TP53, and p16 overexpression, resembles that of HPV-driven disease at other locations with implications for future therapy.Carcinomas arising in the lacrimal drainage system (LDS) are rare but notoriously aggressive tumors, causing substantial morbidity and mortality. The molecular drivers of the disease remain unexplored despite being a prerequisite for identifying targets for future prognostication and therapy. Therefore, we aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. By detecting transcriptionally active HPV in 80% of LDS papillomas and 67% of LDS carcinomas, we suggest HPV to be an important contributor to carcinogenesis in this location. Further, the genomic profile of the HPV16-positive carcinomas, with activating mutations in the PI3K-AKT signaling cascade, wildtype status of n = 17) and LDS carcinoma (n = 15) were included. There was a male predominance (68%) and a median age at diagnosis of 46.0 years (range 27.5–65.5 years) in patients with papilloma and 63.8 years (range 34.0–87.2 years) in patients with carcinoma. Transcriptional activity of the HPV E6/E7 oncogenes was detected in the whole tumor thickness in 12/15 (80%) papillomas  and 10/15 (67%) squamous cell carcinomas (SCC) (HPV11: 3/15 (20%) and HPV16: 7/15 (47%)). Pathogenic variants in PIK3CA, FGFR3, AKT1, and PIK3R1, wildtype TP53, p16 overexpression, and deregulated high-risk E6/E7 transcription characterized the HPV16-positive SCC. The deregulated pattern of HPV E6/E7 expression, correlating with HPV DNA presence and p16 positivity, supports a causal role of HPV in a subset of LDS papillomas and carcinomas. The viral and molecular profile of LDS SCC resembles that of other HPV-driven SCC.The pathogenesis of squamous cell neoplasms arising in the lacrimal drainage system is poorly understood, and the underlying genomic drivers for disease development remain unexplored. We aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. The HPV analysis was performed using HPV DNA PCR, HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic characterization was performed by targeted DNA sequencing of 523 cancer-relevant genes. Patients with LDS papilloma ( Carcinomas of the lacrimal drainage system (LDS)—hence carcinomas derived from the epithelial lining of the lacrimal canaliculi, lacrimal sac, or nasolacrimal duct—are notoriously aggressive tumors constituting challenges in diagnosis and treatment. Since 1960, approximately 550 cases of LDS carcinomas have been reported in the scientific literature [n = 27 cases) [Besides age, the proposed risk factors for LDS carcinoma include chronic dacryocystitis, human papillomavirus (HPV) infection, and LDS papilloma . The ass7 cases) ,4,5,6. APatients with a histopathological diagnosis of papilloma or a squamous cell carcinoma of the LDS diagnosed in Denmark between January 2000 and January 2020 were included in the study by searching the Danish Pathology Register and manually reviewing the pathology records at the Eye Pathology Section, Department of Pathology, Copenhagen University Hospital Rigshospitalet, Denmark . Two papThe methods for HPV detection, immunohistochemistry, and DNA sequencing have been described previously  and are The Gene Read DNA FFPE kit  was used for DNA extraction using the QIAcube (Qiagen). DNA quantification was performed on a Qubit DNA HS assay kit .L1 gene with an expected amplicon size of 150 base pairs. The amplicons, negative controls (containing H20), and positive controls (containing a pool of HPV-positive tumors) were visualized by gel electrophoresis. All HPV-positive amplicons were genotyped by sequencing using the KAPA HTP Library Preparation Kit . The NEXTflex-96 DNA Barcodes  were ligated. DNA quantity was measured by the Qubit dsDNA BR Kit on a Qubit Fluorometer (Invitrogen). The High Sensitivity D5000 kit (Agilent) was used for quality evaluation of the purified library by automated electrophoreses . Sequencing was performed on a MiSeq . The HPV reference genomes (from the papillomavirus database “PaVe” at https://pave.niaid.nih.gov/ (accessed on 1 March 2022)) were used to map the reads.A PCR of the housekeeping gene GAPDH using the GAPDH-a/GAPDH-b primers was performed in all cases to ensure the integrity of the DNA. We expected a 200 base pairs amplicon size for GAPDH. HPV DNA PCR was performed using the GP5+/6+ primers that target a conserved region of the HPV The evaluation of the transcriptional activity of the HPV oncogenes E6 and E7 was performed using mRNA in-situ hybridization following the same procedure as previously described . We usedAll specimens were evaluated for pan-cytokeratin (CK-AECAM) and p16 expression. We performed immunohistochemical evaluations on 4-µm-thick sections using the following clones: CK-AECAM  and p16  Staining for CK-AECAM expression was performed on a Dako Autostainer Link 48 (Agilent) using the EnVision Flex+ Detection Kit . Staining for p16 expression was performed on a Ventana Benchmark Ultra  using the UltraView/Optiview detection kit (760-500/760-700) following the manufacturer’s instructions. p16 expression is used as a phenotypic marker of the HPV E7-mediated degradation of the retinoblastoma protein, thereby acting as a surrogate marker for HPV E6/E7-transforming HPV infections. p16 evaluation was performed prior to HPV analyses and thereby blinded for HPV status. Cytoplasmic and nuclear staining in ≥75% of the cells were considered p16 positive.https://support.illumina.com/downloads/trusight-oncology-500-reference-guide 1000000067621.html (accessed on 1 March 2022)). The TSO500 panel covers 523 cancer-related genes  preparation kit  following the TSO500 reference guide  was used to convert raw sequencing data to FASTQ files, followed by a quality assessment of the reads by the FASTQC v.0.11.85 . The reap-value of <0.05 was considered statistically significant.The statistical analyses were performed using the statistical software R for Windows version 3.6.1. . Average values were described as the median value (range), and an unpaired t-test was applied to compare continuous variables. The progression-free survival probability, defined as time to recurrence, lymph node or distant metastasis, or death, whichever occurred first, was illustrated by a Kaplan–Meier curve. Only descriptive statistics were applied due to the limited sample size. A n = 12) and inverted papilloma (IP) (n = 5)) and 13 patients diagnosed with a carcinoma (SCC (n = 10) and NKSCC (n = 3)). Two metachronous carcinoma  evolved from papillomas, and the total number of carcinomas in our cohort was therefore 15. The base line characteristics are summarized in p = 0.012). All tumors originated from the lacrimal sac epithelium, except two papillomas originating from the lacrimal canaliculi.A total of 30 patients were identified in the database searches; 17 patients with a lacrimal drainage system papilloma (exophytic squamous papilloma (n = 11) reported epiphora as the initial symptom, with a broad range in duration . Six patients (55%) further reported signs of dacryocystitis with redness, swelling of the medial canthus and palpebra, tenderness, and/or purulent discharge. A medial canthal mass, ulceration, and perforation of the skin were reported in four patients (36%). At diagnosis, six (40%) carcinoma patients had bone invasion and two (13%) had perineural invasion. Ten out of fourteen (71%) had positive surgical margins and were treated with adjuvant radiotherapy, except in one case. Orbital exenteration was performed in two patients. Metastases were reported in three cases . All three patients had local recurrence before developing metastases. The median progression-free survival was 34.1 months  among the papillomas and 22.7 months  among the carcinomas  and was consistently negative in all HPV DNA-negative cases available (4 out of 8). A deregulated expression pattern of HPV E6/E7—hence expressed throughout the epithelium in both benign and malignant tumors . All HPVTwelve SCCs were available for DNA sequencing, including four carcinomas with transitional cell morphology . The medPIK3CA , BCR (p.V1094fs*17), TGFBR2 , FGFR3 (p.S249C), PIK3R1 , and TP53  were altered in more than one case. HPV16-positive cases  were characterized by pathogenic variants in PIK3CA (3 out of 7), FGFR3 (2 out of 7), PIK3R1 (2 out of 7), AKT1 (1 out of 7), and wildtype TP53 . Only two HPV-negative carcinomas were available for analysis. The first harbored a likely pathogenic variant in TP53 (p.S37fs*7) and a double-hit, truncating variant in KMT2D (p.E81* and p.S831*), whereas no somatic pathogenic or likely pathogenic variants were detected in the second HPV-negative sample.We present the clinical, histopathological, and genetic features of 32 consecutive patients with papilloma and carcinoma of the lacrimal drainage system in a Danish nationwide cohort. The most important findings were: (1) Deregulated transcription of HPV E6/E7 is highly prevalent in both papillomas and squamous cell carcinomas—supporting a causal role of HPV in these tumors, and (2) HPV16-positive LDS SCC has a genomic profile resembling their HPV-positive counterparts of other locations.The papillomas and the NKSCC with a transitional cell morphology harbored the low-risk genotypes HPV6 and 11, in one case co-existing with the high-risk genotype HPV16. The detection of transcriptionally active low-risk HPV in the exophytic LDS papillomas is in line with previous studies , and it Despite the designation as a “low-risk” genotype, recent studies support that HPV11 may contribute to malignant transformation ,22,23 anIn contrast to the HPV11-positive carcinomas, which all had a non-keratinizing transitional cell morphology, all HPV16-positive carcinomas were non-keratinizing SCC. The oncogenic properties of HPV16, mainly related to the effects of the E6/E7 oncogenes, are well-documented and are an established cause of carcinoma development in the anogenital and head and neck region ,26. The PIK3CA, FGFR3, and AKT1, and loss-of-function variants in PIK3R1  had activating or loss-of-suppressor variants affecting the PI3K-AKT signaling pathway that causes signaling related to cell growth, cell-cycle progression, and survival, giving rise to a selective growth advantage. All HPV16-positive carcinomas were TP53 wildtype, a general marker of HPV-positive carcinomas across sites. Since the HPV E6 targets p53 for degradation, a gene-level loss-of-function of TP53 is not necessary for a functional blockage of p53. Thereby, the genomic profile of HPV16-positive LDS SCC resembles that of other HPV-positive head and neck SCC (HNSCC) [EGFR wild-type, supporting the mutual exclusivity between high-risk HPV positivity and EGFR mutation observed in the SCC of the neighboring sinonasal cavities [TP53 variants, similar to the keratinizing and HPV-negative sinonasal SCC [The HPV16-positive SCCs exhibited a homogeneous molecular profile and seem to be driven by PI3K/AKT signaling pathway activation due to activating, pathogenic variants in n PIK3R1 . The gaie cervix , orophare cervix , and cone cervix  and are e cervix . Similarrognosis . Further (HNSCC) . All HPVcavities ,34. MoreThe present study was limited by the rarity of the tumors and the formic acid preparation for bone decalcification, which meant only a small number of included samples. This reduced the robustness of our findings and the prognostic role of HPV in these tumors could not be assessed. Furthermore, the lack of normal control tissue to filter germline variants forced us to apply stringent filter criteria in the variant call to avoid spurious calls. All materials used for molecular analyses was formalin-fixed and paraffin embedded, causing potential artifacts in the molecular analyses. A future prospective setup could overcome these challenges. On the other hand, our study is strengthened by the multimodal approach for HPV testing, the deep coverage achieved in the DNA sequencing, and the nationwide setup including consecutive cases with a complete follow-up due to national patient registries.TP53, frequent pathogenic variants in PIK3CA and FGFR3, and expression of HPV16 E6/E7, supporting a causal role of HPV16 in these carcinomas. The similarity across sites may be valuable for the future treatment of these rare carcinomas where large studies are hardly feasible and for inclusion in basket trials. Substantial efforts are put into trials of HPV-positive HNSCC, and these results may be important for future patients with HPV16-positive LDS carcinomas.We present the largest series to date investigating the transcriptional activity of HPV in LDS papilloma and carcinoma and the correlating histopathological and genomic alterations in a consecutive, nationwide cohort. HPV16-positive LDS SCCs harbor a molecular profile resembling other HPV16-positive carcinomas with p16 expression, wildtype status of


At this moment, reconstructing historical models is (mostly) a manual and very time-consuming operation, and it is plagued by inaccuracies in the 2D maps. In this paper, we present a new methodology to reconstruct 3D buildings from historical maps, we developed it with the aim of automating the process as much as possible, and we discuss the engineering decisions we made when implementing it. Our methodology uses extra datasets for height extraction, reuses the 3D models of buildings that still exist, and infers other buildings with procedural modelling. We have implemented and tested our methodology with real-world historical maps of European cities for different times between 1700 and 2000.Historical maps are increasingly used for studying how cities have evolved over time, and their applications are multiple: understanding past outbreaks, urban morphology, economy, etc. However, these maps are usually scans of older paper maps, and they are therefore restricted to two dimensions. We investigate in this paper how historical maps can be ‘augmented’ with the third dimension so that buildings have heights, volumes, and roof shapes. The resulting 3D city models, also known as  Historical maps are often the only information available about the pre-satellite and digital map era; they contain information that cannot be found elsewhere  city models, also known as historical 3D city models, which we refer to as 3D models at different times containing buildings and/or other features in the built environment. Some of these models can be highly detailed and realistic, see for instance Frischer et al.  the processing of the historical maps to extract the building plots, (2) the subdivision of these building plots into individual building footprints and (3) the reconstruction of 3D buildings. The only user interventions that are needed to implement it are the generation of training and testing datasets for the building plot extraction step—this is further detailed in Section The first step of the workflow is the extraction and vectorisation of the building plots from the input historical map. We started by digitalising the historical map using the method of Gobbi et al.  as impl makes usa. The turning function expresses how the accumulated tangent angle changes with the normalised accumulated edge length  perpendicularly along its longest edge.Case 3: Two rows of building footprints fit inside the building plot. The building plot is split along its longest median to create two polygons (i.e. the two building rows), which are individually processed using case 2.Case 4: As the building plot is too big to be split entirely into building footprints, we assume that it contains an interior courtyard or continuous space made of gardens, an arrangement that is common in many European cities. We use the straight skeleton , which are needed to implement the map alignment. User-defined building length and depth can either come from the literature, i.e. using standard building depth and length in the region of interest, or they can be based on the average length and depth of the aligned (historic) building footprints if the map alignment was implemented (or the average length and depth measured on current building footprint data). Similarly, for the area threshold (case 1), one can use the minimum building footprint area in the aligned building footprints .The output from this step is a dataset with at most two types of building footprints: the aligned ones and the ones generated with 2D procedural modelling—which we refer to as ‘2PM building footprints’ from this point.The last step of the methodology deals with the reconstruction of 3D buildings. The final historical 3D city model is reconstructed by: (i) generating height attributes; (ii) reconstructing 3D buildings with 3D procedural modelling; and (iii) generating a valid CityJSON file Ledoux, . CityJSOThe building footprints need to have a ground and a roof height to be reconstructed in 3D. As the aligned building footprints already have such height attributes, only the 2PM building footprints need to be assigned some. For assigning the ground height, we used the method of Dukai et al. , which mcomputer generated architecture (CGA) grammar of Parish & Müller (Computer Generated Architecture for Blender (BCGA) addon, which is an open source implementation of the CGA grammar. We use three types of rules:Rule 1: extrudes the building footprint at its assigned roof height;Rule 2: decomposes the resulting 3D shape into different parts  and,Rule 3: generates a roof above the top face of the 3D shape.The BCGA addon already allows the automatic generation of different types of roofs: flat roofs, hip roofs, gable roofs, and mansard roofs  3D city models from vector data using the & Müller . Their mofs Fig. . Other rofs Fig. .Fig. 9Coth century until the beginning of the 20th century, while flat roofs started to be more widely used in Northern Europe from the 19th century   (in Dutch), which is a register containing the location information and several attributes about all buildings in the Netherlands. We also used the 3D BAGIf available, building footprint datasets enriched with the roof height and the year of construction of the buildings can be used to implement the map alignment (Step 2). For Delft, we used the Actueel Hoogtebestand Nederland 3 (AHN3)3D point cloud datasets are needed for inferring the ground height of the building footprints. We used for Delft the The performance of the building plot extraction was assessed using the F-score metric, which measures both the precision and the recall ; modifying the methodology so that building footprints are used would be a simple task.We have presented our methodology for reconstructing historical 3D city models from historical maps, maximizing the automation process, and our implementation is available as open source code. We have tested our methodology with historical maps of Delft and Brussels at different time periods, showing the suitability of our methodology to be applied for different European cities and historical map collections. Our methodology can be extended to any city in Europe For other users focusing on different European cities, the implementation of the methodology depends on data availability. If all the datasets are available, the entire methodology can be implemented, including the map alignment, such as for historical maps of Delft. In the opposite case, this step is skipped, such as for historical maps of Brussels. This difference in data availability also influences the methodology running time, which can be large depending on the size of the study area. This is essentially due to the 3D procedural modelling step, which is more time-consuming than the other parts of the methodology. Therefore, if remaining historical buildings can be recovered from an existing 3D city model using the map alignment, the processing time is significantly reduced. Furthermore, using or not the map alignment influences the way the roof height is assigned, either making use of the aligned building footprints and their height attributes or using regulated values from the literature. Using the first option might be more accurate at the individual building level as the roof height is defined using a spatial process , but it might also be more prone to interpolation errors or irregularities. Besides, using the information in the neighbours supposes that a sufficient percentage of the historical building remains. When not enough buildings are remaining, the shorter methodology version  might be preferred, as implementing the entire methodology would mean assigning the roof heights relying only on a few  buildings.Two main factors influence the performance of our methodology for other study areas: the quality of the scanning process and the cartography rules of the historical maps. The quality of the scanning process influences whether defects affect the scanned historical maps, such as the aliasing and false colour effects. These two phenomena respectively lead to mixed pixels and high colour variations inside geographic features, which both influence the quality of the map digitalisation. They can greatly influence the building plot extraction, leading to lower precision and recall values. The scanning process also deals with the choice of a spatial resolution for scanning the historical map, and this parameter highly influences the results obtained. A higher spatial resolution means more details in the contour of the geographic features and smoother transitions between pixels of different categories of features (mixed pixels). Moreover, the spatial resolution is an important factor to consider when using an object-based classification because it requires the geographic features to be large enough to be identified as objects. If the spatial resolution is too low, some geographic features may only be represented by a few pixels and are then not identified as objects. The second factor that strongly influences the results is the cartographic rules that were used to create the historical maps. For maps with strict cartographic rules, the building plot extraction performs better because all categories of geographic features are depicted in different colours. Contrarily, when different categories of features are represented using the same symbology, it is less evident for the algorithm to differentiate them.One great advantage of our methodology is its flexibility. First, we tried in this research to find solutions around data availability, and therefore we proposed a methodology with two alternative options depending on data availability. Second, our methodology can be implemented with various collections of historical maps, while many of the existing methods that we have investigated only work for a specific map collection. However, a consequence of this is that the results are not perfect, in the sense that some errors or discrepancies are sometimes introduced. For instance, churches and other special buildings are not always recovered with this methodology as they are often represented in different ways on historical maps , and thus it is difficult to implement a methodology that can recover them automatically regardless of the way they are represented. Therefore, in certain cases, these buildings should be manually added. The amount of manual processing that could be implemented to have the most accurate results depends on the input historical maps. If the map was not properly scanned, more manual work will be required for compensating the low recall and precision values. On the contrary, high recall and precision values would require almost no manual processing.Another consequence of that flexibility is that our methodology relies on user-defined parameters. This is sometimes considered as a roadblock towards automation as it might require lots of trial and error work for finding the most suitable values. However, based on our experiments with different collections of historical maps, we provide recommendations for an optimal choice of values for these parameters. Besides, as our methodology is implemented in different steps, users wanting to adjust these values can proceed step by step, which makes it easier to identify the most suitable values. Our methodology also relies on a series of thoughtful assumptions made in order to reconstruct plausible buildings, as we are limited by the amount of historical information available. These assumptions might fail in specific cases or for certain regions. In particular, the 2D procedural modelling algorithm is based on a set of predefined rules, which might not always hold. First, the algorithm subdivides building plots into terraced houses, which might less suit peri-urban areas organized in detached houses/villas. Also, buildings are created with an alignment on the street side, although neighbouring buildings are sometimes built at varying distances from the roads. Specific to case 4 Fig. , certainFinally, with this work, we provide a methodology that allows the reconstruction of a historical 3D city model, with limited user intervention, that probably looks like what the city looked like at a given time. We reconstruct multipurpose LoD2 building models that can be useful for disease mapping (through population estimation), visibility analyses and other spatial analyses. Although our models are not perfect and are affected by discrepancies , we argue that they are a good mean to automate the reconstruction process, while reconstructing an historical 3D city model manually would be very time and money-consuming. Hence, our models supplement the manually made ones when time and money are limited, in two main ways. First, they can act as a starting point; one could start from our automated model and further modify it by simply replacing the 3D buildings of its choice (which is easily done as we use semantic 3D city models). It is also possible to automatically add façade details  to simple building models like ours, see Zhang et al.  and WangSome parts of our methodology could benefit from additional focus to improve the general workflow. Our methodology could be improved by making it possible to create buildings with non-convex footprints with 2D procedural modelling. The BCGA addon algorithm should then also be extended by adding rules for reconstructing cross hip/gable roofs Fig. .Besides improving our methodology, there are also some areas that we did not explore and that could be interesting to investigate in the future. One such area is the use of other types of historical sources in addition to historical maps, which could bring added value to the historical 3D city models reconstructed. For instance, the actual building height could be extracted from historical aerial images using machine learning techniques  Lánský, , or from


The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV Historiresponse ,4,5.+ and CD8+ T lymphocytes in a tumor invasive margin in colorectal carcinoma was recently introduced as an immunoscore, which serves as a prognostic factor for colorectal carcinoma patients and is now approved as an addition to the conventional tumor-node-metastasis (TNM) staging [One of the factors that can explain the difference in clinical outcomes between patients with different tumor etiology is their immune response. The phenotypes and numbers of tumor-infiltrating lymphocytes (TIL) as one of the components of the tumor microenvironment (TME) were described as a significant prognostic factor in many tumors, including HNSCC ,6,7,8,9.+ and CD8+ T lymphocytes are important TIL subpopulations in the TME. As CD4+ T lymphocytes include diverse functional subpopulations, their effect on prognosis is difficult to decipher. Many studies do not analyze T lymphocyte spatial location in the TME and HPV status of HNSCC tumors, which modify the observed correlations [+ T cells represent the main anti-tumor TIL population and are considered as a positive prognostic factor in the majority of tumors [+ TILs infiltration was also shown to be associated with better survival of patients with OP, hypopharyngeal, and laryngeal SCC [+ regulatory T cells (Treg) activity or by the PD-1 (programmed cell death 1)/PD-L1 (PD-1 ligand) interactions [CD4elations . CD8+ T f tumors ,11,12. Hgeal SCC ,13. The ractions . Moreoveractions . In this+ or HPV−. All patients signed an informed consent form and completed a questionnaire about risk factors for HPV infection and HNSCC induction when enrolled in the study approved by the Ethical Committee of the Motol University Hospital in 2001.Samples of primary HNSCC  and non-malignant tonsillar tissue were provided by the Department of Otorhinolaryngology and Head and Neck Surgery, Motol University Hospital, Prague. They were collected, processed, and characterized in previous studies between 2001–2012 ,16,17. B® Plus microscope slides  and confirmed by a pathologist for the presence of tumor tissue. The slides were deparaffinized at 60 °C for two hours, washed in xylene for 3 × 10 min, hydrated with descending grades of ethanol , and fixed for 20 min in neutral buffered formalin, pH 7.2–7.4 . Heat-induced epitope retrieval (HIER) was carried out by using microwave (96–98 °C) in buffer of pH 9.0  or pH 6.0 . After 10 min of blocking using Antibody Diluent/Block (Akoya Biosciences), the slides were incubated with the primary antibodies listed in From each FFPE tumor tissue block, 2 μm sections were prepared on SuperfrostWe designed four different antibody panels. A staining order for primary antibodies was set with respect to different HIER length requirements and stability of the epitopes after multiple HIER treatments. For matching fluorophores with the primary antibodies, possible colocalization of antibodies in the panel was considered. After each round of staining, the complex of primary and secondary antibodies was removed by HIER using the antigen retrieval (AR) buffer, optimal for the following antibody in the panel. To ensure complete removal of the antibodies complex, stripping quality controls were performed. The final settings for each panel with optimized dilutions of antibodies are specified in All slides were snapped by using the MantraSnap 1.0.3 software included in the Mantra Quantitative Pathology Workstation (Akoya Biosciences) for DAPI, FITC, Cy3, Texas Red, and Cy5 data acquisition. From each tissue section, five different regions of interest were randomly selected with a 20X objective. Images were analyzed using the InForm 2.4.6. software (Akoya Biosciences). Four separated algorithms for trainable tissue and cell segmentation and cell phenotyping were prepared. The tissue was segmented into the tumor parenchyma, stroma, and background compartments and checked by the pathologist. The algorithms were trained to specifically phenotype the cells according to different expression of markers. The optimization workflow of algorithms is listed in + and HPV− groups. Patients were grouped based on HPV E6 mRNA detection as HPV+ or HPV−. The numbers of cells of distinct phenotypes were evaluated separately for the parenchyma and the stroma and counted per Mpx for the five regions of interest. Differences between parenchyma and stroma infiltration were evaluated using the sign test, and the Mann–Whitney U test was used to detect differences between the HPV+, p16−), HPV status , and cell numbers of distinct phenotypes per Mpx separately in the parenchyma and the stroma or in total were evaluated. The zero values in the cell numbers were substituted by the 0.1 value as the lowest non-zero value. The models were analyzed using the Bayesian information criterion (BIC). For all the statistical tests, a p value of <0.05 was considered as a significant difference. The statistical analyses were performed using the GraphPad Prism 8.4.2. software  and R version 4.0.2 (https://www.R-project.org/ accessed on 02 November 2022) [The Cox proportional hazards model was applied for the overall survival (OS) and disease-specific survival (DSS) analyses. The following factors were included: age, gender, education , smoking history , alcohol use , tumor location , pathological tumor extension (pT1–4), pathological nodal status (pN0–3), tumor stage , metastasis (pM1), tumor grade (1–3), and relapse . As the samples were collected between 2001 and 2012, the tumors were classified according to the TNM 7 staging system, hence, the p16 positive tumors do not have a separate category. Furthermore, the p16 status (p16er 2022) .+  and HPV−  groups. Of the 97 patients, 18 relapsed and 43 died. The patient demographics and clinical characteristics are listed in From the previously characterized cohort ,16, 97 p+CD4+ (Th), CD3+CD8+ (Tc), and Treg cells was detected in the stroma, compared to the parenchyma . For all measured populations, we observed a highly or moderately positive correlation of TIL counts/Mpx between the parenchyma and the stroma compartments . The Treg counts did not statistically differ between the two tumor compartments neither in the HPV+ nor in HPV− groups. However, we observed statistically non-significantly higher Treg counts in HPV+ patients . Increased levels of CTLA4+CD4+ T cells were detected in the stroma and parenchyma of HPV− tumors, but the difference was statistically significant only for the stroma (p = 0.0021). No difference was observed in the CTLA4+CD8+ T cell numbers between the two groups in either the parenchyma or the stroma . The ICOS+ Treg subpopulation was more abundant in the HPV− than in the HPV+ tumors  while the ICOS− Treg (ICOS−FOXP3+CD4+) numbers were higher in the HPV+ tumors  (As we did not observe differences in Treg numbers between the HPVenchyma) .+CD8+ T cells related to better OS (+/Treg ratio, which was associated with better survival, along with the positive HPV RNA status and a higher number of stromal PD-1+CD8+ T cells (To evaluate the influence of TIL infiltration on prognosis, we used Cox models. We fitted 38 models for OS and DSS analyses. Models with and without HPV status and with the evaluation of the parenchyma and stroma separately or combined were tested. The models with the best BIC score are listed in etter OS . The bes T cells .+CD8+ T cells, and lower tumor stage or nodal status were connected with better OS and DSS, and low number of all CTLA4+CD8+ T cells was linked to better OS (In the models where the IHC markers were evaluated without tissue segmentation but HPV status was included, HPV RNA positivity, higher number of all PD-1etter OS .+CD8+ T cells, a higher number of all CTLA4+CD8+ T, and tumor size or tumor stage. Similarly, for DSS, a lower number of stromal PD-1+CD8+ T cells, together with the number of stromal CD8+ T cells, and higher age related to worse survival (In models not including the HPV status, the main predictors of worse OS were positive smoking history, a lower number of parenchymal PD-1survival .+CD8+ T cells to be a positive prognostic predictor in all models. Additionally, rising tumor stage or nodal status appeared to be a negative predictor for both OS and DSS (+CD8+ TIL can serve as an independent prognostic marker for HNSCC patients and may improve the current prognosis assessment.We have shown the level of stromal or parenchymal PD-1 and DSS . Our datThe major aim of this retrospective study was to analyze the tumor microenvironment in HNSCC of different etiology in order to explain the better prognosis of patients with HPV-positive tumors and to evaluate non-viral markers in the populations of TILs as a possible independent prognostic tool for HNSCC patients. The prognostic significance of the TIL count was evaluated in several malignancies and recently this parameter has been approved as an additional characteristic to the TNM classification for colorectal carcinomas ,6,7,8,9.+CD4+ and CD3+CD8+ T cells. Higher infiltration by T cells of HPV+ tumors than HPV− tumors may be explained by the presence of viral antigens, which stimulate the immune response more strongly. Despite the differences in their designs, several studies have come to similar results. Badoual et al. found higher CD4+ T cell counts in the stroma of HNSCC tumors [+, CD4+, and CD8+ T lymphocytes in both the parenchyma and the stroma of HPV+ tumors [+ tumors, the difference from HPV− tumors was not statistically significant. The same was shown by IHC [+ cohort, in comparison to HPV− tumors, were only reported by a single study for tonsillar SCC [+ Treg cells were more prevalent in the stroma and parenchyma of HPV− tumors, but the difference was statistically significant only for the stroma. Inversely, ICOS− Treg counts were higher in both compartments of HPV+ tumors with a statistically significant difference for parenchyma location. To our knowledge, this is the first study analyzing the spatial prevalence of ICOS on Treg cells in HNSCC of different etiology, and the results suggest that these cells can have different function in HPV+ and HPV− tumors. In our study, HPV-induced tumors were more infiltrated by CD3C tumors . Other s+ tumors ,26,27,28n by IHC , gene exn by IHC . Signifillar SCC . To analllar SCC . ICOS+ T+CD4+ and PD-1+CD8+ T cells in both the parenchyma and the stroma of HPV+ tumors, compared to HPV− tumors. Higher levels of PD-1+CD4+ and/or PD-1+CD8+ T cells and PD-1 mRNA were also observed in HPV+ than in HPV− HNSCC by others [+ than CD8+ T cells in HNSCC [+CD8+ cells in the stroma, compared to the parenchyma but with no difference in the PD-1+CD8+ counts between HPV+ and HPV− OPSCC [Furthermore, we have detected significantly higher counts of PD-1y others ,30,31. Iin HNSCC ,32, whicV− OPSCC . +CD8+ T cells is associated with both better OS and DSS of OC and OPSCC patients in all of the models. When HPV status was included, the multivariate analysis confirmed HPV positivity as the strongest independent positive prognostic factor for OS and DSS [Thanks to the long-term follow-up, we were able to explore the impact of TILs infiltration on prognosis, which is the main benefit of this study. We fitted several models, including clinicopathological characteristics, HPV RNA status, and tissue compartment. We were also interested if any of the studied population of TILs would be an independent prognostic marker for HNSCC regardless of the HPV association. The influence of TILs on prognosis was also analyzed by others who, unfortunately, did not take account of HPV status and/or spatial distribution was not considered, which makes the comparison of results difficult. We found that a higher number of parenchymal or stromal PD-1 and DSS ,4. + T cells was associated with better OS or DFS [− cohort, it has been suggested that PD1+CD8+ TIL cells act as a tumor-specific population, compared to the PD1−CD8+ TILs and play a key anti-tumor role [+CD4+ T cells in HPV+ tumors and by PD-1+CD8+ T cells in HPV+ and HPV− tumors related to better OS than low infiltration by these cell populations [+CD8+ T cells in tumors suggest that patients contain tumor-specific T cells that can be activated and—before silencing by inhibitory molecules, such as PD-1—exert an antitumor effect, supplementing both radiotherapy and surgery and enhancing survival. Due to the limited number of markers, which can be detected using multispectral fIHC, we are not able to characterize the PD-1+CD8+ T cells in the more detailed manner, which is the limitation of this study. To describe the status of PD-1+CD8+ T cells in the TME, expression of other activation and/or exhaustion markers can be examined on a fresh tumor tissue, using other methods, such as flow cytometry. However, the results of our study and of others indicate that this marker should be included in the prognostic immunoscore for HNSCC [+ T lymphocytes on prognosis, which was also observed by others [+ T cells were analyzed in the context of prognosis, and none of these subpopulations influenced OS or DSS. In OC and OPSCC, a higher number of CD8S or DFS ,33,34,35S or DFS ,36,37,38S or DFS ,39. In tulations ,39. The or HNSCC ,39. The y others ,30. In o+ Treg cells in the TME have higher immunosuppressive capacity than the ICOS− Treg population [+ Treg counts found in the HPV− tumors may relate to worse outcomes of HPV− patients. In our study, despite the findings of higher ICOS+Treg cell counts in HPV− tumors, no influence of this subpopulation on OS or DSS was detected. Similar to Lukesova et al. who observed the phenomenon in the blood, in our models, we found that a low CD8+/FOXP3+ ratio related to improved OS and DSS [+/FOXP3+ ratio measured in HPV− tumor tissue microarray cores did not corelate with improved survival [+/FOXP3+ ratio, connected with better DFS in both HPV+ and HPV− groups [The level of Treg cells itself did not affect OS or DSS but was positively correlated with a better locoregional control in HNSCC . It was pulation . The hig and DSS . In the survival . However− groups . +CD8+ T lymphocytes improved the prognosis of the patients in all our models, which points out the importance of this marker and entitles it to be included in the prognostic immunoscore for patients with OC and OPSCC of different etiology.In this study, we evaluated the level of TILs in different compartments of the HNSCC microenvironment using multispectral IHC. We compared the TILs levels with respect to the presence of an active HPV infection and tumor compartment in a retrospective cohort of patients under long-term follow-up. The impact of different TIL infiltration levels on prognosis was studied. We revealed that higher levels of PD-1


Lactobacillus iners at baseline were less likely to have HPV clearance at month 12. Further analysis revealed a significant negative association between high abundance of L. iners and HPV clearance in patients who received non-operative treatment , but not in those who received operative treatment . Our findings provide new evidence for the potential role of VM in the persistent HR-HPV infections.Although vaginal microbiota (VM) may interact with human papillomavirus (HPV) infection and clearance, longitudinal data remain very limited. We aimed to investigate the association between VM at baseline and the clearance of high-risk HPV (HR-HPV) infection within 12 months. Cervical swabs were collected at diagnosis from 85 patients with HR-HPV infection and histologically confirmed cervical lesions, including cervicitis, low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion. Microbiome analysis was performed using 16S rRNA gene sequencing. Among the 73 women included in the analyses, HPV clearance was observed in 58.9% of the patients within 12 months. No significant difference was observed between the HPV-cleared and HPV-uncleared groups regarding age, disease stage, HPV subtype, VM community state types, and VM diversity (α and β). Women with the depletion of enterococcus ASV_62 and enrichment in  Persistent infection with high-risk human papillomavirus (HR-HPV) is the major cause of cervical cancer and its precursor lesion –3. AlthoMore recently, the upsurge in research on microbiome has shifted attention from known epidemiological risk factors of HPV infection, including parity, tobacco smoking, and oral contraceptive use to mucosal microbiota . AccumulLactobacillus spp. or a polymicrobial state with high diversity. Several cross-sectional studies  and low HPV prevalence is generally supported  is commonly categorized into community state types (CSTs), which were first proposed by Ravel et al. , and are studies –17 have upported . Contrar factors , 19. AddHerein, we conducted a longitudinal study to assess the impact of VM composition at baseline on the clearance of HR-HPV infections at 12 months based on a treatment cohort of 85 Chinese women with a single HR-HPV infection.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Scientific and Ethical Committee of the Shanghai First Maternity and Infant Hospital affiliated with Tongji University (protocol code: K08-018).Patients with histologically diagnosed cervical disease were enrolled between April 2015 and October 2016 at Shanghai First Maternity and Infant Hospital and then routinely followed up every 6 months. Cervical swab specimens at diagnosis were collected from every woman for HPV testing and microbiome analysis using 16S rRNA gene sequencing after inclusion in the cohort. Detailed description of the participants can be obtained from the original publication , and high-grade SIL (HSIL); only infected with one HR-HPV subtype (HPV 16/52/58); follow-up data available; and patients with same diagnostic conditions were treated according to a standardized protocol. Briefly, patients with HSIL received the loop electrosurgical excisional procedure (LEEP), and other patients with a lower pathological grade lesion (cervicitis and LSIL) received conservative management rather than immediate excisional treatment. Patients with a known malignancy disease or current pregnancy were excluded from the study.Based on pathological diagnosis and the treatment undergone, patients were divided into the HPV+/LSIL group and the HSIL group. Cervical swabs and cervical biopsies were collected on each follow-up visit for HPV DNA detection and pathological examination. Clinicopathological and follow-up data, including progress notes, clinical laboratory tests, and drugs and pathological reports were captured from the hospital electronic medical record system. All laboratory examinations and inpatient and outpatient electronic medical records were reviewed.The HPV-cleared group was defined as the presence of HPV at baseline turned negative at follow-up tests and no further positive HPV test and cytological or histological abnormality reports. The HPV-uncleared group was defined as persistent same-type HPV infection or pathological progress within a 1-year follow-up.HPV testing at each follow-up visit was performed using the HPV GenoArray test kit as previously reported . The absMicrobial genomic DNA was extracted from cervical swab samples, collected from the ectocervix and endocervix of the uterus of every woman by baseline cervical scrapings, using the FastDNA Spin Extraction Kit . Then, a nested PCR protocol was employed to amplify the 16S hypervariable region V4–V5 using the 16S universal primers: 515F 5’-GTGCCAGCMGCCGCGGTAA-3’ and 907R: 5’-CCGTCAATTCMTTTRAGTTT-3’. The dual-indexed amplicons were pooled according to the manufacturer’s instructions  and sequenced on the Illumina MiSeq platform to produce 2 × 300 bp paired-end reads as described previously.q-value of 20, and a maximum N base of zero. Thereafter, the processed reads were subjected to quality trimming, denoising, merging, and chimera removal to generate amplicon sequence variants (ASVs) using DADA2 , and those with more than two or five expected errors [maxEE = c] or derived from PhiX (rm.phix = TRUE) were discarded. After DADA2 denoising, the paired-end reads were merged with at least a 12-bp overlap. Chimera checking was conducted on the merged reads, and the recovered ASVs were summarized and used to generate the sequence table for the sequencing run. All ASVs were numbered in order.The raw demultiplexed sequences were firstly trimmed off primers from the paired-end reads, and a preliminary quality trimming was then conducted with Cutadapt v2.10 with the setting of discard untrimmed sequences, a minimum ng DADA2 . In thisLactobacillus spp. are essential for further analysis in VM studies, the classification of the ASVs annotated as Lactobacillus was improved by manually BLAST searching them in the National Center for Biotechnology Information database; a maximum of one mismatch was allowed from the alignment. If ASVs could not be annotated at the species level, they were then reannotated as “genus-level ASVs order.”RESCRIPt  was usedThe ASVs table was filtered to exclude sequences annotated as chloroplasts, plastids, or non-bacterial ASVs. Low-abundance ASVs present in only one sample and with a relative abundance lower than 0.01% across all ASVs were also filtered. To reduce the sampling heterogeneity, the ASV table was rarefied to the same reads per sample 100 times using a q2-repeat-rarefy plugin  on the QHierarchical clustering into CSTs based on VM composition and abundance was conducted according to the methods described by DiGiulio et al. . Specifit-tests, and Wilcoxon rank-sum tests were used for two-group comparisons as appropriate. For further analysis, numerical variables such as α-diversity indexes were categorized by 75th quartile scores.Normality tests for continuous data were assessed by Shapiro–Wilk tests. Chi-square tests, Fisher exact tests, PCoA analysis was performed to interrogate the robustness of group-wise clustering. Comparisons of group-wise β diversity (Bray–Curtis distance matrix) were assessed by permutational multivariable ANOVA using the Adonis function in the vegan package . LogistiP-value was adjusted for multiple tests using the Benjamini and Hochberg method. A p-value of <0.05 was considered statistically significant. Statistical analyses were performed with R statistical programming (R version 3.6.1).Lactobacillus capsule. No immunomodulatory medication usage was reported. At 12 months, 61.6% (45/73) of patients had cleared HPV infection, defined as the “HPV-cleared” group, and the remaining 38.4% (28/73) were classified as the “HPV-uncleared” group. The clearance rate was 54.9% among HPV+/LSIL patients and 77.3% among HSIL patients. This difference of clearance rate between the two groups was not statistically significant (p = 0.123). No differences in HPV clearance by HPV subtype were noted . Pairwise comparisons are shown in Eighty-five patients with a single HR-HPV infection and histologically confirmed cervical disease were enrolled in this study. Patients with incomplete clinical information (n = 2), low-quality sequence data (n = 1), or time to first follow-up visit over 1 year (n = 9) were excluded. Overall, 73 individuals were included in the analysis Figure 1A total of 2,785,739 high-quality sequences were obtained from 73 samples, with an average of 38,160 reads per sample. Following the removal of rare frequency , nonbacterial, unclassified, mitochondrial, and chloroplast ASVs, 530 ASVs were finally generated, and then all samples were rarefied to 8,556 reads 100 times to calculate diversity indexes.L. crispatus dominated, CST III was dominated by L. iners , and CST IV  was typified by a highly diverse microbiome rather than containing a major group.To explore CSTs and reduce dimensionality, a hierarchical clustering analysis was performed based on ASV-level data, and five major groups were identified  and the non–Lactobacillus-dominated group  . Both α-diversity and β-diversity analyses showed no significant difference between the HPV-cleared and HPV-uncleared groups Figure 3Enterococcus:ASV_62  was significantly more prevalent among HPV-cleared patients. Interestingly, patients with higher abundance of L. iners at baseline were more likely to fail to clear HPV infection at 12 months .Analysis of bacterial taxonomic categories of the microbiome associated with HPV-uncleared versus HPV-cleared was performed using DESeq2, which built a generalized linear model to validate the abundance of each taxon with adjustments for disease stage, age group, and HPV subtype. DESeq2 results showed that abundances of the two species (one specie enriched and one specie depleted in HPV-cleared group) presented a significant difference between two groups. The abundance of L. iners abundance and HPV clearance, a multivariable logistic regression model and stratified analyses were performed to assess the effect of L. iners abundance on HPV outcome status. The multivariable analysis revealed a significant risk effect of higher L. iners abundance  on HPV clearance  state, that patients with CST III (L. iners dominated) state at baseline were associated with a lower rate of HPV clearance. Previously, the possible link between L. iners and persistent HPV infection has been reported in several cross-sectional studies  or during menses or episodes of bacterial vaginosis, suggesting that this species is very flexible and has a remarkable ability to adapt to the fluctuating vaginal environment  is well documented . These were the top three subtypes with the highest HPV infection rates according to our previous study  and epidL. iners at diagnosis, rather than overall bacterial diversity, was negatively correlated with HPV clearance over 12 months, particularly in patients who received non-operative treatment. Our findings provide new evidence for the potential role of VM in the persistent HR-HPV infections. Further studies are needed to clarify the mechanisms by which L. iners promotes persistent HPV infection or lesion progression.In conclusion, this study among Chinese women suggested that the relative abundance of https://ngdc.cncb.ac.cn/gsa.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive  in National Genomics Data Center (Nucleic Acids Res 2021), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: HRA003171) that are publicly accessible at This study was reviewed and approved by Scientific and Ethical Committee of the Shanghai First Maternity and Infant Hospital affiliated with Tongji University. The patients/participants provided their written informed consent toparticipate in this study.Conceptualization: WS, HZ, ZL and KW. Methodology: WS and XH. Software: WS and LY. Validation: HZ and XC. Formal analysis: WS and HZ. Investigation, XH, LY and WS. Resources, LY, XH and KW. Data curation: XC and HZ. Writing—original draft preparation: WS, HZ and ZL. Writing—review and editing: WS, HZ, LY, XC, XH, KW and ZL. Visualization: W.S. Supervision: HZ and ZL. Project administration: KW and ZL. All authors contributed to the article and approved the submitted version.This study was supported by grants from the Shanghai First Maternity and Infant Hospital Science Project  and Clinical Research Plan of SHDC . The granters played no role in study design, development, data collection, or generation of this manuscript. There is no other financial support.We thank all the participating staff for their involvement with this project.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.


BAI scores of Groups 1 and 2 were significantly higher than those of Groups 3 and 4 (p < 0.05). FSFI scores of Groups 1 and 2 measured during the sixth-month follow-up were significantly decreased . Conclusions: Our findings suggest that patients with HPV 16 and 18 positivity and abnormal cytological findings are more likely to have high anxiety and sexual dysfunction. Background: The objective of this study is to evaluate the effect of HPV diagnosis on the sexual function and anxiety levels of Turkish women. Methods: A total of 274 female patients who tested positive with HPV were included in the study and categorized into four groups: Group 1 , Group 2 , Group 3 , and Group 4 . All patients filled out the Beck Anxiety Inventory (BAI) and Female Sexual Function Index (FSFI) at the time when they tested positive for HPV and during the two-month and six-month follow-ups. Results: Significant increases were observed in BAI scores in all four groups, whereas significant decreases were observed in total FSFI scores in Groups 1 and 2 only( The human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases observed in almost one quarter of women within the fertile period ,2,3,4. TIt has been reported that psychological well-being may be more of a factor than physical well-being in patients infected with HPV infection ,6,11. PrThis study was designed as a prospective observational study to evaluate the effect of an HPV diagnosis, HPV types, and cervical cytology abnormalities on the sexual function and anxiety levels of Turkish women.The study comprised all female patients who tested positive for HPV using the Papanicolaou  test pluThe majority of women were reached through community health centers, and HPV testing and cervical cytology were applied free of charge in all public hospitals. The national cervical cancer screening program includes only women who are ≥30 years old. Thus, patients under 30 years old were excluded from the study ,14. Addin = 6) were excluded from the study. The patients’ psychological status was assessed three times: when they were notified about their HPV infection (baseline), when they were informed about the results of the colposcopic biopsy, when the decision to proceed to LEEP was made (the two-month follow-up), and during the six-month follow-up , high-grade intraepithelial squamous lesion (HG-SIL), and atypical squamous cells of undetermined significance (ASC-US)) ,16. All ollow-up .All patients underwent follow-up examinations and filled out the BAI and the FSFI during the two-month and six-month follow-ups. The Beck Anxiety Inventory (BAI), which is a brief assessment of anxiety with an emphasis on somatic symptoms, consists of 21 questions. The Turkish validation studies of BAI have been completed ,18. BAI The Female Sexual Function Index (FSFI), a self-administered inventory, was used to quantify the sexual functions of the patients included in the study. The Turkish validation studies of FSFI have been completed ,19. FSFIThe study’s primary outcomes were the BAI and total FSFI scores and the changes observed in these scores over time between the groups categorized based on the HPV status. The descriptive data were expressed as mean ± standard deviation values in the case of continuous variables that were determined to conform to the normal distribution and as median values along with minimum–maximum values in the case of continuous variables that were determined not to conform to the normal distribution. The categorical data were expressed as numbers and percentages. The Shapiro–Wilk, Kolmogorov–Smirnov, and Anderson–Darling tests were used to determine whether the numerical variables conformed to the normal distribution.https://www.jamovi.org, accessed on 17 February 2023), JASP software package , and R-project (version 4.1.2) were used in the statistical analysis. Probability (p) values of ≤0.5 were deemed to indicate statistical significance.In the analysis of more than two independent groups, Pearson’s chi-squared test and Fisher’s exact test were used to compare the categorical variables between the groups. The Kruskal–Wallis test was used to compare the median BAI, total FSFI, and FSFI subscale scores. The Dwass–Steel–Critchlow–Fligner test was used to evaluate the differences between the groups. The study groups were re-categorized according to the HPV status of the patients as HPV 16/18 and other-hr HPV to clarify the effect of the HPV status on the scores. The Mann–Whitney U test was used to compare the median BAI, total FSFI, and FSFI subscale scores between two independent groups with numerical variables determined not to conform to the normal distribution. The relationship between the BAI scores and the changes observed in the total FSFI scores over time between the groups were evaluated by the non-parametric covariance analysis using the “sm. ANCOVA” package in the R software. The Spearman rho correlation coefficient was used to analyze the correlations between BAI and total FSFI scores. “Jamovi project  in Group 1, 57 (20.8%) in Group 2, 74 (27.0%) in Group 3, and 43 (15.7%) in Group 4. Twenty (20.0%) and 19 (33.3%) patients in Groups 1 and 2 were infected with multiple HPV genotypes, including other-hr HPV strains, respectively. The distribution of patients’ HPV genotypes, cytological and pathological findings, and the need for the LEEP procedure are shown in The distribution of patients’ demographic and clinical characteristics by study group is shown in p > 0.05). There was a significant difference in the rate of patients who were active smokers between Group 1 and Group 2 (p = 0.029). The distribution of patients’ BAI and FSFI scores by study group is shown in p > 0.05). In contrast, there were significant differences between the groups in terms of BAI and FSFI scores determined during the two-month and six-month follow-ups and the changes in BAI and FSFI scores over time . The patients in Groups 1 and 2 had significantly higher BAI scores measured during the second-month follow-up than those in Groups 3 and 4 (p < 0.05). There were no significant differences between Group 1 and Group 2 and between Group 3 and 4 in BAI scores measured during the two-month follow-up (p > 0.05). Likewise, as can be seen in p < 0.001 for all cases). Additionally, the patients in Group 1 had significantly higher BAI scores measured during the six-month follow-up than those in Group 3 and Group 4 (p < 0.001 for both cases). There was no significant difference between Group 3 and Group 4 in BAI scores measured during the six-month follow-up (p = 0.983). The median increases in BAI scores between the two-month and six-month follow-ups were higher in Groups 2 and 1 than those in Groups 3 and 4 (In  3 and 4 .p < 0.001 and p < 0001). There were no significant differences between the groups in total FSFI scores determined during the second-month follow-up (p = 0.181). p = 0.004 and p < 0.001, respectively). The total FSFI score determined during the six-month follow-up was lower in Group 2 than in Group 1, albeit not statistically significant (p = 0521). The median percent decrease in the total FSFI score determined during the six-month follow-up compared to the total FSFI scores determined before was higher in Group 2 than in the other groups (As can be seen in r groups . p < 0.001). The covariance analysis of BAI scores revealed significant differences in the total FSFI scores determined during the six-month follow-up between Group 3 and Groups 1 and 2, indicating the differences in the anxiety levels . The total FSFI scores determined during the six-month follow-up were significantly lower in patients with HPV 16/18 positivity (p < 0.001).The patients with HPV 16/18 positivity had significantly higher BAI scores determined during the two-month and six-month follow-ups than those with other-hr HPV results .There were significant correlations between the groups in BAI and total FSFI scores determined during the six-month follow-up in the negative direction. There were also significant correlations between the groups created based on HPV 16/18 positivity and abnormal cytology findings in BAI and total FSFI scores determined during the six-month follow-up in the negative direction (The study findings revealed that the patients who tested positive for HPV 16 and 18 had a higher degree of anxiety than the patients with other-hr HPV positivity. Additionally, it was observed that the anxiety levels of patients with HPV 16/18 positivity and abnormal cytological findings increased as the time elapsed to obtain the final results of consecutive diagnostic interventions was prolonged. The effect of the HPV grouping on sexual dysfunction was not as evident as its effect on anxiety. Patients with HPV 16/18 positivity had lower FSFI scores determined during the six-month follow-up compared to the patients with only other-hr HPV positivity and normal cytological findings. The results of the studies on the reciprocal relationships between HPV infection, anxiety, depressive symptoms, and sexual dysfunction are controversial. Patients with HPV infection may display different psychological reactions depending on the diagnosis and treatment of the infection . Denial There are conflicting results about the effect of HPV infection on sexual function ,4,7,8,9.In contrast, a study conducted in Norway reported that women’s anxiety and depression scores were not affected when the primary screening method was changed from cytology to hr-HPV testing . On the The effect of the diagnosis and the treatment of associated cervical intraepithelial neoplasia were studied in several studies . In one In this study, not only the initial diagnosis of HPV infection but also the associated diagnostic procedures, including colposcopy and LEEP in selected cases, were investigated in terms of any correlation with anxiety and sexual dysfunction. In this context, it is important to consider the differences between the methodologies employed by the studies when comparing their results on sexual dysfunction following HPV infection. Alay et al. reported the outcomes of their cohort, including the patients with hr-HPV infection, within the first two-month follow-up period. The methodology of Alay et al.’s study  was condThe relationship between sexually transmitted infections and adverse psychological outcomes has been addressed in several studies. In one of these studies, Leite et al.  found thIn conclusion, the study findings indicated that HPV 16 and 18 that had positivity along with abnormal cytological findings are critical clinical situations that cause higher anxiety levels in patients infected with HPV. Despite the fact that sexual dysfunction was observed in HPV-infected women to a varying extent, the association between sexual dysfunction and the type of HPV positivity and cytological findings could not be elucidated. Thus, further studies with larger samples and more extended follow-up periods are needed. Raising public awareness about HPV by health professionals, including the HPV vaccine in the national vaccination program, and facilitating access to the treatment of cervical pathologies, will lead to less psychological strain on women, and therefore, less damage to the sexual lives of couples.


We aimed to compare etiopathogenic features of 28 penile squamous cell carcinomas (PSCC) from Mozambique, an African country with a high prevalence of human papillomavirus (HPV) and HIV, with 51 PSCC from Spain, a European country with a low prevalence of HPV and HIV. All tumors underwent HPV testing and p16 and p53 immunohistochemistry. PSCC with either p16 overexpression and/or positivity for HPV were considered HPV-associated. The two sites showed striking etiopathogenic differences. Patients from Mozambique were significantly younger than those from Spain and showed mostly HPV-associated, p53-wild-type PSCC, contrary to older patients from Spain showing mostly HPV-independent, p53-altered tumors. These data may be valuable for primary prevention of PSCC worldwide.p < 0.001). HPV16 was the most frequent HPV type identified in 64.0% (16/25) of the HPV-associated tumors from Mozambique, and 60.0% (9/15) from Spain (p = 0.8). On average, patients from Mozambique were almost two decades younger than those from Spain . In conclusion, significant etiopathogenic differences between PSCC in Mozambique and Spain were observed, with a remarkably high prevalence of HPV-associated tumors in Mozambique and a relatively low prevalence in Spain. These data may have important consequences for primary prevention of PSCC worldwide.Penile squamous cell carcinomas (PSCC) are classified by the World Health Organization into two categories based on their relationship with the human papillomavirus (HPV): HPV-associated and HPV-independent. We compared a cohort of PSCC from Mozambique, a sub-Saharan country in southeast Africa with a high prevalence of HPV and HIV infection, and Spain, a country in southwestern Europe with a low prevalence of HPV and HIV, to study the distribution of the etiopathogenic categories of these tumors in both sites. A total of 79 PSCC were included in the study (28 from Mozambique and 51 from Spain). All cases underwent HPV-DNA polymerase chain reaction (PCR) testing, genotyping, and immunohistochemistry for p16 and p53. Any PSCC showing either p16 overexpression or HPV-DNA in PCR analysis was considered HPV-associated. Overall, 40/79 (50.6%) tumors were classified as HPV-associated and 39 (49.4%) as HPV-independent. The two sites showed marked differences: 25/28 (89.3%) tumors from Mozambique and only 15/51 (29.4%) from Spain were HPV-associated ( Penile cancer is an uncommon malignancy with an estimated age-standardized incidence worldwide of 0.80 per 100,000 person-year in 2018 . In its HPV-associated PSCC frequently arise on an intraepithelial precursor named HPV-associated penile intraepithelial neoplasia (PeIN), a lesion with a basaloid, warty, or warty–basaloid morphology , also caThe incidence of PSCC has marked geographical variability, with the highest incidence centered in low- and middle-income countries (LMIC), especially in southern Africa, South Asia, and South America, and a significantly lower, albeit rising, incidence in most European countries . UnfortuThe aim of this study was to compare the differential etiopathogenic characteristics of PSCC in Mozambique, a sub-Saharan country in South-East Africa with high prevalence of HPV and HIV and Spain, a European country with low ratesof HPV and HIV .The computer records of the Department of Pathology of the Maputo Central Hospital (MCH) and the Hospital Clinic of Barcelona (HCB)-two tertiary referral health institutions in Mozambique and Spain, respectively, were revised looking for all the cases diagnosed with PSCC. The period revised in the MCH was from January 2018 to December 2020, being from January 2000 to December 2020 in the HCB.All PSCC identified during these periods were retrieved, and the material available was histologically reviewed. The inclusion criteria for the study were: (1) the presence of invasive PSCC and (2) the material available for HPV detection by polymerase chain reaction (PCR), and p16 and p53 IHC.The ethics committees of both the Faculty of Medicine of the Eduardo Mondlane University & MCH and HCB approved the study .Sections of all the tumors stained with hematoxylin and eosin were reviewed by two pathologists  to confirm the presence of invasive carcinoma. Following the WHO criteria, several histological types were considered as probably related to HPV  and other histological variants were considered as not related to HPV infection  ,6. AfterDNA extraction was performed on 10µm whole sections of formalin-fixed paraffin-embedded tissue from surgical specimens or penile biopsies. The microtome blade was replaced between cases. No microdissection was performed but, in all cases, the tissue analyzed included the invasive tumor. The highest safety measures were carried out while sectioning and performing sample preparation to avoid cross-contamination. DNA was extracted after overnight incubation in 20 μL of proteinase K solution at 56 °C. Subsequently, the DNA was isolated using a commercial kit  according to the manufacturing instructions. Samples were quantified using the NanoDrop 2000  to determine DNA concentrations for the genotyping test. Samples with less than 10 µL of isolated DNA were excluded from the study.The SPF10 PCR and LiPA25 system were used for HPV-DNA detection and typing . A volume of 10 µL of isolated DNA was PCR-amplified using the INNO-LiPA HPV Genotyping Extra II kit . The same kit was used for HPV genotyping. This system allows the genotyping of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 70, 73, 82, 6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 81, 83 and 89. HPV DNA-positive samples not hybridizing with any of the 32 probes were classified as HPV type X (HPV X or undetermined type).Each run was performed with positive and negative controls to monitor the efficiency of DNA isolation, PCR amplification, hybridization, and genotyping procedures.All tumors were stained with a p16 monoclonal antibody using the CINtec Histology Kit . The tumors were considered as p16 positive if they showed strong and diffuse block-like staining, and were considered as negative when the staining was patchy or completely negative.All IHC stains were conducted at the department of Pathology of the HCB, employing the BenchMark ULTRA platform .In this study any PSCC showing either p16 IHC overexpression and/or HPV-DNA in the PCR analysis were considered as HPV-associated. All PSCC showing a negative result for the two techniques (p16 IHC and HPV-DNA PCR) were considered as HPV-independent.As it has recently been proposed that a second biomarker, such as p16 or E6*I mRNA, should also be used to consider a tumor as etiologically associated with HPV , a seconA monoclonal antibody was used for p53 staining . The interpretation of staining was conducted in the invasive tumor following the pattern-based framework recently developed for vulvar carcinomas ,18. ThisThe SPSS 28.0 statistical package  was used to perform the data analyses. Categorical variables are shown as absolute numbers and percentages. And were analyzed with the chi-square or Fisher exact test. Quantitative variables are shown as mean ± standard deviation, and were compared with Student’s t test or analysis of variance test.Overall, 79 tumors were included in the study, 28 from the MCH (Mozambique) and 51 from the HCB (Spain). The age, HIV status, histological variant, p16 IHC, HPV-DNA PCR, and p53 IHC results of the PSCC from Mozambique and Spain are shown in p < 0.001]). HIV status was available in 8 cases from Mozambique and in all cases from Spain. Six out of 8 (75.0%) of the Mozambican patients were HIV positive in contrast with 1/51 (1.9%) of the Spanish patients.The clinical presentation of PSCC was markedly different in the two sites, with the patients from Mozambique being on average almost two decades younger than patients from Spain (mean age of the patients from Mozambique 50.9 ± 14.9 years and mean age in Spain 69.2 ± 13.3 years [p < 0.001).Overall 40/79 (50.6%) tumors were classified as HPV-associated and 39 (49.4%) as HPV-independent. However, marked differences were observed between the two sites: 25/28 (89.3%) tumors from Mozambique vs. only 15/51 (29.4%) from Spain were HPV-associated. Using stricter criteria  20/28 PSCC (71.4%) from Mozambique and 9/51 (17.6%) from Spain were considered as HPV-associated. In both scenarios the differences were statistically significant (p = 0.8). No differences in the distribution of HPV type were identified between the two sites in any of the scenarios (p = 1). The prevalence of infection by multiple HPV types was slightly higher in Mozambique than in Spain, although the differences did not reach statistical significance .HPV16 was the most frequent HPV type identified in 64.0% (16/25) of the HPV-associated tumors from Mozambique and in 60.0% (9/15) of the tumors from Spain  were of the histological variants defined as related to HPV according to the WHO , but four (10.0%) were of the usual type, one of the variants considered as not related to HPV by the WHO. Contrarily, 33/39 (84.6%) of the HPV-independent PSCC were of the histological variants defined as not related to HPV according to the WHO , but 6 (15.4%) were variants classified as HPV-related by the WHO .p = 0.38). Contrarily, the HPV-independent PSCC showed an abnormal pattern of p53 IHC staining in 24/39 (61.5%) and a normal pattern in 15/39 (38.5%) (p < 0.0001).p53 IHC showed a normal staining pattern suggestive of wild-type protein in 35/40 (87.5%) of the HPV-associated tumors, and only 5/40 (12.5%) showed an abnormal pattern suggestive of a mutated p53. No differences were observed between Mozambique and Spain (p < 0.001). Using the restrictive scenario , the proportion of HPV-associated tumors from Mozambique was still extremely high (71.4%), whereas the percentage of HPV-associated tumors in Spain dropped to 17.6% (p < 0.001). HPV16 was the most frequent HPV type identified in both sites , similar to what has been reported in other studies [In this study we provide relevant information in relation to the epidemiology of PSCC in sub-Saharan Africa, a geographical area in which these data are particularly scant. Our series of PSCC from sub-Saharan Africa and Europe shows relevant etiopathogenic differences between the two sites and highlights the extremely high prevalence of HPV-associated PSCC in Mozambique compared with the low prevalence in Spain. Over 89% of the PSCC in Mozambique but only 29% of the PSCC from Spain were HPV-associated ( studies ,19.The results of our Spanish cohort are in keeping with most studies from high-income countries. In these sites, PSCC is typically a disease of old men, and a significant proportion arise independently from HPV, frequently within the background of inflammatory conditions of the penile skin, such as lichen sclerosus and lichen simplex chronicus ,14,20. CAlthough the high prevalence of HPV infection should be considered the main factor the high prevalence of HPV-associated PSCC in the Mozambican cohort, the high prevalence of HIV in Mozambique  has beenInterestingly, although 90% of the HPV-associated tumors were basaloid, warty, mixed basaloid/warty, or lymphoepithelioma-like, the types of PSCC considered, following the WHO classification, as tumors related to HPV ,6, 10% oInterestingly, abnormal p53 IHC-mutant patterns were identified in a high percentage of HPV-independent PSCC and in only a small percentage of HPV-associated tumors, a phenomenon already reported in studies based on IHC, in addition to studies analyzing TP53 mutations ,38,39.A subset of tumors showed discrepant p16 IHC and HPV-DNA results. It is difficult to draw conclusions on the significance of these findings and on whether these cases are or are not etiologically related to HPV with the data of the present study. It has recently been proposed that in addition to the identification of the DNA of the HPV, a second biomarker, such as p16 or E6 mRNA, should also be required to consider a tumor as etiologically associated with HPV . On the Most PSCC in Mozambique, a country in southeastern Africa, which probably reflects the epidemiological picture of many sub-Saharan African countries, are HPV-associated and arise in young men. In contrast, most PSCC diagnosed in Europe are HPV-independent and affect old men. These data suggest that extending HPV vaccination programs to males in LMIC could, in addition to the benefits of tackling the cervical cancer burden, reduce a significant proportion of penile cancer.


HPV genotypes 16, 31, 52, 66, 53, and 51 are the most frequent types in the studied population. Genotypes 16 and 31 account for nearly one-fifth of the infections of diagnosed HPV infections. HPV 16, 31, and 52 are found in nearly 80% of premalignant HSIL lesions (CIN 2 and CIN 3). That leads to the conclusion that vaccination programs should cover as many types of HPV as possible and shows the urgent need to vaccinate the Polish population with a 9-valent vaccine.The prevalence and distribution of oncogenic human papillomavirus (HPV) genotypes in women who underwent screening for cervical cancer in the Wielkopolska region, Poland, were assessed, and the correlation of genotypes with the histological results was evaluated. Cervical samples were collected from 2969 women for cervical cancer screening. Participants were screened by liquid-based cytology and HPV genotyping ( Human papillomavirus (HPV) plays a proven and undisputed role in cancer development among humans—cervical, anogenital, head and neck, and other locations . The onc+ T-cells [Infection caused by HPV is very common. Current data shows that more than 90% of the population is infected with HPV at some point in their lives, making this pathogen extremely prevalent in the general population . In cerv T-cells , mucosal T-cells .Thanks to the speedup of the screening, standardization, and improvement of the quality of the samples, liquid-based cytology (LBC) replaced the conventional Pap smear in developed countries ,20. In aIn 2018, 570,000 new cervical cancer patients were reported globally, and 310,000 patients died . Data frIn our study, we will answer the question about the epidemiology of HPV genotypes in the observed population. In addition, we are looking for the most sensitive and specific test when assessing the frequency in high-grade intraepithelial lesions. In the future, it may be possible to validate screening programs and plan the diagnosis of pre-cancerous changes of the cervix in the most favorable way, both for patients and in terms of the economy.n = 2969) were offered LBC and HPV genotyping tests, but it was their decision whether to perform them. All subjects from the study group underwent a verification diagnostic of abnormal LBC results, the suspicious clinical picture of the cervix, or the presence of an oncogenic genotype of HPV by punch biopsy. Abnormal LBC result means: ASC-US , AGC , LSIL , HSIL , and cervical cancer whereas the visual assessment of the cervix was performed by an experienced colposcopist.We provide a prospective, ongoing 48-month, non-randomized pilot study in patients reporting to the Individual Specialised Medical Practise in 2018–2022. All subjects (The ® (Roche). Then, the probes were passed to an independent, standardized laboratory. PCR was performed, followed by a DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay for HPV detection. The lab technicians performed sequence analysis to characterize HPV-positive samples. The molecular test detected the DNA of 37 HPV genotypes .We collected liquid-based cytology and molecular assessment samples with an endocervical Cyto-Brush preserved in PreservCyt Further validation of abnormal screening results was performed on all patients with an abnormal smear above the ASCUS , a positive HPV test for types 16, 18, 31, and a clinically suspicious cervical image. The Polish Society of Colposcopy and Cervical Pathophysiology recommended the International Federation of Cervical Pathology and Colposcopy classification.p-value set at 0.05. Dependencies between categorical variables were analyzed with the chi-square test. The sensitivity and specificity of different HPV types and cytology were analyzed using MedCalc statistical software.Analysis was conducted in SPSS, version 27, with the n = 233). The remaining distinguished types were observed among 67 to 85 subjects.The study group included 1654 patients aged 18–86 (average age is 36). About 45% of the patients were nulliparous, and more than half had at least one child. The largest group was the group of patients aged 25–34 (675 subjects) and 35–44 (592 subjects). There were 242 women aged 45–54. The youngest and oldest groups were the least numerous and were represented by 76 and 65 women, respectively. HPV was detected in 781 subjects . The most common types of HPV diagnosed among the sample were: 16, 31, 52, 66, 53, and 51. p < 0.001). HPV type 31 was more common among the youngest and oldest groups (p < 0.001). HPV 51 was observed among 9% of the youngest group, 4% of subjects aged 25–44, 2% of subjects aged 45–54; (p = 0.031), which presents in There was a statistically significant dependency between age and HPV type 16, 31, and 51. HPV 16 was observed more often among subjects aged between 25 and 34 (n = 616). Among subjects from HSIL and LSIL groups, there were greater proportions of patients with those types of HPV than among patients from the remaining groups , as shown in HPV 16, 31, and 51 were also related to biopsy results. Among all histopathological diagnoses, we observed: no pathology (246/616), koilocytosis—a microscopic image of HPV infection (46/616), LSIL (151/616), HSIL (166/616), and several diagnoses of cervical cancer and focal atypia. Due to the small number of patients diagnosed with focal atypia (3/616) and cervical cancer (4/616), we did not include the groups mentioned above in the statistical analysis. However, the calculations regarding the sensitivity and specificity of the test determining individual genotypes for detecting SIL changes were applied to all patients who underwent cervical biopsy (The probability that HPV was positive when HSIL was positive (sensitivity) equaled: 54.22% for HPV 16, 16.87% for HPV 31, 7.83% for HPV 52, 5.42% for HPV 66, 4.22% for HPV 53, and 9.04% for HPV 51. Specificity of different types of HPV detecting HSIL equaled respectively: 85.33% for HPV 16, 94.22% for HPV 31, 93.78% for HPV 52, 94.00% for HPV 66, 94.22% for HPV 53, and 96.44% for HPV 51, as shown in The sensitivity of the different combinations of HPV types  was also increasing as more HPV types were added in the case of HSIL, and the specificity of those combinations was decreasing . The lowest accuracy of detecting HSIL was observed for HPV 16, 31, 52, 66, 53 (68.18%), and the highest for HPV 16, 31 (75.32%), as shown in The probability that LBC results or HPV were positive when HSIL was positive (sensitivity) equaled: 91.57% for LBC and HPV 16, 85.54% for LBC and HPV 31, 82.53% for LBC and HPV 52, 83.13% for LBC and HPV 66, 83.13% for LBC and HPV 53, and 83.13% for LBC and HPV 51. Specificity of LBC results and different types of HPV detecting HSIL equaled respectively: 14.22% for LBC and HPV 16, 16.89% for LBC and HPV 31, 17.56% for LBC and HPV 52, 16.44% for LBC and HPV 66, 17.56% for LBC and HPV 53, and 17.56% for LBC and HPV 51, as shown in Our study presents the prevalence of HPV in the Polish population in the Wielkopolskia Voivodeship and covers a group of 2969 subjects, from whom 1654 underwent HPV genotyping tests. The study showed the highest frequency of genotypes 16, 31, 52, 66, and 53 in the general population and 16, 31, and 52 in patients with histopathologically confirmed HSIL, respectively. There was a statistically significant dependency between age and HPV type 16, 31, and 51. HPV 16 was observed more often among subjects aged between 25 and 34 than among other groups. HPV type 31 was more common among the youngest (13%) and oldest group (9%) than among the rest of the subjects. HPV 51 was also the most common in the youngest group. These three HPV genotypes were also related to biopsy results. Among subjects from the HSIL group, there were a greater proportion of patients with those types of HPV than among patients from the remaining groups.Modern knowledge and the rapid development of molecular testing techniques make it necessary to modify the current programs of both primary and secondary prevention. The impact of the implementation of HPV vaccination programs can already be seen in many European countries, and above all in Australia and New Zealand, where the highest decrease in the incidence of HPV-related diseases has been recorded. According to a 2018 publication by Patel et al., the use of the 9-valent vaccine might reduce the incidence of cervical cancer by 90% . Our resThe incidence of certain highly oncogenic HPV genotypes may vary depending on demographics. For the purpose of the discussion, we have collected comparative data from the most extensive publications from different countries in What draws attention at the beginning is the prevalence of HPV genotype 16—it is the most common in each observed population, and the prevalence ranges from 3.9 to 63.7%. The data should be analyzed carefully because the study group in Russia was evaluated not based on histopathological diagnoses but only on cytology and included healthy women as well as those with pathology. Already data on the second most common HPV genotype are very divergent. The second most frequently observed type was 31 , which is consistent with the data obtained in our research group. However, in China, Russia, and New Zealand, the second most frequent genotype was 52, in Venezuela—HPV genotype 18, in Mexico—HPV genotype 51, and in Portugal—HPV genotype 58. The frequency of subsequent genotypes is even more divergent, which confirms the thesis that the above analysis has a cognitive value. Each latitude may differ in the frequency of occurrence of individual HPV genotypes and thus have a cervical cancer screening model based on other genotypes.p = 0.30, p = 0.19, respectively) [Not in all geographical areas, the proportion of histological HSIL+ in women infected with HPV 33 or HPV 31 was significantly different compared to women infected with HPV 16 (ctively) . AccordiThis type of trend is also observed when comparing the results of meta-analyses from Poland. In the 2010 publication of women from Central and Western Poland diagnosed with HG SIL, the most common HPV genotypes were HPV 16, HPV 33, HPV 18, HPV 31, and HPV 56 . ObservaThe recommended screening tests for HPV infection are tests detecting DNA of highly oncogenic HPV types with the possibility of genotyping or phenotyping. Material for molecular testing should be collected on certified, validated liquid media.The obtained results indicate a new epidemiological distribution of human papillomaviruses, which only partially overlaps with global data and thus may be used in the future to better construct criteria for the diagnosis of squamous intraepithelial lesions. Going forward, perhaps as part of secondary prevention, this analysis and subsequent ones involving a wider population may contribute to the development of new vaccines against HPV.HPV infection is one of the most common sexually transmitted infections in the study group; it was close to 47%. HPV genotypes 16, 31, 52, 66, 53, and 51 are the most common types. Genotypes 16 and 31 account for nearly one-fifth of the infections of diagnosed HPV infections. HPV 16, 31, and 52 are found in nearly 80% of premalignant HSIL lesions (CIN 2 and CIN 3). Vaccination programs should cover as many types of HPV as possible. The above study confirms the need to vaccinate the Polish population with a 9-valent vaccine.