Women with anal HPV infection treated for HSIL/CIN2-3 should be re-tested for anal HPV after treatment. Women treated for HSIL/CIN with persisting anal HPV infection following treatment are at high risk of HSIL/AIN, suggesting that this subset of patients would benefit from anal exploration. Women with anal HPV infection treated for HSIL/CIN might be at higher risk of recurrent HPV cervical infection even after successful treatment. p = 0.306). In conclusion, patients with persisting anal HPV after HSIL/CIN treatment are at high risk of HSIL/AIN, suggesting that these women would benefit from anal exploration. The study also suggests that women with anal HPV infection treated for HSIL/CIN might be at higher risk of recurrent cervical HPV even after successful treatment. Women with high-grade squamous intraepithelial lesions/cervical intraepithelial neoplasia (HSIL/CIN) are at high risk of anal human papillomavirus HPV infection, and it has also been suggested that self-inoculation of the virus from the anal canal to the cervix could explain HPV recurrence in the cervix after treatment of HSIL/CIN. We aimed to evaluate the bidirectional interactions of HPV infection between these two anatomical sites. We evaluated 68 immunocompetent women undergoing excisional treatment for HSIL/CIN. Immediately before treatment, samples from the anus and the cervix were obtained . Cervical HPV clearance after treatment was defined as treatment success. The first follow-up control was scheduled 4–6 months after treatment for cervical and anal samples. High resolution anoscopy (HRA) was performed on patients with persistent anal HPV infections or abnormal anal cytology in the first control. Baseline anal HPV was positive in 42/68 (61.8%) of the women. Anal HPV infection persisted after treatment in 29/68 (42.6%) of the women. One-third of these women  had HSIL/anal intraepithelial neoplasia (AIN). Among women achieving treatment success, cervical HPV in the first control was positive in 34.6% and 17.6% of the patients with positive and negative baseline anal HPV infection, respectively ( Human papillomavirus (HPV) has been described as the main etiological factor for the development of premalignant and malignant lesions in the anogenital tract . The focThe first change in this situation started a couple of decades ago, when a significant prevalence of HPV infection and lesions was observed in some risk groups, such as men who have sex with men, people living with HIV, and other immunosuppressed individuals ,7. InterIn addition to the risks related to the development of anal precancer and cancer, HPV infection of the anal canal may also be a risk factor for the persistence of HPV in the genital area. The uterine cervix is by far the most frequent site of HPV-associated infections, premalignant lesions (HSIL) and cancer, and women with HSIL/cervical intraepithelial neoplasia (HSIL/CIN) undergo large-loop excision of the transformation zone (LLETZ) to prevent progression to cancer . HoweverUnderstanding the natural history of HPV infection in the anal and genital areas in women with HSIL/CIN is a crucial first step to elucidate the possible bidirectional interactions of the infection between the different anatomical areas that may contribute to persistence/recurrence of the infection. Proper understanding of these possible interactions would also be key to developing clinical recommendations on who should be screened for anal HPV infection/lesions, and when. This study is an exploratory analysis to evaluate the interactions between cervical and anal HPV infection in immunocompetent women treated for HSIL/CIN. We aimed to examine the prevalence of anal HPV before and after cervical treatment and to estimate the effect that the infection in one area might have on the other anatomical site. All immunocompetent women undergoing a LLETZ procedure due to HSIL/CIN from December 2019 to December 2020 were eligible for the study. According to the clinical protocols and the national guidelines , the criDuring the study period, 68 women underwent a LLETZ procedure at our institution and were eligible for the study. Following the current recommendations of the Ministry of Health of Spain, HPV vaccination was offered to all women undergoing cervical treatment. The 68 women included in the present study accepted vaccination and were referred for free administration of the nine-valent vaccine. Due to the COVID-19 outbreak in this period, the number of women included was significantly lower than expected. The study was approved by the ethics committee of the hospital clinic (HCB/2020/0126). All women were informed of the study and signed the informed consent.Immediately before the cervical LLETZ procedure, a sample from the anal canal was obtained for HPV testing  and liquid-based cytology (LBC). The anal sample was collected by a gynecologist using a cytobrush inserted ∼1.5–2.0 cm into the anus and rotated 360° clockwise for one minute. Subsequently, a second sample from the cervix was obtained for HPV testing  and LBC. After having obtained the cervical and anal samples, the LLETZ procedure was performed using colposcopy vision. A detailed description of the procedure has been previously reported ,25,26. BSubsequently, a cytobrush was used to collect an endocervical sample of the remaining cervix, which was kept in PreservCyt  for intraoperative HPV testing and LBC. The intraoperative HPV testing result was considered as evidence of HPV elimination or persistence, as it allows early identification of HPV status after treatment ; a negatFinally, endocervical curettage was performed with a Kervokian curette by systematically scraping the whole endocervical surface. Cervical and anal samples were stored in PreservCyt solution  for HPV testing and LBC. In all cases, the samples collected were first submitted to HPV testing. The remaining material (if any) was then processed and evaluated using cytology. We used the Cobas HPV test , based on a real-time polymerase chain-reaction (PCR) system for HPV testing. Using this method, 14 high-risk HPV types can be detected, and it also performs specific genotyping for HPV16 and HPV18.A ThinPrep T2000 slide processor  was used to prepare thin-layer cytology slides stained using the Papanicolaou method. The Bethesda system was used to evaluate cytology slides . Following the current guidelines ,29, the All patients with a positive cervical HPV testing, or an abnormal result of the cytology in this follow-up control, were scheduled for a new colposcopy with histological sampling, if required. All patients with a positive anal HPV test or an abnormal cytology result were scheduled for HRA with histological sampling, if required. If all tests performed in the first follow-up control were negative, a second follow-up visit was scheduled 12 months after the first control.HRA was scheduled in all patients with persistent anal HPV infection or abnormal anal cytology in the first follow-up control. The anoscopy was performed by a gynecologist from the colposcopy unit of our center. Before anoscopy, we performed a digital anorectal examination to discard abnormalities suggestive of anal cancer. An Olympus Evis Exera II CV-180 anoscope  was used to perform the HRA. Acetic acid at 5% was used to perform careful anal evaluation, and the anal canal was stained with an iodine-based solution to highlight areas of abnormal epithelium. When suspicious lesions were detected, an HRA-directed biopsy was performed. Women with anal biopsy results showing HSIL were referred for treatment with trichloroacetic acid. All biopsy and surgical specimens were submitted for histological assessment. The specimens were fixed in 10% formalin and routinely embedded in paraffin. Four-micron sections were stained with hematoxylin, eosin and p16 immunohistochemistry (IHC). All IHC analyses were performed using the Roche platform with the CINtec Histology Kit . Only cases showing continuous “block” staining of the basal and parabasal layers were considered positive for p16 ,31. The The margins of the surgical specimens were identified with ink and carefully examined. The margins were considered as negative if no SIL was detected and positive when SIL of any grade was identified. In the latter situation, the site  of involvement was reported ,22.The SPSS software  was used for data analyses. The sample size was not calculated. However, for obtaining the highest possible sample size, we included all women undergoing excisional treatment in our institution from December 2019 to December 2020.In the analysis of HPV genotyping, HPV 16 and/or HPV 18, if present, were considered as responsible for the lesions. These cases were included as “HPV16/18 positive”. Coinfections by other high-risk HPV types were disregarded in these patients. Women showing a negative result for HPV16/18 and a positive result for high-risk HPV types other than 16/18 were classified as “HPV other than 16/18”. Thus, correlations for the HPV genotype could only be calculated for HPV16/18, since infection by HPV other than 16/18 would have been underestimated.Absolute numbers and percentages were used to present categorical variables, which were compared using the Chi-squared or Fisher exact test, while continuous variables were shown as mean and standard deviation (SD). The analysis of variance test was used to compare means.Clinical characteristics of the women included in the study and baseline cervical and anal HPV testing.The mean age of the 68 women included in the study was 42.0 years (SD 9.4). Forty-nine women (72.0%) were treated due to HSIL/CIN2, fifteen (22.1%) due to HSIL/CIN3, and four women (5.9%) due to a repeated cytological result of HSIL with a histological diagnosis of LSIL/CIN1. Fifty-seven women (83.8%) had only cervical lesions, whereas, besides cervical involvement, eleven (16.2%) showed HPV-associated lesions in other areas of the lower genital tract: three had lesions in the vulva, two in the vagina, one had vulvar and vaginal lesions and six showed lesions in the perineal area. p = 0.301). Baseline anal HPV infection was identified in 90.1% (10/11) and 56.1% (32/57) of the women with multicentric and isolated cervical HPV-associated lesions, respectively (p = 0.030). Among the 42 women with positive baseline anal HPV testing, 19 (45.2%) had HPV16/18 (17 being HPV16) and 23 (54.8%) had HPV other than 16/18. Baseline anal HPV testing was positive in 61.8% (42/68) of women. Baseline cervical HPV testing was positive in 95.6% (65/68) of the women. p = 0.772). Treatment success (an immediate post-treatment HPV negative result in the cervix) was achieved in 63.3% (43/68) of the women: 61.9% (26/42) of the women with a positive baseline anal HPV and 65.4% (17/26) of the women with a negative baseline anal HPV status (p = 0.049). The mean time to the first follow-up control was 6.3 months (SD 2.0). p = 0.306). The correlations of the anal and cervical HPV genotype of the 12 women who showed treatment success and positive cervical HPV in the first control are shown in Cervical HPV testing in the first control was positive in 34.6% (9/26) of the women with treatment success (an immediate post-treatment HPV negative result in the cervix) but with a positive baseline anal HPV status. This percentage was lower  among the women who achieved treatment success and had a negative baseline anal HPV status, although the differences did not reach statistical significance (Ten of the 29 (34.5%) women in whom anal HPV persisted after treatment  showed an HSIL/AIN: six women had HSIL/AIN2 and four had HSIL/AIN3. Our study is novel in its approach of looking at the relationship between the natural history and the bidirectional interactions between cervical and anal HPV infection in women treated for cervical HSIL/CIN2-3. The results of this study show that women treated for HSIL/CIN2-3 are not only at high risk of harboring anal HPV infection but also of developing high-grade lesions in the anal canal. Our study also yields information on which patients are at higher risk of HSIL/AIN2-3 and should undergo HRA to detect these lesions, providing some insight on possible screening strategies for anal lesion detection in this subset of women. Finally, our study also shows that women with anal HPV infection are at higher risk of HPV re-infection of the cervix after successful clearance of the cervical infection due to treatment. Although it has been suggested that immunocompetent women with HSIL in the genital area  are at high risk of anal HPV infection and disease, very few reports have analyzed anal HPV infection in these patients . In the However, none of these former studies have analyzed which patients with HSIL/CIN and anal HPV infection are at higher risk of HSIL/AIN. This is relevant data, as these women are the subset of patients who could benefit from anal cancer screening and anal canal examination via HRA. In the present series, we found that all HSIL/AIN arose in women with persistent anal infection . Remarkably, none of the women who were negative in their baseline anal HPV testing, including four women who became positive in the first control, developed HSIL/AIN. These results confirm the low risk of lesion that acute (or short-term) HPV infection represents . Thus, aInterestingly, one-third of the women with a positive baseline anal HPV testing result became spontaneously negative for HPV in the anal canal after removal of the cervical lesion through excisional treatment. This finding suggests that the anal HPV testing result could be due to HPV contamination or self-inoculation from the cervical HPV infection that may disappear after removal of the cervical lesion. Indeed, it has been shown that self-inoculation from cervix to anus and from anus to cervix is quite common ,40. HoweInterestingly, in this study anal sex was reported by more than half of the women, including those with a negative baseline anal HPV infection. This prevalence is higher than the prevalence described in previous reports, which suggests potential underreporting of this sexual practice in some studies ,40. DespAlthough this study was not designed to evaluate genotype concordance between cervical and anal infection, 70% of the women with a baseline anal HPV16/18 infection also had HPV16/18 in the cervix. A high degree of genotype concordance between the genital area and the anal area has been previously reported ,40,41. TAnother interesting result from the present study is that the baseline anal HPV status provides information on the risk of persistent cervical HPV infection after treatment. In a previous study, we showed that an HPV-negative result immediately after cervical treatment was early evidence of treatment success in women treated for HSIL/CIN . InteresThis study has several strengths. First of all, this is the first study to evaluate the natural history of HPV infection in the cervix and the anal area in women treated for HSIL/CIN and its timeline correlation. Another important strength is that all the women were prospectively recruited, and all cases were very well characterized through cervical and anal HPV testing via genotyping and LBC. Finally, an important novelty of the present series is the knowledge of treatment success . The HPV status immediately after LLETZ allows accurate follow-up of the HPV infection before and after cervical treatment and its correlation with anal HPV infection. However, there are also some limitations that should be noted. The most important limitation is the small number of patients included in the study, which may have impaired reaching statistical significance in some of the analyses. The number of women included in the study was lower than initially planned due to the COVID-19 outbreak in 2020, which coincided with the scheduled inclusion period of the study. The pandemic stopped the activity of screening programs and management of lesions for several months, making the inclusion of patients slower and more difficult than initially expected. Another possible limitation of the study is that the HPV testing method did not allow extensive HPV genotyping, and thus the concordance between cervical anal HPV genotypes could not be extensively assessed; however, this was not the objective of the present study, and this lack of information would not modify the relevance of the results presented. In conclusion, the present series provides evidence about the importance of knowing HPV status in both the anus and the cervix in women treated for HSIL/CIN. This knowledge allows for stratifying the risk of anal HPV lesions in women with HSIL/CIN and also helps in assessing the risk of HPV persistence after treatment in women with anal HPV infection due to self-inoculation. Larger longitudinal studies are necessary to confirm these results, which would help in the establishment of anal cancer screening algorithms in high-risk women, and also in the risk stratification of women after HSIL/CIN treatment.


The role of human papillomavirus (HPV) status for the prognosis of oropharyngeal cancers (OPCs) is discussed controversially. Here, we present an analysis of 144,969 head and neck cancer cases (ICD-10 codes: C00–C13) with a sub-cohort of 62,775 tumor cases of the oropharynx . To this end, de-identified data from electronic health records of about 60 healthcare organizations from 30 different countries were used. Odds ratios, hazard ratios (HRs), and Kaplan–Meier analyses were used to compare outcomes between different cancer entities of neoplasms of the base of the tongue (C01), of tonsils (C09), and of the oropharynx (C10) of women and men with and without HPV infection. To avoid the bias from different age distributions, the cohorts were balanced using propensity score matching. The 5-year survival rate for HPV-positive patients is somewhat better than that for HPV-negative patients, but for age- and sex-balanced cohorts, there remains no significant advantage for HPV-positive patients . Looking at the different entities and HPV status for age-matched male and female patients separately, HPV is a significantly positive prognostic factor for female patients in some entities, whereas for male patients, it is only a positive prognostic factor for malignant neoplasms of oropharynx (C10) . Head and neck cancers, most of which are histologically squamous cell carcinomas, consist of cancers of the oral cavity, nasopharyngeal, hypopharynx, and oropharynx. Oropharyngeal cancers (OPCs) are one of the most common subtypes of head and neck cancers . AlthougIn the United States and Western Europe, 70%–80% of all OPCs are associated with HPV . OPCs inGenerally, patients are younger and healthier and have a significantly lower incidence of traditional risk factors such as smoking and alcohol abuse , 7.Clearance of the virus was observed for 90% of the patients within 1 to 2 years . In the More than 200 genotypes of HPV have been identified, of which 15 are classified to have oncogenic potential . HPV hasThe diagnosis of HPV is challenging as most OPCs have small primary tumor sizes. Although it is extensive and, in many cases, involves the lymph node, the diagnosis is usually made in progressed stages due to swollen lymph nodes .On the one hand, according to the prevailing view in literature, HPV–associated oropharyngeal carcinoma is linked with a more favorable prognosis when compared to HPV–negative disease . On the As sex has been identified as an independent parameter in the onset of HPV–positive OPC, up to date, no studies about the influence of sex on long–term survival rates are available, this current study contains an analysis of HPV–positive OPC according to age and sex with respect to survival rates during 5–year follow–up.via the ICD–10 code (C00–C13), a sub–cohort for OPC was generated using ICD–10 codes C01, C09, and C10. In addition, cohorts of HPV–positive and HPV–negative patients were carefully defined on the basis of criteria, which can be found in the This work distributes a retrospective analysis of an international head and neck cancer cohort including 144,969 cases. Sub–cohorts were defined by HPV status and male or female cases. Patients with head and neck cancer were identified The TriNetX platform assures the quality of data to be controlled by processes and procedures triggered in response to questions about the data provided. TriNetX combines longitudinal clinical data and analytics to help in generating RWE. Datasets do not leave hospitals: queries are executed in a federated manner, and only aggregated results are visible on TriNetX.TriNetX analytics tools were used to obtain baseline characteristics, to balance cohorts with propensity score matching, and to analyze outcomes of interest in the final cohorts. The index event for each analysis was selected as the diagnosis of head and neck cancer (ICD 10: C00–C13) within the last 5 years excluding adenocarcinomas. Baseline characteristics, including demographics and diagnoses, were obtained. To compare the outcomes of different cancer entities for men and women with and without HPV infection, we applied hazard ratios (HRs) with 95% confidence intervals (CIs) and generated Kaplan–Meier curves. Propensity score matching was used to balance cohorts in address confounders that could bias analysis. Cohorts were balanced for age, alcohol, and nicotine dependence. Propensity scores matched cohorts 1:1 using a nearest neighbor greedy matching algorithm with a caliper of 0.25 times the standard deviation. This 1:1 matching was conducted to replicate randomized conditions as closely as possible. The primary outcome was defined as death.We analyzed 144,969 cases of head and neck cancer and selected 62,775 cases of OPC  for a detailed analysis. The consolidated standard of reporting trial (CONSORT) flow diagram, shown in In The sum of cases amounts to 144,969 with a percentage distribution of 70% male and 30% female patients.The prominence of malignant neoplasm of base of tongue (C01) is notable, with a frequency of 18% and a total of 26,345 patients, it is the most common carcinoma in this study, followed by mouth cancer (C06) with a frequency of 16%.In terms of the distribution of men and women, it is noticeable that the percentage for each ICD–10 code of male patients is higher compared to the proportion of female patients.C12, malignant neoplasm of pyriform sinus, has the highest proportion of male patients with 82%, with a total frequency of only 2%.The largest distribution of female individuals can be seen in C03, malignant neoplasm of the gum, in which both sexes are almost evenly distributed .The sex inequality in the 5–year survival rate with and without HPV–positive oropharyngeal carcinoma is illustrated in Comparing cancers with varying localization  within male patients, it can be found that the survival rates between patients tested HPV positive and HPV negative differs. Individuals with oropharynx carcinomas (C10) who tested HPV negative have a survival rate after propensity score matching of 66.3%, in turn, patients who tested HPV positive occurred to have a significantly higher survival rate of 81.5%. The HR of 1.077 after age/sex matching shows that, in this cancer entity, HPV infection is associated with a survival advantage.For C09, neoplasms of the tonsils, it occurs that HPV–negative male patients have an 87.8% chance of surviving, which is 4% higher than male HPV positively tested C09 patients.In the sub–cohorts of female patients, tonsil (C09) and oropharynx (C10) carcinomas in the HPV–positive cohorts have a 15%–20% higher survival rate in comparison with the HPV–negative female patients, with a HR larger than 2.The disparities do not only appear within one sex, differences in survival rate and HR are also noticeable between both sexes. Malignant neoplasms of tongue base (C01) and tonsils (C09) have an almost 6%–7% higher survival rate for female patients with HPV. Only in malignant neoplasms of the oropharynx male and female survival rates did hardly differ from each other.A further analysis of all cohorts with 1:1 matching regarding alcohol and nicotine dependence Figure 2Looking at Kaplan–Meier analysis For HPV–positive OPC , Kaplan–Meier analysis for For tongue base neoplasms (C01), male (green) or female (purple) individuals display a similar trend up to 2.5 years. After this time, a divergency between the survival rate of female and male patients can be seen. Female patients are more likely to survive a neoplasm of tongue base than HPV–positive male patients.Whereas the divergence for C01 firstly appears after 2.5 years, the survival rate of the sub–cohort of C09 of the two sexes departs after 114 days. Kaplan–Meier analysis C exhibits quite a significant disparity, the curve of female patients shows a slow decline and keeps a steady level of 0.9 after 1,211 days, whereas the male sub–cohort continuously decreases down to 0.83.Kaplan–Meier analysis D, neoplasms of the oropharynx (C10), depicts a similar development between the survival rate of female and male patients.In conclusion, HPV is a better prognostic marker for female tongue base and tonsil carcinoma patients than it is for HPV–positive male patients.Traditional risk factor for the onset of head and neck squamous cell carcinomas (HNSCCs), which comprise of cancers of the oral cavity (OSCC) as well as OPC, larynx, and hypopharynx, is the use of nicotine and alcohol. Nevertheless, this study compared a number of factors influencing the survival (not as hazard factor) and found a lower influence of nicotine and alcohol dependence compared to HPV status. An exemplary list of confounders  and their influence on survival is given in the An increasing number of HNSCCs can be associated with infection with the human papillomavirus (HPV). In the United States alone, the incidence of HPV–positive OPC increased by about 225% between 1988 and 2004, as is considered to be responsible for up to 90% of all new OPCs nationwide . HPV–posAs an additional parameter in OS analyses of HPV–positive HNSCC, several studies have identified the racial background of the patients. Not only the rates of HPV infections differ between black and white study populations, but also the survival rates are supposedly better in African Americans with HPV–positive OPC , 25. TheBecause of the large cohorts, RWE studies tend to provide useful data to recognize trends, but their validation by prospective clinical trials as the gold standard is crucial to eliminating weaknesses in data like the unclear origin of HPV test samples.Sex disparities, differences between tumor entities, and HPV diagnoses result in varying survival rates and reveal the necessity of customized, patient–specific prognoses and therapies. Causes for these disparities demand further investigation and warrant a re–evaluation of therapy strategies to improve clinical practical guidelines and enable optimal treatment for patients with oropharyngeal cancer.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/TriNetX is compliant with the Health Insurance Portability and Accountability Act (HIPAA), the US federal law that protects the privacy and security of healthcare data. TriNetX is certified to the ISO 27001:2013 standard and maintains an Information Security Management System (ISMS) to ensure the protection of the healthcare data it has access to and to meet the requirements of the HIPAA Security Rule. Any data displayed on the TriNetX Platform in aggregate form, or any patient–level data provided in a dataset generated by the TriNetX Platform, only contain de–identified data as per the de–identification standard defined in Section §164.514(a) of the HIPAA Privacy Rule. The process by which the data is de–identified is attested to through a formal determination by a qualified expert as defined in Section §164.514(b)(1) of the HIPAA Privacy Rule. This formal determination by a qualified expert, refreshed in December 2020, supersedes the need for TriNetX’s previous waiver from the Western Institutional Review Board (IRB). The TriNetX network contains data provided by participating Healthcare Organizations (HCOs), each of which represents and warrants that it has all necessary rights, consents, approvals, and authority to provide the data to TriNetX under a Business Associate Agreement (BAA), so long as their name remains anonymous as a data source and their data are utilized for research purposes. The data shared through the TriNetX Platform are attenuated to ensure that they do not include sufficient information to facilitate the determination of which HCO contributed which specific information about a patient. This study was reviewed and approved by Ethikkommission der Charité - Universitätsmedizin Berlin, EA4_064_18. Written informed consent was obtained from all participants for their participation in this study.SHP analyzed and interpreted data, wrote the first version of the manuscript, and designed the figures. SN analyzed and interpreted data. SP conceived the study, performed the analysis, and wrote the manuscript. MH analyzed and interpreted data. SK analyzed and interpreted data and wrote the manuscript. All authors read and approved the final version of the manuscript.This work was funded by the German Research Foundation (DFG: PR 1562/1–1).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lorazepam is a benzodiazepine derivative that is globally used for the therapy of anxiety and insomnia.The objective of our work was to show that Lorazepam can be a cause of unexpected liver injury even though it is a rare entity.We reported the case of a patient who had a Drug-Induced Liver Injury (DILI) under Lorazepam. We performed a literature review based on a PubMed search with the following keywords: “Lorazepam,DILI”.A 20 year-old-Tunisian woman was hospitalized in the psychiatry department of the hospital of Nabeul in Tunisia for a brief psychotic episode.She had a DILI under Olanzapine, Chlorpromazine and Lorazepam, which conducted us to interrupt her treatments except for the Lorazepam(5mg/day). The hepatic tests went back to normal even under Lorazepam. Few days later, the liver enzymes increased again to reach very high levels. Extensive workup was negative for other causes of liver injury, including viral hepatitis A, B, C and E.; capillary electrophoresis of serum proteins was normal; Exhaustive immunological tests were performed searching for auto immune hepatitis primary biliary cholangitis and other antibodies like antinuclear antibodies were negative. Liver biopsy showed polymorphic inflammatory infiltrate including some eosinophilic polynuclear cells and rare vaguely epitheloid macrophages, with necrotico-inflammatory foci in the lobules, all of which were consistent with DILI. Lorazepam was discontinued and within 10 days her liver enzymes decreased and completely normalized.Lorazepam, with an unknown action mechanism, can be a cause of DILI.No significant relationships.


Some anti-malarial drugs often cause haemolytic anaemia in glucose-6-phosphate-dehydrogenase deficiency (G6PDd) patients. This study aims to analyse the association of G6PDd and anaemia in malaria patients receiving anti-malarial drugs.A literature search was performed in major database portals. All studies searched using keywords with Medical Subject Headings (MeSH) were included, without date or language restriction. Pooled mean difference of haemoglobin and risk ratio of anaemia were analysed using RevMan.2 5%, p = 0.39), regardless of the type of malaria and dose of drugs. In particular with primaquine (PQ), mean difference of haemoglobin in G6PDd/G6PDn patients with dose < 0.5 mg/kg/day was − 0.04 . The risk ratio of developing anaemia in G6PDd patients was 1.02 .Sixteen studies comprising 3474 malaria patients that included 398 (11.5%) with G6PDd were found. Mean difference of haemoglobin in G6PDd/G6PD normal (G6PDn) patients was − 0.16 g/dL  and weekly PQ (0.75 mg/kg/week) did not increase the risk of anaemia in G6PDd patients. In 2021, according to the World Health Organization (WHO), approximately 241 million malaria cases occurred worldwide. Although there was a significant decrease of 20 million malaria cases compared to 2010, data from the last three years showed a trend of increasing incidence and relative to the 2019 data, a steep increase of fourteen million malaria cases occurred in 2021.Data of mean difference of haemoglobin level before and after anti-malarials administration in G6PDd and G6PDn patients were only available in seven studies comprising nine groups that could be further analysed in the forest plot. The mean difference of haemoglobin in G6PDd patients is higher than the G6PDn patients [− 0.70  vs. 0.27 ]. When both groups were further analysed, the gap of mean difference between G6PDd and G6PDn group was lower in the G6PDd group -0.16 ; however, it was not statistically nor clinically significant  in Fig. Figure Three studies –22, 27 eIn the study protocol, many anti-malarials were included to evaluate their effect on the G6PDd population, however, most of the studies were done using primaquine and only a small number of studies used other anti-malarials, such as tafenoquine, chlorproguanil–dapsone–artesunate (CDA), chlorproguanil–dapsone (CPG–DDS), sulfadoxine-pyrimethamine (SP), chloroquine; therefore, we only examined the pooled effect of primaquine. 16 studies on malaria patients treated with anti-malarial agents were analyzed. Among 3,474 patients, 11.5% of them are G6PD deficient. Most studies evaluated PQ as the investigational drug and excluded severe malaria infection. PQ is one of the drugs to be wary of due to the high risk of haemolytic anaemia .Plasmodium species and the administered anti-malarial drugs, forest plot of seven studies showed a small mean difference of haemoglobin level between G6PDd and G6PDn group (− 0.16 g/dL)  Fig. . Other ssubjects , 24.A study by Llanos-Cuentas compared the haemolytic effect of primaquine and tafenoquine. The results showed that the decrease in haemoglobin levels occurred in the normal G6PD group and did not occur in the G6PDd group, although the number of subjects in the G6PDd group was very low. Decreased haemoglobin levels occurred in the tafenoquine and primaquine groups but were self-limited without requiring special treatment .Based on these studies, it was presumed that anti-malarial treatment in non-severe malaria patients would be unlikely to cause a clinically significant side effect of anaemia. Analysis of three studies on PQ also did not show an increased risk ratio of developing anaemia in G6PDd patients, hence, confirming the safety of PQ , the result of the forest plot for the mean difference of haemoglobin level between G6PDd and G6PDn groups was not statistically nor clinically significant. Results from other studies with administration of PQ dose < 0.5 mg/kg/day) (not included in the forest plot) also disclosed a consistent outcome [Instead of the type of the  outcome , 23. Whe outcome , 17. Stu outcome , 26. TheThe change of mean difference of haemoglobin level also showed no significant decrease during seven or 28-days follow-up period, indicating that anaemia as a side effect in the administration of anti-malarial was not hazardous in both short and long-term evaluation. Haemolytic anaemia caused by G6PDd is usually a self-limited condition, and anaemia is usually resolved when the precipitating drugs are discontinued.Among sixteen studies in this review, half were classified as low risk of bias. Some pooled analyses showed high heterogeneity and, therefore, subgroup analysis was performed which exhibited significant results and lower heterogeneity. The funnel plot could not be arranged because of the lack of studies in meta-analysis.This review is the first systematic review of the effect of G6PD status, which includes only malaria patients receiving anti-malarial agents. A particular strength of this study is that all of the included study was high-quality cohort and randomized controlled trials with before and after endpoints, thus, the authors are confident in this study’s ability to demonstrate a causal relationship between drug administration and haemoglobin level. In the table displaying the summary of findings , and weekly PQ was not statistically and clinically significant, and therefore, a massive screening of G6PD status might not be relevant in all populations. Several studies on the safety of anti-malarial regimens concerning G6PDd patients are ongoing .Single low dose (0.25 mg/kg) or daily standard low dose of PQ (0.25 mg/kg/day) and weekly PQ (0.75 mg/kg/week for eight weeks) does not increase the risk of anaemia in G6PDd patients.


The relative risk (RR) to develop CIN2+ during follow-up vs. the the CIN1 persistence was 1.005  vs. 0.994 (95% CI 0.994–1.018) for age, 3.472 (95% CI 1.066–11.320) vs. 1.266 (95% CI 0.774–2.068) for non-vaccinated, 0.299 (95% CI 0.088–1.018) vs. 0.518 (95% CI 0.242–1.109) for HIV status negative, respectively. Analyzing the time to negativity, the odds ratio (OR) was 1.012 (95% CI 1–1.024) for age and 1.591 (95% CI 1.223–2.069) for vaccination; on the other hand, considering the relationship between the time to negative and the HPV genotypes contained in the 9-valent HPV vaccines, the OR was 1.299 (95% CI 1.026–1.646) for at least one of these at recruitment and 0.631 (95% CI 0.471–0.846) at follow-up. Furthermore, the presence of at least one of the HPV genotypes targeted by the HPV nonavalent vaccine is a key indicator of the risk of progression to CIN2+: OR was 3.443 (95% CI 1.065–11.189) for the presence of at least one HPV genotype at enrollment and 5.011 (95% CI 1.899–13.224) for the presence of at least one HPV genotype at follow-up, respectively. Conclusions: We reported in a retrospective study the benefit of anti-HPV vaccination in promoting negativity and increasing low-grade cervical lesions regression.Background: Low-grade cervical lesions have a high percentage of clearance in young women, even if 71–82% of low-grade intraepithelial lesion/atypical squamous cells of undetermined significance (LSIL/ASCUS) reported a High-Risk Human Papillomavirus (HR-HPV) infection, which correlates with an increased risk of Cervical Intraepithelial Neoplasia (CIN)2+. The immunogenic effect of the anti-HPV vaccine appears to be significant. The aim of the study is to evaluate the effect, two years after the diagnosis, of the anti-HPV preventive vaccination on patients with low-grade cervical lesions. Methods: We collected clinical, colposcopic, histological, and virological data from patients aged 21–45 years who attended the colposcopy service of the department of Obsetrics and Gynecology of IRCCS Foundation Policlinico San Matteo, Pavia, Italy. In the 2005–2019 period and had a low-grade pap-smear. Results: We enrolled 422 women consecutively, divided into two groups (vaccinated and not vaccinated) for the retrospective analysis. The rate of persistence and progression of CIN were higher in the not-vaccinated group ( The human papillomavirus (HPV) plays a causal role in nearly all cervical cancers and in certain subsets of head and neck, anal, penile, and vulvar cancers . The maiIn 2012, the Lower Anogenital Squamous Terminology (LAST) classified low-grade cervical intraepithelial neoplasia (LSIL/CIN1) as a common finding in cervical specimens. This lesion is typically self-limited with spontaneous regression . For thiIn addition, a key role is played by the presence of multiple infections from HR-HPV genotypes, which correlate with an increased risk of high-grade CIN (CIN2+) ,16,17,18Since the introduction of the anti-HPV prophylactic vaccine, this mechanism of multiple infections has been antagonized, resulting in a reduction of HPV-related diseases and cervical cancer. Previous data reported that anti-HPV vaccines appeared to associate with the development of significant immunogenicity, reporting efficacy and safety, especially in the naïve population ,21,22,23In recent years, the literature has demonstrated that HPV vaccination could have a significant protective effect in women surgically treated for HPV disease and could also impact disease recurrence. The HPV vaccine’s protective role in women with prevalent HPV infection, on the other hand, is unknown. The SPERANZA project, a prospective case-control study, found a reduction in disease recurrences (CIN2+) in the vaccinated group following surgical treatment and hypothesized two possible long-term outcomes: prevention for patients who had not previously been exposed to HPV vaccine types and reactivation or reinfection when the immune system is ineffective to provide long-term protection .Regarding the possibility of administering an anti-HPV vaccine in L-SIL/CIN1, literature data support the hypothesis that the vaccine allows the development of an immune response and, therefore, facilitates the viral clearance of HPV also in this category of cervical lesions, allowing the clearance of HPV and the regression of the lesion. The trial conducted by De Vincenzo et Al. suggests that anti-HPV vaccination could have a promising role in reducing the time required to eliminate HR-HPV or Pap test positivity in adult women, with a significant reduction in time of negativization compared to subjects who were not vaccinated .The aim of the study is to evaluate the effect of anti-HPV vaccination in a population with a diagnosis of low-grade cervical intraepithelial neoplasia (LSIL/CIN1) two years after the diagnosis. In particular, we seek to determine the relationship between anti-HPV vaccination and the possibility of cervical lesion persistence or progression to CIN2+ in women who did not receive anti-HPV vaccination.We analyzed clinical, colposcopic, histological, and virological information, from women aged 21 to 45 years, admitted to the Colposcopic Service of Department of Obstetrics and gynecology Obstetrics and Gynecology, IRCCS Foundation Policlinico San Matteo, Pavia, Italy, due to an abnormal screening pap smear. Women were recruited between 2005–2019 with low-grade abnormal pap smears, according to the cytological classification in use during the study period. The patients were referred by the cytological screening service, external institutions, and private practice. Exclusion criteria were the following: ongoing pregnancy, previous HPV infection or treatment for CIN or total hysterectomy before enrollment, biopsy suggestive for CIN2+, and HPV vaccination before enrollment. We divided the patients into two periods, in accordance with the vaccine availability. The anti-HPV vaccine (Gardasil 9) schedule was approved by the European Medicines Agency (EMA) and Agenzia Italiana del Farmaco (AIFA) in 2015. The patients enrolled before 2015 were considered the only control group, while the other patients who required voluntary vaccination were assigned to the study group. The patients who met the inclusion criteria received the vaccine after the colposcopic examination; all patients signed an informed consensus document. Institutional Review Board (IRB) approval was obtained by the hospital ethical committee .The database was composed of a series of anamnestic items filled in after structured interviews at entry and during follow-up, as well as clinical, colposcopic, histological, and virological items.In all patients, we performed HPV-DNA detection and genotyping, colposcopy, and targeted biopsies, according to an established protocol. Cervical samples for HPV genotyping were obtained immediately before colposcopy. After speculum examination, scrapes were taken with a cervix brush, suspended in ThinPrepPreservCyt Solution , and stored at 4 °C. DNA extraction was performed by lysis and digestion with proteinase K . HPV seqThe risk of the HPV type being associated with the development of cancer is based on the data from the International Agency for Research on Cancer (IARC). We classified HPV types into the following categories: high-risk with proven carcinogenicity , low-risk , and untypable  .A standardized colposcopic examination was performed immediately after cervical brushing for HPV genotyping by three different gynecologists  certified by the Italian Society of Colposcopy. The colposcopic examination was based on international colposcopy nomenclature . MultiplPatients underwent cytological and colposcopic examination every six months for two years, and HPV-DNA sampling and histological biopsy were performed at entry and at the end of follow-up.Kruskal–Wallis analysis of variance and the chi-square test to compare continuous and categorical variables, respectively, were applied to carry out univariate statistical analysis. In order to evaluate relative risk (RR) while correcting for potential confounding effects, we included logistic equations for age (continuous), HIV status (yes/no), vaccine administration (yes/no), and LSIL cytology at diagnosis (yes/no) as explanatory variables in CIN outcomes. The analysis of the odd ratio (OR) was conducted by the stepwise COX regression method to test the role of vaccination on the risk of CIN progression during follow-up, adjusting for confounding effects. All the analyses were carried out with Stata 17.0  .A total of 422 women, aged between 21 and 45 years old, who attended the colposcopy service of our department in the 2005–2019 period were included in our study. We divided the population into two groups: 210 patients without any HPV vaccination and 212 patients with voluntary opportunistic vaccines. All women with a low-grade pap-smear and/or histological CIN1 were observed for at least 2 years, as reported in p = 0.613); most of the population came from Europe and, in particular, Italy  (p = 0.432).Regarding the geographical distribution of the population of the study, 10/212 (4.72%) of the vaccinated women and 5/201 (2.38%) of the unvaccinated ones were extra-European (p = 0.030), parity (p = 0.002), and HIV positivity (p = 0.025), while there was no significant difference in contraceptive methods (p = 0.09).There was a statistically significant difference between the two groups regarding smoking  and the type of transitional zone (p = 0.005).The most frequent colposcopic finding during the first visit in both groups was a G1 small lesion with a visible transitional zone (TZ1), but there was a significant difference between the vaccinated and the unvaccinated for the type of colposcopic lesion , the class of risk, or the number of HPV genotypes involved (p = 0.120). In addition, more than half of the women in both groups were infected with one genotype included in the vaccine . At enrollment, CIN1 was found in 147/212 (69.34%) of vaccinated individuals and 209/2010 (99.52%) of unvaccinated individuals (p = 0.001), after cervical biopsy.The analysis of the HPV status showed that there was no significant difference among the groups regarding HPV positivity  was performed and the number of patients treated by LEEP was higher among the unvaccinated . Finallyp = 0.019).The regression of CIN among vaccinated women was reported in 162/212 subjects (76.42%), the persistence in 46/212 (21.70%), and the progression in 4/212 (1.89%). In the control group, however, women experienced regression in 143/210 (68.1%), persistence in 52/210 (24.76%), and progression to CIN2+ in 15/210 (7.14%) (p = 0.807). Instead, 153/212 (72.17%) of vaccinated people maintained HPV16 negativity at follow-up, as opposed to the unvaccinated women, 134/210 (63.81%) (p = 0.046).In addition, an evaluation for HPV-16 was performed at entry and during the follow-up . The ratAs Eventually, analyzing the time to negativity , the oddPrevious studies have reported the benefit of anti-HPV vaccination in patients with low-grade cervical lesions (LSIL/CIN1). For example, the VIVIANE Study highlighted how the vaccine, in women older than 25 years, allows for protection against HPV infection and, consequently, against CIN1+ lesions, especially those related to HPV-16 and HPV-18. In particular, in seronegative subjects for the corresponding HPV types according to the protocol cohort, the vaccine has been proven effective against 6-month-old persistent HPV infection and 6-month-old persistent CIN1+ or related cytological abnormalities (ASCUS or LSIL) sustained by HPV 16/18 genotypes . In addition, the vaccine demonstrated cross-reaction protection against 6-month persistent infection sustained by HPV 31  and HPV 45 . The authors suggested that, in accordance with their results, the vaccine may be useful to prevent reinfection in women with previous HPV-16 or HPV-18 positivity .A recent study underlines that in women with abnormal cytology, the positive predictive value (PPV) for CIN2+ was lower in patients with previous anti-HPV vaccination  compared to unvaccinated ones . In addition, PPV was significantly lower in the patients who underwent vaccination before the age of 21  than in patients with vaccine administration after the age of 21 . According to our data, the vaccine administration may prevent the progression to HSIL/CIN2+. Especially regarding young women, these results, similar to our data, open a new scenario on how to deal with this category of women in the management of low-grade cervical lesions . Moreovep = 0.001 and OR 0.33, 95% CI 0.20 to 0.52; p < 0.0001, respectively). For this reason, adjuvant HPV vaccination is linked to a lower incidence of overall CIN recurrence [2 = 58%, 2 = 0.14, median follow-up 36 months, interquartile range 24–43.5). The effect estimate was significantly greater for the risk of CIN2+ recurrence associated with HPV 16 or HPV 18 . For incidental and persistent HPV infections, there was insufficient proof of benefit [In addition, a systematic review of literature and meta-analysis underlined that CIN1+ and CIN2+ recurrence rates were all lower in the vaccinated group than in the unvaccinated group  is unique.-It is well known that CIN1 is predominantly a reversible lesion, the vast majority reverting over time to normal epithelium. Only a small percentage progress to CIN2+, which is typically irreversible. In the vast majority, unless treated with ablative or excisional methods, the lesion tends to progress to high-grade cervical lesions.-The authors investigated the use of the HPV vaccine in women with CIN1. Indeed they successfully demonstrated that a larger percentage of CIN1 lesions heal or regress, with vaccination than in those who remained unvaccinated.In conclusion, our findings support the use of anti-HPV vaccination in low-grade cervical lesions to promote negativization and the increase of cervical lesions regression. This opens a new scenario for how to deal with the clinical management of CIN1 persistence.


A high prevalence and incidence of head and neck tumors make Brazil the country with the third-highest number of cases of these malignant neoplasms. The main risk factors are smoking and alcohol consumption; however, cases related to the human papillomavirus (HPV) have tripled in number, demonstrating a changing disease profile. Studies have reported the prevalence of HPV in laryngeal squamous cell carcinoma (LSCC) to vary between 8% and 83%. The role of HPV as an important causative factor in LSCC remains unclear.This retrospective study included 82 patients with LSCC diagnosed between 2014 and 2019 at two oncology hospitals in São Luís, Brazil. Sociodemographic and clinical data, and the histopathologic characteristics of the tumors, were collected directly from medical records. Genetic material was extracted from paraffin-embedded samples using nested polymerase chain reaction (PCR) and automated sequencing for HPV detection and genotyping. The results by social and clinicopathologic variables were then compared using the chi-squared test and multivariate analysis.p = 0.002).Sociodemographic analysesdemonstrated that most patients were men (87.8%), brown-skinned (75.6%), and resident in the state capital (53.7%). They generally had a poor education status (53.7%), having only an elementary school education (completed/incomplete), and 51.2% were self-employed in occupations such as farming or fishing. Smoking and alcohol consumption habits were observed in approximately half the patients. With respect to clinical characteristics, 39% of patients exhibited T1/T2 staging, 51.2% had no distant metastasis, and 30.5% had lymph node invasion. HPV DNA was detected in half the samples (50%), with the high oncogenic type 16 being the most prevalent. There was no significant relationship observed between the economic, educational, occupational with the HPV LSCC in the presented data, although multivariate analysis demonstrated that HPV DNA was more likely to be present in T3–T4 tumors ( A high incidence and prevalence of head and neck squamous cell carcinoma make Brazil the country with the third highest number of cases of this malignant neoplasm . The morHead and neck squamous cell carcinoma constitutes a diverse group of cancers with different behaviors and prognoses, necessitating different treatment approaches. The disease can occur at various sites, such as the oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, paranasal sinuses, and salivary glands . ExposurThe role of HPV in squamous cell carcinoma of the oropharynx is well established; however, its role in laryngeal squamous cell carcinoma (LSCC) remains unclear. The reported prevalence of HPV in LSCC varies greatly between studies, ranging from 8% to 83% . The typLSCC is the second most common head and neck squamous cell carcinomas . It is hAs in head and neck cancer, HPV is an important prognostic factor in LSCC, considered to possibly influence prognosis and treatment decisions . There aIn this retrospective study, we analyzed formalin-fixed, paraffin-embedded laryngeal squamous cell carcinoma specimens collected at two public oncology referral hospitals in Maranh ao, a state in northeastern Brazil. The study included 82 samples from patients diagnosed with laryngeal carcinoma between January 2014 and December 2019.Patient information and the histopathologic characteristics of the tumors were obtained from medical records. Because the samples consisted of paraffinized tumors, written informed consent was not required to be obtained from the patients. Patient identities were not disclosed in the study, which was approved by the Ethics in Research Committee of the Federal University of Maranhao (register number 3.023.486).Patients were included when paraffin blocks and histology slides of laryngeal carcinoma, whether from biopsy or surgical treatment at any follow-up stage, were available in the archives of the pathology services.Patients were excluded if their medical records and/or paraffin tumor blocks were not found or were not sufficient for laboratory analysis in the study.The samples were reviewed by a pathologist, and blocks representative of tumor  were selected.For DNA extraction, 15 sections of approximately 5 µm thickness containing biologic material were used. The sections were stored in 2.0 mL tubes at 4 °C until the DNA extraction step.Genomic DNA was extracted from the samples using the ReliaPrep FFPE gDNA Miniprep kit from Promega , according to the extraction protocol suggested by the manufacturer. To assess the concentration and quality of the biologic material, the samples were quantified using a Nanodrop Lite spectrophotometer. Concentrations were reported in nanograms per microliter, and the DNA quality was verified by reading the 260/280 nm wavelength ratio. When the ratios were in the range 1.7–1.9, the material was considered pure.Nested PCR reactions were performed using a Veriti 96-Well Thermal Cycler , with primers PGMY09 and PGMY11 for the first round and primers GP + 5 and GP + 6 for the second round .The samples were purified using the GenElute PCR Clean-Up  Kit according to the manufacturer’s instructions and were subsequently quantified. Automated sequencing was performed by ACTGene Análises Moleculares company which currently provides DNA sequencing services, using the AB 3500 platform . Samples were diluted in 6 µL of a solution containing 30–60 ng purified amplification product, 5 picomol of primer, and double-distilled water.To confirm and identify the HPV type, the nucleotide sequences of sequenced samples were compared and submitted to the World Nucleotide Database (GenBank) using the BLAST program.p < 0.05. After the univariate analysis, significant variables (p < 0.05) were selected for binary logistic regression analysis to assess their relationship with HPV infection.Categorical variables are presented as percentages, and the chi-squared test was used for comparisons between groups. Statistical significance was set at The analysis showed that most patients were men (87.8%), brown-skinned (75.6%), and from the capital of the state (53.7%). Furthermore, 53.7% had only an elementary school education (completed/incomplete), and 51.2% were self-employed in occupations such as farming or fishing. Laryngeal cancer staging data showed that 29.3% had stage N0 disease; 39%, stage T1/T2; and 51.2%, stage M0. Finally, 51.2% were nonsmokers, and 57.3% were nonalcoholic .HPV analyses by nested PCR demonstrated that half the samples were positive for the virus. The patients were therefore divided into two groups: HPV+ and HPV–.p < 0.05, In univariate chi-squared analysis between the two groups, only residential location, T stage, and smoking status were statistically significant .According to recent publications, the mean age of patients with laryngeal cancer is 65 years, with a higher proportion of men than of women . Our patAnalyzing geographic distribution, patients with HPV+ laryngeal cancer came mainly from the capital, while patients with HPV–laryngeal cancer came primarily from the interior of the state. The difference was statistically significant by the chi-squared test, but that significance was lost when binary logistic regression was applied. Differences between patients depending on the geographic regions in which they live are possibly attributable to different social and sexual habits .p < 0.05). However, this variable’s statistical significance was lost when binary logistic regression was applied.Smoking is an important risk factor in the pathogenesis of laryngeal tumors . GeneralIn other studies, no significant relationship between smoking and the presence of HPV has been found; both variables should therefore be regarded as independent factors in the oncogenic process of the larynx .Alcoholism is also described as a risk factor for this type of tumor; however its use was not related to HPV infection which was observed by others .Unfortunately, TNM classification data were not available for all patients. N0 and M0 staging were prevalent among the analyzed tumors. No statistical association with HPV infection was observed, as in other studies .T1–T2 staging was prevalent, but a higher index of T3–T4 tumors was observed in the HPV+ group compared with the HPV–group. By applying binary logistic regression, we observed that patients staged as T3–T4 had a greater chance of having HPV DNA. Similar results were observed in a Turkish population . Duray aIn fact, it is currently uncertain whether HPV positivity in LSCC could be used as a prognostic factor. There is strong support in the literature for the association between HPV-positive carcinomas and better prognoses, especially in oropharyngeal carcinomas, but for LSCC the results are still controversial. Generally, author found no difference at all or a slight but nonsignificant improvement in terms of overall or disease-free survival for HPV-positive cases.Epidemiology studies reported that HPV infection rates in laryngeal cancer range from 8% to 83% . These dIn our study, half the samples contained HPV DNA. For HPV detection, we used formalin-fixed samples and nested PCR with PGMY09/11 and GP5+/6+ primers . The senRegarding geographical distribution, systematic reviews and meta-analysis study demonstrated that HPV prevalence is significantly higher in South America, compared to Asia, North America and Europe . Two TurA North American study demonstrated that a poor education status and a low income level were related to the presence of HPV DNA in head and neck tumors . HPV infOf the total HPV+ cases, 68.3% had a high risk HPV genotype like those found by HPV 6, a low-oncogenic genotype, was observed in one patient in our study. Other researchers have also found HPV 6 in laryngeal tumor samples . HPV 6 iProphylactic HPV vaccination could lead to a worldwide decline in HPV-driven head and neck squamous cell carcinoma . In BrazTNM Classification for Head and Neck Cancer, HPV status is an important prognostic factor that can influence treatment decisions (According to the 8th edition of the ecisions . The higA strength of our study is that it is the first to include a northeastern Brazilian population, whose socioeconomic status is significantly lower than that of the better known southeastern regions of Brazil, including S ao Paulo and Rio de Janeiro states .Overall, the sample size was small to run multivariable models, and this is a limitation of our study. Many patients were excluded from the study because the blocks containing the tumor samples were not found and/or the data from the medical records were insufficient. Only recently have the medical records of patients become electronic and easy to find. Formerly, they were kept manually, with poor filling and difficult accessibility, especially for patients who were deceased.HPV infection may play an important role in the initiation and progression of laryngeal cancer, but the subject still needs clarification, given that HPV infection rates vary greatly between studies.Our findings demonstrate an important prevalence of HPV in a sample of laryngeal epidermoid tumors in a population in northeastern Brazil, with the highly oncogenic type 16 being the most prevalent HPV genotype. In addition, patients with HPV+ LSCC had a higher index of T3–T4 tumors. Our study is the first in Brazil to include only laryngeal tumors in a northeastern population. These data are important for improving our knowledge about the role and consequences of HPV infection in laryngeal tumors. Limitations include clarification of the effect of HPV on prognosis in LSCC, given that some important data were absent from the medical records. Further studies for the evaluation of molecular markers such as p16 and p53 expression are needed to clarify the protein modifications that HPV may cause in laryngeal SCC.10.7717/peerj.13684/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13684/supp-2Supplemental Information 2Click here for additional data file.


Oropharyngeal squamous cell carcinomas (OPSCCs) are the only head and neck malignancy with a clear increase in prevalence in Western countries, due to the HPV epidemics with an increasing proportion of HPV-related OPSCCs. Such figures, however, are extremely variable around the globe. The present report is the first to assess the prevalence of HPV-related OPSCC in Sardinia, a relatively isolated population in the West. The rate of HPV-driven OPSCC in such a population is close to that of less developed areas, with clear implications on epidemiology, prognosis, and reliability of methods for assessing HPV-related carcinogenesis. In fact, in the present setting, the specificity of p16 IHC alone in diagnosing HPV-related carcinogenesis is only 75% with a 25% false positive rate.HPV infection is a clear etiopathogenetic factor in oropharyngeal carcinogenesis and is associated with a markedly better prognosis than in smoking- and alcohol-associated cases, as specified by AJCC classification. The aim of the present work is to evaluate the prevalence of HPV-induced OPSCC in an insular area in the Mediterranean and to assess the reliability of p16 IHC (immunohistochemistry) alone, as accepted by AJCC, in the diagnosis of HPV-driven carcinogenesis in such a setting. All patients with OPSCC consecutively managed by the referral center in North Sardinia of head and neck tumor board of AOU Sassari, were recruited. Diagnosis of HPV-related OPCSS was carried out combining p16 IHC and DNA testing on FFPE samples and compared with the results of p16 IHC alone. Roughly 14% (9/62) of cases were positive for HPV-DNA and p16 IHC. Three more cases showed overexpression of p16, which has a 100% sensitivity, but only 75% specificity as standalone method for diagnosing HPV-driven carcinogenesis. The Cohen’s kappa coefficient of p16 IHC alone is 0.83 (excellent). However, if HPV-driven carcinogenesis diagnosed by p16 IHC alone was considered the criterion for treatment deintensification, 25% of p16 positive cases would have been wrongly submitted to deintensified treatment for tumors as aggressive as a p16 negative OPSCC. The currently accepted standard by AJCC  harbors a high rate of false positive results, which appears risky for recommending treatment deintensification, and for this aim, in areas with a low prevalence of HPV-related OPSCC, it should be confirmed with HPV nucleic acid detection. In recent decades, a dramatic increase in oropharyngeal squamous cell carcinoma (OPSCC) incidence has been observed in Western countries. This burden of OPSCC appears to be related to the human papillomavirus (HPV) epidemic ,2, whichHPV-driven OPSCC has been clearly demonstrated to be a different entity compared to the traditional smoking-/alcohol-associated OPSCC, as has been acknowledged in the eighth American Joint Committee on Cancer (AJCC) staging system edition since 2016 . A largeThe global pooled prevalence of HPV-driven OPSCC has been estimated at ~45%, however, most data describe a huge geographic heterogeneity of such figure, with rates of HPV-induced OPSCC approaching 80% in the US and other Anglosaxon Countries , as well as Scandinavia, and markedly lower rates in other countries and areas of the world: lower than 20% in highly populated countries as India and Brazil, and in some areas of China ,4,5,6,7.This may have huge implications on overall prevalence of OPSCC, but also on the prognosis and on the clinical approach to the disease in different areas of the world.The aim of the present work is to evaluate the prevalence of HPV-induced OPSCC in a small, relatively isolated insular area in the Mediterranean, by a validated assay combining p16 immunohistochemistry (IHC) and DNA testing ,10. A sePatients with a diagnosis of OPSCC, managed at the University Hospital of Sassari , between 2017 and 2021, were enrolled. TNM classification and staging of tumors according to AJCC (8th ed. 2017)  were assPersonal , clinical , and follow-up data  were prospectively collected in an ad-hoc electronic form.All the available data were analyzed in order to assess the prevalence of HPV-driven OPSCC and the histopathological and molecular-associated findings.According to the algorithm described by VUMC group ,10,11, wThe patients were managed and parameters collected following the current standard described by the main international guidelines ; therefoUpon diagnosis of OPSCC, p16 IHC and GP5+/GP6+ were performed on formalin-fixed paraffin-embedded (FFPE) archives of the local Pathological Anatomy Unit. A total of 10 consecutive sections of 3 µm were cut and used for histological and molecular analyses. P16 expression was assessed using the CINtec Histology kit , a qualitative immunohistochemistry test using mouse monoclonal anti-p16 antibody, following the manufacturer’s instruction. Samples were considered positive if a diffuse, continuous staining of basal and parabasal cell layers of the oropharyngeal squamous epithelium (>75%) was observed in a sequence of proliferating cells. Otherwise, negative status was defined as either focal p16 staining  or no p16 staining .After the deparaffinization step, the nucleic acid extraction was performed using the commercial AllPrep DNA/RNA FFPE kit , following the manufacturer’s instruction . DNA andHPV-DNA detection was performed using the generic primers GP5+/GP6+ , with the following thermal cycling conditions: 2 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 48 °C and 30 s at 72 °C, followed by 5 min at 72 °C. Real-time PCR was performed on CFX96 Real-time PCR system (BioRad). CCR5 housekeeping gene was used as an internal control of amplification.HPV-positive samples were tested for the detection of E6-mRNA by RT-PCR. The retro-transcription of RNA was performed by the commercial kit Quantitect Reverse Transcription Kit (QIAGEN). The amplification step was performed using the protocol designed by Cocuzza et al., which allows the identification of E6 mRNA of HPV-16, -18, -31, -33, -35, -45, -58 and U1 gene, as internal control, as previously reported ,16. QualStatistical analysis was performed using the JMP in software, release 7.0.1, from the SAS Institute .2 tests.The correlations between p16 IHC and HPV status, and generally among the other categorical variables, were evaluated using ΧThe correlation between detection methods was also determined by calculating the kappa value, a measure that takes a value of zero if there is no correlation between the samples. To score the significance, the kappa values were ranked. Values less than 0.4, between 0.4 and 0.75, or higher than 0.75 represent poor, fair-to-good, or excellent correlations, respectively.Sixty-two OPSCC patients were observed in the period under analysis. Descriptive statistics of demographic, molecular, and clinico-oncological characteristics are reported in Nine out of 62 cases (14.5%) turned out to be HPV-driven according to the previously defined criteria (p16 staining at IHC and hr-DNA amplification). All positive cases were confirmed at RT-PCR, six (66.7%) were associated with HPV16, one (11.1%) to HPV18, and two (22.2%) to HPV35.The analyses for the detection of E6-mRNA, carried out only for HPV-16 and -18 positive samples, confirmed the presence of the previously diagnosed genotype and the expression of oncogenic genes.In the present series, 12/62 cases were p16 positive, 3 of them (25%) tested negative for HPV DNA. In turn, all samples positive for hr-HPV DNA were also p16 positive. Sensitivity of p16 IHC alone in detecting HPV-induced carcinogenesis is therefore 100%, and specificity is 75%. It also means that 3/53 (5.66%) HPV-negative cases stained positive for p16.In our previous works ,18, the Disease-specific survival (DSS) in the present whole series is 68% at 3 years, HPV-driven carcinogenesis is associated with longer DSS (100% vs. 64% at 3 years), and statistical significance is lacking because of the small number of positive cases. If HPV-driven carcinogenesis were assessed by p16IHC alone, the possibility to predict DSS would have been clearly compromised, as shown in The present data demonstrate a prevalence of HPV-induced carcinogenesis among OPSCC in North Sardinia of 14.5%. It is useful information for a number of reasons. This is the first estimate, to our knowledge, of such a figure in the Sardinian population, and it is obtained using a widely validated, reliable, and conservative assay (combining p16 IHC and HPV nucleic acid detection) ,19. SardMore interestingly, 14.5% is one of the lowest figures of HPV prevalence in OPSCC reported in Italy ,25,26,27In general, when dealing with HPV-driven oropharyngeal carcinogenesis, a recurrent bias of clinical studies is the diagnostic tool . Even ifA cut-off should be established for the specificity, below which p16 IHC alone should not be considered sufficient anymore to diagnose HPV-driven oropharyngeal carcinogenesis. It means that the current TNM classification for the oropharynx, which considers p16 immunohistochemistry sufficient for diagnosing HPV-related OPSCC, loses reliability among populations with lower rates of HPV-driven cancers, which is probably the majority of the world population, including Sardinia.Notably, if the HPV-related OPSCC rate is extremely variable, in the series where p16 IHC and nucleic acid detection are both performed, the false positive rate (FPR) of p16 IHC among HPV-negative cases is between 5 and 20% ,44,45,46The present report assesses the prevalence of HPV-related OPSCC in Northern Sardinia, a relatively isolated population showing a different epidemiology from other Italian regions and European trends, closer to less developed areas. This scenario leads to clear implications on prognosis and reliability of methods for assessing HPV-related carcinogenesis. In fact, in the presence of low prevalence of HPV-induced tumors, a high rate of false-positive cases diagnosed by p16 IHC is systematically reported. For this reason, p16 alone cannot be considered an adequate surrogate marker for HPV detection. We believe that in such cases, integration through hr-HPV nucleic acid detection should be mandatory.


The present update is a reassessment of the 2018 ‘Guidelines for HPV-DNA Testing for Cervical Cancer Screening in Brazil’  Although cervical cancer is the fourth most common cancer among women worldwide, mortality rates are decreasing, mainly in high-income countries.According to the World Health Organization (WHO), eliminating cervical cancer depends on multiple efforts, including prevention through vaccination, screening and treatment of precursor lesions, and treatment and palliative care for invasive cervical cancer.In 2016, Instituto Nacional do Câncer  updated its guidelines for cervical cancer screening.There is a relevant discussion among clinicians if the guidelines should consider the management based on risk instead of primarily on test-based algorithms. The risk-based approach means that the probability of finding a case of cervical intraepithelial neoplasia 3 or more severe lesion (CIN 3 + ) would guide the practice, not solely the combination of test results.The present review aims to reassess the 2018 recommendation for HPV-DNA testing in cervical cancer screening in Brazil, according to the changes observed in the new international guidelines and updates on HPV screening knowledge. The review considered the social circumstances of health care access in the country. Topics not addressed in the first recommendation are now discussed due to the increase in the use of HPV testing.For the present critical review, relevant guidelines were identified through a search on the PubMed (MEDLINE), CENTRAL (Cochrane), and Embase databases; the reference sections of the retrieved publications; and the websites of relevant organizations launched during the past five years. The following guidelines were accessed:‘National Cervical Screening Program: Guidelines for the Management of Screen-Detected Abnormalities, Screening in Specific Populations, and Investigation of Abnormal Vaginal Bleeding’ ;The guidelines were accessed to answer questions regarding the clinical practice from the Brazilian perspective. The reference sections of the publications and the medical literature databases were used to develop the answers. Access to care and relevant cultural practices were considered from the perspective of the Brazilian health system framework.In the present review, we have considered HPV tests positive (HPV + ) when the woman gets a positive result for oncogenic types. The acronyms CYTO+ meant abnormal cytology and COLPO+ meant abnormal colposcopy. The question mark ('?') meant the topic regarded.A summary of the recommendations is presented inShould HPV testing alone replace cytology for women older than 30 years?1.The WHO recommends screening based on HPV testing instead of cytology, when resources are available, since 2014.Testing for HPV is more likely to detect adenocarcinoma precursor lesions than cytology-based screening.In high-income countries, HPV testing is cost-effective because of its higher negative predictive value combined with extended testing intervals.For women older than 30 years in Brazil, HPV testing alone should replace cytology. Cytology should be used as a triage test for cases of positive result on the HPV test = .Should cytology be offered at the same time as the HPV test in primary screening (co-testing)?2.When comparing the HPV test alone and combined with cytology (co-testing), the evidence shows that cytology does not substantially increase the detection of precursor lesions; therefore it does not provide extra reassurance.The HPV test alone is as effective as co-testing, and it has a lower cost. Co-testing is not recommended for primary screening.Should women between 25 and 29 years of age be screened by HPV testing?3.There is a consensus about the superiority of HPV testing in women older than 30 years of age. Many consider offering cytology alone for younger women due to the low specificity of the HPV test in women under the age of 30. Most of the positive results in young women reflect transient infections instead of precursor lesions.The critical issue is that, when offering cytology for women aged ≤ 30 years and HPV-testing for those older than that, both technologies would have to be available simultaneously, which would require more resources and induce their misuse. Thus, recent guidelines have updated the recommendation to start screening in women aged 25.As for starting the screening before the age of 25 years, although the US Preventive Services Task Forces recommend starting at the age of 21,Women between 25 and 29 years of age should be screened by HPV testing. To reduce the risk of overdiagnosis, genotyping tests should be preferred in this situation. For those concerned about the effects on the outcomes of future pregnancies, a shared decision should be considered for the treatment when the risk of developing invasive lesions is low.Should cytology still be performed when an HPV test is positive? (HPV+ CYTO?)4.The HPV test is more sensitive than cytology, and should be used for primary screening. However, if screening were only based on HPV testing, patients would be refered to unnecessary colposcopies since most HPV infections are solved immunologically. Regarding cost-effectiveness, a second test before the colposcopy would improve the specificity. The recommendation is for a second test, or ‘triage test’, by cytology, preferably by liquid-based cytology using the same sample (reflex test).In low- and middle-income countries, the reflex test can optimize the time and costs in screening, avoiding follow-up losses. Moreover, the reduction in unnecessary referrals to colposcopy is even more important in these countries.Cytology should be performed after a positive HPV test, to avoid unnecessary colposcopies. Liquid-cytology using the same sample (reflex test) is preferable, to avoid follow-up losses.What is the recommendation when the HPV test is positive and the cytology shows atypia? (HPV+ CYTO + : ?)5.The current recommendation is to perform colposcopy for all women. However, in women under the age of 30, for those with a previous negative HPV test and a new positive HPV test with triage cytology showing minor abnormalities , it is possible to consider a more conservative management.When the HPV test is positive and the cytology shows atypia, women should be referred to colposcopy. In women under 30 years of age, when follow-up is not a problem, a new HPV test in one year can be considered if the cytology shows ASC-US or LSIL.What is the recommendation when an HPV test is positive, the cytology shows atypia, but the colposcopy is normal? (HPV+ CYTO+ COLPO negative: ?)6.It is a consensus in the clinical practice that biopsies should assess any colposcopy changes.When the colposcopy is normal and the cytology shows ASC-US or LSIL, there is no well-established recommendation yet. Cancer Council AustraliaWhen the colposcopy is normal but the cytology result is atypical squamous cells, cannot exclude a high-grade lesion (ASC–H), atypical glandular cells (AGCs), or a highgrade squamous intraepithelial lesion or more severe (HSILþ), further assessment might be necessary.Two additional procedures can improve diagnosis when the cytology is abnormal and the colposcopy is normal: Careful exclusion of vaginal intraepithelial neoplasia and the performance of an endocervical assessment by cytobrush or curettage, more importantly in type-3 transformation zone.There is no sufficient evidence to state a firm recommendation regarding discordant colposcopic impressions and other test results. Despite the lack of consensus, the cytology result can guide the management: if the cytology result shows ASC-US or LSIL, it is possible to repeat the HPV test in 12 months; if the cytology shows ASC-H or HSIL + , it is possible to refer the patient to an excisional procedure, waiting or not for the results of an endocervical assessment.It is possible to recommend an excision treatment bypassing a biopsy when the HPV test is positive, and cytology and colposcopy are abnormal? (HPV+ CYTO+ COLPO + : BIOPSY?)7.Biopsy results can guide the adequate management, to avoid overtreatment and detect lesions in early stages. For those cases in which the risk of HSIL is high, the colposcopy can be used without the biopsy confirmation to guide the excision of the transformation zone.In the Brazilian setting, this approach is suitable due to the high estimation of follow-up losses. The 2016 INCA guidelines already stated that the biopsy can be skipped before the excision treatment when cytology and colposcopy are abnormal (screen-and-treat approach).In women older than 30 years of age, biopsy can be skipped before the excision treatment when the HPV test is positive and cytology and colposcopy are abnormal. In younger women, biopsy is recommended to avoid overtreatment.What should be the recommendation when the HPV test is positive, but the cytology is negative? (HPV+ CYTO negative: ?)8.When the HPVtest is positive and the cytology result is negative, the past tests should be accessed. If there is a history of previous positive or abnormal result, the recommendation is to refer to colposcopy due to the higher risk of persistent HPV infection.When the HPV test is positive and the cytology is negative, follow-up on one year with a new HPV test is recommended. If there is previous history of positive HPV test or cytology, the women should be referred to colposcopy even when the cytology is negative.What is the recommendation when an HPV genotyping test is positive for types 16 or 18? (HPV 16/18 + : ?)9.Although genotyping is not crucial, it is well defined that HPV subtypes 16 and 18 present the highest risks for the development of high-grade lesions or occult cancers.When an HPV genotyping test is positive for types 16 or 18, the women should be immediately referred to colposcopy, regardless the result of the cytology test. If the colposcopy shows mild abnormalities, a biopsy should be performed. If the colposcopy suggests a high-grade or a more severe lesion, the excision treatment is preferable, except in women under 25 years of age, when a biopsy is mandatory.What is the recommendation when the HPV genotyping test is positive for types 16 or 18, but the colposcopy is normal? 10.The cytology result should be confirmed in this situation, even if by a reflex test or by cytology collected during the colposcopy visit. The risk of HSIL+ is high for positive results for types 16 or 18, so treatment can be expedited if the cytology shows ASC-H or HSIL + , even when the result of the bipsy is not available.When the HPV test is positive for types 16 or 18 and the colposcopy is normal, the result of the cytology test should be confirmed to guide the subsequent approach. If the cytology result is negative (ASC-US or LSIL), follow-up in one year with an HPV test is recommended. If the cytology result shows HSIL + , excision should be considered. In case of a negative cytology result, follow-up should include new cytology and colposcopy examinations in six months and a new HPV test in one year.When to stop screening when HPV testing is available?11.An observational cohort studyScreening should be discontinued in patients older than 65 years of age if there has been adequate surveillance for the past 10 years. If there is a history of HSIL + , screening should be continued until the life expectancy or at least 25 years of the treatment.How should we perform screening in pregnant women?12.The risk of precursor lesions does not increase during pregnancy, neither do the rates of progression to cancer. Therefore, screening, either by an HPV test or by cytology, should follow the regular surveillance.Pregnant women should be managed as non-pregnant women regarding screening. Biopsies are only accepted if cancer is suspected.How should we perform the screening in immunocompromised women?13.There is sufficient evidence that immunocompromised women have higher risks of developing precursor lesions.The screening in immunocompromised women should start within 1 year of the first sexual intercourse, each 6 to 12 months, with cytology for women under the age of 25 and HPV tests in women older than 25 years. Management should consider that this group has a high risk of developing HSIL + .How should we perform the screening in vaccinated women?14.According to the proportion of uptake among the population, vaccination against oncogenic HPV genotypes can significantly decrease the rates of precursor lesions and cervical cancer.In Brazil, vaccines have been available in private clinics since 2006, but were only incorporated in the Brazilian Unified Health Care System in 2014. As soon as vaccination coverage increases, the expectations are to combine the screening and vaccination strategies. Some countries have recently updated their guidelines, suggesting that the vaccinated population can start the screening later.The screening in vaccinated women should be the same as in non-vaccinated women, since the coverage rates are still low and have not reached safe levels among the screened populations to suggest changes on the recommendations.


Cells infected with high-risk human papillomaviruses (HPV) can accumulate DNA damage and eventually transform into HPV-driven cancers. Genome instability, or the progressive accumulation of DNA alterations , in HPV-infected cells is directly induced by the HPV genes and indirectly promoted by HPV infection through the consequences of chronic infection maintenance, increased cell growth, and accumulation of damaging mutations in genes that themselves affect genome instability. While the HPV genome typically exists as a separate entity within cells, genome instability increases the chances of HPV integrating within the host (human) genome, which is common in HPV-induced cancers. The DNA regions surrounding HPV integrations are unstable and can undergo complex alterations that affect both human and HPV genes. This review discusses HPV-dependent and -independent drivers and mechanisms of genome instability in HPV-driven cancers, both globally and around sites of HPV integration, and describes the changes induced in the tumour genome.Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV’s ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration. Loss of genome stability, either through mutation of a critical tumour suppressor or chronic carcinogenic exposures, is a central hallmark of cancer ,2,3. VirGenomic instability refers to an increased prevalence of alterations that get passed down to daughter cells upon cell division . These aE6 and E7 oncogenes cause differentiated keratinocytes to re-enter the cell cycle, resulting in an induction of proliferation . Th. Th56].  tumours . The p53 tumours . The absE7 genes with protein products that have a higher binding affinity to a wider range of Rb-related proteins  than non-oncogenic HPV types, resulting in a more robust induction of proliferation . Th. Th33]. feration . Indepenferation ,67. E7 aferation . E7 drivferation ,70,71,72feration . Abrogatferation ,75. G1/SE5 gene is not consistently expressed in HPV-driven cancers, but its presence may aid in perpetuating uncontrolled cell cycling in tumours . I. IE1, E2pression .Chromosome ends are protected from shortening after DNA replication by telomeric repeats, which naturally shorten with ageing . The enzTERT expression and excessive telomere shortening in E6/E7-transfected cells correlated with an increased frequency of anaphase bridges a marker of unsuccessful chromosome segregation ,2119,21]mplified ,201,202.The HPV genome may integrate as a single full copy, a portion of the genome, or as multiple repeating or rearranged segments. Multiple neighbouring integration sites may exist as a single locus. Kadaja et al. first showed that E1 and E2 drive a re-replication of the HPV genome that leaves a heterologous population of intermediate integrant structures . This “oRAD51 (which is recurrently targeted by HPV integration) can reduce tumour growth, slow cell cycle progression, and sensitize the tumours to radiation and cisplatin [BRCA1 and FOXO3; therefore, loss or inhibition of TGF-β signalling led to the inhibition of HR and increased tumors’ reliance on other, error-prone repair methods [E6 and E7 oncogenes into HNSCC cells also induces sensitivity to WEE1 inhibition [Genome instability may drive tumour evolution, but too much genome instability can lead to cell death. This double-edged sword can be exploited in cancer therapy by driving mechanisms that induce genome instability in tumours that are unable to repair the damage (such as HR deficiency). For instance, the DDR is important for both HPV infection and carcinogenesis, and has been targeted by therapy to treat both contexts. HPV infection was modelled in HPV18 organotypic raft cultures, and inhibition of the ATR/Chk1 and ATM/Chk2 pathways was shown to inhibit re-entry into the cell cycle and reduce viral amplification by 90–99% . The inhisplatin . Targetiisplatin . Alsahafisplatin . Anotherisplatin . The aut methods . A commo methods . In HNSC methods ,211. Ano methods ,213. WEE methods ,214,215.hibition . The autGenome instability is one of the central hallmarks of cancer; however, the mechanism by which cells generate genome instability varies between tumours and cancer types. HPV-driven cancers have the element of a ubiquitous exogenous virus that creates a specific mutagenic environment. The HPV genes are able to dysregulate human pathways that are important for maintaining genome integrity; eventually, this leads to further genetic mutations and the onset of cancer. A consequence of the increase in genome instability is the integration of the HPV genome into the host genome. Once integrated, the HPV genome and the region around it is prone to significant rearrangements. Targeting the pathways that are commonly utilized for generating genome instability in HPV-driven cancers may be a novel therapeutic avenue in this context. All together, the mechanisms in which genome instability is initiated and maintained within HPV-driven cancers is multifaceted. Identifying the DNA damage within the HPV-driven tumours and understanding the mechanisms that lead to it will help in our understanding of how these cancers develop and progress.


Specific adult populations known to be at high risk for human papillomavirus (HPV)-related disease, such as men who have sex with men, are inconsistently included in national immunization programs. No compilation of the evidence on the real-world impact and effectiveness of HPV vaccines across these populations exists. This systematic literature review identifies and synthesizes the evidence of the real-world impact and effectiveness of the quadrivalent and nonavalent HPV vaccines in high-risk populations: women with prior/current HPV-related anogenital disease, men who have sex with men, immunocompromised/immunosuppressed individuals, female sex workers, transgender and non-binary individuals, and patients with recurrent respiratory papillomatosis (RRP). The outcomes included anogenital precancers/cancers, head and neck cancers, genital warts, and RRP recurrence. From the 2216 records identified, 30 studies (25 effectiveness and 5 impact studies) were included in this systematic literature review. The results, quantity, and quality of these studies were highly variable. The evidence for effectiveness was of high quality only in women with prior/current cervical disease and in individuals with RRP, the most frequently studied populations. No studies of transgender/non-binary individuals or female sex workers were identified. The real-world evidence supports HPV vaccination among women with prior cervical disease and individuals with RRP. Significant real-world data gaps remain in these high-risk populations. Us. Us19]. p < 0.05) results.The data extracted for each study included study characteristics , subject characteristics  vaccination information  and disease outcomes by vaccination status for vaccine effectiveness studies and by time period for vaccine impact studies. The risk of bias in each included study was assessed using the Newcastle-Ottawa scale ,21. The A total of 2200 records were identified from the literature search in Embase/MEDLINE, and 16 additional records were included for review . The PICVaccine use was reported in all studies. 4vHPV was the sole vaccine provided in 11 of the 12 RRP studies ,41,49,51The results specific to each of the four risk groups for which studies were identified are presented below from most studied (RRP) to least studied (immunocompromised/immunosuppressed). Because no studies were identified for transgender/non-binary individuals or female sex workers, no results are presented for these populations.p = NS), and the intersurgical interval was not significantly different between the groups  .,42.30,42p = 0.01) and 50% at two years (p = 0.05) [p = 0.01) and 53% at 2 years (p = 0.05). Swedish 2014 described 4vHPV vaccine effectiveness in HIV(–) MSM with no history of anal warts or history of treated warts, now recurrence-free [The effectiveness of HPV vaccination in MSM was assessed in three studies: two in HIV(–) MSM ,47 and o = 0.05) . Among pThompson 2018 also looked at anal precancer endpoints, but in HIV(+) MSM. The overall study population (N = 314) included seven (2%) transwomen although it is unclear if any of these transwomen were included in the analyzed sample  . As vaccFive retrospective studies assessed the 4vHPV impact in MSM, all with anogenital warts as the sole reported disease outcome  23,25,2,29,36. AThe effectiveness of HPV vaccination on abnormal cytology and cervical precancer in immunosuppressed/immunocompromised individuals was described in two studies from the United States, one in perinatal HIV-infected youth  and one This SLR found that HPV vaccination effectiveness varied across high-risk populations. Notably, study design issues and limited data contributed to this variance and the evidence strength was graded as high only for RRP patients and women with prior cervical disease . In HIV MSM and immunocompromised individuals were weakened by study design and reporting issues noted above relating to the lack of HPV genotyping, the lack of reporting of vaccination timing or follow-up time, and the insufficient characterization of outcomes. Previous SLRs have reported similar issues with the quantity and/or quality of evidence for HPV vaccine effectiveness in these populations ,16. FurtBalancing these limitations are the strengths of this SLR, including its focus on real-world evidence and clinical outcomes rather than HPV test positivity alone, as test positivity does not necessarily correlate with disease outcomes of interest to patients and public health decision makers. We also included a more diverse set of disease outcomes than most previous SLRs. A third strength is the compilation of data on different high-risk populations, which have heterogeneous characteristics, needs, and prevalence of various HPV-related diseases, which are currently included in or under consideration for inclusion in national or regional immunization programs.In conclusion, the findings of this SLR support the effectiveness of 4vHPV/9vHPV vaccination, primarily 4vHPV, in preventing subsequent cervical precancer in women with pre-existing cervical disease, and in improving clinical outcomes in patients with RRP. In HIV(–)MSM, there was also evidence of moderate effectiveness of 4vHPV in preventing subsequent HGAIN and new or subsequent anal warts in those with and without pre-existing disease. Conspicuous data gaps in populations and in HPV-related outcomes were uncovered. The evidence for the effectiveness and impact of HPV vaccination in several vulnerable high-risk populations was either absent or unclear, although a number of these populations  are either covered under some immunization programs or under consideration for such funding. Further investigation is warranted to understand the public health implications of HPV vaccination for these groups and to bolster public health decision making for populations at an elevated risk of preventable HPV-related disease.