Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease that is the result of tobacco and/or alcohol abuse or infection with high-risk Human papillomaviruses. Despite the fact that HPV positive HNSCC cancers form a distinct clinical entity with better treatment outcome, all HNSCC are currently treated uniformly with the same treatment modality. At present, biologic basis of these different outcomes and their therapeutic influence are areas of intense investigation. In this review, we will summarize the molecular basis for this different outcome, novel treatment opportunities and possible biomarkers for HPV positive HNSCC. In particular, the focus will be on several molecular targeted strategies that can improve the chemoradiation response by influencing DNA repair mechanisms. Head and neck cancers comprise a group of cancers that are anatomically located in the oral cavity, the oropharynx, the nasal cavity, paranasal sinuses, the nasopharynx, the hypopharynx and the larynx. Most of these (90%) cancers have squamous cell carcinoma histology and are called head and neck squamous cell carcinoma (HNSCC) ,2.HNSCC is classified as the seventh most common cancer worldwide with around 600,000 new diagnosis each year . In the HNSCC develops mostly via one of the two primary carcinogenic routes, namely the chemical carcinogenesis through exposure to tobacco and alcohol abuse, which are known to be synergistic, and high-risk human papillomavirus (HPV) induced carcinogenesis ,2,5,6,7.Interestingly, epidemiological studies demonstrated a decrease or stabilization of laryngeal, hypopharyngeal and oral cavity cancers. This decrease is ascribed to the gradual decrease of the use of primary exogenous risk factors . In contrast, there is a clear increase in the incidence rates of oropharyngeal cancers mostly located at base of the tongue (BOT) and tonsillar region, which is ascribed to the increased incidence of HPV infections ,10,11.et al. shows that HPV infections in other less common sub-sites are also clinically relevant .,29.28,29Normal HPV infections are not associated with malignant transformation since the majority of HPV infections are spontaneously cleared and most patients show an effective immune response against subsequent HPV infections ,21,30. HE7 binds to cullin 2 ubiquitin ligase complex and results in the disruption and ubiquitination of pRB and other members of pRB pocket family ,29. DegrThe function of E6 complements the function of E7. E6 binds to E3 ubiquitin ligases and results in degradation of TP53, which leads to cell cycle deregulation, due to loss of p21 function (CDK inhibitor) and loss of TP53 mediated apoptosis ,29.Although the HPV oncogenes stimulate cancer formation by promoting limitless replication potential and genomic instability, cancer formation is stimulated by secondary genetic events.Before the knowledge of HPV infections and widespread use of (next generation) sequencing, HNSCC progression was also seen as an accumulation of stepwise (epi)-genetic alterations such as loss of chromosome 9p21 (CDKN2A loss), acquisition of TP53 mutations, 11q13 amplification (CCDN1), EGFR overexpression and PTEN inactivation .et al. being HNSCC cancer containing the transcriptionally active HPV that were classified as HPV+ tumors, HPV− tumors accompanied by high number of genetic changes (high chromosome instability (high CIN)), and HPV− tumors characterized by low CIN [Later on, HPV detection in the DNA of HNSCC patients resulted in a novel classification of head and neck cancers. It was clear that HNSCC are not only heterogeneous in means of their pathology and anatomical localization but also showed heterogeneity in regards to their biology. In 2012 three genetic subclasses were suggested by Leemans Recent progress in molecular technologies (the next generation sequencing and the omics era) gives definitely an in-depth picture of the molecular aberration in HNSCC. However, further classification of the heterogeneous group of HNSCC according to their predictive values (therapies) or even prognostic values is still in its infancy ,32.Nonetheless, it was shown that HPV+ tumors have less chromosomal copy number alterations compared to the HPV− HNSCC. The former is characterized by enrichment of 3q24-27 chromosomal amplifications, this region is coding for oncogene PIK3CA. The latter is characterized by gain of 11q13, a region encoding for cyclin D1 protein (encoded by CCDN1) . Moreoveet al. classified the HNSCC in 4 groups according to their expression profile: the classical, basal, mesenchymal and atypical group. The HPV+ group was classified in atypical group and was characterized by up-regulation of cell cycle and DNA replication genes [Also on expression level, several studies are performed and Chung on genes ,34. Of non genes .Initial mutational studies revealed that HNSCC have a relatively significant mutational overload with ranking 9th highest among tumors from 27 anatomical sites. These studies showed a 2 to 5 fold increase in mutation rates between HPV− and HPV+ HNSCC. However, the latter could not be verified by the recently published TCGA database, where the mutation rates between the two groups did not differ. It was noted that mutational profiles of HPV− HNSCC resembled the smoking-associated lung and esophageal SCC and was characterized with frequent transversions at CpG regions. The HPV+ HNSCCs meanwhile closely resembles the mutational profile of cervical cancers and showed higher mutation frequencies in PI3K pathway components and DNA repair genes ,32,35.HNSCC treatment is based on combination of three major treatment arms, namely surgery, chemotherapy and radiotherapy (RT) ,2. For mpost-hoc. Of note, no data are available about the prognostic value of HPV on solely chemotherapy for primary HNSCC, since this is not the standard treatment modality. However, HPV status did show a correlation with outcome in metastatic and recurrent HNSCC patients treated with chemotherapy [Interestingly, several retrospective and prospective trials have shown that HPV+ HNSCC patients have better overall survival (OS), disease free survival (DFS) and locoregional control (LRC) compared to the HPV− patients and this is independent of the treatment modality. In general the 5-year OS for HPV− HNSCC is around 50% while for HPV+ HNSCC patients values around 80% can be reached ,42,43,44otherapy ,46,47,48et al. demonstrated a better 3-year OS (83.4% vs. 57.1%) and a 58% reduction of risk of death  in HPV+ HNSCC patients treated with (chemo)radiotherapy. What makes this study interesting is that the risk of death increased with each additional pack year of tobacco smoking. The authors suggested a novel classification system of HNSCC patients on basis of four factors: HPV status, pack-years of tobacco smoking, tumor stage and nodal stage [One of the landmark studies conducted by Ang al stage . The infal stage . One of al stage .These data suggest that the outcome of HPV+ HNSCC patients is strongly determined by radiation biology. Possible mechanisms influencing this response are highlighted in the following paragraphs.As previously mentioned, HPV+ HNSCC patients are associated with better outcome after treatment with RT ,26,43,44Over the last couple of years several hypotheses have been put forward correlating RT response to micro-environmental (immune system and hypoxia) and tumor intrinsic factors ,50. It het al. claimed that HPV related head and neck cancer showed lower expression of hypoxia related genes and which they relate to the ability to adapt to hypoxia [One of the most studied environmental factors in relation to RT response is hypoxia, which is known to result in radiation resistance ,13. The  hypoxia . HoweverSeveral preclinical studies in HPV+ HNSCC indicated the importance of tumor intrinsic factors to the RT response. These studies can be classified in two categories according to the influenced pathways namely the influence of HPV on cell cycle and cell death pathways and the influence of HPV on DNA damage response (DDR) and DNA repair ,60.et al. They demonstrated that expression levels of residual wild-type TP53 protein is enhanced by IR and resulted in prolonged G2/M phase arrest and cell death [et al. demonstrated that indeed introduction of E6 expression in HPV− HNSCC resulted in increased RT response by cell cycle regulation and cell death, but in a TP53 independent manner [et al [One of the first studies that not only showed the increased radiation sensitivity of HPV+ HNSCC cells but also investigated the possible influence of HPV on cell cycle progression and apoptosis was performed by Kimple ll death . Interest manner . The accr [et al .et al. showed that the expression of HPV oncogenes in HPV− cells results in promoter methylation and decreased expression of serine/threonine-protein kinase-1 (SMG-1), which resulted in radiation sensitization. Moreover, SMG-1 seems to correlate with HPV status and improved survival in HNSCC patients [et al. ascribed the radiosensitivity of HPV+ cells to E7 oncogene induced delay in sub-lethal DNA damage repair [et al. showed that HPV+ tumors have not only impaired DNA damage response, but also showed that this response was related to the expression of p16INK4A. Namely, they demonstrated that p16INK4A expression resulted in defective homologous recombination repair (HRR) by impairing the recruitment of RAD51 to the site of DNA damage. This function of p16INK4A was independent from its cell cycle regulatory function [Gubanova patients . A seconpatients . Interese repair . In anotfunction .An important and currently lacking aspect of targeted or personalized medicine is the stratification of patients who will benefit from novel treatment options or treatment adaptions, as it is currently tested in the de-intensification trials for HPV+ HNSCC patients. In this regard, the establishment of molecular markers is of utmost importance. The marker should be reliable, well-validated and easy to perform and interpret. The establishment of such robust therapeutic biomarkers has several challenges such as the heterogeneity of the tumors, role of clinical characteristics and additive influence of conventional risk factors such as tobacco and alcohol exposure ,6,13.Currently, HPV positivity in HNSCC cancers is accepted to be a prognostic marker for outcome and is currently assessed in several institutions. However, assessment of HPV infections has ongoing challenges with influence of classic risk factor such as tobacco exposure on the favorable outcome of HPV+ patients and the limitations in the diagnostic testing methods ,13.While HPV specific testing seems logical to use, the implementation in practice is complicated. Currently HPV testing can be divided into two categories, namely detecting the presence of the virus (direct methods) and using p16INK4a expression as biomarker for viral infections (indirect method) . Viral DThis also highlights a major limitation of HPV-based DNA detection techniques namely these techniques detect the presence of HPV but not all HPV infections result in cancer formation. In other words, the clinically relevant HPV detection method is the one that is able to detect the transcriptionally active form of HPV ,68.in-situ hybridization (ISH), RT-PCR for E6 and E7 mRNA and next-generation sequencing technologies. All of these techniques have the advantage that they have a high specificity and acceptable sensitivity, while the implementation in practice and the high costs are major disadvantages [There are several direct methods that correlate with biologically active HPV infections, including vantages ,68.An alternative, which also detects transcriptionally active HPV, is the assessment of p16INK4a expression by IHC. P16INK4a expression correlates well with direct HPV detection methods as was shown in a pooled analysis comparing direct HPV detection methods with p16INK4a IHC with only in about 13% of cases discrepancies ,68. One However, one of the major disadvantages, that also prevent the general acceptance of p16INK4a IHC as gold standard, is the low specificity. The low specificity is systematically seen in several studies as 10%–20% of p16INK4a+ tumors are HPV−. Although several papers have shown the prognostic significance of p16INK4a even in the absence of HPV positivity there are contradictory studies showing significant poor survival rates for HPV−/p16INK4a+ HNSCC patients compared to HPV+/p16INK4a+ HNSCC patients ,73,74,75The current debate around the specificity and correlation with outcome is the reason why several groups are cautious in using p16INK4a IHC as a standalone marker and are suggesting p16INK4a IHC as an initial screen for direct HPV detection methods. Of note, the cause of p16INK4a expression in HPV− cases is still unclear but likely to be due to mutations in p16INK4a/RB pathway ,16,68,72The current treatment options are still suboptimal for both groups of HNSCC patients due to high resistance and recurrence (HPV−) and high toxicity (HPV− and HPV+) issues ,60,76,77Since HPV+ HNSCC patients have better therapy response rates, clinical trials assessing the possibility to de-intensify the current standard treatment options are ongoing ,13,20. THowever, caution must be taken with this kind of trials since the significantly better response seen in HPV+ HNSCC patients can be a consequence of the received aggressive treatment. On top, development of de-escalation strategies can be detrimental in approximately 10% of HPV+ HNSCC patients with high risk of developing distant metastasis ,80.Although several laboratory studies show that targeting of aberrant oncogenic/mitogenic signal transduction pathways can result in radiation sensitization of tumors, translation of this combination treatment strategy to clinical trial settings is rare. Furthermore, it is noteworthy that multiple recent attempts to use molecular targeted agents for treatment of cancer patients have failed due to suboptimal dosing and scheduling as well as the lack of biomarkers that predict response to these targeted therapies ,60.Until recently, EGFR amplification or overexpression was seen as one of the most important aberrations in HNSCC patients leading to the development of EGFR inhibitors including monoclonal antibodies (mAb) as well as tyrosine kinase inhibitors (TKI) ,6,20,32.Cetuximab was one of the first developed and the only Food and Drug Agency (FDA) and European Medicines Agency (EMA) approved targeted agent for HNSCC patients in combination with radiotherapy in locally advanced disease or in combination with platinum-based chemotherapy in recurrent or metastatic disease ,4,81. Hovs. 6% HPV+), which suggest that these inhibitors are less likely to work in HPV+ HNSCC patients [Additionally, genetic studies show that alterations in the EGFR pathway are rare (21%) and predominant in the HPV− population  with similar concepts make use of cetuximab in combination with RT as a treatment arm and cisplatin in combination with RT as the standard arm . The ratDespite these discrepancies, there are still several EGFR inhibitors, mAb as well as TKI, in clinical trials. To increase the success rate of these kinds of therapies, the inhibition should not only be targeted but also biomarker driven ,37,38,60Aberration and subsequent activation of PI3K pathway is one the most frequent events seen in HNSCC patients (34% in HPV− and 56% in HPV+) . ActivatTherefore, a tremendous interest has been shown for the inhibition of this pathway by mTOR/PIK3CA inhibitors. Although, the activity of PI3K inhibitors as single agents in lung squamous cell cancer patients with PTEN/PIK3CA mutations are disappointing. A preclinical study performed in HNSCC mouse model showed selective efficiency of PI3K inhibition in PIK3CA mutated samples ,87,88. MIn conclusion, although limited targetable oncogenic alterations show potential for the development of novel strategies for HPV+ HNSCC patients . DDR on one hand initiates cell cycle arrest by checkpoint activation, giving the cell the opportunity to repair damaged DNA and on the other hand activates the DNA repair mechanisms. Especially double-strand breaks (DSB) have a high lethality when left unrepaired ,89,90,91To understand the importance and possibilities of DNA repair, one should know the major DNA repair mechanisms in mammalian cells. The major repair mechanisms can be divided into five pathways. Most of the direct DSB are repaired by non-homologous end-joining (NHEJ), whereas replication associated DSB are repaired by HRR. Thus, the choice of repair is cell cycle dependent with NHEJ preferred in G0/G1 phase of cell cycle and HRR taking place during S/G2/M cell-cycle phases ,90,91.Characteristic for NHEJ is the lack of use of homologous sequences as repair templates, leading to an error-prone repair. The NHEJ is divided into two pathways being the classical pathway (c-NHEJ) and alternative pathway   and the nucleases. The nucleases are necessary for free end-processing and will lead to more efficient ligation. Subsequent phosphorylation and activation of DNA-PKcs will result in the dissociation of the complexes from DNA-ends. This in turn will enhance the access of the Ligase IV/XRCC4/XLF protein complex, which completes the ligation reaction ,90,91.Defects in classic NHEJ proteins channels DSB toward the alternative NHEJ  pathway. This pathway requires micro-homology and is regulated by PARP1, which will bind to the free DNA-ends instead of the Ku complexes. PARP1 binding will stimulate single-strand end-resection by the MRE11/RAD50/NBS1 (MRN) and CtIP protein complexes. Hereafter ligation will take place as is described for the classical NHEJ pathway. Since this pathway involves limited end-processing and micro-homology, it will result in even more inaccurate repair ,90,91.In contrast, HRR uses homologous DNA sequences as a template for reparation of DNA damage (see Both of the DNA repair processes start with single-strand DNA end-resection that is divided into two phases starting with limited end-resection initiated by MRN/CtIP complexes and is followed by extensive end-resection by helicases (BLM) and nucleases (EXO1 and DNA2) ,90,91.The accurate or the classical HRR pathway involves the binding of Replication protein A (RPA) to single-stranded DNA (ssDNA) that will result in stabilization of ssDNA which is necessary for binding and invasion of the homologous template strand, a process mediated by RAD51 nucleoprotein filaments. The invading strand is extended by newly synthesized DNA, which can subsequently anneal with the other resected-end. Finally, additional synthesis and ligation will result in high-fidelity repair. The inaccurate HHR or the single strand annealing (SSA) is characterized by the exposure of long complementary ssDNA repeats, which flank the DSB. Annealing of DNA is mediated by RAD52 leading to the deletion of one of the repeats and the DNA sequences between the repeats or to translocations when 2 DSB occur in different chromosomes ,90,91.The three other pathways operate on repair of single strand breaks (SSB) after induction with DNA damaging agents. These are termed base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) and they use the complementary strand as a repair template ,90,91.It is becoming clear that these pathways do not act as separate entities and that there is a functional overlap between the pathways. This not only shows the complexity of DNA repair processes but also provides the opportunities to target these pathways to improve the radiotherapeutic index ,89,91.Since RT results in DNA damage inhibition of DNA repair pathways can be exploited for radiation sensitization strategies. As mentioned before, deficiencies in DSB repair pathways are thought to be the most lethal lesions induced by IR. However, it has become apparent that acquired secondary DBS through deficient SSB repair is also important for the survival of cells after IR ,89,91.This knowledge led to the development of a range of novel compounds that influence DNA repair. For example inhibitors of important molecules in DSB repair, such as DNA-PKcs and PARP inhibitors have been shown to sensitize cancer cells to RT. Both strategies block DNA repair, thereby increasing damage in the treated cells and resulting in an increased cell death. Noteworthy is that such approaches do not necessarily provide selective eradication of cancer cells as they also influence normal cells ,89,91.Although cancer cells need repair mechanisms to survive they are also often defects in one or more aspects of DNA repair, which lead to an addiction and reliance to the other/back-up DNA repair pathways. This overreliance or addiction of cancer cells for specific DNA repair pathways can be therapeutically exploited by inhibiting the back-up DNA repair pathways and is called synthetic lethality. The best example of a synthetic lethality approach is the use of Poly (ADP ribose) polymerase (PARP1) in BRCA1/2 mutated breast and ovarian cancers. The inhibition of PARP1 in these cancers resulted in the accumulation of single strand breaks (SSB) leading to DSB and eventually cell death upon cellular replication ,89,91,92In line with this, cancer cells with aberrations in their repair mechanisms are expected to shift their repair to less common used back-up DNA repair pathways in response to IR, this compared to normal cells where the common used DNA repair mechanism is still intact. Inhibition of the back-up DNA repair pathway used by the tumor cells can result in a relative tumor selective radiation sensitization ,91.Another advantage of combining synthetic lethal drugs with DNA damaging agents, like RT instead of using as a single therapy modality, is the possibility to avoid resistance. Since synthetic lethal drugs inhibit DNA repair components, it will also result in an increased mutation rate of the repair pathways and as a consequence the cancer cell can activate the repressed DNA repair pathways by additional mutations as it is noted for BRCA2 and PARP inhibition ,91,93.It is clear that HPV related HNSCC form a distinct entity and that current treatment options are not answering the need of these patients. De-intensification of current therapeutic schemes with molecular targeted agents in combination with standard treatment forms an interesting strategy to increase the (radio)-therapeutic index. However, these strategies also highlight the importance and need for prognostic and predictive biomarkers for stratification of patients. Emerging data generated by high-throughput technologies will give us valuable information in this regard but it will also bring novel challenges regarding to interpretation of clinical relevance of these data and feasibility to clinical translation. This highlights the importance of molecular validation of biological data, but also indicates the importance of understanding and anticipating the possible interaction between different treatments.


The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK) family. p38MAPK signaling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer. MAPK14), p38β (MAPK 11), p38γ (MAPK 12), and p38δ (MAPK 13)  is one of the main signal transduction mechanisms by which the cell adapts to changes in the environment. There are four p38MAPK isoforms in mammalian cells encoded by different genes: p38α , which play important roles in the innate immune response, in inflammation and inflammation-related diseases such as cancer or arthritis.One of the hallmarks used for the division of p38MAPK into two subgroups is the protein sequence similarity. p38γ and p38δ sequences are highly similar to each other (~70% identity), whereas p38α and p38β display higher similarity (75% identity). In contrast, p38γ and p38δ are more divergent in sequence to p38α  , which binds to PDZ domains. p38γ associates with PDZ-domain containing proteins, such as α1-syntrophin, SAP (synapse-associated protein) 90/PSD (post-synapse density) 95, hDlg  and the protein tyrosine phosphatase PTPH1 and under stress conditions it is able to phosphorylate them and modulate their activity  showed substantially lower levels of TPL2 protein, and therefore lower MKK1-ERK1/2 activation and inflammatory cytokine production  mouse model. Combined p38γ and p38δ deficiency markedly reduced arthritis severity by suppressing clinical disease and bone destruction, compared with that in wild type mice anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model (Schindler et al., During the last few years the role of p38γ and p38δ in cancer associated with chronic inflammation has been studied. Chronic inflammation is linked with an increase in malignant disease. Almost 20% of human cancers are related to chronic inflammation caused by infections, exposure to irritants or autoimmune diseases (Hanahan and Weinberg, in vivo has been achieved. It is now clear that p38γ and p38δ are crucial in innate response, inflammation, and inflammatory diseases. Therefore, they deserve to be studied in greater depth as they represent pharmacological target for the development of drugs that might be useful for the treatment of inflammatory pathologies. In fact, development of more specific p38δ inhibitors has been shown to reduce mucus production in human airway epithelial cells (Alevy et al., The role of p38γ and p38δ has been some times ignored since most of the studies to date have focused on p38α, which is the most abundant p38MAPK isoform. Nonetheless, in the last years significant progress in understanding the functions of p38γ and p38δ AC wrote the manuscript. AE, AR, DA made substantial contributions to conception and design, and acquisition of information. All authors contributor to the revision of the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease  is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively. The cancer stem cell (CSC) hypothesis Worldwide, nearly all cervical carcinomas are caused by known high risk (oncogenic) types of Human Papilloma Virus (HR-HPVs) with the majority attributed to persistent infection by HPV-16 or HPV-18 Where implemented, cervical cancer screening programmes have reduced the incidence of invasive disease by recognising and treating pre-invasive lesions. Nevertheless there is still room for improvement, particularly in the selection of cases which require treatment. Although most screening programmes involve cytological examination of exfoliated cervical epithelial cells, molecular HPV testing is being widely introduced to enhance sensitivity. A paradigm shift is imminent whereby the primary screening modality for the future is likely to be HPV testing. However, one significant issue even with clinically validated HPV tests is their inability to determine which infections will result in significant disease. Further, while the clinical sensitivity of HPV testing consistently exceeds that of cytology, the specificity is less optimal. Thus there is a need to develop adjunct triage tests for biomarkers that are more specific for detection of HPV related high grade disease. To this end, we investigated whether there was an association of CSC markers and/or cell proliferation that would differentiate HR-HPV+ve high grade pre-neoplastic cervical disease from insignificant lesions in cervical samples routinely taken for liquid based cytology (LBC) and for which biopsy data was available. We further investigated expression of stem cell related proteins in biopsies of HPV+ve squamous carcinoma of the cervix.Ethical approval was obtained from Scotland A Research Ethics Committee (REF 12/SS/0034). All cervical samples were collected into ThinPrep-preservcyt liquid based cytology transport medium . For this study, 95 anonymised, curated, cervical smear samples were obtained from the Scottish National HPV archive, which holds Generic Scotland A Research Ethics Committee approval for Research Tissue banks (REC Ref 11/AL/0174) for provision of samples for HPV related research after approval from an Independent Scottish HPV Archive Steering Committee. For this study our HPV Archive Application was Reference 0004. The cervical samples used in this study were obtained from different collections within the Scottish HPV Archive. Cytology normal samples were from residual samples from National HPV Surveillance Programme (Opt-out consent through the screening programme approved by Tayside Research Ethics Committee Ref 11/AL/0174) and the abnormals from two previously collected research projects . Additionally, sections from 10 anonymised, formalin fixed cervical biopsies from women with SCCC and 10 normal cervical biopsies from women who had undergone hysterectomy for benign reasons were obtained from the Royal Infirmary of Edinburgh Pathology Department Archive, via the South East Scotland (Lothian) SAHSC Bioresource. The NHS Lothian SAHSC Bioresource holds RTB approval from the East of Scotland REC, (reference number 13/ES/0126) and provides samples with approval of the Tissue Governance Committee.For most samples with abnormalities, information on both cytology and histology was available . DNA was extracted from 10µm sections cut from the biopsies using QIAamp DNA Mini Kit  following the manufacturer's instructions. As we have previously published Since the collection medium ThinPrep preservcyt contains 50% methanol we used a methanol/chloroform based protein extraction method. Briefly, 100µl ice cold chloroform was added to 900µl of sample in an Eppendorf tube on ice, vortexed, incubated on ice for 5min and spun for 5min at 10,000rpm at 4°C in a benchtop microfuge. The aqueous top layer was discarded, 300µl methanol was added and the tube vortexed, and spun for 10 min at 10,000rpm at 4°C in a benchtop microfuge. The supernatant was removed and the pellet resuspended in 100µl Tris Buffered Saline. Protein concentration was determined by Pierce BCA assay . Antibody-based, pluripotent stem cell proteomic arrays  were carried out as per manufacturer's instructions. To identify proteins of interest, for the arrays, pools of samples were compared. Each pool contained 10µg of protein from each individual sample see . DevelopA) Staining protocol: -1 ml aliquots from LBC samples in Preservcyt were passed through a BD Falcon filter-top 5 ml FACS tube (BD Biosciences), to produce a filtered cell suspension. This was then centrifuged at 300×g in a bench top microfuge, the supernatant removed, and the pellet re-suspended in 400µl MACSQuant running buffer  (“buffer”). 200µl of cell suspension was stained with either isotype control (PE—IgM), or anti-TRA-1-60-PE (both Miltenyi Biotec). Additionally, 5µl of the DNA chelating fluorescent dye, DRAQ5  at 1∶20 dilution was added to all samples. Samples were incubated for 15 minutes at room temperature in the dark and then directly analysed (with no further washing) using a MACSQuant Vyb or MACSQuant 10 flow cytometer (Miltenyi Biotec). The entire 200µl of sample was acquired per tube. Samples were analysed using MACSQuantify software v2.4. Area and height were collected for all parameters, antibody stained samples were acquired on a hyper log  scale, and DRAQ5 was acquired on a linear scale.B) Analysis protocol: - RNA was extracted from 1.5 ml aliquots of LBC samples using miRNeasy mini kit  following the manufacturer's instructions. RNA was quantified on a Nanodrop spectrophotometer and stored at −80°C until use. cDNA was made from 500 ng RNA using the Quantitect Reverse Transcription Kit  as per manufacturer's instructions and stored at −20°C until use. Duplex qPCR was carried out in Lightcycler Nano (Roche) realtime PCR system in duplicate in 8 well PCR strips using plates, primers, probes and reagents from Life Technologies Applied Biosystems, UK. Wells contained 2µl cDNA, 1µl gene of interest primer mix, 0.12µl each of 18S forward and reverse primers, 1.6µl 18S probe, and 13µl mastermix. The PCR programme was 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C.Sections (4µm) from formalin fixed, paraffin embedded cervical biopsies were dewaxed in xylene and rehydrated through alcohol to water. Antigen retrieval was performed in 0.9 M citrate buffer in a pressure cooker in an 800W microwave oven for 15 minutes at full power. After cooling, slides were blocked with avidin-biotin block followed by protein block  and stained with goat-anti-SOX2 antibody  or mouse monoclonal anti-TP63 antibody  overnight at 4°C. Sections were then washed, incubated with rabbit polyclonal biotinylated anti-goat or anti-mouse IgG , washed, incubated with streptavidin-HRP, washed and colour developed using DAB solution (Vector Labs). Sections stained with only secondary antibody (no primary control) were included in each run. Parallel sections from 11 cases were stained with SOX2 and TP63. Tumour cells were evaluated for their nuclear expression of the transcription factors. There was no significant difference between TP63 and SOX2 levels (Wilcoxon ranked pairs test). For each section stained, five fields were photographed at ×200 magnification. Using the ImageJ software “cell counter” plug-in, each picture was overlaid with a grid and nuclear +ve and –ve tumour cells counted. At least 150 cells per field (750 per section) were counted.Data were analysed with Graphpad Prism software. The Kruskall Wallis test with Dunn's multiple comparison post-test was used to compare normal and all disease grade groups. To compare two groups the Mann-Whitney test or Wilcoxon Ranked Pairs test was used as appropriate. Significance was assumed if p was <0.05.To determine whether cancer stem cell related proteins were detectable in LBC samples, we used a proteomic microarray for human stem cell proteins. The arrays were carried out on pooled HR-HPV –ve samples from 9 women with no disease and on pooled HR-HPV+ve samples from 9 women with CIN3. Arrays were developed as described in We next investigated whether flow cytometry would be a practical methodology for the detection of markers associated with cancer stem cells in cervical samples. Most of these proteins are intracellular and preliminary attempts to stain for NANOG and OCT4 revealed that background staining is a problem with intracellular proteins (not shown) in these samples. For this reason, we concentrated on detection of the cell surface carbohydrate epitope of podocalyxin detected by the antibody TRA-1-60. Cell cycle was detected by analysing staining with the DNA chelating dye DRAQ5. Cycling cells were determined to be in the S+G2M phase of the cell cycle as described in To determine whether or not the 3 samples with the highest number of TRA-1-60+ve cells represented a subset of the CIN3 cases with potentially higher cancer stem cell presence, an equal amount of protein from each of these samples was pooled and compared to an equivalent pool of proteins from 3 samples with HR-HPV+ve CIN3 that showed no increase in TRA-1-60 using the same proteomic array. Flow cytometry results for the samples selected are shown in To further validate the observation that enhanced SOX2 and/or TP63 protein in cervical samples, from women with high grade pre-neoplastic cervical disease, might relate to tumour progression, we stained a series of parallel sections from cervical biopsies from 11 women with HR-HPV+ve SCCC and 11 women with normal cervical tissue with anti-SOX2 and anti-TP63 antibodies. Up-regulation of hCG is reported in a number of cancers including SCCC SOX2 is a core transcriptional regulator of pluripotency in mouse and human embryonic stem cells Our data support the previous findings that increased expression of both SOX2 and TP63 are associated with dysregulated cell growth in the epithelial compartment of the cervix. Furthermore, our data suggest identifiable changes in their expression in cervical samples from women with high grade pre-neoplastic disease.We also showed by flow cytometry that some HR-HPV positive samples from patients with CIN3 have increased numbers of cells that are positive for the cell surface stem cell marker TRA-1-60. Also, these samples demonstrate further enhancement of HCG, TP63 and SOX2 relative to samples from patients with CIN3 that did not show enhanced TRA-1-60. This indicates that samples from patients with CIN3 contain detectable cancer stem cell markers. SOX2, HCG and TP63 mRNAs were also shown to be upregulated in cervical samples with HR-HPV+ve high grade dyskaryosis and SOX2 and TP63 immunohistochemistry identified cancerous cells, in HR-HPV+ve biopsies, from women with cervical squamous cell carcinoma. Flow cytometric analysis of numbers of cycling cells in cervical samples also differentiated normal from high grade disease, regardless of the HPV status of the normal samples, indicating that the increase was disease and not simply HPV-infection related. The increase in numbers of cycling cells was seen whether high grade disease was based on histological examination of a biopsy or on cytology only. This indicates that this testing modality might be applicable in a wider screening context as a laboratory triage test for significant disease. Although there are disturbances in cell cycle control associated with cervical cancer TRA-1-60 is a carbohydrate epitope of the cell surface sialylated keratan sulfate proteoglycan podocalyxin We chose to stain SOX2 protein in these biopsies as it has been reported to be upregulated in human cervical cancer stem cells Our results indicate that detection of cancer stem cell associated proteins by flow cytometry or western blotting and/or flow cytometric detection of numbers of cycling cells may be useful laboratory tests for triage of HR-HPV+ve cervical samples for significant cervical disease. They further indicate that SOX2 and TP63 immunohistochemistry may have value in diagnosis of HR-HPV related SCCC. It is not possible to follow up any of the patients in this study but it would be of obvious interest in the future to determine the longterm outcome of patients with pre-neoplastic lesions whose cervical samples show increased CSC marker expression and/or numbers of proliferative cells.


Reported human papillomavirus (HPV) vaccination coverage in England is high, particularly in girls offered routine immunisation at age 12 years. Serological surveillance can be used to validate reported coverage and explore variations within it and changes in serological markers over time.Residual serum specimens collected from females aged 15–19 years in 2010–2011 were tested for anti-HPV16 and HPV18 IgG by ELISA. Based on these results, females were classified as follows: seronegative, probable natural infection, probable vaccine-induced seropositivity, or possible natural infection/possible vaccine-induced seropositivity. The proportion of females with vaccine-induced seropositivity was compared to the reported vaccination coverage.Of 2146 specimens tested, 1380 (64%) were seropositive for both types HPV16 and HPV18 and 159 (7.4%) positive for only one HPV type. The IgG concentrations were far higher for those positive for both HPV types than those positive for only one HPV type. 1320 (62%) females were considered to have probable vaccine-induced seropositivity. Among vaccine-induced seropositives, antibody concentrations declined with increasing age at vaccination and increasing time since vaccination.The proportion of females with vaccine-induced seropositivity was closest to the reported 3-dose coverage in those offered the vaccination at younger ages, with a greater discrepancy in the older females. This suggests either some under-reporting of immunisations of older females and/or that partial vaccination (i.e. one- or two-doses) has provided high antibody responses in 13–17 year olds. A national HPV immunisation programme was introduced throughout the UK in September 2008 with routine vaccination offered to all girls aged 12–13 years and a catch-up programme in the first two years offering the vaccination to all girls up to the age of 18 years. From 2008 to 2011, the bivalent vaccine was offered with a change in September 2012 to the quadrivalent vaccine. HPV vaccination is offered free of charge to all girls. The immunisation programme is primarily delivered in schools but also in General Practitioners (GPs) and other health care services, particularly for the older catch-up cohorts. Reported vaccination coverage has been high with over 80% of girls in the routine cohorts completing the three dose schedule . ReporteIn females, following a natural infection with HPV, a detectable antibody response is only detected around 50–70% of the time  and thisAccurate, validated knowledge of HPV vaccination coverage is important to assess the likely direct impact of the HPV immunisation programme as well as the potential indirect effect of herd protection among the unvaccinated. Monitoring of serological markers can also enable vigilance for potential lower levels of direct protection from the immunisation programme within certain sub-groups, and for changes in immunogenicity over time, i.e. antibody waning, which may presage reductions in protection.We have used distributions of anti-HPV 16 and HPV 18 IgG concentrations to classify sera from a sample of young females in England as probable vaccine-induced seropositive or probable natural infection. We compare the resulting estimates of coverage derived from anti-HPV IgG concentrations to reported vaccination coverage, and explore associations between antibody levels and age at vaccination and time since vaccination.National Research Ethics Service (NRES) approval for the sero-epidemiological surveillance of the National Immunisation programme of England and Wales (Research Ethics Committee number 05/Q0505/45) was granted by the Joint University College London/University College London Hospital (UCL/UCLH) Committees on the Ethics of Human Research.Patient consent was not required as this study made use of anonymised specimens (with no patient identifiable data) which were collected and tested as part of Public Health Surveillance conducted to monitor the HPV vaccination programme.Serum specimens from females aged 15–19 years were obtained from the PHE Seroepidemiology Unit (SEU). The SEU routinely collects residual serum specimens after diagnostic microbiological tests for seroepidemiological studies of infections of public health importance for which vaccines are available or under development. Contributing laboratories in England provide anonymised specimens with age at collection, sex, and date of collection. Sera from immunocompromised individuals and repeat sera from the same individuals are excluded . Where pSpecimens were tested at the PHE Vaccine Evaluation Unit (VEU), Manchester for IgG to HPV types 16 and 18 using a type-specific ELISA and all assay critical reagents, including Virus Like Particles (VLPs), transferred from GlaxoSmithKline . BrieflyAntibody concentrations are presented as geometric mean concentrations (GMCs) among seropositive specimens. Whilst average antibody levels following vaccination are far higher than those following natural infection, the ranges overlap. Using the range of concentrations for types 16 and 18 seropositives we classified each result as, (i) “high” seropositivity if the result was above the 95% range of concentrations among those with a single antibody ; (ii) “low” seropositivity as below the lower 95% range of concentrations among those seropositive for both HPV types ; (iii) “moderate” seropositivity as between these two values. Using this grading, we then classified “probable” vaccine-induced seropositivity as seropositive for both types with high concentration for at least one type or moderate concentrations for both types and “probable” natural infection as seropositive for one type only. Specimens with low seropositivity for both types or low seropositivity for one type and moderate for the other were classified as “possible” natural infection or vaccine-induced seropositivity . SerologData on reported coverage for each birth cohort included in our seroprevalence data were obtained from published tables . BrieflyFor comparison with serological coverage, vaccination coverage was estimated using the published coverage for the 10 geographical areas  of the laboratories submitting serum specimens, as well as using national level data. Published coverage, reported by academic year (September to August), was used to estimate coverage by age and calendar year. As estimates using Strategic Health Authority data and national data for reported vaccination coverage were similar, the estimates from national data were used.A valid result for both HPV 16 antibodies and HPV 18 antibodies was available for 2469 of the 2484 specimens (99.4%). 323 specimens with date of collection during January to March of the year following the due date of first vaccine dose were excluded: 2146 specimens were included in the analysis .The mean age of females providing a specimen was 17.9 years (SD 1.4 years). Around one-third of specimens were known to have originated from GUM clinics (ranging from 0% to 93% by contributing laboratory). The mean age was similar for specimens from GUM clinics and those from unspecified source clinics .Across all ages, 64% (1380) of specimens were seropositive for both HPV 16 and HPV 18. Seropositivity for HPV 16 only and for HPV 18 only was found in 5.5% (119) and 1.9% (40) of specimens, respectively. 28% (n = 607) were seronegative for both HPV types . The GMCOverall seropositivity for HPV16 and/or HPV18 was higher in specimens known from GUM clinics (76.3% vs. 69.0% for HPV 16 and/or 18) and specimens from younger ages .Within this sample of serum, using the methods described, probable vaccine-induced seropositivity (VIS) was defined as sera with antibody concentrations above 546 EU/mL for HPV 16 or above 334 EU/mL for HPV 18 , or above 87 EU/mL for HPV 16 and above 42 EU/mL for HPV 18 . The oveAmong the probably VIS, GMCs for HPV16 were higher than GMCs for HPV18 at all ages. For both HPV 16 and HPV 18, GMCs declined with increasing time since vaccination (up to 3-years data available). Specimens estimated to have been taken at equal times after vaccination tended to have higher GMCs if vaccinated at younger ages  and 1296 EU/mL (95%CI 632–2656) for types HPV16 and HPV18 respectively, whereas in 14–17 year olds these were lower at 1631 EU/mL (95%CI 1422–1871) and 669 EU/mL (95%CI 581–770) respectively) .The GMCs for those with probable natural infection (i.e. seropositive for only one HPV type) were 81.1 EU/mL (95%CI 66.6–98.7) for HPV 16 and 61.7 EU/mL (95%CI 44.2–86.0) for HPV 18. GMCs for those seropositive for both types but with possible natural infection were similar (72.9 EU/mL (60.1–88.5) and 45.1 EU/mL (37.8–53.8) for HPV 16 and 18, respectively). The proportion of females with probable or possible natural infection was similar in specimens known to be submitted from GUM clinics . The probable and possible natural infections increased with increasing age .Serological surveillance confirms high coverage of the HPV vaccination programme in young females in England, particularly in those offered the vaccine at school age. The higher proportion with vaccine-induced seropositivity compared to reported three-dose coverage, particularly evident in the older females (offered HPV vaccination at an older age), suggests that three-dose coverage in the catch-up cohorts could be higher than reported, or that two-dose coverage at these ages is associated with high antibody responses, or both.We used the results from serological testing to determine the vaccination status of females. Previous studies have shown only a relatively small proportion of females have natural seropositivity for both HPV types 16 and 18 . ConversThe average antibody concentrations declined with both increasing age at vaccination and increasing time since vaccination . This isQuantitative antibody concentrations from the study are not comparable to those from studies which use different assays/cut-offs, because currently no international HPV standard reference serum exists, and different laboratories therefore use ‘in-house’ standard sera making direct comparisons nonviable. One point for consideration is that the ELISA methodology we applied used an increased lower limit of quantification compared to previous reports (we use a lower limit of 19 EU/mL for HPV16 and 18 EU/mL for HPV18 whereas 8 EU/mL and 7 EU/mL respectively have been used previously) . Using tResidual serum specimens for this surveillance are taken from females attending for diagnostic and screening tests, hence may not be representative of the general population. The reason for the initial test where the serum sample was taken is not provided to SEU along with the sample. Where it was known that a sample originated from a GUM clinic, this was indicated. Analyses were performed separately for known GUM samples and other samples and results were very similar (data not shown). No data are collected on social deprivation, ethnicity or country of birth of females although since England has free access to health care this reduces the potential bias associated with health-seeking behaviour. One previous paper suggested that the comparable results between coverage data and other vaccine seroconversion rates provide some assurance of the representativeness of these specimens .Vaccination coverage may have been slightly under-reported for the older catch-up vaccination cohorts. In this group, vaccination was largely performed outside of schools and revised estimates of coverage to include data on immunisations given late in ‘mop-up’ sessions were not readily available in all areas hence the quality of these data was less certain (2). This would be consistent with the greater differences between reported coverage and vaccine-induced seropositivity observed in females offered vaccination at older ages. However, we also consider that the proportion reported to have received only one or two doses of the vaccine is far greater in older ages (just under 5% in those vaccination in the routine cohorts compared to around 15% in the older catch-up cohorts). The majority of females who had received two-doses would have received the first two doses of the vaccine (i.e. with just one or two months between doses). Limited data are available on antibody responses following such a schedule. Data from trials have shown 100% of females seroconvert after two-doses given six months apart with non-inferior antibody concentrations compared to females receiving three-doses . We wereFuture studies using these methods will explore serological coverage in certain demographic and behavioural subgroups to identify those relatively lacking in direct vaccine-protection and will monitor antibody levels over longer times since vaccination. This will be an important part of assessing longer term protection.S1 DataField names: gender; yearofcollection: Year sample taken; age_year: Age at sample date; result_16: HPV16 antibody concentration (EU/mL); result_18: HPV18 antibody concentration (EU/mL).(XLSX)Click here for additional data file.


Knowledge of the natural history of human papillomavirus (HPV), in particular the role of immunity, is crucial in estimating the (cost-) effectiveness of HPV vaccination and cervical cancer screening strategies, because naturally acquired immunity after clearing an infection may already protect part of the risk population against new HPV infections.We used STDSIM, an established stochastic microsimulation model, quantified to the Netherlands. We explored different assumptions regarding the natural history of HPV-16 and HPV-18, and estimated the transmission probabilities and durations of acquired immunity necessary to reproduce age-specific prevalence.A model without acquired immunity cannot reproduce the age-specific patterns of HPV. Also, it is necessary to assume a high degree of individual variation in the duration of infection and acquired immunity. According to the model estimates, on average 20% of women are immune for HPV-16 and 15% for HPV-18. After an HPV-16 infection, 50% are immune for less than 1 year, whereas 20% exceed 30 years. For HPV-18, up to 12% of the individuals are immune for less than 1 year, and about 50% over 30 years. Almost half of all women will never acquire HPV-16 or HPV-18.Acquired immunity likely plays a major role in HPV epidemiology, but its duration shows substantial variation. Combined with the lifetime risk, this explains to a large extent why many women will never develop cervical cancer. Infection with human papillomavirus (HPV) is a necessary cause for developing cervical cancer , the fouThe bivalent vaccine Cervarix offers protection against HPV-16 and HPV-18, which account for 70–76% of the cervical cancers . The vacet al. (2006), who estimated the median transmission probability at 40% per sex act, but ranging from a lower limit of 5% to an upper limit of 100% [Several mathematical models have been developed to study the spread of HPV and immunity processes responsible for clearing an HPV infection –17. Some of 100% . This wiWe explored the transmission dynamics of HPV-16 and HPV-18, especially regarding the process of acquired immunity, by reproducing the age patterns of type-specific HPV prevalence in the Netherlands ,20, usinSTDSIM simulates the life course of individuals in a dynamic network of heterosexual contacts, in which STIs, such as HPV, can spread. Each individual has its own characteristics that are either constant  or subject to change . Events are determined by probability distributions, and can lead to new events  or a cancellation of future events . More information on the model structure can be found in the S1A Supporting Information.We first used generally available information to quantify our model regarding demography and sexual behavior. Age- and sex-specific life expectancy and migration data  and age-We simulated the transmission of HPV-16 and HPV-18. We assumed equal transmission probabilities per sexual contact for male-to-female and female-to-male transmission ,15,17. TTwo important aspects distinguish the transmission of HPV from other STIs in our model. First, transmission can also occur through genital skin , and preWe further assumed that the durations of infection and acquired immunity follow a Weibull distribution. The Weibull distribution is a continuous probability distribution, defined by a shape and scale parameter. If the shape parameter is set to 1, the distribution reduces to the exponential distribution. The shape parameters were varied with pre-set values of 0.25, 0.50, 1, 2 and 4. This allowed us to explore whether large  or little (high shape parameter) individual variation in the average duration of infection and acquired immunity would be consistent with the available data.et al. [et al. [et al. [et al.[HPV in Men (HIM) Study of Giuliano et al. [The mean duration of an HPV infection in women was based on the studies of Goodman et al.  and Trot [et al. , which m [et al.  into mea. [et al. was theno et al. , which iIn the base case analysis, we assumed that everyone acquires full immunity for some (Weibull distributed) time immediately after clearing an infection. During this period, an individual cannot acquire a new HPV infection, irrespective of whether this is due to the development of antibodies or other components of the adapted immune system. We varied the immunity duration from no immunity to effectively lifelong, and the transmission probability from 0% to 100%, to find parameter values that made the model most accurately reproduce the observed HPV prevalence trends with age  for each HPV-type, and the 5 pre-set shape parameters of the Weibull distributions of the durations of infection and acquired immunity that result in the best fit to the type- and age-specific HPV prevalence. For each parameter combination, we ran the model 100 times. The best fit was based on the binomial log-likelihood of the observed prevalence data and model estimates summed over the age categories. The log-likelihood ratio is the difference between log-likelihoods of two different models . To compet al. [For the combinations of Weibull shapes with the best fit, we used a regression model of the transmission probability and acquired immunity duration as a metamodel ,40. Firset al. . We thenth lowest value as lower bound and 5th highest value as upper bound for each age group.Finally, we used the optimal model to estimate the age-specific proportion of women who are immune for HPV-16 and HPV-18, respectively. We accounted for uncertainty in the type-specific transmission probability and the duration of acquired immunity by re-running the model with 200 alternative parameter combinations . We randet al.  = 15.26, p =.009; S1F Supporting Information, 2 (4) = 43.88, p<.001), and the model with exponential distributions (χ2 (4) = 53.24, p <.001). This best fitting model had Weibull shape parameters of 0.50 and 0.25 for the duration of infection and immunity, respectively, corresponding with half of the men and over 70% of women clearing their infection within 1 year effectiveness and optimal delivery strategies of HPV vaccination in preventing cervical cancer.We also explored an alternative mechanism for acquired immunity after clearing an HPV-16 or HPV-18 infection (S1E Supporting Information). In this mechanism, women are not fully immune for variable duration, yet their susceptibility to re-infection reduces cumulatively after each infection. For both HPV-types, we found similar individual variation in infection duration and transmission probabilities compared to the base case mechanism. It was again necessary to assume acquired immunity after clearing an infection. Also under this mechanism, about half of the women will never acquire HPV-16 or -18. The alternative mechanism did show a worse fit to the observed prevalence data.In conclusion, we show that, using a mathematical model, acquired immunity likely plays a major role in HPV epidemiology. While our model suggests that most people are only immune for a short period of time after clearing an HPV-16 infection, acquired immunity is long-term for most people after clearing an HPV-18 infection. The proportion of women being immune for HPV-16 and HPV-18 and lifetime risk for an HPV-16 and HPV-18 infection already explains to a large extent why most women will never develop cervical cancer.S1 Supporting Information(DOCX)Click here for additional data file.S1 Fig(A) shows the results for HPV-16; (B) for HPV-18.The estimated prevalence is given by the best fittings model when assuming no immunity  or different scenarios when assuming cumulatively decreasing susceptibility to re-infection after each infection (58% for HPV-16 and 80% for HPV-18). These scenarios include an exponentially distributed duration of infection and a Weibull distributed duration of infection (Weibull shape 0.50). (TIF)Click here for additional data file.S2 FigThe proportion of women with no lifetime infections slightly increased in women aged 65+ compared to women aged 50–64 years. This results from a cohort effect due to a combination of historical data on fertility rates and timing of an increase in migration (1965), and will only have a minimal effect on our estimates.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tableet al. and Goodman et al. Bold numbers indicate the Weibull shape and corresponding duration of infection for the best fitting models.The distribution is a weighted average based on the studies of Trottier (DOCX)Click here for additional data file.S4 Tableet al. and Bulkmans et al. By applying the fractions observed in Coupé et al., we obtained the type-specific HPV-16 and HPV-18 prevalence.The observed high-risk HPV prevalence is based on the studies of Lenselink (DOCX)Click here for additional data file.S5 TableThe scenarios include no acquired immunity; exponentially distributed durations (Weibull shape = 1); and Weibull distributed durations.(DOCX)Click here for additional data file.S6 TableScenarios include no acquired immunity; exponentially distributed duration (Weibull shape = 1); and Weibull distributed duration.(DOCX)Click here for additional data file.


Three different trends were identified: one for HPV16, another for HPVs18/45/39, and a third for the rest of HPVs. The frequency trend of HPV16 shows two peaks. The first (63.2%) was found in the youngest women (≤35 years), followed by a decreasing trend until the age of 55–60 years (31.1%). The second peak arose at 61–65 years (52.5%), followed by a decreasing trend. The trend for HPVs18/45/39 declined from the youngest (19.3%) to the oldest  women. In contrast, the trend for the remaining HPVs increased from the youngest (15.8%) to the oldest (46.2%) women. Unlike other life-style factors, low-risk sexual behavior was associated with late onset of CC independent of low-oncogenic HPV types . The data indicate that most CCs in young women depend on the presence of high-oncogenic HPVs. In contrast, almost half of CCs in older patients had low-oncogenic HPVs, suggesting they could depend on the presence of other factors.Despite numerous human papillomavirus (HPV) frequency studies in women with cervical cancer (CC), little is known of HPV frequency trends according to patient age. In this work, we compare the mean age and frequency distribution by age of CC patients positive for different HPVs. This study included 462 CC patients. HPVs were detected by PCR and typed using DNA sequencing. A total of 456 patients (98.7%) were positive for HPV: 418 (90.5%) had single and 38 (8.2%) had double HPV infections. HPV16 (46.5%), HPV18 (10.4%), HPV45 (6.7%), and HPV31 (4.1%) were the most frequent viral types in single-infected patients. The mean ages of single-infected patients with HPV16 (49.2±13.3), HPV18 (47.9±12.2), HPV45 (47.9±11.7), or HPV39 (42.6±8.9) were significantly lower than the mean ages of patients singly (53.9±12.7;  Cervical cancer (CC) is the third most common cancer in women worldwide, affecting 500,000 individuals each year, and it is the leading cause of cancer death among women in developing countries Age varied widely among CC patients, ranging from 22 to 89 years old . The frequency distribution into 5-year intervals shows one peak at 46–50 years interval. However, the distribution did not follow a normal distribution, but was slightly skewed to the right . Most CCOf the 462 CC cases analyzed, 456 (98.7%) were positive for at least one HPV, 418 (90.5%) had single, and 38 (8.2%) had double HPV infections. HPV16 was the most frequent viral type in single infections (46.5%), followed in decreasing order by HPV18 (10.4%), HPV45 (6.7%), HPV31 (4.1%), HPV59 (3.2%), HPV58 (2.8%), HPV33 (2.8%), HPV51 (2.6%), HPV56 (1.7%), HPV52 (1.5%), HPV35 (1.1%), HPV39 (1.1%), HPV11 (1.1%), and other HPVs with a frequency lower than 1% . There w−4, t-test).The mean age of the patients with single HPV infections was 50.2±13.0 years; however, a great range of variation was seen among the patients according to viral types: from 42.6 years for HPV39 to 58.4 years for HPV56 . AlthougRemarkably, the mean age of the patients with double infections was higher than those with single infections  and this difference was statistically significant  . HoweverThe HPV frequencies were studied in patients divided into three age groups: young patients (≤40 years), middle-aged patients (41–55 years), and older patients (>55 years) . InteresTo observe these trends in more detail, the viral type frequency was also investigated in patients grouped by age into 5-year intervals . In the The HPV type frequencies were very similar between the SCC and ACC . HoweverWhereas in FIGO stage I/II tumors, the frequency trends of all HPV groups were very similar to the trends in the whole CC sample set , in stagWe investigated whether the patients' life-style factors were associated with the distribution of the disease by age. The factors studied were age at first sexual intercourse, lifetime number of sexual partners, marital status, contraceptive use, smoking, age at first pregnancy and previous Pap screening. Each of these factors were divided into two groups, according to the risk reported for CC, the lower-risk or reference variable  model with dichotomous outcome (age group) and explanatory variables (HPV types and life-style factors). Since the most notorious changes in the frequency of HPV types and risk factors occurred between the first two groups of age (≤40 yrs and 41–55 yrs), for this analysis we divided the patients in two age groups: ≤40 yrs and ≥41 yrs. The group of patients ≤40 yrs was taken as the reference group and the high-risk variables as the reference variables see . When thAlthough there are numerous studies of HPV frequencies in women with CC, little is known about HPV frequency according to age. In this study, we analyzed, in detail, HPV infection according to age in CC patients. The mean ages of the patients singly infected with HPV16, HPV18, HPV45, and HPV39 were at least 5 years lower than the mean age of the patients singly or doubly infected with the other HPVs. Even more interesting was the finding that the frequency trends of these groups of HPVs become completely different as the patients' ages increase. Three different trends could be identified: one for HPV16, another for HPVs 18, 45, and 39, and a third for the rest of HPVs. HPV16 follows the typical trend with two peaks as has been reported in healthy women in many studies, especially from Latin American and Asian countries There is a great contrast in the frequency of HPVs between the extreme age groups. Whereas the pooled frequency of HPVs 16, 18, 45, and 39 is 64.7% in the whole sample, the frequency in the younger women increases to 81.5% and decreases to 54.5% in the older women. This difference (27 years) was similar, but in the opposite direction, for the frequency of the rest of HPVs; it increases from 16.7% in patients younger than 41 years to 44.2% in patients older than 55 years. The numbers in the whole sample contrast with the reported frequencies for those HPV groups in most studies, mainly from developed countries, in which the pooled frequency of HPVs 16, 18, and 45 is between 75–80% and between 20–25% for the rest of HPVs It has been shown that the most oncogenic HPVs are HPV16 and HPV18, followed by HPV45 In this work we found that 2.4% of CCs only had low-risk HPVs, predominating HPV11 and HPV6. Similar percentages have been found in large samples of CC explored for HPV types Co-infections of the HPVs16, 18 or 45 with other high-risk HPVs have been frequently reported. The high-risk HPVs other than HPV16, 18, and 45 have lower oncogenic potential. In fact, just a small percentage (<3%) of those infections progress to CINIII in a time of 10 years On the other hand, as expected, low-risk sexual behavior  Finally, CC is a rare complication of the viral infection, most infections are transient and do not evolve into neoplastic lesions Therefore, these data collectively suggest that in a fraction of CC in women older than 55 years, especially SCC with advanced clinical stages, the role of HPV seems to be secondary, and other factors could play the principal role in the process of carcinogenesis.The study protocol was approved by the Scientific and Ethics Committees of the Hospital General de Mexico  and was performed in accordance with the ethical principles described in the 1964 Declaration of Helsinki. Informed written consent was obtained from all participants prior to their inclusion in the study.A cross-sectional study was done including a total 503 patients diagnosed with CC at the Department of Oncology of the Hospital General de Mexico in Mexico City. Patients were referred from the Outpatient, Emergency or Gynecology Departments, and from the CC screening program. Most of the patients come from the Metropolitan area (Mexico City and State of Mexico) and nearby states. All patients are Mexican-mestizo and have an important Amerindian genetic background MY09/MY11, GP5+/6+, and L1C1, as described previously HBB gene was used as an internal control to assess the quality of the DNA. The HPV types were identified by sequencing the amplified bands in positive samples using the fluorescent cycle-sequencing method . Sequence analysis was performed using an ABI PRISM 3130×l Genetic Analyzer system . Each band sequenced from the HPV-positive samples was analyzed with the FASTA sequence similarity tool DNA was extracted and purified from cervical scrapes and biopsy specimens using the PureLink genomic DNA kit  and maintained at −20°C until analysis. HPV detection was performed on all cervical scrapes, and when the internal control or HPV signal was weak (n = 44), or the samples yielded negative results for HPV (n = 21), the DNA extracted from the biopsy sample was also examined. In these cases, the DNA results obtained from the biopsy sample determined the presence or absence of HPV. In addition, to validate the analyses done on the scrapes, HPV detection and typing were carried out on another 100 biopsy samples, in which the signals of the paired scrapes were of good quality. The agreement between these parallel experiments was 98% . The HPV detection was performed by PCR using universal primers located in HPV L1 genes 5′-ATTAGGTGTATTAACTGTCAAA -3′ and reverse 5′-TTCTGCTTGTCCAGCTGG-3′) and HPV18 (forward 5′-CGACGATTTCACAACATAGC-3′ and reverse 5′-TCACACTTACAACACATACAC-3′), setting the annealing temperature 5°C below the Tm. Briefly, 500 ng of sample DNA was added to achieve a final volume of 25 µL containing 20 mM Tris-HCl (pH 8.8), 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 200 µM of dATP, dTTP, dGTP and dCTP, 1 µM of each oligonucleotide, and 1 U of Taq DNA polymerase . After denaturing for 2 minutes at 94°C, the reaction was carried out over 40 cycles in a DNA Thermal Cycler . Station 1 was set at 94°C for 30 seconds, station 2 at 52°C for 30 seconds, and station 3 at 72°C for 30 seconds. After this treatment, the reaction was kept at 72°C for another 7 minutes. After the PCR was completed, 5-µL aliquots were electrophoresed for 1 hour at 120 V in 2% agarose gels with 0.3 µg/mL ethidium bromide. The specific fragments were detected using a UV transilluminator  according to the positions of the positive controls and the molecular weight markers in the gel. The HPV16 and HPV18 bands were sequenced. In addition to HPV16 and HPV18 types, other viral types phylogenetically related to them could also be detected . When different viral types were identified with the specific and universal primers, those samples were considered doubly infected.In addition, all samples were assayed using E6/E7-specific primers for HPV16  and explanatory variables (HPV types and life-style factors). Differences were considered significant with a p value <0.05. All statistical tests were carried out using SigmaPlot, XLSTAT, and SPSS ver. 17 software.


Human papilloma virus (HPV) accounts for the most common cause of all virus-associated human cancers. Here, we describe the first graphic user interface (GUI)-based automated tool ‘HPVDetector', for non-computational biologists, exclusively for detection and annotation of the HPV genome based on next-generation sequencing data sets.http://www.actrec.gov.in/pi-webpages/AmitDutt/HPVdetector/HPVDetector.html.We developed a custom-made reference genome that comprises of human chromosomes along with annotated genome of 143 HPV types as pseudochromosomes. The tool runs on a dual mode as defined by the user: a ‘quick mode' to identify presence of HPV types and an ‘integration mode' to determine genomic location for the site of integration. The input data can be a paired-end whole-exome, whole-genome or whole-transcriptome data set. The HPVDetector is available in public domain for download: E7, a known oncogene, at known 17q21, 3q27, 7q35, Xq28 and novel sites of integration in the human genome. Furthermore, co-infection with high-risk HPVs such as 16 and 31 were found to be mutually exclusive compared with low-risk HPV71.On the basis of our evaluation of 116 whole-exome, 23 whole-transcriptome and 2 whole-genome data, we were able to identify presence of HPV in 20 exomes and 4 transcriptomes of cervical and head and neck cancer tumour samples. Using the inbuilt annotation module of HPVDetector, we found predominant integration of viral gene HPVDetector is a simple yet precise and robust tool for detecting HPV from tumour samples using variety of next-generation sequencing platforms including whole genome, whole exome and transcriptome. Two different modes (quick detection and integration mode) along with a GUI widen the usability of HPVDetector for biologists and clinicians with minimal computational knowledge. Epidemiological studies indicate that about 90% of cervical cancers, 90–93% of anal canal cancers, 12–63% of oropharyngeal cancers, 36–40% of penile cancers, 40–64% of vaginal cancers and 40–51% of vulvar cancers are attributable to HPV infection . CurrentHPV detection involves a computational subtraction-based approach, where NGS data are used for alignment against custom-made HPV multi-reference genome sequences to detect the traces of multiple HPV types using an automated pipeline .As a first step of the pipeline, HPV genomes in fasta format is required. We have acquired GenBank (.gb) files of 143 types of HPVs from a web resource Papillomavirus Episteme (PAVE) (http://broadinstitute.github.io/picard/). The alignment files were parsed using UNIX shell program to detect the type of HPV as well as number of reads that align to a particular HPV type. Number of HPV reads were normalised to the total depth of coverage per sample and with respect to different HPV gene sizes.Evaluating the HPV type and HPV-aligned reads is crucial to find HPV in the respective sample. For HPV type detection, we indexed the multi-fasta HPV reference file using BWA aligner followed by alignment of reads to indexed genome . The alihttps://trace.ddbj.nig.ac.jp/DRASearch/; study: SRP048769). The data were converted from SRA to FASTQ using SRAtoolkit. The resulting FASTQ files represents >36 × genome coverage which was further downsampled to 1 × , 2 × , 3 × , 4 × , 5 × , 10 × , 15 × , 20 × , 25 × and 30 × using Picard Toolkit's DownsampleSam function (http://broadinstitute.github.io/picard/). The resulting FASTQ files were used for testing HPV detection using HPVDetector.We downloaded SiHa whole-genome sequence from Sequence Read Archive database of DDBJ . E6 (HPV-16) and GAPDH expression were checked as described previously  as per the manufacture's instruction and later resolved on 1.2% agarose gel to confirm the RNA integrity. DNase treatment was done using the DNase Free kit  followed by first-strand cDNA synthesis taking 2 μl volume containing 10 μl (2 × ) Biomix-Red master mix , 5 μM each primer, 50 ng gDNA. PCR condition was: initial denaturation: 95 °C, denaturation: 94 °C for 1 min, annealing: 55 °C, , extension: 72 °C for 1 min and final extension at 72 °C for 5 min on a PCR machine . Primers were designed to amplify 122-, 126- and 120-bp read sequences of HPV16 identified by HPVDetector in SiHa cell line. Primers flanking the human reads were designed to amplify 119 and 290 bp, respectively. These sequences were further validated by Sanger sequencing. All the experiments were repeated at least twice independently. The details of the primers used in the study are provided in MY09/11 primer sequences were taken from previously reported literature  and number of HPV reads.This mode of HPVDetector determines the genomic location of HPV integrant, annotate with HPV gene, human chromosomal loci and human gene. This mode of HPVDetector pipeline starts with alignment of raw reads against a custom-made reference including a pseudochromosome such as the multi-fasta reference genome containing 143 HPV reference sequences and the HG19 human reference genome. Computational subtraction was carried out to retain discordant read pairs where the sequences are aligned to both human as well as HPV genomes. Finally, HPVDetector outputs a result file, which enlists HPV integration loci on the human genome, annotation of HPV genes, human genes and human genome cytobands.We analysed 22 cervical cancer exome sequencing data  to detect the presence of HPV. Among the 22 samples analysed, HPV was detected in 18 cervical samples, with maximum number of reads supporting the HPV16 sequence  Das et . Co-infeHPV is an independent risk factor in head and neck squamous cell carcinoma (HNSCC), in particular for oral and oropharyngeal carcinomas . At the We analysed 13 gall bladder cancer whole-exome, 1 gall bladder cancer whole-transcriptome and 1 liposarcoma whole-genome sequence data . No trace of the HPV sequence was detected in these samples.SiHa cell line developed from a cervical squamous cell carcinoma patient represents single-copy integration of HPV16 . The inthttp://broadinstitute.github.io/picard/)  . In addiHaving benchmarked the HPVDetector against SiHa for sensitivity, next we tweaked the SiHa whole-genome sequence data to test specificity of the tool by taking reverse (not complement) of the SiHa genome to simulate as a random sequence but retaining composition of nucleotides and genome complexity, using an in-house perl script. We found no spurious HPV reads when the SiHa whole-genome sequence was reversed, suggesting the HPVDetector is specific to detect true HPV traces. Further, to address the issue of specificity among primary tumours, we performed another round of functional validation on tongue squamous tumours that were found HPV negative based on HPVDetector  and valiE7 was found to be predominantly represented among the cervical tumours infected with HPV16, followed by E4, E5 and E6, in decreasing order (To enable accurate gene annotation of the HPV genome sequenced, we prepared a gene annotation database of 143 HPV types from PAVE database (ng order . Of thesng order .We identified 55 integration sites in 7 cervical cancer tumour samples T1099, T1123, T755, T887, T938, T1094, and T959 and 1 head and neck tumour sample using the HPVDetector . In thisIn total, we analysed 116 exome, 23 transcriptome and 2 whole-genome sequencing data, out of which we have detected presence of HPV in 20 exome and 4 transcriptome data .per se—for example, lacks information to annotate the region of the HPV genome to predict the integrated viral gene, of which some are known to function as oncogenes.HPV accounts for the most common cause of all virus-associated human cancers. However, despite large-scale genome-wide DNA sequencing efforts of the cancer genome, there is no dedicated informatics tool to rapidly detect the presence of HPV in these genomes, in an exclusive manner. There are indeed a variety of gene integration finding tools available that can detect different pathogen insertions in the human genome, such as ViralFusionSeq, VirusSeq, VirusFinder, Path-Seq, RINS, and ReadSCAN. These sophisticated tools although have their specific third-party needs, necessitate intense computational infrastructure, cannot be run without specialised and advanced computational expertise of the researcher, and more importantly are not specific for HPV detection, in silico tool ‘HPVDetector' as a unique tool to analyze NGS data to detect HPV sequences for non-computational biologists. Using the HPVDetector tool, we have detected 55 integration sites from the cervical exome and head and neck transcriptome data set. The tool allowed us to perform a comprehensive analysis to generate the information for co-occurrence of HPV subtypes across cervical cancer patients that is known to affect the clinical outcome of the disease. In addition, our finding of significant enrichment of viral gene E7>E4>E5>E6 reads among the cervical tumour samples, using the inbuilt annotation module of the HPVDetector, is consistent with the known biology of HPV genes and their role in carcinogenesis, as E6 and E7 are known viral oncogenes. This unique feature of the HPVDetector with an inbuilt HPV annotation module could potentially be helpful to understand the function of other HPV ORFs with unknown function by studying their incidence against varying tumour stage and types. Although the analysis of cervical tumours were restricted to its exome data set, a complete spectrum of the load of viral genes present in a sample can similarly be determined using the whole-genome data as input to the HPVDetector.We present a new user-friendly HPVDetector demonstrate a low false-negative and false-positive rate that can detect HPV reads at as low as 1 × genome coverage. Reads supported by even two paired reads were found to be credible. No viral reads were detected across 54 head and neck primary tumour samples of Indian origin, as reported earlier (Another critical feature of the HPVDetector is determination of HPV integration sites at the host genome. These integrations are known to occur at preferred regions of the genome .


The human papillomavirus (HPV) prevalence in males has been reported to be between 3.6% and 84%, depending specially on the socioeconomic status. HPV infection has been related as a risk factor for penile cancer. This is a rare tumor, and other risk factors include lack of personal hygiene and men who have not undergone circumcision. Penile cancer is less than 1% of cancers in men in the United States, however, is much more common in some parts of Asia, Africa, and South America, where it accounts for up to 10% of cancers in men. This study aimed to determine the prevalence of HPV-DNA in penile cancers in São Luís, Brazil and to correlate the virus presence to histopathological factors.Tumor paraffin samples of 76 patients with penile carcinoma were tested in order to establish the prevalence and distribution of genotypic HPV using PCR/Nested and automated sequencing. To evaluate the association between HPV types and other clinical and morphological variables, a nonparametric ANOVA was performed using a Kruskal Wallis test, and statistical significance was determined to a value of p < 0.05.The average age of patients at the time of diagnosis was 66 years ± 17.10. Regarding location, 65.79% of the tumors were located in the glans, and the most common types were vegetative (34.21%) and squamous (98.68%). Most of the lesions ranged in size from 2.1 to 5.0 cm, presenting Jackson I stage and Broders II degree. It was observed that 32 patients had at least one invaded and/or infiltrated structure. Lymph node involvement was observed in 19.76% of the patients, and 21.05% showed an inflammatory process. In the molecular evaluation, HPV infection was observed in 63.15% of the lesions, and the most common type was HPV 16.From the statistical analysis, it can be verified that the variables were not associated with infection by the HPV virus. Although penile cancer can result from various risk factors that act in synergy, an HPV virus infection is important for the development of such neoplasm. The human papillomavirus (HPV) is a DNA-virus from the Papoviridae family - genre Papillomavirus, with more than 100 types currently recognized, 20 of which can infect the genital tract; the man is the main disseminator [Penile infection by HPV may be clinical, subclinical or latent. In clinical presentation, the diagnosis is simpler, because it is determined from a good clinical examination to uncover existing lesions. In subclinical and latent forms, other methods, such as peniscopy, are necessary to aid in detection, as it is not possible to detect changes  with the naked eye. In men, there is a higher frequency of the subclinical form .Penile cancer mainly affects men over 50 years old, but approximately 19% of patients are 40 years of age or younger, and 7% are below the age of 30 [The prevalence of the virus in males has been reported to be between 3.6% and 84%, depending on socioeconomic status ,6. PenilAccording to Nardi et al.  the highThe aims of this study were to detect and perform HPV genotyping in biological specimens of penile tumors and to determine the existing associations between viral presence and histopathological clinical aspects.This was a retrospective study performed in paraffined penile tumors collected at two public reference hospitals in Maranhão. A total of 76 samples were included in the study from patients diagnosed with penile cancer between the years 2001 and 2011. Patient information as well as the histopathological characteristics of the tumors obtained from medical records. As the samples consisted in paraffined tumours, there was no written informed consent from the patients. The patient identity was not disclosed in this research. This work was approved by the Ethics in Research Committee of the University Hospital of the Federal University of Maranhão (HU/UFMA).Paraffin blocks and histological slides of penile tumors as a result of biopsy or surgical treatment, with or without lymphadenectomy at any follow–up in the archives of the Pathology Services.Histological slides and/or paraffined blocks not found in the archives of the Pathology Services of referral hospitals and reports did not provide complete information.The samples were reviewed by the pathologist, and blocks with tumor representativeness  were selected. After microtomy, sections suffered a process of deparaffinization. The sections were stored at 4°C, awaiting DNA extraction.The extraction of the genomic DNA from the samples was performed using the QIAamp DNA FFPE Tissue Purification Kit (QIAGEN®) according to the extraction protocol suggested by the manufacturer.The Nested PCR reactions were performed by using primers PGMY09 and PGMY11 for the first round, and primers GP + 5 and GP + 6 for the second round [The sequencing reactions were performed in the Laboratory of Genetics of the National Cancer Institute (INCA) with ET Dye Terminator Cycle Sequencing kit  according to the manufacturer’s suggested protocol.All data were collected and prospectively input in an EpiInfo 3.4.3 and Microsoft Office 2007® were used for the statistical analysis.To evaluate the association between HPV types and other clinical and morphological variables, a nonparametric ANOVA was performed using the Kruskal Wallis test with a statistical significance level of 5% probability (p < 0.05).Tumor biopsies of penile cancer were evaluated in 76 patients aged 26 to 97 years with a mean of 60.7 years and standard deviation of ±17.10, presenting a higher prevalence in the over 66 age group. The clinical representation and pathologic characteristics distribution is shown on Table These results correspond with those obtained in the literature -14. The Regarding the location of the lesions, the glans, in an isolated form or associated with other regions, was the most affected structure as in the research by Delgado et al. , Wanick Studies have shown that the lesions on the glans are directly linked to poor hygiene. This occurs due to the formation of a mass, called smegma, followed by likely irritation of the site and onset of injury, facilitating various infections and future neoplasia if left untreated .Regarding the clinical morphology, the predominantly found lesion was the vegetating type followed by ulceration. The occurrence of both types of lesions in the same patient was observed in 22.37% of the cases. In a study performed in Spain, researchers observed that the vegetative lesion was also more present, in 66% of the cases . On the The dimensions of the lesions were similar to those observed in the Wanick et al. results , with a Unlike other studies, the moderately differentiated tumors (grade II) identified in this work, according to Broder’s classification, were the most prevalent. Fonseca et al.  identifiThe findings indicated that invasion was present in 23.68%, and infiltration occurred in at least one of the structures, with the highest prevalence in the corpus cavernosum. Koifman et al.  reportedRegarding lymph node involvement, a percentage of 19.73% was observed. According to Sacoto et al. , patientThe DNA of the HPV was detected in 63.15% (48/76) of the samples. The oncogenic risk distribution is shown on Table In our study, among the high-risk viral types present were the 16, 18, 45 and 69 types. The HPV of type 11 was the only low oncogenic risk found. Type 16 was the most prevalent, found in 10 cases, followed by type 11 of low risk with 6 cases, type 18 with 4 cases, type 69 with two cases and type 45 with 1 case. The automated sequencing technique was not effective for viral genotyping, because in more than 50% of the samples it was not possible to achieve. This may be due to the presence of co-infections in these samples, which prevents the device from detecting the virus, as described by Gharizadeh et al. 2005) and Verteramo et al. (2009) 05 and Ve. The mosAs shown in Table Infections by HPV are strongly associated with the development of penile cancer; however, the role of viruses in the etiology is not very clear . AlthougHPV DNA was found in 48 of the 76 analyzed samples . The high-risk type HPV 16 was observed in 21.28% (10/48) of the lesions followed by low-risk type HPV 11 in 12.76% (6/48) and high-risk types HPV 18 in 8.51% (4/48), HPV 69 in 4.25% (2/48) and HPV 45 in 2.13% (1/48). In 51.06% of the cases, genotyping was indeterminate, suggestive of co-infection.The average age of the patients in the study was 60.6 years old. Prevalent lesions were larger than 2 cm, in the glans region, in general vegetating, and with Broder’s grade II (moderately differentiated). The clinical and histopathological variables did not tend to have an association with infection by the HPV virus.


HPV vaccination has the potential to prevent the vast majority of cervical cancer cases but cervical cancer screening remains the only prevention strategy in adult unvaccinated women. The range of primary screening tests has expanded to include, in addition to cytology, visual inspection with acetic acid (VIA) and HPV-testing. HPV-testing has the best sensitivity and negative predictive value, and a low-cost HPV test may make large-scale high-quality cervical cancer screening possible in low- and medium-income countries (LMICs). However, on account of its low specificity, HPV-testing would impose the additional burden of triaging HPV+ women using cytology, colposcopy, VIA, or other not yet affordable tests. If minimally invasive treatments that have proven efficacious in HPV+ women with cervical intra-epithelial (CIN) grade 2 and 3 lesions also reduced future cervical cancer risk in lesion-free HPV+ women, the treatment of all HPV+ women would become attractive in LMICs in which screening should be performed as infrequently as possible.In the pre-mass screening era, gynaecologists widely practiced prophylactic ablation of the columnar epithelium visible on the ectocervix (ectopy) and squamocolumnar junction (SCJ) in the hope of preventing cervical cancer. Favourable outcomes were reported, especially from Finland, but conclusive results were not reached on account of weaknesses in the study methods. Indirect support for prophylactic ablation is provided by the hypothesis that some cells of embryonic origin derived from the SCJ are the source of high-grade CIN and cervical carcinoma, and that they do not regenerate after SCJ ablation. SCJ cell elimination may avoid neoplastic transformation though not HPV reinfection. Randomised controlled trials are gold-standard, but if not feasible, an evaluation of the impact of prophylactic ablation could be done in the framework of large HPV-based screening programmes. Careful follow-up of lesion-free HPV+ women would provide much needed information on the risk-to-benefit ratio of prophylactic ablation. Cervical cancer was the first malignancy to become the object of screening programmes. As early as the 1950s, Papanicolaou . Of noteOver the last decade there has been an increase in the variety of tests available for cervical screening, e.g. human papillomavirus (HPV) testing and visual inspection with acetic acid (VIA), thus overcoming some of the limitations of cytology and addressing more closely the needs of the different regions of the world. A global expansion of HPV vaccination may curb the risk of cervical cancer by more than 70% in the cohort of women who are now being vaccinated in their early adolescence. However, no therapeutic vaccines or anti-HPV medical treatments are available and early detection and treatment of precancerous lesions is the only way to avoid cervical cancer in HPV+ women. Between the 1920s and the 1970s, gynaecologists widely performed prophylactic ablation of the squamocolumnar junction (SCJ) and columnar epithelium visible on the ectocervix in an attempt to diminish a woman’s susceptibility to infection and cervical cancer , 4.In the present report, I will briefly outline a few large observational studies of prophylactic ablation and discuss possible synergies between the introduction of HPV-based primary cervical screening, notably in low- and middle-income countries (LMICs), and the possibility of assessing the value of prophylactic ablation.in situ or invasive carcinoma (27 per 1000 women). In contrast, three in situ and no carcinoma were detected in treated women (0.5 per 1000 women). Peyton et al [A large amount of studies have demonstrated the efficacy and safety of cryotherapy  and excion et al  reportedA little later, prophylactic ablation was also widely used in Italy, especially by a team in Milan , 11. Marin situ carcinoma and severe dysplasia , is currently endorsed as a method to triage hrHPV+ in low-income countries in national protocols  and by Wet al [A new population of cells of embryonic origin that derive from the SCJ of the cervix (henceforth referred to as SCJ cells) have recently been characterised , 35. Theet al , 35 haveet al [et al [In an effort to confirm their earlier findings, Herfs et al  assessedl [et al  suggest,in situ and invasive cervical cancer in the participants of two randomised clinical trials on the effect of raloxifene and tamoxifen  were inconclusive [Alternative and less invasive ways than prophylactic ablation may exist to prevent or reverse the HPV-driven malignant transformation process in the cervix, e.g. a therapeutic HPV vaccination  or hormone modulation. An important knowledge gap in the natural history of SCJ cells and by and large the HPV-driven cervical carcinogenicity is the role of female hormones . The cernclusive  on accouet al [et al [If the findings by Herfs et al  that abll [et al  ends witAs in the past, a large randomised trial of prophylactic ablation would be very challenging especially because of concerns about adverse pregnancy outcomes in women who still wish to have children. A meta-analysis  and larget al [et al. [et al [A possible alternative to randomised trials may be a careful evaluation of the impact of prophylactic ablation in the framework of large screening programmes . The newet al  showed t [et al.  and the . [et al  that thepost-hoc diagnostic confirmation. Prophylactic ablation would not be performed in women with mature metaplasia or in women in whom the SCJ were not visible. Of note, the distinction of mature from immature cervix is difficult using VIA/VILI pointing to the additional usefulness of colposcopical examination and the desirability of colposcopical skill improvements in LMICs [In order to advance current knowledge on prophylactic ablation, a study could be envisaged in which cryotherapy is performed in a sufficiently large subset of hrHPV+ women showing columnar epithelium on the ectocervix with or without metaplasia but no evidence of cervical precancerous or cancerous lesions at VIA/VILI or colposcopic examination. The ideal age target for prophylactic ablation needs to be defined but it would likely be from age 30 or 35 to 40 or 45. Women who have evidence of cervical precancerous or cancerous lesions would be managed according to well-established protocols . In womein LMICs . Follow-CINcervical intraepithelial neoplasiaHPVhuman papillomavirushrhigh-riskLEEPloop electrosurgical excision procedureLMICslow- and middle-income countriesPRprevalence ratioSCJsquamocolumnar junctionVIAvisual inspection with acetic acidVILIvisual inspection with Lugol’s iodineNo potential conflict of interest is disclosed by the author.