Classification of high-grade cervical intraepithelial neoplasia by p16ink4a , Ki-67, HPV E4 and FAM19A4/miR124-2 methylation status demonstrates considerable heterogeneity with potential consequences for management.

High-grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a , Ki-67 and host-cell DNA methylation) could provide guidance for clinical management in women with high-grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0-3) and Ki-67 (Scores 0-3), referred to as the "immunoscore" (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124-2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0-2 (6.0%), 151 lesions within IS group 3-4 (30.4%) and 316 lesions within IS group 5-6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). Methylation positivity increased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5-6 (38/316). Notably, in a minority (43/497, 8.7%) of high-grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high-grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a "wait and see" policy to reduce overtreatment of high-grade CIN lesions.

Effectiveness of HPV vaccination against the development of high-grade cervical lesions in young Japanese women.

BACKGROUND: Although more than 10 years have passed since HPV vaccination was implemented, first as an interim programme (Emergent vaccine promotion programme) in November 2010, followed by incorporating into the National Immunization Programme in April, 2013 and suspended in June 2013, limited studies have investigated the HPV vaccine effectiveness against high-grade cervical lesions in Japan. METHODS: We collected the matched data of the results of cervical biopsy and history of vaccination from the Japan Cancer Society database. The subjects were women aged 20 to 29 years screened for cervical cancer between April, 2015 and March, 2017, and with information on HPV vaccination status. We estimated the relative risk of developing high-grade cervical lesions in vaccinated subjects using Poisson regression as compared to unvaccinated subjects. RESULTS: Among the 34,281 women screened, 3770 (11.0%) were vaccinated. The prevalence of CIN2+ was statistically significantly lower in the vaccinated women as compared to the unvaccinated women (Vaccine Effectiveness (VE) =76%; RR = 0.24, 95% CI:0.10-0.60). High VE against CIN3+ was also observed (91%; RR = 0.09, 95% CI:0.00-0.42). CONCLUSION: Women aged 20-29 years who received at least one dose of HPV vaccine had a significantly lower risk of high-grade cervical lesions than those not vaccinated. In Japan, HPV vaccination should be resumed in order to reduce the incidence of cervical cancer.

Risk of High-Grade Histopathology Diagnosed by Cervical Conization in Endocervical Curettage Cervical Intraepithelial Neoplasia 1: A Case-Control Study.

OBJECTIVE: The aim of the study was to estimate risks of cervical intraepithelial neoplasia 2+ (CIN 2+) on loop electrosurgical excisional procedure (LEEP) specimens with the diagnosis of endocervical curettage (ECC) CIN 1 compared with biopsy CIN 1. MATERIALS AND METHODS: We performed a retrospective computer-based search for subjects enrolled in the Obstetrics and Gynecology Hospital of Fudan University. The case group comprised women with an ECC CIN 1 (ECC results of CIN 1 with colposcopy-directed biopsy results ≤CIN 1), and the control group comprised women with a biopsy CIN 1 (colposcopy-directed biopsy results of CIN 1 with negative ECC findings) diagnosis. Variables, including age, cytology, high-risk human papillomavirus, and ECC results, were included in univariate and multivariate logistic regression analyses. p < .05 was defined statistically significant. RESULTS: Overall, 1,195 women with ECC CIN 1 and/or biopsy CIN 1 diagnosis who underwent LEEP participated in the study. ECC CIN 1 comprised 400 women, with LEEP histopathology results revealing 104 (26.00%) CIN 2+. Biopsy CIN 1 comprised 795 women, with LEEP histopathology results showing 150 (18.87%) CIN 2+. Univariate logistic regression showed that cytology (p < .001) and ECC (p = .005) results differ significantly between less than CIN 2+ and CIN 2+. Multivariate logistic regression revealed that the cytology of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesions (OR = 4.73, 95% CI = 2.78-8.05, p < .001) and high-grade squamous intraepithelial lesions or worse (HSIL+, OR = 4.88, 95% CI = 3.00-7.94, p < .001), and ECC CIN 1 (OR = 1.80, 95% CI = 1.33-2.44, p < .001) were risk factors for CIN 2 + . CONCLUSIONS: Endocervical curettage CIN 1 has a greater risk of CIN 2+ diagnosis than biopsy CIN 1, but high-grade cytology has a higher risk than ECC CIN 1.

The newly proposed International Endocervical Adenocarcinoma Criteria and Classification and its relevance to cervical cytology screening assessed in a prospective 2-year study of 118 cases.

BACKGROUND: It is generally acknowledged that interobserver variability for the histological diagnosis of endocervical adenocarcinoma (EA) subtypes is suboptimal. The recently proposed International Endocervical Adenocarcinoma Criteria and Classification (IECC) system is based on the presence of associated human papilloma virus (HPV) infection. It recognises HPV-associated EAs and non-HPV-associated EAs. METHODS: This prospective cytology-histology and molecular genetics-based study investigated the potential effect of IECC being applied to Papanicolaou (Pap) test with regard to the diagnostic accuracy of severe glandular lesions reported at least as adenocarcinoma in situ (AIS). RESULTS: Out of 118 liquid-based cytology Pap tests with AIS+ lesion, complete information on follow-up biopsy and HPV status was available in 51 cases. AIS and EA category correlated with histologically confirmed AIS/EA in 88.5% (23/26) and 70.5% (12/17) of cases, respectively. Interestingly, 93% (40/43) of cases diagnosed as AIS/EA were HPV positive and 7% (3/43) were HPV negative (originating in the cervix, endometrium and adnexa). CONCLUSIONS: Our findings suggest that this approach could possibly divide Pap tests containing severe glandular lesion into two groups: (a) robust diagnosis of HPV-associated EA and (b) non-HPV associated glandular lesions of heterogeneous origin, requiring further clinical preoperative diagnostic workup.

Relationships of p16 Immunohistochemistry and Other Biomarkers With Diagnoses of Cervical Abnormalities: Implications for LAST Terminology.

CONTEXT.­: Lower Anogenital Squamous Terminology (LAST) standardization recommended p16INK4a immunohistochemistry (p16 IHC) for biopsies diagnosed morphologically as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2) to classify them as low-grade or high-grade squamous intraepithelial lesions (HSILs). OBJECTIVE.­: To describe the relationships of p16 IHC and other biomarkers associated with cervical cancer risk with biopsy diagnoses. DESIGN.­: A statewide, stratified sample of cervical biopsies diagnosed by community pathologists (CPs), including 1512 CIN2, underwent a consensus, expert pathologist panel (EP) review (without p16 IHC results), p16 IHC interpretation by a third pathology group, and human papillomavirus (HPV) genotyping, results of which were grouped hierarchically according to cancer risk. Antecedent cytologic interpretations were also available. RESULTS.­: Biopsies were more likely to test p16 IHC positive with increasing severity of CP diagnoses, overall (Ptrend ≤ .001) and within each HPV risk group (Ptrend ≤ .001 except for low-risk HPV [Ptrend < .010]). All abnormal grades of CP-diagnosed biopsies were more likely to test p16 IHC positive with a higher HPV risk group (Ptrend < .001), and testing p16 IHC positive was associated with higher HPV risk group than testing p16 IHC negative for each grade of CP-diagnosed biopsies (P < .001). p16 IHC-positive, CP-diagnosed CIN2 biopsies were less likely than CP-diagnosed CIN3 biopsies to test HPV16 positive, have an antecedent HSIL+ cytology, or to be diagnosed as CIN3+ by the EP (P < .001 for all). p16 IHC-positive, CP-diagnosed CIN1 biopsies had lower HPV risk groups than p16 IHC-negative, CP-diagnosed CIN2 biopsies (P < .001). CONCLUSIONS.­: p16 IHC-positive, CP-diagnosed CIN2 appears to be lower cancer risk than CP-diagnosed CIN3. LAST classification of "HSIL" diagnosis, which includes p16 IHC-positive CIN2, should annotate the morphologic diagnosis (CIN2 or CIN3) to inform all management decisions, which is especially important for young (<30 years) women diagnosed with CIN2 for whom surveillance rather than treatment is recommended.

Long-term CIN3+ risk of HPV positive women after triage with FAM19A4/miR124-2 methylation analysis.

OBJECTIVE: This study evaluates the long-term risk for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) among HPV positive women triaged with FAM19A4/miR124-2 methylation analysis. METHODS: In a post hoc analysis, data on FAM19A4/miR124-2 methylation, cytology, and HPV16/18 genotyping of HPV positive women (n = 1025) from a large population-based screening cohort with 14-year follow-up were evaluated. Cumulative CIN3+ incidences over 3 screening rounds (5-year intervals) of 4 triage strategies were compared: FAM19A4/miR124-2 methylation analysis, cytology, HPV16/18 genotyping with FAM19A4/miR124-2 methylation, and HPV16/18 genotyping with cytology. RESULTS: Kaplan-Meier estimates of 14-year cumulative CIN3+ incidence of HPV positive women with a negative methylation and a negative cytology triage test were comparable (16.3% and 15.6%, respectively). The cumulative CIN3+ incidence of methylation positive and cytology positive women were 39.8% and 46.5%, respectively. HPV16/18 genotyping with methylation and HPV16/18 genotyping with cytology resulted in the lowest 14-year cumulative CIN3+ incidence among triage negative women (10.7% and 10.0%, respectively), but cumulative CIN3+ incidence among triage positive women was lower (33.4% and 35.7%, respectively) compared with triage by methylation alone and cytology alone. CONCLUSIONS: Among HPV positive women of 30 years and older, a negative FAM19A4/miR124-2 methylation triage test provides a similar long-term CIN3+ risk compared with a negative cytology triage test. Because of their high CIN3+ risk, women with a positive methylation triage test could be referred for colposcopy. Therefore, FAM19A4/miR124-2 methylation analysis is a promising alternative to cytology for triage of HPV positive women.

Human papillomavirus genotype-specific risk in cervical carcinogenesis.

OBJECTIVE: To evaluate the risk of genotype-specific human papillomavirus (HPV) infections for the spectrum of cervical carcinogenesis and the distribution of HPV types according to age and different cervical lesions. METHODS: This study included HPV-positive women who underwent cervical biopsy at the Cheil General Hospital & Women's Healthcare Center between July 1, 2011 and December 31, 2017. HPV genotyping was conducted using a Cheil HPV DNA chip kit. RESULTS: The study sample consisted of 400 normal, 399 cervical intraepithelial neoplasia (CIN) 1, 400 CIN 2, 400 CIN 3, and 389 cervical cancer cases. HPV 16 was the most common type found with a prevalence of 9.5% in normal, 6.8% in CIN 1, 15.0% in CIN 2, 44.5% in CIN 3, and 64.3% in cervical cancer. The most common HPV types were 16, 52, 58, 53, 51, 56, 68, and 18 in all study samples. HPV 16, 31, 33, and 58 were more common in CIN 2/3 and cancer, and HPV 39, 51, 53, 56, 66, and 68 were more common in CIN 1 and normal cases (p<0.001). In CIN 3 and cervical cancer, HPV 16 was the most common type in all age groups. HPV 52 was the most common type in CIN 2 (all age groups) and in CIN 1/normal (age ≤30 years) cases. Among the high-risk HPV types, 16, 31, 33, 52, and 58 showed significant risk for high-grade disease. CONCLUSIONS: HPV 16, 31, 33, 52, and 58 showed the significant risk of high-grade disease for cervical carcinogenesis.

Three-Tiered versus Two-Tiered Classification of Squamous Dysplasia in Cervical Cytology: Results of a Follow-Up Study.

OBJECTIVE: Regarding cytological findings of squamous dysplasia, a comparison was made between a three-tiered classification - low-grade squamous intraepithelial lesion (LSIL), high-grade SIL/cervical intraepithelial neoplasia 2 (HSIL/CIN2), and HSIL/CIN3 - and a two-tiered classification - LSIL and HSIL. The respective risk for CIN2+ and CIN3+ was calculated to make decisions regarding management. METHODS: A total of 2,949 women with first-time cytologic findings of squamous dysplasia (LSIL, HSIL/CIN2, or HSIL/CIN3) between January 2013 and June 2016 were enrolled. Subsequent cytological findings and histological diagnoses were evaluated until August 2018. For each category of findings, the risk for CIN2+ and CIN3+ was determined by Kaplan-Meier estimates. The differences in risk between the cytological categories were checked for significance using the log-rank test. RESULTS: For the categories LSIL, HSIL/CIN2, and HSIL/CIN3, the risk for CIN2+ after 12, 24, and 60 months was 3.4, 9.4, and 23.3%; 35.2, 44.8, and 59.8%; and 95.5, 97.8, and 98.9%, respectively. For CIN3+ the risk was 2.0, 5.5, and 13.5%; 28.6, 35.6, and 48.3%; 91.3, 95.6, and 97.9%, respectively. The differences in risk between the categories are highly significant, respectively (p < 0.001). CONCLUSION: A three-tiered classification of squamous dysplasia such as the Munich Nomenclature III for cytology is suitable for risk-adapted clinical management, especially to avoid overdiagnosis and overtreatment.

Detection of high-grade neoplasia in air-dried cervical PAP smears by a microRNA-based classifier.

Recent studies have shown that changes in the expression levels of certain microRNAs correlate with the degree of severity of cervical lesions. The aim of the present study was to develop a microRNA-based classifier for the detection of high-grade cervical intraepithelial neoplasia (CIN ≥2) in cytological samples from patients with different high-risk human papillomavirus (HR-HPV) viral loads. For this purpose, raw RT-qPCR data for 25 candidate microRNAs, U6 snRNA and human DNA in air-dried PAP smears from 174 women with different cervical cytological diagnoses, 144 of which were HR-HPV-positive [40 negative for intraepithelial lesion or malignancy (NILM), 34 low-grade squamous intraepithelial lesions (L-SIL), 57 high-grade squamous intraepithelial lesions (H-SIL), 43 invasive cancers], were statistically processed. The expression level changes of various individual microRNAs were found to be significantly correlated with the cytological diagnosis but the statistical significance of this correlation was critically dependent on the normalization strategy. We developed a linear classifier based on the paired ratios of 8 microRNA concentrations and cellular DNA content. The classifier determines the dimensionless coefficient (DF value), which increases with the severity of cervical lesion. The high- and low-grade CINs were better distinguished by the microRNA classifier than by the measurement of individual microRNA levels with the use of traditional normalization methods. The diagnostic sensitivity of detecting high-grade lesions (CIN ≥2) with the developed microRNA classifier was 83.4%, diagnostic specificity 81.2%, ROC AUC=0.913. The analysis can be performed with the same nucleic acid preparation as used for HPV testing. No statistically significant correlation of the DF value and HR-HPV DNA load was found. The DF value and the HR HPV presence and viral DNA load may be regarded as independent criteria that can complement each other in molecular screening for high-grade cervical intraepithelial neoplasia. Although it has several limitations, the present study showed that the small-scale analysis of microRNA signatures performed by simple PCR-based methods may be useful for improving the diagnostic/prognostic value of cervical screening.

Spectrum of HPV types before and after treatment of cervical intraepithelial neoplasia grade 2 and 3.

BACKGROUND: To monitor residual disease after treatment of high grade cervical intraepithelial neoplasia (CIN), cytology together with human papillomavirus (HPV) testing are commonly performed. OBJECTIVES: To analyse the spectrum of HPV types before and after treatment. STUDY DESIGN: This register-based study included 446 women treated for CIN2 or 3, where cytology samples had been HPV-tested before and after treatment by the use of the MGP-PCR Luminex HPV L1-DNA-assay identifying 39 HPV types, including 12 high risk (HR) HPV types. RESULTS: Before and after treatment, 706 and 248 HPV isolates were detected of 36 and 34 different HPV types, respectively. Among all the HR HPV isolates, type-specific persistency was observed among 14% (76/542) after treatment, compared to 34% (31/92) of low-risk (LR) HPV isolates (p<0.001). Among the potential high risk (PHR) HPV isolates, 8.3% (6/72) persisted. Totally, 99% (440/446) and 40% (179/446) of the women were HPV-positive before and after treatment, respectively. At least one of the 12 HR HPV types was present in 91% (404/446) and 24% (109/446) of the women before and after treatment, respectively (p<0.0001). HR HPV types were present both before and after treatment among 23% (102/446) of the women, and 16% (71/446) manifested at least one persistent HR HPV type. The sensitivity, specificity and negative predictive value of HR HPV testing for detection of residual high grade squamous intraepithelial lesion (HSIL) was based on the first cytology after treatment, and was 91.7% (95% CI: 61.5%-99.8%), 84.1% (95% CI: 80.0%-87.7%) and 99.7% (95% CI: 98.2%-100.0%), respectively. CONCLUSIONS: About one out six treated women (16%) manifested at least one persistent HR HPV type, that was associated with recurrent or residual HSIL disease (odds ratio 58.1, 95% CI 7.4-457) (p=0.0001). Testing for HR HPV demonstrated high sensitivity (92%) for residual HSIL. The higher persistency rate of LR HPV types suggests that they are more likely to be outside the treated area.

Targeted, Deep Sequencing Reveals Full Methylation Profiles of Multiple HPV Types and Potential Biomarkers for Cervical Cancer Progression.

Background: Invasive cervical cancer (ICC) and its premalignant phase (cervical intraepithelial neoplasia; CIN1-3) are distinguished by gynecologic and pathologic examination, yet no current methodologies can predict precancerous lesions that are destined to progress to ICC. Thus, development of reliable assays to assess patient prognosis is much needed.Methods: Human papillomavirus (HPV) DNA methylation is significantly altered in cervical disease. Using an HPV enrichment approach and next-generation DNA sequencing, methylation status was characterized in a case-case comparison of CIN (n = 2 CIN1; n = 2 CIN2; n = 20 CIN3) and ICC (n = 37) samples. Pyrosequencing validated methylation changes at CpGs of interest in a larger sample cohort (n = 61 CIN3; 50 ICC).Results: Global viral methylation, across HPV types, was significantly higher in ICC than CIN3. Average L1 gene methylation in 13 different HPV types best distinguished CIN3 from ICC. Methylation levels at individual CpG sites as a quantitative classifier achieved a sensitivity and specificity of >95% for detecting ICC in HPV 16 samples. Pyrosequencing confirmed significantly higher methylation of these CpGs in E1 of HPV 16 in ICC compared with CIN3.Conclusions: Global HPV methylation is significantly higher in ICC than CIN3, with L1 gene methylation levels performing best for distinguishing CIN3 from ICC. Methylation levels at CpGs in the E1 gene of HPV 16 (972, 978, 1870, and 1958) can distinguish between CIN3 and ICC.Impact: Higher methylation at specific E1 CpGs may associate with increased likelihood of progression to ICC in HPV 16-positive CIN3 lesions. Cancer Epidemiol Biomarkers Prev; 26(4); 642-50. ©2017 AACR.

Serial measurement of type-specific human papillomavirus load enables classification of cervical intraepithelial neoplasia lesions according to occurring human papillomavirus-induced pathway.

This retrospective study examined whether human papillomavirus (HPV) type-specific viral load changes measured in two or three serial cervical smears are predictive for the natural evolution of HPV infections and correlate with histological grades of cervical intraepithelial neoplasia (CIN), allowing triage of HPV-positive women. A cervical histology database was used to select consecutive women with biopsy-proven CIN in 2012 who had at least two liquid-based cytology samples before the diagnosis of CIN. Before performing cytology, 18 different quantitative PCRs allowed HPV type-specific viral load measurement. Changes in HPV-specific load between measurements were assessed by linear regression, with calculation of coefficient of determination (R) and slope. All infections could be classified into one of five categories: (i) clonal progressing process (R≥0.85; positive slope), (ii) simultaneously occurring clonal progressive and transient infection, (iii) clonal regressing process (R≥0.85; negative slope), (iv) serial transient infection with latency [R<0.85; slopes (two points) between 0.0010 and -0.0010 HPV copies/cell/day], and (v) transient productive infection (R<0.85; slope: ±0.0099 HPV copies/cell/day). Three hundred and seven women with CIN were included; 124 had single-type infections and 183 had multiple HPV types. Only with three consecutive measurements could a clonal process be identified in all CIN3 cases. We could clearly demonstrate clonal regressing lesions with a persistent linear decrease in viral load (R≥0.85; -0.003 HPV copies/cell/day) in all CIN categories. Type-specific viral load increase/decrease in three consecutive measurements enabled classification of CIN lesions in clonal HPV-driven transformation (progression/regression) and nonclonal virion-productive (serial transient/transient) processes.

[Categorization of uterine cervix tumors : What's new in the 2014 WHO classification]. / Kategorisierung der Tumoren der Cervix uteri : Neues in der WHO-Klassifikation 2014.

In the 2014 WHO classification, squamous cell precursor lesions are classified as low-grade and high-grade intraepithelial lesions. LSIL corresponds to CIN1, HSIL includes CIN2 and CIN3. Only adenocarcinoma in situ (AIS) is accepted as precursor of adenocarcinoma and includes the stratified mucin-producing intraepithelial lesion (SMILE). Although relatively rare, adenocarcinoma and squamous cell carcinoma can be mixed with a poorly differentiated neuroendocrine carcinoma. Most cervical adenocarcinomas are low grade and of endocervical type. Mucinous carcinomas show marked intra- and extracellular mucin production. Almost all squamous cell carcinomas, the vast majority of adenocarcinomas, and many rare carcinoma types are HPV related. For low grade endocervical adenocarcinomas, the pattern-based classification according to Silva should be reported. Neuroendocrine tumors are rare and are classified into low-grade and high-grade, whereby the term carcinoid is still used.

[Nomenclature of squamous cell precursor lesions of the lower female genital tract : Current aspects]. / Nomenklatur der plattenepithelialen Präkanzerosen des unteren weiblichen Genitales : Aktuelle Aspekte.

The majority of precancerous lesions of the lower female genital tract (intraepithelial neoplasia, IN) are caused by human papillomavirus (HPV) infections resulting in cellular atypia and in turn an altered tissue architecture. Depending on the pathogenesis, a distinction is made between vulvar intraepithelial neoplasia (VIN) classified as classical VIN associated with high-risk HPV infections (u-VIN) and differentiated VIN (d-VIN), which is associated with lichen sclerosus et atrophicus and p53 alterations. In the current World Health Organization (WHO) classification a novel grading system for squamous cell precancerous lesions of the lower female genital tract has been proposed, differentiating low grade squamous intraepithelial lesions (L-SIL) including condyloma and HPV-associated alterations plus VIN 1, vaginal intraepithelial neoplasia (VaIN 1) and cervical intraepithelial neoplasia (CIN 1) from high grade squamous intraepithelial lesions (H-SIL) with VIN 2 and 3, VaIN 2 and 3 as well as CIN 2 and 3. The use of p16 immunohistochemistry can assist the differentiation. The new binary classification, however, contradicts the German cytological nomenclature (Munich nomenclature III), which differentiated three grades of dysplasia in order to avoid overtreatment of patients with moderate IN. The individual nomenclatures are compared to each other. It is recommended to report the grade of precancerous lesions in addition to the SIL classification of the WHO.

[Modern biomarkers for precancerous lesions of the uterine cervix : Histological-cytological correlation and use]. / Moderne Biomarker bei Präkanzerosen der Cervix uteri : Histologische-zytologische Korrelation und Einsatz.

The correlation between the cytological findings from the PAP smear and the histological outcome in cases where the cytological findings must be histologically verified, is an integral component of the German screening program for cervical cancer. These data are collected nationwide as part of a benchmarking process by the individual Associations of Statutory Health Insurance Physicians (KV) in the federal states and reported to the National Association of Statutory Health Insurance Physicians (KBV) in Berlin. In most cases there is a good correlation between cytology and histology but in some cases either a different grade of severity of cervical intraepithelial neoplasia (CIN) is found or the histological findings are negative. The reasons for a lack of correlation can be insufficient sampling in the cytology or the biopsy or a misinterpretation of the individual findings. Although the findings from H&E sections are considered to be the gold standard in the histological evaluation, it has long been known that the interobserver agreement in these preparations is only moderate. A significant improvement becomes apparent, firstly by the classification of cervical cancer precursors into low-grade and high-grade groups and secondly by the targeted application of biomarkers, in particular p16 and Ki-67, according to the recommendations of the lower anogenital squamous terminology standardization (LAST) project. The biomarkers p16 and Ki-67 should be used in the differential diagnostics between reactive and reparative alterations and for further differentiation of a CIN grade 2 but not to confirm a CIN grade 3. It is still unclear whether p16 is suitable as a prognostic marker for low-grade lesions.

Raman spectroscopy for cytopathology of exfoliated cervical cells.

Cervical cancer is the fourth most common cancer affecting women worldwide but mortality can be decreased by early detection of pre-malignant lesions. The Pap smear test is the most commonly used method in cervical cancer screening programmes. Although specificity is high for this test, it is widely acknowledged that sensitivity can be poor mainly due to the subjective nature of the test. There is a need for new objective tests for the early detection of pre-malignant cervical lesions. Over the past two decades, Raman spectroscopy has emerged as a promising new technology for cancer screening and diagnosis. The aim of this study was to evaluate the potential of Raman spectroscopy for cervical cancer screening using both Cervical Intraepithelial Neoplasia (CIN) and Squamous Intraepithelial Lesion (SIL) classification terminology. ThinPrep® Pap samples were recruited from a cervical screening population. Raman spectra were recorded from single cell nuclei and subjected to multivariate statistical analysis. Normal and abnormal ThinPrep® samples were discriminated based on the biochemical fingerprint of the cells using Principal Component Analysis (PCA). Principal Component Analysis - Linear Discriminant Analysis (PCA-LDA) was employed to build classification models based on either CIN or SIL terminology. This study has shown that Raman spectroscopy can be successfully applied to the study of routine cervical cytology samples from a cervical screening programme and that the use of CIN terminology resulted in improved sensitivity for high grade cases.

The Application of Classification and Regression Trees for the Triage of Women for Referral to Colposcopy and the Estimation of Risk for Cervical Intraepithelial Neoplasia: A Study Based on 1625 Cases with Incomplete Data from Molecular Tests.

OBJECTIVE: Nowadays numerous ancillary techniques detecting HPV DNA and mRNA compete with cytology; however no perfect test exists; in this study we evaluated classification and regression trees (CARTs) for the production of triage rules and estimate the risk for cervical intraepithelial neoplasia (CIN) in cases with ASCUS+ in cytology. STUDY DESIGN: We used 1625 cases. In contrast to other approaches we used missing data to increase the data volume, obtain more accurate results, and simulate real conditions in the everyday practice of gynecologic clinics and laboratories. The proposed CART was based on the cytological result, HPV DNA typing, HPV mRNA detection based on NASBA and flow cytometry, p16 immunocytochemical expression, and finally age and parous status. RESULTS: Algorithms useful for the triage of women were produced; gynecologists could apply these in conjunction with available examination results and conclude to an estimation of the risk for a woman to harbor CIN expressed as a probability. CONCLUSIONS: The most important test was the cytological examination; however the CART handled cases with inadequate cytological outcome and increased the diagnostic accuracy by exploiting the results of ancillary techniques even if there were inadequate missing data. The CART performance was better than any other single test involved in this study.

Diagnostic imaging of cervical intraepithelial neoplasia based on hematoxylin and eosin fluorescence.

BACKGROUND: Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues. METHODS: Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450-490 nm with emission captured at 515-565 nm. The 32-bit grayscale fluorescence images were used for image analysis. RESULTS: We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different. CONCLUSION: Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis.

The Pap Test and Bethesda 2014. "The reports of my demise have been greatly exaggerated." (after a quotation from Mark Twain).

The history of 'The Bethesda System' for reporting cervical cytology goes back almost 3 decades. This terminology and the process that created it have had a profound impact on the practice of cervical cytology for laboratorians and clinicians alike. The Bethesda conferences and their ensuing output have also set the stage for standardization of terminology across multiple organ systems, including both cytology and histology, have initiated significant research in the biology and cost-effective management for human papillomavirus-associated anogenital lesions, and, finally, have fostered worldwide unification of clinical management for these lesions. Herein, we summarize the process and rationale by which updates were made to the terminology in 2014 and outline the contents of the new, third edition of the Bethesda atlas and corresponding website.