Arnold-Jan Kruse

Ki-67 immunoquantitation in cervical intraepithelial neoplasia (CIN): a sensitive marker for grading

The aim of this study was to assess the value of Ki‐67 immunoquantitation with a computerized image analysis system for grading support in cervical intraepithelial neoplasia (CIN). Sixty‐five ‘blind’ consensus biopsies (23 CIN 1, 22 CIN 2, and 20 CIN 3) were used as a learning set. Measurements were done in the carefully selected most severely dysplastic part of the epithelium of each CIN case. The resulting discriminating combination of quantitative features was then prospectively applied on 121 new biopsies (test set) and compared with the classical CIN grade assessed routinely by six different pathologists and with the blind review grades assessed by two experienced pathologists. In the learning set of 65 cases, a jack‐knifed stepwise discriminant analysis showed that the 90th percentile of the stratification index and the number of positive nuclei per 100u2009µm basal membrane are the best discriminating set of features to distinguish the three CIN grades at the same time. With these features, two CIN 1 cases were ‘misclassified’ as CIN 2 and nine CIN 2 cases as CIN 3. Overall agreement, therefore, was only 83%. However, recut of the paraffin blocks in the two ‘misclassified’ CIN 1 cases revealed CIN 2 in the first and CIN 3 in the other, while the other CIN 1 cases that were correctly classified with Ki‐67 quantitation remained CIN 1. Likewise, nine CIN 2 cases were misclassified as CIN 3, but in two of these nine cases histological follow‐up clearly indicated CIN 3. Agreement may thus be higher than the 83% in the learning set suggests. In the subsequent prospective evaluation on 121 routine CIN cases (test set), agreement between routine CIN grades (by six independent different pathologists) and quantitative Ki‐67 classification was 78%. However, when compared with the blind review CIN grades of two expert pathologists, agreement was 97% and sensitivity, specificity, and positive and negative predictive value were very high. It is concluded that Ki‐67 immunoquantitation is a useful diagnostic adjunct to distinguish different CIN grades and may also be a sensitive biological indicator of progression of seemingly low‐grade CIN. Copyright

Quantitative molecular parameters to identify low-risk and high-risk early CIN lesions: role of markers of proliferative activity and differentiation and Rb availability.

Summary:In early cervical intraepithelial neoplasia (CIN), the Ki67 stratification index 90th percentile (Si90) is a strong predictor of progression. This study was designed to further investigate the mechanisms leading to elevated Ki67 levels in lesions that progress and to try to improve the prognostic accuracy of Ki67-Si90. We studied 90 CIN lesions in which consensus existed regarding the grade between two experienced gynecologic pathologists. All CINs were p16-positive and showed Ki67 cell clusters above the lower third of the epithelium (both features diagnostic for CIN). Ki67 parameters, cell cycle regulators (Rb, p53, Cyclin A, E and D, p16, p21, p27, and telomerase), and cellular differentiation products (involucrin, CK13, CK14) were compared in the basal zone as well as the deeper and upper halves of the epithelium. Fifteen CIN cases (17%) progressed to a higher CIN grade, including 2 of 25 CIN1 (8%) and 13 of 65 CIN2 (20%) (these proportions of progressing CINs are similar to those in a large meta-analysis). Ki67 quantitation effectively predicted CIN progression as 0 of 40 “Ki67 low-risk” and 15 of 50 (30%) “Ki67 high-risk” lesions progressed. CIN progressors showed decreased Rb, CK13, CK14, and involucrin, but increased p21 and p27 expression. Ki67-Si90 and Rb in the deeper half of the epithelium (RbDeep) were the strongest multivariate independent predictors of progression. Ki67-Si90>0.57 was unfavorable, but only if it coexisted with RbDeep <45% (progression risk = 47%). All early CINs with combined Si90>0.57+RbDeep>45% or any Ki67-Si90 value below 0.57 were nonprogressors. In the high-risk progression subgroup (Ki67-Si90>0.57+RbDeep<45%), all cases with combined CK14<50% and CK13<80% (both in the basal cell layer) (4% of all lesions) progressed. We hypothesize that onco-HPV E7 expression reduces Rb, causing increased and upward proliferation (Ki67-Si90>0.57). Increased RbDeep can reduce proliferation, including its upward spread. Combined quantitation of Ki67, Rb, CK13, and CK14 gives accurate information about the progression risk of early CIN lesions.

Evaluation of MIB-1-positive cell clusters as a diagnostic marker for cervical intraepithelial neoplasia

The objects of the study were to evaluate MIB-1-positive cell clusters (MIB-C) for distinguishing normal, reactive, and cervical intraepithelial neoplasia (CIN) biopsies and to determine possible pitfalls. Seventy-seven consecutive cervical specimens routinely diagnosed (Dx_orig) as CIN 1 or 2, or no-CIN, were revised independently by two expert gynecopathologists. MIB-1 staining and oncogenic human papillomavirus (HPV) assessment (by polymerase chain reaction) were performed. Independent diagnoses (plus oncogenic HPV status, in case of disagreement between the experts) were used to obtain a final diagnosis (Dx_final) and compared with MIB-C. Four of the 27 (15%) Dx_final = normal were HPV positive. Agreement between the gynecopathologists was 72 of 77 (94%). There were 30 (39%) discrepancies between Dx_orig and Dx_final (23 = 30% downgrades and 7 = 9% upgrades). All 23 downgrades were HPV negative and all seven upgrades were HPV positive. Overall agreement between Dx_orig and MIB-C was 73%, and with Dx_final 99%. Sensitivity, specificity, and positive and negative predictive values of MIB-C were very high without false negatives. Tangential cutting of MIB-1-positive parabasal cells and inflammatory cells can erroneously be overdiagnosed as a MIB-C. One single false positive of the 48 non-CIN cases (an immature squamous metaplasia) showed a special, easily recognizable MIB-1 pattern, different from CIN because the MIB-1 staining in the nuclei is not diffuse (as in CIN) but clumped. Moreover, positive nuclei are somewhat less densely packed than in CIN. When tangentially cut parabasal cells and inflammatory cells are carefully excluded, MIB-C is a strong diagnostic adjunct in distinguishing CIN from normal or benign cervical squamoepithelial lesions.

Low- and high-risk CIN 1 and 2 lesions: prospective predictive value of grade, HPV, and Ki-67 immuno-quantitative variables

The aim of this study was to evaluate in small cervical biopsies (non‐cone, non‐large loop excision of the transformation zone, LLETZ) the prognostic value of both routinely assessed and reviewed cervical intraepithelial neoplasia (CIN) grades 1 and 2, oncogenic human papillomavirus (onco‐HPV) DNA (HPV status) and Ki‐67 immuno‐quantitative features for the prediction of progression. In biopsies from 44 CIN patients (the learning set), subjective CIN grade, onco‐HPV by PCR, and Ki‐67 immuno‐quantitative features were assessed. We followed development of the lesions by colposcopy and cytology, but the final endpoint was the histological grade (again in small biopsies). The outcome was defined as progression (histological (CIN 1 to (CIN 2 or 3)) or CIN 2 to CIN 3) or not (all other cases). Single and multivariate (Cox regression) and survival analyses were applied. The resulting predictive combination of quantitative features was then applied to a new test set of 35 consecutive CIN 2 (small) biopsies followed by large (cone or LLETZ) biopsies. In the learning set, mean follow‐up of non‐progression cases was 18.8 months (range 4.7–35.9), and of progression cases 13.1 months (range 6.4–32.9) (p = 0.18). Five cases progressed (11%). Of the 16 CIN 1 and 28 CIN 2 lesions, 31 cases (70%) were onco‐HPV positive (5 of the CIN 1 and 26 of the CIN 2). The age of women with progression or not did not differ (p = 0.68). All 5 progression cases were CIN 2 (on review, one of these was reclassified as CIN 1), and positive for onco‐HPV. Cox regression analysis showed that the percentage of Ki‐67‐positive cells located in the middle third layer of the epithelium (MIDTHIRD) and the 90th percentile of the stratification index (SI90) was the best combination to predict progression (log rank = 5.1, p = 0.02). Furthermore, sensitivity (100%), specificity (56%), positive predictive value (23%), negative predictive value (100%), and overall percentage correctly classified cases (61%) of this Ki‐67 combination were higher than that of subjective CIN grade or HPV status, either single or combined (both for routine and review CIN grades). Adding CIN grade or HPV status did not improve the Ki‐67 prognostic results. Application of the prognostic Ki‐67 combination to the test set of 35 small biopsies followed by large (cone or LLETZ biopsies) gave comparable results. Analyses on homogeneous subgroups (CIN 2 only, onco‐HPV+ only, or CIN2/onco‐HPV+ only) gave similar results. In conclusion, Ki‐67 immuno‐quantitation of small biopsies showing CIN 1 or CIN 2 has strong independent prognostic value for progression. Copyright

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.

Ki67 predicts progression in early CIN: Validation of a multivariate progression-risk model

This study of early CIN biopsies (25 CIN1 and 65 CIN2) with long follow-up was done to validate, in a new group of patients, the value of Ki67 immuno-quantitative features to predict high CIN grade in a follow-up biopsy (often denoted to as “progression”), as described in a previous study. Each biopsy in the present study was classified with the previously described Ki67-model (consisting of the stratification index and the % positive nuclei in the middle third layer of the epithelium) as “low-risk” or “high-risk”, and matched with the follow-up outcome (progression-or-not). Furthermore, it was studied whether subjective evaluation of the Ki67 sections by experienced pathologists, who were aware of the prognostic quantitative Ki67 features, could also predict the outcome. Thirdly, the reproducibility of routine use of the quantitative Ki67-model was assessed. Fifteen cases progressed (17%) to CIN3, 2/25 CIN1 (8%) and 13/65 CIN2 (20%), indicating that CIN grade (as CIN1 or CIN2) is prognostic and that the percentage of CIN1 and CIN2 cases with progression in the present study is comparable to many previous studies. However, the quantitative Ki67 model had stronger prognostic value than CIN grade as none of the 40 “Ki67-model low-risk” patients progressed, in contrast to 15 (30%) of the 50 “Ki67-model high-risk” patients (p < 0.001). In multivariate analysis, neither CIN grade nor any of the other quantitative Ki67 features added to the abovementioned prognostic Ki67-model. Subjective analysis of the Ki67 features was also prognostic, although quantitative assessments gave better results. Routine application of the quantitative Ki67-model in CIN1 and CIN2 was well reproducible. In conclusion, the results confirm that quantitative Ki67 features have strong prognostic value for progression in early CIN lesions.

Relationship between the presence of oncogenic HPV DNA assessed by polymerase chain reaction and Ki-67 immunoquantitative features in cervical intraepithelial neoplasia

The aims of this study were firstly to determine which Ki‐67 immunoquantitative parameters correlate with the presence of oncogenic human papillomavirus (HPV) in cervical intraepithelial neoplasia (CIN) lesions; and secondly to compare prospectively the routinely assessed CIN grades with the Ki‐67 quantitative pathological CIN grade, the expert revised grade, and the presence of oncogenic HPV DNA. HPV polymerase chain reaction (PCR) and Ki‐67 immunoquantitation were performed on 90 consecutive biopsies (16 CIN 1, 35 CIN 2, and 39 CIN 3). CIN grade was assessed routinely by six different pathologists. The presence of the lesion was confirmed in a histological section following the material used for PCR and Ki‐67 analysis. In a second prospective routine test set analysis, 66 more CIN lesions (14 CIN 1, 15 CIN 2, and 37 CIN 3) were routinely graded (also by six different pathologists, routine CIN grade=CINROUT), studied for oncogenic HPV DNA, and graded by quantitative Ki‐67 features (quantitative pathological CIN grade=CINQP). These latter cases were blindly revised by one of the authors (reference CIN grade=CINREF). Eight of the nine Ki‐67 immunoquantitative features showed a significant difference between the oncogenic HPV‐positive and ‐negative cases. The best single discriminator was the 90th percentile of the stratification index (SI90). All 61 cases with Ki‐67 SI90>0.60 were HPV‐positive (68% of the total group studied). Of the 29 cases with SI90≤0.60, 16 were negative and 13 positive for oncogenic HPV and none of the Ki‐67 features (either single or combined) could distinguish them. Using stepwise multivariate analysis, the best discriminating combination of features was SI90 and the percentage of Ki‐67‐positive nuclei in the deep third layer of the epithelium (PERC DL). The combination of SI90 and the percentage of Ki‐67‐positive nuclei per 100u2009µm basal membrane was nearly as strong as that of SI90 and PERC DL. With these two features, 86% of the cases were correctly classified. The subjective estimate of SI90 (>0.60 or ≤0.60) by two independent observers was not accurate and not reproducible. In the prospective routine test set analysis of 66 cases, the 37 CINROUT=3 all had CINQP and CINREF=3 and all these cases were oncogenic HPV‐positive. Eight of the 14 original CINROUT=1 grades were oncogenic HPV (=HPV)‐positive and five of these eight were upgraded by CINQP to CIN 2 and CIN 3. These upgrades were in agreement with the blind reference revisions. The six HPV‐negative CINROUT=1 cases were CIN 1 both by CINQP and by CINREF. Thirteen of the 15 original CINROUT=2 grades were HPV‐positive and seven of these were CINQP=3. All six HPV‐positive CINROUT=2 cases that were CINQP=2 were also CINREF=2 at blind revision. In conclusion, this study has shown firstly, that in CIN lesions, Ki‐67 immunoquantitative features and the presence of oncogenic HPV are highly correlated, and also within one subjective CIN grade; secondly, that subjective impressions of SI90 are not as accurate or reproducible as quantitative image analysis results; and thirdly, that the routine application of QP CIN‐grading gives results that are in very good agreement with CIN grades assessed by an expert. Thus, routine QP‐grading may be used to correct the subjective grade assessed by non‐expert pathologists. Copyright

Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks

Aims: To evaluate whether in situ biomarkers Ki67, mitotic activity index (MAI), p53, mean area of the 10 largest nuclei (MNA10), and whole genome DNA ploidy by flow and image cytometry (FCM and ICM, respectively) have independent prognostic value in urinary bladder urothelial cell carcinomas (UCs). Methods: Ki67 and p53 immunoquantitation was performed in TaT1 consensus diagnosis UCs. MAI and MNA10 were also determined. Single cell suspensions were stained (DAPI for FCM; Feulgen for ICM). There was enough material for all measurements in 171 cases. Kaplan-Meier curves and multivariate survival analysis (Cox) were used to assess the prognostic value of all features (including classic clinicopathological risk factors, such as stage, grade, multicentricity, carcinoma in situ). Results: Thirteen (7.6%) patients progressed. Of the classic factors, grade was strongly prognostic in univariate analysis, as were all the biomarkers. In multivariate analysis, the strongest independent combinations for progression were MNA10 (threshold (T) = 170.0 μm2) plus MAI (T = 30), or MNA10 (T = 170.0 μm2) plus Ki67(T = 25.0%). p53 (T = 35.2%) plus Ki67 (T = 25.0%) also predicted progression well, with high hazard ratios, but p53 measurements were not as reproducible as the other features. The prognostic value of the quantitative biomarkers exceeded that of the classic risk factors and DNA ploidy. The sensitivity, specificity, positive, and negative predictive values of MNA10/MAI or MNA10/Ki67 at the thresholds mentioned were 100%, 79%, 57%, and 100%, respectively. These feature combinations were also strongest prognostically in the high risk treatment subgroup. Conclusions: The combined biomarkers MNA10/Ki67 or MNA10/MAI are more accurate and reproducible predictors of stage progression in TaT1 UCs than classic prognostic risk factors and DNA ploidy.

Imiquimod in cervical, vaginal and vulvar intraepithelial neoplasia: A review

Human papillomavirus (HPV) infection is in the vast majority of patients accountable for the development of vulvar, cervical and vaginal intraepithelial neoplasia (VIN, CIN, VAIN); precursors of vulvar, cervical and vaginal cancers. The currently preferred treatment modality for high grade VIN, CIN and VAIN is surgical excision. Nevertheless surgical treatment is associated with adverse pregnancy outcomes and recurrence is not uncommon. The aim of this review is to present evidence on the efficacy, safety and tolerability of imiquimod (an immune response modifier) in HPV-related VIN, CIN and VAIN. A search for papers on the use of imiquimod in VIN, CIN and VAIN was performed in the MEDLINE, EMBASE and Cochrane library databases. Data was extracted and reviewed. Twenty-one articles met the inclusion criteria and were analyzed; 16 on VIN, 3 on CIN and 2 on VAIN. Complete response rates in VIN ranged from 5 to 88%. Although minor adverse effects were frequently reported, treatment with imiquimod was well tolerated in most patients. Studies on imiquimod treatment of CIN and VAIN are limited and lack uniformly defined endpoints. The available evidence however, shows encouraging effect. Complete response rates for CIN 2-3 and VAIN 1-3 ranged from 67 to 75% and 57 to 86% respectively. More randomized controlled trials on the use of imiquimod in CIN, VAIN and VIN with extended follow-up are necessary to determine the attributive therapeutic value in these patients.

Cervical intraepithelial neoplasia grade 3 lesions can regress

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.