Analytical Cellular Pathology - Volume 24, issue 2-3

Authors: Umesh Adiga, P.S.

Article Type: Research Article

Abstract: In this article we have proposed an integrated system for measurement of important features from 3D tissue images. We propose a segmentation technique, where we combine several methods to achieve a good degree of automation. Important histological and cytological three‐dimensional features and strategies to measure them are described. Figures can be viewed in colour on http://www.esacp.org/acp/2002/24‐23 /adiga.htm.

Keywords: Segmentation, watershed, active model, features

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 47-58, 2002

Authors: Smolle, Josef | Gerger, Armin | Weger, Wolfgang | Kutzner, Heinz | Tronnier, Michael

Article Type: Research Article

Abstract: Background: Tissue counter analysis is an image analysis tool designed for the detection of structures in complex images at the macroscopic or microscopic scale. As a basic principle, small square or circular measuring masks are randomly placed across the image and image analysis parameters are obtained for each mask. Based on learning sets, statistical classification procedures are generated which facilitate an automated classification of new data sets. Objective: To evaluate the influence of the size and shape of the measuring masks as well as the importance of feature selection, statistical procedures and technical preparation of slides on the performance …of tissue counter analysis in microscopic images. As main quality measure of the final classification procedure, the percentage of elements that were correctly classified was used. Study design: H&E‐stained slides of 25 primary cutaneous melanomas were evaluated by tissue counter analysis for the recognition of melanoma elements (section area occupied by tumour cells) in contrast to other tissue elements and background elements. Circular and square measuring masks, various subsets of image analysis features and classification and regression trees compared with linear discriminant analysis as statistical alternatives were used. The percentage of elements that were correctly classified by the various classification procedures was assessed. In order to evaluate the applicability to slides obtained from different laboratories, the best procedure was automatically applied in a test set of another 50 cases of primary melanoma derived from the same laboratory as the learning set and two test sets of 20 cases each derived from two different laboratories, and the measurements of melanoma area in these cases were compared with conventional assessment of vertical tumour thickness. Results: Square measuring masks were slightly superior to circular masks, and larger masks (64 or 128 pixels in diameter) were superior to smaller masks (8 to 32 pixels in diameter). As far as the subsets of image analysis features were concerned, colour features were superior to densitometric and Haralick texture features. Statistical moments of the grey level distribution were of least significance. CART (classification and regression tree) analysis turned out to be superior to linear discriminant analysis. In the best setting, 95% of melanoma tissue elements were correctly recognized. Automated measurement of melanoma area in the independent test sets yielded a correlation of r=0.846 with vertical tumour thickness (p<0.001), similar to the relationship reported for manual measurements. The test sets obtained from different laboratories yielded comparable results. Conclusions: Large, square measuring masks, colour features and CART analysis provide a useful setting for the automated measurement of melanoma tissue in tissue counter analysis, which can also be used for slides derived from different laboratories. Show more

Keywords: Melanoma, image analysis, tissue counter analysis, classification and regression tree, texture analysis, colour analysis

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 59-67, 2002

Authors: Aubele, Michaela | Werner, Martin | Höfler, Heinz

Article Type: Research Article

Abstract: The hypothetical multistep model of breast carcinogenesis suggests a transition from normal epithelium to invasive carcinoma via intraductal hyperplasia (without and with atypia) and in situ carcinoma. These presumptive precursor lesions are currently defined by their histological features, and their prognosis is imprecisely estimated from indirect epidemiological evidence. Cytogenetic and molecular‐genetic analysis of these lesions give evidence for an accumulation of various genetic alterations during breast tumorigenesis. Using immuno‐histochemistry overexpression of the c‐erbB‐2 oncogene was found in ductal carcinoma in situ (DCIS), but not in atypical intraductal hyperplasia (AIDH) and intraductal hyperplasia (IDH). An expression of mutant p53 tumor …suppressor gene as well as expression of cyclin D1 was identified in DCIS. In IDH lesions loss of heterozygosity (LOH) at various loci could be identified, and comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies delivered evidence for DNA amplification on chromosomal region 20q13 in the early stage of IDH. However, little is currently known about genetic alterations in those premalignant lesions, and the chronology of genetic alterations and histopathological changes during carcinogenesis is mainly undiscovered. Figure 1 can be viewed in colour on http://www.esacp.org/acp/2002/24‐23 /aubele.htm Show more

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 69-76, 2002

Authors: Aubele, Michaela | Auer, Gert | Braselmann, Herbert | Nährig, Jörg | Zitzelsberger, Horst | Quintanilla‐Martinez, Leticia | Smida, Jan | Walch, Axel | Höfler, Heinz | Werner, Martin

Article Type: Research Article

Abstract: Multiple chromosomal imbalances have been identified in breast cancer using comparative genomic hybridization (CGH). Their association with the primary tumors' potential for building distant metastases is unknown. In this study we have investigated 39 invasive breast carcinomas with a mean follow‐up period of 99 months (max. 193 months) by CGH to determine the prognostic value of chromosomal gains and losses. The mean number of chromosomal imbalances per tumor was 6.5±0.7 (range 2 to 18). The most frequent alterations identified in more than 1/3 of cases were gains on chromosomes 11q13, 12q24, 16, 17, and 20q, and losses on 2q …and 13q. A significantly different frequency of chromosomal aberrations (p≤0.05) was found between DNA‐diploid and non‐diploid tumors (gain on chromosome 17). Differences were also noted between tumors progressing to distant metastases within the period of follow‐up and those which do not (gains on 11q13 and 12q24; loss on 12q). Significant univariate correlations (p≤0.05) with the metastasis‐free survival of patients were found for lymph node status, the cytometrical determined DNA ploidy (diploid/non‐diploid) and anisokaryosis, and for DNA gains on 11q13, 12q24, 17, and 18p. An unexpected inverse correlation was found between clinical outcome and gains on 11q13 and 12q24. In multivariate analysis independent prognostic value, in addition to lymph node status, was found for chromosomal gains on 11q13, 12q24, 17 and 18p. Amplification on 20q, which did not correlate with metastasis‐free survival in a univariate analysis, showed weak prognostic significance in combination with the nodal status. The prognostic value of chromosomal alterations – some of them by inverse correlation – suggests an interaction and/or compensation of the involved amplified genes and their effects on the occurrence of distant metastases in breast cancer patients. Show more

Keywords: CGH, prognosis, breast cancer, chromosomal imbalances, metastasis‐free survival

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 77-87, 2002

Authors: Perner, P. | Rapp, A. | Dressler, C. | Wollweber, L. | Beuthan, J. | Greulich, K.O. | Hausmann, M.

Article Type: Research Article

Abstract: The response of single breast cancer cells (cell line T‐47D) to 17β‐estradiol (E2 ) under different concentrations was studied by using an instrument that allows to combine far‐field light microscopy with high resolution scanning near‐field (AFM/SNOM) microscopy on the same cell. Different concentrations of E2 induce clearly different effects as well on cellular shape (in classical bright‐field imaging) as on surface topography (atomic force imaging) and absorbance (near‐field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle‐like shape at un‐physiologically high concentrations. The surface topography …of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E2 concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un‐physiological high E2 concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near‐field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co‐localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers. Show more

Keywords: Light microscopy, scanning near‐field optical microscopy (SNOM), transmission light SNOM, breast cancer cells, cell surface response, estrogen (17β‐estradiol) treatment

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 89-100, 2002

Authors: Wählby, Carolina | Lindblad, Joakim | Vondrus, Mikael | Bengtsson, Ewert | Björkesten, Lennart

Article Type: Research Article

Abstract: Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. …By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation. Show more

Citation: Analytical Cellular Pathology, vol. 24, no. 2-3, pp. 101-111, 2002