Analytical Cellular Pathology - Volume 27, issue 4

Authors: van der Bij, Gerben J. | Oosterling, Steven J. | Meijer, Sybren | Beelen, Robert H.J. | van Egmond, Marjolein

Article Type: Research Article

Abstract: Macrophages constitute a large proportion of the immune cell infiltrate, which is present in many tumors. Activation state of macrophages is greatly influenced by their environment, leading to different macrophage subsets with diverse functions. Although previously regarded as potent immune cells that are capable of destroying tumor cells, recent literature focuses on the ability of macrophages to promote tumor development due to secretion of mediators, like growth and angiogenic factors. It is now becoming increasingly clear that a complicated synergistic relationship exists between macrophages and malignant cells whereby tumor cells can affect macrophage phenotype, and vice versa. As such, macrophages …and their contribution in cancer development are currently subject of debate. Show more

Keywords: Cancer, macrophages, metastases, tumor associated macrophage, cytotoxicity

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 203-213, 2005

Authors: Yang, Xu-Yu | Ren, Cai-Ping | Wang, Lei | Li, Hui | Jiang, Chun-Jie | Zhang, Hong-Bo | Zhao, Ming | Yao, Kai-Tai

Article Type: Research Article

Abstract: Background & objective: Nasopharyngeal carcinoma (NPC) is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH) was performed to isolate differentially expressed clones …between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16), two were predicted genes (c9orf74 and MDS006), and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The remaining up-regulated genes identified in this study have not been reported to be markers of metastasis and may represent new candidates of NPC metastasis-related genes. The results of this study may provide novel points of therapeutic intervention for NPC. Show more

Keywords: Nasopharyngeal carcinoma (NPC), suppression subtractive hybridization (SSH), tumor metastasis, reverse northern blot

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 215-223, 2005

Authors: Ploeger, Lennert S. | Huisman, André | van der Gugten, Jurryt | van der Giezen, Dionne M. | Beliën, Jeroen A.M. | Abbaker, Abdelhadi Y. | Dullens, Hub F.J. | Grizzle, William | Poulin, Neal M. | Meijer, Gerrit A. | van Diest, Paul J.

Article Type: Research Article

Abstract: Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and …fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions. Show more

Keywords: Confocal laser scanning microscopy, 3D, DNA ploidy, image analysis

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 225-230, 2005

Authors: Morrison, M.L. | Williamson, K. | Arthur, K. | Price, G.J. | Hamilton, P.W. | Maxwell, P.

Article Type: Research Article

Abstract: A large body of evidence has implicated mitochondria in control of cell death, where key apoptotic mechanisms involve change in mitochondrial membrane permeability and depolarisation of mitochondrial membrane potential (Δψm ). Assessment of Δψm is traditionally conducted using the lipophilic cation JC-1 on the flow cytometer or by fluorescent microscopy. Here we assess JC-1 aggregation using the novel tool of digital texture analysis to establish mitochondrial phenotypic changes induced by the K+ ionophore, valinomycin in a unique model comprising SW480 and SW620 cell lines. This provides an opportunity to study these phenomena in the context of colorectal cancer. …Valinomycin-induced apoptosis was detected using morphology and analysis of DNA content. Cells were treated with valinomycin, images digitally recorded on a calibrated video photometer and subjected to high resolution digital texture analysis. This demonstrated that the HARAM texture features (Mean of the Haralick texture features) were highly valuable in describing the transition of Δψm as the cell undergoes apoptosis. In conclusion this study illustrates the potential of texture analysis as a novel and additional technique for quantifying JC-1 aggregation and revealing the spectrum of collapse of Δψm during apoptosis. Show more

Keywords: Mitochondrial membrane potential, apoptosis, colon cancer

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 231-236, 2005

Authors: Hwang, Hae-Gil | Choi, Hyun-Ju | Lee, Byeong-Il | Yoon, Hye-Kyoung | Nam, Sang-Hee | Choi, Heung-Kook

Article Type: Research Article

Abstract: Multi-resolution images of histological sections of breast cancer tissue were analyzed using texture features of Haar- and Daubechies transform wavelets. Tissue samples analyzed were from ductal regions of the breast and included benign ductal hyperplasia, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (CA). To assess the correlation between computerized image analysis and visual analysis by a pathologist, we created a two-step classification system based on feature extraction and classification. In the feature extraction step, we extracted texture features from wavelet-transformed images at 10× magnification. In the classification step, we applied two types of classifiers to the extracted features, …namely a statistics-based multivariate (discriminant) analysis and a neural network. Using features from second-level Haar transform wavelet images in combination with discriminant analysis, we obtained classification accuracies of 96.67 and 87.78% for the training and testing set (90 images each), respectively. We conclude that the best classifier of carcinomas in histological sections of breast tissue are the texture features from the second-level Haar transform wavelet images used in a discriminant function. Show more

Keywords: Multi-resolution, wavelet-transformed, breast cancer, texture features, statistics-based multivariate analysis, neural network

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 237-244, 2005

Authors: Zalata, Khaled R. | Nasif, Wesam A. | Ming, Si-Chun | Lotfy, Mahmoud | Nada, Nadia A. | El-Hak, Nabil Gad | Leech, Stephen H.

Article Type: Research Article

Abstract: Background and aims: Oncogenes and tumor suppressor genes expression are well described in bladder cancer associated with schistosomiasis especially in Egypt. Scarce studies were directed to colorectal cancer (CRC) associated with Schistosoma mansoni (S. mansoni). Apoptosis (programmed cell death) and the genes regulating this process (e.g., Bcl-2) have recently become a focus of interest in the study of cancer development and progression. In the present study, we aimed to investigate the expression pattern of p53, Bcl-2 and C-Myc in CRC tissues obtained from Egyptian colorectal cancer patients divided in two different groups, one associated with Schistosoma mansoni (CRC-Sm) and the …other without Schistosoma mansoni (CRC-NSm). Methods: Seventy-five CRC tumors containing 36 draining lymph node metastatic tumors were immunohistochemically stained using specific monoclonal antibodies for p53, Bcl-2 and C-Myc, in addition the apoptotic activity of these tumors were analyzed. Results and conclusions: Regardless of the S. mansoni infection, the obtained results showed that the apoptotic activity was more evident in p53 diffuse positive tumors (P=0.021). There was a significant correlation between p53 diffuse positive staining and Bcl-2 positive immunostaining (P=0.011). Signet ring cell carcinoma and mucinous adenocarcinoma exhibited both intense C-Myc expression than non-mucinous carcinoma (P=0.001). When adjusting for S. mansoni infection, 58.3% of CRC-Sm cases were Bcl-2 positive compared to only (33.3%) of CRC-NSm (P=0.046). Apoptotic activity was more evident in the latter group than of CRC-Sm tumors (P=0.009). p53 and C-Myc expressions were found insignificantly different in CRC-Sm compared with CRC-NSm (P>0.05). These observations suggest that the genotoxic agents produced endogenously through the course of schistosomiasis mansoni may play a role in CRC-Sm pathogenesis through the dysregulation of apoptosis by alteration the expression pattern of Bcl-2 protein differently from CRC-NSm suggesting a different biological behavior. Show more

Keywords: Colorectal carcinoma, Schistosoma mansoni, p53, Bcl-2, c-Myc

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 245-253, 2005

Authors: Osterheld, M.C. | Andrejevic Blant, S. | Caron, L. | Braunschweig, R. | Dorta, G. | Bouzourene, H. | Mihaescu, A.

Article Type: Research Article

Abstract: Background: Cytologic evaluation of the biliary tract strictures is nowadays widely used for distinguishing between benign and malignant lesions but remains a challenge for some problematic cases. Digital Image cytometry (DNA-cytometry) helps cytopathologists to resolve some unclear situations. Methods: We have analysed 41 specimens of bile duct brushings obtained from patients during ERCP (11 benign cases, 7 suspicious for malignancy cases and 23 malignant cases) by DNA-cytometry and correlated them with the histological biopsy counterpart. Results: All eleven cytological and histological benign cases were DNA-diploid and among 22 patients with malignant cytological and histological diagnosis 21 were DNA-aneuploid. …One case considered malignant by the cytopathologist revealed DNA-aneuploid but malignancy could not be confirmed by histology. The analysis of the suspicious for malignancy cases revealed that all DNA-aneuploid cases were malignant and all DNA-diploid cases were benign referring to the follow-up of the patients. The comparison between cytology alone and cytology combined with DNA-cytometry related to the histological diagnosis (gold standard) resulted in a sensitivity of 100% and a specificity of 79% for cytology alone; a specificity of 94% and a sensitivity 92% for DNA-cytometry and a specificity of 93% and a sensitivity of 100% with combined analyses. The positive predictive value was 90% for cytology, 96% for DNA-cytometry and for both analyses. The negative predictive value showed 100% for cytology, 89% for DNA-cytometry and 100% for combined studies. Conclusions: Despite the limited number of patients involved in the study, the results obtained indicate an increased of specificity and of positive predictive value using DNA-cytometry. These results confirm the pertinence of these method for challenging cases, in conjunction with other available diagnostic tools. Show more

Keywords: Image DNA cytometry, biliary strictures, cytology

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 255-260, 2005

Authors: Westers, Theresia M. | Houtenbos, Ilse | van de Loosdrecht, Arjan A. | Ossenkoppele, Gert J.

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 261-266, 2005

Authors: Carvalho, Beatriz

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 267-267, 2005

Authors: Giaretti, W. | Malacarne, D.

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 27, no. 4, pp. 269-271, 2005