Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV); however, factors such as HPV genotype and individual immune response may also contribute to its development. The loop electrosurgical excision procedure (LEEP) is a treatment for high-grade cervical intraepithelial neoplasia (CIN), as approximately 30% of these cases may progress to cancer. However, 20–40% of cases will regress spontaneously. HPV16 infection constitutes the highest risk for progression to cervical cancer and a lower probability of regression. Knowledge regarding the regression of lesions caused by other high-risk genotypes alone or in association with biomarker expression and lesion length has been limited. In the present study, the regression rates of high-grade squamous intraepithelial lesions were calculated. Twenty-one percent of the 161 women diagnosed with CIN2-3 on colposcopy-directed biopsies exhibited regression (defined as CIN1 or less) in the subsequent cone excisions. The mean interval between biopsy and treatment was 113 days (range of 71–171). High-grade lesions of the squamous epithelium caused by HPV16, together with lesions caused by HPV31, 52 and 58, showed significantly lower regression rates (HR 0.54, 0.22–0.75; low-regression group) than lesions caused by HPV18, 33, 35, 39, and 45 (HR 2.85, 1.54–5.28; high-regression group). A multivariate analysis of HPV genotypes, epithelial expressions of pRb and p53, immune cell proportions in the stroma (CD4/CD25 and CD4/CD8), and lesion lengths correctly predicted regression in 78% (Harrell‘s C). A Harrell‘s C value of 82% for the low-regression group indicates that different HPV genotypes or groups, together with divergent patterns of tumor suppressors, immune cells, and lesion size, can give prognostic information regarding the outcome of CIN2-3.

Human papilloma virus (HPV) infections vary in their oncogenic potential, and whether an infection progresses to cervical intraepithelial neoplasia (CIN) also depends on the immune response. Therefore, the aim of the present study was to explore biomarkers related to the immune system and cell proliferation, in combination with HPV classified as having high (HOP) or low oncogenic potential (LOP), that can possibly guide a more accurate identification of women following cervical cancer screening programmes in need for immediate follow-up with a biopsy. A next-generation sequencing transcriptomic immune profile analysis applied to 28 persistent CIN3 lesions and 14 normal biopsies identified four genes, the immune markers ARG1 and HLA-DQB2 and the tumour markers CDKN2A and KRT7, as possible markers for differentiating between CIN3 and normal tissue. To validate these findings, analysis of the relative gene expression of these markers by use of reverse transcriptase real-time quantitative polymerase chain reaction was performed in an independent cohort of 264 (82 normal, 64 CIN1, and 118 CIN2/CIN3) biopsies, and the data were combined with information on the HOP- or LOP-HPV identified in the biopsies. Statistical analysis was performed with receiver operating characteristic curves, reporting area under the curve (AUC) with 95% confidence intervals (CIs), and logistic regression. Statistically significantly higher median expression levels of CDKN2A (p < 0.001) and KRT7 (p = 0.045) and significantly lower expression of ARG1 (p = 0.012) were found in biopsies with HOP-HPV infections, with no difference detected for HLA-DQB2 (p = 0.82). Models using expression levels of CDKN2A (AUC, 0.91; 95% CI, 0.86-0.95), KRT7 (0.86, 0.81-0.91), or ARG1 (0.78, 0.70-0.85) together with HOP/LOP-HPV class were significantly better than HPV class alone (0.72, 0.66-0.79) in discriminating CIN2/3 versus CIN1 (p < 0.001, p < 0.001, and p = 0.014, respectively).

Background Cervical cancer screening participation is suboptimal in most settings. We assessed whether human papillomavirus (HPV) self-sampling may increase screening participation among long-term non-attenders in Norway. Methods A pragmatic randomised controlled trial with participation as the primary outcome was initiated in the national cervical screening programme in March 2019. A random sample of 6000 women aged 35–69 years who had not attended screening for at least 10 years were randomised 1:1:1 to receive either (i) a reminder to attend regular screening (control), (ii) an offer to order a self-sampling kit (opt-in) for HPV testing or (iii) a self-sampling kit unsolicited (send-to-all) for HPV testing. Results Total participation was 4.8%, 17.0% and 27.7% among control, opt-in and send-to-all ( P < 0.0001; participation difference (%) send-to-all vs. control: 22.9 (95%CI: 20.7, 25.2); opt-in vs. control: 12.3 (95%CI: 10.3, 14.2); send-to-all vs. opt-in: 10.7 (95% CI: 8.0, 13.3)). High-risk HPV was detected in 11.5% of self-samples and 9.2% of clinician-collected samples ( P = 0.40). Most women (92.5%) who returned a positive self-sample attended the clinic for triage testing. Of the 933 women screened, 33 (3.5%) had CIN2 + (1.1%, 3.7%, 3.8% among control, opt-in, and send-to-all, respectively), and 11 (1.2%) had cervical cancer (0%, 1.2%, 1.3% among control, opt-in, send-to-all, respectively). Conclusion Opt-in and send-to-all self-sampling increased screening participation among long-term, higher-risk non-attenders. Clinical trial registration ClinicalTrials.gov NCT03873376.

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

The purpose of this study was to establish a gene signature that may predict CIN3 regression and that may aid in selecting patients who may safely refrain from conization. Oncomine mRNA data including 398 immune-related genes from 21 lesions with confirmed regression and 28 with persistent CIN3 were compared. L1000 mRNA data from a cervical cancer cohort was available for validation (n = 239). Transcriptomic analyses identified TDO2 (p = 0.004), CCL5 (p < 0.001), CCL3 (p = 0.04), CD38 (p = 0.02), and PRF1 (p = 0.005) as upregulated, and LCK downregulated (p = 0.01) in CIN3 regression as compared to persistent CIN3 lesions. From these, a gene signature predicting CIN3 regression with a sensitivity of 91% (AUC = 0.85) was established. Transcriptomic analyses revealed proliferation as significantly linked to persistent CIN3. Within the cancer cohort, high regression signature score associated with immune activation by Gene Set enrichment Analyses (GSEA) and immune cell infiltration by histopathological evaluation (p < 0.001). Low signature score was associated with poor survival (p = 0.007) and large tumors (p = 0.01). In conclusion, the proposed six-gene signature predicts CIN regression and favorable cervical cancer prognosis and points to common drivers in precursors and cervical cancer lesions.

Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81 mg/mL (range: 0.55-1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3.

Objective. Cervical intraepithelial neoplasia grades 2-3 (CIN2-3) are usually treated by cone excision, although only 30% progress to cancer and 6-50% regress spontaneously. Biomarkers predicting CIN2-3 regression would be of great clinical value and could reduce unnecessary cone excision and associated complications. The aim of this study was to investigate whether punch-biopsy derived immunohistochemical biomarkers, local immune response, CIN lesion size and condom use are independently correlated to regression of CIN2-3.Methods. A prospective population-based cohort study of 162 women aged 25-40, with first-time onset diagnosis of CIN2-3 in colposcopy-directed biopsies was carried out. The median biopsy-cone interval was 16 weeks. Regression was defined as CIN1 or less in the cone biopsy.Results. The regression rate was 21% (34/162). pRb>30% in the lower epithelial half was the strongest predictor for regression (30% regression, p<0.0001). If additionally a CIN-lesion was smaller than 2.5 mm and CD4 + lymphoid cells in the subepithelial stroma <= 195 per 1.04 mm basal membrane, the regression rate was 53%. In CIN-lesions > 2.5 mm and CD4 + - stroma <= 195, consistent condom use increased the regression rate from 13% to 67% (p = 0.003). If pRb was <= 30%, the regression rate was low (6%).Conclusion. Biomarkers and CIN lesion length can predict CIN2-3 regression, and might be helpful to identify patients who can increase the regression rate of CIN lesions by consistent condom use. (C) 2012 Elsevier Inc. All rights reserved.

OBJECTIVE:Cervical intraepithelial neoplasia grades 2-3 (CIN2-3) are usually treated by cone excision, although only 30% progress to cancer and 6-50% regress spontaneously. The aim of this study was to examine the influence of clinical factors like smoking habits, number of lifetime sexual partners, age at first sexual intercourse, sexual activity span and hormonal versus non-hormonal contraception type on the regression rate of CIN2-3.METHODS:In this prospective population-based cohort study 170 women aged 25-40 with abnormal cytology and colposcopy-directed biopsies showing first time onset CIN2-3 were consecutively included. The interval between biopsy and cone excision was standardized to minimum 12 weeks. Regression was defined as ≤ CIN1 in the cone biopsy.RESULTS:The regression rate was 22%. Consistent condom use, defined as those women whose partners used condoms for all instances of sexual intercourse, was infrequent (n=20, 12%). In univariate analysis consistent condom use, hormonal contraception and age at first sexual intercourse significantly predicted regression. In a multivariate analysis only consistent condom use remained as an independent predictor of regression (regression rate 55%, p=0.001, hazard ratio=4.4).CONCLUSION:Consistent condom use between punch biopsy and cone excision in first-time onset CIN2-3 patients significantly increases the regression rate.

Background The major cause of cervical intraepithelial neoplasia (CIN) is persistent infection with human papillomavirus (HPV). Most CIN grade 2 and 3 lesions are treated with cone excision, although a substantial proportion (6-50%) of CIN2-3 lesions will regresses spontaneously. Predictors for regression of CIN2-3 are desirable in order to reduce this overtreatment. Methods In this prospective cohort study, 145 consecutive women with first-time onset CIN2-3 in colposcopy-directed biopsies and standardized biopsy-cone excision interval were included. The genotype of the high-risk human papillomaviruses (= hr HPV) and clinical factors including sexual behaviour, parity, contraception and smoking were assessed. Patients were divided into two groups according to lesions containing HPV16 ( hr HPV16+) and high-risk non-HPV16 ( hr HPV16-) genotypes. Results Women whose partners consistently used condoms showed a significantly higher regression rate than women using other types of contraception (53% versus 13%, p<0.0001). However, this effect was only seen in hr HPV16- patients (73% regression rate versus 13%, p<0.0001). Hr HPV16+ patients had a significantly higher number of sexual partners and more current smokers compared to hr HPV16- patients. The regression rate was not significantly different in CIN2-3 lesions containing HPV16 ( hr HPV16+) versus hr HPV16- genotypes. Conclusions Heterogeneity among hr HPV genotypes excists. HPV-genotype analyses can identify women who significantly increase their chance of regression by consistent condom use.

Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.

Objective. Different Human Papilloma Virus (HPV) tests are currently used. An integrated comparison of the Amplicor, Cobas4800, PreTect HPV-Proofer and APTIMA HPV tests has not been done.Methods. We compared the high-risk HPV detection power of these HPV tests in 528 consecutive population-based follow-up Liquid-Based Cytology samples (LBC) after ASCUS/LSIL index cytology. Their sensitivity and specificity to detect HPV in LBC, their predictive values of histopathologic CIN2-3 in follow-up punch biopsies and CIN2-3 regression in the subsequent cones was assessed. The HPV subtypes detected by the Linear Array genotyping-test (LA), PreTect HPV-Proofer and Cobas4800 were also compared. The follow-up histopathology was consensus expert-reviewed and Ki67/p16-supported. The predictive values of the HPV results in LBC by the different tests for presence of CIN2-3 in follow-up biopsies, and regression in subsequent cones, was assessed.Results. Amplicor, Cobas4800 and APTIMA show good agreement for HPV-positivity/negativity. PreTect HPV-Proofer has many discrepancies versus any of the other methods. The sensitivities for Amplicor, Cobas4800 and APTIMA to detect CIN2-3 were very high (96-100%), but rather low for PreTect HPV-Proofer (53%). Specificity in case of CIN1 or less in follow-up biopsies of Amplicor and Cobas4800 is lower than APTIMA and highest for PreTect HPV-Proofer. HPV subtyping by LA agreed in 90% with Cobas4800 but 70% with PreTect HPV-Proofer.Conclusions. The Amplicor, Cobas4800 and APTIMA give comparable results but PreTect HPV-Proofer differs from the other tests, with low sensitivity but higher specificity. None of the methods predicted regression of CIN2-3. (C) 2011 Elsevier Inc. All rights reserved.

Background Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value. Aim To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3. Methods Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications. Results The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification. Conclusions 114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.

Background and aims 15-30% of cases of cervical intraepithelial neoplasia grades 2-3 (CIN2-3) detected in punch biopsies regress spontaneously (ie, show CIN1 or less in the follow-up cone). Epithelial retinoblastoma protein (pRb), tumour suppressor protein (p53), HPV genotype and immunoreactive cells have been reported to be helpful in predicting regression but their interaction in regression prediction is unknown.Material and methods 55 cases of CIN2-3 in cervical biopsies with subsequent cervical cones were studied retrospectively to assess how epithelial biomarkers, immunoreactive cells (with immunohistochemistry) and high-risk (hr) HPV genotypes (by the AMPLICOR and linear array tests) prognostically interact with epithelial pRb and p53.Results 18% of CIN2-3s regressed (median biopsy-cone interval of 12.0 weeks, range 5.0-34.1 weeks). CIN2-3s that regressed had higher epithelial pRb and p53, lower stromal CD25(+) and CD138(+), and higher CD8 cells than persistent lesions. They also had higher ratios of CD4(+)/CD25(+) and CD8(+)/CD25 in stroma and epithelium. HPV16 correlated with low pRb and low CD8(+). With multivariate analysis a combined high ratio of CD8(+)/CD25(+) in the stroma, high epithelial pRb and p53 expression had independent value to predict the regression.Conclusions CIN2-3 lesions with a non-hrHPV16 infection, high ratios of stromal CD8(+)/CD25(+) and high epithelial expression of pRb or p53 are associated with spontaneous regression.

Clinically, it is important to distinguish cervical intraepithelial neoplasia grade 3 (CIN3) lesions with and without coexisting adenocarcinoma in situ (AIS), but endocervical curetting can be false negative. The frequency of high-risk human papillomavirus genotypes in CIN3 patients with and without AIS differs. CIN3 epithelial cell cycle regulator expression may reflect these differences and thereby indicate coexistent AIS. G1 pathway epithelial cell cycle regulators (pRb, p53, cyclin D, p16) and Ki-67 were analyzed by quantitative immunohistochemistry in CIN3s with and without AIS. Compared with the normal cervical squamous epithelium, the CIN3 epithelium in small punch biopsies showed strong diffuse p16 and Ki-67 expression. CIN3s with coexistent AIS had a significantly lower percentage of pRb (P = .03)- and p53 (P = .03)-positive nuclei in the lower half of the epithelium than CIN3s without coexistent AIS. None of the 10 cases with values of either pRb-positive nuclei 30% or greater or p53-positive nuclei 15% or greater in the lower half of the epithelium had coexistent AIS, contrasting 8 (24%) of the 33 cases with both low values of p53- and pRb-positive nuclei. Combined low p53 and pRb expression in the lower half of the epithelium in punch biopsies is associated with coexistent AIS in the cone. Despite the fact that the results of the current study are interesting and potentially clinically relevant, it should be emphasized that they must be confirmed according to Good Laboratory Practice in independent patient groups, preferably also in a prospective study.

Up to 30% of cervical intraepithelial grades 2-3 (CIN2-3) lesions regress, but some believe that "regression" is due to "curative" punch biopsies. If this is true, CIN2-3 in the resection margins of the biopsies would be associated with more frequent "persistent" CIN2-3. If, however, immunology-related regression exists, regression would increase with increasing biopsy-cone interval. In 61 punch biopsies diagnosed as CIN3 at careful review by two independent gynaecological pathologists, CIN3 in the resection margins and duration of the biopsy-cone interval was evaluated in relation to CIN2-3-or-not in the cones (again after independent review by expert pathologists). 10 of 61 (16%) patients with CIN3 showed CIN1 or less in the follow-up cones. CIN3-or-not in the resection margins, size of the lesion in the punch biopsy, and presence or absence of CIN2-3 in the cones were not correlated with regression-or-not. However, the number of cones without CIN2-3 increased with longer biopsy-cone interval, 5% in patients with a punch-cone biopsy interval under 9 weeks and 38%> or =9 weeks (p<0.001). These results favour the hypothesis that CIN3 can regress, and do not support the "curative punch biopsy" theory.

Up to 30% of cervical intraepithelial grades 2–3 (CIN2–3) lesions regress, but some believe that “regression” is due to “curative” punch biopsies. If this is true, CIN2–3 in the resection margins of the biopsies would be associated with more frequent “persistent” CIN2–3. If, however, immunology-related regression exists, regression would increase with increasing biopsy-cone interval. In 61 punch biopsies diagnosed as CIN3 at careful review by two independent gynaecological pathologists, CIN3 in the resection margins and duration of the biopsy-cone interval was evaluated in relation to CIN2–3-or-not in the cones (again after independent review by expert pathologists). 10 of 61 (16%) patients with CIN3 showed CIN1 or less in the follow-up cones. CIN3-or-not in the resection margins, size of the lesion in the punch biopsy, and presence or absence of CIN2–3 in the cones were not correlated with regression-or-not. However, the number of cones without CIN2–3 increased with longer biopsy-cone interval, 5% in patients with a punch-cone biopsy interval under 9 weeks and 38%≥9 weeks (p<0.001). These results favour the hypothesis that CIN3 can regress, and do not support the “curative punch biopsy” theory.