Analytical Cellular Pathology - Volume 22, issue 3

Authors: Planz, Bernhard | Synek, Christian | Deix, Thomas | Böcking, Alfred | Marberger, Michael

Article Type: Research Article

Abstract: DNA‐image‐cytometry and antibodies directed against the Lewis X‐ and the 486p 3/12 antigen were applied to improve diagnostic accuracy of urinary cytology for the detection of bladder cancer. Cytology, immunocytology and DNA‐image‐cytometry were performed in spontaneously voided urine samples and barbotage bladder washings from 71 patients. The DNA content was determined using the CM‐1 Cytometer according to the recommendation of the ESCAP Consensus Report on Standardization of DNA‐image‐cytometry (1995). For immunocytological examination we used the monoclonal anti Lewis X antibody P‐12 and antibody 486p 3/12. All patients underwent subsequent cystoscopy and for any suspicious lesion biopsy or transurethral resection was …done. Histological findings revealed 31 patients with transitional cell carcinomas of different stages and grades of malignancy. 40 patients had various benign diseases of the urinary bladder. Cytology yielded a sensitivity of 68% and a specificity of 100%. DNA aneuploidy was detected in 81% of cancer patients with a specificity of 100%. By combination of these two methods the overall sensitivity increased to 87%. Immunocytology with Lewis X and 486p 3/12 antibodies showed reactivity in 84% and 87% in combination with a specificity of 80% and 70%, respectively. By combining urinary cytology, immunocytology and/or DNA‐image‐cytometry the overall sensitivity increased to 94% with no change in specificity. DNA‐image‐cytometry should be used to evaluate particularly urothelial cells suspicious for malignancy in urinary specimens. Because of low specificity the monoclonal antibodies against Lewis X‐ and 486p 3/12 antigens are not helpful in screening for bladder cancer. Nevertheless, their high sensitivity may justify their use in case DNA image cytometry is not available and in the follow up of patients with transitional cell carcinoma. Show more

Keywords: Lewis X antigen, 486p 3/12 antigen, DNA‐ and flow cytometry, urinary cytology, bladder cancer

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 103-109, 2001

Authors: Petersen, Iver | Petersen, Simone

Article Type: Research Article

Abstract: Lung cancer is a highly aggressive neoplasm which is reflected by a multitude of genetic aberrations being detectable on the chromosomal and molecular level. In order to understand this seemingly genetic chaos, we performed Comparative Genomic Hybridisation (CGH) in a large collective of human lung carcinomas investigating different tumor entities as well as multiple individual tumour specimens of single patients. Despite the considerable genetic instability being reflected by the well known morphological heterogeneity of lung cancer the comparison of different tumour groups using custom made computer software revealed recurrent aberration patterns and highlighted chromosomal imbalances that were significantly associated with …morphological histotypes and biological phenotypes. Specifically we identified imbalances in NSCLC being associated with metastasis formation which are typically present in SCLC thus explaining why the latter is such an aggressive neoplasm characterized by widespread tumor dissemination. Based on the genetic data a new model for the development of SCLC is presented. It suggests that SCLC evolving from the same stem cell as NSCLC should be differentiated into primary and secondary tumors. Primary SCLC corresponding to the classical type evolved directly from an epithelial precursor cell. In contrast, secondary SCLC correlating with the combined SCLC develops via an NSCLC intermediate. In addition, we established libraries of differentially expressed genes from different human lung cancer types to identify new candidate genes for several of the chromosomal subregions identified by CGH. In this review, we summarise the status of our results aiming at a refined classification of lung cancer based on the pattern of genetic aberrations. Show more

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 111-121, 2001

Authors: Blegen, Harald | Ghadimi, B. Michael | Jauho, Annukka | Zetterberg, Anders | Eriksson, Elina | Auer, Gert | Ried, Thomas

Article Type: Research Article

Abstract: In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki‐67, cyclin A, p27KIP1 , p53 and p21WAF1 , and chromosomal gains and losses. The results show that T1b carcinomas (6–10 mm, n=17) were frequently near‐diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11–20 mm), however, was aneuploid, and proliferative activity and p53 …expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential. Show more

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 123-131, 2001

Authors: Macville, Merryn V.E. | Van der Laak, Jeroen A.W.M. | Speel, Ernst J.M. | Katzir, Nir | Garini, Yuval | Soenksen, Dirk | McNamara, George | de Wilde, Peter C.M. | Hanselaar, Antonius G.J.M. | Hopman, Anton H.N. | Ried, Thomas

Article Type: Research Article

Abstract: We have investigated the use of spectral imaging for multi‐color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier‐transform spectroscopy and digital imaging. A pixel‐by‐pixel spectrum‐based color classification is presented of single‐, double‐, and triple‐color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki‐67 and TP53 in paraffin‐embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright‐field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference …library. We conclude that spectral imaging of multi‐color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell‐ and tissue specimens. Figures on http://www.esacp.org/acp/2001/22‐3/macville.htm. Show more

Keywords: Spectral imaging, Fourier‐transform spectroscopy, multicolor, in situ hybridization, immunocytochemistry, bright‐field microscopy, absorption, optical density, pathology

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 133-142, 2001

Authors: van Hensbergen, Yvette | Luykx‐de Bakker, Sylvia A. | Heideman, Daniëlle A.M. | Meijer, Gerrit A. | Pinedo, Herbert M. | van Diest, Paul J.

Article Type: Research Article

Abstract: This study was done to arrive at a fast and reliable protocol for assessment of fractional volumes of immunohistochemically stained dendritic cells in lymph nodes. Twenty axillary lymph nodes of patients with locally advanced breast cancer were immuno‐histochemically stained with an S100 antibody. Fractional volumes of dendritic cells were assessed by stereology based quantitative immunohistochemistry using an interactive video overlay system including an automated microscope. The gold standard percentage of dendritic cells was the fractional volume of S100 stained cells in 500 fields systematically spread over the whole lymph node. Then, in a computer simulation, different sample sizes (1–200 fields …of vision) were tested and the coefficient of variation (CV) for each sample size was calculated. The CV dropped with increasing sample size. A sample size of 100 fields of vision appeared to be optimal. Intra‐ and interobserver reproducibility appeared to be good (correlation coefficients of 0.95 and 0.86, respectively) when re‐analyzing the cases with the established protocol. In conclusion, a fast and reliable assessment of the fractional volume of dendritic cells in lymph nodes is possible with semi‐automated quantitative immuno‐histochemistry. This method will form the base for further clinical studies into the immunological response in lymph nodes of patients with locally advanced breast cancer. Show more

Keywords: Sampling, stereology, reproducibility, dendritic cells, S100, lymph nodes

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 143-149, 2001

Authors: Menschikowski, Mario | Vogel, Margot | Eckey, Rolf | Dinnebier, Gerd | Jaross, Werner

Article Type: Research Article

Abstract: In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications …of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′ ‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids. Show more

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 151-158, 2001

Authors: Jee, Kowan J. | Kim, Young Tak | Kim, Kyu Rae | Aalto, Yan | Knuutila, Sakari

Article Type: Research Article

Abstract: DNA copy number changes were studied by comparative genomic hybridization on 10 tumor specimens of squamous cell carcinoma of cervix obtained from Korean patients. DNA was extracted from paraffin‐embedded sections after removal of non‐malignant cells by microdissection technique. Copy number changes were found in 8/10 tumors. The most frequent changes were chromosome 19 gains (n=6) and losses on chromosomes 4 (n=4), 5 (n=3), and 3p (n=3). A novel finding was amplification in chromosome arm 9p21‐pter in 2 cases. Gains in 1, 3q, 5p, 6p, 8q, 16p, 17, and 20q and losses at 2q, 6q, 8p, 9q, 10p, 11, 13, 16q, …and 18q were observed in at least one of the cases. Show more

Keywords: Cervical cancer, CGH, amplification at 9p21‐pter

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 159-163, 2001

Authors: Kimmig, Rainer | Wimberger, Pauline | Kapsner, Thomas | Hillemanns, Peter

Article Type: Research Article

Abstract: Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK …17, respectively) prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean) following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated. Show more

Keywords: Flow cytometry, DNA cell cycle analysis, cytokeratin

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 165-178, 2001

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 179-179, 2001

Article Type: Correction

Citation: Analytical Cellular Pathology, vol. 22, no. 3, pp. 181-181, 2001