre cervical cancer grade outcomes of LSIL, HSIL and AGC/AIS were entered as the dependent variable, screening positive for HIV1/2 and HPV 16/18 as the predictor group while screening negative for the detection of both these organisms being entered as the reference. The confounding effect of age, birth control method choice, cervical cancer disease awareness, marital status and education level were controlled for in the regression model. Data are presented as odds ratios (OR) and 95% confidence interval (CI). β coefficient indicates the degree of association differences for model) illustrated that only HPV16/18 was associated with increased risk of having pre cancer grade of AGC/AIS ; HSIL  and LSIL . Surface marker antigen positivity for N. gonorrhea was only detected in HSIL study group (5.1%) whereas antigen positivity for C. trachomatiswas obtained in both HSIL (6.8%); LSIL (2.9%) study groups, Consistent with previous studies showing higher rates of HR HPV16/18 in cervical cancer patients, , 16-18 aaThis study group were merged subjects presenting with both pre cervical cancer grade AGC and AGC/AIS since only one participant was found to have pre cancer of AIS. bAnalysis of continuous variable of age medians was analyzed using Kruskal-Wallis test. cStatistical comparisons for categorical variables were performed using the chi-square test for proportions. χ2, chi-square statistic; df, degrees of freedom) revealed that age medians differed significantly across the study groups (P<0.0001). In between group comparisons showed that women presenting with high grade lesions of both glandular and squamous type were older  compared to LSIL and controls  with 73% in HSIL and 63% in AGC/AIS study groups, being women without education or with primary school education levels as compared to LSIL (48.6%) and controls (52.5%). Although marital status did not reveal statistical difference , most of the study participants were married . There were no statistical differences in the types of occupations undertaken across the study groups . However, the occupations varied among the study participants, with a majority of the women engaging in informal sector occupations such as farmhands, quarry miners, pastoralists, peasant farmers, or in small businesses . There was significant statistical difference obtained on the perception of cervical cancer cause across all the study groups  with varied responses obtained. At least 11% attributed its cause to infections while ~50% responded that they did not know the cause, Evaluation of socio-demographic characteristics and practices, HR HPV exposure may not be sufficient enough to cause abnormal transformation of cervical epithelia. Other factors in addition to HR HPV exposure such as having advanced age in women, low immunity, poor chronic ill-health, HIV/AIDS infection, lifestyle and nutritional factors for example organic diet inadequacies, including multiplicity of sexual partners , 19-25 aMoreover, the findings of higher prevalence of abnormal epithelial cells having high grade lesions (HSIL) is attributable to increasing HPV-driven cellular transformation of koilocytic cells to cells of severer levels of abnormality. These assertions are consistent with previous studies showing that persistence of HR HPV especially high risk HPV like serotypes 16 and 18 infection promotes transformation of koilocytic cells to a degree of higher cellular abnormality , 36. AmoHigh median age which was significantly associated with the pre cervical cancer grades (P<0.0001) was encountered, In this study, koilocytosis, ASCUS and HR HPV16/18 infection are predominant features of pre-cancer grades in pre- and post-menopausal women with low education. Eminent manifestation of koilocytosis and cervical cell atypia in all study subject smears signified that productive and ongoing high risk HPV infection was present. HR HPV persistence is paramount to the transformation of cervical epithelia. Unresolved viral infection arises from protracted immunosuppression influenced by indirect aspects ranging from low education including ignorance, illiteracy and poverty, leading to diminished opportunities to health-seek due to the lack of funds for attainment of sustained good nutrition to build a strong protective immunity. Therefore, the government should intensify the roll out of HR HPV screening in the public hospitals as well as enmass vaccination of young women to avert the trigger of HR 16/18 HPV related pre cervical cancer genesis. Success of this program will be an improvement and upscale the cervical cancer prevention policy.Limitations: liquid based cytology (LBC) techniques over the utility of conventional cytology may have reduced microscopic diagnostic overlaps and pitfalls from mimicry with non-diagnostic finding. DNA methods to distinguishing HPV 16 and 18 genotypes as well as Herpes simplex virus (HSV) and Cytomegalo virus (CMV) strains are recommended for incorporation in the STI screen panel.Cervical cytomorphologic atypia detection is conducted from Pap smear preparation examinations;The Bethesda system is the system used in grading various cellular atypical forms for cervical cancer diagnosis;Koilocytosis signifies productive HPV infection.Cytomorphologic feature examination in Pap smears revealed raised koilocytic atypia amongst the study participants;Elevated koilocytic atypia detected in examined participant smears signified productive HR HPV infection was present which corresponded with high frequencies of screened HR HPV 16/18 infection amongst the study participants;High frequency of participants with low education implies that ignorance, illiteracy and lack of knowledge may lead to diminished opportunities to health-seek early. This may possibly lead to progression of cervical ill health as a result of non-detection of atypical cellular forms and non-screening of persistent HR HPV infections.The authors declare no competing interests.


Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90–99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy. The prevalent human papillomaviruses (HPVs) infect mucosal or cutaneous epithelia. The mucosotropic HPVs can be sexually transmitted and are of major medical importance. The low-risk (LR) HPV-6 and -11 cause 90% of benign anogenital warts, whereas the high-risk (HR) HPVs such as types-16, -18 and closely related genotypes have oncogenic potential . Early sThe amplification of the circular double-stranded HPV DNA genome of 7.9 kb and the assembly of the progeny virus requires a differentiating squamous epithelium . Viral DIn normal mammalian cells, the DNA damage response (DDR) pathway is required to maintain replication fork stability and inhibit firing of dormant replication origins during S-phase ,15,16,17We showed that in PHK raft cultures productively infected with HPV-18, DDR target proteins Myt1 and Wee1 were phosphorylated and activated. In turn, Cdc25C was inactivated by phosphorylation on S216, unable to activate the cdc2/cyclin B1 complex, leading to their accumulation in the cytoplasm and a prolonged G2 phase. It is during this protracted G2 phase, the great majority of viral DNA amplification takes place ,21. InteHPV-31 genome amplification is also dependent on viral E7-dependent activation of ATM DNA damage response pathway in high calcium-promoted differentiation of submerged cultures of CIN612 cells derived from cervical dysplasia, or organotypic raft cultures of stably transfected and possibly immortalized human foreskin keratinocytes . The autIn this study, we examined effects of inhibitors of the ATM/Chk2 and ATR/Chk1 pathways on productive HPV-18 DNA amplification in PHK raft cultures. In general, each inhibitor reduced cells in S-phase and cells in protracted G2 phases. Consequently, these compounds reduced viral DNA amplification. Among them, the Chk1 inhibitor MK-8776 was most effective and virtually abrogated HPV-18 DNA amplification because it also consistently induced DNA damage and apoptosis. We showed that the expression of E7 alone in PHK raft cultures conferred the sensitivity to the Chk1 inhibitor. Since cervical cancer cell lines continuously express HR HPV E6 and E7 oncogenes, we also examined the effects of MK-8776 on SiHa and CaSki cell lines. Our results showed that this Chk1 inhibitor induced extensive cell death when the cells were grown as submerged cultures or as raft cultures. Importantly, the combination of MK-8776 and cisplatin, a conventional therapeutic agent to treat advanced cervical cancer, is lethal to the cancer cell lines.The ATR-Chk1 pathway is a key regulator of replication checkpoint and DNA damage repair . In receIndirect Immunofluorescence (IF) assay showed that days 6–12 exposure of MK-8776 reduced S-phase cells as revealed by BrdU incorporation in the suprabasal strata of infected cultures C. SimilaWe then analyzed HPV-18 DNA amplification. In cultures treated with vehicle, Fluorescent In-Situ Hybridization (FISH) revealed that high signals were observed predominantly in the upper differentiated strata while low signals were detected in lower spinous layers E. Six daNext, we analyzed the expression and levels of viral and host proteins in raft cultures to explore the underlying mechanism of the inhibitory effects of MK-8776 on virus DNA amplification. Steady state levels of HPV E6, E7, their primary target proteins, p53, pRB and p130 and virus induced DDR host proteins from the raft culture lysates were analyzed by immunoblot analyses. Upon exposure from days 7–12, 20 µM MK-8776 neither reduced steady state levels of HPV-18 E6 and E7 proteins, nor increased their target host proteins, A,B. ThusSince the DDR/Chk1 pathway regulates replication fork fidelity, Chk1 inhibition would induce accumulation of DNA damage leading to replication stress and abortive DNA replication. Phosphorylation of H2AX (γ-H2AX) is a signature of DNA damage. Elevated γ-H2AX protein was indeed detected by immunoblot in HPV-18 raft cultures following the exposure to 20 µM MK-8776 from days 7 to 12 C. In conIn mammalian cells, γ-H2AX is induced by ATM as a result of double-stranded DNA break (DSB) , by actiThese results are at variance with the induction of γ-H2AX and cleaved caspase-7 in raft cultures of HPV-31 infected human foreskin keratinocytes (HFK) upon extensive passage . In our We also examined the effects of inhibitors of ATR (VE-821), ATM (KU-60019) and Chk2 (BML-277). Each was investigated in two independent experiments. In general, the inhibitors reduced S-phase cells in the suprabasal strata of HPV-18 infected raft cultures (data not shown) and suppressed viral DNA amplification A–C. HoweSince the toxic effects of the Chk1 inhibitor on the HPV-18 infected raft cultures is preferentially observed in the differentiated cells that reentered the S-phase, we surmised that the expression of E7 or E6 and E7 is responsible for sensitizing the cells. To test this hypothesis, raft cultures of PHKs infected with retroviruses expressing HPV-18 E6, E7 or the empty vector were prepared. Consistent with our previous observations , nuclei In combination with DNA damaging agent, Chk1 inhibitor, MK-8776 was tested in clinical trials against some solid tumors, lymphoma and myeloid leukemia ,32. MK-8We then evaluated the effect of MK-8776 in raft cultures of SiHa cells on days 7–13 and CaSki cells, another HPV-16 positive cervical cancer cell line, on days 6–12. The cultures were harvested on last day of treatment. With increasing concentrations, MK-8776 exposure induced extensive cytotoxic effects in both raft cultures A. In ourWe then examined CaSki cell killing by MK-8776 in combination with cisplatin B, a chemHPV E7 induces S-phase reentry in the post-mitotic differentiated epithelial strata. It also induces ATM and ATR, as well as other DDR proteins, causing prolonged G2 and enabling viral DNA amplification. Thus, DDR proteins play a direct role during HPV DNA amplification ,23,24,44Our observations in the current study highlight the importance of DDR proteins, particularly Chk1, in the context of HPV infection. Intriguingly, the ATR inhibitor VE-821, though inhibited Chk1 activation, it elevated phosphorylated ATM and Chk2 , possiblWe further show that cervical cancer cells caused by the HR HPV-18 and HPV-16 are more sensitive to the Chk1 inhibitor MK-8776 than productively infected PHK raft cultures. MK-8776 and cisplatin, the standard chemotherapeutic agent to treat metastatic cervical cancer, are synthetic lethal to these cells. The elevated sensitivity of cervical cancer cell lines may be attributed to high level of the viral oncoprotein expressions or some additional defects in DNA damage repair pathways in these cancer cells. The fact that cervical cancers are highly deficient in the two major tumor suppressor proteins, p53 and pRB family, that regulate cell cycle control, may make them more susceptible to Chk1 inhibitor than other solid tumors caused by dysregulation of other signaling pathways.In summary, our data suggest that ATR/Chk1 signaling is a critical component not only for the cellular DNA replication, it also has a major impact on viral DNA amplification and the viability of cervical cancer cell lines. Further studies will be needed to determine whether Chk1 inhibitor is a candidate agent in combination therapies to improve treatment of advanced HPV cancers.Primary human keratinocytes were isolated from neonatal foreskin discarded from elective circumcision surgery under UAB approved IRB protocol. Only early passages (P0 or P1) were used. A Cre-recombinase encoding plasmid, pCAGGS-nlsCre  and a reRaft cultures of PHKs transduced with retroviruses containing the HPV-18 URR-driven HPV-18 E6, HPV-18 E7 or the empty vector (pLC) were prepared as described ,50 and eMK-8776 (chk1 inhibitor) and BML-277 (chk2 inhibitor) were purchased from ApexBio . KU-60019 (ATM inhibitor) was sourced from LC-Laboratories , and VE-821 (ATR inhibitor) was bought from Sellekchem . Inhibitors were dissolved in DMSO per the manufacturer’s direction to prepare 1000× concentrated stocks. Inhibitor concentrations were selected based on published reports in various cancer cells lines (that do not contain HPVs) and on their low toxicity in submerged PHK cultures (Four µm sections of FFPE raft cultures of uninfected or productively infected with HPV-18 and of cervical cancer cell lines, Caski or SiHa cells were deparaffinized. Tissue histology was revealed by standard hematoxylin and eosin staining. Images were captured under Olympus BX-63 microscope fitted with DP-73 camera using cellSens software. Image processing to construct composite images was conducted with Adobe Photoshop and Illustrator.t-test was performed with Microsoft Excel to determine standard error and p values.Effect of ATM and Chk1 inhibitors on HPV-18 infected and uninfected PHK raft cultures were performed in three independent experiments. Raft cultures exposed to VE-821 or BML-277 were assessed in two independent experiments. Experiments conducted with cervical cancer cells presented in Frozen PHK and HPV-18 infected raft cultures were homogenized in mammalian cell lysis buffer and protein quantified as described . Fifty o