Analytical Cellular Pathology - Volume 26, issue 1-2

Authors: Reith, Albrecht

Article Type: Research Article

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 1-2, 2004

Authors: Rosati, Anna | Candussio, Luigi | Crivellato, Enrico | Klugmann, Fiora Bartoli | Giraldi, Tullio | Damiani, Daniela | Michelutti, Angela | Decorti, Giuliana

Article Type: Research Article

Abstract: Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV), have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1 /ADR) or MRP (multidrug resistance‐related protein) (PANC‐1) and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied. …BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings. Show more

Keywords: Bodipy‐FL‐verapamil, multidrug resistance, P‐glycoprotein, MRP, cytofluorimetry

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 3-11, 2004

Authors: Kruse, Arnold‐Jan | Baak, Jan P.A. | Janssen, Emiel A. | Kjellevold, Kjell‐Henning | Fiane, Bent | Lovslett, Kjell | Bergh, Johan | Robboy, Stanley

Article Type: Research Article

Abstract: 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. Show more

Keywords: Cervix, CIN, Ki67, image analysis, progression

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 13-20, 2004

Authors: Osterheld, Maria‐Chiara | Caron, Liette | Demierre, Mireille | Laurini, Ricardo | Bosman, F.T.

Article Type: Research Article

Abstract: This analysis of DNA‐ploidy heterogeneity in advanced gastric carcinomas is consistent with the hypothesis of the emergence of a single aneuploid cell clone as a crucial mechanism in the progression from early gastric carcinoma to advanced gastric cancer. The prognostic value of DNA‐ploidy in gastric cancers has been a matter of controversy. Tumour DNA‐ploidy heterogeneity, the presence within the same tumour of multiple stemlines differing in DNA content, has been described in various tumours including gastric cancers. The occurrence of such heterogeneity has been accepted as an explanation for the divergent DNA‐ploidy results in this type of tumours. …A previous study of early gastric cancers suggested that in pure diploid superficial carcinomas, genetic instability might lead to a cell clone which has undergone a ploidy shift and is more aggressive. If so, this would initially result in DNA‐ploidy heterogeneity. Proliferative dominance of the aneuploid clone could eventually evolve to a homogeneous aneuploid tumour. In order to test this hypothesis, we studied DNA‐aneuploidy and DNA‐ploidy heterogeneity in advanced gastric carcinomas. We performed DNA cytophotometry on multiple samples collected from 16 advanced gastric carcinomas and found 15 DNA‐aneuploid tumours (94%) and one diploid tumour. Multiple DNA‐stemlines were found in 4 cases (26%). Analysis of proliferative activity performed on the same samples revealed higher proliferation rate in DNA‐ploidy homogeneous tumours than in aneuploid heterogeneous tumours. Heterogeneous tumours did not overexpress p53. These results confirm that DNA‐aneuploidy is frequent in advanced gastric cancer and demonstrate that a majority of these aneuploid tumours are not DNA‐ploidy heterogeneous. Furthermore, the higher proliferative activity in homogeneous‐aneuploid carcinomas and their more frequent overexpression of p53 support the hypothesis that in gastric cancer tumour progression implies the development of a dominant and more aggressive (higher proliferative activity, p53 overexpression) aneuploid cell clone. Show more

Keywords: Gastric adenocarcinoma, heterogeneity, DNA ploidy, prognostic factors

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 21-29, 2004

Authors: Guillaud, Martial | Cox, Dennis | Malpica, Anais | Staerkel, Gregg | Matisic, Jasenka | Van Niekirk, Dirk | Adler‐Storthz, Karen | Poulin, Neal | Follen, Michele | MacAulay, Calum

Article Type: Research Article

Abstract: Objectives: As part a Program Project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analysis of biopsies from 1800 patients. Several methodological issues have arisen with respect to this analysis: (1) Finding the most efficient way to compensate for staining intensity variation with out losing diagnostic information; (2) Assessing the inter‐ and intra‐observer variability of the semi‐interactive data collection; and (3) the use of non‐overlapping cells from the intermediate layer only. Methods: Non‐overlapping quantitatively stained nuclei were selected from 280 samples with histopathological characteristics of normal (199), koilocytosis (37), CIN 1 (18), CIN …2 (10) and CIN 3 (16). Linear discriminant analysis was used to assess the diagnostic information in three different feature sets to evaluate and compare staining intensity normalization methods. Selected feature values and summary scores were used to evaluate intra‐ and inter‐observer variability. Results: The features normalized by the internal subset of the imaged cells had the same discriminatory power as those normalized by the control cells and by both normalization methods seem to have additional discriminatory power over the set of features which do not require normalization. The use of the internal subset decreased the image acquisition time by ∼50% at each center, respectively. The intra‐ and inter‐observer variability was of a similar size. Good performance was obtained by measuring the intermediate layer only. Conclusion: The use of intensity normalization from a subset of the imaged non‐overlapping intermediate layer cells works as well as or better than any of the other methods tested and provides a significant timesaving. Our intra‐ and inter‐observer variability do not seem to affect the diagnostic power of the data. Although this must be tested in a larger data set, the use of intermediate layer cells only may be acceptable when using quantitative histopathology. Show more

Keywords: Semi‐interactive quantitative histopathology, stoichiometric staining, stain intensity normalization, inter‐ and intra‐observer variability, CIN, SIL, morphology

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 31-43, 2004

Authors: Mattfeldt, Torsten | Trijic, Danilo | Gottfried, Hans‐Werner | Kestler, Hans A.

Article Type: Research Article

Abstract: The subclassification of incidental prostatic carcinoma into the categories T1a and T1b is of major prognostic and therapeutic relevance. In this paper an attempt was made to find out which properties mainly predispose to these two tumor categories, and whether it is possible to predict the category from a battery of clinical and histopathological variables using newer methods of multivariate data analysis. The incidental prostatic carcinomas of the decade 1990–99 diagnosed at our department were reexamined. Besides acquisition of routine clinical and pathological data, the tumours were scored by immunohistochemistry for proliferative activity and p53‐overexpression. Tumour vascularization (angiogenesis) and epithelial …texture were investigated by quantitative stereology. Learning vector quantization (LVQ) and support vector machines (SVM) were used for the purpose of prediction of tumour category from a set of 10 input variables (age, Gleason score, preoperative PSA value, immunohistochemical scores for proliferation and p53‐overexpression, 3 stereological parameters of angiogenesis, 2 stereological parameters of epithelial texture). In a stepwise logistic regression analysis with the tumour categories T1a and T1b as dependent variables, only the Gleason score and the volume fraction of epithelial cells proved to be significant as independent predictor variables of the tumour category. Using LVQ and SVM with the information from all 10 input variables, more than 80 of the cases could be correctly predicted as T1a or T1b category with specificity, sensitivity, negative and positive predictive value from 74–92%. Using only the two significant input variables Gleason score and epithelial volume fraction, the accuracy of prediction was not worse. Thus, descriptive and quantitative texture parameters of tumour cells are of major importance for the extent of propagation in the prostate gland in incidental prostatic adenocarcinomas. Classical statistical tools and neuronal approaches led to consistent conclusions. Show more

Keywords: Artificial neural networks, bioinformatics, classification, immunohistochemistry, incidental carcinoma, learning vector quantization, logistic regression, pathology, pattern recognition, prostatic cancer, stereology, support vector machine

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 45-55, 2004

Authors: Schönherr, Alexandra | Bayer, Mary | Böcking, Alfred

Article Type: Research Article

Abstract: The Ki67 proliferation rate of mesothelial cells was determined in 20 effusions due to malignant mesotheliomas and in 20 non‐neoplastic effusions, to investigate if this marker may be useful to identify neoplastic mesothelioma cells and if there is a correlation between proliferation rate and survival time. Using the ABC‐method, effusions were immunostained and the marker Ki67 was evaluated quantitatively. Ki67 proliferation fraction showed rates from 2.3% to 70% in malignant mesothelioma cells and from 1.8% to 25.5% in reactive mesothelial cells. A significant difference was found (p=0.05) between those two groups. Assuming a threshold at 26%, a sensitivity …of 25% and specificity of 100% resulted. Yet, due to its low sensitivity this marker seems not to be useful for differential diagnosis. Plotting surviving period against Ki67 proliferation fraction a correlation was observed which was not significant. Long term survivors (>28 month) showed proliferation rates below 3.8%. Unexpectedly a highly significant difference (p=0.001) between Ki67 proliferation rates of mesothelial cells from patients with malignant tumors other than mesothelial origin (7.0% to 25.5%) and mesothelial cells of patients without any malignant disease (1.8% to 16.3%) were observed. Setting a threshold at 10% for identification of a malignant disease, a sensitivity of 77.8% and specificity of 90.9% resulted. Show more

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 57-62, 2004

Authors: Lorand‐Metze, Irene | Pereira, Fernanda Gonçalves | Costa, Flávia Pereira Silva | Metze, Konradin

Article Type: Research Article

Abstract: In malignant lymphomas, cell kinetics has shown to be related with histologic type as well as with the clinical behaviour. The aim of our study was to investigate the relevance of cell proliferation parameters on overall survival in non‐Hodgkin's lymphomas as well as their relationship with prognostic factors such as International Prognostic Index (IPI). We performed DNA‐flow‐cytometry (S‐phase fraction and detection of DNA‐aneuploidy) as well as cytologic examination and the AgNOR technique in material obtained by fine needle aspiration of lymph nodes at diagnosis. The majority of the patients were stage IV by Ann Arbor and intermediate risk by IPI …(42/55). When analyzing all patients together, histologic type by the WHO classification, IPI and the presence of a DNA‐aneuploid clone could not separate well patients with a different survival. For all patients, univariate Cox analysis revealed S‐phase (SPF) and AgNOR parameters to be of prognostic value. In the multivariate analysis, however, only SPF remained in the final model. Yet, when stratifying for DNA‐ploidy, only the total number of AgNORs/nucleus was an independent parameter. Looking only at the DNA‐diploid cases, the AgNOR pattern remained the most important parameter, whereas for the DNA‐aneuploid cases this was true for SPF. When studying patients with B large cell lymphoma separately, only DNA‐ploidy was a prognostic factor. In summary, cell kinetic parameters reveal important prognostic information in NHL patients. Furthermore, DNA‐aneuploidy seems to interfere with the analysis of the AgNOR pattern. Show more

Keywords: Lymphoma, cell proliferation, DNA‐aneuploidy, prognosis, AgNORs

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 63-71, 2004

Authors: Böcking, A. | Stockhausen, J. | Meyer‐Ebrecht, D.

Article Type: Research Article

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 73-79, 2004

Authors: Raatz, H. | Böcking, A. | Hauptmann, S.

Article Type: Research Article

Abstract: Establishing prognosis proves particularly difficult with neuroendocrine tumours (NETs) as a benign looking histology can be associated with a malignant behaviour. In order to identify prognostic factors we examined 44 gastrointestinal and pulmonary, paraffin‐embedded NETs histologically and immunohistochemically. DNA‐image‐cytometry was used to examine 40 of these. We found that poor differentiation (corresponding to a Soga and Tazawa type D) and infiltrative growth correlated with a poorer prognosis. Moreover, parameters determined by diagnostic DNA cytometry like the 5c‐exceeding rate, the 2c‐deviation index, DNA‐grade of malignancy, DNA‐entropy and the type of DNA histogram were found to be of prognostic relevance. Morphometric parameters …like the form factor and the mean nuclear area were relevant for survival, tumour recurrence and metastasis. However, in the multivariate analysis the only independent risk factor was the histological differentiation. The 5c‐exceeding rate is a good objective risk factor, which can be used particularly in cases in which only a fine needle biopsie is available. Direct comparison of the histology and the 5c‐exceeding rate in the multivariate analysis suggests that the 5c‐exceeding rate taken as sole prognostic factor might be of higher prognostic relevance than the histology but larger studies are needed to confirm this. Show more

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 81-88, 2004

Authors: Michels, J.J.

Article Type: Research Article

Citation: Analytical Cellular Pathology, vol. 26, no. 1-2, pp. 89-91, 2004