Tumor Susceptibility Gene 101 (TSG101) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer. The multidomain protein encoded by the  Tumor Susceptibility Gene 101 (Tsg101). Designating this gene as a TSG seemed appropriate at the time since the exogenous expression of the Tsg101 cDNA in both antisense and sense orientation resulted in a transformed phenotype. In 1997, TSG101 had become more widely known as a tumor suppressor when it was reported that this gene is mutated, or its expression is lost in a significant subset of sporadic breast cancers [In the mid-1990s, the laboratory of Stanley Cohen at Stanford University conducted an insertional mutagenesis screen on immortalized mouse embryonic fibroblasts NIH3T3 cells) to identify candidate tumor suppressor genes [ cancers . These fT3 cells  cancers ,5,6, andTSG101 mRNAs are 86% identical on the nucleotide level [Tsg101 locus is highly conserved between mouse and human [TSG101 implied that the majority of truncated transcripts that were observed in cancer and nonmalignant tissues are true alternative splice products that originate from exon skipping, rather than aberrant transcripts from cryptic splice sites as proposed earlier [TSG101 is expressed in all tissues and cell types [Tsg101 mRNA is already present in 1-cell and 2-cell stage mouse embryos. The expression of Tsg101 in germ and stem cells may also explain the origin of a processed Tsg101 pseudogene in the mouse genome, which made it challenging to identify the actual Tsg101 locus and isolate genomic DNA sequences for the construction of gene targeting vectors to generate knockout mice [Tsg101 mRNA levels were observed in the brain and the lactating mammary gland [The coding sequences of the mouse and human de level . Sanger  earlier ,9,10,11. earlier . The 5′ ll types ,8, and tout mice ,14,15. DTsg101 as a housekeeping gene had several implications. First, a complete knockout of Tsg101 might cause early embryonic lethality [mRNA expression levels from microarray and RNA-sequencing data are frequently being used to judge the importance of genes in cancer development and patient survival. As discussed later in this review, a tight post-translational control of the TSG101 protein level that balances variations in mRNA expression also imposes challenges for the generation of genetically engineered models to assess the effects of TSG101 gain- or loss-of-function in normal organogenesis and cancer development.The designation of ethality . More imTSG101 transcripts encode proteins of approximately 50 kDa in size with more than 94% similarity. The TSG101 protein contains several conserved domains  in Saccharomyces cerevisiae, which is also known as STP22. Subsequent studies revealed that TSG101/VPS23 is a central member of a subset of VPS class E proteins  that form the Endosomal Sorting Complex Required for Transport I (ESCRT-I). As part of this complex, TSG101 is suggested to selectively bind ubiquitinated cargo proteins and direct their sorting into multivesicular endosomes [Two reports from the laboratories of Duane Jenness and Scott Emr ,34 were ndosomes . In linendosomes ,35,36,37ndosomes , and, asndosomes ,15,39.Insight into the multifaceted functions of TSG101 as a member of ESCRT-I was gained from a series of studies on the association of TSG101 with retroviral proteins and their intracellular movement . TSG101 The specific functions of TSG101 in retroviral particle assembly and budding might be exemplary for a broader role in the biosynthesis and release of exosomes or, as now generally defined, secreted extracellular vesicles (EVs). Using a proteomic approach, Thery et al.  were firTSG101 locus have not been widely employed. As reviewed in the next section, the phenotypes of defined TSG101 genomic knockout models deviate from observations in cell lines where TSG101 is only knocked down with siRNAs. It is unclear to date which of the known molecular mechanisms, if any, are responsible for the indispensable functions of TSG101 for the proliferation and survival of mammalian cells.In summary, the TSG101 protein is suggested to mediate a variety of intracellular processes, but the majority of published articles only highlight its proposed roles in endosomal trafficking. Notably, many of the reports that solely focus on the molecular, biochemical, and intracellular functions of TSG101 emphasize the importance of their specific findings for diseases like cancer and neurodegeneration, but the proposed mechanisms are rarely validated in genetically defined in vivo disease models or in primary human tissue samples. Consequently, the biological relevance of these multifaceted functions of TSG101 for normal development, differentiation, and tissue homeostasis, as well as potential roles of TSG101 in cancer are poorly defined. On the experimental level, most published studies that examined the intracellular roles of TSG101 relied exclusively on RNA interference, overexpression of TSG101 mutants, or the use of chemical compounds that disrupt entire cellular processes. CRISPR/Cas-based targeted knockouts or other gene editing methods to introduce mutations into the endogenous Tsg101 gene promoter sequence and expression profiling, we predicted that a conventional knockout of this gene causes embryonic lethality [Tsg101 in germ cells and differentiated tissues during embryonic and postnatal development as well as in cultured cells that are primary, immortalized, or tumorigenic [p53 did not extend the survival of TSG101-deficient cells [Tsg101. More interestingly, we noted that individual rescue clones expressed exogenous TSG101 at the same levels as the endogenous protein in parental controls [TSG101 cDNA can fully restore normal growth of mouse cells that lack endogenous TSG101, suggesting that both mammalian proteins function in an identical manner. In contrast to the full-length cDNA, expression of TSG101 mRNA splice variants or deletion mutants lacking individual domains of TSG101 did not rescue the deleterious knockout phenotype  [From the analysis of the ethality . Indeed,ethality  were firorigenic ,15,39. Torigenic . As contnt cells ,39. Re-econtrols , supportcontrols . We haveblished) .Tsg101 in the germline resulted in an earlier embryonic lethality prior to or during implantation [Whey Acidic Protein gene promoter (WAP- Cre) demonstrated that TSG101 is equally required for cell survival and tissue homeostasis in adult animals. Secretory epithelial cells that have completed their functional differentiation show a significantly reduced proliferation rate and it is therefore evident that crucial functions of TSG101 for cell survival are independent of its suggested roles in cell division. Re-expression of exogenous TSG101 in the lactating mammary gland restores normal mammary gland development and lactation [In contrast to mutants that lack exons 8 and 9, the Cre-mediated excision of the promoter and exon 1 of antation . A tissuactation . Similaractation .TSG101 conditional knockout mice were also utilized to examine biologically relevant functions of TSG101 for normal tissue homeostasis in organ systems other than the mammary gland. Using a cardiac-specific and tamoxifen-inducible knockout of TSG101, Essandoh et al.  recentlyLike the mammary epithelium and cardiomyocytes, the deletion of TSG101 in oligodendroglia was reported to cause apoptosis, demyelination, vacuolation, and severe spongiform encephalopathy. Mice lacking TSG101 in parts of the central nervous system developed a tremor and had significantly reduced body weight by 8 weeks of age . In a paCip1 is a downstream mediator of the p53-dependent cell cycle arrest in response to the deletion of Tsg101 [Cdkn2a (p16Ink4a/p19Arf knockout) did not alter the ability of MDM2 to sequester p53 and restore the p21-mediated G1 arrest. The collective results obtained from the analysis of Tsg101/p53, Tsg101/p21, and Tsg101/Cdkn2a conditional double-knockout cell lines that were generated in our team suggested that a biologically significant interaction of TSG101 with the MDM2/p53 circuit seems unlikely. The p53/p21-controlled G1 arrest might, therefore, be an indirect consequence of an activation of stress response pathways that are being triggered by the knockout of TSG101 [In summary, the complete loss of TSG101 triggers an accumulation of p53 and a cell cycle arrest at the G1 phase followed by cell death, which is independent of p53 function and proliferation. This finding is not unique to mammals and can be extended to other vertebrate species. A knockdown of TSG101 with antisense morpholinos causes a growth delay and lethality of zebrafish embryos [f Tsg101 ,53. Althf Tsg101 , we demof TSG101 .Fibroblasts with a conditional knockout of TSG101 exhibit several characteristics of stressed cells: a) phosphorylation of mitogen-activated protein (MAP) kinases independent of growth factor stimulation, b) widespread redistribution of actin filaments, and c) induction of autophagy prior to cell death . TSG101 Tsg101 gene causes a substantial decline in the steady-state levels of the EGFR and ERBB2 [Although the precise cellular process that causes TSG101 knockout cells to die is still unknown, one important consequence that may trigger an intense stress-response is growth factor deprivation. Unlike previous observations that a siRNA-mediated knockdown of TSG101 resulted in enhanced recycling or retention of the EGFR within early endosomes , the defnd ERBB2 . This mand ERBB2 , and thind ERBB2 . It remaTsg101 alleles in surviving knockout rescue clones was confirmed by Southern blot (fl/flTsg101) as well as knockout rescue clones expressing HA-tagged exogenous, wildtype (wt) TSG101 AP motif-containing proteins at different sites. Pornillos and coworkers  demonstr) TSG101 B. Given Tsg101 gene causes cell cycle arrest and cell death instead of accelerated growth and neoplastic transformation, which would support its proposed function as a tumor suppressor. This is also the case for other vertebrate animal models such as zebrafish [p53 and Cdkn2a. TSG101 is also essential for the growth of neoplastic cells that are capable of forming tumors in mice [Tsg101 alleles in somatic tissues of genetically engineered mice, such as the mammary epithelium, led to the development of benign neoplasms or cancer [Tsg101 was deleted in the mammary epithelium that lacked both copies of the tumor suppressor p53 [Tsg101 in established, ERBB2-transformed mammary cancer cells causes cell death  [Despite a wealth of information about multifaceted intracellular functions of TSG101, it is still a conundrum why a knockout of the mammalian ebrafish . The ess in mice ,15,39. Nr cancer ,57. Moressor p53 . This wablished) . These fblished) ,61,62. Iblished) . Silenciblished) , which, blished) . On the blished) .TSG101 ortholog Erupted in Drosophila causes excessive cell growth, albeit in a non- cell- autonomous manner through the expression of the Notch target gene Unpaired, which is the secreted ligand that stimulates JAK/STAT signaling in the fly. It is obvious that a mechanism of a non-cell-autonomous role of TSG101 in growth suppression in the fly does not correspond to the phenotypes of a knockdown of TSG101 in normal and neoplastic cells of human and mouse origin. Although the proposed function of Erupted as a paracrine growth suppressor seems interesting, it needs to be explained why the phenotype of the Erupted-deficient fly is very different from a knockout of dVps28, which results in early embryonic lethality [Erupted and dVps28 in mutant flies might suggest that the mechanism that controls the post- translational cross-regulation of the levels of TSG101 and VPS28 in mammalian cells may not exist in Drosophila. Regardless of the nature of these discrepancies, we and others have never observed any palpable tumors in TSG101 conditional knockout mice where one or both copies of Tsg101 were deleted in a mosaic fashion in selected cell types with Cre recombinase. The same was true for a morpholino-mediated knockdown of TSG101 in zebrafish [In support of a tumor-suppressive role of TSG101, Moberg and coworkers  reportedethality . TSG101 ethality , and morethality ,69. The ebrafish . Hence, TSG101 gene in human cancers could not be confirmed. Moreover, alternative splice variants of TSG101, which have been subject to intense investigations in the late 1990s, can only be detected by nested RT-PCR, and they are also present in normal tissues [In conclusion, the various genetically engineered animal models, as well as observations from human cancer cell lines where TSG101 was knocked down with siRNAs, do not support a tumor- suppressive role of its encoded protein, either in a cell-autonomous manner or by means of juxtracrine or paracrine signaling. As stated in the opening paragraph of this review, initial reports about a loss of the  tissues ,6,8. As www.proteinatlas.org), most human cancers exhibit a moderate cytoplasmic and membranous immunoreactivity against TSG101, and the intensity of the staining might serve as a prognostic marker for selected cancer types such as hepatocellular and renal tumors. Using immunohistochemistry staining on a limited set of primary human breast cancers and normal breast tissues, our team observed that TSG101 is elevated in 50% of invasive breast cancers [TSG101 across all cases may not serve as a prognostic marker . In contrast, higher levels of TSG101 transcripts correlate significantly with reduced survival in luminal type A  and in luminal type B  breast cancers . This implies that any studies that are designed to examine the biological significance of TSG101 and associated molecular mechanisms for a given tumor type  should be conducted on cell lines and animal models that resemble the main characteristics of a particular cancer-intrinsic subtype .Investigations from various research teams including our own have shown that TSG101 is overexpressed rather than lost in a significant subset of malignancies, including breast, lung, thyroid, ovarian, and colon cancers ,71,72,73 cancers . HoweverSimilar to breast cancer, it has been reported that TSG101 is upregulated in human ovarian epithelial cells that express oncogenic HRAS or KRAS as well as in 70% of human ovarian carcinomas analyzed on tissue arrays ,72,74. WTSG101 as a transforming oncogene when an expression library of a highly metastatic lung adenocarcinoma cell line was introduced into mouse NIH3T3 cells. To our knowledge, the transforming ability of TSG101 overexpression has not been assessed in any other cell lines, in particular, those of human origin. As mentioned earlier, TSG101 protein levels are being maintained within a relatively narrow range of a given cell type, which may complicate the generation of suitable overexpression models. A minor or moderate expression of exogenous TSG101 results in a compensatory downregulation of the endogenous protein on the post-translational level [Whey Acidic Protein (Wap) promoter can be achieved in functionally differentiated epithelial cells of the lactating mammary gland [The elevated expression of TSG101 in cancer compared to normal tissues may support the notion that TSG101 plays a role at particular stages of cancer initiation or progression. Experimental evidence that TSG101 overexpression might contribute to neoplastic transformation was provided in the inaugural paper by Li and Cohen , who isoal level . Possiblal level . It is ary gland . TSG101 ry gland . In anotry gland  achievedWap gene promoter developed mammary tumors after a relatively long latency [Wap gene promotor is suitable to drive a tissue-specific expression of genes almost exclusively to the mammary epithelium, a high activation occurs predominantly in functionally differentiated alveolar cells during pregnancy and lactation, and the majority of these cells die during post-lactational mammary gland remodeling following the weaning of the offspring. As such, exogenous TSG101 is not persistently overexpressed in most epithelial cells throughout the lifetime of a female mouse. It is therefore not known whether a persistently high expression of TSG101 in diverse epithelial subtypes of the mammary gland may cause an earlier onset of mammary cancer. To our knowledge, the WAP-TSG101 transgenic mouse strain is currently the only published in vivo model that demonstrated that TSG101 can function as a transforming oncogene that causes a sporadic occurrence of preneoplastic lesions and mammary cancer. Given the long latencies of tumor formation, it is evident that secondary genetic or epigenetic changes must occur that drive the progression of bona fide cancers in this model.In contrast to the heart-specific overexpression model, a subset of transgenic females (approximately 20%) that express exogenous TSG101 under control of the  latency . At the TSG101 possesses characteristics of a housekeeping gene, and significant variations in the expression of its encoded protein between diverse cell types are, in part, a consequence of post-translational mechanisms. Given the stringent control of the TSG101 protein levels in a particular cell type, it is likely that the deregulated expression of TSG101 before or during neoplastic progression is primarily caused by an interference with its post-translational control mechanisms. The precise molecular processes that regulate the amount of TSG101 within cells are less well defined and have not been associated with any biologically relevant functions of TSG101 as an oncoprotein.As reviewed earlier, Arf. Evidently, TSG101 may not function as an oncoprotein under these physiological conditions. In contrast, TSG101 may only exert oncogenic activities in the MDM2/p53 circuit when it is upregulated and able to stabilize MDM2 without being ubiquitinated and degraded by this ubiquitin ligase.MDM2, LRSAM1/TAL, and MGRN1 are three E3 ubiquitin ligases that have been reported to control TSG101 levels in mammalian cells. Li and coworkers  have shoUsing a yeast two-hybrid system, Amit et al.  identifiMGRN1 locus has been reported to be amplified in osteosarcomas [Like the PTAP–PSAP double motif in TAL, the Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase possesses a single, evolutionarily conserved PSAP amino acid sequence that can bind to the UEV domain of TSG101 . MGRN1 msarcomas  and, accAlthough TSG101 is suggested to function in a variety of cellular processes, the vast majority of published reports over the past 20 years have highlighted specific molecular mechanisms by which TSG101 may control the intracellular trafficking of cargo proteins such as RTKs and viral particles. Key observations from these studies are often highlighted as proof of the molecular underpinnings by which TSG101 may function as a growth suppressor. Then again, the validity of the proposed mechanisms is rarely, if at all, tested in genetically defined disease models or in primary human cancer tissues. At present, there is no conclusive evidence from experimental models or human tissues in support of the notion that deficiency in TSG101 or a functional loss of the protein could contribute to the genesis and progression of cancer. It has been established that TSG101 may function as a protooncogene in certain cell types. In the context of a whole organism, the biological significance of the proposed multifaceted roles of TSG101 in developmental processes and normal tissue homeostasis is still unclear. None of the proposed molecular roles sufficiently explains why cells that completely lack TSG101 enter a cell cycle arrest and die. Many of the critical functions in the intracellular trafficking of cargos as well as the binding of factors that control the post-translational modification and functionality of the TSG101 protein have been attributed to two structural features, i.e., PTAP amino acid motif-binding and ubiquitin-binding domains. Future studies using mouse models with CRISPR/Cas9-mediated targeted knock-in mutations might show whether these two domains or additional amino acid residues of TSG101 have a biologically significant role in normal development and disease.


There is an oncogenic role of human papillomavirus (HPV) in the development of premalignant and malignant skin cancers, especially squamous cell carcinomas (SCCs). Some of the major risk factors for SCC include older age, fair skin types, immunosuppression, ultraviolet radiation (UVR), history of epidermodysplasia verruciformis (EV), and co-carcinogenesis by the HPV. Our case report exemplifies a unique case of a low-risk, 34-year-old female who developed an HPV-associated squamous cell carcinoma in situ (SCCIS) on her left palmar hand, despite having none of the contributing risk factors. The biopsy also showed full-thickness keratinocyte atypia and increased mitotic activity throughout all the layers of the epidermis. Immunohistochemical stains showed strong and diffuse nuclear staining of p16 and Ki-67 throughout the SCCIS, confirming HPV etiology. We speculate that tumor development in our patient relied on the combined effects of UVR exposure, localized immunosuppression, and the co-carcinogenic effects of HPV infection. Non-melanoma skin cancers (NMSCs) are the most common types of cancer in the United States, surpassing lung, breast, prostate, and colon cancers combined . AdditioCurrently, the human papillomavirus (HPV) has been the focus of the etiological agent in SCC. HPV is a non-enveloped DNA virus that infects and depends on keratinocytes to replicate . It is pIn the context of skin cancer, HPV infections can be divided into three categories: cutaneous, mucosal, and epidermodysplasia verruciformis (EV). EV is a rare cutaneous disorder that significantly increases the susceptibility of cutaneous HPV infections and subsequent skin malignancies . The cliWe present a unique case of a 34-year-old female with no prior history of Verruca vulgaris or known environmental exposures, such as working with children, immunosuppression, or EV, who presented with an HPV-associated squamous cell carcinoma in situ (SCCIS) on her left palmar hand. To the best of our knowledge, this is the only reported case of an HPV-associated cutaneous SCCIS in a patient this young and with none of the contributing risk factors.We report a 34-year-old female with no comorbidities or previous history of malignancies, presenting with a moderately painful, progressive skin lesion on her left palmar hand that present for 5+ years. Physical examination revealed a 2.5-cm hyperkeratotic, erythematous, asymmetric plaque with well-defined, rolled borders and fissures Figure . Shave biopsy revealed SCCIS with viral features, extending to the deep and lateral margins. The biopsy also showed full-thickness keratinocyte atypia and mitotic activity throughout all the layers of the epidermis Figure . PAS stain was negative for fungal organisms. Immunohistochemical (IHC) stains showed strong and diffuse nuclear staining of Ki-67 and p16 throughout the squamous cell in situ, confirming HPV etiology Figure . Mohs micrographic surgery was performed utilizing frozen tissue sections and H&E stains. The first two stages revealed residual carcinoma. The residual carcinoma was excised and submitted for processing, revealing no carcinoma on the third stage. Due to the large size of the defect, primary, complex, or adjacent tissue transfer closure was unachievable, and the wound was left to heal via secondary closure for six weeks. Subsequently, a skin graft was taken from the patient’s left medial arm and placed on the recipient site. The donor site was reconstructed via complex closure, and the recipient site defect measured at 38 mm x 85 mm. 0 period, and thus an indication of the progression through the cell cycle of proliferating tumor cells. Additionally, HPV-associated SCCs are histologically characterized by full-thickness dysplasia of the epidermis, sometimes with the involvement of follicular epithelium.There is increasing evidence supporting the oncogenic role of HPV in the development of premalignant and malignant skin cancers ,8,10. AlIn our case report, the patient’s histological results were consistent with an SCCIS, and the p16 staining in the IHC confirmed an HPV component. However, despite the strong histopathological picture of an HPV-associated SCCIS, the patient exhibited no contributing risk factors, which include a history of EV, immunosuppression, or older age.EV is an extremely rare inherited skin condition that makes a person highly susceptible to infections caused by HPV. Symptoms can arise anywhere from infancy to puberty and consist of verrucae-like lesions distributed over sun-exposed areas of the body, such as hands, feet, face, and ears . After tAnother group of patients who are prone to SCCs is immunosuppressed patients. The HPV virus has been shown to elicit an antibody and cell-mediated immune response, with the T-cell response, specifically, playing a major role against HPV immunity . StudiesOne risk factor that may have played a role in the patient’s development of cSCC is exposure to UVR. The relationship between UVR and cSCC development is not fully understood yet. Studies have shown patients with a history of painful sunburns had an increased risk of developing HPV-associated cSCCs . The curStudying HPV as a potential trigger in SCC carcinogenesis is important because it introduces the possibility for novel therapeutic and preventative measures for HPV infected patients. Currently, there are HPV vaccines that protect against cervical cancer, but they only cover nine of the many HPV serotypes. Additionally, current treatment options also do not eradicate active HPV infections. Future therapies should expand to include preventative vaccines for other HPV-induced cancers, including skin cancer, and enable virus eradication. This could lead to complete clearance of lesions and prevention of reoccurrences. Our case report exemplifies a unique case of a low-risk patient who developed an HPV-associated SCCIS on her left palmar hand, despite having none of the contributing risk factors. At 34 years of age, she was well below the median age of developing SCCIS, and she presented with no history of immunosuppression or EV. Considering these findings, we speculate that tumor development in our patient relied on the combined effects of UVR exposure, localized immunosuppression, and the co-carcinogenic effects of HPV infection.


High-risk papillomavirus (HR-HPV) testing combined with cytology improves the detection of cervical lesions and increases length of screening intervals. For a population-based HR-HPV survey, testing automation is in great need. The Cobas 4800 HPV Test System is a fully automated assay that can simultaneously detect HPV16, HPV18, and other 12 pooled HR-HPV genotypes. This system has been employed for HR-HPV screening in a number of countries; however, such application in a large population in China has not been documented. In this study, we employed the Cobas 4800 HPV Test System to detect HR-HPV in cervical cytology specimens collected from a total of 5650 asymptomatic women from a region of South China. We reported the following: (1) the prevalence of the 14 genotypes of HR-HPV was 12.96%; (2) for those with HR-HPV infection, 2.25% were positive for HPV16, 0.50% for HPV18, 9.15% for pooled 12 HPV types, and 1.06% for multiple HPV infection; and (3) there was no significant difference in the HR-HPV prevalence among different age groups. HPV16 and HPV18 have been shown to be the predominant HPV types found in cervical cancer patients in some regions in China, indicating that a fully automated assay like the Cobas 4800 HPV Test System is especially valuable for population-based HR-HPV screening in these regions as this assay can concurrently detect HPV16 and HPV18. Human papillomavirus (HPV) is a double-stranded DNA virus of the Papillomaviridae family . There aTo date, various methods have been reported for HPV genotyping and detection , 10. ForThis study was approved by the Medical Research Review Committee of Zhongshan Hospital of Traditional Chinese Medicine. Informed consent was obtained from all participants.Asymptomatic women aged 20-65 years were enrolled from January 2018 to December 2018 for this study with the testing cost covered by study subjects or their employers or in combination. The inclusion criteria include being 20 years or older, being not pregnant, having intact uterus, being sexually active, or having history of sexual activity. All subjects were informed of not using vaginal medications, vaginal contraceptives, or lubricants for 48 hours before cervical sampling. Cervical cytology specimens were collected using the Cervex-Brush Combi device  as follows: the endocervical sampling part of the device was inserted into the endocervical canal to allow the lateral bristles to fully contact the ectocervix, and the device was then rotated two full turns clockwise. After withdrawal, the device with the harvested cells was immediately inserted into a vial containing PreservCyt Solution  and vigorously swirled. Samples were used for HPV DNA detection as described below.β-globin, to monitor cell adequacy, and each run included a set of HPV Positive and Negative Controls. DNA extraction and purification were done with the Cobas x 480 Instrument according to the manufacturer's instructions. Briefly, the PreservCyt Solution samples were vortexed and placed on the sample carrier, and the reagents, e.g., Lysis Buffer, Wash Buffer, and Elution Buffer, were loaded in respective reagent reservoir carriers. After sample and reagent loading, DNA preparation was completed automatically and final DNA products were collected into a microwell plate. Subsequently, the microwell plate containing DNA was manually sealed and loaded on the Cobas z 480 Analyzer, and the amplification and hybridization were completed automatically.Lysis Buffer, Wash Buffer, Elution Buffer, HPV Positive and Negative Control Kits, and all other reagents were purchased from Roche Molecular Systems Inc. Each sample had an internal control, χ2 test was used to assess categorical variables. All statistical tests were 2-sided, and P values less than 0.05 were considered statistically significant.Statistical analysis was performed using SPSS 13.0 . The A total of 5650 asymptomatic women aged 20-65 years with a mean age of 38.8 ± 12.2 years met the study requirement and were included in this study. Of these participants, 732 (12.96%) were detected as HR-HPV-positive, including 127 (2.25%) with HPV16 infection, 28 (0.50%) with HPV18, 517 (9.15%) with pooled 12 HPV types, and 60 (1.06%) with multiple HPV infection  .According to the age, participants were divided into 4 groups: 20-30, 31-40, 41-50, and >51 years. As shown in In this study, we used the Cobas 4800 System to detect HR-HPV in 5650 asymptomatic women and found the following: (1) the prevalence of the 14 genotypes of HR-HPV was 12.96%; (2) of the 732 HR-HPV-positive women, 2.25% were positive for HPV16, 0.50% for HPV18, 9.15% for pooled 12 HPV types, and 1.06% for multiple HPV infection; and (3) there was no significant difference in the HR-HPV prevalence among different age groups.Both the RealTime High Risk HPV Test from Abbott Molecular Inc.  and the Cobas 4800 HPV Testing System are clinically validated, fully automated PCR-based assays for the detection of HR-HPV . One advIntegrating HPV Testing in Cervical Cancer Screening Programs-a Manual for Program Managers . These tests consist of hybridization-based assays, i.e., the Hybrid Capture 2 technique and the CareHPV test, and PCR-based assays, e.g., the Cobas HPV test, the Abbott RealTime High Risk HPV test, the Cervista HPV HR test, and the BD HPV assay. All PCR-based assays require DNA isolation while hybridization-based assays do not. Of all the methods, the Cervista HPV HR Test takes 6-7 hours and the Abbott RealTime High Risk HPV assay takes 6-8 hours to run, which may not fulfill the goal of “see and treat” the same day. In contrast, all the rest methods including the Cobas 4800 System, however, require a maximal run time of approximately 5 hours, suitable for patients to complete “see and treatment” in one visit.Several clinically validated molecular systems for the detection of HR-HPV DNA have been included by the World Health Organization in 2016 in the manual Globally, HR-HPV prevalence varies from continent to continent . In ChinTwo studies with the largest sample size conducted in China have compared HR-HPV prevalence rates among age groups. Zeng et al. retrospectively analyzed 671,163 HPV tests performed using the Hybrid Capture 2 method from 2007 to 2014 and demonstrated the highest HR-HPV prevalence of 36.9% in the age group of <20 years while the HR-HPV-positive rates for the age groups of 20-30, 30-40, and 40-50 years were 24.4%, 19.7%, and 19.9%, respectively . HR-HPV HPV16 is the most common type of HR-HPV found in cervical samples of women from North , Southeast (Shanghai and Zhejiang Province), and South China (Guangdong Province) regions, with a positive rate ranging from 1.5 to 3.6% –25, whic


This study aimed to estimate the prevalence of genital, anal and oral HPV infection in Brazil through systematic review and meta-analysis.We searched EMBASE, LILACS, MEDLINE, Web of Science and SciELO from inception to December 2018. Original research articles that assessed the prevalence of genital , anal and oral HPV infection in Brazil were selected in pairs by independent authors. No sex, age, HPV vaccination, language or date restrictions were applied. HPV prevalence was estimated and stratified according to risk factors population and by geographic area throughout the country. The study prevalence was pooled using a random effects model. Analysis was performed using R (version 3.5.2), packages meta version 4.9–4 and metaphor 2.0–0. This review is registered on PROSPERO under protocol number CRD42016032751.We identified 3,351 references. After the screening process, 139 of them were eligible for this systematic review . Prevalence of cervical HPV was 25.41% (95% CI 22.71–28.32). Additionally, prevalence was 36.21%  in the penile region, 25.68%  in the anal region, and 11.89%  in the oral region. Subgroup analysis showed prevalence in each anatomic site was higher in high-risk populations.The prevalence of HPV is high in the Brazilian population and varies by population risk and anatomic body site, with lower rates in the oral cavity compared to that in the cervical, penile and anal region. Studies on HPV have primarily been developed to evaluate infection and cancer in the cervical region. There is a profound lack of HPV data in many geographic regions of Brazil and for different anatomic sites. Human papillomavirus (HPV) is a DNA virus from the Papillomaviridae family, comprising over 170 identified types . The gloHPV is the most common sexually transmitted infection, with sufficient evidence of its carcinogenic effects at different sites ,4. WorldHPV prevalence varies widely between genders as well as among anatomic sites . The worThe HPV National Vaccination Program in Brazil started in 2014, and it will take time to determine the effect of vaccination on the HPV rates . UnderstTherefore, the aim of this systematic review and meta-analysis was to assess the prevalence of genital, anal and oral HPV infection in Brazil to provide support for the Ministry of Health to establish health policies and programs.This review has been registered on PROSPERO under protocol number CRD42016032751. This protocol has also been published in a peer reviewed open access journal .www.greylit.org). Data from conference proceedings were included (even without an author response) if the abstract provided enough information to assess its eligibility and to extract at least the overall prevalence and number of participants.We performed the search of the following databases through December 28, 2018 : EMBASE,The inclusion criteria for articles were as follows: (1) randomized controlled trials, cohort studies, cross-sectional studies, or prevalence studies; (2) studies evaluating the prevalence of genital , anal, and oral HPV infection in Brazil; and (3) HPV assessment via polymerase chain reaction (PCR), hybrid capture or any well-described genotyping methodology. We excluded studies evaluating only pregnant women or HIV-positive participants as well as studies that analyzed material such as blood, sperm and urine. No sex, age, HPV vaccination, language or date restrictions were applied.The evaluation of titles and abstracts was conducted by either VC and ABB or NK and FSF, always in pairs, and using standardized forms. Discrepancies were solved through consensus. We collected the following data: publication title, authors, publication year, study design, population characteristics , the number of HPV-positive and HPV-negative, HPV type, HPV detection and genotyping methodology. For cohort and randomized studies, we extracted only the baseline characteristics of the population. For studies with no data regarding the geographic area where the samples were collected, we contacted the authors to obtain this information. If duplicate studies were discovered, either the study published earlier or the study that provided more information was included. An email was sent to the authors of the eligible duplicate studies to obtain more details about these articles.We stratified the study population according to risk factors. The population samples/community were defined as low-risk when including asymptomatic healthy individuals who were enrolled in a cancer screening program, attended primary care clinics or did not have any clinical lesions, whereas the high-risk population was defined as partners of women positive for a lesion , women with cervical lesion or recruited from a sexual health clinic or key populations .The included studies were assessed for quality using an adapted version of the NIH’s ‘Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies . RevieweCochran’s Q test and the I2 statistic. Sensitivity analysis was conducted by meta-regression analyses to investigate the effect of the genotyping method. We performed subgroup analyses for the population risk factor (low- or high-risk), geographic area , anatomic site , high-risk HPV (HR-HPV) type, and presence of HPV-16 and 18. Studies in which the HPV type (high- or low-risk) was either not assessed or not reported were excluded from the HPV type-specific analyses. We also estimated prediction intervals to provide a range of expected HPV prevalence among Brazilian population [Random effects models were used to calculate the pooled prevalence measures and the corresponding 95% confidence intervals (CI). The heterogeneity of the findings was assessed using both the pulation . PublicaWe identified 3,351 abstracts from the selected databases, and 16 additional studies were included from the listed references of the identified manuscripts as well as other sources. From the 1,562 unique citations, 1,024 were excluded, and the full text of the remaining 538 publications were read. We excluded 394 articles in the subsequent full-text assessments for reasons such as absence of full text, same population of articles already included, incomplete data, different designs form the inclusion criteria, different methods from the inclusion criteria, different anatomic site from the inclusion criteria, without specific data from Brazilian population, or without Brazilian population . After tA summary of the studies characteristics is presented in The majority of the included studies were not population-based and did not have information regarding either the sample size power or the participation rate. The overall quality of evidence for HPV prevalence was rated as very low according to GRADE, mainly because the studies had selection bias, inconsistency and provided indirect evidence. The quality of evidence assessment is shown in Funnel plots were symmetrical for the cervical HPV prevalence analysis, and Begg’s test was not significant for any region of the body, except for the anal region which could not be assessed by the asymmetry test due to the small number of articles .2 = 98%; 2 = 96%; 2 = 94%, 2 = 91%, The overall prevalence of cervical HPV was 25.41%  in low-risk populations (2 = 97%) in high-risk populations  as shown in 2 = 89% 2 = 97%; Among the analyzed studies, the prevalence of anal HPV was 25.68%   , and the2 = 96%; . The preThis systematic review included more than 50,000 people and showed that Brazilian population have a high prevalence of HPV infection as compared to women with normal cytology in different world regions such as Central America 13%), Northern Africa (9.2%), Western Europe (9%) and Southern Asia (7.1%) %, Northe. The higOverall prevalence of HPV and the prevalence of high-risk HPV differs slightly across regions in Brazil. Most studies included in this review were carried out in the South and Southeast regions. This was an expected result, as these are highly populated and the most economically developed regions of Brazil . CervicaOur study showed a prevalence of cervical HPV of 24.11% and 38.01% in low- and high-risk populations, respectively. According to a meta-analysis of one million women, the estimated global HPV prevalence in women with normal cytological was 11.7%, but it was 16.1% in Latin America . With thIt was not possible to pool data for specific genotypes beyond HPV-16 and HPV-18. Our results show a relatively high prevalence HPV-18 in females when compared to data from the USA  and otheThe HPV prevalence in the penile region was 25.49% in the people without clinical lesions and even higher among high-risk populations (54.24%). According to a previous systematic review, genital HPV infection in men ranges from 1.3% to 72.9% , and theThe prevalence of HPV in the anal region was similar to that of the cervical region and comprised 25.68% of population. Anal HPV prevalence is typically higher among high-risk groups, which is in accordance with previous studies . There wPrevalence of cervical HPV in Brazil is higher than in other developing countries. A past review has estimated generally lower prevalence of HPV in the cervix of healthy females in most developing and developed countries, but some of this discrepancy could be explained by the broader inclusion criteria of our study . While tThis meta-analysis shows an oral HPV prevalence of 6.69% among the population without clinical lesions and 22.42% within the high-risk population. As is commonly known, oropharyngeal cancers are associated with tobacco and alcohol intake, but the incidence of HPV-induced cancers increased and HPV infection is now recognized as an important etiological factor in this disease ,169. A pIn terms of public health policy, our results suggest that while some efforts could be targeted towards regions and populations with higher HPV prevalence, broad approaches  seem appThis study is a comprehensive systematic review that used a broad search strategy, including large studies that analyzed different anatomic sites and represented different geographic regions throughout the country. Additionally, the findings of this systematic review increase our understanding of overall and HR-HPV prevalence within the general population in Brazil and by geographic region. We understand that the included studies did not accurately represent a sampling of Brazilian cities; however, this review provides the best estimated data on HPV prevalence to date.Our study has some limitations. Brazil is a vast country, and the prevalence of HPV in some regions was either reported in a small number of studies or not reported at all. Thus, the population included in our review may not be representative of the general population throughout Brazil. The high heterogeneity among studies may impact in the exact prevalence measured. In contrast with randomized trials, non-controlled studies, such as the included in this systematic review, have smaller sample size and consequently higher imprecision and statistical inconsistency. Given that the estimates of individual studies are included in the prediction interval, we hypothesize that most of the heterogeneity are result of the different settings . Furthermore, participants of all ages were included in the present study, and it is known that HPV genital infection decrease with increasing age . On thisPrevalence studies are often used to devise public health actions and improve health services as well as serve as the first step to evaluate national control programs. The overall quality of the evidence for HPV prevalence was low or very low for all anatomic sites. In 2014, Brazil launched a national HPV vaccination program that includes girls between 9 and 13 years old and since 2017 the program started including boys of 12 and 13 years of age. Robust evidence is important for adequate monitoring of the populational impact of the program across Brazil. In view of the lack of proper national evidence regarding the HPV prevalence and databases tracking HPV infection rates, as well as to meet the needs of the Brazilian Ministry of Health, we conclude that is necessary to develop a nationwide study to investigate HPV presence among the Brazilian population. A nationwide HPV prevalence study (POP-Brazil study) with young adults between 16–25 years is in development, and it is predicted that the results of this study will fulfill the lack of epidemiological information detected in this review .In summary, the prevalence of HPV in Brazilian population is high and varies by anatomical body site, with lower rates in the oral cavity compared to that in the cervical, penile and anal region. Studies on HPV have been primarily developed to evaluate infection and cancer in the cervical region. There is a profound lack of HPV data in many geographic regions of Brazil and for different anatomic sites.S1 PRISMA checklist(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


The multi-subunit structural maintenance of chromosomes (SMC) 5/6 complex includes SMC6 and non-SMC element (NSE)3. SMC5/6 is essential for homologous recombination DNA repair and functions as an antiviral factor during hepatitis B (HBV) and herpes simplex-1 (HSV-1) viral infections. Intriguingly, SMC5/6 has been found to associate with high-risk human papillomavirus (HPV) E2 regulatory proteins, but the functions of this interaction and its role during HPV infection remain unclear. Here, we further characterize SMC5/6 interactions with HPV-31 E2 and its role in the HPV life cycle. Co-immunoprecipitation (co-IP) revealed that SMC6 interactions with HPV-31 E2 require the E2 transactivation domain, implying that SMC5/6 interacts with full-length E2. Using chromatin immunoprecipitation, we found that SMC6 is present on HPV-31 episomes at E2 binding sites. The depletion of SMC6 and NSE3 increased viral replication and transcription in keratinocytes maintaining episomal HPV-31, indicating that SMC5/6 restricts the viral replicative program. SMC6 interactions with E2 were reduced in the presence of HPV-31 E1, suggesting that SMC6 and E1 compete for E2 binding. Our findings demonstrate SMC5/6 functions as a repressor of the viral replicative program and this may involve inhibiting the initiation of viral replication. Human papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. Infection with certain “high-risk” HPV types  causes nearly all cervical cancers and an increasing number of oropharyngeal cancers . WiSMC5/6 has previously been reported to inhibit the life cycle of hepatitis B (HBV) by repressing transcription of the non-replicating, covalently closed circular DNA (cccHBV) intermediate form of HBV ,9,10,11.While our findings are in agreement with others describing an antiviral function for SMC5/6 ,9,10,11,HPV E2 proteins have been reported to influence the gene expression and protein stability of host factors ,29. NeitIn conclusion, our study has identified a novel role for the SMC5/6 subunits SMC6 and NSE3 in the life cycle of HPV-31. Our observation that transient SMC6 and NSE3 depletion results in increased HPV-31 replication and transcription implies that SMC5/6 represses the replicative program of HPV-31. The function of SMC5/6 in hepatitis B virus and herpes simplex-1 viral infections appears to be the repression of viral DNA transcription; however, we have not determined whether the antiviral functions of SMC6 and NSE3 during HPV infection are specific to transcription, replication, or both. While we have demonstrated that SMC6 is present on episomal HPV-31 genomes and interacts with HPV E2 proteins, the mechanism by which SMC5/6 represses the viral replicative program remains unclear. SMC5/6-mediated repression may involve inhibiting the E2 recruitment of E1 or other replication factors to the viral ori.Plasmids, siRNA, and Antibodies. The plasmids used were FLAG-SMC6/pcDNA3 , HPV-31 FLAG-E2/pcDNA3, HPV-31 FLAG-E2mt , HPV-31205-372; ), and raCell Culture. HEK293 and HEK293TT cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1X penicillin/streptomycin  at 37 °C and 5% CO2. HaCaT/HPV-31 FLAG-E2 and HaCaT/pcDNA3 were cultured in DMEM/10% FBS and 200 μg/mL G418 . CIN612-9E cells  were cultured in E-media (3:1 [v/v] DMEM/Ham’s F-12/5% FBS/0.4 μg/mL hydrocortisone/0.1 nM cholera toxin/5 μg/mL transferrin/5 ng/mL hEGF/5 μg/mL insulin/adenine μg/mL/1X penicillin/streptomycin) with mitomycin C-treated J2 3T3 fibroblast feeders (J2s). NIKS/HPV-31  were cultured in F-media (3:1 [v/v] Ham’s F-12/DMEM/5% FBS/0.4 μg/mL hydrocortisone/0.1 nM cholera toxin/5 ng/mL hEGF/5 μg/mL insulin/24.3 μg/mL adenine/1X penicillin/streptomycin) with 250 μg/mL G418 and mitomycin C-treated J2s. N/TERT and N/TERT/HPV-16 E2 cells were cultured in KSFM supplemented with bovine pituitary extract, hEGF, and 4 μg/mL hygromycin B .Co-IP and immunoblotting. HEK293 or HEK293TT cells were transfected using polyethylenimine  and then lysed 48 h later in low-salt NP-40 lysis buffer (0.5% NP-40/150 mM NaCl/50 mM Tris-HCl (pH 8)/10% glycerol) and protease inhibitor cocktail . Lysates were rotated for 1 h at 4 °C with benzonase (Millipore) and spun and the supernatant was then collected. For FLAG co-IP experiments, samples were rotated overnight at 4 °C with M2 agarose bead slurry . SMC6 co-IPs were performed as in V5 and E1 co-IPs, but using either rabbit anti-SMC6  or rabbit IgG control  antibodies. Beads were washed four times, alternating high-salt (0.5 M) and low-salt (150 mM) NP-40 lysis buffer washes, and boiled, and SDS-PAGE with the semi-dry transfer of proteins to PVDF membranes was then performed. Membranes were blocked in 5% milk/1X PBS/Tween-20 (0.1%) and then probed overnight at 4 °C with primary antibodies. Protein detection was performed using HRP-conjugated 2° antibodies and chemiluminescence substrates (GE). Total protein was isolated by lysis in 10% SDS/150 mM NaCl lysis buffer, samples were boiled, and Western blot analysis was performed as described above.Chromatin immunoprecipitation (ChIP). ChIP was performed using the ChIP-IT Express Enzymatic kit , according to the manufacturer’s protocols. qPCR was performed using SsoFast Evagreen Mastermix  with primers listed in t values normalized to input samples and fold change calculated relative to control IgG samples (set to 1). Statistical significance was determined by a comparison of fold change values against negative DNA binding sequences or control IgG using a one-tailed Student’s t-test.qPCR DNA replication assay. The transient depletion of SMC5/6 subunits was performed using CIN612-9E and NIKS/HPV-31 cells reverse-transfected with 15 or 20 nM siRNA, respectively, using Lipofectamine 2000 , as per the manufacturer’s protocol. The following day, media were changed and cells returned to incubation for an additional 24 h. DNA was then isolated by phenol/chloroform extraction and qPCR performed as described above with Ct values normalized to β-actin and fold change calculated relative to control groups. All experiments were performed in triplicate and statistical significance was determined by a two-tailed Student’s t-test.RT-qPCR. RNA was isolated using PureLink RNA Micro kits . RQ1 DNase  was used to remove genomic DNA contaminants. Reverse transcription was performed using SuperScript III reverse transcriptase , qPCR was performed as described above with Ct values normalized to GAPDH or β-actin, and fold change was calculated relative to control groups and primers listed in t-test.


The Human Papillomavirus (HPV) is the causative agent of different kinds of tumors, including cervical cancers, non-melanoma skin cancers, anogenital cancers, and head and neck cancers. Despite the vaccination campaigns implemented over the last decades, we are far from eradicating HPV-driven malignancies. Moreover, the lack of targeted therapies to tackle HPV-related tumors exacerbates this problem. Biomarkers for early detection of the pathology and more tailored therapeutic approaches are needed, and a complete understanding of HPV-driven tumorigenesis is essential to reach this goal. In this review, we overview the molecular pathways implicated in HPV infection and carcinogenesis, emphasizing the potential targets for new therapeutic strategies as well as new biomarkers. The Human Papillomavirus (HPV) is the causative agent of more than 90% of cervical cancers, but it is also implicated in the development of other malignancies, such as non-melanoma skin cancers, anogenital tumors, and squamous cell carcinoma of the Head and Neck (HNSCC) . DespiteIn this review, we will give a broad overview of the molecular mechanisms and pathways affected by HPV as well as which of them has potential features for new biomarkers or targeted therapy.Papillomaviridae family . M. M98]. MAutophagy is a self-consumption mechanism used by cells to maintain homeostasis. It balances sources of energy in response to nutrient stress. It is involved in degradation of long-lived proteins, misfolded proteins, and damaged mitochondria, as well as the elimination of intracellular pathogens . It has HPV16 E5 down-regulates the mRNA expression of autophagic genes, such as ATG4a, ATG5, LC3, ULK1, ULK2, Beclin 1, and ATG7, suggesting a downregulation of phagophore assembly (reviewed in ). With aEpithelial-mesenchymal transition (EMT) is a phenotypical change occurring in the epithelial cells of many malignant tumors, and it is a shift in polarity corresponding to greater invasiveness and metastatic potential ,116,117.At the molecular level, many changes are associated with and lead to EMT in cancer cells. Their increased invasive potential is supposedly due to HPV-16 E6 and E7 oncoproteins’ activation of Slug, Twist, and ZEB1/2 transcription factors. The action of E7 leads to actin reorganization and mutation of important cell adhesion molecules. For example, mesenchymal-related Vimentin (VIM), N-Cadherin, and Fibronectin (Fn) overcome epithelial cell-cell adhesion complexes such as E-cadherin, occludin, claudin, and β-catenin a 116,12,123. Sev2+) dependent membrane proteins further divided into cadherin, cadherin-related proteins, and protocadherin families whose role is mainly related to cell adhesion and various developmental differentiation processes [Among the various changes induced by HPV, the cadherin switch is considered an important indicator of cell transformation, which, as mentioned, is associated with invasiveness and metastatic potential ,117,124.rocesses ,126. Cadrocesses . Thus, Erocesses . E-cadherocesses . Uncontrrocesses . Their uInterestingly, several studies observed how oncogenic viruses such as HPV-16, can induce E-cadherin downregulation in epithelial cells in favor of N-cadherin and EMT upregulation, favoring the formation of dysplastic lesions and cancer ,130. A sThe Wnt signaling pathway is a mechanism by which a range of glycoproteins called Wnt transduce signals from the outside to the inside of the cell through the action of β-catenin, a protein originally thought to be only involved in cell adhesion but now known to be also implicated in cell signaling and regulation of developmental and homeostatic processes ,133. ThiAs previously mentioned, EMT is associated with several oncoviruses including HPV-16 and leads to the transformation of cells ultimately favoring cancer progression. A study by Rampias et al. reported that upon knock down of E6/E7 oncoproteins, there was a significant upregulation of Siah-1, a protein which normally promotes the degradation of β-catenin through the ubiquitin/proteasome system . The effAs introduced in the previous section, it is well established that most tumors present an immunosuppressive microenvironment facilitating their growth and development . This doNatural killer (NK) cells, which are a potent cytotoxic defense against pathogens and tumor cells and which respond to the downregulation of HLA antigens and MHC (major histocompatibility complex) class I induced by tumor cells to evade recognition, are rarely found in the TME . This isSaloura et al. investigated CTL infiltration in HPV-positive and negative HNSCC and analyzed the presence of immune checkpoints according to HPV status . First, Nuclear factor kappa B (NF-κB) is a family of transcription factors which bind the immunoglobulin (Ig) κ light chain enhancer element of B cells and hold a very important role in a range of physiological and immune functions. They were found to control cell growth and survival but also regulate immune responses and inflammatory processes . Five meIn cervical cancer, HPV E6/E7 oncoproteins downregulate NF-κB through a mutated IκB, preventing an immune response . In thisThe Janus Kinase/Signal Transducer and Activator of Transcription pathway (JAK/STAT), similarly to NF-κB, mediates signaling pathways controlling cell proliferation and survival (mainly STAT3 and 5) and also has a role in immune responses (STATs 1 and 2). JAK/STAT mediates signals  from transmembrane type I and II receptors directly into the nucleus, allowing a quick response to immune stimuli. Receptor dimerization induces phosphorylation of JAKs and of the receptor’s cytoplasmic tyrosine residue sequentially. STATs then bind and are phosphorylated to form dimers which translocate to the nucleus ,155. ThiThis pathway allows an anti-viral response which blocks its replication and spread, facilitating a response. HPV seems to interfere with this, and E6 and E7 may be implicated in a mutated pathway favoring the transcription of mutated genes and the development of cancer ,157. HPVAs seen, various cytokines are involved in the oncogenesis of several tumors, starting from the infection of cells by high-risk HPV (in the case of HPV-positive tumors) up to the dysregulation of many of the pathways involved . Tumor nThe exact role of TNF-α in tumor development is not entirely clear. Some studies do indeed suggest a beneficial role of the Th1 polarization in HPV infections, suggesting that polarization prevents replication and the transformation of infected cells through apoptosis. Other studies involving HPV-16 and 18, however, suggest a viral resistance and thus an irrelevant effect of TNF-α ,165. SomMicroribonucleic acids (miRNAs) are small (19–25 nucleotides long) single stranded RNAs, also found in cervical cancer and HNSCC, that are both HPV-positive and HPV-negative The DNA damage response (DDR) is a repair mechanism found in cells which detects damaged DNA, adjusting it and preventing its duplication . The majOncogenic viruses have been found to interfere with and modulate various components of these DDR pathways in order to survive and replicate in host cells. HPV uses E2 to identify an origin of replication and recruit the E1 helicase. E6 and E7 then allow the virus to degrade important regulatory proteins in order to interfere with the cell-cycle; re-entry into the S-phase allows amplification of the viral DNA . SeveralDNA methylation is an epigenetic change consisting in the covalent modification of a strand of DNA through the addition of methyl groups. This is accomplished and maintained through the action of several enzymes called DNA methyltransferases. In humans, this involves the 5′ position of cytosine rings placed at 5′ from guanine bases, thus forming CpG islands (reviewed in ). Like oDNA hypermethylation was reported in both host and HPV genes of tumor cells and was recently proposed as a biomarker for cervical cancer as well as for other HPV-related cancers such as HNSCC ,189. TheIn a screening of various populations of HPV-positive women, in which all HR-HPVs were included, CpG islands of L1 and L2 genes were analyzed in several cervical cancer types. The results showed a strong correlation between positivity to HPV (independent of HPV type), methylation, and cancer risk, suggesting the possible future application of methylation assays in diagnostic cancer screens for HPV-positive patients . SimilarHPV tumorigenesis is quite complex. Understanding all the events which take place during this process is important to help future research towards the development of effective therapies against HPV-tumors. Here we showed a variety of mechanisms by which HPV can impinge cellular pathways for its own needs. It can interact with and change the activity of many proteins.Processes altered by HPV ranged from its most known targets, p53 and pRb inhibition dependent pathways, to signaling pathways such as EGFR and MAPKs. HPV can also tailor energy and metabolism by targeting Akt, mTOR, and autophagy. DNA damage response is pushed by HPV to obtain an efficient viral replication. Epithelial-mesenchymal transition is achieved by HPV, as well as deregulation of immune and inflammatory responses. We also reviewed many miRNAs that are regulated by HPV and interfere with a plethora of cellular pathways. HPV also upregulates DNA methylation, leading to inhibition of tumor suppressors.Among these cellular processes, we highlighted several potential targets for HPV therapies, but further studies are needed to understand which can evolve from a potential target to a real targeted therapy for HPV-tumors. We have also discussed potential biomarkers involved in pathways that are differentially affected or modified during the onset of HPV-related cancers, such as Akt, mTOR, miRNAs, TNF-α, TGF-β, BRCA1, and FANCD2. However, the translation to the clinics of potential prognostic and/or diagnostics biomarkers remains a challenge. In this respect, in order to advance our understanding, it will be crucial in the next years to develop strong preclinical models and increase the number of clinical studies.Importantly, HPV vaccination is the most important prevention effort towards the eradication of HPV-driven diseases. These campaigns are already ongoing worldwide (reviewed in ).


Women completed a postal questionnaire ~2 weeks after receiving their screening result. Unadjusted mean anxiety scores ranged from 32.9 (standard deviation [SD] = 12.2) in HPV‐negative women to 42.1 (SD = 14.9) in women who were HPV‐positive with abnormal cytology. In adjusted analyses, anxiety was significantly higher in women testing HPV‐positive with either normal cytology  or abnormal cytology , than the control group. Distress was slightly higher in women who tested HPV‐positive with abnormal cytology , than the control group. We also found increased odds of very high anxiety in women who tested HPV‐positive with normal or abnormal cytology compared to the control group. This pattern of results was only observed among women receiving their first HPV‐positive result, not among women found to have persistent HPV at 12‐month follow‐up. Testing HPV‐positive with normal cytology for the first time, is associated with elevated anxiety despite carrying very low immediate cervical cancer risk. However, receiving the same test result at 12‐month early recall does not appear to be associated with higher anxiety, suggesting anxiety may normalise with repeated exposure and/or over time.We used a cross‐sectional survey to examine short‐term anxiety and distress in women receiving different results following routine human papillomavirus (HPV) primary testing at cervical screening. Participants were women aged 24–65 ( What's new?While a positive test for human papillomavirus (HPV) with negative cytology is associated with low cervical cancer risk, recall for additional HPV testing may have undesirable psychological impacts. In this study, involving women who participated in HPV primary screening in England, women who were informed of an HPV‐positive result for the first time experience increased anxiety and concern. Meanwhile, positive HPV testing at 12‐month recall had little adverse psychological impact, suggesting that anxiety concerning a positive HPV result is relatively short‐lived. Nonetheless, strategies should be in place to mitigate anxiety in women receiving a positive result for the first time. ANOVAanalysis of varianceGHQGeneral Health QuestionnaireHPVhuman papillomavirusHRAhealth research authorityIMDindex of multiple deprivationMDmean differenceNHSNational Health ServiceNHSCSPNational Health Service Cervical Screening ProgrammeORodds ratioSDstandard deviationS‐STAIshort‐form state‐trait anxiety inventoryOver 3 million women take part in the National Health Service Cervical Screening Programme (NHSCSP) in England every year.Psychological considerations are central to the successful implementation of HPV primary screening. Under the primary screening protocol, all women who attend will be told whether they test positive or negative for high‐risk HPV. Testing positive for HPV can lead to elevated anxiety, fear and concern related to the possible development of cervical cancer.We aimed to compare anxiety and distress between women receiving the different possible test results in HPV primary screening. The partial conversion of the pilot screening laboratories to HPV screening allowed us to compare these women with those receiving a normal result as part of the current cytology‐based programme.A cross‐sectional between‐groups design was employed to assess women's psychological responses shortly after receiving their cervical screening test results (baseline), as well as 6 months, and 12 months later. This article reports baseline findings.Participants were women aged 24–65 years who had been screened in one of five NHS sites in England using HPV primary testing as part of the NHSCSP pilot: North West London, Sheffield, Norfolk and Norwich, Liverpool and Central Manchester. The NHS pilot sites had catchment areas with broad geographical coverage across England. Baseline recruitment to our study commenced on 18/11/2016 and ceased on 14/10/2017 with approximately 3–5 months of active recruitment per site. Health Research Authority (HRA) approval was granted on 26/09/2016 .Women were eligible if they had received one of six possible combinations of HPV and/or cytology test results within the last 2 weeks, as indicated by their NHS clinical records. The sampling strategy aimed to recruit roughly equal numbers of women from each test result group. Five of the groups were recruited from women included in the HPV primary screening pilot. We also included a control group who had received a normal cytology result at standard cytology‐based screening within the same geographical areas and processed by the same laboratories. Table via a web link which was printed in HPV primary screening information leaflets and sent alongside their screening invitation letters. The link directed women to our university departmental website which provided study information as well as details of how to opt‐out of being approached to take part. No women opted out.Prior to participant identification, women were notified about the study Eligible women were identified by staff in NHS cytology and virology departments at each of the five participating sites. Staff allocated each potential participant a unique identity number, which they recorded and linked to patient name, address, age, screening history, NHS site and test result; these outcomes will collectively be referred to as “NHS data.”Potential participants were mailed invitation packs to their home, which contained an invitation letter, participant information sheet, consent form, questionnaire booklet and prepaid return envelope. To maximise the response rate, a reminder pack containing the same documents was mailed 3 weeks later.n = 5,494) in nonidentifiable format  to allow for demographic comparisons between responders and nonresponders.Women opted to take part by returning their completed consent form and questionnaire to the university. All documents were preprinted with unique identity numbers which allowed questionnaire data to be linked with NHS data. At the end of recruitment, UCL received NHS data on all of the women approached , NHS site and Index of Multiple Deprivation score and quintile .f = 0.14) in anxiety (as measured by the S‐STAI‐6) at the 12‐month time‐point.n = 5,494). We estimated that approaching approximately 5,500 women would yield a total sample size of 1,210 at baseline, 908 at 6‐months and 681 at 12‐months. Our baseline sample was 1,148 at baseline.The study was powered to detect a small‐to‐medium between‐group difference . Demographic characteristics were compared between the six groups using one‐way ANOVA and Chi‐squared tests as appropriate.We compared the demographic characteristics of responders and nonresponders  which revealed small variations. See Supporting Information Table For each of the primary outcomes (anxiety and distress), we compared the mean scores between the six groups using univariate regression analysis. Further to this, multiple regression analysis was performed to adjust for confounding factors: age, IMD score, ethnicity, marital status, education, number of previous cervical screens and NHS site. Results are presented as mean difference (MD) compared to the control group, along with 95% confidence intervals. We also present descriptive mean values and standard deviations for each of the six groups. For the secondary outcomes , we fitted both univariate and multiple logistic regression models adjusting for the same confounding factors. Results are presented as odds ratios, indicating the odds of the outcome for each of the groups relative to the control group, with 95% confidence intervals. Due to skewed responses to the concern and reassurance items in the control group, we used the HPV positive with normal cytology group as the reference category for analyses of these outcomes.Data completeness was >96% for the majority of outcomes and factors, with the exception of anxiety (89%) and IMD (93%). We used multiple imputation assuming data were missing at random to account for missing data. The imputation model included primary outcomes and all sociodemographic factors, which we assumed included all predictors of missingness. The final models were derived by fitting a regression model including all confounders, and estimates were combined using Rubin's rules.p‐value <0.05 was considered statistically significant.Demographic characteristics have been presented using nonweighted data. All primary and secondary results have been adjusted using the weights described above and using the imputed data. Supporting Information Table Five‐thousand‐four‐hundred‐ninety‐four women were invited to take part and 1,148 returned a questionnaire (response rate of 21%). Thirteen participants were excluded from the study due to returning a questionnaire over 90 days after date of identification and eight due to ineligible age (>65); 1,127 participants were included in the analyses. See Figure Table p = 0.02, and MD = 7.2, 95% CI: 3.7–10.6, p < 0.001, respectively). There were no differences in mean anxiety scores between the other groups and the control group.Regression analysis revealed statistically significant differences in anxiety between test result groups. Women who tested HPV positive with normal cytology or abnormal cytology had higher mean anxiety scores than women in the control group . There were no differences in general distress between the other groups and the control group. Tables Regression analysis also revealed a higher mean general distress score for the HPV positive with abnormal cytology group compared to the control group  between the results groups. We found significantly increased odds of very high anxiety in the HPV positive with normal cytology group  and HPV positive with abnormal cytology group , compared to the control group (no HPV test). None of the other groups differed significantly from the control group.Logistic regression was also performed to determine the effects of test result group on case‐level general distress (GHQ‐12 score > 3/12). None of the groups differed significantly from the control group.We used logistic regression to ascertain the effects of receiving different test results on the likelihood that women scored highly for worry about developing cervical cancer in the next 10 years . After adjusting for potential confounding factors, all three HPV positive groups were found to be at significantly increased odds of high worry when compared to the control group (no HPV test), all with odds ratios over 4  and highly for reassurance . After adjusting for potential confounding factors, the odds of high concern in the HPV positive with abnormal cytology group was 1.8 (95% CI: 1.2–2.9) when compared to the HPV positive with normal cytology group. The odds of high concern were significantly lower for the three normal results groups  when compared to the HPV positive with normal cytology group. All three normal results groups had significantly higher odds of high reassurance compared to the HPV positive with normal cytology group. Reassurance was similarly high across these three normal results groups. Tables Informing women that they test positive for high‐risk HPV accompanied by any cytology result appears to be associated with some adverse psychological effects at the population level, at least in the short‐term. Our findings are consistent with previous studies showing that testing positive for HPV with abnormal cytology is associated with raised anxiety and distress.Reassuringly, women with persistent HPV and normal cytology at 12 months did not have significantly higher anxiety than the control group, although descriptively they displayed slightly higher anxiety than would be expected in the general population . This suggests that raised levels of anxiety and distress associated with an initial HPV positive result may normalise with repeated exposure to the result and/or over time, which is consistent with previous research.HPV primary screening has a high negative predictive value and therefore has the potential to reassure the majority of women who are at extremely low immediate risk of cervical cancer. Our findings indicate that testing HPV negative at any point (including 12 months after an HPV positive result) is associated with high levels of reassurance. However, although an HPV negative result offers better protection from cervical cancer than normal cytology,n = 66); however, this group had similar scores to the other normal and HPV negative groups. Our response rate was 21% which raises uncertainty regarding the extent to which our sample is representative of the wider screening population. We were, however, able to statistically weight our data to the wider screening population for age and IMD quintile as well as compare demographic characteristics between responders and nonresponders. For consistency with the previous NHSCSP evaluation of HPV triage methods,To the best of our knowledge, this is the first major study to evaluate the short‐term psychological impact of primary HPV testing within a routine national programme. Participant recruitment linked to routine clinical management through the NHSCSP HPV primary screening pilot ensured accurate data collection and broad geographical coverage across England. A control group with primary cytology allowed additional between‐group comparisons, strengthening our cross‐sectional design. Our sample size was smaller than we anticipated for the group who cleared HPV at 12‐months  should help minimise adverse psychological responses. Reasons for the switch to HPV primary screening should also be clearly communicated to the public ahead of implementation, to reduce the risk of a public backlash like the one recently observed in Australia,vs. second HPV positive with normal cytology test result suggests that anxiety is likely to be short‐lived and does not persist for women on 12‐month early recall.Testing positive for HPV with normal or abnormal cytology was associated with short‐term adverse psychological effects in routine HPV primary screening, although it is unlikely that this will lead to significant disruption of daily functioning for most women. Our cross‐sectional comparison of women receiving their first Study concept: Waller J, Marlow L, Forster A, Kitchener H, Patnick J. Measurement development: Waller J, Marlow L, McBride E, Forster A. Project management: McBride E, Waller J. Experimental analyses: Ridout D, McBride E. Drafted the manuscript: McBride E, Waller J. Final version of the manuscript: all authors. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.Table S1 Demographic characteristics of nonresponders vs. responders (no weights or adjustments applied)Table S2. Results of univariate analysis for primary and secondary outcomes by test result groups (unweighted and using raw data only)Click here for additional data file.


Despite the availability of Q methodology as a qualitative research alternative that seemingly circumvents the limits of standard qualitative methods across various fields, a recent review of qualitative research literature in leading health-related South African journals indicated that Q methodology is hardly a method of choice in South Africa.This article demonstrates the application of Q methodology, a qualitative research option, in psychological research. The methodology is suitably designed to investigate and clarify diverse subjective experiences, attitudes, opinions and/or beliefs held by a group of people on a given topic.A study on the subjective understandings and perceptions of epilepsy is used to illustrate how Q methodology works. In this particular study, a diverse group of participants, comprising students, traditional healers, doctors, nurses, pastors, high school teachers, laypeople domiciled in rural and urban areas, and speakers of two of the dominant African dialects in the area, was used.Analysis produced three distinctive factors that are appositely named the scientific, the moderated traditionalist and the community-oriented stances. Each factor, constituted on the basis of close resemblance and statistical association between the rank orderings, represents an identifiable understanding of epilepsy by an exclusive grouping of participants.Concluding remarks about Q methodology are provided. Curationis, Health SA Gesondheid, South African Journal of Psychology and the South African Medical Journal, did not find a single study using Q methodology as a method of choice. Health SA Gesondheid published 313 articles in the said period, of which 43% were qualitative. Yet, none of the latter featured Q methodology. A study by Oberholzer et al. (A 10-year (2007–2017) period inspection of entries in leading health-related South African journals, namely, r et al. :4 used ar et al. :185–186.Q methodology is a subjective procedure used to explore existing perspectives on a topic that can potentially elicit a range of distinct points of view. Unlike other standard qualitative procedures, it blends qualitative and quantitative techniques to portray operant subjectivity Brown . The proThe product of reordering stimuli is, according to Brown :18, operThere are essentially two aspects to Q methodology: the qualitative element is in plain view as the individual configures and forms a standpoint, and the quantitative element is in the background, out of sight, yet offering substantive support to the qualitative element. The entire process takes place in a series of logical steps. In this contribution, the steps followed in conducting a Q methodology study will be described. A study of different viewpoints, attitudes and beliefs about epilepsy will be used as an example.Epilepsy is a common syndrome of different cerebral disorders of the central nervous system. It is characterised by chronic recurrent but unpredictable seizures arising from an underlying brain abnormality, in particular spurts of electrical discharge in either specific or general areas of the brain. It affects approximately 69 million people worldwide, 10–15 million of whom are from Africa  area among African individuals from different walks of life. Before proceeding with the study, it was approved by the University of Limpopo’s Faculty of Humanities research and ethics committee  relating to the topic of interest, and the sum total is called a concourse or Q-population. It is from the concourse that the final, representative set of items, the Q-set/Q-sample, will be drawn. In the epilepsy study, initial interviews were conducted with diverse individuals to gather statements pertaining to the concept of epilepsy. Of course, a concourse can never be complete. The aim in putting it together is to ensure that at least the most common and diversely representative ideas are covered. The transcribed interviews were carefully scanned to locate any expression of opinion, attitudes and beliefs about epilepsy. The epilepsy literature was also consulted to add any pertinent perspectives that may not have arisen from the interviews. In this way, 137 statements were developed. The statements were hybrids because they were constructed from more than one source, including naturalistic and non-human sources ; a quasi-normal distribution grid  was used for the purpose. Analysis was carried out for persons rather than by variables , which means that it was persons’ viewpoints that were correlated rather than the conventional Pearson R standard of correlating tests. Thus, factor analysis in Q methodological analysis goes beyond the mere analysis of a transposed correlation matrix Brown :19. It iQ-methodological analysis can be performed on dedicated software. PQMethod 2.35 Schmolck  is a freA correlation analysis was carried out to produce a by-person matrix, consisting of all paired correlations of the 33 Q sorts.en vogue and presumably more precise minimum average partial (MAP) test  and the indirect involvement of supernatural forces in the cure of epilepsy , and appeared to reject genetic factors in the disease . Yet, they are open to education about epilepsy  and are negative about any type of supernatural causal explanations . Their position has to do with the cures rather than the causes of the disease. Regardless of their traditionalist viewpoint, they would discourage alcohol intake among people with epilepsy . Although the taking of medication was not a distinguishing characteristic in this factor, it nevertheless was endorsed . Participants in the ‘moderated traditionalist viewpoint’ do not seem to react with typical bigotry such as being shocked and overreacting to witnessing an epileptic attack . Interestingly, traditional healers belong to this factor.The third and final factor is called the ‘community-oriented viewpoint’ because its focus is on the socio-cultural aspects of epilepsy. Participants in this factor are of the view that epilepsy is traumatic to patients and their caregivers  and they are concerned that people with epilepsy are not well understood by community members . They strongly disagree with the ideas that epilepsy is a contagious mental disturbance , that those afflicted can seek attention by faking seizure attacks  and that its cure is linked to supernatural events. All the Q sorts completed by pastors  belong to this array. However, the array also had a nurse, a health sciences student and a teacher.There were only two clear consensus statements in this study. Participants in all factors agreed with the statement pertaining to the institution of programmes to educate the community about epilepsy  and disagreed with the description that people with epilepsy are mad . Nonetheless, a distinguishing feature about the group of participants who took part in this study is that they tended to share a number of characteristics. For instance, factors 2 and 3 participants agreed with the explanation that epilepsy can be caused by head trauma, a distinguishing feature of factor 1. It is clear from the results of the present study that the context in which this study was conducted is one where the modern, scientific view of epilepsy is making inroads. It is likely that awareness programmes and other forms of intervention have eroded the traditionalist view of epilepsy, where the causes and interventions of the disease are linked exclusively to supernatural causes.Taenia solium tapeworms , which was formed on the basis of the role of neurocysticercosis and other parasitic infections of the brain in epilepsy (Reddy & Volkmer II There are weaknesses related to Q methodology. McKeown :3 did obAnother limitation has to do with how far the results can be generalised beyond the context in which they were made. Q methodology findings have the same generalisability limitations as conventional qualitative research methods. Research data produced from Q methodology are limited because of its somewhat exploratory nature and its data not being based on random samples. Nevertheless, the latter is not a goal of the method Brown :192. It On the positive side, Q methodology is unique in that it is able to characterise the gestalt of each individual participant’s response, and then integrate it into a configuration common to other respondents sharing the viewpoint. Thus, it is a suitable and powerful methodology for exploring and explaining patterns in subjectivities, generating new ideas and hypotheses, and identifying contrasts in views, opinions and preferences. Q methodology offers an innovative approach to qualitative analysis through the ‘quantification of patterned subjectivities’ (Shemmings Q methodology is available as an approach that seemingly circumvents the limits of standard qualitative methods. In particular, the important element of the subjectivity of the researcher is present. Yet, unlike in other methods, Q methodology acknowledges its presence, and the techniques of the methodology are comparatively less amenable to it. The strength of the method lies in its combination of both qualitative and quantitative techniques to harness subjectivity.