The Human Papillomavirus FOr CervicAL cancer (HPV FOCAL) trial is a large randomized controlled trial comparing the efficacy of primary HPV testing to cytology among women in the population‐based Cervix Screening Program in British Columbia, Canada. We conducted a cost‐effectiveness analysis based on the HPV FOCAL trial to estimate the incremental cost per detected high‐grade cervical intraepithelial neoplasia of grade 2 or worse lesions (CIN2+). A total of 19,009 women aged 25 to 65 were randomized to one of two study groups. Women in the intervention group received primary HPV testing with reflex liquid‐based cytology (LBC) upon a positive finding with a screening interval of 48 months. Women in the control group received primary LBC testing, and those negative returned at 24 months for LBC and again at 48 months for exit screening. Both groups received HPV and LBC co‐testing at the 48‐month exit. Incremental costs during the course of the trial were comparable between the intervention and control groups. The intervention group had lower overall costs and detected a larger number of CIN2+ lesions, resulting in a lower mean cost per CIN2+ detected ($7551) than the control group ($8325), a difference of ‐$773 [all costs in 2018 USD]. Cost per detected lesion was sensitive to the costs of sample collection, HPV testing, and LBC testing. The HPV FOCAL Trial results suggest that primary HPV testing every 4 years produces similar outcomes to LBC‐based testing every 2 years for cervical cancer screening at a lower cost. The Human Papillomavirus FOr CervicAL cancer (HPV FOCAL) trial is a large randomized controlled trial comparing primary HPV testing to liquid‐based cytology for cervical cancer screening. We conducted a cost‐effectiveness analysis alongside the HPV FOCAL trial. Results showed the intervention group yielded a higher rate of detected cervical intraepithelial neoplasia of grade 2 or worse lesions (CIN2+) at a lower mean cost per CIN2+ detected ($7551) than the control group ($8325), a difference of −$773. Findings suggest that primary HPV testing is cost‐effective compared to liquid‐based cytology. This analysis involves only the results of the intervention and control groups (the safety group was closed early once safety was demonstrated). Women randomized to the intervention group received baseline HPV testing. Baseline HPV‐negative women were recalled for exit at 48 months where they received HPV and cytology co‐testing. If baseline results were HPV positive, reflex LBC testing was done. If atypical squamous cells of undetermined significance (ASCUS) or greater, they were referred to colposcopy; if the LBC was negative, they were recommended to return in 12 months for repeat HPV and LBC testing. Women randomized to the control group received baseline LBC testing and if LBC negative were recalled at 24 months for screening with LBC and if negative at the 24‐month screen, were then recalled for exit screening at 48 months with HPV and LBC co‐testing. Those who were ASCUS at baseline received reflex HPV testing and if HPV positive, were then referred for colposcopy; and if cytology negative, were requested to return in 12 months for repeat LBC testing. Those with baseline LBC results low‐grade squamous intraepithelial lesions or greater (≥LSIL), or those with three consecutive LBC unsatisfactory results were referred for colposcopy and biopsy and managed based on the results. Women with CIN2+ were referred for management according to BC Cancer Cervix Screening Program provincial guidelines.Resources utilized by participants in both study groups were collected throughout the course of the trial. At each screening appointment, participants underwent a pelvic examination and collection of a cervical specimen. The sample underwent LBC and/or HPV testing . Women with positive samples could be re‐tested, and depending on study arm and result, women with positive HPV and/or LBC results were either recommended for colposcopy or to return for repeat testing in 12 months. If a CIN2+ lesion was detected at colposcopy, the woman was referred for standard of care management which typically includes excision therapy  to remove the lesion. Not all women referred for colposcopy attended; accordingly, colposcopy resources were counted on an ‘intent to treat’ basis.Unit costs were estimated for each resource used in the trial. The cost of a screening appointment was directly derived from reimbursement rates from the BC Medical Services Plan (MSP), which is the provincial insurance provider for medically necessary services in BC.Total costs were estimated for each group, as well as the cost per detected lesion in each arm and the cost per trial participant in each group. It is important to note, the primary end‐point for this study was pre‐determined by the design of the HPV FOCAL Trial, as we are reporting within trial results here. The FOCAL Trial included exit co‐testing in the design of both arms and in its reporting of primary end‐points, which means our primary end‐points also include exit co‐testing.That said, since the exit co‐testing  at the 48‐month appointment is not representative of how screening would be conducted outside the trial, a secondary analysis was performed that excluded the results of 48‐month exit co‐testing. In this scenario, 48‐month results included only CIN2+ that were detected via the primary screening method  in the denominator. Resource use and costs attributable to the secondary screening method were excluded from the numerator. Costs attributable to a reflex secondary screening (due to positive primary) were included, while co‐testing costs that would not otherwise have occurred (due to negative primary) were excluded.Ninety‐five per cent confidence intervals (CIs) around detected CIN2+ lesion counts were estimated from a Poisson distribution. Univariate threshold analyses were conducted to estimate the change in unit costs that would be necessary for the cost per lesion detected to be equivalent between the two screening strategies. All costs are expressed in 2018 US Dollars (USD), converted from Canadian Dollars using the annual exchange rate for 2018.3Within‐trial resource use from the FOCAL Trial is presented in Table Total cost within the intervention group was estimated at $1,472,445 or $154 per enrolled participant. Total cost within the control group was estimated at $1,581,750 or $167 per enrolled participant. The mean cost per detected CIN2+ lesion was $7551 versus $8325 for the intervention and control groups, respectively—a difference of ‐$773. The intervention protocol was less costly and more effective than the control protocol.When analyzing trial results without co‐testing, the two arms had similar total costs , with a higher number of CIN2+ lesions detected in the intervention group (193) than in the control group (165). Cost per detected CIN2+ lesion was $6976 in the intervention group, and $8078 in the control group. The proportion of total cost attributable to co‐testing was lower in the intervention group, representing 8% of total costs vs. 15% of total costs in the control group . Results are presented in Table Univariate analysis suggests that the cost per detected CIN2+ results are sensitive to the cost of sample collection, HPV testing, and LBC testing, due in large part to the nearly‐equivalent costs and number of lesions found between the two groups over 48 months. Full results for univariate threshold analyses across the three different analytic perspectives are found in Table 4Our analysis suggests that despite the higher test cost, HPV‐based screening every four years is associated with lower costs and superior rates of CIN2+ detection compared to liquid‐based cytology screening every 2 years among women who regularly attend screening. This improvement in relative cost‐effectiveness was due primarily to the longer interval between tests, but also in part to a reduced need for re‐screening due to unsatisfactory samples, and a lower false positive rate at each screening appointment. The 48‐month exit co‐testing costs played a larger role in the control group, due to higher rates of CIN2+ detection from the HPV co‐test that was not detected by LBC. Results were sensitive to the costs of sample collection and testing. This suggests that changes in the cost and/or specificity of HPV testing could have an impact on the cost‐effectiveness of a system‐wide screening program.The costs of LBC and HPV testing were estimated from internal FOCAL Trial costs. It is possible that the cost of HPV testing may come down if it is introduced on a population level, meaning HPV based‐screening may result in further cost savings to the public. Data on the costs of annual cytology follow‐up or periodic co‐testing following treatment for cases of CIN2+ (including LEEP) were not collected within the trial. As more cases of CIN2+ were detected and treated in the intervention arm, this may result in a slight over‐estimation of cost savings.The consensus of CanadianOur results were based on a large population of screening participants within a centralized screening program. Nevertheless, there are some limitations to our findings. First, our screening population was observed in the context of a clinical trial, which included reminder calls and follow‐up that exceeds what would be current standard of care at the population level. This may slightly increase the costs and number of CIN2+ cases detected in both arms, but we do not anticipate it would affect our incremental cost‐effectiveness findings. Second, our comparator (LBC‐based screening every two years with reflex HPV testing for ASCUS results) is based on the FOCAL Trial protocol, not current standard of care in BC. At the time the FOCAL trial was developed and conducted, cytology testing every two years was the standard of care. This may slightly increase the number of CIN2+ cases detected in the control arm, and suggests our results may provide a conservative estimate of the incremental benefits of HPV testing when compared to standard care. Third, this evaluation was based on a two‐year screening interval for LBC cytology and a 4‐year interval for HPV testing. The US Preventive Services Task Force recently released a recommendation, based in part on FOCAL Trial results, that primary HPV testing every five years was preferable to conventional screening every 3 years.It is important to note that the number and timing of detected CIN2+ lesions differ between the trial groups. A larger number of lesions were detected in the HPV group in round one (baseline up to 12 month follow‐up), with a smaller number at 48‐month exit; this was reversed for the control group, with more CIN2+ detected in the control group at exit due to the HPV co‐test. This suggests our results may not fully reflect the impact of regular HPV testing in the long term .Our results are also limited to the number of CIN2+ lesions detected within the FOCAL Trial because this was the end‐point for the RCT itself, and assume that women who are lost to follow‐up did not go on to develop a lesion. Consequently, our analysis may underestimate the total number of lesions detected. Given the similar event and dropout rates between the two arms, it is unlikely that this underestimation meaningfully affects the overall findings presented in this analysis.Analysis of the HPV FOCAL Trial results suggests that a population‐level, cervical cancer screening using primary HPV testing with reflex cytology is equivalent or superior to primary LBC testing with reflex HPV testing, at detecting CIN2+ lesions among women who are regular participants in cervical cancer screening. Future investigations, using the HPV FOCAL Trial results, into the cost‐effectiveness of screening according to the USPTF guideline recommendation are also planned, as is the cost‐effectiveness of using a higher threshold  for colposcopy referral, and modeling incremental quality‐adjusted survival beyond the time horizon of the trial as the outcome of interest.As part of its review and approval of the funding application, CIHR approved the design, analysis, and conduct of the study. The funder had no role in the collection, management, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.


To investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).We performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.The results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.Hsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis. Oral squamous cell carcinoma (OSCC) is the most common cancer in the oral maxillofacial region, and the incidence rate of OSCC has remained on an upward trend . Due to Oral leukoplakia (OLK) cannot be pathologically or clinically defined as any other diseases with white plaques or patches that cannot be rubbed off . It is wCircular RNAs (circRNAs) are a large portion of endogenous non-coding RNAs with a covalently closed loop structure with no 5′ cap or 3′ polyadenylation tails , 10. Comin vitro and in vivo  tissue specimens were used to perform high-throughput sequencing in a total of eighteen cases, with six samples in each group; all of those samples were selected from patients who underwent surgery and treatment in Shanghai Ninth People’s Hospital. The clinical manifestation of OLK and OLK canceration is shown in in situ as clusters and finally sequenced for 150 cycles on an Illumina HiSeq 4000 Sequencer .Total RNA was extracted from the experimental tissue samples using TRIzol . RNA concentrations and purity were tested by NanoDrop ND-1000 . Spectrophotometer OD260/OD280 values were used for RNA purity indexes. Quality control results indicated a range of OD260/OD280 between 1.8 and 2.1. Under the guidance of the manufacturer’s instructions, the rRNAs were eliminated by Ribo-Zero rRNA Removal Kits . RNA libraries were constructed by TruSeq Stranded Total RNA Library Prep Kit . The BioAnalyzer 2100 system  was applied for controlling the quality and quantified of libraries. CloudSeq Biotech  provided the high-throughput sequencing service. Libraries (10 pM) were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified via cutadapt software (v1.9.3). The eligible trimmed reads were aligned to a reference genome/transcriptome with STAR software (v2.5.1b). The high-quality circRNAs were detected and identified by DCC software (v0.4.4). Identified circRNAs were annotated using circBase and circ2Traits databases. According to sequencing depth and degree of variation, we normalized the data and screened for altered circRNAs between OSCC and OLK using edgeR software (v3.16.5). The profile of differentially expressed circRNAs between OLK and OSCC was generated by Cluster and TreeView software. Based on the expression levels of all identified circRNAs in OLK and OSCC, the hierarchical clustering analysis proceeded and the significant differential circRNAs were selected. Unprocessed and analyzed sequencing data, after standardization, were uploaded to the National Center for Biotechnology Information Gene Expression Omnibus (GEO). The number of the dataset that was successfully uploaded to GEO is GSE131182 and GSE131568.To filter high-quality trimmed reads for analyzing circRNAs, Q30 was applied for quality controlling, and trim 3’ adaptors and low-quality reads were removed Reverse transcription was applied for total RNA by SuperScript III Reverse Transcriptase . According to the manufacturer’s instructions, qRT-PCR reactions involved using qPCR SYBR Green master mix  with QuantStudio 5 Real-Time PCR System (Thermo Fisher). Divergent primer pairs designed for target 10 circRNAs were selected and are summarized in −∆∆Ct measurement.RNeasy MinElute Cleaning Kit (Qiagen) was used for RNase R assay, and total RNA was extracted from experimental tissues and then incubated with RNase R . Next, the back-spliced junctions of hsa_circ_0060927 were verified by Sanger sequencing. Data analysis for relative expression of circRNAs was performed by 2p-value < 0.05 and FC ≥ 2.0 were selected from the result of sequencing data. Step 2: According to the degree of the differentially expressed circRNAs, the characteristic of circRNAs and the correlation of disease annotation, ten circRNAs were elected from the 389 circRNAs selected in step 1. The ten circRNAs were validated by qRT-PCR. Step 3: Given that the aim was to detect whether there were molecules involved in the progress of OLK carcinogenesis, the comparison of differentially expressed circRNAs in normal versus OLK (N-K) and OLK versus OSCC (K-S) groups was intersected. Hsa_circ_0060927 was the intersection between the two groups and was also the most significant upregulated circRNA expression in K-S. Step 4: RNAase R and Sanger sequencing assay were used to validate the structure of hsa_circ_0060927. The candidate circRNA, hsa_circ_0060927, was further validated in an independent cohort that contained 24 OSCC, 24 OLK, and 24 healthy oral mucosa tissue samples, and ROC curve analysis was depicted after expanding the sample size for verification of hsa_circ_0060927. Cell localization and cell experiment were performed further to explore the expression and function of hsa_circ_0060927. Step 5: Mechanism analyses were performed to determine the importance of the hsa_circ_0060927/miR-195-5p/TRIM14 axis.Step 1: A total of 389 circRNAs with a criterion of in situ hybridization (FISH) to locate hsa_circ_0060927 and miR-195-5p in OLK cells. In brief, FAM-labeled probes were specific to hsa_circ_0060927 and cy3-labeled probes were specific to miR-195-5p. Nuclei were stained by DAPI. All the procedures were conducted according to the manufacturer’s instructions . All images were acquired on an upright fluorescence microscope system .The OLK tissue slices were used to perform fluorescence 2.The Leuk1 cell line was derived from the University of Texas MD Anderson Cancer Center, USA –17, provWe constructed an hsa_circ_0060927-overexpressed vector. Briefly, the sequence with a full length of 1,106 bp was subcloned into a pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector  to generate pLCDH-circ_0060927 constructs. The subcloned sequence containing a front circular frame (SA), a back circular frame (SD) of circRNA biogenesis, and a full length of hsa_circ_0060927 and 5’-ATTTAAATCGGATCCGGCCACACCCTCCCATCAAA-hsa_circ_0060927-ATCCTTCGCGGCCGCTCAGAACACAGCCTTTGTAGG -3’ was directly synthesized. The pLCDH-ciR empty vector was used as the control group. The procedure of transfection was conducted using Lipofectamine 2000 (Invitrogen) kits.Cell proliferation was tested using Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instruction. After 48 h of cell transfection, the cells were seeded in 96-well plates after cell counting, and the plates were cultured in the incubator. After cell attachment, 10 μl of CCK-8 solution  was added to each well on days 1–4. After incubation for 1 h, the absorbance was measured at 450 nm using a microplate reader . All results were expressed as the mean ± SD. Each experiment was proceeded at least three times independently.6 cells/ml, and cell apoptosis assay was detected by flow cytometry  after cell staining.Cell apoptosis assay was performed using an Annexin V–FITC/PI kit . After 48 h of cell transfection, cells were collected after trypsin digestion. Mixing resuspended cells using the Annexin V–FITC/PI kit was according to the manufacturer’s guidance. The cell concentration was 1 × 10After transfection for 48 h, LEUK1 cells were suspended in growth factor-free Defined keratinocyte-SFM. One hundred microliters of suspension cells was added to the Transwell chamber  after cell counting. Defined keratinocyte-SFM containing 0.5% growth factor was added to the lower chamber. After 24 s, nonmigratory cells were removed. The cells migrating through the membrane were counted under a microscope  after fixing by formaldehyde and staining by 0.1% crystal violet.CircRNA–miRNA-coding gene interactions were predicted by target prediction software, Target scan and miRanda, and the construction of the ceRNA network was used by the bioinformatics software Cytoscape (v2.8.0). In order to select the downstream target molecules in which hsa_circ_0060927 may regulate the process of OLK carcinogenesis, the miRNAs related to oral diseases that have been reported corresponded to the above predicted miRNAs. The combining capacity between circRNA and miRNA was predicted by bioinformation software.p-value indicates the statistical significance of GO terms enrichment in the host genes using Fisher’s exact test . KEGG analysis was used to annotate host genes of differentially expressed circRNAs using the KEGG database. The Fisher p-value denotes the significance of each pathway involved, and p < 0.05 was deemed to indicate statistically significant differences.The functional mechanism analysis of the differentially expressed circRNAs between OLK and OSCC was predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. GO terms were screened according to the source gene with significantly different circRNAs and GO annotation list. The RIP assays were performed in OLK tissues. Resuspended cells were collected from tissue homogenate. RNA lysis buffer was used to lyse the cells. Then, the cells were incubated in RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (AGO2) antibody  or negative control Rabbit IgG . Proteinase K was added to the RIP sample and incubated at 55°C for 30 min. Then, immunoprecipitated RNA was isolated and analyzed by qRT-PCR to quantify the enrichment of hsa_circ_0060927.Biotin-labeled hsa_circ_0060927 probe (positive probe) and oligo probe  (negative probe) were synthesized. OLK tissues were lysed with lysis buffer and incubated with specific hsa_circ_0060927 probes. Then, OLK tissues were lysed with lysis buffer and incubated with probe-coated beads at 4°C overnight. The beads were washed, the RNA complexes were extracted with TRIzol , and qRT-PCR was performed to detect miR-195-5p.The wild-type hsa_circ_0060927 (hsa_circ_0060927-WT) vector, which contained miR-195-5p-specific binding sites, and the mutant fragment (hsa_circ_0060927-Mut), which contained the mutant miR-195-5p binding sites, were constructed by annealing double-stranded DNA and inserting them into the pSI-Check2 vector  . Lipofect-test was performed according to actual conditions. Statistical significance of difference between groups was determined by unpaired t-test and one-way ANOVA . Correlations were analyzed using Pearson’s linear correlation analysis. p < 0.05 was considered statistically significant. Student’s receiver operating characteristic (ROC) was performed to assess diagnostic and prognostic values of hsa_circ_0060927. Quantitative data were shown as mean ± standard error of the mean (SEM), and all the data were obtained through three times repetition at least.Statistical analysis proceeded using SPSS 19.0  and GraphPad Prism version 8.0 software. p-value  new circRNAs were identified with the rest found (95.9%) in the circRNA database . Most weTop eight upregulated and two downregulated circRNAs were selected for verification by qRT-PCR. Validation was first performed on the six pairs of OSCC and OLK tissue Figure 2In order to better compare the differential circRNAs in OSCC, OLK,and NOM groups, we performed an intersection analysis on the upregulated, downregulated, and total differentially expressed gene of the three groups respectively; Venn diagrams were drawn (in situ hybridization (FISH) revealed that hsa_circ_0060927 localized in cytoplasm of OLK  assay was then performed in OLK tissues to determine the association between hsa_circ_0060927 and AGO2. AGO2 protein was a key molecule for circRNA to sponge miRNA.The quantitative real-time PCR results showed that the expression of hsa_circ_0060927 pulled down with anti-AGO2 antibodies was significantly higher compared to the anti-IgG group  can be found at: NCBI, GSE131182, and GSE131568.The studies involving human participants were reviewed and approved by the Ethics Committee of Shanghai Ninth People’s Hospital. The patients/participants provided their written informed consent to participate in this study.HZ conceptualized the study. SX, YHS, and YXS developed the methodology. SX was in charge of the software. SX, YHS, and YXS validated the study. SX, YHS, and YXS conducted the formal analysis and the investigation. HZ provided the resources. SX, YHS, and YXS conducted the data curation. SX wrote and prepared the original draft. SX, YHS, YXS, and HZ wrote, reviewed, and edited the manuscript. SX and HZ conducted the visualization. HZ supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the National Key R&D Program of China (2017YFC0840100 and 2017YFC0840110) and Biobank Program of Shanghai Ninth People’s Hospital (YBKA201912).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prognostic associations of p16- and HPV-status were analyzed separately and combined.In contrast to p16, the HPV-status resulted in no significant survival discrepancies  HPV-positive 64.9%, HPV-negative 78.7%). Combining both markers, p16-positive  and p16-negative/HPV-negative groups demonstrated comparable high survival (OS 78.1% vs. 85.6% vs. 73.6%). Lowest survival was observed for patients with p16-negative/HPV-positive OPSCCs (OS 40.8%).Never smoking patients with p16-positive OPSCCs demonstrated the highest survival, whereas within former/current smokers with p16-positive and p16-negative disease it was comparable low (OS 90.0% vs. 63.0% vs. 57.4%).p16- and HPV-status should not be considered as equivalent markers for a better prognosis. Furthermore, they should not generally predominate patient associated factors like smoking.The online version of this article (10.1007/s00405-020-06262-7) contains supplementary material, which is available to authorized users. Oropharyngeal cancer constitutes 11.3% of all head and neck malignancies , a geogrIn the course of HPV-infection, integration of HPV into the host genome results in stabilization and upregulation of the viral oncoproteins E6 and E7, E7 binds and leads to an inactivation and degradation of tumor suppressive retinoblastoma protein, presumably resulting in an upregulation of the protein p16 –15. The 2-laser microsurgery (TLM) with curative intent was performed. The study was conducted at an academic tertiary referral center. Time span of inclusion was 01/2000 and 10/2015 with systematic follow-up until 10/2017. Cases were identified and data were obtained from the hospital’s cancer database and the original medical records. This study was approved by the institutional review board  in accordance with the national regulation and with the Helsinki Declaration of 1975, as revised in 1983. Prior to treatment, all patients gave their written informed consent for anonymized analysis of their individual data.A retrospective analysis of patients with advanced stage OPSCCs (III-IVa) treatable by transoral COExclusion criteria comprised non-OPSCC, previous or simultaneous secondary malignancies, T4b primaries, N3 metastasis, simultaneous distant metastases, previous treatment of the OPSCC in a different institution, treatment of the primary tumor by conventional surgery, flap reconstruction, primary (chemo-)radiotherapy and palliative treatment intent. Of 161 identified patients, 125 had sufficient paraffin embedded tissue material available for p16-immunohistochemistry as well as HPV-genotyping. Thereof 23 patients were diagnosed with early stage I–II OPSCCs and therefore omitted from analysis, resulting a final cohort of 102 patients with advanced disease.Preoperative staging procedures were performed as described previously  accordinThe therapeutic strategy was TLM-resection of the primary tumor with/without neck dissection (ND), with/without postoperative (chemo-)radiotherapy ((C)RT) as described previously . The theThe postoperative examination and follow-up was performed as previously described . After 5p16 immunohistochemistry was performed with monoclonal p16 antibodies   in combination with diaminobenzidine , and the EnVision Flex + System . A strong diffuse (≥ 75%) nuclear and cytoplasmic p16 immunostaining was categorized as p16-positive. No, weak, or moderate staining was considered as p16-negative , 29.For multiplex fluorescent-PCR based detection of DNA from different HPV genotypes a commercial assay developed for diagnostic purposes was applied. Formalin-fixed paraffin-embedded (FFPE) tissue samples of the oropharyngeal primaries were used. A 5 µm FFPE slice was enriched for tumorous tissue by microdissection and DNA was isolated with the innuPREP FFPE DNA Kit—IPC16  in combination with the InnuPure® C16 touch . Quantification and purity of the isolated DNA was measured by a NanoDrop™ 2000 Spectrophotometer . DNA was amplified using the f-HPV-typing™ Multiplex Fluorescent-PCR Kit according to manufacturer’s protocol . The PCR reagent contains 16 fluorescently labelled primers designed to recognize E6 and E7 regions of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 that are most likely present after viral integration. The f-HPV-typing™ also contains a human short tandem repeat, which serves as an internal control . HPV ampInformation about alcohol and tobacco consumption was extracted from the original medical records. Both variables were categorized dichotomously. Patients self-reporting former/current versus no tobacco consumption were assigned to the respective groups. Heavy drinkers were defined as patients who had reported at least weekly strong or daily other alcohol consumption and/or who had a disease related to heavy alcohol consumption (e.g. Wernicke–Korsakow syndrome). Patients self-reporting occasional, limited or no alcohol consumption were assigned to the group of never/social drinking patients. Cases without information on alcohol and/or tobacco consumption were omitted from the subanalysis.Descriptive analysis was stated by the respective mean value, corresponding standard deviation (SD), median and/or absolute and relative frequencies. Analysis of frequency distributions was conducted with the Pearson’s Chi-squared test. Five-year overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), and the local control rate (LCR) were calculated by the Kaplan–Meier method . Date ofn = 41) tonsils, 25.5% (n = 26) tongue base, 3.9% (n = 4) soft palate, 12.7% (n = 13) vallecula, 3.9% (n = 4) posterior wall, 3.9% (n = 4) lateral wall, 5.9% (n = 6) glossotonsillar sulcus and palatal arch, 2.9% (n = 3) uvula, and 1% (n = 1) tonsillar fossa. All patients were treated primarily by TLM. It was completed with neck dissection in 96.1% (n = 98) and postoperative (chemo)radiotherapy in 73.5% (n = 75).102 patients with advanced stage OPSCC  were included in the study. The site of primary origin of oropharyngeal tumors was 40.2% (n = 50) of all tumors. HPV-DNA was detected in 40.2% (n = 41) of all OPSCCs. Distribution of p16-status and HPV-typing results are depicted in Table n = 33), HPV18 in 7.3%, (n = 3), HPV33 in 26.8% (n = 11), HPV51 in 2.4% (n = 1) and HPV59 in 19.5% (n = 8). In most of the tumors one single HPV-type was identified . Most commonly it was HPV16 . The majority of these OPSCCs with selective HPV16 infection showed p16-overexpression . Multiple HPV infections were detected in 24.4% (n = 10) of all HPV-positive specimens. Worthy of note, all of them also exhibited HPV16, while only 30.0% (n = 3) of them showed a p16-overexpression.p16-overexpression was seen in 49.0% (n = 65), presenting either as p16-positive and HPV-DNA-positive  or p16-negative and HPV-DNA-negative . Contradictory results were found in 36.3% (n = 37). p16-positive with no presence of HPV-DNA (p16-positive/HPV-negative) were 22.5% (n = 23) of the OPSCCs. Vice versa 13.7% (n = 14) were p16-negative and positive for HPV-DNA (p16-negative/HPV-positive). Among p16-positive OPSCCs the presence of HPV-DNA and, therefore, concordance of p16- and HPV-positivity was 54.0% (n = 27 of 50).Concordance between p16-immunostatus and presence of HPV-DNA was observed in 63.7% (P > 0.1 respectively). However, T-categories were significantly differently distributed between p16-positive and p16-negative OPSCCs (P < 0.01). p16-negative tumors were more often diagnosed in a higher T-category  compared to p16-positive tumors . A significant different distribution of T-categories was also observed, when taking both markers combined into account (P = 0.02). Concordant p16-negative/HPV-negative cases were more often locally advanced tumors  and concordant p16-positive/HPV-positive cases presented more often as locally circumscribed tumors . Furthermore, histopathological differentiation was significantly differently distributed between p16-positive and p16-negative tumors (high/moderate vs. poor: P = 0.01). Most of p16-negative OPSCCs had a high or moderate differentiation . Most of the poorly differentiated tumors were constituted by p16-positive tumors .Patients’ and disease characteristics, treatment details and follow-up data of the whole study group as well as stratified by p16-status, HPV-status and the resulting subgroups with both markers combined are depicted in Table P < 0.01; alcohol P < 0.01). Never smoking patients had more often p16-positive tumors , whereas former/current smokers had more often p16-negative ones . Furthermore, no/social drinking patients had predominantly p16-positive disease , whereas those with heavy alcohol consumption had more p16-negative cancers . With regard to the HPV-status, no significant different distribution was observed for smoking (P = 0.49) or alcohol consumption (P = 0.11).Tobacco and alcohol consumption were significantly differently distributed between patients with p16-positive and p16-negative disease, respectively (smoking n = 19) of all patients. The occurrence of treatment failures was not differently distributed between patients with p16-positive versus those with p16-negative (P = 0.24) or HPV-positive versus HPV-negative (P = 0.48) OPSCCs. No significant difference was observed including both markers combined (P = 0.08).Local and/or regional treatment failures were diagnosed in 18.6% . The 5-year estimates of the complete cohort were as followed: OS 73.3%, DSS 78.3%, RFS 69.8% and LCR 85.6%.P = 0.46), DSS 69.3% vs. 84.0% (P = 0.19), RFS 68.2% vs. 71.5% (P = 0.60) and LCR 88.8% vs. 84.0% (P = 0.65) for HPV-DNA-positive vs. HPV-DNA-negative tumors, respectively (Online Resource 1). Summarized, prognostic impact has been demonstrated for the p16-status, whereas stratification based on the presence of HPV-DNA did not result in significant discrepancies in survival rates.Comparing the survival of patients with p16-positive or negative tumors showed superior estimates for p16-positive cases . Within n = 27, 26.5%), p16-positive/HPV-negative , p16-negative/HPV-positive , and p16-negative/HPV-negative . Among patients with p16-positive OPSCCs, no differences between the estimates of HPV-DNA positive or negative tumors were observed . Within patients with p16-negative tumors, those with p16-negative/HPV-negative disease demonstrated significant higher estimates for both, DSS and RFS, compared to the group of patients with p16-negative/HPV-positive disease . The OS was 73.6% vs. 40.8% (P = 0.15), and the LCR was 83.9% vs. 78.6% (P = 0.50), for the group of p16-negative/HPV-negative vs. p16-negative/HPV-positive tumors, respectively. Considering all four subgroups, OS and DSS were the highest in the group with discordant p16-positive/HPV-negative  tumors and the lowest for those with a p16-negative/HPV-positive disease  of all patients. These patients were considered for a further subset analysis.Information about tobacco and alcohol consumption was documented for 83.3%  had reported either current or former smoking, whereas 30.6% (n = 26 of 85) had negated any smoking history. Most of the never smoking patients had p16-positive tumors , whereas the group of former/current smokers exhibited predominantly a p16-negative disease . HPV-DNA status was not differently distributed between never smoking patients  and those with a positive history for tobacco consumption .In reference to tobacco consumption, the majority of patients (n = 32) of the patients were categorized as heavy drinkers, whereas 62.4% (n = 53) had reported no or occasional alcohol consumption and therefore were assigned to the group of never/social drinking patients. This group predominantly consisted of patients with p16-positive tumors , whereas heavy alcohol consumption was predominantly reported by patients with a p16-negative disease . Again, HPV-DNA status was not differently distributed between never/social drinking patients  and those with heavy alcohol consumption .With regard to alcohol consumption, 37.6% (P < 0.01). Again, HPV-DNA detection was not differently distributed between never smoking and never/social drinking patients  and smokers and/or heavy drinkers . In summary, the distribution of tobacco and/or heavy alcohol consumption differed only significantly between patients with p16-positive and p16-negative disease.Considering both factors combined, 71.8% (61 of 85) had a positive history of smoking and/or heavy drinking. Most of these patients had p16-negative OPSCCs , whereas the group of never/social drinking patients without a history of tobacco consumption had predominantly p16-positive tumors . The estimates of RFS and LCR for never smoking patients and those with a smoking history showed no significant difference . Further stratification based on p16-status or HPV-status within the two groups of never or former/current smoking patients demonstrated that never smoking patients with p16-positive tumors had favorable OS, DSS and RFS compared to former/current smokers with a p16-negative disease . In contrast, the group of p16-positive cancer patients that shared the history of former/current smoking demonstrated comparable low survival .Summarized, never smoking patients with p16-positive disease showed the highest survival, whereas former/current smokers demonstrated a comparable inferior survival, despite the p16-status.P = 0.741; DSS: 79.0% vs. 70.7%, P = 0.242; RFS: 66.5% vs. 73.8%, P = 0.768; LCR: 88.0% vs. 89.9%, P = 0.953). Therefore, this factor was not considered for further subgroup analysis stratified on p16- or HPV-status.With regard to drinking habits, there was no statistical survival difference between never/social drinking patients and heavy drinkers  and extracapsular spread  were included. For DSS, extracapsular spread was included in the final model when considering p16-status and HPV-status separately . For RFS and LCR no significant covariate was identified, respectively.For adjustment of potential cofounding factors, also a Cox multivariate regression analysis (forward stepwise) was conducted. Regarding the complete cohort, in the final equation of OS the p16-status , P=0.036, p16-positive/HPV-positive: HR=0.525, 95% CI 0.182-1.516, P=0.234, p16-positive/HPV-negative: HR=0.534, 95% CI 0.183-1.559, P=0.251, p16 negative/HPV positive: HR=2.151, 95% CI 0.854-5.417, P=0.104) and likewise DSS , P=0.017; p16-positive/HPV-negative: HR=0.657, 95% CI 0.157-2.757, P=0.566; p16 negative/HPV positive: HR=0.837, 95% CI 0.198-3.529, P=0.808; p16-negative/HPV-positive: HR=3.923, 95% CI 1.239-12.416, P=0.02). For RFS, T-categorization was in addition to the combined p16-HPV-status included in the final equation , P=0.011 p16-positive/HPV-positive: HR=0.365, 95% CI 0.105-1.26, P=0.111; p16-positive/HPV-negative: HR=0.695, 95% CI 0.239-2.021, P=0.504; p16-negative/HPV-positive: HR=2.676, 95% CI 1.03-6.949, P=0.043). For LCR no significant covariate was identified.       Considering the combination of p16 and HPV, results of the multivariate analysis demonstrated that the combined p16/HPV-status and ECS had an impact on OS  as well as DSS . For RFS and LCR no significant covariate was identified.Considering the cohort with information about alcohol and tobacco consumption neither p16-nor HPV-status or both combined were associated with one of the prognostic endpoints in the univariate analyses (Online Resource 3). Consistent with this, in the multivariate Cox regression analysis only extracapsular spread as well as tobacco consumption were identified to effect OS  or base of tongue  were identified for complementary subset analyses. The detailed results are provided in the Online Resources 4–6 (Tables) and 7–8 .The classification algorithms of the current (8th) edition of the AJCC/UICC cancer staging manual differentiate between p16-positive and p16-negative OPSCCs but not between tonsillar/tongue base OPSCC vs. other OPSCC sublocations , 18. NevP < 0.01). In contrast the HPV was not significantly differentially distributed between tonsillar/tongue base OPSCC compared OPSCCs of other sublocations . There was no significant prognostic difference between patients with tonsillar/tongue base OPSCC compared to patients with OPSCCs of the other sublocations . Consistent with results the complete cohort, exclusively the p16-status , but not the HPV-status  was associated with significant prognostic discrepancies in the univariate analyses of the cohort of tonsillar/tongue base OPSCC. Combining both markers, the p16-positive  and p16-negative/HPV-negative groups demonstrated comparable superior estimates . Lowest survival was observed for patients with p16-negative/HPV-positive OPSCCs .p16 positive tumors were significantly more often localized in the tonsillar region or base of tongue .This study presents a retrospective analysis of the oncological outcome of patients with advanced stage p16-positive or p16-negative OPSCCs with further examination for HPV-DNA. All patients received a TLM-based treatment strategy with/without neck dissection, with/without postoperative (chemo-)radiotherapy between 2000 and 2015 as described previously . ThroughDrawing conclusions based on research about HPV-associated tumors is still challenging, since various and inconsistent definitions for a HPV-positive tumor are used . For insInk4a gene [Within the present study for the distinction between p16-positive and p16-negative tumors the criteria suggested for the application of the classification algorithms of UICC/AJCC 8th edition was applied . Even thk4a gene ) or due k4a gene ). p16-ovk4a gene . Thus, oAs previously shown, a positive p16-status was associated with a superior prognosis of patients with advanced stage OPSCCs . Based oNotwithstanding, several additional factors could be responsible for this heterogenous results of the prognostic impact of HPV-presence leading to an aggravated comparability. On the one hand, methodical differences in HPV-detection, definition of “HPV-positivity” and HPV-genotype distribution could have an impact. For instance, a lower survival for non-HPV16-genotype positive tumors compared to HPV16-positivity ones was described , 45. ThiPrevious studies already indicated that different constellations of the p16- and HPV-status can define further prognostic divergent subgroups within OPSCCs –24. NeveIn the current study, survival estimates  of the two p16-positive groups that were either HPV-positive or negative showed no significant difference. This indicates the same prognostic level of patients with p16-positive OPSCCs despite the presence of HPV-DNA. This is in line with the results of Yamamoto et al. for a group treated with definitive chemoradiotherapy, even though for their group treated with definitive radiotherapy alone, they observed a significantly superior OS and PFS for patients with p16-positive/HPV-positive disease compared to those with p16-positive/HPV-negative tumors .In the present study no significant differences among patients with p16-negative tumors with or without positive HPV-status were observed with regard to OS and LCR. The p16-negative/HPV-positive subgroup presented the lowest estimates. This is in line with the results of Yamashita et al. that also reported higher OS for the group with p16-negative/HPV-negative status compared to the one with p16-negative/HPV-positive disease, even though the difference was not statistically significant . In the In contrast to the current study and the one by Yamashita et al. that observed the lowest outcome for the subgroup of p16-negative/HPV-positive tumors , HoffmanPatients with HPV/p16-associated HNSCCs are described to have different risk profiles compared to those with HPV/p16-negative disease . In partIn the current study, most of never smoking patients had p16-positive disease, whereas the majority of former/current smokers exhibited p16-negative tumors. This is in accordance with other studies demonstrating a significant differing frequency of tobacco history between patients with a p16-positive and p16-negative disease , 47. HPVWith regard to survival rates, never smoking patients demonstrated superior estimates compared to former/current smokers. Considering the tumor’s p16-status, the highest survival estimates were observed in never smoking patients with p16-positive tumors, whereas patients with p16-positive disease and a history of smoking had a comparable inferior prognosis like p16-negative tumors of former or current smokers. This indicates that the superior prognosis of p16-positive tumors is abrogated in tobacco consumers. In the present study stratification based on HPV-status exhibited no significant differences between the oncological results of never and former/current smoking patients. This appears to be conflicting with the results of Hoffmann et al., demonstrating a negative impact of smoking in patients with HPV-positive OPSCC and > 10 py on overall survival. A comparable analysis based on p16-status was not mentioned in this study .The relevance of the present study is emphasized by the lack of a standardized definition of HPV-association in OPSCCs as well as a lack of detailed high-evidence data about the implications on the prognosis of the different p16- and HPV-status constellations. This is reflected by the paucity of studies, addressing this topic with a focus on long-term oncological outcome of patients with primary OPSCC treated with curative intent –23. The The impact of different HPV-genotypes on prognosis and their association with p16-overexpression was not further evaluated due to their limited frequency in the present study. This topic should be investigated by multicentered studies performed prospectively or at least by pooling the already existing data of well-described retrospective cohorts.The current data indicate that p16- and HPV-status should not be considered as equivalent markers for a better prognosis in OPSCC. Furthermore, they should not outweigh patient-associated prognostic factors like smoking. Thus, further studies focusing on therapeutic regimes and prognosis of OPSCC should consider both markers and provide detailed information on tobacco consumption.Online Resource 1 Five-year Kaplan-Meier estimates for overall survival (a), disease-specific survival (b), recurrence-free survival (c) and local control rate (d) stratified by HPV-status. Patients at risk are shown below the diagram. P values are calculated by log-rank test (PDF 355 kb)Online Resource 2 Five-year Kaplan Meier estimates for overall survival (a), disease-specific survival (b), recurrence-free survival (c) and local control rate (d) stratified by tobacco consumption and HPV-status combined. Patients at risk are shown below the diagram. P values are calculated by log-rank test (PDF 567 kb)Online Resource 3 Clinicopathological characteristics and prognosis of patients with OPSCCs (PDF 261 kb)Online Resource 4 Results of HPV-typing stratified by p16-status for OPSCCs of the tonsillar region or base of tongue (n = 74) (PDF 261 kb)Online Resource 5 Patient’s disease characteristics and follow-up data of patients with OPSCCs of the tonsillar region or base of tongue (n = 74) stratified by p16-status and HPV-status (PDF 261 kb)Online Resource 6 Treatment failures of OPSCCs of the tonsillar region or base of tongue (n = 74) stratified by p16- and HPV-status (PDF 261 kb)Online Resource 7 Five-year Kaplan-Meier estimates of overall survival (A), disease-specific survival (B), recurrence-free survival (C) and local control rate (D) stratified by p16- and HPV-status combined within the cohort with oropharyngeal squamous cell carcinoma of the tonsillar region or base of tongue (n = 74). Patients at risk are shown below the diagram. P values are calculated by log-rank test (PDF 523 kb)Online Resource 8 Five-year Kaplan-Meier estimates of overall survival (A), disease-specific survival (B), recurrence-free survival (C) and local control rate (D) stratified by tobacco consumption and p16- status combined within the cohort with oropharyngeal squamous cell carcinoma of the tonsillar region or base of tongue with available information on alcohol and tobacco consumption (n = 58). Patients at risk are shown below the diagram. P values are calculated by log-rank test (PDF 619 kb)Below is the link to the electronic supplementary material.


We studied the incidence of HPV genotypes in mostly Black women with cervical carcinoma and correlated histopathologic tumor characteristics, immune markers and clinical data with survival. Disease-free survival (DFS) and overall survival (OS) were recorded for 60 months post-diagnosis. Fifty four of the 60 (90%) patients were Black and 36 (60%) were < 55 years of age. Of the 40 patients with typeable HPV genotypes, 10 (25%) had 16/18 HPV genotypes, 30 (75%) had one of the non-16/18 HPV genotypes, and 20 (50%) had one of the 7 genotypes  that are not included in the nonavalent vaccine. Mixed HPV infections (≥ 2 types) were found in 11/40 (27.5%) patients. Patients infected with non-16/18 genotypes, including the most common genotype, HPV 35, had significantly shorter DFS and OS. PD-L1 (p = 0.003), MMR expression (p = 0.01), clinical stage (p = 0.048), histologic grade (p = 0.015) and mixed HPV infection (p = 0.026) were independent predictors of DFS. A remarkably high proportion of cervical cancer cells in our patients expressed PD-L1 which opens the possibility of the use of immune checkpoint inhibitors to treat these cancers. Exclusion of the common HPV genotypes from the vaccine exacerbates mortality from cervical cancer in underserved Black patients. The incidence in non-Hispanic blacks is significantly higher  than that in non-Hispanic whites 1. Mortality rate is also higher in non-Hispanic blacks  than that in non-Hispanic whites 2. Five-year survival rates among affected Black women decreased from 64 to 59% between 1974 and 1994, whereas the corresponding survival rates among Caucasian women increased from 70 to 72%3. Disparities in cervical cancer outcomes have been a cause of concern amongst the healthcare community, and has been linked to cultural, socioeconomic and genetic factors1. Studies adjusted for cultural and socioeconomic factors have revealed a biological basis of higher mortality in cervical carcinoma in Black women4. Although the evidence is overwhelming, few studies have focused on Black patients to identify the causes that lead to these disparities. This study was undertaken to explore the HPV genotypes and biological differences in cervical carcinoma in women of different racial groups.Cervical cancer is the third most common gynecologic malignancy and cause of death among women in the United States even though a vaccine against HPV, its etiologic agent, has been in use for a decade. Most cervical cancers are squamous cell carcinomas and their epidemiology reveals considerable disparity among races5. HPV DNA is identified in 99.7% of cervical cancers, with over 70% being HPV oncogenic types 16 or 18 (16/18)1. Women with cervical tumors infected with HPV types 16/18 have poor survival as compared to those infected with other HPV types. The clinical outcome of infection with HPV types other than HPV 16 and 18 is variable, but large cohort studies have shown better survival and less persistent infection with non-16/18 HPV types6. As new HPV genotypes associated with cervical cancer are identified, their clinical implication is of value in the development of HPV vaccine. In the last 6 years the number of HPV genotypes included in the US Food and Drug Administration (FDA) approved HPV vaccine (Gardasil 9) has increased from 4 to 9, which has the potency to target 70% of the HPV infections. Black patients have a preponderance of non-16/18 HPV infections, including some rare genotypes with less known pathogenesis and outcomes. More studies on the distribution of HPV genotypes in racially and ethnically diverse patients and a better understanding of pathogenicity of these HPV genotypes are required for a more complete HPV vaccine for better coverage of the vulnerable populations.Infection with high-risk human papilloma virus (HPV) is one of the definitive events linked to the complex process of cervical carcinogenesis8. PD-L1 expression has been studied for determining its therapeutic and prognostic role in cervical cancer. PD-L1 expression in cervical squamous cell carcinoma cells has ranged from 34.4 to 70.1%11. PD-L1 overexpression in tumor cells has been found to be associated with lymph node metastasis, vascular invasion, higher International Federation of Gynecology and Obstetrics (FIGO) stage and poor survival12. PD-1/PD-L1 inhibitors have been approved by the FDA for patients with recurrent or metastatic cancer unresponsive to chemotherapy. However, it can be a potential therapeutic option in patients with early-stage recurrent or locally advanced cervical cancer and who are unresponsive to chemotherapy13. Because of sparse data the efficacy of immune-checkpoint inhibitors has not been established as a first line therapy in primary cervical carcinoma, especially in Black patients, who commonly present with advanced stage disease.The PD-1/PD-L1 axis is one of the immune-checkpoint pathways that have been found to help cancer cells evade the immune response15. Loss of function of one of the MMR proteins  leads to high rates of mutations that accumulate in repetitive nucleotide regions (microsatellites) inactivating the mismatch repair pathway. MMR-deficient tumors have 10–100 times more somatic mutations than MMR-proficient tumors leading to increased tumor antigen burden and immunogenicity. We speculate that MSI may have predictive and prognostic value in cervical cancer along with its co-expression with PD-1/PD-L1 and HPV genotype.Another biomarker for identifying patients for immunotherapy is the Mismatch Repair (MMR) or Microsatellite Instability (MSI) pathway which was recently approved by the FDA for use in different tumor types including cervical carcinomaIn this study we have attempted to characterize the clinicopathological features of cervical carcinoma in our patients and analyzed the expression of PD-1, PD-L1, MMR and HPV genotyping to explore their pathogenic and therapeutic potential.Patients (n = 198) diagnosed with squamous cell carcinoma of the cervix over a 12-year period at a tertiary care hospital were identified. Sixty of these were found to have complete clinical data and adequate histologic specimens, and were grouped based on their race (Black and Other). Although the population we serve is heterogeneous and includes both African American and African Caribbean individuals, our electronic medical records based on a pre-determined classification permit self-reporting only as Black or African American. Histopathological specimens were reexamined to confirm the diagnosis. Paraffin blocks were retrieved, and tissue microarrays were prepared using two 3-mm punches. Clinical data including patient survival were recorded for up to 60 months post-diagnosis. The study was approved by the Institutional Review Board & Privacy Board (IRB) of the State University of New York, Downstate Health Sciences University, and patient consent was not required (IRB #1230970-3). The study methods complied with the local and federal government guidelines and regulations. The IRB determined that the study met the requirements for Waiver of Informed Consent as per the Office of Human Research Protections (OHRP) guidelines. This category includes materials previously collected for either non-research or research purposes. As this is a retrospective study, the clinical data and tissue samples included in the study were collected in the past and the patients were not available for consent at the time of the study.http://www.ncbi.nlm.nih.gov/BLAST/)  (Supplementary Information: Table ST 16. A housekeeping gene (β-globin) common to all high-risk HPV types was utilized as internal control. Specimens that did not yield adequate DNA for HPV typing were retested. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide, band sizes were estimated by comparison with a 100 bp molecular weight marker (Invitrogen 100 bp DNA Ladder), and gels were photographed in a UV transilluminator  . Thirty-three primers designed for high-risk HPV types  were used, and their unique specificity was confirmed by BLAST analysis  and p16 was characterized by immunohistochemistry, and the results were validated by using appropriate positive and negative controls. At least 100 viable tumor cells were evaluated for each of the proteins. Tumor viability was assessed by the presence of intact nuclear, cytoplasmic and membranous features, and devoid of any histologic sign of cell death .Immunophenotyping was performed by using a standard protocol starting with deparaffinization of tissue microarrays, antigen retrieval , cell conditioning (64 min) and application of pre-primary antibody peroxidase. The specimens were then treated with the primary antibody (pre-diluted by manufacturer) . The affinity of the antibody was enhanced by OptiView HQ Linker and Multimer (8 min each). Hematoxylin was used as counterstain (4–8 min), followed by bluing reagent (4 min). A matched negative control reagent was used for quality control.PD-L1 expression was evaluated by using SP263 rabbit monoclonal antibody and OptiView DAB IHC detection kit on Ventana BenchMark Ultra  following the manufacturer’s protocol. Human term placenta was used as the positive control. Positive control staining was deemed acceptable if there was moderate to strong uniform membrane staining of trophoblast-lineage cells, and no staining of placental stroma and vasculature. PD-L1 expressing tumor cells (moderate to strong membranous staining) were quantified as negative (< 1% positive cells), low expression (> 1 to 49% positive cells), or high expression (> 50% positive cells)  was evaluated by using PD-1 monoclonal antibody and OptiView DAB IHC detection kit on Ventana BenchMark Ultra  following the manufacturer’s protocol. Lymph nodes were used as positive control tissue. The percentage of immune cells with strong membranous staining under the light microscope was recorded. Strong membranous PD-1 expression in TILs was categorized as low or high based on values below or above the 50th percentile of PD-1 + TILs/mm17.MMR proteins were analyzed by using the Ventana MMR IHC monoclonal antibody panel for MSH2 (G219-1129), MSH6 (SP93), PMS2 (A16-4) and MLH1 (M1) and OptiView DAB IHC detection kit on Ventana BenchMark Ultra  following the manufacturer’s protocol. The specimens were evaluated for the presence or absence of specific nuclear staining in the tumor cells. Colon cancer tissue with intact MMR expression was used as a positive control. An “intact” status was assigned to specimens with unequivocal nuclear staining in viable tumor cells. “Loss” of expression was assigned to complete loss of nuclear staining or focal weak or equivocal staining in the viable tumor cells in the presence of internal positive controls 18.p16 was evaluated by using CINtec histology antibody, anti-p16INK4a (E6H4) and OptiView DAB IHC detection kit on Ventana BenchMark Ultra . p16-expressing squamous cell carcinoma tissue was used a positive control. Immunohistochemical expression of p16 was defined as “positive” if there was strong diffuse nuclear positivity in tumor cells, while “negative” included those with patchy, wild-type p16 staining patternAge was categorized using 55 years as cut-off for childbearing age. Clinical staging was determined after surgical management of cases and was based on International Federation of Gynecology and Obstetrics (FIGO) 2009 classification. Clinical stages I and II were combined into “early” clinical stage and stages III and IV as “late” clinical stage. Histologic grades were based on the extent of cellular differentiation, and categorized into well, moderate and poor. Patients were followed for up to 60 months after initial diagnosis. Disease-free survival (DFS) was computed based on the number of months from diagnosis to recurrence, and overall survival (OS) was the number of months from diagnosis to death. Cases with incomplete follow up were censored.The results of the HPV PCR analysis were recorded as positive or negative Fig. . Nine caCorrelations between different parameters were determined by Pearson Chi-Square or Fisher's exact test. All reported p values are two sided. A p < 0.05 was considered significant. Cumulative 60-month survival was calculated by the Kaplan–Meier method and analyzed by log rank test .Sixty patients diagnosed with squamous cell carcinoma of the cervix were included in this study. All of the patients received standard treatment protocol as per the National Comprehensive Cancer Network (NCCN) guidelines based on clinical stage (FIGO classification). Treatment ranged from loop electrosurgical excision to radical hysterectomy with lymph node dissection. The mean age at diagnosis was 52.8 (range: 27–86) years. 54 (90%) patients were Black and 6 10%) were Other . 44 (73.3%) patients were diagnosed at an early clinical stage (I & II). About half of the patients’ tumor histologic grade was poor (51.7%), followed by moderate (36.7%) and well (11.7%)  patients; 9 patients had nontypeable HPV genotypes. Thirteen high-risk HPV types  were identified. HPV non-16/18 were present in 27 of 60 (45%) patients, HPV 16/18 in 10 (16.7%) and 3 (5%) patients had both types 16/18 and non-16/18 HPV . The most frequently isolated HPV types were 35 , followed by 18 and 16 . Mixed HPV infections (> 1 type) were found in 11 of 40 (27.5%) patients, and all mixed cases had at least one non-16/18 HPV type. None of the patients had both HPV 16 and 18. HPV type 58 was detected exclusively in mixed infections (5/11). Twenty of 40 (50%) of the patients were infected with at least 1 non-vaccine HPV genotype, which included 35, 39, 51, 53, 56, 59 and 68  patients with 34 (56.7%) showing high and 22 (36.7%) showing low expression. PD-1 expression in TILs was high in 33 of 60 (55%) and low in 27 (45%) patients. MMR was intact in 55 of 60 (91.7%) and lost in 5 (8.3%) patients . Tissue was lost for 5 patients during immunohistochemical analysis for p16; it was positive in 50 of 55 (90.9%) patients, and negative in 5 (9.1%). Nine out of the 11 patients with negative HPV PCR results had adequate amount of tumor tissue for p16 analysis; 8 of them had positive p16 expression and 1 was negative for p16  patients. Of these patients 7/10 (70%) were < 55 years of age, 9/10 (90%) were diagnosed at an early clinical stage, and 6/10 (60%) had poor histologic tumor grade. Patients with non-16/18 HPV genotypes were present in 27/40 (67.5%) patients. They presented later in life (> 55 years), mostly diagnosed at an early clinical stage 21/27 (77.8%), and 15/27 (56%) had well to moderate histologic grade tumor. Both groups have a similar recurrence (30%) and death rate (20%). It is noteworthy that, although the non-16/18 HPV tumors had a lower histological grade, patients had a poor prognosis.Poor histologic grade was most commonly seen in patients with HPV 18  followed by HPV genotype 35 , non-16/18 HPV  and HPV 16   as compared to mixed infections. Mixed HPV infections were present in 11/40 patients. As compared to patients with single HPV infection those with mixed infection had a higher recurrence (54.5% vs. 29.2%) and death (45.5% vs. 20.8%). Mixed HPV infections with 16/18 and non-16/18 HPV genotypes were present in 3 patients; all of these patients had a recurrence and died subsequently. Mixed non-16/18 HPV infections were seen in 8 patients and had a lower recurrence  and death  as compared to the mixed 16/18 and non-16/18 HPV genotypes  as compared to those with non-vaccine covered HPV .More patients with negative HPV PCR had poor histologic grade (63.6% vs. 49%) as compared to patients with HPV PCR positive tumors. 2 of 10 (20%) with negative HPV PCR results had recurrence and subsequently died of the disease. There was no significant difference in the prognosis with HPV PCR positive results. 6/9 patients (66.7%) with non-specific HPV PCR had late clinical stage cancer (p = 0.016).5/55 (9.1%) patients lacked p16 expression in the tumor cells, and they were all Black. Loss of p16 correlated with higher recurrence (p = 0.022) and death (p = 0.003). Patients with negative p16 immunoreactivity showed loss of MMR (40%) (p = 0.011) and negative to weak PD-L1 expression , as compared to those with positive p16.6/60 (10%) patients were Other and had favorable outcomes. All Other patients were diagnosed at an early clinical stage. Only one patient had recurrence and none died during the 60-month follow-up. Most of the Black patients had poor histologic tumor grade at diagnosis . More Black patients were diagnosed at late stage , and about a third of them  had recurrence, 14 of whom died subsequently . Younger women tend to have early stage cancer (80.6%) at diagnosis as compared to older patients (62.5%). At 5-year follow-up, the mortality rate among older patients  was slightly higher than that observed among younger patients .Recurrence was more frequently observed in patients who presented at a late clinical stage p = 0.028); more than half of the patients (57.1%) diagnosed in late clinical stage and only a quarter (25%) of patients in early clinical stage had recurrence. Similarly, greater number of patients diagnosed at a late clinical stage (42.9%) died as compared to those diagnosed at an early stage (20%). Patients with poor histologic grade tumors had higher recurrence (p = 0.022) and death (p = 0.046). No recurrence or death was noted in patients with well-differentiated tumors. Most of those patients who had recurrence (77.8%) subsequently died (p < 0.0001)  than those > 55 years (37.5%) p = 0.026). It was higher in patients with mixed HPV infections (81.8%) than single infection (54.5) and specifically more in HPV 35 (75%) and other HPV non-16/18 types (66.7%) than HPV 16 (28.6%) and HPV 18 (57.1%). PD-L1 expression was higher on tumor cells in non-black patients (83.3%) as compared to black patients (53.7%). MMR expression was lost in 2/11(18.2%) patients with mixed HPV infections as compared to those with single HPV type , but the difference is not statistically significant (p = 0.300). There was no correlation between PD-1 and PD-L1 and MMR protein expression. PD-L1 and MMR did not correlate with age, clinical stage or histologic grade  of 46.1 and 25.0 months (log rank test p = 0.009) and mean overall survival (OS) of 49.99 and 35.78 months, respectively (log rank test p = 0.049).PD-L1 expression showed a positive correlation with DFS and OS. Negative, low and high expression of PD-L1 were associate with mean DFS of 6.1, 27.0 and 36.5 months, and mean OS of 24.9, 28.1 and 40.3 months, respectively. In early clinical stage, high PD-L1 expression was associated with longer DFS (log rank test p = 0.006) and OS (log rank test p = 0.025)  and OS (p = 0.019)  and OS (30.1 months) than those with HPV 16/18 (29.8 and 35.3 months) . Patients with mixed HPV infections had a shorter mean DFS and OS  as compared to those with single HPV type , and in patients with negative HPV by PCR   and OS (18.6 months) compared to those infected with other types in early clinical stage . In late clinical stage, however, patients with HPV genotype 18 had significantly shorter DFS and OS (log rank test p = 0.025).When all other variables are controlled using Cox regression analysis, high PD-L1 expression (p = 0.003), MMR retention (p = 0.01), clinical stage (p = 0.048), histologic grade (p = 0.015) and mixed HPV infection (p = 0.026) remained independent predictors of DFS.The results of this study confirm reported higher prevalence of non-16/18 HPV genotypes among Black women with cervical cancer. However, in contrast to published data, our patients with non-16/18 HPV types had worse prognosis. Those with mixed HPV type infection had even poorer outcome. Also, patients who tested negative for HPV by PCR and negative for p16 on immunohistochemistry (IHC) had poor survival. We also found high rate of PD-L1 expression in cervical cancer cells, which was surprisingly associated with better survival. Our findings suggest a unique tumor microenvironment in squamous cell carcinoma in Black women which could offer therapeutic opportunities leading to survival benefit.21. In our predominantly Black population, we identified 13 of these high-risk HPV types of which type 35 was most frequent (20.0%) followed by types 16 and 18 (each 17.5%). HPV non-16/18 (75%) were more frequent than HPV 16/18 (25%) types. Distribution of 16/18 and non-16/18 has been found to be variable in different parts of the world. In the US a recent population-based study of 693 patients with invasive cervical carcinoma showed a predominance of HPV 16 (51.4%) followed by non-16/18 (24.2%) and HPV 18 (15.9%), while 8.5% patients were negative for HPV21.Of the 120 known HPV genotypes, about 40 infect the human genital mucosa and 15 have been classified as high-risk types 22. In Korea HPV 16 and 58 were found to be in almost equal frequency in a cervical cytology study of 1158 women; patients with HPV 58 had a significantly higher progression rate to high grade dysplasia23.Outside of the US a higher prevalence of non-16/18 HPV high-risk types have been reported than HPV 16/18 genotypes, and the former were found to be associated with a higher grade of cervical disease24. Studies from the Middle East have also shown higher prevalence of non-16/18 HPV genotypes, with HPV 31 most prevalent in Yemen and HPV 33 in Kuwait followed by 35, 39, 45, 52 and 5826. These studies indicate that the prevalence of non-16/18 HPV types differs globally and their impact in different patient populations has been largely under-estimated.A meta-analysis of HPV types in invasive cervical cancer in Asia found that types 16, 18, 52 and 58 had similar prevalence28. In light of these encouraging results, the findings of our study are striking because the currently available 9-valent HPV vaccine  does not include seven HPV types  that were detected in about one-third (33.3%) of our patients, mostly of Caribbean origin. Vaccines currently available to prevent the most common strains of HPV, may not offer the same level of protection in our Black population and other racial and ethnic groups globally. The diversity of patients and the prevalence of different HPV genotypes in the US and globally should be acknowledged as scientists guide further HPV vaccine development. To ensure successful cervical cancer prevention, physicians should continue to perform cervical cancer screening in women until complete high-risk HPV virus coverage is available in the vaccine.Studies on HPV vaccine effectiveness have highlighted protection against HPV-related pre-cancers and cancers, with a recent meta-analysis estimating 51% reduction in high grade cervical dysplasia among 15–19-year-olds and 31% reduction among 20–24-year-olds30.Our patients with non-16/18 types had worse prognosis than those infected with 16/18 in early clinical stage. This finding fails to validate earlier studies which found that women with HPV 16/18 positive tumors had worse survival than those with other HPV typesOne fifth of our patients had a mixed HPV infection and they were all found to have higher grade carcinomas (moderately to poorly differentiated) and a lower DFS and OS than those with a single HPV type infection. These tumors also had a higher PD-1 expression in TILs making them high-risk for aggressive course of disease although they could also prove to be targets for checkpoint inhibitor therapy.35. Only 9% of our patients had negative p16 expression, a finding similar to those reported in other studies on patients with invasive squamous cell carcinoma of the cervix. Among our patients with negative HPV PCR results, 8 of 9 had positive p16 expression. This contradictory pattern has been described in other studies which found the initial HPV PCR negativity—with p16 positive IHC—reduced from 19/209 to 5/209 cervical carcinomas after expanding molecular studies (targeting E6/E7 genes)36. The p16 positivity is therefore considered a more sensitive indicator of HPV-mediated carcinogenesis in cervical cancers. Loss of the frequently targeted viral L1 gene after viral integration could be a reason for HPV-negative test results37, while other studies have identified methodological limitations  as probable causes38.Our patients with negative HPV PCR results and non-typeable HPV types had high tumor grade at diagnosis and were also diagnosed at a late clinical stage. These findings suggest that patients with non-typeable oncogenic HPV types should be treated with higher circumspection because of their aggressive behavior and limited knowledge about their genotype making them hard to include in the vaccines. Patients with negative p16 expression had significantly shorter DFS and OS as compared to those with p16-positive tumors. Although this observation is based on a small sample in our study, similar observations in other population studies have recorded poor survival among women negative for both HPV PCR and p16 expression11. Expression of PD-L1 has been reported to be of variable prognostic information in neoplasms of different organs. It is a poor prognostic indicator in solid tumors, including esophageal, gastric, colorectal, and pulmonary adenocarcinoma, and a good prognostic indicator in lung squamous cell carcinoma40. Our study found PD-L1 to be a predictor of longer survival in early stage cervical carcinoma. Presence of higher PD-L1 increases the possibility of using targeted immunotherapy in these patients. Racial difference in PD-L1 expression of tumor cells had not been widely investigated. A recent single institution study of 114 cases, 36% of which were Black, concluded that PD-L1 expression in non-small cell lung cancer did not differ by race, clinical stage or smoking history41. Findings of a higher PD-L1 expression on cervical carcinoma cells in our population should be explored further in a larger multiracial study to identify the association of race with PD-L1 expression.PD-L1 expression in cervical cancer in our patients was 93% which far exceeds the reported frequency of 35–70%45, and increased survival in melanoma, glioblastoma, and ovarian, breast, and primary HPV-positive head and neck cancers50. Although not of prognostic value in our study, higher expression of PD-1 may be helpful to guide immunotherapy in the treatment of cervical carcinoma in patients with HPV infection of more severe types like non-16/18 and mixed, and patients with high stage carcinoma with limited treatment options.PD-1 expression in TILs surrounding cervical carcinoma showed no association with clinical stage, histologic grade, or HPV type, although PD-1 expressing TILs were greater in younger patients and patients with mixed and non-16/18 HPV infections. The prognostic significance of PD-1 expression in cervical carcinoma is still not fully elucidated, however, increase in PD-1 expression has been associated with poor prognosis in gastric, renal, nasopharyngeal and non-small cell lung carcinomas15. Loss of MMR proteins has been shown to correlate with tumoral PD-L1 expression in colorectal and endometrial carcinomas, but this association has not been examined in cervical carcinoma, where MMR deficiency is less common. In the current study 8.3% (5/60) patients were MMR-deficient whereas other reports have documented 25.8% (17/66) cervical carcinoma patients with MMR deficiency and significant correlation with PD-L1 expression on tumor cells51. All of our five MMR-deficient patients expressed PD-L1.MMR deficiency in solid tumors has recently been linked to susceptibility to immunotherapies targeting the PD-1/PD-L1 axis21. Blacks have worse survival rates likely due to more advanced-stage tumors at diagnosis and less opportunity likely to receive adequate treatment as compared with white women54. This trend was similarly reflected in our study where more than half of the Black patients had poor histologic tumor grade at diagnosis, about a third were diagnosed at late clinical stage and a third had recurrence who subsequently died. On the other hand, all non-Black patients, albeit in small number, had favorable prognosis; all of them were diagnosed at an early stage and all survived.Race, specifically the Black race, has been identified as one of the most common demographic factors associated with cervical squamous cell carcinomasMost Black patients in our study were infected with non-16/18 HPV genotypes, which were associated with a lower disease-free and overall survival. HPV 35 was the most common type isolated in our patient population, and this HPV genotype is not included in the commercially available nonavalent vaccine. Exclusion of HPV 35 and other common genotypes  from the vaccine exacerbates mortality from cervical cancer in these underserved communities. Patients with HPV 35 and those with mixed HPV infection had shorter DFS and OS, especially in early clinical stages. A remarkably high percentage of PD-L1 expression in cervical cancer cells was observed in our predominantly Black patient population and a high PD-1 expression on TILs in younger patients. These findings suggest that immune checkpoint inhibitors could be given consideration in the management of primary invasive squamous cell carcinoma of the cervix among Black patients.Supplementary Figures.Supplementary Tables.


Having a 30-year follow-up of a cohort of women tested for HPV is a unique opportunity to further study long-term risk of CIN3+. The study objective was to compare HPV status at baseline with the risk of CIN3+ in the follow-up period of 30 years.n = 642) referred to the HPV outpatient clinic at the University Hospital of North Norway (UNN) in 1990–1992, with an HPV test at baseline, were included in a prospective cohort. HPV-testing was performed by two different HPV-DNA tests, and genotypes 6, 11, 16, 18, 31 and 33 were identified. High-risk (HR) HPV genotypes  were classified as HPV positive, whereas low-risk (LR) genotypes (6 and 11) in addition to absent HPV were classified as HPV negative. A single cohort in which women were classified for their HPV status underwent follow-up prospectively to the last time-point of observation of 30 years.All women  cases of CIN3+ were detected, of whom 70.3% (104/148) were HPV positive and 29.7% (44/148) were HPV negative at baseline. The proportions of women who developed CIN3+ following a positive and a negative test were 46.6% (104/223) and 10.5% (44/419), respectively. Most cases of CIN3+ were seen shortly after the baseline HPV test, with 112 cases of CIN3+ diagnosed within the first year. In total, 48.6% (72/148) with HPV 16 and 57.6% (19/33) with HPV 33 developed CIN3+. Within the first year, CIN3+ was detected in 37.8% (56/148) with HPV 16, and 51.5% (17/33) with HPV 33. The long-term risk of CIN3+ was significantly lower than the short-term risk, and mainly associated with HPV 16. Overall, eight cases of cervical cancer were detected. Five were HPV positive, harboured HPV 16 at baseline and developed cervical cancer after 3, 4, 5, 11 and 24 years of follow-up.HPV status at baseline is predictive for the subsequent risk of developing CIN3+. Women with a positive HPV test in 1990–1992 had a significantly higher risk of CIN3+ during 30 years of follow-up than those with a negative test. HPV 16 was associated with the greatest long-term risk of cervical cancer. All patients with a positive HPV test at baseline should be followed up until negative.ISRCTN10836802. Registered 14 December 2020 - Retrospectively registered.ISRCTN, The online version contains supplementary material available at 10.1186/s13027-021-00386-z. The importance of Human Papilloma Virus (HPV) in the evolution of cervical intraepithelial neoplasia (CIN) and cancer is well-established , 2. PersThe Norwegian Cervical Cancer Screening Program (NCCSP), established in 1995, recommended primary screening by cervical cytology in the age group 25–69 years . TraditiA single positive test for HR-HPV in women with normal cytology is predictive of her subsequent risk for developing CIN2+ –19. HaviFrom July 1st 1990 to October 15th 1992, all women referred to the HPV outpatient clinic at UNN Tromsø, having an HPV test at baseline, were included in a prospective study. They were referred from their primary care physician, mainly within Troms and Finnmark counties. The reason for referral was symptoms associated with genital condylomas, cervical- and vulvar dysplasia, resistant vaginal discharge, vulvodynia, vulvar lichen planus or pruritus. Six hundred forty-two women were eligible for study participation. Age at inclusion was between 16 and 70 years. A clinical and colposcopic examination was performed in all patients. Paired samples for HPV testing were taken from cervix and vulva (cytology and biopsy) and visible lesions. The cervical scrapings and biopsy for HPV testing were taken from the endocervical canal and the transformation zone on the ectocervix. Patients with a histological confirmed CIN2+ were offered treatment. HPV results were not used to direct patient management.HPV-testing was performed by a two-step nonradioactive Southern blot DNA hybridization method using biotinylated HPV-specific probes . HPV-types identified were 6, 11, 16, 18, 31 and 33 causing 78.3% of all cervical cancer [2O). PCR-products were further identified with separate HPV 6, 11, 16, 18, 31 and 33 dot blot hybridizations using specific alkaline phosphatase-labelled oligonucleotides as earlier described [PCR amplifications were done in a GeneAmp PCR System 9600 , with the following modifications. PCR reaction condition with each primer at 0.25 μM of each primer, 100 μM of each deoxynucleotide and 1 U Taq polymerase in a final reaction volume of 50 μl were used. To obtain reproducible HPV PCR-results it was necessary to use hot-start PCR to avoid primer-dimer artefacts, adding Taq-polymerase after heating the samples at 80 °C for 2 min. The cycling conditions were 1 min at 94 °C, 30 s at 55 °C, 30 s at 72 °C; and a final three-minute extension interval at 72 °C. An aliquot of 15 μl of the PCR-products was analysed on ethidium bromide-stained 2% agarose gels. All experiments were done in parallel with positive (diluted cloned HPV-DNAs) and negative controls . During the follow-up period, all incidents of CIN3+ were detected and compared with the HPV status at baseline.Statistical package SPSS, version 22.0, was used for the statistical analyses. The Chi-square test was used to determine whether there was a statistically significant difference within our cohort. Univariate analyses were done using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log rank test. To measure the uncertainty associated with the results we calculated the 95% confidence interval (CI). The Kaplan-Meier survival curves with 95% CI in the supplemental are computed using the survfit-function in R (version 4.0.3).Two hundred twenty-three women (34.7%) were HPV positive at baseline Table . Five weAs illustrated in Fig. Finally, the overall cumulative incidence of CIN3+ for each HPV genotype was analysed. HPV 33 shows a high risk of CIN3+ at baseline, but the long-term risk is mainly associated with HPV 16. This is further supported using 4 years of quarantine from baseline, having few cases of CIN3+ associated with other HPV types  of the women in our cohort. The risk of CIN3+ among the HPV positive and HPV negative was 46.6 and 10.5%, respectively. Castle ollow-up . Most caollow-up . A studyet. al discovered that HPV 16, 18, 31 and 33 infection, and especially HPV16 persistence, were associated with high absolute risks for progression to CIN3+. They also found that 2 years persistence of HPV16 carries a 50% risk of CIN3+ [The incidence of CIN3+ was significantly higher among the HPV positive, compared to the negative. The majority of CIN3+ was detected at, or within 3 years after, baseline. Using 4 years of quarantine from baseline, the incidence of CIN3+ was considerably lower. This is as expected because the first HPV infection often occurs soon after the first sexual intercourse and women with prevalent CIN3+ at baseline were treated. It is still likely that some women with a negative HPV test at baseline still acquired an HPV infection during 30 years of follow-up. The majority of CIN3+ followed a positive HPV test at baseline. More specifically, most of them had HPV 16. The long-term risk of CIN3+ was further quite similar for women with HPV 31 or 33 and those HPV negative. A study by Kjaer When removing the quarantine, women with HPV 33 and 16 had the highest incidence of CIN3+ (Additional file The strengths of this study include the long follow-up time: curves for HPV 16/18 diverging even after 20 years. All women had a colposcopic examination done at baseline, and follow-up was ensured by the call-recall system initiated by the NCCSP. Another strength is the virtual absence of loss to follow-up because of the existence of the National Cancer Register. This study also has some limitations. Participant had no second colposcopic examination unless cytology findings suggested so, leading to several women being followed-up by cytology only. Because we measured HPV only once, we have no information about persistent infection and its role in the risk of progression to high-grade cervical lesions. Due to a small number of events, it is difficult to conclude on the long-term risk for other HPV than HPV 16.HPV status at baseline is predictive for the subsequent risk of developing CIN3+. Women with a positive HPV test in 1990–1992 had a significantly higher risk of CIN3+ during 30 years of follow-up compared to those with a negative test. HPV 16 provided the greatest long-term risk of cervical cancer. All patients with HPV at baseline should be followed up until negative.Additional file 1 : Table S1. HPV genotypes at baseline, cases of CIN3+ and PPV CIN3+ during follow-up. PPV = positive predictive value. Figure S1: HPV status at baseline by age. Figure S2. Cases of CIN3+ during follow-up by age. Figure S3. Cumulative incidence of CIN3+ by years of followup comparing HPV 16/33, HPV 18/31 and HPV negative. Figure S4. Cumulative insidence of CIN3+ by years of follow-up comparing HPV 16/33, HPV 18/31 and HPV negative, with 95% CI. Figure S5. Cumulative incidence of CIN3+ using 4 years quarantine from baseline, comparing women with HPV 16/33, HPV 18/31 and HPV negative. Figure S6. Cumulative incidence of CIN3+ by years of follow up using 4 years quarantine from baseline, comparing HPV 16/33, HPV 18/31 and HPV negative, with 95% CI. Figure S7. Cumulative incidence of CIN3+ by years of follow-up comparing women with a positive HPV test and a negative HPV test at baseline, with 95% CI. Figure S8. Cumulative incidence of CIN3+ by years of follow-up comparing women with HPV 31/33, HPV 16/18 and HPV negative, with 95% CI. Figure S9. Cumulative incidence of CIN3+ by years of follow-up using 4 years quarantine from baseline, comparing women with a positive HPV test and women with a negative HPV test, with 95% CI. Figure S10. Cumulative incidence of CIN3+ by years of follow-up using 4 years quarantine from baseline, comparing women with HPV 16/18, HPV 31/33 and women with a negative HPV test, with 95% CI. Figure S11. Cumulative incidence of CIN3+ by years of follow-up comparing all HPV genotypes. Figure S12. Cumulative incidence of CIN3+ by years of follow-up using 4 years quarantine from baseline, comparing all HPV genotypes.