Using a proxy for HPV status, the objective was to evaluate differences in incidence and survival rates of OCPC anatomic sub-sites identified as: 1) HPV-like; 2) non-HPV-like ; and 3) “other”-like , and rurality/urbanity  codes). Although HPV status was unavailable in this dataset, OCPC anatomies and histologies were classified into three sub-categories, based on potential risk factors. Frequencies, average annual, age-adjusted incidence rates, five-year relative survival rates, and 95% confidence intervals were examined across and within OCPC categories.HPV-like OCPC incidence rates sharply increased from 1975 through 2015 while non-HPV-like and “other”-like OCPC rates decreased, all converging to similar rates from 2016 through 2018. Increasing over time for both categories, survival was highest for HPV-like and lowest for non-HPV-like OCPCs; survival for “other”-like OCPCs remained stable. Generally, across OCPC categories, incidence and survival rates were significantly higher among males vs. females, Whites vs. African Americans, and non-Hispanics vs. Hispanics. “Other”-like OCPC incidence decreased with increasing nSES tertiles, while no nSES differences were observed for HPV-like and non-HPV-like OCPCs. Incidence rates were significantly lower among urban  residents. For all OCPC categories, survival rates were significantly higher with increasing nSES and variable across RUCA categories.HPV-like and non-HPV-like OCPC cases had distinct sociodemographic differences; “other”-like OCPC cases were a sociodemographic blend of HPV-like and non-HPV-like OCPC cases, resembling more of the sociodemographic makeup of non-HPV-like OCPC cases. To prevent new OCPCs, additional studies are needed to epidemiologically and clinically differentiate between OCPC categories so that high-risk groups can be better targeted in future public health interventions. In 2022, it was estimated that 54,000 incident cases of oral cavity and pharynx cancer (OCPC) would be diagnosed in the United States (US) and 11,230 people would die from the disease  and tobacco and alcohol use  have consistently been identified as common OCPC risk factors , 7–10. TAs confirmed by HPV testing, HPV has been associated with more than 70% of all US cancers found in the oropharyngeal region of the head and neck , namely Tobacco use and alcohol consumption are primary non-HPV-related OCPC risk factors . These Onot predominantly associated with HPV positivity nor tobacco/alcohol use. For instance, HPV-related and non-HPV-related  OCPCs are less frequently diagnosed in the hypopharynx investigated. The OCPC region is expansive and only cancers from very well-defined specific anatomies have been routinely HPV-tested and associated with, and categorized by, known risk factors . Cancers among these additional specific anatomies are likely indicative of other OCPC risk factor(s) and therefore are opharynx , 24.There is limited recent epidemiological information on OCPC anatomic sites that are well-established as associated with HPV, tobacco/alcohol use, and/or unidentified risk factors. This is especially concerning given the rising incidence rates of some OCPC specific anatomies, including  tonsils and tongue , 16, 25.The Surveillance, Epidemiology and End Results (SEER) Program is a collection of population-based central cancer registries capturing facts from 22 geographic areas representing 48.0% of the US population . The folChaturvedi et al.’s specific anatomic and histologic methodology  based o. MoreoveHere, cancers listed within the OCPC category of the SEER variable ‘Site Recode’ were investigated. Given that the SEER datasets used in these analyses did not include HPV status for the OCPC diagnoses, Chaturvedi et al.’s methodology  was utilHPV-like OCPC sites included the specific anatomies of base of tongue (ICD-O-3: C019),  tonsil , and oropharynx (ICD-O-3: C100-109). Non-HPV-like OCPCs included the specific anatomies of the tongue , gum (ICD-O-3: C030-039), floor of mouth (ICD-O-3:C040-049), hard and soft palates (ICD-O-3: C050-059), and other/unknown sub-anatomies of the mouth (ICD-O-3: C060-069). HPV-like and non-HPV-like OCPCs were further restricted to specific histologies  using the variable ‘ICD-O-3 Histology/Behavior’ . “Other”-like OCPCs included those not included in the HPV-like and non-HPV-like categories, either because they were not one of the specific anatomies , nasopharynx (ICD-O-3: C110-119), salivary gland (ICD-O-3: C080-089), hypopharynx (ICD-O-3: C130-193), and other uncategorized OCPCs (ICD-O-3: C140-148)) nor histologies  described as being predominantly associated with either HPV or tobacco/alcohol use  or ‘non-Spanish-Hispanic-Latino’ , using the variable ‘Origin Recode NHIA’. Stage at diagnosis was characterized using ‘SEER Combined Summary Stage’ and was categorized as ‘The nSES, or Yost, Index in SEER is a time-dependent composite score, including census tract-level information about education index, household income, percent below 150% of poverty line, median house value, percent unemployed, median rent, and percent working class variables . nSES scRecent (2014-2018) average annual, age-adjusted incidence rates, per 100,000 persons, frequency distributions, and 95% confidence intervals (CI) were calculated using SEER*Stat software (version 8.3.9). Age-adjustment of incidence rates were calculated using the 2000 U.S. standard population . For exaSurvival rates and associated 95% CIs were also calculated using SEER*Stat software for cases diagnosed from 2011 through 2017, with follow-up through 2018. For survival rates according to nSES and RUCA categories, cases diagnosed from 2009 through 2015, with follow-up through 2016, were used, as these represented the most recent years available. Survival rates are based on relative survival, a net survival measure representing cancer survival in the absence of other causes of death; it is the ratio of the proportion of observed survivors in a cohort of cancer patients to the proportion of expected survivors in a comparable set of cancer-free individuals.For both incidence and survival rates, non-overlapping 95% CI’s were used to indicate statistically significant differences between levels of sociodemographic factors. Additionally, late-stage only cancers were examined for variations in incidence and survival rates according to nSES and RUCA categories.OCPC incidence rates have shifted over time Figure 1There were 65,538 invasive OCPCs diagnosed from 2014 through 2018; of these, 39.9% were HPV-like, 29.2% were non-HPV-like, and 30.9% were “other”-like OCPCs Table 1.As an additional analysis, incidence rates were restricted to late-stage diagnoses  for nSES and RUCA. HPV-like OCPCs had the highest incidence rates for each nSES tertile. Incidence rates decreased with higher nSES for non-HPV-like and “other”-like OCPCs alike. Data is not shown as no differences were statistically significant. For RUCA late-stage analyses, incidence rates were largely similar to those including all stages (data not shown).From 2000 to 2013, OCPC survival rates have varied by OCPC category Figure 3in situ non-HPV-like OCPCs had a significantly higher survival rate than any other staged non-HPV-like OCPC  for nSES and RUCA. Those residing in census tracts in the highest nSES tertile for each of the three OCPC categories maintained a significantly better survival rate than any other tertile within each OCPC category . Comparing the survival rates of the highest nSES tertile across OCPC categories, HPV-like OCPCs had the highest rate, followed by “other”-like OCPCs, and then non-HPV-like OCPCs  variables while better survival rates were shared among the same categories of sex and stage. Uniquely, “other”-like OCPC incidence rates decreased as nSES increased; patterns were not seen for HPV-like and non-HPV-like OCPCs. Across all OCPCs, survival was significantly higher for those in ‘all rural’ census tracts.Many of these HPV-related and non-HPV-related OCPC findings were supported by previous research. Prior studies found that HPV-related OCPCs were more common among younger ages , 38, 39,The current study found that OCPC incidence rates did not vary by nSES for HPV-like OCPCs; incidence rates decreased with increasing nSES for late-stage non-HPV-like OCPCs. Prior research reported that males with HPV-positive head and neck squamous cell carcinomas were more likely to live in zip codes with higher median household incomes and educational attainments than males with HPV-negative cancers . AnotherThe current findings concerning higher incidence rates of HPV-like OCPCs in non-urban census tracts are supported by county-based incidence rates reported by others for oropharyngeal cancer , 28. FurThis study was strengthened by SEER Program data, which includes many US OCPC cases, representing complete and quality data from a large racially and ethnically heterogeneous population . FurtherThis study also had limitations. A primary limitation in these analyses pertains to the potential for misclassification in the categorization of HPV-like and non-HPV-like OCPCs as HPV status was unknown in these SEER datasets and there are no universally accepted HPV status specific categories. Supported by national-level (US) evidence of HPV-tested OCPC sub-anatomies , an acceEspecially when OCPC HPV status is unavailable as in this investigation, every effort to coalesce varying OCPC terminology and related categories into one coherent list is vital for ensuring that OCPC incidence rates do not go unchecked and that higher risk groups do not go unrecognized. This is especially concerning given that the current study found incidence and survival rates of OCPCs differed significantly by aetiological risk factor category and reported considerable variation over time and across sociodemographic characteristics, including nSES and RUCA categories. Medical providers and public health personnel can incorporate the current study’s findings into their ongoing efforts to minimize the OCPC burden by tailoring and targeting, respectively, their OCPC education approaches to high risk groups. For example, public health can create targeted posters and brochures aimed at males, Whites, non-Hispanics, and rural residents to make them aware of their greater OCPC vulnerability. Additionally, they should make concerted efforts to inform females, minority races and ethnicities, and poorer populations of their potentially lower likelihood of survival should they be diagnosed with an OCPC, emphasizing the importance of routine screening in these groups to detect their cancers earlier. Similarly, medical providers can utilize these risk factors to individually assess their patients’ risk so they may further tailor their patient education efforts and intervene appropriately. This study’s consideration of “other” HPV-like OCPCs gives health-related professionals a jumpstart at beginning to understand and inform higher risk groups of the lesser-known cancers, potentially preventing them from ever becoming an area of public health concern. Ideally, public health and medicine will work in unison to educate any OCPC high risk populations with clear and consistent messaging of the aforementioned OCPC incidence- and survival-based risk factors to make the greatest impact on OCPC rates. The recent availability of a SEER dataset containing HPV status for a subset of head and neck cancers may help further clarify which specific anatomies and histologies should be classified into the categories the current study estimated herein. A better understanding of the epidemiological composition of these multiple OCPC aetiological risk factor-based categories will allow physicians and public health professionals to continue to develop more appropriately and effectively tailored and targeted practices to minimize the OCPC burden for individuals and communities alike.www.seer.cancer.gov.Publicly available datasets were analyzed in this study. This data can be found here: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors approved the final manuscript version and are accountable for all research-related activities herein. KJ: conceptualization, formal analysis, visualization, writing-original draft, writing-review & editing, project administration. JF: data curation, formal analysis, validation, visualization, writing-original draft, writing-review & editing. EP: conceptualization, resources, writing-review & editing, supervision.KJ (P01 CA229143-S1) has National Cancer Institute (NCI) funding through the National Institutes of Health (NIH).EP receives grant funding through the university from Pfizer and Merck Foundation.The remaining 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.


The incidence of anal cancer is increasing globally. Evidence‐based improvement in early detection and management of this morbid cancer is thus required. In other cancers associated with Human Papillomavirus (HPV), viral status and dynamics, including viral load (VL) has been shown to influence clinical outcome. Our aim was to determine the influence of HPV status and HPV16 VL on the clinical outcomes of anal cancer patients.A total of 185 anal cancer lesions were genotyped for HPV. Of the HPV16 positive component, VL was determined using a digital droplet PCR assay. The association of qualitative HPV status and VL (low (<12.3), medium (12.3–57) and high (>57 copies/cell)) on overall survival and hazard of death was assessed.p = 0.01]. When adjusted by age, sex, stage and response to treatment, the association of positive HPV status with improved survival remained (HR 0.24 [0.11–0.55] p < 0.001). High VL was associated with improved overall survival in the univariate analysis with a HR of 0.28 . When adjusted only by age and sex, high VL was associated with better overall survival .Of the 185 cases, 164 (88.6%) samples were HPV positive. HPV16 was detected in 154/185 samples (83.2%). HPV positive status was associated with improved overall survival in the univariate analysis [hazard ratio (HR) of 0.44, 0.23–0.82, HPV status appears to be independently associated with improved outcomes in anal cancer patients. Moreover, HPV viral load quantification may be informative for further risk stratification and warrants further investigation. While human papillomavirus (HPV) status has been linked to clinical outcome in oropharyngeal cancer, there are sparse data on how viral presence and dynamics affect the trajectory of anal cancer. This work suggests HPV positive status is independently associated with improved outcomes in anal cancer patients and that viral load measurement shows potential for further risk stratification. Given the rise in anal cancer, identification of biomarkers that can delineate discrete prognostic groups are welcome. Treatments include chemoradiotherapy (CRT) or resection of the affected tissue, depending on the area affected .Various studies have assessed HPV viral load (VL) in the different HPV‐driven cancers and its association with overall survival and prognosis and/or as a biomarker for lesion progression. In cervical cancers, low HPV viral load may implicate a worse prognosis  and generally, anal lesions are detected and managed, clinically. Moreover, as HPV testing is not currently included in the diagnostic work up of anal disease, there is no routinely collected information on HPV‐associated epidemiology in anal disease in Scotland. Proactive efforts are therefore needed to understand the nature and implications of HPV in anal cancer to inform prevention and management strategies of the future.Our aim was to determine the HPV‐attributable fraction of anal cancers, that is, the fraction associated with HPV in Scotland, and to use this data to ascertain the influence of qualitative HPV status on clinical outcomes. Then, using this well annotated data set we also aimed to determine whether VL (in the HPV16 positive component) exerted an influence on clinical outcomes.22.1Squamous cell carcinoma of anus or anal canal samples (ICD‐11 coding 2C00.3) preserved in formalin fixed paraffin embedded (FFPE) blocks diagnosed between 2009 and 2018 were collated. These samples were originally obtained as part of standard of care for the management of patients with anal disease.Clinico‐demographic information was obtained on age, sex, stage of cancer (using the American Joint Committee on Cancer [AJCC] TNM system),Cases categorised according to the various clinical and demographic variables are summaried in Table 2.2A total of 185 anal cancer samples were annotated for HPV type‐specific prevalence, initially, using a PCR‐based assay: the Anyplex II 28 assay  centrally at the Scottish HPV Reference Laboratory, Edinburgh, UK. One 10 μm section per sample was obtained and incubated in Seegene Univeral Lysis Buffer (LB) at 65°C overnight. DNA extraction was performed using the Microlab Nimbus IVD (Hamilton) with the StarMAg Universal cartridge Kit (Seegene). Mastermix was prepared with the Nimbus and PCR on the CFX Real‐time PCR instrument (Biorad).This initial result allowed determination of type‐specific prevalence and examination of the association between qualitative HPV status (any HPV vs. no HPV) and survival, which we subsequently term the ‘qualitative analysis’. Given the dominance of HPV16 in the cohort, viral load analysis was restricted to samples that tested HPV16 positive, either as a mono infection or within a mixed infection. Once viral load was obtained , it was classified in three different groups: low, medium and high and linked to overall survival. A diagram illustrating the process followed can be found in Appendix 2.3Absolute quantification of viral load was performed on HPV16 + ve cancer samples (145 mono and nine mixed infections) using a droplet digital assay (ddPCR). This element of our analysis is referred to as the quantitative analysis group. Nucleic acid was extracted from HPV16 + ve samples  using the QIAamp DNA Mini Kit (Qiagen) and sample concentration measurement was performed with the Qubit dsDNA High sensitivity kit (Thermo Fisher Scientific).EcoRI and HindIII in 1x NEB Cutsmart buffer ) were used for the mix. Reactions were mixed with Droplet Generation Oil on DG8 cartridges in the QX200 droplet generator (Bio‐Rad) to generate droplets. Thermal cycling conditions were: 95°C for 10 min followed by 40 × 30s at 94°C and 60°C for 1 min prior to final extension at 98°C for 10 min.ddPCR was performed as described in Stevenson et al. 2020n = 9).Post‐amplification, droplets were analysed on a QX200 Droplet Reader (Bio‐Rad), and output data files were analysed using QuantaSoft analysis software v1.7.4 (Bio‐Rad). The viral load for each sample was calculated relative to the endogenous RRP30 cellular gene internal control, with two copies present per cell. Any initially invalid results were repeated using a new FFPE section and fresh DNA extraction. After retesting, consistent invalids were not included in the analysis  for L1 low viral load was <12.3, medium between 12.3 and 57 and high viral load above 57 copies/cell.2.5Overall survival by qualitative status of HPV  and HPV VL  was analysed using the Kaplan–Meier method. The univariate and multivariate hazard ratios of HPV status (negative vs. positive) and virus load (low vs. medium and high) for all cause death were derived using Cox proportional hazard model. Two multivariate models were derived––age  and sex adjusted model as described in Stevenson et al.For the qualitative detection HPV, the types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 were considered as high risk. HPV types considered as low‐risk HPV types include: 6, 11, 40, 42, 43, 44, 54 and 61.33.1Overall HPV status stratified by demographic and clinical characteristics are presented in Table Of the female cases 90.8% were HPV positive; of the male cases, 84.6% were HPV positive. The majority of HPV positive cases were diagnosed in the 60–69 (31.1%) age range and were stage II (37.8%). Overall, 76.2% responded to treatment and 70.1% were alive at censoring.With respect to the cases assessed for the quantitative analysis, 69.7% of samples were from females, 30.3% from males. The majority were diagnosed aged 60–69 years 31.0%), at stage II (37.2%). A total of 76.6% responded to treatment and 71.7% were alive at time of censoring. A higher proportion of those with high VL responded to treatment compared to those with low/medium VL (81.2% for the high VL group vs. 74.5% and 74.0% for the low and medium VL group). A higher proportion of those with high VL were alive at the time of censoring  samples were positive for at least one HPV type. High‐risk (hr) types were detected in 87.03% of the samples. Monoinfection of HPV16 was present in 145 (78.4%) samples. Seven samples (3.8%) had a combination of HPV16 and other hr‐HPV type(s). Two samples (1.1%) were co infected with HPV16 and a low‐risk type. HPV18 was the second most dominant type, present in three samples (1.6%). HPV 33 and 68 were detected in two samples (1.1%), HPV 35, HPV 51 and HPV 52 in 1 (0.5%) while HPV 39 was detected in three (1.6%). The presence of low‐risk types without any other hr‐HPV was detected in three samples (1.6%).3.3Of the 185 cases included in the qualitative analysis, 61 (33.0%) patients died during follow‐up.p value 0.0077 and 0.006, respectively).Kaplan–Meier curves were produced and stratified by HPV status (positive and negative) Figure , and HPVp = 0.01) (Table p = 0.003 and Stage IV HR 25.6 (6.0–109), p < 0.001 versus stage I and response to treatment 0.12 (0.07–0.33) p < 0.001 versus non response to treatment. After adjusting for age, gender, stage and response to treatment, HPV status continued to influence the overall survival, HR 0.24 (0.11–0.55) p < 0.001. When adjusting for age and gender alone, HR for HPV positive status was 0.41(0.21–0.82) p = 0.011.HPV + ve status was associated with improved overall survival in the univariate analysis with a hazard ratio (HR) of 0.44  were associated with valid reads in the ddPCR for HPV16 L1 sequences. Nine samples were excluded from the analysis because they generated less than 10,000 droplets  even after repeat testing.Viral loads ranged from 0.021 to 710 copies of the HPV L1 gene per cell with a mean of 60.57 L1 copies. Those who were deceased at time of analysis  had a median L1 VL of 33.11 (IQR 3.6–43.5); while those still alive  had a median L1 VL of 74.57 (IQR 8.2–104.5) , Figure For the Kaplan–Meier estimator, overall survival was calculated by classifying viral load in three groups, low, medium and high. Overall survival in those with medium and high viral load was higher than in those with low VL p = 0.06, Figurep = 0.007) compared to low viral load. Variables associated with worse overall survival in the univariate model were Stage IV vs. stage I with HR of 25.2 (5.65–113), p < 0.001 and response to treatment [HR of 0.13 (0.064–0.27) p < 0.001] versus non response to treatment.Table p = 0.924, high VL 0.39 (0.12–1.24) p = 0.111 compared to low VL. In the age/gender adjusted Cox model, high viral load was still associated with improved overall survival compared to low VL  p = 0.006.After adjustment for age, gender, stage and response to treatment, viral load did not significantly influence the overall survival; medium VL HR 1.04 (0.45–2.40) 4To our knowledge this is the first study looking at viral load in anal cancer samples and its association with overall survival. Most anal cancers included in this study were positive for HPV (88.6%), with HPV16 being the clear dominant type (93.3%) in the positive cases. This is consistent with the high positivity of HPV in anal cancer and the high prevalence of HPV16 reported by Desanjosé et al.We have identified that HPV status (HPV positive) was associated with improved overall survival in the univariate analysis compared to HPV negative cases. When adjusted, HPV status continued to influence overall survival. This aligns with the systematic review by Urbute et al., where the authors found HPV DNA positive anal cancers have significantly better OS compared with HPV negative cancers.The ddPCR assay indicated that high viral load as measured by quantifying HPV16 L1 gene copies was associated with a better clinical outcome than low copies of L1 in the univariate analysis, when compared with low and medium VL. However, when Cox HR was adjusted, viral load did not influence the overall survival. This could be due to the relatively small sample size; as the confidence interval just exceeds one, it is possible that a larger study may tip into significance. If we consider the unadjusted analysis, our observations with viral load and survival are similar to those seen for oropharynx, where high viral load correlated with improved survival using the same ddPCR technology applied in the present study.The reason as to why higher viral load may plausibly confer a better prognosis (notwithstanding any association with stage) in HPV associated disease is not fully understood. It is feasible that a higher viral load in epithelial cells may maximise exposure to immune effector cells. Additionally, in cervical cancer cases, high viral load, mediated by integrated or episomal genomes has been shown to link with high levels of viral oncoprotein expression. For integrated genomes, epigenetic drivers and changes in stability of transcribed E6 E7 mRNAs are the main mechanisms of increased viral oncogene expression.We decided to use ddPCR given the relative lack of data on the implications of VL in the anal disease context and the fact that ddPCR delivers a high precision,There are limitations to the study––although the sample set was well annotated it was still relatively small. A larger study would have conferred greater power to investigate the impact of viral load, particularly in view of the various adjustments. Also, we did not account for differences between margin versus canal tumours or percentage of tumour captured as this information was not available to us. Additionally, due to the relationship of HIV with anal cancer development, knowledge of HIV status and markers of immune status/competence would have been a valuable addition to our data set.Notwithstanding the above limitations, we would argue that assessment of HPV status in anal cancer cases and viral load is worthy of further investigation. High viral load of HPV in anal cancer serves as a proxy for improved survival and may have potential as biomarker––particularly at an early stage in clinical management where response to treatment is unknown. Larger series and studies which investigate this relationship further are welcome.DG: Received gratis consumables from Seegene to support the HPV genotyping of the anal cancer samples of this study. KC: KC's institution has received research funding or gratis consumables to support research from the following commercial entities in the last 3 years: Cepheid, Euroimmun, GeneFirst, SelfScreen, Hiantis, Seegene, Roche, Abbott and Hologic. All other authors have nothing to declare.DG was involved in the lab testing, analysis of data, and drafted the manuscript. RG performed the data retrieval, and critical appraisal of the manuscript. AK and AS assisted with lab experiments and critical appraisal of the manuscript. JP assisted with the statistical analysis and critical appraisal of the manuscript. KK assisted with the statistical design. MTGH and SVG assisted in critical appraisal of the manuscript. KC was involved in planning, draft supervision and assisted in drafting and critical appraisal of the manuscript.Favourable ethical opinion to conduct the research was provided by University of St Andrews Teaching and Research Ethics Committee, reference MD 14482. This was further supported by approval for use of samples through the National Research for Scotland Bioresource (20/ES/0061), application reference SR1283.Not applicable.


Patients with inborn errors of immunity (IEI) are prone to develop infections, either due to a broad spectrum of pathogens or to only one microbe. Since skin is a major barrier tissue, cutaneous infections are among the most prevalent in patients with IEI due to high exposures to many microbes. In the general population, human papillomaviruses (HPVs) cause asymptomatic or self-healing infections, but, in patients with IEI, unusual clinical expression of HPV infection is observed ranging from epidermodysplasia verruciformis (EV) (a rare disease due to β-HPVs) to profuse, persistent, and recalcitrant warts  or even tree man syndrome (due to HPV2). Mutations in EVER1, EVER2, and CIB1 are associated with EV phenotype; GATA2, CXCR4, and DOCK8 mutations are typically associated with extensive HPV infections, but there are several other IEI that are less frequently associated with severe HPV lesions. In this review, we describe clinical, immunological, and genetic patterns of IEI related to severe HPV cutaneous infections and propose an algorithm for diagnosis of IEI with severe warts associated, or not, with lymphopenia. Human papillomaviruses (HPVs) are DNA viruses with a specific tropism to keratinocytes, which are the main component of stratified epithelia, including skin, genital, and laryngeal mucosa. There are more than 200 different genotypes of HPVs classified in five  genera. HPV subtypes of all genera infect the skin, and only some HPVs of α-genus infect the mucosal epithelia. Some α- and β-HPV types are oncogenic and are associated with benign genital condyloma, cervical and anogenital cancers, and non-melanoma skin cancers, respectively ,2.In the general population, HPVs cause asymptomatic or self-healing infections, with spontaneous clearances reported: 23% at 2 months and 66% by 2 years . The traInborn errors of immunity (IEI) are characterized by an impaired immune response, affecting tissue-intrinsic immunity that is either, innate, adaptive, or both. IEI could be associated with higher susceptibility to infections, auto-inflammation, and/or autoimmunity. Unusual clinical expression of HPV infection is frequently observed in patients with IEI. The spectrum of the clinical phenotype is large from epidermodysplasis verruciformis (EV) (a rare disease due to β-HPV) to profuse, persistent and recalcitrant warts  . Very raThere are some published reviews and case reports that describe clinical, immunological, and genetic patterns of IEI related to severe HPV cutaneous infections, but there are too many aspects of these issues that are still unknown and are being discovered continuously. Here, we present an up-to-date review of the major clinical, immunological, and genetic patterns of IEI related to severe HPV cutaneous infections, and we propose an algorithm for diagnosis of IEI with severe warts in order to help clinicians who may encounter patients with recurrent and recalcitrant warts due to an underlying inherited immunodeficiency.Depending on the HPV genera, there are different characteristics of HPV skin lesions. Macroscopy and histology analyses could help with an appropriate diagnosis. With less than 250 cases reported worldwide, epidermodysplasia verruciformis (EV) is a rare disease that appears at young ages: infancy (7.5% of cases), childhood (61.5% of cases), and adolescence (22% of cases). Lesions are characterized by progressive onset hyperpigmented or achromic flat verrucous lesions, irregular patches of a reddish-brown color, keratotic seborrheic lesions, and pityriasis versicolor-like macules. The lesions are found mainly on sun-exposed areas, such as the face, trunk, neck, forearms, hands, and feet . AlthougEV can be isolated  or syndromic  associated with other clinical manifestations, infectious, or not. . Among 4Profuse warts (PWs) are defined as more than 20 lesions in more than one area of the body. If they do not disappear after 6 months of treatment, they are also classified as recalcitrant . PW caulIn exceptional cases, the warts can also transform into exophytic cutaneous lesions and giant horns, resulting in tree man syndrome . TMS preTMC6 and TMC8, which encode EVER1 and EVER2, two endoplasmic reticulum plasma membrane proteins, respectively, and in CIB1, which encodes calcium and integrin binding protein . For. For6].  and IgE .More recently, patients with CARMIL2 and CD28 deficiencies were associated with HPV susceptibility ,17. ThesCXCR4 gene, encoding a chemokine receptor. The immunological phenotype is characterized by neutropenia, low counts of dendritic cells (DC), memory B cells, and naïve CD4+ and CD8+ T cells [+ T cell, and NK cell counts are the most common immunological features of the patients [This category includes warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, and classical CID and SCID syndromes . The war T cells . In GATApatients ,24.When an HPV-related clinical manifestation is severe, meaning profuse, chronic or recalcitrant and resistant to treatment, an IEI should be suspected especially if there are other infections, atopy, autoimmunity, or malignancy. Together with familial and patient history and physical examination, a guided differential diagnosis hypothesis should be formulated. Afterwards, focused laboratory testing should be investigated starting with immunoglobulin levels, T cell counts, and T cell subpopulation counts . In FiguHPV skin lesions are a common symptom during infancy to childhood. Although recalcitrant warts, or even EV, are a rare clinical manifestation, physicians, including dermatologists and pediatricians, should consider IEI for a patient with recurrent or disseminated HPV skin lesions. The diagnosis strategy is crucial for a prompt and appropriate treatment of those patients. Furthermore, investigations of patients with EV or PW will increase our understanding of skin-intrinsic host immunity against HPVs.


Human Papillomaviruses (HPV) are highly prevalent in the sexually active populations, with a significant burden in terms of health and psychological cost in all class ages. High-risk (HR) HPV genotypes are associated with anogenital dysplasia and cancers, and anal HPV-induced cancer is increasingly observed in women. The interactions of HPV genotype’s between the anus and the cervix, and the subsequent occurrence of dysplasia remains unclear. This clinical study set out to test the hypothesis that risk factors for anal HR-HPV and dysplasia may differ in women with or without cervical dysplasia or in HIV-positive women.Cervical and anal HPV genotypes and cytology testing will be performed prospectively in a cohort of women recruited in a tertiary university hospital in Switzerland. Women will be allocated to three groups: 1) normal previous cervical smear; 2) high-grade cervical dysplasia (H-SIL) at previous cervical smear; 3) HIV+, independently of previous cervical smear result. General inclusion criteria comprised the followings: Female—Age > = 18 years; Satisfactory understanding of French; No objection to HIV testing. Specific inclusion criteria are: Group 1, no past or current gynecological dysplasia and HIV negative; Group 2, Gynecological dysplasia (H-SIL) or carcinoma in situ demonstrated by histology  and HIV negative; Group 3: HIV-positive (regardless of viremia or CD4 count) with or without gynecological dysplasia. General exclusion criteria are: Pregnancy; History of anal dysplasia/cancer; Status after pelvic radiotherapy; Absence of anus and anal canal. Estimated prevalences of anal dysplasia are: in group 1, 1% (0–2%); in group 2, 15% (5–27%), and in group 3, 30% (19–45%). With a 10% margin error, a sample size of 120 women per group is required to reach 90% power for detecting statistical significance .The primary endpoint is the prevalence of anal and cervical dysplasia, and description of the respective HPV genotypes in each group. The results of this study could improve the standard of screening of cervical and anal dysplasia in women through evidence of concomitant presence of HPV’s and/or dysplasia in anus or cervix to support vaccination for instance. Beginning of recruitment started in September 2016. Results should be presented in end of 2022. Preliminary analysis for first 100 patients reveals that the mean age of the population is 39.6 (± 10.9) years with mean age of first sexual intercourse of 18.5 (± 3.9) years. In this cohort, 12% are vaccinated and 38% having had anal intercourse. Overall, 43% of the studied population had cervical HR-HPV in the studied population, and 53% had normal cytology. Anal LR HPV and HR HP were found in 27.6% and 38.4% of all patients respectively. Eighty percent had normal anal cytology. Groups 1,2 and 3 had a significant difference in terms of age, gestity, parity, age of first sexual intercourse, systematic use of condom, number of cervical LR HPV and HR HPV and abnormal cervical cytologies.CER-VD#2015–00200—on the 29th of June 2016 and is registered on the Swiss National Clinical Trials Portal (SNCTP), SNCTP000002567, Registered 29 June 2016, https://www.kofam.ch/en/snctp-portal/study/40742/The study was approved by the institutional review board— In cervical lesions, IARC group 1 include HPV 16 and 18 (hereafter high risk HPV (HR-HPV), whereas potential high-risk HPV (herafter indeterminate risk HPV (IR-HPV) and low risk (LR-HPV) include others HPV types , Gestity 2.1+/-2.1 [0 – 16], Parity 1.4+/- 1.2 [0 – 4], with higher education being secondary school for 26.3% of patients, and university for 24.2%. Mean age for first sexual intercourse 18.5+/-3.9 years old [Preliminary analysis of first 100 patients is reported hereafter. Demographic of patients are shown in ears old  – 41. 38Table 2A and 2B. Cervical LR HPV was found in 21% of all patients, and Cervical HR HPV in 43%. 53% had normal cytology, 14.1% LSIL, 4.3% HSIL, 18.5% ASCUS and 9.8% ASC-H. 37% of patients had cervical biopsies, of which 2.7% were normal, 13.5% were LSIL and 83.8% HSIL. Anal LR HPV was found in 27.6% of all patients, and anal HR HPV in 38.4%. 80% had normal cytology, 5.9% LSIL, 5.9% HSIL, 5.9% ASCUS and 2.4% ASC-H. 6% of patients had anal biopsies, of which 16.7% were normal, 66.7% were LSIL and 16.7% HSIL.PCR genotyping, cytology and pathology in the overall population, respectively for the cervix and the anus are presented in Table 3 displays sub-groups univariate analysis of factors associated with cervical HPV-HR. Groups 1,2 and 3 were significantly different in terms of age, gestity, parity, age of first sexual intercourse, systematic use of condom, number of cervical LR HPV and HR HPV and abnormal cervical cytologies. There were no differences in terms of anal intercourse, HPV vaccination, number of previous sexual partners, smoking status, anal LR HPV and HR HPV and anal cytologies.Anal dysplasia is the precursor of anal cancer but is not subject to systematic screening despite its growing incidence in the last decade. By analogy to cervical cancer screening, systematic screening for anal dysplasia would be the most sensitive option for anal cancer prevention . ConsideThe identification of certain risk groups of patients such as those with HIV or severe gynecological dysplasia and the calculation of their prevalence within these groups will probably allow us to argue in this direction, as they can be confounding factors. These patients could benefit from screening for anal dysplasia and hopefully reduce the number of anal canal cancers. Association of tobacco use and anal dysplasia through HPV prevalence will be studied. Anal sex, probably through a mechanism of micro-lesions of the anal mucosa, is most likely a risk factor even if the current data are unclear. Our data may show an association between the presence of anal dysplasia and anal sex. If so, it would justify that patients should be informed of this risk during the consultations about sexuality. The counseling provided in consultation could thus be individualized according to the reported sexual behaviors and target the indications for anal screening, taking into account the distribution of the different HPV genotypes in the genital and anal spheres.The preliminary analysis of first 100 included patients gives interesting and promising insights, although those data should be carefully considered, as the power for significant results has not been met yet.The present study has some limitations and inherent bias, beyond its cross-sectional design that does only enhance an overview of the situation and does not follow patients in time. The resources allocated to a cross sectional study from a single center limits the maximal number of patients that we can include. This may induce a limited representativity of the population, since the percentage of cervical HR HPV (43%) does not reflect the general population. Nevertheless, group 1  was created to ensure a sample size that could be representing the average population consulting in a day-to-day clinic of GYN/OBS in a tertiary hospital. The number of patients included does also limit the power HPV genotype’s sub-group analysis. A population selection bias is possible since anogenital HPV infection could also be managed in clinics and private practice and not only in tertiary center. Recall bias are possible, as the disease examined is slowly evolving through time and accuracy of recall regarding prior exposures can vary. The duration of the study will depend on the number of patients recruited in each of the three groups. We do not expect one group to be faster to complete than another, since all three groups of patients are recruited from three dedicated clinics in an academic center with a large caseload. Regarding the proportion of patients recruited from the total screened during the consultation, as there is no additional time during the consultation for the study and some patients do not speak/understand enough French, we might face some delay in recruitment.The study of the different HPVs in the gynecological and anal areas will allow us to compare HPV carriage at both sites and to evaluate if the same genotypes, in our population, are associated with dysplasia whether they are found at the gynecological or anal level. If HPV 16 and 18 are involved in an increased prevalence of anal dysplasia, we will have an additional argument in favor of the HPV vaccination program in young women.S1 File(DOCX)Click here for additional data file.


The aim of this study was to determine the incidence of infections and cytological abnormalities and to investigate possible predisposing factors such as sociodemographic characteristics, sexual behavioral habits, and gynecological and obstetric backgrounds. Between 2013 and December 2016, a cross-sectional study was conducted among 429 consenting women, from whom cervical samples were tested for the presence of Human papillomavirus (HPV) by polymerase chain reaction (PCR). Susceptibility to HPV infection was assessed by binary logistic regression in light of possible predisposing factors, which were collected using a questionnaire. In our sample population, the prevalence of HPV infection was 49%; high-risk types had a higher prevalence of 89.1%. A larger proportion of HPV-infected women were under 25 years of age, were single, and had monthly incomes up to minimum wage. Multivariate binary logistic regression analysis showed that age younger than 25 years increased the odds of infection fivefold, while a monthly income of one to three minimum wages provided protection against HPV infection, even if the women were married or had a cohabiting partner. In the HPV-positive group, squamous intraepithelial lesions (SIL) occurred more frequently in women who earned up to one minimum wage monthly, but a monthly income of one to three minimum wages protected against the development of SIL. The results suggest that age, marital status, and monthly income are important cofactors for HPV infection and the development of SIL. Chlamydia trachomatis (CT), are associated with disease progression [Cervical cancer (CC) is the third most common cancer in women worldwide, with an estimated 570,000 new cases in 2018, of which more than 85% occurred in less developed regions . It has The HPV prevalence in cervical specimens in Brazil determined in a systematic review and meta-analysis study was 25.41% with a prediction interval of 7.17% to 60.04% depending on the population and geographical area studied . EpidemiThe aim of this cross-sectional study was to determine the incidence of HPV infection and cytological abnormalities and to investigate possible predisposing factors such as sociodemographic characteristics, sexual behavioral habits, and gynecological and obstetric history.The women participating in the study were from the city of Londrina and surrounding small towns. This large city is located in the northern region of the Brazilian state of Paraná (southern region of the country) and is more than 300 km from the respective capital (Curitiba).Cervical cell samples were randomly collected between 2013 and December 2016 from 429 women who presented for outpatient appointments at the colposcopy outpatient clinic of the Intermunicipal Health Consortium of Middle Paranapanema and the College Hospital and Clinical Center of the College of Londrina, and who had undergone cervical cancer screening cytology at two primary healthcare facilities . The women included in the present study were participating in cervical cancer prevention programs, since, according to the Brazilian guidelines for cervical cancer screening, routine cytological examination is recommended for women aged 25 to 64 years who are sexually active . After sCervical cancer smears obtained during screening in healthcare units were sent to the public health laboratory. They were evaluated and reported according to the diagnostic criteria of the Bethesda system (2014). SIL was defined as low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSILs), atypical squamous cells of undetermined significance (ASC-US), HSILs that could not be excluded (ASC-H), or cervical carcinoma (CC), while controls were negative for intraepithelial lesions or malignancy and all SIL types were excluded .Genomic DNA from cervical cells was extracted from Cytobrush samples using DNAzol  according to the manufacturer’s instructions. DNA concentrations were measured at 260 nm using the NanoDrop 2000c™ spectrophotometer , and purity was assessed by the 260 nm/280 nm ratio. DNA samples were then stored at −20 °C.HPV was detected by PCR using primers MY09 (5′–CGTCCMAARGGAWACTGATC–3′) and MY11 (5′–GCMCAGGGWCATAAYAATGG–3′), which amplify a conserved region of approximately 450 base pairs (bp) of the L1 HPV gene . ReactioPCR assay for detection of CT was performed using primers specific for the gyrA gene: forward primer C2 (5′–TGATGCTAGGGACGGATTAAAACC–3′) and reverse primer C5 (5′–TTCCCCTAAATTATGCGGTGGAA–3′), as described previously . Co-ampl® Terminator v3.1 Cycle Sequencing Kit , 50 ng of DNA template, and 5 µM primer (forward or reverse) in a final volume of 10 µL. PCR conditions were as follows: 10 s at 95 °C, 30 cycles of 20 s at 95 °C, 20 s at 50 °C, and 60 s at 60 °C. Amplicons were sequenced in a 24-capillary 3500xl Genetic Analyzer . Percentage identity was determined using the program BLAST by comparing the DNA sequences of the amplicons to known HPV L1 or gyrA nucleotide sequences in the GenBank databases.To confirm primer specificity, some amplicons of both HPV and CT DNA were purified using the PureLinkTM PCR Purification Kit (Invitrogen) according to the manufacturer’s instructions. The sequencing reaction was performed using the BigDye2) or Fisher’s exact probability test, where appropriate, and expressed in absolute numbers (n) and percentages (%). Differences between categories of the same variables were assessed using the Mann–Whitney test and expressed as the median and interquartile range (IQR) 25–75%. The odds ratio (OR) and 95% confidence interval (95% CI) were determined. Binary logistic regression analysis was used to determine the significant predictors of HPV infection compared with controls (uninfected), and multinomial logistic regression was used to analyze the risk factors for the presence of SIL compared with controls  in HPV-infected women. In both analyses, sociodemographic and sexual behavioral, gynecologic, and obstetric factors associated with HPV infection or a SIL diagnosis in the bivariate analysis were each included as explanatory variables. All tests were two-sided, and a significance level of α = 0.05 was assumed. Analyses were performed using IBM SPSS Statistics 22.0 software .Associations between categorical variables were analyzed with the chi-square test , schooling (p = 0.83), knowledge of HPV (p = 0.50), and viral transmission (p = 0.63). However, HPV-infected women had a lower mean age, 33 (25–46) years, than uninfected women, 43 (32–52) years (p < 0.001). To determine in which age range HPV infection was more common, the continuous variable “age” was divided into “age ranges”, as shown in p < 0.001), were single (p < 0.001), had a reported monthly income of up to minimum wage (p = 0.018), and were smokers (p = 0.014).The sociodemographic characteristics of the HPV-negative and -positive women are shown in p = 0.21), number of sexual partners in the past 6 months (p = 0.051), oral contraceptive use (p = 0.13), condom use (p = 0.44), spontaneous abortion (p = 0.27), and CT infection (p = 0.056) were not statistically different between the HPV-positive group and the control group. However, HPV infection was associated with first sexual intercourse before age 18 (p = 0.012), at least four lifetime sexual partners (p < 0.001), and pregnancy that did not occur (p = 0.008).p = 0.056 value may indicate a tendency toward a higher CT prevalence in HPV-infected women.The occurrence of CT infection was more common in women who tested positive for HPV infection. Although this difference was not statistically significant, the C. trachomatis was 4.7%, and the proportion of coinfections with HPV and C. trachomatis in cervical lesions was 7.2% and in healthy individuals was 2.2% in the population sample studied. The presence of cervical lesions was 25.2%. When cervical lesions were stratified according to the degree of involvement, a prevalence of 7.3% for LSIL, 17.7% for HSIL, and 1.2% for cancer could be observed.Considering the number of existing cases of the analyzed conditioning variables in relation to the total population sample analyzed in this study, the characterization of the sample in terms of prevalence of HPV infection was 49%, with a higher prevalence of these high-risk types (89.1%) compared with low-risk types (8.0%) and undetermined risk (2.9%). The prevalence of infection with p = 0.004), whereas both married and lifetime partners  and a monthly income of one to three minimum wages  provided protection against HPV infection  were independently associated with HPV infection, a binary logistic regression analysis was performed with a control group as the reference category. A direct association was found for age less than 25 years, which increased the odds of acquiring the virus approximately fivefold , oral contraceptive use (p = 0.010), and spontaneous abortion (p = 0.010) (data not shown). The presence of LSIL was associated with <25 years (p = 0.002), a monthly income of up to minimum wage (p < 0.001), and more than four lifetime sexual partners (p = 0.019). In multinomial logistic regression analysis with normal cytology as the reference category, all age categories >25 years and spontaneous abortion  were independently associated with the risk of developing LSIL (p = 0.009) was an independent risk factor  in Londrina, Paraná State, Brazil. To our knowledge, this is the first epidemiological and molecular report to investigate HPV prevalence in this region.This study found a prevalence of 49.0% HPV infection in the women studied, of which 89.1% were high-risk types, 8.0% were low-risk types, and 2.9% were of undetermined risk (0.9%). However, we were unable to perform it in all our sampling, configuring a limitation of our study. HPV infection was associated with four sociodemographic characteristics , two sexual behavior variables , and one gynecologic and obstetric aspect (parity).Only those younger than 25 years, marital status with a married or civil partner, and monthly income of one to three minimum wages  were indThe association between young age and HPV infection is well established in the literature as an independent factor, showing that HPV prevalence is age-specific worldwide for both low-risk types (LR-HPV) and high-risk types (HR-HPV) . TeenageSingle women were significantly more likely to have HPV in the HPV group , which iIt is also known that multiparity, which becomes more likely in women who start having sex earlier, is associated with a higher risk of exposure of women to HPV infection and other cofactors ,20.Tobacco smoking was also associated with HPV infection in this study . SeveralWomen who reported a monthly income at or below the minimum wage were more likely to be infected with HPV . These rWe found an independent association between monthly income of one to three minimum wages and protection from SIL among women infected with HPV . These rRegarding sexual behavior and gynecologic and obstetric aspects, an association was found between HPV infection and first sexual intercourse before the age of 18 years and at least four sexual partners during life . As mentChlamydia trachomatis (CT) (Our study showed a tendency for association between HPV infection and the intracellular bacterium tis (CT) . This teHaving more than four sexual partners over a lifetime was associated with the development of HSIL, and spontaneous abortion was associated with the development of LSIL, which is consistent with other meta-analysis of epidemiological studies demonstrating the involvement of these factors from HPV infection to tumor occurrence ,32.Cervical cancer screening is effectively performed in Brazil through the Pap smear, a method that has led to a decrease in CC incidence in the country over the last five decades. Nevertheless, new cases of CC are detected every year, and mortality from this cancer is still alarming worldwide . It is nAlthough this was a cross-sectional study that did not allow us to draw conclusions about causal relationships, we would like to use our cohort and several reports published in recent years to draw attention to the fact that diagnosis by HPV DNA using polymerase chain reaction in combination with cytologic analysis allows more accurate detection of HPV and lesions, even when abnormalities are not detected in the Pap smear. In this article, we described the HPV occurrence in the northern region of the Brazilian state of Paraná and important cofactors for HPV infection and the development of SIL in our population-based study. In this sense, we hope to contribute to a better characterization of HPV epidemiology and to the implementation of public health policies in Brazil.


Human papillomavirus (HPV) infection, which often includes high-risk genotype infection, is one of the leading causes of cervical cancer. This cross-sectional research included 503 Iranian women referred to the gynecology clinic of Kamali Hospital in Karaj, Iran, for routine cervical cancer screening between 2020 and 2021. Cervical specimens were collected from all participants with a special brush and transported to the laboratory for cervical cytology diagnosis. Overall HPV incidence among Iranian women was 39.96%, of which 23.06% had high-risk HPV genotypes and 9.7% had low-risk HPV types. The risk associated with HR-HPV types was considerably associated with employment and marital status. In 2018Papillomaviruses are double-stranded deoxyribonucleic acid (DNA) viruses detected in animals and humans. Over 200 human papillomavirus genotypes (HPV) have been recognized so far, and nearly 40 genotypes infect genitalia . GenitalUnprotected sexual intimacy is the major risk factor related to the attainment and durability of HPV infection and the growth of HPV-associated cancers . These uIt is important to study the predictors of HPV infection in high-risk areas to address the gaps in healthcare, giving special consideration to the differences in sexual behavior among men and women and people from various racial origins. Previous studies have focused on the incidence and genotype distribution of HPV infection among Iranian females , 12. AltThis cross-sectional research included 503 Iranian women referred to the Gynecology Clinic of Kamali Hospital in Karaj, Iran, for routine cervical cancer screening between 2020 and 2021. Sexually active, 15–70 years old, from the Iranian population, having clinical symptoms including genital warts, intermenstrual bleeding, vaginal discharge, pain during intercourse and bleeding after intercourse, and or having come for annual visits were considered as inclusion criteria. Exclusion criteria included abnormal vaginal bleeding, obvious mass in the cervix, hysterectomy or management of potential cervix cancer, obstetrical/gynecological emergency, pregnancy, and mental impairment. Women who agreed to participate were invited to a brief meeting and a gynecological examination, comprising collecting samples for cytology diagnoses and HPV tests.Interviews were done in a private room to obtain the medical history, demographic and behavioral characteristics of participants. Questions included demographic profile and HPV-related risk factors, including employment status, level of education, breastfeeding status, marital status, menstrual cycle status, gravida status, post-coital bleeding, number of sexual partners, hookah use, cigarette status, and reason for referral , 9, 13. Participants completed the routine gynecological examination after the administration of the questionnaire. A trained gynecologist collected cervical samples using cytobrush at the intersection of the endocervix and the ectocervix. Two midwives participated in observing the collection of samples. The samples were kept in a protective solution abiding by the manufacturer's guidelines and transported to the laboratory for Papanicolaou (Pap) smear or cervical cytology diagnosis. Cervical cytology smears were examined by a cytopathologist who did not know the participant's HPV DNA test results. Cytological results were defined as high-grade intraepithelial lesions (HSILs), low-grade squamous intraepithelial lesions (LSILs), atypical glandular cells (AGCs), atypical squamous cells of undetermined significance (ASCUS), cervical intraepithelial neoplasia (CIN), koilocytic change, and chronic cervicitis , 15.Samples for HPV analysis were collected using cervical swabs, placed in a sterile tube, and transported to the test center for HPV DNA testing using a PCR-based method. The presence of 36 HPV genotypes including 18 LR–HPV genotypes , 18 HR-HPV  were evaluated , 16.After obtaining the HPV DNA test and cytology results, women with positive HR-HPV DNA or a Pap result of ASCUS or higher were sent to a gynecologist for further assessment or colposcopy examination.Statistical analysis was done using SPSS 21.0 statistical software package  and GraphPad Prism v.7.0 . P-values<0.05 were considered statistically significant.607 participants completed the inclusion criteria and were included in this research, out of which 503 participants completed the study, and their data were analyzed. The results of PCR demonstrated that the overall HPV prevalence among Iranian women was 39.96% (201/503), from which 23.06% (116/503) had HR-HPV genotypes, 9.7% (49/503) had LR-HPV genotypes and 7.2% (36/503) had both HR-HPV and LR-HPV genotypes.A logistic regression analysis was done to identify the risk factors for HPV. Hookah use (7.986 times), cytology result (CN1) (3.802 times), employment status (3.331 times), cytology result (CN2) (2.309 times), breastfeeding (2.506 times), and early age at first pregnancy (1.068 times) increased the chance of being HR-HPV positive .In this study, the overall incidence of HPV among Iranian women was 39.96%, of which 23.06% had HR-HPV genotypes, 9.7% had LR-HPV genotypes, and 7.2% had both HR-HPV and LR-HPV genotypes. In addition, our results revealed that the risk of HR-HPV genotypes was considerably associated with employment status, level of education, gravida status, breastfeeding status, marital status, number of sexual partners, hookah use, cytology test result, colposcopy test result, and reason for referral.th leading cause of cancer mortality in women 15 to 44 years of age in Iran [Cervical cancer is the 10 in Iran . It is b in Iran . HPV pre in Iran . Herein, in Iran . However in Iran , 20. It One of the effective methods for cervix cancer screening for many years has been Pap smear or cervical cytology. The benefits of cytology are its low cost and simplicity . Based oIt is of great importance to recognize distinctive risk factors of HR-HPV genotypes, along with ways to minimize the associated risk factors and take necessary actions against them. In the absence of a complete study that combines all the risk factors in an Iranian population, we studied the association between employment status, level of education, gravida status, breastfeeding status, marital status, menstrual cycle status, post-coital bleeding, and many sexual partners, hookah use, cigarette status, Pap smear test result, colposcopy test result and reason for referral. The results revealed that the overall risk for HPV infection among Iranian women was associated with employment status, level of education, gravida status, breastfeeding status, marital status, number of sexual partners, hookah use, Pap smear test result, colposcopy test result, and reason for referral. Among these risk factors, hookah use (7.986 times), Pap smear test result (CN1) (3.802 times), employment status (3.331 times), Pap smear test result (CN2) (2.309 times), breastfeeding (2.506 times), and early age at first pregnancy (1.068 times) increased the chance of being HR-HPV positive.Based on our results, hookah was the most common risk factor for HR-HPV positive. Traditional use of hookah is common in Iran. Even though society is well aware of the disadvantages and dangers of hookah, bearing in mind the tradition of smoking hookah in Iran, many see it as healthy and harmless. Finding nicotine and carcinogenic properties of tobacco (used in a hookah) in cervical mucus has led to the hypothesis that the mixture of tobacco and HPV occurs in the development and resistance of cervical lesions . It has The socioeconomic status of a person affects the occurrence of health-related activities. Individuals with low socioeconomic status may lack the ability to afford essential health care or may live in regions with limited access to health services, which can generate susceptibility to the growth of various diseases. We used employment status and level of education as indicators of the socioeconomic status of the women. Bad health-related quality of life in individuals who suffer from HPV-positive can be mostly related to employment status. Herein, the obtained results showed that employment status is related to the rate of HPV. As shown, the rate of HPV in working women was greater than in homemaker women. One explanation for this result may be wearing clothes for outdoor work and less hygiene during office hours. However, Kulhan et al. in Turkey showed no relationship between employment status and HPV infection in women .Early sexual debut is related to a higher risk of HR-HPV infection . Age at Finally, this research revealed that the incidence of HPV was greater in women who were not breastfeeding. It was suggested that the incidence of HPV was greater in people with breast cancer . TherefoThis study has several strengths. Firstly, according to our understanding, it remains the only research to report the relationship between socioeconomic status, especially employment status, hookah use, and HR-HPV infection in Iranian women. Secondly, this study included several females from the overall population. Given its size, we could analyze the relationship between numerous risk factors and the prevalence of HR-HPV infection with a relatively strong statistical power. However, many limitations exist that require attention. Firstly, this research was performed only on a case group of individuals who attended the gynecology clinic of Kamali Hospital in Karaj, Iran. Due to this, we are confident that our findings should not be generalized to all Iranian women. Secondly, a lack of data regarding males' sexual behavior is also an important risk factor for HPV infection and cervical infections in females.In conclusion, we observed that HR-HPV is highly prevalent among Iranian women. The main risk factors for HR-HPV disease are employment status, level of education, parity, breastfeeding status, civil status, figures of sex partners, hookah use, and Pap smear test result. Since HPV is a great risk factor for cancer of the cervix, considering the diagnostic methods, prevention and identification of individuals with this infection is a useful way to prevent this cancer. Hence, determining the HPV prevalence and related risk factors in major inhabitants of the Iranian community can develop and advance health policies applied by the government and health agencies.


Immune checkpoint inhibitors (ICI) are approved for treatment of recurrent or metastatic oropharyngeal head and neck squamous cell carcinoma in the first- and second-line settings. However, only 15–20% of patients benefit from this treatment, a feature increasingly ascribed to the peculiar characteristics of the tumor immune microenvironment (TIME).Immune-related gene expression profiling (GEP) and multiplex immunofluorescence (mIF) including spatial proximity analysis, were used to characterize the TIME of 39 treatment-naïve oropharyngeal squamous cell carcinomas (OPSCC) and the corresponding lymph node metastases. GEP and mIF results were correlated with disease-free survival (DFS).HPV-positive tumors disclosed a stronger activation of several immune signalling pathways, as well as a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells, than HPV-negative patients. Accordingly, mIF revealed that HPV-positive lesions were heavily infiltrated as compared to HPV-negative counterparts, with a higher density of T cells and checkpoint molecules. CD8+ T cells appeared in closer proximity to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-positive primary tumors, and related metastases. In HPV-positive lesions, PD-L1 expression was increased as compared to HPV-negative samples, and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. Considering the whole cohort, a positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-positive patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of HPV-negative patients.Our results suggest that checkpoint expression may reflect an ongoing antitumor immune response. Thus, these observations provide the rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combination of ICI with tumor-specific T cell response inducers or TAM modulators for the “cold” OPSCC counterparts.The online version contains supplementary material available at 10.1186/s13046-022-02481-4. RM) CD8+ T cells co-expressing the CD103 marker has recently emerged as a favorable prognostic indicator in many cancer types, included HNSCC [Among head and neck squamous cell carcinomas (HNSCC), oropharyngeal squamous cell carcinomas (OPSCC) comprise cancers of the palatine tonsils, base of tongue, soft palate and posterior pharyngeal wall , and areed HNSCC –14.Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 pathway have been approved for recurrent and metastatic HNSCC patients in the first- and second-line settings –17. HoweOverall, new immune-based therapies increasingly rely on an in depth characterization of the tumor-immune cell interactions . NotwithHere, using a combination of immune-related gene expression profiling (GEP), quantitative multiplex immunofluorescence (mIF) and spatial proximity analyses, we provide insights about the TIME characterization of HPV-positive and HPV-negative OPSCC, both on primary and metastatic lesions. We advance a potential new rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combined use of ICI and tumor-specific T cell response inducers or tumor-associated macrophages (TAM) modulators for the non-inflamed counterparts.Thirty-nine consecutive patients undergoing up-front surgery with simultaneous neck dissection for N-positive OPSCC  at Treviso Regional Hospital and Trieste University Hospital from August 2010 to January 2021, were included in the study. Patients with previous cancer history or recurrent cancer or distant metastases, and those previously undergoing chemotherapy or radiotherapy were excluded. Clinicopathological information including gender, age, year of diagnosis, cancer sub-site, pTNM stage, histological grading, extracapsular extension, margins status (R), adjuvant treatment, tobacco smoking, alcohol drinking were retrieved from the electronic medical records. Pathologic staging was based on the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. All patients underwent a regular follow-up until death or 31 December 2021. The study was approved by the ethic committees for clinical experimentation of Treviso and Belluno provinces and Friuli Venezia Giulia region, and all patients signed an informed consent.INK4a status was evaluated from FFPE sections by immunohistochemistry using an anti-human p16INK4a antibody (clone G175–405) and the BD Pharmingen™ IHC Detection Kit. p16INK4a positivity was based on a strong and diffuse nuclear and cytoplasmatic staining in at least 70% of tumor cells. A tumor was defined as HPV-positive by double positivity for HPV DNA and p16INK4a.Search and typing of HPV DNA sequences were carried out from genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) sections by QIAamp DNA Mini kit (Qiagen), according to the manufacturer’s instructions. DNA was tested by PCR with MY09/MY11 primers, followed by restriction fragment length polymorphism analysis of the amplification products. The DNA quality of the samples was verified by amplification of the β-globin gene. p16Total RNA was extracted from 2 consecutive 10 μm-thick FFPE primary tumor sections, using the RNAesy FFPE kit (Qiagen), according to the manufacturer’s instruction. RNA quantification was performed with Nanodrop 1000 spectrophotometer (ThermoFisher scientific), and the RNA integrity and quality were evaluated with the Agilent 2100 Bioanalyzer System, using the RNA 6000 nano kit (Agilent). The PanCancer Immune Profiling panel (NanoString Technologies) was used to measure the expression of 770 immune-related genes covering innate and adaptive immune responses. The panel included 40 housekeeping genes, 8 negative controls and 6 synthetic positive controls. Samples were processed according to the manufacturer’s instructions provided by NanoString Technologies. RNA (300 ng) from each sample was hybridized with panel probes for 19 hours at 65 °C, and then complexes were processed on the nCounter FLEX platform (NanoString Technologies). Cartridges were scanned at 555 fields of view. Gene expression data were analysed with the nSolver 4.0 Software (NanoString Technologies), and a quality check was performed in the gene expression analysis. Raw data were normalized using a ratio of the expression value to the geometric mean of housekeeping genes on the panel; then, normalized data were Log2 transformed. The nCounter Advanced Analysis module V.2.0.134 software (NanoString Technologies) was used for differential expression analysis and to obtain scores for cell type profiling and pathway analysis, based on the expression of predefined genes -based method (Akoya Biosciences), as previously reported –24. Two At least 20 fields of each multiplex-stained slide were imaged using the Mantra Quantitative Pathology Workstation (Akoya Biosciences) at X20 magnification. For each sample, only areas comprising tumor cells were considered to avoid the acquisition and analysis of normal-like tonsil and lymph node tissues. The inForm Image Analysis software  was used to unmix multispectral images using a spectral library built from acquisition of single fluorophore-stained control tissues, and containing fluorophores-emitting spectral peaks. A selection of representative multispectral images was used to train the inForm software to create algorithms, as previously described . Brieflyp-values were used to decrease the false discovery rate. Differentially expressed genes (DEGs) were defined by Log2 fold difference of > 1 or < − 1, and an adjusted p value < 0.05. Disease-free survival (DFS) was defined by the time from the last treatment to death for any cause or relapse, whichever occurred first, or to the last follow-up date. A binary value (low vs. high) was assigned to each patient based on the median score, cell density or percentage cut-off for each gene set or marker. The Kaplan-Meier method was used to generate DFS curves and statistical differences were evaluated using the log-rank Mantel-Cox test. Moreover, univariate Cox regression modelling for proportional hazards was used to calculate hazard ratio (HR) and 95% confidence interval (CI) for the association of dichotomized immune variables and patient outcome. For the correlation analyses, the non-parametric Spearman’s correlation coefficient (r) was calculated. All reported p-values are two-sided and p ≤ 0.05 was considered statistically significant.Statistical analyses were carried out using GraphPad Prism software (version 8.0) and IBM SPSS Statistics (version 28). Patient characteristics were evaluated according to the HPV status by using Fisher’s exact test. For continuous variables, median, quartiles and range were described and statistical analyses were performed with the non-parametric two-tailed Mann-Whitney test between two groups. Linear regression analysis was used to investigate differential gene expression using HPV status as a covariate. The Benjamini-Hochberg adjusted n = 32; 82.1%). Twenty-four (61.5%) patients harbored a transforming HPV infection defined by HPV DNA and p16INK4a double positivity, while the remaining 15 cases were double negative. Prevalence of HPV-positive tumors was higher among never smokers. HPV16 was the most prevalent type , with the remaining subjects being positive for HPV33 (2 cases) and HPV18 (1 case). Twenty-seven/39 patients (69.2%) underwent adjuvant post-operative (chemo)radiation. No significant difference in DFS was observed between HPV-positive and HPV-negative patients (p = 0.34).Patient characteristics according to the HPV status are reported in Table To determine the immune signatures of HPV-positive and HPV-negative OPSCC, we used the NanoString PanCancer Immune Profiling Panel on the entire patient cohort. Gene expression analysis revealed 30 DEGs between HPV-positive and HPV-negative OPSCC Fig. A. ConverThe assessment of the impact of immune signatures and pathways on DFS in the entire study cohort revealed that patients with a higher expression of CD45, T cells and total TILs signatures, as well as a higher ratio between M1/M2 macrophage signatures, had a longer DFS  and CD4 for T helper lymphocytes. Moreover, the expression of the checkpoint molecules PD-1, PD-L1 and CTLA-4 on T cells, macrophages and tumor cells was also investigated.As recapitulated in the representative Fig. r = 0.857, p < 0.0001 in HPV-positive; r = 0.8315, p = 0.0002 in HPV-negative). Accordingly, CD8 + CD103+ TRM cells, CD8 + PD-1+ CTL and triple positive CD8 + CD103 + PD-1+ T cell subsets were more represented in HPV-positive than in HPV-negative tumors . The analysis of CD8+ T cell subsets revealed that CD8 + CD103+, CD8 + PD-1+ and CD8 + CD103 + PD-1+ T lymphocytes were present at higher densities in HPV-positive than HPV-negative metastases, both in the stroma and within the tumor cell nests .Although striking differences in terms of immune infiltrate were detected between HPV-positive and HPV-negative samples, within the primary tumors and the matched lymph node metastases of either groups of patients no significant differences in immune cell densities were observed , or stratifying the cohort by HPV status . However, recent evidences highlight the importance of patient sex in anti-tumor immune response, which in turn impinges on the efficacy of ICI [Patient sex did not impact DFS of our cohort, even considering the overall population  and melanoma upon anti-PD-1 administration [RM gene program, which is characterized by tissue residency, cytotoxicity, effector functions and inhibitory receptors including PD-1 [RM cells in “hot” HPV-positive tumors, but also to induce the expansion of anti-tumor T cell clones in “cold” HPV-negative lesions.Additionally, the correlation found between the increased tumor-infiltrating CD8+ T cells and PD-1 expression may indicate that the local immune response also induces the PD1/PD-L1 checkpoint pathway, which in turn might limit the capacity of TILs to ultimately eliminate the tumor. Paradoxically but in agreement with Badoual et al. , a high  effects . All thestration , 45. Inting PD-1 . Moreoveing PD-1 . OverallMore than 50% of HNSCC patients present with metastasis to regional lymph nodes at the time of diagnosis, a feature associated to poor survival and a major prognostic factor for determining the appropriate treatment . Since TPD-L1 is considered a predictive marker for response to PD-1/PD-L1 blockade therapies ; howeverThe myeloid cell compartment constitutes another major player in the TIME, and the inflammation associated with tumor and metastasis recruits high amounts of macrophages in the stroma, forming a sort of barrier to obstacle lymphocyte infiltration within the tumor mass . In thisRM cells as well as by the higher percentage of contacts between tumor cells and CTL. On the other hand, OPSCC arising in women apparently develop also complex mechanisms of resistance to counteract such more efficient initial immune recognition and response, as revealed by the higher abundance of Treg cells, the higher expression of multiple checkpoint molecules, and the enrichment of interactions between inhibitory elements and CD8+ lymphocytes. Conversely, we found that the TIME of men secondary lesions was characterized by lower amounts of tumor cells expressing HLA class I, which could lead to a less efficient presentation of tumor neoantigens and potentially explain the poorer immune infiltration. Overall, our results are in line with previous studies performed in different type of malignancies [Sexual dimorphism has been recently ascribed as a relevant factor for cancer incidence and survival , even thgnancies –66, and gnancies , 66, 67,Finally, some considerations about the limitations of this work. First, there are potential biases due to the retrospective nature of the study, and results must be considered as hypothesis-generating only. Second, the sample size of our cohort is relatively small, and therefore further studies with more OPSCC patients stratified by HPV status are warranted to fully validate our findings. In this regard, however, we want to stress that such patient cohort is quite homogenous in terms of HPV status, sex and anatomical sub-sites of primary and secondary lesions. The third limitation is that the quantity of smoking/drinking is not defined, since we collected data from electronic medical records that usually do not report such data. Fourth, since the main aim of the study was to characterize the TIME both in primary lesions and related lymph node metastases, the present series consists only of patients undergoing upfront surgery that allowed to collect both types of specimens. While this makes the study population more homogeneous, the results relating to the impact of TIME on the outcome can not be extended to patients undergoing upfront (chemo)-radiotherapy. Finally, since only FFPE material was available, the evaluation of the HPV status was based on the double positivity for high-risk HPV-DNA and p16. Although detection of E6 and E7 mRNA would have provided more robust data, double positivity for HPV-DNA and p16 was observed to be the strongest surrogate marker for transforming HPV infection , 69.Our study clearly establishes that remarkable differences exist in the immune infiltrate between “T-cell-inflamed” HPV-positive and “T-cell-non-inflamed” HPV-negative OPSCC, and that these features are conserved both in primary and metastatic lesions. Moreover, although TAMs and checkpoint molecules expression are generally regarded as immunosuppressive elements, they are not necessarily synonymous of tumor immune evasion and may reflect an ongoing antitumor immune response. Furthermore, we highlight sex-specific differences in the TIME composition of OPSCC and in their prognostic value. Based on these observations, our study provides the rationale for the integration of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and at the same time advances the notion that approaches combining ICI with tumor-specific T cell response inducers or TAM modulators might be beneficial also for the HPV-negative “cold” counterparts.Additional file 1: Supplementary Table S1. Automatic definition of cell types by NanoString nSolver Software. Supplementary Table S2. Automatic definition of immune pathways by NanoString nSolver Software. Supplementary Fig. S1. Representative images of cell-to-cell distance analyses. (A) For mean distance between different cell subtypes, the nearest neighbors analysis was used. The mean distance between tumor cells (light blue dots) and the nearest CD8+ cells (red dots) is represented in the figure as an example. (B) The count within analysis was employed to calculate the percentage of reference cells, among the total number of reference cells, which are present within a 20 μm radius from at least one cell of a different phenotype. The percentage of tumor cells (light blue dots) within a 20 μm radius from a CD8+ T lymphocyte (red dots) is represented in the figure as an example. Original magnification X20. Supplementary Table S3. Differentially expressed genes (DEGs) between HPV-positive and HPV-negative (used as baseline) OPSCC patients. Supplementary Fig. S2. Differential expression of immune-related pathways and cell type genes in HPV-positive and HPV-negative OPSCC. Trend plots depicting differential expression of predefined (A) pathway genes and (B) gene expression-based cell types in HPV-positive and HPV-negative OPSCC. Supplementary Table S4. Correlation analysis between immune cell populations in HPV-positive and HPV-negative primary tumors and metastases. Supplementary Fig. S3. Immune cells in primary tumors and related metastases. Density (number of cells/mm2) of different immune cell populations in HPV-positive and HPV-negative primary tumors and metastases. Supplementary Fig. S4. The immune cell contexture of metastases correlates with patient outcome. (A-C) Kaplan-Meier survival curves for disease-free survival according to the immune cell composition of (A) the entire cohort (n = 39), (B) HPV-positive (n = 24) and (C) HPV-negative (n = 15) lymph node metastases. The median cut-off of each immune cell subset density was used to separate high and low infiltrated groups. Log-rank p values, hazard ratios (HR) and 95% confidence intervals (CI) are reported in each graph. Supplementary Fig. S5. The density of immune cells differs between females and males with HPV-positive lesions. Density (number of cells/mm2) of different immune cell populations in females and males with HPV-positive (A) primary tumors and (B) metastases.