Analytical Cellular Pathology - Volume 33, issue 5-6

Authors: Antonacopoulou, Anna G. | Floratou, Konstantina | Bravou, Vasiliki | Kottorou, Anastasia | Dimitrakopoulos, Fotinos-Ioannis | Marousi, Stella | Stavropoulos, Michalis | Koutras, Agelos K. | Scopa, Chrisoula D. | Kalofonos, Haralabos P.

Article Type: Research Article

Abstract: Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated. Methods: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein …levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. Results: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the −31G/C snp may be related to CRC development and improved overall survival. Conclusion: Our results support a role of survivin in colorectal carcinogenesis while the −31G/C snp may constitute a marker of survival. Show more

Keywords: Survivin, splice variant, polymorphism, colorectal cancer, survival

DOI: 10.3233/ACP-CLO-2010-0537

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 177-189, 2010

Authors: Marchán, S. | Pérez-Torras, S. | Vidal, A. | Adan, J. | Mitjans, F. | Carbó, N. | Mazo, A.

Article Type: Research Article

Abstract: Background: Pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration. Methods: In view of these findings, we examined the role of β3 in pancreatic cancer by generating two stable β3 -expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo. Results: Transduction of β3 selectively augmented the functional membrane αv β3 integrin levels, as evident from …the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected in β3 -overexpressing cells. Moreover, tumors expressing β3 displayed reduced growth. Interestingly, treatment of mice with an αv -blocking antibody inhibited the growth of β3 -expressing tumors to a higher extent. Conclusions: Our results collectively support the hypothesis that αv β3 integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors. Show more

Keywords: Pancreatic cancer, integrins, cell migration, tumor growth, α_vβ_3

DOI: 10.3233/ACP-CLO-2010-0538

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 191-205, 2010

Authors: Zatelli, Maria Chiara | Tagliati, Federico | Amodio, Vincenzo | Buratto, Mattia | Pelizzo, Mariarosa | Pansini, Giancarlo | Bondanelli, Marta | Ambrosio, Maria Rosaria | degli Uberti, Ettore C.

Article Type: Research Article

Abstract: Background: Pituitary tumour transforming gene 1 (PTTG1) is over-expressed in a variety of endocrine-related tumours. We aimed at evaluating PTTG1 expression and function in human neoplastic parafollicular C-cells, represented by medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH) samples and by the TT cell line. Methods: TT cells and tissues derived from human CCH (8 samples) and MTC (12 samples) were analyzed by northern blot, furthermore TT cells were subjected to PTTG gene silencing and cells were analyzed for DNA synthesis. Results: PTTG1 expression was significantly higher (p<0.01) in CCH (3-fold), in papillary thyroid cancer and in MTC …(5-fold) than in normal thyroid, and in MTC lymph-node metastases as compared to primary lesions (~2-fold; p<0.05). PTTG1 mRNA and protein correlated with tumour diameter and TNM status (p<0.05). In TT cells, PTTG1 silencing did not completely block DNA synthesis, but significantly reduced [3 H]Thymidine incorporation (~50%; p<0.01) for up to 3 days. Conclusions: PTTG1 levels correlate with tumour aggressiveness. PTTG1 silencing causes reduced MTC cell proliferation, supporting the hypothesis that PTTG1 might have an important role in C-cell neoplastic proliferation. Show more

Keywords: Pituitary tumour transforming gene 1, medullary thyroid carcinoma, TT cells, siRNA

DOI: 10.3233/ACP-CLO-2010-0536

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 207-216, 2010

Authors: Costa, Ana Flávia | Altemani, Albina | Vékony, Hedy | Bloemena, Elisabeth | Fresno, Florentino | Suárez, Carlos | Llorente, José Luis | Hermsen, Mario

Article Type: Research Article

Abstract: Background: ACC can occasionally undergo dedifferentiation also referred to as high-grade transformation (ACC-HGT). However, ACC-HGT can also undergo transformation to adenocarcinomas which are not poorly differentiated. ACC-HGT is generally considered to be an aggressive variant of ACC, even more than solid ACC. This study was aimed to describe the genetic changes of ACC-HGT in relation to clinico-pathological features and to compare results to solid ACC. Methods: Genome-wide DNA copy number changes were analyzed by microarray CGH in ACC-HGT, 4 with transformation into moderately differentiated adenocarcinoma (MDA) and two into poorly differentiated carcinoma (PDC), 5 solid ACC. In addition, Ki-67 …index and p53 immunopositivity was assessed. Results: ACC-HGT carried fewer copy number changes compared to solid ACC. Two ACC-HGT cases harboured a breakpoint at 6q23, near the cMYB oncogene. The complexity of the genomic profile concurred with the clinical course of the patient. Among the ACC-HGT, p53 positivity significantly increased from the conventional to the transformed (both MDA and PDC) component. Conclusion: ACC-HGT may not necessarily reflect a more advanced stage of tumor progression, but rather a transformation to another histological form in which the poorly differentiated forms (PDC) presents a genetic complexity similar to the solid ACC. Show more

Keywords: Adenoid cystic carcinoma, high-grade transformation, dedifferentiation, microarray CGH

DOI: 10.3233/ACP-CLO-2010-0547

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 217-228, 2010

Authors: Pennarun, Bodvael | Kleibeuker, Jan H. | Oenema, Tjitske | Stegehuis, Janet H. | de Vries, Elisabeth G.E. | de Jong, Steven

Article Type: Research Article

Abstract: Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis in tumor cells upon binding to its cognate agonistic receptors, death receptors 4 and 5 (DR4 and DR5). The activity of the insulin-like growth factor 1 (IGF-1) survival pathway is often increased in cancer, influencing both cell proliferation and apoptosis. We hypothesized that inhibiting the IGF-1 receptor (IGF-1R) using NVP-AEW541, a small molecular weight tyrosine kinase inhibitor of the IGF-1R, could increase death receptor (DR)-mediated apoptosis in colon cancer cells. Methods: The analyses were performed by caspase assay, flow cytometry, Western blotting, immunoprecipitation and fluorescent microscopy. Results: Preincubation with …NVP-AEW541 surprisingly decreased apoptosis induced by recombinant human TRAIL (rhTRAIL) or an agonistic DR4 antibody while sensitivity to an agonistic DR5 antibody was increased. NVP-AEW541 could inhibit IGF-1-induced activation of the phosphatidylinositol 3-kinase (PI3K) pathway. The effects of the PI3K inhibitor LY294002 on TRAIL-induced apoptosis were similar to those of NVP-AEW541, further supporting a role for IGF-1R-mediated activation of PI3K. We show that PI3K inhibition enhances DR5-mediated caspase 8 processing but also lowers DR4 membrane expression and DR4-mediated caspase 8 processing. Inhibition of PI3K reduced rhTRAIL sensitivity independently of the cell line preference for either DR4- or DR5-mediated apoptosis signaling. Conclusions: Our study indicates that individual effects on DR4 and DR5 apoptosis signaling should be taken into consideration when combining DR-ligands with PI3K inhibition. Show more

Keywords: DR4, DR5, TRAIL, PI3K, colon cancer

DOI: 10.3233/ACP-CLO-2010-0549

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 229-244, 2010

Authors: Steinbakk, Anita | Malpica, Anais | Slewa, Aida | Gudlaugsson, Einar | Janssen, Emiel A.M. | Arends, Mark | Kruse, Arnold Jan | Yinhua, Yu | Feng, Weiwei | Baak, Jan P.

Article Type: Research Article

Abstract: Objectives: To analyze the prognostic value of microsatellite instability (MSI) in a population-based study of FIGO stage 1–4 endometrial endometrioid adenocarcinomas. Study design: Survival analysis in 273 patients of MSI status and clinico-pathologic features. Using a highly sensitive pentaplex polymerase chain reaction to establish MSI status, cases were divided into microsatellite stable (MSS), MSI-low (MSI-L, 1 marker positive) and MSI-high (MSI-H, 2–5 markers positive). Results: After 61 months median follow-up (1-209), 34 (12.5%) of the patients developed metastases but only 6.4% of the FIGO 1. MSI (especially as MSI-H vs. MSS/MSI-Lcombined) was prognostic in FIGO 1 but not …in FIGO 2–4. The 5 and 10 year recurrence-free survival rates were 98% and 95% in the MSS/MSI-L vs. 85% and 73% in the MSI-H patients (p=0.005). Conclusions: MSI-H status assessed by pentaplex polymerase chain reaction is an indicator of poor prognosis in FIGO 1, but not in FIGO 2–4 endometrial endometrioid adenocarcinomas. Show more

Keywords: Endometrial cancer, endometrioid, FIGO stage 1, MSI, prognosis

DOI: 10.3233/ACP-CLO-2010-0550

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 245-255, 2010

Authors: Hadi, Awal M. | Mouchaers, Koen T.B. | Schalij, Ingrid | Grunberg, Katrien | Meijer, Gerrit A. | Vonk-Noordegraaf, Anton | van der Laarse, Willem J. | Beliën, Jeroen A.M.

Article Type: Research Article

Abstract: Background: Fibrosis is associated with various cardiac pathologies and dysfunction. Current quantification methods are time-consuming and laborious. We describe a semi-automated quantification technique for myocardial fibrosis and validated this using traditional methods. Methods: Pulmonary Hypertension (PH) was induced in adult Wistar rats by subcutaneous monocrotaline (MCT) injection (40 mg/kg). Cryosections of myocardial tissue (5 μm) of PH rats (n=9) and controls (n=9) were stained using Picrosirius red and scanned with a digital microscopic Mirax slide scanner. From these sections 21 images were taken randomly of each heart. Using ImageJ software a macro for automated image analysis of the amount …of fibrosis was developed. For comparison, fibrosis was quantified using traditional polarisation microscopy. Both methods were correlated and validated against stereology as the gold standard. Furthermore, the method was tested in paraffin-embedded human tissues. Results: Automated analysis showed a significant increase of fibrosis in PH hearts vs. control. Automated analysis correlated with traditional polarisation and stereology analysis (r2 =0.92 and r2 =0.95, respectively). In human heart, lungs, kidney and liver, a similar correlation with stereology (r2 =0.91) was observed. Time required for automated analysis was 22 and 33% of the time needed for stereology and polarisation analysis, respectively. Conclusion: Automated quantification of fibrosis is feasible, objective and time-efficient. Show more

Keywords: Collagen, fibrosis, slide scanner, whole slide image, image analysis, open source, Picrosirius red, polarised light, stereology

DOI: 10.3233/ACP-CLO-2010-0548

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 257-269, 2010

Authors: Wang, Yinhai | McCleary, David | Wang, Ching-Wei | Kelly, Paul | James, Jackie | Fennell, Dean A. | Hamilton, Peter

Article Type: Research Article

Abstract: Background: Tissue MicroArrays (TMAs) are a valuable platform for tissue based translational research and the discovery of tissue biomarkers. The digitised TMA slides or TMA Virtual Slides, are ultra-large digital images, and can contain several hundred samples. The processing of such slides is time-consuming, bottlenecking a potentially high throughput platform. Methods: A High Performance Computing (HPC) platform for the rapid analysis of TMA virtual slides is presented in this study. Using an HP high performance cluster and a centralised dynamic load balancing approach, the simultaneous analysis of multiple tissue-cores were established. This was evaluated on Non-Small Cell Lung Cancer …TMAs for complex analysis of tissue pattern and immunohistochemical positivity. Results: The automated processing of a single TMA virtual slide containing 230 patient samples can be significantly speeded up by a factor of circa 22, bringing the analysis time to one minute. Over 90 TMAs could also be analysed simultaneously, speeding up multiplex biomarker experiments enormously. Conclusions: The methodologies developed in this paper provide for the first time a genuine high throughput analysis platform for TMA biomarker discovery that will significantly enhance the reliability and speed for biomarker research. This will have widespread implications in translational tissue based research. Show more

Keywords: Cluster, dynamic load balancing, high performance computing, parallel processing, Tissue MicroArray, TMA, virtual slide

DOI: 10.3233/ACP-CLO-2010-0551

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 271-285, 2010

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 33, no. 5-6, pp. 287-291, 2010