Bianca van Diermen

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.

Microsatellite instability and DNA ploidy in colorectal cancerx: Potential implications for patients undergoing systematic surveillance after resection

Appropriate stratification tools for targeted surveillance after resection for colorectal cancer (CRC) are lacking. The objective of the current study was to investigate the effect of microsatellite instability (MSI) and DNA ploidy on surveillance after surgery.

Prospective Multicenter Validation Confirms the Prognostic Superiority of the Endometrial Carcinoma Prognostic Index in International Federation of Gynecology and Obstetrics Stage 1 and 2 Endometrial Carcinoma

PURPOSE To validate the prognostic value of the endometrial carcinoma prognostic index (ECPI; combined myometrium invasion, flow cytometric DNA ploidy, and morphometric mean shortest nuclear axis [MSNA]) versus classic prognosticators. PATIENTS AND METHODS Prospective multicenter ECPI analysis was conducted in 463 endometrial carcinomas with a median of 6.5 years (range, 1 to 10 years) follow-up, review of pathology features, and univariate (Kaplan-Meier) and multivariate (Cox) analyses. RESULTS Initial routine and review diagnoses varied considerably (invasion depth, 11%; type, 20%; grade, 34%; vessel invasion, 72%); the review diagnoses were stronger prognostically. In International Federation of Gynecology and Obstetrics stage 1 (after histopathologic examination; pFIGO-1; n = 372; 38 deaths occurred as a result of disease [10.2%]), DNA ploidy was prognostic in hysterectomies (P <.00001) but not in curettages (P =.06). ECPI was a stronger prognostic indicator than other features. ECPI, MSNA, and DNA ploidy were also prognostic in pFIGO-1B and -1C subgroups. Multivariate analysis in pFIGO-1 showed that uterine MSNA < or = versus > 7.93 microm (hazard ratio [HR], 3.4) and grade (as 1 + 2 v 3; HR, 2.6) added to the ECPI (HR, 32), but only in patients with an unfavorable ECPI of > 0.87. Adjuvant radiotherapy was not an independent prognostic factor in any of the subgroups. In pFIGO-2 (n = 46), ECPI, DNA-ploidy, and age (< or = 64, > 64 years) were significant. In FIGO-3 (n = 31) and FIGO-4 (n = 14), none of the classic or other features analyzed was of prognostic value, which explains why in previous studies using different mixtures of FIGO stages, DNA ploidy prognostic results varied. CONCLUSION In endometrial carcinoma, DNA-ploidy is prognostic in hysterectomy and not in curettage samples. The ECPI is prognostically much stronger than the classic features widely used for therapy triage in pFIGO-1 and -2.

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.

DNA cytometric features in biopsies of TaT1 urothelial cell cancer predict recurrence and stage progression more accurately than stage, grade, or treatment modality

OBJECTIVES To compare retrospectively the predictive value for recurrence and stage progression of DNA ploidy and S-phase fraction by flow cytometry and highly automated ultrafast image cytometry (ICM) in biopsies of TaT1 urothelial cell carcinomas (UCCs) of the urinary bladder with stage, grade, other pathologic features, and treatment. METHODS Three experienced pathologists reviewed the stage and grade of 228 UCCs; 193 (85%) consensus cases were analyzed further. We had enough material for single-cell suspensions for both flow cytometry and ICM in 183 cases (94.8%). The 2001 European Society for Analytical Cellular Pathology standards for DNA ICM were followed. The predictive value of DNA features, classic prognosticators (stage, grade, carcinoma in situ, multicentricity), and treatment modality for recurrence and stage progression were analyzed with univariate (Kaplan-Meier) survival and multivariate (Cox model) regression analysis. Ta and T1 cases were analyzed separately. RESULTS Of the 228 cases, 88 (51.5%) recurred and 13 (7.6%) progressed. On univariate analysis, most of the DNA features studied were statistically significant. Treatment modality and grade were only prognostic for progression (not for recurrence) and only in Ta cases. On multivariate analysis, DNA ICM features performed best; the strongest recurrence predictor for Ta UCC was a DNA index (DI) of 1.0 versus all others, and for T1 UCC, a DI of less than 1.3 versus 1.3 or greater. The best stage progression predictor for Ta UCCs was a DI of 1.0 plus an S-phase fraction of less than 10%, and for T1 UCCs, a DI of less than 1.3 versus 1.3 or greater. With multivariate analysis, sex, age, grade, carcinoma in situ, multicentricity, and treatment modality were excluded once the DNA ICM features were selected. CONCLUSIONS DNA image cytometric features predict recurrence and stage progression in TaT1 UCC biopsies more accurately than classic prognostic factors, independent of treatment modality.

Predictive Testing of Early CIN Behaviour by Molecular Biomarkers

Each year, 330,000 new Cervical Intraepithelial Neoplasias(CIN) occur in the European Union (EU) of which 120,000 are early CIN where grade (1, 2) indicates the progression-risk to CIN-3 and therefore determines the treatment choice. However, the Positive Predictive Value (PPV) of CIN grade to predict progression is low (10% and 20% for CIN-1 and -2 respectively, 16% on average) resulting in an enormous number of over-treatments indicating worrisome grade reproducibility.Certain molecular biomarkers such as Ki-67 have a higher PPV (30%, an improvement of 14%), which in Europe alone could improve treatment for many thousands of women per year with considerable cost reduction for the health care system. The quantitative Ki-67 prognostic model has been validated in independent retrospective and prospective studies from different laboratories. Moreover, the PPV of Ki-67 alone can be improved by additional molecular biomarkers (retinoblastoma protein = Rb, cytokeratins= CK-14/-13). Combined Ki67-Rb allows a 2-tiered progression-risk subgroup assignment as very low ( approximately 0% progression, 71% of all CIN-I/II patients)and high risk (48% progression risk, incidence 32%), leaving a small (7% of all) prognostically undetermined group (17% progression). Additional CK-14 and -13 analysis can sub-classify the high-risk in an intermediate and very high risk subgroup(with 40% and 100% progression risks respectively).Thus, molecular biomarkers are potentially important determinators of early CIN lesion behaviour. Important factors for widespread acceptance of molecular biomarkers are (1) market penetration by user-friendly equipment, (2) (inter)national keeping of GLP conditions (reproducibility, independent validation), requiring customer-driven industrial efforts,governmental measures, and additional PPV improvement to further reduce over-treatment.

A monotonous population of elongated cells (MPECs) in colorectal adenoma indicates a high risk of metachronous cancer.

Accurate predictors for metachronous colorectal cancer (CRC) development after polypectomy are lacking. We evaluated the prognostic value of classical clinicopathologic features and a monotonous population of elongated cells (MPECs) in colorectal adenomas from 171 consecutively selected population-based patients with long-term follow-up. Quantitative image analysis, and univariate and multivariate regression analysis were applied. Ten of 171 adenomas (5.8%) developed metachronous CRC (defined as >24 mo interval and >5 cm from the index adenoma to the cancer). Median follow-up of adenomas with metachronous CRC was 68.4 and without cancer 149.7 months (range: 25 to 192 and 25 to 256, respectively). The most prognostic classical features were the localization of the marker adenoma as proximal (ie, in the cecum through transverse colon) versus distal from the transverse colon [P=0.0003, hazard ratio (HR)=8] and the number of polyps found during colonoscopy (≤2 vs.>2, P=0.002, HR=6). Quantitative features of the MPECs included the longest nuclear axis and variance of the number of nuclei with 2 neighbors (higher and lower in cancer cases, respectively). Of the 171 adenomas, 50 (29%) had MPECs, of which 9 (18%) patients developed metachronous CRC at follow-up, contrasting 1/121 (0.8%) without MPECs (P=0.0003, HR=23). MPECs occurred in both low-grade and high-grade dysplasia, and in tubular and (tubulo) villous adenomas. MPECs had the strongest predictive value for metachronous CRC development. Adenomas proximally located had additional value but only if they were MPEC positive (which only occurred in 5 adenomas, 3 of which (60%) developed cancer). Having more than 2 polyps also had additional prognostic value but only in MPEC-negative adenomas [10 cases; 1 (10%) developed cancer]. Dysplasia grade and histologic growth pattern had no additional value. Thus, colorectal adenomas with subsequent metachronous cancer development can be identified more accurately with MPECs than with classical prognostic factors.

Prognostic comparison of proliferation markers and World Health Organization 1973/2004 grades in urothelial carcinomas of the urinary bladder ☆

European treatment guidelines of non-muscle-invasive urothelial carcinoma of the urinary bladder are strongly dependent on grade, but grading reproducibility is wanting. Protocolized proliferation features such as Mitotic Activity Index (MAI), Ki-67, and phosphohistone H3 are prognostic and reproducible. The objective of this population-based study was to compare proliferation biomarkers with each other and with World Health Organization (WHO) 1973/2004 grades with regard to prediction of stage progression. A total of 193 primary non-muscle-invasive urothelial carcinomas were analyzed using WHO73/04 grades and measurement of the proliferation markers mentioned above. Sensitivities, specificities, and positive and negative predictive values with confidence intervals (CIs) were estimated with regard to progression prediction. Kaplan-Meier survival curves were made, and the hazard ratio and Harrells C-index with 95% CIs, P values, and adjusted C-index for stage progression or not of WHO73, WHO04, and the proliferation markers were calculated. The median follow-up time was 75 months (range, 1-127). A total of 111 patients (52%) experienced recurrence within 5 years, and 14 patients (7%) progressed. High values of MAI predicted stage progression with a positive predictive value of 0.22 (95% CI, 0.12-0.37). The positive predictive value of Ki-67 and phosphohistone H3 were 0.15 (both 95% CIs, 0.07-0.29) and comparable to that of the WHO04. The prognostic value of MAI was strongest, exceeding that of the other proliferation markers and the WHO grading systems. In conclusion, in non-muscle-invasive urinary bladder urothelial carcinomas, proliferation biomarkers have prognostic value, possibly exceeding that of the WHO classifications.

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.

Correlation of grade of urothelial cell carcinomas and DNA histogram features assessed by flow cytometry and automated image cytometry

Objective: To analyse how DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and an optimised fully automatic DNA image cytometer (ICM) correlate with grade in TaT1 urothelial cell carcinomas (UC) of the urinary bladder. Materials and methods: Two-hundred-and twenty-eight consensus cases were analysed. Single cell suspensions were stained (DAPI for FCM, Feulgen for ICM). There was enough material for both FCMand ICMin 202 of these cases. FCMand optimised ICM measurements were performed on the 202 UCs. To discriminate between different grades, single- and multivariate analyses was performed on DNA histogram features obtained with the MultiCycle program (using DNA index (DI) and SPF). Results: Overall measurement time of the adapted ICM method was 10.7 minutes per case (range 5.9–29.8 min.) and required little additional interactive object rejection (average 152 objects (84–298) on 3000 objects per case measured, which took 9.9 minutes on average, range 8.3–15.5 minutes). The ICM histograms looked much “cleaner” with less noise than the FCM graphs. The coefficient of variation (CV) of the diploid peak for ICM(5.4%) was significantly lower than for FCM(5.9%) (p < 0.0001). ICM features were more strongly correlated to grade than FCMfeatures. In multivariate analysis, the best discriminating set of features was DNA ploidy and SPF (both by ICM). Conclusions: The adapted fully automated DNA ICM works very well for UCs. Low CV DNA ICM histograms are obtained in a time comparable to FCM. The DNA ICM results have stronger discriminative power than DNA FCM for grade in TaT1 UCs. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-3/bol.htm.