In the era of cervical cancer elimination, accurate and validated pipelines to detect human papillomavirus are essential to elucidate and understand HPV association with human cancers. We aimed to provide an open-source pipeline, “HPV-meta”, to detect HPV transcripts in RNA sequencing data, including several steps to warn operators for possible viral contamination. The “HPV-meta” pipeline automatically performs several steps, starting with quality trimming, human genome filtering, HPV detection (blastx), cut-off settlement  and finishing with fasta sequence generation for HPV positive samples. Fasta sequences can then be aligned to assess sequence diversity among HPV positive samples. All RNA sequencing files  present in the cancer genome atlas (TCGA) were analyzed. “HPV-meta” identified 25 different HPV types being present in 488/10,904 specimens. Validation of results showed 99.98% agreement . Multiple alignment from fasta files warned about high sequence identity between several HPV 18 and 38 positive samples, whose contamination had previously been reported. The “HPV-meta” pipeline is a robust and validated pipeline that detects HPV in RNA sequencing data. Obtaining the fasta files enables contamination investigation, a non very rare occurrence in next generation sequencing. About 12 HPV types are nowadays classified as oncogenic, high-risk HPV genotypes, and persistent infection of these oncogenic HPV types is known to be a necessary cause for cervical cancer5. Furthermore, other cancers as oropharyngeal, anal, penile, vaginal and vulvar carcinomas, have also been associated and are known to be caused by high-risk HPV types in the majority of their cases6.Human papillomaviruses (HPVs) are a group of double-stranded DNA viruses which comprise up to 222 different types, with new types being continuously identifiedTraditionally, tests for HPV detection are based on broad-spectrum signal amplification assays followed by rapid high-throughput target-amplification assays. Nowadays, next generation sequencing (NGS) has enabled scientists to move beyond simply detecting presence/absence of HPVs and provide a more detailed insight of the HPV sequence and infection.11. Another cancer type, non-melanoma skin carcinoma, has also revealed a plethora of human papillomavirus types when skin tumors have been DNA sequenced12. However, detection of HPV DNA does not necessarily mean that an HPV infection has been detected13 and contrary to the other cancer types, whole transcriptome studies have rarely detected HPV in skin tumors15. Investigation of viral transcription is important, as transcription of viral genes is necessary for viral pathogenicity, as demonstrated in head and neck cancers17. Considering the global aim set by the WHO towards elimination of cervical cancer, it seems essential to understand the biology of human papillomavirus by performing metatranscriptomic analysis.Metagenomics studies of viral DNA have systematically found HPVs in the described-above tumors, even in cases where HPV had been reported as “apparently negative” when tested with non-NGS methods18. The International HPV Reference Center already reported existence of 114 possible chimeras and 13 taxonomy/naming errors when performing a GenBank screening analysis of HPV deposited sequences in 202119 and several authors have reported presence of HPV contamination among different studies 21. Well-validated HPV pipelines are needed to prevent database contamination and false calling to continue with quality and order in HPV research.Although NGS has been extensively used in HPV research, there is a higher risk of possible cross-contamination among specimens when compared to non-sequencing methods (due to the high throughput achieved when sequencing), as well as a lack of standardization and quality guidelines when considering sequencing analysishttps://github.com/hpvcenter/HPV-meta) to detect HPV transcripts in RNA sequencing data which could further provide information to operators and warn for possible HPV contamination. To achieve this aim, a well-known and validated public database, the cancer genome atlas  was used for pipeline development and validation purposes. TCGA is a collaboration between the National Cancer Institute and the National Human Genome Research Institute and contains molecularly characterization of over 20,000 primary cancers and matched normal specimens spanning 33 cancer types .The present study aimed to develop a robust open-source HPV pipeline, “HPV-meta”  datasets from the TCGA portal belonging to primary tumors (n = 9774), metastases (n = 394), recurrent tumors (n = 47) and solid normal tissues (n = 693) to develop and validate “HPV-meta”, a pipeline aiming to detect HPV transcript in RNA sequencing data. All research was performed in accordance with relevant guidelines/regulations.By using the GDC transfer tool, we downloaded 10,908 bam files comprising around 90 terabyte of sequencing data. For each of the bam files, we downloaded the corresponding metadata, including the specimens and clinical data .In order to process the large amount of sequencing data, we developed an automated pipeline called “HPV-meta” for HPV detection in RNA sequencing specimens. “HPV-meta'' performs several steps including quality trimming, human genome filtering, HPV detection, cut-off settlement and fasta sequence generation Fig. . The fashttps://github.com/hpvcenter/HPV-meta). For this study, the code was integrated onto the Spark and Hadoop environment but it can definitely be used in other environments as well.The pipeline and all the tools used for the data processing and analysis are openly accessible in the github repository , this step must be omitted, and files should be subjected to quality filtering directly.A close look at the original bam files included in the TCGA database  revealed that all bam files were mapped to databases containing a human reference genome as well as other viral sequences. HPV was included in some of those databases. Therefore, the “HPV-meta” pipeline starts by using pysam v. 0.16.1 (htslib/bcftools v. 1.10.2 and samtools v. 1.10) with a bed file containing human chromosomes and genomic regions to filter out the mapped human reads but not the viral sequences. Non-human reads remain in the newly created bam file (file_name_NH.bam), which is then sorted and converted to a fastq file (file_name_NH.fq.gz) using pysam.https://github.com/usadellab/Trimmomatic)22. Parameters for Trimmomatic are as follows: -SE -phred 33 -leading 3 -trailing 3- slidingWindow:4:15 and-minlen:18.To obtain high quality non-human reads, “HPV-meta” uses Trimmomatic v. 0.39 , it is required to trim/remove the first 3 nucleotides from every R2 read for the RNA sequencing fastq-files24. The program is run under default settings, except for -i 0.95, -R 0.75 and–silent-clip. Reads that map to the human reference genome  are filtered out, and the corresponding bam files with no human sequences are then sorted and converted to fastq files again . We highly recommend filtering human sequences twice when users start with bam/sam files coming from public databases as the human reference genomes used previously may not be the latest update.High quality reads are afterwards re-screened against the human reference genome version GRCh38 using Nextgenmap v. 0.5.5https://pave.niaid.nih.gov/, accessed on 2021-08-17), including all protein sequences from HPV reference and non-reference genomes), using the open source software Diamond v. 2.0.725 using blastx with default parameters and –top 1.High quality non-human reads are queried against all HPV protein sequences included in the PaVE database , in order to maintain a similar % of minimum coverage for positivity calling. The “HPV-meta” pipeline calculates the coverage for each sequencing file and reports the total coverage as well as the coverage for each HPV gene. HPV detection will only be reported for one HPV type per specimen. In the case of multiple HPV infection within a sample, the HPV type with the highest number of unique reads will be reported. In the case that 2 HPV types present the same number of unique reads, the type which presents a higher coverage is selected.Positivity of HPV is only considered if at least 10 reads are present for one HPV type and 690 bp of its genome are covered. The cut-off is based on previous validated cut-offs used for HPV detection on total nucleic acid extracted specimens https://www.hpvcenter.se, accessed on 2021-08-17), together with complete genome sequences from HPV types that are not officially established yet , using NextGenMap24 under the same settings as described above. For each specimen, reads mapping to the HPV genotype previously detected with diamond are filtered for further analysis.The “HPV-meta” pipeline generates a consensus fasta file for each HPV type classified as positive within a specimen. High quality non-human reads fastq files  are queried against a database of known HPV sequences including all HPV genomes officially established by the International HPV Reference Center  for each consensus was saved as a bed file using covtobed v. 1.3.1 (https://github.com/telatin/covtobed). The bed file was used later to generate a fasta file using bedtools v. 2.30.0, in which the regions of the genome not covered (< 5 reads) were marked with “N”s (instead of having the reference nucleotides in not covered positions).Resulting BAM files are then processed through a quality control and left aligned using the The Genome Analysis Toolkit (GATK) v. 4.2.3.0, LeftAlignIndels module. The HPV genomes are then genotyped by GATK HaplotypeCaller28. With the generated fasta sequences, operators can for example compare the sequence identities obtained for one HPV type in one sample, with the rest of sequences presenting the same HPV type but detected in other specimens. The present study includes an analysis of sequence identity for all HPVs that were present in at least 30 samples .It is well reported in literature that for each HPV type, extensive sequence diversity is observed when analyzing HPV positive samples. Around 84.5% of women infected with HPV isolates do carry unique HPV sequences within their cervical samplesWe would like to note that 100% identity is not a final proof of contamination, as different women may be infected with the same HPV type and identical sequence. However, isolates showing 100% nucleotide identity should be considered as “flagged” for the operator in order to have a closer look if needed.A total of 10,908 bam files were downloaded from the TCGA database, and subjected to HPV detection using the “HPV-meta” pipeline. Four sequencing files  could not be processed due to failure/corruption in the original bam files and therefore the analysis was made in 10,904 sequencing files .All bam files belonged to specimens located at 137 different organs, with breast (n = 1203), kidney (n = 1019), lung -upper lobe- (n = 642), thyroid gland (n = 564) and endometrium (n = 561) being the organs presenting the most number of specimens.The “HPV-meta” pipeline identified 488 HPV positive specimens among the 10,904 bam files (4.5%) according to pipeline cut-offs. A total of 25 different HPV types were detected among the studies included within the TCGA, with HPV 16 (261/488), HPV18 (72/488) and 38 (41/488) being the types most commonly found .All HPV types detected by “HPV-meta” were confirmed within the specimens, except for 2 samples where the genotypes detected with both approaches did not agree. One specimen  showed HPV 52 in the original bam file while the “HPV-meta” revealed HPV 58. Further analysis on original bam files revealed presence of both genotypes, with HPV 52 showing 23 reads and HPV 58 19 reads. The “HPV-meta” pipeline revealed presence of HPV 52 (16 unique reads) and HPV 58 (16 unique reads) and HPV 58 was selected as the dominant HPV type due to higher coverage (690 nt). The other specimen (ovarian primary tumor) showed HPV-mCG3 in the original bam file (15 reads) while the “HPV-meta” revealed presence of HPV-mSK041 . All specimens classified as negative by the “HPV-meta” pipeline have also been reported negative in the TCGA studies.The “HPV-meta” pipeline provided HPV coverage for all HPV positive specimens, revealing that HPV16 showed the highest median coverage when detected in gum, upper gum, oropharynx, base of tongue and tonsil specimens  followed by HPV 6 detected in anterior wall of bladder , when compared to other HPV types and locations  for all HPV types detected at these locations: brain and cerebrum, liver, colon , corpus uteri, mouth and floor of mouth, posterior wall of oropharynx, cecum, cardia, thyroid gland, skin, pleura and lower lobe of lung, head of pancreas, prostate gland, ovary and connective, subcutaneous and other soft tissues of lower limb and hip . Within the HPV 16 sequences, a total of 245/274 sequences were aligned (29/274 sequences were excluded due to fasta generation failures) and 30,012 pairwise comparisons were done (245 × 245/2). Up to 2078.5/30,012.5 comparisons showed 100% identity, translating into 6.93% sequences showing 100% identity to other HPV 16 sequences.The minimal identity detected among sequences was 94.59% . Clear clusters with 100% identity were detected for this HPV type  Using TCGA database, a well validated (as well as exploited) database, that provided no less than 10,904 RNA sequencing files from 137 different organs, spanning 33 cancer types. Open science, data sharing and collaboration is essential for understanding any research question; (b) screening against an updated and complete HPV database including all officially established HPV types as well as the non-official types; and (c) the possibility of generating fasta files (obtain the HPV sequence) that can be used to compare sequence identity among samples presenting the same HPV type within a study. This is due to the singularity of the HPV genome, mostly covered by transcribing ORFs .Weakness of the study may include the single HPV result obtained from the pipeline. The pipeline was designed to report only one HPV type in case of multiple HPV infection. However, the information about the different HPV types detected, the number of unique reads and the coverage is saved for every positive HPV. The operator may choose to change the code in order to report all HPV types detected. Furthermore, “HPV-meta” targets only HPV (no other viruses and/or microorganisms), however, changing the database and including the desired microorganisms´ sequences may enable the pipeline to potentially detect other microorganisms.“HPV-meta” identified HPV in 488/10,904 specimens and HPV was present in 46/137 organs. For some cancer forms, like cervical cancers, it is already known that they will regularly contain HPV transcripts from the E6 and E7 viral oncogenes and these cancer forms did serve as a positive control in this project. Organs with HPV-associated cancers showed the highest number of specimens positive for HPV as expected (cervix uteri (n = 291/309), endometrium (n = 45/561) and tonsils (n = 34/41)), but HPV was also detected in non-HPV associated tumors (e.g. 7/683 cerebrum/brain tumors). To validate the results, we examined all original bam files from the TCGA as they had the HPV mapping information and compared those results with the results from “HPV-meta”. There was almost a 100% agreement (99.98%) with only 2/488 HPV positive samples not reporting the same genotype. The disagreement of one of them  could be explained due to a multiple infection of 2 genotypes presenting the same number of reads in the same sample. The second disagreement occurred in one ovarian tumor where 2 different non-officially HPVs were reported. While the type detected in the original bam files corresponded to HPV-mCG3 , the “HPV-meta” revealed presence of HPV-mSK041 . It can be thus confirmed that “HPV-meta” could detect all HPVs with no obstacle.21. In agreement with these authors, HPV 38 was detected in 41 samples, and all of them belonged to the same study (endometrium), which should cause at least suspicion about possible contamination. Fasta generated alignments and pairwise comparisons showed total sequence identity among most of the sequences (> 80%) for HPV type 38, raising a flag for possible contamination. The same scenario occurred in some TCGA studies for HPV 18. Use of positive controls may be necesary when performing NGS sequencing, but negative controls are a must.Possible sequencing contamination is a weakness from NGS and operators must be highly experienced when performing sequencing protocols. Possible contamination within samples has already been reported for TCGA specimens In conclusion, the “HPV-meta” pipeline has shown to be an open-source pipeline which accurately detects HPV in RNA sequencing data with validated cut-offs. Obtaining the fasta files adds the possibility of contamination investigation, a non-very rare occurrence in next generation sequencing which should always be considered.Supplementary Information.


Background: Blue spaces have been a key part of human evolution, providing resources and helping economies develop. To date, no studies have been carried out to explore how they may be linked to paediatric oncological diseases. Objectives: To explore the possible relationship of residential proximity to natural and urban blue spaces on childhood leukaemia. Methods: A population-based case–control study was conducted in four regions of Spain across the period 2000–2018. A total of 936 incident cases and 5616 controls were included, individually matched by sex, year of birth and place of residence. An exposure proxy with four distances  to blue spaces was built using the geographical coordinates of the participants’ home residences. Odds ratios (ORs) and 95% confidence intervals (95%CIs) for blue-space exposure were calculated for overall childhood leukaemia, and the acute lymphoblastic (ALL) and acute myeloblastic leukaemia (AML) subtypes, with adjustment for socio-demographic and environmental covariates. Results: A decrease in overall childhood leukaemia and ALL-subtype incidence was found as we came nearer to children’s places of residence, showing, for the study as a whole, a reduced incidence at 250 m (odds ratio (OR) = 0.77; 95%CI = 0.60–0.97), 500 m , 750 m , and 1000 m . AML model results showed an increasing incidence at closest to subjects’ homes . Conclusions: Our results suggest a possible association between lower childhood leukaemia incidence and blue-space proximity. This study is a first approach to blue spaces’ possible effects on childhood leukaemia incidence; consequently, it is necessary to continue studying these spaces—while taking into account more individualised data and other possible environmental risk factors. In the present moment, more than half of the world’s population lives in cities that are undergoing rapid changes, and natural spaces are becoming more relevant . Blue anHowever, this is not really anything new. Blue spaces have been a key part of human development . They arAs the population and urban areas grow rapidly, the number of researchers trying to establish relationships between blue spaces and health has increased. As cities’ urban areas have expanded, blue spaces are generating more interest due to their possible restorative and recreational potential for the surrounding population. According to the available literature, these spaces have been positively associated with improved well-being, mental health, and physical activity promotion . AlthougLeukaemia represents 30% of all childhood cancers, and is the most common type of cancer in children . AlthougA childhood leukaemia case–control population study was conducted in four different Spanish regions: Vizcaya, Barcelona, Valencia, and Murcia, covering the period 2000–2018 . These fInstituto Nacional de Estadistica, INE). They were individually matched to cases in a 6:1 ratio by sex, year-of-birth, and region-of-residence to address possible geocoding errors in the controls chosen. The data used for the study were from children aged 0 to 14 with leukaemia diagnoses between 2000 and 2018. The Spanish Registry of Childhood Tumours (RETI-SEHOP) registered the incident cases. RETI-SEHOP collects information from cases of childhood cancer from hospitals’ paediatric oncology units all over Spain . The aveAll cases’ home addresses at the time of diagnosis were successfully geocoded, and were included in the RETI-SEHOP database. Controls’ coordinates were obtained from the Birth Registry, and were extracted from the mother’s home address as listed on the birth certificate. They were given a 30 m random error to preserve participants’ anonymity. Cases and controls’ residence coordinates were projected into the ETRS89/UTM zone 30N(EPSG:25830) using QGIS software. We excluded controls whose geocoded coordinates were outside the selected regions. We selected blue spaces from the Spanish Land Use Information System (SIOSE) database provided by the Spanish National Geographic Institute website (IGN) . This daFor this study, the levels and sub-levels that were directly related with natural and urban blue spaces were selected: waterways, marine and continental wetlands, lakes, ponds, reservoirs, coastal lagoons, estuaries, and seas. Artificial blue spaces built for agricultural purposes were not included. Then, buffers for distances of 250 m, 500 m, 750 m, and 1 km from the participants’ home residences were constructed. Subsequently, we designed three exposure groups according to the presence of blue spaces in those buffers: (a) the “reference group” was defined as those subjects who did not have any identifiable blue space in their 1 km exposure buffers, (b) the “exposure group” was established for those children that had blue space at the studied distances, (c) and finally, those children not accounted for in one of the previous groups for each distance were classified as an “intermediate group”. We included, as potential confounders, sex, birth year, degree of urbanisation (DGUR), socio-economic status (SES), and activity rate in the analysis. Although SES data were not available at an individual level, data at census-tract level were taken. SES combines information regarding the activity, professional situation, and occupation of the heads of families in each census tract ranging from 0.46 (worst) to 1.57 (best) . In addi2 population grid cells). As the selected regions have higher-density population compared to most Spanish regions [Indicators of urbanisation were assigned using data from the European DGUR data-set . It clas regions , we re-s10) around each child’s home levels was assigned. This agency has produced European interpolated air-quality maps since 2006. These maps are derived primarily from database stations that monitor data across the entire continent. Each grid of the map covers an area of 100 km2 [10 levels for the chosen regions for all possible study years and we estimated mean PM10 level for each child.Applying data available from the European Environment Agency (EEA), particulate matter with a diameter of less than 10 µm  and 95% confidence intervals (95%CIs) associated with blue-space exposure were estimated using fit mixed multiple unconditional logistic regression models that include all the above-mentioned covariates as potential confounders. These all-independent regression models were estimated for overall childhood leukaemia, ALL and AML subtypes, and the sensitivity groups (explained in the following methodology sub).® , R® version 4.1.1, STATA® version 15, and the geographic information system QGIS® version 3.18.2 were used. The statistical programs Microsoft Excel 365We decided to perform two different sensitivity analyses. The first was carried out to evaluate a possible misclassification problem due to residential mobility. A matching strategy to find cases with the same address at the time of birth (birth certificate) and diagnosis (included in the RETI-SEHOP registry) was developed. The aim of the second sensitivity analysis was evaluate possible prenatal period exposure. To this end, we decided to select all cases younger than 3 years old in this group.The analysis included 936 cases and 5616 controls. 10 levels at place of residence and surrounding greenness values were also slightly different.Regarding the sensitivity analysis, for the first analysis we were able to identify more than half of the cases  with the same address at birth and diagnosis and less than half younger than 3 years old . Compared to the main group of cases, for the <3-years-old sensitivity group we found a difference between ALL and AML distribution. PMIn this study, we explored the possible association between urban and natural blue spaces and childhood leukaemia incidence in different Spanish regions, taking into account several exposure distances. Our findings showed a reduced OR of childhood leukaemia in zones close to blue spaces. To our knowledge, this study is the first to explore this relationship; consequently, we should be cautious with its interpretation. On the one hand, the oceans are essential to human health and well-being. The benefits to the general population are related to wellbeing, mental health, and physical improvement thanks to being places where people can interact socially or do physical activities, for example . FocusinOn the other hand, there are some risks related with water. With climate change and increasing pollution of oceans and rivers as a consequence of human activity, all these factors increase the risk of disease vectors ,31 and mBlue-space studies on their possible effects agree only that there is no well-established methodology for how these spaces can be studied. Some researchers have only analysed coastal and/or salt-water blue spaces ,40 whileRegarding spatial measurement tools, the most used are land-use maps. Land-use maps have been used in many spatial studies to explore green spaces and crops ,43. TheiReturning to the heterogeneity exposure measurement, it was difficult to establish a buffer size selection properly. Previous blue-space studies have used a great variety of distances, ranging from 100 m to 50 km ,45. As yIt has been hypothesised that blue spaces may follow similar causal pathways to those identified for urban green spaces including physical activity increment, stress reduction, promotion of social contacts, or reducing extreme temperatures . HoweverPrevious studies have introduced socio-economic status as a potential modifier ,49,50. IThere are studies that show that blue spaces are a significant part of natural and urban environments and are correlated with outdoor green spaces and their outcomes . We deciFocusing on sensitivity analysis, our first analysis objective was to explore if there were cases whose home address was different between birth and diagnosis time. The change of residence could bias the results; however according to official data in Spain, approximately only 1% of children change their residence to a different province . To reduThe second sensitivity analysis’s aim was to evaluate the possible effect of these spaces during pregnancy itself. We decided to select those cases younger than 3 years old for this (43.8%). Despite both sensitivity analyses showing similar results to the cases in the main group, the <3-years-old group have a higher overall OR for the 250 m exposure buffer. This could be derived from the histologic subtype distribution, in which AML represented 20% of the cases versus 15% in the total sample. Regarding AML, this leukaemia subtype showed the opposite relationship with blue spaces to overall leukaemia. Previous studies, stratifying by leukaemia type, indicate that exposure to benzene and low-aromatic hydrocarbons are most strongly correlated with AML . AlthougAs far as we are aware, this is the first study exploring the effects of blue spaces on childhood leukaemia incidence. Our results could point to a possible link between blue-space proximity and this disease. International cooperative transdisciplinary research is needed to identify and weigh up the effect of the prevention and control of blue-space pollution on cancer risk. Despite this, this study can be taken into account for further research to promote blue spaces as a potential health tool for children, so we would encourage a focus on their potential benefits.


INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard, was assessed in HPV-DNA-positive formalin-fixed paraffin-embedded samples from 29 countries. The concordance of p16INK4a and E6*I mRNA among 78, 257, and 51 HPV-DNA-positive OCC, OPC, and LC, respectively, was moderate to substantial in OCC and OPC but only fair in LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. We concluded that the diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven head and neck carcinoma.The utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC), and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16Background: Tests or test algorithms for diagnosing HPV-driven oral cavity and laryngeal head and neck carcinomas (HNC) have not been yet validated, and the differences among oral cavity and laryngeal sites have not been comprehensively evaluated. We aimed to assess the utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC) and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. Methods: Formalin-fixed paraffin-embedded OCC, OPC, and LC carcinomas were collected from pathology archives in 29 countries. All samples were subjected to histopathological evaluation, DNA quality control, and HPV-DNA detection. All HPV-DNA-positive samples  were also tested for p16INK4a immunohistochemistry and E6*I mRNA. Three different cutoffs of nuclear and cytoplasmic staining were evaluated for p16INK4a: (a) >25%, (b) >50%, and (c) ≥70%. The concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was assessed. Results: A total of 78 OCC, 257 OPC, and 51 LC were HPV-DNA-positive and further tested for p16INK4a and E6*I mRNA. The percentage of concordance between p16INK4a (cutoff ≥ 70%) and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was 79.5% (95% CI 69.9–89.1%), 82.1% (95% CI 77.2–87.0%), and 56.9% (95% CI 42.3–71.4%), respectively. A p16INK4a cutoff of >50% improved the concordance although the improvement was not statistically significant. For most anatomical locations and p16INK4a cutoffs, the percentage of discordant cases was higher for HPV16- than HPV-non16-positive cases. Conclusions: The diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC, including the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC. Apart from oropharyngeal carcinomas (OPC), oral cavity (OCC) and laryngeal carcinomas (LC) are the predominant subtypes of head and neck carcinomas (HNC), where a fraction of cases are driven by human papillomavirus (HPV) . HoweverThe prognostic advantage of HPV-driven OPC versus non-HPV-driven OPC is well established. On the other hand, the clinical implications of HPV-status in OCC and LC are not clear . Thus, wThe WHO campaign for eliminating cervical cancer  raises tINK4a, a cell surrogate marker of HPV carcinogenic transformation, using immunohistochemistry is easy to implement and the most widely used standalone technique for HPV-driven OPC diagnosis in the clinical setting. A nuclear and cytoplasmatic 70% cutoff of stained cells is recommended [The identification of p16ommended .However, to diagnose HPV-driven HNC at non-oropharyngeal sites, tests and test algorithms have not been validated so far, nor has there been an in-depth evaluation of the differences among non-oropharyngeal sites. While HPV E6/E7 mRNA detection is widely accepted as the reference standard test to elucidate the oncogenic role of HPV in the tumor, it is still challenging to implement in specific settings due to RNA fragmentation and degradation in paraffin-embedded tissue [INK4a immunohistochemistry alone to assess HPV status in OPC, a fraction of p16INK4a-positive OPCs are HPV-DNA-negative, and HPV-DNA-negative/p16INK4a-positive OPCs do not show a prognostic advantage with respect to HPV-DNA/p16INK4a double-negative cases [INK4a immunohistochemistry to confirm HPV causality in OPC tumors, but no consensus for HPV testing has been reached yet. Moreover, most studies analyzing the discordance between p16 and HPV used an HPV-DNA-based test rather than the reference standard, an mRNA-based test.Moreover, despite the well-proven prognostic impact and clinical implications of HPV status in OPC and the wide use of p16ve cases ,9. TheseINK4a double testing is increasingly used for diagnosing HPV-driven OPC. In a meta-analysis of 11 studies [INK4a double positivity were estimated. Yet, there is still limited information about the accuracy and prognostic value of this combination of biomarkers in OPC, and even less in non-oropharyngeal HNC.HPV-DNA/p16 studies , a sensiINK4a were 22.4%, 4.4%, and 3.5% for OPC, OCC, and LC, respectively [The ICO international study estimated the fraction of cases attributable to HPV in 3680 formalin-fixed paraffin-embedded (FFPE) HNC tissues collected from pathology archives in 29 countries . Estimatectively ,11.INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. For that, we evaluated the concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases from the ICO international study.Our aim in the present study was to assess the utility of a diagnostic algorithm for the detection of HPV-driven OCC, OPC, and LC of HPV-DNA testing followed by p16INK4a immunohistochemistry and HPV E6*I-mRNA detection in the ICO international study, a large cross-sectional international study coordinated by the Catalan Institute of Oncology (ICO)  in collaboration with DDL Diagnostic Laboratory  [We conducted a statistical reanalysis of HNC cases positive for HPV-DNA, tested for p16erlands) . The EthINK4a detection.Details of the protocol were described elsewhere . BrieflyHPV determination was performed using PCR with the SPF-10 primer system followed by DEIA (DNA Enzyme Immuno Assay). DEIA-positive HPV-DNA samples were genotyped using the Line Probe Assay LiPA25 ; DNA quality was evaluated in all HPV-DNA-negative samples by testing for the tubulin-β gene. All DEIA and LiPA25 assays were performed at ICO.In all HPV-DNA-positive HNC, E6*I mRNA detection was performed by RT-PCR . The E6*I mRNA assay targets 20 HPV types (HPVs 16/18/26/31/33/35/39/45/51/52/53/56/58/59/66/67/68/70/73/82). For each case, type-specific E6*I mRNA PCR was performed for all types detected by the SPF-10 PCR/DEIA/LiPA25 system that had at least one of the mRNA targeted types, in addition to HPV16. Quality control for mRNA detection was performed by detection of ubiquitin C mRNA. All E6*I mRNA assays were performed at DKFZ, Heidelberg, Germany.INK4a as previously described [All HPV-DNA-positive cases were evaluated for p16INK4a and E6*I mRNA among OCC, OPC and LC cases that were HPV-DNA-positive, HPV16-DNA-positive, and HPV-DNA-positive for types other than HPV16. Concordance percentage was estimated according to whether the condition np (1 − p) > 5 was met or not. If it was met, a normal distribution approximate method was applied [p-value was also calculated to evaluate the distribution among the discordant cases. All statistical tests were two-sided, and statistical significance was set at a p-value of less than 0.05. All analyses were performed with STATA software, version 16.0 .Concordance percentage and kappa statistics were calculated to evaluate the agreement between p16 applied ; if not, applied . The kap applied : <0: pooINK4a and E6*I mRNA. Most HPV-DNA-positive cases came from Europe (237) and Central and South America , kappa 0.59 (PABAK = 0.59)) and substantial among HPV-DNA-positive OPC (percentage of concordance 82.1% (95% CI 77.2–87.0%), kappa 0.36 (PABAK = 0.64)). Among LC cases, the concordance was only fair (percentage of concordance 56.9% (95% CI 42.3–71.4%), kappa 0.23 (PABAK = 0.14))  for HPV-DNA-positive LC was 42.1% when considering a p16INK4a cutoff ≥70%, around two times higher than for HPV-DNA-positive OPC and OCC . The same was also observed for p16INK4a cutoffs of >25% and >50% ) . Lowerinnificant . The perand >50% . For allA− cases .INK4a, the percentage of HPV16-positive cases increased for OCC  and OPC  and decreased for LC (41.0%). These differences in HPV type distribution by HPV relatedness definition were statistically significant.HPV16 was the most common type among HPV-DNA-positive cases for all HN sites, although with lower proportions in OCC (69.0%) and LC (51.0%) than in OPC 83.0%, . The nexINK4a  and E6*I mRNA decreased for all HN sites when only considering those cases that are HPV16-DNA-positive as compared to cases HPV-DNA-positive for any type, although this decrease was not statistically significant  triple-positive , whereas, for OCC, they decreased to 43.6%, and the percentage of HPV-DNA/p16INK4a/E6*I mRNA triple-negative cases increased from 7.0% in OPC to 35.9% in OCC, meaning that a much lower proportion of OCC than OPC cases are truly HPV-driven. The reduction in p16INK4a and E6*I mRNA concordance in LC was mainly due to a decrease in the triple positivity. Noteworthily, the proportion of cases HPV-DNA0positive/p16INK4a0negative/E6*I mRNA0positive was much higher in LC than in OPC and OCC , strengthening the argument that p16INK4a testing, even in combination with another HPV test, is not useful to diagnose an HPV-driven LC.We found that the concordance between E6*I mRNA and p16INK4a but are HPV-negative [INK4a and E6*I mRNA discrepancies were substantially higher for LC than OCC. A possible higher misclassification of OPC cases as OCC than LC could partially account for those observed differences. Other aspects such as different type of tissue or different biology of the virus by anatomical site within the head and neck could also explain the observed differences [INK4a expression pattern was observed in those cases as compared to HPV16-positive or HPV-positive for any type.It has been previously reported that a higher proportion of both OCC and LC than OPC cases overexpress p16negative ,16. In oferences . The higRegarding studies comparing p16 performance for diagnosing an HPV-driven oral cavity, laryngeal, and oropharyngeal HNC, the group of Dr. Maura L. Gillison in the USA assessed the accuracy of two different criteria for p16 positivity in HPV-related OCC, OPC, and LC ,19,20. TINK4a cutoffs for any site, although a non-statistically significant higher concordance between p16INK4a and E6*I mRNA was observed for all HN sites when lowering the p16INK4a cutoff from ≥70% to >50%. At the time the main study was published, the internationally agreed recommendations for testing all new OPC for high-risk HPV using a nuclear and cytoplasmatic 70% cutoff of stained cells [INK4a immunohistochemistry test were used across studies [INK4a accuracy by cutoff were not observed for any head and neck site. Thus, when considering HPV-DNA/p16INK4a double test for diagnosis of HPV-driven OCC or LC, cutoffs other than 70% for p16 immunohistochemistry could be applied. Other authors have also pointed out that the 70% p16 cutoff used for HPV-OPC diagnosis may not be fully transferable to other HNC since the squamous epithelium is different within the HN area [We did not observe differences between p16 studies . Moreove studies . A recen studies . It foun HN area .INK4a−/E6*I mRNA+ cases than for p16INK4a+/E6*I mRNA− cases. The observation was consistent for all HN sites and p16INK4a cutoffs. Noteworthily, around 70%, 31%, and 52% of HPV-DNA+/p16INK4a− OPC, OCC, and LC cases, respectively, were E6*I mRNA+ and 2.5%, 8.1%, and 14.3% of HPV-DNA+/p16INK4a+ OPC, OCC, and LC cases, respectively, were E6*I mRNA−. These results may indicate the need for using and optimizing an mRNA-based test rather than a DNA-based test in combination with p16INK4a for diagnosing an HPV-driven HNC, as also pointed out by other authors [INK4a−/E6*I mRNA+ cases have different survival outcomes than p16INK4a+/E6*I mRNA− cases. A study from the US in OPC found that patients who were p16-negative but HPV mRNA-positive had a prognosis somewhat closer to double-positive patients, while those who were p16-positive, but HPV mRNA-negative had a prognosis closer to that of double-negative patients [We found that, among HPV-DNA-positive HNC cases, the percentage of discordant cases was much higher for p16 authors . It woulpatients .INK4a double-positive cases, the percentage of HPV16-positive cases increased for OCC and OPC, but decreased for LC. Moreover, the percentage of discordant cases was higher for HPV16 than HPV-non16-positive cases. These results, if confirmed in other larger studies, may have implications when designing strategies of primary prevention of HPV-driven HNC with HPV vaccination. Moreover, current clinical practice algorithms for HPV testing make no effort to discern the impact of genotypes in HNC patients. A recent study found that HPV genotypes were unevenly distributed across anatomic sites of the head and neck, with an association of certain genotypes with small cell transformation [When considering only cases where HPV was the truly triggering carcinogenic agent  or HPV-DNA/p16ormation .The clinical significance of HPV status in OCC and LC is unclear, and previous studies have shown mixed results ,26. WhilINK4a double test, both in oropharyngeal and in non-oropharyngeal HNC sites, one could consider algorithms also involving tumor morphology [In order to improve the diagnostic accuracy of the HPV-DNA/p16rphology , as alrerphology . HoweverINK4a and E6*I mRNA, and the limited sample size did not allow assessing differences between anatomical subsites within the oral cavity, oropharynx, and larynx or between histological diagnoses. Of note, the current clinical guidelines on HPV testing in HNC do not recommend HPV testing in non-oropharyngeal HNC or in non-squamous OPC [Our study had several limitations. Not all cases were tested for p16mous OPC . Lastly,INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases, was observed. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC including which are the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC.In conclusion, the diagnostic algorithm of HPV-DNA testing followed by p16


Laryngeal papillomatosis is the most frequent benign neoplasia in children. It is caused by HPV 6 and 11. The lesions are exophytic and highly recurrent, compromising the airway mucosa, mainly the larynx. Study design - clinical prospective.to show morphologic alterations of the epithelium (light and electron microscopy) in the HPV-6 lesions.specimens of laryngeal lesions obtained during surgery of four children  were submitted to HPV typing (PCR), light microscopy and electron microscopy.in all specimens, HPV type 6 was found. Epithelial projections were found by electron microscopy with superficial cells in desquamation. Light microscopy showed exophytic projections of the keratinized stratified squamous epithelium overlying a fibrovascular core. Koilocytes (vacuolated cells), suggesting the viral infection by HPV, were identified. No alterations were seen in the basement membrane and corion. Ultraestrutural analysis showed vacuolated cells with clear cytoplasmic inclusions, intercellular injuries and widening intercellular spaces.morphologic alterations of the epithelium in the HPV-6 lesions are superficial, and additional studies including the others HPV types are needed to show the more aggressive and extensive aspect of the disease. Laryngeal papillomatosis is a benign neoplasm characterized by exophytic proliferative lesions of connective tissue covered by epithelium. It usually starts in the commissure and anterior third of the vocal folds, and may eventually affect all of the larynx, and even the trachea, bronchii and lung parenchyma in some cases. Lesions are generally multiple, rarely solitary.3The juvenile form affects children in their first years of life; symptoms include fixed hoarseness that becomes progressive dyspnea an eventually dramatic clinical pictures of respiratory discomfort and stridor. In these cases, there are multiple vegetating confluent lesions occluding the glottic lumen, often extending to supra- and subglottic structures. The adult form affects adolescents and adults; in these cases there are fewer lesions that are focal and less recurring, but the malignancy potential is increased.4Ullman was the first to describe the viral etiology of laryngeal papillomatosis, in 1923.11Notwithstanding these advances in HPV DNA studies, its transmission mode remains unclear. HPV is positive in the airways of neonates and in maternal genital condylomas, which suggests that it is transmitted in the birth canal, from mothers with genital condylomas.Some studies have found HPV in benign laryngeal lesions such as polyps, Reinke's edema, leukoplasia, lichen planus, and even in the normal mucosa of the mouth.19Laryngeal papillomatosis is still a major challenge to pediatricians, infectologists, immunologists and otorhinolaryngologists, even with recent advances in diagnosis. New research on HPV has added precious information to help elucidate the mysteries around this disease. However, there have been few morphological studies in this area.The purpose of this study was to analyze morphological alterations seen in laryngeal papillomatosis caused by HPV subtype The Research Ethics Committee of the institution in which the investigation was done approved the study (protocol number 411/2006). Specimens collected during surgery of four children with a diagnosis of laryngeal papillomatosis were analyzed, and these children were monitored in the Speech Therapy and Voice Disorder Unit where the study was done. The children included in this study consisted of one boy and three girls; three of them were aged between 3 and 6 years, and one was aged 15 years. All children presented with hoarseness and respiratory discomfort, mostly upon significant effort; all had already undergone various surgical procedures in the past. None of their mothers reported the presence of papillary lesion or prior treatment of condyloma.At the time of surgery, fragments of laryngeal lesions were reserved for light and electron (transmission and scanning) microscopy and for defining the HPV subtype using molecular techniques.Light microscopy - fragments were first fixated in 10% formaldehyde for at least 48 hours, then included in paraffin, sectioned, and hematoxyllin-eosin (HE) stained. The specimens were also PAS (periodic acid-Schiff) stained for clearer imaging of the basement membrane. Next, the histological slides were examined under light microscopy at different magnification, and the images were taken using a digital camera (Leica Q-Win software version 3.1).Electron microscopy - scanning and transmission electron microscopy were done on new fragments fixated in 2.5% glutaraldehyde, according to the procedures described by the Electron Microscopy Center of the Bioscience Institute (Centro de Microscopia Eletrônica do Instituto de Biociencias). Following preparation, specimens were examined and photographed under scanning electron microscopy , at 15KV. The exposed surface was tracked quadrant by quadrant, and then photographed at increasing magnification. Sections were also examined and photographed under transmission electron microscopy .The results of morphological analysis were presented descriptively.HPV typing - viral DNA was extracted using a CTAB (cetyltrimethylammonium bromide) solution and tested with the polymerase chain reaction (PCR). Viral DNA was transferred to the membrane using the Dot Blot technique, for typing. Marked probes from the kit AlkPhos Direct (Amersham) were used to detect the types of HPV. The amplification efficiency was monitored by 1.5% agarose gel reaction electrophoresis (Gibco BRL). The size of amplified products were compared with the 123 pb standard (Gibco BRL) and photographed under ultraviolet transillumination.In this study all of the specimens that were examined belonged to the HPV subtype 6. The vocal fold mucosa that was affected by HPV was covered by pale exophytic papilliferous growths located mostly in the anterior glottic region . Light mScanning electron microscopy revealed that vocal folds were covered by epithelial projections with many desquamating superficial cells. The growths varied in size; similar smaller growths appear among larger ones. Cell desquamation appeared more intense in certain regions .Figure 3Transmission electron microscopy showed that cell nuclei contained decondensed chromatin and evident nucleoli. Various organelles were found in the cytoplasm, especially mitochondria, suggesting intense cell metabolism. Usually interdigitated intercellular spaces were widened, distorting the desmossomal structure. Perinuclear vacuolization was evident in light and transmission electron microscopy .Figure 4Papillomatosis may present with severe involvement of the laryngeal mucosa; there may be recurring lesions in the vocal folds, the pharynx and the trachea. In extreme cases this may cause acute respiratory failure. This condition is the most common laryngeal benign neoplasm in children. Although it was described decades ago, its pathophysiology and treatment have been the target of much research.21HPV is a DNA virus that parasitizes epithelial cell nuclei. It is difficult to identify the virus with light and electron microscopy, as our study showed. Molecular biology is required to confirm the parasite and establish its subtype. In our study, a marked epithelial change that was seen both under light and transmission electron microscopy, was perinuclear vacuolization, which is typical of HPV infection.All HPV types taken from laryngeal lesion in our sample belonged to the subtype 6. It is known that HPV-6 and 11 are frequent in children and teenagers, and that they do not malignize despite frequent recurrences. On the other hand subtypes 16 and 18, which may occasionally be found in laryngeal papillomatosis, do have a malignant potential. Padayachee and PrescottThere have been few morphological studies showing the ultrastructural changes caused by each HPV subtype. Most studies do not even inform the HPV type. This study of the HPV-6 morphology enabled us to conclude that epithelial changes are superficial, and that deeper structures such as the basement membrane and the adjacent chorion are preserved. However, the more aggressive behavior of subtypes 11, 16 and 18 may open the possibility for identifying deeper epithelial changes, as well as atypias, since subtypes 16 and 18 have been related to airway malignancies.Morphological changes caused by the HPV-6 demonstrate its non-invasive nature. Additional morphological studies are needed to investigate the ultrastructural alterations caused by other HPV subtypes that are considered as more aggressive.


The results may suggest an etiologic role of HPV in bladder cancer. HPV vaccine administration in both sexes could be key to prevent the infection caused by high-risk genotypes.The etiology of bladder cancer is known to be associated with behavioral and environmental factors. Moreover, several studies suggested a potential role of HPV infection in the pathogenesis with controversial results. A systematic review was conducted to assess the role of HPV. A total of 46 articles that reported the prevalence of HPV infection in squamous (SCC), urothelial (UC), and transitional cell carcinomas (TCC) were selected. A pooled prevalence of 19% was found, with a significant difference in SCC that was mainly driven by HPV-16. Moreover, infection prevalence in case-control studies showed a higher risk of bladder cancer in HPV-positive cases (OR: 7.84;  Bladder cancer (BCa) is the 10th most prevalent cancer globally, with >572,000 and >212,000 incident cases and deaths . MoreoveWhile the role played by human papillomavirus (HPV) in the development of cervical, anogenital, and oropharyngeal cancers was proven ,8,9, theA systematic literature review aimed at retrieving papers focused on the prevalence of HPV infection in BCa was carried out from its inception to 31st December 2021. The literature search was performed using PubMed and Scopus, selecting the key words “Human Papillomavirus”, “HPV”, or “Papillomavirus” and “bladder cancer” or “bladder carcinoma”, combined in different strings. No restrictions related to age of patients, setting, or time of the study were chosen.Lists of references of all selected articles were screened to find other eligible studies not included in the above-mentioned databases.Case-control, cross-sectional, and cohort studies reporting HPV infection prevalence were selected.(1)Studies dealing with patients with primary BCa;(2)Studies describing molecular and non-molecular HPV detection methods on fresh or FFPE  bladder biopsies;(3)Studies focused on the following medical conditions: SCC, urothelial carcinoma (UC), and transitional cell carcinoma (TCC);The following inclusion criteria were considered:(1)Review articles, abstracts, letters, commentaries, correspondences, case-reports, and case-series enrolling <10 subjects;(2)Use of languages other than English;(3)Secondary malignancies located in the bladder.Articles were excluded for the following reasons:Article selection and data extraction were performed by two Authors and double-checked (M.N. and D.B.), while discrepancies of opinions or disagreement were resolved by a third investigator (S.G.).Qualitative and quantitative variables were collected in an ad hoc electronic form.The following variables were collected: first author’s last name; title of the article; year and country/countries of the study; period of the study; epidemiological study design; sample size; sex and age; type of samples; HPV detection methods; histological subtypes; tumor grading; HPV prevalence.No ethical approval was needed given the anonymized and aggregated nature of the data.Inter-rater agreement was ~100% for the phases of study selection and data extraction, and the few inconsistencies were resolved by consensus and with the support of a third investigator (G.S.).Guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) were followed to guide the process of the systematic review .The Newcastle–Ottawa Scale  was usedQualitative and quantitative variables were summarized with absolute and relative (percentage) frequencies and means/medians  respectively.Forest plots were used to show pooled risk differences of the selected outcomes and interval  estimates, as well as the weight of the sample size of the recruited studies. The I2 indicator  showed the association between true variability and overall variation.p-value less than 0.05 was deemed statistically significant. The statistical software Stata version 17  and StatsDirect version 3.1.12 .Fixed and random-effects models were chosen depending on the estimated between-study heterogeneity. A two-tailed A total of 637 articles were identified through electronic database searches; 162 (26.2%) were excluded for being duplicates, and then, a total of 475 studies were screened by titles and abstracts. Fifty-six (11.8%) full texts were evaluated, and ten (17.9%) were excluded for the following reasons: tissue samples did not include bladder tissues (n = 5), full text was not available (n = 2), case-report (n = 1), review (n = 1), and dataset described in another study (n = 1). A total of 46  manuscripts were included in the review .Twelve (34.3%) cross-sectional studies were deemed at moderate risk of bias, whereas 23 (65.7%) were classified as low-risk . Nine 8.Studies were published during the period 1995 ,35,36,38The sample size ranged from 10  to 689  patientsThe majority of the samples were FFPE 2706/3518; 76.9%)  : −0.0451 ; p-value: 0.37542; I2: 75.9%] : 0.0046 ; : 13.5%] . No stat: 75.9%] .p-value: 0.0002) and vs. UC .Pooled prevalence stratified by histological subtypes was 36.5% , 32.5% , and 18.5%  for SCC, TCC, and UC, respectively , with a Also, 69/479 (6.9%) were multiple infections ,52,55,57The most prevalent genotype was HPV-16 , followed by HPV-18 , HPV-6 , and HPV-11 . Moreover, 68 (14.2%) infections were caused by other HR-HPV genotypes .p-value < 0.00001; I2: 34.7%) for the association between HPV infection and occurrence of BCa (The studies with a case-control design numbered 11/46 (23.9%) in the selection, with a total of 611 cases vs. 227 controls ,50,53,54e of BCa .This systematic review was performed to evaluate the prevalence of HPV infection in BCa, keeping into consideration confounding demographic, histological, and diagnostic variables.An overall HPV prevalence of 19% was found in 46 studies, in line with previous meta-analyses which reported a prevalence of 16.88%  and 14.3A statistically significant difference in the prevalence of infection was found in different histological subtypes, with higher estimates in SCC than those in TCC and UC. The high affinity of HPV to differentiating squamous epithelium, previously demonstrated in cervical, head and neck, and anogenital carcinomas , as wellThe assessment of the role of HPV as causative agent in different histological subtypes could have been affected by the adoption of the new WHO BCa classification . This reAlthough several studies suggested a relationship between advanced stage  and HPV prevalence ,43,44, tSimilarly to other HPV-related cancers, HPV-16 was the most prevalent genotype in BCa, supporting previous findings on the increased risk of BCa in cases of infection caused by high-risk HPV genotypes, mainly by HPV-16 . The higFinally, our study showed a significant association between HPV and BCa (OR: 7.84), confirming the findings of recent meta-analyses on the risk of BCa ,62,65. HPrevious systematic reviews investigated the role of HPV infection in BCa and described a moderate association. Our study selection and analysis showed an important role of HPV infection in SCC BCa. However, some limitations should be acknowledged. More stratified analyses related to demographic  and clinical  confounders should be performed. The cross-sectional assessment does not help prove the temporal relationship between an exposure and the development of cancer.The present systematic review highlights a potential role of HPV in the development of bladder cancer, indirectly supporting the adoption of primary preventive strategies in both sexes, as recommended by international authorities. Further epidemiological studies are needed to confirm those findings and assess the role of diagnostic and preventive strategies for HPV-related bladder cancer.


The incidence and mortality rates of cervical cancer are rising among young women in Japan. In November 2021, the Japanese Ministry of Health, Labour, and Welfare reinstated the active recommendation for the human papillomavirus (HPV) vaccine, which was discontinued in June 2013 due to reports of adverse reactions, including chronic pain and motor dysfunction, following vaccination. However, vaccine hesitancy among the younger generation remains, and it is essential to identify the barriers in vaccination uptake. Therefore, we aimed to conduct a randomized study using different methods of providing educational contents to improve health literacy regarding cervical cancer and HPV vaccination among female students in Japan. Here, we present the results of our preliminary report and discuss current topics related to HPV vaccination in Japan. Data were collected from 27 female students—divided into three groups: no intervention, print-based intervention, and social networking service-based intervention—using the health literacy scale and communicative and critical health literacy scale. Our primary results indicate that participants’ knowledge and health literacy improved post-intervention. Therefore, medical professionals must provide accurate scientific knowledge regarding routine HPV vaccination and the risk of cervical cancer to young women to improve their health literacy and subsequently increase the HPV vaccination rates. The incidence and mortality rates of cervical cancer among young women are increasing in Japan . Human pAlthough the vaccine coverage rate varies among countries, studies conducted in countries with national HPV vaccination programs have demonstrated clear benefits of mass vaccination in reducing viral prevalence and associated disease burden . In addiThe Japanese Ministry of Health, Labour, and Welfare  reinstated the official active recommendation for HPV vaccination in November 2021. Despite this, we believe that unless health literacy about cervical cancer and the HPV vaccine among young women who are eligible for vaccination improves, it is likely that vaccination uptake will remain sluggish ,14,15. MA total of 15,450 female students from 17 departments of institutes of a private university group, including Teikyo University , Teikyo Heisei University (5000 students), and Teikyo Institute of Advanced Nursing (450 students) have been enrolled in the ongoing study . At the The inclusion criteria were young female students (1) aged 18–26 years (2) who had received less than three doses of the HPV vaccine or were unvaccinated, and (3) who could access and use SNS-based programs and follow-up questionnaires. Exclusion criteria were students (1) who faced mental and physical challenges using SNS-based programs and (2) who had completed three doses of the vaccine. All students and their parents provided written informed consent. The study was approved by the institutional review board of Teikyo University. A data monitoring committee was not deemed necessary for this feasibility study as we did not anticipate any adverse events; nonetheless, any unintended consequences of the interventions were recorded.A cluster-randomized parallel-group trial with three groups will be conducted: Group 1, no intervention (control); Group 2, educational intervention (print-based education); and Group 3, educational intervention (SNS-based education). The presence or absence of an educational effect will be determined by comparing Groups 1 and 2 with Group 3 for discussion purposes .A faculty or department and the allocation unit are considered as clusters. Allocators prepare an allocation table using a stratified block randomization method with two stratification factors: medical and non-medical institution  + Teikyo Institute of Advanced Nursing Studies  and Teikyo Heisei University ). This is an open-label study, and both the allocators and participants know the allocation results.The medical information and educational tools on cervical cancer and HPV vaccination developed by the principal investigator, which are distributed to female students every six months, are randomly assigned to three arms: Arm 1, no intervention (control); Arm 2, distributed by mail; and Arm 3, distributed through SNS  on websites . All thrA questionnaire is sent to three randomly allocated groups at the time of enrolment (baseline). The research secretariat delivers the allocation results to the enrollees when sending the baseline questionnaire, either by post in a booklet form or via an SNS. The survey questionnaires are emailed to all the groups, and along with the final questionnaire at 15 months, they receive a reminder to complete the questionnaires. The questionnaires inquire about the participants’ faculty affiliation, age, diet, HPV vaccination history, and whether they had a family health care provider, smoked, exercised voluntarily, ever visited an obstetrician, received routine vaccinations, as well as their knowledge regarding cervical cancer, using the health literacy scale. At the time of questionnaire delivery, students in the no-intervention control group are informed that they will not receive any medical information or educational tools on cervical cancer and HPV vaccination and will be sent the same questionnaire, omitting basic information, thrice during enrolment.The primary outcome is (1) increasing self-reported HPV vaccine uptake rates via SNS-based educational contents: from no intention to at least taking one dose or all three doses and (2) increasing knowledge of susceptibility to cervical cancer, its severity, and benefit from HPV vaccination as measured by the reliable and valid health belief model . We investigate vaccination barriers on self-reported HPV vaccine uptake and behavioral intention or attitude changes toward HPV vaccine uptake in support of educational contents between unvaccinated and vaccinated participants who had previously received a single or double dose of the HPV vaccine.Participants answer five questions on the communicative and critical health literacy (CCHL) scale ranging from 1 (very easy) to 5 (very difficult) regarding the HPV vaccine and cervical cancer screening. The mean within-individual health literacy score is the mean of the five questions on CCHL; a score of 4 or more is considered “highly health literate.” The primary endpoint is the proportion of participants with high health literacy scores immediately after the third delivery of educational contents (12 months in the study). The null hypothesis is that the proportion of participants with high health literacy in Groups 1 and 2 is equal, while the alternative hypothesis is that the proportion of participants with high health literacy in Group 2 is higher than that in Group 1.The proportion of high health literacy under the competing hypotheses was P1 (intervention group) = 0.60 and P2 (control group) = 0.30, implying that the meaningful difference in the proportion of high health literacy between the two groups was 0.30, the within-class correlation was 0.100, the mean cluster size was 200, the two-sided significance level was 0.05, and the power was 0.80. The required number of clusters for each group was calculated as 4.4. If there are five clusters in each group, the total number of required participants is assumed to be 5 × 3 × 200 = 3000, and assuming a dropout rate of about 15%, 3500 participants are required.We use a logistic regression mixed-effects model, with the dichotomous variable high or low health literacy as the outcome variable, to calculate the odds ratio of the effect of group on high health literacy with group (control and intervention), institution, medical and non-medical as population effects, and cluster as a variable effect. If the odds ratio is significantly higher than 1, we can conclude that the intervention affected the participants’ health literacy. Adjustment factors such as age and prior knowledge can be included in the statistical model upon the discretion of the statistical analyst and principal investigator. A cluster-randomized study with three arms of groups will be delivered in the following clusters (17 schools): law, economics, literature, foreign language, education, medicine, pharmaceutical sciences (2 schools), medical informatics and technology, nursing (3 schools), science and engineering, modern life, health care, health and medical science, and medical sports.We conducted a preliminary investigation between 2019 and 2020 in Japan. Due to the coronavirus disease (COVID-19) pandemic, only a few students (27 applicants) participated in the study, which was conducted in our laboratory at Teikyo University, after seeing the participation invite posted in the teaching department. Information pertaining to students’ faculty affiliation, family background, smoking status, attitudes toward health, and routine vaccination status was collected. Furthermore, the extent of their knowledge regarding cervical cancer and the HPV vaccine was noted. Although one of the inclusion criteria for study participants was being unvaccinated for HPV, six HPV-vaccinated students were included in this preliminary study .The participants were divided into three randomly assigned groups and sent a questionnaire at enrolment (baseline). As this was a preliminary investigation, the number of participants was small to allow comparison between the three groups; therefore, we summarized the results for all the participants. Two rounds of the survey were conducted, and the preliminary results are shown in The results of the CCHL scale are presented in Currently, national HPV vaccination programs are publicly funded in more than 120 countries worldwide . In JapaSurveillance after HPV vaccination must continue, but we should also consider the rigorous treatment that patients need to undergo on contracting cervical cancer. The efficacy of HPV vaccine outweighs the risk of adverse symptoms. Therefore, the HPV vaccination rate is on the rise as Japanese MHLW resumed actively recommending the HPV vaccine program in November 2021. In addition, due to the COVID-19 pandemic, people’s physical and emotional barriers to vaccination to prevent transmission of the virus gradually eased . Thus, aWe conducted a preliminary investigation that compared the knowledge of cervical cancer and the HPV vaccine among female students before and after the distribution of print-based and SNS-based educational contents. Due to the small number of students included in the analysis, it was impossible to draw any firm conclusions; however, the results showed a general tendency for improvement in knowledge. Although non-medical students may have found it difficult to understand the educational contents, their participation in the study could have led them to seek more knowledge through the Internet. In the future, we would like to analyze communication failures regarding information on HPV from medical, social-scientific, and behavioral perspectives. Furthermore, participants’ health literacy improved as indicated by the results of the second survey. However, as the data were not adequate, we were unable to compare the three groups and comment on the impact of the educational interventions on knowledge; nonetheless, the motivation to participate in the study alone could likely indicate a behavioral change in the participants.In the future, we hope to reach our target of 3500 participants. The study aimed to include female students aged 18–26, who missed their routine vaccination periods and were not yet eligible for vaccination after the Japanese MHLW resumed active vaccination. Currently, there is an ongoing debate in Japan about subsidizing catch-up vaccinations, and there has been no decision on introducing the more promising 9-valent vaccine as a routine vaccine. Students who wish to be vaccinated prior to the catch-up vaccination scheme must pay for them; moreover, many of them have been self-inoculated. Therefore, it is crucial that we, especially medical professionals, inform them about routine HPV vaccination and the risk of cervical cancer, which often affects women in their 20s and 30s, based on accurate scientific knowledge. Some studies have analyzed knowledge and awareness about cervical cancer and HPV vaccines in other countries ,38,39,40There is a need for continued grassroots efforts to improve knowledge of cervical cancer and HPV vaccination among female students in Japan. Medical professionals, including obstetricians and gynecologists, need to take responsibility and provide accurate scientific knowledge regarding cervical cancer to female students and their parents. Furthermore, efforts need to be made to eradicate cervical cancer. Finally, we continue to recruit participants for this study and hope to publish comprehensive results after completing the study.


In-situ hybridization provides a convenient and reliable method to detect human papillomavirus (HPV) infection in formalin-fixed paraffin-embedded tissue. Cases of conjunctival papillomas, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma in situ (cCIS), and invasive squamous cell carcinoma (SCC), in which low-risk (LR) and/or high-risk (HR) HPV types were evaluated by RNA or DNA in-situ hybridization, were retrospectively identified. LR HPV types were frequently detected in conjunctival papillomas , including 17/18 (94%) with RNA probes, compared to 8/12 (75%) with DNA probes. None of the CIN/cCIS or SCC cases were positive for LR HPV by either method. HR HPV was detected by RNA in-situ hybridization in 1/16 (6%) of CIN/cCIS cases and 2/4 (50%) of SCC cases, while DNA in-situ hybridization failed to detect HPV infection in any of the CIN/cCIS lesions. Reactive atypia and dysplasia observed in papillomas was generally associated with the detection of LR HPV types. Collectively, our findings indicate RNA in-situ hybridization may provide a high-sensitivity approach for identifying HPV infection in squamous lesions of the conjunctiva and facilitate the distinction between reactive atypia and true dysplasia. There was no clear association between HPV infection and atopy in papillomas or dysplastic lesions. The ocular surface barrier, predominately comprising the conjunctival epithelium, is uniquely vulnerable to chronic environmental exposure and microbial infection ,2. As a Atopy has also been reported as a risk factor in OSSN development. Several proposed mechanisms for atopy-associated tumorigenesis have been hypothesized, including pruritus-induced epithelial microtrauma with secondary HPV infection, type I hypersensitivity-mediated inflammation and tissue remodeling, and competitive binding of IgE receptors by allergen-specific IgE, limiting anti-tumor immune responses ,24,25,26It has been estimated that as many as 400 types of HPV, a small, spherical, non-enveloped, double-stranded DNA virus, exist, and approximately 200 have been fully characterized, with mucosal types grouped into low- (LR HPV) and high-risk (HR HPV) subtypes based on potential for malignant transformation ,31,32,33A number of studies suggest HPV, alone, is sufficient as a causative agent for conjunctival lesions; however, others propose a potential two-hit etiology, requiring prior UVR damage, chemical mutagenesis, or immunosuppression in addition to HPV infection ,43,44,45RNA in-situ hybridization permits direct visualization of intralesional transcriptionally active virus, with probes capable of detecting E6 and E7 mRNA, and provides morphologic evaluation of nuclear HPV localization within virally infected neoplastic cells ,65,66,67HPV in-situ hybridization was performed on 53 lesions, including 33 papillomas, 16 CIN/cCIS, and 4 invasive SCCs. Demographic and clinicopathologic information for these patients is provided in Representative clinical photographs and photomicrographs of each type of lesion are presented in In-situ hybridization using DNA probes was performed in 14/33 (42%) of papillomatous lesions and 4/16 (25%) CIN/cCIS lesions. In-situ hybridization using RNA probes was performed in the remaining lesions: (19/33 (58%) papillomas, 12/16 (75%) CIN/cCIS, and 3/3 invasive SCCs). Low-risk types were detected in 25/30 (83%) papillomas, 0/11 CIN/cCIS, and 0/1 invasive SCCs. High-risk types were detected in 2/28 (7%) papillomas, 1/16 (6%) CIN/cCIS, and 2/4 (50%) invasive SCCs. Detection of LR and HR HPV types was mutually exclusive in papillomatous lesions, and four lesions (12%) were negative for both LR and HR HPV.HPV signal presented in epithelial cells as dot-like foci visible at 100× magnification in the nucleus. Multiple dots in at least a subset of cells were required to call a case positive. In some cases, the small intracellular signals were quite numerous, and could become almost confluent within a single nucleus. Staining was restricted to epithelial cells, and stromal elements, including blood vessels and mesenchymal cells, were negative in all cases examined. In cases containing normal conjunctiva adjacent to a lesion, the non-neoplastic epithelium was also always negative.With respect to the extent of staining within individual lesions, HPV was present diffusely in most papillomas. Focal HPV positivity was observed in 8/25 (32%) LR HPV-positive papillomas and 1/2 (50%) HR HPV-positive papillomas, while diffuse staining was noted in 17/25 (68%) and 1/2 (50%) LR and HR HPV-positive papillomas, respectively. In contrast, HPV expression was more localized in the OSSN cases that contained virus. Focal HPV positivity was observed in the single HR HPV-positive CIN (Case 39) and both HR HPV-positive invasive SCC lesions. Further details of HPV in-situ hybridization are summarized in Conjunctival papillomas can become quite inflamed, leading to reactive epithelial atypia, in which epithelial cells show generally mild nuclear enlargement and pleomorphism, along with some chromatin changes and more prominent nucleoli. Reactive atypia can sometimes be difficult to distinguish from dysplasia, although the latter process is often less uniform and more severe. Detection of LR HPV could potentially be helpful in such cases and support a less aggressive biological potential if positive. In 18 of 33 (55%) papilloma cases, intraepithelial inflammation and epithelial atypia diagnosed as reactive were detected. Dysplasia was rarer, and noted in 4/25 (16%) LR HPV-positive papillomas, 1/2 (50%) HR-positive papillomas (Case 29), and none of the HPV-negative papillomas. In three LR HPV-positive papillomas diagnosed with dysplasia, the focal in-situ hybridization signal corresponded to the foci of dysplastic epithelium, while in Case 33 and Case 39, discrete foci of epithelial dysplasia were observed, but more diffuse LR and HR HPV signal was noted. An inflamed papilloma with reactive atypia and LR HPV-positive epithelium is demonstrated in Koilocytic cells were identified in all lesion types but were relatively rare, with focal change observed in 2/6 (33%) of HPV-negative papillomas, 10/25 (40%) LR HPV-positive papillomas, 1/15 (7%) HPV-negative CIN/cCIS, and 1/2 (50%) HR HPV-positive invasive SCCs. More diffuse koilocytic-appearing cells were observed in 1/25 (4%) LR HPV-positive papillomas, as shown in Patients returned for follow-up care in 17/33 (52%) papilloma cases, 8/16 (50%) CIN/cCIS cases, and none of the invasive SCCs. The length of follow-up (mean ± SD) was 605 ± 1219 days and 887 ± 1534 days for papilloma and CIN/cCIS cases, respectively. Recurrence was noted in only one case (Case 14) following an initial diagnosis of an HPV-negative papilloma. The recurrent lesion was originally treated with excision and subsequently observed.History of atopic disease, including asthma, eczema, or allergic rhinitis or conjunctivitis, was also recorded for patients when possible. Electronic medical records were available for review for 18/33 (56%) of all papillomas, including 3 HPV-negative lesions, 14 LR HPV-positive lesions, and 1 HR HPV-positive lesion. Atopic disease, including asthma (1/3), rhinitis/conjunctivitis (1/3), and a combination of the two (1/3), was reported in patients with HPV-negative papillomas. A history of asthma was present in 9/14 (64%) patients with LR HPV-positive papillomas, 2 of whom also had additional atopic disease, including eczema and rhinitis/conjunctivitis, respectively. Two additional patients with LR HPV-positive papillomas had histories of eczema and rhinitis/conjunctivitis, alone. There was no reported history of atopic disease in the single HR HPV-positive papilloma. For cases of CIN/cCIS, there was no history of atopic disease in the HR HPV-positive lesion that had medical records available for review.The prevalence of HPV infection in conjunctival epithelial proliferations has been difficult to establish, due, at least in part, to the range of detection methods used ,72. To sConjunctival papillomas can occasionally become inflamed and show prominent reactive atypia, which can be difficult to distinguish from clinically worrisome dysplasia. Among the 33 papillomas we examined, generally mild dysplasia was identified in 5 cases (15%). This number may be elevated due to selection bias, as papillomas with concerning microscopic features were more likely to have had HPV status analyzed during the study period. LR HPV was detected in four of five potentially dysplastic papillomas, while HR HPV was only detected in one. This suggests that the majority of conjunctival papillomas with atypia or dysplasia are associated with LR HPV, and that in-situ analysis could be useful in supporting limited malignant potential in many of these cases. Similar to these findings at the ocular surface, LR HPV was detected in the majority of papillomas, including those with atypical features of the nasopharynx, oral cavity, and larynx, while HR types are more commonly detected in invasive SCCs of these tissues ,82,83,84The reported prevalence of HPV in CIN and other more aggressive OSSN is more variable, with positive cases almost always containing high-risk types ,34,35,85Immunohistochemical approaches, including direct detection of HPV-encoded proteins or alterations in p16 expression, have been investigated, but more recent studies suggest that these methods are prone to false-positive results ,58,59,82Many demographic and clinical associations in our study were similar to prior reports. Papillomas of the conjunctiva reportedly occur more commonly in younger patients, while more dysplastic OSSN lesions tend to present in older ones, and our cases had a mean age of 43, 68, and 74 years at the time of presentation for papillomas, CIN/cCIS, and invasive SCCs, respectively ,77,87. PLimitations of the current study include the relatively small number of invasive SCC cases, precluding the possibility of statistical comparisons between groups. In addition, detailed review of electronic medical records was not possible for all cases in this series, as many were older pathology consultations submitted by outside physicians. While RNA in archived formalin-fixed paraffin-embedded tissue is labile and sensitive to degradation, potentially affecting the sensitivity of in-situ hybridization, we do not believe this is of concern in our study, as all in-situ hybridization assays were performed around the time of initial histologic review ,65.A retrospective review of the Ophthalmic Pathology Archives of The Johns Hopkins Hospital for cases matching keyword searches including “conjunctiva,” “in-situ hybridization,” and “human papillomavirus” between 2007–2021 was conducted. Electronic medical records were reviewed for the following clinical and pathologic information: age at presentation, gender, lesion type and location, clinical treatment modalities, length of clinical follow-up, history of recurrence, history of atopic disease, HPV in-situ hybridization assay details, and details regarding any additional immunohistochemical stains. Any case for which in-situ hybridization was performed to detect LR and/or HR HPV in a conjunctival papilloma, CIN/cCIS, or invasive squamous cell carcinoma was included. Cases were reviewed to confirm the initial diagnosis, and intralesional koilocytic or dysplastic features were noted.HPV in-situ hybridization was performed by the Johns Hopkins Hospital clinical laboratories at the time of initial review of histology using standard protocols. Lesions assayed prior to 2015 were subject to DNA ISH , and those evaluated from 2015 and onward were analyzed using RNA probes . Both DNA and RNA probes for LR HPV detect the same two types , while DNA probes for HR HPV detect 12 types  and RNA probes detect 18 HR HPV types . While 37/53 lesions (70%) were evaluated for both LR and HR types, evaluation by either DNA or RNA approaches was mutually exclusive. The commercially available probes utilized in this study were designed to hybridize with E6/E7 in the various HPV types. All determinations of in-situ hybridization positivity were performed by an ocular pathologist (CGE).We confirmed that LR HPV is frequently detected in conjunctival papillomas while HR HPV is rarely detected in these lesions. The fact that most papillomas with histopathological dysplasia were positive for LR HPV suggests that their malignant potential is limited, and that in-situ hybridization may help with prognostic assessment, although analysis of more cases with long-term follow-up will be needed to confirm this. HR HPV was detected by RNA in-situ hybridization in a relatively small number of CIN and SCC, but was not clearly linked to atopy or other clinical and histopathological features.


In conclusion, only one in five adolescents in the Ilala municipality received a second dose of HPV vaccine. Such low uptake was associated with attitude towards the HPV vaccine and low awareness of HPV-2 vaccines. Regular community sensitization and awareness campaigns by relevant authorities and implementers may help to increase the HPV vaccine uptake.Cervical cancer represents the most common neoplastic pathology among women, with a high burden of morbidity and mortality globally. Tanzania is no exception. The human papillomavirus (HPV) vaccine remains the most effective intervention to address such a burden. However, the uptake of the second dose to confer full immunity remains a challenge. This study aimed to assess the uptake and factors associated with the second dose of the HPV (HPV-2) vaccine uptake among adolescents in the Ilala municipality of Dar es Salaam, Tanzania. Using a quantitative cross-sectional study, data of 389 adolescent girls was collected using a self-administered structured questionnaire. Analyses were conducted using Statistical Package for the Social Sciences (SPSS) software through descriptive and multivariate logistic regression methods to determine uptake, characteristics, and factors associated with the uptake of the second dose of the HPV vaccine. Among the 389 adolescents, the uptake of the HPV-2 vaccine dose was only 21.3%, a lower level compared with the first dose of HPV vaccine (35.2%). Factors associated with the uptake of the HPV-2 vaccine were age (AOR 0.14,  Cervical cancer is one of the leading causes of cancer death among women and the fourth most frequently occurring malignancy among women globally ,2. It haEast Africa has the highest age-standardized incidence rate for cervical cancer at 42.7 per 100,000 women per year. Evidence suggests that Uganda harbors an estimated 33.6% of women with human papillomavirus (HPV) in the general population with 44 per 100,000 women developing the disease yearly . In TanzCervical cancer is associated with HPV infections, with serotypes 16 and 18 of HPV being responsible for about 70% of all invasive cervical cancer cases worldwide [Vaccination against HPV is one of the major public health strategies for preventing cervical cancer in the general population. The vaccines are bivalent (CervarixTM) targeting HPV types 16 and 18, and quadrivalent (GardasilTM), targeting HPV 16 and 18 and HPV 6 and 11 that cause genital warts. Additionally, a nine-valent HPV vaccine Gardasil 9TM) is available and targets HPV types 31, 33, 45, 52, and 58 in addition to HPV types 6, 11, 16, and 18 TM is ava.To ensure maximum protection, the HPV vaccination requires two doses within a six-month interval for adolescent girls aged 9–14 years as per the World Health Organization (WHO) recommendation . Evidence from Tanzania indicates that only one in two adolescent girls aged 14 years were covered by the second dose of the HPV vaccine, while more than two in three received the first dose in 2019. Such vaccines are being given for free by the government of Tanzania. Factors for such low vaccine uptake remain unknown. Evidence on discrepancies pose a challenge for policy and planning to provide targeted interventions for cervical cancer prevention in Dar es Salaam and Tanzania as a whole. A cross-sectional quantitative study was conducted using self-administered questionnaires  in IlalaThe required sample size of 388 respondents including a 5% non-response rate was determined using a formula given by Fisher et al. (2003). A three-stage sampling procedure for sampling school girls was applied in the study. At the first sage, three wards  were selected in the Ilala municipality by simple random selection whereby the sampling frame was a list of all wards in the Ilala municipality. At the second stage, two secondary schools from each pre-selected ward were randomly selected, making a total of six secondary schools which were involved in the study . A list of all secondary schools in the pre-selected wards formed a sampling frame. At the third stage, adolescent girls were randomly selected from their respective classes for participation in the study. This was carried out for each pre-selected secondary school until the required number of respondents was realized, with the attendance registers of the students forming the sampling frame.The study enrolled all adolescent girls aged 12–14 years in the pre-selected secondary schools. This is the age range group of adolescent girls that were thought to be in secondary schools, taking into account that some of them start schools earlier, especially in urban regions such as Dar es Salam.The dependent variable was uptake of the second dose of the HPV vaccine. Independent variables included factors such as socio-demographic characteristics, knowledge about the HPV-2 vaccine, perception, attitude towards the HPV vaccine, awareness on the second dose of the HPV vaccine and household wealth index. Knowledge of the respondents about the HPV vaccine was measured using fifteen questions, each with the response of “yes” or “no”, and the correct response scored one point. A score of more than or equal to 7 out of 15 points were considered good knowledge. Respondents who heard about the HPV-2 vaccine were considered as being aware of the second dose of the HPV vaccine . Adolescp < 0.2 were included to obtain adjusted odds ratio (AOR) with 95% (CI) to test factors independently associated with an uptake of the second dose of the HPV vaccine. Categorical variables were summarized as percentages and frequencies and presented in tables and figures. Data was reviewed for completeness, and coded and analyzed using SPSS  version 26. Summary statistics such as frequencies and percentages of different variables were computed, then univariate Logit mode was conducted to measure the association between explanatory variables and outcome variables. At multivariate analysis, all independent factors with Ethical approval of the study was obtained from the Muhimbili University of Health and Allied Sciences (MUHAS) Research and Ethics Committee with an IRB number MUHAS-REC-05-2021-602 before conducting data collection. Permission to carry out the study in the region was obtained from the regional and district council. The purpose of the study was explained to participants and written informed consent was obtained from school adolescent girls aged 18 years and above before the interview was conducted. Heads of selected secondary schools consented on behalf of the girls aged less than 18 years who could not consent. Information provided by the participants was disclosed only for research purposes.Out of 389 respondents, only 83 (21.3%) had received the second dose of HPV vaccine, 137 (35.2%) had received the first dose of HPV vaccine shot which was high compared to the second shot. The majority of the respondents had not received both first and second doses of the HPV vaccine . Therefop < 0.001) compared to those whose caregivers had secondary education. A total of 53 (24.7%) adolescents whose parents were self-employed received a second dose, a proportion higher than those whose parents were either employed or unemployed (p = 0.032). There was no significant difference in second dose uptake with regards to household wealth level.  adolescents were between 12 and 13 years, and of them, only 2 (3.5%) had received a second dose of the HPV vaccine. Of the older adolescents, 81 (24.4%) had received the second dose. Adolescents whose parents had no formal education were less likely to receive a second HPV vaccine  were found to be independently associated with second HPV vaccine uptake with p-value = 0.023, and were 2.04 times more likely to receive the HPV-2 vaccine compared to those with a negative attitude.A total of 219 (56.3%) respondents with a positive attitude towards HPV vaccine were positively associated with the uptake of HPV-2 , p-value = 0.003. Therefore, positive attitude and awareness on HPV-2 vaccine were independent predictors of HPV-2 uptake. A total of 322 (82.8%) respondents with awareness about HPV-2 vaccine were 9.16 times more likely to receive a second HPV vaccine shot compared to the group without awareness , p-value-0.124. Those with good knowledge of the HPV vaccine were 186 in total (48.2%), and were 1.05 times more likely to receive the HPV-2 vaccine compared to those with poor knowledge  with p-value 0.878  respondents had good perception about the HPV vaccine, and such respondents were 1.58 times more likely to receive the HPV-2 vaccine compared to those with poor perception  ue 0.878 . HoweverThe study examined the uptake of the second dose of HPV vaccine and factors that may be associated with its uptake in Dar es salaam, Tanzania. The study findings provide recommendations to policy makers and other stakeholders to improve strategies for increasing the second dose coverage, to provide full protection to women from cervical cancer.In this study, findings revealed that the uptake of the second dose of the HPV vaccine was lower compared to the first dose. Age, attitude, and awareness were independent predictors of HPV vaccine second-dose uptake.Only one in five adolescent girls (21.3%) had taken a second dose of the HPV vaccine in Dar es salaam, Tanzania. This is a lower uptake compared to the reported 35.2% for the first dose. This finding suggests a low uptake trend, similar to another study in Uganda which revealed 17.6% of adolescents had taken the second HPV vaccine dose . The lowAge was associated with an uptake of the second dose of HPV vaccine in this study. Adolescent girls aged between 12 and 13 years were 86% less likely to receive the second HPV vaccine shot compared to their elders, similar to other contexts ,19. In aAttitude was significantly associated with an uptake of the second dose of the HPV vaccine in this study. Adolescent girls with positive attitude towards HPV vaccine were positively associated with the second dose of the HPV vaccine uptake, similar to other studies from low- and high-income countries ,21,22,23Awareness about the HPV vaccine second dose among school adolescent girls in this study was also found to be an independent predictor of second HPV-vaccine uptake. Public awareness programs on HPV vaccination are of paramount in increasing the second dose of HPV vaccination among adolescent girls . This fiPerception on the HPV vaccine second dose in this study was not an independent predictor of uptake. Comparatively, other studies in particular showed no significant association between perception and second dose of HPV vaccine uptake . ContrarHPV vaccine knowledge promotes coverage in various contexts. Media, medical practitioners, teachers, and parents play a significant role in fostering HPV vaccine knowledge which in turn leads to increased HPV-2 vaccination coverage . In thisThe current study showed that the socio-economic status of individual adolescent girls was not significantly associated with the uptake of the second HPV vaccine. This was contrary to other studies which showed an association of HPV vaccine uptake with lower economic status and middle-wealthy quantiles ,32. ThisThe evidence presented in this study is not short of limitations. The study assessed level of uptake for the second dose but the context may not be the same as in the first dose owing to massive campaigns, awareness, and communications made to adolescents and their caregivers. The study design was cross-sectional, which has its own limits in making causal inferences between the main outcome and independent variables.In conclusion, this study showed that the uptake of second dose of the HPV vaccine among adolescent girls in the Ilala municipality was low. Factors associated with the second dose of the HPV vaccine uptake included good attitude towards the HPV vaccine and awareness about HPV vaccines. This evidence calls for a regular HPV vaccination community sensitization about HPV and its vaccine to increase awareness, and therefore improve attitudes towards vaccination itself.