The aim of this study was to visualize the tumor propagation and surrounding mucosal field in radiography-based 3D model for advanced stage HNSCC and combine it with HPV genotyping and miRNA expression characterization of the visualized area. 25 patients with T1-3 clinical stage HNSCC were enrolled in mapping biopsy sampling. Biopsy samples were evaluated for HPV positivity and miR-21-5p, miR-143, miR-155, miR-221-5p expression in Digital Droplet PCR system. Significant miRNA expression differences of HPV positive tumor tissue biopsies were found for miR-21-5p, miR-143 and miR-221-5p compared to the HPV negative tumor biopsy series. Peritumoral mucosa showed patchy pattern alterations of miR-21-5p and miR-155 in HPV positive cases, while gradual change of miR-21-5p and miR-221-5p was seen in HPV negative tumors. In our study we found differences of the miRNA expression patterns among the HPV positive and negative tumorous tissues as well as the surrounding mucosal fields. The CT based 3D models of the cancer field and surrounding mucosal surface can be utilized to improve proper preoperative planning. Complex evaluation of HNSCC tissue organization field can elucidate the clinical and molecular differentiation of HPV positive and negative cases, and enhance effective organ saving therapeutic strategies. Oral HPV positivity and the number of viral-induced oropharyngeal tumors have significantly higher frequency in males2. In the oropharyngeal region the highest HPV positivity can be detected in the palatine tonsils. The HPV positivity ratio of these tumors can amount to 70% compared to the other sub regions3. The mucosal surface of lacunas and crypts constitute a strong conjunction between the epithelial mucosa and the lymphatic tissue underneath and is often revealed in the background of tonsillar predilection of HPV initiated carcinogenesis4. Clinical investigations have shown revealed remarkable differences between tumors induced by HPV and chemical carcinogenesis: HPV induced tumors particularly emerge at younger ages, and in many cases, the first clinical sign of the malignancy is the lymph node metastasis. The size of primary tumor is frequently small and thereby, it is difficult to explore in the early stage, however, later it often increases in tumor size compared with those HPV negative tumors in the same stage. HPV positive cancers respond much better to oncological and surgical therapy, thus prognosis is generally, far more favorable8. Considering the differences from aspects of prognosis, behavior and development, a separate TNM classification was released in 2017 for HPV positive oropharyngeal tumors10.In addition to the widely-known etiological factors including various chemical carcinogens and poor oral hygiene, the human papilloma viruses (HPV) are major risk factors in the development of head and neck squamous cell carcinomas (HNSCC)Papillomaviridae and the HPV Reference Center has classified more than 120 types (Papillomavirus Episteme – NIH). The viral genome in this family is a circular, double-stranded DNA (deoxyribonucleic acid). Regarding the most conserve sequence of the viral genome, which is the L1 open reading frame (ORF) region, the HPV types belong to seven taxonomical genera . Protein products translated from L1 and L2 ORFs constitute the viral capside, while E6 and E7 ORFs encode oncoproteins that modulate the viral transformation process. HPV infects the basal cells of the multilayer epithelium. The modification of the epithelial cell differentiation, through regulation of the cell cycle, results in the generation of the continuous reactivation of DNA replication and leads to the replication of the genome of the HPV virus14 Cutaneous and mucosal invasive types of the HPV can be distinguished, accordingly. Based on oncological risk groups mucosal types can be assigned according to low, intermediate and high risk HPV. The identification of multiple viral infections is frequently observed and the resulting virulence might be influenced by the different genotypes. However interference between different HPV types and the impact on their virulence are events that have not been demonstrated yet. Due to the fact, that clinical observations about the behavior of the HPV positive oropharyngeal cancer differ so much from HPV negative HNSCC, a new TNM classification was needed to be introduced with a specific staging guideline. This statement is underlined by the The Cancer Genome Atlas (TCGA) publication on comparative molecular characteristics of the HPV positive and negative HNSCCs. The difference between the HPV associated and non-viral groups was clearly confirmed15. The TCGA study was not able to determine sufficient number of oncogenic mutation HNSCC that can drive the mechanisms of carcinogenesis; beside of this fact many epigenetical alterations were proved to characterize this cancer type. Linking to this concept, a recent publication of Masuda et al. stated that HNSCC is strongly linked to changes in epigenetic regulatory16. miRNAs as translator molecules between the environmental exposures and genomic regulation might reflect the carcinogenic process through their expression profile changes.HPV belongs to the family of 18.Chemical and viral carcinogens constitute a genetically and epigenetically modified field in the intact cells of the mucosal surface as the first step of carcinogenesis. The expression, “cancer field“ or “tissue organization field” is described in the literature. Later, followed by precursor changes, a primary tumor, and recurrence of a tumor or a second primary tumor can develop within this field. The behavior of the oral cavity, oropharyngeal, hypopharyngeal and laryngeal squamous cell carcinoma can be represented via this model of tumor development20. The comparison of cancer and healthy cells originating from the same tissue presents differences in the expression patterns of miRNA. The analysis of the miRNA expression profile proved to be an appropriate method towards differentiating between normal and tumorous tissues, and selecting subgroups specifically in relation to tissue regarding several tumorous diseases21. Previous reviews have observed a discrepancy of miRNA findings across studies focusing on cancers of the head and neck, and there has been no consensus on specific miRNAs that are associated to HPV positivity and HNSCC.micro RNAs (miRNA) are small, 19–25 mer regulator molecules. Interestingly, miRNAs can behave either as oncogenes (oncomirs) or tumor suppressors according to which target mRNA they regulateIn our study, we aimed to analyze the miRNA profile of oropharyngeal squamous cell carcinomas through a cancer field model in tumors and surrounding healthy tissues. We collected mapping biopsy series of tissue samples from the tumors and from the peritumoral mucosa 1-2-3 cm away from the macroscopic tumor margin. We also aimed to visualize the mucosal field and the tumor propagation in 3D model based on radiography and to combine it with clinical and genomic results. In our experimental design we selected a set of miRNA targets- miR-21-5p, miR-143, miR-155, miR-221-5p- which satisfy the causality criteria for carcinogenesis in HNSCC. Considering the behavioral differences of HPV positive and negative oropharyngeal tumors, we investigated the miRNA pattern alterations among the two groups with highly quantitative droplet digital PCR technique.Tissue samples (10–60 mg) were collected from patients undergoing surgical intervention and treatment at the Department of Otorhinolaryngology, Head and Neck Surgery, Clinical Center, University of Pécs. During our study period between 2017 and 2018, 48 patients were diagnosed and received multimodal therapy for oropharynx, hypopharynx and larynx HNSCCs. From this patient cohort 25 patients were selected for miRNA analysis based upon the presence of stage T2-3 squamous cell carcinoma of the oropharynx. Tumor classification and clinicopathological evaluation were carried out by a pathology expert according to the “8th Edition TNM Classification for Head and Neck Cancer, American Joint Committee on Cancer (AJCC), 2017”. Clinicopathological staging, HPV P16 immunohistochemistry and HPV genotyping were performed in all HNSCC cases. Our sample collection strategy followed a strict scheme towards effectively mapping the biopsy originating from the cancer area. We obtained our first sample from the non-necrotic tumor tissue, and next, we collected peritumoral macroscopically normal tissue samples at 1 cm, 2 cm and 3 cm distance from the edge or perimeter of the tumor. Following collection, tissue samples were snap frozen at −80 °C and stored until processing. The clinical and experimental study protocol described here was performed under the license number 2014/5341 . The methods were carried out in accordance with the relevant guidelines and regulations. Informed consent was obtained from all participants who were enrolled in this study.TM 2000 . 5 ng total RNA from each sample was reverse transcribed using the miRCURY LNA Universal RT microRNA PCR Kit .Cell free total RNA was isolated from 60 µg fresh frozen tissue samples using the Aurum Total RNA mini kit . Isolated RNA quality was checked using the Thermo Scientific NanoDropRegarding quantitative PCR analysis, we procured the Qiagen miRCury LNA miRNA PCR assays , in accordance with the following miRNA targets: hsa-miR-21-5p, hsa-miR-143, hsa-miR-155 and hsa-miR-221-5p. Master mixes were prepared to include 1 µl of miRCury LNA miRNA assay containing the target-miRNA specific forward and reverse primer pair, 12 µl of QX200 ddPCR EvaGreen Supermix  and 9 µl PCR grade water and 2 µl (~100 pg) of cDNA sample. Application onto the plate was perform using approximately 100 pg/µl template concentration. Regarding the negative controls, we prepared a master mix, in which the template cDNA was substituted with PCR grade water . From each master mix, droplets were generated in the automated droplet generator unit of the QX200 automated ddPCR system using Droplet Generation Oil for EvaGreen (Bio-Rad). PCR amplification was carried out in the QX200 Thermo cycler with the following thermic conditions .22. Furthermore, the recently published the 8th edition of TNM-staging handbook also acknowledged the relevance of differentiating HPV and non-HPV related cases23.At present, HPV association of head and neck SCC is determined using p16 immunhistochemistry, and it is widely considered as an important marker with respect to pathogenesis and prognosisINK4a, the expression of which is thought to be up-regulated due to pRb degradation induced by high-risk HPV E7 protein, a marker which is able to identify tumors with relevant viral oncogene expression25.Some consider p16INK4a Detection System . The immunostaining was performed according to the recommended manufacturer’s protocol.All HNSCC cases were evaluated based on FFPE tissue samples using the standard H&E staining and p16 immunhistochemistry. Regarding the later model, we used the CINtec p16Additionally, the p16 positive staining was concluded based on the identification of strong nuclear and cytoplasmic staining in at least 70% of malignant cells. No staining, faint nuclear and cytoplasmic staining with background marking, small foci of staining or staining in non-malignant cells were considered negative results. The positive control was a cervical squamous cell carcinoma previously proven to be p16 positive, as part of the standard clinical laboratory procedure.60 µg of fresh frozen tumor tissue sample was used for the total DNA extraction using the HighPure template preparation kit . DNA concentration and quality was examined using NanoDrop spectrophotometry.23.Linear array HPV genotyping test  was used to determine specifically, which HPV genotypes were present in the tissue samples. The test detects 37 human papillomavirus genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58,59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP610826. Testing was performed in accordance with the recommended manufacturer’s instructions.The Linear Array is a qualitative test which uses biotinylated primers sets PGM09/PGMY11 and PC04/GH20 for simultaneous amplification of a 450 bp and 268 bp fragments of the HPV L1 gene and human beta-globin gene, respectively. Following PCR amplification, genotyping is performed using a single nylon strip coated with HPV type-specific and human beta-globin-specific oligonucleotide probesConcentration data acquired from QuantaSoft software (BioRad) were next exported and analyzed for quantitative differences between the tissue samples according to histopathology, distance from the tumor, and, the location and HPV status were analyzed using IBM SPSS Statistics 21.0 for ANOVA with statistical significance level p ≤ 0.05.28. Based on contrast enhancement, anatomy and asymmetry, tumor mass was delineated in axial plane slices by a radiologist using 3DSlicer segment editor draw tool. Tumor contours were revised and modified if necessary by a board-certified neuroradiologist with over 10 years of clinical experience.CT DICOM data including arterial and venous phase contrast-enhanced high resolution series  of the patient above were converted to nifti format using MRIConvert 2.1.0  and loaded in 3DSlicer 4.1Using the segment editor module of 3DSlicer 4.1 software 3D segmentation of the tumor was created according to the delineation. Using the same module, the 3D segmentation of the air inside the pharynx was created as well. Using the margin and hollow tools a 1 mm thick boundary was created from the segmentation of the air in order to model the mucous membrane. Using the margin tool on the 3D segmentation of the tumor 10 mm, 20 mm and 30 mm offset volumes were created according to the sample collection site distances. Using a logical operator tool, intersections were created using the segmentations of the mucous membrane and the offset volumes applied to the tumor.In the study period our clinical site diagnosed 48 patients with HNSCC 34 men and 14 women. After clinicopathological classification of the tumors we enrolled to this study only the patients with stage T2-3 HNSCCs with oropharyngeal location, based on the “8th Edition TNM Classification for Head and Neck Cancer, American Joint Committee on Cancer (AJCC), 2017”  with p16 immunohistochemical staining , miR-143 (F = 5.627 p = 0.021), and miR-221-5p (F = 5.065 p = 0.028). The statistical analysis did not confirm miR-155 to be consequently correlated to HPV infection in the tissue biopsy series . Based on our results, we were not able to identity a single miRNA marker specific for HPV positivity, but patterns of the miRNA expression profiles correlated with the presence of HPV  was found to be statistically significant on the ANOVA test Fig. . miR-143In the tumors and their neighboring intact mucosa, miR-21-5p showed gradually decreasing values correlating with distance from the tumor propagation. Average miR-21-5p values do not differ significantly among the tumor samples and in the 1 cm peritumoral normal mucosa. We found a significant decrease in miR-21-5p expression levels in tissues at 2 and 3 cm distance from the tumor margin.The concentration of miR-221-5p was found to be high in tumor tissues while it decreased rapidly in the hystopathologically normal peritumoral mucosa. The standard deviation of the miRNA-221-5p expressions were found to scatter in wider range among the tumor biopsies, while we detected more integrated expression levels in the mucosa biopsies. Mean miR-221-5p expression levels did not significantly differ from the site of the 1 cm peritumoral mucosal region. miR-221-5p levels were equally found to be low in 2 and 3 cm distant biopsies.Although we noticed gradual down-regulation of miR-143 expression in our cancer field detection model, it was not confirmed to be statistically significant on the ANOVA.Regarding miR-155, we were not able to detect correlation in the mapping biopsy samples with the tumor propagation field.The patient’s HR CT DICOM files were uploaded to 3D slicer version 4.1. The 3D model was generated based on a HPV negative right side exulcerated tonsillar tumor (stage T3) with the anatomical extension into the vallecula and deeply infiltrating the lateral pharyngeal wall. The area pointed red on the high resolution CT shows the primary tumor. It is visualized by 3D slicer program in an anatomical model also appearing with deep orange color Fig. . In this33.Growing interest has focused on the development of novel treatment strategies based on HNSCC cell biology. Within this trend, recent identification of human papilloma virus (HPV) positive HNSCC is a milestone discovery, because it is has become apparent that this type of cancer has a distinctive genetic and epigenetic profile, which can be cured within the framework of conventional organ-preserving treatment34. In our study we applied a genotyping platform which is able to recognize a broad spectrum of mucosal HPV genotypes.HPV genotyping experiments, have demonstrated that HPV 16 has the highest percentage in oropharyngeal tumors and in a smaller proportion, the presence of HPV 33 and 35 genotypes have also been confirmedWe could confirm HPV 16 as the most frequent genotype in all our clinical samples (7 cases out of 48) and we also detected HPV 52 besides HPV 16 in a single case. HPV 52 belongs to the high cancer inducing risk category (HR) HPV group and it was found to be the third most prevalent genotype- after HPV 42 and HPV 16- during the routine cervical HPV genotyping at our laboratory site. In the head and neck region the oropharynx presents the highest ratio of HPV associated HNSCC. The mucosal surface of lacunas and crypts constitute a strong conjunction between the epithelial mucosa and the lymphatic tissue underneath and is often revealed in the background of tonsillar predilection of HPV initiated carcinogenesis. During our study period between 2017 and 2018, 48 patients were diagnosed with oropharynx, hypopharynx and larynx HNSCCs. From this patient cohort 25 patients had orophyaryngeal tumor and were selected into the study for HPV genotyping and microRNA expression characterization. Due to the very prospective manner of the study  whilst we gained HPV negative and HPV positive case numbers according to the general HPV incidence in our region. Although the quantity of HPV positive samples used in our statistical analysis restrict the interpretation of our results due to its smaller number compared to the HPV negative tumors . To fulfill the need for group equalizing we should have waited a two-year period to be able to complete sample size for HPV positive tumor group. Since our study is a proof of concept for the 3D model combined with miRNA expression analysis we presume that case number would not influence much on the outcome. Also it is hard to reflect the current results on HPV positivity of the HNSCCs found in our region, because it is known that geographic distribution and the prevalence of certain HPV genotypes may differ, which can cause differences in tumor induction.35. In our study we found differences in miRNA expression patterns in the HPV positive and negative tumor tissues as well as in the surrounding mucosal fields.The molecular pathways of viral and chemical carcinogenesis are different resulting in alterations in miRNA profiles38. However, these studies lacked the clinical and histopathological homogeneity of their samples. Sample enrollment was heterogenic for the clinical stage. Furthermore, tumor negative samples were collected close to the tumor itself; consequently, these studies neglected to take in consideration the tumor organization field. Our study samples were all taken from homogeneous, advanced stage  tumor propagations for both HPV negative and positive tumors, so our results reflect miR-21-5p characteristics of clinicopathologically homogenous tumor groups. Our tumor negative samples were collected according to a complete mapping biopsy strategy taking into consideration the TOF (tissue organization field).We found miR-21-5p expression to reach higher levels in HPV positive tumors and 1 cm distant peritumoral area. In study evidences published so far, miR-21 showed significantly lower expression in HPV positive head and neck tumors, compared to HPV negative ones39. It is associated with a better prognosis, hence its higher expression was observed in HPV positive oropharyngeal tumors40. We have also found a high miR-143 expression in HPV positive tumors compared to the negatives, but the difference was more characteristic for the peritumoral mucosa, which showed significantly higher concentrations of miR-143.miR-143 is a tumor suppressor miRNA, which has been identified as having a lower expression level in HPV associated head and neck tumors. It inhibits the proteins E2F (E2 transcription factor) and survivin and their protein translation, which contributes to tumor proliferation and neoangiogenesisAs well as miR-21-5p, we recognized miR-221-5p with significantly higher expression levels in the HPV positive cancers compared to the negatives, which may also be explained by the fact that HPV positive tumors were large in size. On our mapping biopsy evaluation the highest miRNA values were measured for the HPV positive tumor tissues and miR-221-5p expression. In conclusion, our findings confirm that miR-221 is involved in HPV positive HNSCC tumor growth.33. Many factors seem to contribute to their development, such as the accumulation of genetic and epigenetic alterations, e.g. chromosomal abnormalities, inactivation of tumor suppressors, and increased activity of oncogenes. Signaling pathways in antitumor mechanisms are involved in tumor initiation and progression correlating with biological aggressiveness: TP53 (tumor protein p53), RB1/INK4/ARF  and NOTCH1 (neurogenic locus homolog protein 1) signaling pathways are recognized. Data from DNA sequence analysis implies that the phosphatidylinositol 3-kinase PI3K-Akt-mTOR pathway is also frequently involved in the cancer development41.Regarding the HPV negative HNSCCs despite the exponentially expanding information on the cellular biology, research evidence has not lead to a holistic understanding of the dynamically evolving molecular circuitry of this type33. Thereby, miRNA behaves as a morphostatic factor, linking epigenetic to genetic factors, it is characterized by distinct genetic attributes and plays a key role in the regulation of the mucosal landscape and has been proven to be useful in the molecular characterization of several tumor types41. In our study, we utilized the benefits of this molecule to detect and characterize the HNSCC tissue organization field. Considering the quantitative differences in the miRNA expression, we found many alterations between tumors and phenotypically intact tissues at defined distances. We revealed significant changes of miR-21-5p and miR-221-5p in HPV negative tumors. Tumor surrounding normal mucosa showed gradual concentration depletion of these markers.Recent rapid progress in epigenetic approach has revealed that noncoding RNAs (ncRNA), especially miRNA, have a fine-tuner role within gene transcription. Through interaction with mRNA and chromatin modulators, miRNA orchestrates the stoichiometric equilibrium of gene expression on dynamically changing cellular context42. An elevated expression of miR-21 was also reported in squamous cell carcinomas of the head and neck44, including larynx45, pharynx and tongue tumors, primarily metastatic squamous cell carcinomas of the tongue in the latter46. The increased expression of miR-21 is closely related to a low five-year survival rate and lower response to chemoradiotherapy, which can be an important tool for treatment planning and prognosis estimation in the future37. Currently, there is no evidence in relation to mapping biopsy in literature, but during the progress of our examinations, we found high values of miR-21-5p in the healthy tissue at a distance of 1 cm from the tumor, which correlates with the surgical safety zone.In previous studies, the role of miR-21 has been demonstrated in the inhibition of protein translation of the mRNA of the p53 gene responsible for cell cycle regulation in malignant melanoma and oral squamous cell carcinoma47. It can be closely associated with a local recurrence in the case of oropharyngeal cancer48, and its expression is higher in laryngeal tumors49. It also correlates with tumor proliferation, tendency for metastasis and poor prognosis53. The changes in biomarker values in miR-155, measured at different biopsy distances, could not prove the role of miR-155 in the peritumoral field in our study.miR-155 is a type of miRNA, identified in squamous cell cancers of the larynx, it promotes tumor invasion by blocking the SOC  and STAT  transcription factorsHussein et al. confirmed decreased miR-143 expression within the HNSCC tumor tissue, compared to the values measured in the mucosal controls55. In our study, we found a higher expression in tumor samples than in normal tissue, however the tissues taken at 1 cm distance in the intact mucosa showed similar, and in some cases higher expression levels compared to the tumor samples themselves. Changes were found to be very moderate. The expression results decreased with the distance in the peritumoral field.A previous study by 56 and head and neck squamous cell carcinoma57, while low expression has been reported in tumor-free tissue. We can confirm these results for miR-221-5p as our tissue organization field model, showed a significant gradual change on the tumor surrounding mucosal surface.High expression of miR-221 has been demonstrated by several previous studies in the case of laryngeal squamous cell carcinomaOverall, it can be said that the differing patterns of HPV positive tumors can explain the prognostic and behavioral differences compared to HPV negative tumors. In order to perform a comprehensive examination regarding epigenetic differences, additional investigations are needed with more cases involved. We plan to examine regulation of the miRNAs, we have analyzed thus far, through their target mRNAs in a future research project.In the CT based 3 D model we tried to visualize the tumor mass and the peritumoral tissues considering our mapping biopsy strategy and the tissue miRNA expression results. In the 3D model the clinical tumor mass and the peritumoral tissue locating at 1 cm distance from the macroscopically tumorous margin - where similar miRNA patterns were proved - presents in red tone colors with precise compilation Figs. ,7. SurgiHPV positive cases, where organ preservation and chemo-irradiation therapy are usually preferred, could also be managed in a more precise way based on the 3D models and genomic cancer field evaluation.Our ultimate goal was to optimize the diagnostic steps in HNSCC management, to estimate and minimalize the chances of tumor recurrence and to achieve better survival with higher life quality of HNSCC patients.The clinical and experimental study protocol described here was performed under the license number 2014/5341 . The methods were carried out in accordance with the relevant guidelines and regulations. Informed consent was obtained from all participants who were enrolled in this study.Supplementary Information


Human papillomavirus (HPV)‐based cervical cancer screening requires triage of HPV positive women to identify those at risk of cervical intraepithelial neoplasia grade 2 (CIN2) or worse. We conducted a blinded case–control study within the HPV FOCAL randomized cervical cancer screening trial of women aged 25–65 to examine whether baseline methylation testing using the S5 classifier provided triage performance similar to an algorithm relying on cytology and HPV genotyping. Groups were randomly selected from women with known HPV/cytology results and pathology outcomes. Group 1: 104 HPV positive (HPV+), abnormal cytology ; Group 2: 103 HPV+, normal cytology with HPV persistence at 12 mo. ; Group 3: 50 HPV+, normal cytology with HPV clearance at 12 mo. (assumed <CIN2), total n=257. For the combined groups, S5 risk score CIN2/3 relative sensitivity, specificity and positive predictive value (PPV) were compared with other triage approaches. Methylation showed a highly significant increasing trend with disease severity. For CIN3, S5 relative sensitivity and specificity were: 93.2% (95%CI: 81.4–98.0) and 41.8% (35.2–48.8), compared to 86.4% (75.0–95.7) and 49.8% (43.1–56.6) respectively for combined abnormal cytology/HPV16/18 positivity ; adjusted PPVs were 18.2% (16.2–20.4) and 19.3% (16.6–22.2) respectively. S5 was also positive in baseline specimens from eight cancers detected during or after trial participation. The S5 methylation score had high sensitivity and PPV for CIN3, compatible with US and European thresholds for colposcopy referral. Methylation signatures can identify most HPV positive women at increased risk of cervical cancer from their baseline screening specimens. What's new?DNA methylation testing could simplify the triage process for screening HPV+ women for cervical cancer, according to new results from a case‐control study. Most pre‐cancerous cervical lesions do not progress to cancer, so triage is done to identify those lesions more likely to become cancerous and boost screening specificity. Here, the authors tested women in the HPV FOCAL study for baseline methylation using the S5 classifier. Methylation signatures, they found, performed with 93% sensitivity and 18% PPV for CIN3, comparable to the combination of cytology and HPV genotyping (86% sensitivity and 19% PPV). HPVhuman papillomavirusPPVpositive predictive valueCINcervical intraepithelial neoplasiaLBCliquid‐based cytologyNILMnegative for intraepithelial lesions and malignancyRCTrandomized controlled trialHC2hybrid capture 2 high‐risk HPV testASCUSatypical squamous cells, undetermined significanceSTMspecimen transport mediumROCreceiver operating characteristicRLUrelative light unitsAUCarea under the ROC curvePersistent high‐risk human papillomavirus (HPV) infection is the primary cause of cervical cancer.Widespread adoption of primary HPV cervical screening has supported the search for a triage test which retains high sensitivity but increases specificity and positive predictive value (PPV), while accurately identifying women at high risk for CIN3+. Reflex liquid‐based cytology (LBC) is commonly used,EPB41L3. HPV FOr CerviCAL Cancer Screening (HPV FOCAL) is a population‐based Canadian randomized controlled trial (RCT) comparing HPV versus LBC for primary cervical cancer screening.The S5 DNA methylation classifier was developed in a London UK colposcopy referral populationThe HPV FOCAL RCTWe focused on baseline HPV positive women detected by the HC2 test. Women were classified into three groups based on their HC2 and reflex LBC results Table . Group 1HPV16/18 genotyping was done by the cobas® 4800 HPV test . The Linear Array HPV Genotyping Test (Roche) was used to genotype cobas “other high risk positive” specimens. A HPV16/18 genotype was assigned if the specimen was cobas positive for one or both of HPV16 or HPV18, regardless of the detection of any other HPV type(s).DNA was extracted from 200μL of each STM sample , eluted into 40μL, and used for methylation testing. DNA concentrations were estimated and DNA was shipped on dry ice to the Wolfson Institute laboratory where methylation testing was done as previously described.All analyses were based on a pre‐specified statistical analysis plan. The main hypothesis was that S5 methylation triage at baseline had equivalent sensitivity and PPV to triage by baseline LBC ≥ASCUS or LBC NILM and HPV16/18 positivity (LBC ≥ASCUS/HPV16/18). Histopathologically confirmed CIN2/3 versus <CIN2 was used as the reference standard.EPB41L3 together with HPV16L1, HPV16L2, HPV18L2, HPV31L1 and HPV33L2. The PCR‐based assay was followed by quantitative pyrosequencing to measure methylation levels of each assay component. The S5 risk score was calculated as: S5 = 30.9(EPB41L3) + 13.7(HPV16L1) + 4.3(HPV16L2) + 8.4(HPV18L2) + 22.4(HPV31L1) + 20.3(HPV33L2); a score of ≥0.8 indicated a positive methylation test.The S5 risk score is based on methylation levels of the human gene HC2 relative light unit (RLU)/cutoff ratios, where a positive test was ≥1.0, were used as a surrogate for HPV viral load; a higher ratio indicated higher viral load.Relative sensitivity and specificity  for cumulative round 1 CIN2/3 and CIN3 during the trial were calculated for S5 performed at baseline; 95% non‐parametric bootstrapped CIs were obtained from 10,000 bootstrap replicates. Unadjusted PPVs were calculated by dividing the number of women with CIN2/3 or CIN3 cervical lesions (true positive screens) by the number with a positive triage test in the methylation study subset.S5 ROC curves were generated for CIN2/3 and CIN3 by re‐weighting the sampled groups as described above, from which area under the ROC curve (AUC) with 95%CI was calculated from a non‐parametric empirical bootstrap. The combined ROC estimated the classification performance of S5 and its components for all HC2+ women in the HPV arms of the trial.Statistical calculations were performed using R version 3.3.1.p=0.170; CIN3: p=0.248) than the sensitivity of combination triage by LBC ≥ASCUS/HPV16/18. S5 relative specificities for <CIN2 and <CIN3 [44.0% (95%CI: 36.1–52.2) and 41.8% (95%CI: 35.3–48.4) respectively] were similar to LBC ≥ASCUS/HPV16/18 triage, but were lower than both LBC ≥ASCUS and HPV16/18 triage , which was used as the reference standard, the S5 classifier had sensitivities for CIN2/3 and CIN3 of 75.7% (95%CI: 67.3–83.7) and 93.2% (95%CI: 84.8–100.0) respectively  was higher than for HPV16/18 positive and LBC ≥ASCUS triage, but was similar to the combined strategy of LBC ≥ASCUS/HPV16/18 triage (4.2%), which was our most sensitive and main comparison. The highest referral rate was for the full FOCAL trial triage approach (5.9%) which detected all 107 CIN2+ cases.Of the 107 CIN2/3 cases, 81 (76%) were S5 positive at baseline. FOCAL triage identified 54 (50%) at baseline and the remaining 53 (50%) cases after 12 month re‐screening. For the 44 CIN3 cases, 41 (93%) were S5 positive at baseline. FOCAL triage identified 27 (61%) at baseline and the remaining 17 (39%) cases at 12 months (Table ptrend<0.0001) and with HPV viral load (Supporting Information Table ptrend=0.0001). Women with <CIN2 and LBC ≥ASCUS, LBC NILM or non‐HPV16 positivity had median scores near the S5 cutoff, while HPV16 positive women had a higher median S5 score, similar to some of the women with high‐grade disease and cancer  for CIN3 and cancer among HPV positive women. S5 specificities were lower but PPVs were comparable to other accepted triage methods. Compared to the FOCAL trial triage of colposcopy referral for HPV positive women with baseline abnormal reflex cytology or NILM baseline cytology with 12‐month HPV persistence, methylation triage can provide objective and more timely identification of most women with high‐grade cervical lesions at baseline screening. Of women with CIN3, S5 detected 93% of cases at baseline, compared to 61% for the FOCAL trial baseline triage. For CIN2/3 the percentages were 76% for S5 triage and 50% for FOCAL triage, respectively.S5 methylation testing had similar triage performance for detection of CIN2/3 at baseline compared to a triage approach based on immediate colposcopy referral for women with LBC ≥ASCUS, or LBC NILM with HPV16/18 positivity, a triage approach used predominantly in the US.An earlier study of S5Performance characteristics for methylation studies (not including those with self‐collected samples) using a variety of genesEPB41L3 or other host genes has been reported to have comparable performance to cytology for HPV positive women,Sensitivity and specificity was not reported for the FOCAL trial as there was no verification performed for negative screens. We used the FOCAL triage approach as the reference method; thus, the sensitivities for other single and combination triage approaches reported in this paper are relative to those based on the FOCAL trial, which were assumed for comparison purposes to be ~100%. The relative CIN3 sensitivity for the S5 classifier (93.2%) was similar to FOCAL while that for CIN2/3 was lower (75.7%). This might be expected given that most of the S5 negative CIN2+ cases were associated with non‐HPV16/18/31/33 genotypes. Targeting additional HPV genotypes in the S5 classifier might improve sensitivity, but could result in lower specificity. Methylation triage including the Of eight women who developed cervical cancers during or after FOCAL trial participation, two were HC2 negative on the baseline specimen. All eight cancers were S5 positive at the baseline screen, but the median S5 score for the baseline samples for women with cancers was lower than for women with CIN3 (5.8 vs. 9.3). Some of the tested samples from subjects with cancer were obtained several years prior to the cancer diagnosis, which could have resulted in lower S5 scores than if samples had been tested closer to their cancer diagnoses. This is likely the case, as the four women who had a subsequent sample tested had substantially higher S5 scores than for their baseline samples. Moreover, six of the cancers tested were adenocarcinomas and it has been reported that these tend to display lower methylation levels compared to squamous cell carcinomas.EPB41L3 human gene, which was found to have the best performance in an earlier credentialing study of a number of human genes in the Predictors 1 and 2 studies.At least two methylation assays based on human genes are commercially available for HPV positive triage. The GynTect® assay is based on ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671,A strength of our study is that the samples were obtained from a RCT embedded within an organized cervical screening program, with high compliance to colposcopy recommendations, standardized colposcopic examinations with biopsy, and centralized blinded pathology review. An important limitation of our case–control study is that it was retrospective because the trial was not designed specifically to assess prospectively additional molecular triage methods in HPV positive women. In addition, although women with CIN2/3 and <CIN2 were randomly selected from the population of women meeting those criteria, it is possible that the methylation‐tested sub‐population is not representative of all women in the trial with CIN2/3 and <CIN2. Optimal ethnic and geographically representative validation of S5 triage will require additional studies designed to directly compare S5 with established strategies, preferably with colposcopy referral for all women with a positive triage test. An intriguing question is whether S5 classifier negative CIN2+ reflects lesions destined to regress spontaneously, or result from the S5 classifier not including targets for some high‐risk genotypes. To understand this phenomenon would require systematic follow up of CIN2+ women who are undergoing assessment for CIN progression or regression.In conclusion, DNA methylation assessed by the S5 classifier correlates strongly with aggressive cervical disease, showing high sensitivity for CIN3 and cancer, the raison d’être for a cervical screening program. S5 PPV for CIN3 is compatible with both US and European colposcopy referral thresholds.Study conception and design: M. Krajden, A. Lorincz, D. Cook, A. Brentnall. Acquisition, analysis and interpretation of data: D. Cook, A. Brentnall, L. Smith, L. Gondara, M. Krajden, A. Lorincz, D. van Niekerk, G. Ogilvie, A. Coldman. Writing, review, and revision of the manuscript: D. Cook, A. Lorincz, A. Brentnall, M. Krajden, J. Cuzick. Laboratory analyses: J. Law, T. Chan, R. Warman, C. Reuter. All authors reviewed the manuscript for critical intellectual content and agreed to submission of the manuscript for publication.Supplementary Figure 1. HPV FOCAL Trial SchematicThe yellow highlighted area illustrates the trial subset used for the methylation case‐control study.Click here for additional data file.Supplementary Table 1. Median baseline S5 scores by reflex cytology grade and HPV genotypeSupplementary Table 2. Median baseline S5 scores by HPV viral loadSupplementary Table 3. HPV, cytology and viral load results for S5 negative CIN2+ casesClick here for additional data file.


To test the effectiveness of a comprehensive team-based intervention to improve human papillomavirus (HPV) vaccination completion rates and reduce missed opportunities to vaccinate in rural Oregon.Stepped-wedge cluster randomized trial.Forty family physicians and pediatricians who are members of the Oregon Rural Practice-based Research Network.Tailored to individual practice needs, components will include (1) practice facilitation with clinicians, nurses, front office staff, and others who have patient contact to redesign patient care and communication strategies to optimize HPV vaccine series completion; (2) workflow mapping adapted to practice context to support HPV vaccine delivery; (3) a practice improvement model designed to firmly establish reminder and recall systems and then standing orders; (4) education for patients and parents that underscores HPV vaccination is safe, effective, and an important approach for reducing cancer risk; and (5) partnering with community organizations to plan and implement a social marketing campaign on HPV vaccination.Initiation and completion of the HPV vaccine series as well as reduction in rates of missed opportunities to vaccinate derived from Oregon Immunization Program data.NCT03604393: .Trial was registered on July 11, 2018. The first participant was enrolled on September 11, 2018.ClinicalTrials.govPRS,  Given the low rates and wide range of HPV vaccine initiation and completion in rural settings, a critical need exists to support primary care practices and their communities toward improving HPV vaccination rates and identify successful strategies for future intervention.The human papillomavirus (HPV) types in the current 9 valent vaccine account for about 73% to 88% of cancers of the cervix, vagina, and anus and for approximately 55–65% of cancers of the vulva, penis, and oropharynx , 2. AbouIn 2006, the US Food and Drug Administration approved a vaccine to protect against HPV-16 and HPV-18 , which aUnfortunately, the US National Immunization Survey (NIS) indicates many fail to complete the vaccine series . Among fMany barriers have contributed to low HPV vaccination rates, including factors at the system  and individual levels . One systematic review of 76 published studies  identifiMore rigorous research is needed to understand how to optimize HPV vaccination rates, especially in rural underserved communities. Toward this end, we developed a research proposal funded by the American Cancer Society (RSG-18-022-01-CPPB) to test, using a stepped-wedge cluster randomized design, a multi-component intervention to improve HPV vaccine completion rates and reduce missed opportunities to vaccinate. Table Oregon Rural Practice-based Research Network (ORPRN) is based at Oregon Health & Science University (OHSU) with a mission of improving the health of rural Oregonians by promoting knowledge transfer between communities and clinicians . ORPRN h> 40,000 people) that see adequate numbers of patients aged 11–17 years. Based on our sample size estimate, we will need 40 practices for the study. We will overenroll to 45 to account for an attrition rate of 11%. We received over 60 letters of support from rural practices that expressed interest in participating. Practice enrollment is currently underway. Community organizations established for at least 1 year and co-located in communities of participating primary care practices will also be eligible to take part. These will include Accountable Care Organizations (ACOs), which are healthcare organizations tasked with providing high-quality, low-cost care to their Medicaid and Medicare beneficiaries. In Oregon, the ACOs for the Medicaid population are 16 Coordinated Care Organizations (CCOs) [In this study, we will enroll eligible ORPRN primary care clinics located in rural communities  .The study has four specific aims described below. Figure To address aim 1, we will use data on vaccine initiation and completion rates from OIP’s immunization registry, ALERT Immunization Information System (IIS), to identify 12–15 practices with both high and low HPV vaccine initiation and completion rates in rural Oregon family medicine and pediatric practices. After practices are enrolled, we will conduct direct observations using expert qualitative investigators (authors MD and RG) to assess practice systems and workflows associated with high and low vaccination rates. An iterative sampling framework will inform practice recruitment—we anticipate enrolling, using a 2:1 ratio, higher performing clinics relative to lower performing clinics. Additional study measures for aim 1  Community Advisory Council (CAC), patient and family advisory groups, or local public health programs. Together, the practice and community group will plan and implement an evidence-based community-level intervention , 27 desiStudy measures for aim 3 will include pre- and post-community partner group interviews on the acceptability of HPV vaccination and patient/parent surveys to assess child and parents’ knowledge about cancer risk reduction and the HPV vaccine series, where and how information about HPV and cancer risk reduction via vaccination was sought, as well as demographic information.To address aim 4, we will create a practice and community HPV vaccine improvement toolkit for use by practices and community partners. The toolkit will be disseminated throughout Oregon by ORPRN, OIP, and the American Cancer Society through publication and as an online resource. Determining the toolkit’s direct impact on HPV vaccination completion rates is beyond the scope of this study. However, OIP will track the number of electronic downloads of the toolkit and be able to link this to rates of HPV vaccine completion as an indirect assessment of improvements in vaccine completion rates due to the toolkit. Our national dissemination plan will include five key activities: (1) presenting the final toolkit and project at the National Immunization Conference, which brings together a variety of partners to explore science, policy, education, and planning issues related to immunization; (2) distributing the toolkit on the National VFC/AFIX Group listserv through OIP’s partnership. Subscribers to this listserv represent more than 260 state and local immunization program and immunization registry staff from the USA; (3) distributing the toolkit on the Oregon Immunization Partners listserv, which currently has 2900 public and private providers; (4) distributing the toolkit on the Oregon Public Clinic listserv, which includes more than 1100 public providers from health departments, federally qualified health centers, school-based health centers, and tribal practices; and (5) Putting the toolkit on the OIP website hosted by the state health department. The OIP website is a critical resource utilized by over 2000 providers, ACOs, Health Systems, payers, and members of the public.With OIP, we identified specific HPV series initiation and completion rates from 2017 at the level of the county as well as the practices, and we have obtained county and practice-weighted population estimates for 13–17 year-olds , MenACWY , and HPV. As shown in Fig. Rural areas experience unique challenges regarding HPV vaccination and this phenomena is understudied in the current literature. Access to primary care can be challenging in rural areas and preventive care in these regions is often underutilized , 31. LowOur proposed study is innovative in at least nine important ways: 1) It is based in one of only two practice-based research network in the USA with a priority focus on rural primary care research; (2) it will involve tailored interventions for both pediatric and family medicine practices, allowing for specialty comparisons of staffing, workflow, and community linkages to deliver HPV vaccine; (3) ORPRN and OIP will collaborate to provide a descriptive baseline assessment of Oregon immunization rates and missed opportunities within rural practices, fostering a robust understanding of the gaps in meeting population health goals at practice and community levels (aim 1); (4) the practice-based intervention (aim 2) is designed to reduce burdens that busy primary care providers face using a team-based care approach with engaged, activated parents and adolescents; (5) the practice-based intervention (aim 2) is flexible enough to allow practices to tailor efforts that work best for them and they will not be burdened by data collection as the ALERT IIS system will provide vaccination data on all enrolled practices at 3-month intervals; (6) our interventions are designed to change primary care practice structure, communication strategies, and behavior through team-based care quality improvement (QI) efforts  the use of the ALERT IIS as a data source for intervention studies, (2) comparing Medicaid and ALERT IIS data sources, (3) rates of HPV vaccinations of males and females in rural pediatric and family medicine practices, (4) urban/rural differences based on the practice location to see if these differences exist within practices, (5) urban/rural differences at the population and practice levels, (6) facilitators and barriers for both initiation and completion of HPV vaccines in rural pediatric and family medicine practices, (7) rates of missed opportunities to vaccinate for HPV according to youth gender, (8) effectiveness of a multi-component primary care-based intervention on completion of the HPV vaccine series and reduce missed opportunities to vaccinate, (9) characteristics of practices with high uptake and low uptake of the primary care-based quality improvement intervention, (10) contribution of evidence-based social marketing campaigns as a supplement to practice-based interventions to improve HPV vaccination, and (11) components and dissemination of a toolkit designed to help primary care practices improve HPV vaccination. In conclusion, there is a critical need to conduct this study, which started in July of 2018. We look forward to undertaking this important work and what we will learn from it.


The presence of human papilloma virus in benign oral lesions has been studied by different techniques obtaining extremely variable results. The objective of this study was to determine the presence of human papillomavirus in 83 cases of benign hyperplastic epithelial oral lesions.Eighty-three oral lesions with clinical or histopathological features suggestive of HPV infection were retrieved from the files of four oral pathology services. Demographic data were obtained from patient´s medical charts. All cases had available clinical image, H&E preparations and paraffin blocks with enough tissue for HPV detection by in situ hybridization, and immunohistochemical reactions for Ki67.Episomal positivity for wide spectrum HPV was observed in 24% of the cases; most of them (70%) HPV 6/11 positive. HPV 16/18 was not detected. Condyloma acuminatum was the most common lesion associated with HPV (75%), followed by verruca vulgaris (15%), squamous papilloma and multifocal epithelial hyperplasia, 5% each. Koilocytes were identified in all the HPV positive cases. Ki67 showed an abnormal proliferation pattern in 90% of the HPV positive cases; most of them (70%) showing groups of proliferating cells in focal superficial regions, and in 20% positivity was seen almost in the whole thickness of the epithelium. HPV negative cases showed Ki67 positive cells restricted to the basal layer.Regarding oral lesions associated with HPV, condyloma is the most common lesion expressing low-risk subtypes. The etiology of squamous papilloma remains controversial as HPV was found in 1.9% of the cases. The identification of koilocytes and the pattern of expression of Ki67 reflect HPV infection and are helpful for classification. Papillary oral lesions not associated to HPV deserve further studies to better clarify its etiology. Key words:Human papillomavirus, condyloma, papilloma. The World Health Organization (WHO) recognizes four human papillomavirus (HPV) related oral lesions; squamous cell papilloma (SP), condyloma acuminatum (CA), verruca vulgaris (VV) and multifocal epithelial hyperplasia (MFEH). These lesions are benign hyperplastic exophytic proliferations of the oral epithelium associated with different subtypes of HPV. Papilloma tend to be solitary and pedunculated, the percentage of HPV positive lesions is highly variable, ranging from 0% to 100%, with an average of 34% depending on the detection technique. Low-risk HPV 6 and 11 are the most commonly found subtypes. Histologically SP show sharp-pointed digitiform projections of the epithelium without evident koilocytes or keratohyaline granules. VV is uncommon intraorally, it affects mainly lip vermillion, clinically, a white rough surface is seen, often presenting as multiple or clustered lesions. Features that help distinguishing VV from SP are a thick layer of orthokeratin, prominent keratohyaline granules and presence of koilocytes. VV is associated with HPV 2, 4, 40 and 57. Oral CA corresponds to the counterpart of genital condyloma, usually HPV 6/11 positive, and it can be differentiated from SP by its bulbous, rounded and short projections  probe that includes genotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51 and 52, and two specific probes; HPV 6/11 and HPV 16/18 . The Catalyzed Signal Amplification System  was used for visualization. All procedures were performed following the manufacturer´s protocol. Cases of uterine cervix and tongue squamous cell carcinoma were used as positive and negative controls, respectively. The presence of a complete nuclear  or punctate/dots (integrated) dark brown signals within the epithelial cells were considered positive . Solitary lesions prevailed, and palate, tongue and lip vermilion were the most affected sites. Twenty (24%) from 83 cases of hyperplastic epithelial oral lesions were positive for the wide spectrum probe, and 14 (70%) out of these were of the 6/11 subtype. In all the cases positive cells had an episomal pattern. The high-risk specific probe 16/18 was not positive in any case. Regarding to diagnosis, cases classified as condyloma were the most associated with HPV, 75% were positive for the 6/11 specific probe Fig. .Ki67 highlighted proliferating cells restricted to the basal layer in 67.4% of the cases, mainly corresponded to SP. In 27 cases 32.5%) a more diffuse pattern was observed, in 22 cases besides the basal layer, focal groups of superficial cells expressed Ki67 corresponding to condyloma and VV, while in 5 cases positivity was observed in the whole thickness of the epithelium , this raises the question whether these lesions belong to the same spectrum with different clinical presentation . We founCases positive for the wide-spectrum probe and negative for the specific probes, half of them with diagnosis of VV were in some way expected because these probes did not include the genotypes typically associated with this lesion: 2 and 57 . These cAbundant koilocytes and an abnormal expression of Ki67 are features identified in most of the HPV associated lesions. In CA these viral altered cells were seen in the upper layers of epithelial crevices, as reported previously. Koilocytes have been identified in approximately 87% of oral condyloma and in a range from 27% to 45% of oral papilloma ,17. We fKi67 showed to be interesting to indicate association with HPV, as almost 70% of the cases showing a non-restricted basal layer positivity, resulted to be HPV associated. It is important to consider that these lesions should be distinguished from epithelial dysplasias not associated with HPV.One final point that deserves further consideration is that the squamous papillomas were mostly HPV negative. This is probably associated to the fact that from the more than 170 genotypes described, we used in this study only a limited number of probes, and it is unknown with certainty what percentage of the genome is common for those genotypes not commercially available. High discrepancies are found when using methods such as ISH or PCR, as the latter possess a higher sensitivity but lower specificity, and studies using PCR shows higher percentages of positivity . In conc


Almost 25% of US military veterans live in rural areas and over 70% of these are age 55 years or older. The physical, cognitive, and functional declines common to aging coupled with accumulated physical and psychological traumas often incurred through military service make caring for older Veterans an especially difficult challenge. Furthermore, the geographic distances associated with rural living pose a significant barrier to timely access to care for aging Veterans and caregivers living in remote communities. Significant gaps remain in VA’s understanding of the needs of this population, which is essential for developing adequate models of care. This symposium highlights various aspects of the rural older Veteran experience and will suggest opportunities for improving access to care for this population. The first presentation estimates the differential impact of rural status on mortality rates among older Veterans using mortality data from the General Social Survey . A second study is an analysis of urban/rural differences in PTSD symptoms as function of multiple co-factors and accounting for geographic location . Seaman et al present qualitative data regarding the experience of Veterans with head and neck cancer to understand how rurality impacts cancer survival . The final presentation will highlight a model to address mental health problems in older Veterans in primary care  and will specifically highlight how this VA program may be adapted to address such needs the needs of older Veterans in rural areas.


Background: Undergoing a timely Pap smear, high-risk human papilloma virus (HPV)- and colposcopy-based testing can reduce HPV-associated cervical cancer (CC) development in women. However, in rural areas, women and minorities without insurance do not undergo periodic assessment and remain at greater risk of HPV infection and CC. Methods: In this study, 173 women from rural East Texas with various ethnic backgrounds were examined thorough HPV/Pap-based testing and colposcopic assessment. Results: Of the 113 informative cases, 77% (87/113) were positive for high-risk HPV infection and 23% of subjects (26/113) were negative. Associations between HPV positivity with young age (p = 0.002), and a low number of pregnancy (p = 0.004) and births (p = 0.005) were evident. Women with long-term use of contraceptives  were associated with increased risk of HPV infection. African-American women had a higher risk of abnormal Pap outcome compared to Caucasians . HPV seemed to be a predictor of abnormal Pap outcome  in these subjects. Unmarried/widowed/divorced women had an increased abnormal Pap test outcome compared to married women or women living with a partner (p = 0.01), with over 278% increased odds . Insured women undergoing periodic checkups were detected early with high-risk HPV infection and abnormal Pap test/colposcopic outcome. Conclusions: Comprehensive and timely screening of uninsured women and minorities in rural East Texas are warranted, which could potentially prevent the onset of HPV-associated CC. Cervical cancer (CC) is one of the leading causes of death among women ,4,5,6,7.The present study has undertaken an initiative to address this problem and evaluated outcomes from 173 women in rural areas of East Texas for CC risk determination through hrHPV detection, Pap test and colposcopy-based examination. Through this evaluation, we have observed an association between HPV positivity and increased risk with young age, low number of pregnancies and births and long-term contraceptives use. Unmarried/widowed/divorced women had an increased abnormal Pap test outcome compared to married women or women living with a partner. On the other hand, African-American women exhibited higher risk of abnormal Pap outcome compared to Caucasian American women. Tyler is located in East-Central Texas within the Smith County. In Smith County, CC incidence has an age-adjusted rate of 7.5 per 100,000 ,14. In cAll the Pap tests were liquid-based either with ThinPrep or SurePath and were evaluated by relevant Pathologists using the 2001 Bethesda reporting system . Human pRoutine examination of cervical health and HPV infection status of these subjects was done by expert clinicians and the resident team at The University of Texas Health Science Center at Tyler. Biopsies were taken as indicated and evaluated by relevant pathologists. Appropriate treatment interventions were also employed as necessary per standard guidelines.Biopsy tissue samples were fixed in formaldehyde and then paraffin embedded (FFPE) following standard protocol. Five micron sections were stained with the standard hematoxylin and eosin method and assessed by a histopathologist under a compound microscope. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 . Descriptive statistics were generated for all variables as reported in In this study, 173 women, aged 18–65 (mean 34.3) were examined in 2007–2017. In terms of medical coverage, 72% (123/171) of these women did not have any health insurance. For Ethnic distribution, out of the 166 informative cases, 102 (61%) were Caucasian (W), 34 (20%) were African-American (AA) and 32 (19%) were Hispanic/Latino (HIS). Of the informative cases, 34% (58/170) had normal BMI, 28.2% (48/170) overweight and 37.6% (64/170) obese. Of the 152 informative cases, 52 (34%) were married or living with a partner and 100 (66%) were unmarried, widowed or divorced. Of the 173 informative cases, 94 (54%) used contraception and 79 (46%) did not. The mean age of menarche of these subjects was 12.7 years and the mean number of births was 2.3. Of the 170 informative cases, 66 (39%) had a smoking history and 104 (61%) were non-smokers. Thirty percent (35/115) had a history of alcohol consumption, whereas 70% (80/115) did not have any history of alcohol consumption; 52% (55/105) had a family history of cancer and 48% (50/105) did not.p = 0.002), and low number of pregnancies (p = 0.004) and births (p = 0.005) was observed  were not available for 60 women and, as a result, they were regarded as non-informative. Of the 113 informative cases, 87 (77%) were positive for HPV and 26 (23%) were negative for any HPV infection A. An assobserved . Prolongobserved . p = 0.01) with over 278% increased odds  (p = 0.04) for an abnormal Pap test outcome with about 67% reduced odds for those who reported the consumption of some form of alcohol compared to those who did not  , high-grade squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells, cannot exclude HSIL (ASCUS-H) and atypical glandular cells of undetermined significance (AGCUS) using the Bethesda Nomenclature . Of the 9-11.10) . Notably11,0.99) . A 77% iThe colposcopy outcome was normal in 38% (61/162) of the cases, mild dysplasia was observed in 46% (75/162), moderate dysplasia in 7% (11/162) and severe dysplasia was evident in 9% (11/162) C. There A total of 162 women received colposcopic examination. Moderate to severe dysplasia was detected in 12 subjects. A standard loop electrosurgical excision procedure (LEEP) was employed in these 12 subjects to prevent further progression of these lesions. One patient had progressed to invasive squamous cell carcinoma of the cervix and one subject was detected with grade I adenocarcinoma. One subject had undergone both LEEP followed by hysterectomy due to severe dysplasia.Cervical cancer develops and progresses through high-risk HPV infection and associated preneoplasitc changes in the uterine cervical epithelium. Importantly, timely screening and appropriate interventions can prevent progression of the preneoplasitc lesions to CC. Timely interventions through HPV/Pap testing and colposcopic evaluations are critical for early detection and prevention of CC, as demonstrated by other studies ,19,20,21The study has limitations regarding the sample size. Tyler is an underserved rural area with an estimated population size of around 105,729 people . MoreoveIn summary, the present study identified a high frequency of relatively young women, including minorities, with hrHPV positivity and associated preneoplastic changes. Regular cervical health checkups in concert with the implementation of appropriate educational programs could be beneficial to encourage women to participate in CC screening programs, particularly in rural communities with low socioeconomic and weak educational backgrounds .


In a Perspective, Sarka Lisonkova and Michael Kramer discuss the accompanying study by Kathryn Fitzpatrick and co-authors on management of amniotic fluid embolism. PLOS Medicine, Kathryn Fitzpatrick and colleagues [Owing to its uncertain etiology, varying symptoms, rapid onset, and high fatality rate, amniotic fluid embolism (AFE) is one of the most challenging obstetric emergencies. In a new international study published in lleagues  provide lleagues . The clilleagues .Because AFE is a diagnosis of exclusion, a precise case definition is difficult to establish. For these reasons, Fitzpatrick and coauthors examined risk factors, prognosis, and clinical management of AFE using three different definitions: the most liberal definition proposed by the United Kingdom Obstetric Surveillance System (UKOSS) , a conseIn previous studies, the case fatality of AFE varied between 11% and 48%, depending on the study design  and case definition ,6. UsingThe AFE presentation that dominates the main clinical picture—e.g., coagulopathy (with or without massive bleeding), refractory pulmonary hypertension, or neurological symptoms—should dictate the therapeutic approach. Even before the diagnosis of AFE is established, high-quality cardiopulmonary resuscitation (CPR) should be the first response to cardiac arrest, followed by ongoing cardiorespiratory support. Optimal volume management in response to changing hemodynamics is also crucial, although it is difficult to strike the correct balance between maintaining cardiac output and preventing fluid overload and pulmonary edema.To some degree, the varying treatment modalities reported by Fitzpatrick and colleagues reflect the variation in presenting symptoms among the AFE cases included in their study. Because most of the cases had coagulopathy and bleeding, coagulation management and surgical interventions to stop the bleeding appeared to improve overall survival. The interventions included the administration of concentrated fibrinogen, platelets, and transfusion of whole blood or red blood cells. All these interventions, however, are contingent upon successful CPR in cases with initial cardiopulmonary arrest, a sine qua non for survival. Even though the authors’ analyses of AFE management modalities were statistically adjusted for cardiac arrest (as a marker of AFE severity), the strength of the protective association with these modalities is highly likely to be biased by reverse causality. Receipt of any intervention depends on survival until the time it is administered, and thus survival may be a cause, rather than a consequence, of the intervention. Nevertheless, Fitzpatrick and colleagues are correct in drawing attention to early detection and correction of coagulation deficiencies in suspected AFE cases, as well as to potentially important newer treatments such as tranexamic acid.Concerns for fetal well-being add to the gravity of AFE. Acute fetal distress often accompanies the underlying triggers of AFE  and hypoxia caused by maternal respiratory or cardiac arrest or severe hemorrhage. Expedited delivery is important in women with AFE at 23 weeks’ gestation or later to stabilize the mother and prevent fetal demise. In most severe cases, emergency cesarean delivery should occur within 5–10 minutes after cardiac arrest to prevent fetal or neonatal death, but that is seldom possible. Rates of stillbirth and early neonatal death after AFE are as high as 40% [AFE is one of the leading causes of maternal death in industrialized countries, but unanswered questions remain about its etiology, prediction, and optimal management. Multicenter international data with a high level of clinical detail, as reported by Fitzpatrick and colleagues, are essential for further progress. Additionally, a common definition and uniform set of collected data are needed to establish a large pool of AFE cases for future surveillance and research. The “strict definition” proposed by Clark and colleagues  is more From the clinical standpoint, AFE does not match the typical picture for embolism and more closely resembles conditions such as anaphylaxis or systemic inflammatory response syndrome . Future


ТР53 Arg72Pro polymorphic marker with cervical cancer (CC) in Kyrgyz women. The aim of this study was to ascertain the magnitude of association of gene ТР53 gene Arg72Pro polymorphism were identified using PCR-RFLP assay.We identified and included 205 women of Kyrgyz ethnicity for this case-control study, of whom N=103 were women (mean age 53.5 ± 10.0 years) with histologically confirmed CC and N=102 controls (mean age 46.5 ± 8.5 years). We detected human papilloma virus (HPV) DNA types 16 and 18 using polymerase chain reaction (PCR) with hybridization/fluorescent detection. Genotypes of  ТР53 gene showed no difference between CC and control groups . However, when CC cases associated with HPV were tested against controls, Arg72 allele and Arg72Arg genotype prevalence were greater compared to controls . In HPV-positive women, Arg72Arg genotype of ТР53 gene was associated with a 1.85-fold increase in the likelihood of CC  1.03-3.32]), whereas Arg72 allele increased this likelihood 1.94-fold (OR=1.94 [95% CI 1.20-3.15]). Eighty-eight percent (90/103) women with CC had HPV, of whom 43.4% (39/90) had HPV type 16, 24.4% (22/90) had HPV type 18, whereas 32.2% (29/90) carried both types. The univariate analysis of allele and genotype distribution of Arg72Pro polymorphic marker ofТР53 gene in Kyrgyz women increase the risk of HPV-associated CC. Arg72Arg genotype and Arg72 allele of  Cervical cancer (CC) incidence across the globe varied dramatically from somewhat 3.5 to 21 cases per 100 thousand women . In developed countries, this cancer incidence shows the trend for reduction, which may associated with better awareness of risk factors, active prevention and advanced pre-cancer diseases recognition and treatment. On the contrary, in developing world, CC is the leading type of cancer among all malignant tumors in women. Some small, but persistent CC incidence increase is reported in Kyrgyzstan since mid-90s. In Kyrgyzstan, CC incidence in 2011 was 11.0 cases per 100,000, including 60.5% with stage I–II; 30.6% with stage III and 8.9% with stage IV. Mortality rate was 3.3 per 100,000, one-year mortality was 46.9%; and five-year survival was 39.7%. The mean age of CC patients was 40 years . CC prevention is one of prioritized directions in public health and health promotion. CC is to large extent associated with human papilloma virus (HPV). HPV DNA was found in more than 99.5% of biopsy material of CC patients . At present, there exist more than 120 HPV types, whereas 30 of those infect anogenital area. Some HPV types exhibit high oncogenic risk including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. HPV type 16 is considered as most prevalent in CC and found in up to 50-70% CC cases, followed by type 18 (confirmed in 10-20% CC cases) . Vaccination against HPV was initiated in 2007 in countries with efficient public health system. On the contrary, such vaccine is not yet available in low-income countries such as in Kyrgyzstan.Usually it takes ten years and more for malignant transformation from the initial contamination. Viral genome may remain latent for a very long time, and its activation is one of the pivotal stages in oncogenesis, however, this process remains poorly understood. Recent studies have shown that aside from HPV, suppressor genes may also play role in CC pathogenesis. р53 gene is one of the few most studied suppressor genes, important for induction and progression of carcinogenesis associated with HPV . Few studies have confirmed allele status of Р53 gene is associated with CC in HPV-positive women . ТР53 (rs1042522) gene in CC of HPV-positive women in Kyrgyz Republic. This work is the first genetic study of HPV in Kyrgyz Republic. The aim of this study was to assess the role of Arg72Pro polymorphic locus of Study Outline and Sample Population This study was designed as case-control. Cases were 103 CC women, treated in the in-patient department of gynecology of the National Center of Oncology and Hematology in Bishkek from 2015 to 2016. In all included cases the CC diagnosis was based on morphology tests, clinical and ancillary examinations, including ultrasonography and computer tomography (CT), oncogynecologist visit, smear cytology using Papanikolau method and colposcopy. In all cases the diagnosis was verified with biopsy of smears obtained at gynecological consultation and after surgery.One-hundred and two more women were controls, who were unrelated with the women in the main group. Women with the history of any cancer were excluded from controls at a stage of screening. The mean age of CC women was 53.5 ± 10.0 years and 46.5 ± 8.5 years in controls. Only women of Kyrgyz ethnicity constantly residing on the territory of Kyrgyz Republic were included in cases and controls. All participants of both groups signed informed consent to participate. The study protocol was approved by the local institutional review board of the Institute of Molecular Biology and Medicine in Bishkek Kyrgyz Republic .Molecular methods of cervical smears tests Epithelial cell smears from the cervix were the material for molecular tests in this study. High carcinogenic risk HPV type 16 and 18 were detected and differentiated using reagents set «AmpliSense, HPV 16/18-FRTA» via PCR with hybridization/fluorescent detection in real time. This method is based on a simultaneous HPV DNA segments amplification along with the segment of β-globin gene DNA, using for the purpose of endogenous internal control. All test stages, such as nucleic acid separation, amplification, analysis and interpretation, were completed as of the producer’s manual (High-Risk Human Papilloma virus Infections). DNA extractionDNA was extracted from the white cells of peripheral blood, using the standard method of phenol-chloroform extraction. Determination of ТР53 Genotype ТР53 gene using PCR with subsequent restriction of its products and restriction fragments length polymorphism (PCR-RFLT) assay. PCR was conducted in Hybaid (UK) amplifier. We amplified ТР53 gene Arg72Pro locus with direct 5`TTGCCGTCCCAAGCAATGGATGA–3` and reverse 5`TCTGGGAAGGGACAGAAGATGAC–3` primers. Twenty µL of amplification mixture contained 1x buffer for Go-Taq-polymerase, 1.5 мМ MgCl2, 1.6 мМ of dNTP mixture, 5 pmol of each primer, 100 nanogram of genome DNA and 1 unit of Go-Taq- polymerase . Amplification protocol included preliminary denaturation at 94°С for 5 minutes with 35 subsequent cycles of denaturation at 94°С (30 sec), annealing at 60°С (30 sec) and chain elongation at 72°С for 30 sec. The program was finalized with elongation at 72°С for 5 min. Amplification products were then hydrolyzed with BstUI endonuclease  at 37о for 16 hours. Restriction products were separated usng elecroforesis in 3% agarose gel and the fragments were visualized by means of GelDoc IT system  in ultraviolet light. Arg72 allele has fragments 133 and 86 b.p. long, where as 72Pro minor allele is 199 b.p. long . Differences were considered significant with р<0.05.We used previous report from Chinese population to calculate the current study sample size using the built-in formula in Equation 6.10. In the preceding study , the odds ratio (OR) of CC in Arg72Pro carriers was 3.83. Given the number of exposed controls in our study 35%, case:control ratio 1:1 and Z-alpha 1.96, the minimum required number of cases for OR 3.83 should be 36 with the corresponding number of controls 36 also to attain 80% power. Taking stricter OR 2.38 for Pro72Pro from the same study, the minimum required number of cases would be 86. Therefore, our study sample size of 102 cases ensures sufficient study power (100% and 84% correspondingly). We analyzed data in GraphPad Prism v 5.04. We tested the observed distributions vs. the expected Hardy-Weinberg equilibrium via χSubjects’ profileAt the time of diagnosis, the age of CC patients ranged from 20 to 77 years. Forty-five women (44%) in the CC group (N=103) were in the age range from 51 to 60 years, whereas younger women with the age range 41 to 50 years were less prevalent . There were very patients in the age group of 20 to 30 years , 15 patients (14.5%) were 31-40 лет years old, 9 women (8.9%) were 61-70 years old and only 5 patients (5%) were older than 70. Among the range of existing CC stage classifications, we used FIGO scale to grade patients. In the current sample, clinical stage Ι was found in 3 patients (3%), stage ΙΙ in 35 patients (34%), stage ΙΙΙ was identified in 62 cases (60%), and stage ΙV was found in 3 (3%) patients. Squamous cancer was diagnosed in 98 (95%) women, whereas adenocarcinoma was found in 5 (5%) patients. G2 tumor was verified in 100 (97%) included women, whereas G3 in 3 (3%).Ninety women (87%) of the entire pool of 103 CC patients were infected with HPV, of whom 43.4% (39/90) had HPV type 16; 24.4% (22/90) had type 18 and 32.2% (29/90) exhibited HPV of both types (16/18). HPV was found in one subject of the control group. Distribution of genotypes and allele frequencies among subjectsТР53 Arg72Pro polymorphic locus in CC women and in control group reflected Hardy-Weinberg equilibrium . In both group we found that Arg allele and Arg72Arg genotype prevailed over the alternative variants. Arg72Arg, Arg72Pro and Pro72Pro genotype prevalence in the group of women with CC were 58%, 33% and 9% correspondingly, whereas Arg and Pro alleles’ frequency was 0.74 and 0.26.Genotype distribution of gene 2 = 1.24, р=0.54).The corresponding frequency of Arg72Arg, Arg72Pro and Pro72Pro genotypes in the control group was 52%, 35% and 13%, whereas Arg and Pro allele’ frequency was 0.70 and 0.30. We did not find significant differences in the prevalence of genotypes and alleles of gene Р53 Arg72Pro polymorphic locus when comparing CC patients with controls  . In HPV-The association of CC stage and differentiation with the genotypes of TP53 gene Arg72Pro polymorphic locus Patients were divided into three groups as of their genotypes. The first group consisted of patients with Arg72Arg homozygous genotype; the second group was formed from Arg72Pro heterozygous genotype; finally, Pro72Pro minor genotype made the third group .The association of genotype with tumor malignancy (G)In our sample, more than half of all CC had II degree of differentiation. Moderate differentiation (G2) was present in 97% cases (n=100), whereas low differentiation (G3) was identified in 3% (n=3) . As TablThe association of genotype with the disease stage (FIGO)We also analyzed the distribution of tumor stages in a sample of CC women. Clinical stage Ι was found in 3 patient (3%), stage ΙΙ in 35 patients (34%), stage ΙΙΙ was identified in 62 cases (60%), and stage ΙV was found in 3 (3%) patients. In a comparative analysis, we could not associate the stage with alleles or genotypes of P53 gene’s Arg72Pro polymorphic locus .The association of genotype with tumor histological type We also tested the association of genotypes of TP53 gene’s Arg72Pro polymorphic locus with tumor histological types. Histological examination revealed squamous cancer in 97% women with CC, whereas adenocarcinoma was found in 3%. There was no statistically significant difference in the distribution of squamous cancer or adenocarcinoma in various genotype combinations  .ТР53 gene with CC in HPV-positive women of Kyrgyz ethnicity. CC is believed to be a HPV-associated disease , and in our study, 87% (90 out of 103) women with CC had confirmed HPV. Our analysis showed that CC risk in HPV-positive women was associated with Arg allele carriage  and Arg/Arg genotype . On the contrary, Pro allele was protective against CC , as was Pro/Pro genotype . Our data also confirm the association of Pro72Arg marker of ТР53 gene is located on a short shoulder of the 17th chromosome (17p13.1) , contains 11 exons and transcribes 2.8 kbmRNA. Phosphopotein р53 is a protein product of the gene and contains 393 amino acids. р53 protein regulates cell cycle, induces apoptosis in case DNA impairment cannot be repaired, and stabilizes genome. Cell cycle regulation impairment, which is caused by р53 activity change, will promote cancer progression. Such defect can be caused both by mutations and polymorphism .ТР53 has around 200 known single nucleotide polymorphisms (SNP) [http://www-p53.iarc.fr/]. Guanine replacement with cytosine in position 215 of the fourth exon (Arg72Pro (rs1042522)) is considered one of the most meaningful polymorphisms of ТР53 gene. Such mononucleotide replacement results in two versions of р53 protein, including the one with arginine (Arg) and one with proline (Pro) in the 72nd codon . Biochemical properties of these two protein variants differ. Data from the literature show that 72Arg ТР53 protein may have a greater potency to initiate apoptosis compared to 72Pro ТР53, because 72Arg protein penetrates mitochondria quicker, and that entails cytochrome C release into the cell cytoplasm and subsequent apoptosis induction .ТР53 gene may promote CC, associated with papilloma virus. They also reported that Е6 oncoprotein, coded by HPV-18 and HPV-16, may interact with р53 protein, and that induces its degradation . Storey et al.  found that p53Arg allele is destroyed by Е6 HPV-18 much faster than p53Pro. Mutant variant of this protein loses this property, and that leads to cancer transformation. The fact that all cancer cells exhibit the mutant variant of р53 inclined the researchers to conclude that the normal variant of this protein may act as a carcinogenesis suppressor. Based on that, the authors hypothesized that allele status of Storey et al., (1998) found a 7-fold increase of CC risk in HPV-positive women with Arg72Arg genotype. Min-min et al., (2006) also found higher CC risk in HPV-positive Chinese women with Arg72Arg genotype when compared to their Pro72Pro counterparts. Other studies in India , South Africa  and Santiago  also found greater CC risk in HPV-positive women with Arg72Arg genotype. Population-based studies in the European region, including Italy , Lithuania  and Germany  identified similar association of higher CC risk in HPV-positive women with Arg72Arg genotype when compared to two other variants. On the contrary, other authors reported no association of Arg72Pro polymorphic locus with CC risk in HPV-positive women . Our findings are quite consistent with a number of other studies showing higher susceptibility of HPV-positive women to CC when having Arg72Arg genotype and 72Arg allele. ТР53 gene Arg72Arg polymorphism variants may depend on the geographical location. Thus, Arg72 allele prevalence in the Irish was 0.88; 0.72 in Italians; 0.63 in Germans; 0.65 in Russians; 0.52 in our sample of Kyrgyz; 0.42 in Koreans; 0.44 in Japanese; 0.34 in Jews; 0.33 in South Africans and 0.27 in Indians . In other words, Arg72 allele prevalence lessens closer to the equator. This may be important to verify the distribution and frequency of selected neoplasms associated with HPV 16 and 18 in the given population. Of note, the prevalence of Ethnicity is an important contributor to allele distribution in the general population since numerous studies have shown different prevalence of specific alleles, including mutations, between ethnic groups. Therefore, selection of ethnically homogenous groups is pivotal for molecular and genetic studies. In this analysis, we report genotyping data of CC patients along with controls from one laboratory and using standardized methodology. The groups were compiled from one population, which prevents inter-population variations.The strength of this report is the first presentation of the association of Arg72Pro polymorphic locus with CC in HPV-positive women in Kyrgyz population. The limitations of this study are small sample size, inclusion of only one gene for the polymorphic locus evaluation.In conclusions, given the high prevalence of papilloma viral infection among women combined with HPV etiology in CC, identification of the population with high genetic risk of CC is of significant importance. This will allow reducing such risk, since timely vaccination against HPV will be targeted. The authors declare no conflicts of interest. This study protocol was approved by the local committee on bioethics of the Institute of Molecular Biology and Medicine , Bishkek, Kyrgyz Republic.