Cervical cancer caused by persistent infection with HR HPV genotypes is the second leading cause of death in women aged 15 to 44 in Serbia. The expression of the E6 and E7 HPV oncogenes is considered as a promising biomarker in diagnosing high-grade squamous intraepithelial lesions (HSIL). This study aimed to evaluate HPV mRNA and DNA tests, compare the results according to the severity of the lesions, and assess the predictive potential for the diagnosis of HSIL. Cervical specimens were obtained at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia, during 2017–2021. The 365 samples were collected using the ThinPrep Pap test. The cytology slides were evaluated according to the Bethesda 2014 System. Using a real-time PCR test, HPV DNA was detected and genotyped, while the RT-PCR proved the presence of E6 and E7 mRNA. The most common genotypes in Serbian women are HPV 16, 31, 33, and 51. Oncogenic activity was demonstrated in 67% of HPV-positive women. A comparison of the HPV DNA and mRNA tests to assess the progression of cervical intraepithelial lesions indicated that higher specificity (89.1%) and positive predictive value (69.8–78.7%) were expressed by the E6/E7 mRNA test, while higher sensitivity was recorded when using the HPV DNA test (67.6–88%). The results determine the higher probability of detecting HPV infection by 7% provided by the mRNA test. The detected E6/E7 mRNA HR HPVs have a predictive potential in assessing the diagnosis of HSIL. The oncogenic activity of HPV 16 and age were the risk factors with the strongest predictive values for the development of HSIL. It is estimated that approximately every fourth malignancy can be linked to an infectious agent, that is, its contribution to various stages of cancer development (reviewed in ). About HPV vaccination is a crucial prevention tool against HPV infection and HPV-related precancers and cancers . If vaccStrong evidence for HPV as a causative aetiology of cancers of various locations was provided by the IARC, which classified HPV according to its potential to cause malignant cell alteration as follows :Group 1  includes HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59;Group 2A (probably carcinogenic) includes HPV genotype 68;Group 2B  includes HPV genotypes: 26, 53, 66, 67, 70, 73, 82, 30, 34, 69, 85 and 97;Group 3  includes HPV genotypes 6 and 11.Persistent HPV infection is the most critical risk factor for the development of cervical cancer, which is confirmed by the presence of HR HPV in over 99% of cervical cancer samples. Concerning the oncogenic potential, infection with a particular HR HPV genotype carries a specific risk for cellular transformation and malignancy (reviewed in ). NamelyDuring viral genome integration into the host cell genome, E1 or E2 are usually disrupted . This geGiven the above, this research aimed to determine the oncogenic activity of the most commonly diagnosed HR HPVs in cervical smear samples using the mRNA test and compare the results according to the severity of the cervical intraepithelial lesion. Furthermore, it aimed to examine the clinical characteristics and predictive potential in assessing the diagnosis of high-grade cervical intraepithelial lesions of HPV DNA and mRNA tests.From 2017 to 2021, cervical smears were obtained from a sample of 365 female patients (age 20–74 years) with normal and abnormal results of cervical cytology who were undergoing gynecological exams at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia. All of the women in the study did not receive any prior treatment for cervical dysplasia or cancer, and all were unvaccinated against HPV infection. The samples were collected using the ThinPrep Pap test (Hologic Inc.) according to the manufacturer’s instructions and sent for further analyses to the Center of Virology, Institute of Public Health of Vojvodina, Novi Sad, Republic of Serbia.The classification of cytological findings was performed according to the criteria of the Bethesda System 2014. It was categorized into negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL). All of the women enrolled in the study were informed about the research objective and signed an informed written consent form. The study protocol was reviewed and approved by the Medical Ethical Committee of the Institute of Public Health of Vojvodina, Novi Sad, Serbia .g for 5 min. The formed pellet was dissolved in 200 µL of nuclease-free water and used for nucleic acid extraction. According to the manufacturer’s instructions, DNA extraction was carried out using the SaMag STD DNA Extraction Kit . The extracted DNA was eluted in 100 μL elution buffer. The detection and genotyping of 12 HR HPV genotypes , marked as the HPV DNA test, were performed using the High Risk Typing Real-TM Kit  following manufacturer’s instructions. The E7 gene of specific HPV genotypes was amplified using primers and TaqMan probes in the multiplex reaction performed in a total of 13 μL. The β globin gene is used as an internal control. Real-time PCR was performed on the SaCycler-96 . After the initial activation of the DNA polymerase at 95 °C for 15 min, five cycles of amplification were performed under the following conditions: 95 °C/5 s, 60 °C/20 s, and 72 °C/15 s, and 40 amplifications were performed under the following conditions: 95 °C/5 s, 60 °C/30 s (fluorescence detection), and 72 °C/15 s. The kinetics of the detected fluorescence signals were monitored using the SaCycler-96 software package .The ThinPrep cervical smear samples were stored at 4–8 °C for up to 3 days from the sampling day. The 2 mL of collected samples were transferred to nuclease-free tubes and centrifugated at 8000× g). The supernatant was carefully transferred to a nuclease-free tube. The real-time reverse transcription PCR (RT-PCR) analysis, marked as the HPV mRNA test, was performed using specific primers and TaqMan probes to detect the E6/E7 mRNA of individual HPV genotypes. The sequences for the primers and probes  following the manufacturer’s instructions. The total RNA was eluted in 50 µL ultrapure water free from nucleases. Following the manufacturer’s recommendations, potentially present contaminants were removed using the TURBO DNA-free Kit . The routine procedure for removing contaminants using the kit above included the addition of 5 µL of 10× TURBO DNase Buffer and 1 µL of TURBO DNase enzyme into each sample of extracted total RNA, with incubation for 30 min at a temperature of 37 °C. After the action of the enzyme, 5 µL of inactivation reagent (Dnase Inactivation Reagent) was added, with incubation for 5 min, at room temperature and occasional vortexing. After that, centrifugation was performed  test of independence and quality of the match. The Student’s t-test was used to compare values between the two age groups, a numerical characteristic. A one-way analysis of variance (ANOVA) and the Bonferroni post-hoc test were applied to compare values between three or more data groups. Frequencies were used to present the analysis of the oncogenic activity of multiple HR HPV infections. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and their 95% confidence intervals (CIs) of HR HPVs DNA and E6/E7 mRNA HPVs detection and cytology test were calculated. To quantify the diagnostic capabilities of the selected test and evaluate its significance, the receiver operating characteristics (ROC) curve was used, which enables testing the significance of differences in the discriminating potential of different variables for the same binary outcome. It is based on a graphical presentation of pairs of sensitivity and specificity that can be obtained by estimating the threshold value for all values of discontinuous variables of the sample. Univariate and multivariate logistic regression were used to analyze the connection between two or more features, generating adequate statistical models. Multivariate logistic regression analysis was applied to all of the analyzed factors to construct a predictive model and named the most relevant predictors for the development of HSIL. A p-value of less than 0.05 defined as statistically significance.All of the statistical analyses were performed using SPSS statistics software Version 21.0 . Testing the difference in frequencies of attributive features was performed using the Chi-square  were classified based on the Bethesda System 2014 into four categories by cytological criteria.n = 274) in the study population . The most prevalent HPV genotype is HPV 16 which makes up 38.3% (105/274) of the total HPV-detected genotypes in 42.7% (105/246) of HP- DNA-positive samples. HPV 31 takes second place with 17.2% (47/274) of total HPV-detected genotypes in 19.1% (47/246) of HPV-DNA-positive samples. Equally represented are HPV 33 and HPV 51, each with 8.8% (24/274) of total HPV-detected genotypes in 9.8% (24/246) of HPV-DNA-positive samples (n = 172) for further examination of oncogenic activity, according to the study’s aim. Multiple HPV infections were found in 15.7%. The most common co-infections were those with HPV 16 and 31, found in 7.6% (13/172) of cases with multiple infections  had HPV-DNA-positive results, which indicates that the overall prevalence of HPV in the study population was 67.4%. All of the HPV genotypes covered by the HPV DNA test were identified ( samples . The resfections .n = 29), had normal results, whereas 83.1% (n = 143) showed different abnormalities. A total of 26.7% (n = 46) of the examined women had ASCUS; in 25.6% (n = 44), LSILs were found, whereas HSILs were detected in 30.8% (n = 53). The mean age of the patients was 36.7 years. Among the specimens, the number of Serbian women who were ≤30 years, 31–44 years, and ≥45 years old accounted for 36.5% (n = 68), 36.0% (n = 62), and 24.4% (n = 42) of the samples, respectively , including multiple infections. A minority of women, 16.9%  and HPV 31  was determined, depending on the degree of severity of the cytological findings, which was not determined for HPV 33 and 51  (The distribution of the most frequently detected HPVs concerning cytology is shown in ctively) .2 test; χ2 = 29.500; p = 0.000) (p < 0.001). The Bonferroni post-hoc test determined that the female patients with HSIL are statistically significantly older than those with ASCUS (p < 0.001), NILM (p < 0.001), and LSIL (p = 0.012) (p = 0.022). The average age of the female patients with confirmed HPV DNA 16 was 36.9 years; with HPV DNA 33, it was 34.1 years, while the average age of patients with HPV DNA 51 was 40.5 years. The statistical analyses show that the proportion of HPV 16 positivity maintained at the same level as age. Conversely, the proportion of HPV 31 positivity decreases with age. The detection of HR HPV 33 genotypes decreases with age, while HR HPV 51 increases  . The sta= 0.012) , Table 5n = 172) and HR-HPV-negative samples (n = 119), were tested by the HPV mRNA test. The E6/E7 mRNA HPV was detected exclusively in HR-HPV-DNA-positive samples . E6/E7 mRNA HPVs are the most prevalent in patients with HSIL cytological findings (88.9%). In the group of patients with LSIL cytological findings, it was demonstrated in a lower percentage (60%). A two-fold lower prevalence is observed in patients with ASCUS (29.4%). Oncogene activity in women with normal cytological findings is present in 10.9% of samples . This result singled out the HR HPV 16 genotype for further analyses. The presence of E6/E7 mRNA HR HPV 31 was the least detected in HSIL (7.5%) compared to other cytological groups (22.7–26.1%). The distribution of the oncogenic activity of the remaining genotypes (HR HPV 33 and 51) is approximately the same across different cytological groups and remains at a low level (2.2–11.4%)  .The analyses of the oncogenic activity of multiple HR HPV infections are presented by frequencies. The overall oncogenic activity, including both single and multiple HR HPV infections detected using the E6/E7 mRNA HR HPV test, increases with the degree of cervical lesion severity (60–100%). The oncogenic activity detected in single-genotype infections (20.0–85.7%) is higher compared to the oncogenic activity of multiple genotypes (0–40%) .2 test; χ2 = 7.331; p = 0.026), wherein individuals with positive E6/E7 mRNA HPV results were older than the others. The detection of E6/E7 mRNA HR HPV 31 and 33 decreases with the increasing age of the patient, while E6/E7 mRNA HR HPV 51 increases  to analyze the prevalence of E6/E7 mRNA HPV in the specific age groups. The lowest percent of E6/E7 mRNA HR HPV 16 was detected in the younger group and the highest percent was detected in patients over 44 years. A statistically significant difference was observed in patients with E6/E7 mRNA HR HPV 16 expression , similar in middle-aged women (31–44 years), and lower in women 45 years and older. The prevalence of oncogenic activity of the HPV genotypes increases with the severity of the cervical intraepithelial lesion. Compared to the prevalence of the examined HR HPV genotypes, the same parameter is lower in women with normal and undefined cytological findings, approximately the same in low-grade lesions, and significantly higher in high-grade lesions .The calculated clinical characteristics of the DNA and E6/E7 mRNA HR HPV tests are shown in To quantify the diagnostic capabilities of the E6/E7 mRNA HPV test and evaluate its significance, an ROC curve was used to assess the assays for detecting HSIL. The area determined by the ROC curve (AUC) of E6/E7 mRNA HR HPV is 0.812 (CI (95%): 0.752–0.871), while the area under the ROC curve formed by the parameters of the HR HPV DNA test is 0.740 (CI (95%): 0.680–0.799) .p < 0.001) (p < 0.001)  correlation . There i< 0.001) A. The on< 0.001) B.Univariate multinomial logistic regression examined the individual factors  that indicated an increased probability of developing HSIL .For the influence of HR HPV 16 on the probability of HSIL, a statistically significant predictive value was determined in the NILM and LSIL cytological groups. Patients with the confirmed presence of the HPV 16 genotype through a DNA test have a higher probability of being diagnosed with HSIL than NILM (3.8-fold), while they will have a 2.6-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with LSIL  Table S1The influence of the oncogenic activity of all tested of the HR HPV genotypes (E6/E7 mRNA HR HPVs) on diagnosing high-grade lesions of the cervical epithelium has a statistically significant prediction found in all cytological groups, NILM, ASCUS, and LSIL. If patients have confirmed indicators of oncogenic activity, they will have an almost seven-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with NILM. The same patients have a 19-fold higher probability of being diagnosed with HSIL compared to the probability of detecting ASCUS and a 5-fold higher probability of detecting HSIL compared to LSIL  Table S2E6/E7 mRNA HR HPV 16 is an indicator for diagnosing HSIL, and a statistically significant predictive value was determined for all types of cytological findings, NILM, ASCUS, and LSIL. In a patient with confirmed HPV 16 oncogenic transcripts, the probability of diagnosing HSIL is 50-fold higher than the probability of diagnosing NILM. Patients with a positive result of E6/E7 mRNAs HR HPV 16 are 12-fold more likely to be diagnosed with HSIL compared to the detection of ASCUS, while in patients with the same result, the probability of detection of HSIL is 6-fold higher than the probability of detection of LSIL  Table S3A statistically significant predictive value for the age category was determined in all cytological groups, NILM, ASCUS, and LSIL. The probability of detecting HSIL concerning normal results in HR-HPV-positive patients is 6.3-fold higher if they are ≥ 45 years of age than women under 30. A similar prediction for the detection of HSIL was shown concerning ASCUS (6.5-fold). Patients from the oldest age category have a 3.4-fold higher probability of detecting HSIL concerning LSIL than the youngest  Table S4The predictive model contains four independent variables: HR HPV DNA 16, E6/E7 mRNA HR HPVs, E6/E7 mRNA HR HPV 16, and age category. The HSIL lesion, as a representative of a high degree of cervical atypia, represented a dependent variable concerning all of the analyzed relevant factors. Multivariate logistic regression analysis was applied to all of the analyzed factors to construct a predictive model and named the most relevant predictors for the development of HSIL .p = 0.022). A statistically significant but almost three-fold weaker predictor for the detection of HSIL compared to NILM is the patient belonging to the oldest age category : 1.66–26.60; p = 0.007), while female patients from the age category 31–44 years have a slightly lower statistically significant probability : 1.36–21.30; p = 0.016) for the detection of the same lesion. The age category was determined as the strongest statistically significant predictor by analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detecting ASCUS. HR HPV DNA-positive patients belonging to the oldest age category (≥45 years) have an 8.7-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with ASCUS compared to patients belonging to the younger age category : 2.15–35.57; p = 0.002). Female patients with the proven oncogenic activity of HPV 16 have a probability of detecting HSIL 6.4-fold higher compared to the probability of being diagnosed with ASCUS : 1.22–33.54; p = 0.029). By analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detection of LSIL, the strongest statistically significant predictor was determined to be the oncogenic activity of HPV 16 : 1.09–23.83; p = 0.038), while weaker statistically significant predictability is shown by the patient’s belonging to a specific age category. Female patients belonging to the oldest age category (≥45 years) have a 3.7-fold greater probability of detecting an HSIL change compared to the detection of LSIL compared to the younger age category (≤30 years) : 1.16–11.92; p = 0.027) : 1.54–236.98; = 0.027) .Persistent infection caused by HR HPV is the leading risk factor for developing cervical intraepithelial lesions and cervical cancer (reviewed in ). Many cIn our study, 365 female patient samples from Serbia were tested for the twelve HR HPV genotypes. It should be emphasized that the study has limitations. Used HPV tests are non-validated according to European Guidelines for use in primary screening (Meijer HPV test criteria) or according to the VALidation of HPV GENoyping Tests (VALGENT) protocols . The comHPV genotype 16 38%) emerged as the most frequently detected genotype in the study group, in concordance with previous reports ,27. The % emergedThe presence of HR HPVs determined further examination of their oncogenic potential. In our study, the expression of E6/E7 mRNA HR HPV was identified in 67% of the HPV-positive samples . The perIn our study, HPV 16 constituted 29.7%, followed by genotype 31 (19.2%), genotype 33 (9.3%), and genotype 51 (8.7%). The results showed that approximately every second HPV 16 genotype is oncogenically expressed (48.6%). Our data agree with the results reported in other studies. Rossi et al. (2017) observed a positivity rate for E6/E7 mRNA HR HPV, ranging from 58% to 77% in HPV-DNA-positive women . A studyTo analyze the oncogenic activity of the four most frequently diagnosed HR HPVs related to the age categories, a statistically significantly higher prevalence of positive E6/E7 mRNA HPV 16 findings was observed in the older age groups . In agreThe growing interest in molecular diagnostics methods has led various authors to compare the characteristics of the HPV DNA test with the mRNA test, evaluating the diagnostic accuracy in identifying high-grade cervical lesions. Our data showed that the specificity (89%) and PPV (70–79%) of the mRNA test are statistically significantly higher, while the same test has a statistically significantly lower sensitivity than the HPV DNA test. These results agree with the statements of different authors, who emphasized slightly lower values of the sensitivity of the mRNA test compared to the sensitivity of the HPV DNA test. At the same time, the specificity is expressed at a higher level ,49  in patients with high-grade lesions is higher than the possibility of diagnosing them with the HPV DNA test . The obtained results are in line with the other studies emphasizing the potential usefulness of this test. Sun et al. (2021) also found higher AUC values for the mRNA assay compared to the DNA HPV (0.929 vs. 0.833) , while aAccording to the previously established degree of influence on diagnosing HSIL, relevant factors were selected in our research. Factors that represent an increased risk for more severe cervical changes were gradually examined .Firstly, the HPV-DNA-16-positive results have a statistically significant predictive value for diagnosing HSIL . In the Secondly, the oncogenic activity of all of the tested HR HPVs has a statistically significant predictive value for diagnosing HSIL . The obtFurthermore, in patients with positive results of indicators of the oncogenic activity of HPV 16, a statistically significantly higher probability of diagnosing a high-grade lesion was determined in all types of cytological groups . AccordiWithin the results of this research, a statistically significant final model with all of the predictors was constructed, which shows the strength of the potential of the examined factors, that is, biomarkers for diagnosing precancerous lesions in women with HR HPV infection. Looking at each cytological group individually, two independent variables made a statistically significant contribution to the model and were thus named as the strongest predictors. These are the oncogenic activities of HR HPV 16 and the age category (≥45 years). It is known that the presence of HPV infection is confirmed in all age categories. However, belonging to a particular age group is a determinant significantly associated with the risk of acquiring this infection, depicting the peak in prevalence, which generally takes place around 20–25 years of age. For lesions to progress to more severe forms of cervical disease, a period of 5–14 years is necessary, during which the infection persists and the process of oncogenesis takes place ; the resIn summary, this study describes the detection rates of the most common HR HPVs  and E6/E7 mRNA HR HPV expression in 365 Serbian women who showed normal and abnormal cytological findings. Those HR HPV genotypes are oncogenically active in more than half of the examined cases, and the detected oncogenic activity has predictive potential in diagnosing high-grade cervical intraepithelial lesions. According to the constructed predictive model, the oncogenic activity of HPV 16 and age are risk factors with the strongest predictive values for developing those lesions. Thus, our data indicate that mRNA testing may be more relevant than HPV DNA for assessing lesion grade and diagnosing and monitoring women at risk of progressive cervical disease. This way, the mRNA test as a tool for better risk stratification of HPV infection could overcome unnecessary examinations, increased costs, and patient anxiety. However, further follow-up studies are needed to determine the clinical utility of the mRNA HR HPV test.


Laminin Subunit Beta 3 (LAMB3) is a transcription factor and participates in the coding of laminin. It plays an important role in cell proliferation, adhesion, and transfer by regulating various target genes and signaling pathways. However, the role of LAMB3 in human pan-cancer immunology and prognosis is still poorly understood. The TCGA, GTEx, CCLE, and HPA databases were utilized for the analysis of LAMB mRNA and protein expression. The expression of LAMB3 in various immune and molecular subtypes of human cancer was examined using the TISIDB database. The prognostic significance of LAMB3 in various cancers and clinical subtypes was investigated using Kaplan-Meier and Cox regression analysis. The relationship between LAMB3 expression, various immune cell infiltration, immune checkpoints, tumor mutational load, microsatellite instability, and DNA methylation was examined using the TCGA database. Clinical samples of four lung cancer cell lines and eight lung cancer cases were collected to confirm the expression of mRNA in lung cancer. In 17 cancers, the mRNA for LAMB3 is expressed differently and has good diagnostic and prognostic value in 22 cancers. Cox regression and Nomogram analysis show that LAMB3 is an independent risk factor for 15 cancers. LAMB3 is implicated in a variety of tumorigenesis and immune-related signaling pathways, according to GO, KEGG, and GSEA results. LAMB3 expression was associated with TMB in 33 cancer types and MSI in 32 cancer types, while in lung cancer LAMB3 expression was strongly associated with immune cell infiltration and negatively correlated with all seven methylated CpG islands. Cellular experiments demonstrated that LAMB3 promotes malignant behavior of tumor cells. Preliminary mechanistic exploration revealed its close association with PD-L1, CTLA4, cell stemness gene CD133 and β-catenin-related signaling pathways. Based on these findings, it appears that LAMB3 could be a potential therapeutic target for immunotherapy and tumor prognosis. Our findings reveal an important role for LAMB3 in different cancer types. The global incidence and mortality of cancer are increasing year by year, seriously affecting the safety of public health and the quality of human life . DespiteLaminin plays an important role in regulating cell migration and signal transduction and is necessary for the formation and operation of the basement membrane . Tumor iBe that as it may, most exploration on the job of LAMB3 in growths is restricted to explicit kinds of disease, LAMB3 the job in human pan-cancer prognosis and immunology is rarely systematically analyzed. We used multiple databases, including TCGA, CCLE, genotype tissue expression (GTEx), human protein mapping (HPA), and cBioPortal to analyze LAMB3 expression levels and prognosis in pan-cancer. We also discussed the potential correlation between LAMB3 expression and the immune checkpoint (ICP) gene, immune cell infiltration level, microsatellite instability (MSI), and tumor mutation load (TMB) in 33 cancers. In addition, to investigate the biological function of LAMB3 in tumors, we carried out an enrichment analysis of LAMB3 co-expressed genes. Finally, we explored differences in LAMB3 expression and prognosis in lung cancer to further verify their outcomes in human cancers. Our results show that LAMB3 are prognostic and a risk factor for multiple cancers. LAMB3 plays an important role in tumor immunity by affecting tumor infiltration immune cells, TMB and MSI. This study provides new insights into the role of LAMB3 in anti-cancer immunotherapy.https://portal.gdc.cancer.gov/) and the GTEx database . Data on tumor cell lines are downloaded from the CCLE database . The BioGPS database (http://biogps.org) is used to analyze the expression of LAMB3 in different cancers and paired normal cell lines. Use R software v3.6.3 for statistical analysis and visualization, and ggplot2 package [version 3.3.3] for visualization. The Mann-Whitney U test detects two sets of data and p < 0.05 is considered statistically significant.RNA sequencing data and clinical follow-up information for 33 tumor types and normal tissues were downloaded from the TCGA database (http://www.proteinatlas.org/) with the top 5 protein expressions and differential gene expression.To assess the differences in LAMB3 expression at the protein level, we downloaded immunohistochemical images of LAMB3 protein expression in normal tissues and their corresponding tumor tissues, including THCA, LUADLUSC, HNSC, SKCM, and STAD, from the HPA database (http://cis.hku.hk/TISIDB/). The database integrates multiple types of data to assess interactions between tumors and the immune system.The correlation between the expression of LAMB3 and the molecular or immune subtype of pan-cancer is discussed in the TISIDB database , and 95% confidence interval. Statistical analysis and visualization using R software v3.6.3. The survminer package [version 0.4.9] is used for visualization. The survival package [version 3.2-10] is used for the statistical analysis of survival data. Statistical testing using Cox regression. p < 0.05 is considered statistically significant.The receiver operating catalytic (ROC) curve is used to assess the diagnostic value of LAMB3 in pan-cancer. We only screened for diseases with AUC >0.7. The relationship between LAMB3 expression and cancer prognosis was evaluated using the Kaplan-Meier diagram . In addition, we further studied the relationship between LAMB3 expression and prognosis  in different clinical subgroups of lung cancer. The forest diagram shows the Cox regression analysis  was used to examine the prognostic value of LAMB3 in pan-cancer and its clinical subtypes. Column line plots were constructed using the R package “rms.”p < 0.05 was considered statistically significant.RNAseq data for cancer (level 3) and corresponding clinical information were obtained from the TCGA database. For reliable immune correlation assessment, we used immunedeconv, which is an R package that integrates six state-of-the-art algorithms including TIMER, xCell, MCP-counter, CIBERSORT, EPIC, and quantized. The expression values of SIGLEC15, IDO1, CD274, HAVCR2, PDCD1, CTLA4, LAG3, and PDCD1LG2 were extracted to observe the expression of immune checkpoint-associated genes. These eight genes are the transcripts associated with immune checkpoints. TMB is derived from the article The Immune Landscape of Cancer published by The first 20 proteins that may bind to the LAMB3 were screened from the STRING website. Cytoscape (version 3.9.0) is applied to the visualization of PPI networks.GO, KEGG, and GSEA are performed using proteins or differential genes interacting with LAMB3 to analyze the biological and molecular functions of LAMB3 in different cancer types. All of the above analyses were performed and visualized using the R package (version 3.6.3). The ggplot2 package and the cluster Profiler package [version 3.14.3] were used for the above analysis. The reference gene set for GSEA analysis is c5. all.v7.5.1. symbols.gmt [Gene ontology], c6. all.v7.2. symbols.gmt [Oncogenic signatures] and c7. all.v7.5.1. symbols.gmt [Immunologic signatures].2.Four lung cancer cell lines  A549, H226, H1299, SK-MES-1, and normal bronchial epithelial cells BEAS-2B. The above cells were routinely cultured in DMEM high sugar medium containing 10% FBS at 37°C and 5% COLung cancer tissues and their paracancerous tissues from eight patients were collected at the First Affiliated Hospital of Kunming Medical University. All patients signed informed consent and this study was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University.RNA was extracted using the TRIzol method of isolation according to the manufacturer’s instructions (Invitrogen), and template cDNA was synthesized using a reverse transcription kit . Each gene is amplified with 1 μL template cDNA and 0.6 μL primers (20 μ L system). Denaturate the PCR reaction mixture at 95°C for 10 min and amplify the cDNA template as follows: Stage1 (pre-denaturation): 1 cycle, 95°C 15 min; Stage2 (PCR) Reaction): 95°C denaturation 10 s, 60°C annealing extended 32 s, repeating 40 cycles.https://www.cbioportal.org/).Genetic alterations of LAMB3 in lung cancer were obtained using the cBioPortal database (http://bio-bigdata.hrbmu.edu.cn/diseasemeth/index.html). RNAseq data in level 3 HTSeq-FPKM format and Illumina human methylation 450 methylation data were downloaded from the lung cancer project of the TCGA database to visualize LAMB3 in relation to multiple CpG loci using ggplot2 [version 3.3.3].The DNA methylation status of LAMB3 gene CpG sites was analyzed in the TCGA dataset for lung cancer disease using the DiseaseMeth database |>2.0 and P adj<0.05. Then, we performed statistical analysis with the cluster Profiler package for GO and KEGG enrichment analysis of DEGs. In addition, a PPI network of DEGs with log2 FC > 1.5 or log2 FC < −2 as the threshold is established by using the STRING network, and the hub gene is analyzed by CytoHubba’s MCC algorithm in Cytoscape (version 3.7.2).We explored DEGs between different LAMB3 expression groups  in lung cancer using the deseq2 package. The ggplot2 package was used for visualization analysis with a threshold of |log2 fold-change (FC)|>1.0 and To comprehensively assess patient survival, we constructed a nomogram integrating distinct clinicopathological information using the “rms” package. ROC curves were used to assess the accuracy of the signature prediction. 32 hub genes interacted with LAMB3 in lung cancer. The above 32 genes were subsequently subjected to one-way Cox regression analysis to obtain 7 genes associated with prognosis in lung cancer. Then the least absolute shrinkage and selection operator (LASSO) regression analysis was performed. The LASSO regression could improve the accuracy and interpretability of the model, and eventually build a risk factor model to predict survival.Cells or exosomes were lysed in RIPA buffer containing 1% PMSF, and the protein concentration was determined using the BCA method. Protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies and HRP-coupled secondary antibodies. The membranes were subjected to ECL luminescent solution. The grey-scale values were analyzed using ImageJ software.A549 and H1299 were routinely cultured in DMEM high sugar medium containing 10% FBS at 37°C and 5% CO2. Cells at 40%–60% confluence were transfected with inhibitor LAMB3 for 48 h. Transfection efficiency was observed using fluorescence microscopy and WB. The cells  were incubated in 96-well plates for 0 h, 24 h, 48 h, and 72 h respectively, and performed with Cell Counting Kit-8 (CCK8) assay. Cells  were inoculated into Culture-Insert with scratches up to 500 μm wide. The length of scratches was observed and recorded under the microscope after culturing for 0, 6, 12, and 24 h. The area of scratched area was quantified using ImageJ software. The matrix gel was diluted at a ratio of 1:8, and 100 μL of the diluted matrix gel was added to the upper chamber of the Transwell plate, and incubated for 30 min at 37°C. Cell suspension of 200 μL (5 × 104 cells) is added to the upper chamber, and 500 μL of complete medium containing 20% FBS was added to the lower chamber. The plates were incubated for 48 h. The cells were gently wiped off the surface of the matrix gel and the transwell was fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Six random fields were counted under the light microscope.Combined analysis by the GTEx database and TCGA database showed that LAMB3 mRNA levels were significantly higher in most cancers compared to their normal tissues in unpaired samples, such as CESC, CHOL, ESCA, HNSC, LIHC, LUAD, LUSC, STAD, THCA, UCEC. In contrast, LAMB3 mRNA expression was lower in BRCA, GBM, KICH, KIRC, KIRP, PCPG, and PRAD than in the corresponding normal tissues . The aboIn addition, we also analyzed the HPA database’s IHC results and compared them to the gene expression data previously mentioned to assess LAMB3 expression at the protein level. In most cancers, LAMB3 protein expression showed an upward trend over normal tissues . We screThe correlation between LAMB3 differential expression and pan-cancer molecular subtype is explored from the TISIDB database. LAMB3 was found to be expressed differently in 10 different molecular subtypes, including STAD, LUSC, LIHC, LGG, KIRP, HNSC, ESCA, BRCA, UEEC, and PCPG . MoleculAt the same time, we observed that the expression of LAMB3 was significantly correlated with the different immune subtypes of 20 cancer types . Cancers containing five or more immune subtypes were STAD, READ, PCPG, PAAD, LUSC, LUAD, KIRP, KIRC, BLCA, THCA, BRCA, and COAD . In addiThe ROC curve was used to evaluate the diagnostic value of LAMB3 in pan-cancer. The results showed that LAMB3 was extremely accurate (AUC>0.9) in predicting 11 cancer types, including LAML (AUC = 0.995), CHOL (AUC = 0.985), OV (AUC = 0.977), PAAD (AUC = 0.969), UCS (AUC = 0.969), CESC (AUC = 0.950), READ (AUC = 0.942), THYM (AUC = 0.923), OSCC (AUC = 0.918), HNSC (AUC = 0.910), and LUAD (AUC = 0.905) . Meanwhip < 0.001), PFI , and DSS . Higher LAMB3 expression was linked to a worse prognosis for LUSCLUAD, according to Cox regression results (p < 0.001), PFI , and DSS . For KIRC, Cox regression results show that the higher the LAMB3 expression, the worse the prognosis (p = 0.01), PFI , and DSS . Higher LAMB3 expression was linked to a worse prognosis for LUAD, according to Cox regression results (p < 0.001), PFI , and DSS . For KIRC, Cox regression results show that the higher the LAMB3 expression, the worse the prognosis (p = 0.001), PFI , and DSS . Higher LAMB3 expression was linked to a worse prognosis for HNSC, according to Cox regression results (p = 0.002), PFI , and DSS . For GBMLGG, Cox regression results show that the higher the LAMB3 expression, the worse the prognosis (p = 0.007), PFI , and DSS . Higher LAMB3 expression was linked to a better prognosis for BLCA, according to Cox regression results (p = 0.006), PFI , and DSS . For UVM, Cox regression results show that the higher the LAMB3 expression, the worse the prognosis (p = 0.002), PFI , and DSS . Additionally, Cox regression results in COAD, KICH, LIHC, OS, OV, PRAD, READ, SKCM, STAD, TGCT, THYM, CESC, GBM, SARC, BRCA, UCEC, and UCS reveal that some aspects of the prognosis for cancer are correlated with the level of LAMB3 expression.It is critical to investigate the relationship between LAMB3 expression levels and prognosis, and we performed a survival correlation analysis for each malignancy, including OS, DSS, and PFI. Through forest diagrams, we showed tumors whose LAMB3 expression levels were significantly correlated with OS, DSS, and PFI, including PAAD, LUSCLUAD, KIRP, LUAD, KIRC, HNSC, GBMLGG, BLCA, and UVM . For PAA results . This inrognosis . This in results . This inrognosis . This in results . This inrognosis . This in results . This inrognosis . This inAn emerging method of treating tumors is immunotherapy. ICP genes have been shown to have a significant impact on immunotherapy and the infiltration of immune cells. We originally investigated the relationship of LAMB3 expression with ICP qualities in human malignant growths and the capability of LAMB3 in immunotherapy . The LAMThe relationship between TMB and MSI, which are both intrinsically linked to the sensitivity of immune checkpoint inhibitors, and LAMB3 expression levels was the next topic of our investigation. MSI has a significant positive correlation with TGCT, LUSC, UVM, STAD, SARC, DLBC, OV, BRCA, READ, GBM, THCA, UCEC, KIRC, LIHC, LAML, HNSC, LGG, and SKCM, according to the MSI correlation results. MSI has a significant negative correlation with KIRP, MESO, ACC, KICH, PAAD, BLCA, COAD, CESC, LUAD, ESCA, PCPG, THYM, PRAD, CHOL, and UCS . TMB hasFollowing that, we investigated the connection between the levels of infiltration of 26 immune-related cells and the expression levels of LAMB3. Immune cell infiltration levels were significantly correlated with LAMB3 expression in most types of cancer . LAMB3 e20 proteins that interact with LAMB3 proteins were found using the STRING database . The 20 In conclusion, our pan-cancer analysis revealed that LAMB3 is an independent risk factor for multiple cancers and is closely linked to their development and prognosis  Supplem. In partLAMB3 genomic alterations in lung cancer were explored via the cBioPortal website. The findings demonstrated that 5% of LAMB3 genomic changes occurred . ChangesThese factors were identified as highly significant prognostic predictors based on multivariate Cox regression analysis. Columnar plots showed significantly higher clinical value in predicting the probability of survival at 1, 3, and 5 years in patients with lung cancer carcinoma. We further used ROC curves to assess the accuracy of LAMB3 in predicting OS in lung cancer patients. As shown in We investigated the connection between the amount of infiltration of 26 immune-related cells and the level of LAMB3 expression in lung cancer. LAMB3 gene expression was positively correlated with Natural killing (NK) cells, NK CD56dim cells, Neutrophils, Tcm, Th2 cells, Tgd, Treg, DC, and Th1 cells. LAMB3 gene expression was negatively correlated with aDC, B cells, CD8 T cells, Cytotoxic cells, Eosinophils, iDC, Macrophages, Mast cells, NK CD56bright cells, pDC, T cells, T helper cells, Tem, TFH and Th17 cells . After tThe DiseaseMeth tool was used to look at the relationship between the levels of DNA methylation and CpG islands in the LAMB3 gene . All sevetc. MF primarily participates in hormone activity and receptor-ligand activity, etc. KEGG pathway enrichment was mainly associated with Spliceosome, Salivary secretion, and RNA transport  and TMB are important factors in good immunotherapy outcomes and pan-cancer prognosis . Our stuWe examined the LAMB3 co-expression network at the end. In terms of immunity, we discovered that antigen processing and presentation, B/T cell activation, and immune response regulation are all performed by LAMB3 and its co-expressed genes. We have also found LAMB3 is equally closely related in terms of oncogenic signatures and gene ontology. Furthermore, at the cellular level, we found that reducing LAMB3 expression in lung cancer cell lines A549 and H1299 inhibited the proliferation, migration, and invasive ability of tumor cells. Preliminary mechanistic exploration showed that it can regulate PD-L1 and CTLA4 expression, echoing the above findings that LAMB3 regulates T cell and tumor immunity. CTLA-4 inhibits T cell activation by competing with CD28 receptors to bind to B7 ligands on antigen-presenting cells . PD-L1 iin vitro or in vivo experiments to confirm these findings are lacking. Third, despite our findings that immune cell infiltration and associated immune targets in human cancers are closely linked to LAMB3 gene expression, we lack direct evidence that LAMB3 influences prognosis by participating in immune infiltration. The mechanism by which LAMB3 is involved in immune regulation is still unknown, and the effect of LAMB3 on tumor immunity also varies depending on the type of tumor. The mechanisms of related immunotherapy require additional cellular and animal research. In conclusion, further investigation of LAMB3 in lung cancer and its significance in pan-carcinoma diagnosis and prognosis can contribute new perspectives to a comprehensive comprehension of its crucial role in tumor progression. Furthermore, this research provides a comprehensive analytical foundation for the in-depth validation of molecular biology experiments and even the future clinical application of cancer therapy.In summary, we have performed the first comprehensive and systematic analysis of LAMB3 and cross-validated it using different databases, patient samples, and tumor cell lines. The factor was expressed differently between most tumors and normal tissues and revealed the correlation between LAMB3 expression and clinical prognosis, immune microenvironment, and DNA methylation. Although these bioinformatics are carried out by collecting information from various databases, clinical samples, and tumor cell lines, there are some limitations to this study. First, distinct databases contain cancers that are at odds with one another. To better understand LAMB3’s expression and function, additional research in this area would require a larger number of clinical and tumor cell lines. Second, despite the investigation of the gene’s potential signaling pathways and its prognostic and diagnostic value in pan-cancer, more in-depth


Military culture relies on hierarchy and obedience, which contradict the implementation and use of collaborative care models. In this commentary, a team of lived experience, clinical and research experts discuss, for the first time, cultural, communication and policy considerations for implementing collaborative care models in military mental healthcare settings. For example, patients may advocate to return to a previously beneficial mental health programme when symptoms recur or to realign treatment based on personal goals outside of standard symptom remission. To actualise these ideals fully, CDM addresses not only patient–provider decisions but also patient–system decisions; whether and how a treatment is considered ‘evidence-based’; and admissions and discharge policies.6 The CDM model builds on the shared decision-making approach7 and seeks to overcome common implementation barriers through ‘designed for dissemination’ strategies that increase generalisability and feasibility across settings and decisional contexts.8Military veterans and current military service members are at a heightened risk for mental health disorders, including suicide.9 and attitudes towards active duty service members and veterans tend to be positive across most groups,10 dynamics within and without the military have an impact on veterans’ mental health and on treatment engagement and success.11 US veterans have unique mental health needs compared with the general US population, including relatively high rates of suicide, post-traumatic stress disorder (PTSD), depression and substance use, among other concerns.13 Service members are frequently dissuaded from speaking up across settings and in particular from disclosing mental health symptoms and engaging in mental healthcare.13 Although US veterans are beneficiaries of the only nationalised healthcare programme in the USA, the Department of Veterans Affairs (VA) has undergone considerable scrutiny, which has decreased some veterans’ willingness to seek services.14 Therefore, this vulnerable and specialised group face specific barriers to getting their mental health needs met during active duty and after.Military veterans from the USA may find the CDM model particularly important. Although patriotism in the USA is high compared with other countries19 Facing the growing need to improve veterans’ mental healthcare, this commentary illuminates clinical challenges for implementing CDM in military settings, focusing on veterans in the USA with mental illness.CDM facilitates disclosure and empowerment, identifying a promising method to increase help-seeking and effective care for veterans. Although there is an increased appreciation of CDM and person-centred mental healthcare by military bodies, it has been mostly related to PTSD, most of the work was conducted within the USA and implementation remains challenging.20 Veterans often prefer to silence themselves or disengage entirely from mental health treatment rather than state a differing opinion.Military practice and culture are rooted in hierarchy and obedience to authority. Veterans have been trained to believe in the importance of hierarchy and encouraged to obey and comply with perceived authority figures. Valuing hierarchy clashes with the CDM process, where clinicians and patients are equal contributors to the decision-making process. Our ongoing research suggests that when veterans are presented with the principles of CDM, they fear the consequences of ‘disrespecting’ or ‘disobeying’ clinician judgement if they disagree with treatment recommendations.21 Such beliefs easily translate to expectations that one should handle any mental health issues independently, reducing willingness to engage in mental healthcare.21 It also could lead to difficulty being vulnerable, disclosing symptoms or other signs of perceived ‘weakness.’21 The deep comradery developed among service members who serve together can exacerbate this problem, as veterans may struggle to disclose issues that could reflect poorly on other service members .Many veterans held leadership positions in the military, meaning they expect themselves to be strong, competent and able to take care of their own problems.22 which differs from civilian communication,23 leading to a communication mismatch that significantly interferes with CDM. Civilian clinicians who lack appropriate training in military culture may interpret the normative military communication style as aggressive or offensive, focusing clinical efforts on changing the veteran's communication style (as it is perceived as aggressive) rather than on the veteran's treatment needs and goals.20 Some veterans with severe mental illness (SMI) have described fear of being perceived as too aggressive by clinicians, including being disrespectful, malicious, dangerous or ‘crazy.’20 Such fear can decrease veterans’ willingness to assert preferences and reduce disclosures of important experiences and symptoms – key components of CDM.Veterans have been trained to use a direct and clear communication style in the military,24 However, military healthcare often has only a few treatment options, and sometimes only one. While the VA's investment in evidence-based practices is a benefit in many ways, there is not an appropriate evidence-based practice for every person and clinical situation. Clinical trials represent about 20% of people with schizophrenia-spectrum disorders.25 People from minority racial/ethnic groups, those experiencing comorbid substance use, somatic disease and suicidality are (among others) less likely to be included, decreasing applicability of these treatments for many veterans with SMI.25 Therefore, owing to lack of treatment options, true CDM is limited within the VA setting, even when veterans and clinicians are fully committed to it.Implementation of CDM requires a participatory decision-making process that considers all possible options based on the patient's needs, preferences and values.26 among clinicians, including ability to increase treatment-seeking.27 It is crucial to understand a veteran's beliefs about their military service and how these beliefs shape their relationship to decision-making in mental healthcare. Attention to challenges of readjustment to civilian life have also been identified as particularly important when communicating with veterans.28 Clinicians should familiarise themselves with normative military communication. If needed, structured training should be completed to address the communication mismatch between veterans and civilian clinicians, so that clinicians adapt their clinical response appropriately and ensure that veterans’ treatment needs and goals remain the target of treatment rather than the clinician's perception of rudeness or impaired social skills.Civilian clinicians working with veterans should engage in training to increase their understanding of military culture and its impact on veterans’ interactions with mental healthcare. Engaging in mental healthcare and CDM means adapting processes to fit each individual's needs and values, including considering cultural identity. Significant literature supports the importance of military cultural competence7 The continual development of rapport, trust and transparency is fundamental to creating a therapeutic space supportive of CDM.4 A valid, CDM-aligned choice is for a veteran to decline to participate in a treatment decision.4 Clinicians are encouraged to become familiar with power-sharing in clinician–veteran dyads and consider how they can effectively empower veterans and create an egalitarian relationship. Positioning decision-making as shared risk-taking can help develop this understanding.24Clinicians should encourage and empower veterans to participate in CDM, including psychoeducation about CDM, transparency about available treatment options, even if very few exist, jointly setting recovery goals and asking the veteran about preferred treatment options.4 For example, one CDM-aligned intervention adapted for the VA, collaborative decision skills training,20 includes fundamental CDM psychoeducation, communication and problem-solving skills, and brief coping skills to increase comfort and confidence. Interventions that address how CDM and self-advocacy broadly benefit veterans may also be helpful.29CDM is a significant change from past interactions with providers, as veterans are often unaware that they have the option to collaborate in treatment decision-making. Veterans may need support to access CDM and effectively engage in it. We recommend empowerment-oriented, patient-centred strategies focused on increasing veterans’ knowledge, skills and confidence with engaging in CDM.Military bodies must identify policies and changes that increase veterans’ access to options and consider how civilian clinicians and veterans communicate and share power. Although the specific actions will vary by country, the fundamental principles of prioritising veterans’ power over their care is essential: for example, broadening access to a variety of care options; expanding accessibility, including in rural areas and through telehealth, in-home and in-community options; investing in culturally tailored and trauma-informed approaches; and engaging veterans at all levels of decision-making, including policy decisions. For example, the USA's VA MISSION Act of 2018 intends to increase access to care for veterans under certain conditions (e.g. if a needed service is unavailable at the VA). Expanding community care can increase veterans’ power in decision-making by increasing choice over whether to receive care within the VA or the community. Such change can increase access for veterans who prefer community care and for veterans seeking culturally tailored care that the VA currently does not provide.Military bodies should also invest in broad training and implementation of CDM. More intensive training is needed to customise to specific clinic and population needs and provide education about military culture and communication. More extensive training would also increase clinicians’ ability to assess and reflect on ways to step back from power and build trust.Finally, investment in scholarships and infrastructure to diversify the clinician workforce is needed. The existing structure for licence-eligible clinical training can be difficult for veterans to access, especially veterans with lived experience of mental health concerns. A diverse clinical workforce facilitates the ability of veterans to work with clinicians with important shared experiences, improving effective tailoring for each veteran's needs and preferences and potentially increasing trust and collaboration for CDM. It also may increase awareness of military culture, and other lived experiences, in the clinician workforce, facilitating culturally informed care regardless of an individual clinician's background.


In addition, the various HPV diagnostic methods were analyzed. Although there is a vast amount of literature on HPV, this review is unique in its ability to present the key information in an organized and accessible way and enables healthcare professionals to gain a better understanding of the relationship between HPV and oropharyngeal cancer. This, in turn, can contribute to the prevention of various cancers caused by the HPV virus, including oropharyngeal cancer.Oropharyngeal squamous cell carcinomas (OPSCC) represent a major public health challenge. In 2020, the international agency for research on cancer (IARC) recorded 98,421 cases of OPSCC worldwide. Over the past decade, the epidemiological profile of patients with OPSCC has shifted, mainly due to a change in etiological factors. Previously, alcohol and tobacco were considered the primary contributors, but the human papillomavirus (HPV) is now recognized as the leading cause of these tumors. This study aimed to conduct a literature review on the relationship between OPSCC and HPV for the general practitioner. The review examined the primary clinical differences between HPV Oropharynx squamous cell carcinomas (OPSCC) represent a major public health challenge. In 2020, the international agency for research on cancer (IARC) recorded 98,421 cases of OPSCC worldwide .Over the past decade, the epidemiological profile of patients with OPSCC has shifted, mainly due to a change in etiological factors. Previously, alcohol and tobacco were considered the primary contributors, but the human papillomavirus (HPV) is now recognized as the leading cause of these tumors .HPV-related OPSCC have different clinical presentation and prognoses when compared to non-HPV-related tumors. Thus, knowledge of the relationship between different epidemiological and clinical variables and HPV positivity in these tumors is crucial for better understanding and knowledge of this disease.+ and HPV− OPSCC patients. The number of sexual partners is the primary determinant of anogenital HPV infection in both men and women  AND  AND (“cancer” OR “tumor” OR “neoplasms”) to identify articles reporting on HPV and its relation to oropharyngeal cancer. Articles published in English, Portuguese or Spanish were included, regardless of study design or publication date. The studies considered most relevant were selected for inclusion in this review and additional articles on HPV vaccination were also included to complement the analysis. HPV represents the most common sexually transmitted infection caused by a virus in the world . HPV infMore than 200 types of HPV are known, and the eight most common types of HPV worldwide are HPV 16, 18, 31, 33, 35, 45, 52, and 58. Type 16 is the most common of all, followed by type 18 in various parts of the world .The prevalence of HPV in the oropharynx and oral cavity varies widely across different populations. A study by In a meta-analysis performed by Smoking and alcohol consumption are the most important risk factors for head and neck cancer. However, many patients with OPSCC do not have any of the traditional risk factors. Many studies have correlated HPV as an etiological agent in many of these patients .HPV is present in head and neck mucosal regions at a rate of 7% in healthy individuals . Most HPIn 2007, the international agency for research on cancer (IARC) considered HPV as a known carcinogenic for the oral cavity and oropharynx. However, the evidence linking HPV to cancer in the larynx was found to be limited .+ , with a higher predominance of caucasian, more years of education, higher family income and more likely to be married.In a study carried out by The ICON-S study found that the mean age of HPV-positive OPSCC patients was 57 years, which is younger than the mean age of 61 years for HPV-negative patients. This difference in age may be attributed to a decrease in smoking and alcohol use among HPV-positive patients, which is associated with better treatment tolerance and improved prognosis .+ patients have lower rates of smoking and alcoholism when compared to HPV− patients and generally report a greater number of sexual partners and/or frequency of oral sex , a comprehensive medical history is crucial to identify risk factors such as smoking, alcohol consumption, and sexual behavior. A physical examination is also necessary, including inspection of the oropharynx, palpation of all lymph node levels in the neck, and nasolaryngoscopy or laryngoscopy to evaluate the extent of the tumor. Biopsy of the suspicious lesion in the oropharynx and fine needle aspiration biopsy of cervical masses should be performed. For accurate staging, a CT scan of the neck and thorax is required. Additionally, it is important to determine whether the tumor is positive for human papillomavirus (HPV). In general, immunohistochemistry for p16 is performed on the biopsy to detect the presence of HPV .in situ hybridization found that 10% of p16-positive patients were HPV-negative, while 7% of p16-negative patients were HPV-positive , recommend the clinical use of p16 immunohistochemistry to characterize cases as HPV-positive and HPV-negative cases . The 8thIt is therefore recommended that all patients undergoing biopsy of tumors in the oropharynx region be asked to carry out a p16 immunohistochemistry test in addition to an anatomopathological examination of the specimen. This will help to ensure accurate diagnosis and appropriate treatment selection for OPSCC patients.HPV-positive OPSCC has a better prognosis compared to HPV-negative OPSCC . The 8th+ group and 57.1% in the HPV-negative group, while the 3-year disease-free rate was 73.7% in the HPV+ group and 43.4% in the HPV− group. Furthermore, the study revealed that the 3-year survival rate was 83.6% for the HPV+ group and 51.3% for the HPV− group, and the 3-year disease-free rate was 74% for the HPV+ group and 38.4% for the HPV− group. The study identified an intermediate-risk group of patients with OPSCC who were HPV+ and had more than 10 pack-years of tobacco smoking, which had an intermediate prognosis (rognosis .in situ hybridization. HPV DNA was positive in 38 patients (40%). The study showed that disease-free survival in HPV+ patients was 91% in the first year and 86% in the second year, while it was 69% in the first year and 53% in the second year for HPV- patients. Patients with HPV+ tumors had higher response rates after induction chemotherapy (82% vs 55%) and after radiochemotherapy treatment (84% vs 57%) compared to those with HPV− tumors. Overall 2-year survival was 95% for HPV+ patients compared to 62% for HPV− patients.In a study conducted by The risk behavior most associated with HPV-related OPSCC is oral sex. Thus, prevention should promote actions such as the use of condoms and a reduction in the number of sexual partners.Prevention for HPV-related OPSCC includes vaccination against HPV, which is recommended for people aged 9 to 26 years old. Currently, there are three types of HPV vaccines approved by the FDA: Gardasil-9, approved in 2014 , Cervarix, approved in 2009 (which prevents types 16 and 18), and Gardasil, approved in 2006  . The recvs 1.61% in non-vaccinated individuals (The HPV vaccine has demonstrated a reduction in the prevalence of oral HPV infection. In a study of 2,627 participants aged 18 to 33 years, both men and women were analyzed for the presence or absence of HPV in mouthwashes 4 years after vaccination. Of the participants, 18.3% received one or more doses of the vaccine. The study observed a prevalence of HPV types 16, 18, 6 or 11 in the oral cavity of 0.11 in vaccinated individuals ividuals . It has ividuals .Another important point is the potential for health professionals to be contaminated with HPV during procedures that emit aerosols, such as excision or electrocoagulation of HPV-related lesions. Although the level risk is still uncertain, evidence suggest a real risk may exist .The treatment of oropharyngeal cancer requires a multidisciplinary team. Treatment options may include radiotherapy, surgery and/or chemotherapy and the choice of treatment depends on the clinical stage of the tumor and the clinical condition of the patient. Typically, radiotherapy or surgery are indicated for early-stage T1 and T2 tumors without cervical metastasis, and radiotherapy associated with chemotherapy is recommended for more advanced cases.Although assessing the patient’s HPV status is important for staging and predicting prognosis, there is still insufficient support to carry out different treatment options. Therefore, the treatment for OPSCC associated with HPV should be the same for HPV-negative cases except in clinical trials.Some studies have investigated the de-escalation of RT and/or chemotherapy treatment showing encouraging results in phase II non-randomized studies . Due to + and HPV− OPSCC were also discussed, including their impact on prognosis and treatment. This information is crucial as OPSCC is a significant public health issue and preventive measures can be taken in most cases if the virus is identified early.This review described the relationship between the HPV virus and OPSCC. The various HPV diagnostic methods were analyzed and an explanation was given for p16 being used as a marker for HPV infection. The clinical differences between HPVWhile there is a wealth of literature on HPV, this review presents key information in an organized and accessible way that can help increase awareness and understanding among health professionals on the relationship between HPV and oropharyngeal cancer. This can lead to a reduction in the incidence of HPV-related cancers, including OPSCC, and improve patient outcomes.


This association was slightly significant, and furthermore, high risk HPV was not found to be associated with PCa. Higher body mass index (BMI) , more total meat consumption , exhibited association to PCa. However, PCa family history only presented a statistically significant difference by forward stepwise analysis . While much focus has been on the association between HPV and PCa, the results of this study indicate that more efforts should be directed towards investigating dietary habits, personal lifestyle and family history as factors for PCa. These results could serve as a basis for designing PCa prevention strategies.The rising incidence rate of prostate cancer (PCa) worldwide has become a public health concern. PCa has a multifactorial etiology, and the link between human papillomavirus (HPV) and PCa has been widely investigated by numerous case–control studies. This age-matched, case–control study included 143 PCa patients and 135 benign prostatic hyperplasia (BPH) patients, with prostatic specimens testing negative for malignancy, as control. Study participants were recruited from four major hospitals in Taoyuan City, Taiwan, period 2018–2020, looking into HPV infection and other PCa risk factors, including dietary habits, family history, personal lifestyle, and sexual behavior. Multiple logistic regression analysis and forward stepwise selection analysis were conducted to identify potential risk factors for PCa. HPV DNA was found in 10 of the 143 PCa cases (7%) and 2 of the 135 BPH controls (1.5%) (OR = 6.02, 95% CI = 1.03–30.3,  The relatively slow progression of PCa, along with developments in early detection and treatment, has led to a decreasing trend in mortality rates. USA cases diagnosed between 2001 and 2016 showed five-year survival rates of 98%2.In recent decades, with incidence rates of prostate cancer (PCa) on the rise worldwide, PCa has become an emerging public health issue in both developed and developing countries. PCa is now the second leading malignancy in men worldwide, with 1.4 million new cases and over 375,000 deaths in 2020 (GLOBOCAN)4. In USA, age-adjusted incidence rates increased throughout the 1980s , peaked in the early 90 s , and has been in gradual decline since, to 107 per 100,000 (2017)6. Median age of diagnosis in USA is 66 years of age, whereas in Taiwan, it is 74. In USA, at time of initial diagnosis, only 13% had cancer in regional lymph nodes and 6% had developed distant metastasis. Comparable figures for Taiwan showed 18.35% of patients were stage III at initial diagnosis, while an astounding 33% were stage IV7. Bone metastases, lower urinary tract symptoms, asymptomatic pelvic lymphadenopathy, or asymptomatic liver/lung metastasis are commonly observed in such advanced stage PCa.Comparing Taiwan and USA, epidemiological data of PCa for the two countries tell different stories. From 2000 to 2017, age-adjusted incidence rates in Taiwan doubled from 15.83 to 31.87 8 published in 1991 showed that prostate specific antigen-based (PSA) screening could provide better PCa detection rates. Subsequent widespread adoption of PSA-screening in the early-90 s led to a spike in PCa incidence detection rates in USA. The improved early detection and ensuing treatment have been credited for the steady decline of incidence and mortality rates since the late-90 s. While many European countries have since enacted PSA-screening programs, Taiwan has yet to follow suit. The increasing rates of PCa, with relatively high rates of late-stage cases, may aggravate the financial burden of Taiwan’s National Health Insurance System, and should merit greater public awareness.Catalona et al.11. Perdana et al. conducted a literature review study concluding that age, family history, insulin like-growth factors, dietary habit, lifestyle, environmental and occupational exposures were risk factors, while selected dietary products and supplementation may help in prevention9. Malik et al. had a multifactorial case-control study which demonstrated that age and smoking were risk factors, while fruits and vegetables could prevent PCa10. Pernar et al. provided descriptive epidemiology statistics and summarized that older age, family history, ethnicity, and genetics were strong risk factors10. Since the early-1950’s, sexually transmitted diseases (STD) have been linked as possible risk factors for PCa, with possible rationale being the repetitive inflammations caused by STD increasing cancerous growth and invasion of prostate cells. Initially, these studies were small, self-reported, or case-controlled with retrospective cohort, and generally focused on gonorrhea and syphilis. As new STD have been recognized, new resources for PCa research have been implemented to allow more prospective study designs, well-developed questionnaires, and serological/pathological assessments12. While several studies have explored relationships between STD and PCa, findings were inconsistent. Some studies have linked STD to cancer risks with increased odd ratios (OR): 1.2–1.515, while other studies found no statistically significant difference17.PCa is a multifactorial disease, and numerous epidemiologic studies have been conducted to survey various possible PCa risk factors22, with plausible speculation being that serology substantiates presence of HPV infections in human body, but is unable to identify the definite source of infection. In studies where polymerase chain reaction (PCR), radical prostatectomy (RP), transrectal ultrasound (TRUS) biopsy, or transurethral resection of the prostate (TURP) were performed on prostate pathology samples, correlation of high risk HPV to PCa carcinogenesis or progression was disclosed, but overall findings were inconclusive27.Human papillomavirus (HPV) is the most common viral STD worldwide. While cervical cancer was once the most common cause of cancer mortality for women in USA, advancements in screening (Pap and HPV) have allowed for early detection and treatment. Some studies have further investigated HPV in relation to PCa, with benign prostatic hyperplasia (BPH) patients as control group, and the results have been mixed. Studies that used seroprevalence of HPV antibodies in blood samples have mostly observed null results14. Another meta-analysis of 25 eligible studies did not find increased PCa risk for HPV-16  nor HPV-18 28. However, results of secondary analysis indicated that only HPV-16 DNA prevalence was higher in PCa . A similar study including 24 case-control studies involving 971 patients and 1,085 controls showed that the pooled estimate for OR was 2.27 (95% CI: 1.40–3.69)29. However, there were several limitations for these meta-analyses. Firstly, these individual case-control studies lacked power, as sample sizes were limited in number, and did not provide detailed calculation methods. Also, as is case with most case-control studies, possible information bias, selection bias, and difficulties in establishing temporal relationship, were factors to be considered. Secondly, some major confounders such as age, comorbidity, dietary habit, hygiene, occupational exposure, and sexual lifestyle were not adjusted for, leaving room for skepticism. Thirdly, previous studies were mostly conducted in Australia, Europe, and USA, lacking data and evidence from East Asian countries.Results from several meta-analyses on HPV-PCa subjects have also been published. One such report analyzed 29 eligible studies (PCR or serologic), demonstrating that HPV slightly elevated risk of PCa Aiming to overcome the previously observed shortcomings, we conducted a case-control study by including complete sample size calculation, age-matching between groups, and examining test–retest reliability. The intent of this case-control study was to identify potential PCa risk factors including detection of HPV infection, as well as dietary factors, family history, personal lifestyle, and sexual behavior.7, whilst concerning incidence rates of PCa and BPH was relatively low for ages 55 years and younger. Patients aged 86 and over, were excluded due to perceived slow reaction times, poor physical performance statuses, and difficulties in comprehension of interview problems. Age was matched ± 5 years range between case and control groups. Exclusion criteria (assessed via questionnaire interview) included HPV-vaccinated as well as patients not initially diagnosed as PCa.This study utilized a prospective hospital-based, matched case-control design, at a 1:1 ratio. The case group consisted of 143 PCa patients, while the control group consisted of 135 BPH patients with prostatic biopsies testing negative for malignancy. Study participants were recruited between February 2018 and December 2020, from four major hospitals in Taoyuan City, Taiwan, upon approval from with each’s respective institutional review board (IRB). Patient age at enrollment was between 55 and 86. According to Taiwan’s Cancer Registry Annual Report for 2017, the median diagnostic age of PCa in Taiwan was 7323: (39/95) 41% versus (11/55) 20%, Carozzi et al.24: (14/26) 53.8% versus (5/25) 0%, Leiros et al.25: (17/41) 41.5% versus (0/30) 0%, and Serth et al.26: (10/47) 21% versus (1/37) 3%. Three sample size calculators  were utilized to determine number of subjects for enrollment, with results of 74, 78, and 85. Patient drop-outs, refusals, or incomplete data, were forecasted to be 20–25%. As a result, 100 cases and 100 controls were determined to be the ideal sample size, and target recruitment was set at 120 for each group.A two-sided significance level  was set at 0.05, and power (% chance of detection) at 80%. Given that the sample size in most previous studies numbered only a few dozen, or lacked information on sample size calculation, these individual case-control studies were likely insufficiently powered. To overcome this limitation, we reviewed four related case-control articles which had clear HPV prevalence data in both groups: Singh et al.We collected laboratory data from patients who agreed to provide prostate specimens at the urology departments of any of the four hospital sites. The main surgical procedures were radical prostatectomy (RP), transurethral resection of the prostate (TURP), and transrectal ultrasound biopsy (TRUS biopsy). RP was only performed in the PCa group. Informed consent forms were required. Potential PCa risk factors analyzed include detection of HPV infection, as well as dietary habits, family history, personal lifestyle, and sexual behavior.A structuralized questionnaire was devised to identify for possible covariates, such as risk factors and exposures, during which 15 questionnaires and two experts were consulted. Based on an IRB request for patient privacy, the option to refuse answering for individual questions was allowed. The collected information included age, body mass index (BMI), diet, family history of cancer, occupational record, race, history of sexual activity and STDs, as well as use of tobacco, alcohol, and betelnut chew. The questionnaire was administered by qualified interviewers in-person. Only records of patients who provided both pathological specimens and completed the questionnaire were included in the data analysis.Cases ascertained were those pathologically proven for PCa, aged 55 to 86. The majority of these cases had biopsies due to elevated-PSA and were suspected of PCa. A small number of cases were incidentally found to be malignant PCa during TURP for BPH symptoms. Selection of controls, or confirmed non-cases, were defined as BPH patients with clinically apparent lower urinary tract symptoms (LUTS), and were refractory to oral medicines, urethral catheterization, and received TURP from urologists. Another source of non-cases were patients with incidental PSA-elevation and highly suspected of PCa, whose TRUS biopsies detected no cancer cells.HPV detection was performed in formalin-fixed, paraffin-embedded tissue (FFPE) samples by two commercial kits, Cobas 4800 HPV Test (Roche Molecular Systems) and DR HPV Genotyping IVD Kit (DR. Chip Biotech), at the Taipei Institute of Pathology. The Cobas 4800 HPV test individually detects HPV16 and HPV18, as well as pooled-detection of 12 other high risk HPV types  by real time PCR. The DR HPV Genotyping IVD Kit (HPV-27) individually identifies 27 common HPV types, including 15 high risk  and 12 low risk types .® Genomic DNA Tissue kit .The FFPE tissue specimen blocks were collected from the participating hospitals, deriving from different types of surgical procedures including RP, TRUS biopsy, and TURP. These FFPE blocks were sent to the Taipei Institute of Pathology where H&E staining slides had been prepared for pathologist review to confirm the diagnosis of malignant or benign disease. A minimum of three tissue sections, 5 um in thickness, per patient were collected for DNA extraction. DNA was extracted by using MagCorep < 0.05), further multiple logistic regression analysis was conducted to determine the effect size of such single independent variables, in the presence of actual or potential confounding. Forward stepwise selection is a variable selection method, starting with a model that contains no variables (null model), then gradually adding the most significant variables one at a time, until it either reaches the stopping rule (criterion) or includes all of the variables into the model. The stopping rule (criterion) in this study is when p ≥ 0.05. The most significant variables are chosen from the result of simple logistic regression, including BMI, HPV detection, occupation exposure, etc.Control and case groups were matched for age, and chi-square test was utilized to verify that there was no difference in distribution by age groups. Twenty patients from each group were selected for a second interview, within 30 days of each patient’s first interview, to validate the reliability of the questionnaire. Test–retest reliability coefficient was computed to measure the correlation between results from the first and second interview. Intraclass correlation coefficients (ICC) value of > 0.7 was deemed to be acceptable. As for the exposures or potential risk factors for PCa, independent t-test and chi-square test were adopted for univariate analysis of all variables and PCa status. If any individual variables reached statistical significance  and one assistant per hospital site, whereby the assistant was trained by the PI to conduct interview in similar manner.The consistency of responses to the questionnaire was examined as follows: 20 cases and 20 controls, interviewed twice within 30 days, conducted by the same interviewer. Test–retest reliability was estimated by calculating the (CC) of the measured values at two separate time points. A higher CC between measured data denotes greater test–retest reliability, whereas Pearson’s This study was performed in line with the principles of the Declaration of Helsinki. We obtained IRB approval from National Yang Ming Chiao Tung University, Min Sheng General Hospital, Landseed International Hospital, Taoyuan General Hospital and Ten Chen Hospital. Informed consent was obtained from all individual participants included in the study.p = 0.161). Median age (with interquartile range) was 71.8 (11.2) and 71.3 (11.1) for case and control groups, respectively. Recruited patients were stratified into six subgroups . The chi-square test showed that the age distribution between groups were not different, with p value of 0.826, substantiating the age-matching method. Mean subject height was 164.8 cm (± 5.9 SD) for case group, and 166.7 cm (± 6.0 SD) for control group, with significant difference (p = 0.007). Mean subject weight was 68.2 kg (± 9.4 SD) in case group and 66.2 kg (± 10.1 SD) in control group, with no significant difference (p = 0.091). The mean BMI was 25.7 kg/m2 (± 3.2 SD) in cases and 23.8 kg/m2 (± 3.3 SD) in controls, with significant difference (p < 0.001). In terms of occupational exposure, i.e., chemical solutions/materials, plastic materials, metal dust, machinery, electroplating, printing ink, dyes, paints, radiation, and electromagnetic field, subjects in PCa group had a significantly higher proportion than patients in BPH group . BPH was more remarkable in control group than in case group, 94.8% versus 79.0% (p < 0.001), as selection for control group was largely from BPH patients with clinically apparent LUTS, as well as patients with incidentally determined PSA elevation.From Feb 2018 through Dec 2020, 278 subjects were recruited, with 143 in the case group and 135 in the control group (Tablep = 0.024), which demonstrates greater HPV correlation in the PCa group. Revealed HPV types were 18 (2), 52 (1), 53 (3), 62 (1), non-16/18 (3) in case group, while type 52 (2) was only noted in control group, with number in parenthesis denoting number of HPV cases. HPV types 18, 52, 58, 62 were high risk while non-16/18 was low risk. However, the association between total HPV detection and PCa was slightly significant in further logistic regression analysis. If low risk types were not included, then high risk HPV detection was not associated with PCa.We detected 10 out of 143 PCa patients as well as 2 out of 135 BPH patients to be HPV positive, with positive detection rate of 7% versus 1.5% , urinary tract infection (p = 0.174), bladder disease (p = 0.833), pelvic trauma (p = 0.859), pelvic surgery (p = 0.709), autoimmune disease (p = 0.486), pelvic cancer (p = 0.531), and extrapelvic cancer (p = 0.061) between the groups (Table History of radiation therapy (RT), dietary supplement, and medication history was illustrated in Table ps Table .p < 0.001). Meat consumption was categorized as white meat, seafood, and red meat. The majority of subjects in both groups ate red meat primarily, but red meat consumption was significantly higher in case group than control group . We further explored meat consumption by questioning subjects as to percentage of total meat consumed being red meat, as stratified into three groups: less than 25%, between 25 and 50%, and greater than 50%. Case group was 22.7%, 31.8%, and 45.5%, respectively, whereas control group was 45.7%, 32.3%, and 22.0% (p < 0.001). Case group also reported greater update of dairy products, although not statistically significant (p = 0.052). Case group also consumed fewer vegetables (p = 0.01), fewer fruits (p = 0.027), and fewer soybean products than the control group. An interesting observation was that all vegetarian subjects belonged to the control group, while none were found in the case group (p < 0.003), in line with the possibility that vegetarian diets are a preventative factor for PCa.Differences in personal lifestyles, especially in dietary habit, were noted Table . Meat cop = 0.173).Other non-significant variables were shown in Table 30. This study also explored family history of PCa via questionnaire, and results revealed positive correlation between PCa and family history of PCa, with 11.4% in case group and 3.7% in control group, with family history of PCa (p = 0.016) (Table p < 0.001), and score summation 8–19 was deemed as moderate BPH. Circumcision before first sexual intercourse was associated with a slightly lower reduction in the relative risk of PCa in some studies31, but inconclusive. Our study showed contradictory results, with more patients in case group than control having received circumcision   Table . However6) Table  into whi32, but this issue remains controversial. In our study, 3.6% of case group and 7.4% of control group have received vasectomies, without significant difference (p = 0.161). The condition of genital beading and tattoo over lower abdomen or genital organ, and the experience of HPV vaccination was not different between groups. International Index of Erectile Function (IIEF-5), consisting of 5 questions, has evolved to be the standard for evaluating the severity of erectile dysfunction (ED). The 5 domains are erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction, with score ranging from 5 to 25. IIEF-5 was 8.1 (± 9.5 SD) in PCa group and 8.3 (± 9.5 SD) in BPH group, without difference (p = 0.842), and score summation 8–11 was defined as moderate ED.The two groups were not different in regard to history of androgen replacement therapy (for hypogonadism) or anti-alopecia agent (such as finasteride) Table . Vasectop = 0.007), with 46.2% of PCa patients having had ≧ 4 partners throughout their life, while figure was only 28.9% in BPH patients (Table p = 0.003. Table p = 0.372), number of marriages , number of children , and sexual partners in recent one year (p = 0.709). As to the prevalence of condom use, control group had greater proportion of using condom than case group (56.3% vs 43.7%), but without significance (p = 0.094). The case group showed greater patronage of prostitutions throughout their life, with 32.9% having ≧ 3 prostitutes vs 20.7% in control group, although not statistically significant (p = 0.057).PCa patients had more lifetime sexual partners than BPH patients (p = 0.029) and sexual partners (p = 0.037). History of STD in spouses (p = 1.000) and cancer history (p = 0.904) was not different between two groups. HPV vaccines were launched in Taiwan circa 2006, and generally applied to adolescents and young adults, so almost none of the wives in either group had received vaccination for HPV (p = 0.235).We also questioned subjects regarding sexual behavior and reproductive status of their wives Tables  and S88.p < 0.001 and p = 0.003), HPV positivity (p = 0.040 and p = 0.046), and meat consumption (p < 0.001 and p = 0.011) in both simple and multiple LR. IPSS summation score of PCa was lower than that of BPH, which was due to essence and definition of controls selection. PCa-associated risk factors, in only simple LR but not in multiple LR, were occupational exposure (p < 0.001), eating red meat as main protein (p = 0.009), higher red meat consumption (p < 0.001), PCa family history (p = 0.022), circumcision history (p = 0.018), ≧ 4 lifetime sexual partners (p = 0.002), suffering 1 type of STD (p = 0.002), wife’s lifetime spouse ≧ 2 (p = 0.036), and wife’s lifetime sexual partners ≧ 2 (p = 0.044). Potential protective factors for PCa, in only simple LR but not in multiple LR, were daily consumption of more vegetables (p = 0.006), fruits (p = 0.011), and soybean products (p = 0.002). Further forward stepwise LR (Table p < 0.001), BMI , positive HPV infection , and PCa family history .Results of simple and multiple logistic regression (LR) of risk factors associated with PCa were shown Table . PCa risTest–retest reliability of the questionnaire was assessed by calculating the correlation coefficient (CC), and all the information was collected in supplement Table . Twenty To our knowledge, this is the first age-matched case-control study in East Asia to look at HPV infection, dietary habit, and other potential risk factors for PCa, using pathology specimens with questionnaires, sample size calculation, logistic regression analysis, and test–retest method.33. Our data showed total meat consumption ≧ 1 serving per meal was at higher PCa risk than those < 1 serving per meal , but interestingly, the risk was not observed for red meat consumption. Methods of meat preparation were not investigated in this study. People who prefer high calorie intake of saturated animal meat and fat have often been associated with an increased risk of obesity and PCa, as subsequent increased testosterone levels are involved in prostate tissue proliferation9. Obesity may not only increase PCa incidence, but also increase risk of PCa mortality and recurrence28. Another mechanism is that obesity, especially when combined with physical inactivity, causes a decreased tissue response to insulin, notably in terms of glucose absorption. Insulin resistance causes chronically high blood insulin levels, which is a growth-promoting hormone and hence a scientifically reasonable risk factor for PCa. Obesity, deemed to have more metabolically active adipose tissue, is usually measured by higher BMI or larger waist circumference11. The BMI of subjects were also assessed in our study, noting higher BMI yielded slightly higher risk of PCa . Family history of PCa influences the risk of developing the disease14. Five single-nucleotide polymorphisms have been identified as having a statistically significant association with PCa when present in conjunction with a family history of PCa, according to Zheng et al.12. From 1986 to 2004, the Health Professional Follow-Up Study followed 3695 PCa patients and discovered increased PCa risk by 2.3 times, with a family history of PCa in both the father and a brother (95% CI: 1.76–3.12)9. Further evidence from twin studies elucidated that shared genetic factors were responsible for a large portion of familial aggregation of PCa, with a heritability estimate of 75%11. The data obtained from the forward stepwise logistic regression method in our study also revealed that those with a PCa family history had a higher risk of PCa . Sometimes, collinearity happens when different independent variables we use in a regression model are highly correlated with one another. Collinearity will inflate the variance and standard error of coefficient estimates. In other words, these independent variables with high correlation explain some of the same variance in the dependent variable, which reduces statistical significance. As a result, the model will become less reliable. One of the solutions to reduce or remove collinearity is to carefully select the variables or consider using other regression methods such as stepwise selection analysis. Collinearity may explain why p-value of PCa family history (0.027 and 0.074) was lower in forward stepwise selection analysis than in multiple logistic regression model.Clinical studies have suggested that certain eating habits and dietary supplements may prevent or promote PCa. Our research did not find correlation between any specific dietary supplement and PCa, but multiple logistic regression analysis uncovered that BMI and total meat consumption was correlated with PCa. A growing body of literature has linked high intake of meat, particularly red meat and processed meat, to an increased risk of cancer. Evidence showed that this danger was not caused by meat per se, but rather by high-fat consumption or mutagenic heterocyclic amines produced by the ways of high-temperature cooking  and processing procedures p = 0.003). Tantamango–Bartley et al.34 reported that vegan diets may confer a lower risk of PCa , since vegans consumed a diet heavy of antioxidant-rich foods, reversing chronic inflammation induced by oxidative stress and free radical production that are known to damage genomic DNA and hasten PCa development. Another study from Taiwan conducted by Chen et al.35 recognized a beneficial association with vegan diets against PCa .All vegetarians were found in control group (8/135), while none were in PCa group (0/143), indicating that vegetarian lifestyle might have a protective effect for PCa  but a more notable OR (6.02 to 1.79). Singh et al.23 demonstrated HPV infection in 41% (39/95) of tumor biopsies and 20% (11/55) in BPH that served as disease controls, and a total of 92% HPV were type 16 and 18. Carozzi et al.24 found HPV DNA in 65.3% cancer and 48.0% benign biopsies without significance (p = 0.33), but high risk HPV type positivity in 53.8% (14/26) cancer and 20.0% (5/25) benign biopsies (p = 0.03), showing high risk types to be mainly 16, 18, 52, and 58. Leiros et al.25 detected HPV DNA in 41.5% (17/41) carcinoma samples, whereas all 30 hyperplasia samples were HPV-negative, but most HPV types were low risk or undefined. Serth et al.26 detected significantly higher copy numbers of HPV16-E6 sequences in the prostate tumors with 21% (10/47) when compared to the control tissue with 3% (1/37).While numerous studies worldwide have indicated the presence of HPV infection in PCa, we wanted to take a deeper look into Taiwan’s population. The study revealed the presence of HPV in two age-matching study groups, with case group comprising of PCa patients and control group comprising of BPH patients. Compared to the global averagep = 0.046), suggesting association between HPV and PCa. However, CI of 95% was wide, and the difference observed was slightly significant. Additionally, if low risk types were not included, then high risk HPV detection would be deemed to be not associated with PCa. HPV frequencies in case group observed in the studies, from which sample size calculation was derived , showed great discrepancy to that observed on our study (7%). Discrepancies in distribution and prevalence of HPV genotypes vary greatly across ethnic groups, geographic areas, and lifestyle groups, may also factor here. This is an observed limitation to our study, and future studies should re-calculate sample size for more robust results.In our study, HPV DNA PCR amplification tested positive in 12 of total 278 patients (4.3%), of which 10 were amongst the 143 PCa patients (7%) and 2 amongst the 135 BPH patients (1.5%), with significant difference  of PCa patients had STD experience, which was higher than the 8.2% (11/135) in control group (p = 0.003), only a few had multiple lifelong STD episodes . Furthermore, almost all STD patients had sought prior medical treatment and achieved remission of the infection. These reasons may explain the insignificant results in the multivariate analysis.Sexual activity and STDs have been hypothesized to play a role in PCa development via a variety of pathophysiological routes, but the studies have thus far reported mixed resultsCalculation of sample size, test–retest method, matching and adjustment for confounders are common techniques and an important part of study design in epidemiologic research. To our knowledge, these methods were not commonly used in prior case-control studies investigating correlation between HPV and PCa. Complete study design with these methods could be considered as strength of this study. Test–retest method is used to ensure the stability and reliability of survey scores across two time-points over a short period. If reliability is found lacking, there is little confidence that the data produced is an actual representation of the respondents. Assessment of personal or medical history is relatively problematic in elderly men due to age-related changes in performance, and we found the test–retest reliability crucial for our study. One drawback of the test–retest method is that it may be subject to recall bias, since respondents may just repeat what they previously claimed. Socially undesirable items like STDs or sexual behaviors are often under-reported, causing non-differential misclassification. In conclusion, our study uncovered additional protective factors for PCa, including regulating BMI by maintaining good physical activity and reducing total meat consumption. Middle-age and elderly men with PCa family history should stay highly vigilant about the occurrence of PCa. All these results could serve as a basis for designing PCa prevention strategies. It is suggested that clinicians educate patients on up-to-current findings in order to lower risk and promote effective prevention of PCa.Supplementary Information.