Analytical Cellular Pathology - Volume 30, issue 1

Authors: Veenendaal, Liesbeth M. | Kranenburg, Onno | Smakman, Niels | Klomp, Annemarie | Borel Rinkes, Inne H.M. | van Diest, Paul J.

Article Type: Research Article

Abstract: Background: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFβ signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases. Methods: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related …to markers of Wnt (β-catenin), Notch (HES1) and TGFβ (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed. Results: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear β-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear β-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear β-catenin. Conclusions: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFβ signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFβ pathways is reduced in secondary colorectal tumors. Show more

Keywords: Notch, vimentin, HES1, colorectal, metastasis, epithelial mesenchymal transition, KRAS

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 1-11, 2008

Authors: Barboro, Paola | Rubagotti, Alessandra | Orecchia, Paola | Spina, Bruno | Truini, Mauro | Repaci, Erica | Carmignani, Giorgio | Romagnoli, Andrea | Introini, Carlo | Boccardo, Francesco | Carnemolla, Barbara | Balbi, Cecilia

Article Type: Research Article

Abstract: Introduction: Although several molecular markers for bladder cancer have been identified, at present little information on prognostic biomarkers is available in the literature. Prognostication of this tumor is largely based on clinicopathological characteristics. Our aim was to identify nuclear matrix (NM) proteins that might serve to better characterize the phenotype of the invasive bladder cancer and to investigate their diagnostic and prognostic roles. Methods: NM proteins expressed in normal (n=3) or non-tumoral (n=9) tissue specimens and muscle-invasive bladder cancer (n=21) specimens were analyzed by two dimensional (2D) gel electrophoresis. PDQuest image analysis software was used to generate a comparative …NM proteome analysis. Selected spots were characterized by liquid chromatography coupled to tandem mass spectrometry and Western blot. Results: We detected over 800 protein spots in each 2D map and 43 spots were identified. 30 proteins were differentially expressed by bladder tumor cells; among these, 19 proteins were detected in bladder tumoral tissues but not in normal and non-tumoral tissues and seven proteins correlated with tumor stage. One protein (p54nrb ) was strongly correlated with vascular invasions and appeared to be also significantly (P<0.0001) associated with a decreased probability of survival. Conclusion: Important alterations in NM proteins occur in muscle-invasive bladder cancer. The differentially expressed proteins include biomarkers potentially useful for disease diagnosis, progression and prognosis. Our findings beyond improving the understanding of the biology of bladder cancer, could help to stratify patients into different prognostic subgroups and to select those who might be better candidate to multimodal therapeutic approaches. Show more

Keywords: Bladder carcinoma, nuclear matrix proteins, proteomics, biological markers, diagnosis, prognosis

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 13-26, 2008

Authors: Jansen, Corine | Hebeda, Konnie M. | Linkels, Marianne | Grefte, Johanna M.M. | Raemaekers, John M.M. | van Krieken, Johan H.J.M. | Groenen, Patricia J.T.A.

Article Type: Research Article

Abstract: Non-Hodgkin's lymphoma comprises many related but distinct diseases and diagnosis and classification is complex. Protein profiling of lymphoma biopsies may be of potential value for use in this lymphoma classification and the discovery of novel markers. In this study, we have optimized a method for SELDI-TOF MS based protein profiling of frozen tissue sections, without dissection of tumour cells. First we have compared chip surfaces and lysis buffers. Also, we have determined the minimal input using laser dissection microscopy. Subsequently, we have analyzed and compared protein profiles of diffuse large B-cell lymphoma (n=8), follicular lymphoma (n=8) and mantle cell lymphoma …(n=8). Benign, reactive lymph nodes (n=14) were used as a reference group. CM10 chip surface in combination with urea lysis buffer and an input of approximately 50,000 lymphocytes allowed the detection of many differential peaks. Identification of the diffuse large B-cell lymphoma cases was reliably made in the supervised classification. Unsupervised clustering showed segregation into a benign/indolent cluster predominantly formed by benign, reactive lymph nodes and follicular lymphoma cases and into a more aggressive cluster formed by diffuse large B-cell lymphoma and mantle cell lymphoma cases. In conclusion, our protocol enables protein profiling of protein lysates derived from small histological samples and the subsequent detection of many differentially expressed proteins, without the need of tumour cell dissection. These results support further evaluation of protein profiling of small lymphoma biopsies as an additional tool in pathology. Show more

Keywords: Classification, non-Hodgkin's lymphoma, protein profiling, proteomics, SELDI-TOF MS

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 27-38, 2008

Authors: Friedrich, Katrin | Weber, Theresa | Scheithauer, Jens | Meyer, Wolfdietrich | Haroske, Gunter | Kunze, Klaus Dietmar | Baretton, Gustavo

Article Type: Research Article

Abstract: The purpose of this study was to compare the chromosomal genotype between breast cancers with and without secondary manifestations and between primary tumors and their secondary manifestations. Eighty six breast cancers, twenty lymph node metastases, ten distant metastases and ten local recurrences were analyzed by comparative genomic hybridization. Tumors with local recurrences showed significant more frequent losses at 2q32 than the tumors without recurrences. Lymph node positive cases showed significant more frequent losses at 9p21 than node negative cases. Lymph node metastases exhibited significant more frequent losses at 7q11, 14q24.3–q31 and 17q22–q24 than their primary tumors. In cases with distant …metastases, losses at 5q23 were more frequent than in those without, but not reaching the significance level. The distant metastases showed significant more frequent losses at 5p15, 12q24 and 17q22–q24 than the primary tumors. These results reveal strong evidence that the potential for progression is determined in the primary tumor and that different ways of the development of local recurrences, lymph node and distant metastases exist. After confirmation of the results by interphase FISH on tissue micro arrays, the detection of these specific chromosomal imbalances may contribute to a more individual prediction of prognosis in breast cancer. Show more

Keywords: Breast cancer, local recurrences, metastases, comparative genomic hybridization

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 39-50, 2008

Authors: Mannello, Ferdinando | Fabbri, Laura | Ciandrini, Eleonora | Tonti, Gaetana A.

Article Type: Research Article

Abstract: Background: Erythropoietin (Epo) is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non-erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia-induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells. Methods: Epo was determined by ELISA, immunoprecipitation, western blot and immunocytochemical analyses in 130 Nipple Aspirate Fluids (NAF) from 102 NoCancer and 28 Breast Cancer (BC) patients, comparing results with those found …in 10 milk, 45 serum samples and breast cancer cell lines. Results: Epo levels in NAFs were significantly higher than those in milk and serum. No difference in Epo electrophoretic mobility was found among NAF, milk and serum samples, and conditioned cell culture medium. Immunolocalization of intracellular Epo in ductal cells floating in BC NAFs was similar to those of cancer cell lines. No significant correlation between TNM classification and Epo in NAFs from BC patients was found. Significantly higher Epo concentration was found in NAF from BC patients compared to NoCancer. Conclusion: We demonstrate that breast epithelial cells are a source of Epo in breast microenvironment, suggesting the presence of a paracrine/autocrine Epo function in NAFs, triggering off intracellular signaling cascade with subsequent BC initiation. Show more

Keywords: Erythropoietin, breast cancer, nipple aspirate fluid, benign breast disease, milk, serum, immunocytochemistry

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 51-61, 2008

Authors: van Baal, Jantine W.P.M. | Milana, Francesco | Rygiel, Agnieszka M. | Sondermeijer, Carine M.T. | Spek, C. Arnold | Bergman, Jacques J.G.H.M. | Peppelenbosch, Maikel P. | Krishnadath, Kausilia K.

Article Type: Research Article

Abstract: Esophageal adenocarcinoma (EA) and esophageal squamous cell carcinoma (ESCC) are the two main types of esophageal cancer. Despite extensive research the exact molecular basis of these cancers is unclear. Therefore we evaluated the transcriptome of EA in comparison to non-dysplastic Barrett's esophagus (BE), the metaplastic epithelium that predisposes for EA, and compared the transcriptome of ESCC to normal esophageal squamous epithelium. For obtaining the transcriptomes tissue biopsies were used and serial analysis of gene expression (SAGE) was applied. Validation of results by RT-PCR and immunoblotting was performed using tissues of an additional 23 EA and ESCC patients. Over 58,000 tags …were sequenced. Between EA and BE 1013, and between ESCC and normal squamous epithelium 1235 tags were significantly differentially expressed (p<0.05). The most up-regulated genes in EA compared to BE were SRY-box 4 and Lipocalin2, whereas the most down-regulated genes in EA were Trefoil factors and Annexin A10. The most up-regulated genes in ESCC compared to normal squamous epithelium were BMP4, E-Cadherin and TFF3. The results could suggest that the BE expression profile is closer related to normal squamous esophagus then to EA. In addition, several uniquely expressed genes are identified. Show more

Keywords: Barrett's esophagus, SAGE, gene expression profile, esophageal squamous cell carcinoma, esophageal adenocarcino- ma

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 63-75, 2008

Authors: de Wilde, Jillian | De-Castro Arce, Johanna | Snijders, Peter J.F. | Meijer, Chris J.L.M. | Rösl, Frank | Steenbergen, Renske D.M.

Article Type: Research Article

Abstract: Background: Previous studies demonstrated a functional involvement of the AP-1 transcription factor in HPV-induced cervical carcinogenesis. Here, we aimed to obtain further insight in expression alterations of AP-1 family members during HPV-mediated transformation and their relationship to potential regulatory (Notch1, Net) and target (CADM1) genes. Methods: mRNA expression levels of c-Jun, JunB, junD, c-Fos, FosB, Fra-1, Fra-2, Notch1, Net and CADM1 were determined by quantitative RT-PCR in primary keratinocytes (n=5), early (n=4) and late (n=4) passages of non-tumorigenic HPV-immortalized keratinocytes and in tumorigenic cervical cancer cell lines (n=7). In a subset of cell lines protein expression and AP-1 complex …composition was determined. Results: Starting in immortal stages c-Fos, Fra-2 and JunB expression became up regulated towards tumorigenicity, whereas Fra-1, c-Jun, Notch1, Net and CADM1 became down regulated. The onset of deregulated expression varied amongst the AP-1 members and was not directly related to altered Notch1, Net or CADM1 expression. Nevertheless, a shift in AP-1 complex composition from Fra-1/c-Jun to c-Fos/c-Jun heterodimers was only observed in tumorigenic cells. Conclusion: HPV-mediated transformation is associated with altered AP-1, Notch1, Net and CADM1 transcription. Whereas the onset of deregulated expression of various AP-1 family members became already manifest during the immortal state, a shift in AP-1 complex composition appeared a rather late event associated with tumorigenicity. Show more

Keywords: Cervical cancer, HPV, AP-1, Fra-1, c-Fos, Net, Notch1, CADM1

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 77-87, 2008

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 30, no. 1, pp. 89-89, 2008