In Japan, recommendations for HPV vaccines were suspended in 2013 due to unfounded safety fears. Although vaccine opponents claim modifying sexual behavior can prevent cervical cancer, no comprehensive data exist on sexual behavior and the risk of high-grade cervical disease in a Japanese population. This study investigates sexual behavior and the risk of HPV infection and cervical disease in 3968 women aged 20–41 yrs undergoing cervical screening between April 2014 and March 2016. Mean age at first intercourse was 18.4 yrs ± 2.8 and 32% of women reported ≥ 6 lifetime sexual partners. In regression analyses, number of partners was a significant risk factor for HPV infection. However, for high-grade disease (CIN2+), when HPV genotype was adjusted for, number of partners was not statistically significant. The greatest risk factor was an HPV16/18 infection . In conclusion, we found that having an HPV16/18 infection and not sexual behavior was the most significant risk factor for high grade cervical disease in young Japanese women. These infections can be prevented by a highly effective vaccine and we recommend that the Japanese government resume proactive recommendations for the HPV vaccine immediately. Risk factors for HPV infection include: early onset of sexual activity, multiple sexual partners, or partners with multiple sexual partners4. Risk factors for viral persistence and progression to high grade cervical precancers and invasive cervical cancer include exogenous factors such as tobacco smoking, high parity and coinfection with other sexually transmitted infections5; viral factors such as HPV genotype, particularly HPV 16 and HPV 18, viral load and HPV variants7; and host cofactors such as factors related to immune response8.Cervical cancer is highly preventable and if diagnosed and treated early, highly curable. Yet it remains the fourth most frequent cancer in women globally. Almost all cases are caused by persistent infection with one or more sexually transmitted high-risk human papillomavirus (hrHPV) type9. Therefore, HPV vaccination is an important public health intervention to prevent cervical cancer by routinely targeting adolescent girls before they become sexually active.Following the global call to action for the elimination of cervical by the World Health Organization’ s (WHO) Director General in 2018, a global strategy to accelerate cervical cancer elimination was drafted with clear targets for the period 2020–2030. These include that 80–100% of girls get vaccinated against HPV by the age of 15 yrs10. Incidence of cervical cancer and high-grade cervical intraepithelial neoplasia (CIN) is increasing in women of reproductive age11. Biennial screening with cytology is recommended for women aged ≥ 20 yrs as secondary prevention12. However, screening is opportunistic and coverage is lower than most OECD countries (42.4% in women 20–69 yrs)13. Bivalent and quadrivalent HPV vaccines were approved in Japan in October 2009 and July 2011, respectively. Public funding became available from 2010 and from April 2013, the HPV vaccine was included in the National Immunization Program (NIP) for girls aged 12–16 yrs. However, after unfounded sensational reports of so-called ‘adverse events’ were published by the Japanese media and pressure by a ‘Victims’ Support Group, the Japanese Ministry of Health, Labour, and Welfare (MHLW) suspended proactive recommendations for the HPV vaccine in June 201315. After that, coverage dramatically decreased, from > 70% in those born between 1994–1999 to < 1% in those born in 2000 and later16. The national education materials for schoolchildren recommend cancer screening for cervical cancer prevention in Japan, however, HPV vaccination are not described in the materials17.In Japan, there are over 10,000 cases of cervical cancer annually and more than 2700 women die from this diseaseOne reason often given by groups opposing the HPV vaccine is that only women who engage in ‘risky’ sexual behavior are at risk for cervical cancer and by modifying behavior cervical cancer can be prevented.Since there is a close correlation between sexual activity, HPV infection, and cervical cancer risk, but almost no comprehensive data that investigates a combination of all these factors in a Japanese population. We investigated the correlation between sexual behavior , HPV infection, including genotype-specific infection and high-grade cervical disease in sexually active Japanese women of reproductive age for a better comprehensive approach to cervical cancer prevention in Japan.In total, 3968 women were enrolled in the study when attending for publicly funded cervical cancer screening in Niigata city between April 2014 and March 2016. Of those, 188 women were excluded due to age, duplicate results, missing results for HPV testing or equivocal HPV test results, leaving a total of 3780 women enrolled. The response rate to the questionnaire on sexual history was 87.4% (3304/3780). Seventy-three women were excluded from the analysis because they reported no previous sexual activity. Therefore, 3231 sexually active women were included in the final analyses.p < 0.01, Fig. p < 0.01, polyserial correlation = 0.55, Fig. Number of participants in each age-group was 2179, 725 and 327 for groups A (20–30 yrs), B (35–36 yrs) and C (40–41 yrs), respectively. Mean overall age of participants was 29.8 yrs ± 5.7 and 26.3 yrs ± 2.9 in group A. Mean overall age at first intercourse was 18.4 yrs ± 2.8 yrs. For age at first intercourse, 3.4%, 24.1%, 44.6% and 27.9% of women had their first sexual experience at 14 yrs or younger, 15–16 yrs, 17–19 yrs or ≥ 20, respectively, which indicates 72.1% of women (2329/3231) became sexually active as a teenager. Among these women, mean age at first intercourse was significantly earlier in group A (16.8 ± 1.5), compared to groups B (17.2 ± 1.4) and C (17.9 ± 1.2) (p for trend < 0.01). A similar trend was observed for HPV 16/18 genotypes (p for trend < 0.01) and HPV16/18/31/33/45/52/58 (p for trend < 0.01) respectively. Prevalence of HPV type-specific infections is shown in supplementary Table p for trend < 0.01. Regarding histology, for CIN 1 and CIN2, prevalence decreased with age; p for trend was 0.04 and 0.01, respectively. For CIN3, there was no significant trend. Prevalence was highest in women 35–36 yrs, after which levels in woman 40–41 yrs decreased to levels similar to those in woman aged 20–30 yrs. There was one invasive cervical cancer in a woman aged 29 yrs.In group A, 15.2% of women had a hrHPV infection. This was significantly higher than in groups B and C, at 6.9% and 5.8%, respectively, (p for trend < 0.01). Statistical significance remained after adjusting for age and smoking history (model 2), but the trend was lost after making an additional adjustment for number of sexual partners (model 3). A similarly trend was seen for the prevalence of HPV16/18, HPV16/18/31/33/45/52/58 and CIN2 or worse (CIN2+) (p for trend < 0.01). Prevalence of CIN2+ was highest in women who became sexually active at 14 yrs or younger (4.6%), followed by a gradual decline in prevalence at 2.2%, 1.3% and 0.7% in the women whose sexual debut was at 15–16 yrs, 17–19 yrs and > 20 yrs, respectively (p for trend < 0.01). Statistical significance remained after adjusting for age and smoking history (model 2), but the trend was lost after further adjustment for number of sexual partners (model 3).Prevalence of hrHPV peaked in women who became sexually active at 14 yrs or younger (26.6%), followed by a gradual decline in prevalence at 16.4%, 12.2% and 7.4% in women whose sexual debut was at 15–16 yrs, 17–19 yrs and ≥ 20 yrs, respectively (p for trend < 0.01). A similar trend was observed for HPV16/18, HPV16/18/31/33/45/52/58 and CIN2+ (p for trend < 0.01). In the fully adjusted model (model 3), the odds of having an HPV 16 or HPV 18 infection in women who had ≥ 6 lifetime sexual partners was 78-fold greater (p for trend < 0.01). For CIN2+ , the reference was 2–5 partners, since no woman with one partner had CIN2+ . Despite this, the odds of having CIN2+ in women who had 6–9 or ≥ 10 partners were still 2 to 4 times higher.Prevalence of hrHPV peaked in women who had ≥ 10 lifetime sexual partners (25.1%), followed by a gradual decline in prevalence at 20.2%, 9.0% and 3.6% in women who had 6–9, 2–5 and one, respectively (p < 0.01), even after adjusting for sexual history, age and smoking history. The risk was 66-fold with a non-HPV 16/18 Hybrid Capture (HC)2 infection (p < 0.01).Finally, we investigated risk factors for CIN2+ . Unlike HPV infection, lifetime number of sexual partners and age at sexual debut were not statistically significant factors for having CIN2+ when HPV genotype was adjusted for. Compared to women who were HPV negative women, women with a vaccine type HPV 16/18 lesion had a 114-fold risk for CIN2+ , HPV infection (including genotype) and high-grade cervical disease (CIN2+) in sexually active Japanese women of reproductive age. As a results, number of sexual partners was not an independent risk factor for CIN2+ when HPV genotype was adjusted for in the multivariate analyses. The greatest risk factor for CIN2+ was an HPV 16/18 infection (aOR 113.7).19. One national study reported that 23% of female high school students (16–18 yrs) were sexually active in 2011 compared to only 9% in 1981. Similarly, in 2011, 46% of female university students (aged ≥ 18 yrs) reported being sexually active, compared to only 19% in 198118. Regarding number of previous sexual partners, there is a paucity of robust Japanese data. There is only one national study on HIV by the MHLW in 1999 that reports such data. In this study, only the percentage of males and females by age-groups who had had ≥ 5 sexual partners is reported. Furthermore, the number of participants in each age-group is not given19. A more recent study by Imai et al. only reports numbers for university students20. In the present study, we found that 72% of women become sexually active as a teenager. Similar to the past national study, the age of sexual debut is becoming earlier in younger cohorts. In the present study of the general population, almost half of participants had 2–5 sexual partners and approximately 30% of participants had more than ≥ 6 sexual partners in their lifetime. There was a significant correlation between lifetime number of sexual partners and lower age at first intercourse.Two previous studies have reported cohort differences in sexual behavior for Japanese populations21. In our univariate analysis we also found this to be true. However, after adjusting for number of sexual partners, we found that statistical significance disappeared.Several cross-sectional studies have shown that early age at sexual debut is a risk factor for HPV infection22. In the present study, we also found that even after adjusting for age, smoking history and age at first intercourse, the risk of a hrHPV infection significantly increased as the number of partners increased. For the two most oncogenic types, HPV 16 and HPV 18, risk of infection was 78-fold once the number of partners reached 6 or more. In the present study of women attending for screening, 32% of participants reported ≥ 6 sexual partners. In the national HIV study by Kihara et al., it was also reported that the younger one is, the likelihood of having ≥ 5 partners increases. 19. While it was common in the past for women to marry at an early age, in 2016, the average age of marriage for Japanese women was 29.4 yrs, which means women could be in several monogamous relationship before choosing to settling down and that having more than one sexual partner does not reflect any particularly risky sexual behavior.Rate of acquisition of sexual partners, as well as the association between number of sexual partners and hrHPV infection is consistent and strong23. HPV 16/18 also have a faster time of progression to precancerous lesions6. Similar data have been reported for Japan24. In the present study, the greatest risk factor for CIN2+ was an HPV16/18 infection , even after adjusting for sexual history, age and smoking history.While many women may get cervical HPV infections, most infections do not persist or progress to high grade cervical disease. HPV genotype is not only the strongest viral cofactor, but also the strongest absolute risk for persistence and progression. HPV 16 is highly carcinogenic and responsible for over half (53.4%) of cervical cancers worldwide25. A recent systematic review of 10 year real-world use reported that HPV vaccines have dramatically decreased the incidence of vaccine-type HPV infection and high-grade cervical disease26.HPV vaccines have been available since 2006/2007. These vaccines induce a high-level antibody response to HPV 16 and HPV 18, responsible for over 70% of cervical cancers globally and over 90% of cervical cancers in Japanese women 20–29 yrs15.Another study demonstrated that for cohorts eligible for free HPV vaccination, compared to those not eligible, the incidence of CIN2+ or CIN3+ in women attending cervical screening was significant lower, p = 0.014, p = 0.01627. However, the Japanese MHLW suspended proactive recommendations for the vaccine in June of 2013, and uptake plummeted from > 70% to,1%16.This means young Japanese women are once again at risk for HPV 16 and 18 related high-grade cervical disease28. This risk could be mitigated if the Japanese MHLW resumed proactive recommendations for the HPV vaccine. Furthermore, a recent modelling study has shown that rapid restoration of HPV vaccination coverage in girls aged 12 years, plus catch-up of those girls who missed vaccination due to the suspension of proactive recommendations using the nine-valent HPV vaccine, could mitigate much of the health impact of the HPV vaccine crisis in Japan to-date29. In the present study, the three most prevalent HPV types were HPV52, 16 and 58, which are covered by the nine-valent vaccine 31. As part of Japan’s national cancer Control Plan, educational activities to increase school children’s awareness of factors to prevent cancers should be undertaken. To facilotate this, teaching materials were made available by the Ministry of Education, Culture, sports, Science and Technology32. However, with regards to cervical cancer prevention, only screening is recommended, no mention is given about the crucial role HPV infection plays in the etmology of cervical cancer and the fact that a highly safe and effective vaccine exists to prevent this infection is also omitted17. More education about HPV and the role it plays in the development of cervical cancer is urgently needed for young adolesents.For a comprehensive approach to cervical cancer prevention, education also plays an important role. The WHO states that “national educational campaigns for HPV vaccine introduction should be used to increase awareness about cervical cancer and its prevention”33. A second limitation is that the number of women who had received the HPV vaccine was small (3.5%) which meant vaccination status could not be controlled for in the multivariate analyses. A third limitation is that due to a limited budget, the number of women that could be recruited in their thirties and forties was limited. Finally, our study has no information of male’s sexual behavior which is also a significant risk factor for HPV infection and cervical disease in women.This study has several strengths. Firstly, it is one of the few studies to report sexual age at sexual debut and number of sexual partners in Japanese women. Secondly, to our knowledge, it is the only study to report the relationship between sexual behavior, type-specific HPV infection and high-grade disease status in a Japanese population. There are also several limitations that must be addressed. Firstly, our results may not reflect the population of Japan as a whole, since our study was only performed in one region of Japan, However, regarding age at sexual debut, our results were similar to a recent Japanese internet study on sexual behavior, conducted in all 47 prefectures of JapanIn conclusion, we found that > 30% of sexually active Japanese women had ≥ 6 previous sexual partners, that sexual debut is becoming earlier and associated with a greater number of sexual partners. We showed that the number of previous sexual partners is a significant risk factor for hrHPV infection, but HPV 16 and 18 infection and not number of sexual partners, is the most significant risk factor for CIN2+ . Educating adolescents about the HPV infection and achieving high uptake with the HPV vaccine, especially when given before the age of sexual debut would help mitigate these risk factors. Consequently, it is imperative that the Japanese government resumes proactive recommendations for the HPV vaccine and educates teenagers about the risks of HPV infection so that Japan women can be afforded the same level of protection against the disease as their global peers.The cross-sectional study was conducted in Niigata city between April 2014 and March 2016. Niigata city has a population of around 790,000 and is the 16th most populous city in Japan. Women aged 20–30 yrs (group A), 35–36 yrs (group B) and 40–41 yrs (group C) were recruited when attending for public cervical cancer screening. Birth cohort years were 1984–1995, 1978–1980 and 1973–1975, respectively. Public funding for HPV vaccination started in 2010 for girls aged 13–16 yrs in Niigata city. Therefore, participants born after 1994 were eligible for free HPV vaccination. The present study protocol was approved by the institutional review board of Niigata University Graduate School of Medical and Dental Science and registered at the UMIN Clinical Trials Registry, trial number UMIN000030718. Written informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.Information about sexual history was obtained through a short self-administered questionnaire which asked about age at sexual debut and number of previous sexual partners. For the latter, participants had to choose from the following 5 categories: none, 1, 2–5, 6–9 and ≥ 10. Age at first intercourse was categorized into four groups: 14 yrs or younger, 15–16 yrs, 17–19 yrs and ≥ 20 yrs. Information on HPV vaccination status was also obtained from the questionnaire. As a potential confounder for persistence and progression of HPV infection, we also obtained information on smoking history from personal cervical cancer screening records. Responses were classified as: never, former or current smoker.34. This assay detects 13hrHPV genotypes; HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.Residual liquid-based cytology  specimens from cervical screening were used for HPV testing. All samples were tested with HYBRID CAPTURE (HC) 2  for pooled infection with one or more 13 hrHPV genotypes; HPV types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Only those samples positive for the HC2™ test underwent HPV genotyping with the MEBGENTM HPV kit 35. Abnormal categories included cases of atypical squamous cells of undetermined significance (ASC-US) or worse. A result of negative for intraepithelial lesion or malignancy (NILM) was considered normal. All women with abnormal cytology, except for those cases that were ASC-US HPV negative, were referred for colposcopy. Information on cervical histology was obtained using data from Niigata city health center. For cervical histology, the three-tiered cervical intraepithelial neoplasia (CIN) classification  was used. CIN2 or worse (CIN2+) was defined the high-grade cervical disease36.Cervical cytology diagnoses were made at Niigata University and classified according to the Bethesda Systemp value of < 0.05 was considered to indicate statistically significant.Characteristics of study participants were presented as means and SDs for continuous variables and as percentaged for categorical variables. Welch’s ANOVA test and polyserial correlation were used to investigate the correlation between age at first intercourse and number of sexual partners. Welch’s ANOVA test was also used to assess the difference in age at first intercourse between the three age-groups. Univariate and multivariate logistic regression analyses were conducted to investigate the correlation between sexual behavior , HPV infection status, HPV type-specific infection and cervical disease. Three models were used: model 1 adjusted for age; model 2 adjusted for age and smoking history; and model 3 adjusted for age, smoking history, age at first intercourse or number of sexual partners. Finally, we performed a multivariate analysis to investigate independent risk factors for high-grade cervical disease (CIN2+ vs. NILM). The model was adjusted for age, smoking history, sexual behavior, and HPV type-specific infection. We used IBM SPSS Statistics Version 25.0  and R version 3.4.1 . A two-sided Supplementary Information.


The diagnostic efficiency of AUC by applying HPV16/18 viral load was 0.682 for the diagnosis of CIN II+. The optimal HPV16/18 viral load for predicting CIN II+ was 6.80 RLU/CO (relative light units/cut‐off), with corresponding sensitivity of 48.6%, specificity of 79.7%, and Youden index of 0.283. In the ASCUS population, viral loads were statistically different in HPV16/18 and the other 12 HR‐HPV when compared cervicitis group with CIN I group and CIN II+ group . Statistical differences were detected concerning HPV16/18 viral load, contact bleeding status, and smoking status when compared cervicitis group with CIN I group and CIN II+ group (p < 0.05), with a corresponding odds ratio of 1.004, 1.533, and 5.513, respectively. Our findings suggest that HR‐HPV viral load can be regarded as a useful tool to predict the grade of cervical lesions for ASCUS triage.This study aims to evaluate HR‐HPV viral load in the cervical lesion assessment and its diagnostic value on the triage of ASCUS. The three‐step protocol for cervical cancer screening was carried out in 5171 patients from June 2017 to August 2019, and 1620 histopathological results were obtained. The positive rate of HR‐HPV and TCT increased with the aggravation of pathological grades of cervical lesions. The sensitivity and specificity of HR‐HPV (DH3) to detect CIN II+ were 91.91% and 84.46%, respectively. In comparison, the corresponding results of the cytology test were 80.51% and 83.12%. HPV16/18 viral load was positively correlated with the grade of cervical lesions (ClinicalTrials.gov ID: NCT03178136. This study which involves 5171 patients is focusing on high‐risk HPV viral load and cervical lesions. It is the first research on the diagnostic value of high‐risk HPV viral load by using DALTONbio hybrid capture 3. Our findings suggest that HPV16/18 viral load has good diagnostic efficacy and specificity in predicting CIN II + lesions. HR‐HPV viral load detection can be used to screen the high‐risk population with CIN II + lesion to the greatest extent. In China, cervical cancer has been the second most common female malignant tumor, which is characterized by younger onset and annual increase in its incidence.According to prior research, human papillomavirus (HPV) infection is intimately associated with the occurrence and development of cervical lesions. HPV infection is mostly a transient or intermittent phenomenon, 90% of which can be resolved spontaneously within two years, or develop into persistent infection.The internationally recognized three‐step protocol for cervical cancer screening includes primary screening, colposcopy, and histopathology. At present, the primary screening scheme is widely applied in China by HPV detection and Thinprep cytologic test (TCT) alone or jointly. HPV genotyping, HPV‐RNA detection, and HPV‐DNA viral load detection are commonly used in Chinese hospitals. For those with abnormal primary screening, the scope and extent of the lesions can be further determined by colposcopy. Therefore, histopathological examination is still the gold standard for diagnosis and treatment.HPV infection is the most common reproductive tract infection in women of childbearing age. The 14 internationally recognized high‐risk HPV (HR‐HPV) types are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. HPV16 and HPV18 are known as the most serious types, with 39%‐78% of cervical cancer reported to be attributed to these two types.In view of these problems, there are many researches committed to identifying the precise screening indicators of cervical cancer with clinical application value in recent decades. HR‐HPV viral load is so far one of the most controversial indicators. Unlike previous studies of HPV viral load, the present study applied DALTONbio hybrid capture 3 (DH3) to detect the viral load of HPV16/18 and the other 12 types of HR‐HPV viruses. This study is expected to investigate the correlation between the viral load of high‐risk HPV and cervical lesion grades as well as its diagnostic value for the shunt management of ASCUS.22.1From June 2017 to August 2019, eligible subjects were selected from 10 hospitals, including Shengjing Hospital of China Medical University, Anshan Cancer Hospital, The Second Hospital of Chaoyang, Dalian Maternal and Child Care Hospital, Shenyang Women's and Children's Hospital, Women's and children's hospital of Yingkou City, The Second People's Hospital and Gynecology and Obstetrics Hospital of Fuxin City, The First Affiliated Hospital of Jinzhou Medical University, Dalian Municipal Women's and Children's Medical Center, The First Affiliated Hospital of China Medical University. The inclusion criteria were women aged 25–65 years old, with a medical history of sexual behavior, which voluntarily underwent cervical cancer screening. Those with a history of other systemic tumors and hysterectomy were excluded from this study. Samples of cervical exfoliated cells were collected for HR‐HPV viral load test and TCT. Basic information of enrolled patients was collected to establish medical records. The study was reviewed and approved by the hospital ethics committee, and all the subjects gave written informed consent.2.2Non‐menstrual samples were collected from subjects without intercourse and other vaginal operations three days prior sampling. Cervical brush and sample preservation solution were used for sampling in each test, and the test steps were strictly operated in accordance with the instructions for each reagent.HR‐HPV viral load test: the 14 types of HR‐HPV  were detected quantitatively by DH3 . The test result of the viral load value was calculated as relative light units/cut‐off (RLU/CO) ratio. The cut‐off value is 1.0 pg/mL. The viral load value ≥1 indicated a HPV positive result.TCT: the TBS (The Bethesda System) grading system was used to evaluate the results by two pathologists. The abnormal results included atypical squamous cells undetermined significance (ASCUS), atypical squamous cells‐cannot exclude HSIL (ASC‐H), low squamous intraepithelial lesion (LSIL), high squamous intraepithelial lesion (HSIL), squamous of cervical carcinoma (SCC), etc. Subjects with ASCUS and above were determined as TCT positive results.Patients with positive HR‐HPV viral load and TCT results were further treated according to the routine diagnosis and treatment procedures. Pathological examination was further performed in patients with abnormal results of colposcopy, if necessary. The final diagnosis was based on pathological results.2.32 test for comparison of rates, multivariate logistic regression analysis, receiver operator characteristic (ROC) curve, etc. p < 0.05 meant that the difference is statistically significant.The database is established by EpiData 3.1 software. All of the patient's relevant information and test results are inputted into the final database after a duplicate check. By SPSS 22.0 software, analysis of data is achieved by various statistical methods, including spearman rank correlation analysis, χ33.1A total of 5171 patients were enrolled in this study, including 1620 patients with histopathological findings, with an average age of 41 ± 10 years old. As for the pathological results, there were 472 cases of chronic cervicitis, 604 cases of CIN I, 299 cases of CIN II, 215 cases of CIN III, and 30 cases of cervical cancer. Table 3.2p < 0.001, r = 0.275). The sensitivity and specificity of TCT to detect CIN II+ were 80.51% and 83.12%, respectively..The positive rates of TCT in chronic cervicitis, CIN I, CIN II, CIN III, and cervical cancer were 66.53% (314/472), 77.32% (467/604), 78.60% (235/299), 82.79% (178/215), and 83.33% (25/30), respectively, showing an increased trend. Besides, there was a grade correlation between the results of TCT and the grade of cervical lesions (p < 0.001).A total of 1,219 cases were positive for the detection of HR‐HPV viral load. The sensitivity and specificity of HR‐HPV (DH3) to detect CIN II+ were 91.91% and 84.46%, respectively. The distribution of HR‐HPV viral load in cervical lesions is shown in Table p < 0.001, r = 0.321). In HPV16/18 positive cases, the HPV16/18 viral load was positively correlated with cervical lesion grades, regardless of the other 12 types of HR‐HPV results . In HPV16/18 negative and the other 12 types of HR‐HPV positive cases, the other 12 types of HR‐HPV viral load have a weak positive correlation with cervical lesion grades .Spearman rank correlation analysis showed that HPV16/18 viral load was correlated positively with the grade of the cervical lesion . Spearman rank correlation test showed that HPV16/18 viral load was weakly correlated with the abnormal grade of cervical cytology . Meanwhile, the other 12 types of HR‐HPV viral loads were positively correlated with abnormal grade of cervical cytology .Among 1620 subjects with biopsy results, there were 495 cases of ASCUS, 132 cases of ASC‐H, 397 cases of LSIL, 177 cases of HSIL, and 18 cases of AGC according to the results of TCT. There were significant differences in the distribution of HPV16/18 viral load and other 12 types of HR‐HPV viral loads in cervical lesions of different grades based on cervical cytology  and CIN II + lesion group (p < 0.001). Besides, the sensitivity of HR‐HPV viral load was 94.87% to diagnose CIN II+lesions.Among 495 ASCUS cases, there were 172 cases of chronic cervicitis, 206 cases of CIN I, 76 cases of CIN II, 37 cases of CIN III, and 4 cases of cervical cancer. Table 3.4p < 0.2 in univariate analysis (Table p < 0.05), with corresponding OR were 1.004, 1.533, and 5.513, respectively. However, no statistical difference was found in the viral load of the other 12 HR‐HPV, education level, cervical cancer screening, columnar ectopy, age of sexual initiation, age of first pregnancy, and medical history of CIN (p > 0.05).On the basis of prior literature guideline and professional knowledge, the factors that may be strongly correlated with the occurrence and development of cervical lesions were screened for logistic regression analysis. Relevant factors included 10 variables such as educational level, cervical cancer screening, smoking, columnar ectopy, contact bleeding, age of sexual initiation, age of first pregnancy, medical history of CIN, and two HR‐HPV viral loads. Logistic regression analysis was carried out for the variables with is Table . Accordi4Massive studies have confirmed that HR‐HPV infection is a necessary condition for the presence of cervical precancerous lesions and cervical cancer.Currently, TCT combined with HPV test is used in Chinese cervical cancer screening program. Abundant evidenceIn virus infection‐related tumors, high viral load has been reported to be significantly related to the risk of carcinogenesis. For example, Epstein‐Barr virus DNA load was intimately correlated with the occurrence and development of nasopharyngeal carcinoma.ASCUS is the most common abnormal diagnosis result in TCT, and its proportion is much higher than other results. ASCUS is defined as more obvious changes in cell abnormality than the reactive change, but not reaches the degree of squamous intraepithelial lesions. It can be a benign change or a potential malignant change with active hyperplasia. In a study carried out by Yarandi F et al.,p < 0.05), with corresponding OR of 1.004, 1.533 and 5.513, respectively. However, the viral load of the other 12 types of HR‐HPV, education level, cervical cancer screening history, columnar ectopy, age of sexual initiation, age of first pregnancy, medical history of CIN were not risk factors of high‐grade cervical lesions. It is known that multiple factors seem to intervene in cervical lesions.According to the logistic regression analysis results, there were statistical differences in HPV16/18 viral load, contact bleeding, and smoking when compared cervicitis group and CIN I lesion group with CIN II + lesion group  viral load value may be considered as a potential indicator to predict cervical lesion grade and ASCUS triage management. HR‐HPV viral load is expected to be applied in clinical cervical cancer screening, which will make the screening and ASCUS triage more effective and precise.5There is no conflict of interest in this study.


Sweden has 10.2 million inhabitants and more than 2.4 million have a foreign background. A substantial number of immigrants come from countries where glucose-6-phosphate dehydrogenase deficiency (G6PDD) is frequent. The total birth rate annually in Sweden is approximately 117,000 and newborn screening is centralized to one laboratory. We determined glucose-6-phosphate dehydrogenase (G6PD) activity in 10,098 dried blood spot samples (DBS) from the whole country with a fluorometric assay . The first 5451 samples were anonymised and run as singletons, whilst the following 4647 samples were coded. Enzyme activity ≤40% of the mean of the day was found in 58 samples (1/170) and among these, 29 had activities ≤10% (1/350). Twenty-nine samples with residual activities between 2–39% in the coded cohort were subjected to Sanger sequencing. Disease-causing variants were identified in 26 out of 29 infants, of which six were girls. In three patients, we did not find any disease-causing variants, although two patients were hemizygous for the known polymorphisms c.1311T>C and c.1365-13C>T. The most common disease-causing variant found in 15 of the 29 samples  was the Mediterranean mutation, c.563C>T (p.(Ser188Phe)) in exon 6. G6PDD is thus a surprisingly prevalent disorder in Sweden. It is a housekeeping enzyme important for the control of oxidative stress, especially in red blood cells, which lack nucleus and mitochondria. Patients with glucose-6-phosphate dehydrogenase deficiency  are asymptomatic unless they have a very low residual activity or are exposed to some specific triggers, including certain medicines, infections or fava beans (Vicia faba), the latter being the most common trigger ;[=]. In three patients, we did not find any disease-causing variants, although two were hemizygous for the known polymorphisms c.1311T>C and c.1365-13C>T .The incidence of G6PDD in Sweden was unknown before this study. Our results indicate that 1/350 newborn infants have a G6PDD with an enzyme activity of ≤10% of normal. With the present annual number of births in Sweden of 117,000, approximately 330 patients would be recalled with a suspected severe G6PDD if newborn screening for G6PDD was to be implemented in the Swedish NBS programme. If all newborn infants with residual activity of ≤40% of normal were to be recalled, this number would increase to almost 700 or 0.6% of all newborn infants.The proportion of boys and girls among the 58 infants with an enzyme activity below the cut-off (≤40%) in this study is unknown. NBS screening programmes detect hemizygous boys and homozygous girls, missing out most of the heterozygous girls . In the With the immigration to Sweden from areas in the world with a known high prevalence of G6PDD, it is not surprising that the incidence of newborn infants with an enzyme activity less or equal to 40% is as high as 1/170. This is much higher than indicated by clinically diagnosed patients. In a recent article where the incidence of hazardous hyperbilirubinaemia  and kernicterus  in newborn infants was studied, the authors conclude that the incidence of the two diagnoses is increasing . This isScreening for G6PDD has been recommended if the incidence in newborn boys is more than 4–5% . This isG6PDD is becoming a more common disorder in Sweden. It is important for clinicians to be aware of the disorder when patients originating from areas where malaria was or still is common and G6PDD frequent, are diagnosed with acute haemolytic anaemia. This is also the case when newborn infants develop hyperbilirubinaemia rapidly or develop hyperbilirubinaemia not responding to traditional treatments. NBS for G6PDD ought to be kept in mind in the future since an increase in the incidence among newborn infants is to be expected.


The eradication rate differed among patients with primary low-risk and high-risk HPV types—76.92%  and 50%  respectively. Conclusion: ALA-PDT is an effective treatment even after the failure of previous modalities. The persistence of clinical lesions and high oncological risk HPV types should be an indication for treatment prolongation.Background: Genital warts are the manifestation of the human papillomavirus (HPV) infection, which may last for weeks or months before the clinical presentation. The primary aim of the study was the correlation of the DNA HPV genotypes eradication with the treatment response in male patients with persistent genital warts. Methods: Twenty-one male patients (age range: 22–58) after failure of cryotherapy and podophyllotoxin treatment were enrolled in the study. Genetic tests  analyzed the presence of DNA-HPV before and 6 months after four sessions (4 weeks apart) of photodynamic therapy with 5-aminolaevulinic acid (ALA-PDT). The treatment efficacy was evaluated before each PDT session and at the end of the study. Results: The single HPV DNA type was present in 15/21 of the patients (13/15 HPV6). The high-risk HPV types were found in 8/21 subjects, of which 6/8 had several types. Six months after four sessions of PDT, complete response was found in 16/21 (76.19%;  Genital warts are a common symptom of human papillomavirus (HPV) infection. They develop within weeks or months after the infection, but in most cases, the virus can be latent for months or years. Risk factors for HPV infections include early onset of sexual activity, multiple sexual partners, high-risk sexual practices, and poor hygiene ,2,3. HPVThe primary aim of the study was the correlation of the DNA HPV genotypes eradication with the treatment response in vaccine-naive patients. The secondary aim was the assessment of PDT treatment efficacy of persistent genital warts in adult male patients.The method of analysis was chosen from the prospective single arm observational study. The study was approved by the Ethical committee of Military Institute of Medicine (Number 100/WIM/2018) and was conducted in accordance with Declaration of Helsinki. The study was conducted in Dermatology Department of the Military Institute of Medicine in Warsaw.As eligible for enrollment, we invited male patients >/= 18 years old who were initially diagnosed by a dermatology specialist with genital warts and underwent two unsuccessful therapies with cryotherapy (at least 2 sessions) and podophyllotoxin (used twice daily for 3 consecutive days repeated for up to 5 weeks). Before enrollment, each patient had to sign the Informed Consent approved by the Ethical Committee.The exclusion criteria regarded the contraindications to ALA-PDT procedure.At the baseline visit, the clinical examination was performed by a dermatology specialist and genital swabs used for HPV DNA testing were collected from all the lesional skin areas. The sterile polyester swabs were individually packaged and a specimen collection vial contained 1 ml of specimen transport media. The swabs were taken with a brush that was rubbed gently but firmly several times on the genital warts. After collection, the swab was placed in the sterile transport media vial; then, the end of swab was cut of to permit capping. The closed vial was kept at 4 °C and shipped to Synevo/Nucleagena laboratory on dry ice on the collection day. The swabs were repeated after 6 months follow-up after the 4th PDT session on primary areas of lesions’ manifestation and from new areas in case of the wide spreading of the genital warts.The Laboratory Synevo/Nucleagena is regularly certified by INSTAND and QCMD and guaranteed that all real-time PCR procedures were performed in accordance with manufacturer’s instructions. The test detected and identified the 37 most common HPV genotypes  and 25 low oncogenic types ) as well detected the co-existence of several types of the virus, e.g., low and high oncogenic.2 and 100Hz. Clinical evaluation was performed before each of the four ALA-PDT procedures as well as 4 weeks and 6 months after the last exposure. The clinical outcomes, adverse reactions, and recurrence rates were assessed. The photographic documentation was performed after receiving an agreement signed by the patient. Based on the photographic documentation performed on the baseline visit, the areas for the collection of the swabs after 6 months follow-up after the 4th PDT session were precisely specified  ointment freshly prepared by the hospital pharmacy was applied for three consecutive hours. Irradiation was performed with Waldmann PDT 1200L lamp with energy density of 100J/cmpecified .p < 0.05 was considered statistically significant.All data were analyzed by Statistica 13.3. The numerical data were compared with the T-test, and the classified data were compared by the Chi-square test. A total of 21 male patients fulfilled the inclusion criteria and were enrolled in the study. The median age was 30.0 years, the mean age was 34.7 years, and the age ranges were between 22 and 58 years. All patients presented genital warts in the penis area, and 7 participants additionally presented warts at the perianal area . None ofp = 0.0007)  A.p = 0.03) (HPV DNA 6 months after treatment was not found in 14/21 (66.67%), and in 7/21 (33.33%), there was no eradication of HPV DNA. The difference was statistically significant ( = 0.03) B. HPV DN = 0.03) C.p = 0.05)  (1–6 ± 1.6) and in the 6th month of observation, the average number of viruses was 0.95 (0–7 ± 1.77) ( = 0.05) D.p < 0.001) [p = 0.05, p = 0.0003). Thus, the HPV DNA detection is the indicator of the infection severity and the prognostic factor of treatment duration [The reported frequency of genital HPV DNA in men ranged from 1.3% to 72.9% (most studies report ≥20%), with the majority of HPV infections asymptomatic ,16. The < 0.001) . Blomber< 0.001) . Hasanza< 0.001) . Wang et< 0.001) . Those fduration . Wang etduration . Based oduration . In our duration . In the duration . The mosduration . Median duration . On the duration . Hoffmanduration . The autduration .2 laser for the treatment of cervical condylomata acuminate, the eradication rate of HPV infection after 3 months was 90.20% and 65.80%, respectively, but the CR rate in the group treated with ALA-PDT was also significantly lower (90.2%) than in the CO2 laser group (96.2%) [Various treatment techniques are used to treat genital warts including ablation, immunotherapy, PDT, or a combination of all of the above ,24,25. T (96.2%) . Hu et a (96.2%) . The aut (96.2%) . In our  (96.2%) ,12,13,14 (96.2%) ,12,13,14Nevertheless, this study proved that ALA-PDT is the effective option for about 66% of male patients with persistent genital warts previously treated ineffectively. As the clinical complete response did not indicate the DNA HPV eradication, the objective response should be confirmed by the RT-PCR evaluation, which might influence the further decision about the prolongation or modification of treatment.ALA-PDT may be an important method for the treatment of genital warts, especially after the failure of previous treatments. The presence of high-risk DNA HPV types may be associated with a weaker response to PDT and the indication for the therapy prolongation.


A relatively large number (11/52) of mouthwash waste fluids tested positive for HPV . These results surpassed the sensitivity observed testing the same specimens using the conventional method . Our method (pyro-genotyping) was developed using nine HPV genotypes targeted for vaccination and the results were highly sensitive compared to those of the conventional method. This less expensive, high-throughput, and simple method can be used for detecting oral HPV infection with fewer socio-psychological barriers.Human papillomavirus (HPV) is a common sexually transmitted infection worldwide, which spreads via contact with infected genital, anal, and oral/pharyngeal areas  owing to diverse manners of sexual intercourse. In this study, we devised an oral HPV detection method using mouthwash waste fluids that causes less psychological resistance to visiting the outpatient otolaryngology departments. We successfully detected only the specific unique reverse sequencing probe (using pyro-genotyping) and identified the nine genotypes of HPV targeted for vaccination by pyrosequencing the mouthwash waste fluids of non-head and neck cancer patient volunteers ( More than one million people are infected with sexually transmitted diseases every day worldwide. Human papillomavirus (HPV), one of the causes of sexually transmitted infections (STIs), differs from the human immunodeficiency virus and herpes simplex virus genetically and clinically. Globally, more than 290 million women are infected with HPV ,2.HPV is clinically divided into 14 cancer high-risk genotypes  whose epidemiological association with cancer has been indicated, and 5 cancer low-risk genotypes , which lead to benign lesions such as warts, genital condylomata, and laryngeal papillomata .A conventional genotyping analysis using a restriction fragment length polymorphism  assay  was performed according to the manufacturer’s instructions using the same clinical mouthwash samples as those used for pyrosequencing analysis .p values were 2-sided, and p < 0.05 was considered statistically significant.All statistical analyses were performed using the SPSS for Windows, version 12  and the PRISM software for Windows, version 7 . Tree view analyses were performed using the Geneious prime 1.8.0_171-b11 sequence analysis software . The categorical variables were compared using the Chi-square test and Fisher’s exact test, as appropriate. All reported


Numerous studies have shown that human papillomavirus (HPV) infection is one of the important risk factors for head and neck squamous cell carcinoma (HNSCC) progression and affects the expression of multiple genes, which might serve as new biomarkers. This study examines the effects of HPV infection on long non-coding RNA (lncRNA) expression and the immune system, particularly PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress). The Cancer Genome Atlas (TCGA) expression data for lncRNA genes and clinical data were analyzed by GraphPad Prism 5/7. The expressions of PRINS, CDKN2B-AS1, TTTY14, TTTY15, MEG3, and H19 were significantly different in HPV-positive and HPV-negative patients. HPV-positive patients with high PRINS expression demonstrated significantly better overall survival (OS) and disease-free survival (DFS). HPV-positive patients with high PRINS expression showed changes in gene expression associated with immune and antiviral responses. A majority of HPV-positive patients with high PRINS expression demonstrated a high number of immune cells within tumors. PRINS expression was significantly associated with HPV-infection HNSCC tumors. Validation of these results using data set from Gene Expression Omnibus (GEO) indicated that PRINS is upregulated in HPV active infections and in “atypical 1 (IR)” HNSCC clusters, negatively influencing patients’ overall survival. Patients with high PRINS expression display different immunological profiles than those with low expression levels. For instance, they have active HPV infection status or are clustered in the “atypical 1 (IR)” subtype of HNSCC which influences both viral infection and patients’ survival. It is likely that PRINS could be used as a potential biomarker for HNSCC patients, but its role is dual. On the one hand, it stimulates patients’ immune response, while on the other it can be favorable in virus replication. Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer by incidence worldwide . It is eIt is essential to emphasize that HPV(+) and HPV(−) cancers represent different molecular and clinic-pathological characteristics. The prognosis for HPV(+) patients is significantly better than for those with tobacco-related HPV(−) carcinomas in cases of similar treatment . RecentlThe HPV virus has double-stranded circular DNA that encodes several proteins which affect the host’s cellular program . The infVirus-associated epigenetic alterations are likely to be associated with the modulation of long non-coding RNAs (lncRNAs) . lncRNA HPV is an important factor that affects miRNA modulation and oncogenesis ,20,21. HThis study focuses on lncRNAs associated with HPV infection: CDKN2B-AS1 (ANRIL), TTTY14 (NCRNA00137), TTTY15 (NCRNA00138), PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress) (NCRNA00074), XIST (NCRNA00001), MEG3 (NCRNA00023), H19 (NCRNA00008), MALAT1 (NCRNA00047), and CYTOR (NCRNA00152) . Their ehttp://www.cbioportal.org), gene expression profiles, and clinical data of 522 HNSCC samples from the TCGA project  [http://ualcan.path.uab.edu/index.html) [This study applied available cBioPortal tools (summary)  and UALCex.html) . Due to The selected samples were divided into two subgroups using the median expression as a cutoff: low lncRNA and high lncRNA, overall survival (OS), as well as disease-free survival (DFS) were analyzed in both groups. The expression level of lncRNAs was correlated with the following clinicopathological parameters: age (<55 vs. >55), gender , smoking category (1+3 vs. 2+4), alcohol consumption (yes vs. no), grade (G1+G2 vs. G3+G4), disease stage (I+II vs. III+IV), T stage (T1+T2 vs. T3+T4), N stage (N0 vs. N1+N2 N3), angiolymphatic invasion (negative vs. positive), and perineural invasion (positive vs. negative).The expression profiles of genes associated with antiviral and inflammatory responses were downloaded from cBioPortal. Next, levels were analyzed in the high and low lncRNA expression groups. UALCAN resources were used to identify genes that are positively and negatively correlated with selected lncRNAs  in both expression groups. The correlated genes were then analyzed using the PANTHER Classification System and placed into specific biological processes and cellular pathways . To detehttps://bioinformatics.mdanderson.org/estimate/disease.html. These scores were used to define the infiltration of immune cells into tumor tissues and to infer tumor purity. Statistical analyses were then carried out.The immune scores and ESTIMATE  scores were downloaded from To validate the obtained results from the TCGA database, the Gene Expression Omnibus (GEO) data repository, with GSE112026  and GSE6p < 0.05 was considered as statistically significant.All statistical analyses were performed using GraphPad Prism 5/7 . The Shapiro-Wilk normality test, t-test, Mann–Whitney U test or one-way ANOVA test with post-test were used for lncRNA and gene expression according to specified subgroups. All TCGA and GEO data are presented as the mean with standard error of the mean (SEM) or as the median for graphs from UALCAN with lower and upper quartiles. DFS and OS were analyzed using the Log-Rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. The hazard ratio  and 95% confidence interval (CI) of the ratio were calculated. In all analyses, In total, 81 HPV(−) and 40 HPV(+) HNSCC samples from TCGA  with clinical data were included in the analysis. The characteristics of the study groups are presented in The analysis of selected lncRNA expression levels in all patients revealed that four of the lncRNAs are up-regulated in HPV(+) patients: CDKN2B-AS1, TTTY14, TTTY15, and PRINS. However, two lncRNAs were down-regulated in HPV(+) patients in comparison to HPV(−) patients: MEG3 and H19. No significant differences were observed in XIST, MALAT1, or CYTOR as shown in Receiver operating characteristic (ROC) curve analyses showed the high discrimination potential of the following to identify HPV(−) and HPV(+) HNSCC patients: CDKN2B-AS, TTY14, TTY15, PRINS, MEG3, and H19. There were no differences in XIST, MALAT1, and CYTOR, as shown in p = 0.0073). This relation was observed in the group of high PRINS in comparison to low PRINS patients without taking into consideration HPV status (p = 0.0327), as well as in HPV(+) compared to HPV(−) patients according to HPV status only (p = 0.0220). Differences in OS were not observed in patients with HPV(−) cancers with high or low PRINS expression (p = 0.4947) (p = 0.0104) and in HPV(+) patients with high versus low PRINS expression levels (p = 0.0343), The OS of the subgroups with low and high expression of CDKN2BAS1, TTTY14, TTTY15, MEG3, and H19 among HPV(−) and HPV(+) patients showed no significant differences . However 0.4947) . Signifip > 0.05) in the expression level of PRINS in the HPV(+) HNSCC patients. However, in all patients, (HPV(−) and HPV(+)), a significant difference in the tumor grade was observed (p = 0.0156), as shown in Analysis of the clinicopathological parameters  revealed no significant differences  Toll-like receptors and chaperones: CTSS, TLR8; (ii) signaling downstream of Toll-like receptor: IRF5; (iii) Toll-like receptor signaling response genes: CCL5, CD40; (iv) NOD-like receptors and signaling molecules: CARD9, PYCARD, PSTPIP1; (v) signaling downstream of RIG-I-like receptor: MAVS, and (vi) type I interferon signaling: IFNAR1 .Analysis of the inflammatory response genes revealed significant changes in the following genes: (i) growth factors: IGF1; (ii) immune-stimulatory factors and pro-inflammatory genes: IL2; (iii) chemokines: CXCL5; CCL21; (iv) chemokine receptors: CCR2, CCR7; and enzymatic modulators of inflammation and immunity: GZMA. All of these genes except CCL21 were up-regulated, as shown in p = 0.0099). There were no OS differences associated with immune score in HPV(−) and all patients , but in the HPV(+) group there was slightly better OS for patients with higher immune scores (p = 0.0841). Moreover, most HPV(+) patients with high PRINS expression (72%) had higher immune scores than the other groups (Immune scores indicated significantly higher infiltration of tumor tissues by immune cells in HPV(+) samples than HPV(−) samples  was indicated (p = 0.0985) as well as between HPV(−) vs. HPV16 vs. other types of HPV (p = 0.2549) were found. However, expression levels of PRINS were upregulated in the group of HPV+(DNA+/RNA+) vs. HPV(−) (p = 0.0401) and HPV+(DNA+/RNA+) vs. HPV+(DNA+/RNA-) (p = 0.0247) (p = 0.001). For analysis of HPV(−) vs. all HPV(+), no significant differences were observed . However, when the HPV(+) group was divided due to the presence of virus RNA, the specificity and sensitivity, as well as AUC, were improved: HPV+(DNA+/RNA+) vs. HPV(−)  and HPV+(DNA+/RNA+) vs. HPV+(DNA+/RNA+)   HNSCC samples compared to normal levels (ndicated A. No dif 0.0247) B. The RO 0.0160) C.p > 0.05) in the expression level of PRINS in specific clinicopathological parameters groups in the HPV(−), HPV(+), HPV+(DNA+/RNA+) nor HPV+(DNA+/RNA-) of HNSCC patients, apart from the N-stage in the HPV+(DNA+/RNA+) group, where PRINS was upregulated in N1+N2+N3 vs. N0   .All patients (HPV(+) and HPV(−)), HPV(+), HPV+(DNA+/RNA+) and HPV+(DNA+/RNA-) HNSCC patients were divided into two subgroups based on the median of PRINS expression as a cutoff and the patients’ OS between them was assessed. No significant differences between low and high-PRINS expressing groups were observed .An analysis of antiviral and inflammatory response genes was also conducted. To validate the previous observations, the same set of genes was investigated. In the HPV+ (DNA+/RNA+) group, a significant difference was observed only for CTSS, TLR8 and GZMA genes . Surprisp < 0.0001) (p = 0.8010)   1” and “classical 2” the PRINS expression was the highest ( 0.0001) A. In the 0.8010) B. Moreov 0.8010) C. Finallctively) D.In the last decade, HPV has been identified as an independent risk factor for HNSCC . MoreoveCompared to normal corresponding tissue, TCGA data showed that HNSCC cases had higher expression of CDKN2B-AS1, MALAT1, and CYTOR and lower expression of TTTY14 and H19. Differences in TTTY15, PRINS, XIST, and MEG3 were not found. Analyses of HPV(+) and HPV(−) HNSCC patients indicated that the HPV(+) group had up-regulation of CDKN2B-AS1, TTTY14, TTTY15, and PRINS and down-regulation of MEG3 and H19. Moreover, our ROC analyses showed that CDKN2B-AS, TTTY14, TTTY15, PRINS, MEG3, and H19 could distinguish HPV(+) from HPV(−) samples.A previous gene expression microarray analysis of HNSCC had pointed out that CDKN2B-AS1, TTTY14, TTTY15, and PRINS are up-regulated in HPV(+) compared to HPV(−) samples, while XIST and CYTOR are down-regulated . In vitrUnfortunately, the available results on lncRNA in HPV(+) HNSCC are limited. Most of the results come from cervical cancer models. Only Nohata et al. showed that 27 and 140 lncRNAs are significantly up-regulated in HPV(+) in comparison with HPV(−) tumors, especially LINC01305, LINC01089 (LIMT), and PTOV1-AS1 lncRNAs . These oNext, we checked the OS of HPV(−), HPV(+), and all patient subgroups with low and high expression of CDKN2BAS1, TTTY14, TTTY15, PRINS, MEG3, and H19. Significantly better OS was observed only in HPV(+) patients with high expression of PRINS. No differences were found between low- and high PRINS-expressing patients with HPV(−) cancers. Lower DFS was parallel to OS in the same HPV(+) patients. This clearly indicates that PRINS could prove to be a prognostic factor for HPV(+) patients. These results and clinicopathological parameters emphasize that PRINS features could be an independent prognostic biomarker for HPV(+) HNSCC patients.The lncRNA PRINS was first identified by Sonkoly et al. and referred to as Psoriasis susceptibility-related RNA Gene Induced by Stress (PRINS). It is transcribed by RNA polymerase II at different levels in various human tissues, including oral mucosa, and its sequences have homology to stress-induced transcripts, such as heat shock-inducible RNA, G8, and Alu elements. The RNA level of PRINS proved to be increased by stress signals such as UV-B, viral infection (herpes simplex virus), and translational inhibition .The expression of PRINS is also regulated by the proliferation and differentiation states of keratinocytes, and the knock-down of PRINS impairs cell viability after serum starvation in vitro. Moreover, T-lymphokines reduce PRINS expression in the uninvolved psoriatic epidermis, but not in the healthy epidermis. Sonkoly et al. hypothesized that this lncRNA functions as a ribo-regulator and modifies the expression of genes involved in the proliferation and survival of cells exposed to stress .In HeLa cells, the down-regulation of PRINS resulted in changes in cell morphology and altered the gene expression profile . Moreover, the up-regulation of PRINS in psoriatic non-lesional epidermis induced the expression of anti-apoptotic G1P3 protein and caused the hyperproliferation of keratinocytes . PRINS iPRINS is also involved in the immune mechanisms, and its expression level is related to chemokines . In a raWe found that PRINS is involved in mounting an antiviral response by affecting Toll-like receptors and chaperones, signaling downstream of Toll-like receptors, signaling response genes, NOD-like receptors, signaling molecules, and signaling downstream of RIG-I-like receptors. It is also connected with type I interferon signaling. Moreover, PRINS modulates inflammation by affecting the expression of growth factors, immune-stimulatory factors, pro-inflammatory genes, chemokine receptors, and enzymatic modulators of inflammation and immunity. Our observations indicate that PRINS is up-regulated during stress and influences local inflammation through the regulation of CCL5, which is well known as a key pro-inflammatory chemokine and is associated with viral infection. It also induces the in vitro migration and recruitment of T cells, dendritic cells, eosinophils, NK cells, mast cells, and basophils, and it supports angiogenesis .Consequently, the ESTIMATE tool was used to elucidate PRINS’ association with the immune state of HNSCC patients. Patients with high immune scores tended to have better survival but for HPV(+) only. Importantly, this observation was not indicated for HPV(+) with low PRINS expression. A previous report based on TCGA and GEO databases analyzed tumor-infiltrating immune cells in HPV(+) and HPV(−) HNSCC patients. The results showed that HPV(+) patients have more infiltrating B and T cells and fewer neutrophils. T cell subtypes revealed that cytotoxic T cell subtypes predominated in HPV(+) HNSCC, and the ratio of M1/M2 macrophages was much higher in this group. Moreover, enhanced infiltration of B cells and CD8+ T cells was identified as an independent protective factor, while high neutrophil infiltration was a risk factor for HPV(+) HNSCC patients . Our anaThe classification of HPV(+) HNSCC patients into better- and worse-prognosis subgroups is already known. However, there is still a lack of biomarkers to stratify patients for the implementation of more precise treatment. Zhang et al. indicated that HPV(+) patients could be divided into two distinct subtypes: HPV-KRT (with high expression of genes in keratinocyte differentiation and the oxidation-reduction process) and HPV-IMU . The HPV-IMU patients exhibit better prognosis, but the OS analysis did not show significant differences between HPV-IMU and HPV-KRT HNSCC patients . Our stuTo validate our results, we used available data from GEO and observed that PRINS is indeed upregulated in both HPV(+)  and in HPV+(DNA+/RNA+), which is assessed as active HPV infection  in opposThe available data and our results are intriguing in terms of the implementation of PRINS lncRNA in clinical practice as a biomarker. PRINS lncRNA is upregulated in HPV(+) patients and connected with active HPV infection. The lack of evidence of the variable PRINS expression among clinical parameters could suggest a connection to better or worse patient survival. We postulate that PRINS could be responsible for the regulation of the immune system through the modulation of inflammation as well as the stimulation of viral replication. However, this hypothesis needs experimental verification based on in vitro and in vivo models.In HPV(+) HNSCC patients’ tumors four lncRNAs  and two (MEG3 and H19) are up- or down-regulated, respectively as compared with HPV(−) patients. The specified profiling allows for HPV(+) and HPV(−) differentiation. Patients with high PRINS expression display different immunological profiles than those with low expression and have active HPV infection status or are clustered in the “atypical 1 (IR)” subtype of HNSCC.


We evaluated the value of adding DNA Fifty patients with OPSCC were analyzed. Concordance between HPV–DNA ISH and p16 IHC was measured by Gwet's agreement coefficient.p16 IHC was positive in 35/48 (72.9%), negative in 8/48 (16.7%) patients. Wide spectrum DNA–ISH was positive in 9/23 (39%) and negative in 14/23 (60.9%) patients. High-risk 16/18 (HR) HPV DNA–ISH was positive in 11/23 (47.8%) and negative in 12 (52.2%) patients. The agreement between HPV DNA–ISH and p16 IHC is fair (Gwet's AC1 = 0.318).The agreement between p16 IHC and HPV–DNA ISH was fair. However, ISH sensitivity was low. Our findings add to the current data that p16 IHC testing is reliable and may be enough as a stand-alone test for HPV detection in OPSCC. in situ hybridization (ISH) to p16 IHC. Fifty patients with OPSCC were analyzed. p16 IHC was positive in 72.9% of patients and DNA–ISH was positive in 39% of patients when wide spectrum probe was used and positive in 47.8% of patients when high-risk probe was used. The agreement between the two tests was fair. However, DNA–ISH sensitivity was low. p16 IHC may be enough as a stand-alone test for detection of HPV in OPSCC.Current testing guidelines recommend p16 immunochemistry (IHC) for detecting human papillomavirus (HPV) in oropharyngeal cancer (OPSCC). We evaluated the value of adding HPV DNA  Head and neck cancers are the sixth most common malignancy worldwide with a global incidence of over 650,000 cases annually and over 330,000 deaths . Over 92HPV-associated OPSCC is biologically and clinically distinct from tobacco and alcohol-related OPSCC. Histologically, HPV-associated OPSCC are characteristically nonkeratinizing and basaloid whereas HPV-negative OPSCC is classically keratinizing ,5. HPV-aBecause of the clear prognostic importance HPV status in patients with OPSCC, the eighth edition of the AJCC Cancer Staging Manual has separated HPV positive OPSCC from HPV negative OPSCC. Clinical trials are looking at de-escalation strategies  for HPV positive disease and it is possible that treatment algorithms in the near future may heavily depend on HPV status. Therefore, it is of paramount importance to accurately categorize OPSCC as HPV positive or negative. However the optimal method(s) for testing HPV status is still not completely standardized. The current guidelines from the College of American Pathologists (CAP) as well as American Society of Clinical Oncology (ASCO) recommend testing for HPV in tumor samples of OPSCC using surrogate marker p16 IHC . PerformIn our study, we evaluated the utilization and value of using HPV–DNA ISH as an adjunct to p16 IHC to detect HPV status of oropharyngeal carcinoma in our practice. We also assessed the level of concordance between two types of tests with a special focus on never-smokers, in whom majority of the tumors can be considered HPV positive  Figure .A retrospective analysis of 50 patients with OPSCC diagnosed between 2012 and 2014 was conducted. The study was approved by Institutional Review Board of North Shore University Hospital. Study subjects were identified through our hospital cancer registry. Data points included age, gender, smoking history, stage of disease at diagnosis, primary site of disease and HPV status. All p16 IHC tests were performed at our own institution and were reviewed by board certified pathologists. Immunohistochemical staining for p16 was performed using Ventana BenchMark Autostainer . Selected paraffin blocks from each case were cut at 5-μm thickness and dried at 60°C for 1 hour. Following deparaffinization, rehydration and blockage of endogenous peroxidase, slides were treated with antigen retrieval solution followed by incubation with primary antibodies against p16 . Antibody binding was visualized using the iVIEW DAB Detection Kit (760-091). Only samples with strong, diffuse nuclear and cytoplasmic staining greater than 70% were regarded as positive. HPV-specific tests, HR DNA–ISH or wide-spectrum (WS) DNA ISH probes that target the genomic DNA of HPV types 6, 11, 16, 18, 30, 31, 33, 35, 45, 51 and 52, were performed at GenPath Diagnostics and Integrated Oncology. The probe used was Leica-Kreatech HPV ISH probes on Leica III BOND autostainer. The HPV positivity was defined as punctate and diffuse nuclear staining of tumor cells. There were variabilities in the selection of HPV–DNA ISH tests, however all of tests included probes against HR HPV ISH types 16/18. The variability in the selection of HPV ISH probe is because of unclear standardized testing guidelines, multiple tests available on the test directory made it difficult to maintain consistency among different ordering professionals and referring sites with less experience ordering the tests.Descriptive statistics were used to summarize the data . The level of agreement between p16 IHC and HPV–DNA ISH (WS and HR) was measured by calculating Gwet's agreement coefficient (AC1). All analyses were carried out using SAS Version 9.4 Fifty patients were analyzed. Thirty-nine were male (78%) and median age was 61.5 years. Most common primary site was base of tongue  followed by tonsil . Most patients had advanced disease: 16 (32%) stage III, 31 (62%) stage IVA, 1 (2%) stage IVB. Patient demographics are shown in p16 IHC testing was performed in 48 (96%) and ISH in 43 (86%). Wide-spectrum (WS) ISH was utilized in 23 (46%) and HR HPV ISH in 23 . There were two patients who did not have p16 IHC or HPV–DNA ISH testing performed. Thirty five out of 48 patients (72.9%) tested positive for p16 IHC, eight (16.7%) tested negative and five (10.4%) were equivocal. For WS HPV–DNA ISH, 9/23 (39%) tested positive and 14/23 (60.9%) tested negative. For HR HPV–DNA ISH, 11/23 (47.8%) patients tested positive and 12/23 (52.2%) tested negative. p16 IHC was positive in 18/20 (90%) of positive HPV–DNA ISH (WS and HR) tumors and 2/20 (10%) were equivocal whereas out of positive p16 IHC tumor samples, 18/35 (51.4%) were positive for HPV–DNA ISH (WS and HR). The results of p16 IHC and HPV ISH are shown in Among never-smokers (n = 20), p16 IHC was diffusely positive in 19/19 (100%) tested, whereas HPV ISH was positive in 8/17 (47%) as shown in The level of agreement between p16 IHC and HPV–DNA ISH (WS and HR) was assessed using Gwet's AC1 which divides the strength of agreement into poor, fair, moderate, good and very good. In our study, the level of agreement based on Gwet's AC1 was found to be 0.318  which is fair (AC1 0.2–0.4).It has now been established beyond any doubt that HPV positivity connotes excellent response to treatment and overall prognosis when compared with HPV-negative OPSCC. Several trials are underway to determine if de-escalation strategies would be appropriate for these patients with goals to reduce toxicity/morbidity from chemotherapy and radiation in these patients while maintaining similar survival rate. With evolving treatment strategies based on results of HPV status of oropharyngeal cancer, standardization and optimization of testing methods are of paramount importance. Indeed, a majority of currently enrolling clinical trials  utilize p16 IHC as a stand-alone test as part of eligibility criteria. However, there are some that require HPV specific testing to be positive for inclusion . Moreover, the current eighth edition of AJCC Cancer Staging Manual classifies OPSCC into p16-associated (HPV associated) and p16-negative disease but does not specify the tests that should be utilized for assessing HPV status.As our study demonstrates, p16 IHC testing was used fairly consistently (96%) at our institution and our referring sites for assessing HPV status of oropharyngeal cancer while the type of HPV DNA ISH testing varied considerably. Overall, the level of agreement between p16 IHC and HPV–DNA ISH tests was fair but p16 IHC positivity was substantially higher than that of DNA–ISH.p16 overexpression is widely accepted as a very sensitive surrogate marker for transcriptionally active HPV. However, the main critique for using p16 IHC as a stand-alone test is that p16 is a surrogate marker and the IHC does not directly measure the presence of transcriptionally active HPV. When using p16 IHC as a stand-alone test for risk stratification, there is concern for patients with positive p16 but truly HPV negative tumor with aggressive disease course can be wrongly stratified into lower risk category. However, several studies have shown that there is strong correlation of p16 expression and improved outcomes in multivariate analyses after controlling for variables. Recent observations suggest that expression of p16 in OPSCC portends a favorable outcome regardless of HPV status. Hazard ratios for overall survival in patients with p16 positive OPSCC in recently published studies ranges from 0.21 to 0.42 when compared with p16 negative cases . AdditioIn our study, p16 IHC was positive in 100% of the non-smokers, a cohort of patients who are highly likely to be HPV positive . HPV–DNAAs we move closer toward new HPV-specific treatment paradigms in OPSCC, accurate assessment for HPV status is essential. The discordance among currently available HPV testing methods makes it necessary to incorporate not only the HPV status of OPSCC patients but also to specify optimal testing guidelines into the decision-making algorithm. Our findings add to the currently available data that p16 is an easily available, highly sensitive and reliable surrogate marker for HPV positivity. The utilization of p16 IHC as a stand-alone test is reasonable for HPV detection in OPSCC until other testing modalities such as HPV E6/E7 mRNA ISH or detection of E6/E7 mRNA by RT-PCR become widely available. While treatment strategies for oropharyngeal cancer are evolving, it is also imperative that we accurately test and document HPV status since this will hugely impact treatment decision-making.Due to the markedly improved outcome of HPV positive OPSCC, many studies with de-intensification treatment strategies are under investigation. The results of these de-intensification trials are highly anticipated and the general hypothesis is that milder treatment strategies offer similar benefit as standard regimens for patients with HPV positive OPSCC while producing fewer treatment-related side effects. It is therefore extremely important to optimize and standardize HPV testing methodologies that are easy to perform, broadly available and cost effective. Addition of HPV specific test, mRNA–ISH as it becomes widely available, to p16 IHC may help optimize testing for HPV status by maximizing sensitivity and specificity. Additional validation studies and cost analyses are needed to standardize testing guidelines for detecting HPV in OPSCC.The incidence of human papillomavirus (HPV) associated oropharyngeal cancer (OPSCC) is increasing in the Western countries while the incidence HPV-negative  OPSCC is declining. Today, 70% of OPSCC cases are HPV-associated.Compared with HPV-negative OPSCC, HPV-associated OPSCC confers better prognosis despite often diagnosed in advanced stages due to small primary tumor and early metastasis.pRb and increases the transcription of p16 which leads to tumor cell survival.Expression of HPV viral protein E6 and E7 leads to oncogenesis. E6 promotes the degradation of tumor suppressor p53 preventing apoptosis when cells enter unscheduled S-phase. E7 promotes the degradation of tumor suppressor College of American Pathologists (CAP) and American Society of Clinical Oncology (ASCO) recommend testing for HPV in tumor samples of OPSCC using surrogate marker p16 immunohistochemistry (IHC). Performing subsequent confirmatory HPV-specific tests is at the discretion of the pathologist.in situ hybridization (ISH) and p16 IHC are widely used testing methods due to their availability and ease of use as they can be performed on the standard formalin-fixed, paraffin-embedded (FFPE) specimens. mRNA–ISH can also be performed on FFPE samples and has high concordance rate with p16 IHC but not yet widely available. The gold standard test is detection of HPV E6/E7 mRNA by RT PCR. However, the test requires cumbersome sample preparation, cannot be performed on FFPE tissue samples and not widely available.DNA–Accurately diagnosing HPV positivity in OPSCC is important as it confers better prognosis. Numerous de-intensification treatment strategies are under investigation for HPV-associated OPSCC that can potentially reduce treatment related toxicities and morbidities while maintaining similar progression-free survival and overall survival.Our study showed that p16 IHC testing was used consistently in 96% of patients whereas the use of HPV DNA–ISH varied (46% wide spectrum probes and 46% high risk probes).p16 IHC was positive in 90% of positive HPV–DNA ISH (WS and HR) tumors whereas out the positive p16 IHC tumor samples, 51.4% were positive for HPV–DNA ISH (WS and HR).Amongst never-smokers with OPSCC, who have high likelihood of having HPV-associated disease, p16 IHC was diffusely positive in 100% of patients, whereas HPV ISH was positive in 47%.The level of agreement between p16 IHC and HPV–DNA ISH (WS and HR) was fair .Our study adds to the currently available data that p16 IHC is highly sensitive and reliable surrogate marker of HPV positivity.The utilization of p16 IHC as a stand-alone test is reasonable until more sensitive HPV-specific test such as E6/E7 mRNA–ISH becomes more widely available.


Ensuring that routine immunization services for adolescents are maintained or reinitiated is essential to continuing progress in protecting persons and communities from vaccine-preventable diseases and outbreaks.Three vaccines are recommended by the Advisory Committee on Immunization Practices (ACIP) for routine vaccination of adolescents aged 11–12 years to protect against 1) pertussis; 2) meningococcal disease caused by types A, C, W, and Y; and 3) human papillomavirus (HPV)-associated cancers . Differences were observed in vaccination coverage by race and ethnicityIn 2019, compared with adolescents living in MSA principal cities, coverage with ≥1 dose of HPV vaccine among those living in non-MSA areas was 9.6 percentage points lower, the percentage who were HPV UTD was 9.8 percentage points lower, and coverage with ≥1 dose of MenACWY was 5.1 percentage points lower. These disparities were only observed among adolescents living at or above the poverty level . Coveraghttps://stacks.cdc.gov/view/cdc/91796). Being HPV UTD by age 13 years increased an average of 3.4 percentage points for each consecutive birth year, from 8.0% among adolescents born in 1998 to 35.5% among those born in 2006.HPV vaccination initiation by age 13 years increased an average of 5.3 percentage points for each consecutive birth year, from 19.9% among adolescents born in 1998 to 62.6% among those born in 2006  in 2019, suggesting that more parents are making the decision to protect their teens against HPV-associated cancers. Efforts from federal, state, and other stakeholders to prioritize HPV vaccination among adolescents, and reducing the number of recommended HPV vaccine doses from a 3-dose to a 2-dose series  to request the adolescent’s vaccination history were mailed in early 2020. A lower response rate was observed for those requests, likely because of the effect of the COVID-19 pandemic on health care provider operations.The COVID-19 pandemic has the potential to offset historically high vaccination coverage with Tdap and MenACWY and to reverse gains made in HPV vaccination coverage. Orders for adolescent vaccines have decreased among VFC providers during the pandemic. A recent analysis using VFC provider ordering data showed a decline in vaccine orders for several VFC-funded noninfluenza childhood vaccines since mid-March when COVID-19 was declared a national emergency .Adolescent vaccination coverage in the United States continues to improve for HPV and for meningococcal vaccines, with some disparities. Among adolescents living at or above the poverty level, those living outside a metropolitan statistical area (MSA) had lower coverage with HPV and meningococcal vaccines than did those living in MSA principal cities.Ensuring routine immunization services for adolescents, even during the COVID-19 pandemic, is essential to continuing progress in protecting individuals and communities from vaccine-preventable diseases and outbreaks.


Since their discovery in the 1990’s, microRNAs (miRNA) have opened up new vistas in the field of cancer biology and are found to have fundamental roles in tumorigenesis and progression. As head and neck squamous cell carcinoma (HNSCC) with positive human papillomavirus (HPV+) is significantly distinct from its HPV negative (HPV−) counterpart in terms of both molecular mechanisms and clinical prognosis, the current study aimed to separately develop miRNA signatures for HPV+ and HPV− HNSCC as well as to explore the potential functions. Both signatures were reliable for the prediction of prognosis in their respective groups. Then Enrichment analysis was performed to predict the potential biological functions of the signatures. Importantly, combining previous studies and our results, we speculated that HPV+ HNSCC patients with low signature score had better immunity against the tumors and enhanced the sensitivity of therapies leading to improved prognosis, while HPV− HNSCC patients with high signature score acquired resistance to therapeutic approaches as well as dysregulation of cell metabolism leading to poor prognosis. Hence, we believe that the identified signatures respectively for HPV+ and HPV− HNSCC, are of great significance in accessing patient outcomes as well as uncovering new biomarkers and therapeutic targets, which are worth further investigation through molecular biology experiments. Based on the latest GLOBOCAN data (September 2018), head and neck squamous cell carcinomas (HNSCC), further classified as oral cavity, oropharynx, nasopharynx, larynx, and hypopharynx according to their primary site of origin, had the top eight combined incidence rate and was the 5th leading cancer by combined 5-year prevalence worldwide . Apart fMicroRNAs (miRNAs) are non-coding RNA molecules with about 22 nucleotides in length, which mainly act as negative regulators of target genes and have been shown to play an essential role in a range of biological functions, including cell proliferation, apoptosis, tumor growth, and metastasis . NeverthIn the current study, we integrated miRNA-Seq data and corresponding clinical follow-up information of HNSCC patients from The Cancer Genome Atlas (TCGA) database and Genome Data Analysis Centers (GDAC) server to identify features of miRNAs associated with prognosis. As it has been required to accurately discriminate between HPV-related and HPV-unrelated HNSCC, we then developed distinct miRNA signatures by dividing samples into HPV+ group and HPV− group. Enrichment analysis was further performed to investigate the potential functions, of which the results were truly distinguishable compared the two groups. The exploration procedure of the current study was illustrated in http://cancergenome.nih.gov/) on March 9, 2020. The matched patients’ clinical information was downloaded from the Broad Genome Data Analysis Centers Firehose server (https://gdac.broadinstitute.org/). The annotation of mRNAs was performed based on GENCODE (Release 33) and all miRNAs were renamed according to miRBase database v22 (www.mirbase.org) (The level three miRNA isoform expression quantification data and level three RNA-sequencing data (RNA-Seq-HTSeq-FPKM) of HNSCC patients was download from The Cancer Genome Atlas  . The expase.org) . The “DEase.org)  was furtExpression (miRNAi) × Coefficient (miRNAi). Patients were divided into high risk and low risk groups according to the median risk score. Kaplan-Meier analysis was conducted using R package “survminer” and “survival.” The receiver operating characteristic (ROC) curve was obtained by R package “survivalROC.” Univariate and multiple Cox regression analysis were used to identify independent prognostic factors (p < 0. 05).Cases with follow-up less than 90 days were excluded in advance and the prognostic value of each miRNA was calculated in the univariate Cox regression analysis using R package “survival” (p<0. 01). After primary filtration, Cox Least Absolute Shrinkage and Selection Operator (LASSO) regression with 10-fold cross-validation was built to pick out candidate miRNAs. Multivariate Cox regression was further performed using R package “survival” based on miRNAs disclosed in the above steps and constructed a prognostic risk score model. Risk score = Target genes of prognosis-related miRNAs were predicted by three databases  simultaneously. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the target genes was conducted by R packages “org.Hs.eg.db” and “clusterProfiler”  (p < 0.0vs 61.7 years); and the difference was statistically significant.A total of 515 HNSCC samples and 44 normal-adjacent control tissues with corresponding clinic data were included in this study. The cases were further grouped by HPV status and the basic characteristics of the patients were summarized in vs normal, HPV− vs normal, HPV+ vs HPV−), respectively. The lists of significantly deregulated miRNAs in each group were compared with Venn Diagram  for three groups  pathway analysis was performed to assess the functional involvement of the survival-related miRNAs Figure 6FH) and T regulatory cells (Tregs).Then, we further calculated the fractions of immune-related cells in the tumor micro-environment (TME) through CIBERSORTx and uncovered the relationships between the signature and these cells Table 1.FH and activated memory CD4+ T cells. These findings implied that these miRNAs in the signature had potential for serving as biomarkers and therapy targets in HNSCC and deserved further investigation.For the signature of HPV− group, miR-605-5p related to unfavorable prognosis was found negatively associated with CD8+ T cells, macrophages M1, and activated memory CD4+ T cells, which acted as tumor suppressors. miR-135b-3p related to good prognosis was found negatively correlated with macrophages M2 which could promote tumor progression. And the signature score was negatively correlated with TWe have further found targeted mRNAs of survival-related miRNAs for HPV+ and HPV− HNSCC patients by TargetScan, miRTarBase, and miRDB. TCGA has been used to choose them and the negative correlation of miRNA and mRNA was applied to validate them. Then 1 mRNA in HPV− miRNA group and 10 mRNAs in HPV+ miRNA group were obtained and shown in Over the past decade, the role of HPV has attracted increasing attention in HNSCC, and the significant differences of HPV+ HNSCC in prognosis and etiologic mechanisms from its HPV− counterpart have made it critical to describe and discuss the two subgroups separately whenever possible. Although this vital distinction mainly referred to oropharyngeal tumors within HNSCC, as this was the best-studied entity, HPV was also involved in 23.5% of oral cancer and 24% of laryngeal cancer cases . HoweverIn the present study, we separately developed distinct miRNA signatures associated with OS of patients for the two groups. Both signatures were reliable for the prediction of prognosis in their respective groups according to the ROC curve. The lists of significantly deregulated miRNAs and survival-related miRNAs in each group were compared with Venn Diagram. Noteworthy were no survival-related miRNA in common compared HPV+ to HPV− groups, reinforcing their differences at the molecular level. Further, KEGG pathways analysis displayed that there were 11 common pathways between HPV+ group and HPV− group, and the gene ratio of HPV− group was generally higher than that of HPV+ group. In addition, GSEA of the signature score showed that HPV+ samples with low risk score were mainly enriched in immune-related pathways and HPV− group with high risk score appeared to be associated with metabolism and other vital oncogenic pathways. Previous studies have shown pronounced advances achieved in cancer immunotherapy. Despite the success, a relative lower overall clinical response of HNSCC was observed compared to other types of tumor treated with similar approaches, highlighting the need to gain better understanding of the complex immune landscape within TME of HNSCC , 31.FH. As components of the immune system, CD8+ T cells and NK cells have important roles in suppressing tumors by killing tumor cells with cytotoxic molecules, and the presence of tumor dense CD8+ T cells infiltration possibly represents pre-existing anti-tumor immune responses , National Science Foundation of Sichuan Province (No. 2020JDRC0018 and 2020YFS0171), Clinical Project of West China College of Stomatoloy, Sichuan University (LCYJ2019-8), and Exploration and research projects of West China College of Stomatoloy, Sichuan University (LCYJ2020-YJ-1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.