Analytical Cellular Pathology - Volume 16, issue 4

Authors: Takahashi, Hiroshi | Fujita, Shuichi | Yamabe, Shigeru | Moriishi, Takeshi | Okabe, Haruo | Tajima, Yoshifumi | Mizuno, Akio

Article Type: Research Article

Abstract: Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S phase of the cell cycle, and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. PCNA expression was studied in odontogenic keratocysts (n=15 ) and ameloblastomas (n=46) using an avidin–biotin–peroxidase complex method on routinely processed paraffin sections. The percentage of PCNA‐positive cells determined by point counting was significantly lower in the ameloblastomas (mean 9.4%, standard deviation (SD) 11.0) than in odontogenic keratocysts (mean 29.9%, SD 24.0). In ameloblastomas, the mean percentage of …PCNA‐positive cells was lowest in the acanthomatous pattern and highest in plexiform pattern. The mean percentage of PCNA‐positive cells in plexiform pattern was non‐significantly higher than that in follicular pattern. The mean percentage of PCNA‐positive cells in plexiform and follicular patterns was significantly higher than that in cyctic and acanthomatous patterns. The frequency of PCNA‐positive cells was significantly higher in the peripheral cells of follicular and plexiform patterns than in the central cells of both patterns (p<0.01) . Therefore, peripheral cells were regarded as reserve cell of central cells. The mean percentage of PCNA‐positive cells in the epithelial lining of odontogenic keratocyst was not significantly different from those in the peripheral cells of follicular and plexiform patterns of ameloblastoma. In contrast, the odontogenic keratocyst exhibited a mean percentage of PCNA‐positive cells which was statistically higher than that in other histological elements of ameloblastomas. The present study suggests that odontogenic keratocyst is regarded as benign odontogenic tumour. Show more

Keywords: Ameloblastoma, immunohistochemistry, odontogenic keratocyst, proliferating cell nuclear antigen

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 185-192, 1998

Authors: Barbisan, Luís Fernando | Russo, Jose | Mello, Maria Luiza S.

Article Type: Research Article

Abstract: Changes in nuclear and nucleolar morphometric parameters were investigated by image analysis procedures in human breast MCF‐10F epithelial cells expressing different stages of the tumourigenic progression after benzo[a]pyrene (BP) transformation (BP1, BP1‐E, and BP1‐E1 cell lines), and additionally transfected with the c‐Ha‐ras oncogene (BP1‐Tras cell line). Nuclear pleomorphism was evident in all the transformed cells. The analysis of different morphometric parameters did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of the tumourigenesis, with the exception of the nucleolar size, which could be associated to the expression of the tumourigenic …phenotype, and a nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumourigenic phenotypes from one another. The nuclear morphometric data established for the BP‐transformed cells and for the cells additionally transfected with the c‐Ha‐ras oncogene were suggestive of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF‐10F cells. The nuclear changes found in the BP1‐Tras cell line were assumed to be related to the additional effects and/or enhanced genomic instability induced by transfection with the ras oncogene. Show more

Keywords: Human breast epithelial cells, benzo[a]pyrene, c‐Ha‐ras oncogene, image analysis, nuclear/nucleolar morphometry

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 193-199, 1998

Authors: Öztürk, Melek | Bolkent, Sema | Yilmazer, Selma | Kaner, Gültekin | Ünal, Hilal

Article Type: Research Article

Abstract: Amplification and overexpression of the c‐erbB‐2 oncogene are of prognostic significance in human breast cancer. Overexpression of c‐erbB‐2 is the result of gene amplification. However, increased transcript levels of c‐erbB‐2 are also detected in the absence of gene amplification. In this study for the detection of the overexpression mRNA in situ hybridisation (ISH) and immunohistochemistry (IHC) were used. Our aim was to develop the suitable mRNA ISH protocol for formalin‐fixed paraffin‐embedded material and to compare the localisation of transcripts and protein products in 20 primary breast carcinomas. Sections were immunostained with monoclonal c‐erbB‐2 antibody. In ISH method digoxigenin‐labelled oligoprobe was …used for the detection of c‐erbB‐2 mRNAs. We determined optimal condition for the ISH procedure (e.g., probe concentration, digestion, post washing). c‐erbB‐2 protein overproduction was detected in 11/20 cases with IHC. The mRNA signals were observed in malignant cell cytoplasm in 6/20 cases by ISH. ISH positive signals were found in only one case without detected overexpression of the protein. There were cell to cell variations in the hybridisation signals even within individual tumours. The ISH and IHC positive signals for c‐erbB‐2 was observed mostly in infiltrating ductal carcinomas that belong to aggressive lesions. Show more

Keywords: c‐erbB‐2, breast cancer, in situ hybridisation, immunohistochemistry

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 201-209, 1998

Authors: Esa, Arif | Trakhtenbrot, Luba | Hausmann, Michael | Rauch, Joachim | Brok‐Simoni, Frida | Rechavi, Gideon | Ben‐Bassat, Isaac | Cremer, Christoph

Article Type: Research Article

Abstract: A new fluorescence in situ hybridization (FISH) technique called Fast‐FISH in combination with semi‐automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available \alpha ‐satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast‐FISH protocol, the microscopic images of the fluorescence‐labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively …by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast‐FISH was found. Automated spot counting after Fast‐FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast‐FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast‐FISH technique requires a hybridization time as low as 1/ 20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours. Show more

Keywords: Fast‐FISH, image analysis, spot counting, chromosomal aberrations, hematological malignancies

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 211-222, 1998

Authors: Abad, Mar | Ciudad, Juana | Rincon, Manuel R. | Silva, Isabel | Paz‐Bouza, José I. | Lopez, Antonio | Alonso, Alberto G. | Bullon, Agustin | Orfao, Alberto

Article Type: Research Article

Abstract: In the present study the prognostic value of both DNA ploidy and the proliferative activity of tomour cells were studied in a series of 76 consecutive patients suffering from gastric tumours. DNA ploidy and the proliferative index (as measured by the percentage of S‐phase cells) were determined by flow cytometry using fresh tumour specimens. The presence of DNA aneuploid clones by flow cytometry was detected in 62% of the cases (mean DNA index of 1.63\pm 0.46 ; range 1.08–2.92), the mean proportion of S‐phase cells being of 18.4\pm 11.5\% . In comparison with diploid cases, aneuploid tumours …showed a higher proliferative activity (cases with more than 15% S‐phase cells: 18.4% versus 6.1%, p=0.0001 ) as well as a higher incidence of node involvement (95% versus 68%, p=0.001 ). By contrast, no significant differences were detected with respect to sex, age, histologic grade and type, clinical stage, tumour size and the incidence of extranodal involvement. Upon grouping the patients according to the proportion of S‐phase cells no significant differences were observed for the clinical and biological parameters explored except for an association between a high percentage of S‐phase cells and the presence of DNA aneuploidy (40% versus 96%, p=0.0001 ). Regarding survival the presence of DNA aneuploidy was significantly associated with poor outcome as compared to the diploid cases (median of 15 versus 26 months, p=0.005 ). By contrast, the proportion of S‐phase cells did not predict patients’s outcome. Multivariate analysis of prognostic factors showed that the presence of DNA aneuploidy (p=0.003) together with the histologic type (p=0.03) and the existence of extranodal metastases (p=0.05) were the best combination of prognostic factors for survival prediction. Show more

Keywords: DNA aneuploidy, flow cytometry, gastric cancer, prognosis

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 223-231, 1998

Authors: Alvarez‐Riesgo, José Antonio | Sampedro, Andrés | Hernández, Radhamés | Folgueras, María Victoria | Salas‐Bustamante, Ana | Cueto, Antonio

Article Type: Research Article

Abstract: Flow Cytometry (FC) has been incorporated into cancer research in relation to its prognostic value together with histological parameters and TNM stages. We have studied by means of FC the cell cycle of 132 samples from male patients with Squamous Cell Lung Carcinoma (SQCLC). All of the patients received curative surgery and the clinical follow‐up was 60 months. The clinical and cytometric parameters were evaluated in order to predict the patients’ outcome. The presence of tumoural recurrence and the tumoural stage showed statistical significance associated with survival. The multivariant analysis reveals radiotherapy (p=0.004 ) as protective variable and the …high S‐phase fraction (SPF) (p=0.001 ) and stage IIIA (p=0.012 ) as risk factors. The SPF appears as an independent prognostic factor for overall survival time. We can build a prognostic index representative of different prognostic groups, which allows us to improve the individual monitoring of these patients. Show more

Keywords: Squamous cell lung carcinoma, flow cytometry, cell proliferation, DNA ploidy, prognosis

Citation: Analytical Cellular Pathology, vol. 16, no. 4, pp. 233-242, 1998