TNM8 staging for oropharyngeal squamous cell carcinomas (OPSCC) surrogates p16 immunohistochemistry for HPV testing. Patients with p16+ OPSCC may lack HPV aetiology. Here, we evaluate the suitability of TNM8 staging for guiding prognosis in such patients.HPV status was ascertained using p16 immunohistochemistry and high-risk HPV RNA and DNA in situ hybridisation. Survival by stage in a cohort of OPSCC patients was evaluated using TNM7/TNM8 staging. Survival of p16+/HPV− patients was compared to p16 status.p = 0·0190 for TNM8 compared with p = 0·0530 for TNM7) in p16+ patients. Patients who tested p16+ but were HPV− (n = 20) had significantly reduced five-year survival (33%) compared to p16+ patients (77%) but not p16− patients (35%). Cancer stage was reduced in 95% of p16+/HPV− patients despite having a mortality rate twice (HR 2.66 [95% CI: 1.37–5.15]) that of p16+/HPV+ patients under new TNM8 staging criteria.TNM8 staging was found to improve on TNM7 (log rank Given the significantly poorer survival of p16+/HPV− OPSCCs, these data provide compelling evidence for use of an HPV-specific test for staging classification. This has particular relevance in light of potential treatment de-escalation that could expose these patients to inappropriately reduced treatment intensity as treatment algorithms evolve. The American Joint Committee on Cancer and the Union for International Cancer Control eighth edition TNM staging guidelines (TNM8), effective January 2018, recommend stratification of OPSCC by HPV status to improve staging.5 Due to insufficient evidence supporting the clinical need for a second line HPV test and for ease of clinical adoption, use of p16 immunohistochemistry (IHC) alone as a surrogate marker of HPV status has been recommended.7 p16 IHC is a highly sensitive test surrogate for transcriptionally active HPV+ OPSCC; however, recent analyses have identified that as many as 20% of p16+ OPSCC patients may lack transcriptionally active HPV with potential relevance to clinical outcomes.11 A recent meta-analysis for head and neck cancers determined incidence was approximately 6.7%.3 Hence, the United Kingdom (UK) Royal College of Pathologists recommend use of a second line in situ hybridisation test (ISH) for confirmation of HPV status in p16 positive (p16+) tumours.12Transcriptionally active human papillomavirus (HPV) is the causal agent in 13–60% of oropharyngeal squamous cell carcinoma (OPSCC), with increasing incidence rates globally.13 At present, patients with OPSCC are only stratified by HPV status if they are enrolled in a clinical trial evaluating de-intensification of conventional treatment modalities or new agents.13 The outcomes of ongoing clinical trials for de-escalation of treatment based on p16 and/or an ISH test (to confirm HPV status) will make accurate classification and staging of patients increasingly important to ensure appropriate clinical management and prognostic predictions.11 The new TNM8 staging guidelines, reduces stage allocation of HPV+ tumours by using p16 IHC alone and has set the precedent for stratification of OPSCC. This has been reflected in the most recent recommendations on HPV testing guidelines published by the College of American Pathologists.6Despite early indications of improved outcomes, current clinical practice still dictates that all OPSCC patients are managed based on stage and other clinical parameters.The aim of this study was to assess the prognostic relevance of p16 positivity with and without transcriptionally active HPV in a large, non-biased, combined UK cohort of OPSCC patients. Three independent cohorts with similar baseline characteristics were combined to evaluate the staging capacity of TNM8 for p16+ patients and to assess the impact of TNM8 staging in p16+/HPV− patients.All consecutive OPSCCs diagnosed in Northern Ireland from 2000 to 2011 were included under ethical approval from the Northern Ireland Biobank (NIB 11/0001). Two distinct OPSCC cohorts diagnosed within the Liverpool Head and Neck Oncology Service from 1998 to 2009 and 2002 to 2011, respectively, were accessed under prior ethical approval . Clinical data were retrieved from individual patient electronic clinical records. Data recorded for each cohort included sex, age, tumour stage, nodal status, distant metastases, treatment received, smoking status, alcohol history, date of diagnosis and death. Overall survival was defined as the time from diagnosis until time of death as recorded on the death certificate. Data were right censored for patients still alive using the following censor dates . Clinicians were unaware of HPV status at the time of treatment for patients in the cohort. Treatment decisions were made in accordance with contemporaneous UK national guidelines.Formalin fixed paraffin embedded (FFPE) specimens  arising from the oropharynx were identified from three independent UK OPSCC cohorts and used to construct tissue microarrays (TMAs) wherever possible. Tumour site of origin was determined in patients identified by clinical, pathological and radiological review prior to inclusion in the study. Inclusion criteria were based on confirmed diagnosis of an untreated primary OPSCC; here defined as either base of tongue (C01), soft palate (C05·1), uvula (C05·2) tonsils (C09), or oropharynx not otherwise specified (C10·9).14 For each patient, triplicate cores were independently assessed by two investigators for all assays. Patients were considered p16 positive by p16 IHC if there was strong and diffuse nuclear and cytoplasmic staining in at least 70% of tumour cells.15TMA sections were assessed for p16 IHC as previously described using a Ventana Benchmark Autostainer with a commercial kit (CINtec® p16 Histology); all assays were validated on full face sections.PPIB or UBC) and negative (DapB) reagent controls were employed across TMAs and assessed prior to HPV RNA-ISH detection in order to determine integrity of mRNA within the TMA.16 Patient FFPE material positive by PPIB or UBC but negative for DapB were considered suitable for analysis by the test probe for HPV. For the Northern Ireland cohort, high-risk HPV RNA-ISH was carried out in the Northern Ireland Molecular Pathology Laboratory using RNAscope 2·0 manual assay with a pooled HPV genotype detection kit . RNA-ISH for the Liverpool cohort was carried out in the Newcastle upon Tyne Hospitals National Health Service Foundation Trust using the RNAscope 2·0 manual assay with a pooled HPV genotype detection kit . For both cohorts, patients were considered HPV RNA-ISH positive if the tumour tissue demonstrated brown reaction product specifically localising within the malignant nuclei.Positive -.Patients were initially staged according to TNM7 guidelines with subsequent clinical restaging to TNM8 using p16 status. Cohort baseline characteristics were compared between p16+ and p16− OPSCC patients using Pearson’s chi-square test for independence.Overall five-year survival analysis using the Kaplan–Meier method was conducted. Survival curves were compared using the log-rank test. The missing indicator method was used to handle missing clinical data. Cox proportional-hazards models were used to calculate hazard ratios (HR) and associated 95% confidence intervals (CI). Multivariable models were developed by backwards selection of age, sex, smoking status, alcohol history and treatment received.19The reporting standards of the current study fulfil recommendations set by the STROBE statement for reporting of observational studies and the REMARK guidelines for tumour biomarker prognostic studies.p < 0.0009) in addition to p16/HPV status these were included in the multivariable survival analyses. Mean follow-up for the combined cohort was 5.04 years (Range 0.01–25.06 years).The three cohorts included 521 primary OPSCCs in previously untreated patients for which HPV status was determined by p16 IHC and HPV RNA-ISH in 482 (93%) individuals. Of these, 446 (86%) patients had complete treatment and staging information for TNM7 and TNM8 and were therefore included in subsequent analyses. The proportion of p16+/HPV+ patients in the Liverpool cohorts was significantly higher than observed in the Northern Irish epidemiological cohort , Table p < 0.05), Table Approximately half 52%) of patients were p16 IHC positive. Of the 232 p16+ patients assessed, 20 (9%) did not test positive for HPV RNA. These 20 patients were also negative for HPV DNA-ISH confirming there was no bias due to differences in the type of RNA ISH probe used between the two study sites. Incidence of p16+/HPV+ and p16+/HPV− OPSCC was more common in males, previous smokers and those presenting with advanced stage according to TNM7 and stage II when adjusted using TNM8  of p16+ patients staged III/IV under TNM8 alive after five years compared to 67% (95% CI: 52.63–85.38) and 78% (95% CI: 71.71–85.39) of stage I and II patients, respectively, Fig. Inclusion of solitary p16 IHC testing with TNM8 improves capability to appropriately stage OPSCC for prognostic stratification but it critically fails to recognise patients who lack HPV aetiology and carry significantly inferior clinical outcome. Interrogation of HPV status using RNA-ISH within p16+ OPSCC identified a subpopulation of p16+ patients who had a two-fold increased risk of death following adjustment for potential confounders. These p16+/HPV− patients were more similar to patients with demonstrably HPV− disease and would stand to benefit from current standards of treatment intensity. Importantly, when the newly proposed TNM8 staging guidelines were applied we found that 95% of these p16+/HPV− patients would be down-staged in circumstances where p16 IHC alone was used with no additional confirmatory ISH test. Due to selection bias in the cohorts utilised only 9% of p16+ OPSCC patients were HPV− in the combined cohort. However, when considered in the context of a population-based cohort, this subpopulation was equivalent to 11% of p16+ OPSCC patients diagnosed in Northern Ireland over a ten-year period. These findings carry significant implications for evolving treatment algorithms if HPV testing guidelines for patient management follow precedent set by TNM8 for oropharyngeal cancers; failure to confirm true HPV status could result in inappropriate de-escalation or sub-optimal therapy for these p16+/HPV− patients.20 TNM8 guidelines were developed to take account of variations in prognosis within OPSCC based on HPV status. Use of p16 IHC alone, as per TNM8 staging guidelines, demonstrated improved prognosis in our study but with overlap of stage I and II cancers as reported in some but not all studies.25 As part of an ‘organ preservation’ strategy the primary treatment modality for the majority of patients with OPSCC has been radiotherapy with and without concurrent chemotherapy and as such TNM8 staging criteria has been evaluated primarily in these patients.20 Notably our study and other studies demonstrating overlap of stages I and II included cohorts where the majority of patients received surgery and where use of pathologic TNM8 staging was recommended instead of clinical.26 Use of clinical TNM8 staging in retrospective cohorts of surgically managed patients may result in bias producing the overlap of stage I and II patients.TNM7 staging guidelines did not take into account prognostic differences between HPV positive and negative cancers arising in the oropharynx. Indeed, the use of TNM7 was found to be insufficient for staging patients with p16+OPSCC in our study, a finding substantiated within the literature.6 p16 IHC was chosen for use in TNM8 as it is more cost effective and accessible to use without specialist molecular tests however its use in isolation does not meet the UK Royal College of Pathologists’ recommended guidelines, under which our study is modelled.12 At present, HPV assessment diagnostically for OPSCC is based on FFPE samples. As the samples available were not fresh frozen, use of RT-PCR for HPV detection could not be reliably used to confirm HPV status in p16+/HPV− samples. The HPV RNAscope assay has been demonstrated to be both optimally sensitive and specific for HPV detection in FFPE tissue but does not have fully regulatory approval for clinical use.27 We thereby eliminated potential bias in HPV RNA-ISH detection between Northern Ireland and Liverpool samples by testing all patient FFPE material that were not positive for p16 IHC and HPV RNA-ISH with clinically approved HPV DNA-ISH testing for FFPE samples. This established if any HPV DNA from 12 high-risk genotypes was present in the tumour tissue. No patient who was p16+/HPV RNA− was found positive for HPV DNA thereby eliminating any potential bias in our study because of the probes utilised.Following the precedent set by the UICC, the College of American Pathologists have updated their clinical recommendations for HPV testing and now advocate the use of p16 IHC as a standalone test, citing insufficient evidence of clinical need for a second line test in oropharyngeal primaries.28 The College for American Pathologists finds use of p16 IHC in patients with primary OPSCC to be sufficient for molecular stratification of HPV in the head and neck.6 Interestingly, the majority of p16+/HPV+ and p16+/HPV− tumours in our study originated in either base of tongue or tonsil, supporting a recent meta-analysis, but we found the oropharyngeal sub-site did not confer survival advantage when adjusted for covariates.29 These data demonstrate that p16 IHC appropriately classified 91% of HPV+ patients, a finding substantiated by recent meta-analyses within the literature for head and neck cancers.3 Critically, we find that the resulting 9% of patients who are p16+/HPV− are of increasing clinical importance and concern as these patients have significantly reduced survival compared to p16+/HPV+ patients and would not benefit from TNM8 staging guidelines; as 95% of patients with p16+/HPV− tumours were found to be down-staged when assessed using clinical TNM8 as HPV+ OPSCC.We found the strategy of using p16 IHC followed by HPV RNA-ISH has allowed identification of all clinically relevant incidences of transcriptionally active HPV+ tumours within our cohort and is supported by recent meta-analyses which have demonstrated that a two tiered test is essential for a reliable diagnosis.31 These findings are supported in a recent study of OPSCC and stage, which utilised HPV DNA-ISH and p16 IHC.33 In recognising the importance of a second line test to prevent sub-optimal treatment of transcriptionally inactive HPV tumours we find confirmation of HPV status would result in more appropriate prognostication of p16+/HPV− patients and a better staging tool overall for OPSCC.Based on the results of the current study we find that ongoing clinical trials for de-intensification of treatment in OPSCC patients, which utilise p16 IHC only to stratify patients, carry potential for p16+/HPV− patients to receive sub-optimal treatment as treatment algorithms evolve.To conclude, our study provides compelling evidence that lack of a standardised two-tiered HPV testing process, when using p16 IHC will negatively affect the clinical management of p16+/HPV− patients as treatment algorithms evolve. When p16 IHC is utilised as a first line-screening test, subsequent confirmation of HPV status based on definitive HPV detection using ISH must follow.


HPV-16 genotyping, viral load, and physical status  in the saliva samples of OPC patients were determined using the qPCR method. A good inter-rater agreement (k = 0.612) was found between tumor p16 expression and oral HPV-16 infection in OPC. Importantly, according to the eighth edition staging system, HPV-16 DNA viral load (>10 copies/50 ng) was significantly associated with the advanced stages of OPC. In concordance with previous studies, a mixed HPV-16 form  was predominately found in OPC patients. Taken together, our data support HPV-16 detection in saliva as a screening biomarker to identify people within the community who are at risk of developing OPC.The incidence of human papillomavirus (HPV)-positive oropharyngeal cancer (OPC) is rising in high-income countries, including Australia. Increasing evidence suggests that accurate HPV testing is pivotal for clinical decision making and treatment planning in these patients. Recently, the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor–node–metastasis (TNM) staging system for OPC (based on the p16INK4a (p16) status) was proposed and has been implemented. However, the applicability of this new staging system is still far from clear. In our study,  Oropharyngeal cancer (OPC) usually arises from the tonsillar area, the base of tongue, and the oropharynx, and is one of the most common subtypes of head and neck cancers (HNC), accounting for 97,000 deaths annually worldwide ,2. ThereThe clinical diagnosis of OPC is challenging when compared to other cancer types such as prostate and breast, as the tumors are usually tiny in the early stages of disease, and are located in the regions of the mouth that are not easily visible and accessible ,9. As a In January 2018, the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor–node–metastasis (TNM) staging system for OPC was released and adopted by the clinicians. In this edition, OPC patients are stratified based on the tumor p16INK4a (p16) status, which is a common surrogate marker for HPV . HoweverThere is solid evidence to support the use of saliva as a nexus diagnostic testing fluid for the detection of HPV DNA in OPC patients ,18. Furtn = 121) who could provide a sufficient amount of DNA are summarized in The demographics of OPC patients . All of the samples were positive for beta-goblinSalivary HPV-16 DNA (E2 or/and E6/7) was detected in 71 out of 89 (80%) p16-positive OPC patients, as shown in When tumors were classified by the seventh edition staging system, early stages (I and II) of OPC showed a higher viral load with a median value of 475.4 copies of HPV-16 E6/7 DNA per 50 ng when compared to advanced stages (III and IV) (266.5 copies/50 ng). Conversely, when the eighth edition staging system was used, the HPV-16 viral load was elevated in advanced stages of OPC (774.1 copies/50 ng) when compared to early stages (232.0 copies/50 ng) A. More iThe oral HPV-16 physical status  in 71 OPC patients was examined by qPCR-based E2/E6/7 ratios, as shown in The incidence of HPV-positive OPC, particularly in younger men, is accelerating in high-income countries as well as in Queensland and Australia ,21. ReceIncreasing evidence supports the notion that an accurate HPV test is crucial for clinical decision making and treatment planning for OPC patients ,23,24. HAt present, there is no standard or routine HPV DNA testing for OPC; therefore, it is pivotal to develop a non-invasive and low-cost test. The results of several studies support the idea of using salivary HPV DNA as a biomarker to monitor disease progression and tumor recurrence in OPC patients ,25,26. PThe persistence of high-risk HPV infections (HPV-16 and 18) is strongly associated with the development of cervical neoplasia and progression toward cervical carcinoma ,28. In aPersistent high-risk HPV infections can also trigger genomic instability and subsequently promote the integration of viral DNA into the host genome ,33. ThisThis study was approved by the University of Queensland (UQ) Medical Ethical Institutional Board (HREC No: 2014000679 and 2014000862); Queensland University of Technology (QUT) (HREC No: 1400000617 and 1400000641); the Princess Alexandra Hospital (PAH) Ethics Review Board (HREC/12/QPAH/381), and the Royal Brisbane and Women′s Hospital (RBWH) (HREC/16/QRBW/447). A total of 127 patients who have been diagnosed with OPC from the PAH and RBWH were recruited to this study . All of Saliva and oral rinse samples of participants were collected as previously described . BrieflyTotal DNA was isolated from saliva/oral rinse samples using the QIAamp DNA Mini Kit , following the manufacturer’s instructions. Briefly, cell pellets were resuspended with 200 μL of phosphate-buffered saline (PBS) before adding the 10 μL of proteinase K and 200 μL of lysis buffer to the mixture. After 10 min of incubation at 56 °C, 200 μL of 100% ethanol was added to the mixture, which was then transferred to QIAmp DNA mini spin columns as per manufacturer protocol.For the detection of HPV-16 positively in saliva samples, qPCR was carried out with the specific primers targeted against the HPV-16 E2  and E6/7 . Human β-globin  was used as an internal control. For the determination of HPV-16 E2 and E6/7 viral loads, HPV-16 DNA standard calibration curves were generated by plotting the threshold cycle  against the logarithm of the copy number of seven-fold serially diluted Caski DNA with spiked HPV-16 negative HNC cell line DNA (SCC-25). The qPCR used the following conditions: 50 °C for 10 min; 95 °C for 10 min; 40 cycles at 95 °C for 15 s and 60 °C for 60 s; and a final melting curve analysis with following conditions: 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.n = 71) was determined as described previously [HPV-16 physical status in saliva samples collected from HPV-16 DNA positive OPC patients . Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and their 95% confidence intervals were calculated. The potential association between HPV-16 DNA viral load/physical status and the disease stage of OPC patients was examined by the Fisher’s exact test, and p values less than 0.05 were considered significant. All of the statistical analysis were performed using GraphPad Prism 7 software version 7 .The inter-rater agreement between tumor p16 expression and oral HPV-16 infection was determined by the Cohen’s kappa coefficient with a 95% confidence interval (Taken together, our data supports the use of salivary HPV DNA as a non-invasive biomarker in OPC patients, and indicates that disease staging could be based on viral load in combination with an eighth edition staging system.


INK4a is used as an indicator of the presence of HPV and as a prognostic marker for squamous cell carcinomas in various sites. However, the role of this marker in penile carcinoma has not yet been completely elucidated. The aim of this study was to analyze whether the expression of p16INK4a is associated with the presence of HPV, histological parameters, and survival in penile cancer.Infection with human papillomavirus (HPV) is reported to be present in 30–50% of penile cancer cases. The immunohistochemical test for p16INK4a protein using paraffin-embedded tissue. Evaluation of histological parameters was performed following complete embedding of the tumor tissue in paraffin.A study was conducted from 2014 to 2016 that included 55 patients with penile carcinoma. HPV DNA was detected through PCR using fresh tumor tissue, and immunohistochemistry was performed for analysis of p16INK4a, HR-HPV DNA was present in 21 (95.5%) (p = 0.032). Regarding histological parameters, p16INK4a and HR-HPV were significantly associated only with tumor subtype ; all carcinomas with basaloid characteristics were positive for p16INK4a. Although HPV+ patients had a higher disease-free survival (p <0.001), p16INK4a expression was not associated with patient survival.HPV DNA (low-risk and high-risk genotypes) was found in 49 (89.1%) cases, and 46/49 (93.9%) showed high-oncogenic risk HPV (HR-HPV). Of the 22 cases positive for p16INK4a protein was significantly associated with the presence of HR-HPV and this expression may serve as a marker for the presence of the virus. The p16INK4a protein was not associated with the histological prognostic parameters, with the exception of tumor subtype, nor with patient survival. In the results, we showed that the objective of the present study was reached.Our study, using fresh tissue samples, showed the highest incidence of HPV compared to that observed in the literature. Expression of the p16 Penile cancer (PC) is considered a rare neoplasm in developed countries , 2. HoweINK4a protein overexpression by negative feedback in infected cells [Among several risk factors for the development of PC, infection with human papillomavirus (HPV) is one of the most highly associated factors reported in the literature. The incidence of HPV infection in penile carcinoma ranges from 30 to 50% –9, partied cells , 16.INK4a is often used as an alternative marker for the presence of high-oncogenic risk HPV in cervical neoplasias as well as squamous cell carcinoma in other organs [INK4a expression in patients with PC. Thus, the objective of the present study was to analyze if there was an association between the expression of P16INK4a protein and: 1) histological parameters present in tumors that were totally included in paraffin; 2) the presence of HPV in patients with PC; and 3) patient survival.Immunohistochemical expression of p16r organs –19. Thisr organs –24. HoweThe present study included analysis of 55 cases of penile carcinoma. Diagnosis was confirmed by anatomopathological diagnosis and all patients underwent a surgical procedure at the Presidente Dutra University Hospital or the Aldenora Bello Maranhense Institute of Oncology between 2014 and 2016.Data were collected from an interview with the patients, analysis of histopathological reports, and review of patient medical records. Samples of paraffin-embedded tumor tissues were selected for immunohistochemistry, and fresh tissue samples were used for the polymerase chain reaction (PCR) analysis.th edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual was used to classify tumor staging [The histological diagnosis of HPV was defined by the presence of koilocytosis, with three obligatory criteria: a) perinuclear halo, b) nuclear atypia, and c) binucleation . To incr staging .To determine the presence or absence of HPV DNA in the PC samples, DNA extraction with phenol-chloroform was performed according to the protocol of Sambrook et al. (1989) . SubsequHPV genotyping was performed for the samples identified as HPV-positive by the sequencing reaction with the DYEnamic ET Terminator Cycle Sequencing Kit, according to the protocol suggested by the manufacturer .INK4a was performed according to the manufacturer's protocol using a mouse monoclonal primary antibody p16INK4a Clone G175-405  diluted at a ratio of 1:75 μc. Blade evaluation and image acquisition were performed using a Zeiss optical microscope (AxioCam MRC) and image acquisition software (Zen 2012) . To define the expression patterns of p16INK4a, the classification of Cubilla et al. (2011) was adapted [INK4a expression patterns were found and were categorized as follows: pattern 0, complete absence of staining in all neoplastic cells; pattern 1, irregular and discontinuous individual staining in some of the neoplastic cells; pattern 2, a more extensive, although discontinuous, staining pattern with small clusters of positive neoplastic cells; and pattern 3, continuous and complete cytoplasmic and nuclear staining in all neoplastic cells. Only pattern 3 was considered positive for p16INK4a expression  and analyzed with SPSS V22 . Relative and absolute frequency analyses were performed to verify the distribution of the categorical variables. A Kolmogorov-Smirnov test was used to analyze the normality of the quantitative variables with these variables expressed as mean and standard deviation. The association between HPV status and p16The study was approved by the Scientific Commission—COMIC—HUUFMA with the opinion no. 2457/2014-60 and by the Research Ethics Committee of the University Hospital/CEP-HUUFMA with approval number: 1.093.435. To meet the regulations for research with human subjects, established by Resolution no. 466/12 of the National Health Council, all duly guided participants provided a signed Free and Informed Consent Form (FICF).We analyzed 55 patients with PC, with a mean age of 61 ± 17.87 years at the date of surgery. Among these patients, 44 (80%) were married or in a stable relationship, 26 (47.3%) were farmers, 50 (90.9%) had a low educational level (illiteracy or elementary school), and 29 (52.7%) were smokers. Phimosis was present in 40 (72.7%) cases.Among the cases included, 28(50.9%) were of subtypes of cancer associated with the presence of HPV, which include warty, basaloid, or mixed carcinomas with warty or basaloid components.Pathological cancer staging with high metastatic potential (pT1b to pT3 stage) was observed in 44 cases (80%), and 43 (78.2%) were classified as tumors of low degree of differentiation (Broders degree GI-II). Perineural or lymphatic invasion was present in 19 (34.5%) and 16 (29.1%) cases, respectively. Histological evaluation revealed the presence of koilocytosis in 41 cases (74.5%). A more detailed description of the histological characteristics is presented in All patients underwent penectomy; 78.2% underwent partial penectomy and 21.8% total penectomy. The average time from onset of symptoms until the search for treatment was 21.3 months, and the follow-up period was 17.3 months. During the follow-up period, five patients died from cancer, eleven had lymph node metastases, 28 remained alive without disease, and four did not undergo follow-up, while seven experienced recurrence. Regarding the type of complementary treatment, two patients were administered chemotherapy and seven received radiotherapy.HPV DNA was present in 49 cases (89.1%), of which 46 (93.9%) were classified as high-oncogenic risk HPV (HR-HPV) and 3 (6.1%) as low-oncogenic risk HPV (LR-HPV). Among the high-risk subtypes, the most frequent was HPV 16 (69.5%). The remaining HPV subtypes associated with high oncogenic risk were either observed alone or together with other subtypes. The low-risk subtypes included subtype HPV 11, HPV 6 and HPV 30. In addition, two cases of multiple infection with the HPV subtypes 35 and 59 was observed .INK4a protein is shown in INK4a protein and 22 (40%) were positive. Among the p16INK4a positive cases, 11 (50%) were usual carcinomas and the other 11 (50%) were basaloid, warty, or mixed with warty or basaloid components. HR-HPV DNA was present in 21 (95.5%) of the p16INK4a-positive cases. Among the LR-HPV cases, only one was considered p16INK4a-positive.The distribution of expression patterns of the p16INK4a positivity and tumor subtype (p = 0.036); 100% of basaloid or mixed basaloid carcinomas were p16INK4a positive. When basaloid or mixed basaloid carcinomas were excluded from analyses, no association with p16INK4aprotein overexpression was found. Other histological characteristics of the tumors were not found to be significantly associated with p16INK4a . A significant association was also identified between HR-HPV DNA and p16INK4a (p = 0.032), with 21/46 cases of HR-HPV positive for p16INK4a.INK4a status, it was observed that HPV-negative patients had a lower disease-free survival (log rank p< 0.001), whereas in relation to p16INK4a expression, there was no statistical difference in the analysis of overall survival and disease-free survival  were found to be high-oncogenic risk HPV. According to a study by Ferrándiz-Pulido et al., the usual HR-HPV-related penile carcinomas seem to have better prognoses compared to the carcinomas related to HR-negative HPV . In our HPV-negative patients had a lower disease-free survival compared to HPV-positive patients. This result agrees with the findings of Ferrándiz-Pulido et al. , and supINK4a protein was only associated with tumor subtype, with p16INK4a overexpression observed in all cases. However, no association was observed with other histological tumor characteristics such as size, clinical stage, histological grade, or lymphatic or perineural invasion. A study of 202 cases of penile carcinomas showed that p16INK4a expression was more likely in tumors with basaloid morphological characteristics [As seen in other neoplasms, several histological parameters have been associated with PC prognosis. These include HPV status, histological grade, tumor thickness, lymphatic or venous embolization, tumor subtype, clinical stage, and lymph node involvement –42. In tINK4a positivity in PC is strongly related to the presence of the high-risk oncogenic HPV genotype [INK4a and the presence of HR-HPV. Among the LR-HPV cases, one of them was p16INK4a positive. This fact may be explained by the possibility of non-HPV-related oncogenesis involving the pRb pathway, or the capability of some LR-HPV genotypes to induce complete malignant transformation in infected epithelial cells acting as HR-HPV [INK4a negative, which may be related to inactivation of the gene encoding p16INK4a (CDKN2A), loss of heterozygosity, or promoter hypermethylation. In addition, studies on cervical carcinomas associated with HPV show maximum p16INK4a expression in carcinoma in situ, and a reduction in expression as the carcinomas become invasive [According to Cubilla et al., p16genotype . Therefogenotype . In the invasive .INK4a staining pattern should be considered as a cutoff point for p16INK4a positivity. Another limitation was the inability of some patients to undergo follow-up, because most included subjects lived in rural areas far from capital cities and may not have returned for follow-up after the surgery.As in other studies, some limitations to this research were noted. One of the challenges of this study was to identify which p16INK4a protein was found to be associated with the presence of high-risk oncogenic HPV in penile carcinoma samples. Therefore, this protein may serve as an alternative marker for the presence of the virus. Results also showed that the p16INK4a protein is associated with tumor subtype but not with other histological prognostic parameters. Although HPV+ patients exhibited a higher disease-free survival, p16INK4a was not associated with patient survival. In addition, we found the highest incidence of HPV ever recorded in PC, with an absolute predominance of high-risk oncogenic HPV. This finding may be explained by the fact that this study was the first to use fresh tissue samples instead of paraffin-embedded tissues.In this study, the expression of p16S1 Datasethttps://doi.org/10.17605/OSF.IO/3MUV4.(XLSX)Click here for additional data file.S1 Protocolhttps://doi.org/10.17605/OSF.IO/V4SU2.(DOCX)Click here for additional data file.S2 Protocolhttps://doi.org/10.17605/OSF.IO/MHFD9.(DOCX)Click here for additional data file.


HIV-infected individuals are at increased risk of anal cancer; in the majority of cases this is linked to human papillomavirus (HPV) infection. Anal cancer screening is not routinely offered in Zimbabwe.A cross-sectional study was performed on 152 patients  attending Opportunistic Infection Clinics at 2 tertiary hospitals between November 2014 and June 2015. Demographic data, immunological parameters and behavioural characteristics were collected. An anal swab was collected from each patient for HPV genotype testing. HPV testing was performed using MY09/MY11 PCR, followed by typing using the dot blot method.The mean age was 39.6 years . Median CD4 count was 375 cells/μL. 96% were on antiretroviral therapy. Only one patient identified as a man who has sex with men. Of 122 samples tested for HPV, 54 were positive (44%). HPV was three times more common in females (60%) than males (20%). Being HPV-positive was associated with history of perianal warts, history of cervical intraepithelial neoplasia and having more than ten lifetime sexual partners. The most commonly detected high-risk HPV genotypes were HPV-58 (13%), HPV-31 (11%) and HPV-16 (9%). Nine patients harboured multiple high-risk HPV types. The two most commonly detected low-risk genotypes were HPV-11 (17%) and HPV-53 (11%).Overall anal HPV prevalence was 44% in this mostly heterosexual HIV-positive population. Oncogenic HPV types accounted for almost half of infections, supporting the need for surveillance of anal cancer in this population. Anal cancer is rare in the general population but is significantly more prevalent in high-risk groups such as men who have sex with men (MSM) and those infected with human immunodeficiency virus (HIV). In up to 90% of cases anal cancer is linked to human papillomavirus (HPV) infection . HIV preA substantial amount of data are now available from studies on anal HPV infections and consequent disease in MSM. There are numerous HPV genotypes and anal cancer in this population is most strongly linked to persistent HPV-16 and HPV-18 infection . HoweverAnecdotal observation and crude cancer registry data suggest an upward trend in the number of cases of anal cancer in Zimbabwe in the past few years. Anal cancer screening is not routinely offered in Zimbabwe. Against this backdrop we sought to establish the prevalence of anal HPV infection in asymptomatic HIV-positive patients presenting at two Opportunistic Infections Clinics (OIC) in two large urban hospitals in Harare.A cross-sectional study was performed on 152 consecutive patients  attending OIC at Parirenyatwa Group of Hospitals and Harare Central Hospital between November 2014 and June 2015. Eleven patients opted not to be included in this study with the main reason given for refusal being lack of time. A total of 9000 adult patients are enrolled at the two OIC, with a male:female ratio of 1:1.5. The OIC serve a low to middle-income urban population of Harare, Zimbabwe. Patients visit the OIC for HIV testing and counselling services, routine follow up of HIV disease, and fulfilment of prescribed antiretroviral drugs. 85% of patients enrolled at the OIC are on highly active antiretroviral therapy (HAART). Ethical approval for this study was obtained from the University of Zimbabwe, College of Health Sciences Joint Research Ethics Committee and the Harare Central Hospital Ethics Committee. The study was registered with the Medical Research Council of Zimbabwe.Males and females age ≥ 18 years were included in the study after providing informed signed consent. The source test document was checked to verify that each participant was HIV-positive. Demographic data and behavioural characteristics were collected through a face-to-face structured interview. CD4 counts and viral loads were verified by inspection of the participant’s clinical record.The participant was positioned on a standard examination couch in the left lateral position with knees drawn up to the chest and buttocks at the edge of the bed. The perianal region was inspectedafter gently parting the buttocks. A Dacron-tipped swab was moistened in 0.9% normal saline, then introduced blindly into the anal canal as far as possible . Using tHPV testing was done using primer pair MY09/MY11 polymerase chain reaction (PCR), followed by typing using the dot blot method, as described previously . For intSamples were thawed to room temperature. HPV DNA was extracted using the conventional ammonium acetate method. AmpliTaq Gold Polymerase enzyme was added to the samples to amplify target DNA. The PCR cycles were at: 95 °C for 9 min, 40 cycles of , 72 °C for 5 min and then held at 4 °C. After PCR, the DNA amplification mixture was denatured using an alkaline buffer then, applied to a Biodyne B membrane. The DNA was fixed to the Biodyne B membrane by baking at 80 °C for 1 h and then exposed to an HPV L1 consensus probe mixture. A sample was defined as HPV-positive if it reacted positively to the consensus probe mixture. Hybridisation was then carried out between the sample DNA and specific biotin-labelled HPV probes for 14 high-risk HPV genotypes  and 15 low-risk HPV genotypes . The hybridization products were then detected using enhanced chemiluminescence (ECL). Commercial HPV standards were blotted onto each membrane as positive controls for their respective genotype.p < 0.05.Data collection forms were inspected for completeness and accuracy by trained study personnel. Patients with missing data for a particular analysis were excluded only from that analysis. Data was analysed using IBM SPSS Statistics for Windows, version 22 . Comparison between groups of frequencies was performed with Chi-square test or Fisher’s exact test of association. All results were evaluated at a 95% confidence interval; significance was set at Data from 122 patients were analysed after excluding 30 whose samples tested negative for beta-globulin; 73 (60%) were female and 49 (40%) were male. The mean age was 39.6 years . All patients were Black Africans. A summary of the study population is shown in Table Whilst we set out to probe for 14 high-risk and 15 low-risk HPV genotypes, we chose not to report results for genotypes 26, 70 and 82 (intermediate risk) and genotypes 32, 54, 55 and 61 (low-risk) due to lack of availability of working standards for those particular genotypes. These genotypes were therefore excluded from analyses and are not reported. Fifty-four of 122 samples tested HPV positive (44%). Of the 54 HPV-positive samples, 49 (91%) had specific HPV genotypes detected. Anal HPV was detected in 10/49 males (20%) and in 44/73 females (60%). High risk HPV types were detected in 25 patients (20%); low-risk HPV types were detected in 24 patients (20%). Nine patients (7%) had multiple high-risk HPV types i.e. more than one specific high-risk HPV type detected per sample. All high-risk HPV genotypes that we sought except HPV-39 were detected in this study population.Table The most commonly detected high-risk HPV genotypes were HPV-58 (13%), HPV-31 (11%) and HPV-16 (9%). HPV-18 was detected in 2.5% of samples. HPV-11 (17%) and HPV-53 (11%) were the two most commonly detected low-risk genotypes.Figures p < 0.0001). It was not significantly associated with marital status, HAART  use, cigarette smoking, alcohol use, number of sex partners, condom use, current CD4 count nor nadir CD4 count. For males and females combined, detection of any high-risk HPV was significantly associated with ≥5 lifetime partners (p = 0.021). When data were analysed for males and females separately, there was no change noted in the above associations. A summary of statistical associations between a positive HPV result and selected socio-demographic and immunological factors is shown in Table A positive test for any HPV was significantly associated with female gender  was 44%. This is the first paper describing prevalence of anal HPV infection in African heterosexual men, and one of the first in African women.The anal HPV prevalence in females was three-fold higher than in men (60% vs. 20%). Most studies on females note that prevalence of anal HPV is actually higher than cervical HPV . SimpsonHigh-risk HPV prevalence was 21% being found in 10% of males and 27% of females. Overall HPV-16 was found at a prevalence of 4% which is important because HPV-16 is one of the most consistent genotypes associated with anal cancer . To dateOverall, low-risk HPV infections were as common as high-risk infections in our study (20% vs. 21%). Though low-grade types are much less oncogenic by definition, they cause a significant burden of genital warts. The majority of these warts are attributable to infection by HPV-6 and 11 . These aThere are several limitations to our results. The study was cross-sectional yet many HPV infections are transient . We usedIn conclusion the prevalence of anal HPV was 44% in this group of largely heterosexual HIV-positive males and females. Oncogenic HPV genotypes accounted for almost half of infections, supporting the need for surveillance of anal cancer in this population. These data represent a baseline upon which future studies may be built. Specifically, longitudinal studies can potentially reveal the pattern of persistence of anal HPV and chronicle the development of cytopathological changes over time.


The potential HPV transmission route includes horizontal transmission “in utero” and vertical transmission from parents. Less is known about the role of child’s father as a potential source of HPV infection and involved in the pathogen’s epidemic chain. A possible consequence of perinatal infection includes HPV-related childhood diseases and carrying the risk of cervical cancer development in female offspring. In view of the evidence, studies of HPV co-occurrence in one or both parents and their offspring seem vital for the implementation of respective preventive measures. Consequently, the aim of this study was to determine the incidence of common HPV 16/18 infections in newborns and their parents, and to assess its role of the periconceptional transmission.To determine the incidence of common HPV infections in newborns from single pregnancies and their parents. The study included 146 pregnant women, as well as their partners, and newborns. They were tested for the presence of HPV 16/18 DNA using the PCR method. HPV types 16 and/or 18 were identified using type-specific PCR primers. The quality of the extracted DNA was evaluated by PCR using PC03/PC04 β-globin-specific primers. The relationship between the presence of neonatal and parental HPV infection was analyzed using a multivariable regression model. Calculations were carried out with the Statistica 10.p < 0.001), 12 descendants of HPV-positive fathers , and 10 children originating from two infected parents . Those findings points to a increase risk of an acquired infection in newborns with HPV-positive parents.The presence of HPV DNA was detected in 19  newborns, 28  mothers, and 20  fathers. The viral DNA was detected in 14 newborns delivered by HPV-positive mothers (OR = 26,08; CI: 8,07-84,31, Our findings suggest the possible role of the periconceptional transmission in the mode of acquired HPV 16/18 infections. The potential HPV transmission route includes horizontal transmission “in utero” and vertical transmission from parents. Data presented in various researches show the possibility of perinatal or intrauterine mode of infection –4. Less This research was conducted between March 2009 and July 2011. The studied group comprised pregnant women who delivered at the Clinic of Obstetrics and Pathology of Pregnancy, Medical University of Lublin (Poland), and their partners. For the study we recruited 146 consecutive parental couples. The gestational age varied from 33 to 41 weeks. Individuals selected for this study met specific inclusion criteria. Among those criteria were: 1) singleton pregnancy, 2) no HPV infection symptoms present in any of the parents, and 3) physiological cervical smear in pregnant woman. Among the exclusion criteria were: 1) previous HPV infections, 2) abnormal cervical smear in pregnant woman, and 3) multiple pregnancy.The protocol of this study was performed in accordance with the Declaration of Helsinki and approved by Local Bioethical Committee at the Medical University of Lublin (No. KE-0254/143/2007). Before participation in any of the procedures the studied group expressed their consent by completing a written form.Posterior vaginal fornix smear samples as well as peripheral blood samples were collected from the women in the onset of labour. Additionally, buccal mucosal smears were drawn from both parents and their children. Discharge gathered in newborns upper airways was extracted in order for buccal smears to be collected directly after birth. A suitably trained member of the personnel managed the collection of all material. Subsequently the samples were transferred to sterile tubes awaiting for HPV DNA testing . The material was then subject to freezing at − 70 °C pending additional analyses.2, 1% Triton X-100), the investigated DNA (10 ng/μl), and Tag polymerase at a final concentration of 40 U/ml. After preliminary denaturation (15 min at 94 °C), samples were amplified for 31 cycles in a thermal cycler, consisted of a 30 s period of denaturation at 94 °C, annealing at 59 °C for the same amount of time, followed by primer extension at 72 °C for 1 minute. In the last PCR cycle, the stage of complementary DNA synthesis at 72 °C was extended to 420 s. Agarose gel electrophoresis in the presence of pBluescript DNA digested with HindI was utilized to analyze PCR products. A further HPV DNA detection, and HPV genotypes identification by direct sequencing from PCR reaction tube (without purification from MY09 and MY11 oligonucleotides was performed). The obtained results were subsequently analyzed with BLAST database (http://blast.ncbi.nlm.nih.gov).PCR testing was utilized as means of detecting HPV DNA in the collected samples. HPV types distinction was made possible by using PCR primers for L1: MY09: 5′-CGTCCMARRGGAWACTGATC-3′ and MY11: 5′-GCMCAAGGWCATAAYAATGG-3′, where M = A + C, R = A + G, W = A + T, Y=C + T. This set of primers amplifies DNA from at least 33 different HPV genotypes. HPV types 16 and/or 18 were identified using the following type-specific PCR primers: HPV16/L1A/HPV16/L1B, 5′-GCCTGTGTAGGTGTTGAGGT-3′ and 5′-TGGATTTACTCCAACATTGG-3′ product size: 264 bp; HPV18/L1A/ HPV18/L1B, 5-’ GTGGACCAGCAAATACAGGA-3’and 5′- TGCAACGACCACGTGTTGGA-3′, product size: 162 bp; HPV18ME12/HPV18ME50/E6, 5′-CACGGCGACCCTACAAGCTACCTG-3′, and 5′-TGCAGCACGAATTGGCACTGGCCTC-3′, product size: 404 bp. The quality of the extracted DNA was evaluated by PCR using PC03/PC04 β-globin-specific primers. The reaction described above was developed with reference according to Tucker et al. . The tot*p ≤ 0.05.To verify the normal distribution of continuous variables we use the Kolmogorov-Smirnoff test. Statistical characteristics of these variables were presented as medians and ranges, and their intergroup comparisons were performed using the Mann-Whitney U-test. The distributions of qualitative variables were compared amongst studied groups with the Pearson’s chi-square test and Fischer’s exact test. We use a multivariable regression model to analyze the relationship between the presence of neonatal and parental HPV infection; odds ratios (ORs) were determined. All calculations were carried out with Statistica 10  package, with the level of significance set at The examined group consisted of women ages 17 to 43 with a median age of 29. Twenty-eight (19.18%) cases of HPV 16/18 infection were detected amongst the pregnant women participating in this study. In 10 cases, HPV DNA was detected solely in vaginal smears, and in 5 cases solely in the buccal smears; 13 women tested positive for both samples. In two cases, HPV DNA was also detected in the peripheral blood of the pregnant women. There was no significant difference between participants with positive and negative results on HPV DNA testing as regards their demographical and obstetrical features (Table Twenty (13.7%) cases of HPV 16/18 infection were detected amongst fathers participating in this study as confirmed by the examination of buccal smears. The offspring of study participants included 80 female and 66 male newborns. Neonatal birth weight ranged from 1740 g. to 4670 g. with a median weight of 3405 g. In case of 19 (13.01%) newborns HPV 16/18 DNA was found in the collected samples. In 6 cases the infection was confirmed based on a buccal smear test and in 3 patients through examination of the upper respiratory airway discharge; 10 newborns had positive results of both samples. In one case, HPV was also detected in the umbilical blood sample. Newborns positive and those negative for HPV 16/18 DNA did not differ significantly in terms of their obstetrical characteristics Table .Table 2p < 0.001), 2) infection of both parents , 3) maternal infection confirmed by a positive vaginal smear test , and 4) maternal infection confirmed by a positive buccal smear test . In contrast, the risk of neonatal infection was not modulated by the detection of HPV DNA in maternal blood  and by the number of HPV-positive maternal samples   proved to be significant risk factors for the acquiring of a neonatal HPV infection on the multivariate analysis of logistic regression: 1) maternal infection confirmed by a positive result of at least one examined sample , an infection in the mother co-existed with that of the father, and in nearly 70% of cases the infection of both parents corresponded to the presence of HPV genetic material in neonatal tissues. These findings and that of the logistic regression analysis (OR = 24.20) point to the possible role of the periconceptional transmission of the pathogen in the pathogenesis of acquired HPV infections. This substantiates further research in this direction, as well as the consideration of HPV testing of both future parents in the protocol of preconception care.p = 0.000 in either case) can be more useful for the risk assessment of intrauterine HPV transmission than the virology examination of peripheral blood . This finding seems particularly attractive in the context of the popularization of non-invasive methods of virology diagnosis. The identification of parents in whose swabs of the oral mucosa HPV DNA was detected can help emerge a group of children who run a relatively significant risk of being infected.Aside from this important conclusion, our findings suggest that testing vaginal or buccal smears in pregnant women (OR = 21.54 and OR = 10.64, respectively; Our findings suggest the possible role of the periconceptional transmission in the mode of acquired HPV 16/18 infections.


Aim: To explore the mechanism by which long non-coding RNA (lncRNA) TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the microRNA miR-134-5p/malignant brain tumour domain containing 1 (MBTD1) axis.Results: The lncRNA TTN-AS1 was highly expressed in osteosarcoma and was associated with poor prognosis. The lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. MiR-134-5p targeted MBTD1, which was regulated by lncRNA TTN-AS1. MBTD1 was highly expressed in osteosarcoma and was associated with poor prognosis. MBTD1 promoted cell viability and inhibited apoptosis, and knockdown of MBTD1 reversed the cancer-promoting effects of lncRNA TTN-AS1. Downregulation of lncRNA TTN-AS1 reduced drug resistance.Conclusion: In osteosarcoma, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance.Methods: The expression characteristics of genes in osteosarcoma patients were analysed using bioinformatics. Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect protein and RNA, respectively. A cell counting kit 8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis. The effects of lncRNA TTN-AS1 and MBTD1 on osteosarcoma in vivo were studied by using a tumour burden assay. Osteosarcoma is a musculoskeletal malignancy that originates in the interstitial cell line . The incLong non-coding RNA (lncRNA) typically has a length of more than 200 nucleotides . LncRNAsIn this paper, we found that the lncRNA TTN-AS1 and malignant brain tumour domain containing protein 1 (MBTD1) were overexpressed in osteosarcoma, and they were associated with the poor prognosis of osteosarcoma patients. Based on this, we revealed a novel mechanism by which lncRNA TTN-AS1 regulated proliferation and apoptosis of osteosarcoma cells via miR-134-5p/MBTD1. This may provide new ideas for the discovery of strategies for osteosarcoma treatment.Bioinformatics analysis revealed that lncRNA TTN-AS1 was upregulated in osteosarcoma . ClinicaQPCR results showed that the TTN-AS1 level in the sh-TTN-AS1-1 and sh-TTN-AS1-2 groups was lower than that in the sh-NC group , while vector-TTN-AS1 significantly upregulated the level of TTN-AS1 , indicating successful transfection –2D. Sh-TThe predicted results from the bioinformatics analysis showed that lncRNA TTN-AS1 targeted miR-134-5p . ClinicaThe TargetScan website was used to predict the target gene of miR-134-5p . The lucThe results also showed that upregulation of lncRNA TTN-AS1 promoted MBTD1 protein expression  –4K. In aThe level of MBTD1 in cancer tissues was significantly higher than that in normal tissues , 5B. OstThe relative cell viability of the vector-TTN-AS1 + sh-NC group  was significantly higher than that of the vector-NC group; the cell viability of the Vector-TTN-AS1 + sh-MBTD1 group  was lower than that of the Vector-TTN-AS1 + sh-NC group –6B. The The cell viability of the osteosarcoma cell lines Saos-2 and U2OS treated with different concentrations of cisplatin was examined. The cell viability decreased by approximately 50% when the concentration of cisplatin was 10 μM –7B. The In this study, we discovered the role of lncRNA TTN-AS1 and MBTD1 in osteosarcoma and analysed the mechanism by which lncRNA TTN-AS1 regulates apoptosis and drug resistance of osteosarcoma cells through the miR-134-5p targeting axis.The role of lncRNA in osteosarcoma has been confirmed, and both lncRNA and miRNA can be used as markers for the diagnosis and prognosis of osteosarcoma –23. ThisTo further explore the mechanism by which lncRNA TTN-AS1 regulated osteosarcoma cell growth and apoptosis, we found through bioinformatics prediction and experimental studies that lncRNA TTN-AS1 targeted miR-134-5p levels as ceRNA and that miR-134-5p inhibited MBTD1 expression by directly binding to the 3′UTR region of MBTD1 mRNA. Studies have found anti-tumour mechanisms of miR-134-5p in non-small cell lung cancer and nasopharyngeal carcinoma –31. MiR-MBTD1 belongs to the polycomb group (PcG) and regulates the transcription process. This study found the role of MBTD1 in tumourigenesis . MBTD1 iIn conclusion, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance in osteosarcoma. However, further research is needed on the mechanisms by which TTN-AS1 participates in drug resistance and by which MBTD1 promotes the development of osteosarcoma.https://www.ncbi.nlm.nih.gov/geo/). A difference analysis was performed on the data. The miRcode website (http://www.mircode.org/) performed a biosignal analysis to predict miRNAs downstream of lncRNA TTN-AS1. The TargetScan website (http://www.targetscan.org/vert_72/) was used to predict downstream target proteins of miR-134-5p.The osteosarcoma patient gene data (GSE12865) were downloaded from the GEO database . Quantitative polymerase chain reaction (qPCR) was used to detect lncRNA TTN-AS1, miR-134-5p and MBTD1 mRNA expression levels in cancer tissues and normal tissues. The 5-year survival of patients with high or low expression of lncRNA TTN-AS1 and MBTD1 mRNA was compared. The correlation between lncRNA TTN-AS1 and pathological features of patients with osteosarcoma was analysed. Approval was obtained from the Tongren Hospital Ethics Committee.Human osteoblast hFOB 1.19 and osteosarcoma cell lines Saos-2 and U-2OS were purchased from American Type Culture Collection  and were grown in RPMI-1640 medium containing 10% foetal bovine serum , 50 U/mL penicillin and 50 μg/mL streptomycin . The plasmid constructs and the corresponding negative control (NC) were built by Shanghai Genepharma Company (China). Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. The cells were transfected with 100 nM RNA oligonucleotides using LipofectamineTM 2000 . Cells were harvested 48 h after transfection for subsequent experiments.2O. QPCR was performed using the following procedure: 2 min at 95°C, 40 cycles of 15 sec at 95°C, 25 sec at 60°C and 60 sec at 72°C. U6 acted as an internal reference for miR-134-5p, and GAPDH served as an internal reference for lncRNA and mRNA. The primers were synthesized by Sangon Biotech  and are shown in Total RNA was acquired using TRIzol . The cDNA was generated from 1 μg isolated miRNA/mRNA by using an iScriptTMcDNA Synthesis Kit . For qPCR processing, the FastStart Universal SYBR Green Master kit  was used. The reaction system was prepared as follows: 2.5 μL cDNA template, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 10 μL 2 × SYBR Green master mix, 5.5 μL dHThe whole-cell protein was extracted by using ice-cold RIPA buffer mixed with protease inhibitors. The protein lysate was separated by SDS-PAGE at 110 V for 100 min and transferred to PVDF membranes at 90 V for 90 min. The PVDF membrane was blocked in 5% non-fat milk for 1 h at room temperature, and antibody  was added at 4°C overnight. The membrane was further probed with the secondary antibody IgG H&L (HRP)  after washing the membrane with PBST (PBS with 0.2% Tween 20). The protein bands were detected by Pierce ECL Plus western blotting substrate  on a ChemiDoc MP instrument .4 cells / mL, 100 μL / well), and the blank controls  were set. After hypoxia-reoxygenation, 10 μL of CCK-8  reagent was added to each well, and the culture was continued for 2 h at 37°C. The absorbance values were measured at 450 nm on a microplate reader , and the corresponding optical density ratio was expressed as cell vitality.The cells were seeded in a 96-well culture plate  was added, gently mixed and incubated at room temperature for 5 min. The cell apoptosis rates were detected using an Annexin-V-FITC detection kit  and flow cytometry .The 2×10The sequences containing the wild-type (WT) or mutated (Mut) region MBTD1 were synthesized by Sangon  and inserted into a pGL3 vector . For the luciferase reporter assay, miR-134-5p mimics and the respective reporter plasmids were transfected into cells using Lipofectamine 2000 according to the manufacturer’s protocol. After 24 h, the Renilla and Firefly luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega Corp.) and a luminometer .6 cells correspondingly. Twenty-eight days after injection, the mice were sacrificed, and tumours were removed to be weighed and photographed. The animal experiment protocol was approved by the Animal Experimentation Ethics Committee of Tongren Hospital.Specific pathogen-free (SPF) 4-week-old BALB/c nude mice were purchased from the animal centre of Air Force Medical University . Saos-2 and U2OS cells were divided into three groups: vector-NC + sh-NC, vector-TTN-AS1 + sh-NC and vector-TTN-AS1 + sh-MBTD1. The mice in each group were injected with 1×10All analyses were conducted using Prism GraphPad 7.0 software. All variables were shown as the mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.


Studies comparing self-samples and clinician-collected samples for high-risk human papillomavirus (HPV) detection using clinically validated PCR-based HPV DNA assays are limited. We measured the concordance of HPV detection between home-based self-sampling and general practitioner (GP) sampling using the Cobas 4800 HPV DNA test and studied women’s accept of home-based self-sampling.Paired GP-collected samples and cervico-vaginal self-samples were obtained from 213 women aged 30–59 years diagnosed with ASC-US within the cervical cancer screening program. After undergoing cervical cytology at their GP, the women collected a self-sample with the Evalyn Brush at home and completed a questionnaire. Both samples were HPV-tested using the Cobas 4800 test. Histology results were available for those who tested HPV positive in GP-collected samples.We observed good concordance for HPV detection between self-samples and GP-collected samples . No underlying CIN2+ cases were missed by self-sampling. Women evaluated that self-sampling was easy  and comfortable .Home-based self-sampling using the Evalyn Brush and the Cobas 4800 test is an applicable and reliable alternative to GP-sampling. DNA tests for HPV testing in cervical cancer screening have been developed due to the strong causal relationship between persistent cervical infection caused by high-risk human papillomavirus (HPV) types and cervical cancer and its pre-cancer lesions , 2.Screening based on HPV testing is more sensitive in detecting cervical intraepithelial neoplasia of grade 3 (CIN3) and cancer than cytology-based screening , and womStudies comparing self-samples and clinician-collected samples for HPV detection show moderate to very good concordance in referral populations , 8, wherA systematic review demonstrated that self-sampling is a well-accepted screening method. The main reported concerns were pain, discomfort touching themselves, and uncertainty as to whether the sample was collected correctly . HoweverThis study aimed to measure concordance in HPV detection between paired self-samples collected at home using the Evalyn Brush device and general practitioner (GP)-collected samples, when using the clinically validated Cobas 4800 HPV DNA test. Further, we wanted to measure women’s accept of home-based self-sampling.This study was conducted in the Central Denmark Region (CDR), which covers 23% of the Danish population . Like thCurrently, 23–49-year-old women are invited for cervical cancer screening every third year, 50–64-year-old women every fifth year. A liquid-based cervical cytology sample is taken at their GP (GP-collected sample). The primary screening method is microscopic examination of the cytology sample for 23–59-year-old women; a primary HPV-DNA check-out test for 60–64-year-old women .In the CDR, the Department of Pathology at Randers Regional Hospital handles all cytology and HPV analyses. As per routine, GPs obtain the sample using a cervical brush and the brush head is placed in 10 ml SurePath medium  and mailed to the Pathology Department for further processing and testing according to guidelines, which for women aged 30–59 is microscopic examination. Women with high-grade cytological lesions  are referred directly for colposcopy. Women diagnosed with ASC-US undergo routine reflex HPV DNA triage testing using the cell pellet from 1 mL SurePath medium. ASC-US/HPV-positive women are referred for colposcopy within 3 months. ASCUS/HPV-negative women are referred back to the routine screening program. Women with LSIL are monitored by repeated cytology testing .For HPV DNA analyses, the Cobas 4800 assay  is used. This test is a real-time PCR fully automated method separately detecting HPV16, HPV18, and 12 other high-risk HPV types  using the β-globin gene as an extraction and amplification control .Paired GP-collected samples and cervico-vaginal self-samples were obtained from 30 to 59-year-old women diagnosed with low-grade cytological lesions (ASC-US) within the screening program. As per routine, GP-collected samples from these women are all analyzed by both microscopy and HPV, which is not the case for younger and older women, or women with other cytological diagnoses .Between June 2015 and December 2016, eligible women were consecutively identified daily through patient lists provided by the Department of Pathology. They received written information about the study and then contacted the investigator for oral information if they wanted to participate. A written and signed informed consent form had to be mailed back to the investigator before inclusion.The exclusion criteria were pregnancy, giving birth less than < 3 months previously, and collecting the self-sample after colposcopy.® Brush, Rovers Medical Devices B.V., Oss, Netherlands), written and picture-based user instructions showing how to collect the cervico-vaginal sample using the device, a questionnaire, and a pre-addressed return envelope.The women were sent a self-sampling kit comprising a brush device , stored overnight at 4 °C, and then vortexed for 5 min. A 6.4 ml volume of the self-sample material was centrifuged at 3000 x RPM for 20 min at room temperature. After centrifugation, with supernatant removed, the cell pellet was placed in 1 mL 25% ethanol-buffered (TRIS) and stored at -80 °C, until further processing. A volume of 6.4 ml was chosen to adjust for the material volume used for cytology examination performed on the GP-collected samples.The self-samples were thawed overnight at 4 °C before the day of analysis. For analysis, the self-sample (1 ml volume) was vortexed for 15 s before being placed in test tubes, being the starting point for the HPV testing. Each run included four water samples to measure contamination. All HPV testing was performed following the manufacturers’ instructions using a protocol without a sample pre-heating step which was also the case for the GP-collected samples. The investigator and the laboratory personnel performing the HPV testing were blinded to the HPV results of the GP-collected samples.The questionnaire included three questions on self-sampling experience and one on the clarity of the user instructions. To avoid low frequencies, the responses were grouped into three groups “Agree” , “Disagree” , and “Do not know”. Multiple response answers were not allowed. The women were asked to record the date they had taken the self-sample, whether they had had sexual intercourse in between the two samplings, and their partner status . Open feedback was possible. The data were double-entered into REDCap .Information on the HPV self-sample test results was obtained through patient lists provided by the Department of Pathology. Data on the GP-collected HPV test results and the histological results were retrieved from the nationwide Danish Pathology Data Bank (DPDB), which has been complete since the late 2000s . HistoloThe study was designed to estimate a 95% confidence interval (CI) with a width of +/− 5%. With an expected 86% sensitivity and 85% specificity of HPV detection (high-risk HPV types) using the Evalyn Brush and a PCR-based HPV DNA test, a minimum of 198 women had to be included .The HPV concordance between the paired samples was assessed using the Kappa statistic  and defined as “Poor” (κ ≤ 0.20), “Fair” (0.21 ≤ κ ≤ 0.40), “Moderate” (0.41 ≤ κ ≤ 0.60), “Good” (0.61 ≤ κ ≤ 0.80), or “Very good” (κ ≥ 0.81) . The oveP-values < 0.05 were considered statistically significant. All statistical analyses were performed using STATA, version 14 (STATA College).The study was approved by the local Ethical Committee of the Central Denmark Region  and by the Danish Data Protection Agency .From a total of 1110 eligible women, 216 (19.5%) returned a self-sample. Three women (0.3%) were excluded because the self-sample was taken after the biopsy, leaving 213 (19.2%) women for analysis.n = 113, 53.0%) followed by women aged 30 to 39 years  and 50 to 59 years . All paired GP-collected samples and self-samples were valid for HPV testing.The included women’s median age was 44 years (IQR: 38–49 years). The majority were aged 40 to 49 years . Histological results were available for 46 women, of whom 19 (41.3%) had a normal biopsy, 4 had CIN (not specified) (8.7%), 11 had CIN1 (23.9%), and 12 (26.1%) had CIN2+ .For self-samples, the HPV prevalence (any type) was 24.4% 95% CI: 18.8–30.8%); for GP-collected samples 22.1% (95% CI: 16.7–28.2%) (Table 8.8–30.8%n = 38)  (p = 0.20). None of these nine women reported having trouble taking the self-sample, although one woman responded feeling insecure if she had collected the self-sample correctly. Histological results were available for eight out of the nine women, of whom five (62.5%) had no CIN, one (12.5%) had CIN (not specified), and two women (25.0%) had CIN1. The 12 women diagnosed with CIN2+ all had HPV detected in their self-sample.A total of 23 women (10.7%) had disconcordant results. In nine cases, the women were GP-collected sample HPV-positive/self-sample HPV-negative. For these women, the median number of days between the two samples was insignificantly higher than in women with concordant sample results  reported no sexual intercourse in the time span separating the two samples.The HPV prevalence was insignificantly higher in self-samples than in GP-collected samples for HPV16/18  versus  and HPV of other types  versus  (Table p = 0.33) for any of the questions.A total of 212 out of 213 women answered the questionnaire. Self-sampling with the Evalyn Brush was recorded as easy by 97.2% (95% CI: 93.9–98.9%)  was comparable with the mean k (k = 0.71) reported for brush devices combined with PCR-based HPV DNA tests in the review by Schmeink et al. 0 was com. This diIn our study, the sensitivity and specificity of HPV detection (of any type) was 80.9% and 91.6% in self-samples, respectively, when using the GP-collected samples as reference standard. These results are comparable to those by Van Baars et al.  who founKetelaars et al.  found noMost importantly, we showed that no underlying CIN2+ cases were overlooked by self-sampling. Some of the women in the GP-collected HPV negative/self-sample HPV-positive group might possibly have had underlying CIN2+, but this cannot be explored further in this study since referral for colposcopy was based on the result of the GP-collected sample.The self-sampling device and user instructions must be acceptable if we wish to improve screening participation by this method. In our study, more than 85% expressed confidence in having collected the self-sample correctly, and only 5% expressed discomfort with collecting the sample. Hence, home-based self-sampling using the Evalyn Brush appeared to be a well-accepted screening method. Yet, almost 10% reported uncertainty regarding sample collection and some stated the lack of a click when rotating the brush as their reason. Despite small numbers, this finding is higher than reported by Van Baars et al.  (0.8%) wWe report a high acceptability of home-based self-sampling and good concordance between self-samples and GP-collected samples in terms of HPV detection using the clinically validated Cobas 4800 HPV DNA test. Our findings demonstrate the feasibility of offering self-sampling as an alternative to GP-sampling in cervical cancer screening.


TSG101 (Tumor susceptibility 101) gene and its aberrantly spliced isoform, termed TSG101∆154-1054, are tightly linked to tumorigenesis in various cancers. The aberrant TSG101∆154-1054 mRNA is generated from cancer-specific re-splicing of mature TSG101 mRNA. The TSG101∆154-1054 protein protects the full-length TSG101 protein from ubiquitin-mediated degradation, implicating TSG101∆154-1054 protein in the progression of cancer. Here, we confirmed that the presence of TSG101∆154-1054 mRNA indeed caused an accumulation of the TSG101 protein in biopsies of human nasopharyngeal carcinoma (NPC), which was recapitulated by the overexpression of TSG101∆154-1054 in the NPC cell line TW01. We demonstrate the potential function of the TSG101∆154-1054 protein in the malignancy of human NPC with scratch-wound healing and transwell invasion assays. By increasing the stability of the TSG101 protein, TSG101∆154-1054 specifically enhanced TSG101-mediated TW01 cell migration and invasion, suggesting the involvement in NPC metastasis in vivo. This finding sheds light on the functional significance of TSG101∆154-1054 generation via re-splicing of TSG101 mRNA in NPC metastasis and hints at its potential importance as a therapeutic target. TSG101 was initially found in a screen for potential tumor-suppressor genes in mouse [The multi-complex machinery, termed the ‘endosomal sorting complex required for transport’ (ESCRT), regulates the sorting of ubiquitinated proteins to endosomes, facilitating receptor traffic and turnover, and it has been implicated in normal development, cell differentiation, and growth, as well as the budding of certain enveloped viruses . The in mouse , this prin mouse ,9. TSG10in mouse . In addiin mouse ,12. We din mouse .TSG101 is an established cancer-associated gene and aberrantly spliced TSG101 mRNAs have been reported in various kinds of cancers . TSG101iewed in ,16). Besiewed in ,24,25,26iewed in ,22,27. Aiewed in ,19,21,24iewed in . Our disiewed in .via re-splicing of TSG101 mRNA, i.e., the protection of full-length TSG101 protein from its ubiquitin-mediated proteasomal degradation [Detailed examination of TSG101∆154-1054 in pre-neoplastic lesions, as well as biopsies of cervical cancer, revealed a significant correlation between the expression of this transcript and neoplastic progression . In addiradation . Becausep < 0.005).Using reverse-transcription, followed by nested polymerase chain reaction (RT-nested-PCR), a shortest isoform (around 250-bp marker) among the various TSG101 isoforms was most frequently observed besides the full-length TSG101 transcripts in almost half of the NPC tissues . To verify this finding, a plasmid encoding TSG∆154-1054 was constructed and used for transient transfection assays in an NPC cell line, TW01. Mirroring the finding from NPC biopsies, the amount of endogenous TSG101 protein in TW01 cells increased with the expression of TSG∆154-1054 , although the TSG∆154-1054 expression is only marginally correlated with advanced tumor stage .The demographics of our NPC patient cohort are shown in TSG101 gene was first identified, genomic mutations and alternatively spliced mRNA isoforms were detected in human breast cancer [When the human t cancer . Since tt cancer ,21,22,24t cancer , the detWe previously demonstrated that the generation of TSG∆154-1054 is a consequence of tumorigenic activation of a re-splicing of TSG101 mature full-length mRNA, and thus the completion of the normal splicing pathway activates the distant weak alternative splice sites . InteresUsing NPC biopsies, we first observed that the generation of aberrant TSG∆154-1054 mRNA correlates with increasing amounts of the full-length TSG101 protein. This observation was recapitulated in transiently and stably transfected cancer cells, in which TSG∆154-1054 expression indeed increases full-length protein, but not mRNA levels, of TSG101. This effect could be inhibited by the specific degradation of TSG∆154-1054 mRNA, indicating a direct correlation between the full-length TSG101 protein levels and the splicing variant TSG∆154-1054.In mammalian cells, the amount of TSG101 protein is strictly counterbalanced by a post-translational process involving its ubiquitin-proteasome proteolysis ,33. A coDespite these alternative degradation routes in controlling the TSG101 amount, increased levels of the TSG101 protein are nevertheless detected in a variety of cancers ,37,38. TTsg101-knockout mice revealed that the TSG101 protein is essential in cell proliferation and survival [Conditional survival ,10. Regasurvival . In ovarsurvival ,40.PEG10  gene [It was reported that siRNA-mediated downregulation of TSG101 caused partial cell cycle arrest and reduced the colony formation capacities of prostate and breast cancer cells, and notably, it diminished the migratory activity of breast cancer cells . A recen10) gene . Here, w10) gene . It is o10) gene  through in vivo [Our results first highlighted the impact of TSG∆154-1054-mediated stabilization in the functional activities of TSG101. Moreover, subcutaneous inoculation of a cell line stably expressing TSG∆154-1054 into athymic nude mice substantially increases the efficiency of tumor formation in vivo . This rovia the stabilization of TSG101, which has recently been suggested to be used as a cancer biomarker [In summary, we provide novel evidence of the TSG∆154-1054 protein function in regulating TSG101 stability during NPC tumorigenesis. TSG∆154-1054 plays a key role in promoting the metastasis of NPC iomarker ,43,44. TThirty-eight NPC biopsies were used, with the primary tumors staged from I to IV, classified according to American Joint Committee on Cancer (AJCC) staging system. Thirty lymphoid hyperplasia (LH) biopsies from nasopharynx that had been pathologically diagnosed with an absence of cancer cells were recruited as the control group. The Departments of Otolaryngology and Pathology of National Taiwan University Hospital provided all of these fresh frozen-sectioned specimens. All of the procedures were conducted in accordance with the ethical standards of the committee on human experimentation of the College of Medicine, National Taiwan University . TSG∆154-1054 expressing TW01 stable lines were previously established in our laboratory [TW01 cells, which are a human NPC cell line, were grown in Dulbecco’s modified Eagle’s medium , supplemented with 10% fetal calf serum , L-glutamine, and penicillin-streptomycin. TW01 cells stably expressing TSGΔ154-1054 and the control empty vector (pcDNA3.1) were established in complete DMEM under G418-selection.The expression plasmids encoding TSG101 and TSG∆154-1054 were generated by the PCR technique and were subcloned into the pcDNA3.1 vector . To yield fusion proteins, TSG101 was fused either with an N-terminal HA- or FLAG-tag. The construction of the pSuper-siE7 plasmid was described in our previous publication . The siTThe siTSG∆154-1054 template (5′-GACCTAACTCTCCCTTATA-3′) is designed complementary to the TSG101 sequence nucleotides 145 to 153 (GACCTAACT) joined to nucleotides 1055 to 1064 (CTCCCTTATA). ON-TARGETplus SMARTpool siRNAs against human TSG101  and non-targeting siRNA  were purchased from GE Healthcare Life Sciences.Transfection of expression plasmids and ON-TARGETplus SMARTpool siRNAs was carried out using TransFast Transfection reagent  and DharmaFECT , respectively, following the manufacturers’ instructions. siRNAs were transfected twice into TW01 cells and, at 48 h post-transfection, transiently or stably transfected culture cells were harvested for the following experiments.Total RNAs were isolated from cultured cells with TRIzol reagent (Thermo Fisher Scientific), as described by the manufacturer. Complementary DNA was generated from total RNAs and with randam hexmers and SuperScript III reverse transcriptase (Thermo Fisher Scientific), according to the manufacturer’s instructions. Nested-PCR analyses targeting TSG101 transcripts were carried out as previously described, with the primers P1 (5′-CGGTGTCGGAGAGCCAGCTCAAGAAA-3′) and P2 (5′-CCTCCAGCTGGTATCAGAGAAGTCAGT-3′) for the first-round PCR, and P3 (5′-AGCCAGCT CAAGAAAATGGTGTCCAAG-3′) and P4 (5′-TCACTGAGACCGGCAGTCTTTCTTGCTT-3′) for the second-round PCR ,17. To v48 h post-transfection, transiently or stably transfected culture cells were harvested and cell lysates were prepared using lysis buffer containing 3% SDS, 2 M urea, and 2% 2-mercaptoethanol. Immunoblotting was performed with antibodies directed against TSG101 , HA , and GAPDH , as described previously . ExpressTW01 cells that were seeded onto six-well plate were incubated with cycloheximide (300 μg/mL) at 18 h post-transfection prior to harvesting at serial time points. The stability of HA-tagged TSG101 (HA-TSG101) was examined by immunoblotting and quantified by densitometry (see above). The cycloheximide chase assay was also performed with TSG∆154-1054 expressing TW01 stable cell lines , which wg0h − g20h)/g0h, where g0h and g20h are the inter-scratch distances at time 0 h and 20 h, respectively.Transfected TW01 confluent cells were maintained in DMEM containing 1% FCS and they were scratched using a sterilized micropipette tip. The monolayer cells were washed extensively with DMEM and then cultured in DMEM with 1% FCS for another 20 h. The migration of TW01 cells was observed under a bright-field microscopy . The migration rate (%) is calculated as , and the stained cells were counted. Quantification of the invasion ability was determined by the invaded cell number.24-well transwell chambers with 8 μm pore-size PET membranes  were covered with 50 μL of Matrigel (Corning) and then incubated at 37 °C for 2 h. Transfected TW01 cells were cultured for 24 h, 2.5 × 10t-test. The correlations between TSG∆154-1054 positivity and TNM stage or metastasis were assessed by the Chi-square test.Aberrantly spliced TSG101 products in cancer and control tissues were assessed while using the chi-square test and Mann–Whitney (Wilcoxon) rank sum test. In the scratch-wound assay to test cell migration activity, each image was a representative result from three independent experiments that were performed in duplicate and the effect of TSG∆154-1054 was evaluated by a two-tailed