Analytical Cellular Pathology - Volume 32, issue 1-2

Authors: Walenkamp, Annemiek M.E. | Bestebroer, Jovanka | Boer, Ingrid G.J. | Kruizinga, Roeline | Verheul, Henk M. | van Strijp, Jos A.G. | de Haas, Carla J.C.

Article Type: Research Article

Abstract: Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5) was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with …the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies. Show more

Keywords: Leukemia, HL-60, Staphylococcus aureus, SSL5, PSGL-1

DOI: 10.3233/CLO-2009-0486

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 1-10, 2010

Authors: Kaarbø, Mari | Mikkelsen, Øyvind Løveseter | Malerød, Lene | Qu, Su | Lobert, Viola H. | Akgul, Gulcan | Halvorsen, Thomas | Mælandsmo, Gunhild M. | Saatcioglu, Fahri

Article Type: Research Article

Abstract: Background: Androgen receptor (AR) and the phosphatidylinositol-3 kinase (PI3K) signaling are two of the most important pathways implicated in prostate cancer. Previous work has shown that there is crosstalk between these two pathways; however, there are conflicting findings and the molecular mechanisms are not clear. Here we studied the AR–PI3K pathway crosstalk in prostate cancer cells in vitro as well as in vivo. Methods: Quantitative PCR, Western analysis, reporter assays, and proliferation analyses in vitro and in vivo were used to evaluate the effect of PI3K pathway inhibition on AR signaling and cell growth. Results: Transcriptional activity of …AR was increased when the PI3K pathway was inhibited at different levels. In the androgen responsive prostate cancer cell line LNCaP, androgen and the mTOR inhibitor rapamycin synergistically activated androgen target genes. Despite increased androgen signaling, rapamycin treatment reduced LNCaP cell growth; the AR antagonist bicalutamide potentiated this effect. Furthermore, the rapamycin derivative CCI-779 reduced the growth of CWR22 prostate cancer xenografts while increasing AR target gene expression. Conclusion: These findings suggest that inhibition of the PI3K pathway activates AR signaling. Despite the increase in AR signaling which has proliferative effects, the result of PI3K pathway inhibition is antiproliferative. These findings suggest that the PI3K pathway is dominant over AR signaling in prostate cancer cells which should be considered in developing novel therapeutic strategies for prostate cancer. Show more

Keywords: Androgen receptor, PI3K pathway, mTOR

DOI: 10.3233/CLO-2009-0487

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 11-27, 2010

Authors: Heikaus, Sebastian | Pejin, Igor | Gabbert, Helmut Erich | Ramp, Uwe | Mahotka, Csaba

Article Type: Research Article

Abstract: Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs) of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro. Methods: The analyses were performed by semiquantitative real-time PCR, Western Blot and Caspase-2 …Assay. Results: Our in vivo results showed an overall decrease in proapoptotic caspase-2L expression during tumour progression due to an increase in the relative share of caspase-2S mRNA in total caspase-2 mRNA expression. Furthermore, an increase in the expression of PIDD and RAIDD could be observed. In contrast, antiapoptotic BCL-2 expression increased only during early tumour stages, whereas expression decreased in pT3 RCCs. In vitro, caspase-2 activation in RCC cell lines coincidenced with sensitivity of tumour cells towards Topotecan-induced apoptosis. However, inhibition of caspase-2 could not prevent Topotecan-induced apoptosis. Interestingly, Topotecan-resistance could be overcome by the apoptosis-sensitizing drug HA14-1. Conclusion: Our study confirms the concept of a shift towards a more antiapoptotic transcriptional context during tumour progression in RCCs. Furthermore, it shows that caspase-2 participates in chemotherapy-induced apoptosis in RCCs although it is not mandatory for it. Additionally, inhibition of antiapoptotic BCL-2 family members might provide a possible way to overcome chemotherapy resistance of RCCs. Show more

Keywords: Renal cell carcinoma, caspase-2, PIDD, RAIDD, PIDDosome, BCL-2, HA14-1

DOI: 10.3233/CLO-2009-0492

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 29-42, 2010

Authors: Scholten, Kirsten B.J. | Ruizendaal, Janneke J. | Graf, Marcus | Schoedl, Thomas | Kramer, Duco | Meijer, Chris J.L.M. | Man, Stephen | Hooijberg, Erik

Article Type: Research Article

Abstract: Background: T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRαβ open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms. Methods: We isolated the HVP16E6 specific TCRα and TCRβ open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains …to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRα and TCRβ chains. Results: Careful analysis of recipient T cells carrying the HPV16E6 TCRβ and endogenous TCR chains revealed the transgenic TCRβ chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-γ production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay. Conclusion: Promiscuous behavior of the HPV16E6 specific TCRβ chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains. Show more

Keywords: Immunotherapy, cervical carcinoma, adoptive transfer, T cell receptors

DOI: 10.3233/CLO-2009-0493

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 43-56, 2010

Authors: Grabsch, Heike | Sivakumar, Shivan | Gray, Sally | Gabbert, Helmut E. | Müller, Wolfram

Article Type: Research Article

Abstract: Background: Patients with gastric cancer (GC) have a poor survival and biologicals such as Trastuzumab have not been used routinely in these patients. Existing data on HER2 expression and its clinical relevance in GC are still limited and controversial. Methods: HER2 expression was investigated by immunohistochemistry in 418 GC from Germany and 506 GC from England. Results were compared to clinicopathological parameters and patient survival. Results: Less than 10% of all GC showed HER2 expression in more than 5% of tumour cells and 91% of these were intestinal type GC. In both series, no relationship was found between …HER2 expression, patient survival or TNM stage. Marked intratumoural heterogeneity was noted. Conclusions: This is the largest study to date demonstrating in two independent series that HER2 expression is not related to gastric cancer patient prognosis and that only a very small subgroup of intestinal type GC may potentially respond to HER2 targeting therapy. Due to prominent intratumoural heterogeneity of HER2 expression in GC, HER2 testing in endoscopic biopsies before treatment will be prone to false negative results. Show more

Keywords: Gastric cancer, HER2, prognosis, immunohistochemistry

DOI: 10.3233/CLO-2009-0497

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 57-65, 2010

Authors: Koo, Vincent | El Mekabaty, Amgad | Hamilton, Peter | Maxwell, Perry | Sharaf, Osama | Diamond, Jim | Watson, Jenny | Williamson, Kathleen

Article Type: Research Article

Abstract: Background: Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-β1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis. Methods: AY-27 rat bladder cancer cells incubated with 3 ng/ml TGF-β1 or control media for 24 or 48 h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was …measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed. Results: TGF-β1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48 h, the mean Spindle Index of TGF-β1 treated cells was significantly higher than Mock (p=0.02, Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p=0.02, Chi-square). TGF-β1 significantly increased matrigel invasion. Conclusion: The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis. Show more

Keywords: Matrigel, TGF-β_1, chemoattractant, invasion, motility, migration, EMT, bladder cancer, AY-27, spindle phenotype

DOI: 10.3233/CLO-2009-0501

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 67-76, 2010

Authors: Corrias, Maria Valeria | Gambini, Claudio | Gregorio, Andrea | Croce, Michela | Barisione, Gaia | Cossu, Claudia | Rossello, Armando | Ferrini, Silvano | Fabbi, Marina

Article Type: Research Article

Abstract: Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse …patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P=0.044 and P<0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusion: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up. Show more

Keywords: Neuroblastoma, ALCAM/CD166, immunohistochemistry, prognosis

DOI: 10.3233/CLO-2009-0494

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 77-86, 2010

Authors: Barraclough, Dong Liu | Sewart, Susan | Rudland, Philip S. | Shoker, Balvinder S. | Sibson, D. Ross | Barraclough, Roger | Davies, Michael P.A.

Article Type: Research Article

Abstract: Background: A major challenge of cancer research is to identify key molecules which are responsible for the development of the malignant metastatic phenotype, the major cause of cancer death. Methods: Four subtracted cDNA libraries were constructed representing mRNAs differentially expressed between benign and malignant human breast tumour cells and between micro-dissected breast carcinoma in situ and invasive carcinoma. Hundreds of differentially expressed cDNAs from the libraries were micro-arrayed and screened with mRNAs from human breast tumor cell lines and clinical specimens. Gene products were further examined by RT-PCR and correlated with clinical data. Results: The combination of subtractive …hybridisation and microarray analysis has identified a panel of 15 cDNAs which shows strong correlations with estrogen receptor status, malignancy or relapse. This panel included S100P, which was associated with aneuploidy in cell lines and relapse/death in patients, and AGR2 which was associated with estrogen receptor and with patient relapse. X-box binding protein-1 is also an estrogen-dependent gene and is associated with better survival for breast cancer patients. Conclusions: The combination of subtracted cDNA libraries and microarray analysis has thus identified potential diagnostic/prognostic biomarkers and targets for cancer therapy, which have not been identified from common prognostic gene signatures. Show more

Keywords: Suppression subtracted hybridisation, cDNA microarray, patient survival, breast cancer, quantitative RT-PCR

DOI: 10.3233/CLO-2009-0499

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 87-99, 2010

Authors: Ivanyi, Philipp | Morgan, Michael | Piao, Wenji | Ukena, Sya N. | Steube, Klaus | Ganser, Arnold | Franzke, Anke

Article Type: Research Article

Abstract: Background: The pTα/preTCR regulates the β-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56Lck . Methods: To define the relevance of pTα in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTα and p56Lck mRNA and protein expression as also the tyrosine-phosphorylation. The p56Lck specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC50 value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines. Results: pTα expression patterns were markedly different …in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G1/0 phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56Lck and showed a corresponding P-Tyr signal. Conclusion: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTα mediated proliferation in this leukemic sub-type. In addition, p56Lck represents also independently of pTα a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases. Show more

Keywords: T-ALL, pTα, PP1, molecular targeted therapy, tyrosine kinase inhibitor

DOI: 10.3233/CLO-2009-0500

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 101-108, 2010

Authors: Grabe, Niels | Lahrmann, Bernd | Pommerencke, Thora | von Knebel Doeberitz, Magnus | Reuschenbach, Miriam | Wentzensen, Nicolas

Article Type: Research Article

Abstract: Background: Although cytological screening for cervical precancers has led to a reduction of cervical cancer incidence worldwide it is a subjective and variable method with low single-test sensitivity. New biomarkers like p16 that specifically highlight abnormal cervical cells can improve cytology performance. Virtual microscopy offers an ideal platform for assisted evaluation and archiving of biomarker-stained slides. Methods: We first performed a quantitative analysis of p16-stained slides digitized with the Hamamatsu NDP slide scanner. From the results an automated algorithm was created to reliably detect cells, nuclei and p16-stained cells. The algorithm's performance was evaluated on two complete slides and …tiles from 52 independent slides (11,628, 4094 and 25,619 cells/clusters, respectively). Results: We achieved excellent performance to discriminate unstained cells from nuclei and biomarker-stained cells. The automated algorithm showed a high overall and positive agreement (99.0–99.7% and 70.9–83.4%, respectively) with the gold standard and had a very high sensitivity (89.1–100.0%) and specificity (98.9–100.0%) to detect biomarker-stained cells. Conclusion: We implemented a virtual microscopy system allowing highly efficient automated prescreening and archiving of biomarker-stained slides. Based on the initial results, we will evaluate the performance of our system in large epidemiologic studies against disease endpoints. Show more

Keywords: Virtual microscopy, internet, whole slide scanning, image processing, p16, cytology, cervical cancer

DOI: 10.3233/CLO-2009-0508

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 109-119, 2010

Authors: Weber, Achim | Chung, Hye-Jung | Springer, Erik | Heitzmann, Dirk | Warth, Richard

Article Type: Research Article

Abstract: Background: The general transcription factor II H (TFIIH), comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB) and p80 (XPD), cause the hereditary cancer-prone syndrome xeroderma pigmentosum. Methods: The TFIIH subunit p89 was monitored during interphase and cell division by immunofluorescence staining, GFP-fusion constructs including deletions, live cell imaging and immuno-precipitations. Results: Here we demonstrate that during cell division, from prophase until telophase, the TFIIH core subunit p89, but not other subunits of TFIIH, associates with the centrosomes and the adjacent …parts of the mitotic spindle. With overall constant levels throughout mitosis, p89 re-localizes to the newly formed nuclei by the end of mitosis. Furthermore, p89 interacts with the centrosomal protein γ-tubulin. Truncations of p89 result in an abnormal subcellular distribution during interphase and abolished centrosomal association during mitosis. Conclusions: Our observations suggest a so far unappreciated role for p89 in cell cycle regulation, and may be the structural basis for a long known, but hitherto unexplained interaction between p89 and tubulin. Show more

Keywords: TFIIH, mitosis, centrosome

DOI: 10.3233/CLO-2009-0509

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 121-130, 2010

Authors: Yang, Nan | Nijhuis, Esther R. | Volders, Haukeline H. | Eijsink, Jasper J.H. | Lendvai, Ágnes | Zhang, Bo | Hollema, Harry | Schuuring, Ed | Wisman, G. Bea A. | van der Zee, Ate G.J.

Article Type: Research Article

Abstract: Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings. Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-β2 at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest …increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05). Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions. Show more

Keywords: Methylation, cervical (intraepithelial) neoplasia, DAPK, CCNA1, CADM1

DOI: 10.3233/CLO-2009-0510

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 131-143, 2010

Authors: Meijer, Gerrit

Article Type: Other

DOI: 10.3233/CLO-2010-0513

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 147-148, 2010

Authors: Moelans, Cathy B. | Kibbelaar, Robby E. | van den Heuvel, Marius C. | Castigliego, Domenico | de Weger, Roel A. | van Diest, Paul J.

Article Type: Research Article

Abstract: Background: Testing for HER2 amplification and/or overexpression is currently routine practice to guide Herceptin therapy in invasive breast cancer. At present, HER2 status is most commonly assessed by immunohistochemistry (IHC). Standardization of HER2 IHC assays is of utmost clinical and economical importance. At present, HER2 IHC is most commonly performed with the HercepTest which contains a polyclonal antibody and applies a manual staining procedure. Analytical variability in HER2 IHC testing could be diminished by a fully automatic staining system with a monoclonal antibody. Materials and methods: 219 invasive breast cancers were fully automatically stained with the monoclonal antibody-based Oracle …HER2 Bond IHC kit and manually with the HercepTest. All cases were tested for amplification with chromogenic in situ hybridization (CISH). Results: HercepTest yielded an overall sharper membrane staining, with less cytoplasmic and stromal background than Oracle in 17% of cases. Overall concordance between both IHC techniques was 89% (195/219) with a kappa value of 0.776 (95% CI 0.698–0.854), indicating a substantial agreement. Most (22/24) discrepancies between HercepTest and Oracle showed a weaker staining for Oracle. Thirteen of the 24 discrepant cases were high-level HER2 amplified by CISH, and in 12 of these HercepTest IHC better reflected gene amplification status. All the 13 HER2 amplified discrepant cases were at least 2+ by HercepTest, while 10/13 of these were at least 2+ for Oracle. Considering CISH as gold standard, sensitivity of HercepTest and Oracle was 91% and 83%, and specificity was 94% and 98%, respectively. Positive and negative predictive values for HercepTest and Oracle were 90% and 95% for HercepTest and 96% and 91% for Oracle, respectively. Conclusion: Fully-automated HER2 staining with the monoclonal antibody in the Oracle kit shows a high level of agreement with manual staining by the polyclonal antibody in the HercepTest. Although Oracle shows in general some more cytoplasmic staining and may be slightly less sensitive in picking up HER2 amplified cases, it shows a higher specificity and may be considered as an alternative method to evaluate the HER2 expression in breast cancer with potentially less analytical variability. Show more

Keywords: HER2, Oracle, HercepTest, CISH

DOI: 10.3233/CLO-2010-0514

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 149-155, 2010

Authors: Meijer, Gerrit A.

Article Type: Other

DOI: 10.3233/CLO-2010-0512

Citation: Analytical Cellular Pathology, vol. 32, no. 1-2, pp. 156-156, 2010