Objective: The purpose of this study was to evaluate the effect of different combination models of high-risk human papilloma viruses (HPV) genotyping in triaging Chinese women with atypical squamous cells of undetermined significance (ASCUS).Methods: We established a screening cohort of 3,997 Chinese women who underwent cervical cytology and HPV genotyping test. Women with ASCUS cytology underwent punch biopsy under colposcopy/endocervical curettage. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of different combination models of HR-HPV genotyping calculated that cervical intraepithelial neoplasia 2 or higher (CIN2+) on histology were endpoints.Results: Of the full sample, 393 women had ASCUS. Among ASCUS women with a CIN2 lesion, the prevalence for HPV were 40.0% (type 16), 10.0% (type 18), 0.0% (type 33), 30.0% (type 52), 40.0% (type 58), and 30.0% (other nine types). For ASCUS women with a CIN3 lesion, the prevalence for HPV were 68.4% (type 16), 15.8% (type 18), 10.5% (type 33), 31.6% (type 52), 15.8% (type 58), and 36.8% (other nine types). Combination model including HPV16/18/33/52/58 for predicting CIN2+ lesion in women with ASCUS had relatively higher sensitivity [93.1% ], specificity [75.8% ], PPV [23.5% ], and NPV [99.3% ] than other combination models. Moreover, the referral rate of HPV16/18/33/52/58 (29.3%) was lower than HR-HPV (36.1%).Conclusions: The study demonstrates that specific HR-HPV types HPV16/18/33/52/58 may be an effective strategy in ASCUS triage. This improves the subsequent selection of ASCUS patients. Cervical cancer is the third most common cancer among women worldwide, the second most common cause of cancer death, and causes 300,000 deaths a year . NeverthMost women with ASCUS do not have clinically significant diseases, nonetheless, a substantial proportion (15–20%) of them do have cervical intraepithelial neoplasia (CIN) in a follow-up histopathological diagnosis . ResearcPersistent infection of high-risk (HR) genotypes of human papillomavirus (HPV) may lead to the development and progression of cervical cancer , 12. In The ideal triage strategy of HPV genotyping for ASCUS cases is one where sensitivity and specificity can be maximized, which can reduce the rate of missed diagnosis and avoid unnecessary referral for colposcopy. The present study was designed to evaluate the effectiveness of HR-HPV genotype testing for triaging Chinese women with ASCUS, and to provide a new parameter for formulating the best triage strategy of ASCUS.A cervical cancer screening cohort of 3,997 women was established in Jiyuan City, Henan from April to May 2017. Participants underwent cervical cytology and HPV genotype testing. Women were eligible for the study if they were >21 years of age; had sex; were not currently pregnant; had no history of hysterectomy, cervix surgery, or cervical cancer treatment; and were able to provide informed consent. This study was approved by the Institutional Review Board of Affiliated Cancer hospital of Zhengzhou University.Prior to study enrollment, a trained health care worker obtained informed consent and administered a questionnaire in a confidential interview with each eligible woman to assess medical and surgical history of the cervix uteri and cervical cancer; marital status; educational levels; smoking; drinking history; and reproductive information.Each eligible woman had a gynecological examination of their vulva, vagina, and uterine neck. A physician performed speculum exams, collected specimens of cervical exfoliated cells by broom brush, and transferred the cells to a PreservCyt liquid , stored at 4°C for liquid-based cytology classification and HPV DNA testing.Liquid-based cytology (LBC) testing results were ASCUS women who had been required recall for colposcopy examination with 12 weeks after collecting specimens of cervical exfoliated cells. An experienced gynecologist performed colposcopy-directed biopsies on women with ASCUS. If the squamo-columnar junction was completely visible, it was a satisfactory colposcopy examination. Women with normal colposcopy examination were not needed to undergo biopsies. Conversely, women with abnormal colposcopy examination underwent directed cervical biopsies where lesions were visible. If the colposcopy examination was unsatisfactory (the squamo-columnar junction was not completely visible), physicians performed endocervical curettage (ECC) .We performed HPV genotyping using an HPV DNA Genotyping Kit  according to the manufacturer's instructions. The HR-HPV test was based on multiple nucleic acid amplification Polymerase Chain Reaction (PCR) method with fluorescence detection. Primers and probes were designed specifically for target sequences of the L1 gene for high-risk HPV. PCR procedure could detect 14 types of HPV DNA  from the cervical exfoliative cells and distinguish HPV types 16, 18, 33, 52, and 58. Each experiment had positive and negative quality control and blank control. We synchronously measured reference gene β-Globin for judging a false negative either due to insufficient sampling or operation error.We used the Thinprep liquid-based cytologic test (TCT). The Bethesda system (TBS) was used for cytology and the CIN classification systems for histology. The worst reading across all histological findings was the final diagnosis for each woman. A woman was assessed as negative for CIN if a biopsy had not been indicated or the histology finding was negative. Cyto-pathologists and pathologists from the Zhengzhou University Affiliated Cancer Hospital prepared and read the cytological and histological slides. Before the study began, the Hospital held a training meeting on colposcopy and histopathology diagnosis to standardize the protocol.P ≤ 0.05 (two-sided) were considered statistically significant.We analyzed the data using SPSS version 20.2 . All women with ASCUS underwent colposcopy and/or ECC. The referral rate was calculated as number of ASCUS patients with positive HR-HPV type divided by the total number of ASCUS patients. We estimated the mean and standard deviation of the quantitative variables and numbers and percentages for categorical variables. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of HPV genotyping for detecting cervical lesions CIN2+ were calculated and compared to the pathological diagnosis as the gold standard. From these results, we could evaluate the accuracy and effectiveness of HPV genotyping in triaging women with ASCUS. Of the overall cervical cancer screening cohort , a total of 393 (9.8%) women had cytologically confirmed ASCUS. The prevalence of the screening cohort was 18.2%  . Table 2Cervical lesions CIN2+on histology was as the endpoint, the sensitivity and NPV of different combination models of HR-HPV increased with HPV16/18, HPV16/18/52, HPV16/18/52/58, HPV16/18/33/52/58, and HR-HPV . HoweverAn ASCUS result is the most common non-normal cytologic finding in cervical cancer screening, which is either an actively proliferated benign lesion or a potentially malignant lesion; the histopathology results of which are very different. Therefore, the establishment of management standards for patients with ASCUS is urgently needed. In recent years, HPV DNA testing has been incorporated into screening programs, which is firstly a means of triaging patients with ASCUS , 18, andIn the previous studies, the percentage of ASCUS in cervical cytology was found to range between 3 and 10% –23. SimiA meta-analysis comparing the accuracy of HR-HPV testing against that of repeated cytology for detection of underlying CIN2+ or CIN3+ in women with ASCUS showed that HR-HPV-triage had significantly higher sensitivity, but not significant specificity than repeated cytology in ASCUS triage . Our resThis study has several limitations. First, we did not test and classify all HPV genotypes, so every combination of all HPV genotypes can't be assessed. However, the infection rates of HPV16/18/33/52/58 are highest in Chinese women and this study showed the model with these types was best at triaging ASCUS women. Second, this study was conducted in a single region, which may reduce generalizability to other regions. We did not perform external validation in additional datasets. In addition, this study lacked a significant follow up in our cases. As a result, the study population may not be reflective of a true screening population.In summary, this is the first study in China to assess the effect of different models of HR-HPV genotyping in triaging Chinese women with ASCUS cytology. Our study demonstrates that genotyping for HPV16/18/33/52/58 genotypes serves as a robust triage system for ASCUS women based on its high sensitivity, specificity, and the minimal number of HR-HPVs needed for genotyping. The development of a specific HPV genotyping assay may significantly improve the cost-effectiveness of screening. In the next step, we plan to externally validate our novel HPV genotyping assay with a different dataset.All datasets generated for this study are included in the manuscript and/or the This study was approved by the Institutional Review Board of Affiliated Cancer Hospital of Zhengzhou University. Each participant was informed and signed an informed consent.ZG and S-KZ contributed to the design and write wrote the manuscript. ZG, QC, and P-PC performed statistical analysis. ZG, M-MJ, and P-PC contributed to investigation and HPV test. H-MC, D-MZ, and L-YR contributed to cytology and histology examination. X-BS helped conceiving the study and assisted with the statistical analyses. All authors read and approved the final manuscript.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.


E6 and E7 region-specific gene polymorphisms of HPV-33 and HPV-58 in Southwest China and to identify ideal epitopes for vaccine design. Both HPV-33 and HPV-58 belong to α-9 genus HPV and are highly homologous, so their correlations are included in our research.Human papillomavirus (HPV) E6 and E7 oncoproteins play a crucial role in HPV-related diseases, such as cervical cancer, and can be used as ideal targets for therapeutic vaccines. Human leukocyte antigen (HLA) participates in the immune response to block HPV infection and invasion by its target/recognition function. HPV-33 and HPV-58 are highly prevalent among Chinese women. Therefore, it is of great significance to study the E6 and E7 variations and polymorphisms of HPV-33 and HPV-58 in Southwest China, we collected samples, extracted and sequenced DNA, and identified variants. Nucleotide sequences were translated into amino acids by Mega 6.0 software. The physical/chemical properties, amino acid-conserved sequences and secondary structure of protein sequences were analysed by the Protparam server, ConSurf server and PSIPRED software. The T and B cell epitopes of the E6/E7 reference and variant sequences in HPV-33 and HPV-58 were predicted by the Immune Epitope Database (IEDB) analysis server and the ABCpred server, respectively.To study the 41-58EVYDFAFADLTVVYREGN of HPV-33 E6 and 40-60SEVYDFVFADLRIVYRDGNPF of HPV-58 E6. Six optimal HLA-I-restricted T cell epitopes were selected from HPV-33 and HPV-58 E7, and these epitopes are mainly located in 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7.Five and seven optimal HLA-I restricted T cell epitopes were selected from HPV-33 and HPV-58 E6, respectively, and these optimal epitopes are mainly located in E6/E7 gene polymorphisms and T/B cell epitopes of their reference and variant sequences were studied, and candidate epitopes were selected by bioinformatics techniques for therapeutic vaccine design for people in Southwest China. This study was the first to investigate the correlation of epitopes between HPV-33 and HPV-58. After experimental validation, these selected epitopes will be employed to induce a wide range of immune responses in heterogeneous HLA populations.HPV-33/HPV-58 The online version of this article (10.1186/s12985-019-1168-y) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) is a double-stranded type of DNA virus of approximately 8 kb in size and is widely spread in humans . HPV infE6 and E7 proteins are the main virus-transforming proteins of high-risk HPV that participate in inducing cell proliferation and causing human epithelial cell immortalization and transformation . E6 and + cytotoxic T lymphocytes (CTLs). HLA-II assists in identification of exogenous antigen and stimulates CD4+ helper T lymphocytes (HTLs). In cell-mediated immune responses, CTLs are considered the major eradicators of both HPV-infected and cervical cancer cells [+CTLs activated after proliferation can directly kill tumour cells or secrete cytokines to inhibit tumour cells. Lymphatic factors produced by the activated CD4+ HTLs enhance the function of CTLs and natural killer (NK) cells and activate macrophages or other antigen-presenting cells (APC), which have antitumour effects [Human leukocyte antigen (HLA) has a self-recognition function to regulate the body’s immune response and control HPV infection and virus removal by presenting antigen proteins –11. HLA er cells . CD8+CTL effects . CTL epi effects , 17. The effects , 18. B cE6/E7 gene diversity of HPV-33/HPV-58 in Southwest China was chosen as the subject of this study [The treatment of HPV-related diseases by antigen epitopes was previously proposed . Therefois study . To impr6H5CO2Na, 1 L H2O).Samples were collected from hospitals in Sichuan and Chongqing, including the Angel of Maternal and Child Health Hospital and the Reproductive Health Research Center of Chengdu. This study was approved by the Ethics Education and Research Committee of Sichuan University and the Ethics Committee of Sichuan University . A total of 16,793 cervical specimens were collected from January 1, 2009 to December 31, 2015. All methods were performed in accordance with the relevant guidelines and regulations. Specimens were collected from participants using cervical swabs and stored at − 20 °C in buffer solution . Subjects over 14 years old with visible cervical lesions or HPV-related diseases  were included .HPV DNA was extracted and evaluated using the Human Papillomavirus Genotyping Kit For 23 Types  according to the manufacturer’s guidelines.E6/E7 genes were amplified by polymerase chain reaction (PCR) using a thermal cycler . Primers for HPV-33 E6 and E7 were as follows: 5′-AAAAAAGTAGGGTGTAACCGA-3′ and 5′-TGCCACTGTCATCTGCTGT-3′ (melting temperature 54 °C) as well as 5′-ACGGTGCATATATAAAGCAAACATT-3′ and 5′-CTTCTACCTCAAACCAACCAGTACA-3′ (melting temperature 60 °C) when a second round of amplification was needed. HPV-58 E6 and E7 fragments were amplified using specific primers described previously [2 and dNTPs, 2 U of Pfu DNA polymerase , and 0.25 μmoL of each primer. Conditions were as follows: 95 °C (10 min); 35 cycles of 94 °C (50 s), 54 °C (60 s) (different for each gene), 72 °C (60 s); and a final step of 72 °C for 7 min. PCR amplification products were stained with Gene-Green nucleic acid dyes and visualized on 2% agarose gels under ultraviolet light WFH-202. Target products were sequenced at Sangon Biotech . Data were confirmed by performing the PCR amplification and sequence analysis at least twice.Primers were designed according to the HPV-33/HPV-58 reference sequence. HPV-33/HPV-58 complete eviously . The totP < 0.05 was considered statistically significant. Compared with the reference sequence, mutations with a frequency ≥ 10% were considered as major mutations [Data were analysed by SPSS version 19 . Pearson chi-square test was employed to confirm the results. utations . Mega 6.utations . The Proutations . Moreoveutations . The PSIutations .According to the average frequency of HLA alleles from the major histocompatibility complex database (dbMHC) in the Chinese population , 13 HLA-T cell epitopes of HPV-33/HPV-58 E6/E7 reference and variant sequences were predicted by Immune Epitope Database (IEDB) analysis server. According to the method recommended by IEDB, the lower percentile rank (PR) of antigen epitopes represented a better affinity. Thus, we selected candidate epitopes based on PR . In our The ABCpred server was used to predict B cell antigen epitopes of HPV-33 and HPV-58 E6/E7 reference and variant sequences according to the default parameters . The higE6/E7 variant sequences are KX354744-KX354775).The datasets generated during the current study are available in GenBank (accession codes for HPV-33 and HPV-58 E6 Open Reading Frame (ORF) was 450 bp and that of E7 was 294 bp. Compared with the HPV-33 E6/E7 reference sequence (GenBank: M12732.1), mutations in HPV-33 E6 (450 bp) and HPV-33 E7 (297 bp) nucleotide sequences are presented in Table E6 sequences and 96 (44.45%) E7 sequences have nucleotide mutations. In these mutant E6 sequences, eight nucleotide mutations were observed, including six non-synonymous mutations and two synonymous mutations. Non-synonymous mutations include A213C, G329C, A364C, A387C, A446G, and G542 T, leading to the amino acids substitution of K35 N, S74 T, N86H, K93 N, Q113R and R145I, respectively. Among them, the most common mutations occurred at the 213th and 387th site of E6 sequence with a 19.44% mutation rate, and both amino acid substitutions are from lysine to aspartate. Finally, seven HPV-33 E6 variant types were determined. In the E7 mutant sequences, four single nucleotide changes were identified, which were all non-synonymous mutations, including G658C, C706T, C706A, and A862T that lead to the amino acids substitution of S29 T, A45V, A45E and Q97L, respectively. The most common mutation was C706T (A45V) with a 16.67% mutant rate. In addition, seven HPV-33 E7 variant sequence types were determined.A total of 216 HPV-33 samples were sequenced. Data showed that the total length of the HPV-33 E6 (450 bp) and HPV-58 E7 (297 bp) are presented in Table E6/E7 reference sequence, 356 (87.90%) E6 variant sequences and 326 (80.50%) E7 variant sequences were detected. In these E6 variant sequences, eight variant types and eight nucleotide mutations were found, including 4 non-synonymous mutations and 4 synonymous mutations. Non-synonymous mutations were G203C, C367A, A388C and G543A, resulting in the amino acids substitution of E32Q, D86E, K93N and R145K, respectively. A338C (K93N) is the most common non-synonymous mutation with a 27.41% mutation rate. In 326 E7 variant sequences, twelve variant types were determined, and thirteen nucleotide mutations were found, including 10 non-synonymous mutations and 3 synonymous mutations. Non-synonymous mutations include G599A, C632T, G694A, C755A, G760A, G761A, A763G, A793G, C801A, and T803C, which lead to the amino-acid substitutions of R9K, T20I, G41R, T61N, G63S, G63D, T64A, T74A, D76E and V77A, respectively. G761A (G63D) was the most common non-synonymous mutation with a 40.25% mutation rate.In this study, 405 HPV-58 samples were sequenced, and the mutations in HPV-58 In this study, amino acids composition analysis of HPV-33 E6 and E7 was performed, and the residue numbers of HPV-33 E6 and E7 were 149 and 97, respectively. In the reference sequence of HPV-33 E6, Arg (10.10%) and Leu (10.10%) represented the highest residue proportion followed by Glu (8.10%) and Cys (6.70%). In reference sequence of E7, the top 3 residues with the highest content were Thr (11.30%), Leu (10.30%) and Asp (9.30%). An additional file shows more details [see Additional file The secondary structure prediction of HPV-33 E6 showed that E6 reference contained 27.5% helix, 20.8% sheet, and 51.7% coil, whereas the E6 variant was composed of 28.2% helix, 16.1% sheet, and 55.7% coil. The result suggests that the mutation of E6 mainly affects the secondary structure of the residues 87–88 and 132–134, resulting in an increase of coil and a decrease of β-sheet. The prediction results of E7 revealed that E7 reference consists of 12.4% helix, 16.5% sheet, and 71.1% coil, and the substitutions had no influence on its secondary structure  and Leu (10.70%) represented the highest proportion of residues followed by Glu (7.40%), Cys (7.40%) and Lys (7.40%). In the E7 reference sequence, the top 3 residues with the highest content were Thr (13.30%), Leu (10.20%) and Cys (9.20%). More details are shown in Additional file Secondary structure prediction of HPV-58 E6 demonstrated that E6 reference is composed of 27.5% helix, 16.8% sheet, and 55.7% coil, similarly, E6 variant is 28.2, 16.8, and 55%. The above fact indicated that the substitutions merely cause the change of secondary structure in the residue 87. The prediction results of E7 illustrated that both E7 and its variant possess the similar secondary structure , suggesting that the substitutions are less important for secondary structure  optimal HLA-I-restricted epitopes in HPV-33 E6 and E7 were selected from the common segment with a concentrated distribution of HLA-I and HLA-II restricted epitopes. If no common segment was present, the optimal epitopes were selected based on prediction results of HLA-I. In this step, if a peptide was contained in another longer segment and the PR values were equal, we would choose the longer segment. If there were more predicted epitopes that have the same PR as the fifth selected, these epitopes would be included. Following this principle, we selected 5 and 6 optimal epitopes from HPV-33 E6 and E7, respectively  in the reference sequence. All of the predicted epitopes without mutation sites were integrated into four different segments, which was similar to the integration results of HPV-33 E6 . NumberHPV is an important pathogenic virus that is closely associated with diseases of the human skin and mucosa . High-riIn order to select optimal epitopes for therapeutic vaccine design, we integrated bioinformatics techniques to investigate the influence of mutation on the secondary structure and epitope affinity. Polymorphism analysis demonstrated that multiple identical non-synonymous mutation sites exist in E6 of HPV-33 and 58, including the 86th, 93rd and 145th residue. The 93rd residue is the most common non-synonymous mutation in E6 of HPV-33 and 58, and both substitutions are from lysine to aspartate. Substitution of the 86th residue in HPV-33/58 E6 resulted in a change of secondary structure in the adjacent position, increasing the number of residues on the α helix after this site. In addition, based on previous studies in our lab, R145I in HPV-33 E6 and R145K in HPV-58 E6 are positive selection sites , and the41-58EVYDFAFADLTVVYREGN, and five of the seven optimal epitopes selected from HPV-58 E6 are located at 40-60SEVYDFVFADLRIVYRDGNPF. These two segments have high sequence similarity and are ideal for designing therapeutic vaccines. Four of the six optimal epitopes selected from HPV-33 and 58 E7 correspond to the same position. Here, 77-84RTIQQLLM, 77-90RTIQQLLMGTVNIV, 81-90QLLMGTVNIV and 82-90LLMGTVNIV in HPV-33 E7 correspond to 78-85RTLQQLLM, 78-91RTLQQLLMGTCTIV, 82-91QLLMGTCTIV, and 83-91LLMGTCTIV in HPV-58 E7, respectively. These 4 optimal epitopes are located in 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7. These two segments also exhibit high sequence similarity and are ideal for designing therapeutic vaccines. The above analysis reveals that most of the optimal epitopes from HPV-33 E6/E7 were located in the same segment as the optimal epitopes from HPV-58 E6/E7 and exhibit similar predicted affinity. This finding reflects the similarity of immunogenicity between HPV-33 and HPV-58.Accordingly, there are some significant differences between HPV-33 and HPV-58 in the epitope prediction results. DQB1*05:01 is considered to be a protective gene . All mutE6/E7 affect the composition and secondary structure of protein sequences and contribute to the affinity and immunogenicity of the peptide epitopes. In our study, optimal epitopes for therapeutic vaccines were selected from identical regions in reference and variant sequences. In addition, whether an epitope is included in the predicted results of HLA-II-restrictive epitopes is also considered. The final selections are based on the PR of predicted epitopes. The 6 optimal epitopes from HPV-33 E6 are located at 41-58EVYDFAFADLTVVYREGN, and five of the seven optimal epitopes selected from HPV-58 E6 are located at 40-60SEVYDFVFADLRIVYRDGNPF. The 4 optimal epitopes are located at 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7. This information reflects the similarity of immunogenicity between HPV-33 and HPV-58. Using immune-informatics technologies to provide a new approach to access ideal epitopes, these results are valuable for the development of therapeutic vaccines and cancer immunotherapies.Nucleotide mutations in HPV-33 and HPV-58 Additional file 1:Table S1. Amino acid composition and hydrophilicity of HPV-33 E6 reference and variant sequence. Table S2. Amino acid composition and hydrophilicity of HPV-33 E7 reference and variant sequence. Table S3. Amino acid composition and hydrophilicity of HPV-58 E6 reference and variant sequence. Table S4 Amino acid composition and hydrophilicity of HPV-58 E7 reference and variant sequence. (XLSX 16 kb)Additional file 2:Figure S1. Secondary structure prediction of HPV-33 E6 reference and variant sequence. Figure S2. Secondary structure prediction of HPV-33 E7 reference and variant sequence. Figure S3. Secondary structure prediction of HPV-58 E6 reference and variant sequence. Figure S4. Secondary structure prediction of HPV-58 E7 reference and variant sequence. (PDF 754 kb)Additional file 3:Table S5. The diversity of predicted binders of HPV-33 E6 for HLA-I alleles between reference and variant sequences; Table S6. The diversity of predicted binders of HPV-33 E7 for HLA-I alleles between reference and variant sequences; Table S7. Predicted epitopes binders for HLA-II alleles of HPV-33 E6; Table S8. Predicted epitopes binders for HLA-II alleles of HPV-33 E7. (XLSX 42 kb)Additional file 4:Table S9. The diversity of predicted binder of HPV-58 E6 for HLA-I alleles between reference and variant sequences; Table S10. The diversity of predicted binders of HPV-58 E7 for HLA-I alleles between reference and variant sequences; Table S11. Predicted epitopes binders for HLA-II alleles of HPV-58 E6; Table S12. Predicted epitopes binders for HLA-II alleles of HPV-58 E7. (XLSX 42 kb)Additional file 5:Table S13. Predicted linear B cell epitopes of HPV-33 E6; Table S14 Predicted linear B cell epitopes of HPV-33 E7. (XLSX 13 kb)Additional file 6:Table S15. Predicted linear B cell epitopes of HPV-58 E6; Table S16. Predicted linear B cell epitopes of HPV-58 E7. (XLSX 12 kb)


Helicobacter pylori (H. pylori) detection in endoscopy-obtained esophageal and gastroesophageal junction biopsies and in oral cell specimens taken at the time of esophageal cancer diagnosis in a case–control study in Addis Ababa, Ethiopia.Ethiopia lies in the high-risk corridor of esophageal squamous cell carcinoma in East Africa, where individuals with this malignancy often do not report established risk factors, suggesting unidentified etiologies. Here, we report the prevalence of mucosal human papillomavirus (HPV) and of H. pylori DNA was detected using highly sensitive assays that combine multiplex polymerase chain reaction and bead-based Luminex technology. The proportions of specimens testing positive were expressed as percentages, with binomial 95% confidence intervals. Agreement of results between tissue biopsy and oral cell specimens was estimated using the kappa statistic. Comparison of study participants’ characteristics by test results was done using the Pearson chi-square test.DNA extraction was performed from fresh frozen tissue and oral cell pellets obtained with saline solution gargling subsequently fixed with ethanol. Mucosal HPV and H. pylori DNA was 55% (95% CI: 42–68%), and 20 of 34 H. pylori-positive specimens  were positive for the cagA gene. Agreement of detection rates between tissue and oral cells in cases was poor for HPV and for H. pylori.HPV DNA was detected in 1 of 62 tumor specimens : 0–9%), corresponding to HPV16 type. HPV DNA was detected in the oral cavity of 7 cases  and 4 of 56 matched healthy controls , with multiple HPV types detected. Detection of H. pylori was more commonly detected, with a high proportion testing positive for the pro-inflammatory gene cagA. These novel findings remain to be replicated in larger studies and with the addition of serological determinations to better understand their biological significance in the context of esophageal and gastroesophageal junction cancers.The prevalence of mucosal-type HPV was very low, whereas  Helicobacter pylori [H. pylori]) could also contribute etiologically to the occurrence of EC, and biological specimens were therefore collected to explore this possible link.Ethiopia forms part of the East Africa high-risk corridor for esophageal squamous cell carcinoma (ESCC) . WhereasH. pylori as carcinogenic to humans (IARC Group 1) in patients with chronic infection [H. pylori is involved in up to 89% of non-cardia gastric cancer cases worldwide [H. pylori infection and gastric lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) [H. pylori strains can be subdivided based on the presence or absence of a gene that encodes for the cytotoxin-associated gene A (CagA) protein: cagA-positive and cagA-negative strains, respectively. It is well established that cagA-positive strains are associated with an increased risk of developing gastric cancer [H. pylori is linked to its ability to deliver the pathogenicity factor CagA protein into the host gastric epithelial cells [A subgroup of HPV types referred to as high-risk (HR) HPV types have been identified as the etiological agents of anogenital cancers and a subset of head and neck cancers; HPV16 is the most oncogenic type . HR HPV nfection , 6. H. porldwide , and a lymphoma) , 8. H. pc cancer . The caral cells , where ial cells .H. pylori infection and risk of ESCC are not consistent in indicating an association, or its direction [H. pylori infection is associated with decreased risk of adenocarcinoma of the esophagus [Based on evidence available up to 2009, the IARC Monographs Working Group concluded that the epidemiological evidence was inadequate to support a role for HPV infection in EC etiology, recognizing the very large geographical variation in the rate of HPV infection detected in cancer cases . A more irection –26. In csophagus .H. pylori infections in the upper aerodigestive tract (UADT), including the esophagus, is unknown in the general population as well as in patients with UADT cancer undergoing upper endoscopy. Therefore, the main objective of this study is to report the prevalence of HPV and of H. pylori detection in endoscopy-obtained esophageal and gastroesophageal junction biopsy specimens and in buccal cells taken at the time of EC diagnosis, in participants in a case–control study undertaken to inform the planning of a large-scale etiological study of UADT cancers in Ethiopia [In Ethiopia, the prevalence of HPV and Ethiopia ., tobacco use, or alcohol consumption from the wards of TAH, and healthy individuals attending TAH to visit inpatients and not related by blood to study cases . Controls were matched to cases based on sex, age, and zone of residence in the country. A total of 133 controls were enrolled and matched to EC and gastroesophageal junction cancer cases (73 cases). Although at least one healthy control (n = 93) was individually matched to each cancer case, this was not possible for hospital-based controls because a reduced number were enrolled (n = 40). Details on data collection via questionnaire are described elsewhere [The biological specimens tested in this study were collected in a hospital-based case–control study conducted in Addis Ababa, Ethiopia (May 2012–May 2013), considered to be a pilot study because it was a first study, but with complete case–control methodology as previously described [Eligible study participants provided written consent indicating whether they accepted or declined each of the study components: completion of a risk-factor questionnaire administered by face-to-face interview, blood drawing, buccal cell collection by oral gargling and spitting, and in addition, for cancer cases, a biopsy of tumor tissue obtained at endoscopy. The study protocol and consent forms were reviewed and approved by the Institutional Review Boards of Addis Ababa University in Ethiopia and the Morehouse School of Medicine in Atlanta, USA, and the IARC Ethics Committee in Lyon, France.Biopsies of suspected tumor tissue at Adera Higher Clinic and Mexico Higher Clinic were obtained through endoscopy after sedation. Biopsy tissue punches were placed in cryotubes and fully immersed in RNA/DNA Allprotect Tissue Reagent (Qiagen) for fresh tissue preservation, and initially stored at + 4 °C until transport to the Armauer Hansen Research Institute (AHRI), for storage at − 80 °C, on the day of collection, unless the specimen was collected during a weekend, in which case it was transported to AHRI on the Monday.Although this pilot study enrolled 61 EC cases and 12 gastroesophageal junction cancer cases (73 cases), 11 of these cases were enrolled at TAH, where endoscopy with biopsy was not performed because these patients had been diagnosed elsewhere and referred to TAH for cancer treatment, with no fresh tissue specimens available. Therefore, 11 (15%) of the enrolled cancer cases were excluded from the testing reported here.g for 10 min at + 4 °C. The supernatant was discarded, and the pellet was re-suspended in 1 mL of fixative solution (96% ethanol) with repeated pipetting and vortexing, and distributed into two aliquots kept at + 4 °C until shipment to IARC. In the present investigation, a priori, we opted to test the buccal cell specimens of only one control for each case, and among controls, we selected healthy individuals.The gargling spit was collected with saline solution, fixed using 96% ethanol (2:1 ratio), and temporarily stored at + 4 °C. At the laboratory, the spit-containing tube was centrifuged at 3,000 DNA extraction was performed from frozen tissues and oral cell pellets using the Qiagen BioRobot EZ1 with the EZ1 DNA Tissue Kit according to the manufacturer’s instructions . Briefly, frozen tissues and oral cell pellets were incubated in proteinase K and Buffer G2  at 56 °C until the tissue was completely lysed. The DNA was stored at − 20 °C until further use.H. pylori DNA was detected using highly sensitive assays that combine multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described previously [ureA , and cagA  genes from H. pylori were used. Two primers for the amplification of beta-globin were also added to provide a positive control for the quality of the template DNA. The cutoff was calculated as previously reported by Schmitt et al. [The presence of mucosal HPV and eviously –29. The t et al. . After Pt et al. .H. pylori  were expressed as percentages with binomial 95% confidence intervals. Among H. pylori-positive specimens, the proportion testing positive for the virulence gene cagA was also calculated. Differences in prevalence by type of specimen or between cases and controls in oral specimens were determined by the extent of overlap of the 95% confidence intervals around the contrasted proportions . Agreement of results between tissue biopsy and oral cell specimens available in cases was estimated using the kappa statistic. Comparison of study participants’ characteristics by HPV or H. pylori test results was done using the Pearson chi-square test.The proportions of specimens testing positive for HPV DNA  or Of 73 enrolled cancer cases, 62 (85%) had tumor tissue stored for testing. The mean age at diagnosis was 57 ± 12 years, and 29 (47%) of the included samples were from male patients. Squamous cell carcinoma was the most common histology (74%), and more malignancies were detected in the lower third of the esophagus than in other segments (Table Fresh frozen tissue specimens were available for testing in 62 cases (100%). All specimens were beta-globin-positive. HPV DNA was detected in 1 tumor specimen . All specimens were beta-globin-positive. HPV DNA was detected in the oral cavity of 7 cases . HPV31 and HPV56 genotypes were each detected in the buccal cells of 2 cases , and HPV16, HPV18, HPV35, HPV51, and HPV66 genotypes were detected in 1 case each  had oral cells available for testing. Of these specimens, one was beta-globin-negative and was excluded from further consideration. HPV DNA was detected in the oral cavity of 4 of 56 matched healthy controls . The following genotypes were identified in the oral cavity: HPV16, HPV18, HPV35, HPV39, HPV53, and HPV66. Specimens from 2 controls had dual detection, with the following combination of genotypes: HPV18 and HPV39 in 1 control and HPV35 and HPV66 in 1 control. Low-risk HPV types were not identified in the oral cells of healthy controls. The proportion of controls with HR HPV DNA-positive buccal cells was similar to the proportion observed in cases.H. pylori DNA was common: 55% (95% CI: 42–68%). An equal proportion tested positive for H. pylori 16S and for H. pylori ureA: 30 of 62, or 48% (95% CI: 35–61%). Of 34 H. pylori-positive specimens, 20 were positive for the cagA gene  .All 62 fresh frozen tissue specimens (100%) were beta-globin-positive. Detection of H. pylori-positive oral specimens (95% CI: 6–24%)  than by the 16S test . One quarter of H. pylori-infected oral specimens tested positive for the cagA gene .Among the 61 cases with buccal cells, 8 (13%) had H. pylori detection in buccal cells of controls was 7% (95% CI: 2–17%). Of 4 H. pylori-positive samples, 2 tested positive for the cagA gene , based on 62 specimens, with only a single biopsy positive for HPV16. Relying on overall findings or restricting results to those from ESCC specimens , the prevalence of mucosal HPV detection in esophageal tissue in our study differs from that in similar testing conducted elsewhere in Africa. A study in Malawi, the country with the highest EC incidence in East Africa, reported a HPV16 positivity of 15% among 40 patients with ESCC undergoing endoscopy with formalin-fixed, paraffin-embedded tissue biopsy specimens tested using multiplex quantitative PCR and in situ hybridization . HoweverThe different findings among published studies across East Africa could reflect true differences in the rate of HPV infection in tumor specimens and/or differences in the rate of detection, possibly associated with the methods used in the selection of patients and the testing of specimens and the different types and quality of specimens used . None of the adenocarcinoma tissue biopsies in our study tested positive for HPV, in agreement with the findings of a study in Australia that included both EC and gastroesophageal junction adenocarcinomas .In our study, the diversity of HPV types seen in the oral cavity of cases was not informative of the diversity of HPV types detected further down in the esophagus. For cancer sites for which HPV is an established causal agent, the prevalence and agreement of detection across different types of specimens is larger. For instance, higher prevalence of HR HPV types and agreement of HPV detection between tumor tissue (20%) and oral cells (23%) in US cases of head and neck cancer has previously been reported . In contThe majority of the HPV genotypes detected in the oral cells of cases and controls have been found to be present in the cervical cytology of women with normal and also with abnormal findings in Ethiopia –39. The H. pylori testing of esophageal and gastroesophageal junction tumor tissue specimens as opposed to gastric tissue. Detection of H. pylori was rather common in the Ethiopian biopsy specimens: 55% (95% CI: 42–68%). H. pylori is commonly found in the superficial mucosal layer of the stomach [H. pylori, perhaps as a superficial contaminant of the esophageal biopsies originating from the stomach. H. pylori infection has been commonly detected in patients undergoing endoscopy for gastrointestinal problems in Ethiopia and in other African countries, and also in symptomatic patients screened using stool specimens [This is one of the very few studies that documents  95% CI: 2–68%. H.pecimens –44.H. pylori-positive specimens tested positive (59%) for the cagA gene. The presence of this gene is considered to be associated with H. pylori virulence and contributes to neoplastic transformation in the gastric mucosa [cagA gene in EC and gastroesophageal junction cancer biopsies in the East Africa EC high-risk corridor.The IARC in-house CagA-based PCR assay determined that more than half of the c mucosa , 45, 46;H. pylori in EC and gastroesophageal junction cancer cases may be partly explained by contaminants from the stomach, thus representing a challenge in the investigation of the role of H. pylori in EC and a potential weakness of the present study. Further studies are required to determine whether H. pylori plays a direct role in esophageal carcinogenesis.The findings of our study, and their interpretation, are based on a small number of cancer patients with reduced sampling of the malignant tissue seen during endoscopy; these study characteristics may have affected the reported results. Also, the presence of H. pylori detected in EC biopsies was much higher (55%), with a high proportion containing the pro-inflammatory gene cagA.The prevalence of mucosal-type HPV was very low in EC biopsies collected in cancer cases enrolled in a study in Addis Ababa. Single infection with HR HPV16 was detected in the tissue specimen of 1 case (2%), and 7 HR HPV types were identified in the oral cells of 7 cases (11%), including multiple types detected in 2 cases. The prevalence of HPV in oral cells was similar in cases and controls, with considerable overlap of types. In contrast, the prevalence of


What is the prognostic significance of esophageal tumor human papillomavirus (HPV) status?In this case-control study involving 142 patients with Barrett high-grade dysplasia and esophageal adenocarcinoma, HPV positivity was associated with a significantly improved disease-free survival compared with viral negativity. Recurrence and progression were reduced in the HPV-positive cohort as were distant metastasis and death from esophageal adenocarcinoma.Barrett high-grade dysplasia and esophageal adenocarcinoma in patients who are HPV positive have a favorable prognosis compared with viral-negative esophageal tumors and may benefit from treatment de-escalation. This case-control study compares disease-free and overall survival among patients with Barrett high-grade dysplasia or esophageal adenocarcinoma with vs without underlying human papillomavirus (HPV) infection demonstrated on biopsy. High-risk human papillomavirus (HPV) has been associated with Barrett dysplasia and esophageal adenocarcinoma. Nevertheless, the prognostic significance of esophageal tumor HPV status is unknown.To determine the association between HPV infection and related biomarkers in high-grade dysplasia or esophageal adenocarcinoma and survival.INK4A and p53. Sequencing of TP53 was also undertaken. The study took place at secondary and tertiary referral centers, with 151 patients assessed for eligibility and 9 excluded. The study period was from December 1, 2002, to November 28, 2017.Retrospective case-control study. The hypothesis was that HPV-associated esophageal tumors would show a favorable prognosis . Pretreatment biopsies were used for HPV DNA determination via polymerase chain reaction, in situ hybridization for E6 and E7 messenger RNA (mRNA), and immunohistochemistry for the proteins p16Disease-free survival (DFS) and overall survival (OS).INK4A expression, low p53 expression, and wild-type TP53. There were more Tis, T1, and T2 tumors in HPV-positive patients compared with HPV-negative patients . Mean DFS was superior in the HPV-positive group  as was OS . Recurrence or progression was reduced in the HPV-positive cohort  as was distant metastasis  and death from esophageal adenocarcinoma . Positive results for HPV and transcriptionally active virus were both associated with a superior DFS . Positivity for E6 and E7 mRNA, high p16INK4A expression, and low p53 expression were not associated with improved DFS. On multivariate analysis, superior DFS was demonstrated for HPV , biologically active virus , E6 and E7 mRNA , and high p16 expression .Among 142 patients with high-grade dysplasia or esophageal adenocarcinoma , 37 were HPV positive and 105 were HPV negative. Patients who were HPV positive mostly had high p16Barrett high-grade dysplasia and esophageal adenocarcinoma in patients who are positive for HPV are distinct biological entities with a favorable prognosis compared with viral-negative esophageal tumors. Confirmation of these findings in larger cohorts with more advanced disease could present an opportunity for treatment de-escalation in the hope of reducing toxic effects without deleteriously affecting survival. Intriguingly, these high rates of EAC have occurred against a backdrop of progressive reduction in the risk estimate of malignancy associated with BE.5 In this regard, our discovery of a strong association of transcriptionally active high-risk human papillomavirus (HPV) with a subset of Barrett dysplasia (BD) and EAC5 may be relevant.Esophageal adenocarcinoma (EAC) is one of the fastest-growing and deadliest cancers in the Western world,6 Moreover, treatment failure after endoscopic ablation of BD or EAC is predicted by persistent high-risk HPV infection and overexpression of the p53 gene (TP53).7 We also discovered that HPV-positive EAC is molecularly distinct from HPV-negative EAC, indicating different biological mechanisms of tumor genesis.8 Hybrid sequences containing HPV-16 and the human genome have been identified, indicating a host-viral interaction.8 Aberrations of the retinoblastoma protein (pRb) pathway, ie, upregulation of p16INK4A and downregulation of pRb, as well as wild-type TP53 are hallmarks of active HPV involvement in BD and EAC. Additionally, the transformation-specific combination of high p16 expression and low pRb expression, which is a feature of HPV-driven lesions, has been identified in a significant proportion of virally positive BD and EAC.9Increasing high-risk HPV viral load and integration status has been linked with more severe disease along the Barrett metaplasia-dysplasia-adenocarcinoma sequence.14 Interestingly, a systematic review has reported HPV prevalence rates of 35% in 174 patients with EAC, similar to our findings.15 Another systematic review that included 19 studies concluded that the pooled prevalence of HPV in EAC was 13%, suggesting the low prevalence rate may have been caused by small sample sizes and compromised detection methods.16Ability to detect HPV in earlier negative studies and more recent investigations may have been hampered by poor tissue classification and suboptimal testing methods. This is further exacerbated by the low HPV viral load in esophageal tissue. Some of these misclassified negative studies only used nondysplastic BE. Metaplastic tissue is not associated with HPV. Racial or geographic variations can also account for this anomaly.17 A meta-analysis reported a 74% improved disease-free survival (DFS) and 53% better OS in HPV-associated HNSCC vs HPV-negative HNSCC.18 We therefore hypothesized that HPV-associated EAC and high-grade dysplasia (HGD) would show a similar favorable prognosis compared with HPV-negative esophageal lesions.It is well documented that patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) have an improved rate of overall survival (OS)  and a reduced risk of recurrence  compared with viral-negative tumors.STROBE) reporting guideline. Eligible patients were those with HGD or EAC (Siewert classification type I or II) deemed suitable for treatment, ie, for endotherapy  or esophagectomy with or without neoadjuvant chemotherapy and/or radiotherapy. The study period was from December 1, 2002, to November 28, 2017, and patients were enrolled from a tertiary referral center, Bankstown-Lidcombe Hospital, Sydney, Australia (n = 139), and a regional health care center, Launceston General Hospital, Launceston, Tasmania, Australia (n = 3). Demographic and clinical data were obtained from a prospectively maintained database. Inclusion and exclusion criteria have been previously documented.9 Pretreatment tissue was prospectively collected in 94 patients  and retrospectively retrieved (FFPE only) from the remaining 48 patients. Oral and written informed consent were obtained from participants prior to the investigation. This study was approved by the Human Research Ethics Committee, Tasmania, and South Western Sydney Local Health Network.This retrospective case-control study followed the Strengthening the Reporting of Observational Studies in Epidemiology  were excluded from endotherapy and underwent esophagectomy with D2 lymph node dissection. Siewert type I tumors were subjected to Ivor Lewis esophagogastrectomy and Siewert type II cancers to either radical total gastrectomy including lower esophagus or Ivor Lewis esophagectomy depending on the patient’s build.Cancer Staging Manual by the American Joint Committee on Cancer.19 Patients with locally advanced disease (T3 or T4 N0 or any T stage with N1) were potential candidates for neoadjuvant or adjuvant treatment. After treatment, repeat staging was done to exclude metastatic disease prior to proceeding to esophagectomy. As such, 14 patients did not undergo esophagectomy due to tumor-related factors or patient refusal. Nevertheless, they were included in the analysis as they had undergone radiotherapy and/or chemotherapy.Staging was performed according to the seventh edition of the + and GP6+ primers for both high-risk and low-risk HPV as previously published.5 To minimize contamination, separate rooms were used for reaction preparation, template handling, performing nested reactions, and post-PCR analysis. Routine decontamination by UV irradiation was performed in the DNA-free PCR hood before each run. To guard against systematic contamination of PCR reagent, appropriate positive  and negative (deionized water and PCR master mix without template) controls were included in each step of the PCR process. Genotypes of HPV were determined by sequencing.5 Real-time PCR assays measuring HPV E6 and E7 copy numbers were performed to determine viral load using genotype-specific HPV-16 and HPV-18 primers.6Detection of HPV in genomic DNA extracted from fresh frozen or formalin-fixed biopsy tissue was performed by nested polymerase chain reaction (PCR) amplification of a conserved viral L1 gene using MY09 and MY11 and GP59 Negative and positive controls were probes recognizing the bacterial gene dapB and the endogenous ubiquitin C mRNA, respectively. In addition, cervical cancer tissue, HNSCC samples, BE and BD, EAC, and esophageal squamous cell carcinoma (ESCC), which all had detectable transcriptionally active HPV , served as positive controls. We used HNSCC, BE and BD, EAC, and ESCC devoid of virus as negative controls. Positivity was defined as the presence of punctuate cytoplasmic and/or nuclear staining that exceeded the dapB (negative control) signal. Expression of the p16INK4A and p53 proteins was assessed by immunohistochemistry (IHC) on FFPE tissue using EnVision FLEX Mini Kits and CINtec Histology Kits  (mtm laboratories), respectively, using appropriate positive and negative controls.9 All IHC scoring of slides was independently performed by 2 experienced gastrointestinal pathologists blinded to the virological status and the clinical outcome of patients. For p16INK4A, at least moderate staining of both nucleus and cytoplasm in more than 25% of esophageal dysplastic or tumor tissue was considered as p16 overexpression. Staining of 25% or less was deemed low expression. Using this criterion, we have demonstrated that high p16 expression is associated with HPV-associated BD and EAC with reasonable sensitivity and specificity as others have in HNSCC.20 In the case of p53, intense nuclear staining of more than 10% of esophageal cells was considered overexpression.9 Mutations of TP53 were confirmed by sequencing of the gene using the semiconductor-based Ion Torrent sequencing platform (ThermoFisher) according to the manufacturer’s instructions as previously published.9In situ hybridization (ISH) of RNA for high-risk HPV-16 and HPV-18 E6 and E7 messenger RNA (mRNA) was performed manually using the RNAscope 2.5 High-Definition Assay (Advanced Cell Diagnostics Inc) with a cocktail of probes targeting 18 high-risk HPV types  according to the manufacturer’s instructions.The primary end points were DFS from the time of diagnosis to the date of first local, regional, or distant failure and OS, defined as time between diagnosis to the date of death or last follow-up.t test for comparing the mean values between the 2 groups in regard to all numerical data. A χ2 analysis was used for evaluating the association between the binary measurements in the viral-positive and viral-negative groups. Survival analysis was conducted using the Kaplan-Meier method to estimate the DFS and OS of the 5 HPV variables, ie, viral DNA status, transcriptionally active HPV, E6 and E7 mRNA, p16, and p53. The log-rank test was used to analyze the association between the HPV variables with DFS and OS. Cox proportional hazards regression models were used to estimate the importance of these biomarkers for DFS and OS after adjusting for age, sex, body mass index , history of smoking, excess alcohol use, medications , T stage, N stage, surgical or endoscopic mucosal resection margin status, degree of tumor differentiation, and neoadjuvant or adjuvant treatment (model 2). Because viral status itself was associated with disease severity, adjusting for stage and treatment (chemotherapy and/or radiotherapy) may have prevented identification of prognostic effects derived exclusively from the 5 HPV variables. Therefore, additional Cox regression models were applied with all of the above covariates but excluding T and N stages as well as chemotherapy and radiotherapy (model 3). All statistical tests were performed using SAS statistical software version 9.4 (SAS Institute Inc), and the level of significance was set at .05 (2-sided).Differences between HPV-positive and HPV-negative cases in regard to baseline characteristics were assessed using the 2-sample INK4A IHC, and E6 and E7 mRNA ISH  (97.1%) had detectable viral load. Among the 37 DNA-positive lesions, 18 (48.7%) had E6 and E7 mRNA detection by ISH and 19 (51.4%) overexpressed p16INK4A. Eleven samples (29.7%) were positive for all 3 markers.Polymerase chain reaction analysis of HPV DNA was performed on all 142 patients with esophageal lesions (38 HGD and 104 EAC). Thirty-seven patients (11 with HGD and 26 with EAC) had positive results for HPV DNA; 33 had HPV-16, 1 had HPV-18, and the remaining 3 had low-risk types 6 and 11. No multiple genotypes were detected in the same patient. Amplifiable β-globin gene was present in all specimens, and median (range) viral load was 0.1 copy per 10 cell genomic DNA (0-1.12 copies per 10 cell genome). All specimens with measurable viral load revealed coherence between genotypes found on MY09 and MY11 and GP5INK4A and E6 and E7 mRNA presence were significantly greater in the HPV-positive group compared with the viral-negative cohort. Of note, HPV-positive patients had more early-stage  esophageal lesions compared with viral-negative patients , but nodal stage was similar between the groups. Most were stage N0 or N1 in both the HPV-positive (89.2%) and viral-negative (89.5%) patients with HGD and EAC. No significant differences were detected in any of the other clinical or pathological baseline characteristics. In the transcriptionally active HPV-positive group , the patients were significantly younger than the biologically inactive virus group (HPV positive without p16INK4A and/or E6 and E7 mRNA)  . Similarly, mean (SD) OS was 43.7 (32.9) months in the viral-positive group compared with 29.8 (25.3) months in the HPV-negative patients . As expected, recurrence or progression was much reduced in the HPV-positive cohort compared with the HPV-negative cohort . Recurrence per se was almost a third less in the HPV-positive patients in comparison with HPV-negative patients . There appeared to be better  local and regional control for the HPV-positive patients compared with viral-negative individuals . Significantly, reduced distant metastasis was observed in the HPV-positive group  as were deaths from EAC .P = .002) and 56% , respectively , biologically active virus , E6 and E7 mRNA , and high p16 expression   , presence of transcriptionally active virus , and E6 and E7 mRNA detection   had wild type. In the 73 specimens with TP53 mutations, 50 (68.5%) were missense mutations present in the DNA binding domain , 2 (2.7%) were missense mutations in the oligomerization domain (exons 9-10), and 2 contained frameshift mutations in exons 5 and 8 that induced a loss of both DNA binding and oligomerization domains. In 35 of 37 HPV-positive patients with HGD and EAC with successful sequencing, TP53 mutations were detected in only 9 patients (25.7%) compared with 64 of 97 (66.0%) in the viral-negative group .Of 142 HGD and EAC specimens, 132 (93.0%) were successfully sequenced, and in 104 of 132 (78.8%) p53 IHC and sequencing data matched. Seventy-three (55.3%) harbored mutated INK4A (surrogate marker) are associated with improved DFS in patients with HGD and EAC. It was mainly due to a reduction in distant metastasis and possibly better local and regional control in HPV-positive patients compared with HPV-negative patients. This resulted in a lower mortality from EAC. Mean duration of OS was again significantly improved in the HPV-positive group compared with the HPV-negative group. Nevertheless, the association of HPV status with OS failed to reach significance by the log-rank test. Although 26 of 37 HPV-positive individuals (70.3%) were alive at the end of the follow-up period compared with 58 of 105 HPV-negative individuals (55.2%), this was not statistically significant, possibly because of the modest sample size and associated comorbidities. These findings are somewhat similar to the data in head and neck cancers. Human papillomavirus–positive HNSCCs have improved survival and lower rate of local and regional recurrence compared with HPV-negative head and neck cancers. No significant differences were detected in distant metastases, possibly because the studies were insufficiently powered.23 In ESCC, it has been reported that patients with p16-positive cancers had superior 5-year OS and PFS rates compared with patients with p16-negative cancers.24 Similarly, Kumar and colleagues25 found that ESCC patients with p16-positive tumors subjected to neoadjuvant chemotherapy had better complete remission rates than the p16-negative group. Conversely, in a recent publication, HPV, p16, and p53 were not found to be prognostic factors in ESCC.26This is the first study, to our knowledge, to show improved survival associated with HPV-positive HGD and EAC. Esophageal lesional HPV status and associated viral transcriptional markers, ie, E6 and E7 mRNA (gold standard) and p168 Moreover, TP53 aberrations are less frequent in HPV-positive BD and EAC compared with viral-negative esophageal lesions.9 Prior studies from our group have also demonstrated that HPV-positive BD and EAC are mostly wild-type TP53.9 In this study, we similarly found that only a quarter of HPV-positive HGD and EAC lesions harbored TP53 mutations vs two-thirds of HPV-negative HGD and EAC having the same molecular aberration. Although patients with EAC who have p53 mutations have been shown to have reduced OS in a recent meta-analysis, we could not confirm the same in our study.27 Possible reasons include the small sample size and other nonviral interactions involving p53 function, including smoking (which in this study was high in both HPV-positive and HPV-negative patients) and alcohol use.28 Not surprisingly, comorbidity, especially smoking related, also influences survival negatively.29 Response to chemotherapy in the presence of other antiapoptotic proteins not analyzed in this study  could be another reason for this discrepancy.32The results of this study are consistent with our previous work that demonstrated HPV-positive and HPV-negative EAC are distinct diseases just as in HNSCC. A previous study found that HPV-positive EAC harbored approximately 50% fewer nonsilent somatic mutations than HPV-negative EAC.8 Interestingly, three-quarters of HPV-positive esophageal lesions were early T stage  compared with slightly more than half of the HPV-negative group. Nevertheless, the vast majority of both cohorts consisted of nodal stage N0 or N1 , which is probably indicative of patient selection for endotherapy or esophagectomy with curative intent. Our results concur somewhat with data from HNSCC, another malignant neoplasm in which a subset are HPV driven. Patients with HPV-positive HNSCC are generally 10 years younger33 and have more early T-stage but advanced N-stage disease, although they respond better to treatment and have a better outcome than patients with HPV-negative HNSCC.35Patients whose HGD and EAC harbored transcriptionally active HPV (n = 30) were significantly younger  compared with patients with esophageal lesions devoid of biologically active virus (n = 112) . This is in keeping with findings from our previous small study on exome sequencing of HPV-positive and HPV-negative patients.36 Central reporting of biopsy specimens by experienced gastrointestinal pathologists was an added strength.The determination of HPV presence in BD or EAC can be difficult using FFPE specimens. While PCR is more sensitive than ISH for HPV DNA detection, it risks false-positivity. Thus, in addition to PCR for viral DNA, we also included E6 and E7 mRNA transcript analysis by ISH, which is a reliable marker of HPV involvement, and IHC for p16 overexpression, a surrogate marker for HPV infection. Sequencing and IHC for p53 were also undertaken given their importance in BD and EAC progression.This investigation was retrospective in nature and the study sample was small. The case-control nature of the study introduces biases pertaining to selection, information, and observation as well as confounding. As HPV status was not known at the time of enrollment and treatment decision, it minimized both selection and observer bias. Measurement bias was addressed by blinding the scientist and pathologists to the clinical and virological status of the patients and to treatment outcome. Confounding was mitigated with adjustment for potential confounders in the multivariate statistical analysis. The independence of prognostic effects of the 5 dichotomous variables, HPV DNA positivity, transcriptionally active HPV, E6 and E7 mRNA detection, and p16 and p53 overexpression, is important. Nevertheless, we could not include these variables in a single multivariate model as they were intercorrelated. Thus, we were unable to identify the independent effects of these factors. Some of the specimens analyzed were more than 10 years old, which increases the risk of DNA and RNA invalidity. Moreover, IHC analysis is subjective and lacks uniform scoring systems, which can hamper reproducibility between studies.If these findings of a favorable prognosis of HPV-positive HGD and EAC are confirmed in larger cohorts with more advanced disease, it presents an opportunity for treatment de-escalation in the hope of reducing toxic effects without deleteriously affecting survival.


CIITA and RFX5—regulators of MHC-II—were significantly upregulated in HPV+ tumors. This coordinated upregulation of MHC-II genes was correlated with higher intratumoral levels of interferon-gamma in HPV+ carcinomas. Furthermore, genes that encode various co-stimulatory molecules involved in T-cell activation and survival were also significantly upregulated in HPV+ tumors. Collectively, these results suggest a previously unappreciated role for epithelial cells in antigen presentation that functionally contributes to the highly immunogenic tumor microenvironment observed in HPV+ HNSCC.High-risk human papillomaviruses (HPVs) are responsible for a subset of head and neck squamous cell carcinomas (HNSCC). Expression of class II major histocompatibility complex (MHC-II) is associated with antigen presenting cells (APCs). During inflammation, epithelial cells can be induced to express MHC-II and function as accessory APCs. Utilizing RNA-seq data from over 500 HNSCC patients from The Cancer Genome Atlas, we determined the impact of HPV-status on the expression of MHC-II genes and related genes involved in their regulation, antigen presentation, and T-cell co-stimulation. Expression of virtually all MHC-II genes was significantly upregulated in HPV+ carcinomas compared to HPV− or normal control tissue. Similarly, genes that encode products involved in antigen presentation were also significantly upregulated in the HPV+ cohort. In addition, the expression of  High-risk human papillomaviruses (HPVs) are small, non-enveloped, double-stranded DNA viruses that are responsible for an estimated 5% of all human cancers ,2. TheseCD39 and multiple T-cell exhaustion markers including LAG3, PD1, TIGIT, and TIM3 compared to HPV− HNSCC. Importantly, patients with higher expression of these exhaustion markers—indicative of a T-cell-inflamed tumor—exhibited markedly improved survival in HPV+, but not HPV−, HNSCC [We and others have noted significant differences in the immune landscape between the tumor microenvironments of HPV+ and HPV− HNSCC ,17,18,19In order for an effective T-cell-specific anti-tumor response to occur, a tumor associated antigen must be presented in either the context of MHC-I or class II MHC (MHC-II) . This prAlthough the ability of HPV to suppress MHC-I expression in cell culture systems is well known ,26, thisclass II major histocompatibility complex transactivator (CIITA) and regulatory factor X5 (RFX5)—essential master regulators of the MHC-II transcriptional control system—were significantly upregulated in the HPV+ cohort. This coordinated upregulation of the mRNA levels of genes involved in the MHC-II antigen presentation pathway and their regulation were correlated with the higher intratumoral levels of IFNγ observed in HPV+ carcinomas. In addition, genes that encode various T-cell co-stimulatory molecules involved in T-cell activation and survival were found to be significantly upregulated in HPV+ tumors compared to HPV− tumors and normal control tissue. Taken together, these results suggest a previously unappreciated role for epithelial cells in antigen presentation that functionally contributes to the highly immunogenic tumor microenvironment observed in HPV+ HNSCC. This further illustrates the profound differences in the immune landscape between the tumor microenvironments of HPV+ and HPV− HNSCC and may in part contribute to the superior clinical outcomes that are associated with HPV+ HNSCC.In this study, we used RNA-seq data from over 500 human head and neck tumors from The Cancer Genome Atlas (TCGA) to determine if the presence of oncogenic HPV is associated with altered expression of all classical MHC-II genes and other key genes involved in their regulation, antigen presentation, and T-cell co-stimulation. We found that expression of virtually all classical MHC-II genes was significantly upregulated in HPV+ tumors compared to their HPV− counterparts or normal control tissue. Similarly, genes that encode products that are fundamental to proper antigen loading and presentation were also significantly upregulated in HPV+ tumors. Importantly, the relative level of expression of these inducible MHC-II genes was far beyond those genes associated with professional APCs. Furthermore, the expression of HLA-DPA1, -DPB1, -DQA1, -DQA2, -DQB1, -DQB2, -DRA, -DRB1, -DRB5, and -DRB6 genes, encoding the various α- and β-chains for all three classical isotypes . As the majority of the HPV+ samples are from the oropharynx subsite, we repeated this analysis with HPV+ and HPV− samples that occur only in the oropharynx. Similarly, to the analysis including all subsites, HPV+ oropharyngeal tumors expressed significantly higher levels of MHC-II α- and β-chain genes compared to their HPV− oropharyngeal tumor counterparts (CD19 (B-cells) [CCL13 (DCs) [CD84 (macrophages) [CDH1) [Constitutive expression of classical MHC-II molecules is typically restricted to professional antigen presenting cells—DCs, macrophages, and B-cells . Howeverisotypes . Uniformterparts . CollectB-cells) , CCL13 (13 (DCs) , and CD8ophages)  (Supplem) [CDH1)  (SupplemHLA-DR antigens-associated invariant chain or Cluster of Differentiation 74 (CD74) gene [CD74 was found to be significantly upregulated in HPV+ HNSCC compared to their HPV− counterparts or normal control tissues (In the endoplasmic reticulum (ER), newly synthesized MHC-II α- and β-chains form a trimeric complex with a non-polymorphic protein called the invariant chain (Ii), which is encoded by the 74) gene . This as74) gene ,42. Once tissues .HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB genes. Like the classical MHC-II genes, all four genes encoding the class II-like MHC molecules are similarly upregulated in HPV+ HNSCC versus HPV− tumors or normal control tissues   . As expeic genes . However tissues . In addi tissues .To further illustrate the IFNγ-specific coordinated upregulation of MHC-II genes and related genes that encode products essential for antigen processing and presentation, we generated a correlation matrix for both HPV+ .CIITA, RFX5, and subsequent expression of all the classical MHC-II genes and related genes required for antigen loading and presentation observed in HPV+ head and neck carcinomas are likely a consequence of IFNγ exposure. Furthermore, the correlation matrix illustrates the unique simultaneous coordination of the MHC-II transcriptional control system that has been shown to be dictated by the master transcriptional regulator CIITA [Thus, the upregulated expression of or CIITA ,47. CD28 in HPV+ tumors compared to HPV− or normal control tissues  was significantly upregulated in HPV+ tumors compared to HPV− or normal control tissues  was found to be significantly downregulated in HPV+ tumors compared to HPV− and not significantly different compared to normal control tissues (ICOS) and its ligand ICOSLG and found that both were significantly upregulated in HPV+ tumors compared to HPV−, but only ICOS was significantly upregulated in comparison to normal control tissues  and its ligand OX40L  and found that both genes were significantly upregulated in HPV+ tumors compared to their HPV− counterparts and normal control tissues . The normalized, gene level Firehose dataset was utilized for all the genes analyzed. RSEM-normalized RNA-Seq data was extracted into Microsoft Excel and the HPV-status was determined based on published datasets [Level 3 RNA-Seq by Expectation Maximization (RSEM)-normalized Illumina HiSeq RNA expression data for the TCGA head and neck cancer (HNSC) cohort was downloaded from the Broad Genome Data Analysis Centers Firehose server  utilizing packages ggplot2 and reshape2. Correlation matrix figure was assembled into final form using Adobe Illustrator.Level 3 RSEM normalized RNA-seq data for the genes listed above were extracted from the TCGA database and processed as detailed in Materials and Methods 4.1. For the HPV+ (upper triangle) and HPV− samples (lower triangle), pairwise spearman correlation was performed for each gene involved in the MHC-II transcriptional control system. Hierarchical clustering was utilized to group genes based on strength of correlation. Correlations and clustering were performed using R statistical environment (version 3.4.0; HPV+ HNSCC tumors are remarkably different from their HPV− counterparts in that they express high levels of all components of the MHC-II antigen presentation apparatus. Importantly, while some of this signal can be attributed to professional APCs, the extremely high relative level of expression supports a model whereby the T-cell inflamed environment in HPV+ HNSCC induces a functionally effective MHC-II presentation system based on tumor epithelial cells. As MHC-II-dependent antigen presentation is critical for CD4+ help in CD8+ T-cell responses, which are essential for the control and clearance of cancerous cells, it is likely that the expression of non-self-derived viral antigens or tumor derived neoantigens, combined with intact MHC-II presentation and appropriate co-stimulation contributes to the markedly better patient outcomes for HPV+ versus HPV− head and neck carcinomas. As immune checkpoint inhibition therapy in other cancers has been reported to be most effective for tumors with high MHC-II levels, suggesting that stratification based on MHC-II levels may help predict those likely to respond to checkpoint inhibition therapy.


However, among nonalcohol drinkers, there was no association between smoking or secondhand smoking status and these risks. These findings suggest that women exposed to secondhand smoking at home or in the workplace might be at high risk of HR-HPV persistence when it is combined with alcohol drinking, even though neither active smoking nor secondhand smoking independently affects the risk.Tobacco smoking is established as a cofactor of human papillomavirus (HPV) for cervical cancer risk. However, the role of secondhand smoking in cervical carcinogenesis is controversial. We aimed to assess the association between secondhand smoking and high risk- (HR-) HPV persistence, a pivotal event in development of cervical cancer. In total, 9,846 women who underwent health-screening examinations from 2002 to 2011 at the National Cancer Center, Korea, were included. Secondhand smoking was defined as being exposed to secondhand smoke at home or in the workplace. Multivariate logistic regression analysis was used to estimate the odds ratios (OR) and 95% confidence intervals (CIs) for risks of HR-HPV infection at baseline , 1-year persistence , and 2-year persistence . Active smoking, secondhand smoking, and secondhand smoking in nonactive smokers had no association with these risks. Among alcohol drinkers, secondhand smoking in nonactive smokers had higher risks of HR-HPV infection at baseline (OR = 1.25, 95% CI = 1.05–1.48, Several studies, including a large meta-analysis and several cohort studies, have shown an association between cigarette smoking and cervical cancer or its precursor lesions –3. CigarIn the Republic of Korea, cigarette smoking among women is rare 7% in 2009), but the proportion of men aged ≥ 15 years who smoke is the second highest among the Organization for Economic Cooperation and Development (OECD) countries . Similar% in 2009Although most studies have investigated cervical cancer precursors, we have focused on the initiation of cervical carcinogenesis  in order to aid efforts at primary prevention of cervical cancer. In the present study, we aimed to assess the associations between smoking status  and the risks of HR-HPV infection at baseline, 1-year HR-HPV persistence, and 2-year HR-HPV persistence as well as the same associations among alcohol drinkers.This study was a part of the Korean Prospective Study for the Transition of Human Papillomavirus into Cervical Carcinoma , 15. AmoThis study received approval from the Institutional Review Board of the National Cancer Center, Korea (NCCNCS-11-433), and all of the study participants provided informed consent.supplement). All of the participants were asked if they had ever smoked a cigarette. Nonsmokers were defined as having smoked <100 cigarettes in their lifetimes . Smokers were defined as having smoked ≥100 cigarettes in their lifetimes (n = 825). Regarding SHS, all of the participants were asked, “Do you live with anyone in your household who regularly smokes cigarettes or other tobacco products (other than yourself)?” and “Do you work with anyone in your workplace who regularly smokes cigarettes or other tobacco products (other than yourself)?” (1) Exposure to SHS at home = No/Yes, how many family persons who smoked regularly, time of exposure to SHS; (2) exposure to SHS in the workplace = No/Yes, time of exposure to SHS. We did not account for exposure to SHS in public places. Secondhand smoking was defined as being exposed to SHS in either the home or the workplace, regardless of active smoking status, while non-SHS was defined as not being exposed to SHS at home or in the workplace. In addition, SHS in nonsmokers was defined as having never smoked in life but having been exposed to SHS.Information on smoking and secondhand smoking behaviors was collected using a standardized questionnaire . A chemiluminescent HPV DNA test was used to measure relative light units (RLU) with a probe designed for 13 types of HR-HPV. The test results were read as positive at concentrations of 1 pg/ml or greater than the RLU/cutoff ratio (RLU of specimen/mean RLU of two positive controls). The cytological grades for Pap smear reports were based on the Bethesda classification system . t-test were used to assess the differences in the distributions of categorical and continuous variables between groups, respectively. Multivariate logistic regression models were used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). A multivariate logistic regression analysis was run to assess the associations between smoking status and risk of HR-HPV infection at baseline, 1-year persistence, and 2-year persistence after adjustment for confounding factors. We checked for confounding by calculating the Mantel-Haenszel summary measure for the effect of alcohol drinking status. After separating data for strata defined for alcohol drinkers and nonalcohol drinkers, a multivariate logistic regression analysis was again performed to assess the association between smoking status and risk. All of the analyses were adjusted for age and BMI continuously and for marital status, parity, menopausal status, oral contraceptive use, education level, and alcohol drinking status as well. Risk estimates were calculated for nonactive smoking, non-SHS smoking, and non-SHS smoking in nonactive smokers as reference categories. All of the analyses were performed with SAS 9.3  and the episensr packages in R language (version 3.3.2) [Descriptive statistics were used to summarize the overall distribution of characteristics by study group. The chi-square test andn 3.3.2) .2. Most of the subjects were married (97%) and had two or more children (87.3%). Approximately 60% of the subjects were postmenopausal women and 20% had used oral contraceptives (currently or in the past). Approximately one-half of the subjects had consumed alcoholic beverages (currently or in the past), and 8 and 37% were active cigarette smokers and secondhand smokers, respectively. At enrollment, the majority of the subjects (97%) had normal cytological results, but 186 (2.0%) had atypical squamous cells of undetermined significance (ASCUS), 58 (0.6%) had low-grade squamous intraepithelial lesions (LSIL), and 29 (0.3%) had high-grade squamous intraepithelial lesions (HSIL) or more severe abnormalities. There were wide variations in the distributions of age, BMI, menopausal status, education level, alcohol drinking status, and active smoking status. These confounding factors were adjusted in a subsequent multivariate logistic regression analysis.Of the 9,846 women, 980 (9.9%) were infected by HR-HPV at enrollment  and the risks of HR-HPV infection at enrollment, 1-year HR-HPV persistence, and 2-year HR-HPV persistence . Only alp = 0.038, homogeneity of the OR). The subjects were stratified by alcohol drinking status (nondrinkers/current or past drinkers), and the associations between smoking status and the risks of HR-HPV infection and its persistence were assessed among alcohol drinkers and nondrinkers, separately ( p for multiplicative interaction = 0.005), 1-year HR-HPV persistence , and 2-year HR-HPV persistence . Secondhand smoking in nonactive smokers also had higher risks of HR-HPV infection at baseline , 1-year HR-HPV persistence , and 2-year HR-HPV persistence . Among alcohol nondrinkers, active smoking had no association with these risks, but SHS had lower risks of HR-HPV infection at baseline  and 1-year HR-HPV persistence . SHS in nonactive smokers also had lower risks of HR-HPV infection at baseline  and 1-year HR-HPV persistence .Previously, alcohol drinking behaviors and their combination with high HR-HPV viral load were associated with risk of HR-HPV persistence , 15. Theparately . Among a Ours is the first report of an association between SHS and HR-HPV persistence and the combined effect of SHS and alcohol drinking on that risk. Whereas most of the previous studies focused on cervical cancer precursors [Our findings showed that the combination of exposure to SHS and alcohol drinking was associated with higher risks of HR-HPV infection and its persistence in women who had never smoked in their lives but had been exposed to SHS.ecursors , our preTobacco smoking has been established as an important risk factor in cervical carcinogenesis , 18–21 aAs these findings suggest, exposure to SHS might be related to the development of cervical neoplasia in Korea women. A previous study showed that alcohol drinking is associated with a higher risk of HR-HPV persistence in Korean women . It is cThere are plausible mechanisms explaining the impact of SHS on risk of HPV persistence. Tobacco includes carcinogens that could have a direct transformational effect on the cervix, leading to immune suppression and HPV persistence , 30. ActOur study has several limitations. First, although sexual behaviors are critical confounding factors of HPV infection , bias duIn conclusion, alcohol drinkers exposed to SHS at home or in the workplace had a higher risk of persistent HR-HPV infection than did alcohol drinkers not so exposed. This combined effect was observed also in women who had never smoked in their lives. Reducing both exposure to SHS and consumption of alcoholic beverages could be a prudent approach to the prevention of cervical cancer.


The incidence of cervical cancer in Kenya is among the highest in the world. Few Kenyan women are able to access screening, thus fueling the high cervical cancer burden. Self-collected human papilloma Virus (HPV) tests, administered during community-health campaigns in rural areas may be a way to expand access to screening.careHPV Test Kits. Women were notified of results through their choice of short message service (SMS), phone call, home visit or clinic visit. HPV positive women were referred for cryotherapy following a screen-and-treat strategy.In December 2015, we carried out a four-day community health campaign (CHC) to educate participants about cervical cancer prevention and offer self-administered HPV screening. Community enumeration, outreach and mobilization preceded the CHC. Samples were sent to Migori County Hospital for HPV DNA testing using p = 0.046). Just over three-quarters of all women (195/255), were notified of their results. Those who were unable to be reached were more likely to prefer receiving results from clinic  and were less likely to have mobile phones . Although 76% of HPV positive women were notified of their results, just half (51%) of those testing positive presented for treatment. HPV positive women who successfully accessed the treatment facility did not differ from their non-presenting counterparts by demographics, health history, desired route of notification or access to a mobile phone.Door-to-door enumeration identified approximately 870 eligible women in Ngodhe Community in Migori County. Among the 267 women attending the campaign, 255 women enrolled and collected samples: 243 tests were successfully resulted and 12 were indeterminate. Of the 243 resulted tests, 47 (19%) were positive for HPV, with young age being the only significant predictor of positivity. In multivariate analysis, each additional year of age conferred about a 4% decrease in the odds of testing positive (95% CI 0.1 to 7%, Nearly a third of eligible women in Ngodhe Community attended the CHC and were screened for cervical cancer. Nearly all women who attended the CHC underwent cervical cancer screening by self-collected HPV tests. Three-quarters of all participants received results, but just half of HPV positive participants presented for treatment in a timely fashion, suggesting that linkage to treatment remains a major challenge.NCT02124252, Registered 25 April 2014. While cervical cancer is the fourth most-common cancer among women worldwide, there is dramatic disparity in incidence globally . CervicaKenya experiences a high cervical cancer burden. The crude incidence rate is 22.4 per 100,000 women . The lacCytology-based screening is not practical for wide-spread use in sub-Saharan Africa due to its high cost, low sensitivity, inherent need for a laboratories and trained technicians and complex follow-up protocols . TestingThe impact of any successful screening program depends on widespread uptake and linkage to treatment for those who screen positive. Despite the potential for overtreatment, “screen-and-treat” approaches, ideally with HPV testing as the sole screening test or HPV followed by VIA triage, are recommended over approaches involving colposcopy and/or cytologic or histologic confirmation of high-grade dysplasia , 10. WheAn alternative to clinic-based screening is periodic, high-volume community health campaigns (CHCs) offering self-collected HPV testing with referral for treatment when necessary. Taking screening outside of the clinic and utilizing self-collected samples overcomes the need for a pelvic exam, which Kenyan women have described as unacceptable,  and otheWe carried out a community health campaign (CHC) in Ngodhe Community in Migori County to inform a two-phase cluster randomized trial of implementation strategies for cervical cancer prevention. The aim of the present study was to assess the uptake and acceptability of the CHC and subsequent self-collected HPV test, as well as to report the HPV prevalence and proportion of screen-positive participants who successfully obtained treatment.In December 2015, we conducted a four-day community health campaign (CHC) offering education about cervical cancer precursors and prevention, as well as self-administered screening for HPV. This CHC was used to inform a two-phase cluster randomized trial of implementation strategies for cervical cancer prevention in Western Kenya.The CHC took place in the Ngodhe Community of Kanyadeto sub-county, in Migori County. Ngodhe community, with a population of roughly 5590 was selected as the pilot site for the larger study as it is representative of the community size targeted for the cluster-randomized trial. Kenya Ministry of Health community health workers enumerated all women in the community aged 25–65 through door-to-door home visits prior to the campaign.Community engagement activities and stakeholder meetings were carried out for two weeks before the CHC. Community health workers and study staff provided information in public places including markets, churches and women’s groups meetings about cervical cancer screening and described the upcoming campaigns as an opportunity to learn more about and undergo HPV-based testing. Women were also approached about the upcoming campaign through door-to-door outreach. Fliers and posters were also displayed to advertise the campaign. All women in Ngodhe aged 25–65 were invited to participate in the CHC and screening. Participation was voluntary and subjects incurred no cost.Upon arrival at the CHC, women were screened for eligibility. Women aged 25–65 living in Ngodhe with no prior total hysterectomy or history of cervical cancer were eligible to participate. Women outside the age-range or with a history of cervical cancer or total hysterectomy for any indication were excluded. Women then participated in a group education module on cervical cancer, followed by a description of study procedures and participation. Individual informed consent was then obtained and followed by a brief survey collecting demographics, basic health information and reproductive health history. Women were then provided the testing kit, given self-collection instructions with diagrams for clarification and directed to private areas within the tent for the self-collection. In private spaces created for the campaign, participants inserted test kit brush into the vagina and placed the brush tip into a specimen cup before sealing. Sampling instructions and diagrams were also displayed in these private areas.After collection, specimens were given to study staff, who conducted a post-test survey to assess acceptability of self-testing and to obtain phone numbers and preferred route of notification of results. Four notification options were offered: short message service (SMS), phone call, home visit, or collection of results from their nearest health facility. Study staff provided participants with their results via their preferred notification option. SMS notification was considered successful if transmission of text message was confirmed by the Frontline SMS™ program . Phone and home visits were successful if the participant was reached in person, and given their results directly by study staff. Clinic notification was considered successful if the participant returned to her nearest clinic to pick up her results by clinic staff.careHPV Test Kit . The laboratory was staffed by one certified laboratory technician and a research assistant with spent approximately 30% of her time there. The careHPV Test Kit, specifically designed for use in LMICs, is a signal-amplification test for high-risk HPV DNA detection. Antibodies bind to magnetic beads, rapidly capturing specific target HPV nucleic acid sequences, which are then detected using a chemiluminescence signal. This method can qualitatively detect 14 types of high-risk HPV  in cervical or vaginal specimens. More than 80 specimens can be processed in 2.5 h [Specimens were transported to Migori County Hospital for processing and batch analysis with the in 2.5 h . If sampResults were sent to the data team, who supplied them to the research assistants and community health workers, along with the standard follow-up plan. The community health workers then notified participants of their results by their preferred route. The study coordinator oversaw all the results. Follow-up guidelines were provided per Ministry of Health protocol. HIV negative women who tested negative for HPV were instructed to repeat screening in five years. If women were HIV-infected and tested HPV negative, they were told to repeat screening in one year. Women who tested positive for HPV were instructed to attend Migori County Hospital for treatment according to a screen-and-treat model.All women who tested HPV positive were offered treatment at the hospital, with a pre-treatment visual inspection with acetic acid (VIA) to determine mode of treatment. Cryotherapy was the standard treatment for those women with an entirely visible lesion and squamocolumnar (SC) junction, with lesions covering less than 75% of the cervix, no extension to the endocervix or vagina and no evidence of cancer. According to a screen-and-treat strategy, women who had an otherwise satisfactory VIA with no identified lesions were also offered cryotherapy. Cryotherapy was performed by nurses who had specific cervical cancer screening training, including VIA and cryotherapy. Loop Electrosurgical Excision Procedure (LEEP) would have been offered to those with lesions not otherwise amenable to ablative therapy. Should LEEP have been necessary, the medical superintendent  would have provided it. Women with lesions highly suspicious for cancer on visual inspection were offered biopsy and referred to a Provincial Hospital for further management.P values less than 0.05 were considered statistically significant. Factors known or theorized to be associated with HPV infection or those found to be significantly associated with high-risk HPV in the bivariate analysis were considered for multivariate analysis.To test for association between HPV positivity and categorical explanatory variables, we performed chi-squared tests and Fisher’s exact tests. For continuous explanatory predictors, we used two independent-sample t-tests to compare sample means by outcome. Subjects for whom the outcome variable (HPV test result) was missing (test result was “indeterminate”) were compared to the other participants to evaluate potential bias. We used logistic regression to explore the association of demographic and reproductive health variables with this outcome. We also considered two other outcomes: successful notification of results  and successful presentation for treatment (among HPV positive participants). We tested these two outcomes for association with demographic and health history variables. Association with categorical explanatory variables was explored with chi-squared tests and Fisher’s exact tests. For continuous explanatory predictors, we used two independent-sample t tests to compare sample means by outcome. We also used logistic regression to look at unadjusted odds ratios of successful presentation for treatment by aforementioned predictors. All data were analyzed using Stata version 14.2 .During the door-to-door enumeration, community health workers identified approximately 870 women aged 25–65 in Ngodhe Community. During the four-day campaign, 267 31%) attended the health campaign education component. Of these, 255 consented to participate in the survey and obtain a vaginal swab for high-risk HPV testing. All 255 participants collected a sample, which was sent to the lab for processing: 243 tests were successfully resulted and 12 tests were read as “indeterminate.” The 12 women with indeterminate tests were compared to the 243 women with HPV tests yielding a result. The women with indeterminate samples were not different from the women with sufficient samples in terms of age, history of prior screening, HIV positivity, use of family planning, and/or current pregnancy. The 243 participants with valid HPV results were included in this analysis . Nearly one in five (19%) women self-reported prior cervical cancer screening: of those 63% had been screened using VIA. About one-quarter (24%) of participants self-reported as HIV positive. Family planning was used by 44% of the respondents, with the most common method being implants. Five percent of the participants were currently pregnant.Forty-seven 19%) women had positive HPV tests. In unadjusted analysis, none of the previously described variables  differed by HPV result (Table 9% women p = 0.034). No other predictor variable  was independently associated with HPV-positivity , each additional year of age conferred about a 4% decrease in the odds of testing positive  felt they had adequate privacy. Despite 16% of participants reporting that the test caused some pain, nearly all (98%) said they would test again via self-collection, and 99% would recommend the procedure to a friend.p < .001). Otherwise, women reached with results were not different from those unreachable in terms of age, HIV status, history of prior cervical cancer screening, use of a family planning method, current pregnancy or HPV test results , incorrectly not offered treatment for lack of visible lesion (N = 1), and two were found to be ineligible for the study secondary to reported-age (N = 1) or a history of a hysterectomy (N = 1). All the HPV positive women treated were appropriate candidates for ablation and underwent cryotherapy.Twenty-four (51%) of the 47 HPV-positive women sought treatment within 4 weeks of receiving their results, with the median time interval between notification of results and presentation for treatment being 7 days (interquartile range: 4–15). Twenty (43%) were able to successfully obtain treatment on this first visit. Those who did not complete treatment were either pregnant  and collecting results at a nearby clinic was associated with lower odds of accessing the treatment facility compared to receiving results by text message (the most commonly preferred route). Moreover, almost all of the 60 women who were not even reached to deliver test results (90%), had elected to pick up their results in a nearby clinic. These women were also significantly less likely to have access to mobile phones. These trends suggest that more resource- and personnel-intensive home visits, maybe be a better platform for delivering results in this screen-and-treat model.careHPV test kits are theoretically possible, but samples are usually batched, allowing for quick, but not point-of-care immediate results. Running the test assay would not have been feasible at our community-based screening sites. Thus, improving linkage to treatment is the only way to improve adherence in a two-step screen-and-treat model. Minimizing the time from test to receipt of results will be an important consideration as this model is scaled up. Home visits by Community Health Workers have been shown to improve adherence to scheduled follow-up visits in cytology-based cervical cancer screening programs in South Africa [Decreasing loss to follow-up can be accomplished through fewer visits or improved linkage to treatment. One-day screen-and-treat visits using the h Africa , 38. HomcareHPV support staff and the Migori-based laboratory. Investigators are also striving to minimize delays in results and notification in preparation for the planned two-phase cluster randomized trial of implementation strategies for cervical cancer prevention in Western Kenya.A limitation of this study was our sample size. While the reported prevalence of HPV (19%) is similar to that reported by other studies from the region, , 36, 39 This study evaluating a CHC as the planning step in a cervical cancer screen-and-treat strategy shows that high-volume, short-duration campaigns can be a successful way to increase screening and to successfully deliver results to participants. However, observed follow-up rates in this study of just 51% suggest the need for improved linkage to treatment. Extrapolating from data from Uganda, loss to follow-up at this level likely indicates that HPV testing is still probably more effective than VIA in decreasing lifetime risk of cancer, but at a much higher cost than VIA. Decreasing loss to follow-up could bridge the cost disparity. Given trends in our data and findings from other studies, , 38 home