Human papillomavirus (HPV) entry into epithelial cells is independent of canonical endocytic pathways. Upon interaction with host cells, HPV establishes infection by traversing through an endocytic pathway that is clathrin- and caveolin-independent, but dependent on the annexin A2/S100A10 heterotetramer (A2t). We examined the contribution of monomeric annexin A2 (AnxA2) vs. A2t in HPV infection and endocytosis, and further characterized the role of these molecules in protein trafficking. We specifically show that cell surface A2t is not required for HPV attachment, and in the absence of A2t virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. These findings suggest that A2t is a central mediator of high-risk HPV intracellular trafficking post-entry and pre-viral uncoating. HPV-associated diseases inflict a significant disease burden on the global population, yet there remain unanswered questions about HPV cellular entry. As such, initial establishment of HPV infection remains an active area of investigation. HPV is a non-enveloped double-stranded DNA virus composed of major capsid protein L1 (HPV L1) and minor capsid protein L2 (HPV L2)4. Although structurally simple, HPV infection depends on the exploitation of complex host cell machinery and endocytic processes. HPV type 16 (HPV16) is the most common oncogenic genotype and is widely used to study the infectious lifecycle of HPV. Since 1995, HPV entry has been thought to be receptor-mediated; nevertheless, a consensus HPV receptor has still not been identified5. Although many HPV entry-associated molecules and co-factors have been recognized in what is shaping up to be an incredibly complex and unique endocytic pathway (recently reviewed in6), a central mediator has yet to be described.Persistent infection with mucosal-tropic high-risk human papillomavirus (HPV) causes cervical, vaginal, anal, penile, and oropharyngeal cancers9. Trafficking of HPV from the cell surface to the nucleus can be broken down into five key stages: cell surface binding, entry, viral vesicle trafficking, capsid uncoating, and transporting of the viral genome (vDNA) through the trans-Golgi network (TGN) to the nucleus. HPV binds to the cell surface through two distinct attachment events. First, HPV capsid proteins interact with heparan sulfate proteoglycans (HSPGs) found on the plasma membrane of basal keratinocytes or within the surrounding extracellular matrix13. The binding of HPV to HSPGs induces conformational changes in both HPV L1 and L216, exposing the amino terminus of HPV L2 which contains a furin/proprotein convertase cleavage site17. These conformational changes in the capsid reduce HSPG-affinity and the virion is then transferred to the elusive secondary uptake receptor/receptor complex located within tetraspanin enriched microdomains (TEMs)19. Candidate receptors to date have included α6 integrin21, epidermal growth factor receptor23, and the protein complex studied herein – the annexin A2 heterotetramer (A2t)25. After handoff to this secondary receptor/receptor complex, HPV is internalized through a non-canonical endocytic mechanism and trafficked through the degradative endosomal system. While it has been shown that in optimal conditions viral trafficking may be rapid, bulk internalization is relatively slow and asynchronous due to the time it takes for extracellular structural modifications of the capsid and a hypothesized limited availability of the secondary receptor/receptor complex27. Internal trafficking is dependent on endocytic mediators including, but not limited to Rab GTPases, certain components of the ESCRT machinery, sorting nexin 17, and the cytoskeletal adapter protein obscurin-like 1 protein (OBSL1)31. Through this process, early HPV-containing endosomes are delivered to multivesicular endosomes (MVEs) where the majority of capsid uncoating occurs through compartment acidification and cyclophilin-mediated dissociation of the viral genome (vDNA) and capsomeres33. Delivery of HPV to MVEs is dependent on CD63, a tetraspanin that has been shown to facilitate HPV trafficking and directly interact with the viral capsid34. The vDNA, concealed within a vesicle, then escapes lysosomal degradation by transport to the TGN via interaction of cytosolically exposed HPV L2 with the retromer complex37. The vDNA-containing vesicle eventually infiltrates the nucleus during the nuclear envelope breakdown step of mitosis, completing intracellular trafficking and establishing infection39.The literature to date has shown that HPV16 endocytosis into host basal epithelial cells is independent of canonical clathrin-, caveolin-, flotillin-, lipid raft-, cholesterol-, and dynamin-mediated endocytosis25. However, the function and necessity of A2t at the cell surface and the precise endocytic steps mediated by the heterotetramer and/or its individual subunits in HPV infection are not well understood. A2t is a multifunctional membrane-associated protein complex composed of two annexin A2 (AnxA2) monomers bridged by an S100A10 homodimer42. AnxA2 and S100A10 are expressed in many tissues, have been studied in the context of diverse cellular processes, and are linked to multiple aspects of human health and disease45. The AnxA2 monomer and A2t, however, have independent functions and should therefore be considered biochemically distinct. In the mucosa, A2t is expressed in the basal layer of the epithelium47, which is the initial site of HPV infection. On a cellular level, A2t is localized to the inner and outer leaflets of the plasma membrane, while AnxA2 can be found in the cytosol or membrane-bound. More specifically, these proteins have been implicated in membrane domain organization, membrane trafficking events, membrane-cytoskeletal connection through F-actin binding, MVE biogenesis, and membrane curvature53. S100A10 is a small dimeric helix-loop-helix protein found in close association with AnxA2 (complexed together to form A2t) and implicated in the trafficking of a variety of membrane-resident proteins41.Previous evidence suggests a role for A2t at the cell surface and in the intracellular trafficking of HPV55. A large proportion of studies investigating AnxA2 utilize anti-AnxA2 knock-down strategies, effectively removing both monomeric AnxA2 and heterotetrameric A2t. In the past, researchers have reported findings about either moiety without truly distinguishing between the two. Therefore, to resolve the ambiguity between AnxA2 and A2t in the context of HPV endocytosis, this study employs complete protein knockout of either S100A10 or AxnA2/S100A10 (A2t) in epithelial cells (HeLa) to distinguish the specific contributions of these host factors in HPV entry and intracellular trafficking. Here, we provide evidence supporting the role of A2t in HPV intracellular trafficking, and further characterize the involvement of S100A10 in protein trafficking. Our data suggest that AnxA2 and A2t are not required for cell surface attachment nor non-infectious internalization of HPV. Instead, these data reveal a role for A2t in post-entry intracellular trafficking and show that AnxA2 interacts with CD63, a known mediator of HPV trafficking. We show that in the absence of S100A10 alone or complete A2t, cellular entry of HPV is not reduced, however, progression of early HPV-containing endosomes to MVEs is significantly inhibited. Consequently, HPV capsid disassembly is repressed, and the virus is redirected to the lysosome for degradation. Taken together, these findings suggest that A2t is required for the infectious post-entry intracellular trafficking of HPV. Additionally, we reveal a previously underappreciated role for S100A10 in endosomal transport.S100A10 is rapidly ubiquitinated and degraded in the absence of AnxA256. It has also been suggested that the A2t subunits, AnxA2 and S100A10, may play separable roles in HPV16 entry and intracellular trafficking25. Therefore, we sought to decipher the specific contributions of AnxA2 and A2t in HPV infection and endocytosis. To remove A2t, we knocked out either S100A10 or the full A2t complex via CRISPR/Cas9 gene editing and generated clonal HeLa cell populations. Given that S100A10 is rapidly degraded in the absence of AnxA255, the AnxA2 knockout (KO) effectively serves as a full A2t subunit KO. Western blot analysis confirmed the absence of protein expression in the KO clones nes Fig. .Figure 157, differences in cell proliferation between clonal populations and WT cells were analyzed to exclude altered proliferation rates as a potential confounding factor for the reduction in HPV infection that we observed. Cell counts and proliferation were measured via trypan blue exclusion and DNA-based quantification respectively  containing a GFP reporter plasmid, we then screened at least two S100A10 KO and two A2t KO clones and found consistent inhibition of infection  was used as model cargo for pinocytosis63, and uptake was analyzed over time. We observed a significant decrease in FITC-BSA uptake in both the S100A10 KO and A2t KO cells compared to WT , and further posits that A2t is an HPV L2-specific cell surface receptor for HPV1671. The second, based on detailed studies from Ozbun’s laboratory, emphasizes an interaction between AnxA2 and HPV16 capsid protein L1 (HPV L1) at the cell surface, and suggests that S100A10 is involved in post-entry intracellular trafficking25. In the absence of S100A10 and A2t, we were surprised to find no difference in the quantity of HPV bound to the cell surface. Given that HPV exposure increases AnxA2 translocation to the cell surface, and because A2t and HPV colocalize at the cell surface25, it is reasonable to hypothesize that A2t functions as an HPV receptor. However, we show quantitative evidence that A2t is not a sole requirement for the binding of HPV to the host cell surface. These data do not negate the involvement of A2t at the plasma membrane, but rather indicate that a direct HPV-A2t interaction is not required for cell surface binding. On a larger scale, these data are in agreement with the emerging consensus that HPV cell surface binding involves multi-protein complexes and warrants further work defining an HPV-entry associated membrane platform72.Two primary models for the involvement of A2t in HPV infection have been previously described25. Dziduszko and Ozbun interrogated this pathway using an anti-AnxA2 antibody targeting the N-terminus to inhibit interactions at the surface, and it is difficult to discern whether the observed kinetic effect is due to functional silencing of AnxA2 or antibody-triggered endocytosis. Nonetheless, work from the same study showed an increased colocalization of HPV16 with late endosome/lysosome marker LAMP1 following anti-S100A10 antibody treatment. These data suggested a role for A2t in HPV intracellular trafficking but fell short of giving a complete picture of the discrete post-endocytic steps that A2t and/or its subunits may be regulating. To directly interrogate these differences, we used our KO cells to investigate at which points S100A10 and A2t are required in HPV intracellular trafficking. Our results revealed the same phenotype in the S100A10 and A2t KO cells with respect to HPV binding, endocytosis, and intracellular trafficking events. These findings indicate that the AnxA2 monomer alone is not capable of shuttling the virus to the MVE.Following HPV binding to the cell surface, we found no significant difference in HPV internalization into S100A10 KO and A2t KO cells compared to WT HeLa cells. These results are inconsistent with previous findings that negative regulation of AnxA2 results in delayed HPV16 internalization8. Conversely, some investigators have hypothesized that association of HPV with various cell factors may be indicative of the utilization of multiple entry routes depending on receptor engagement25. Our data show that targeting AnxA2 and S100A10 does not lead to aberrant HPV internalization through alternative endocytic pathways, and support the previous hypothesis that HPV employs a specific and potentially novel endocytic pathway. Furthermore, our data directly demonstrate that both the AnxA2 monomer and A2t are involved in pinocytosis but not receptor-mediated, clathrin-dependent endocytosis.An important feature of this study was analyzing the effect that knocking out S100A10 and A2t has on non-HPV endocytic mechanisms. HPV endocytosis has been shown to be pinocytosis-like, but independent of canonical endocytic pathways such as clathrin-mediated endocytosis, and suggested to be novel34), and that monomeric AnxA2 alone is not sufficient to facilitate this process. Subsequently, capsid disassembly was dramatically inhibited in the absence of S100A10 and A2t, confirming previous reports and corroborating our intracellular trafficking results. These results are strikingly similar to those published by Gräßel et al. in 2016. In this study, knockdown and knock-in techniques were utilized to demonstrate the requirement of the CD63-syntenin-1 complex in HPV infection and trafficking to MVEs34. Not surprisingly, we found that colocalization of AnxA2 and CD63 is increased upon HPV infection. Consistent with observations in the literature, we also observed a notable increase in AnxA2 intensity in HPV-treated cells25. Throughout the intracellular trafficking of HPV, AnxA2 and CD63 appear to be members of a complex that likely includes other members (e.g. syntenin-1 and S100A10) and is critical for efficient HPV infection. Taken together, these data suggest that successful infiltration and unchecked descent of HPV into the host nucleus requires A2t-dependent vesicle trafficking from early endosomes to MVEs.By tracking HPV via confocal microscopy, we found that A2t is required for the progression of HPV from early endosomes to CD63+ MVEs (the late endosome stage for HPV41), but in contradiction to the data supporting these conclusions, another group claims that S100A10 is dispensable for the endosomal transport functions of AnxA274. As a result of our study design, we were able to directly test if S100A10 is involved in AnxA2-dependent vesicle trafficking. Utilizing both HPV as model cargo and BSA as pathway-specific canonical cargo in our uptake experiments, we provide clear evidence for a role for S100A10 in AnxA2-associated trafficking. Furthermore, a study investigating MVE biogenesis did not discern between monomeric AnxA2 and A2t by examining the role of S100A1053. Our findings support previous work defining a role for AnxA2 and provide the first evidence that heterotetrameric A2t is functioning in MVE biogenesis.Previous studies have implicated S100A10 in the trafficking of a variety of membrane-resident proteins  derived from cervical carcinoma were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% FBS, 1x BME, and 1x gentamycin at 37 °C with 5% CO83. Non-target plasmid was used as negative control and pMSCVpuro vector (a gift from Ling Shao) was used for selection. HeLa cells co-transfected with Lipofectamine2000 (Thermo Fisher) were selected with puromycin for 72 h, and clonal populations were generated through a limited dilution series. Clonal populations were then sequenced to verify gene disruption and analyzed for protein expression via western blot.Addgene plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)  was used for CRISPR/Cas9-targeted ANXA2 and S100A10 gene disruption via mutant Cas9 nickase (D10A variant). The following target sequences were used: 5′-CAGGAAGCACGAACATCAGC-3′ and 5′-ACAGGGGCTGGGAACCGACG-3′ for ANXA2; and 5′-AATGGTGAGGCCCGCAATTA-3′ and 5′-GTAGTACACATGAAGCAGAA-3′ for S100A10. Guide RNA (gRNA) were cloned using Feng Zhang Laboratory protocolsKnockout clones were analyzed for differences in cell proliferation via cell counting (trypan blue exclusion method) as well as DNA quantification using the CyQUANT Cell Proliferation Assay Kit (Invitrogen). Briefly, WT, S100A10 KO, and A2t KO cells were grown for 48 h, collected with trypsin-EDTA, diluted 1:1 with trypan blue stain (Invitrogen), and viable cells were counted. To quantify DNA via CyQUANT kit, cells were cultured for 48 h, frozen at −80 °C overnight, thawed at room temperature, and lysed using CyQUANT cell lysis buffer with RNAse and GR dye according to manufacturer protocol. Fluorescence of GR dye-bound DNA was measured using a SpectraMax M5 plate reader (Molecular Devices) and DNA was quantified by comparison to DNA standard curve.84 (http://home.ccr.cancer.gov/LCO/ripcord.htm). Briefly, 293TT cells were co-transfected with codon-optimized L1 and L2 plasmids for HPV16, -18, -31, and -45, and pCIneoGFP reporter plasmid. For bulk PsV preparations, the self-packaging p16L1L2 plasmid was utilized. HPV L1 and L2 expression vectors used were p16L1L2 (bulk), p16sheLL, p18sheLL, p31sheLL, and p45sheLL . Infectious titer was determined by flow cytometric analysis of GFP+ 293TT cells 48 h post-treatment and calculated as infectious units/mL. Bulk PsV preps were quantified via coomassie blue staining utilizing known BSA concentrations.HPV16, -18, -31, and -45 PsVs were prepared as previously described85. Western blot analysis confirmed the presence of HPV L1 and L2, while a neutralizing antibody ELISA confirmed the presence of intact particles. Coomassie Blue staining was performed to determine protein purity and standardize the concentration of HPV L1 content of the VLP preparations.HPV16 virus-like particles (VLPs) were produced using a recombinant baculovirus expression system in insect cells as previous described87. Anti-HPV16 L1 (H16.56E) used in immunofluorescence experiments was a gift from Martin Sapp88. Anti-AnxA2 (610069), anti-S100A10 (610071), and anti-HPV16 L1 (550840) were purchased from BD Biosciences. Mouse IgG isotype control (ab37355), rabbit IgG isotype control (ab37415), anti-CD63 (ab118307), and goat-anti-rabbit TRITC (ab6718) were purchased from Abcam. Anti-β actin (4970), anti-clathrin (4796), and anti-EEA1 (3288) were purchased from Cell Signaling Technology. Anti-mCherry (677702) and goat-anti-mouse DyLight 488 (405310) were purchased from BioLegend. Antibodies used in AnxA2-CD63 colocalization immunofluorescence microscopy were anti-AnxA2 (11256-1-AP) and anti-CD63 (ab8219) from Proteintech and Abcam, respectively. Goat-anti-mouse IRDye 800CW (925-322) and goat-anti-rabbit (H + L) Alexa Fluor 680 (A27042) secondary antibodies used for infrared western blot imaging were purchased from Li-Cor and Invitrogen, respectively.Previously described anti-HPV16 L1-specific neutralizing antibody H16.V5 and internal epitope-recognizing 33L1-7 (L1-7 Ab) were gifts from Neil Christensen and Martin Sapp, respectively50) the following day. TCID50 was determined by titrating the multiplicity of infection (MOI) for each genotype to result in approximately 50% infected cells, 48 h post-infection. Infection is defined in this manuscript as gene transduction of the GFP reporter plasmid. In infection assays, %GFP-positive cells were analyzed 48 h post-virus addition via flow cytometry . For rescue experiments, cells were seeded in 6-well plates, grown to 80% confluency, and transfected with pEGFP-N3_ANXA2-mCherry (a gift from Volker Gerke) or an empty vector using Lipofectamine 2000 (Thermo Fisher). Utilizing HPV16, cells were subjected to the HPV-infectivity assay as described above and infection rescue was analyzed by first gating mCherry-positive cells and then measuring %GFP-positive cells within that population using CXP software (version 2.2)  and then treated with either buffer (vehicle control) or 2 U/mL heparinase I (Sigma-Aldrich) for 1 h at 37 °C to remove HSPGs. Plates were transferred to ice and washed thoroughly with ice cold PBS + CaCl2. PBS supplemented with 1 mM CaCl2 was used for all wash steps as AnxA2 binds membranes in a calcium-dependent manner. Cells were then treated with 10 μg/1E6 cells HPV16 in cold serum-free media for 1 h at 4 °C to saturate binding. Cells were collected on ice via scraping, stained with H16.V5 (1:100) for 30 min at 4 °C, and fixed with 2% PFA in order to ensure measurement of particles that might internalize at room temperature during analysis. Mean fluorescent intensity (MFI) was measured via flow cytometry.2.5E5 cells were seeded in 6-well plates and grown overnight. Cells were washed with ice cold buffer  from MFI at 0 h . MFI of WT HeLa cells was set to 100% and KO cells were normalized to that value. For pHrodo-HPV16 uptake assays, HPV16 VLPs were labeled with pH-dependent rhodamine fluorophore  in a 10:1 (dye:HPV L1 protein) ratio, purified with 2% agarose beads (sized 50–150 µm) (Gold Biotechnology), and treated as described above. Briefly, after 30 min at 4 °C cells were treated with HPV16 PsVs for 4 h at 37 °C and MFI of pHrodo-HPV16 was measured via flow cytometry. MFI was measured from 0–12 h and trends remained consistent.2.5E5 cells were seeded in 6-well plates and grown overnight. Plates were cooled to 4 °C for 30 min and 5 μg/1E6 cells of HPV16 PsVs were added for 1 hr at 4 °C. Cells were collected and surface-bound HPV was measured as described in cell surface binding assay. Unbound virus was washed away with PBS + CaCl2, and then incubated for 4 h at 37 °C to promote internalization. Cells were collected by in-plate lysis using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher) and lysates were analyzed via western blot for cleaved HPV L1 (CAMVIR-1 antibody) and β-actin using Odyssey Infrared blot imager (Li-Cor).2.5E5 cells were seeded in 6-well plates, allowed to adhere, and then treated overnight (16 h) with 50 μM chloroquine diphosphate crystalline (Sigma-Aldrich) or vehicle control. Plates were cooled to 4 °C for 30 min, treated with 5 μg/1E6 cells HPV16 PsVs for 1 hr at 4 °C, thoroughly washed with PBS + CaCl2.5E5 cells were seeded in 6-well plates and grown overnight. Plates were cooled to 4 °C for 30 min and either Alexa Fluor 488-conjugated transferrin from human serum  or fluorescein isothiocyanate (FITC)-conjugated bovine serum albumin (BSA)  were added, and cells were transferred to 37 °C. Cells were collected at 0.5, 1.5, and 3 h after the temperature shift, and MFI was measured via flow cytometry.90. Using the JACoP plugin91, thresholds were automatically set, and the extent of colocalization was measured using Mander’s colocalization coefficient. Batch image analysis was achieved through an application-specific script in Fiji macro language.For HPV colocalization with EEA1 and CD63, and for L1-7 Ab staining experiments, cells were seeded on 8-well chamber slides (1E4 cell/well) and grown overnight. Slides were cooled to 4 °C for 30 min prior to treatment with 0.5 μg/1E6 cells of HPV16 PsVs for 1 h or 7 h at 37 °C. At 1 h, cells were stained for HPV16  and either EEA1 (1:100) or clathrin (1:50). At 7 h, cells were stained for HPV16 (H16.56E) and either CD63 (1:100), or L1-7 Ab (1:10). For AnxA2-CD63 colocalization, cells were seeded on 3-well chamber slides (2.5E4 cells/well) and grown overnight. Slides were treated with HPV16 PsVs at 0.5 μg/1E6 cells for 4 h or 7 h at 37 °C. Cells were stained for AnxA2 (1:100) and CD63 (1:100). For imaging in non-permeabilized conditions, Triton X-100 was omitted from the immunostaining procedure. All slides were fixed with 2–4% PFA prior to staining and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Images were visualized on a Nikon Eclipse Ti-E laser scanning confocal microscope. For colocalization analysis, 10–20 images were analyzed with Fiji Background from control groups was subtracted in all experiments and KO cells were normalized to WT cells for comparison. Statistical analyses were performed using GraphPad Prism (version 6) and detailed statistical tests for each figure are provided in legends.The datasets generated during the current study are available from the corresponding author upon reasonable request.Supplementary Figures 1 and 2


Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on genital high-risk human papilloma virus (HR-HPV) clearance and quality of cervical smear.Probiotics has been shown to be effective in reducing vaginal colonization of pathogenic organisms. The aim of this study was to investigate the influence of probiotic strains This was a randomized, double-blinded, placebo-controlled trial. Women with genital HR-HPV infection were randomized into study and control groups. A probiotic or placebo preparation was administered orally (one capsule daily) until negative HR-HPV testing. A cervical smear and HR-HPV tests were performed at the beginning of the study and every 3 months thereafter until a negative result was obtained.p = 0.006). Twenty-two women had a mildly abnormal initial cervical smear and nine had an unsatisfactory smear. At 6 months follow-up, both mildly abnormal cervical smear and unsatisfactory smear rates had decreased significantly in the study group compared to the control group (p = 0.017 and 0.027).A total of 121 women with genital HR-HPV infection were enrolled (62 in the study group and 59 in the control group). There was no significant difference in HR-HPV clearance rate between the two groups (58.1% vs. 54.2%). The only factor predicting HR-HPV clearance was a lower initial viral load (HR 3.214; 95% CI: 1.398, 7.392; Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 did not influence genital HR-HPV clearance, but may have decreased the rates of mildly abnormal and unsatisfactory cervical smears.The application of probiotic strains NCT01599416, May, 2012. Retrospectively registered.Clinicaltrials.gov  A healthy vaginal microbiome, mostly containing lactobacilli microorganisms, can aid in the prevention of bacterial vaginosis (BV), fungal infections and other possible problems by maintaining an acidic pH < 4.5) that is unfavorable for the growth of common pathogens. The reduction or absence of vaginal lactobacilli has been shown to be a major cause of BV, and to increase the risks of vaginitis and recurrent urinary tract infections by 2–4 times , 2. In a.5 that iEpidemiologic studies have indicated that HR-HPV is the main etiological agent in the development of cervical cancer. However, only 10% of HR-HPV infections persist and potentially progress to cervical cancer . It is uLactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Reid et al. had reported evidence that these two probiotic strains can be delivered to the vagina following oral intake via morphology identification and molecular typing [The quality and sensitivity of a cervical cytological diagnosis can be affected by the presence of vaginal infections due to the accumulation of numerous microorganisms, white blood cells, and degradation products. In order to improve the reliability of cervical cytology, treatment of vaginal infections and maintaining a healthy vaginal environment are necessary. U-relax® , an oral probiotic used to restore vaginal flora, contains two patented and clinically proven probiotic strains: r typing . Clinicar typing –11. HoweThe study was a randomized, double-blinded, placebo-controlled clinical trial. Participants were recruited at Kaohsiung Chang Gung Memorial Hospital from January 2010 through June 2013. After Institutional Review Board approval, informed consent was obtained from all patients. The inclusion criteria were 1) females aged 30 to 65 years with HR-HPV infection, 2) cervical smear test with negative for intraepithelial lesion or malignancy results, 3) not pregnant. The exclusion criteria included 1) cervical cancer patients, 2) cervical intraepithelial neoplasia before conization, and within 6 months after conization, 3) gastrointestinal dysfunction or prior history of gastrointestinal surgery. All women received cervical smears and HR-HPV testing to confirm the HR-HPV infection. A colposcopic biopsy was performed in patients with cervical cytology of ASCUS and above.http://www.randomization.com/) using a computerized, balanced (1:1) method. Random numbers were generated by a computer, and the randomization code was inserted into numbered, sealed, opaque envelopes. A single envelope was opened by the patient when they were included. Study coordinators, patients, gynecologists and members of the panel were masked to the intervention after assignment. The study group was treated orally with one capsule a day of U-relax® . Each capsule contains 180 mg of a standardized, light beige fine powder  consisting of freeze-dried cultures (50% Lactobacillus rhamnosus GR-1 and 50% Lactobacillus reuteri RC-14). Quality control on the product showed that each capsule of U-relax has a minimum potency of 5.4 billion (5.4E+ 9) CFU (Colony Forming Units) and can be stored in room temperature 5-30C without dramatic change in CFU. The control group received the same capsule contains 180 mg of a standardized, light beige fine powder but without any probiotic bacteria. The treatment was discontinued until negative HR-HPV testing.In order to achieve 75% power at a 0.05 significance level, an overall sample size of at least 180 subjects (90 in the control and 90 in the treatment group) should be enrolled. However, enrollment was slow therefore a decision of closing accrual was made 2 years after study commenced. Randomization was performed through application program provided at the website . The preparations were then analyzed for abnormal cells by cytopathologists, and the results were classified according to the Bethesda System. Inflammation was defined as an increased number of polymorphonuclear leukocytes or neutrophils and parabasal cells with generalized eosinophilia of the cells. Unsatisfactory result was defined as more than 75% of the epithelial cells were obscured or could not be clearly visualized. For HR-HPV testing, we used Hybrid Capture 2 (HC2) test kits  according to the manufacturer’s protocol. Samples with a relative light unit (RLU) ratio higher than 1.0 were recorded as positive, and viral load was defined as RLU/positive controls.t-test. Hazard ratios (HRs) were estimated from multivariate logistic regression models to identify the independent factors predicting HPV clearance. The Kaplan-Meier method was used to estimate the time to clearance of HPV infection. All data were analyzed using SPSS statistical software version 22 . Results with a p value < 0.05 were considered to be statistically significant.Age, parity, menopausal status, educational level, employment status, prior hysterectomy, intrauterine device (IUD) use, viral load, HPV clearance, and cervical smear results were compared between the two groups. Data obtained from this study were analyzed using the chi-square test, Fisher’s exact test and Student’s Finally a total of 141 women were assessed for eligibility. Five were excluded due to not meeting inclusion criteria or declined to participate, and the remaining 136 women with HR-HPV infection were enrolled and randomly allocated to the study (68 women) and control (68 women) groups. A two-sided log rank test achieved 55.8% power at this sample size. During followed up period, 6 and 9 women in the study and control groups, respectively, were further excluded for analysis due to lost follow-up or stopped treatment. The CONSORT flow diagram is shown in Fig. p = 0.741). Age, menopausal status, educational level, employment status, prior hysterectomy, intrauterine device (IUD) use, and the use of probiotics were not associated with viral clearance. However, the women with HR-HPV clearance had significantly lower mean parity (p = 0.03) and lower initial viral load (p = 0.019). In multivariate logistic regression analysis, a lower initial HR-HPV viral load was the only independent factor predicting viral clearance after adjusting for confounding factors   and control (54.2%) groups. Figure p = 0.017). The unsatisfactory cases also dramatically dropped from 5 to 2 in the study group while there was no change in the control group (p = 0.027). The cytological findings at 9 and 12 months remained similar between the two groups. No progression of cytological results was noted throughout the study period.Cervical cytological findings before and after therapy are presented in Table The rationale of using probiotic strains in HPV clearance is via three proposed mechanisms. First, a synergistic environment is created. The anti-microorganism effects of probiotic strains are through space competition, nutrition competition, and the production of inhibitory compounds  . Thus an10CFU/day) for 6 months [Two studies investigating the association between probiotics and HPV had been reported. In a recent trial, Palma et al. reported that HPV clearance was higher in the treatment of metronidazole plus 6 months vaginal Lactobacillus implementation than that with 3 months use, no control arm was included in that trial . However6 months . Our repLactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 could decrease the presence of unsatisfactory and/or borderline satisfactory cytological findings and thus provide a more reliable cytological diagnosis [Cervical smear screening is an effective method to detect cytological abnormalities. However, the quality of a smear is sometimes compromised by inflammatory cells and exudate, inadequate cellularity or failure to sample the transformation zone leading to an unsatisfactory result. A high unsatisfactory rate can increase the number of patient revisits, thereby increasing the overall cost of the screening program. Therefore how to lower the unsatisfactory rate has become an important issue before adopting cervical smear testing protocols into a large-scale screening program. The use of liquid-based cytology has been shown to be effective in reducing the unsatisfactory rate compared with conventional smears as it provides a cleaner smear though removing obscuring elements such as blood and inflammation . The rediagnosis . In our iagnosis . The higAnother finding of this study was a significant decrease in the cervical smears showing mildly abnormalities (ASCUS/LSILs) in the women after taking probiotics. Although most of these mild abnormalities will regress without treatment, some may signal a precancerous condition or rarely cancer, especially in those positive for HR-HPV , 28. VerSeveral studies have shown that viral load measurements of HR-HPV types in cervical specimens can be a suitable indicator of viral clearance or persistence , 31. In There are several limitations to this study. First, substantial risk factors for cervical cancer such as smoking, number of sexual partners, and the use of oral contraceptives were not included for analysis, and this may have led to some form of bias. Second, status of BV and vaginal pH, which are thought to be important variables determining HPV clearance, were not evaluated in our study. Third, the sample size was small after adjustment because of slow recruitment and also the follow-up period was short. Fouth, HPV typing and quantitative real-time PCR assays were not applied since viral clearance may differ among various HPV types and the accuracy of quantitative PCR in viral load measurement is greater than that of HC-II.Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 provided no influence on genital HR-HPV clearance but may have decreased the mildly abnormal and unsatisfactory rates of cervical smears. A larger well-designed randomized study is warranted to confirm these results.We demonstrated that the application of the probiotic strains


These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-β in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF- Cervical cancer (CeCa) is the fourth cause of cancer death in women worldwide, accounting for more than 300,000 deaths per year, of which more than 80% occur in developing countries , 2. PersMost HPV infections are eliminated by the immune system in approximately 1 or 2 years after exposure . HoweverRecent reports have shown that the adenosinergic pathway plays an important role in the pathogenesis of gynecological cancer . In thisThis was a cross-sectional, descriptive, and observational pilot study.The study population was selected among patients who visited the Department of Gynecology of HGZ No. 2-A Troncoso, Mexican Social Security Institute (IMSS), Mexico City, Mexico. Cervical cytology samples were obtained between February 2016 and February 2018 after obtaining informed consent endorsed by the local bioethics committee. All women underwent cytological and histopathological analyses by means of a directed colposcopy. Two experienced cytotechnologists independently examined all Papanicolaou tests. Samples with an inconsistent diagnosis were excluded from the study. The cytology diagnoses were classified according to the Bethesda system, while a CIN-1 histopathological diagnosis was classified according to Richardt [3 cervical cells fixed in ThinPrep PreservCyt® solution were deposited onto positively charged glass slides . Subsequently, the cells were permeated with 0.01% Triton X-100 in PBS and incubated for 2 h with 2% (w/v) bovine serum albumin (BSA) in PBS . Next, the cells were incubated for 1 h with the antibodies. After two washes with PBS, the slides were incubated for 1 h using goat anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase . The development was carried out with a substrate-chromogen solution, 3,3'-diaminobenzidine dihydrochloride (DAB) , for 3-5 min. Cells incubated with the correspondent secondary antibody were included as a control. Nuclei were stained with Mayer's hematoxylin . The slides were scanned through an Aperio CS digital pathology device  to obtain electronic files. The presence of proteins CD73 and CD39 in the cervical cells was determined by densitometric analysis considering the total expression density (TED) from 100 cells per sample using Image-Pro Plus version 6.0.The expression of ectonucleotidases CD39 and CD73 in cervical cells was determined by immunocytochemical staining with a mouse anti-CD39 monoclonal antibody (clone eBioA1 (A1), 14-0399-82, eBioscience)  and an anti-CD73 rabbit-derived polyclonal antibody (Cat. NBP1-85740)  according to protocols previously described , 18. Briμg of total protein  placed in triplicate in 96-well flat-bottomed plates for the ELISA/RIA  was incubated for 1 h at 37°C and then overnight at 4°C. Next, the plates were washed with wash solution (PBS-0.1% Tween-20) and then incubated with blocking solution (2% BSA w/v in PBS-0.1% Tween-20) for 2 h at 37°C. Once the plates were washed, anti-CD39 and anti-CD73 antibodies were added at a dilution of 1 : 500 in blocking solution and incubated for 2 h. The plates were washed six times and incubated with goat anti-mouse or anti-rabbit IgG antibodies bound to alkaline phosphatase  at a dilution of 1 : 5000. The plates were incubated at 37°C for an additional 2 h, and after eight washes, the alkaline phosphatase substrate  was added to diethanolamine solution  at 10% (pH 9.8). The reading was performed at a wavelength of 405 nm using an ELISA plate reader.The content of ectonucleotidases CD39 and CD73 solubilized in supernatants of cervical samples was determined by an enzyme-linked immunosorbent assay (ELISA). Data were interpolated in type curves of recombinant enzymes CD39 and CD73  using different concentrations (1-100 ng/mL) diluted in PBS. To detect the content of soluble CD39 and CD73 in cervical samples, we previously determined the total protein concentration in each supernatant by using the Bradford reagent . Afterwards, 2 μg of total protein from the supernatants of the samples collected was incubated with adenosine triphosphate (ATP) or adenosine monophosphate (AMP) at a final concentration of 5 mM. After incubation for 72 h, Ado production was evaluated. To inhibit the enzymatic activity of CD39 and CD73, the specific inhibitors sodium polyoxotungstate (POM-1)  and adenosine 5′-diphosphate (APCP) , respectively, were added at a final concentration of 5 mM as previously described [μL. The amount of Ado produced by each sample incubated with ATP or AMP was evaluated through high-performance liquid chromatography, applying 25 μL of each reaction to a chromatograph  using a mobile phase composed of 0.5% acetonitrile, 5% methanol, and 94.5% sodium acetate (0.25 M and pH 6.3). Prior to the reading, the samples were filtered in Amicon filters of 3000 Daltons . An Ado standard curve was prepared to evaluate the Ado content in the different samples using Empower 3 .To determine the hydrolytic activity of ectonucleotidases CD39 and CD73 solubilized in cervical samples, 2 escribed . The totμL of each supernatant on fluorescent gel-coated plates . Samples were eluted for 1 h using a mobile phase composed of isobutanol : isoamyl alcohol : ethoxyethanol : ammonia : water (9 : 6 : 18 : 9 : 15) [In some assays, different concentrations  of POM-I or APCP were used to inhibit the capacity of supernatants of cervical samples to hydrolyze ATP or AMP. The presence of the products (AMP and Ado) was detected by thin-layer chromatography (TLC) by placing 1  9 : 15) , and 5 mβ1 content in serum samples, the human TGF-β1 Quantikine ELISA Kit (R&D Systems) was used according to the manufacturer's protocol.To quantify TGF-All numerical data are presented as the mean value ± standard error of the mean (SEM) of three independent experiments. Comparisons and correlations were evaluated by multivariate statistical analysis using GraphPad Prism version 7 . Values < 0.05 were considered statistically significant.The present study was performed using 50 cervical samples from women who visited the Department of Gynecology of HGZ Troncoso, IMSS, and according to the clinical assessment and histopathological study. Twenty-five samples corresponded to women whose colposcopy was positive for CIN-1 and 25 ND samples that were negative for HPV infection and whose colposcopy was negative for CIN-1. All samples and clinical data from the participants were taken after obtaining informed consent, in accordance with the ethics and confidentiality requirements related to working with human samples from the institutions involved. The mean age of the CIN-1 patients was 30.4 (range 19-43) years , and thaP < 0.001) (P < 0.001) groups (The presence of CD39 and CD73 was visible mainly at the membrane and in the cytoplasm . The TED< 0.001)  and CIN-) groups .P < 0.05) of soluble CD39 and CD73 than those of ND samples. The contents of CD39 and CD73 in the supernatants of ND samples were 2.87 ± 0.98 and 1.71 ± 0.86 ng/μg of total protein, respectively. Cervical samples from patients with CIN-1 positive for HPV-16, HPV-16+CI, and HPV-16neg had 8.84 ± 1.65 and 5.32 ± 0.99, 6.14 ± 0.92 and 4.2 ± 0.41, and 4.3 ± 0.76 and 3.24 ± 0.39 ng/μg of total protein, respectively  of POM-1 or APCP in supernatants of patients positive for HPV-16, which showed the highest contents of CD39 and CD73, suppressed in a dose-dependent manner the conversion from ATP to AMP or AMP to Ado  (P < 0.001) groups   and CIN-) groups .β), whose levels in tissues and plasma are directly correlated with the severity of the infection [The development of CeCa is followed by several mechanisms of suppression and evasion of the immune response . For exanfection , 33, maknfection , 35. Addnfection  and redunfection  and cytonfection  and IL-1nfection , which anfection .β increases the levels of CD39 and CD73 in activated T cells and suppressive myeloid cells in both mice [β1 in CeCa has been directly related to the degree of disease progression [β1 promoter by recognizing the Sp1 sequence [β, which is important for maintaining the expression of CD73 in tumor cells. We showed that the Ado product of the enzymatic activity of CD73 induced the production of TGF-β in tumor cells by interacting with A2AR and A2BR, suggesting an important connection between the adenosinergic pathway and TGF-β production in cells infected with HPV [α) [β1 stabilizes HIF-1α [α [β1; in consequence, it will be very interesting to know whether these contents of TGF-β are capable to inducing and maintaining CD39 and CD73 expression in the cervical microenvironment of CIN-1 patients HPV-16+. On the other hand, these patients exhibited significantly a greater number of sexual partners (averages 3.4 and 2.5) and pregnancies (averages 2.4 and 2.5), respectively, than NDs, whose averages were 1 and 1.2, respectively  . Therefos HIF-1α . LikewisIF-1α [α . In the ectively . It is iectively . In factresponse , 41–43, response , it woulβ than those of CIN-1 patients negative for HPV-16 and ND.In this study, we provide evidence that cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to cells from samples of CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-β in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.These results suggest that persistent infection with HR-HPV, mostly HPV-16, which is present in approximately 50% of CeCa cases , 56, may


We carried out a retrospective study on the prevalence of HPV and genotype distribution by nested PCR and nucleotide sequencing analysis, in formalin-fixed, paraffin-embedded biopsies of 135 head and neck cancers (HNC) and 29 cervical cancers received between 2009 and 2017 for diagnosis at the Laboratoire National de Biologie Clinique et de Santé Publique of Bangui, the capital city of the Central African Republic. One oropharyngeal squamous cell carcinoma sample was positive for HPV type 16. The overall HPV prevalence in HNC biopsies was 0.74% (95% CI: 0.0–2.2). Among the 29 cervical cancer samples, 19  were positive for HPV. These results indicate that HNC are infrequently associated with HPV infection in the Central African Republic. Head and neck cancers (HNC), excluding nasopharyngeal, most frequently squamous cell carcinoma, are among the most aggressive tumours worldwide . In addiAlthough HPV contribution to HNC is substantial, high heterogeneity by cancer site, region and sex has been reported . PopulatThe aim of our research was to provide original data on the role of HPV in invasive HNC in the Central African Republic, a country of Middle Africa, where cervical cancer is the second most frequent cancer in women between 15 and 44 years of age, suggesting high prevalences of oncogenic HPV among general population . The cruLaboratoire National de Biologie Clinique et de Santé Publique (“LNBCSP”) of Bangui, the capital city of the Central African Republic, is the reference national laboratory in laboratory medicine, including pathology laboratory. The LNBCSP received all biospies and other pathological investigations for the whole country, and also implement the cancer registration.The During 2009 to 2017, the LNBCSP received 135 invasive biopsies from HNC , following manufacturer’s instructions. Finally, the total DNA was eluted with 100 μl of DNase-free elution buffer.. The amplification of albumin DNA by in house real-time PCR was used as marker of DNA integrity. Finally, a 1 μg amount of extracted DNA was amplified by “hot start” PCR with degenerate HPV consensus primers MY09 and MY11, as described previously , followehttps://blast.ncbi.nlm.nih.gov/Blast.cgi) to be aligned and matched with all HPV sequences available within this database. HPV type was identified on the basis of ≥95% sequence homology in L1 region from HPV sequences available in HPV online databases. The assigned genotype was further confirmed by phylogenetic assay using Mega 2.1 software (www.megasoftware.net).FFPE biopsies of 25 squamous cell carcinoma and 4 adenocarcinoma of the cervix were also analyzed in parallel. Caskie cells (ATCC® CRM-CRL-1550) were used to obtain HPV DNA-positive control. Each isolate was sequenced in both directions. HPV genotypes were determined by direct sequencing of PCR products. Briefly, amplicons were subjected to cycle sequencing with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit . The sequencing reactions were then run on the ABI Prism 3700 Genetic Analyzer (Applied Biosystems). All clinical sequences were submitted to the BLAST server   biopsy samples from patients suffering from HNC, suggesting that HPV prevalence in HNC in the Central African Republic is very infrequent. In contrast, the majority (65.5%) of cervical cancer biopsy samples collected and analyzed under the same conditions were positive for high-risk HPV.Our observations appear very different from what has been reported previously in the other regions of the world, where HPV DNA can be detected overall in 26% of HNC . SimilarOur study has limitations. The retrospective analysis and the small size of biopsy specimens may have introduced selection bias. In addition, 60.7% of study specimens were HNC from oral cavity or larynx, which are driven more by alcohol and smoking than HPV . Further


HPV) has been identified as a major factor that leads to cervical cancer, although HPV infection alone cannot cause the disease. In fact, HPV‐driven cancer is a small probability event because most infections are transient and could be cleared spontaneously by host immune system. With persistent HPV infection, decades are required for progression to cervical cancer. Therefore, this long time window provides golden opportunity for clinical intervention, and the fundament here is to elucidate the carcinogenic pattern and applicable targets during HPV‐host interaction. In this review, we discuss the key factors that contribute to the persistence of HPV and cervical carcinogenesis, emerging new concepts and technologies for cancer interventions, and more urgently, how these concepts and technologies might lead to clinical precision medicine which could provide prediction, prevention, and early treatment for patients.Cervical cancer is the third most common cancer in women worldwide, with concepts and knowledge about its prevention and treatment evolving rapidly. Human papillomavirus ( Cervical cancer remains the third most common cancer in women worldwide, with approximately 529,800 new cases and 275,100 deaths annually HPV infection is recognized as a major causative factor in the development of cervical cancer Like many other types of malignancies, cervical cancer is a chronic complex disease caused by a combination of inherited genetic factors and external environmental influences Previous researches on host susceptibility to cervical cancer have focused on the genes that are involved in the human leukocyte antigen (HLA), which is also known as the major histocompatibility complex (MHC) system Additional studies also reported the potential roles of common gene variants that are involved in the cellular cycle and apoptosis, cell proliferation and differentiation, DNA repair, and immune responses α, β, γ, μ, and ν), 48 species, and 206 types Human papillomaviruses are a big family with the systematic classification of five genera , and A4 (Asian) sublineages; B (African 1); C (African 2) and D, including Asian American (AA) and North American (NA). In the same way, HPV18 variants have been defined into three major lineages  and additional sublineages (A1 to A5 and B1 to B3) with translation According to the largest to date oncogenic risk analysis of HPV16 sublineages at Kaiser Permanente Northern California, A4, C, D2, and D3 had higher risk of cervical precancer and cancer compared to extensive A1/A2 sublineages In contrast to HPV16, a worldwide study revealed that HPV18 sublinages showed no significant association with cervical cancer risk or histological types Besides, to provide comprehensive guidance for clinical application, it is worth further research that how oncogenic difference among HPV intratypes was influenced by ethnic genome variability, and geographical or behavioral factors.Integration of the HPV genome into the host chromosome is a key genetic step in cervical carcinogenesis The first experiment to identify HPV integration in the human genome was published in 1987, when a single copy of HPV16 was detected in the intergenic region between KLF5 and KLF12 on chromosome 13q22 in the SiHa cell line With the development of next‐generation sequencing, genomewide profiling of HPV integration sites is becoming feasible and cost‐effective In addition to HPV integrations into the human genome of the host genes, somatic mutations of the host genome during HPV‐induced carcinogenesis have also been an important aspect of studying cervical carcinogenesis. DNA mutations analysis plays an important role in identifying differences between cancer tissue and noncancer tissue, and in guiding diagnostic and therapeutic regimens.To date, the most comprehensive genomic landscape paper was published in Nature Journal; using NGS analysis, it revealed both known and novel high frequent mutations. The authors showed that the common mutations in SCC were EP300 (16%), FBXW7 (15%), PIK3CA (14%), HLA‐B (9%), and p53 (9%) while PIK3CA (16%), ELF3 (13%), KRAS (8%), and CBFB (8%) were in ACC Besides these novel driver mutations, some important common mutations including the oncogenes PIK3CA, EGFR,KRAS, and the gene suppressor PTEN, p53, STK11, and MAPK genes An important application of particular genomic mutations is that they may act as potential early screening biomarkers of cervical cancer. Therefore, numerous studies have explored the somatic mutations spectrum in CINs to cervical cancers and found potential early diagnostic gene mutation markers, such as oncogene EGFR, and PIK3CA, and the gene suppressors TP53 and PTCH1 Studies also showed that gene mutations can have an impact on the prognosis of cervical cancer. CHEK1, EI24, PTCH1, and ATM belong to the PI3/PI4‐kinase family, and the tumor suppressor gene CADM1 is a predictor of the worst prognosis Another prevalent epigenetic mechanism during HPV‐induced carcinogenesis is DNA methylation, which is a methyl ‐CH3) covalent addition to cytosine in the DNA sequence known as CpG dinucleotide  covalentAs exogenous pathogen, HPV epigenomic pattern has been thought to have prominent significance. Although the CpG islands on HPV genome are underrepresented, high density of CpG sites and characterized difference in E2/E4 region and alpha 9/7 clades still exist, indicating the underlying value of HPV DNA methylation mechanism To identify specific methylation alteration of HPV DNA, bisulfite‐based methylation‐specific PCR (MS‐PCR) and sequencing were used and demonstrated hypermethylation of LCR region of HPV 16 correlated with oncogenic progression in cell lines and cervical lesion Besides, high‐risk HPV E6 and E7 are directly related to functions of DNMTs(DNA methyltransferase), which are the key enzymes responsible for DNA methylation n = 149, n = 121), using Illumina 450K methylation arrays, Lando indicated that promoter methylation events at 3p11‐p14 could become a positive progression marker and a prognostic marker for cervical cancer In accordance with the data, DNA methylation was found to be a common event in cervical carcinogenesis. The level of methylation was positively correlated with the severity of both CINs and cervical cancer. Hesselink and Bierkens developed an objective nonmorphological molecular method of CADM1/MAL methylation as a cervical cancer screening marker with high sensitivity (ranging from 100% to 60.5%) for CINIII and patients with cervical cancer All above showed that altered DNA methylation pattern of high‐risk HPV types and host genome has the potential application in the risk‐based cervical cancer screening and diagnose, suggesting further study involving large‐scale population experiment to test the effect.Along with accumulation NGS data of cervical cancer, molecular characterization of different levels from genome to metabolism becomes gradually clear. However, achieving management of NGS data by integrative analysis proposes huge challenge but inevitable task to classify virus‐induced carcinogenetic subgroups by comprehensive molecular features, which is the guidance of ultimate clinical application of unbiased NGS strategy.Recently, the Cancer Genome Atlas Research Network conducted a multi‐omic analysis of invasive cervical cancer on 228 extended samples β pathway, also indicating potential therapeutic agents Of note, the most important value of this article is the intention of serving clinical translation and reasoning. By structural variation analysis, the TCGA researchers identified BCAR4, CD274 (PD‐L1), and PDCD1LG2 (PD‐L2) linked rearrangement events. PD‐L1 and PD‐L2 are two important immune checkpoints as well as promising immunotherapy targets In summary, with epigenomic and genomic data on cervical cancer and precancers being accumulated, a fundamental ground for the molecular classification of any HPV‐associated carcinogenesis has been established, as well as personalized biomarkers for each stage Practice Bulletin published, recommended HPV genotyping test alone as primary screening combined with cytology triage, which has been implemented in some developed countries Pap cytology has brought up as standard secondary cervical cancer screening for more than half a century, with standardized evaluation Bethesda System established in 1988 and other modified technique like liquid‐based cytology (LBC) and process automation developed in 2000s However, given that HPV test is accompanied by high false positive, the unavoidable increasing consequences of referral to colposcopy would bring psychological side effect and downstream overdiagnosis and overtreatment. Besides, in developing country where >80% cervical cancer incidence was observed, the screening strategy was not performed the best effect due to complex reasons including socioeconomic disparity, low screening coverage and limited funding of government, high personal cost and lack of related health knowledge and crucially, low quality of screening services, and unsound follow‐up management Novel screening strategies of cervical cancer in development focus on the utility of biomarkers and other molecular tests on account of the objectivity and reliability, including p16/ki67, E6/E7 mRNAs, microRNAs, and methylation of HPV and host genome Except for HPV test, followed great invention was prophylactic HPV vaccine approved in 2000s. The FDA has approved bivalent vaccine Cervarix against HR‐HPV16/18, responsible for 70% cervical cancer, quadrivalent vaccine Gardasil preventing infection with HPV6/11 added and 9‐valent vaccine Gardasil9 against additional five types 31, 33, 45, 52, and 58. The primary prevention of HPV‐related cervical cancer with HPV vaccine was strikingly promising and many countries have in succession initiated the national immunization program for girls aged 9‐25 before sexual behavior onset. However, with the vaccinated girls reaching screening age, the current prevention and screening guideline is not fully ready for the differentiation between who has been HPV‐vaccinated and who has not. A recent study using model‐based analysis suggested that for HPV‐vaccinated women, the screening should start later, take place less frequently and involve primary HPV testing rather than cytology, much different from current guideline routines As persistent infection with high‐risk types of human papillomavirus (HPV) is a necessary condition for cervical carcinogenesis, in recent years HPV detection has gradually become the primary screening method for cervical cancer, instead of cervical cytology. The development of HPV tests is still growing rapidly. To date, at least 193 different HPV tests are available for the detection of HPV in cervical specimens The Hybrid Capture 2 (HC2) HPV DNA test has been widely adopted to determine HPV infections and to detect HSIL and cervical cancer. Generally, sensitivities for detection of CIN2 or greater ranged from 84.9% to 100%, and specificities ranged from 69.5% to 95.8% Recently, detection of the HPV E6/E7 oncogene mRNA has become an alternative to the HPV DNA test. Studies show that little expression of E6/E7 mRNA may be detected in transient infections, but in persistent infections, E6/E7 mRNA is overexpressed. Therefore, detection of upregulated expression of E6/E7 mRNA can be directly related to disease progression and may further reduce the number of colposcopy referrals To date, there are numerous HPV tests, including PCR‐based MY09/11 and CPI/II systems However, data of NGS‐based HPV testing are still lacking, and the technology clearly has both advantages and disadvantages. First, with the knowledge that the NGS‐based HPV test is highly sensitive, the data of this method must be interpreted with care, because high sensitivity also means a high false‐positive rate and possible overtreatment of patients. Second, costs and turnaround time are the major problems with diagnostic methods based on NGS technologies Overall, it is quite possible NGS‐based assay will become even more cost‐effective than the classical HPV testing assays such as Cobas 4800. Nevertheless, full automation and standardization of protocols for library preparation and sequencing as well as user‐friendly and mature interpretation tool are urgently required for the implementation of this technology in routine diagnostics Along with the prevalence of HPV vaccination and cervical cancer screening, the treatment of early stage cervical disease also advanced, together will continue decreasing the incidence and mortality of cervical cancer The current immunotherapy methodology with clinical trials undergoing or accomplished includes therapeutic vaccine , targeted antibodies , T‐cell immune checkpoint inhibitor , and adoptive T‐cell transfer .As a paper commented, the targeted therapies of cervical cancer revealed encouraging though limited effect Overall, there are still modified immunotherapy researches and clinical trials in progress hopefully to improve the survival of the patients who have limited choices.Currently, there is no effective treatment for HPV persistence. Previous researchers (including those from our laboratory) have shown that targeting HPV E6/E7 mRNAs with siRNA could effectively knock down their expression and induce apoptotic cell death in HPV‐positive cell lines. However, siRNAs only block HPV E6/E7 mRNAs temporarily, and they do not attack HPV DNA in the nuclei, which serves as a store of escape mutants that cause resistance to siRNA application. In recent years, artificially engineered genome editing techniques such as zinc finger nucleases (ZFNs), Tal‐effector nucleases (TALENS), and the RNA‐guided engineered nucleases (RGENs or CRISPR/Cas9) were used to cleave the specific DNA sequence of HPV. Generally, the double‐stranded DNA breaks (DSBs) introduced by these custom‐engineered endonucleases should trigger DNA repair pathways (NHEJ repair pathway in most of the cases), resulting in the disruption of target viral oncogenes and the elimination of HPV infections Figure .In the early stage of development of HPV‐targeted genome editing tools, ZFNs were designed to disrupt the E6 or E7 genes of the high‐risk HPV types 16 or 18. These reports showed ZFNs could disrupt HPV genomes in HPV‐positive cell lines and in vitro culture models. Of note, one study demonstrated that injected ZFNs may even inhibit growth of HPV‐positive tumor xenografts, implicating their potential as anticancer drugs. However, ZFNs also have some limitations. First, construction of ZFNs is very labor‐intensive and may be hard to replicate in laboratories that do not have sophisticated skills molecular engineering skills. Second, the specificity of ZFNs varies greatly in different studies. Generally, the lengths of the recognition sequences are determined by the number of zinc fingers for the corresponding nuclease (often 18–24 nucleotides for 6 to 8 fingers). Although increasing the number of zinc fingers would improve their specificity comparable to that of the TALENs, extensive experiments would be required to screen the best combinations of ZFN monomers with different numbers of zinc fingers Another comprehensive study has demonstrated that TALENs as well as ZFNs can also be used as a therapeutic strategy to treat HPV‐related cervical lesions. In the study, the author achieved comparable or better efficacy to cleave E6 and E7 genes of HPV‐16 and HPV‐18 using artificially engineered TALENs. Interestingly, through topical application of TALENs directly onto the cervix of the HPV‐driven malignant phenotype of K14‐HPV16 transgenic mice, cervical intraepithelial neoplasms regressed to normal cervical tissues. Mutations of the E7 gene and reduction in the HPV16 DNA load were further confirmed. One potential advantage of TALEN is that its longer DNA‐binding sequence (approximately 30–40 nucleotides or more) may determine its improved specificity over clustered regularly interspaced short palindromic repeat (CRISPR) RNA‐guided nuclease With the emergence of CRISPR, this widely adopted technique has also been developed to target the E6 or E7 genes of HPV types 6, 11, 16, or 18. The antiviral efficacy of CRISPR/Cas9 has been shown in a number of in vitro cell culture models and also in HPV‐positive tumor xenograft models. Disruption of HPV E6 and E7 led to downregulation of their corresponding viral proteins and resulted in restoration of the tumor suppressor p53 and pRb. The primary concern of the CRISPR technique is its off‐target effects when it is applied in human subjects. The specificity of the newly developed and RNA‐guided CRISPR endonuclease is mainly determined by the protospacer adjacent motif (three nucleotides) and seed sequences (approximately 12 nucleotides), which are located at the 3′‐end of its recognition sequence GN20GG Genome editing‐based antiviral therapy may play valuable roles in HPV‐related cervical carcinogenesis. If this therapy is appropriately combined with HPV testing, patients in precancerous stages, such as persistent HPV infections and their related CINs, would be able to take HPV testing first to determine the HPV subtype(s) and then select the corresponding type‐specific genome editing tools to treat the appropriate HPV infection(s) and their related precancerous diseases. These patients would benefit from this new “screen‐and‐immediately‐treat” strategy, instead of potential overtreatment including repeated screenings, colposcopy‐directed biopsies, and cold knife conization. Consequently, additional costs, patient anxiety, and adverse effects that can result from repeated screenings, colposcopy‐directed biopsies, and cold knife conization  could be avoided.In the future, more study still needs to be performed. When applied to human subjects, the length of time that the complexes need to stay resident in the vagina must be further determined; to extend this time, polymer‐complexed artificially engineered nucleases could be directly applied to the anogenital tract using devices such as the CerviPrep drug delivery system With the emerging new concepts and technologies for cancer interventions, the precise prevention, diagnosis, and treatment of cervical are not only necessary, but now http://gco.iarc.fr/today/online-analysis-map?mode=cancer&mode_population=continents&population=900&sex=2&cancer=16&type=1&statistic=0&prevalence=0&color_palette=default&projection=natural-earth#modalTakeATourVideoFigures None declared.


Squamous papillomas are exophytic proliferations of surface oral epithelium. Human papillomavirus (HPV) infection is widely accepted as the etiology of squamous papillomas however the virus cannot be detected in a significant percentage of lesions.Using polymerase chain reaction (PCR), we tested 35 formalin-fixed paraffin-embedded (FFPE) squamous papillomas for the presence of HPV DNA.Six papillomas (17%) tested positive for HPV DNA; four contained HPV-6 and two contained HPV-11. Given that β–globin DNA was only identified in half of the samples, DNA degradation appears to have significantly impacted the results.The results likely represent an underestimation of the true number of HPV-positive specimens in our study. Potential explanations for HPV-negative squamous papillomas include transient HPV infection, failure of the experiment to detect HPV if present, or the possibility that some lesions may not result from HPV infection. Key words:HPV, PCR, FFPE, papilloma, oral. Squamous papilloma represents the most common benign epithelial neoplasm of the oral cavity . The cliThe etiology of squamous papilloma has been attributed to infection with low-risk human papillomavirus (HPV) genotypes such as 6 and 11. HPV DNA is detected in an average of 34% of oral squamous papillomas however a wide range of detection rates have been reported using various experimental methods  and 86% In situ hybridization (ISH) is labor intensive and lacks sensitivity, therefore polymerase chain reaction (PCR) has been the preferred method in recent studies  tissue specimens represent a vast source of materials for use in research studies. FFPE tissues are the most common specimen stored in pathology laboratories. Analysis of these specimens can allow us to determine the prevalence of HPV genotypes in multiple clinical lesions. There is no gold standard or FDA-approved method for HPV detection in FFPE tissues . In situ studies ,8. LargeThirty-five FFPE squamous papillomas submitted to the LSU Oral Pathology Biopsy Service archives between 2014 and 2015 were selected. All specimens were reviewed microscopically by two oral pathologists (MR and KC) to confirm each was an ideal histopathologic representation of a squamous papilloma without significant overlapping features with condyloma acuminatum, verruca vulgaris, or multifocal epithelial hyperplasia. Six specimens diagnosed as fibroma were selected to serve as negative controls, and a previously confirmed HPV DNA-positive cervical biopsy specimen was used as a positive control. This study was approved by the Louisiana State University Health Sciences Center Institutional Review Board (IRB# 3974).Using a microtome, five 10μm-thick sections were obtained from each specimen. Genomic DNA was extracted using the QIAmp FFPE Tissue Kit following the manufacturer’s instructions (Qiagen). The DNA extraction was performed immediately following tissue sectioning. Quantity of DNA was determined by spectrophotometry (NanoDrop). Quality of DNA was determined by polymerase chain reaction (PCR) for the human β-globin gene. If a sample contained detectable β-globin DNA, then it was assumed that HPV DNA should be intact and amplified during PCR. PCR was performed on the extracted specimen DNA using three separate HPV-specific primer sets: MY09/11, FAP59/64, and SPF1/2 according to published methods . The average patient age was 52 years old (range 10-85). The papillomas were harvested from a variety of intraoral areas; soft palate and tongue were the two most frequent locations . The HPVp=.1167), DNA quantity (p=.095), or formalin fixation time (p=.736) (The presence of β-globin DNA was detected in 17 of 35 papillomas (49%). Of the six HPV-positive samples, two (33%) were positive for β-globin. Two of the 6 fibromas were positive for β-globin and no fibromas were positive for HPV. When comparing HPV-positive and HPV-negative squamous papillomas in our sample, there were no statistically significant differences in DNA quality ((p=.736) .In this report, we detected HPV 6 or 11 in 6 out of 35 FFPE tissues diagnosed as oral squamous papillomas using a combination of HPV-specific PCR assays. Previous studies have identified HPV 6 and/or 11 in 13-35% of squamous papillomas by ISH ,11 and iet al. found no statistically significant difference in HPV detection between freshly frozen samples and paraffin embedded tissue  compared to biopsy specimens . Again, Oral squamous papillomas have been attributed to low-risk HPV infections however viral DNA cannot be detected in a significant proportion of lesions. This contrasts with other papillary oral lesions such as condyloma acuminatum in which nearly all cases are HPV-positive. We consider the possibility that some papillomas may not be caused by HPV infection although there are multiple alternative explanations. Oral HPV infection could be transient in nature and therefore no longer detectable a certain time after having induced a clinical lesion. It is unlikely that the specimens contained genotypes different from the probes we used in our experiment. In agreement with previous reports, we detected no high-risk HPV genotypes within our papilloma specimens.Technical challenges with DNA extraction from FFPE tissues are well known. Inadequate DNA extraction underestimates the prevalence of HPV. We believe this occurred in our study given the low percentage of β–globin positive samples. When we consider only the β–globin positive samples, 36% (6 of 17) contained HPV DNA; this may be a more accurate representation of true HPV prevalence in papillomas.