CIN Grade Research Articles

P-B14 Methylation as a triage marker in a hHPV+ population in Nigeria

Objectives: To assess the feasibility of the Precursor-M methylation test as a triage marker on self-collected and hospital-collected samples amongst a hrHPV+ population in a resource limited setting. Methods: A total of 400 women were invited to participate through flyer recruitment in Abuja, Nigeria. After randomization, 200 were asked to self-collect and deposit cervical samples at designated collection points and 200 were invited to the hospital for a hospital-collected sample (HCS). A dry flocked swab was used in both groups. DNA was isolated with the nucliSENS easyMAG, tested with the GP5+/6+-PCR-EIA. A qPCR (RNaseP) to assess the level of DNA was performed. EIA+ samples with a Cq<28 (human genomic DNA concentration >1 ng/μL) were tested with the Precursor-M methylation kit. Cut-offs for positivity were applied as described by the test manufacturer. Results: Samples from all 298 responding women (74.5% response) were included. The mean age of the women was 41.1 (SD 7.8, range 30–62). A total of 29 samples were found hrHPV+ by GP5+/6+; 28 (96.6%) contained enough DNA for Precursor-M testing (11 HCS, 17 self-samplers). 28.6% (8/28), including 3 HCS and 5 self-samplers, were methylation positive. Conclusions: The percentage of 28.6% methylation positive samples corresponds with previous findings (De Strooper et al, 2014). In order to assess the feasibility of methylation testing as a triage marker in the detection of high grade CIN lesions in an all-molecular screening setting, a follow-up study collecting a colposcopy directed biopsy will be performed. Results are expected by September 2015.

Expression of Daxx and HPV16 E6 and its significance in cervical lesions

To investigate the expression of death domain associated protein (Daxx) and human papillomavirus 16 E6 protein (HPV16 E6) in cervical lesions and analyze their significance. Immunohistochemical SABC method was used to detect the expression of Daxx and HPV16 E6 in 194 cases of cervical tissues with different lesions. (1) The positive expression rate of Daxx was 28.57% (12/42), 40.00% (18/45), 65.91% (29/44), 66.67% (42/63), respectively, in chronic cervicitis, cervical intraepithelial neoplasia I-II (CIN I-II), CIN III and cervical squamous cell carcinoma. The positive expression rate of HPV16 E6 was 15.38% (6/39), 36.17% (17/47), 46.30% (25/54), 100.00% (24/24) in the above four groups. The positive rates in cervical cancer group and high grade CIN group were significantly higher than these in low level of CIN group and chronic cervicitis group (P<0.05). (2) The expression of Daxx was stronger in HPV16 E6 high positive group than that in HPV16 E6 low positive group (P<0.05). (3) There was no significant relationship between the expression of Daxx and pathological classification, clinical stage, situation of lymph node metastasis or patients' age in cervical squamous cell carcinoma (P>0.05). (4) There was significant positive relationship between Daxx and HPV16 E6 expression (r=0.695, P<0.05). The expression of Daxx and HPV16 E6 gradually increases with cervical lesion degree aggravating. Here might be synergy between them, and both could promote the development of cervical lesions.

Human leukocyte antigen class II DRB1*1302 allele protects against cervical cancer: At which step of multistage carcinogenesis?

We investigated the role of human leukocyte antigen (HLA) class II alleles in multistage cervical carcinogenesis. Cross-sectional analysis for HLA association with cervical cancer included 1253 Japanese women: normal cytology (NL, n = 341), cervical intraepithelial neoplasia grade 1 (CIN1, n = 505), CIN grade 2 or 3 (CIN2/3, n = 96), or invasive cervical cancer (ICC, n = 311). The HLA class II allele frequencies were compared by Fisher’s exact test or the χ2-test. The Bonferroni adjustment corrected for multiple comparisons. Among the study subjects, 454 women with low-grade squamous intraepithelial lesion cytology were prospectively monitored by cytology and colposcopy every 3–4 months to analyze cumulative risk of CIN3 within the next 10 years in relation to HLA class II alleles. HLA class II DRB1*1302 allele frequency was similar between women with NL (11.7%) and CIN1 (11.9%), but significantly decreased to 5.2% for CIN2/3 and 5.8% for ICC (P = 0.0003). Correction for multiple testing did not change this finding. In women with low-grade squamous intraepithelial lesion cytology, the cumulative risk of CIN3 diagnosed within 10 years was significantly reduced among DRB1*1302-positive women (3.2% vs. 23.7%, P = 0.03). In conclusion, the two different types of analysis in this single study showed the protective effect of the DRB1*1302 allele against progression from CIN1 to CIN2/3.

PAX1 methylation as an auxiliary biomarker for cervical cancer screening: A meta-analysis

ObjectiveSeveral studies have implicated PAX1 epigenetic regulation in cervical neoplasia. The aim of this meta-analysis was to assess PAX1 gene methylation as a potential biomarker in cervical cancer screening. MethodsA systematical search of all major databases was performed, in order to include all relevant publications in English until December 31st 2014. Studies with insufficient data, conducted in experimental models or associated with other comorbidities were excluded from the meta-analysis. Summary receiver operating characteristics (SROC) for Cervical Intraepithelial Neoplasia grade 2 or worse (CIN2+) versus normal, and CIN grade 3 or worse (CIN3+) versus normal, were estimated using the bivariate model. ResultsOut of the 20 initially included studies, finally 7 (comprising of 1385 subjects with various stages of CIN and normal cervical pathology) met the inclusion criteria. The sensitivity of CIN2+ versus normal was estimated to be 0.66 (CI 95%, 0.46-0.81) and the specificity 0.92 (CI 95%, 0.88-0.95). On the other hand, the sensitivity of CIN3+ versus normal was 0.77 (CI 95%, 0.58-0.89) and the specificity 0.92 (CI 95%, 0.88-0.94). Moreover, the area under the curve (AUC) in the former case was 0.923, and in the latter 0.931. ConclusionThe results of this meta-analysis support the utility of PAX1 methylation as an auxiliary biomarker in cervical cancer screening. PAX1 could be used effectively to increase the specificity of HPV DNA by detecting women with more advanced cervical abnormalities.

HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening

ObjectiveCervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women. MethodsWe developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C. ResultsHPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5–24months later had progressed to ≥CIN2. ConclusionsWe successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression.

The Interpretive Variability of Cervical Biopsies and Its Relationship to HPV Status.

Diagnostic interpretation of a cervical biopsy is a key element in the decision to treat or not to treat a woman with an abnormal screening test. This study assesses the variability of these diagnostic interpretations on a population basis using the New Mexico HPV Pap Registry database. An experienced panel of gynecologic pathologists reviewed a stratified random sample of 6272 biopsies, which was then extrapolated to the entire population of 21,297 biopsies read by the community pathologists. Diagnoses by the community and panel pathologists were compared, and paired diagnoses were correlated with positivity for human papillomavirus 16 (HPV16) and any high-risk HPV as objective measures of progressive potential. Panel agreement with the community diagnosis was 38.2% for cervical intraepithelial neoplasia grade 1 (CIN1), 38.0% for CIN grade 2 (CIN2), 68.0% for CIN grade 3 (CIN3), and 70.6% for cancer. The κ value was 0.46 overall but higher for dichotomous categorization based on CIN2 or CIN3 cutoff points (0.68 and 0.67, respectively). On a population basis, there were fewer CIN1 and more negative diagnoses in the panel review but similar proportions of CIN2 and CIN3. HPV16 and high-risk HPV positivity increased with disease severity, but panel review did not improve the correlation of higher-grade disease with these objective measures. In this population-based study of the variability in cervical diagnoses, we noted significant variability in the community and panel diagnoses, especially for CIN2, the threshold for excisional treatment. New biomarkers are needed to more accurately stratify precursor lesions according to their malignant potential.

Accuracy of Colposcopically Directed Biopsy: Results from an Online Quality Assurance Programme for Colposcopy in a Population-Based Cervical Screening Setting in Italy.

Purpose. To report the accuracy of colposcopically directed biopsy in an internet-based colposcopy quality assurance programme in northern Italy. Methods. A web application was made accessible on the website of the regional Administration. Fifty-nine colposcopists out of the registered 65 logged in, viewed a posted set of 50 digital colpophotographs, classified them for colposcopic impression and need for biopsy, and indicated the most appropriate site for biopsy with a left-button mouse click on the image. Results. Total biopsy failure rate, comprising both nonbiopsy and incorrect selection of biopsy site, was 0.20 in CIN1, 0.11 in CIN2, 0.09 in CIN3, and 0.02 in carcinoma. Errors in the selection of biopsy site were stable between 0.08 and 0.09 in the three grades of CIN while decreasing to 0.01 in carcinoma. In multivariate analysis, the risk of incorrect selection of biopsy site was 1.97 for CIN2, 2.52 for CIN3, and 0.29 for carcinoma versus CIN1. Conclusions. Although total biopsy failure rate decreased regularly with increasing severity of histological diagnosis, the rate of incorrect selection of biopsy site was stable up to CIN3. In multivariate analysis, CIN2 and CIN3 had an independently increased risk of incorrect selection of biopsy site.

P16INK4a immunohistochemistry in cervical biopsy specimens: A systematic review and meta-analysis of the interobserver agreement.

The interpretation of cervical biopsy specimens guides management of women with suspected cervical cancer precursors. However, morphologic evaluation is subjective and has low interobserver agreement. Addition of p16(INK4a) immunohistochemistry may improve interpretation. We performed a systematic review and meta-analysis of published data on interobserver agreement of p16(INK4a) positivity using p16(INK4a) immunohistochemistry and of cervical intraepithelial neoplasia grade 2 (CIN2+) and CIN grade 3 (CIN3+) classification using H&E morphology in conjunction with p16(INK4a) in comparison with H&E morphology alone. The literature search revealed five eligible articles. The results show strong agreement of pathologists' interpretation of cervical biopsy specimens as p16(INK4a) positive or negative (pooled κ = 0.90; 95% confidence interval [CI], 0.88-0.92) and significantly higher agreement for a CIN2+ diagnosis with H&E morphology in conjunction with p16(INK4a) (κ = 0.73; 95% CI, 0.67-0.79) compared with H&E morphology alone (κ = 0.41; 95% CI, 0.17-0.65). Also, a slightly higher agreement for CIN3+ can be observed (κ = 0.66; 95% CI, 0.39-0.94 for H&E morphology in conjunction with p16(INK4a) and κ = 0.61; 95% CI, 0.44-0.78 for H&E morphology alone), but this difference was not statistically significant. The published literature indicates improved interobserver agreement of the diagnosis of CIN2+ with the conjunctive use of H&E morphology with p16(INK4a) immunohistochemistry compared with H&E morphology alone.

Folate and vitamin B12 may play a critical role in lowering the HPV 16 methylation-associated risk of developing higher grades of CIN.

We previously reported that a higher degree of methylation of CpG sites in the promoter (positions 31, 37, 43, 52, and 58) and enhancer site 7862 of human papillomavirus (HPV) 16 was associated with a lower likelihood of being diagnosed with HPV 16-associated CIN 2+. The purpose of this study was to replicate our previous findings and, in addition, to evaluate the influence of plasma concentrations of folate and vitamin B12 on the degree of HPV 16 methylation (HPV 16m). The study included 315 HPV 16-positive women diagnosed with either CIN 2+ or ≤CIN 1. Pyrosequencing technology was used to quantify the degree of HPV 16m. We reproduced the previously reported inverse association between HPV 16m and risk of being diagnosed with CIN 2+. In addition, we observed that women with higher plasma folate and HPV 16m or those with higher plasma vitamin B12 and HPV 16m were 75% (P < 0.01) and 60% (P = 0.02) less likely to be diagnosed with CIN 2+, respectively. With a tertile increase in the plasma folate or vitamin B12, there was a 50% (P = 0.03) and 40% (P = 0.07) increase in the odds of having a higher degree of HPV 16m, respectively. This study provides initial evidence that methyl donor micronutrients, folate and vitamin B12, may play an important role in maintaining a desirably high degree of methylation at specific CpG sites in the HPV E6 promoter and enhancer that are associated with the likelihood of being diagnosed with CIN 2+.

Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer

ObjectivesMethylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping. Methods1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33–63years, n=46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3+) of 50%, 60%, 70%, and 80%. ResultsThe CIN3+ sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3+ sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3+ sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%). ConclusionsCombined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3+ sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.

Evaluation of p16, human papillomavirus capsid protein L1 and Ki-67 in cervical intraepithelial lesions: Potential utility in diagnosis and prognosis

Cervical dysplasia, a potentially precancerous lesion, has increased in young women. Detection of cervical dysplasia is important for reducing morbidity and mortality in cervical cancer. This study analyzes the immunohistochemical expression of p16, HPV L1 capsid protein and Ki-67 in cervical intraepithelial lesions, and correlates them with lesion grade to develop a set of markers for diagnosis and detect the prognosis of cervical cancer precursors. Seventy-five specimens were analyzed, including 15 cases of CIN 1, 28 cases of CIN 2, 20 cases of CIN 3, and 12 cervical squamous carcinomas, besides 10 normal cervical tissues. They were stained for p16, HPV L1 and Ki-67. Sensitivity, specificity, predictive values and accuracy were evaluated for each marker. p16 expression increased during progression from CIN 1 to carcinoma. HPV L1 positivity was detected in CIN 2 and decreased gradually as the CIN grade increased but disappeared in carcinoma. Strong Ki-67 expression was observed in high grades CIN and carcinoma. p16, HPV L1 and Ki-67 were sensitive but with variable specificity in detecting CIN lesions. p16, HPV L1 and Ki-67 are useful markers in establishing the risk of high-grade CIN. They complete each other to reach an accurate diagnosis and to detect the prognosis.

Management of women with low grade cytology: how reassuring is a normal colposcopy examination?

To determine the rate of cervical intraepithelial neoplasia grade 2 (CIN2+) in women with low grade cervical cytology and a normal colposcopy examination over 3 years of follow-up. Cohort study within a randomised controlled trial. NHS Cervical Screening Programmes in Grampian, Tayside and Nottingham. Eight hundred and eighty-four women aged 20-59 years with borderline nuclear abnormalities (BNA) or mild dyskaryosis with a normal and adequate colposcopy examination. Samples at baseline were tested for 14 high-risk (Hr) types using GP5+6+ methodology and for HPV 16 and 18 using type-specific primers. Women were followed up post-colposcopy by cervical cytology at 6-month intervals in primary care. After 3 years, women were invited for an exit colposcopy examination and underwent LLETZ if any colposcopic abnormality was identified. Absolute and relative risks of CIN2+ during follow-up and/or at exit colposcopy. The median age was 36 years. The absolute risk of developing CIN2+ within 3 years was 1.86 per 100 woman years and for CIN3+, 0.64 per 100 woman years. One microinvasive cancer was identified. The relative risk (RR) was highest for women with initial mild dyskaryosis who were HrHPV-positive (RR 5.86, 95% confidence interval 2.53-13.56) compared with women with BNA who were HrHPV-negative. For women with low grade cervical cytology, the risk of a high grade CIN within 3 years of a normal colposcopy examination is low. Women can be reassured that, even with a positive HPV test, the risk of developing CIN2 or worse is sufficiently low to return to the routine 3-year recall.

Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression

Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers.

Age and geographic variability of human papillomavirus high-risk genotype distribution in a large unvaccinated population and of vaccination impact on HPV prevalence

BackgroundThe prevalence of infections with human papillomavirus (HPV) specific genotypes differs by age and areas. Knowledge of these differences will help predicting how prophylactic HPV vaccination and screening program could best be integrated. ObjectivesTo investigate variations in the HPV distribution between areas and ages in Italy and the impact of vaccination on HPV prevalence. Study design37,367 women aged 25–60 years who attended cervical screening in eight different areas in Northern and Central Italy were tested for HPV infection with the high-risk hybrid capture (hr-HC2) assay. hr-HC2 positive samples were genotyped by an intensive integrated strategy. Resultshr-HPV types were detected in 79.1% of HC2 positive women. HPV16 was the most frequent type, followed by HPV31, HPV18 and HPV56. A statistically significant variability in HPV type distribution between centres (overall χ84df2=195.86p<0.001) was observed. No significant overall difference in the HPV type distribution was observed in the age groups 25–34, 35–44 and 45–60 years. Considering cross-protection, overall 57.6% (95%CI 56.0–59.3) of all infections by hr-HPV types was preventable by vaccination with the bivalent vaccine and 49% (95%CI 46.9–51.1) with the quadrivalent vaccine. The variability between centres was statistically significant with both bivalent (χ7df2=43.8, p<0.0001) and quadrivalent vaccine (χ7df2=32.9, p<0.0001). ConclusionsWe observed differences in HPV genotype distribution according to centres but not to age. Results suggest that the higher proportion of HPV16/18 related high grade CIN in younger women could be the result of faster progression and not of earlier infection by these types.

HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade

Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.

Expression of HPV‐related biomarkers and grade of cervical intraepithelial lesion at treatment

New human papillomavirus (HPV)-related biomarkers may allow better identification of clinically significant lesions that warrant excision and, conversely, identification of the false positive cases that have been overreported by traditional techniques. The aim of this study was to investigate how the expression of several HPV-related biomarkers correlates to the severity of the lesion at treatment. Prospective observational study. University Hospital (2009-2011). All women submitted for excisional treatment. A liquid-based cytology sample was obtained before treatment and was tested for HPV typing, mRNA E6 & E7 with NASBA or flow cytometry and p16. All women had histological diagnosis in the form of excisional cone (gold standard). Correlation of HPV biomarker positivity rates to the grade of the lesion at treatment histology. Two hundred women were recruited: 23 were found to have negative histology (11.5%), 79 (39.5%) CIN1, 50 (25.0%) CIN2 and 48 (24.0%) CIN3. All biomarkers (HPV DNA typing, HR HPV, single HPV 16/18, mRNA E6 & E7 expression and p16) revealed an increased linear positivity rate with increasing severity and grade of the lesion (chi-squared test for trend p < 0.05). This was stronger for HPV (all and high-risk) followed by mRNA with NASBA, flow cytometry, HPV 16/18 and ultimately p16 immunostaining. The linear correlation between various HPV-related biomarkers and the grade of the lesion suggests that these biomarkers may prove to be useful in the prediction of CIN grade and, as a result, the need for treatment.

Accuracy of human papillomavirus testing on self-collected versus clinician-collected samples: a meta-analysis

Screening for human papillomavirus (HPV) infection is more effective in reducing the incidence of cervical cancer than screening using Pap smears. Moreover, HPV testing can be done on a vaginal sample self-taken by a woman, which offers an opportunity to improve screening coverage. However, the clinical accuracy of HPV testing on self-samples is not well-known. We assessed whether HPV testing on self-collected samples is equivalent to HPV testing on samples collected by clinicians. We identified relevant studies through a search of PubMed, Embase, and CENTRAL. Studies were eligible for inclusion if they fulfilled all of the following selection criteria: a cervical cell sample was self-collected by a woman followed by a sample taken by a clinician; a high-risk HPV test was done on the self-sample (index test) and HPV-testing or cytological interpretation was done on the specimen collected by the clinician (comparator tests); and the presence or absence of cervical intraepithelial neoplasia grade 2 (CIN2) or worse was verified by colposcopy and biopsy in all enrolled women or in women with one or more positive tests. The absolute accuracy for finding CIN2 or worse, or CIN grade 3 (CIN3) or worse of the index and comparator tests as well as the relative accuracy of the index versus the comparator tests were pooled using bivariate normal models and random effect models. We included data from 36 studies, which altogether enrolled 154 556 women. The absolute accuracy varied by clinical setting. In the context of screening, HPV testing on self-samples detected, on average, 76% (95% CI 69-82) of CIN2 or worse and 84% (72-92) of CIN3 or worse. The pooled absolute specificity to exclude CIN2 or worse was 86% (83-89) and 87% (84-90) to exclude CIN3 or worse. The variation of the relative accuracy of HPV testing on self-samples compared with tests on clinician-taken samples was low across settings, enabling pooling of the relative accuracy over all studies. The pooled sensitivity of HPV testing on self-samples was lower than HPV testing on a clinician-taken sample (ratio 0·88 [95% CI 0·85-0·91] for CIN2 or worse and 0·89 [0·83-0·96] for CIN3 or worse). Also specificity was lower in self-samples versus clinician-taken samples (ratio 0·96 [0·95-0·97] for CIN2 or worse and 0·96 [0·93-0·99] for CIN3 or worse). HPV testing with signal-based assays on self-samples was less sensitive and specific than testing on clinician-based samples. By contrast, some PCR-based HPV tests generally showed similar sensitivity on both self-samples and clinician-based samples. In screening programmes using signal-based assays, sampling by a clinician should be recommended. However, HPV testing on a self-sample can be suggested as an additional strategy to reach women not participating in the regular screening programme. Some PCR-based HPV tests could be considered for routine screening after careful piloting assessing feasibility, logistics, population compliance, and costs. The 7th Framework Programme of the European Commission, the Belgian Foundation against Cancer, the International Agency for Research on Cancer, and the German Guideline Program in Oncology.

Immunodetection of phosphohistone H3 (PHH3) in cervical squamous neoplasia

<h3>Background</h3> Phosphohistone H3 (PHH3) is a core protein histone detected during mitosis and therefore used to evaluate the mitotic activity of tumors. Immunohistochemical detection depends on its phosphorylation at serine 10 or serine 28. Intrae-pithelial levels of mitotic figures are helpful in grading cervical intraepithelial neoplasia and are of prognostic relevance in cervical squamous carcinoma. <h3>Design</h3> PHH3 immunoexpression was evaluated in formalin-fixed, paraffin-embedded archival cervical epithelia consisting of normal squamous epithelia (NE, n=54), low-grade squa-mous intraepithelial neoplasia (CIN1, n=25), high-grade squamous intraepithelial neoplasia (CIN2/3, n=44) and invasive squamous cell carcinoma (SCC, n=64). A standard immunohis-tochemical technique was performed using an automated immu-nostainer with a prediluted rabbit polyclonal antibody to PHH3. PHH3 indices were grouped in three categories of low (0–9), intermediate (10–24) and high (>24) numbers in ten high power fields. <h3>Results</h3> Immunostaining for PHH3 was seen in nuclear patterns. Mitotic figures were consistently decorated as were occasional nuclei without morphological features of mitosis, which may be attributed to pre- or post-mitotic states. Staining was either diffuse or coarsely granular. Exclusively low PHH3 indices with scarce staining signals in the basal/parabasal epithelial compounds were noted in NE and CIN1. They were increased in a stepwise manner to CIN2/3 and to SCC (<i>p</i><0.0001, chi-square test). In CIN2/3, staining was observed up to the middle and upper epithelial levels. In SCC, immunoreactivity was mainly detected in cells next to central horn pearls at invasive margins, and was seen diffusely in areas of non-keratinizing tumor cells. In the individual cohorts, significantly increased indices were recorded between CIN1 and CIN2/3 (<i>p</i><0.0001) as well as CIN2/3 and SCC (<i>p</i>=0.0005). In SCC, indices were increased between FIGO stage I and stages II to IV (<i>p</i>=0.0028). There was no significant relation of PHH3 indices and prognosis. <h3>Conclusions</h3> Immunohistochemical detection of PHH3 as a mitosis-associated factor is a useful aid in determining the grade of CIN, and reflects the local progression of SCC of the uterine cervix by its relation to increased FIGO stage.

Vaginal evisceration: an unexpected complication of conization.

Background. Large loop excision of the transformation zone (LLETZ) is routinely performed for the management of high grade intracervical neoplasia (CIN). Several uncommon complications have been described, including postoperative peritonitis, pseudoaneurysm of uterine artery, and bowel fistula. We report a unique case of postoperative vaginal evisceration and the subsequent management. Case. A 73-years-old woman underwent LLETZ for high grade CIN. On postoperative day 3, she was admitted for small bowel evisceration through the vagina. Surgical management was based on combined laparoscopic and transvaginal approach and consisted in bowel inspection and reinstatement, peritoneal washing, and dehiscence repair. Conclusions. Vaginal evisceration is a rare but potentially serious complication of pelvic surgery. This case report is to make clinicians aware of such complication following LLETZ and its management.

Comparison between Visual Inspection of Cervix and Cytology Based Screening Procedures in Bangladesh

Cervical cancer continues to be a major problem in Bangladesh with approximately 18,000 new cases annually of which over 10,000 women die from it. Visual inspection of the cervix after 3-5% acetic acid (VIA) application is a simple and easy to learn method for cervical cancer screening, although cytology-based screening is more often applied in developed countries where it has successfully reduced the prevalence of cervical cancer. To compare the efficacy of VIA and cytology-based primary methods for cervical cancer screening in Bangladesh. This hospital based comparative study was conducted at the VIA centre and Colposcopy Clinic of Bangabandhu Sheikh Mujib Medical University (BSMMU) from October 2008 to October 2010. Among 650 women, 74 (11.4%) were VIA+ve and 8 (1.2%) had abnormalities in their Pap smear reports. During colposcopy, 38 (7.7%) women had different grades of CIN and 4 (0.6%) had cervical cancer. The gold standard histology findings proved 20 women had CIN I, 14 had CIN II/II and 4 had cervical cancer. Among the 38 histology diagnosed abnormalities, VIA test could identify 30 abnormalities including two cervical cancers. However, Pap smear could detect only 8 cases of histological abnormalities (2 low grade and 6 had high grade lesion) and it missed all the cervical cancer cases. The sensitivity and specificity of VIA were 88.9% and 52.1%. The positive predictive value (PPV) and negative predictive value (NPV) were 41.0%, and 92.6% respectively. Moreover, the sensitivity, specificity, PPV and NPV of Pap smear were 33.3%, 95.8%, 75.0% and 79.3%, respectively. VIA test should be used as the primary screening tool even with its low sensitivity and specificity in low resource countries like Bangladesh. False positive results may be greater, but overtreatment can be minimized by colposcopy evaluation of the VIA positive women.