Irene Tveiterås Øvestad

Local immune response in the microenvironment of CIN2-3 with and without spontaneous regression

Fifteen to thirty percent of cases with histologically confirmed CIN2–3 in cervical biopsies regress spontaneously (ie, show CIN1 or less in the follow-up cervical cone). The balance between immune-reactive cells from the host and high-risk human papillomavirus (hrHPV) genotypes may provide a biological explanation for this phenomenon. We retrospectively studied 55 cases of CIN2–3 in a cervical biopsy with subsequent cervical cone to assess whether hrHPV genotypes (by AMPLICOR and Linear Array tests) CD4, CD8, CD25, CD138 and Foxp3 cells (by quantitative immunohistochemistry) in the cervical biopsies can predict regression (defined as CIN1 or less in the follow-up cone biopsy). Eighteen percent of the CIN2–3 cases regressed (median biopsy–cervical cone time interval: 12.0 weeks, range: 5.0–34.1 weeks). HPV-16 correlated with low CD8+ and high CD25+. None of the regressing CIN2–3 lesions contained HPV-16. The regressing CIN2–3 lesions had lower numbers of stromal CD138+ and higher numbers of stromal CD8+cells; higher stromal and intra-epithelial ratios of CD4+/CD25+ cells; higher ratios of CD8+/CD25+ cells and lower ratios of CD8+/CD4+, CD138+/Foxp3+ and CD25+/Foxp3+cells in the stroma. With multivariate survival analysis, stromal CD8+ cell numbers, CD4+/CD25+ cell ratios and CD138+ cell numbers are found to be independent regression predictors. In conclusion, in non-HPV-16 CIN2–3 lesions, assessing stromal immune cells can be a useful prognostic indicator of regression or persistence.

Comparing subjective and digital image analysis HER2/neu expression scores with conventional and modified FISH scores in breast cancer

Background: HER2/neu expression and fluorescence in situ hybridisation (FISH) amplification have therapeutic significance. Aims: To compare subjective HER2/neu expression scores with digital image analysis (DIA) and conventional and modified FISH scores in breast cancer. Methods: Sixty HercepTest-immunostained breast carcinomas, prospectively scored as consensus 2+ and 3+ (DAKO protocol) by two observers, were analysed with DIA, and conventional (Vysis) and modified FISH scoring protocols. Results: With consensus scoring, 23 (38%) of the 60 cases were 2+ and 37 (62%) were 3+. Agreement with DIA scores was 100%. With conventional FISH scoring, 4 of the 3+ cases did not show amplification, but all of those negative cases had high HER2/neu copy numbers. With the modified FISH scoring protocol, all HercepTest immunohistochemical 3+ cases were amplified. Of the 2+ cases, 3 were amplified with the modified FISH protocol and 4 with the conventional FISH protocol. Conclusions: Modified FISH scores were better correlated with HercepTest 3+ consensus and DIA scores than were conventional FISH scores. HER2/neu DIA scoring is a cost-effective supplementary tool in surgical pathology.

Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3

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.

The impact of epithelial biomarkers, local immune response and human papillomavirus genotype in the regression of cervical intraepithelial neoplasia grades 2-3.

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.

Digital image analysis improves the quality of subjective HER-2 expression scoring in breast cancer.

AimTo compare HER-2 scoring reproducibility by subjective and digital image analysis (DIA) scores with each other and with fluorescence in situ hybridization (FISH) assessed HER-2 amplification. MethodsHerceptest-stained Tissue Micro Arrays of 219 breast carcinomas were scored (DAKO protocol) by 3 observers (both independent and as consensus), scored by DIA and both scores were compared with FISH amplification results. ResultsInterobserver subjective scores reproducibility was good (κ 0.82 to 0.86) but therapeutically important 3+/2+discrepancies occurred in 11% to 16% of all 3+ cases. Subjective scores and FISH results differed considerably. Consensus scores by 3 pathologists correlated better with FISH, reducing the number of both Immunohistochemical (IHC) negative/FISH positives and IHC 3+/FISH negatives. DIA scores were well reproducible and correlated better with FISH amplification than did subjective scores. ConclusionsDIA scores were comparable with consensus scores between 3 expert pathologists, were very well reproducible and performed better in classifying IHC 3+/FISH+ cases than did subjective scores.

Interaction of epithelial biomarkers, local immune response and condom use in cervical intraepithelial neoplasia 2–3 regression

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 2.5mm and CD4+ lymphoid cells in the subepithelial stroma ≤ 195 per 1.04 mm basal membrane, the regression rate was 53%. In CIN-lesions>2.5mm 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.

LOH at 1p31 (ARHI) and proliferation in lymph node-negative breast cancer.

Background: The mitotic activity index (MAI) is a strong prognosticator in node-negative invasive breast cancer patients. Recently, a correlation between the MAI and specific chromosomal aberrations at chromosome 1p was described. Methods: Analysis of MAI, immunohistochemical staining patterns for proliferation-associated phosphohistone H3 (PPH3), phosphorylated ERK1/2, p21, cyclin E, Ki67 and cyclin D1 proteins; and prognosis in 158 adjuvant chemotherapy-treated T1-2N0M0 invasive breast cancer patients, analysis of LOH at 1p31 (including ARHI) using the dinucleotide repeats D1S207, D1S430 and D1S464 in 76 patients. Single and multivariate survival analysis was used to evaluate the importance of the various markers tested. Results: LOH at 1p31 did not correlate with MAI nor provide prognostic information. Phosphohistone H3 was the best prognosticator for patients in all age groups with 20 year distant metastasis free survival of distant metastases 93% vs. 72% respectively (p = 0.004, HR = 4.5). In multivariate analysis, phosphohistone H3 < 13 vs. ≥13 exceeded the prognostic value of the mitotic activity index. Conclusions: LOH at 1p31 is common in breast cancer, and correlates with loss of proliferation-associated proteins, but not with MAI, PPH3 or prognosis. PPH3 is the best prognosticator in this study group of adjuvant chemotherapy-treated lymph node-negative breast cancer patients.

Consistent Condom Use Increases the Regression Rate of Cervical Intraepithelial Neoplasia 2-3

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. 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.

Consistent condom use increases spontaneous regression in high-risk non-HPV16 but not in HPV16 CIN2-3 lesions, a prospective population-based cohort study

BackgroundThe 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.MethodsIn 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.ResultsWomen 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.ConclusionsHeterogeneity among hr HPV genotypes excists. HPV-genotype analyses can identify women who significantly increase their chance of regression by consistent condom use.

Prediction of Spontaneous Regression of Cervical Intraepithelial Neoplasia Lesions Grades 2 and 3 by Proteomic Analysis

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.