Conclusions: data showing a 12.7% human papillomavirus representation among patients with non-small cell lung cancer were obtained. The presence/absence of a viral component in patients with lung cancer was a clinically significant parameter. HPV types 16, 18, and 56, which are the most oncogenic, were most often detected.The significance of the role of human papillomavirus (HPV) in the development of lung cancer remains an open question. The data from the literature do not provide conclusive evidence of HPV being involved in the pathogenesis of lung cancer. The aim of this work was to detect the presence of HPV infections with a high carcinogenic risk in patients with non-small cell lung cancer (NSCLC). Materials and methods: the study involved 274 patients with stage IIA–IIIB non-small cell lung cancer. We analyzed normal and tumor tissues as well as blood from each patient. DNA was extracted from patients’ specimens, and HPV detection and genotyping was carried out using commercially available kits by PCR. Results: HPV was detected in 12.7% of the patients . We detected nine different types of human papillomavirus in the patients, namely, types 16, 18, 31, 35, 45, 51, 52, 56, and 59. The HPV-positive samples had a clinically insignificant viral load and were predominantly integrated. The relationship between the presence of HPV and its virological parameters and the clinical and pathological parameters of the patients was established. A metastatic-free survival analysis showed that all patients with HPV in the tumor tissue had a higher 5-year survival rate (94%) compared with the HPV-negative patients (78%). The result was not statistically significant ( Many factors play a role in lung cancer (LC), ranging from smoking and unfavorable environmental conditions to various individual molecular genetic parameters of the organism. The possible cause of the development of LC may be polymorphisms in the genes that regulate the metabolism of carcinogenic substances, the cell cycle, and other key processes in carcinogenesis, for example ,2. ThereA few authors have described cases of a 100% spontaneous transformation of papillomas into differentiated squamous cell carcinomas of the lungs (51.1%) and the larynx (42.9%) .HPVs with a low carcinogenic risk (LCR) (types 6 and 11) were previously identified in papillomas in such patients. According to meta-analysis data, the frequency of occurrence of human papillomavirus in LC widely varies, depending on the number of patients examined, from a complete absence of the virus in the tumor tissue to an occurrence in more than 75% of cases ,9. In mop = 0.001). The most prevalent virus genotype in the patients was type 16 (38.8%). A decrease in the p53 and RB expression was shown; the expression of inflammatory cytokines was increased in the HPV-positive lung cancer samples and control tissues compared with the HPV-negative lung cancer and HPV-negative control tissues. As a result, the authors suggested that an HPV infection could cause the induction of inflammation and EMT, which may contribute to the development of lung cancer.In a recent study, Rezaei et al. showed an association between HPV and lung cancer . In partDespite the availability of such work, the question of the role of HPV in the pathogenesis of lung cancer remains open and requires discussion. The aim of this work was to detect the presence of HPV infections with a high carcinogenic risk in patients with non-small cell lung cancer (NSCLC).n = 126) and a group of patients with primary resectable non-small cell lung cancer (n = 148). Patients in the first group received two courses of neoadjuvant chemotherapy according to the vinorelbine 25 mg/m2 (1st and 8th days)/carboplatin AUC 6 (2nd day) regimen. The administration of neoadjuvant chemotherapy is determined by the large size of the primary tumor node and the presence of lymph node metastases. The main objective of neoadjuvant chemotherapy is to damage tumor cells, reduce tumor size, reduce the degree of malignancy, and sanitize the lymphatic channels of the lung root and mediastinum to prevent lymphatic and hematogenous metastases and, therefore, relapse and the possibility of organ-preserving surgery. After NAC, these patients underwent surgery in the form of a pneumonectomy or lobectomy. The second group of patients did not receive NAC and were immediately operated on for pneumonectomy and lobectomy.The study involved 274 patients with primary operable non-small cell lung cancer (IIB-IIIA stage), central and peripheral localization, aged 39–76, with an average age of 60.8 years old, undergoing treatment at the Cancer Research Institute, Tomsk NRMC, from 2010 to 2016. The study was conducted according to the ethical principles suggested in the Declaration of Helsinki (fixed in 2013) and approved by the Ethical Committee of the Cancer Research Institute . The diagnosis was verified histologically. The patients were conditionally divided into two study groups, depending on the treatment tactics: a group of patients with neoadjuvant chemotherapy (NAC) (2 (1st and 8th days)/carboplatin AUC 6 (2nd day); doxorubicin 50 mg/m2/carboplatin AUC 6 (on day 2); gemcitabine 1250 mg/m2 (1st and 8th days)/carboplatin AUC 6 (2nd day); carboplatin AUC6 (on day 2)/paclitaxel 175 mg/m2. The interval between chemotherapy courses was 3 weeks. Chemotherapy was administered to patients who were in good general condition and had normal laboratory parameters. The research was conducted in accordance with the 1964 Helsinki Declaration and the local ethics committee of the institute; all patients signed informed consents for the study. After the operation, all patients received three courses of adjuvant chemotherapy (ACT) with «platinum doublets» according to the following schemes: vinorelbine 25 mg/mIn addition, the control group, which consisted of 30 apparently healthy volunteers (11 women and 19 men), was included in the study. Average age: 58.1 ± 1.05 (within 47–69 years). The main clinical and pathological parameters of patients are presented in 3), taken by bronchoscopy, were surgical samples of normal lung tissue, normal bronchial tissue, tumor tissue (~60–70 mm3), and whole blood. Tissue samples were placed in an RNAlater solution  and stored at −80 °C (after a 24-h incubation at +4 °C) for further DNA isolation. Blood was collected in vacuum tubes with K2 EDTA . The sampling of bronchoalveolar lavage to form a control group was performed using fibrobronchoscopy. Whole blood samples were also taken from this group.The test materials used as biopsy samples of normal and tumor tissue before treatment . DNA concentration and purity of isolation were evaluated on Qubit 4.0  using a DNA quantification kit. The concentration ranged from 10 to 100 ng/μL. The isolated DNA was stored at −80 °C in a low-temperature freezer .® reagent kits ; AmpliSens® HPV HR genotype FL, cat #R-V25  . The presence of HPV high-risk (HR) 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 genotypes was determined. The value of viral load was calculated in genomic equivalents of HPV DNA/105 cells, and the threshold of the relevant amount of the virus was taken to be equal to 3 lg HPV DNA/105 cells. The method is based on simultaneous amplification (multiplex-PCR) and “real-time” detection of DNA regions E1-E2 of HPV genes and a beta-globin gene used as an endogenous internal control. To exclude false-positive and false-negative results, each sample was duplicated during PCR.The detection and genotyping of HPV DNA were performed by real-time PCR on a RotorGene 6000  using AmplisensHBB gene [www.ncbi.nlm.nih.gov (accessed on 1 November 2022), as well as the Primer-BLAST database , digital PCR was used . DNA (100 ng) was cleaved using HaeIII . The mastermix for ddPCR included 1× ddPCR Supermix for probes , 0.9 μM primer, and 0.25 μM probe , together with 5 μL cleaved sample DNA. The PCR designs were duplexed, combining each HPV genotype (16 and 18) with the human control HBB gene . For thedatabase . The entp < 0.05.Statistical data processing was carried out using the Statistica 8.0  software package. The incidence of HPV-positive and HPV-negative patients in each group was assessed in absolute values and as a percentage of the total number of patients studied. Fisher’s two-sided test was used to compare the incidence of HPV-positive patients in the groups depending on clinical and pathological parameters and response to NAC, as well as comparison with the control group of patients. Survival was assessed using the Kaplan–Meier method. The reliability of differences between the groups was compared using the log-rank test. The difference was considered reliable at HPV was identified in 35 patients (12.7%) regardless of the group of patients and the type of material A. In theThe HPV high-carcinogenic risk typing in the study group of patients detected nine types of the virus in patients with NSCLC: 16, 18, 31, 35, 45, 51, 52, 56, and 59. The frequencies of the types in HPV-positive patients, depending on the test material, are shown in Mono- and mixed infections (the presence of two or more types of HPV at the same time) were observed in 23 and 12 patients, respectively (65.7% and 34.7%) .The analysis of the viral load of HPV-positive patients showed a predominantly insignificant viral load: 25 cases out of 35 were positive (74.3%). Six HPV-positive patients (17.1%) had a significant viral load, and there were four patients with an increased viral load (11.4%). The division into groups also showed that an insignificant viral load prevailed in the group of patients with NAC (66.7%) and the group of patients with primary operable LC (78.3%) . Next, pp = 0.03). A similar result was shown in the group of patients with primary operable LC: 26% of HPV-positive patients versus 10.2% (p = 0.01). For the first group of patients, with NAC, there was a relationship between the presence of HPV and the tumor size. Interestingly, the presence of HPV was associated with a larger primary tumor node size (11 of 81 cases (13.6%) vs. 1 of 45 cases (2.2%) in the group with T1–2; p = 0.05). An association with a strong tendency was also demonstrated: HPV-positive patients were predominantly male , which can be explained by the predominance of men in our sample of patients (88.7%). An analysis of the relationship between viral load and the main clinical and pathological parameters of patients showed that in 85.7% of cases (18 out of 21 patients) with a peripheral, clinical, and anatomical form of the tumor, an insignificant viral load prevailed (p = 0.05)   with the presence of HPV and its virological parameters in patients with LC was assessed . The inc = 0.05) . In addi = 0.02) .p = 0.83).It is interesting to assess the association of HPV with the efficacy of neoadjuvant chemotherapy . Data onn = 30), HPV type 18 was found in only one patient with a significant viral load and a mixed form. The association of HPV with lung cancer was then assessed using a two-tailed Fisher test (In the control group (her test A.There was no association between HPV and LC .p = 0.49).Thus, the incidence of HPV-positive specimens in the control group was 3.3% (1 of 30 cases), compared with the study group, where the rate was 12.7% (35 of 274 cases). The analysis performed showed no statistically significant differences in the presence of HPV in the study group compared with the control group (p = 1.00). In addition, no statistically significant relationship was shown for the blood of the studied patients (p = 0.37).Many authors use normal tissue from the same patients as a control group when evaluating the association of HPV with lung cancer. Based on this, we performed an additional analysis of the association of HPV with lung cancer with the results of the analysis of normal lung tissue used as a control group . It was p = 0.08). The presence of HPV is not associated with overall survival  of patients with lung cancer was assessed for HPV infection with a high carcinogenic risk. It was found that HPV-positive patients had a higher 5-year metastatic-free survival rate (94%) compared with HPV-negative patients 78%; A. It canIn addition, a multivariate regression analysis was performed to identify prognostic factors for metastasis-free survival .p = 0.83). A small tumor size (T2) in patients is a favorable factor for metastasis-free survival rates .It was found that the presence of HPV in patients with lung cancer is not a factor that increases or reduces the risk of tumor metastasis   B.Thus, HPV-positive patients with NSCLC had a high level of metastatic survival in comparison with the survival rates of HPV-negative patients. It is important to note that the data obtaining a favorable outcome for patients with HPV-positive lung cancer, despite the small group size, not only agree with the global literature data but are also important for the prognosis of the disease.The etiological role of HPV in the development of anogenital carcinomas has been widely studied and is well known ,14. HoweIn this work, 12.7% of the cases of NSCLC were HPV-positive (35/274 patients). The data obtained were consistent with the results of our earlier meta-analysis, according to which the frequency of HPV in tumor tissues in LC was 28.7% and the frequency of HPV in normal lung tissues was 7.6% [Regarding the determination of the prevalence of the desired virus in normal and tumor tissues, we obtained figures of 31.4% and 22.9%, respectively. This result was consistent with the literature data, in particular in a 2020 study that included 140 patients with primary lung cancer, where it was shown that the total infection rate of the sample included in the study was 20.7%. The virus was detected in 9.3% of the tumor samples (13 out of 140) and in 16 paired samples of normal and tumor tissues (11.4%) . p < 0.0001; 30.9% vs. 5.2% in the control group for HPV 18, p < 0.0001) [It is important to note that for the samples of normal tissue, tumor tissue, and blood obtained from each individual patient, the highest percentage of virus identification was observed in the blood (40.0%). These data are unique thus far; the Russian literature does not mention the determination of HPV in the blood of patients with NSCLC. It is believed that HPV is spread through respiratory secretion through close contact with mucous membranes or the skin . There ip < 0.0001) and HPV type 18  [When determining the types of HRC HPV in the study sample, it was shown that the highest frequency of occurrence was characteristic of HPV types 16 and 18, with their predominance in the material of normal lung tissue and blood, respectively. A high frequency of HPV type 56 in the tumor tissue was also established. However, the leader in the frequency of occurrence was HPV genotype 18 (36.8%), which was consistent with the world literature data on the definition of HRC HPV among the oncopathologies of various localizations ,20. In a 0.0001) .An important limitation of the presented work was the lack of information on the smoking status of the studied patients, since it has been shown that smoking is one of the main factors in the development of lung cancer. Although a recent study showed that HPV is found in 20% of non-smokers with lung cancer .Regarding the analysis of HPV DNA concentrations in LC samples, one of the first studies of the significance of the viral load as a predictor of the development of this oncopathology was conducted by Coissard et al. The authors showed a low viral load of fewer than one copy of the virus per cell . A few aE2F1 (20q1). Another important structural change in HPV infections is the deletion of the TRAF3 gene, which occurs in approximately 20% of HPV-associated tumors. This gene plays an important role in antiviral immunity and is a regulator of NF-κB (cell growth and proliferation) [NF-κB, and the secretion of numerous pro-inflammatory cytokines and metabolites, including TGF-β, IL-1, IL-6, IL-11, and TNF-a. This pro-inflammatory tissue microenvironment leads to the suppression of anti-humoral immunity and promotes tumor development and metastasis [NF-κB activation [NF-κB activity is increased. Previous studies have shown that NF-κB acts as a tumor promoter in NSCLC. Elevated levels of NF-κB expression were found in NSCLC tissues in contrast to the corresponding normal tissues. NF-κB overexpression has been associated with metastasis and a poor prognosis for NSCLC patients [Several mechanisms have been proposed for HPV oncogenesis, including the presence of mutations and structural changes in genes. For example, several HPV-associated tumors contain an amplification of the proliferation and cell cycle regulator gene eration) . In resptastasis . Presumativation . In non-patients . Unfortup = 0.83). The data available in the literature are consistent with the results obtained in this work. For example, Wang et al. showed that patients with HPV types 16 and 18 and lung adenocarcinomas had higher survival rates compared with patients without the virus in the tumor [Regarding the prediction of the outcome of patients with HPV-associated carcinomas, differences in the survival of patients with tumor pathologies of the reproductive organs ,29,30, hhe tumor . Ragin ahe tumor . It is ihe tumor ,36.To date, the role of HPV in the pathogenesis and outcome of patients with NSCLC remains debatable. This study investigates potential associations between HPV and NSLC in a cohort of patients with the presence of three study points for each patient, namely, normal tissue (control), tumor tissue, and blood. The only limiting factor was the small size of the total sample and the sample of HPV-positive patients. This study was designed and conducted to obtain data on the prevalence of HPV in patients with NSCLC. In the future, we aim to conduct a large-scale multicenter study to confirm the role of HRC HPV in the development and prognosis of NSCLC.


A biomarker that is useful for the detection of human papillomavirus (HPV)‐related oropharyngeal cancer (OPC) and cancer of unknown primary (CUP) is indispensable. We evaluated the diagnostic performance of HPV DNA and mRNA in oral gargle samples and circulating tumor HPV16 DNA (ctHPV16DNA) in blood samples. Oral HPV DNA and mRNA were analyzed using commercially available HPV assays of the GENOSEARCH HPV31 and Aptima, respectively. ctHPV16DNA was analyzed using in‐house droplet digital polymerase chain reaction. Seventy‐four patients with OPC and eight patients with CUP were included. The sensitivity and specificity of oral HPV DNA, oral HPV mRNA, and ctHPV16DNA were 82%  and 100% (95% CI = 88‐100), 85% (95% CI = 69‐94) and 94% (95% CI = 73‐100), and 93% (95% CI = 81‐99) and 97% (95% CI = 84‐100), respectively, for HPV16‐related OPC, while those were 20% (95% CI = 1‐72) and 100% (95% CI = 3‐100), 0% (95% CI = 0‐52) and 100% (95% CI = 3‐100), and 100% (95% CI = 54‐100) and 100% (95% CI = 16‐100), respectively, for HPV16‐related CUP. The sensitivity of ctHPV16DNA for HPV16‐related OPC was higher than that of oral biomarkers, though the difference was not statistically significant. ctHPV16DNA remarkably correlated with the anatomic extent of disease, total metabolic tumor volume and HPV16 copy number per tumor genome in patients with HPV16‐related OPC/CUP, whereas oral biomarkers did not. In conclusion, ctHPV16DNA is a potentially promising biomarker for HPV16‐related OPC, while further studies are required for HPV16‐related CUP. What's new?A minimally‐invasive biomarker that allows the detection of human papillomavirus (HPV)‐related oropharyngeal cancer and cancer of unknown primary is indispensable. Here, the authors show that circulating tumour HPV DNA (ctHPVDNA) correlates with the tumour burden and HPV copy number per tumour genome in HPV‐related oropharyngeal cancer and cancer of unknown primary. Neither oral HPV DNA nor mRNA exhibits such a correlation. ctHPVDNA outperforms oral HPV DNA and mRNA in detecting HPV‐related oropharyngeal cancer and in distinguishing HPV‐related cancer of unknown primary from HPV‐unrelated cancers. ctHPVDNA emerges as a potentially useful biomarker for HPV‐related oropharyngeal cancer and cancer of unknown primary. Among high‐risk HPVs, HPV16 is a predominant driver in the pathogenesis of HPV‐related OPSCC.Liquid biopsies that analyze tumor materials, such as cells or nucleic acids, which are obtained in a minimally invasive or noninvasive manner from the blood or other body fluids, have emerged as novel modalities for cancer management.22.1Patients with newly diagnosed OPSCC or node‐positive SCCUP, who first visited the Osaka University Hospital between 2017 and 2020, were prospectively enrolled in the study. Pathological diagnosis was based on a biopsy of the primary site for OPSCC and on fine needle aspiration or open biopsy of the neck for SCCUP. Biopsy specimens of OPSCC were examined to ascertain p16 and HPV DNA status, as detailed below. Fine needle aspiration specimens of SCCUP were also assessed to determine HPV DNA status. p16 status of SCCUP was assessed only when surgically excised node specimens were available. OPSCC that was positive for both p16 and any high‐risk HPV DNA and SCCUP that was positive for any high‐risk HPV DNA were defined as HPV‐related. Likewise, OPSCC that was positive for both p16 and HPV16 DNA and SCCUP that was positive for HPV16 DNA was defined as HPV16‐related. OPSCC and SCCUP that were related to high‐risk HPV except for HPV16 were defined in the same manner. Overexpression of p16 was not considered for the definition of HPV‐related SCCUP. This is because we previously found that SCCUP with metastatic node that was high‐risk HPV DNA‐positive was also always p16‐positive.2.2http://blast.ncbi.nlm.nih.gov/Blast.cgi). HPV genotypes of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and 69 were classified as high‐risk. Formalin‐fixed paraffin‐embedded biopsy or surgically excised specimen sections were immunohistochemically examined to ascertain p16 status using the antibody clone p16 CINtec E6H4 . p16 was scored positive if ≥70% of the tumor cells exhibited robust and diffuse nuclear and cytoplasmic staining results.DNA was extracted from fresh frozen biopsy or fine needle aspiration specimens using the DNeasy Blood & Tissue Kit  or QIAamp DNA Mini Kit (Qiagen). Extracted DNA was used for nested polymerase chain reaction (PCR) followed by direct sequencing to detect and genotype HPV, as per previously reported methods.2.3Both oral and blood samples were collected prior to any treatment. Participants were advised to gargle with 10 mL of phosphate‐buffered saline for 30 seconds. After gargling, expectorated fluid samples  were obtained using a sterile collection tube. A volume of 2 mL of the oral sample was transferred to an Aptima Urine Specimen Transport Tube . Both oral samples remaining in the collection tubes and those transferred to the Aptima Urine Specimen Transport Tube were immediately shipped at 4°C to the LSI Medience Corporation  and stored at 4°C until HPV DNA and mRNA testing. Blood samples (8.5 mL) were collected using a Cell‐Free DNA Collection Tube (Roche). After centrifugation at 3500 rpm for 10 minutes, the plasma was retrieved and stored at −80°C until use. The process from blood collection to storage was completed within 2 to 3 hours.2.4For the assessment of oral HPV DNA, DNA was extracted from a 400 μL aliquot of the oral sample in the collection tube using the QIAsymphony SP/AS Instruments (Qiagen). Extracted DNA was then eluted in a final volume of 60 μL and was analyzed using the GENOSEARCH HPV31 kit  to detect and genotype HPV. The GENOSEARCH HPV31 kit allows the identification of 31 HPV genotypes, including high‐risk types  and low‐risk types , using reverse sequence‐specific oligonucleotide PCR. Briefly, DNA samples were amplified using multiplex PCR. The resultant amplicon mixtures were hybridized with genotype‐specific probes, which were detected using the Luminex System . β‐globin was used as an internal control to verify the presence of sufficient cellular components. For the assessment of oral HPV mRNA, the Aptima HPV assay kit (Hologic) that allows the detection of E6/E7 mRNA from 13 high‐risk HPVs  and HPV66 without providing genotype‐specific information was used. The Aptima HPV assay includes a control for monitoring the entire process. Aliquots (2 mL) of the oral samples in the Aptima Urine Specimen Transport Tube were automatically analyzed using the Panther system (Hologic) according to the manufacturer's instructions. Assessment of oral HPV DNA and mRNA was completed within a week after sample collection.2.5Cell‐free DNA was extracted from 3 mL of cryopreserved plasma and was suspended in 100 μL using the QIAamp Circulating Nucleic Acid Kit (Qiagen). The amount was measured using the Qubit 2.0 fluorometer and Qubit dsDNA HS Assay Kit . HPV16 E6 and E7 copy numbers were quantified through the use of our in‐house optimized droplet digital PCR (ddPCR) using the QX200 Droplet Digital PCR System , as per methods reported previously.2.6To evaluate HPV16 copy number per tumor genome, genomic DNA extracted from fresh frozen biopsy or fine needle aspiration specimens was analyzed with quantitative PCR using the HPV Genotypes 14 Real‐TM Quant kit  and Bio‐Rad CFX96 qPCR instrument (Bio‐Rad) according to the manufacturer's instructions. HPV16 copy number per tumor genome was calculated with reference to the β‐globin copy number.2.718F‐fluorodeoxyglucose‐positron emission tomography/computed tomography (FDG‐PET/CT) data with a standardized uptake value (SUV) threshold of 2.5, using PETSTAT Viewer Version 2.2 , as per methods reported previously.Metabolic tumor volume (MTV) was measured from pretreatment attenuation‐corrected 2.8P < .05.The sensitivity of oral HPV DNA for HPV‐related tumor was estimated, on the presupposition that the genotype of oral HPV DNA had to be consistent with that of tumor HPV DNA so that the test results were considered positive. The specificity of oral HPV DNA for any high‐risk HPV‐related tumor was calculated as the number of patients who had negative test results for any high‐risk HPV out of patients with HPV‐unrelated tumor. The specificity oral HPV16 DNA for HPV16‐related tumor was calculated as the number of patients who had negative test results for HPV16 out of patients with HPV16‐unrelated tumor, including HPV‐unrelated tumor and tumor that was related to high‐risk HPV except for HPV16. The specificity of oral HPV DNA for tumor that was related to high‐risk HPV except for HPV16 was calculated as the number of patients who had negative test results for high‐risk HPV except for HPV16 out of patients with HPV‐unrelated tumor because otherwise the specificity had to be calculated for each high‐risk HPV genotype. The sensitivity of oral HPV mRNA for HPV‐related tumor was calculated as the number of patients who had positive test results out of patients with HPV‐related tumor, while the specificity was calculated as the number of patients who had negative test results out of patients with HPV‐unrelated tumor. This is because HPV genotype was unidentifiable with the oral HPV mRNA test. The sensitivity and specificity of ctHPV16DNA for HPV16‐related tumor was calculated in the same manner as those of oral HPV DNA for HPV16‐related tumor. We calculated 95% confidence interval (CI) for sensitivity and specificity with the ClopperPearson method. The Fisher's exact test was used to compare categorical variables, while the exact Wilcoxon rank‐sum test or Kruskal‐Wallis test was used to compare continuous variables, as appropriate. Spearman's rank correlation was used to assess the relationship between two continuous variables. Trends were evaluated using the Jonckheere‐Terpstra test. All statistical analyses were performed using JMP Pro 14  or IBM SPSS Statistics 26 , and statistical significance was set at 33.1We had a total of 100 and 14 patients with newly diagnosed OPSCC and node‐positive SCCUP, respectively, between 2017 and 2020 at Osaka University Hospital. As shown in Figure 3.2ρ = 0.97, P = 2.7e−30), as shown in Figure The correlation between the copy numbers of circulating HPV16 E6 and E7 DNA was excellent among patients with HPV16‐related tumors . Importantly, ctHPV16DNA was detectable in all six patients with HPV16‐related SCCUP. Increasing trend of ctHPV16DNA levels was also observed along with the progression of N classification     Figure . As HPV ) Figure . We furt) Figure , suggestSpecifically, ctHPV16DNA was undetectable in three patients with OPSCC classified as T1N0M0, T2N0M0 and T2N1M0 (the seventh edition of the UICC TNM classification system). One patient with T1N0M0 disease presented with an HPV16 copy number per tumor genome of 1.4 and a total MTV of 6.2 mL, which yielded a small product of 8.68, thus providing an explanatory approach for the observation of undetectable ctHPV16DNA levels. In contrast, the other two patients presented with higher HPV16 copy number per tumor genome  and higher total MTVs , yielding products of 50.16 and 48.32, respectively. Considering that ctHPV16DNA was detectable in other patients with products of the same levels, it was suggested that ctHPV16DNA was undetectable due to certain unspecified factors in these two patients. Conversely, ctHPV16DNA was detectable in two patients whose product of HPV16 copy number per tumor genome and total MTV was minimal (0.23) or zero. One patient with T2N1M0 OPSCC (the seventh edition of the UICC TNM classification system) presented with an HPV16 copy number per tumor genome of 0.03, and a total MTV of 7.8 mL, yielding a minimal product. The proportion of tumor cells to nontumor cells in his biopsy specimen was most likely small, which might have resulted in seemingly lower HPV copy number per tumor genome than the actual number. The other patient harbored locally advanced OPSCC classified as T1N2bM0 according to the seventh edition of the UICC TNM classification system, but the total MTV was immeasurably small because SUVmax of the tumor was 2.4.3.3Table P = .59). Neither T classification, N classification nor disease stage showed correlation with oral HPV DNA‐positivity, irrespective of the seventh and eighth editions of the UICC TNM classification system. This suggests that oral HPV DNA‐positivity was independent of the anatomic extent of the disease in patients with HPV16‐related OPSCC. Additionally, neither total MTV nor HPV16 copy number per tumor genome exhibited correlation with oral HPV DNA‐positivity in patients with HPV16‐related OPSCC. Oral HPV DNA‐positivity according to the characteristics observed in patients with any high‐risk HPV‐related OPSCC is shown in Table As primary tumor status, OPSCC or SCCUP, had a major impact on the sensitivity of oral HPV DNA, we focused on patients with HPV‐related OPSCC to assess the association of oral HPV DNA with tumor burden. Table 3.4P = .30, Table Oral HPV mRNA test showed results that were similar to those of the oral HPV DNA test. Oral HPV mRNA was detectable in 41 of the 50 patients with any high‐risk HPV‐related OPSCC and was undetectable in 17 of the 18 patients with HPV‐unrelated OPSCC, indicating that the sensitivity and specificity of oral HPV mRNA for any high‐risk HPV‐related OPSCC were 82% (95% CI = 69‐91) and 94% (95% CI = 73‐100), respectively  of ctHPV16DNA for HPV16‐related OPSCC seemed higher than, or equivalent to, that reported previously, which ranged from 71% to 96%.We observed a significant correlation between ctHPV16DNA levels and the product of HPV16 copy number per tumor genome and total MTV in patients with HPV16‐related tumors. Undoubtedly, ctHPVDNA is likely undetectable in patients with tumors with low HPV copy number per tumor genome, especially when the tumor volume is small. In this regard, we should recognize the pitfalls of ctHPVDNA‐guided surveillance for recurrent disease after definitive treatment. Chera et al reported that longitudinal monitoring of ctHPVDNA would facilitate early identification of recurrent disease in patients with HPV‐related OPSCC.We found previously that whenever SCCUP with metastatic node was HPV DNA‐positive, it was also p16‐positive.Oral HPV DNA has been previously examined in patients with HPV‐related OPSCC using commercially available assays, such as DNA ELISA kit HPV SPF10, version 1 ,D'Souza et al previously reported the efficiency of oral HPV mRNA in detecting HPV‐related OPSCC.It is reasonable to assume that the number of tumor cells in oral samples that are exfoliated from primary tumor during gargling would affect the detection efficiency for oral HPV DNA and oral HPV mRNA. Oral HPV DNA and oral HPV mRNA are likely detectable in patients with HPV‐related OPSCC, while these oral biomarkers are hardly detectable in patients with HPV‐related SCCUP. Actually, our findings substantiated the assumption: the sensitivity of oral HPV DNA was 82% for HPV16‐related OPSCC, but only 20% for HPV16‐related SCCUP. Likewise, the sensitivity of oral HPV mRNA was 85% for HPV16‐related OPSCC, but 0% for HPV16‐related SCCUP. Moreover, considering that neither oral HPV DNA nor oral HPV mRNA detection showed any correlation with the extent of the local disease, such as T classification, in patients with HPV‐related OPSCC, other unspecified factors seem crucial for exfoliating cells from tumor. We speculate that patients who could not gargle well likely presented with negative results in the oral HPV tests. While the skill of oral gargling cannot be easily and objectively evaluated, the number of exfoliated cells may be considered a surrogate measure of oral gargling efficiency. Unfortunately, however, we did not determine the number of exfoliated cells.HPV‐related OPSCC is a slow‐growing entity and ctHPV16DNA exhibits satisfactorily high sensitivity and specificity in detecting HPV‐related OPSCC, suggesting the utility of ctHPV16DNA‐guided screening for HPV16‐related OPSCC. However, considering a low lifetime riskHPV DNA detection is relevant to HPV infection but not always to HPV‐driven transformation, whereas HPV mRNA detection is relevant to HPV‐driven transformation.The performance of HPV16 E6 serum antibody for HPV16‐related OPSCC has been previously explored in several studies that have reported high sensitivity (88%‐96%) and specificity (96%‐98%).This study has several limitations. The sensitivity of ctHPV16DNA for detecting HPV16‐related OPSCC was higher than that of oral HPV DNA or oral HPV mRNA. However, the advantage of ctHPV16DNA over oral HPV DNA or oral HPV mRNA should be interpreted with caution because the difference in sensitivity was not statistically significant. Moreover, the sample size of patients with SCCUP was especially small. Thus, the results for SCCUP were preliminary. ctHPVDNA analysis was performed using our in‐house ddPCR system and was limited to HPV16. The development of a commercially available multiplex ddPCR assay that widely covers high‐risk HPV genotypes is required to familiarize ctHPVDNA‐guided management of HPV‐related OPSCC. Oral HPV DNA was measured using GENOSEARCH HPV31, a commercially available HPV DNA assay, which is not globally distributed, although it is common in Japan. Neither oral HPV DNA nor mRNA was subjected to quantitative measurements, and these biomarkers were not considered as continuous variables.In conclusion, ctHPV16DNA is a potentially promising biomarker for the detection of HPV16‐related OPSCC. Further studies are required to establish the usefulness of ctHPV16DNA in distinguishing HPV16‐related SCCUP from HPV16‐unrelated SCCUP.The authors declare no conflicts of interest.The study received ethical approval from the institutional review boards of Osaka University. Written informed consent was obtained from all patients.Appendix S1: Supplementary InformationClick here for additional data file.


Hypoxylon sp. CO27-5, 36 highly sequence-similar  stwintrons are extant (sister stwintrons). An additional 81  sequence-unrelated stwintrons are described here. Most of them are located at conserved gene positions rooted deep in the Hypoxylaceae. Absence of exonic sequence bias at the exon–stwintron junctions and a very similar phase distribution were noted for both groups. The presence of an underlying sequence symmetry in all 117 stwintrons was striking. This symmetry, more pronounced near the termini of most of the full-length sister stwintrons, may lead to a secondary structure that brings into close proximity the most distal splice sites, the donor of the internal and the acceptor of the external intron. The Hypoxylon stwintrons were overwhelmingly excised by consecutive splicing reactions precisely removing the whole intervening sequence, whereas one excision involving the distal splice sites led to a frameshift. Alternative (mis)splicing took place for both sister and uniquely occurring stwintrons. The extraordinary symmetry of the sister stwintrons thus seems dispensable for the infrequent, direct utilisation of the distal splice sites.Introns are usually non-coding sequences interrupting open reading frames in pre-mRNAs . Stwintrons are nested spliceosomal introns, where an internal intron splits a second donor sequence into two consecutive splicing reactions leading to mature mRNA. In  Originally, introns were defined as non-coding sequences interspersed with exons in primary transcripts (pre-mRNA), which when accurately excised generated the open reading frame (ORF) in mature mRNAs. Spliceosomal introns U2 introns) are a hallmark of Eukaryota and widespread in nuclear transcriptomes, with average densities of three to four introns per gene in Pezizomycotina . Group- introns Euglena gracilis ( sister stwintrons of high-sequence similarity over the complete width of the intervening sequence and another 13, named sheared sister stwintrons, with high similarity restricted to a section thereof. Most of these 36 sequence-related stwintrons are exclusive to the narrow taxon of and EC38 .Hypoxylon sp. CO27-5 DNA sequence contigs. The existence of 117  stwintrons was established and two-thirds of these were stwintrons that had been not identified earlier by sequence similarity. The overwhelming majority of the newly identified UO  stwintrons are located at gene positions that are also occupied by “orthologue” stwintrons in the orthologue genes in 16 other taxa of Hypoxylaceae, resulting in the identification of more than 1500  stwintrons.We also found 12 canonical introns in that taxon derived from an ancestor sister stwintron, ten of which exhibited very strong internal symmetry, as there is no spacer between the terminal inverted repeats so typical for the sister stwintrons. During the analysis of two of the genes carrying such “multiple occurring” (MO) intervening sequences, we found two  stwintrons, the sequences of which showed no obvious similarity with those of the sister stwintrons. In the current work, we employed a simple motif search tool to identify additional “uniquely occurring” (UO)  stwintrons while screening Hypoxylon sp. CO27-5 genome sequences  stwintron in the ubiquitous reticulon-like gene was present across almost all Pezizomycotina classes  analysis. To keep the search results manageable, we thus precluded the use of less-canonical intron sequence elements, such as the 5′-GC2 for donor, 5′-RCC3AAY for BP or 5′-G1AG for acceptor, which are rarely extant in filamentous fungal U2 introns.We analysed the equences  with a sequences  and invo classes , and it Hypoxylon sp. CO27-5 (master accession number: MDCL01) consist of 580 sequence contigs. The contigs were downloaded from the National Center for Biotechnology Information (NCBI) servers and screened with the stwintron sequence motif search model A using the DNA Pattern Find program from the Sequence Manipulation Suit  stwintron is part of the split donor (5′-G1|T2) of its external intron). The numbers between accolades are the space ranges set between the five conserved sequence motifs in  stwintrons.Furthermore, we defined distance ranges between the five short  stwintron sequence motifs at the 5′- and 3′-splice sites, and the conserved sequence elements, including the branch point As (adenosines) from our statistical analysis of the  stwintrons in Pezizomycotina reticulon-like genes see , Model Aion Suit . The exaG(GTAAGT|GTATGT|GTGAGT|GTACGT|GTAAGC|GTATGC|GTAGGT|GTAAGA|GTGCGT|GTACGC|GTGAGC|GTATGA|GTAAGG|GTTAGT|GTCAGT|GTGAGA|GTGTGT|GTTTGT|GTACGA|GTATGG|GTTCGT|GTGGGT|GTAGGC|GTACGG).{25,80}[AGT][CT]T[AG]A[CT].{4,20}[ACT]AGT(AAGT|ATGT|GAGT|ACGT|AAGC|ATGC|AGGT|AAGA|GCGT|ACGC|GAGC|ATGA|AAGG|TAGT|CAGT|GAGA|GTGT|TTGT|ACGA|ATGG|TCGT|GGGT|AGGC|ACGG).{25,110}[AGT][CT]T[AG]A[CT].{4,20}[ACT]AGHypoxylon sp. CO27-5, most of the gene model predictions could be corroborated by RNA sequence reads (the six relevant SRA accessions listed in Hypoxylon sp. CO27-5 using 60 nt long query sequences containing the predicted exon/exon fusion site at its centre. To detect the stwintron splicing intermediate (splinter)—the RNA species that lacks the predicted internal intron of the  stwintron but still contains its external intron—we used query sequences (60 nt) containing in their centre, the predicted fusion site of the upstream exon and the external intron of the stwintron with its functional donor sequence intact. SRAs that confirm putative stwintrons and their predicted splinter are listed in Hypoxylon sp. EC38 (the six relevant SRA accessions listed in In Hypoxylon sp. CO27-5 genome as well as in other Hypoxylaceae genomes by TBLASTN screening of the NCBI’s Whole Genome Shotgun contigs (WGS) database  stwintrons were encountered are listed and referenced in The coding sequences (ATG–stop) of the orthologue genes carrying  stwintrons were mined in the database  to trackdatabase  occur onHypoxylon sp. CO27-5  stwintrons is shown in Hypoxylon sp. CO27-5 orthologue genes  were routinely collected from the other Hypoxylaceae genomes. The second sheet of We manually predicted the intron–exon structure of each gene guided by comparative genomics of coding sequences, taking the intron phase into account. The sequence-derived information thus collected for each of the 117 Note that automated annotation software does not recognise  stwintrons, complex intervening sequences that start with the noncanonical 5′-GGU  at their 5′-splice site. We did not use the results of automated annotation (“Models” or “mRNA” in the nr/nt database), neither did we use deduced protein databases. After translation of our predicted mRNAs, we functionally annotated most of the genes carrying  stwintrons with one or more protein domains categorised in the Protein Families (Pfam) database .Hypoxylon sp. CO27-5, like (stw)intron lengths, stwintron phase, minimal free energy for the optimal predicted secondary structure and AU content were transferred to new spreadsheets for their individual analysis using the Excel tool box , comparing the miscellaneous group of 81 “uniquely occurring” stwintrons (identified in the current work) with the group of the 38 sequence-similar sister stwintrons . Multipns  was maintained on potato dextrose agar (PDA)   stwintron .In a previous communication, we identified a set of repetitive or multiple occurring (MO) intervening sequences, mostly  stwintrons, in eight genomes of species of Hypoxylaceae and suggested how the most symmetrical canonical introns within this set in A breaks . These sHypoxylon sp. CO27-5, we analysed its genome sequences  search protocol when the EC38 genome was screened for  stwintrons .To identify additional  stwintrons in equences  with a sIn addition, three of the 13 sheared sister stwintrons  were noBy contrast, in HCOc263A, the distance between the donor-BP of the external intron was 126 nt, longer than the upper limit set in the primary  search model A. The tool did identify the 5′ end of HCOc016B, but the canonical BP (motif 4) of the external intron of this stwintron associated with the functional 3′-acceptor was 8 nt above the set length range for the distance between the donor–BP; upon experimental verification (described below), the full-length stwintron was found to be 58 nt longer at 3′ than that proposed by the search tool using model A. These observations led us to change the distance range between the donor and the BP elements of the external intron from 25–110 to 111–200 nt in the  stwintron search model  , respectively, ~54% identical at the AA level. Moreover, we found a third paralogue gene encoding a protein of 246 AAs, ~56% and ~59% identical to the two aforementioned paralogues, with a  stwintron at the very same position. This latter stwintron (named number no-301) was not detected with the  search model A, as the BP sequence of its external intron was the less conventional 5′-CHypoxylon sp. CO27-5 using three approaches. The first approach involved a BLASTN search for short RNA sequence reads deposited in the Sequence Read Archives (SRA) database at NCBI , which  related . When SR related ) for reaSecondly, we assessed the relevant sequences of the fully spliced RNA species (mRNA) as well as the predicted splinter of selected  stwintrons by targeted RT-PCR and cDNA sequencing, providing more sensitive means when the expression levels of the stwintron-containing gene are limiting. For the experimental details, see 2 instead of 5′-GU2) for either the internal or external intron, for instance, in the orthologue of stwintron number no-071 in Entonaema liquescens for the split external donor sequence (5′-G|C2AAGU).Finally, we identified and collected the orthologues of the stwintron-containing CO27-5 gene in the genomes of 19 other species/strains of the Hypoxylaceae family accessible at NCBI see  by targeHypoxylon sp. CO27-5 or the absence of the orthologue gene in 16 columns near the right side of the Excel table. In the column at the right of In Hypoxylon sp. CO27-5, experimentally or by comparative genomics, allowed, for the first time, for the statistical analysis of the sequences of more than one hundred different stwintrons of the same type present in one organism. Below, we compare a sizable group of sequence-related and terminally symmetrical stwintrons from Hypoxylon sp. CO27-5 and EC38, the 38 so-called sister stwintrons, with the 81 seemingly unrelated stwintrons identified with our  stwintron search tool (see above).In our current work, we showed the existence of more than 1500  stwintrons in species of the Hypoxylaceae, a family of endophytic fungi in the Xylariales order of the Sordariomycetes class (Ascomycota phylum). The identification of  stwintrons in Hypoxylon sp. CO27-5 and/or EC38 , the large majority of the UO  stwintrons (68 out of 81: ~84%) were present in orthologue genes in all or most of the 17 assessed species/taxa of the Hypoxylaceae family. Amongst the 13 “atypical” UO stwintrons, five were located in genes that did not have orthologues in most of the other assessed species/taxa. These large differences in the occurrence of orthologue stwintrons  strongly suggest that the large majority of the group of 81 UO stwintrons are much older than the highly symmetrical sister stwintrons unique to the CO27-5/EC38 taxon, as the former were likely generated in a common ancestor before divergence of all 20 taxa. The group of the 13 sheared sister stwintrons appears to be composed of mixed constituents: six of them were restricted to a narrow taxonomical clade that consists of or includes both Hypoxylon sp. CO27-5/EC38 and H. pulicicidum/Hypoxylon sp. E7406B.The comparative approach immediately showed a substantial distinction between these two groups . While 2H. pulicicidum/E7406B). On the other hand, three sheared sister stwintrons identified by BLASTN screening with HCOc017A—HCOc016B, HCOc024B and HCOc091A —were pr38 taxon , which sH. rickii, H. fragiforme and H. lienhwacheense—the original  stwintron found in the other Hypoxylaceae species and also present in other Xylariales species presumably morphed into a  stwintron. Insertion of four nt 5′-GTAA or 5′-GTGA directly upstream of the G1 of the external intron of the original  stwintron created (split) external intron donors 5′-GTAAG5|T6 or 5′-GTGAG5|T6 .A peculiar situation was observed for the phase-two stwintron number no-274 in a gene encoding a well-conserved plasma membrane protein of the monovalent cation:proton antiporter-1 family, suggesting that extant [D] stwintrons may change stwintron type by local mutation. In three species—TGAG5|T6 . The intWe analysed the insertion sites of the 81 MO  stwintrons after stacking the five concatenated conserved sequence motives at the 5′- and 3′-splice sites  terminally extended with the first 15 nt of their neighbouring exons and compared with the local exon environments with the same Logo analysis for the “control” group of 38 sister stwintrons .Hypoxylon sp. CO27-5 stwintrons. There were two instances of [TAG] directly upstream of a stwintron terminating with the 3′-acceptor TAG, but [CAG] did not occur directly upstream of any of the Hypoxylon sp. CO27-5 stwintrons.The insertion sites of stwintrons of both groups are seemingly not sequence biased; there were no conserved sequence patterns in the exons directly next to the stwintron–exon junctions. In line with the above, there was no prevalence for alternatively spliced / stwintrons  . The comMeanwhile, [GT] directly downstream of most of the 3′-acceptors of the stwintron occurred in eight of the 117 stwintrons, while [GTA] was present in two of them .All 81 UO stwintrons were seamlessly integrated in continuous exonic sequences, without loss or gain of exonic sequences, very much as it was observed for all but one of the sister stwintrons (exception described in ). Their Collemare and co-workers  formulatHypoxylon sp. CO27-5/EC38 and Hypoxylon sp. E7406B/H. pulicicidum . Their In general, the differences in stwintron size were confined to the core of the constituent intron between the 5′-donor and the lariat branch point sequence (BP). By contrast, we observed a proclivity to conserve a relatively short distance between the canonical branch point sequence (BP) and (associated) 3′-acceptor for both the internal and the external intron in the older UO stwintrons. The distance between the canonical lariat branch point sequence (BP) and 3′-acceptor of the internal intron was seven nucleotides in 34 of the 38 sister stwintrons (89%) as well as in 49 of the 81 UO stwintrons (60%) along with 11 other stwintrons with this distance being six nucleotides and five other stwintrons with this distance being eight nucleotides . For theHypoxylon sp. CO27-5 (not shown). In line with what has been observed in model ascomycete fungi  stwintrons contain more AU than GC  in the 81 UO stwintrons newly identified and found that without exception, some level of sequence symmetry could be detected intron propagation.Almost all of the symmetries shown in -workers  that manDanio rerio) several years ago, where splicing depends on the intron’s RNA secondary structure  stwintrons m-(GT)n introns are spliceosomal introns with secondary structures that appear to be under positive selective pressure to guarantee their proper excision . Howeverwintrons . By defiwintrons .Hypoxylon sister stwintrons by intron definition is by far prevalent over splice-site pairing mediated by the secondary structure of the complete intervening sequence. The exact removal of the stwintron sequence by two consecutive conventional splicing reactions would occur despite the remarkable observation that the internal splice sites of sister stwintrons could mask each other from recognition by the assembling spliceosome. This prevalence would imply that in evolutionary older UO stwintrons that occur in most sequenced species of the Hypoxylaceae family , the secondary structure of the whole intervening sequence is not conserved between species, because the selective pressure bears solely on the correct excision by two consecutive standard U2 splicing reactions. We tested this in nine  stwintrons that were position-conserved in most species of the family  than in the external intron of most of the 81 older stwintrons (5–7 nt in 75% of the latter), albeit essentially similarly short as the latter for the internal introns of both groups of stwintrons. The presence of an underlying symmetry in all 117 stwintrons is surprising, although the symmetry is more pronounced near the extremities of most of the 23 full-length sister stwintrons. This terminal inverted repeat has the potential to form an extensive secondary structure that brings in close proximity the stwintron’s most distal 5′- and 3′-splice sites. This raises the prospect of secondary structure-mediated excision of almost the whole stwintron sequence in one reaction. However, the Hypoxylon stwintrons are overwhelmingly excised by consecutive splicing reactions that precisely remove the intervening sequence, rather than being engaged in one splicing event that creates a frameshift (+1) in the joined exons. Indeed, SRA evidence (cf., For the first time, the existence of stwintrons of nested canonical U2 introns could be studied, analysing more than one hundred  stwintrons present in one fungus, nce cf.,  suggest


Because of its high prevalence and poor long-term clinical treatment effect, liver disease is regarded as a major public health problem around the world. Among them, viral hepatitis, fatty liver, cirrhosis, non-alcoholic fatty liver disease (NAFLD), and autoimmune liver disease are common causes and inducements of liver injury, and play an important role in the occurrence and development of hepatocellular carcinoma (HCC). Tanshinone IIA (TsIIA) is a fat soluble polyphenol of Salvia miltiorrhiza that is extracted from Salvia miltiorrhiza. Because of its strong biological activity , it is widely used in Asia to treat cardiovascular and liver diseases. In addition, TsIIA has shown significant anti-HCC activity in previous studies. It not only has significant anti proliferation and pro apoptotic properties. It can also play an anti-cancer role by mediating a variety of signal pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-B (NF-κB). This review not only reviews the existing evidence and molecular mechanism of TsIIA’s anti-HCC effect but also reviews the liver-protective effect of TsIIA and its impact on liver fibrosis, NAFLD, and other risk factors for liver cancer. In addition, we also conducted network pharmacological analysis on TsIIA and HCC to further screen and explore the possible targets of TsIIA against hepatocellular carcinoma. It is expected to provide a theoretical basis for the development of anti-HCC-related drugs based on TsIIA. Globally, HCC causes a heavy disease burden and is the main cause of cancer-related deaths in many regions of the world. It is estimated that HCC is the fourth most common cause of cancer-related deaths in the world , 2. In CSo far, there are many therapeutic methods for liver cancer, including orthotopic liver transplantation, hepatectomy, ablation technology, interventional therapy, radiotherapy, biotherapy, molecularly targeted therapy, and the use of traditional Chinese medicine. However, these therapies have their own advantages and disadvantages and do not always produce the best patient treatment effect –8. For ein vitro in  and mouse H22 liver cancer cells subcutaneously or in situ into mice with low immune function to establish animal models. It is finally confirmed that TsIIA can effectively inhibit tumor growth in vivo, no matter whether it is injected intravenously, subcutaneously or intraperitoneally. It is worth noting that in the current animal experiments, it has not been observed that TsIIA has toxic effects on the activity of other organs and tissues except tumor tissues. At the same time, we summarized the current in vivo experiments of TsIIA and HCC  inflammation will lead to the loss of a large number of liver cells, thus aggravating the severity of various liver diseases. These include liver ischemia reperfusion injury, nonalcoholic fatty liver disease (NAFLD), alcoholic hepatitis, liver fibrosis, and even HCC . Among t2.1Liver ischemia/reperfusion (I/R) injury is the main manifestation of liver injury after liver transplantation or hemorrhagic shock, with high incidence rate and mortality. The occurrence of I/R involves many molecular mechanisms . Interes2.2in vivo and in vitro experiments conducted by Park , concanavalin A (Con-A), D-galactosamine , acetaminophen, and lipopolysaccharide (LPS). The liver injury caused by toxic chemicals is mainly related to an immune reaction and inflammation. They usually lead to large-area apoptosis of liver tissue, which leads to liver fibrosis and finally leads to cirrhosis and HCC , 70, 71. by Park , they fo by Park  et al. T by Park . In fact by Park , which h by Park , 77. As  by Park . Among t by Park . Therefo by Park  It is wo by Park –82.3The occurrence of liver fibrosis is often mediated by a variety of mechanisms, including hepatitis B and C, alcohol consumption, fatty liver, cholestasis, and autoimmune hepatitis, which can be induced. The liver tissue fibrosis is driven by persistent liver injury and is subsequently characterized by the development of liver cirrhosis , 84. It Activation of hepatic stellate cells (HSC) plays a key role in liver fibrosis; activation of HSC plays a key role in myofibroblasts that produce extracellular matrix (ECM) in the liver , 86. IntLiver precursor cells, also known as oval cells, are the key to liver tissue repair after liver injury. After severe liver injury, liver oval cells proliferate and differentiate into several lineages, including bile duct epithelial cells, liver cells, etc., which drive the repair of damaged liver cells; therefore, liver precursor cells have significant significance for the treatment of liver fibrosis and cirrhosis , 94. PreCholestasis is characterized by decreased bile flow and bile acid accumulation. It is one of the most common but destructive liver diseases and is significantly related to the occurrence of liver fibrosis , 97. Int4In recent years, nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. In some countries, more than 40% of the population is affected. This makes NAFLD a worldwide public health burden and attracts worldwide attention . NAFLD iNAFLD is usually driven by many factors, among which metabolic syndrome is the most common. Metabolic syndrome not only significantly promotes the occurrence and progression of NAFLD but also plays a key role in the occurrence and progression of HCC , 112. In55.15.1.1Apoptosis refers to the process of spontaneous cell death regulated by genes, which plays an important role in maintaining the homeostasis of the internal environment and the normal growth and development of organisms.It is reported that the occurrence and progression of tumors often indicate an imbalance between apoptosis and proliferation. However, the signal molecules and proteins related to apoptosis have been regarded as effective targets for cancer prevention and treatment by scientists and have been extensively studied by the scientific community for a long time , 122. In5.1.2A normal cell cycle is essential for the homeostasis of the internal environment and the normal development of organisms. The disorder of this system often leads to uncontrolled cell proliferation, which leads to the occurrence of tumors.The cell cycle’s normal progression is dependent on the regulation of various cyclins, cell cycle dependent protein kinases, and cell cycle inhibitors. In mammals, cell cycle processes are orchestrated by molecular mechanisms centered on the phase-specific expression of cyclins. They form complexes with specific cyclin-dependent kinases (CDK) and promote the phosphorylation of the retinoblastoma tumor suppressor (Rb) to allow the cell cycle to progress to the next stage –125. The5.2in vitro and in vivo models constructed by Ma et al. with hepatoma cells Bel-7404, SMMC-7721, and Bel-7402. TsIIA promotes the apoptosis of hepatoma cells and inhibits cell proliferation, invasion, and migration by up-regulating the protein expression of SMAD7 mRNA in vitro and in vivo in a time- and dose-dependent manner. Moreover, it also reduced Ki67 (a marker reflecting tumor proliferation and high invasiveness) , the number of tumors visible on the lung surface of mice was observed: 14 ± 5, 12 ± 4, 9 ± 4, 6 ± 3 , dissolving extracellular matrix, promoting angiogenesis, recruiting certain cytokines and chemokines, and activating relevant molecular signals , 130, whiveness) . In factiveness) , 134. Iniveness) , while Tiveness) . This wa4, 6 ± 3 . In addi4, 6 ± 3  , sorafenib and its derivatives SC-1, trans resveratrol (RESv), 5-fluorouracil (5-FU), and TsIIA significantly enhanced the anti-tumor effect of anti-cancer drugs , 136. Iny 17.30% . In addiy 17.30% . As we ay 17.30% . Among ty 17.30% . It has y 17.30% . Howevery 17.30% . In facty 17.30% . Howevery 17.30% , 144.Of course, the combination of TsIIA and anticancer drugs can also significantly inhibit the migration and invasion of HCC. In the research of Qiu et al., 1.5 µg/mLTsIIA combined with 5 µM sorafenib and 1.5 µg/mLTsIIA combined with 5µM sorafenib derivative SC-1 were compared with sorafenib alone or its derivative SC-1. TsIIA in combination with sorafenib reduced Huh7 (21.3% ± 1.3%), and HepG2 (116% ± 13.7%) cell migration when compared to the control group. TsIIA combined with SC-1 also reduced Huh7 (6.1% ± 0.7%) and HepG2 (24% ± 4.6%) cell migration. In terms of inhibiting cell invasion, TsIIA combined with sorafenib reduced Huh7 (27.1% ± 2.5%) and HepG2 (19.7% ± 2.5%) cell invasion compared with the control group. TsIIA combined with SC-1 reduced Huh7 (34.3% ± 4.4%) and HepG2 (12.9% ± 4.4%) cell invasion . Secondl6https://pubchem.ncbi.nlm.nih.gov), we screened drug targets using the Swiss target prediction database(http://www.swisstargetprediction.ch/) and the traditional Chinese Medicine System Pharmacology database(https://www.tcmsp-e.com/), and submitted the collected targets to the UniProt database (https://www.uniprot.org/), limiting the species to “Homo sapiens”, converting the protein targets into official gene names, select gene targets with probability greater than 0 in the Swiss target prediction database, and obtain drug target genes: Tanshinone IIA (145), after excluding duplicate genes. Secondly, we searched the Genecards(https://www.genecards.org/) and Disgenet databases(https://www.disgenet.org/) by using the keyword “Hepatocellular carcinoma” to obtain disease targets, and obtained 9637 disease target genes after excluding duplicate targets in the two databases. Then, we input the drug target genes and disease target genes obtained by the above methods into the online Venny 2.1 mapping platform(https://www.bioinformatics.com.cn/)to obtain the cross target genes of “Hepatocellular carcinoma” and “Tanshinone IIA” 121 to form a protein-protein interaction map. We set the species as “human”and the comprehensive score > 0.4 is the critical value for inclusion in the network. Then we further visualized these results with the help of Cytoscape 3.9.1 (https://david.ncifcrf.gov/tools.jsp) and setting the species as “Homo species”,we further analyzed the enrichment analysis of TsIIA on HCC related biological processes (BP), cell components (CC), molecular functions (MF) and signal pathways. For the obtained information, we meet the p-value<0.05, and select the first 10 enrichment information of BP, CC and MF and the first 20 enrichment information of KEGG according to the sequence of gene number, and use bioinformatics online platform(https://www.bioinformatics.com.cn/)Visualize the analysis results , myc proto-oncogene protein (myc), transcription factor AP-1 (JUN), Proto-oncogene tyrosine-protein kinase SRC(SRC), caspase-3 (CASP3), and epidermal growth factor receptor(EGFR) play a key role in the anti-HCC process of TsIIA. In addition, previous studies have confirmed that TsIIA, as a fat-soluble polyphenol, can enter cells directly through the cell membrane due to its fat-soluble property and rely on the direct combination of the D ring in the molecular structure of TsIIA with the groove in the DNA molecule, thus interfering with the synthesis and transcription of DNA and thus exerting the anti-tumor effect Figure 67As we all know, the occurrence of tumors involves multiple pathways and multiple targets, and blocking a single signal pathway or gene product is usually not enough to prevent or treat malignant tumors. Previous studies have shown that the occurrence of HCC not only involves the abnormal expression and mutation of multiple oncogenes , but also is regulated by multiple abnormally activated signal pathways  . They joMitogen-activated protein kinase (MAPK) is a serine/threonine kinase that plays an important role in regulating cell activity and signal transduction, including cell proliferation, differentiation, survival, death, and transformation, and is closely related to the occurrence and transfer of HCC, especially in HCC caused by HBV infection, because HBV can encode a regulatory protein (HBx). This protein can activate some genes involved in cell proliferation regulation in the MAPK pathway, such as Ras, Raf, ERK, and JNK, to increase proliferation and inhibit apoptosis –155. HowFurthermore, numerous laboratory studies show that TsIIA can treat cancer by regulating the microRNA (miRNA) in tumor cells. It includes regulating the miR30b-p53 PTPN11/SHP2 pathway to induce the death of liver cancer cells , up-regu8Since 1968, Ballie and Thomson et al. have fully synthesized TsIIA for the first time with 7-methoxy-1-tetrahydrozolidone as the starting material. The synthesis and clinical application of TsIIA and its derivatives have been widely discussed by scholars at home and abroad. Therefore, we also derived many synthetic routes for TsIIA, including substituted benzene synthesis, naphthalene derivative synthesis, Diels-Alder addition, and other synthetic routes . Secondl9Although the clinical application of TsIIA as an anti-tumor drug still has a long way to go, with the in-depth research of scientists, we also see the hope of solving this problem. Many derivatives related to TsIIA are also being prepared. Among them, STS is a water-soluble derivative of TsIIA, which has been used in the treatment of coronary heart disease, angina pectoris, etc. . After iIn addition, the development of nanoparticle technology also provides a direction for the clinical transformation of TsIIA. Compared with TsIIA suspension, all kinds of nanoparticles containing TsIIA reported in the existing literature achieve higher concentration and longer retention time in the liver. These nano preparationinclude nano particles formed by encapsulating TsIIA into globins (TA Gb NPs),novel poly (lactic acid) nanoparticles containing TsIIA (TS PLA NPs) were synthesized by a single oil in water lotion/solvent evaporation method.The preparation formed by loading TsIIA into mPEG-PLGA-PLL-cRGD  nanoparticles (TNP) , 44, 19010Up to now, The occurrence of cancer is often a process of multiple targets and pathways. Therefore, highly effective anticancer drugs often need to have multi pathway and multi target biological activities.However, TsIIA is expected to become a candidate drug for HCC treatment due to its ability to inhibit the proliferation, survival and migration of human HCC through multiple targets, links and pathways, and as a synergist of chemotherapy drugs. It can even change their drug resistance.Although TsIIA has been widely reported to be beneficial to health, lead to its clinical application is limited due to its low bioavailability. Fortunately, as described above, scientists are preparing microemulsions, microspheres, solid dispersions, liposomes, nanoparticles and other new dosage forms of TsIIA injection to significantly increase the drug concentration of TsIIA in HCC tissues and improve its bioavailability.However, in order to better understand the mechanism of action of TsIIA, improve the bioavailability, safety, dose effect and stability of TsIIA, and transform TsIIA into a candidate drug for treating HCC, further preclinical and clinical studies are needed in the future.in vivo experiments of TsIIA and HCC, we did not find that TsIIA had toxic effects on other tissues except HCC. But these studies are based on in vitro cell experiments and in vivo animal experiments. Therefore, future research needs us to make clear the metabolism of TsIIA in human liver tissue, its side effects, and safety through well-designed human intervention experiments and pharmacokinetic experiments. In conclusion, all experimental data show that TsIIA can inhibit inflammatory reactions, oxidative stress, mediate apoptosis signal molecules, improve obesity and diabetes, promote stagnation of the hepatocyte cycle, and inhibit angiogenesis of hepatocytes in vivo and in vitro.These biological effects promote that TsIIA is expected to become a new drug or auxiliary drug for anti HCC.Secondly, the safety of the extraction of natural plants will also hinder the clinical application of drugs. Fortunately, the safety of TsIIA has been gradually confirmed. At present, the TsIIA aqueous solvent STS has been widely, safely, and effectively used in clinical practice in China. At the same time, in our review of the ZZ conceptualized the review, analyzed the data, and helped write the manuscript. HL, HP, YZ, XZ, XL, LY, and YX helped to write the manuscript and prepared the figures. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.


The study aims to report on the feasibility and associated adverse events of HPV-Vaccination (HPVV) in a private clinic setting in Lebanon and, when available, the results of subsequent cervical cancer screening.  Opportunistic HPV vaccination is offered at the Women's Health Center of the AUBMC. We retrospectively reviewed the patients' demographic data, the incidence of adverse events, and their cytological screening. n = 1013) aged 26.2 years (12–54 years) were opportunistically vaccinated with one of two HPV vaccines; 845 (83.4%) received the quadrivalent vaccine (Q4V), and 151 (14.1%) received the bivalent vaccine (B2V). The majority (75.8%) received three doses while 16% received two doses. Out of these women, 26.3% (267) became sexually active postvaccination (NS2), whereas 17% (174) were sexually active prior to vaccination (SA) and the rest 57% (572) reported no sexual activity (NS1). Among the SA group, 26% (46/147) presented with abnormal cytology at time of vaccination. As for the NS2 women, 5% (14/267) had subsequently abnormal screening within 37 (12–103) months following vaccination.  A cohort of healthy women ( In this observational study, we report the successful introduction of HPVV with negligible adverse events. The incidence of abnormal cervical cytology was low among our patients. Of all cancers worldwide, 4.5% are attributable to HPV (8.6% in women versus 0.8% in men) , 2. CervSecondary prevention via cytological testing, despite its low sensitivity, and more recently, HPV DNA testing, with its higher sensitivity, have both contributed to the significant reduction in the incidence and mortality of cervical cancer. However, most countries in the EMENA region, including Lebanon, lack organized screening . The timUnlike the rest of the world, the introduction of organized HPVV has been very slow in the EMENA region, except for Abu Dhabi. In Lebanon, data on HPV vaccine uptake is lacking, as well as demographic variables associated with vaccine uptake and the continuation rate for all three doses . In addiWe report on the introduction of HPVV in an adult, private, academic setting in Beirut, Lebanon. We analyze the associated demographic variables, the rate of completion of the three-dose schedule, the incidence of adverse events, and the subsequent incidence of abnormal cytology postvaccination in sexually naïve and sexually active patients.As of 2007, opportunistic HPVV was offered to women and their adolescent daughters attending the Women's Health Center (WHC) of the American University of Beirut-Medical Center (AUBMC). Following nondirective counseling by physicians and nurse practitioners, women or parents of adolescents chose one of the two available vaccines (Q4V or B2V). The initial purpose of this prospectively collected database was to monitor adverse events (AEs) and to send reminders to vaccine recipients to complete their subsequent doses on time. The patients were followed up as part of the standard of care by the gynecology nurse specialist and called consequently to ensure the completion of HPVV doses. We performed a retrospective review of the electronic medical records of these females from 2007–2017 after obtaining the Institutional Review Board (IRB)'s approval in Beirut (IRB code: OGY.MS.11). Informed consent was waived by the IRB of the American University of Beirut as it did not involve any direct contact with the patients for the aims of the study. The collected data included demographic data such as age, medical history, and residence; the type of vaccine used; batch number; the number of doses received; and any AEs that could have led to the discontinuation of the rest of the vaccination doses. We did not collect data on those who were offered vaccines and refused as per our IRB proposal. In addition, for those women who were or became sexually active, we coupled these data with our cytology laboratory data to report on the rates of subsequent abnormal cytological screening. Initially, all women aged 12 years and <26 years were offered to be vaccinated, but later the age was increased to 45 years.Women and girls were offered vaccination if they were healthy with no chronic medical illness, not immunocompromised, and not currently treated for cancer, including any genital cancer. A speculum examination was performed in nonvirginal patients and a Pap smear was collected using either an Ayre's spatula or a cytobrush to exclude any lesions suggestive of cancer. Cytological samples were in alcohol and sent to the AUBMC Cytopathology lab, which is accredited by the College of American Pathologists (CAP). 2%) of patients had unsatisfactory cytological samples and were recalled for repeat testing. The cytology slides were read by cytopathologists who were board-certified in Cytopathology by the American Board of Pathology and were unaware of the patients' vaccination status or prior history.t-test and chi-square, and statistical significance was considered at p < 0.01 and 95% CI.Management of the abnormal screening followed the 2012 ASCCP guidelines . Statistp value <0.001). After 2012, there was a nonsignificant trend toward a two-dose vaccination schedule in both Q4V and B2V (p = 0.022). The mean age at the first Pap smear in the 267 patients who were or became sexually active was 27.7 ± 4.7 (17–45) years. During that same period, the average rate of abnormal cytology in AUBMC ranged from 1.7 to 2.3% .From 2007 to 2017, 1013 females were vaccinated with one of the two available HPV vaccines; 845 (83.4%) received Q4V and 151 (14.1%) received B2V. The average age of the recipients was 26.2 ± 5.8 (12–54) years. The majority (75.8%) completed all three doses while only 16% received two doses. Between 2007 and 2012, 88.3% (481/545) completed three doses, while between 2013 and 2017, 74.6% and 25.4% (98/386) received three and two doses, respectively , 26% (46) had biopsy-proven CIN; seven were CIN2+ and 39 < CIN2  vs. 29 (18–38), 9 < CIN2 . Patient9 < CIN2 . In the p value = 0.50, p-value = 0.53].The time to recurrence of all lesions (<CIN2 and CIN2+) in NS2 and SA groups was not statistically significantly different  that included a major reaction that included edema of the face and inability to breath following the administration of the first dose of Q4V; otherwise, there were no other AEs, namely, syncope or reported local injection site reactions.The prevalence of HPV infection among women in the Middle East and Lebanon is estimated to be around 4–10% , 18–21. According to the World Health Organization (WHO), the highest impact on the mortality of cervical cancer will most likely occur when HPV vaccination is offered in an organized universal fashion where more than 90% of eligible women are vaccinated . HoweverHowever, our experience still reflects a successful introduction of HPVV in a private setting in a country with a relatively low incidence of HPV-related diseases. Because it is a Women's Health Center catering to a mostly adult population and not a pediatric clinic setting, the average age of the vaccinated women is much older than in typical organized school-based vaccination campaigns. It is noteworthy that following the release of the data on the efficacy of the two doses of the vaccine in younger girls in 2013, some older girls and women chose to skip the third dose. Nevertheless, the overall uptake of the three-dose regimen remained high. There were no noticeable or reportable AEs except for only one side effect of vaccination found in our study. The interval from the time of vaccination until the performance of the first Pap smear decreased over time, and the subsequent rate of abnormal Pap smears with both vaccines was rather low. This rate, however, is higher than in the overall population of the AUBMC (1.7–2.0%). These findings may be attributed to an earlier onset of sexual activity after vaccination, or maybe women were getting vaccinated because they were intending to get married or become sexually active, or they may reflect an earlier screening pattern over the years. At our institution, the current high cost of HPV screening ($USD 170) is still a major obstacle to using it as a primary screening tool since it is not covered by most third-party payers and most women have to pay the cost of screening out-of-pocket.It is hard from this small sample and the short follow-up duration of an average of up to 4.5 years, to estimate the impact of vaccination in both the SA and NS2 groups. Thus, a larger sample size is needed to assess the protective effect of the vaccine.Moreover, the incidence of adverse events was very small except for the anaphylactic reaction following the first dose of the Q4V, which was reported with the batch number at that time to the manufacturer.The strength of the study is that it is the first comprehensive review of the introduction of HPV vaccination in adult females and adolescent girls in Lebanon and the region with a relatively high continuation rate and low rate of adverse events. The reporting of the follow-up cytological screening, although over a short period, is also noticeable. The limitations of the study are the unknown HPV status of women at baseline and in a country with limited resources and the high cost of HPV testing. Moreover, the study confirms the low rate of CIN2+ lesions in both the AS and the NS2 patients and the low recurrence rates postvaccination as reported .In summary, HPVV can be safely introduced in a private WHC setting, although it does not replace a universal organized vaccination campaign at a younger age. It can be used as an example of a successful catch-up campaign. The high three-dose-schedule continuation rate reflects the high level of education of our patients and the effectiveness of the WHC recall system to ensure the completion of all three doses of the vaccine.


INK4A expression of 224 participants. Global DNA methylation was evaluated through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).Penile cancer is one of the most aggressive male tumors. Although it is preventable, the main etiologic causes are lifestyle behaviors and viral infection, such as human papillomavirus (HPV). Long-term epigenetic changes due to environmental factors change cell fate and promote carcinogenesis, being an important marker of prognosis. We evaluated epidemiological aspects of penile squamous cell carcinoma (SCC) and the prevalence of HPV infection using high-risk HPV (hrHPV) and p16INK4a. hrHPV was not related to systemic or lymph node metastasis and locoregional recurrence, nor influenced the survival rate. P16INK4a seems to be a protective factor for death, which does not affect metastasis or tumor recurrence. Lymph node and systemic metastases and locoregional recurrence increase the risk of death. An increased 5mC mark was observed in penile SCC regardless of HPV infection. However, there is a reduction of the 5hmC mark for p16INK4a + (P = 0.024). Increased 5mC/5hmC ratio (> 1) was observed in 94.2% of penile SCC, irrespective of HPV infection. Despite the increase in 5mC, it seems not to affect the survival rate .The incidence of HPV was 53.2% for hrHPV and 22.32% for p16INK4a seems to be a good prognosis marker for penile SCC and the increase in 5mC, an epigenetic mark of genomic stability, may support tumor progression leading to poor prognosis.P16The online version contains supplementary material available at 10.1186/s13148-022-01360-1. Penile cancer is a rare tumor type with an increased worldwide incidence, having 36,068 new cases registered in 2020. The majority of cases occur in regions with a low human development index, where India 10,677), China 4,628) and Brazil  are the most affected countries [0,677, Ch,628 and Different histological types are associated with penile cancer, such as sarcoma, melanoma and basal cell carcinoma , 8, neveThe prevalence of HPV in penile neoplasia can vary widely depending on the literature and among different regions of the world, ranging from 11 to 87% , 20. AssEnvironmentally induced epigenetic changes have been recently added as hallmarks of cancer, which contribute to tumor initiation and progression . Global INK4a expression will provide better knowledge about the molecular mechanisms related to penile SCC. Therefore, evaluate the prevalence of HPV infection and 5mC and 5hmC epigenetic marks in penile SCC and its association with clinicopathological alterations. As an important mechanism of genomic stability, aberrant epigenetic reprogramming related to penile SCC pathogenesis and viral infection may be used as a biomarker for prognosis and targeted therapies. Determining the prevalence of HPV, one of the most common infections of the reproductive tract, is important for the development of public health strategies for the prevention and treatment of related diseases.Since HPV infection may drive epigenetic changes, the characterization of the global DNA methylation and its association with hrHPV DNA and p16INK4a immunodepression and reached 53.2% when tested by hrHPV hybridization. Low-risk HPV infection was not identified. Tumor staging based on the primary tumor was mainly corpus spongiosum invasion  followed by the first stages including carcinoma in situ (pTis), noninvasive carcinoma (pTa), subepithelial invasion without lymphovascular invasion (pT1a) and with lymphovascular invasion (pT1b) (30.1%).The participants’ age, HPV infection, pathological classification, and treatments of penile SCC are presented in Table INK4a or hrHPV, a positive correlation was observed between these markers . All other clinicopathological variables were not associated with HPV markers p16INK4a and hrHPV.Systemic metastasis was observed in 16.5% of participants diagnosed with penile SCC, while lymph node metastasis was presented in 40.1% and locoregional recurrence in 26.1%. Intense peritumoral lymphocyte infiltrate (PLI) and intratumoral polymorphonuclear infiltrate (IPI) reached 40.1% and 41.5% of penile SCC, respectively. The main therapeutic strategies were partial amputation 73.2% and total amputation 14.7%, followed by other associated strategies (12.1%) such as prostatectomy, emasculation, and exercise injury. Chemotherapy was the treatment option for 25.8% of men, which consisted of cisplatin associated with 5-Fluorouracil or cisplatin associated with taxol or paclitaxel, as a palliative or neoadjuvant therapy before and after lymphadenectomy. Radiotherapy was used only as a palliative alternative for pain in cases of metastasis (14.1%) (Table P < 0.001), systemic metastasis  and lymph node metastasis  decreased the survival rate in penile SCC. On the other hand, p16INK4a expression seems to be a protective factor for death  and hrHPV  did not affect survival even with a HR < 1. The remaining parameters evaluated did not affect the survival rate. However, on the non-adjusted Kaplan–Meier curves metastasis, recurrence, chemotherapy, radiotherapy .The survival rate was measured by a multivariate-adjusted Cox hazards regression model  was observed , as well as 5hmC .Increased global 5mC (> 60%) mark was observed in 89.2% of penile SCC cases, regardless of HPV infection . Despite the elevated incidence, HPV infection was not related to systemic or lymph node metastasis and locoregional recurrence and, therefore, does not influence the survival rate. Long-term changes in DNA methylation are characteristic of environmentally induced carcinogenesis as penile cancer [Penile cancer is one of the most aggressive male malignancies, although it is one of the easiest to prevent, as the main etiologic causes are lifestyle conditions and viral infection . We obsee cancer . IncreasThe incidence of penile cancer directly impacts public health, as the specialized medical care, psychosocial impact and the risk of cervical cancer in their female partners increase the cost of treatment . DespiteINK4a, the incidence of HPV infection was twofold higher using hrHPV than p16INK4a, and only 17% of all cases were double positive for both markers. Considering that the tumor suppressor protein p16INK4a is upregulated by the HPV oncogene [INK4a expression may be linked to gene expression loss, caused by promoter methylation events and loss of heterozygosity [n = 3772 cases in 47 studies) and 42.1% (95% CI 36.4–47.8) using p16INK4a (n = 1296 cases in 23 studies) in SCC [INK4a must be considered for some types of cancer, such as head and neck SCC [Despite the correlation between hrHPV and p16oncogene , 46, HPVzygosity , 46, impzygosity . A recenneck SCC .INK4a were not related to disease staging, metastasis, or infiltration of the immune system cells. On the other hand, p16INK4a seems to be a protective factor for death, as well as hrHPV (HR < 1), although it was not significant, an increase in sample size would answer this question. In addition, positive cases for p16INK4a seem to be associated with radiotherapy. Although previous studies associate p16INK4a expression and radiotherapy [INK4a was correlated with a good prognosis, as lack of nodal metastasis in head and neck SCC [INK4a was not associated with prognosis parameters or survival rate [Although previously mentioned as a risk factor for penile SCC , 23, ourotherapy , 76, thineck SCC , 77 and neck SCC . In penival rate .The presence of lymph node metastasis in the inguinal and iliac regions increased the risk of death, which is the most common metastatic event  and the Epigenetic mechanisms, like DNA methylation, present great plasticity as the epigenome can be reprogrammed through environmental factors , 69. AbeINK4a negative diagnosis. Decreased level of 5hmC mark was observed in head and neck cancers positive for HPV, and virus presence might influence the oxidation process from 5mC to 5hmC in genes of cell junction pathways [The increased 5mC marker was accompanied by a reduced level of the 5hmc mark in the majority of penile SCC, which was associated with a p16pathways . 5hmC ispathways , which dpathways . Aberranpathways . Furtherpathways  and cervpathways .Given that 5mC is a protective marker against DNA damage and the hydroxymethylation of 5mC is an active demethylation process, the balance between 5mC and 5hmC marks is directly linked to genomic stability, contributing to cancer development and progression . We obseDespite the important findings regarding the HPV incidence and the association with the 5mC/5hmC mark with penile SCC prognosis, other studies should address the epidemiological characteristics of penile cancer and epigenetic reprogramming through lifestyle changes. Our study was limited by the lack of information regarding the participant's background, such as sexual behavior, sociodemographic profile, HPV vaccination, and the identification of other related histological subtypes and tumor topography. Even though we evaluated hrHPV and lrHPV, there is a diversity of viral genotypes that have not been identified individually and should give better knowledge regarding the infection and disease prognosis, as well as the time of exposure to infection that was not tracked. Although partial and total penectomy represents better survival compared to even more invasive ones, these techniques have drastic implications for the individual's personal life, which calls for less invasive therapies. The epidemiologic data, for staging and surgical excision, suggest that medical care is just sought late raising costs in specialized treatment. The elevated incidence of HPV highlights the importance of policies to encourage vaccination to control viral infection in the male population.INK4a negative samples. This information should be further explored as this data may predict potential clinical relevance for penile cancer prognosis and may suggest new treatment strategies, as hypomethylating agents for epigenetic targeted therapies (epidrugs). The poor prognosis of penile cancer and its relationship with environmentally induced changes reinforces the benefits of primary health care and the elevated incidence of hrHPV highlights the importance of vaccination and continued educational strategies for both prevention and treatment of malignant lesions.We reported an incidence of 53.2% of hrHPV infection in men with penile SCC in the State of Ceará, Northeast Brazil, a region with the lowest per capita income in the country. Despite the increased incidence, HPV infection was not associated with poor prognosis, such as systemic or lymph node metastasis, locoregional recurrence, but it seems to increase the survival rate. The current research also indicates, for the first time, that increased global DNA 5mC and reduced 5hmC marks are characteristics of penile SCC. Despite no statistical difference, increased 5mC may contribute to poor prognosis as the HR was 1.06 in SCC. Although viral infection contributes to the loss of aberrant DNA reprogramming, the 5mC/5hmC ratio was not related to HPV infection, and 5hmC levels were increased in p16This is a retrospective study that included 224 participants with penile SCC who underwent partial or total penectomy, and advantage stage with enlarged prostatectomy and emasculation, without any prior history of chemotherapy or radiotherapy. Penile SCC samples were obtained at Hospital Haroldo Juaçaba, Ceará, Northeast Brazil, and detailed clinicopathological and follow-up data were assessed from 2000 to 2018. The study was approved by our Institutional Review Board (process number 2.427.846).INK4a, hrHPV, 5mC, and 5hmC markings, the number of samples available for analysis varied  2017) . Local r7 . LocalThe Tissue MicroArray (TMA) blocks were composed of representative 2.0 mm cores, in duplicate, from the 224 samples from the epidemiological study cases. Blocks were sectioned to 4 μm thick and mounted on glass slides with an organosilane-based adhesive . TMA sections were then used for staining in HPV detection assays and analyses of 5mC/5hmC epigenetic markers. All the experiments were analyzed by a blinded observer.INK4a immunoexpression was carried out using an anti-p16 antibody clone E6H4 . p16INK4a expression must be ≥ 75% in neoplastic cells to be considered positive in immunohistochemistry (IHC) staining, with continuous and complete cytoplasmic and nuclear staining [The p16staining . ChromogThe TMA sections were stained with anti-5-methylcytosine (anti-5mC) and anti-5-hydroxymethylcytosine (anti-5hmC) antibodies using streptoavidin-biotin IHC. Briefly, after deparaffinization and rehydration, TMA slides were submitted to antigenic recovery in citrate pH 6.0 0.01 M. The slides were, then, blocked by endogenous peroxidase in a 3% hydrogen peroxide solution for 30 min, to block unspecified staining. Subsequently, slides were incubated with primary antibodies  in a humid and dark chamber at 4 °C overnight. Secondary incubation took place with the DAKO EnVision™ kit  for 30 min at room temperature and counterstained with Mayer’s hematoxylin. The analysis of 5mC and 5hmC was performed by evaluating the percentage of positivity of 100 cells in the tumor area of each TMA sample, in duplicate, for each target. Counting was made by two independent observers and the percent positivity rate was used to construct the score (% score). As each sample had 5mC and 5hmC scores, the 5mC/5hmC ratio was calculated for all participants. We used brain samples as positive control and the absence of the primary antibody during the incubation as the negative control.INK4a and hrHPV and the Wilcoxon nonparametric test for independent variables was applied to verify the relation between quantitative variables and p16INK4a and hrHPV. A multivariate regression analysis of survival data was based on the Cox proportional hazards model using PHREG procedure. The characteristics with reduced sample size as staging (pT4) were not included in the model. The Student's t-test was used to compare 5mC and 5hmC marks and HPV infection. All statistical analyses were performed using SAS® 9.3 software , and P < 0.05 was considered significant. The Kaplan–Meier curves were predicted by log-rank (Mantel-Cox) test performed in GraphPad Prism version 8.4.2. For survival curves, the sample size evaluated to time to death is presented in Table Qualitative variables were summarized considering absolute and relative frequencies. The Fisher’s exact test was applied to verify which qualitative variables are associated with p16Additional file 1. Study flowchart, immunohistochemistry and survival rate for penile squamous cell carcinoma.