Analytical Cellular Pathology - Volume 29, issue 6

Authors: Agelopoulos, Konstantin | Kersting, Christian | Korsching, Eberhard | Schmidt, Hartmut | Kuijper, Arno | August, Christian | Wülfing, Pia | Tio, Joke | Boecker, Werner | van Diest, Paul J. | Brandt, Burkhard | Buerger, Horst

Article Type: Research Article

Abstract: Background: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. Methods: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA-SSR I). The results were verified and extended by means of immunohistochemistry using the tissue …microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin-1, eps15, EGF, TGF-α, pErk, pAkt and mdm2. Results: We were able to show that the presence of egfr CA-SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin-1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin-1 and eps15 were also significantly correlated with the expression of pAkt (p<0.05), pERK (p<0.05), mdm2 (p<0.01) and EGF (p<0.001 for caveolin-1). Eps15 and pERK were further associated with tumour grade (p<0.01 and p<0.001, respectively). Conclusion: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin-1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin-1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours. Show more

Keywords: Phyllodes tumours, egfr, caveolin-1, eps15

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 443-451, 2007

Authors: Zaffaroni, Nadia | Costa, Aurora | Pennati, Marzia | De Marco, Cinzia | Affini, Emanuela | Madeo, Maria | Erdas, Roberta | Cabras, Antonello | Kusamura, Shigeki | Baratti, Dario | Deraco, Marcello | Daidone, Maria Grazia

Article Type: Research Article

Abstract: Background: The biology of malignant peritoneal mesothelioma (MPM) is largely unknown. In the present study, we assessed the expression of survivin and other members of the inhibitors of apoptosis proteins (IAP) family (IAP-1, IAP-2 and X-IAP) in a series of 32 MPM surgical specimens and investigated the effects of survivin knockdown in an established MPM cell line. Methods: Expression of different IAPs was measured by immunohistochemistry. MPM cells were transfected with a small-interfering RNA (siRNA) targeting survivin mRNA and analyzed for survivin expression, growth rate, and ability to undergo spontaneous and drug (cisplatin, doxorubicin)-induced apoptosis. Results: Survivin expression was observed …in 29 (91%) surgical MPM specimens, whereas the positivity rate for the other IAPs ranged from 69% to 100%. Transfection of MPM cells with the survivin siRNA induced a marked inhibition of survivin protein expression, a time-dependent decline in cell growth and an enhanced rate of spontaneous and drug-induced apoptosis, with a concomitant increase in the catalytic activity of caspase-9. Conclusion: Our results show for the first time that survivin, as well as other IAPs, is largely expressed in clinical MPMs and suggest that strategies aimed at down-regulating survivin may provide a novel approach for the treatment of the malignancy. Show more

Keywords: Survivin, IAPs, peritoneal mesothelioma, siRNA, apoptosis, caspase-9

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 453-466, 2007

Authors: Paredes, Joana | Correia, Ana Luísa | Ribeiro, Ana Sofia | Schmitt, Fernando

Article Type: Research Article

Abstract: Background: P120-catenin is a member of the Armadillo protein family, which is involved in intercellular adhesion and cell signalling. It directly interacts with the classical cadherins juxtamembrane domain and contributes for both junction formation and its disassembly. Accumulating evidences indicate that p120-catenin is important in tumour formation and progression, although the role of their multiple spliced isoforms in the regulation of cadherin-mediated adhesion of malignant cells is still not well understood. We investigated the expression of p120-catenin isoforms in a collection of breast cancer cell lines with distinct molecular profiles and expressing different cadherins. Methods: We assessed the expression by …RT-PCR and Western-blotting analysis. Results: We observed that the expression of p120-catenin isoforms was associated with the genomic and transcriptional phenotype of breast cancer cells. Besides, the recruitment of p120-catenin isoforms was not apparently related with the particular expression of E-, P- or N-cadherin. Conclusion: We demonstrate that mammary tumour cells exhibit a characteristic p120-catenin isoform expression profile, depending from their specific genomic and transcriptional properties. These particular expression patterns, combined with other regulatory proteins and in a specific cellular context, may explain how p120-catenin can either contribute to strength intercellular adhesions or instead to promote cell motility. Show more

Keywords: Breast cancer cells, cadherin, cell–cell adhesion, p120-catenin isoforms

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 467-476, 2007

Authors: Konstantinopoulos, Panagiotis A. | Vandoros, Gerasimos P. | Karamouzis, Michalis V. | Gkermpesi, Maria | Sotiropoulou-Bonikou, Georgia | Papavassiliou, Athanasios G.

Article Type: Research Article

Abstract: Background: COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-κB transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-κB, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN), p-IκB-α (phosphorylated IκB-α), EGF-R, COX-2, NF-κB and VEGF expression in stromal …myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p-IκB-α, NF-κB, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-IκB-α, NF-κB, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-κB transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production. Show more

Keywords: Colon adenocarcinoma, stromal myofibroblasts, EGF-R, AP-1, NF-κB, COX-2, VEGF

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 477-482, 2007

Authors: van der Laak, Jeroen A.W.M. | Siebers, Albertus G. | Aalders, Sabine A.A.P. | Grefte, Johanna M.M. | de Wilde, Peter C.M. | Bulten, Johan

Article Type: Research Article

Abstract: Objective and reproducible assessment of cancer biomarkers may be performed using rare event detection systems. Because many biomarkers are not true ‘rare events’, in this study a semi-rare event detection system was developed. The system is capable of assigning a discriminant score to detected positive cells, expressing the extent and intensity of the immunocytochemical staining. A gallery image is constructed showing the diagnostically most interesting cells as well as quantitative data expressing the biomarker staining pattern. To increase scanning speed, an adaptive scanning strategy is studied in which scanning is aborted when a sufficient number of positive cells has been …identified. System performance was evaluated using liquid based cervical smears, stained with an antibody directed against p16INK4a tumor suppressor protein. Overexpression of p16INK4a in cervix is related to high-risk HPV infection, which is associated with carcinogenesis. Reproducibility of the system was tested on specimens containing limited positivity. Quantitative analysis was evaluated using 10 cases within normal limits and 10 high grade lesions. The system was highly reproducible in detecting positive cells and in calculating discriminant scores (average CV 0.7%). Quantitative features were significantly increased in high grade lesions (p<0.001). Adaptive scanning decreased scanning time with only minor impact on scanning results. The system is capable of automated, objective and reproducible assessment of biomarker expression and may be useful for a variety of applications. Show more

Keywords: Rare event detection, automated image analysis, immunocytochemistry, cervical cytology, p16^INK4a protein

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 483-495, 2007

Authors: van Deurzen, C.H.M | van Hillegersberg, R. | Hobbelink, M.G.G. | Seldenrijk, C.A. | Koelemij, R. | van Diest, P.J.

Article Type: Research Article

Abstract: Background: The need for routine axillary lymph node dissection (ALND) in patients with invasive breast cancer and low-volume sentinel node (SN) involvement is questionable. Accurate prediction of second echelon lymph node involvement could identify those patients most likely to benefit from ALND. Methods: A consecutive series of 317 patients with invasive breast cancer and a tumor positive axillary SN followed by ALND was reviewed. Clinicopathologic features of the primary tumor and the SN were assessed as possible predictors of second echelon lymph node involvement. Results: Second echelon metastases were found in 116/317 cases (36.6%). Frequency of second echelon …lymph node involvement in patients with isolated tumor cells (ITC, N=23), micro- (N=101) and macrometastases (N=193) was 13%, 20% and 48%, respectively (p<0.001). Based on the area % of SN occupied by tumor no subgroup of patients could be selected with less than 20% second echelon lymph node involvement. However, none of the patients with SN ITC or micrometastases and a primary tumor size ≤1 cm (N=12, 3.8%) had second echelon lymph node involvement. Conclusions: Accurately measured SN tumor load predicts second echelon lymph node involvement. However, even in patients with ITC, the second echelon lymph nodes are involved in 13% justifying ALND. Show more

Keywords: Breast cancer, sentinel node, second echelon lymph node metastases, morphometry, tumor load

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 497-505, 2007

Authors: Kerkhof, Marjon | Kusters, Johannes G. | van Dekken, Herman | Kuipers, Ernst J. | Siersema, Peter D.

Article Type: Research Article

Abstract: Barrett esophagus (BE) is caused by chronic gastroesophageal reflux and predisposes to the development of esophageal adenocarcinoma through different grades of dysplasia. Only a subset of BE patients will finally develop esophageal adenocarcinoma. The majority will therefore not benefit from an endoscopic surveillance program, based on the histological identification of dysplasia. Several studies have been performed to find additional biomarkers that can be used to detect the subgroup of patients with an increased risk of developing malignancy in BE. In this review, we will summarize the most promising tissue biomarkers, i.e. proliferation/cell cycle proteins, tumor suppressor genes, adhesion molecules, DNA …ploidy status and inflammation associated markers, that can be used for risk stratification in BE, and discuss their respective clinical application. Show more

Keywords: Barrett esophagus, biomarkers, esophageal adenocarcinoma

Citation: Analytical Cellular Pathology, vol. 29, no. 6, pp. 507-517, 2007