SFRP1, Posible biomarcador en la progresión o regresión de lesiones de cérvix asociado al Virus del Papiloma Humano / SFRP1, Possible Biomarker in the Progression or Regression of Cervical Pre-neoplastic Lesions Associated with Human Papilloma Virus

Resumen Objetivo: Comparar la expresión de mRNA y proteínas de SFRP1, PTPRN, CDO1, EDNRB, CDX2, EPB41L3 y HAND1 en pacientes con lesión intra-epitelial cervical de bajo y alto grado, con posterior progresión o regresión. Material y Método: Se realizó análisis de expresión de genes mediante RT-PCR y análisis de expresión de proteínas por inmunohistoquímica. El análisis estadís tico fue realizado con las pruebas: Wilcoxon, coeficiente de correlación de Spearman e índice de concordancia. Las muestras fueron pareadas en momento 1 y momento 2. Resultados: SFRP1 mostró tendencia de mayor expresión de mRNA en lesión intra-epitelial de bajo grado (momento 2) Vs. alto grado (momento 1). La expresión de proteínas por inmunohistoquímica de SFRP1 en casos de progresión (83,3 %) mostró disminución en su graduación (p = 0,0313*); los demás genes en estudio no tuvieron cambios estadísticamente significativos. Discusión: SFRP1 mostró comportamiento ajustado a resultados de estudios previos donde se encontró hipermetilado en lesiones intra-epiteliales de alto grado; su subexpresión por hipermetilación se reportó en otros canceres, proceso que colabora con su silenciamiento y transición epitelial-mesenquimatosa del cáncer de cuello uterino. Conclusiones. SFRP1 es potencial biomarcador en lesiones preneoplásicas del cuello uterino asociadas al virus de papiloma humano.

Abstract Objective. The aim of this work was to compare the expression of mRNA and proteins of SFRP1, PTPRN, CDO1, EDNRB, CDX2, EPB41L3 and HAND1 in patients with low and high grade cervical intraepithelial lesion, with subsequent progression or regression. Material and Methods: Gene expression analysis was conducted through RT-PCR and protein expression analysis was performed by immunohistochemistry. The statistics analysis were Wilcoxon test, Spearman's correlation coefficient and concordance index. The samples were paired during moment 1 (initial patient diag nosis) and moment 2 (follow-up histological diagnosis). Results: SFRP1 showed a trend of higher mRNA expression in low-grade intra-epithelial lesions (moment 2) Vs. high-grade (moment 1). The expression of proteins by immunohistochemistry of SFRP1 in progression cases (83.3%) showed a decrease in its graduation (p = 0.0313*); the other genes under study had no statistically significant. Discussion: SFRP1 showed a biological behavior adjusted to the results of previous studies where hypermethylation was found in high-grade intra-epithelial lesions; its subexpression by hypermethylation has been reported in other cancers, a process that collaborates with its silencing and epithelial-mesenchymal tran sition of cervical. Conclusions. SFRP1 is a potential biomarker in preneoplastic lesions of the cervix associated with human papillomavirus.

Bioinformatics analysis of the role of CXC ligands in the microenvironment of head and neck tumor.

Chemokines play a significant role in cancer. CXC-motif chemokine ligands (CXCLs) are associated with the tumorigenesis and progression of head and neck squamous cell carcinoma (HNSC); however, their specific functions in the tumor microenvironment remain unclear. Here, we analyzed the molecular networks and transcriptional data of HNSC patients from the Oncomine, GEPIA, String, cBioPortal, Metascape, TISCH, and TIMER databases. To verify immune functions of CXCLs, their expression was analyzed in different immune cell types. To our knowledge, this is the first report on the correlation between CXCL9-12 and 14 expression and advanced tumor stage. CXCL2, 3, 8, 10, 13, and 16 were remarkably related to tumor immunity. Kaplan-Meier and TIMER survival analyses revealed that high expression of CXCL1, 2, 4, and 6-8 is correlated with low survival in HNSC patients, whereas high expression of CXCL9, 10, 13, 14, and 17 predicts high survival. Only CXCL13 and 14 were associated with overall survival in human papilloma virus (HPV)-negative patients. Single-cell datasets confirmed that CXCLs are associated with HNSC-related immune cells. Thus, CXCL1-6, 8-10, 12-14, and 17 could be prognostic targets for HNSC, and CXCL13 and 14 could be novel biomarkers of HPV-negative HNSC.

Detection of viral gene expression in risk-stratified biopsies reveals no active HPV in cutaneous squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) infection is known to promote the development of mucosal squamous cell carcinoma (mSCC), including pathologically high-grade lesions, but its role in cutaneous squamous cell carcinoma (cuSCC) remains unclear, particularly in lesions that are considered high risk. OBJECTIVE: We aimed to determine whether enhanced HPV transcriptional activity can be detected in high-risk cuSCC samples compared with low-grade SCC samples or normal skin. METHODS: We performed RNA sequencing of cuSCC across 23 risk-stratified skin lesions. A subset of samples was tested for the presence of HPV DNA. High-quality, non-human reads from each sample group were used for viral analysis using Microbiome Coverage Profiler. RESULTS: None of the samples analysed had detectable expression of HPV RNA, while 64% of samples tested positive for HPV DNA. All samples were found to have expression of human endogenous retrovirus, and multiple samples showed expression of other viruses. CONCLUSIONS: Viral and prophage gene expression can be monitored in cuSCC or normal skin biopsies, yet no sample in our study showed evidence of active HPV gene expression despite evidence of HPV genome presence. This suggests HPV transcription does not play a role in differentiating high-risk cuSCCs from low-risk cuSCCs or normal skin.

NanoString Technology for Human Papillomavirus Typing.

High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter® platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 104 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 104 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.

The value of p16 and HPV DNA in non-tonsillar, non-base of tongue oropharyngeal cancer.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) is dominated by tonsillar and tongue base carcinomas (TSCC/BOTSCC), but there are carcinomas at other sites, such as uvula/soft palate/pharyngeal wall here defined as other OPSCC. Human papillomavirus (HPV) positive TSCC/BOTSCC have favorable outcome, and the TNM-classification separates OPSCC into HPV mediated (p16INK4a overexpressing, p16+) and HPV unrelated OPSCC (p16INK4a non-overexpressing, p16-) cancer, but the prognostic role of p16+ in other OPSCC is unclear. AIMS/OBJECTIVES: This study therefore aimed to further investigate the prognostic role of p16+, presence of HPV DNA, or both combined in other OPSCC. MATERIAL AND METHODS: 195 other OPSCC, from patients diagnosed 2000-2018 were tested for p16, and/or presence of HPV DNA and the data correlated to outcome. RESULTS: Neither overall survival, nor disease free survival correlated to presence of p16+ or HPV DNA in other OPSCC. p16+ and HPV DNA presence were correlated (p < .0001), but the sensitivity of p16 as a surrogate marker for presence of HPV DNA was low (49%). CONCLUSIONS AND SIGNIFICANCE: The data suggest that p16+ (and p16+/HPV DNA) positive other OPSCC should be analyzed cautiously and possibly separately from the HPV mediated OPSCC staging group.

Correlation between integration of high-risk HPV genome into human DNA detected by molecular combing and the severity of cervical lesions: first results of the EXPL-HPV-002 study.

OBJECTIVES: The aim of the EXPL-HPV-002 study is to evaluate the integration of 14 high-risk HPV as a biomarker of the severity and the progression of cervical lesions. Such a „triage biomarker“ would help to reduce the number of unnecessary colposcopies, to avoid over-treatment of lesions that spontaneously regress and to better target the lesions requiring treatment. DESIGN: EXPL-HPV-002 is a prospective, open-label, single arm, GCP study conducted at 2 clinical sites in the Czech Republic. SETTINGS: Investigations centers: Private Gynecology Center, Brno; Gynecological and Obstetrical Clinic, Brno; Genotyping central lab: NRL for Papillomaviruses and polyomaviruses, IHBT, Prague; Histology Central reading: Aeskulab Pathology, Prague; Molecular combing HPV test: Genomic Vision, Bagneux. METHODS: From June 2016 to May 2018, 688 patients aged 25-65, referred to colposcopy after an abnormal Pap-smear, were enrolled in the study. Among them 60% were found HPV high-risk. The study is divided in two phases: 1. a cross-sectional phase using data collected at first visit (colposcopy images ± histology, pap-smear for HPV genotyping and molecular combing) to study the association between HPV integration status versus colposcopy and histology grades; 2. a longitudinal phase using data collected in follow-up visits: cytology at 6, 18 and 30 months and colposcopy ± histology at 12, 24 and 36 months. A pap-smear collected at 12, 24 and 36 months allows to perform genotyping and molecular combing. HPV integration status is analyzed in comparison with the evolution of lesions, viral clearance and HPV genotype. HPV genotyping and molecular combing were performed on pap-smear samples in central laboratories. Histology data were reviewed by central reading. RESULTS: The transversal phase of the study is achieved and shows that the HPV integration into the human DNA, monitored by molecular combing, can significantly differentiate normal subjects from women with cervical lesions or cancer. CONCLUSION: HPV integration into the host genome, monitored by Genomic Visions technology, is a reliable diagnostic biomarker that will greatly help clinicians to improve their medical decision tree.

Anal Cancer Precursor Lesions in HIV-Infected Persons: Tissue Human Papillomavirus Type Distribution and Impact on Treatment Response.

BACKGROUND: Data on tissue distribution of human papillomavirus types in anal high-grade squamous intraepithelial lesions are limited and the impact on treatment outcomes poorly understood. OBJECTIVE: We aimed to investigate potential predictors of treatment failure after electrocautery ablation, including human papillomavirus type(s) isolated from index lesions. DESIGN: This was a retrospective cohort study. SETTINGS: The study was conducted at a tertiary academic referral center in New York City. PATIENTS: Seventy-nine HIV-infected patients with a diagnosis of anal high-grade squamous intraepithelial lesions between January 2009 and December 2012 were included, and genomic DNA was extracted from biopsy tissue. MAIN OUTCOME MEASURES: The prevalence of human papillomavirus types in index lesions and surveillance biopsies after electrocautery ablation were analyzed to evaluate treatment response. RESULTS: Of 79 anal high-grade squamous intraepithelial lesions, 71 (90%) tested positive for ≥1 human papillomavirus type; 8 (10%) had no human papillomavirus detected. The most common type was 16 (39%), followed by 33 (15%). Human papillomavirus type 18 was seen in 3%. Sixty-one patients (77%) underwent electrocautery ablation and had subsequent surveillance biopsies. Surveillance biopsies yielded benign findings or low-grade squamous intraepithelial lesions in 31 (51%) of 61 and recurrent high-grade squamous intraepithelial lesions in 30 (49%) of 61 patients (mean follow-up: 35 mo). Ablation response did not differ significantly based on baseline demographics, smoking history, history of anogenital warts, mean CD4 T-cell count, antiretroviral-therapy use, and HIV viral load (<50 copies/mL). The recurrence of high-grade lesions was not significantly associated with high-risk human papillomavirus types detected in index lesions. LIMITATIONS: Human papillomavirus genotyping in surveillance biopsies was not performed. CONCLUSIONS: Anal high-grade squamous intraepithelial lesions in HIV-infected patients contain a wide range of human papillomavirus types, and individual lesions commonly harbor multiple types concomitantly. Recurrence of anal high-grade squamous intraepithelial lesions after electrocautery ablation occurs frequently and is not affected by high-risk human papillomavirus types. See Video Abstract at http://links.lww.com/DCR/A833.

Detección del virus del papiloma humano en muestras obtenidas mediante técnica de autotoma en un grupo de universitarias peruanas / Detection of the human papillomavirus in samples obtained by self-collection technique in a group of peruvian college students

RESUMEN Estudio transversal que determinó la frecuencia y genotipos del virus del papiloma humano de alto riesgo (VPH-AR) a través de la técnica de autotoma en un grupo de universitarias de Lima. Participaron 221 estudiantes y se detectó el ADN del VPH-AR con el método de reacción en cadena de la polimerasa (PCR). La frecuencia del VPH-AR en las participantes fue de 43,4%; de este grupo se encontraron los genotipos VPH 16 en el 15,6% y VPH 18 en el 4,2% y otros VPH-AR en el 80,2%. Se concluye que la frecuencia del VPH-AR es mayor en el grupo de universitarias de este estudio en comparación a investigaciones nacionales previas.

ABSTRACT Cross-sectional study that determined the frequency and the genotypes of the (HR-HPV) high-risk human papillomavirus through the self-collection technique in a group of college students of Lima. Two hundred twenty-one (221) students participated and the DNA of the HR-HPV was detected with polymerase chain reaction (PCR). The frequency of HR-HPV in participants was 43.4%; of this group, genotype HPV 16 was present in 15.6%, HPV 18 in 4.2%, and another HR-HPV in 80.2%. We can conclude that the frequency of HR-HPV is greater in the group of college students of this study in comparison with previous national investigations.

Development and validation of a method for human papillomavirus genotyping based on molecular beacon probes.

We describe a new assaying system for the detection and genotyping of human papillomavirus (HPV) based on linear-after-the-exponential-PCR(LATE-PCR) and melting curve analysis. The 23 most prevalent HPV strains (types 6, 11, 16, 18, 31, 33, 35, 39, 42, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 81, 82, and 83) are assayed in two sealed reaction tubes within 2 h. Good sensitivity and specificity was evaluated by testing cloned HPV DNA and clinical samples. The detection limit was 5-500 copies/reaction depending on the genotype. No cross-reactivity was observed with the other HPV types that are not covered by our method or pathogens tested which were commonly found in female genital tract. When compared with the HPV GenoArray Diagnostic kit, the results from 1104 clinical samples suggest good overall agreement between the two methods,(98.37%, 95% CI: 97.44%-98.97%) and the kappa value was 0.954. Overall, this new HPV genotyping assay system presents a simple, rapid, universally applicable, sensitive, and highly specific detection methodology that should be useful for HPV detection and genotyping, therefore, is potentially of great value in clinical application.

Detection of nucleic acids with a novel stem-loop primer rolling circle amplification technique.

This paper presents a new rolling circle amplification (RCA) technique using stem-loop primers (SLP). The technique enables detection of target DNA by either linear or exponential amplification (SLP-lRCA and SLP-eRCA) in both liquid and solid phases. For solid-phase detection, SLP-eRCA detects nucleic acids in four steps: (1) covalently immobilize an array of capture probes (CP) on a solid support; (2) hybridize the CP array with the DNA sample; (3) incubate the CP array with an RCA reaction containing two SLPs; (4) image the CP array. SLP-eRCA detects nucleic acids in liquid phase in one step: a real-time RCA reaction containing the DNA sample and two SLPs. Both liquid- and solid-phase detection methods employ a general rolling circle and an SLP. The other SLP is specific to the target. The technique was verified by detecting synthesized oligonucleotides and six different human papillomaviruses (HPVs), both in liquid phase and on a solid surface. The technique also detected two high-risk HPVs (HPV16 and HPV18) in cervical carcinoma cells (HeLa and SiHa) and clinical samples. This study provides proof-of-concept for the new RCA technique for nucleic acid detection, which overcomes major limitations of current RCA approaches.

[Detection of the human papillomavirus in samples obtained by self-collection technique in a group of peruvian college students]. / Detección del virus del papiloma humano en muestras obtenidas mediante técnica de autotoma en un grupo de universitarias peruanas.

Cross-sectional study that determined the frequency and the genotypes of the (HR-HPV) high-risk human papillomavirus through the self-collection technique in a group of college students of Lima. Two hundred twenty-one (221) students participated and the DNA of the HR-HPV was detected with polymerase chain reaction (PCR). The frequency of HR-HPV in participants was 43.4%; of this group, genotype HPV 16 was present in 15.6%, HPV 18 in 4.2%, and another HR-HPV in 80.2%. We can conclude that the frequency of HR-HPV is greater in the group of college students of this study in comparison with previous national investigations.

Estudio transversal que determinó la frecuencia y genotipos del virus del papiloma humano de alto riesgo (VPH-AR) a través de la técnica de autotoma en un grupo de universitarias de Lima. Participaron 221 estudiantes y se detectó el ADN del VPH-AR con el método de reacción en cadena de la polimerasa (PCR). La frecuencia del VPH-AR en las participantes fue de 43,4%; de este grupo se encontraron los genotipos VPH 16 en el 15,6% y VPH 18 en el 4,2% y otros VPH-AR en el 80,2%. Se concluye que la frecuencia del VPH-AR es mayor en el grupo de universitarias de este estudio en comparación a investigaciones nacionales previas.

HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection.

INTRODUCTION: Persistent high-risk HPV (HR-HPV) infection is associated with a greater risk of cervical cancer. PATIENTS AND METHODS: Statistical data on the prevalence of HR-HPV infections in the Algerian population is lacking. We conducted a prospective study of 300 women aged between 25 and 50 years, screened for cervical cancer from 2012 to 2015 in Sidi Bel Abbès, a western region of Algeria. We aimed to assess the reliability of the repeated use of the HC II test (three longitudinal HPV tests 9 months apart from each other) in diagnosing the persistence of HR-HPV infection. RESULTS: The prevalence of HR-HPV infection was 7.33% and infected women were aged 37.9±3years. For 90.9% of HR-HPV-positive patients, the infection persisted for a mean of 18.5months [95% CI: 16.9-22.1months]. Among these patients, 55.55% developed CIN1 and 11.11% developed CIN2. The sensitivity of the HC II test was 81.74% [95% CI: 71.3-89.6] and its positive predictive value associated with abnormal cervical biopsy was 27.49% [95% CI: 16.0-33.33]. CONCLUSION: Repeating the HC II test is a good predictor for identifying women at high risk of cervical cancer.

Comparison of the cervista HPV HR test and luminex XMAP technology for the diagnosis of cervical intraepithelial neoplasia.

OBJECTIVES: This study was designed to compare the Cervista high risk (HR) human papillomavirus (HPV) test with Luminex XMAP technology for the detection of the relationship between HPV infection and cervical intraepithelial neoplasia. METHODS: In total, 3280 patients in a cervical specialty clinic were divided into two groups for either Cervista (1855 patients) or Luminex (1425 patients). Subsequent colposcopy examinations were performed in 1270 women with cytologic results showing ASCUS or higher level lesions. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were evaluated. RESULTS: The positive rates of the Cervista and Luminex groups were 61.48% and 67.43%, respectively, and occurrence in those 30-40 years old was most common. The typing of HPV showed that A9 positive cases were the most prevalent genotype (33.53%), followed by A5/A6 (16.44%) and A7 (11.37%) in the Cervista group, and HPV-16 was the most prevalent genotype (25.81%), followed by HPV-18 (18.6%) and HPV-31 (8.6%) in the Luminex group. Moreover, the overall concordance rate was 96.26% (95% confidence interval, 93.51-99.00%) in the 187 women with cytologic results of ASCUS or higher. There were no significant differences in the positive rates of HPV between the Cervista and Luminex groups, and both had a high sensitivity and NPV. CONCLUSIONS: The results showed a high good concordance between the two methods in diagnostic accuracy. Among the patients in the cervical specialty clinic, both the A9 group of HPV and HPV-16 showed the highest positive rate. Cervista and Luminex shared similar a clinical value in the detection of CIN2 or higher.

RNA chromogenic in situ hybridization assay with clinical automated platform is a sensitive method in detecting high-risk human papillomavirus in squamous cell carcinoma.

Detection of active human papillomavirus (HPV) is clinically important because its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas. Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 squamous cell carcinomas. Importantly, a clinical automated platform gives (1) consistent and reproducible results for HPV ISH and (2) better standardization across clinical laboratories. Compared with polymerase chain reaction results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%) and more specific (87% versus 74%). When only accounting for 2+-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity, whereas specificity was only 55%. In 3 cases, both RNA and DNA ISH were positive, whereas polymerase chain reaction results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed, paraffin-embedded tissues and has the potential of being a standalone clinical test.

Avaliação do rastreamento do câncer do colo do útero e sua periodicidade em um município de Santa Catarina / Assessment of cervical cancer screening and its periodicity in a city of Santa Catarina state

Modelo do estudo: Estudo de Prevalência. Objetivo do estudo: Conhecer a prevalência dos resultados alterados dos exames preventivos para câncer do colo do útero e a sua regularidade na coleta. Metodologia: Estudo observacional, transversal e retrospectivo. Foram estudadas 3.425 mulheres usuárias do Sistema Único de Saúde e 9.436 exames citopatológicos de novembro de 2003 a janeiro de 2014. Resultados: A mediana da idade foi de 35 anos (25,0 - 46,0, percentil 25 a 75%). No primeiro exame citopatológico, 2,7% das mulheres apresentaram alterações em células epiteliais. As Células escamosas atípicas de significado incerto (ASC-US) (n=54; 1,7%) e as lesões intra-epiteliais de baixo grau (LIEBG) (n=24; 0,7%) foram as mais frequentes. Ao longo do período observado houveram 87 novas alterações citopatológicas, totalizando 173 exames alterados em 9.436 analisados. Quanto a regularidade, 58,5% pacientes repetiram a segunda coleta. Conclusões: Houve um perfil predominante de mulheres jovens, sendo o diagnóstico de ASC-US o mais frequente. A flora bacteriana mais frequente foi Lactobacillus principalmente em mulheres jovens. (AU)

Study Model: Prevalence Study. Study objective: To determine the prevalence of the abnormal cervical cancer screening test (PAP) and regularity in repeat it. Methods: An observational, cross-sectional and retrospective study. 3,425 women, users of the Brazilian Unified Health System, and 9,436 cytopathology, conducted from November 2003 to January 2014, were studied. Results: The median age was 35 years (25.0 to 46.0, 25 to 75% percentile). In the first PAP, 2.7% of women examined had alterations in epithelial cells. The atypical squamous cells of uncertain significance (ASC-US) (n = 54; 1.7%), and low-grade intraepithelial lesion (LSIL) (n = 24; 0.7%) were the most frequent alterations found. During the observed period, 87 new abnormal PAPs were found, totalling 173 altered tests in 9436 analysed. Regarding regularity of test, 58.5% patients repeated the second PAP. Conclusions: There was a preferential profile of young women, and the diagnosis of ASC-US was the most frequently found. Lactobacillus, especially in young women, was the main bacterial flora. (AU)

HPV-based strategy in follow-up of patients treated for high-grade cervical intra-epithelial neoplasia: 5-year results in a public health surveillance setting.

OBJECTIVE: Human papillomavirus (HPV) DNA testing is used increasingly for measuring the outcome of treatment for high-grade cervical intra-epithelial neoplasia (CIN2+). However, there is no international consensus regarding the number of tests and follow-up visits necessary in the post-treatment surveillance. A negative HPV DNA test result may permit relaxing the intensive post-treatment surveillance, but this possibility has not been standardized by all institutions to date. STUDY DESIGN: In 2008, the surveillance programme covering the Emilia-Romagna region in northern Italy adopted the HPV DNA test as a routine tool in the follow-up of women treated for CIN2+. Data from a prospective 5-year study are reported herein. Three hundred and ten patients treated for CIN2+ with a loop electrosurgical excision procedure underwent HPV DNA testing, cytology and colposcopy at 6 months post treatment. If all three tests were negative, women were tested at 18 months with cytology and colposcopy. If any of the three tests were positive, women were tested at 12, 18 and 24 months with cytology and colposcopy. When appropriate, a colposcopy-directed biopsy or CIN2+ retreatment was performed. After 18-24 months, the patients were tested annually with cytology for 3 years. RESULTS: None of the 172 (55%) women who were HPV negative at 6 months were found to have residual/recurrent CIN2+ during the surveillance period. In contrast, among the 138 (45%) HPV-positive women, 17 cases of residual/recurrent CIN2+ (17/138; 12.3%) were identified between 6 and 24 months. CONCLUSION: HPV DNA testing at six months after treatment for CIN2+ effectively identifies women who are disease free (HPV negative), and for whom a single follow-up at 18 months is sufficient.

Performance of a new HPV and biomarker assay in the management of hrHPV positive women: Subanalysis of the ongoing multicenter TRACE clinical trial (n > 6,000) to evaluate POU4F3 methylation as a potential biomarker of cervical precancer and cancer.

The ongoing Triage and Risk Assessment of Cervical Precancer by Epigenetic Biomarker (TRACE) prospective, multicenter study aimed to provide a clinical evaluation of the CONFIDENCE™ assay, which comprises a human papillomavirus (HPV) DNA and a human epigenetic biomarker test. Between 2013 and 2015 over 6,000 women aged 18 or older were recruited in Hungary. Liquid-based cytology (LBC), high-risk HPV (hrHPV) DNA detection and single target host gene methylation test of the promoter sequence of the POU4F3 gene by quantitative methylation-specific polymerase chain reaction (PCR) were performed from the same liquid-based cytology sample. The current analysis is focused on the baseline cross-sectional clinical results of 5,384 LBC samples collected from subjects aged 25 years or older. The performance of the CONFIDENCE HPV™ test was found to be comparable to the cobas® HPV test with good agreement. When applying the CONFIDENCE Marker™ test alone in hrHPV positives, it showed significantly higher sensitivity with matching specificity compared to LBC-based triage. For CIN3+ histological endpoint in the age group of 25-65 and 30-65, the methylation test of POU4F3 achieved relative sensitivities of 1.74 (95% CI: 1.25-2.33) and 1.64 (95% CI: 1.08-2.27), respectively, after verification bias adjustment. On the basis of our findings, POU4F3 methylation as a triage test of hrHPV positives appears to be a noteworthy method. We can reasonably assume that its quantitative nature offers the potential for a more objective and discriminative risk assessment tool in the prevention and diagnostics of high-grade cervical intraepithelial neoplasia (CIN) lesions and cervical cancer.

Laryngeal Cancer, HPV DNA vs E6/E7 mRNA Test: A Systematic Review.

OBJECTIVE: The reported range of involvement of human papillomavirus (HPV) in laryngeal squamous cell carcinoma (SCC) is wide because of the methods used to detect HPV. DATA SOURCES: A computerized Medline study was carried out using the following as key words: "Papillomavirus Infections"[Mesh] and "Laryngeal Neoplasms"[Mesh]. MATERIALS AND METHODS: Studies that were included were written in English and reported results of HPV DNA with RNA in laryngeal SCC. RESULTS: There were six reported HPV mRNA extraction. Among these studies, Lewis et al reported that out of the 31 cases analyzed, only 2 were HPV DNA+ and of these only 1 was mRNA HPV+ (3%). Halec et al reported 102 cases of which 32 were HPV DNA+ cases and of which only 6 were mRNA+ (5%). Chernock et al reported 76 cases of which 13 were HPV DNA+ cases and of which 4 were mRNA+ (5%). Masand et al reported 8 cases of which 1 was HPV DNA+ case and none was mRNA+. Gheit et al reported 43 cases of which 4 were HPV DNA+ cases and of which 2 were mRNA+ (4%). Castellsagné et al reported 1042 cases of which 59 were HPV DNA+ case and of which 51 were mRNA+ (4.8%) CONCLUSIONS: When determining the role of HPV in laryngeal SCC, evidence of HPV DNA warrants further examination for E6/E7 mRNA as simple assays such as p16 are nonspecific in laryngeal SCC. Further studies of HPV and its role in laryngeal SCC are warranted.

[Molecular diagnosis of human papillomaviruses (HPV): What test(s) in clinical practice?] / Diagnostic moléculaire des papillomavirus humains (HPV) : quel(s) test(s) en pratique clinique ?

Prescription of an HPV test in practice will enable the clinician to optimize the monitoring and the management of patients, especially in the context of cervical cancer screening. Numerous HPV tests are available that present different analytical and clinical sensitivity and specificity. International recommendations on clinical performance of HPV tests used for cervical cancer screening have been published by a group of experts, and tests that meet these performance criteria should be used. Apart from the HPV detection kit, the whole circuit from sampling to report of the results must be considered. This implies that the pre-analytical (sampling, quality of sample collection medium, storage condition and sample transportation…) and post-analytical steps (quality of result reporting, providing expert advices…) are also standardized. For this purpose, medical-biology laboratories are subjected to a COFRAC certification, as defined by the international standard ISO 15189 providing quality criteria for any clinical laboratory test and HPV test in particular.

Excellent analytical and clinical performance of a dry self-sampling device for human papillomavirus detection in an urban Chinese referral population.

AIM: To evaluate the analytical and clinical effectiveness of cervicovaginal self-sampling with a dry sampling device (Evalyn Brush) for high-risk human papillomavirus (hr-HPV) testing and detection of cervical disease. METHODS: The study population consisted of 101 patients from a large gynecological outpatient clinic in Shanghai referred for abnormal cervical screening results and 101 women without cervical lesions. Self-samples obtained in the clinic and physician-collected cervical specimens (reference) were stored at -20 °C for 16-18 weeks and then transferred to 20 ml of ThinPrep medium and tested for hr-HPV using a multiplex real time polymerase chain reaction assay. All women had a colposcopic examination with a Pap smear and directed or random biopsies. RESULTS: High risk-HPV was detected in 92 patients (45.5%) with the self-collected cervicovaginal specimens and in 93 (46.0%) with the physician-collected cervical specimens, resulting in an agreement of 97.5% and a Kappa of 0.95 (95% confidence interval 0.91-0.99). Among all of the included women, 46 (22.8%) had cervical intraepithelial neoplasia grade 3 or worse (cervical intraepithelial neoplasia 3+). Hr-HPV was found in 43 of these patients (93.5%) with self-sampling and in 44 (95.7%) with the physician-collected specimens. CONCLUSIONS: Self-collected dry cervicovaginal samples transferred to ThinPrep medium and tested for hr-HPV using a clinically validated polymerase chain reaction assay showed very good agreement with physician-collected cervical specimens and a very high hr-HPV positivity rate for cervical intraepithelial neoplasia 3 +.