Human papillomavirus (HPV) associated cancers are increasingly reported globally, including in sub-Saharan Africa (SSA). However, with the exception of cervical HPV infection, data from SSA on the epidemiology of oral and genital HPV infections are limited. This study assessed the prevalence and concordance of oro-genital and anal HPV genotype specific infections among women in the general population.We conducted a cross-sectional study in sexually active women aged 18–45 years in Ibadan, Nigeria. After a face-to-face interview and clinical examination, oral, cervical, vulvar, and anal samples were collected from participants and tested by the Anyplex II 28 HPV assay. Descriptive and multivariable analyses were used to report prevalence and risk factors associated with HPV infections.The prevalence of any vulva, cervical, anal, and oral HPV infections was 68.0% (210/309), 59.7% (182/305), 56.8% (172/303), and 16.1% (14/286), respectively. There was an inverse relationship between age-group and HPV prevalence of HPV in all anatomic sites except for the oral HPV infections. HPV 35 was the most prevalent high-risk HPV genotype in the vulva, cervix and oral cavity. Associated risk factors for HPV infection in each of the anatomic sites were reported. Overall, 10.0% (31/310) women had concordance of any HPV type in the four anatomic sites.There was a high prevalence of oro-genital and anal HPV infections among sexually active Nigerian women, with concordance of HPV types in the cervix, vulva, anus and oral cavity. We advocate large longitudinal studies that will involve sampling of multiple anatomic sites and inclusion of other women in the community for better understanding of HPV epidemiology in this region.The online version contains supplementary material available at 10.1186/s13027-021-00398-9. Worldwide, human papillomavirus (HPV)-associated cancers are estimated to account for 4.5% of all cancers, with the highest burden occurring in the low-middle income countries (LMIC) , 2. HPV Nigeria has one of the highest burdens of cervical cancer in sub-Saharan Africa after eastern and southern Africa . It is eThis household survey was conducted among sexually active women aged 18–45 years, from the general population in two local government areas (LGA) in Ibadan. Two contiguous peri-urban communities were selected in Akinyele LGA: . Mokola , a high-density urban community in Ibadan North LGA was the second study site. These study sites were selected because they are heterogenous in culture, religion and socio-economic/demographic profile. Each community is comprised of smaller enumeration areas (EA)—this is a small, compact area, carved out of a bigger locality or a group of localities with well-defined and identifiable boundaries. It usually has a unique name and code for identification. EAs represent the primary sampling unit during census.The study excluded young adolescents (< 18 years), pregnant or nursing mothers, women not resident at the study sites and those that declined participation including refusal for biological sample collection. Assuming an alpha of 0.05 and a design effect of 2 owing to the clustered sampling design, a sample size of 300 was calculated to be able to estimate the prevalence of HPV and determine associated risk factors in each of the four anatomic sites with adequate precision among women. The research assistants, nurses and laboratory staff had an intensive three-week training and participated in community engagement before the study commenced.Eligible people were selected through a two-stage sampling technique. The first stage sampling involved random selection of four EAs each at Mokola and Moniya, and one EA at Sasa by probability proportional to size using the 2006 National Population Commission Census lists of the EAs . Each hoEligible participants were randomly enrolled at home by female research assistants who explained the study objectives and provided potential participants with an information leaflet in English or in a local language (Yoruba/Hausa/Igbo). Individuals that agreed to participate were given appointment to be seen in a local primary healthcare centre. Potential participants had reminder phone calls at 72 h and 24 h before and a short message service on the morning of their clinic appointment.At the clinic, participants signed a written or witnessed informed consent following a repeat explanation of study procedures, including collection of biological samples. A face-to-face interview was conducted in English or in Yoruba for participants that could not understand English. The interview covered socio-demographics, sexual behaviours  and hygiene practices, intravaginal practices, alcohol, smoking and stimulant use, previous STI and awareness about HPV vaccine. After the interview, a female nurse collected blood for a rapid diagnostic HIV test and other biological samples. Samples from participants who did not wish to know their HIV were tested by an RDT in the laboratory. This HIV testing protocol was based on the National Guidelines for HIV Prevention Treatment and Care, Federal Ministry of Health, Nigeria . HIV posSamples were also collected by a nurse from the mouth, cervix, vulvar and anal cavity in separate sample bottles. Briefly, an oral sample was collected using a 30 s oral rinse and gargle method with 10mls of Scope mouth wash (Procter & Gamble©). The nurse demonstrated the rinse and gargle procedure to a participant prior to sample collection. The participant sample was then collected into a 10 ml labelled sample bottle. and placed immediately into a cold box (2–4 °C). For the vulvar sample, the tip of a Dacron swab was used to rub the introitus on either side of the vaginal orifice without touching the urethral orifice. The cervical sample was then collected by inserting a sterile Cusco speculum into the vagina to expose the cervix. The tip of a new Dacron swab was inserted into the cervical os and gently rotated 360 degrees with care being taken to avoid trauma to the cervix and potential bleeding before removing it. An anal sample was collected with the participant in a left lateral position. A Dacron swab was inserted into the anal canal  and rotated 360 degrees with gentle pressure around the anal verge before removal. Each of the samples collected with swabs were placed in separate 2 ml cryotubes that were labelled with a barcoded sticker prior to being placed into a cold box (2–4 °C). All samples were subsequently stored in a − 80 °C freezer at the Institute of Advanced Medical Research and Training, College of Medicine, University of Ibadan, Nigeria until shipment to Catalan Institute of Oncology, Spain. Health related incentives , a soft drink and biscuit and 1000 Naira (2.8GBP) to cover transportation.HPV genotyping of the samples was performed at the Catalan Institute of Oncology, Spain using the Anyplex™ II HPV28 assay . This assay detects 28 HPV genotypes, including HR-HPV , LR-HPV  and possibly carcinogenic genotypes .DNA extraction from the mouthwash was performed using the Maxwell® 16 LEV Blood DNA kit . Briefly, mouthwash liquid samples were spin at 12.000xg for 20 min. After discarding supernatant, cell pellet was eluted in 300ul of lysis buffer + 30ul Proteinase K and DNA extraction performed following the manufacture’s instructions. DNA was extracted from cervical, vulvar and anal dry swabs using the Maxwell 16 Buccal swab LEV DNA Purification kit . Briefly, dry swabs were eluted in 300 ul of lysis buffer + 30 ul Proteinase K, incubated for 20 min. at 56 °C and spin at max. speed for 2 min. DNA extraction from eluted samples was performed following the manufacture’s instructions.The Anyplex™ II HPV28 detection test was performed as recommended by the manufacturer. The data recording and interpretation were automated with Seegene viewer software . Beta (β)-globin was used as the internal control to demonstrate the presence of human DNA in each sample and to determine invalid samples. However, any sample that was positive for HPV but negative for Beta (β) globin was considered as valid.Data were double entered into REDCap software . Thereafter, the raw data were exported in CSV format and saved. The data were imported into STATA 16.0  software for analysis.The participants’ selected descriptive variables were summarised using frequencies and proportions for the categorical variables and mean and standard deviations for the continuous variable. The primary outcome was prevalence of any HPV infection. The prevalence of HR-HPV and LR-HPV infection and groups classified according to the 2009 IARC epidemiological oncogenic classification  for each of the anatomical sites were also calculated with their 95% confidence intervals. The trends of association between each classification of HPV infection and the age group of participants were calculated with ANOVA.A conceptual framework for the risk factor analysis of any HPV infection was developed , history of transactional sex, ever had mutual masturbation, history of female genital mutilation, alcohol use, illicit drug use, ever had STIs and ever heard of HPV. Each level 2 variable was added one by one to a model that included level 1 variables that met a p value cut off of ≤ 0.1 after adjusting the ‘core variables’. Any level 2 variable that met a p value cut off of ≤ 0.1 was included with the level 1 core variables in the level 2 adjusted model. Level 3 biological variables were laboratory detection of concurrent HPV infection from the other three anatomical sites apart from the outcome measure. For example, if the outcome measure was to determine risk factor for any cervical HPV, concomitant detection in vulvar, anal and oral sites were included as explanatory variables. Each level 3 variable was added one by one to a model that included level 1 ‘core variables’ and level 2 factors that met a p value cut off of ≤ 0.1. Any level 3 variable that met a p value cut off of ≤ 0.1 was included with the level 1 and level 2 ‘core variables’ in the level 3 adjusted model. This strategy allowed the effects of variables at each level of the framework to be assessed, adjusted for more distal variables.The concordance of HPV between oral, cervical, vulvar and anal samples in individual participant was defined as the presence of the same type of virus across the four sites. The proportion of concordance for specific HPV type was calculated as the number of each HPV type in all the four sites, any of three and any of two anatomical sites.Out of the 930 eligible women listed, 304 in Mokola, Ibadan North LGA and 297 in Moniya/Sasa, Akinyele LGA were randomly selected and visited at home; only 177 and 159 women in Mokola and Moniya/Sasa, respectively, were met at home and invited to the clinic to participate in the study. Of the 310 women that consented and participated, 157 and 153 women were from Mokola and Moniya/Sasa, respectively. The enrolment of participants at the two study sites was summarised in Fig. The overall mean age of participants was 29 years (SD = 7) Table . There wAmong the selected behavioural factors Table , particiThe two biological factors that differed among women in the two communities were proportion of women with any anal HPV (p = 0.010) and oral HPV (p = 0.004). Overall, the proportion of women with any HPV was highest in the vulvar followed by cervical, anal and oral sites.Three hundred and ten samples were collected from each of the anatomical sites. of these, twenty-four (7.7%) oral samples, seven (2.2%) anal samples, five (1.6%) cervical samples and one (0.3%) vulvar sample were considered invalid due to the low amount of β globin portion of the DNA. All invalid samples were treated as missing.Overall, the prevalence of any HPV infection in women was 68.0% 95% CI 62.4–73.1) in the vulvar samples, 59.7% (95% CI 53.–65.2) in the cervical samples, 56.8% (95% CI 51.0–62.4) in the anal samples and 16.1% (95% CI 12.0–12.9) in the oral samples  and 0.44 (95% CI 0.25–0.78) odds of having cervical HPV infection, respectively, compared to those aged 18–24 years. There was 1.54 (95%CI 1.16–2.04) higher odds of having cervical HPV infection for a unit increase in the number of lifetime partner for vaginal sex. The odds of detecting cervical HPV infections was 12.85 times (95%CI 5.70–28.99), 4.37 times (95%CI 1.50–12.71) and 3.48 times (95%CI 1.74–6.96) in women that had concomitant vulvar, oral and anal HPV infections, respectively, compared to those with no HPV infections at these anatomical sites  of vulvar HPV infection among married or living as married women relative to single women. Having 6 or more than lifetime vaginal sex partners was associated with a lower odds  of vulva HPV infection compared to those with smaller age difference. There were 2.03 (95% CI 1.38–3.01) odds of HPV infection for each unit increase in the number of lifetime partner for vaginal sex. Concomitant cervical  and anal  HPV infections were associated with a higher odds of vulvar HPV infection compared to those with no concomitant infections at such anatomical sites  of having anal HPV infection among women whose age difference with her first vaginal sex partner was six years or more compared to those with smaller age differences. The odds of anal HPV infections were 1.92 (95% CI 1.35–2.71) higher for each unit increase in number of lifetime partner for vaginal sex. The odds of having anal HPV was higher in women with concomitant cervical  and vulvar  HPV infection compared with those without HPV infections at these anatomical sites  compared to those in Mokola. The odds of having oral HPV infection was 4.81 (95%CI 1.58–14.62) higher in women with concomitant cervical HPV infection than those with no such infection (Table Thirty-one 10.0%) out of 310 women had concordance of any HPV infection in the four anatomical sites of the cervix, vulvar, anal and oral cavities. Fifteen of these women had concordant HR-HPV genotypes  in Brazil . There iThe data on the prevalence of vulvar HPV infection among women in the general population are limited compared to other anogenital sites. Studies that reported on vulvar HPV infections were typically conducted among women that had vulvar lesions and were not representative of general population prevalence estimates of vulvar HPV infections , 23. Of The prevalence of any or HR oral HPV infections was lower than for the other three anatomical sites. Oral HPV infections including HR genotypes were higher in this study than some previous studies , 28. SpeHPV-35 was the most common HR genotypes in the cervix and vulva, HPV-52 in the anal cavity and HPV-51 in the oral cavity. HPV-42 was the most common LR genotypes in all anatomic sites in this study. Previous studies in Nigeria had also reported HPV35 and 42 as the most prevalent for cervical HPV infection , 11. StuAll the risk factors associated with cervical, vulvar, anal and oral HPV infection had been previously reported in other studies , 34–36. Concomitant cervical HPV infection appeared as a constant risk factor for oral, vulvar and anal HPV infection in this study. In 2019, a meta-analysis of pooled data involving 13,427 women with paired cervical and anal samples showed that cervical and anal HPV infections were highly correlated, particularly in HR-HPV genotype specific infections . ConcomiThere are potential limitations in this study. We were unable to report on the prevalence of adults older than 45 years which is the period when the second peak prevalence of HPV infections is sometimes reported. The exclusion of sexually naïve females could also have contributed to the high prevalence of HPV. The cross-sectional design of the study did not allow for investigation of risk factors for HPV persistence and the clearance. It was not possible to test for association between different types of oral sex (given or received) and oral HPV infection. The risk of oral HPV has been reported to be higher in individuals that received oral sex compared to those that give oral sex , 40. AnaIn conclusion, this study provides a robust estimate of cervical, vulvar, anal and oral HPV prevalence among sexually active women in the general population within the age range for HPV screening in Nigeria and demonstrates how prevalent HPV infection is in these women. The risk factors for specific HPV infection of different anatomical sites were similar to findings in previous studies. Although HPV infection is transient, the relatively high HPV infection in all anatomical sites relative to previous studies may possibly be due to the changing sexual behaviour and increasing sexual risk practices in the general population, in addition to the study design and the characteristics of study population. The detection of HPV-35 and -51 as part of the most prevalent HR-HPV types in the genital (cervix and vulva), anal cavity and oral cavity, respectively, may suggest the need to consider the coverage of these genotypes in future HPV vaccine design, especially so for HPV-35, that is being reported commonly in invasive genital cancers .We advocate adequately powered population level longitudinal studies that can investigate the persistence of HR-HPV infections in different anatomic sites to better understand the epidemiology of HPV and associated malignancies in SSA. This information will help policy makers to make evidence based informed decisions on prevention and management of HPV infections and associated cancers in Nigeria and other countries with similar demographic profiles in SSA.Additional file 1:Fig S1. Conceptual framework for the risk factor analysis of any HPV infection among females in the two communities in Ibadan, Nigeria. Fig S2. Prevalence with 95% confidence intervals of specific HPV genotypes in the four anatomic sites. Table S1. Pattern of HPV concordance by means of anatomical sites among females in Ibadan, Nigeria (n=310).


Immune checkpoint inhibitors (ICIs) effectively treat lung adenocarcinoma (LUAD) with fewer side effects. However, for LUAD patients, the lack of predictive markers for ICIs makes their clinical benefits less than ideal. Despite reports suggesting that a TTN (titin) mutation plays an important role in immunotherapy of solid tumors and gastric cancer, the relationship between the TTN mutation and LUAD immunotherapy has not been determined. We collected a LUAD cohort with whole-exome sequencing (WES) and immunotherapy prognosis. The ICI cohort was used to explore the relationship between TTN mutation status and prognosis. Then, the Cancer Genome Atlas (TCGA)-LUAD and Chen-LUAD cohorts were downloaded from the cbioportal website. We also used CIBERSORT, gene-set enrichment analysis (GSEA), and single-sample GSEA (ssGSEA) to evaluate the proportion of immune cells and the degree of pathway activation in LUAD patients, respectively. DDR signaling pathways obtained from the Molecular Signatures Database (MSigDB), tumor mutation burden (TMB), and NAL were used to evaluate the immunogenicity of LUAD patients. In the ICI cohort, TTN-mutant (TTN-MT) had significantly longer overall survival (OS) than TTN-wildtype (TTN-WT) (P = 0.009). Univariate and multivariate COX models showed that TTN mutation status can independently predict immunotherapy prognosis. Notably, the results of tumor immune microenvironment (TIME) analysis showed that TTN-MT patients had inflammatory TIME, which showed enriched activated immune cells and higher immune scores. Immunogenicity analysis showed higher immunogenicity in TTN-MT patients, which indicated high levels of gene mutations in TMB, NAL, and DDR pathways. GSEA and ssGSEA results showed that TTN-MT was substantially enriched in chemokine secretion, inflammatory factor secretion, and antigen presentation. Some pathways related to immunosuppression and immune depletion were significantly downregulated. TTN-MT is associated with significantly prolonged OS in LUAD patients. Additionally, TTN-MT is related to high immunogenicity and inflammatory TIME, suggesting that TTN-MT may be a potential predictive marker for patients with LUAD to accept ICIs. At present, lung cancer tumor malignancy presents with the highest rates of morbidity and mortality, while lung adenocarcinoma (LUAD) is the main subclass of primary lung cancer (about 85%) . LUAD haIncreasingly more studies have started looking for the predictive markers of ICIs, in an attempt to maximize the clinical benefits of immunotherapy for patients. The research shows that traits such as PD-L1 expression, TMB, microsatellite instability (MSI), DNA damage repair (DDR), tumor-infiltrating lymphocytes (TILs) may be predictors of patients’ responses to immunotherapy. However, the widespread implementation of these markers has numerous limitations. For example, the PD-L1 detection methods lack standardization and consistency, TMB is heterogeneous among different laboratories and platforms, only a small number of patients with lung cancer contain the MSI-H phenotype (< 1%) –9, and rTTN is used to express myosin, which is connected by unstructured peptide sequences and is responsible for maintaining muscle tension , while aAdditionally, Yang and his colleagues found that MUC4, MUC16, and TTN mutations were associated with the immune prognosis of gastric cancer in the immunotherapy cohort . With thhttps://portal.gdc.cancer.Gov/) database of TCGA-LUAD cohort. Using cbioportal , we downloaded another LUAD cohort (Chen-LUAD), which includes expression data and mutation data (WES) (We downloaded a LUAD cohort (ICIs-cohort) with ICIs-based treatment plans from the supplementary materials of a published study . The cohta (WES) . The pathttp://cibersort.stanford.edu/. The CIBERSORT algorithm analyzes the expression data in the TCGA-LUAD cohort according to LM22 (22 immune cells) signature and 1,000 permutations. Additionally, they play corresponding roles in the immune response  obtained from the supplementary materials of published research  method  was usedTo explore TTN-MT’s predictive role in the prognosis of LUAD patients after ICI therapy, we used a univariate COX regression model and multivariate COX regression model for the follow-up analyses. The two forest maps show the results of the univariate COX analysis and multivariate COX analysis, respectively. vs. 4%) and CUX1 (22.7 vs. 0%) in the TTN-MT group were significantly higher than those in the TTN-WT group (P < 0.05).To explore the profiles of mutated driver genes in different TTN mutation statuses, we analyzed the 20 driver genes with the highest mutation frequency in LUAD patients in the ICI-cohort and TCGA cohorts, respectively. Most of the driver gene mutation types in the ICI cohort are mainly missense mutations [such as TP53, MUC16, KRAS, LRP1B, CTNNA2, KEAP1, NF1, CDH10, AKAP9, FAT4, PTPRB, PTPRD, SETBP1, STK11, ATM, and CTNND2 (vs. 24%) in the TTN-MT group was significantly lower than that in the TTN-WT group (P < 0.05). In the TCGA cohort, most driver genes belong to an oncogene, while very few driver genes belong to TSG. We found that the mutation frequencies of most driver genes in the TTN-MT group were significantly higher than those in the TTN-WT group. These frequencies include TP53 (61.9 vs. 36.4%), TTN (100 vs. 0%), MUC 16 (56.8 vs. 25.2%), CSMD 3 (52.9 vs. 23%), LRP1B (47.1 vs. 20%), USH2A (45.1 vs. 18%), ZFHX4 (44.4 vs. 17.7%), FLG (35 vs. 16.4%), XIRP2 (38.1 vs. 13.8%), SPTA1 (33.9 vs. 16.4%), NAV3 (30 vs. 11.8%), FAT3 (29.6 vs. 11.8%), COL11A1 (28.8 vs. 12.1%), ZNF536 (29.6 vs. 11.1%), ANK2 (28.8 vs. 11.1%), CSMD1 (27.2 vs. 12.1%), PCLO (30 vs. 9.5%), and PCDH15 [29.2 vs. 9.8% . Additionally, we also compared some immune scores related to immune response, and the results showed that the TTN-MT group was partly altered. The scores of homologous recombination defects and Th2 cells were significantly higher than those of the TTN-WT group , FGFR, and lipid metabolism. Williams et al. proved that PI3K inhibitor could alleviate the TIME of immune exhaustion and increase the sensitivity of breast cancer to immune checkpoint blocking treatment . Wang etAdditionally, the WNT/β signaling pathway plays an important role in forming immune heterogeneity, such as inhibiting T cell activation and infiltration, promoting T cell apoptosis, inhibiting antigen treatment and degree, and inhibiting the killing effect of the immune system on tumor cells . TGFβ inHowever, this research has some limitations. First, we have only explored the relationship between the TTN mutation status and immunotherapy prognosis and response in one COAD cohort of patients treated with ICIs. We still need to verify these results in several cohorts of LUAD receiving ICIs. Second, this study lacks cell and animal experiments for the verification of follow-up mechanisms. We hope that the potential mechanism between TTN mutation and the prognosis of patients receiving immunotherapy can be verified by cell experiments and animal experiments in the future.In this study, we found that TTN-MT may be a predictive marker of immunotherapy for LUAD patients. TTN-MT is significantly associated with significantly improved immunotherapy prognoses in LUAD patients who also have significantly increased immunogenicity and inflammatory tumor TIME.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization, SL, XY. Formal analysis, ZW. Visualization, ZW. Writing—original draft, CW. Writing—review and editing, SL, XY, ZW, CW. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer XW declared a shared affiliation, with no collaboration, with the authors, to the handling editor at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human papillomavirus-positive (HPV+) cervical cancers are highly heterogeneous in molecular and clinical features. However, the molecular classification of HPV+ cervical cancers remains insufficiently unexplored.Based on the expression profiles of 50 genes having the largest expression variations across the HPV+ cervical cancers in the TCGA-CESC dataset, we hierarchically clustered HPV+ cervical cancers to identify new subtypes. We further characterized molecular, phenotypic, and clinical features of these subtypes.We identified two subtypes of HPV+ cervical cancers, namely HPV+G1 and HPV+G2. We demonstrated that this classification method was reproducible in two validation sets. Compared to HPV+G2, HPV+G1 displayed significantly higher immune infiltration level and stromal content, lower tumor purity, lower stemness scores and intratumor heterogeneity (ITH) scores, higher level of genomic instability, lower DNA methylation level, as well as better disease-free survival prognosis. The multivariate survival analysis suggests that the disease-free survival difference between both subtypes is independent of confounding variables, such as immune signature, stemness, and ITH. Pathway and gene ontology analysis confirmed the more active tumor immune microenvironment in HPV+G1 versus HPV+G2.HPV+ cervical cancers can be classified into two subtypes based on the expression profiles of the 50 genes with the largest expression variations across the HPV+ cervical cancers. Both subtypes have significantly different molecular, phenotypic, and clinical features. This new subtyping method captures the comprehensive heterogeneity in molecular and clinical characteristics of HPV+ cervical cancers and provides potential clinical implications for the diagnosis and treatment of this disease. Cervical cancer is the most common gynecological malignancy , of whicCurrently, surgery, chemotherapy and radiotherapy are three major therapeutic options for cervical cancer, although they have limited efficiency for advanced or recurrent cervical cancers . RecentlIn this study, we identified subtypes of HPV-positive (HPV+) cervical cancers based on gene expression profiles in cervical cancers. Furthermore, we compared molecular and clinical features between the HPV+ cervical cancer subtypes. We also compared molecular features between HPV+ and HPV-negative (HPV-) cervical cancers. This study aimed to explore a new subtyping method for HPV+ cervical cancers and provide potential clinical implications for the diagnosis and treatment of HPV+ cervical cancers.https://portal.gdc.cancer.gov/), and GSE30784 and GSE39366 were from the NCBI gene expression omnibus (https://www.ncbi.nlm.nih.gov/geo/). From GDC, we also downloaded the protein expression, somatic mutation, and somatic copy number alteration (CNA) profiles and clinical data in TCGA-CESC. We took TCGA-CESC as the discovery set and performed main analyses in this dataset. The other two datasets were validation sets. These datasets contained only data of human mRNA expression, but not data of the expression of infecting HPV variants. The data on the mRNA expression of HPV alpha-7 and alpha-9 clades were not available in any of these datasets. A summary of these datasets is shown in We downloaded three datasets of gene expression profiles in cervical cancer, including TCGA-CESC , GSE2957We first identified 50 genes having the largest expression variations across the HPV+ cervical cancers in TCGA-CESC. Based on the expression profiles of the 50 genes, we performed the hierarchical clustering of the HPV+ cervical cancers in the three datasets, respectively.We quantified the enrichment level of an immune signature or phenotypic feature in a tumor sample by the single-sample gene-set enrichment analysis (ssGSEA) of its marker gene set . The ssGt test with a threshold of adjusted P value < 0.05 and fold change of their mean expression levels > 2. By inputting the differentially expressed genes into the GSEA web tool    to identWe used the ESTIMATE algorithm  to calcuWe used Kaplan–Meier curves to display the survival time difference between cervical cancer subtypes, whose significance was evaluated by the log-rank test. The function “survfit” in the R package “survival” was utilized to perform this analysis. In addition, we performed multivariate survival analysis to investigate the correlation between cervical cancer subtypes and survival prognosis after adjusting for confounding variables, including immune score, stemness score, and ITH score. All these variables are continuous variables. The Cox proportional hazards model was utilized to implement the multivariate survival analysis with the function “coxph” in the R package “survival”.The TMB was defined as the total number of somatic mutations in the tumor. We used GISTIC2  to calcuhttps://github.com/WangX-Lab/PreHPVcc).We utilized the Random Forest (RF) algorithm  to prediU test or Kruskal–Wallis (K–W) test for not normally distributed data  and Student’s t test or one-way analysis of variance (ANOVA) test for normally distributed data. We utilized the chi-square test to analyze contingency tables. We used the Benjamini-Hochberg method .In class comparisons, we used the Mann–Whitney g method  to adjusP = 0.01)  (P < 0.001) (P < 0.001) (P = 0.002) (P = 0.005) (P = 0.005)   to calcuP = 0.007; hazard ratio (HR) = 3.76 and its 95% confidence interval (CI)   (Genomic instability often leads to increased TMB and CNAs . We foun= 0.057) . We obta= 0.057) . We founU test, P = 0.011)  . StrikinWe compared gene expression profiles between HPV+G1 and HPV+G2 and identified significantly upregulated genes in both subtypes. Based on these upregulated genes, we identified KEGG pathways highly enriched in HPV+G1 and HPV+G2, respectively, using the GSEA web tool . Many ofWGCNA  identifit test, P < 0.05). We found 22 proteins having significantly higher expression levels in HPV+G1, including NDRG1_pT346, Notch1, EGFR, Annexin-1, CD49b, PI3K-p85, EGFR_pY1068, Caveolin-1, YB-1_pS102, Src_pY416, Bad_pS112, MEK1, Myosin-IIa_pS1943, PAI-1, YAP_pS127, YAP, Bcl-2, Fibronectin, MYH11, GSK3_pS9, Syk, and VHL  > 1.5) (P = 0.04) . Furthermore, we did not observe significantly different OS or DFS rate between the HPV+ subtypes and HPV- .Both ITH and stemness scores followed the pattern: HPV+G1 < HPV+G2 < HPV-, while global methylation levels were higher in HPV+G2 than in both HPV+G1 and HPV-  sensitivity, specificity, and AUC in TCGA-CESC were 99.1%, 95.0%, and 100.0%, respectively. The prediction sensitivity, specificity, and AUC in GSE29570 were 100%, 80.0%, and 90.0%, respectively, and in GSE39001 were 92.0%, 100%, and 96.0%, respectively Figure 5P < 0.001) (P < 0.001) (P = 0.002) (P = 0.002) (P < 0.001). This result supports that HPV-18 infection is an adverse prognostic factor in cervical cancer  Figure 6< 0.001) . The thr= 0.002) . RPPA-ba= 0.002) . We founl cancer , while Hl cancer .Based on the expression profiles of the 50 genes with the largest expression variations across the HPV+ cervical cancers in TCGA-CESC, we identified two subtypes of HPV+ cervical cancers, namely HPV+G1 and HPV+G2. We demonstrated that this classification method was reproducible in two validation sets. Compared to HPV+G2, HPV+G1 displayed significantly higher immune infiltration level and stromal content, lower tumor purity, lower stemness scores and ITH scores, higher level of genomic instability, lower DNA methylation level, as well as more favorable prognosis. The multivariate survival analysis suggests that the survival difference between both subtypes is independent of confounding variables, such as immune signature, stemness, and ITH. It is interesting to observe that HPV+G1 has a better DFS prognosis than HPV+G2, while HPV+G1 is more genomically instable compared to HPV+G2. Genomic instability is a common characteristic of cancers that drives cancer development . NeverthKRT1, KRT4, KRT13, and KRT14. It supports previous findings that keratin expression plays a role in cervical cancer classification . The 47 genes included 34 genes which were more highly expressed in HPV-16 positive HPV+G1 versus HPV-18 positive HPV+G2 tumors and were also more highly expressed in HPV+G1 versus HPV+G2 tumors. In contrast, the other 13 genes were more highly expressed in HPV-18 positive HPV+G2 versus HPV-16 positive HPV+G1 tumors and were also more highly expressed in HPV+G2 versus HPV+G1 tumors. These data suggest that the HPV genotype could exert a significant effect on the expression pattern of most of the 5o genes since the HPV-18 genotype has a significantly different distribution between HPV+G1 and HPV+G2. In fact, previous studies have shown that HPV infection is able to cause global gene expression changes at the precancerous and cancerous stages of cervical cancer  and the NCBI gene expression omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE30784 and GSE39366.Publicly available datasets were analyzed in this study. This data can be found here: TCGA-CESC .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Previously, human papillomaviruses were best known for causing diseases in the genital tract, where high-risk types may cause, e.g., cancer of the cervix uteri, while low risk types could cause condylomas. Recently, more efforts have also been made to disclose their effects in the head and neck region. This Special Issue on “HPV in the head and neck region” includes, one editorial, eight reviews, one brief report and two systematic reviews that contribute to the overall knowledge of HPV in the head and neck region ,10,11,12The editorial describes focal epithelial hyperplasia (FEH) or Heck’s, which is a rare benign condition, primarily associated with HPV13 or 32 or both, usually presented as multiple asymptomatic whiteish to mucosal-colored, soft, papular or nodular lesions in the oral cavity, needing treatment if inconveniently growing [One review covers the issue of HPV vaccination of boys and young men, a vaccine previously mainly considered for females, but, as indicated here, an issue important also for males . The hurA second review covers HPV-associated benign squamous cell papillomas (SCP) in the upper aero-digestive tract, their malignant potential and the varying association to HPV infection depending on sites . A revieA separate review and systematic literature search dealt with the effects of HPV vaccines on oral and oropharyngeal HPV infections and reported a decrease in HPV vaccine-type infections in HPV vaccinated study participants .Another review topic was HPV and squamous cell carcinoma of unknown primary in the head and neck region, and its clinical implications, concluding that the detection of HPV in cytological specimens was indicative of a primary oropharyngeal squamous cell carcinoma (OPSCC) . AdvanceINK4A but also HPV DNA positivity is emphasized. In addition, some drawbacks of the 8th TNM-classification of Malignant tumors system are discussed, and the importance of prophylactic HPV vaccination is underlined. Along a similar line with regard to improved treatment is a review on the attempts to identify additional prognostic and driver genes in HPV positive tonsillar and base of tongue cancer in order to better personalize patient treatment [Striving towards improved treatment options of OPSCC in the era of check point inhibitors was also covered and this review suggests that indeed new options for the future will fuel the next evolution of treatment for OPSCC . Additioreatment .A brief report, with a systematic review on HPV-related multiphenotypic sinonsasal carcinoma (HMSC), a new emerging tumor entity according to the World Health Organization (WHO) was also presented . The autA systematic review on the accuracy of HPV detection in OPSCC was also included . An overFinally, a systematic review on the HPV-positivity in OPSCC worldwide presented variations in HPV occurrences with tendencies for the highest occurrences in Northern Europe, the USA, Lebanon, China, and South Korea .With this, we would like to thank all authors for their valuable contributions to this Special Issue. It has been extremely interesting and rewarding to learn more of this relatively new field. Together, we hope we will continue our endeavors to increase the knowledge in the field in order to both better prevent disease and individualize patient treatment.


Due to lack of statistical power, the 40% relative difference in HR-HPV carriage between treated and untreated women was not significant. In women living with HIV, the treatment of a cervical lesion may be beneficial for clearing HR-HPV infections.Even when receiving combination antiretroviral therapy, women living with HIV are at high risk of human papillomavirus (HPV) infection and/or cervical lesions, including cancer. Using data from the PapilloV prospective cohort, we evaluated the prevalence of high-risk HPV (HR-HPV) infections after cervical lesion treatment and investigated factors associated with their carriage. Women were followed up for three years with annual Pap smear and HPV genotyping. We offered treatment to women presenting either a Pap smear with high-grade squamous intraepithelial lesion or higher, and/or a biopsy showing cervical intraepithelial neoplasia II or III. We compared the prevalence of HR-HPV infection at the time of first treatment indication and at the end of follow-up among women who received treatment and those who did not. Overall, 46 women had treatment indication. HR-HPV prevalence significantly decreased from 67% to 27% ( Women living with HIV are at increased risk of presenting with a human papillomavirus (HPV) infection and/or cervical lesions, including cancer, even when receiving combination antiretroviral therapy ,2,3. In Thailand, among women living with HIV, the prevalence of cervical infection ranges from 25% to 62% for all types of HPV, and from 19% to 43% for high-risk HPV (HR-HPV) ,8.In a study evaluating the effect of cervical lesion treatment on HR-HPV carriage, women who had undergone punch biopsies had faster clearance of HR-HPV infections compared to the natural course of infection . After tAmong women living with HIV with a treatment indication for cervical lesion, we evaluated the impact of treatment on HPV carriage, including HR-HPV, between the time of cervical lesion detection and the end of follow-up, by comparing women who received treatment to those who did not. We also investigated potential factors associated with persistent carriage of HR-HPV.We used data from the PapilloV cohort study, in which 829 women living with HIV in Thailand were screened annually for HPV infection and cervical lesions over three years. The PapilloV study was a prospective, multicenter cohort of women living with HIV, receiving antiretroviral therapy, nested in a larger HIV cohort in Thailand . Women wIn short, women were followed up for three years. Annual visits comprised a gynecological exam, including a Pap smear and HPV testing with genotyping. Colposcopy was performed if an abnormal Pap smear showed atypical squamous cells of undetermined significance (ASC-US) or higher-grade lesions and/or presence of either HR-HPV or potential HR-HPV (pHR-HPV) infections. Biopsy was performed in case of visible lesion at colposcopy.Cervical lesion treatment was indicated in case of a Pap smear showing high-grade squamous intraepithelial lesion (HSIL) or higher and/or in case of a biopsy showing cervical intraepithelial neoplasia (CIN) II or III. Women underwent treatment as per physician decision. The study investigation team had no influence on whether treatment was offered or not to the study participants. HPV genotyping of cervical specimens was conducted using HPV-PapilloCheck . Genotypes considered high-risk HPV were the following: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, while HPV 6, 11, 40, 42, 43, and 44/55 were considered as low-risk HPV genotypes. Due to the limited evidence regarding their potential carcinogenicity, the following genotypes  were classified as potentially high-risk genotypes (pHR-HPV) . Among the 46 women included in the analysis, 14 (30%) had an HSIL on Pap smear, 12 (26%) had a CIN2, and 20 (43%) a CIN3 on the biopsy at the time of treatment indication. Thirty women received cervical treatment while 16 did not. Out of 30 women treated, 24 were treated once: among them, 22 experienced Loop Electrosurgical Excision Procedure (LEEP) and one participant a cold knife conization, while information on treatment type was missing for one participant. Among the six women who received two successive treatments: two had benefited from LEEP twice, while information was incomplete for four women. Apart from Pap smear and biopsy results, treated and untreated women had similar characteristics at the time of treatment indication .At the time of treatment indication, overall, 63% (29/46) of women had at least one HR-HPV infection. The most frequent HR-HPV genotypes were HPV 16 in 10 women (22%) and HPV 52 in seven women (15%), while only three women (7%) had an HPV-18 infection .95%, 48% to 82%) at the time of treatment indication and 27%  at the end of follow-up, a 60% relative decrease of HR-HPV infection prevalence compared to the baseline   at baseline versus 38%  at the end of follow-up, a 32% relative decrease, which was not statistically significant   . Among w= 0.257) .95, 0.2 to 2.2), p value = 0.449).There was no significant association between prevalence of HR-HPV infection by the end of the follow-up and the fact that women had received treatment  Table A1p value < 0.001).When adjusted for having received treatment, none of the women’s characteristics at the time of indication  were associated with HR-HPV carriage at the end of follow-up Table A3We found a significant decrease in HR-HPV carriage among women living with HIV who had been treated for a cervical lesion, while it was not significant among women who had not received treatment. However, the relative difference in decrease of HR-HPV infection prevalence was not significant between the two groups.Interestingly, after adjusting for treatment, there was no significant association between women’s HIV characteristics and the presence of an HR-HPV infection at the end of the follow-up. However, the persistence of abnormal cytology findings (ASC-US+) was correlated with HR-HPV carriage by the end of the follow-up.Some studies have assessed how treatment of cervical lesions might influence HPV clearance, but few have focused on women well-controlled for HIV infection. Also, it should be noted that HPV infections observed at the end of follow-up could be related to persistent infection, but also to new or recurrent infections. It was not possible to differentiate between them and this measurement bias cannot be addressed.3. This may have mitigated the mechanical effect of cervical treatment.In 1997, Bollen et al. showed that HPV prevalence decreased from 98% to 31% when comparing samples before and after cervical treatment . In thatIn a study in Nicaragua including 67 HIV-naïve women receiving LEEP for CIN2+ cervical lesions, 93% had at least one HR-HPV infection with genotypes 16, 31, 52, or 58 before treatment. Two years after treatment, the prevalence of HR-HPV infection was 8.4%, while it was 7.5% for CIN2+ cervical lesions . In our In a large study in China among HR-HPV infected women (HIV uninfected) without high-grade cervical lesions, viral clearance was estimated at 54.3% over a mean duration of 14.5 months, with higher clearance among women aged under 50 . EvidencRegardless of treatment, Travassos et al. showed a 46% clearance HR-HPV carriage after one year, among 62 women living with HIV and receiving antiretroviral therapy for more than a year . This isOur analysis is limited by a lack of statistical power, estimated post hoc at 16%. However, we did find that HR-HPV carriage decreased significantly over time among women who had received treatment.Treatment of cervical lesions in women living with HIV under antiretroviral therapy may reduce the carriage of HR-HPV infections. Larger controlled cohort studies or meta-analyses are needed to assess which factors might be linked to a reduction of HR-HPV carriage among women with cervical lesions. In this population, nonavalent or new L2-based vaccination programs could be considered for preventing HPV-induced cervical cancer.


Prostate cancer affects a significant proportion of men worldwide. Evidence from genetic and clinical studies suggests that there may be a causal association between prostate cancer and the human papilloma virus (HPV). As HPV is a vaccine-preventable pathogen, the possibility of a role in prostate cancer causation may reinforce the importance of effective HPV vaccination campaigns. This is of particular relevance in light of the COVID-19 pandemic, which may have considerable effects on HPV vaccine uptake and distribution. Prostate cancer is the commonest solid-organ malignancy in men worldwide and represents a significant healthcare burden. Although the feasibility of modifying recognised risk factors for prostate cancer is currently low, there is evidence to suggest that the vaccine-preventable pathogen, Human Papilloma Virus (HPV) may be associated with the pathogenesis of prostate cancer . HPV is The potential role of HPV in the causation of prostate cancer has been the subject of considerable debate and the focus of several recent systematic reviews Table . HPV is APOBEC3B, a gene which has been found to have a protective role against the effects of viruses [However, at the genetic level, there appears to be variation in the nature of HPV infection between benign and cancerous tumours. A study by Glenn et al.  demonstr viruses . Mutant  viruses . In addi viruses .Due to the possible role of HPV in prostate cancer causation, it may be argued that it is increasingly important to intensify current HPV vaccination strategies, with a sustained gender-neutral approach. This may be of greater relevance than ever before, as the COVID-19 pandemic has posed a significant challenge to HPV vaccination campaigns. Unfortunately, short-term measures to mitigate the spread of COVID-19, such as school closures and limited clinic visits, have led to a fall in HPV vaccination rates . In the Despite the incongruity of evidence surrounding HPV exposure and prostate cancer causation, extant evidence suggests an association worthy of further investigation. The significance of a confirmed role of HPV in the development of prostate cancer is weighty, and would strengthen calls for current generic vaccination programmes , ultimat


Objectives: The present study aimed to identify the predictive value of inflammatory indexes stratified according to human papillomavirus (HPV) infection status in women with FIGO 2018 stage IB∼IIA cervical cancer. We also explored the influences of HPV infection status on the survival of cervical cancer patients.Methods: We collected data for 583 women with stage IB∼IIA cervical cancer in Sun Yat-sen University Cancer Center between 2009 and 2017. The t-test, chi-squared (χ2) test and Fisher’s exact test were applied to compare the differences of inflammatory indexes and clinicopathological features between HPV-positive and HPV-negative groups. Univariate and multivariate analyses were used to identify clinicopathological factors that were associated with the prognosis of cervical cancer patients.Results: There were no differences in overall survival (OS) and progression-free survival (PFS) between HPV-positive and HPV-negative groups. In HPV-positive group, the maximum tumor size, neoadjuvant chemotherapy and the body mass index (BMI) correlated significantly with C-reactive protein/albumin ratio (CAR). The maximum tumor size and the prognostic nutritional index (PNI) correlated significantly with the platelet-lymphocyte ratio (PLR). The maximum tumor size, neoadjuvant chemotherapy and PLR correlated significantly with PNI. Univariate and multivariate analyses showed that the depth of tumor invasion  and CAR  were independent predictors of poor OS and PFS. PNI was an independent protective factor of OS . PLR was an independent factor of PFS . In HPV-negative group, BMI correlated significantly with CAR. Only depth of invasion  was the independent predictor of poor OS, and no inflammation indexes were independent predictors of prognosis.Conclusion: In patients with HPV-positive cervical cancer, depth of invasion, PNI and CAR are independent factors of OS, and depth of invasion, PLR and CAR are independent factors for PFS. For patients with HPV-negative disease, no inflammation indexes had predictive value for prognosis. The predictive value of inflammation indexes on prognosis is more significant in patients with HPV-positive cervical cancer. Stratification of HPV infection status promotes a more precise clinical application of inflammation indexes, thus improving their accuracy and feasibility. Globally, uterine cervical cancer (UCC) still ranks fourth for both incidence and mortality among cancers, with about 570,000 new cases and 311,000 deaths each year . With thIndexes of a systemic inflammatory response (SIR), such as the C-reactive protein (CRP)-albumin (ALB) ratio (CAR), the platelet-lymphocyte ratio (PLR), the prognostic nutritional index (PNI), the neutrophil-lymphocyte ratio (NLR), and the lymphocyte-monocyte ratio (LMR), have been used not only to determine the nutritional status and complications but also to predict prognosis of patients in various cancers –11. As aAlthough several studies on survival and recurrence outcomes of patients with cervical cancer in various stages have been published, to date, there is no report concerning the impact of HPV infection status on the inflammatory indexes and the prediction of prognosis in patients with cervical cancer. In this study, we firstly divided the patients into HPV-positive group and HPV-negative group, and compared the clinical parameters and prognosis between two groups. We analyzed retrospectively the predictive value of preoperative inflammation markers  on the prognosis of patients with FIGO 2018 stage IB∼IIA cervical cancer with different HPV infection statuses.www.researchdata.org.cn), with the approval RDD number as RDDA2021001939.This retrospective study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Sun Yat-sen University Cancer Center Institutional Review Board (IRB-number B2020-345-01). Informed patient consent was waived in this observational and noninterventional study, in which the patient’s data were kept under strict control. The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform , survival time, and preoperative routine blood examination results  were collected retrospectively from the medical records. In addition, we also collected the postoperative adjuvant treatment including radiotherapy and chemotherapy.Venous blood was drawn from all patients within 1 week before surgery. The CAR, PLR, PNI and body mass index (BMI) were calculated using the following equations:currence .Receiver operating characteristic (ROC) curves were generated for pre-treatment CAR, PLR, and PNI to determine the cut-off values that yielded optimal sensitivity and specificity. The upper right point of the ROC curve was selected, and the Youden index was calculated according to the sensitivity and specificity of each possible point in the statistical results. Then, the largest point was selected to determine the optimal cut-off value of the hematological index, such that the numerical variable could be transformed into a classified variable to analyze the statistical data.Every patient accepted high-risk type HPV test before surgery. Cervical cells sample was collected by the Digene Hybrid Capture 2 (HC2) HPV DNA Collection Device , which consisted of a cervical brush and a digene specimen transport medium. The DNA copy number of the high-risk type HPV was calculated. If the copy number was 0–1 pg/ml, it indicated that there was no HPV infection. When the copy number was greater than 1 pg/ml, it indicated a positive HPV infection.2) and paclitaxel (175 mg/m2), every 3 weeks before surgery.Preoperative neoadjuvant chemotherapy was administered to inhibit some micrometastasis and distant metastasis and to improve the radical curability and safety of surgery. Patients whose tumor could not be removed completely in the beginning were given 2 cycles of cisplatin , and for squamous cell carcinoma patients with two of three risk factors , postoperative radiotherapy was required. Among other patients, those with poorly differentiated grade and tumor size more than 2 cm, or highly maligant pathological type such as clear cell adenocarcinoma, neuroendocrine carcinoma, were given chemotherapy without radiotherapy. All other patients following radical hysterectomy did not need any adjuvant therapy , 26.After the initial treatment, follow-up assessments were performed once every 3 months for the first 2 years, once every 6 months for the next 3 years, and then once every year after 5 years. The main examinations included routine blood tests, tumor markers measurement, gynecological examinations, chest radiography, and computed tomography (CT) or magnetic resonance imaging (MRI) scans. In cases with abnormal findings and suspected tumor recurrence, lesion biopsy was performed to determine whether recurrence had occurred. As study endpoints, OS was defined as duration from the date of initial surgery to the date of death for any causes or the last follow-up , and PFS was defined as the time interval from the date of surgery to progression or relapse.t-test, Pearson’s chi-squared (χ2) test and Fisher’s exact test. Comparison analyses of clinicopathological variables between high- and low-inflammatory index groups were performed as well.Comparison analyses of inflammatory indexes and clinicopathological variables between HPV-positive and HPV-negative groups were conducted using p < 0.05 indicated that the difference was considered statistically significant.The survival curves were plotted using the Kaplan–Meier method, and log-rank tests were carried out to assess survival differences between groups. Significant prognostic variables in the univariate analysis were selected for the multivariate Cox Regression model analysis to identify independent prognostic variables using the forward stepwise method. Analyses were performed using SPSS 23.0 software  and GraphPad Prism 8 software . A two sided 2 = 35.962, p < 0.0001). The proportions of other characteristics were relatively balanced. During the follow-up period, a total of 42 patients experienced recurrence or progression, and 31 of them died.In total, 583 patients with stage IB∼IIA cervical cancer were recruited in this study, of which 501 (85.93%) cases were HPV-positive and 82 (14.07%) cases were HPV-negative. The associations of characteristics between two groups were shown in In this study, 109 HPV-positive cases and 24 HPV-negative cases received neoadjuvant chemotherapy before surgery. Patients with risk factors were required to accept radiotherapy or chemotherapy after surgery. In HPV-positive group, there were 151 cases without postoperative adjuvant therapy, 96 cases with chemotherapy, 111 cases with radiotherapy and 143 cases with chemotherapy and radiotherapy. In HPV-negative group, there were 28 cases without postoperative adjuvant therapy, 17 cases with chemotherapy, 12 cases with radiotherapy and 25 cases with chemotherapy and radiotherapy. We compared the prognosis of different subgroups and found that there was no influence on prognosis of cervical cancer patients no matter what kind of postoperative adjuvant therapy they received .The cut-off values of CAR, PNI, and PLR were determined using ROC curves . The anahi and CARlo, PLRhi and PLRlo, PNIhi and PNIlo groups were analyzed using t-test, chi-squared (χ2) test and Fisher’s exact test in HPV-positive group and HPV-negative group, respectively.Patients with different HPV infection statuses were divided into two groups according to the cut-off values of CAR, PNI, or PLR. The associations of clinical characteristics between CAR2 = 8.360, p = 0.004), higher receipt rate of neoadjuvant chemotherapy  and nonstandard BMI   and lower PNI  , lower receipt rate of neoadjuvant chemotherapy  and lower PLR   correlated significantly with larger tumor size (χ 0.0001) . A highe 0.0001) . While a 0.0001) .2 = 6.786, p = 0.009)  , and theo groups  as well o groups .hi , PLRhi  had worse OS and PFS  had favorable OS and PFS (p > 0.05)  .p = 0.005), the levels of PNI  and CAR  were independent factors for OS (p = 0.012), the levels of PLR  and CAR  were independent predictors of PFS (p = 0.048) was an independent prognostic factor of OS (For HPV-positive group, the multivariate analysis showed that the depth of invasion (HR: 3.651, 95% CI: 1.464–9.103, s for OS . In addis of PFS . For theor of OS , while nor of OS .All the above data suggested that for patients with HPV-positive FIGO stage IB∼IIA cervical cancer, the depth of invasion, the levels of PNI and CAR are independent predictors of OS, and the depth of invasion, the levels of PLR and CAR are independent predictors of poor PFS. For patients with HPV-negative cervical cancer, none of CAR, PNI and PLR is independent factor of prognosis.The immune system, which resists pathogenic microorganisms, repairs damage, and maintains stability of internal environment, mainly relies on normal autoimmunity reactions . Some paPrevious studies have indicated that inflammation is one of the factors influencing the prognosis of patients with cancer , 37. OurIn the present study, we proposed to evaluate the prognostic value of inflammatory indexes based on the patients’ HPV infection status. The same results were not observed in both HPV-positive and HPV-negative groups. Before our analyses, there were many studies that explored the relationships between inflammatory indexes and prognosis in cervical cancer, but none of them paid attention to the HPV infection status of the patients , 48. TheThere are some limitations of this study. First, this study is retrospective. Therefore, there might have been selection bias and analysis bias. However, a large sample size guarantees the sufficient statistical power and reduces the risk of bias in HPV-positive group. In this study, one of the weaknesses is the low number of HPV-negative cases. It is vital to expand the number of HPV-negative group to avoid bias in our future studies. Second, in the present study, there were differences in the pathological types between the HPV-positive and negative groups. Thus, to avoid these problems, it is crucial to carry out prospective researches.In summary, we found that in patients with HPV-positive cervical cancer, the depth of invasion, the levels of CAR and PNI are independent predictors of OS, and the depth of invasion, the levels of PLR and CAR are independent predictors of PFS. In HPV-negative cases, three inflammation indexes had no prognostic value. Interestingly, when the analysis was limited to patients with negative lymph node metastasis, CAR, PLR and PNI still had significant prognostic value. Our research verified that clarifying the HPV infection status will be more accurate for the application of preoperative inflammatory parameters in the prognosis of cervical cancer.


Further, high levels of CD8+ T-cells in TC are associated with a favorable prognosis. Increased understanding of the tumor immune microenvironment is needed to identify prognostic biomarkers and novel treatment targets. In this study, we assess the immune profiles of fresh TC biopsies and contralateral healthy tissue against a detailed HPV analysis, including DNA quantification and RNA expression analysis. We demonstrate a correlation between levels of CD8+ T-cells and antigen-presenting cells in TC, and we further present differentially expressed biomarkers and signaling pathways in CD8HIGH vs. CD8LOW HPV+ TC. Our study thus describes immune phenotypes and the heterogeneity of the immune compartment in HPV+ TC. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions.Human papilloma virus (HPV) infection is the leading cause of tonsillar cancer (TC), and HPV+ TC has a favorable prognosis compared to HPV− disease. In this study, we examined aspects of the tumor microenvironment of TC, focusing on T-cells, dendritic cells (DC), and macrophages. Fresh biopsies of TC and the contralateral healthy tonsil (HT) were obtained from 20 patients, analyzed by multiparameter flow cytometry, and assessed against a detailed HPV-status. Additionally, RNA-sequencing data from 38 TC samples available in the public database, The Cancer Genome Atlas (TCGA), were explored, focusing on the same leukocyte populations. HPV+ TC featured increased levels of CD8+ T-cells and antigen-presenting cells . In HPV+ TC, CD8+ T-cell frequencies correlated to DC levels independently of tumor stage, HPV 16 copy number, and E7 oncogene expression as well as frequencies of other leukocytes. Similarly, RNA sequencing data were explored by dividing the HPV+ TCs according to predefined CD8+ T-cell scores in silico. Higher levels of genes expressed by antigen-presenting cells and effector T-cells, such as immune checkpoints and cytokines, were detected in the CD8HIGH HPV+ TC samples (cf. CD8LOW HPV+ TC). In conclusion, CD8HIGH HPV+ TC displays a unique inflammatory profile associated with increased effector T-cell functions and the presence of antigen-presenting cells in the tumor microenvironment. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions.Human papillomavirus (HPV) is the main causal agent of tonsillar cancer (TC) and HPV For example, patients with detectable levels of antibodies against HPV oncoproteins E6 and E7 in OPC have a more favorable prognosis than those without a humoral immune response [+ TC, as the tumor immune phenotypes may be used to improve clinical protocols, e.g., resulting in escalation or de-escalation of treatment.Tonsillar cancer (TC) is a subset of oropharyngeal cancer (OPC). In addition to tobacco smoking and alcohol abuse, high-risk human papillomavirus (HPV), notably HPV16, is a cause of the disease . Indeed,+ ,4. As foresponse . Such ob+ T-cells in TC, indicating an “inflamed” phenotype, predicted a favorable outcome [+ plasmacytoid DCs (pDC), CD1c− CD141− DCs, as well as CD1c+ and CD141+ conventional DCs (cDC), and indicated subset-specific immune features [+ DCs in HPV+ OPC associated with CD4+ T-cell abundance [+ macrophages as associated with HPV+ disease and proposed an association of CD68+ PD-L1+ cells and survival [A series of studies has addressed the immune composition and its heterogeneity in OPC/TC, including aspects of the main leukocyte populations involved in initiating and sustaining antigen-specific cell-mediated immunity, such as dendritic cells (DC), macrophages, and T-cells. As originally suggested by Galon et al. for colorectal cancer , Näsman  outcome ,8. Subse outcome . In a prfeatures . Moreovebundance . Oguejiosurvival .Taken together, while detailed and of great interest, the above-mentioned studies ,10,11,12+ TC lesions feature high and transcriptionally active HPV-DNA levels . Furthermore, we showed that CD8+ T-cell levels are increased in HPV+ TC (cf. HPV− TC as well as HT), and that CD8HIGH HPV+ TC features specific characteristics regarding immune cell and gene expression profiles (cf. CD8LOW HPV+ TC as well as HT). Moreover, we demonstrated that macrophage and DC levels are high in HPV+ TC (cf. HT) and that CD8+ T-cell levels correlate with DC abundance, but not to macrophage infiltration, CD4+ T-cell presence, or HPV16 E7 DNA CN or E7 mRNA levels.In this study, we examined the TME specifically of TC, focusing on the presence/status of DCs, macrophages, and T-cells assessed by multiparameter flow cytometry. Notably, we compared TME data between biopsies obtained from the TC lesion and the contralateral healthy tonsil (HT) of the same patient and assessed the results in relation to HPV status. In addition, publicly available RNA sequencing data of whole TC tissue were explored in silico. We demonstrated that HPV+ TC samples had levels of E7 DNA over one copy per cell and detectable levels of HPV16 E7 mRNA  (+ T-cells were significantly increased in HPV+ TC (cf. HPV− TC and HT)  B. Furthe and HT) C, while V status D. CD14+ DR+ gate A. Highered to HT E,F, indi HPV− HT .+ T-cell infiltration in HPV+ TC , whereas cDC frequency was not (p = 0.0732) , and pDCs were also significantly more prevalent in the CD8HIGH group (cf. CD8LOW TC)  . In orde8LOW TC) C,D. Simiificance E.+ and HPV− TC. Highly expressed genes in HPV+ TC showed enrichment in T-cell–cell activation and differentiation (cf. HPV− TC), e.g., “T-cell receptor activation’’ as well as “Th1-, Th2-, and Th17-differentiation’’ (− TC (cf. HPV+ TC) corresponded to intracellular signaling and reassembling of the cellular matrix, e.g., “PI3K-Akt signaling pathway’’, “Focal adhesion’’, and “Extracellular matrix receptor interaction’’  and CD8LOW (n = 9) groups . Furthermore, the CD8HIGH HPV+ TC group presented a significantly lower profile score for lymphocyte depletion (cf. CD8LOW HPV+ and HPV− TC) (The CD8BERSORTX  were use) groups A. The trpipeline , with siHPV− TC) B.HIGH HPV+ and CD8LOW HPV+ TC samples revealed 1008 DEGs. Moreover, pathway enrichment analysis revealed a clear profile of immune activation with higher expression of genes in the CD8HIGH HPV+ group involved in pathways such as “Signaling by interleukins’’, “Cytokine signaling in immune system’’, “Interferon signaling’’, “Neutrophil degranulation’’, “Antigen processing-cross presentation’’, “MHC class II antigen presentation’’, “CTLA4 inhibitory signaling’’, and “PD-1 signaling’’ , APC functions , cytokines , cytokine receptors , immune checkpoints  and PDCDLG2 (PDL2)), innate immune response  and general leukocyte functions .The two-group comparison between CD8naling’’ C. On theuction”. D. Furtheproteins E. CD8HIG+ T-cells and DCs within the TME of HPV+ TC, irrespective of other immune populations and HPV status. We indicate that CD8HIGH HPV+ TC patients feature an increased expression of genes related to antigen presentation, T-cell effector profile, and immune checkpoints. Our observations may be relevant for the identification of treatment targets pertinent to TC, as well as for prognostic markers that can potentially permit individualized treatment.In this study, we investigate aspects of the TME of TC, focusing on T-cells, DCs, and macrophages. Fresh tumor material is analyzed, compared to the contralateral HT, and assessed against HPV status. Taken together, we reveal a significant correlation between CD8+ TC lesions as well as in contralateral HTs, the HPV-DNA load was much greater in the former. Furthermore, we observed that a combination of HPV16 E7 DNA and mRNA separated TC lesions (these features present) from HTs (absent). The presence of HPV beyond the cancer lesion has previously been highlighted [HPV is a distinctive factor associated with a good clinical outcome for TC, arguably because it facilitates an antigen-specific inflammatory response. In this study, we observed that while HPV-DNA was present in HPVhlighted , but the+ T-cell infiltration is a favorable prognostic factor in TC [HIGH HPV+ cf. HPV− TC lesions . In this study, data from flow cytometric and transcriptomic analyses support HPV+ TC as predominantly rich in CD8+ T-cells, suggesting an “inflamed” or “immune excluded” rather than a “desert” phenotype [+ TCs displayed a wide spectrum of CD8+ T-cell levels. Further studies are required to investigate the aspects associated with the heterogeneous infiltration of CD8+ T-cells in HPV+ TC, potentially to identify patients who pose a particular therapeutic challenge.In addition to HPV status and activity, a high degree of intralesional CD8or in TC ,8. Using lesions . A trendhenotype . However+ T-cells in HPV+ TC patients. CD8HIGH HPV+ TC patients feature high levels of DCs and macrophages, indicative of an active recruitment of APCs into the TME. We also demonstrate that the frequency of CD8+ T-cells correlates predominantly with DC abundance, specifically with pDCs, rather than with macrophage infiltration, CD4+ T-cell presence, or aspects of HPV-status. Interestingly, increased levels of pDCs were seen in the CD8HIGH HPV+ TC group (cf. CD8LOW HPV+ TC and HT). The precise role of pDCs in tumor immunity is not clear; however, a few studies have correlated the presence of pDCs within the TME with poor prognosis [HIGH HPV+ TC patients might be associated with potential increases in type I IFN activity.This study characterizes changes in the TME composition that underlie the heterogeneous distribution of CD8rognosis ,23. The rognosis . Additiorognosis . TherefoHIGH HPV+ TC (cf. CD8LOW HPV+ TC and HT), which, however, did not reach statistical significance. Even though the role of CD141+ DCs is key to initiate CD8+ T-cell responses through cross-presentation of antigen, their contribution to CD8+ T-cell frequency is less evident, especially considering their low abundance [+ DCs contribute to cytokine production via secretion of IL-12 and IL-10, which displays their ability to trigger inflammation or tolerance in a TME-dependent manner [+ T-cell levels both through priming and subsequent clonal expansion of T-cells at HPV-antigen exposure and through secretion of chemokines inducing the recruitment of CD8+ T-cells from the surrounding lymphoid tissue. Taken together, our data indicate that CD8+ T-cells levels correlate with overall DC abundance, reflecting the complementary functions of DC subsets.We observed an increasing trend of cDC subsets in CD8bundance . In contt manner . Intratu+ T-cell infiltration, whole tissue RNAseq datasets of HPV+ TCs from the TCGA cohort were compared according to CD8 scores. Patients with high CD8 scores displayed an increase in genes involved in antigen uptake, C-type lectins and Toll-like receptors, and antigen degradation/loading on MHC molecules . Together, these observations are indicative of active antigen uptake and processing in the TME of HPV+ TC with a high level of CD8+ T-cells. Additionally, we observed genes involved in antigen presentation (HLA type II molecules), co-stimulation (CD86), and cytokines . Remarkably, among the genes related to antigen presentation, we observed canonical CD1c+ DC marker Clec10a, as well as CD163 and CD14 markers, shared between the newly characterized CD163+ DC subset and macrophages [HIGH HPV+ TC. However, whole tissue RNAseq cannot delineate the activity of single cells, and a more detailed analysis of myeloid APC is required to elucidate their specific role.In order to assess transcriptomic changes in the TME associated with CD8rophages ,25. ThesLOW HPV+ and HPV− TC display lymphocyte depletion. In HPV− TC, lymphocyte depletion can be associated with wound healing response and extracellular matrix deposition, leading to immune-exclusion [LOW HPV+ TC may represent defective lymphocyte chemotaxis into the tumor. Accordingly, the DEGs upregulated in CD8HIGH HPV+ TC (cf. CD8LOW HPV+ TC) suggest that intra-tumoral leukocyte chemotaxis encompasses three axes: CCL3/5-CCR5, CXCL9/11-CXCR3, and XCL2. These chemotactic pathways have been reported in the context of APC and T-cell cross-talk [+ in response to viral particles, stimulates CXCL9/11 expression by a variety of stromal cells, which induce migration of CXCR3+ pDCs and T-cells [+ TC [HIGH and CD8LOW HPV+ TC was not pursued in this study, which is a limitation. Further analyses of, e.g., quantification of identified secreted and cell surface antigens, and identification of the cell types producing these, are thus warranted to fully decipher the TME dynamics in TC.Immunophenotype analysis by signature scores highlights that CD8xclusion . Conversoss-talk ,28,29,30 T-cells . In factls [+ TC . ValidatHIGH HPV+ TC subset (cf. CD8LOW HPV+ TC) included PD1, PD-L2, TIGIT, LAG3, CTLA4, and HAVCR2/TIM3, suggesting an exhausted profile [+ T-cells could benefit from immune-checkpoint therapy [HIGH HPV+ subset, e.g., CCL5, CXCL9/13, CXCR6, and HLA-DRA, which contribute to a pan-cancer signature suggested to predict an optimum response to anti-PD1 immune checkpoint-therapy [+ T-cells, may benefit from strategies aiming to increase intra-tumoral antigen presentation. Targeted antigen delivery in vivo, administration of Flt3-LG, or DC vaccination comprise some examples of immunotherapies pursuing increased antigen presentation [Constant exposure to HPV viral antigens is reported to contribute to T-cell exhaustion by inducing upregulation of immune checkpoint molecules, thus impairing T-cell activity . In our  profile . In this profile . However therapy . Accordi-therapy . In contentation . ArguablThe study was approved by the Swedish Ethical Review Authority (ref. no. 2017/580), and written informed consent was obtained from all participating patients. Paired biopsies from TC lesions and contralateral HTs were obtained from 20 patients at the time of diagnosis . The bioEach sample was transferred to a 500 µL solution of 4 M guanidinium thiocyanate, 22 mM sodium citrate, 5% sarcosyl, and 1% mercaptoethanol  and incubated at room temperature overnight. Extraction of nucleic acids was performed with a Total NA-kit using MagNA Pure LC , while mRNA extraction was performed using an Oligotex Direct mRNA Mini Kit . The mRNA was eluted by adding 45 µL of Oligotex elution buffer (70 °C) to the column, followed by 1 min centrifugation at max speed. Purified DNA and RNA were stored at −20 °C and −80 °C, respectively.Identification of 40 HPV types was carried out by a modified general primer PCR, amplifying a segment of the HPV L1 gene, followed by Luminex analysis (Thermo Fisher Scientific) ,39. Vira+ T-cells were divided into CD8+ and CD4+ T-cells, and antigen-presenting cells (APC) were identified as CD3− Lin − HLA-DR+ cells and further subdivided into CD14− DCs and CD14+ macrophages. cDCs and pDCs were identified as CD11c+ CD13+ and CD11c− CD13−, respectively. pDC identity was confirmed by including CD123 in subsequent staining, with 90% overlap between CD11c− CD13− and CD123+ CD11c− CD13− populations (data not shown). cDCs were subclassified as CD141+ CD1c+, CD141− CD1c+, and CD141− CD1c, accounting for the total number of cDCs. The frequency of an individual population was calculated in relation to the tissue infiltrating CD45+ leukocytes using FlowJo software. Only samples with more than 100 events included in the DC gates were considered for DC subpopulations analysis. Statistical analysis and plotting were conducted in GraphPad Prism 9.0 . Differences in mean percentages of leukocyte populations were assessed by either unpaired Welch’s t-test or Mann–Whitney test, after evaluating normality using the Shapiro–Wilk test. p-values < 0.05 were considered significant. A correlation matrix was built using Pearson’s correlation coefficients to evaluate variables related to the frequency of CD8+ T-cells. Linear correlations were considered significant when r > 0.7 and p-values < 0.05. Further grouping of HPV16+ TC samples into CD8HIGH (3rd and 4th quadrants) and CD8LOW (1st and 2nd quadrants) was performed based on their frequency of CD8+ T-cells.The characterization and frequency assessment of immune cell populations was performed using a panel of commercially available antibodies . Briefly− samples and 31 HPV16+ samples in the present cohort. The RNA-Seq data were normalized using edgeR version 3.30.3 [Clinical and transcriptomic data for 38 TC samples were retrieved from the GDC portal repository of The Cancer Genome Atlas (TCGA) . The infn 3.30.3 ,44.+ and HPV− TC were identified by two-tailed t-test  using Qlucore Omics Explorer 3.6 . The obtained DEGs were enriched for KEGG pathways in Stringdb, available on Cytoscape [Differentially expressed genes (DEGs) between HPVytoscape .+ TC group was further divided into CD8HIGH and CD8LOW subgroups using two previously defined CD8 T-cell signatures [p-value < 0.05 were further considered for analysis (n = 27). To avoid bias from one signature, only those samples overlapping in both signatures were termed as CD8HIGH  and CD8LOW . The remaining TC samples (2nd and 3rd quadrants) were removed from further analyses. Differences among the TC groups were further assessed using six pan-cancer immune subtype signatures [HIGH and CD8LOW HPV+ TC were estimated by two-group comparison using a Mann–Whitney test . DEGs obtained were then subjected to REACTOME pathway enrichment using Stringdb, available on Cytoscape [HIGH and CD8LOW HPV+ TC were further explored using Gene ontology (GO:0002376 and GO:0006955) and Ensembl annotations, accessed through the biomaRt package [HIGH and CD8LOW HPV+, as well HPV−, TC patient groups using Kaplan–Meier analysis and log-rank (Mantel–Cox) test.The HPVgnatures ,19. Aftegnatures . Samplesgnatures  and compytoscape . Bar plo package  (version package . Surviva+ T-cells levels in HPV+ TC cf. HPV− TC and HT. Furthermore, we showed that CD8+ T-cells correlated with DC frequency in HPV+ TC, and indicated genes associated with an inflamed profile in HPV+ TC with high predicted CD8+ T-cell infiltration. This subset of patients displayed an increase of antigen presentation, IFN-γ responses, immune-checkpoint regulation, and chemotactic activity-related genes. Further studies are warranted to assess if this information can be used to identify treatment targets and, on an individual basis, for prognostic information and treatment selection.In conclusion, we evaluated the immune profiles of fresh TC and contralateral HT samples and demonstrated an increase in CD8


Background: To explore the biological and clinical effects of titin-antisense RNA1 (TTN-AS1) in bladder cancer (BC) and the association between TTN-AS1 and activating transcription factor 2 (ATF2) in BC.Methods: The Kaplan–Meier method was performed to analyze the association between the expression of TTN-AS1 and prognosis of BC patients from TCGA data set and our institution. Quantitative real-time PCR (RT-PCR) was conducted to explore the expression of TTN-AS1 between the patients who underwent TURBT and Re-TURBT. MTT, colony formation, and tumor formation assays were conducted to evaluate the effect of TTN-AS1 on the ability of proliferation in BC cell lines. Transwell assay was performed to evaluate the effect of TTN-AS1 on the ability of invasion in BC cell lines. Bioinfomatics and immunohistochemical staining was used to identify the relationship between TTN-AS1 and ATF2.Results: The higher expression of TTN-AS1 was related to poorer disease-free survival (DFS) in patients with BC. The expression of TTN-AS1 was higher in BC patients who underwent Re-TURBT compared with BC patients who underwent TURBT. Knocking down TTN-AS1 resulted in inhibiting the ability of proliferation and invasion of BC cells. ATF2 may serve as a downstream target of TTN-AS1 in BC, and the high expression of ATF2 is also related to adverse DFS.Conclusion: Our study reveals that TTN-AS1 serves as an oncogene by activating ATF2 in BC. The findings suggest that TTN-AS1 may act as a novel therapeutic target for patients with BC. Bladder cancer (BC) is one of the most frequency urinary malignancies in males with high recurrence and poor prognosis . As one in vitro and in vivo by targeting lncRNA TTN-AS1 supports its potential therapeutic effects.Long noncoding RNA (lncRNA), which is a subtype of RNA defined as transcripts >200 nt without protein-coding ability, is explored often and suggested to have a regulatory role in the development and progression of BC . A recenin vitro and in vivo. We also demonstrate that ATF2 and lncRNA TTN-AS1 plays a vital role in the progression of BC.Activating transcription factor 2 (ATF2), a member of the AP1 transcription factor family, is demonstrated to be an inducer for BC cells  proliferation as well as invasion . In thisThis study was approved by the Ethics Committee of Tianjin Medical University General Hospital, Tianjin, China (No. KY2020K09). Written informed consent was obtained from all patients, and the study was conducted in accordance with the Declaration of Helsinki.n = 130). All patients did not receive chemotherapy and radiotherapy before surgery. The fresh paired BC tissues which were recurred (recurrence means that a patient who was diagnosed with BC for the first time has a new tumor in the bladder after undergoing TURBT surgery) were immediately stored in liquid nitrogen after TURBT (n = 6).The bladder tissue specimens were surgical specimens from BC patients with complete clinicopathological data. BC tissues that were embedded in paraffin were acquired by transurethral resection of bladder tumor (TURBT) at the Tianjin Medical University General Hospital  and Ki67 .2.Human BC cell line T24 and 5,637 cells were purchased from the American Tissue Culture Collection (ATCC). T24 and 5,637 cells were incubated in RPMI 1640 . All media were supplemented with 10% fetal bovine serum . All experimental cell lines were cultured at 37°C containing 5% COTransfection was performed using cells in the logarithmic growth phase. The cells are seeded into six-well plates at a density of 5 × 105 cells/well. After the cells grow to 60% of the area of each well, transfection is performed. The specific procedure was carried out with Lipofectamine 3,000 according to the manufacturer’s instructions . The shRNA targeting TTN-AS1 (shTTN-AS1) and the shRNA targeting scramble (shSCR) were obtained from Shanghai GenePharma Co., Ltd .Total RNA was extracted using Trizol and reverse transcribed into cDNA. Using the cDNA as a template, PCR was performed to detect the expression of TTN-AS1 using GAPDH as an internal reference. The primer sequences were as follows: TTN-AS1 forward primer, 5′- GCCAGGTAGAGTTGCAGGTT-3′; reverse primer, 5′- GAAGCTGCTGCGGATGAATG-3′; GAPDH forward primer, 5′-GATTCCACCCATGGCAAATT-3′; reverse primer, 5′-TCTCGCTCCTGGAAGATGGT-3′. Reaction bands were observed using gel electrophoresis.2 at 37°C for culture. After a certain period of time, discard the medium in the well plate, add 100 μl of MTT solution to each experimental well and then place it in a cell culture incubator for 4 h. The supernatant was discarded again, and 150 μl of DMSO was added to each experimental well. Place the 96-well plate on a plate shaker and shake it slowly, and then place the solution to be checked in the microplate reader, measure the optical density at a wavelength of 570 nm, and record the optical density value.Weigh 0.5 g of MTT  order and dissolve it in 100 ml of PBS and pass the solution through a sterile filter membrane with a pore size of 0.22 μm to filter out bacteria in the solution. Add 100 μl of cell culture medium to each well of the 96-well plate and ensure that there are 2 × 103 cells in each well. Place the paved 96-well plate in a constant temperature cell incubator containing 5% COWhen the cells are in the logarithmic growth phase, the BC cells were collected and cultured by a conventional cell culture method. When macroscopic colonies appeared in the Petri dish, the culture was terminated. Discard the supernatant, add 4% paraformaldehyde to fix the cells, and then stain the cells with GIMSA application staining solution. Slowly wash away the staining solution with running water and let the plate dry completely. Finally calculate the number of clones.Take 200 µl of the cell suspension and add it to the Transwell chamber to ensure that each well contains 1 × 104 cells. Perform cell culture in a conventional cell culture method. After 48 h, remove the chamber and discard the supernatant. Fix and stain the cells under the Transwell membrane; observe and count under a microscope.n = 10) were purchased from Beijing HFK Bioscience Co., Ltd . Subcutaneous tumor growth assays were performed with T24 cell lines (1 × 106 T24 cells injected in each mouse). The tumor size was measured every 7 days with a Vernier calliper, and the volume change of the tumor was calculated. The tumors were harvested under standard, institutionally approved processes. The mass of each tumor was measured with an electronic balance. Tumor samples were paraffin-fixed and processed for immunohistochemical (IHC) analysis.Male nude mice  overnight at 4°C. The secondary antibodies (goat antirabbit IgG) were incubated at room temperature for 1 h. The signals were examined under a microscope.t-test or ANOVA for functional analysis. Survival curves were plotted using Kaplan–Meier survival plots and a log-rank test was used to test significance. All statistical analyses were performed using SPSS software and GraphPad Prism7. p < 0.05 was considered statistically significant. p < 0.05 was marked as *, p < 0.01 was marked as **, p < 0.001 was marked as ***, and no significant difference was expressed as n.s.Statistical significance was determined using two-tailed Student’s p ≤ 0.01), high tumor grade (p = 0.02), lymph node metastasis (p ≤ 0.01) and vascular invasion (p = 0.01).LncRNA TTN-AS1 has been stated and functionally confirmed to be involved in a variety of cancer progressions . Althougn = 6) collected in our hospital. PCR results suggest patients received re-TURBT with recurrence may be correlated with local increased expression of TTN-AS1 . The scramble set (shSCR) (n = 5) was injected with lentiviruses carrying control scramble shRNA, and the treatment set (shTTN-AS1) (n = 5) was injected with lentiviruses carrying lncRNA TTN-AS1 shRNA. Injections were carried out every 3 days for 3 weeks lncRNA TTN-AS1 knocked down during this long-term treatment period led to a significant suppression in the growth of the T24 tumors  mice (4 tumors . The eff4 tumors . These rin vivo T24 tumor xenografts (To explore the downstream of lncRNA TTN-AS1 in BC, we first evaluated its similar expression pattern genes upon the TCGA BC data set by the GEPIA online tool . ATF2, anografts .via modulating the miR-218–5p/HS3ST3B1 axis (LncRNA, the length of which was more than 200 nt, plays a crucial effect in modulating proliferation, invasion, and migration of various types of tumors, such BC , renal c3B1 axis . Except 3B1 axis . In brea3B1 axis .in vitro experiments, in vivo experiments, and clinical data analysis.So far, no relevant research has pointed out the role of TTN-AS1 in the occurrence and progression of nonmuscular invasive BC. In this study, we found, for the first time, that TTN-AS1 is abnormally highly expressed in BC and is related to the poor prognosis of BLACA patients. In terms of mechanism, TTN-AS1 can promote the proliferation and invasion of tumor cells by regulating the level of downstream factor ATF2. In short, we find that TTN-AS1 can exhibit the role of oncogenes and serve as a therapeutic target for BC through via the AR signal pathway. Our study analyzes the survival information of BC patients in the TCGA database by bioinformatics and also finds that BC patients in the ATF2 high expression group had adverse DFS. Interestingly, we found that TTN-AS1 and ATF3 were correlated significantly via bioinformatics. Therefore, we propose the hypothesis that ATF2 may be a downstream factor of TTN-AS1 in BC. Furthermore, we used IHC to analyze the protein expression level of ATF2 in tumor tissues in both the knockdown TTN-AS1 and control groups. We find that, after knocking down TTN-AS1, the protein level of ATF2 was also significantly downregulated. Therefore, the oncogenic factor ATF2 of BC may be a potential downstream factor of TTN-AS1. TTN-AS1 promotes the proliferation and invasion of BC cells via inducing the expression of ATF2.LncRNA regulates tumor mechanisms in a variety of ways, including affecting the transcription of the upstream promoter region of the coding protein gene, mediating chromatin remodeling, histone modification, and changing the cellular localization of proteins, etc., . ATF2 isIn summary, TTN-AS1 plays a role as a tumor-promoting factor in the progression of BC and can positively regulate the proliferation and invasion of BC cells by regulating the expression of ATF2. At the same time, TTN-AS1 is related to the poor prognosis of BC patients. Therefore, TTN-AS1 can be used as both a potential therapeutic target for Ponge and a predictor to evaluate the prognosis of patients with BC. In addition, this study contains the following shortcoming: the mechanism through which TTN-AS1 regulates the expression of its downstream factor ATF2 requires further experiments to explore.