Human papillomavirus (HPV) associated malignancies are the leading cause of cancer death in Botswana. We sought to determine causative HPV types in patients with anogenital malignancies in Botswana to inform vaccine strategy.m2000 real-time PCR platform. Tissues with other high-risk types were subsequently analysed using a multiplex qPCR assay that includes 15 validated fluorophore probes.We used formalin-fixed and paraffin-embedded (FFPE) tissue blocks from patients diagnosed with anal, penile and vulvar squamous cell carcinomas between the years, 2014 and 2016. Presence of HPV 16, 18, or other high-risk (HR) types was detected using Abbott A total of 126 tissue specimens, comprising of 21 anal , 31 penile and 74 vulvar were studied. Ninety-three (73.8%) patients had their HIV status documented in the records while the rest did not. Eighty-three (83) out of 93 were HIV positive, a prevalence of 89.4% (95% CI: 81–94). HPV was detected in 68/126 (54%) tissues, of which 69% (95% CI: 54–79) had HPV 16 only, 28% (95% CI: 19–40) had other hr.-HPV types and 2.9%  were co-infected with HPV 16 and other hr.-types. Other high-risk types detected included HPV 26, 31, 33, 35, 39, 45, 51, 52, 66 and 68. HPV 18 was not detected. Multiple-type HPV infection was detected in 44 of 47 (93.6%) HIV positive participants co-infected with HPV. In HIV-negative individuals, only HPV 16 was detected.In our study, anogenital carcinomas were associated with HPV 16 and other hr.-HPV types besides HPV 16 and 18. HIV co-infected patients had multiple hr.-HPV types detected whereas in HIV-negative patients only HPV 16 was detected. Our study suggests that multivalent vaccines may be more suitable in this setting, especially for HIV-infected individuals. Human papillomavirus (HPV) is a common sexually transmitted infection and is one of the major causes of infection-related cancer worldwide . GloballHigh-risk HPV (hr-HPV) prevalence rate remains highest in HIV-infected individuals despite the increasing coverage of combination antiretroviral therapy (cART) –8. HIV iMore than 120 HPV genotypes have been identified and these are categorized as either high-risk/oncogenic or low-risk HPV types, based on their aetiological association with squamous cell carcinoma (SCC) of the cervix , 20. HigIt is also reported that, although HPV types 16 and 18 are the most common, others hr.-HPV types such as HPV 45, 35 and 58 are being detected more frequently in SSA . HPV pre provides cross-protection against HPV types; 26, 18, 31, 33 and 45 [To date, there is no known cure for HPV infection and prevention via vaccination remains the bulwark against some of the hr.-HPV types. The quadrivalent Gardasil vaccine  protects against HPV types 6, 11, 16 and 18. However, Cervarix vaccine 3 and 45 . A new 93 and 45 . The mos3 and 45 . VariatiStudies comparing invasive cervical cancers in Botswana, Kenya and United States have demonstrated differences in HPV type distribution, which may be driven by HIV infection , 27. DatThe incidence of HPV-associated cancers is increasing by an estimated 3.9% per year in Botswana and this increase has been primarily associated with HIV co-infection . TreatmeThis was a retrospective cross-sectional study that used residual Formalin Fixed Paraffin Embedded (FFPE) tissue material from 137 patients diagnosed with anogenital cancers who attended public health facilities for diagnostic and treatment services in the southern part of Botswana during the years, 2004 to 2006 and 2013 to 2016. Anogenital cancer diagnosis was made by the Anatomical Pathologist at the Botswana’s Ministry of Health and Wellness National Health Laboratory: a reference laboratory providing pathology services to southern Botswana. Demographic data, and clinical data on the anatomical site of the tissue sampled, HIV status, histopathology result, and year of diagnosis was obtained from the patients’ pathology reports. The specimens were de-identified for the purposes of this study. The Ethical Review board of University of Botswana, and the Ministry of Health’s Research and Development Committee (HRDC) approved this study. We obtained permission to access the specimens from the National Health Laboratory.Each tissue block was cooled at −3.5 °C before a 20 μm thick section were cut and processed for DNA extraction. The first 20 μm tissue section for each block was discarded to ensure consecutive cleaner sections were collected. To avoid contamination between different participants, specimens during sectioning, the microtome stage and blade were cleaned with xylene followed by absolute alcohol and DNA/RNA AWAY solution after each tissue block section.The tissue samples were incubated overnight in 15 ml of xylene to ensure complete removal of paraffin wax from the tissue, followed by three one-hour washes in absolute alcohol to clear xylene. Gradual re-hydration of the tissue sections was done by successional one-hour incubations in 95%, 80%, and 70% alcohol.me HR HPV assay reaction vessel for DNA isolation using the mSample Preparation System DNA kit on the Abbott m2000sp instrument, following the manufacturer’s instructions.Tissue sections were incubated in distilled water overnight before digestion. Tissue digestion was achieved by adding 600 μL of Nuclisens lysis buffer  and 200 μL of proteinase-K enzyme  to the tissue section. This was mixed by vortexing, followed by pulse centrifugation and overnight incubation at 56 °C. Tissue digests were incubated at 100 °C to de-activate the Proteinase-K enzyme before centrifugation at 3, 500 rpm for 5 min. After centrifuging, 700 μL of the lysate was transferred into Abbott RealTime HR HPV assay  qualitatively detects DNA from 14 high-risk human papillomavirus genotypes using modified GP5+/6+ primer mix consisting of three forward primers and two reverse primers targeting the conserved L1 region of HPV and an internal control (IC) primer pair targets a human β-globin sequence. The Abbott RealTime HR HPV assay provides specific probes that are differentially labelled qualitatively detect of HPV16 and HPV18, and an evaluation of the human beta-globin internal control. Probes for hr.-HPV types 31, 33, 35, 39, 45, 51, 52, 56, 56, 59, 66 and 68 are labelled with the same dye, and presence of any of these genotypes is reported as Other HR detected [The Abbott RealTime, were subsequently analysed using an in-house multiplex qPCR assay. This assay uses 15 validated fluorophoreprobes in order to differentiate specific hr.-HPV types. To validate our methods and results, 26 h-HPV positive and 10 h-HPV negative known samples from previous HPV studies were included as controls from extraction to genotyping. Their previous results were confirmed by our methods. The in-house hr.-HPV testing was performed as a triplex where each well of a 96 well PCR plate contained primers and probes for two HPV types in addition to human genomic DNA control (β-globin). The real-time PCR reactions were setup in 20 μL total volumes containing 10 μL KAPA Probe Force qPCR Master Mix  and HPV primer and probe mix, as well as 10 μL of the extracted DNA template. The PCR reactions were run on the ABI 7500 Real-Time PCR System ; starting with the pre-PCR hold at 98 °C for 10 min, followed by 5 cycles at 95 °C for 10 s, 50 °C for 20 s and 72 °C for another 20 s. The last stage was 95 °C for 10 s, followed by 45 cycles in 1 min at 60 °C before holding indefinitely at 60 °C. Two or three replicates with concordant detection of the HPV types being tested were considered true positives. Results were considered valid only if the human genomic DNA control (β-globin) was positive.All tissues with other hr.-HPV types detected using the Abbott RealTiPrevalence estimates were presented with 95% confidence intervals calculated using the binomial exact methods. Fisher’s exact tests were used to compare prevalence by selected categorical variables such as HIV status, HPV DNA extraction and HPV type. Data was first managed using the Microsoft Excel spreadsheets and all analysis was conducted using STATA version 14.2 .A total of 126/137 (92%) FFPE tissue blocks had amplifiable DNA as measured by the successful detection of the human ß-globin gene. Samples were obtained from individuals with squamous cell carcinoma, of which 21 (16.7%), 31 (24.6%) and 74 (58.7%) were from anal, penile, and vulvar biopsies, respectively. The median age was 44 years  . Overall HPV 16 prevalence was 69% (95% CI: 58–80). The highest HPV 16 prevalence was observed in vulvar SCC at 81% (34/42), followed by anal SCC at 62% (8/13) and the lowest prevalence was observed in penile SCC at 39% (5/13). The presence of HPV 16 was statistically marginally associated with anatomical site , Table C at 81% 4/42, folC at 81% 4/42, folMultiple-type hr.-HPV infection was detected in 44 of 47 (93.6%) HIV-positive participants co-infected with hr.-HPV. In HIV-negative individuals, only HPV 16 was detected. HPV 18 was not detected in this study.To our knowledge this is the first report on the prevalence of oncogenic HPV infection in patients with anogenital squamous cell carcinoma (SCC) in Botswana. As expected, the prevalence of HIV co-infection was high, with 89% of the patients with documented HIV status being positive for HIV. This is higher than the HIV prevalence in the general population, which is estimated to be at 24.3% in 15 to 49 years age group in Botswana. The observed high HIV and HPV co-infection in our study is consistent with findings from other studies that have reported an underlying HIV infection as a predisposing factor to acquisition and persistence of oncogenic HPV infection –14, 16 i, [Although the prevalence of hr.-HPV in this study was high in this population with SCC (54%), it is lower than what has been estimated by cervical cancer studies in Botswana. This could be due to the fact that the previous studies focused on the individuals with HIV infection in Botswana. Ramogola-Masire et al.,  reported, [MacLeod et al.,  reported, .Across all anatomical sites, the most frequently observed hr.-HPV type was HPV 16 at 69% and other hr.-HPVs at 28%. This is in keeping with previous studies that have reported HPV 16 as the most commonly isolated genotype in anogenital cancers. The prevalence of HPV 16 was much higher compared to previous studies , 29, 30.Unlike in the previous studies done on cervical cancer, , 29, 30,, [A similar study conducted in Myanmar reported an overall hr.-HPV prevalence of 34% in anogenital SCC  whereas ,  found thPrevalence of penile carcinomas carrying hr.-HPV has been reported to range from 30 to 100%  and thisA proportion of penile cancer cases have also been found to be not associated with HPV 16, as compared to anal and vulvar cancer cases . This inWomen are reported to have higher anal cancer rates than men in the entire population , 53. ThiHowever, this information does not correlate with earlier findings by  who repoHPV 16 and 18 are reported to be the most common genotypes associated with anogenital cancers worldwide. Our findings show that HPV 16 is the most common but HPV 18 is rare., [HPV 16 remains the most prevalent genotype in sub-Saharan Africa with HPV 18 showing lower prevalence than other hr.-HPVs as compared to developed countries . The ava,  has repoIn our study HPV type 16 was the most commonly isolated genotype at 59% but there were other significant hr.-HPV types, which are not covered by the HPV vaccine currently in use in Botswana. Quadrivalent Gardasil© protects against HPV 6, 11, 16 and 18 is currently in use in Botswana but it is only given to young girls aged 9–13 years . The secOur study had limitations; we used available tissue samples in a national reference laboratory bio-repository serving southern region of Botswana population, and this may limit generalization. However, it contributes the first set data in anogenital SCC in Botswana.HIV status was not available for a small fraction of the samples obtained and although DNA amplification rate was high 92%, using archived FFPE for genotyping requires further investigation. It is plausible that stratifying our data according to age categories could have epidemiological insights. Due to the small sample size per anatomical site, a sub-analysis according to anatomical site and age could not performed.Anogenital carcinomas in our study were associated with HPV 16 and other hr.-HPV types. HIV-positive participants had multiple hr.-HPV types detected as compared to HIV-negative participants where only HPV 16 detected. Our study suggests that multivalent vaccines may be more suitable especially for HIV positive individuals. Further studies on possible other causes of anogenital SCC that is not hr.-HPV-associated should be explored, as there were some patients with hr.-HPV negative penile and vulvar SCC.


Most human papillomavirus (HPV) infections are transient and additional triage approaches should be built after HPV-based primary cervical cancer screening. We evaluated the accuracy of p16/Ki-67 and p16/mcm2 dual staining as biomarkers for triaging HPV positive women in China. 4070 participants aged 35 to 64 years attending ongoing cervical cancer screening were enrolled in 2015-2016. Cervical exfoliated cells were collected for HPV DNA analysis and the residual positive specimens were tested for liquid-based cytology and biomarkers. Women infected with HPV 16/18 type or other 12 high-risk HPV types with abnormal cytology results received colposcopy. We found the positive rates of both biomarkers increased significantly with histology severity. p16/Ki-67 positivity in HPV16/18 group, other 12 high-risk HPV group and HPV negative group was 50.0%, 33.7% and 8.9%, respectively. The corresponding p16/mcm2 positivity was 70.0%, 56.3% and 6.7%, respectively. The sensitivity and specificity of p16/Ki-67 for CIN2+ in all HPV-positive women were 91.7% and 63.5%, with a referral rate of 36.2%, while p16/mcm2 were 87.5% and 42.1%, with a referral rate of 58.4%, respectively. The sensitivity of p16/Ki-67 increased to 95.8% for CIN2+ and 100% for CIN3+ when combined with high-grade cytology, without decrease in specificity. Our studies suggest that p16/Ki-67 is an efficient triaging biomarker for HPV-positive women and could reduce colposcopy workload. p16/mcm2 is more sensitive compared with cytology for identifying cervical lesions. As the world's most populous developing country, China still faces serious disease burden of cervical cancer, especially in medical resource limited rural areas where reside more than 70% population of the whole country . DespiteSeveral studies have demonstrated that cytology provides more sensitivity for detection of precancer when informed of HPV status compared to a single cytology test , 8. TherINK4a (abbreviated as p16) is a cyclin-dependent kinase (CDK) inhibitor and can be strongly over expressed in transforming HPV infections. As a negative regulator of cell proliferation, p16 protein can down-regulate the activity of CDK4 and CDK6 when the retinoblastoma protein has been inactivated [0 [TMC composed of 2 antibodies against mcm2 and topoisomerase IIα (TOP2A) has been selected as promising diagnostic adjunct for cervical squamous intraepithelial lesions [p16ctivated , 11. Prectivated , 13. As vated [0 . Minichrvated [0 , 16. Rec lesions , 18.So far, no studies have combined a cocktail reagent simultaneous containing p16 and mcm2 antibodies just as CINtec PLUS  targeted for p16/Ki-67 co-expression. As cell-cycle related acceleration proteins, there may be potential overlaps in function between Ki-67 and mcm2. With this assumption, we performed this study to evaluate the clinical performance of p16/Ki-67 and a novel p16/mcm2 dual-staining cytology approach with the focus on triaging HPV-positive women.Among 4,070 participating women with complete HPV data, 357 (8.8%) were positive for HPV DNA. Liquid-based cytology was obtained for all HPV-positive women and 252 (70.6%) of 357 were normal, 46 (12.9%) were atypical squamous cells of undetermined significance (ASC-US), 23 (6.4%) were low-grade squamous intraepithelial lesions (LSILs), 4 (1.1%) were atypical squamous cells that cannot exclude high-grade lesion (ASC-H), 4 (1.1%) were high-grade squamous intraepithelial lesions (HSILs), and 28 (7.8%) were unsatisfactory (including 4 HPV 16/18 types and 24 other HR-HPV types) due to insufficient sample material which we decided to exclude. A total of 329 eligible dual-staining tested, 119 (36.2%) were p16/Ki-67 positive, 192 (58.4%) were p16/mcm2 positive.Women infected with HPV 16/18 type (n=50) or other carcinogenic types with abnormal cytology results (n=62) were referred to immediate colposcopy while 32 women refused. In the 80 colposcopies performed, 3 cases of CIN2, 9 cases of CIN3 and 2 squamous carcinoma were found. There were also 5 cases of CIN2, 3 cases of CIN3 in HPV-positive, cytology-negative women who received colposcopy. A total of 169 women had complete histology results. We compared the baseline characteristics of women for compliance and non-compliance to colposcopy (Table p<0.05). Positivity rates of both p16/Ki-67 and p16/mcm2 dual staining increased significantly with histology severity (ptrend<0.001). There were 119 (70.4%) women with inflammation, 26 (15.4%) with CIN1, 8 (4.7%) with CIN2, 14 (8.3%) with CIN3 and 2 (1.2%) with squamous cervical cancer. The corresponding p16/Ki-67 positive rates were 34.4%, 46.2%, 87.5%, 92.9%, and 100%, respectively, while p16/mcm2 were 56.3%, 65.4%, 75.0%, 92.9%, and 100%, respectively.As shown in Table p=0.024), but no differences were found for p16/mcm2 expression rate in these two groups (p=0.063).All participants were divided into three groups based on HPV infection status to compare the positive rates of both biomarkers in different HPV types. Table p<0.05). The specificity of p16/Ki-67 for both endpoints were similar to LBC  and lower than HPV16/18 genotyping . Compared with LBC and HPV16/18 genotyping, p16/mcm2 had a lower specificity for both CIN2+ and CIN3+. Notably, no significant differences of sensitivity were found between p16/Ki-67 and p16/mcm2 dual staining, but p16/Ki-67 had a higher specificity for both endpoints . In addition, the positivity of p16/Ki-67 in HR-HPV positive women was 36.2%, indicating that more than 60% of women needn't receive colposcopy if p16/Ki-67 dual staining was used as a triage test and meanwhile women who performed p16/mcm2 test would also lead a referral rate decline by more than 40%.Sensitivities, specificities, PPVs, NPVs and colposcopy referral rates for all HPV-positive women to detect CIN2+ and CIN3+ were evaluated Table . The senWe also evaluated the clinical performance of biomarkers for triaging women with HPV-positive, cytology-negative results. Positivity rates of p16/Ki-67 and p16/mcm2 among 252 HPV-positive, cytology-negative women were 29.0% and 55.6%, respectively. There were 11 CIN2+ and 6 CIN3+ cases diagnosed during preliminary follow-up  and a 10% random selection of normal slides were reviewed by an expert pathologist. Meanwhile, four-quadrant cervical biopsies and endocervical curettage (ECC) were performed to maximize the diagnosis of disease in case of no visible lesions under colposcopy. Besides, all dual staining tests in our study were performed in the central laboratory according to the same criteria by using the same sample.In our study, we observed a significantly increased positive rates of both p16/Ki-67 and p16/mcm2 with the increasing grade of histology severity, which were similar to previous studies , 24. TheWe found that p16/Ki67 was positive in 36.2% of all HPV-positive women, with a sensitivity of 91.7% for CIN2+ and 93.8% for CIN3+, indicating that it could detect most cervical precancer and meanwhile reduced unnecessary colposcopy referrals. The sensitivity of p16/Ki-67 in our study was significantly higher than LBC and HPV16/18 genotyping, in accordance with previous studies , 33. It X C from previous studies [Interestingly, we evaluated the performance of both biomarkers in women with HPV-positive, cytology-negative results and found that p16/Ki-67 dual staining represented better sensitivity and comparable specificity than former studies , 35, esp studies . It meanOur study demonstrated that combined strategies of cytology and biomarkers have an increased sensitivity and moderate specificity only when cytology adopted a high-grade threshold. Meaning that the biomarkers could increase specificity by triaging women with ASC-US and LSIL results and increase sensitivity by triaging women with NILM results. Our results showed very low sensitivity of HPV16/18 genotyping, similar to previous study , and thuThe main limitation of our study is that we can't have a colposcopy assessment for all HPV-positive women. Since the study was nested in the pragmatic process of an organized national program. According to the study design, only cytology positive women received colposcopy and part of women with NILM results were followed up after 1 year. This differential ascertainment led to a bias in favor of cytology sensitivity. Our study focused on the cross-sectional performance of biomarkers even though cervical precancer includes a certain regressive lesions during natural history, not distinguishable from progressive ones. But the relevance of regression has limited because we recruited only 35 years or older women. Furthermore, we evaluated the prognostic value of biomarkers only in HPV-positive, cytology negative women, without containing the longitudinal data of CIN in women with normal triage results.In summary, p16/Ki-67 had a higher sensitivity and comparable specificity compared with cytology, and could increase sensitivity for both endpoints when combined with high-grade cytology. p16/mcm2 was more sensitive than cytology for identifying cervical lesions. Furthermore, both biomarkers could recognize cervical lesions simply based on dual-staining within one cell which potentially eliminate the morphological criterion, suitable for Chinese rural areas where experienced cytopathologists aren't available. It seems p16/Ki-67 is more promising to reduce colposcopy workload and allows longer intervals in HPV positive, biomarker negative women.From April 2015 to May 2016, 4,070 eligible women in the districts of Wanzhou  and Shuangliu  with a median age of 47.5 years agreed to participate in a HPV-based cervical cancer screening. The screening strategy at each participating center was implemented in accordance with the approved guidelines. All participants have signed up informed consent forms and this study was approved by the institutional review boards of the Cancer Institute/Hospital, Chinese Academy of Medical Sciences (CICAMS) in Beijing and the local ethical committee . All participants should meet the following criteria: aged between 35-64 years; without history of previous cervical diseases and had an intact cervix; were not pregnant; understood the study procedure and were able to provide informed consent.This was a population-based study within the setting of the national cervical cancer program searching for suitable screening technologies for Chinese rural areas. After completing a questionnaire containing various risk factors by well-trained health workers, one cervical cytology specimen was collected for each participant with a cytobrush by physicians and then transferred to PreservCyt solution , stored at 4°C, and transported to central lab for primary HPV DNA testing. Women negative for HPV will return to routine screening with a 3-year interval. For each of the HPV positive women, a thin-layer slide for LBC was prepared and read. Women who tested positive for HPV16/18 type or other 12 HR-HPV types  with ASC-US or worse were referred to colposcopy and biopsy, while those with normal cytology were followed-up and re-tested in 12 months  which detects viral DNA by nucleic acid hybridization with a pooled probe set for 14 HR-HPV types, including HPV16 type and HPV18 type as well as other 12 high-risk types . All PCR reactions were performed in a 40-ul volume using the ABI PRISM 7000 . Each individual reaction contained 36ul mixture of 2 × TaqMan universal PCR master mix with uracil-N-glycosylase  and two fluorescent probes. The amplification profile was initiated by a 2-minute incubation at 94°C, followed by a two-step amplification of 10 seconds at 93°C and 31 seconds at 62°C for 40 cycles. Data were collected at the end of the amplification process and all experiments were performed in triplicate including positive and non-template controls according to the instructions of the manufacturer.Thin-layer cytology slides were prepared with the ThinPrep 2000 System . The preparation process adopted the common gynecology sample procedure: the cell suspension was firstly homogenized after the PreservCyt container placed in the instrument and then cells were collected through a filter membrane to a glass slide. Cells were fixed with 95% ethanol and stained using the Papanicolaou method. Cytology slides were interpreted by two experienced cytotechnologists and confirmed by a third pathologist using the classification of the 2001 Bethesda reporting system. Positive cytology results were defined as atypical squamous cells of undetermined significance or worse, which led to referral to colposcopy.A second and third monolayer slides were prepared from the residual PreservCyt material by ThinPrep 2000 system in parallel to perform p16/Ki-67 and p16/mcm2 immunocytochemistry dual staining. Immunostaining for p16/Ki-67 was performed using the p16/Ki-67 Detection KIT , which contains an antibody cocktail comprising of a mouse monoclonal antibody (clone MX007) directed at p16 protein and a rabbit monoclonal antibody (clone MIB-1) against Ki-67 protein. Secondary antibodies included goat antimouse antigen-binding antibody fragments for detection of p16 with horseradish peroxidase (HRP) and goat antirabbit antibody fragments for detection of Ki-67 with a polymer reagent conjugated to alkaline phosphatase (AP). HRP-mediated conversion of 3,3’-diaminobenzidine (DAB) chromogen, and AP-mediated conversion of Fast Red chromogen emerged the red and yellow-brown staining of p16 and Ki-67, respectively. Finally, we used hematoxylin to counterstain cytology slide with a 3-step mounting procedure including 85%, 95% and pure ethanol medium, each for 1 minute. p16/mcm2 dual staining was performed using antibody cocktail composed of p16 antibody (clone MX007) and rabbit anti-human mcm2 monoclonal antibody (clone SP85) according to the manufacture's instructions. Other procedures were similar to p16/Ki-67 dual staining as the above described. Slides with at least one cervical epithelial cell that simultaneously showed red cytoplasmic immunostaining (p16) and brownish nuclear immunostaining (Ki-67) or moderate to intense yellow-brown nuclear staining (mcm2) were defined as positive results irrespective of the morphological features, while those without any double-stained cells were deemed as negative results. All slides were coverslipped for viewing by a trained cytotechnologist under a light microscope blindly to other testing results.Colposcopy processing was conducted in the local reference clinic of the screening program within one month. Colposcopy-directed biopsy specimens were always collected in case of visible cervical lesions and endocervical curettage was performed if the squamocolumnar junction was invisible or suspicious of endocervical lesion. H&E-stained slides were evaluated using the CIN systems according to the current World Health Organization classification. Histopathological diagnosis was firstly made by local pathologists and then all the CIN and a 10% random sample of negative slides were selected to be reviewed by a panel of senior pathologists from corresponding superior hospitals . Women were regarded as negative histology results if a biopsy need not be taken or the histology diagnosis was normal or CIN1, while those CIN2, CIN3, squamous cell carcinoma (SCC) and adenocarcinoma (ADC) results were defined as CIN2+ cases. The final diagnosis was based on the reading of the panel review regardless of directed biopsies or endocervical curettage.The study targeted the recruited women with complete screening, triage and diagnostic confirmation results. Diagnostic accuracy of p16/Ki-67 and p16/mcm2 dual staining for CIN2+ and CIN3+ were evaluated by sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), with 95% confidence intervals (CIs). Chi-square of trend for proportion was calculated to test linear associations between triaging methods and increasing severity of histological diagnoses. Associations between biomarkers and HPV status were compared using Chi-square test. McNemar tests were used to compare paired matching data. All data was conducted using SPSS version 19.0  with statistically significant level of 0.05 (two-sided).


Women living with HIV (WLWH) are at increased risk of persistent human papillomavirus (HPV) infection, cervical dysplasia and cervical cancer compared with women from the general population (WGP). We assessed the prevalence and distribution of cervical high-risk (hr) HPV infection and cytological abnormalities in WLWH compared with WGP in Denmark. Predictors of HPV and cytological abnormalities were estimated in WLWH.Study on HIV, cervical Abnormalities and infections in women in Denmark (SHADE) in 2011 and were examined for cervical HPV and cytological abnormalities.WLWH consecutively enrolled in the WLWH were matched on age and prior cytological findings with WGP from an earlier study. HIV demographics were retrieved from the nationwide Danish HIV Cohort Study. Logistic regression was used to estimate predictors of hrHPV and cytological abnormalities.p < 0.0001). WLWH had a higher number of multiple infections (>1 h genotype present) . Hr genotypes in descending order of frequency were HPV58 (7.1 %), 52 (5.4 %), and 16 (4.8 %) in WLWH versus HPV16 (4.1 %), 52 (2.8 %) and 58 (2.4 %) in WGP. Predictors of hrHPV in WLWH were short duration of HAART (adjusted OR per year 0.90 (95 % CI 0.84-0.96)), AIDS prior to inclusion (adjusted OR 3.61 (95 % CI 1.75-7.46)), ≥5 lifetime sexual partners (adjusted OR 2.20 (95 % CI 1.08-4.49)), sexual debut <16 years of age (adjusted OR 2.05 (95 % CI 1.03-4.10)) and CD4 < 350 cells/μL (adjusted OR 2.53 (95 % CI 1.20-5.40)). Cytological abnormalities were prevalent in 10.4 % vs. 5.2 % (p = 0.0003) of WLWH and WGP. In WLWH with hrHPV, short duration of HAART predicted cervical dysplasia (adjusted OR per year 0.83 (95 % CI 0.71-0.97)).Of 334 included WLWH 26.4 % were positive for hrHPV as opposed to 16.6 % WGP  contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) is the most common sexually transmitted disease (STD)  with a lComprehensive data on the prevalence and distribution of HPV genotypes are informative in the planning of HPV/CC screening tools and with the rollout of HPV vaccines . The hrHThough, immune reconstitution induced by highly active antiretroviral therapy (HAART) might decrease the prevalence of HPV and cervical dysplasia, the effect of improved immunosurveillance remains controversial , 7. The Compared with other Western countries WGP in Denmark have high incidences of HPV and CC , 18, 19.The aim of the present study was to assess the prevalence and distribution of cervical hrHPV and cytological abnormalities in WLWH in Denmark. Further, we aimed at identifying predictors of hrHPV and cytological abnormalities in WLWH in a setting with free access to CC screening, healthcare and HAART.Study on HIV, cervical Abnormalities and infections in women in Denmark (SHADE) [Denmark has a population of 5.6 million  and an e (SHADE)   and cytology results from WGP included in the Danish Horizon Study, Copenhagen, Denmark, were retrieved from the authors .Cytological evaluation of SurePath samples was undertaken at Department of Pathology, Hvidovre, Copenhagen University Hospital (HVH). The outcomes were reported using the Bethesda 2001 system  and clasCervical samples were examined by the CLART HPV2 assay  at Department of Pathology, HVH. PCR amplification of genotype specific HPV L1 fragments from 35 individual HPV genotypes was performed including 13 hrHPV: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 . SamplesContinuous variables were summarized as median and interquartile ranges (IQR) or mean and ranges and compared using the Wilcoxon rank sum test. Categorical variables were reported as counts and percentages and compared using the chi-square test or Fisher’s exact test.For comparison of HPV status, genotype distribution and cytology results study participants were matched 1:5 to WGP on prior screening history in accordance with the algorithm used in the Horizon study  and age Univariate and multiple logistic regression analyses were used for identifying predictors of hrHPV expressed as odds ratios (OR) and 95 % confidence intervals (CI). Nine candidate predictor variables were chosen a priori due to current knowledge on risk factors of HPV , 30, 31;Predictors of ASCUS or worse (ASCUS+) were estimated by including the aforementioned variables, presence of cervical hrHPV and adherence to the general population CC screening program in the analysis. A subgroup analysis was performed in WLWH with ASCUS+ and cervical hrHPV only.Duration of HAART, AIDS prior to inclusion and CD4 count are dependent covariates and where calculated using two models: A model where all variables but CD4 at inclusion was included and a model where duration of HAART and AIDS prior to inclusion were replaced by CD4. We only presented the OR of the CD4 count from the second model.p-values smaller than 0.05:13 (p < 0.0038) were considered statistically significant. In the remaining analyses p-values <0.05 were considered statistically significant. For category variables with more than two outcome categories (df > 1), we controlled for repeated testing by estimating the combined p-value. Individuals with missing explanatory values were excluded from the multiple regression analyses. The validity of the model was tested using the Hosmer and Lemeshow Goodness-of-Fit Test.To account for multiple testing when comparing the distribution of genotypes, we applied Bonferoni correction. Therefore, in analyses of the 13 hrHPV genotypes SAS statistical software version 9.3  was used for data analysis. The matching of study participants and the HPV genotype distribution figures were performed in R 3.2.0 .p = 0.0015), have higher CD4 counts (p = 0.012), and increased probabilities of being on HAART with a suppressed viral load (p = 0.0042). Moreover, they had a higher uptake of both the annual HIV-and general population CC screening program (p < 0.0001 and p < 0.0001) and a higher probability of the latest cytology result being normal (p < 0.0001)  were included. At inclusion, median age and duration of HIV were 42.5 (IQR 36.8-48.3) and 11.3 (IQR 5.9-16.9) years  had a cervical swab performed. Of these 295 (90.5 %) yielded sufficient DNA for analysis and were matched at 1:5 with 1475 WGP from the Horizon study  (Table p = 0.030 and p = 0.047) , whereas both groups of women would receive a similar degree of protection from the 9-valent HPV vaccine based upon their HPV genotype distribution (p = 0.14)  Table .n = 21, 7.1 %), 52 , 16 , 51 , 18  and 33  versus 16 , 52 , 58 , 31 , 51  and 33  in WGP ). We compared genotype distribution in WLWH of different races and found that Black WLWH had a higher risk of HPV58 and 56 compared with WGP (p < 0.0001 and p = 0.0023) . We cop = 0.0004). HPV58 , 52 , 51  versus HPV16 , 58  and 52  predominated in WLWH and WGP, respectively .In WLWH and WGP with ASCUS+ 61.8 % versus 49.0 % were hrHPV-positive , 16  and 56  versus HPV52 , 16  and 45  ), AIDS prior to inclusion (adjusted OR 3.61 (95 % CI 1.75-7.46)), ≥5 lifetime sexual partners (adjusted OR 2.20 (95 % CI 1.08-4.49)), sexual debut <16 years of age (adjusted OR 2.05 (95 % CI 1.03-4.10)) and CD4 < 350 cells/μL (adjusted OR 2.53 (95 % CI 1.20-5.40)) predicted prevalent hrHPV (Table p = 0.0003). Cytological abnormalities in WLWH and WGP were distributed as follows: ASCUS: 8 (2.5 %) versus 42 (2.6 %), (p = 0.90); LSIL: 20 (6.1 %) versus 23 (1.4 %), (p < 0.0001) and HSIL: 6 (1.8 %) versus 20 (1.2 %), (p = 0.38). No WLWH or WGP presented with carcinoma.Five 1.5 %) of the 332 cytology samples received from WLWH were inadequate for evaluation leaving 327 for interpretation. These were matched with cytology results from 1635 WGP ) (Additional file In all adjusted analyses we checked the effect of missing values on outcome by adding an extra category with missing values. This had no impact on the estimates.In this multicentre, cross-sectional cohort study of WLWH in Denmark, we found a higher prevalence of cervical hrHPV in WLWH compared with WGP matched on prior screening history and age. Further, WLWH had a higher number of hrHPV genotypes and more carried multiple hrHPV infections. Presence of ≥1 genotypes covered by the 9-valent HPV vaccine was higher in WLWH and a higher number of WGP had all present genotypes covered by the 4-valent HPV vaccine. WLWH had a different distribution of hrHPV genotypes and this difference was mainly attributed to WLWH of Black race. There was a higher risk of ASCUS+ in WLWH, due to a higher prevalence of LSIL. Finally, cervical HPV and ASCUS+ were predicted by short duration of HAART.Prevalent HPV infection is dependent on age with a peak prevalence in women in the early 20s followed by a steady decline and a second, but smaller peak in women ≥45 years of age attributed to either new acquisition or viral persistence . TherefoStudies of WGP across all continents have repeatedly identified hrHPV16 and 18 - accounting for about 70 % of all CCs , 6 – to The molecular mechanisms leading to the increased risk of HPV in PLHIV are poorly understood . TugizovOverall, WLWH had more cytological abnormalities, due to a higher prevalence of LSIL. A higher prevalence of LSIL, but not ASCUS and HSIL, in WLWH is also reported by others . Most LSPredictors of cervical dysplasia and CC are those also associated with HPV infection such as smoking, early sexual debut, number of lifetime sexual partners, hormonal contraceptives and STDs other than HPV , 7. We hWe have a very well-described cohort due to the DHCS and DPDB and were able to match on prior cytological outcomes. Compared to other Western countries Danish women represent a CC high-risk population , therefoPossible limitations are that WLWH included in SHADE were more likely to comply with CC screening programs and to have a previous normal cytology result. Therefore estimates regarding cytological abnormalities in WLWH might be conservative. The effect of HAART duration can be confounded by other time-scales such as duration of HIV. Moreover, racial origin of the WGP group was not available for direct comparison to the SHADE cohort. Furthermore, a higher number of inadequate HPV samples were collected in the present study compared to ordinary CC screening samples included in the Horizon study, probably as a consequence of sampling by personnel less trained in gynecological routines. Finally, this is a cross-sectional study giving information on HPV infection at the time of one single sampling. The limitation in this design is that without previous or subsequent sampling results, any HPV infection observed may be a transient infection.WLWH had a higher risk of being cervical hrHPV positive, a higher frequency of multiple infections, a different genotype distribution and more cytological abnormalities than WGP. Cervical HPV and ASCUS+ were predicted by short duration of HAART.


EGFR and rs12034979 in HSPG2 significantly correlated to HPV16 infection; rs2575738, rs2575712, rs2575735 in SDC2 and rs6697265 in HSPG2 significantly correlated to HPV18 infection; rs10510097 in FGFR2, rs12718946 in EGFR significantly correlated to HPV52 infection; rs4947972 in EGFR, rs2981451 in FGFR2, rs2575735 in SDC2 significantly correlated to HPV58 infection. And rs3135772, rs1047057 and rs2556537 in FGFR2, rs12034979 in HSPG2, rs16894821 in SDC2 significantly correlated to cervical lesion progression induced by HPV16 infection; rs6697265 and rs6680566 in HSPG2, rs16860426 in ITGA6 by HPV18 infection; rs878949 in HSPG2, rs12718946 and rs12668175 in EGFR by HPV52 infection; no SNP by HPV58 infection. Our findings suggest that HPV receptor and associated gene variants may influence the susceptibilities to HPV type-specific infection and cervical lesion progression, which might have a potential application value in cervical cancer screening and therapy.Human papillomavirus (HPV) infects cervical epithelial cells through cellular membrane receptors, and then induces the initiation and progression of cervical cancer. Single nucleotide polymorphisms (SNPs) may impact the susceptibility and outcome of diseases, but it's still unknown whether variant in HPV receptor and associated genes is associated with type-specific HPV infection and cervical lesion progression. We examined 96 SNPs in 8 genes which may participate in the HPV infection process in 875 samples with HPV negative or single HPV16, 18, 52, 58 positive from 3299 cervical exfoliated cell samples, by Illumina BeadXpress VeraCode platform, and analyzed the correlation between the SNPs and type-specific HPV infection and cervical lesions progression. We found rs28384376 in  Cervical cancer is the second most common cancer among women, with more than 527,624 new cases and 265,672 deaths , in the The differences of HPV genotype distribution are also found in different grades of cervical lesions. A meta-analysis showed that HPV16 positivity was gradually increased from normal/ASCUS/LSIL/CIN1 (20-28%), through CIN2/HSIL (40/47%) to CIN3/ICC 58/63%)[8/63%[5].CD4, as well as CXCR4 and CCR5, for HIV [NTCP) for HBV [FURIN) mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, virion binds to alpha 6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Therefore, as opposed to a sequential handoff of the virion from one receptor to another, we hypothesize that a receptor complex coalesces and includes HSPGs(HSPG2 and SDC2), CyPB(PPIB), alpha 6 integrin(ITGA6), tetraspanins(TSPAN1), GFR(EGFR and FGFR2) and A2t. Thus, we selected eight genes, which have been identified as mainly molecules involved in HPV infection process, into our study. The eight genes included EGFR, PPIB, HSPG2, FGFR2, FURIN, ITGA6, TSPAN1 and SDC2). Considering the association of individual SNP with pathogen infection and disease outcome, we assume that SNPs in HPV receptor and associated genes may influence the susceptibility to type-specific HPV infection and cervical lesions progression.Previous studies have revealed that viruses infect host cells through the relevant receptors, such as herpes virus entry mediator(HVEM) for Herpes virus,  for HIV , and sod for HBV . Further for HBV , and SNP for HBV . It has  for HBV –16. Acco for HBV –18. GrowThus, we selected 96 single-nucleotide polymorphism (SNP) sites in the eight genes that were reported to be involved in the process of HPV infection using the haploview software, and evaluated the correlation between the distribution frequency of various SNP sites and type-specific HPV infection and cervical lesion progression in four common HPV genotypes (HPV16/18/52/58) in China and East Asia. Our study aimed to find out the genetic susceptibility to type-specific HPV infection and cervical lesion progression and search for a novel strategy for cervical cancer prevention or therapy.The median age of 875 women whose cervical samples were collected was 41 yrs (21-69 yrs), included 40 yrs (22-68 yrs) in 214 controls (HPV negative), 43 yrs(21-69 yrs) in 294 single HPV16 positive, 44 yrs(27-59yrs) in 55 single HPV 18 positive, 40 yrs(21-69 yrs) in 155 single HPV 52 positive, and 41 yrs(23-68 yrs) in 157 single HPV 58 positive. Further, the median age in 502 women with ≤LSIL and 373 with ≥HSIL was 40 yrs(21-63 yrs) and 43 yrs(21-69 yrs), respectively.2 test and fisher's test.All the SNPs in control population of this study were tested by Hardy–Weinberg equilibrium (HWE) as shown in SDC2 gene  and one in EGFR  in HPV16 positive group. There were three significant SNPs in SDC2 gene  and two in HSPG2 gene  and one in TSPAN1 gene  in HPV18 positive group. There was one significant SNP in SDC2 gene , one in HSPG2 gene  and one in PPIB gene  in HPV52 positive group. There were two significant SNPs in EGFR gene  and one in FURIN gene  and one in SDC2 gene  in HPV58 positive group. The detailed data were shown in Compared with HPV negative, there were two significant SNPs in Compared with HPV negative, there was one protective SNP genotype “TA” of rs2651465  in HPV16 positive group. Three protective SNP genotypes “AA” and “AC” of r s2575712  and “AG” of r s3767137  in HPV18 positive group. There was one susceptible SNP genotype “AA” of rs2589205  and one protective SNP genotype “GA” of rs6680566  in HPV52 positive group. There were two susceptible SNP genotypes “AG” of rs11770506  and “GC” of rs4947972 , two protective SNP genotypes “AC” of rs17514846  and “AA” of rs2575712  in HPV58 positive group. The detailed data were shown in HSPG2 gene (Freq (case) =0.057, Freq (control) =0.16, χ2=7.612, Chi square's P value=0.0058, Fisher's P value=0.004508) in HPV18 positive group. There were one protective haplotype “GGG” in HSPG2 (Freq(case)=0.326, Freq (control) =0.403, χ2=4.421, Chi square's P value=0.0355, Fisher's P value =0.036331) and one susceptible haplotype “GGA”  in HSPG2 (Freq (case) =0.295, Freq (control)= 0.228, χ2=4.305, Chi square's P value =0.038, Fisher's P value=0.039358) in HPV52 positive group. There were two protective haplotypes “GG”  in EGFR (Freq (case) =0.578, Freq (control)=0.667, χ2=6.133, Chi square's P value=0.0133, Fisher's P value =0.01617) and “AA”  in FURIN (Freq (case)=0.097, Freq (control)=0.152, χ2=4.724, Chi square's P=0.0297, Fisher's P value=0.043411) in HPV58 positive group. The detailed data were shown in Compared with HPV negative, there was no significant different haplotype in HPV16 positive group. There is one protective haplotype “GGAGA”  in All of the tested SNPs in the table were in Hardy–Weinberg equilibrium (HWE) in the control population from the normal samples (p>0.05), as shown in Table EGFR gene  and one in HSPG2 gene  in HPV16 positive group. There were three SNPs in SDC2 gene  and one in HSPG2 gene  in HPV18 positive group. There was one SNP in FGFR2 gene  and one in EGFR gene  in HPV52 positive group. There was one SNP in EGFR gene  and one in FGFR2 gene  and one in SDC2 gene  in HPV58 positive group. The detailed data were shown in Table Compared with HPV negative in the normal samples, there was one significant SNP in Compared with HPV negative in the normal samples, there were two susceptible SNP genotypes “AA” of 28384376  and “AG” of rs12034979  in HPV16 positive group. Three susceptible SNP genotypes “AA” of rs2575738 , “GG” of rs6697265  and “GC” of rs6697265  in HPV18 positive group. There was one protective genotype “GG” of rs12718946  in HPV52 positive group. There was one protective genotype “AA” of rs2981451  in HPV58 positive group Table .2=4.149, Chi square's P value =0.0417, Fisher's P value =0.059048) and “GGAA” (Freq(case)= 0.037, Freq (control)= 0.006, χ2=4.395, Chi square's P value =0.0361, Fisher's P value =0.096763) in block 10  in FGFR2 gene and one critically protective haplotype “GA” (Freq(case)= 0.161, Freq (control)= 0.287, χ2=3.876, Chi square's P value =0.049, Fisher's P value =0.051813) in block 7  in SDC2 gene in HPV16 positive group. There was one susceptible haplotype “GGG” in block3  (Freq(case)= 0.587, Freq (control)= 0.414, χ2=4.93, Chi square's P value =0.0264, Fisher's P value =0.0386) and two protective haplotypes “GGA” in block2  (Freq(case)= 0.108, Freq (control)= 0.243, χ2=4.209, Chi square's P value =0.0402, Fisher's P value =0.040537) and “GGAGA” in block1  (Freq(case)= 0.043, Freq (control)= 0.157, χ2=4.26, Chi square's P value =0.039, Fisher's P value =0.042397) in HSPG2 gene in HPV18 positive group. There were two susceptible haplotypes “GGGAA” (Freq(case)= 0.225, Freq (control)= 0.148, χ2=4.164, Chi square's P value =0.0413, Fisher's P value =0.043573) in block 1  and “GGA” (Freq(case)= 0.333, Freq (control)= 0.244, χ2=3.958, Chi square's P value =0.0466, Fisher's P value =0.05283) in block2  in HSPG2 gene in HPV52 positive group. There was one susceptible haplotype “GC” (Freq(case)= 0.311, Freq (control)= 0.184, χ2=7.765, Chi square's P value =0.0053, Fisher's P value =0.006766) and one protective haplotype “GA” (Freq(case)= 0.17, Freq (control)= 0.278, χ2=5.001, Chi square's P value =0.0253, Fisher's P value =0.029213) in block 8  in FGFR2 gene in HPV58 positive group = 0.09, Freq (control)= 0.032, χup Table .EGFR and HSPG2 gene to HPV16 infection, SDC2 and HSPG2 to HPV18 infection, EGFR, FGFR2 and HSPG2 to HPV52 infection, and EGFR, FGFR2 and SDC2 to HPV58 infectionTaken above results together, some variants in HPV receptor and associated genes were found to be correlated to type-specific HPV infection, including All of the tested SNPs in the table were in Hardy–Weinberg equilibrium (HWE) in the control population of this study (p>0.05 except for rs878949 and rs12668175 in HPV52), as shown in Table FGFR2 gene  and one  in HSPG2 gene and one  in SDC2 gene in single HPV16 positive with ≥HSIL. There were two significant different SNPs in HSPG2 gene  and one in ITGA6 gene  in HPV18 positive with ≥HSIL. There were two significant different SNPs in EGFR gene  and one in HSPG2 gene  in HPV52 positive with ≥HSIL. There was no significant different SNP site in HPV58 positive with ≥HSIL  and “AG”  of rs3135772; “AG”  of rs12034979 and “GA”  of rs16894821; two susceptible SNP genotypes “GG”  of rs2556537 and “AG”  of rs1047057 in single HPV16 positive with ≥HSIL. There was a susceptible SNP genotype “CC”  of rs6697265 and two protective SNP genotypes “GG”  and “GA”  of rs6680566 in HPV18 positive with ≥HSIL. There were two susceptible SNP genotypes “AG”  of rs878949 and “GG”  of rs12718946 in HPV52 positive with ≥HSIL. There was no significant different SNP genotype in HPV58 positive with ≥HSIL Table .2=5.442, Chi square's P value =0.0197, Fisher's P value =0.022419) in block 6  in SDC2 gene in HPV16 positive with ≥HSIL. There was one protective haplotype “GG” (Freq(case)= 0.333, Freq (control)= 0.594, χ2=6.25, Chi square's P value =0.0124, Fisher's P value =0.021295) in block2  in HSPG2 gene in HPV18 positive with ≥HSIL. There was one susceptible haplotype “CAA” (Freq(case)= 0.271, Freq (control)= 0.131, χ2=7.681, Chi square's P value =0.0056, Fisher's P value =0.00897) in block2  in HSPG2 gene in HPV52 positive with ≥HSIL. There was no significant different haplotype in HPV58 positive with ≥HSIL = 0.209, Freq (control)= 0.319, χIL Table .FGFR2, HSPG2 and SDC2 with HPV16, ITGA6 and HSPG2 with HPV18, and EGFR and HSPG2 with HPV52, but no with HPV58.Taken above results together, some variants in HPV receptor and associated genes were found to be associated with cervical lesion progression induced by type-specific HPV, including It is well known that the occurrence and development of human cervical cancer are related to high-risk HPV infection. HPV 16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with its L1major capsid protein, and afterwards HSPG/growth factor/HPV16 complexes activate growth factor receptors which initiate signaling cascades during early virion-host cell interactions . After HEGFR gene and rs12034979 in HSPG2 gene, and two susceptible genotypes “AA” of rs28384376 and “AG” of rs12034979, and two risk haplotypes “GGAA” and “GGGG” in block10  in FGFR2 gene and one protective haplotype “GA” in block7  in SDC2 gene in HPV16 positive group, suggesting the genetic variants in EGFR, HSPG2, FGFR2 and SDC2 genes may be associated with type-specific HPV16 infection. The differences of haplotype frequency distributions in target genes between cases and controls were significant by χ2 test (p<0.05) while not by fisher's test (p>0.05), suggesting that each kind of haplotype may be susceptible to type-specific HPV16 infection and there is no dominant susceptible or protective haplotype. The OR of rs28384376 and rs12034979 are 2.642 and 3.114 respectively, and there was no protective SNP site except for one critical haplotype. All those phenomenons might be employed as an explanation that HPV16 possesses the highest prevalence among all high-risk genotypes in the worldwide. Again we found three susceptible SNP sites rs2575738 and rs2575735 in SDC2 gene and rs6697265 in HSPG2 gene, and one protective SNP site rs2575712 in SDC2 gene, and three susceptible genotypes “AA” of rs2575738 and “GG”, “GC” of rs6697265, and one susceptible haplotype “GGG” in block3  and two protective haplotypes “GGA” in block2 , “GGAGA” in block1  in HSPG2 gene in HPV18 positive group, suggesting that genetic variants in HSPG2 and SDC2 genes are associated with type-specific HPV18 infection. Similarly, there was one susceptible SNP site rs10510097 in FGFR2 gene and one protective SNP site rs12718946 in EGFR gene, and one protective SNP genotype “GG” of rs12718946; and two susceptible haplotypes “GGA” in block2 , “GGGAA” in block1  in HSPG2 gene in HPV52 positive group, suggesting that genetic variants in HSPG2, FGFR2 and EGFR genes are associated with type-specific HPV52 infection. And again, there was two susceptible SNP sites rs4947972 in EGFR gene and rs2575735 in SDC2 gene and one protective SNP site rs2981451 in FGFR2 gene, and one protective SNP genotype “AA” of rs2981451, and one susceptible haplotype “GC” and one protective haplotype “GA” in block8  in FGFR2 gene in HPV58 positive group, suggesting that genetic variants in EGFR, SDC2, and FGFR2 are associated with type-specific HPV58 infection. Thus, variants of HPV receptor and associated genes in SNP sites, genotypes and haplotypes may influence significantly the susceptibility of the individual to type-specific HPV18, 52 and 58 in Chinese women, but seems unobvious to type-specific HPV16 infection.In an analysis on the susceptibility of HPV receptor and associated gene variants to type-specific HPV infection, we found two susceptible SNP sites rs28384376 in FGFR2 gene, and three protective SNP sites rs3135772 in FGFR2 gene, rs12034979 in HSPG2 gene and rs16894821 in SDC2 gene, and two susceptible SNP genotypes “GG” of rs2556537 and “AG” of rs1047057 and four protective SNP genotypes “AA”, “AG” of rs3135772, “AG” of rs12034979, “GA” of rs16894821, and one protective haplotype “GG” in block 6  in SDC2 gene, suggesting that genetic variants of FGFR2, HSPG2, and SDC2 are related to cervical lesion progression induced by HPV16 infection. In HPV18 positive group, there was one susceptible SNP site rs6697265 in HSPG2 gene and two protective SNP sites rs6680566 in HSPG2 gene and rs16860426 in ITGA6 gene, and one susceptible SNP genotype “CC” of rs6697265 and two protective SNP genotypes “GG” and “GA” of rs6680566, and one protective haplotype “GG” in block2 , suggesting that genetic variants in HSPG2 and ITGA6 are related to cervical lesion progression induced by HPV18 infection. In HPV52 positive group, there were three susceptible SNP sites rs878949 in HSPG2 gene, rs12718946 and rs12668175 in EGFR gene, and two susceptible SNP genotype “AG” of rs878949 and “GG” of rs12718946, and one susceptible haplotype “CAA” in block2 , suggesting that genetic variants in HSPG2 and EGFR are related to cervical lesion progression induced by HPV52 infection. In HPV58 positive group, there was no significant different genetic variant related to cervical lesion progression. Thus, HPV receptor and associated gene variants may be associated with cervical lesion progression induced by HPV16, 18, and 52, but not HPV58, infection in Chinese women.Furthermore, we analyze the correlation between HPV receptor and associated gene variants and cervical lesion progression in each single HPV positive group. In HPV16 positive group, there were two susceptible SNP sites rs2556537 and rs1047057 in Totally 3299 residual cervical exfoliated cell samples were primarily collected from women with various gynecologic disorders who underwent primary HPV testing, HC2 or Cervista test, in Clinic of Gynecologic Oncology of Women's Hospital, Medical School, Zhejiang University from October 2012 to May 2015. Of HPV positive samples, those were excluded just as follows: 1) other than A7/9 positive tested by Cervista, 2) previous surgical or physical therapy to cervix, 3) cervical infection by other pathogens, such as HIV, Syphilis, and Candida, 4) incorporative immune disease or use of immunosuppressive agents, 5) incorporative other malignant tumors, 6) incorporative pregnancy. HPV genotyping was performed for the remaining HPV positive samples using HybriBio's proprietary flow-through hybridization technique, and the single HPV16, 18, 52, 58 positive samples were selected for study. All HPV negative samples were identified by no bands on agarose gel electrophoresis after PCR amplification using HPV L1 consensus PCR primers (primers MY09 and MY11). After the samples with not-enough remaining cells, HPV genotype failure, and unqualified DNA were further excluded, 875 samples were finally enrolled into the study, include 214 HPV negative, 294 single HPV16 positive, 55 single HPV18 positive, 155 single HPV52 positive, and 157 single HPV58 positive. All the samples had the histological diagnosis, and the diagnostic criteria were followed by the American Society for Colposcopy and Cervical Pathology (ASCCP) 2006 guidelines . Sample The HPV test was performed by the Hybrid Capture 2 (HC2) test (Qiagen Digene) or Cervista test (Hologic). HC2 test is used for detecting the pool of 13 high-risk HPV genotypes including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Cervista test is used for detecting 14 genotypes (above 13 types plus 66), with a report of A5/A6, A7, and/or A9  positive or negative. Hybribio Rapid GenoArray test kit (GA) is used for HPV genotyping, including 6 low-risk types , 15 high-risk types . All the tests were performed according to the manufacturer's protocol –26.EGFR, PPIB, HSPG2, FGFR2, FURIN, ITGA6, TSPAN1 and SDC2) were genotyped. Validated tagSNPs were selected with a MAF > 5% in the HapMap Asia population. SNPs that satisfied the following criteria were considered for detection: 1) tagSNPs were preferentially selected, 2) those SNPs were previously reported to be frequent in Chinese population (http://www.ncbi.nlm.nih.gov/snp). The total genomic DNA was extracted from the cervical exfoliated cells using KoningTM Mutisource Genomic DNA Extration Kit-Mini and PureLink® Genomic DNA Kits (invitrogen). The DNA concentration was detected, agarose gel eletrophoresis was run and the final concentration was quantified to 50 ng/μl. All the SNPs were genotyped by Illumina BeadXpress VeraCode platform (USA), according to the manufacturer's protocol.The Haploview software 4.2  was used to analyze the tagSNPs and haplotype block based on the CHB (Chinese Han Beijing) population data of HapMap , a total of 96 SNPs in 8 HPV receptor and associated genes (All statistical analyses were performed using PLINK version 1.07 . All p v


When two identical visual discs move toward each other on a two-dimensional visual display, they can be perceived as either “streaming through” or “bouncing off” each other after their coincidence. Previous studies have observed a strong bias toward the streaming percept. Additionally, the incidence of the bouncing percept in this ambiguous display could be increased by various factors, such as a brief sound at the moment of coincidence and a momentary pause of the two discs. The streaming/bouncing bistable motion phenomenon has been studied intensively since its discovery. However, little is known regarding the neural basis underling the perceptual ambiguity in the classic version of the streaming/bouncing motion display. The present study investigated the neural basis of the perception disambiguating underling the processing of the streaming/bouncing bistable motion display using event-related potential (ERP) recordings. Surprisingly, the amplitude of frontal central P2 (220–260 ms) that was elicited by the moving discs ~200 ms before the coincidence of the two discs was observed to be predictive of subsequent streaming or bouncing percept. A larger P2 amplitude was observed for streaming percept than the bouncing percept. These findings suggest that the streaming/bouncing bistable perception may have been disambiguated unconsciously ~200 ms before the coincidence of the two discs. Well-known examples of this bistable perception include the famous face-vase drawing, Necker cube and binocular rivalry. Additionally, bistable perception occurs not only in the static figures mentioned above, but in moving objects as well. Consider two identical visual targets moving toward each other along the same horizontal line with equal and constant speed in a two-dimensional display: Two visual targets start their motion from opposite sides, coincide at the center of the screen, move apart, and stop at each other’s starting point. In this display, observers typically perceive the motion of the targets after coincidence as either “streaming through” or “bouncing off” each other2.The human visual system often experiences unitary and stable perception by integrating relevant sensory information in the environment. However, on occasions when the available sensory information is fragmented or ambiguous, our perception may alternate between two or more mutually exclusive interpretations5. Interestingly, a large number of behavioral studies have reported various factors that can reverse perceptual dominance from streaming toward bouncing, such as a momentary pause of the two discs5, a brief sound14, a transient visual distractor15, and even a transient tactile vibration16 at the moment of coincidence of the two discs as well as post-coincidence trajectory duration18, pre-coincidence trajectory switches (used to manipulate expectation)19, a partial overlap21 of the two discs, and an orientation difference between the stripes on the discs’ and their path of motion15. Several neuroscience studies have explored the neural mechanisms of the effect of sound on streaming/bouncing motion display . For example, an event-related functional magnetic resonance imaging (fMRI) study has shown that a transient sound induced higher activation in multimodal areas  when subjects perceived bouncing than streaming22. An electroencephalograph (EEG) study found increased beta-rhythm synchronization across frontal, parietal, and occipital cortex and gamma-rhythm synchronization across central and temporal regions for bouncing trials compared to streaming trials when the coincident sound was introduced23. Furthermore, using transcranial magnetic stimulation (TMS) to temporarily deactivate the posterior parietal cortex resulted in an attenuated magnitude of the sound’s effect on improving the bouncing percept24. Combining these neuroscience studies, cross-modal integration between polysensory and unisensory brain cortices may underlie the effect of sound on reversing the perceptual dominance of the streaming/bouncing motion display.Despite the ambiguous nature of the streaming/bouncing motion display, subjects usually show a strong bias to report the streaming percept (~80% streaming response vs. ~20% bouncing response)17. For example, Watanabe and Shimojo argued that moving objects in a natural environment are seldom (but still possibly) aligned at the same depth plane17. In other words, the bouncing event in a three-dimensional world would occur only when the two moving balls exist in the same depth plane . Our perceptual system may compute this probability when facing a visual streaming/bouncing motion display, so the streaming percept could be dominant and the visual streaming/bouncing motion display could potentially be ambiguous. Although this probabilistic inference account is highly reasonable and has been confirmed by several studies20, there is little experimental evidence to support this account in the context of a purely visual streaming/bouncing motion display25.Despite numerous identified bounce-inducing factors, especially evidences based on neural mechanisms for the audiovisual bounce-inducing effect (ABE), the neural basis of disambiguating perception during the streaming/bouncing bistable perception processing in purely visual streaming/bouncing display remains unclear. In other words, it is not understood how one percept (streaming or bouncing) overwhelms the other and eventually becomes a stable percept under an ambiguous context in a visual streaming/bouncing bistable display. Sekuler and Sekuler as well as Watanabe and Shimojo have proposed that the human perceptual system might utilize experience of the three-dimensional world in a probabilistic way to derive a stable percept in a two-dimensional visual streaming/bouncing motion display27. Consistent with this viewpoint, many studies have found neural activity in the primary visual cortex32, lateral geniculate nucleus34, and extrastriate visual cortex39 that correlates with perceptual outcomes or perceptual transitions in ambiguous displays. However, a growing number of fMRI studies have demonstrated that the frontal and parietal cortex play a causal role in initiating perceptual reversals in bistable displays43, which indicates that high-level brain areas could reorganize ambiguous information within the visual cortex and eventually reverse our perceptual interpretations45. On the other hand, a series of event-related potential (ERP) studies have identified two successive ERP components (difference wave) related to perceptual reversals of bistable stimuli, which are the occipital-parietal “reversal negativity” (RN)51 and the central-parietal “late positive complex” (LPC)52. These phenomena were discovered by comparing trials on which perceptual outcome was reversed to trials on which the perceptual outcome remained the same across successive trials. The RN (170–300 ms post-stimulus) is considered to be an early index of switches between neural representations that synthesize the current contents of conscious perception, while the LPC (400–600 ms) is thought to represent post-perceptual processing related to evaluating and reporting perceptual outcomes54. Therefore, the emerging brain model of bistable perception is a dynamic and highly interactive network between low-level (sensory) and high-level  brain regions.In previous bistable perception studies, a traditional view proposed that antagonistic activity within the visual system is a neural consequence of spontaneous perceptual reversalsApart from the reversal-related brain activities that are associated with the perception reversals mentioned above, the percept-related brain activities that are associated with the ongoing stable percept deriving should also be highlighted in bistable perceptions, especially in the case of visual streaming/bouncing display because “streaming through” and “bouncing off” of the two visual discs after coincidence are completely opposite perceptual states in nature. Therefore, exploring the neural basis of streaming and bouncing percepts in the visual streaming/bouncing display can contribute to better understandings of the issue of how and when one perceptual state prevails over the other and eventually becomes a stable percept during an ambiguous situation in bistable perceptions along with the well-known effect of sound on reversing the perceptual dominance of the streaming/bouncing display (ABE). Using high density ERP recordings, the present study investigated the brain dynamics of streaming/bouncing bistable motion processing. A trial-based analysis was performed to investigate neural activities in trials on which subjects reported streaming percepts  with trials on which bouncing percepts  were reported. Surprisingly, the amplitude of the P2 component over the frontal central scalp ~200 ms before the coincidence of two discs was found to be larger for the steaming percept than for the bouncing percept. These results demonstrated that brain activity ~200 ms before the coincidence of two discs was predictive of the subsequent perceptual outcome of the visual streaming/bouncing display.A total of 23 healthy subjects participated in this study after giving informed consent as required and approved by the Human Research Protections Program of SooChow University. All methods were carried out in accordance with the relevant guidelines and regulations. All participants had normal or corrected-to-normal vision and were naive to the purpose of the experiment. Data from 5 subjects were eliminated due to an inadequate number of trials  for one of the perception outcomes (streaming or bouncing), leaving data of 18 subjects  for further analysis.2 of luminance) throughout each block. In 66.7% of trials , two identical black discs (each 1.05° in diameter) were initially presented at the opposite edges of the background. The discs were separated by 18.9°  horizontally and placed 3.46° above the fixation cross on the first frame (the duration for each frame was 50 ms). From frame 2 through 9, the two discs moved toward each other along same horizontal path. Each frame was presented immediately after the offset of the preceding frame . On frame 10 (450 ms after the onset of the first frame), the two discs visually coincided above the fixation cross. From frame 11 through 19, the discs moved apart from each other and stopped at the starting point of the opposite disc  and were presented on a 27-inch LCD monitor . A small red cross  served as a fixation point and was presented at the center of the gray background  in a randomized sequence and the intertrial intervals (ITI) varied from 1200 to 1600 ms . Vertical eye movements and blinks were monitored via two bipolar electrodes above and below the left eye . The left mastoid electrode served as the reference and all electrode impedances were kept below 5 kΩ during data acquisition. The raw EEG and EOG signals were amplified with a gain of 10,000, filtered with an amplifier bandpass of 0.05–100 Hz, and were digitized with a sampling rate of 1000 Hz. The EEG signals on response-required trials were averaged in 1200 ms epochs time-locked to the onset of the two visual discs  was recorded continuously using a 64-channel tin-electrode cap  based on an extended 10–20 system montage. Standard 10–20 sites were FP1, FPz, FP2, F7, F3, Fz, F4, F8, T7, C3, Cz, C4, T8, P7, P3, Pz, P4, P8, O1, Oz and O2To examine the neural activities of the streaming percept and bouncing percept, ERPs recorded on the response required trials were analyzed on the basis of the subjects’ perceptual responses. A trial-by-trial analysis was performed by separating the response required trials on which subjects reported streaming percept  from trials on which bouncing percept  were reported. ERP waveforms were averaged separately for streaming trials and bouncing trials. On average, there were 315 ± 22 (mean ± SE) streaming trials and 197 ± 18 bouncing trials. The main ERP components in both streaming and bouncing trials were quantified as the mean amplitudes with respect to a 200-ms pre-stimulus baseline over the following time windows and with the following electrodes: (1) C1 component ; (2) P1 component ; (3) N1 component ; (4) P2 component ; (5) N400 component ; (6) N600 component ; and (7) P3 component . These time windows and their corresponding measurement electrodes were chosen because each ERP component had its maximal amplitude over its given time windows and electrodes. After quantifying these main ERP components, the mean amplitudes of each ERP component were then subject to a two-way repeated-measure ANOVA with factors of perceptual response (streaming/bouncing) and the electrode (channels listed above for each ERP component), respectively. When appropriate, ANOVA results were corrected using the Greenhouse-Geisser procedure. Post-hoc comparisons for the main effects of electrode were made to determine the significance of pair-wise contrasts when appropriate, using the Bonferroni adjustment for multiple comparisons.5, the percentage of streaming responses in our study was significantly higher compared to the bouncing responses . Although there was a trend of faster reaction times for streaming responses compared to bouncing responses , which appears to be consistent with the observation that the dominant percept (i.e. streaming) in the visual streaming/bouncing display leads to shorter reaction times10, this difference did not reach significant level  in the present study.The percentage of certain responses (streaming or bouncing), and reaction times  between streaming trials and bouncing trials were compared by ANOVA, respectively. Consistent with the findings in previous behavioral studies that streaming percept dominates in the original visual streaming/bouncing motion display56 in both streaming and bouncing trials  = 2.32, p = 0.146, η2p = 0.12; streaming, −0.71 ± 0.27 μV (mean ± SE); bouncing, −0.40 ± 0.19 μV; Fig. F = 2.98, p = 0.062, η2p = 0.15] as well as the response x electrode interaction effect  were also not significant. After C1, the occipitally distributed P1 component  also did not show a significant main effect of perceptual response , and neither the main effect of the electrode  nor the response x electrode interaction effect  was significant. Similarly, the subsequent N1 component peaking at 170–200 ms over occipital electrodes also did not reveal a significant modulation as a function of perceptual response  nor the response x electrode interaction effect  was significant. These results suggest that early visual-evoked ERPs before the coincidence of the two discs were not associated with distinct perceptual states in the visual streaming/bouncing display.Time-locking to the onset of the two discs  = 10.19, p < 0.006, η2p = 0.38], with greater P2 amplitude on streaming trials . The main effect of the electrode was also significant . Pair-wise comparisons for this main effect showed that P2 amplitude was larger on FCz than on FC2 (p < 0.03 after Bonferroni correction), and was larger on C1 and Cz than on C2 (ps < 0.05 after Bonferroni correction). The response x electrode interaction effect for P2 amplitude was not significant . Following the positivity of P2 component, there was also a negative deflection labeled as N400 component  = 6.19, p < 0.03, η2p = 0.27], with greater N400 amplitude for the bouncing trials , and a significant main effect for electrodes , with the N400 amplitude being larger on Cz than on C1 and C2 (ps < 0.03 after Bonferroni correction), as well as a nonsignificant response x electrode interaction effect .As shown in Fig. ent Fig.  over theIt is noteworthy that streaming and bouncing trials were separated based on subjects’ perceptual responses, and when ERP waveforms started to differ significantly in the P2 interval between streaming and bouncing trials, the two visual discs were still moving toward each other Fig. . In otheF = 0.10, p = 0.753, η2p = 0.01; streaming, −4.67 ± 0.73 μV; bouncing, −4.60 ± 0.65 μV], and neither the main effects of the electrode  nor the response x electrode interaction effect  was significant. Following the N600 component, a late slow positivity extended over 800–900 ms for both the streaming and bouncing trials  = 5.75, p < 0.03, η2p = 0.26], with greater P3 amplitude on bouncing trials (4.10 ± 0.70 μV) compared to streaming trials (3.28 ± 0.71 μV). The main effect of the electrode was also significant , with the P3 amplitude being larger on Pz than on CP1 and CP2 (ps < 0.013 after Bonferroni correction). Finally, the response x electrode interaction effect for the P3 amplitude was not significant . These results indicated that the post-coincidence P3 amplitudes could also distinguish different perceptual outcomes in the visual streaming/bouncing display.After the streaming/bouncing event occurred , there was firstly a broad negativity with the maximal deflection during 560–660 ms Fig.  over froals Fig.  is most als Fig. , which wet al. that found the dominant percept  in the visual streaming/bouncing display led to faster RTs10, although there was a tendency of faster RTs for streaming responses  in the present study. A possible reason for this disparity might be that the existence of streaming/bouncing display with a salient sound at the moment of coincidence of the two discs [visual-auditory (VA) condition] in their experimental design influenced the response bias when participants perceived the purely visual streaming/bouncing display [visual-only (V) condition]. Therefore, when both VA and V conditions existed, salient sound in the VA condition would induce more bouncing percepts, which might result in hesitation when the subjects perceived streaming in the VA condition. Conversely, experience with the VA condition might also result in hesitation when subjects perceived bouncing in the V condition. Thus, the pattern that markedly faster RTs for bouncing percepts in the VA condition and faster RTs for streaming percepts in the V condition eventually occurred in study of Sanabria et al.10. However, the present study did not introduce a transient sound at the moment of coincidence, and so there was no response bias effect. Therefore, the results led to no substantial difference in RTs between streaming and bouncing percepts in the present study.The present study investigated the neural basis of the processing of the classic streaming/bouncing motion display using high density event-related potential (ERP) recordings. The behavioral results showed no significant difference on RTs between the streaming and bouncing percepts, which was inconsistent with the findings of Sanabria 60, which suggests that ongoing brain activities before stimulus-driven processes might contribute to how perceptual conflict is resolved by the human brain. The pre-coincidence differences in ERPs in the present study seem to make sense if we assume the motion sequences start at the moment of coincidence. Specifically, if the movement of the two discs started at the exact frame of their coincidence without any motion trajectory before coincidence, it is obvious that subjects in the behavioral task would perceive neither streaming nor bouncing but only two discs moving apart from the coincident point. Thus, the movement of the two discs before their coincidence actually served as a prerequisite for subsequent perceptual outcomes. Indeed, Grove et al. found that manipulating pre-coincidence trajectory switches (used to manipulate expectation) in streaming/bouncing display could significantly bias subsequent perceptual inferences19, which supports this viewpoint. It is also worth mentioning that the fMRI study that focused on the audiovisual bounce-inducing effect (ABE) conducted by Bushara et al. also investigated the purely visual streaming/bouncing display, but their data was time-locked to the coincidence of the two discs and found no difference in brain activity between the streaming and bouncing percepts22. When the fMRI results and our results showing a pre-coincidence difference on P2 and N400 components are combined, an inference can be drawn that subjects processed the motion information of two discs before their coincidence is an essential factor in influencing subsequent perceptual outcomes. In other words, the origin of ambiguity in visual streaming/bouncing processing may come from how subjects processed the motion information of the two discs before their coincidence. That is not to say other factors that occurred after or at the moment of the coincidence of the two discs are not important. In fact, a brief sound presented at the moment of coincidence could reverse the dominance of the streaming percept (i.e. ABE)6, and the post-coincidence trajectory duration was also found to be sufficient to bias subsequent perceptual inferences18. Therefore, based on the present results and the discussion above, the pre-coincidence ERP differences found in the present study may reflect unconscious reduction of the ambiguity of streaming/bouncing display.The ERP results showed a larger positive deflection on the frontal central P2 (220–260 ms) amplitude and N400 (350–440 ms) amplitude  before the coincidence for the streaming trials than bouncing trials. It is noteworthy that brain activities before motion or stimuli onset could bias subsequent perceptual results in ambiguous displays61, and is involved in working memory62 and semantic processing63, its neurophysiological role has not been well characterized partly because it often overlaps with other adjacent ERPs such as N1, N2 and P3 while recording64. However, a recent study conducted by Shu et al. found that the P2 component is sensitive to depth perception, with larger P2 amplitude on three-dimensional (3D) than two-dimensional (2D) images 65. Similarly, Liu et al. found that size perception was modulated by depth cues, with larger P2 amplitude on ball in upper than in lower visual field  for the 3D background condition but no difference for the 2D background condition66, indicating the P2 component is sensitive to depth information as well. If this is the case, the greater P2 amplitude on streaming than bouncing trials in the present study . In other words, when subjects considered the two moving discs as being at different depth planes (indexed by larger P2), they would perceive streaming after the coincidence, but when the two discs were thought to be at the same depth plane , the bouncing percept would be reported. Consistent with this account, Grove and Sakurai found streaming percepts increased as depth disparity between two discs increased in both the visual-auditory and visual-only streaming/bouncing display, although their focus was the audiovisual bounce-inducing effect (ABE)67. Similarly, Matsuno and Tomonaga investigated the visual streaming/bouncing display in chimpanzees and found streaming percepts increased when more depth cues were introduced25. Combined with the evidences and explanations above, the P2 amplitude difference between bouncing and streaming trials in the present study may reflect whether or not the subject perceived the two moving discs before their coincidence as being at the same depth plane. However, further research is still needed to examine whether the perceived depth between the two discs is the determinant factor for the visual streaming/bouncing illusion, because the present study did not test it directly.It has yet to be determined, however, that what aspects of perceptual process are reflected by the pre-coincidence positive deflection  that started ~200 ms before coincidence of the discs. Although the P2 component is typically thought as part of a cognitive matching system that compares sensory inputs with expectations derived from memory64, whereas the stimuli  inducing the streaming and bouncing percepts were always the same in our experiment. Thus, identical stimuli sequences elicited the similar early visual ERPs is exactly what would be expected. In contrast, as previous bistable perception studies demonstrated, due to the same physical inputs of stimuli, any changes in the electrophysiological response between mutually exclusive perceptions could therefore be ascribed to higher level perceptual or cognitive factors, rather than to factors relied on early sensory-input properties49. This point of view fits the results of P2 in the present study and the prominent hypothesis that the P2 component represents part of a cognitive matching system that compares sensory inputs with expectations derived from memory57.As reported above, the pre-coincidence positive deflection that larger positivity was observed for the streaming percept than bouncing percept started ~200 ms before coincidence. However, the early visual-evoked ERPs  were almost identical between the streaming and bouncing trials  task-defined stimulus77. In the present behavioral results as well as previous studies of visual streaming/bouncing display, the streaming percept was dominant and occurred more frequently, whereas bouncing was the inferior percept and occurred occasionally5. Therefore, it is reasonable to infer that the streaming percept is subjectively the default state for subjects, which is considered as a highly probable perceptual outcome. Therefore, the bouncing percept is subjectively a novel perceptual outcome for subjects with a low probability. If that is the case, larger P3 amplitude for bouncing trials in the present study may reflect a novel bouncing percept with a low probability that triggered contextual updates for perceptual representation in subjects’ working memory. On the other hand, the P3 component in the present study seemed to appear so late that a time window of 800–900 ms was used to measure its amplitude. However, it makes sense because previous studies have shown that the latency of P3/P300 depends on the time required for stimuli classification, that is, P3 component should appear after classifying stimuli based on task requests81. Although ERP difference before streaming/bouncing event occurred had already influenced the subsequent perceptual outcomes in the present study, it is apparent that distinct perceptual outcomes must emerge after observing the occurrence of streaming/bouncing event. Specifically, classifying stimuli based on task requests  in the present study actually started from the moment of the coincidence of the discs. Thus, if ERP waveforms were time-locked to the moment of the coincidence (450 ms), the time window of P3 component would become 350–450 ms, and along with its parietal maximal scalp topography , the P3 amplitudes also dissociated streaming/bouncing responses, with greater P3 amplitude on bouncing trials than streaming trials Fig. . Numerouphy Fig. , these c45, and recent findings that the P2 component was sensitive to depth information66, as well as the existing hypothesis that experience in a three-dimensional environment was involved in disambiguating the visual streaming/bouncing display19, we conclude that the pre-coincidence frontal central positive difference between streaming and bouncing percepts in the present study may reflect whether or not the subject perceived the two moving discs before their coincidence as being at the same depth plane. Further studies are still needed to examine directly whether the perceived depth relationship between the two discs is the determinant factor for the visual streaming/bouncing display.In summary, the present study investigated the neural-activity patterns associated with the streaming and bouncing percepts to explore the origin of ambiguity and the neural basis of disambiguating perception during processing of the classic visual streaming/bouncing display. Interestingly, a frontal central positive deflection ~200 ms before the coincidence of the discs was found to be predictive of subsequent perceptual outcomes in the visual streaming/bouncing display. Moreover, P3 amplitudes ~400 ms after the coincidence of the two discs also dissociated streaming/bouncing responses. Base on previous studies of bistable perception that highlighted the role of high-level brain areas on perceptual interpretations


In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed.Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL.γH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state. Infection by HPV is responsible for the development of cervical squamous intraepithelial lesions (SIL) and cervical carcinoma, as well as for other squamous cell carcinomas in the anogenital and the head and neck area. There is an ongoing quest for biomarkers predicting the eventual progression of HPV related SIL into carcinoma.Dysregulation of the DNA damage response (DDR) mechanism has been implicated in the pathogenesis of a multitude of malignancies and is currently investigated, in order to identify predictors of disease progression as well as novel therapeutic targets .Genomic instability is important in the pathogenesis of cervical cancer and it is driven by the integration of DNA from high risk HPV types . The disEarly protein markers of tumorigenesis and their expression in HPV associated premalignant lesions may lead to better understanding of the pathogenesis of malignancy as well as better and earlier therapeutic interventions. Recently, several markers—including p53, Ki67 and γH2AX among others—have been used to this effect in HPV related tumorigenesis ).For γH2AX, slides were immersed in a high pH target retrieval solution [K8004 [DAKO]), boiled in the PTLink (DAKO) at 97For Ki67, antigen retrieval pretreatment with BondTM Epitope Retrieval Solution 2 was performed (pH 8.9 at 25oC), following which immunostaining was performed in the automated Leica Bond-Max system.A light Hematoxylin counterstain was performed for both immunostains.All immunostained slides were simultaneously assessed by two trained (3 years experience) pathologists with comparable experience , as well as the above mentioned qualified pathologist (IP). For controls and SIL cases, lower  and higher (surface) segments of the squamous epithelium were independently assessed; immunopositive cells had only nuclear staining. In every case, the percentage of immunopositive nuclei in 100 consecutive cells was calculated, both for the lower and higher segments of the epithelium. Since discrimination between lower and higher epithelial segments was not possible in carcinoma cases, the percentage of positive nuclei was calculated for a hundred consecutive cells.We analyzed cytology data separately and dichotomized it into cytological high grade  and low grade lesions; we then correlated cytological with histological findings. Histological grading was dichotomized for comparison between low grade and high grade squamous intraepithelial lesions ((LGSIL and HGSIL respectively). Demographic parameters between patients according to histological grading and type of virus were performed by analysis of variance (ANOVA) and Kruskal-Wallis or chi-square test as appropriate. Further, patients were characterized as LGSIL harboring high-risk HPV types (LGSIL-HR) vs LGSIL harboring low risk virus (LGSIL-LR) and by whether they had activated or inactivated infection by means of mRNA positivity for one of the two oncoproteins. Correlation between histological grading, high risk vs. low risk virus presence, activated vs. non-activated status and γH2AX expression was performed using chi-square test and ANOVA as appropriate. In order to find whether a statistically significant difference existed in the γH2AX expression between the lower and higher epithelial segments of SILs, independent samples t-testing was performed. SPSS version 22.0 for Windows software  was used for data analysis. All statistical tests were two-tailed.In total, 275 cases  were included in the study: 112 LGSIL, 99 HGSIL, 24 SCC, 12 AdC and 28 cervical specimens with no essential lesions . The meaLesions were then categorized as harboring viruses at an activated vs. non activated status, depending on mRNA positivity. Out of 108 negative /inadequate samples with Oncotect, 23 (21.3%) tested positive with NucliSens. Differences were then sought in LGSIL harboring a high-risk HPV type, depending on whether they were identified as non-activated (n = 56) or activated (n = 46).  to LGSIL harboring a high risk virus at a non-activated state  and in HGSIL . There wConcerning carcinomas γH2AX expression was significantly higher when compared with most of the other histological lesions .The distribution of HPV types in LGSIL according to HPV type and mRNA positivity is shown in We assessed the expression of the histone 2AX phosphorylated at serine-139 (γH2AX) protein in cervical histological lesions of increasing grade. We found a gradual increase of both basal and surface γH2AX expression up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state. Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. Moreover, there was a higher surface γH2AX expression in LGSIL cases with HR HPV infection at a non—activated state (as identified by the mRNA expression) versus LGSIL cases with HR HPV infection at an activated state.Complex chromosomal changes and DNA damage are observed in high-risk HPV-associated malignancies . Endogenin vitro and in vivo [We attempted to evaluate DNA damage by investigating the presence of cells with nuclei exhibiting positive immunostaining for γH2AX. The histone variant H2AX is an important factor in preserving genome integrity in the mammalian genome . Phospho in vivo  studies  in vivo  and cell in vivo ,21, CHK2 in vivo , p53/p21 in vivo , DNA-PK[ in vivo , MDC1[24 in vivo . The genIt has been suggested that H2AX has both structural and functional roles in chromatin de-condensation and the repair process nearby the DSB ,27. Its Histone phosphorylation produces foci that can be microscopically observed after specific antibody labeling ,32. ThisBased on the above data, we attempted to evaluate the presence of DNA damage by quantifying the percentage of γH2AX immunopositive cells, as a surrogate marker for DNA DSBs, as well as to determine whether γH2AX can be used as a biomarker to identify early HPV lesions with high-risk oncogenic potential.Our data suggest that there is a different expression of the γH2AX between the basal and surface components in SILs of increasing severity. After an initial increase, potentially corresponding to a γH2AX response to inciting cellular events, a decreased expression occurs in LGSIL harboring a high risk HPV type (LGSIL-HR) at an activated state. The observed decrease of γH2AX expression between LGSIL and HGSIL could be attributed to a significantly advanced extent of DNA damage in HGSIL compared to LGSIL, possibly no longer amenable to repair mechanisms, hence a trend towards a lower expression of this protein; the expression range across HGSIL lesions could be attributed both to the extent of the lesion (most biopsies harboring limited areas of HGSIL) as well as to other factors promoting H2AX phosphorylation (vide infra). The same hypothesis could also account for the lower expression of γH2AX in LGSIL cases associated with an activated HPV (LGSIL-HR activated) compared to those associated with a non-activated state (LGSIL-HR non-activated).Although an increased expression of γH2AX was noted in in squamous cell carcinomas and adenocarcinomas compared to LGSIL we cannot use this finding for differentiation between them since there is a significant difference in structure between these lesions. More specifically, whereas in LGSIL, which is an intraepithelial lesion, it is possible to delineate a basal and a superficial half of the epithelium, this is no longer the case for SCC or AdC due to the disordered invasive growth. Therefore, a comparison of γΗ2ΑΧ in basal and superficial parts of the epithelium between various intraepithelial lesions, such as the one we performed in our work, is no longer possible for carcinomas. The fact that the protein expression was significantly higher in carcinomas comparing to other histological lesions reflects, in all probability, the multiple DNA damage events involved in this advanced stage of carcinogenesis.HPV-16, 18, 31, 33 and HPV-45 as a group. Nevertheless, future studies will be necessary for further verification of these findings and evaluation of potential concomitant confounders.Our study is limited by the small number of observations for validating a novel biomarker. Due to the lack of pilot data, power calculations could not be performed. Therefore, we chose to include consecutive samples received over a defined 3 year period, apart from carcinomas, where due to their rarity, all relevant samples from the archives of the Department of Pathology were retrieved. Data presented can be used for power calculations in future studies. Nevertheless, the inclusion of a high number of HGSIL, as well as the combination of two methods ensuring both broad range and high sensitivity of mRNA overexpression, adds power to this study. Furthermore, our results were corroborated when examining lesions associated with the most important high-risk HPV types, such as We believe that the findings of our study are secondary to HPV infection and not due to differences in apoptosis or other regulators of γH2AX. Although γH2AX has been also reported to be induced during apoptotic DNA fragmentation, histological signs of apoptosis, such as nuclear condensation and fragmentation, were not seen in γH2AX positive foci, their absence suggesting that these foci represent cells that have acquired DNA damage.In conclusion, understanding of the mechanism by which repair enzymes contribute to HPV genome amplification may provide further insight in the HPV related DNA damage response that may be involved in the development of malignancy . OverallS1 Dataset(SAV)Click here for additional data file.