Human papillomavirus (HPV) infection, smoking, and excessive alcohol consumption have been established as risk factors for the development of oropharyngeal squamous cell carcinoma (OPSCC). While the HPV epidemic has led to an increasing incidence of OPSCC, HPV-negative OPSCC cases associated with smoking and alcohol remain stable. As HPV-positive and -negative OPSCC present two distinct etiological, clinical, and prognostic entities, different treatment and follow-up strategies are being discussed. Still, specific surveillance strategies for HPV-positive OPSCC are lacking, as the risk of second primary tumors (SPT) in the era of HPV-associated OPSCC has not been comprehensively assessed. Our study investigated the frequency and localization of SPT of HPV-positive OPSCC, as well as their prognostic impact. We find that the SPT of HPV-positive OPSCC are less frequent than those of HPV-negative OPSCC, and they are also associated with higher survival rates. The localization of SPT of HPV-positive OPSCC did not differ from the localization of SPT of HPV-negative OPSCC.p-value 0.01). Smokers showed significantly more SPT in the head and neck area than outside. The development of an SPT led to a significantly lower survival time in HPV-negative patients, while it did not significantly affect the survival time of HPV-positive patients. Conclusions: Patients with HPV-positive index tumors had a significantly lower risk of developing SPT than patients with HPV-negative tumors. If SPT developed, survival was significantly shorter in patients with HPV-negative tumors than with HPV-positive tumors.Purpose: To investigate the frequency, localization, and survival of second primary tumors (SPT) of oropharyngeal squamous cell carcinoma (OPSCC) depending on human papillomavirus (HPV) status. Methods: We performed a retrospective chart analysis of 107 OPSCC patients treated at the Zurich University Hospital from 2001 to 2010. Rate and localization of SPT after an index OPSCC were stratified according to smoking and HPV infection status. Results: In total, 57/91 (63%) included patients showed an HPV-associated OPSCC. Of these, 37/57 (64.9%) patients with an HPV-positive and 32/34 (94.1%) patients with an HPV-negative OPSCC were smokers. The median age at diagnosis of the SPT was 59.54 years (interquartile range 52.7–65.6). In addition, 8/57 (14%) HPV-positive and 13/34 (38.2%) HPV-negative patients developed SPT. The rate of SPT in patients with HPV-positive index tumors was significantly lower than in patients with HPV-negative OPSCC ( Oropharyngeal squamous cell carcinomas (OPSCC) are rare. Major risk factors for OPSCC include excessive tobacco and alcohol consumption . In 1946High-risk human papillomavirus (HR-HPV) infections have been known to cause genital tumors for about 3 decades . About 2HPV-induced OPSCC present an etiologically, molecularly, clinically, and prognostically distinct entity from tobacco- and alcohol-associated OPSCC ,14,15,16The aim of this study was to analyze the frequency and localization as well as the prognostic significance of SPT after index OPSCC in relation to the risk factors HPV infection and smoking. Our research questions were the following: (i) How common are SPT in a consecutive cohort of patients with OPSCC? (ii) Are SPT more common in smokers than in nonsmokers? (iii) Does this frequency depend on HPV status? (iv) Does the location of SPT differ between smokers and nonsmokers or HPV-positive and HPV-negative patients? (v) What is the impact of the development of SPT on survival?A retrospective analysis was performed using medical records of patients who were treated in a curative intent for newly diagnosed oropharyngeal carcinoma (OPSCC) at the Department of Otorhinolaryngology—Head and Neck Surgery at the University Hospital Zurich (USZ), Zurich, Switzerland between 2001 and 2010.Inclusion criteria were an index tumor in the oropharynx. Exclusion criteria on the other hand were a non-evaluated HPV status. The starting point of observation was the diagnosis of the index tumor. Endpoint of observation was either last follow-up examination until January 2019 or death.HPV positivity was determined according to an accepted algorithm , which iFurthermore, age at diagnosis of the index tumor, gender, location of the index tumor, tobacco and alcohol consumption, occurrence of SPT, and disease progression  were obtained from medical records. Death was distinguished as due to index tumor, due to SPT, and independent of cancer. Survival rates were plotted using Kaplan–Meier survival curves, with the start of observation being the time of diagnosis of the index tumor.Smokers were defined as ≥10 pack years, regardless of the time of tobacco use.Alcohol abuse was defined as a history of >2 U/day for women and >3 U/day for men, respectively, with a standard unit (U) corresponding to 10 g of pure alcohol.An SPT was defined as a second carcinoma in the upper aerodigestive tract, lung, or bladder, differing in site from the index tumor, which was unlikely to be a metastasis of the index carcinoma .HPV types 16, 18, 33, 35, 56, and 59 were considered high-risk HPV types (HR-HPV) .Age, sex, history of alcohol abuse, recurrence of index tumor, death from OPSCC-associated cancer, and development of second carcinomas were compared among the following three groups of patients: (1) HPV-positive nonsmokers, (2) HPV-positive smokers, and (3) HPV-negative patients.Time to SPT occurrence was defined as time between diagnosis of index OPSCC and initial diagnosis of SPT.Frequency and location of SPT after an index OPSCC were determined in relation to smoking and HPV status. Among SPT, synchronous tumors (detected simultaneously or within 6 months of the index tumor diagnosis) were distinguished from metachronous tumors (detected >6 months after the index tumor) . In addip < 0.05.Statistical analyses of the data were performed using Stata 13. Patient characteristics were evaluated using T-test and Fisher’s exact test of group means controlled for unequal variances of individual characteristics. For evaluation of statistical significance of continuous variables such as age, the T-test was applied, while for discrete variables, Fisher’s exact test was applied. Statistical significance was defined at the 5% level of significance, i.e., To evaluate the effect of HPV status, UICC stage , and therapy mode, age, sex, tobacco, and alcohol abuse on the development of SPT, HPV status, and death from cancer, multivariate Cox regression analyses were performed.For survival analysis, we used Kaplan–Meier survival curves and controlled for significant differences between groups using log-rank tests. Additionally, a multivariate cox regression analysis was performed to evaluate the influence of age, gender, tobacco and alcohol abuse, HPV status, index-UICC stage, therapy mode of the index tumor, and development of an SPT on survival.Ethical approval by the Zurich Ethics Committee for retrospective analysis of patient data is available (KEK-ZH-No. 2013-0019).After exclusion of 16 patients due to undefined HPV status, the analyzed cohort consisted of 91 patients. In total, 57/91 OPSCC (62.6%) showed p16-overexpression and positivity for HR-HPV DNA, as detected by PCR. Therefore, these tumors were considered as HPV-positive tumors. With 89.5% (51/57 HPV-positive tumors), HPV16 was the most frequently identified HPV type. Other HPV types detected were HPV18, HPV 33, HPV 35, HPV 56, and HPV 59.More than 90% of the index tumors of HPV-positive patients were located in the tonsils, as shown in The mean age of all patients with OPSCC was 59.54 years and comparable among the three groups .p-value 0.002). Only 2/34 patients with HPV-negative tumors were nonsmokers and therefore, they were not separately analyzed.Independent of HPV status, OPSCC patients were more likely to be male (74.7%) and smokers (72.9%). HPV-positive patients smoked significantly less than HPV-negative patients .There were no patients with history of alcohol abuse among nonsmokers, whereas 9/37 (24.3%) of the HPV-positive smokers and 19/32 (59.3%) of the HPV-negative smokers had a history of alcohol abuse. This difference in alcohol consumption between smokers and nonsmokers was statistically highly significant , whereas there was no difference between HPV-positive nonsmokers and HPV-positive smokers.In the overall cohort, 21/91 (23.1%) OPSCC patients developed an SPT during the observation period : 4 (20%)p = 0.012), which was statistically significantly inversely correlated with the development of SPT.Using a multivariate Cox regression analysis, we examined the influence of HPV-positivity, smoking, alcohol, age, gender, UICC stage, and the therapy mode for the index tumor, on the development of SPT. The results of the multivariate Cox regression are shown in Six of 21 (28.6%) SPT appeared synchronously with the index tumor, while 15/21 (71.4%) appeared metachronously. The synchronous SPT were located in the oropharynx (2 SPT), hypopharynx (2 SPT), larynx (1 SPT), and lung (1 SPT).Metachronous SPT appeared after an average of 68.9 months . The majority (60%) of the metachronous SPT was located inside the head and neck area : oral caOne case showed a metachronous SPT in the esophagus, and 5 were located in the lung.The number of SPT and timely distribution of SPT occurrence in relation to the HPV status are depicted in p-value of 0.161, Metachronous SPT of HPV-positive patients occurred after an average of 91.6 months, while the SPT of HPV-negative patients were diagnosed after 49 months on average. Although this difference is striking, it did not reach statistical significance .p < 0.0001, Overall, 17/91 (18.7%) OPSCC patients died: 2 HPV-positive nonsmokers, 5 HPV-positive smokers, and 10 HPV-negative patients. Patients with HPV-positive OPSCC showed significantly better disease-specific survival than patients with HPV-negative OPSCC  OPSCC patients with SPT and 12/70 (17.1%) patients without SPT died. OPSCC patients without SPT had significantly better disease-specific survival than OPSCC patients with SPT  died from the index tumor and eight (14%) developed an SPT from which no one died within the observation period. There was no significant difference in survival in the HPV-positive patient group with SPT compared to HPV-positive patients without .Out of 34 HPV-negative patients, 5/21 (23.8%) died without SPT and 5/13 (38.5%) died with SPT. The disease-specific 5-year survival rate was statistically significantly higher in HPV-negative patients without SPT (83.3%) than in HPV-negative patients with SPT .p-value 0.105).Interestingly, none of the patients with a SPT in the oropharynx died during the time of observation. This stands in contrast to 23.8% deaths among patients with an SPT outside the oropharynx. However, the difference was not statistically significant , base of tongue (14.7%), pharyngeal wall (5.9%), and soft palate (2.9%).In this study, the definition of SPT was based on Warren and Gates , even thAs hypothesized, HPV-positive patients developed significantly fewer SPT 15.79%) than HPV-negative patients (38.24%) ,21,36. S.79% thanContrary to expectations, we did not find a significantly increased incidence of SPT in smokers compared to nonsmokers, even with statistical control for age, sex, HPV status, and alcohol abuse . PuttingIn a literature review of the last 40 years (1979–2019), Coca-Pelaz et al.  found thIn our study, HPV-positive patients mostly showed metachronous SPT. This in agreement with the results of Xu et al. , who shoOn the basis of the clonal relationship of first and second tumors , RietberIn a case report of a 46-year-old patient with three synchronous HPV-associated OPSCC and two cervical metastases, McGovern et al.  also comThe latter theory was supported by the findings of a case report of Joseph et al. , who shoDue to the small number of HPV-positive SPT in our cohort, we could not make robust statements on the frequency of HPV-associated SPT at individual sites. Still, the detected localizations, especially of HPV-positive SPT, are noteworthy.In our cohort, looking specifically at SPT in the head and neck area, tumoral HPV positivity of the index tumor did not show any significant influence on the frequency of SPT, while smoking and age of the patient did. Thus, the risk of SPT in the head and neck area decreased with increasing age, while it increased in smokers.This is consistent with the results of Peck et al.  who demoTumor follow-up was conducted over an average of 4.76 years after diagnosis, which is in line with other studies on the same topic . Still, As Kreimer et al. ,44 foundFrom The Kaplan–Meier plot in Looking at the impact of SPT on the prognosis of OPSCC patients, the consensus in the literature is the following: despite advances in therapy, the survival rate of OPSCC patients remains reduced, especially if SPT are developed ,46. BhatIn contrast, in HPV-positive OPSCC patients, survival with SPT did not significantly differ from survival without SPT . The facThis study is limited by the small number of cases for analysis of SPT. Since SPT are much less frequent in HPV-positive OPSCC than in HPV-negative OPSCC, we only recorded eight SPT of HPV-positive OPSCC. Thus, conclusions about the frequencies of specific SPT localizations are limited, but they provide a lead for larger prospective studies.Retrospective analyses, as performed in this study, must be interpreted with caution due to the limited quality of retrospectively collected data, sample size limitations, and the potential for noncontrolled influencing factors.Although this study focused on SPT of HPV-associated OPSCC, HPV status of the SPT was concluded solely based on the location and the HPV status of the index OPSCC. Therefore, uncertainty remains regarding the HPV status of SPT. The possibility remains, especially with tumors of the lung, that SPT are actually single metastases. Absolute certainty in this regard can only be obtained by genetic sequence analysis, which was not investigated in this study.A strength of our study is the long observation period of up to 17 years.HPV-positive OPSCC showed a lower risk of SPT than HPV-negative OPSCC. SPT of HPV-negative OPSCC occurred in the typical tobacco- and alcohol-associated sites. Significantly more HPV-negative patients died from index OPSCC or its SPT than HPV-positive patients. Thus, HPV positivity is a positive prognostic factor not only in the context of overcoming cancer but also in terms of long-term survival. Since SPT of HPV-positive OPSCC still occurred after more than 10 years, follow-up should not be reduced despite the lower SPT risk of HPV-positive OPSCC.


Similarly, adjuvant vaccination reduced the risk of developing anal intraepithelial neoplasia (p = 0.005) and recurrent respiratory papillomatosis (p = 0.004). No differences in anogenital warts and vulvar intraepithelial neoplasia recurrence rate were observed comparing vaccinated and unvaccinated individuals. Conclusions: Adjuvant HPV vaccination is associated with a reduced risk of CIN recurrence, although there are limited data regarding its role in other HPV-related diseases. Further research is warranted to shed more light on the role of HPV vaccination as adjuvant therapy after primary treatment.Objective: To assess evidence on the efficacy of adjuvant human papillomavirus (HPV) vaccination in patients treated for HPV-related disease across different susceptible organ sites. Methods: A systematic review was conducted to identify studies addressing the efficacy of adjuvant HPV vaccination on reducing the risk of recurrence of HPV-related preinvasive diseases. Results were reported as mean differences or pooled odds ratios (OR) with 95% confidence intervals (95% CI). Results: Sixteen studies were identified for the final analysis. Overall, 21,472 patients with cervical dysplasia were included: 4132 (19.2%) received the peri-operative HPV vaccine, while 17,340 (80.8%) underwent surgical treatment alone. The recurrences of CIN 1+ (OR 0.45, 95% CI 0.27 to 0.73;  The vaccine was initially approved for women, and then expanded also to men in 2009. These vaccines consist of noninfectious, HPV-like particles that elicit the production of neutralizing L1-specific antibodies blocking the viral entry into host cells. Currently, there are three types of HPV vaccines available: 2-valent (Cervarix®), 4-valent (Gardasil®), and 9-valent (Gardasil9®), all targeting the two most oncogenic serotypes, HPV 16 and HPV 18 , 2 0% (95% CI 0–90%). The pooled estimated OR was 1.04   evaluated the recurrence of AGWs within four years after surgical treatment. AGWs recurred in 123 women (18.8%): 55 (24.4%) in the vaccinated and 68 (15.8%) in the unvaccinated group. Heterogeneity for this comparison was I = 0.88) .Two studies ,33 repor2 44% (95% CI 0–89.6%) and the pooled estimated OR was 0.81   evaluated the VIN/VaIN recurrence within seven years after surgical treatment. AGWs recurred in 114 women (15.4%): 36 (14.3%) in the vaccinated and 78 (15.9%) in the unvaccinated cohort. Heterogeneity was I = 0.52) .Only one monocentric, retrospective study   reportedp = 0.005).The study evaluated a total of 202 patients: 88 in the vaccinated group and 114 in the unvaccinated cohort showing a statistically significant reduction of AIN recurrence in vaccinated women  compared with unvaccinated (35: 30.7%). This study suggested that adjuvant HPV vaccination after surgical treatment for AIN significantly reduced the risk of disease recurrence   reportedp = 0.004).Of the 24 patients included, 13 were in the vaccinated group and 11 in the unvaccinated group. Respiratory papillomatosis recurred in 13 patients : 2  in the vaccinated and 11 (100%) in the unvaccinated cohort. The study suggested that adjuvant HPV vaccination after standard treatment for RRP significantly reduced the risk of disease recurrence  by Dion et al.  addressiConfirming the efficacy of the HPV vaccine also in the secondary prevention setting would pave the way to a new era in the management of the spectrum of HPV-related diseases. HPV-related diseases represent a substantial health burden worldwide. Persistent HPV infection causes up to 4.5%  of all new cancer cases worldwide ,48. MoreAdministering the HPV vaccine shortly before or after the standard treatment is a simple and safe intervention with potentially extraordinary outcomes. Last but not least, the therapies for recurrent HPV-related diseases are costly. In 2012, Chesson et al. estimated that HPV-related disease treatments cumulatively account for nearly $8 billion in direct health care costs per year . TherefoAwaiting more consolidated data on these specific points, it should be acknowledged that our meta-analysis has several strengths and limitations. The strengths include the following: (a) a comprehensive evaluation of all currently available data on HPV-related diseases across different specialties providing a large sample size; (b) the quality of the methodology assessment and the strict inclusion criteria. The limitations include the following: (a) heterogeneity between studies in terms of inclusion criteria and methodologies; (b) the analysis of both randomized and non-randomized studies; (c) selection and information bias.The present systematic review and meta-analysis demonstrates that adjuvant HPV vaccination is associated with a reduced risk of CIN recurrence, while reporting scant data regarding its role in other HPV-related diseases. Further randomized trials are needed to shed more light on the post-expositional role of HPV vaccines across different disciplines and potentially drive post-expositional HPV vaccination into daily practice. Rediscovering the role of prophylactic HPV vaccines in the secondary prevention setting could pave the way to a new era in the management of HPV-related diseases.


Human papillomavirus (HPV) infection can cause cervical and other cancers, including vulva, vagina, penis, anus, or oropharynx. However, in China's northern Henan Province, data on the prevalence and genotype distribution of HPV among women attending gynecology clinics is limited. This study aimed to investigate the current prevalence and genotype distribution of HPV among women attending gynecology clinics in northern Henan Province.This study included 15,616 women aged 16–81 years old who visited the Xinxiang central hospital's gynecology department between January 2018 and December 2019. HPV DNA was detected by a conventional PCR method followed by HPV type-specific hybridization, which was designed to detect 17 high-risk HPV (HR-HPV) genotypes and 20 low-risk HPV (LR-HPV) genotypes. HPV prevalence and corresponding 95% confidence intervals (95% CI) were calculated using SPSS 18.0.The overall HPV prevalence was 19.7% among women in northern Henan Province. Single, double, and multiple HPV infections accounted for 13.7%, 4.3%, and 1.8% of the total cases. Most infections were caused by HR-HPV (71.8%), and single genotype HPV infection (13.7%) was the most common pattern. The most common HR-HPV genotype was HPV16 (4.3%), followed by HPV52 (3.5%) and HPV58 (2.0%). The most common LR-HPV genotype was HPV6 (1.4%), followed by HPV61 (1.1%) and HPV81 (1.1%).HPV infection is high among women attending gynecology clinics in northern Henan Province. The highest prevalence was found in women less than 20 years old. In northern Henan Province, the 9-valent HPV vaccine is strongly recommended for regular immunization. Cervical cancer, ranked after breast cancer, colorectal cancer, and lung cancer, is the fourth most common cancer among women worldwide, with approximately 530,000 new cases and 275,000 deaths every year –3. AccorUp to present, three licensed prophylactic vaccines, a bivalent vaccine against HPV16 and 18, a quadrivalent vaccine against HPV 6, 11, 16, and 18, and a 9-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58, are considered as effective and safe for preventing HPV infection. However, the current vaccines offer protection only targeted at a few HPV genotypes and provide cross-protection for certain HPV genotypes. For example, the bivalent vaccine against HPV16 and 18 has a cross-protection against HPV 31, 34 and 45 , 11, undAt present, the three vaccines against HPV can be used in China. However, only the bivalent vaccine and the quadrivalent vaccine against HPV are available in northern Henan Province. The 9-valent vaccines are only used in some provinces in China since May 2018. Lack of epidemiological data on HPV infection and genotype distribution makes it difficult to implement preventative measures such as HPV vaccination in northern Henan Province. The sponsors and local government may support HPV vaccines by using the scientific data of HPV prevalence. Therefore, the prevalence and genotype distribution of HPV among women attending gynecology clinics in Henan Province were identified in this study. The results of this research are of great importance for estimation of the awareness of HPV infection and the introduction of vaccination program in northern Henan Province of China.Women aged 16 and 81 attending regular gynecological outpatient clinics in Xinxiang central hospital between January 2018 and December 2019 in northern Henan Province were invited to participate in this study. They were divided into six age groups. The G1 group meant age < 20; the G2 group meant 20 ≤ age < 30; the G3 group meant 30 ≤ age < 40; the G4 group meant 40 ≤ age < 50; the G5 group meant 50 ≤ age < 60; the G6 group meant age ≥ 60. Women were excluded for: the presence of cervical cancer, pregnancy at the time of enrollment, previous HPV vaccination, without age information, hysterectomy, or immunosuppression. Finally, a total of 15,616 participants were included in this study for analysis.Cervical specimens were collected by the trained clinicians assisted by the speculum in Xinxiang central hospital. The endocervical and ectocervical cells were collected from the cervical canal using a plastic brush . The brush was placed into a 2 mL vial of Hybribio cervical cells preservation solution  for HPV DNA detection. This study was performed strictly following the Declaration of Helsinki and approved by the Ethics Committee in Xinxiang Medical University. Informed consent was obtained from all participants before enrollment.HPV DNA was extracted from the cervical cells using the Hybribio viral DNA extraction kit . Briefly, the cervical cells were first digested by proteinase K. Then, the released DNA was obtained through absorption to magnetic glass particles, washed, and purified from these particles using the automated nucleic acid extraction instrument . The concentration and purity (OD260/OD280 1.6–1.8) of DNA were determined by Nanodrop 2000 . After the concentration was determined, the samples were stored.HPV DNA amplification and genotyping were conducted using a commercial Hybribio HPV genotyping detection kit  for 37 HPV types, including 17 HR-HPV types  and 20 LR-HPV types . The China Food and Drug Administration has authorized the kit for clinical use. The HPV L1 consensus biotinylated primer sets were used in the PCR assay. 1 μL of the DNA was used in the 25 μL PCR master mix. PCR reaction was initiated with denaturation at 95 °C for 9 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 30 s and elongation at 72 °C for 30 s, with a final extension at 72 °C for 5 min.HPV genotyping was performed by the flow-through hybridization method. The flow-through hybridization was performed on a medical nucleic acid hybridization instrument prewarmed at 45 °C prior to usage. A nylon membrane on which 37 HPV genotype-specific oligonucleotide probes secured was placed into the instrument. The biotinylated PCR product was denatured at 95 °C for 5 min and then chilled on ice for 2 min before hybridization. The PCR product was mixed with the hybridization solutions, and the mixture was added into the sample wells to conduct flow-through hybridization for 10 min. The nylon membrane was washed with the hybridization solution three times, and the empty region was blocked without reaction. The hybridizing signal was detected with streptavidin alkaline phosphatase, binding to biotinylated PCR products, and its subtract NBT/BCIP (nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate). The genotyping result was detected by the position of the HPV-genotype probes on the membrane. The blue dot on the membrane, indicating a positive result, was judged by the naked eyes. The experimental procedures, including PCR and flow-through hybridization, followed the manufacturer's manual. Multiple dots showed multiple infections. Quality controls were carried out throughout the experiment, including PCR amplification and hybridization by using positive and negative controls provided by the kit.HPV prevalence and genotype distribution were analyzed. Single, double, and multiple HPV infections were defined as infections with one, two, and more than two genotypes of HPV infections. HPV prevalence in designated groups and corresponding 95% confidence intervals (95% CI) were calculated using SPSS 18.0 for Windows .From January 2018 to December 2019, 15,616 cervical specimens collected for HPV DNA detection were used for statistical analysis. There were 3081 specimens positive for any HPV DNA, and the overall prevalence of HPV was 19.7% (95% CI 19.1–20.4%). The 15,616 participants were divided into six age groups, and the HPV infection rate in each group was calculated as shown in Table Single genotype HPV infection (13.7%) was the most common pattern, and it occurred more frequently than double (4.3%) and multiple HPV infections (1.8%) shown in Table There were 36 different HPV genotypes, including 17 HR-HPV genotypes and 19 LR-HPV genotypes, identified in this study. The prevalence of 17 HR-HPV was demonstrated in Table China has a vast territory and a large population of more than 1.4 billion, and the prevalence of HPV varies greatly in different provinces of China. Some studies reported the prevalence and genotype distribution of HPV in different provinces in China –15. HoweThis present study provided age-specific HPV infection rates among women. Our results showed that women under 20 years had the highest infection rate. Single, double and multiple infections rates were also the highest in women under 20 years. There are two possible reasons for this phenomenon: the first reason is that literature shows that HPV prevalence is higher in the younger age group , 24. TheThe present study found that the prevalence of HPV declined gradually in the middle-aged groups and slightly increased in the G6 group (22.9%), which indicated that women over 60 years suffered severely from HPV infection. For women over 60 years, HPV infections may occur when they are young, and the infections probably are persistent infections instead of new HPV infections. If the HPV cannot be eliminated by the immune system and persist for several years, this will present as a risk factor for neoplasia development. HPV infection rates was 21.1% in women aged 20–29, 18.1% in women aged 30–39, 17.7% in women aged 40–49 and 20.8% in women aged 50–59. According to recent data from China National Cancer Center, there were 98,990 new cases of cervical cancer and 30,500 deaths in China in 2015 . The numOur present study also provided the age-specific distribution of single, double, and multiple HPV infections rates. Except for the G1 group, the single, double, and multiple HPV infections rates of the rest groups were very close. There has not been a consensus on whether numerous HPV infections raise the risk of cervical cancer more than a single HPV infection up until now. Some studies have reported that multiple HPV infections had a higher risk of cervical cancer occurrence and development than single HPV infections , 30. HowIt is important to know the HPV genotypes distribution since data concerning the distribution of HPV genotypes is concerning with the vaccine development. Persistent infection with HR-HPV is the primary etiological factor for cervical cancer. Up to 70% of cervical cancers are caused by HPV16 and HPV18, with the remainder of cervical cancers caused by other HR-HPV genotypes. In the present study, HPV16, HPV52, HPV58, HPV53, and HPV39 were the top five HR-HPV genotypes. HPV16 ranked first in our study, and the infection rate reached up to 4.3%. HPV52 and HPV58 ranked second and third, respectively. Besides HPV16, both HPV52 and HPV58 are also strongly correlated with cervical cancer development . The topFurthermore, the study also investigated the distribution of LR-HPV. Our study showed that the top five most common LR-HPV genotypes were HPV6, HPV61, HPV81, HPV54, and HPV11. For the LR-HPV types, cervical cancer association is very rare in the general women population. LR-HPV mainly causes genital warts, common warts, flats warts, and many other skin lesions , which aA high prevalence of HPV was reported in the study. HPV16, HPV52, and HPV58 were the dominant HR-HPV in northern Henan Province. The results in our study provide important information for cervical cancer screening and vaccination in women in northern Henan Province of China.


Background: This study evaluates the possible effect of 9-valent (9vHPV) vaccination on the results of HPV and cytological tests in a cohort of adult women. Methods: This study is a retrospective, single-cohort, monocentric study. Sexually active women aged 14–70 years, who underwent 9vHPV vaccination, were enrolled. Dose administration dates, side effects and data on Pap smears and HPV tests performed before and after the first vaccine dose were collected. Subjects were considered “unexposed” to the vaccine for all time intervals before the first dose administration, and “exposed” to the first, second and third vaccine doses in all time intervals following each specific dose. Results: A total of 512 women underwent the first 9vHPV dose administration and were enrolled in the study. Median age at vaccination was 30.5 (14–70). Log-rank tests and Cox regression analyses showed a highly statistically significant (p < 0.0001) difference in the time to negativization after the exposure to the third vaccine dose in the 207 women starting with a Pap+ smear (HR (95% C.I.), 2.66 (1.83–3.86)) and in the 198 women starting with an HPV HR+ test (HR (95% C.I.), 7.80 (4.83–12.60)). Conclusions: 9vHPV vaccination may play a role in shortening the clearance time of HPV HR+ or Pap positivity in sexually active adult women.  Human papillomavirus (HPV) is the most common sexually transmitted infection of the lower genital tract worldwide, with an estimated lifetime risk of infection greater than 80%. Persistent infection with high-risk HPV types is associated with precancerous and cancerous lesions ,2. ProphThe World Health Organization (WHO) still recognizes HPV-related disease as a major global public health issue and recommends HPV vaccination in all national public programs ,8. SinceDifferent approaches for population and individual HPV vaccination programs have been chosen according to the specificities of national/regional health care systems ,15.In Italy, the national HPV vaccination program was implemented in 2007 as an active and free campaign for girls in their 12th year of life, even if some regions opted to extend the free of charge offer to further cohorts, such as girls aged 15 or 16 years or girls aged 25 years, linking it to the first call for screening. Moreover, women aged 14–26 years were given the opportunity of a catch-up vaccination with copayment, in local health unit (ASL) or hospital-based outpatient services. After the introduction of the 2017–2019 National Preventive Vaccination Plan (2017–2019 PNPV) . In October 2018, the Food and Drug Administration (FDA) approved the extension of the eligibility range for the new 9vHPV vaccine to both women and men aged 27–45 years . In JuneObviously, the optimal time for HPV immunization lies before to the individual’s sexual debut , but HPVData on the natural history of HPV infection in older women are still controversial. In fact, while the majority of HPV infections are transient and clear within a couple of years following exposure, especially in the young population , 10–20% The likelihood that a newly detected HPV is due to a new infection versus re-detection of a prior infection (latent HPV infection) declines with age . MoreoveHowever, rates of progression from infection to high-grade lesions are similar in young and adult women , and thiAlthough HPV vaccines are, nowadays, still not considered to play a therapeutic role in patients with prevalent HPV infection or disease and vaccine efficacy decreases with the increase in age at vaccination ,35, a poThis observational retrospective study aims to evaluate the possible effect of the vaccination timing on the results of microbiological and cytological tests in a cohort of adult sexually active women who underwent opportunistic 9vHPV vaccination and to evaluate the potential of the 9vHPV vaccine in boosting HPV viral clearance.Study design—This is a retrospective, single-cohort, monocentric study. This study was approved by the local ethics committee . Participants—From our institutional data repositories, we identified sexually active women, aged 14–70 years, who underwent 9vHPV vaccination, between June 2017 and November 2020, in the “PreGIO” gynecologic outpatient clinic, Fondazione Policlinico Universitario Agostino Gemelli, IRCCS, in Rome, Italy. In “PreGIO”, women are vaccinated opportunistically, encouraged by their care provider or by self-decision. Immuno-compromised women  and women with a major abnormal cytological alteration (H-SIL) prior to 9vHPV vaccination are excluded from vaccination. The vaccine used in our outpatient clinic is the 9vHPV vaccine, offered with copayment (at a price for each dose of € 110.00), administered according to the schedule of 0, 2 and 6 months, with an observation of 15 min after dose administrations. Data collected—For each patient enrolled in this study, clinical data including age, dose administration dates, side effects and previous HPV-related treatments were collected from the PreGIO archive, and data on Pap smears and HPV tests performed before and after the first vaccine dose were retrieved by the Gemelli Hospital Information System, or by collecting personal exams brought by the women (during vaccination or follow-up visits).Both conventional Pap smears and ThinPrep samples were considered for the analysis as long as they were reported according to the Bethesda system. Therefore, Pap smear results were reported as negative when negative to intraepithelial lesion or malignancy, or positive if minor abnormal cytological alterations—diagnosis of atypical squamous cells of undetermined significance (ASC-US) or low-grade lesion (LSIL)—were identified. Concerning HPV DNA tests, they were executed following different techniques (Digene HC2 or HPV DNA genotyping tests). Digene HC2 is able to differentiate only between 2 HPV DNA groups, low-risk (LR) and high-risk (HR) types. Where genotyping was performed, we defined HR+, LR+ or negative status according to the Anyplex™ II HPV28 Detection Seegene test—performed in Gemelli Hospital—in which HR types are 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82; and LR types are 6, 11, 40, 42, 43, 44, 54, 61, 70. With the aim to include also the Digene HC2 results in our analysis, we decided to group the HPV results of the genotyping test as HPV HR+ when at least one HR genotype was identified, HPV LR+ when only LR genotypes were identified and HPV-negative when no HPV type was reported. Patients with an HR-HPV test and LSIL were histologically confirmed to be CIN1 or less after colposcopy-guided biopsy.Statistical analysis—For descriptive analysis of subject characteristics and vaccination, Pap smear and HPV test timings and results, medians, IQRs and range values were calculated and presented as single numbers or visualized by box plots. Inferential analysis was focused on the time to negativization of any positive Pap test or HPV HR+ test, at any time before or after vaccine dose administration. In particular, for the analysis of the time to negativization of Pap smears, only women for whom at least 1 positive Pap smear followed by at least 1 other Pap smear (regardless of the result) was retrieved were included in the analysis. On the other hand, for the analysis of the time to negativization of HPV tests, only women for whom at least 1 HPV HR+ test followed by at least 1 other HPV test (regardless of the result) was retrieved were included in the analysis. In this case, HPV LR+ test results were considered as negativizations of a previous HPV HR+ test, such as HPV-negative tests. Exposure to vaccine was considered as a time-changing variable, and subjects were considered “unexposed” for all time intervals before the first dose administration, and “exposed” for the first, second and third vaccine doses in all time intervals following each specific dose. Data on Pap and HPV tests were evaluated following the LOCF (last observation carried forward) criterium for the time intervals among doses, when no further testing was performed in those time intervals. The resulting data were analyzed by survival analysis (time-to-event) methods: Kaplan–Meier, log-rank test and Cox regression for the evaluation of potential confounding factors (age in days at event and conization within 1 y before the vaccination 1st dose). All statistical analyses and graphical data displays were performed by Stata 16.1 MP statistical software ,37. A total of 512 sexually active women underwent the first 9vHPV dose administration during the time period considered for the study . Among them, 499 performed the second dose (at a median distance of 70 days—10 weeks—from the first dose) and 474 the third dose (at a median distance of 203 days—29 weeks—from the first dose). The distribution of dose timings since the date of the first dose is shown in Dates and results of a total of 1006 Pap smears on 421 women and 718 HPV tests on 368 women were collected by the hospital information system platform of Fondazione Policlinico Gemelli or by asking the vaccinated women for the tests performed outside the Gemelli Health Care System.Among the Pap smears, 364 were positive for ASC-US/L-SIL and 642 were negative. Regarding the timing of the collected tests, the oldest one was performed 2975 days (8.1 years) before the first dose administration, and the latest one was performed 1273 days (3.5 years) after the first dose administration. Half of the tests were performed at least 57 days before the first dose (median distance from the first dose), and 50% of the tests (IQR of distance from the first dose) were performed between 229 days before and 233 days after the first dose.Among the HPV tests, 459 were positive for HPV-HR types, 53 showed HPV-LR types and 206 were totally negative. HPV 16 was the most frequent genotype detected in both single and co-infections. Regarding the timing of the collected tests, the oldest one was performed 3445 days (9.4 years) before the first dose administration, and the latest one was performed 1273 days (3.5 years) after the first dose administration. Half of the tests were performed at least 84 days before the first dose (median distance from the first dose), and 50% of the tests (IQR of distance from the first dose) were performed between 308 days before and 233 days after the first dose.The distribution of testing times, stratified for specific results, is shown in the box plots reported in It can be noted that around 75% of the positive Pap and HPV-HR tests were performed before the vaccine first dose, whereas negative Pap or HPV-LR results were more frequently reported in tests performed after the first dose.Since dates for Pap and HPV tests collected by the enrolled women ranged from years before the vaccine first dose to years after the administration of the last dose, and for many women more than one test of the same type was retrieved, we tried to analyze the data to understand if the exposure to vaccine doses could have reduced the time span between a positive test (Pap+ or HPV HR+) and a following negative test of the same type. Therefore, we divided the experience of each enrolled woman for which a Pap+ test (or an HPV HR+ test), followed by at least another Pap test (HPV test), regardless of the result, was retrieved into four time intervals: the pool of days from the date of a test performed before the first dose and the date in which the first dose was administered will become unexposed person-days, and each following negative test will be considered a spontaneous regression; all the time intervals after the first dose and the test negativization after the first, second and third doses may have been influenced (more or less) by the exposure to the vaccine. Numbers of women, cumulative person-days and negative tests following a previous positive test occurring during each individual time span in different vaccination statuses are summarized in Among the 421 women with at least one Pap smear recorded, 207 (49.2%) reported at least one positive Pap smear followed by another Pap smear. A total of 191 women had at least 1 day of follow-up after a positive Pap smear before the first dose , 124 had at least 1 day of follow-up between the first and the second dose , 115 had at least 1 day of follow-up between the second and the third dose  and 93 had at least 1 day of follow-up after the third dose .p < 0.0001). After verification performed by univariate and multivariate Cox regression models, no confounding by age at the test date or vaccine administration date or by conization treatment performed within a year before the first dose (37/207 women were treated) was identified  reported at least one HPV-HR+ test followed by another HPV test. A total of 194 women had at least 1 day of follow-up after a positive HPV HR+ test before the first dose , 151 had at least 1 day of follow-up between the first and the second dose , 147 had at least 1 day of follow-up between the second and the third dose  and 126 had at least 1 day of follow-up after the third dose .p < 0.0001). After verification performed by univariate and multivariate Cox regression models, no confounding by age at the test date or vaccine administration date or by conization treatment performed within a year before the first dose (46/198 women were treated) was identified , and AEs were mainly local reactions (limited to pain and swelling at the injection site) in 50 women (9.8% of the sample). The only event of clinical relevance was one episode of supra-clavicular reactive lympho-adenopathy homolateral to the injection site after the first dose in a 25-year-old, who fully recovered in 15 days after the injection.The rationale for vaccinating adult women comes from the observation that the incidence of HPV infections decreases with age but it remains prominent even years after sexual debut, and that the incidence of HPV infection shows a second peak around 45–50 years . FurtherThus, although viral persistence appears to be the predominant cause of HPV infection in adult women, new infections may occur, and it is impossible to discriminate whether a new infection represents a new acquisition or a reactivation of a latent infection. Even if the vaccine therapeutic properties on pre-existing HPV-related lesions are still doubtful, women already infected with an HPV genotype included in a vaccine may indeed benefit from protection against other HPV genotypes that are not part of the infection . MoreoveWith this background, although the vaccine has maximum effectiveness if administered before exposure to the virus , and it Previous diagnosis and treatment of an HPV-related lesion did not represent a contraindication to vaccination. Long-term observational cohort studies of vaccinated populations ,42 and rThe aim of our retrospective, monocentric study was to evaluate in a time-to-event analysis the possible relationship among the vaccination status and the time to negativization of the commonly used diagnostics tests for HPV infection  in sexually active women who underwent opportunistic 9vHPV vaccination while reporting a positive HPV DNA HR test or showing minor cytological abnormalities.p < 0.0001), with a suggestion of a stronger effect on HPV HR+ than on Pap smears. Neither age at vaccination/testing time nor having been treated for cervical lesions in the year before the first dose played any role as confounders or interaction variables.Our study deals with a population of women who, according to the clinical practice, will normally undergo a follow-up in view of a possible spontaneous regression of HPV infection, with or without cytological low-grade lesions. We observed a good tolerability and a good compliance, with many subjects completing the whole vaccination schedule in the regular time, even if it has to be considered that we are performing an “on-demand” vaccination with a copayment modality, and therefore the participants are generally very highly motivated. Our time-to-event curves for Pap smear and HPV-HR+ test negativization, comparing time periods referring to women in different vaccinal statuses, both show high statistically significant differences among the vaccinal status groups (To our knowledge, as, to date, there are no similar data reported in the literature about the follow-up of commonly used diagnostics tests for HPV infection results in the adult population after 9vHPV vaccination, our results may be of great interest in understanding a possible beneficial role of HPV vaccines in reducing the time for HPV clearance where an HPV infection pre-existed before the first dose. There are few data in the literature on the benefit of HPV vaccination in adult populations with an active HPV infection. The retrospective study of Garolla et al.  in male The study of Ferris et al.  on the pA recent pilot observational study, conducted in Greece , investiA plausible explanation of the biological mechanism underlying our results may be related to the prevention of the reinfection and reactivation of a latent infection or to the reduction in viral shedding, as advised by some immunological studies . These iWhen, in natural infection, the immune system happens to be ineffective in providing a long-lasting protection, HPV vaccination may prevent the loss of immunological effectiveness: in fact, the high antibody levels following vaccination seem to prevent new areas of epithelial infection, whether due to dissemination from existing sites of HPV infection or from new HPV exposure, and thus prevent disease. The importance of HPV vaccination of previously HPV-infected women is also substantiated in a recent publication by Vorsters et al., who consider this practice safe and to generate a high-level immune response . The autThe main methodological limitations of this study are linked to the observational retrospective design, the need to rely on the results of Pap smears and HPV tests spontaneously performed at highly variable time intervals in different laboratories and the lack of data on potentially relevant risk factors for the history of HPV infections, such as sexual habits, smoking status and use of estro-progestins. In particular, regarding cytology and HPV testing interval times, it is worthy of note that while, in Italy, the national cervical screening program offers free cytology every 3 years in women aged 25–29 years and, from 2018, according to European guidelines , high-riOn the other hand, this study addresses IARC’s call for data on the natural history of HPV infection among older women , may helHPV disease prevention guidelines can be informed and improved by data on the prevalence, incidence and persistence of HPV infections at any age, and particularly among older women, who are typically not targeted for HPV immunization. Our study suggests that 9vHPV vaccination may have a promising role in shortening the clearance time of HPV HR+ or Pap smear positivity in sexually active adult women. Further prospective studies may confirm this mechanism which may help in boosting the prophylactic role of HPV vaccination in women older than 26 years of age. Extending HPV vaccination to this population has the potential to be of utmost importance both for current public health policy and for the long-term reduction in HPV-related cancers.


We encountered HPV6‐positive cervical papillary squamous cancer (PSCC) that was difficult to diagnose. The case was initially diagnosed and treated for condyloma. To the best of our knowledge, this is the first report of HPV6 infection in PSCC. We encountered HPV6‐positive cervical papillary squamous cancer (PSCC) that was difficult to diagnose. The case was initially diagnosed and treated for condyloma. To the best of our knowledge, this is the first report of HPV6 infection in PSCC. Subsequently, the patient was re‐diagnosed and treated for non‐invasive cervical papillary squamous cancer (PSCC) with HPV6 infection. To the best of our knowledge, this is the first report of HPV6 infection in PSCC. Papillary squamous cell carcinoma (PSCC) of the uterine cervix is an extremely rare histological type of cervical cancer, which accounts for approximately 1.6% of all cervical cancers.2A 47‐year‐old woman with one pregnancy and one parity, and no relevant medical, family, or menstruation history, presented to our hospital. The patient had been followed up from CIN 1 to 2 at a previous hospital for 8 years. A high‐risk HPV test was negative twice. Subsequently, a papillary mass was found in the cervix, and histological examination of the cervix suggested CIN 3 or condyloma. Conization was performed at another hospital, and the diagnosis was cervical condyloma with a positive margin.Subsequently, the patient returned to the previous hospital and was followed up. However, 3 months later, there was recurrence of papillary tumor in the cervix; a biopsy was performed, which led to the diagnosis of condyloma. Cervical laser ablation was performed to treat the tumor. Two months later, the cervical tumor relapsed. Although laser vaporization was performed, the tumor relapsed again after 1 month. Cryotherapy was performed to treat the relapsed tumor, but the cervical tumor relapsed. Since the tumor recurred repeatedly in a short time, pathohistological examination was performed by a pathologist at another hospital and the possibility of PSCC was indicated. The patient was then referred to our hospital for examination.Colposcopy revealed a condyloma‐like papillary tumor approximately 1.5 cm in diameter Figure . No abno3We encountered a case in which a condyloma‐like papillary tumor was repeatedly treated as condyloma; however, there was repeated recurrence. Therefore, the patient was re‐diagnosed and treated for non‐invasive cervical cancer with HPV 6 infection. In 1986, Randall reported nine cases of PSCC for the first time worldwide. The characteristic pathological finding of PSCC is full‐thickness dysplastic cells in a papillary architecture with fibrovascular cores and an invasive component that is usually deep to the papillary excrescences.In this case, treatment for condyloma was performed several times for non‐invasive PSCC. However, despite inadequate treatment for non‐invasive PSCC, the tumor did not invade or spread, indicating slow progression of PSCC, as previously reported.It is also necessary to pay attention to the difficulty in diagnosis and pitfalls, and to consider PSCC when an intractable condyloma‐like mass is observed. Additionally, it is necessary to consider a re‐diagnosis of the pathology if PSCC is suspected. It has been reported that the positive rate of high‐risk HPV in PSCC is lower than that in cases of conventional SCC (50% vs. >95%). High‐risk HPV positivity in PSCC is often associated with HPV type 16.We performed high‐risk HPV screening tests several times before surgery, but they were negative; however, HPV 6 was identified by HPV DNA genotyping of tumor samples after surgery. Only HPV 6 was specifically detected in cryosurgery specimens using the Multiplex PCR method. The Multiplex PCR method allows for rapid, highly sensitive, and simultaneous measurement of 16 HPV genotypes  in a single step. The primer set is designed to avoid false negatives. The results of the Multiplex PCR method and preoperative high‐risk HPV genotyping suggest that the patient was infected with HPV 6 rather than high‐risk HPV.To the best of our knowledge, this is the first report of HPV 6 infection in PSCC. Although HPV 6 is not a high‐risk HPV, HPV 6 infection was previously observed in CIN 2/3,We have no conflicts of interest to declare regarding this case.KS, FI, AT, YM, and TO analyzed and interpreted data; KS, MH, and MI prepared the manuscript and figures; MT, TI, MMU, and TT reviewed the manuscript; and KS, MM, and YO revised the manuscript for important intellectual content. All authors have read and approved the final version of this manuscript.The study design was approved by the ethics committee of the University of Tokyo.The patient provided written informed consent for the publication of any associated data and accompanying images.Fig S1Click here for additional data file.


TP53, CDKN2A, FAT1, NOTCH-1 and PIK3CA. Numerical alterations involve gains in MYC and EGFR, as well as amplifications in HPV integration loci. A few genes including TP53, CDKN2A, PIK3CA and CCND1 harbor both somatic mutations and copy number alterations. Notch, RTK-RAS and Hippo pathways are frequently deregulated. Nevertheless, the relevance of the identified alterations, their role in signaling pathways or their association with HPV status remain elusive. Combined targeting of different pathways might represent a valid therapeutic approach in PSCC. This work calls for large-scale sequencing studies with robust HPV testing to improve the genomic understanding of PSCC.Penile squamous cell carcinoma (PSCC) is a rare but aggressive neoplasm with dual pathogenesis (human papillomavirus (HPV)-associated and HPV-independent). The development of targeted treatment is hindered by poor knowledge of the molecular landscape of PSCC. We performed a thorough review of genetic alterations of PSCC focused on somatic mutations and/or copy number alterations. A total of seven articles have been identified which, overall, include 268 PSCC. However, the series are heterogeneous regarding methodologies employed for DNA sequencing and HPV detection together with HPV prevalence, and include, in general, a limited number of cases, which results in markedly different findings. Reported top-ranked mutations involve  Malignant tumors of the penis are rare but impose a major challenge due to their high morbidity and mortality . They ocTwo different etiopathogenic pathways have been described in PSCC : one assCD274 (PD-L1) amplifications observed in PSCC [TP53, CDKN2A, EGFR, PIK3CA and MYC) or analyze a limited number of samples and systematic and extensive genomic analyses of PSCC have not taken place yet.Patients with PSCC frequently develop early loco-regional and angiolymphatic spread and have a devastating prognosis . The man in PSCC  hint at  in PSCC . The devSince 2014, increased access to next generation sequencing (NGS) has pushed forward the molecular characterization of prevalent neoplasms such as breast or lung cancer. Unfortunately, molecular progress on rare cancers such as PSCC or vulvar cancer  has beenRelevant studies on genomic alterations  were captured through a search of Pubmed Medline database using the terms “penis”, “penile”, “cancer”, “carcinoma”, “molecular”, “genomic”, “genetic” and “mutation”. We also conducted a manual search of reference lists from the identified papers. Those original articles focused on genome sequencing in PSCC published until 30 June 2021 and having openly published and extractable datasets were deemed eligible. After the first search results, we excluded papers in languages other than English and those with unavailable full text. Then, we screened titles and abstracts excluding misclassified non-original studies , those not focused on human PSCC or not involving DNA sequencing, and other types of documents . After reviewing the full text and examining methodology, we discarded studies not focused on genomic sequencing of PSCC and those assessing less than 10 genes or using non-tissue samples. The results of the eligible studies  were further screened in terms of availability and completeness for each studied gene. The researchers extracted the relevant data including the number of PSCC samples and patients studied, the type of DNA sequencing method, the number and prevalence of genomic alterations and the type and results of HPV testing. The genes involved in copy number alterations were searched among those with somatic mutations to identify genes with both types of abnormalities.The flowchart of the study with the outline of the search results and the study selection process is shown in Four studies (57%) ,19,20,21Four of the five NGS studies included an analysis of non-tumor samples, most commonly blood ,23 or noHPV testing based on PCR was performed in all seven studies, however, only five correlated HPV status and molecular results. The HPV tests included PCR-reverse dot blot assay (two series), unspecified PCR (two series), PGMY9/11 (two series) and Cobas HPV assay (one study). Only three studies, including one of the WES series , additioFour out of seven studies (57%) evaluated the prognostic implications of the genomic alterations identified in PSCC. The follow-up ranged from 27  to 96 moTP53, CDKN2A, NOTCH1, PIK3CA, FAT1, CASP8 and FBXW7. TP53 was mutated in 32% (48/148) of the assessed samples, with frequencies ranging from 10 to 48%. Strikingly, few recurrent somatic alterations were reported in the two WES series (17% and 11%) [and 11%) ,19. MYC locus). Two studies identified copy number variations involving the locus of EGFR in 10 to 70% of cases [EGFR variations with increased tumor size. McDaniel et al. [of cases ,24. The of cases  showed cIncRNAs, ADAM6, LINC00226, LINC00221 and KIAA0125), 2p12-p11.2, 10q26.13 and 8q23.1 were identified with high frequencies (85–100%); however, all of them were reported in a single study [TP53, CDKN2A, PIK3CA, CCND1, ALK, BIRC6, IL7R, PDE4DIP and LAMA1. Amplifications or gains at HPV integration sites 14q32.33 . Regarding copy number alterations, one study  showed lBRCA1, BRCA2, ARID1A, ATR, CHEK2, PARP1, FANCA, PALB2, and RAD51). Transition mutations, including mostly C>T alterations, mediated by the APOBEC family of cytosine deaminases  were repAt least 10 signaling pathways have been identified as disrupted in PSCC in four WES studies. Of them, two ,20 (50%)MYC and CCND1 gains, and those with negative p16 IHC, showed shorter time to progression or survival. The same study reported that high mutational burden in the five most frequently mutated genes  correlated with an advanced stage.McDaniel et al.  noticed MYCN and FAK correlated significantly with worse survival in one study [Amplifications in ne study . Chahoudne study  showed ane study . EGFR and cell cycle kinase CDK4, as well as somatic mutations in KRAS. Ferrándiz-Pulido et al. [PDGFA-mutated tumors. Chahoud et al. [TP53, CDKN2A, NOTCH1, PIK3CA, FBXW7, CASP8, LAMA1 and TTN, among others. Most of the studies suggest potential actionable targets on the basis of the identified genomic alterations. McDaniel et al.  proposedo et al.  indicated et al.  hypothesADAM6 alterations involved in the Notch pathway, although there is little knowledge of their role in cancer. The same authors proposed using EGFR and COX2 inhibitors. The Brazilian series, which mostly included HPV-positive tumors , proposeConsiderable insight on the genomic landscape of PSCC has been acquired over the last six years (2015–2021), as evidenced by the seven studies identified. However, these studies are highly heterogeneous in terms of sociodemographic characteristics, methodology  and include a limited number of samples, which may hamper the validity of some of the conclusions. As a result, the series are also highly heterogeneous in terms of their findings. TP53, CDKN2A, FAT1, NOTCH1 and PIK3CA are consistently identified in PSCC. Copy number alterations have also been reported in three of these genes  [TP53 and CDKN2A are well-known tumor suppressor genes [NOTCH1 and FAT1 mutations are consistently featured in PSCC [Somatic mutations in cancer-related genes  PIK3CA) ,21, whicor genes ,20,24. N in PSCC . However in PSCC . CASP8 and FBXW7 alterations. CASP8 is known for its involvement in apoptosis cascade, whereas FBXW7 acts as a promoter of tumorigenesis through ubiquitin degradation of cell cycle regulators, including p53 [FAT1 mutations, the contribution and clinical relevance of both genes in PSCC remain to be elucidated. Curiously, patients with TP53 wild-type tumors of oral cavity harboring both CASP8 and HRAS mutations showed improved outcomes [NBPF1 gene in PSCC, identified with high frequency but only in a single study. NBPF1 is known to deactivate the PI3K signaling pathway leading to tumor growth inhibition [Another intriguing and frequent finding of the recent studies ,19,20 inding p53 . As occuoutcomes . It is ahibition . MYC, EGFR and CCND1. The high numbers of MYC and EGFR variations in PSCC are in concordance with previous evidence reported in head and neck cancers squamous cell carcinoma, a similar tumor with dual pathogenesis [The genomic profile of PSCC also typically contains numerical alterations in ogenesis . PIK3CA and EGFR alterations [Notch represents the most involved signaling pathway in the studies exploring the whole exome. Curiously, the PI3K pathway is not among the three most frequently involved signaling pathways, despite frequent identification of erations ,20. It iAlthough PSCC has been divided into two different etiologic pathways (HPV-associated and -independent), the overall mutational profile of HPV-associated PSCC is not considerably different from HPV-independent tumors in the published studies. However, a marked variability in HPV prevalence hampers comparability of findings among studies. Indeed, whereas the HPV prevalence ranged from 12 to 37 in six of the studies ,20,23,24NOTCH1 mutations or APOBEC scores. The association of MYC gains with adverse prognosis was also shown in a single study [MYCN and FAK variations described by Yongbo et al. [The prognostic role of most molecular alterations in PSCC also remains elusive. Remarkably, only a single study in this review , based ole study , in accole study . The proo et al.  certainlTP53, CDKN2A, PIK3CA, MYC, and EGFR, have proven to be challenging to target separately [TP53-mutated patients [NOTCH1 and PIK3CA mutations are frequent in PSCC [NOTCH1-mutated patients.Unfortunately, the genes most frequently altered in PSCC, including parately ,35,36. Tpatients . Patientpatients . Lastly, in PSCC , vulvar  in PSCC  and head in PSCC , there iHigh heterogeneity in findings among the studies might be due to methodological differences in DNA sequencing. Indeed, the targeted NGS study , which eIn conclusion, there is still limited understanding of molecular abnormalities involved in PSCC. There is a lack of evidence regarding the association of molecular abnormalities with main clinico-pathological variables. The existing studies are limited in sample size, sociodemographic heterogeneity and variability in DNA sequencing methodology. There is a particular gap of knowledge in the characterization of molecular profiles in relation to HPV status. Given the rarity of PSCC, especially in high-income countries, a number of genomic studies regarding this disease face challenges in acquiring enough samples. Therefore, large multicenter studies are urgently needed to continue on the path of the molecular characterization of PSCC.


Human papillomavirus (HPV) is a virus with the potential to infect human epithelial cells and an etiological agent of many types of cancer, including head and neck cancer. The aim of the study was to determine the prevalence of HPV infection in patients with head and neck squamous cell carcinoma (HNSCC), patients with chronic tonsillitis, and healthy individuals, and to establish high- and low-risk HPV genotypes in these groups. The objectives also comprised the delineation of the relationship between the infection with high- or low-risk HPV subtypes and clinicopathological and demographic characteristics of the study groups. This study was composed of 76 patients diagnosed with HNSCC, 71 patients with chronic tonsillitis, and 168 cases without either of these conditions (the control group). HPV detection and identification of subtypes were performed on isolated DNA using a test which allowed detection of 33 common high-risk and low-risk HPV subtypes. The prevalence of HPV infection was 42.1%, 25.4%, and 37.5% in HNSCC, chronic tonsillitis, and control groups, respectively. HPV 16 was the most prevalent genotype in all groups and the non-oncogenic HPV 43/44 was frequent in HNSCC patients. This analysis provides insight into the prevalence of oral oncogenic and non-oncogenic HPVs in patients with head and neck cancer, patients with chronic tonsillitis and healthy individuals, and leads to the conclusion that further investigations are warranted to examine a larger cohort of patients focusing on high- and low-risk HPV genotypes. Efforts should be focused on screening and prevention strategies, and therefore, it is important to introduce tools for effective detection of HPV genotypes. Furthermore, given the role of vaccines against oral HPV infection, our observations lead to the suggestion that HPV vaccination should be of considerable importance in public health strategies. Human papillomavirus (HPV) is a double-stranded DNA virus with the potential to infect human epithelial cells, an etiological agent of many types of cancer , and theDespite advances in surgical treatments and chemo- and radiotherapy, there are depressing observations regarding morbidity and mortality in head and neck cancer patients. Patients with oral cancer often present with symptoms at a late stage and show high recurrence rates after treatment . A risinChronic tonsillitis may be defined as a sore throat lasting for 3 months and accompanied by tonsillar inflammation ,19. The The aim of the study was to determine the prevalence of HPV infection in patients with HNSCC, patients with chronic tonsillitis, and healthy individuals, and to establish high- or low-risk HPV genotypes in these groups. The objectives also comprised the delineation of the relationship between high- and low-risk HPV infection and clinicopathological and demographic characteristics of the study groups.This study was composed of 315 patients, of whom 76 subjects were diagnosed with HNSCC, 71 patients presented with chronic tonsillitis, and 168 cases were not affected with these conditions (the control group). The cancer samples were obtained from surgical resection and were histologically diagnosed as squamous cell carcinoma. Samples of tonsillar tissue from the chronic tonsillitis group were collected from patients during tonsillectomy and histologically verified. Samples of tonsillar tissue were verified as chronic tonsillitis. No dysplastic changes were found in the analyzed samples in the chronic tonsillitis group. Demographic data of the study groups are shown in All patients were diagnosed, and samples were collected at the Department of Otorhinolaryngology and Oncological Laryngology in Zabrze, Medical University of Silesia in Katowice. Fresh specimens were flash frozen in liquid nitrogen and stored at −80 °C prior to DNA extraction. Written informed consent was obtained from all patients. The study was conducted with the approval of the Bioethics Committee . DNA isolation, HPV detection, and type determination were conducted at the Department of Medical and Molecular Biology, Faculty of Medical Sciences in Zabrze, Medical University of Silesia. The main inclusion criteria for the HNSCC group included a diagnosis of primary squamous cell carcinoma of the oropharynx, no radio- or chemotherapy. Written informed consent to participate in the study and the age above 18 were the inclusion criteria for all groups.The HNSCC group included 55 (72.4%) males and 21 (27.6%) females (mean age 57.8 ± 10.0 years). Most patients  were aged 40 years or more . Fifty-tThe group consisted of 71 patients (mean age 33.4 ± 11.1 years), including 34 (47.9%) men and 37 (52.1%) women. Consumption of tobacco and alcohol was related to 10 (14.1%) and 21 (29.6%) patients, respectively. 6 (8.5%) subjects were both smokers and alcohol users and 25 (35.2%) either smoked or drank alcohol see .Buccal epithelial scrapings were collected from the control group (mean age 39.7 ± 16.1 years), i.e., 112 women (66.7%) and 56 men (33.3%). 121 (72.0%) individuals were alcohol users and 31 (18.5%) were smokers, 29 (17.3%) were both smokers and alcohol users, while 123 (73.2%) individuals either smoked or consumed alcohol see . None of®-24  with ceramic beads . DNA was extracted from buccal epithelial cells of the control group using a GeneMATRIX Swab-Extract DNA Purification Kit  using standard procedures. Qualitative and quantitative analysis of the isolated DNA was performed using a spectrophotometer .DNA was extracted from the tumor specimens and tonsillar tissue samples using a Gene Matrix Tissue DNA Purification Kit  according to the manufacturer’s instructions. Before extraction, samples were slowly thawed and homogenized in FastPrepHPV detection and identification of subtypes were performed by GenoFlow HPV (Human Papillomavirus) Array Test Kits  which use biotin-labeled primers, specific probes, PCR and “flow-through” hybridization technology, which allows detection of 33 common HPV subtypes. The following are considered high-risk  and low-risk  subtypes based on their phylogenetic and epidemiological criteria and the biological niche. The extracted DNA was mixed with PCR reagent mix and DNA Taq Polymerase provided with the kit and amplified using a Mastercycler Personal Thermal Cycler  according to the manufacturer’s protocol. Next, the amplicons were genotyped using the “flow-through” process involving hybridization, enzyme conjugation, and colorimetric detection. Amplification controls, positive and negative controls, and hybridization controls were performed in all runs. Colored dots on the membrane indicated positive reaction and were recorded by scanning the membrane and image analysis . An example of the results is shown in 2 or Fisher exact probability test was used. To measure the association between an exposure and an outcome occurring in the absence of the exposure, the odds ratio (OR) with 95% confidence interval (CI) was calculated. Statistical significance was set at a p value below 0.05 and all tests were two tailed. Statistical analysis was performed using Statistica 13.3 [The distribution of variables was evaluated by the Shapiro–Wilk test and the quantile-quantile plot. The interval data were expressed as a mean value ± standard deviation (M ± SD) in the case of normal distribution. To assess the relationship between qualitative variables, the χica 13.3 .p < 0.05, more in the HNSCC group: 42.1% vs. 25.4%). HPV 16, HPV 43/44 and HPV 18 were detected in all groups with HPV 16 being the most common in all groups and no significant differences were found between the three groups. HPV 43/44 was the second most prevalent type and was significantly more common in the HNSCC group than in the control group  and in the HNSCC group compared with the chronic tonsillitis group  . The HNSCC group had a greater probability of HPV-positive status than the chronic tonsillitis group .More than 35% of all cases were positive for HPV. There were statistically significant differences in the percentage of HPV-positive cases between the chronic tonsillitis and HNSCC groups (.05) see . ConsideIn HNSCC cases, HPV was present in 42.1% of patients. The most common infection in the HNSCC group was HPV 16 (23 patients). HPV 18 and HPV 43/44 were identified in one (1.3%) and eight (10.5%) patients, respectively. Coinfection with HPV 16 and HPV 43/44 or HPV 73 occurred in one case each . The prevalence of HPV in patients with chronic tonsillitis was 25.4%; HPV 16 was the most prevalent type and occurred in 22.5% of patients. Infection with HPV 43/44, HPV 18 and HPV 56 was noticed in one case, respectively. Coinfection with HPV 16 and HPV 56 was noted in 1.4% of subjects. In the control group, the prevalence of HPV was 37.5%, and the most common types were HPV 16 (32.7%), followed by both HPV 43/44 and HPV 18 (2.4%), HPV 35 (1.2%), and one case (0.6%) of HPV 51 and HPV 73. 2.4% of HPV-positive samples had been infected with two types .A summary of the number of HPV-positive patients depending on age, sex and addictions in the HNSCC group is given in 2 = 0.51, p = 0.476), sex , smoking , alcohol consumption , or the use of both stimulants . On the other hand, a relationship was found between the use of a single stimulant  . In the group where stimulants were not used, the probability of a positive HPV status was higher than in the group of smokers or alcohol users . Moreover, smokers showed a decreased HPV 16 infection rate . A detailed summary of the relationships of qualitative variables in individual study groups is presented in In the HNSCC group, no relationship was found between HPV-positive status and age ≥ 40 years (χp = 0.152), (p = 0.091), smoking (p = 0.978), (p = 0.878), drinking (p = 0.846), (p = 0.894), smoking and drinking (p = 0.983), (p = 0.635), smoking or drinking (p = 0.705), (p = 0.686), respectively , with older subjects being HPV-negative. In the remaining groups, differences in age between positive and negative participants were not reported. Due to the limited occurrences of HPV 18, 35, 51, 56 and 73 in the study, the results for those variants were not included.For total HPV status and HPV 16 and HPV 43/44 variants, the age of the study participants considering positive and negative results was analyzed . Statist2 = 17.05, p < 0.001) and between the chronic tonsillitis and control groups . When the chronic tonsillitis and HNSCC groups were compared, alcohol users were predominant in the HNSCC group  and in the control group . The use of both stimulants promoted a higher prevalence of the disease in the HNSCC group compared to the control group .No difference was found between the percentage of alcohol users in the HNSCC group and controls. However, the difference was observed between the HNSCC and chronic tonsillitis groups  and in the control group compared to the chronic tonsillitis group .An additional analysis considering drinking and smoking showed higher HPV incidence rates in the HNSCC group compared to the chronic tonsillitis group .Additionally, the percentages of HPV-positive participants (considering HPV variants) were compared for all groups and no differences were found between sexes in any of the groups or for any HPV type . Moreovep < 0.05, OR = 3.6, 95%CI: 1.1–12.2), lower odds of tumors in the mandible  and higher odds of tumors in the palatine tonsil . The association between HPV 43/44 and T4 stage (p < 0.05) showed that the odds of a T4 tumor were greater in patients with HPV 43/44 in tumor tissue . No association was found for lymph node status (N) or grading for HPV 16 or HPV 43/44. Due to the limited prevalence of HPV 18 and 73 in the study, the results for those variants were not included.Considering tumor localization in HNSCC cases, HPV was located in 42.9% (12/28) cases in the tongue, 23.5% (4/17) in the mandible, 63.6% (7/11) in the left palatine tonsil, 38.5% (5/13) in the floor of the mouth, 33.3% (2/6) in the soft palate, 60.0% (3/5) in the right palatine tonsil, and in 20.0% (1/5) in the cheek. Other localizations were HPV negative. The results showed a statistically significant association between HPV 16 and the T stage. HPV 16 positive patients had lower odds of a T4 tumor  of HPV infection in the normal oral mucosa of healthy individuals. Other studies of cancer-free individuals reported values from 0% to 81.1% in normal oral mucosal cells, exfoliated cells from normal tonsils, or oral gargles and tonsillar washings .5% of HP,91,92,93The limitation of the study is the small size of the groups. Further studies on a larger scale are needed to assess the HPV types in the control group, using non-invasive methods such as oral swabs or salivary rinses. Moreover, further studies are warranted to characterize the route of transmission and time of persistent and latent HPV infection in normal oral mucosa.No associations between HPV status and age, sex, smoking or drinking habits were found in the group with chronic tonsillitis and healthy individuals, which is in line with other studies ,91. HoweThe transmission of HPV is both sexual and non-sexual , and oraIn our study, we detected seven HPV subtypes in total, including high-risk HPV 16, 18, 35, 51, 56, 73, and one low-risk type (HPV 43/44) found in all groups, including HNSCC patients. Discrepancies between our data and other published data ,85,88,91There is a need for educational intervention to increase knowledge about HPV and HPV-associated cancer and transfer effective recommendations for HPV vaccination, which is a complex issue ,103. HPVThe epidemiology of HPV infection is related to many complicated issues and the HPV-related carcinogenesis is still controversial. Our analysis provides insight into the prevalence of oral oncogenic and non-oncogenic HPVs in patients with HNSCC, patients with chronic tonsillitis, and healthy individuals. HPV 16 was the genotype with the highest incidence, and we found that non-oncogenic HPV 43/44 was frequent in HNSCC patients, which was not previously confirmed. An oncogenic potential for HPV 43/44 can be suggested but further and more extensive studies are warranted to confirm it. Our study shows that further investigations are necessary to examine larger number of patients with attention paid to high and low-risk HPV genotypes. Moreover, it seems reasonable that the presence of any HPV type in patients with HNSCC should be considered during treatment planning. There is a need for larger analyses if LR-HPV infection is a risk factor for the survival outcome. In our study, cancer specimens were obtained during a 2-year period. We plan to analyze the prognostic impact of the HPV status on the potential 3- and 5-year overall survival and 3- and 5-year disease-free survival in HNSCC patients.The prevalence of high-risk HPV in the mucous membrane of healthy individuals is high and additional studies are warranted to estimate the progression rate from HPV infection to HPV-associated HNSCC. Despite the undisputed significance of protective HPV vaccination programs, the data from a low percentage of vaccinated people showed that such programs were not sufficient for prevention and efforts should be focused on screening and effective detection of HPV. We believe that HPV infection can be prevented through extensive and free vaccination programs, and the creation of an effective platform for HPV testing. Furthermore, our observations lead to the suggestion that HPV vaccination is of considerable importance in public health strategies. A panel of vaccines for different types of HPV, including low-risk types, should be adopted for vaccination of male and female populations.