Analytical Cellular Pathology - Volume 31, issue 3

Authors: Kramer, D. | Thunnissen, F.B. | Gallegos-Ruiz, M.I. | Smit, E.F. | Postmus, P.E. | Meijer, C.J.L.M. | Snijders, P.J.F. | Heideman, D.A.M.

Article Type: Research Article

Abstract: Background: Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed. Methods: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype. Results: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5–2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay …following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing. Conclusions: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning. Show more

Keywords: HRM, direct cycle sequencing, G12, G13, K-ras, EGFR, genotype, codon, (nested-)PCR, formalin-fixed paraffin-embedded, molecular diagnostics, TKI, receptor tyrosine kinase inhibitors

DOI: 10.3233/CLO-2009-0466

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 161-167, 2009

Authors: Mesker, Wilma E. | Liefers, Gerrit-Jan | Junggeburt, Jan M.C. | van Pelt, Gabi W. | Alberici, Paola | Kuppen, Peter J.K. | Miranda, Noel F. | van Leeuwen, Karin A.M. | Morreau, Hans | Szuhai, Karoly | Tollenaar, Rob A.E.M. | Tanke, Hans J.

Article Type: Research Article

Abstract: Background: For stage I–II colon cancer a significant number (5–25%) of patients has recurrent disease within 5 years. There is need to identify these high-risk patients as they might benefit from additional treatment. Stroma-tissue surrounding the cancer cells plays an important role in the tumor behavior. The proportion of intra-tumor stroma was evaluated for the identification of high-risk patients. In addition, protein expression of markers involved in pathways related to stroma production and epithelial-to-mesenchymal transition (EMT) was analyzed: β-catenin, TGF-β-R2 and SMAD4. Methods: In a retrospective study of 135 patients with stage I–II colon cancer, the amount of …stroma was estimated on routine haematoxylin–eosin stained histological sections. Sections were also immunohistochemically stained for β-catenin, TGF-β-R2 and SMAD4. Results: Of 135 analyzed patients 34 (25.2%) showed a high proportion of stroma (stroma-high) and 101 (74.8%) a low proportion (stroma-low). Significant differences in overall-survival and disease-free-survival were observed between the two groups, with stroma-high patients showing poor survival (OS p<0.001, HZ 2.73, CI 1.73–4.30; DFS p<0.001, HZ 2.43, CI 1.55–3.82). A high-risk group was identified with stroma-high and SMAD4 loss (OS p=0.008, HZ 7.98, CI 4.12–15.44, DFS p=0.005, HZ 6.57, CI 3.43–12.56); 12 of 14 (85.7%) patients died within 3 years. In a logistic-regression analysis a high proportion of stroma and SMAD4 loss were strongly related (HZ 5.42, CI 2.13–13.82, p<0.001). Conclusions: Conventional haematoxylin–eosin stained tumor slides contain more prognostic information than previously fathomed. This can be unleashed by assessing the tumor–stroma ratio. The combination of analyzing the tumor–stroma ratio and staining for SMAD4 results in an independent parameter for confident prediction of clinical outcome. Show more

Keywords: Colon cancer, primary tumor, high-risk patients, stroma, prognosis

DOI: 10.3233/CLO-2009-0478

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 169-178, 2009

Authors: Fontijn, Dennis | Bosch, Linda J.W. | Duyndam, Monique C.A. | van Berkel, Maria P.A. | Janmaat, Maarten L. | Boven, Epie

Article Type: Research Article

Abstract: Background: 1F6 human melanoma xenografts overexpressing either the 18 kD (18kD) form or all (ALL) forms of human basic fibroblast growth factor (bFGF) demonstrate an abundant number of microvessels and accelerated growth. We now examined whether bFGF mediates vascular endothelial growth factor (VEGF) expression. Methods: Quantitative RT-PCR was used to determine bFGF and VEGF mRNA, VEGF protein secretion was measured by ELISA and VEGF promoter activation was assessed by a dual luciferase activity assay. Western blot was carried out to detect phosphorylation of bFGF-regulated target proteins. Results: In 1F6-18kD and 1F6-ALL clones VEGF mRNA was increased 4- to …5-fold and VEGF protein secretion was highly stimulated due to activation of the VEGF promotor. PI-3K, p38 MAPK and ERK1/2 MAPK pathways were activated, while inhibition of PI-3K or p38 resulted in, respectively, 55% and up to 70% reduction of VEGF mRNA overexpression. A concurrent 60% decrease in VEGF protein secretion was mostly apparent upon inhibition of PI-3K. Inhibition of ERK1/2 hardly affected VEGF mRNA or protein secretion. Two unselected human melanoma cell lines with high metastatic potential contained high bFGF and VEGF, while three non- or sporadically metastatic cell lines displayed low bFGF and VEGF. Conclusion: These data indicate that stimulation of VEGF protein secretion in response to bFGF overexpression may contribute to increased vascularization and enhanced aggressiveness in melanoma. Show more

Keywords: Basic fibroblast growth factor, vascular endothelial growth factor, melanoma, PI-3K, p38 MAPK, ERK1/2 MAPK

DOI: 10.3233/CLO-2009-0477

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 179-190, 2009

Authors: Jonsdottir, Asta Björk | Vreeswijk, Maaike P.G. | Wolterbeek, Ron | Devilee, Peter | Tanke, Hans J. | Eyfjörd, Jorunn E. | Szuhai, Karoly

Article Type: Research Article

Abstract: Background: Inherited mutations in the tumour suppressor gene BRCA2 greatly increase the risk of developing breast, ovarian and other types of cancers. So far, most studies have focused on the role of BRCA-pathways in the maintenance of genomic stability. In this study we investigated the potential role of the BRCA2 protein in cytokinesis in unmodified primary human fibroblast carrying a heterozygous mutation in the BRCA2 gene. Methods: Cell divisions were monitored with time lapse live-cell imaging. BRCA2 mRNA expression levels in BRCA2+/− and BRCA2+/+ cells were quantified with quantitative real-time polymerase chain reaction (qRT-PCR). To investigate …the localization of the BRCA2 protein during cytokinesis, immunofluorescence staining using antibody directed against BRCA2 was carried out. Immunofluorescence staining was performed directly after live-cell imaging and cells with delayed cytokinesis, of which the co-ordinates were saved, were automatically repositioned and visualized. Results: We demonstrate that unmodified primary human fibroblasts derived from heterozygous BRCA2 mutation carriers show significantly prolonged cytokinesis. A Subset of the BRCA2+/− cells had delayed cytokinesis (40 min or longer) making the mean cell division time 6 min longer compared with BRCA2+/+ cells, 33 min versus 27 min, respectively. Lower BRCA2 mRNA expression levels were observed in the BRCA2 heterozygous samples compared with the BRCA2 wild type samples. The BRCA2 protein localizes and accumulates to the midbody during cytokinesis, and no difference was detected in distribution and localization of the protein between BRCA2+/− and BRCA2+/+ samples or cells with delayed cytokinesis and normal division time. Conclusions: The delayed cytokinesis phenotype of the BRCA2 heterozygous cells and localization of the BRCA2 protein to the midbody confirms that BRCA2 plays a role in cytokinesis. Our observations indicate that in a subset of cells the presence of only one wild type BRCA2 allele is insufficient for efficient cytokinesis. Show more

Keywords: BRCA2, cytokinesis, live-cell imaging, primary human fibroblasts, heterozygous phenotype

DOI: 10.3233/CLO-2009-0465

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 191-201, 2009

Authors: Savino, Maria | Parrella, Paola | Copetti, Massimiliano | Barbano, Raffaela | Murgo, Roberto | Fazio, Vito Michele | Valori, Vanna Maria | Carella, Massimo | Garrubba, Maria | Santini, Stefano Angelo

Article Type: Research Article

Abstract: Background: The development of non-invasive procedure to determine HER2 status may represent a powerful method for monitoring disease progression and response to the treatment. Methods: Serum samples and RNA from peripheral blood were evaluated in 85 breast cancer patients (49 HER2 positive and 36 HER2 negative) and 22 healthy controls. HER2 mRNA levels were measured by real-time quantitative PCR (QPCR) and serum HER2 protein by immunoenzimatic assay (EIA). ROC curve analyses were used to determine the optimal cut off values. Results: A statistically significant difference was detected for both QPCR and EIA in HER2 positive patients as compared …with both healthy controls and HER2 negative tumours. QPCR showed a 91% (CI95%: 84%–98%) specificity and a 78% (CI95%: 68%–88%) sensitivity for an optimal cut off value of 4.74. The optimal cut off value for EIA was 22 ng/ml yielding a 95% (CI95%: 90%–100%) specificity and a 59% (CI95%: 48%–70%) sensitivity. The QPCR assay was slightly less specific than EIA in discriminating HER2 positive breast cancers from HER2 negative tumours (78% CI95%: 69%–87% versus 86% CI95%: 79%–93%), but it was more sensitive (76% CI95%: 67%–85% versus 55% CI95%: 44%–66%). Conclusions: Our results indicate that QPCR performs better than EIA in the determination of HER2 status of breast cancer patients and could be useful in monitoring the disease during follow up. Show more

Keywords: HER2, breast cancer, QPCR, EIA, RT-PCR, IHC

DOI: 10.3233/CLO-2009-0468

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 203-211, 2009

Authors: Westphal, K. | Akgül, B. | Storey, A. | Nindl, I.

Article Type: Research Article

Abstract: Background: A role for cutaneous human β-papillomavirus (HPV) types as co-factors in the development of non-melanoma skin cancer has been postulated. Here we have investigated the effects of E7 expression on keratinocyte differentiation, proliferation and cell-cycle proteins in organotypic skin cultures. Methods: Recombinant retroviruses containing the E7 genes from cutaneous HPV types 1, 4, 5, 8, 20, 38 and RTRX7 were produced that include types associated with benign and malignant lesions. Adult human primary keratinocytes were transduced with these recombinant retroviruses and differentiated into skin-equivalents using de-epidermalised human dermis. Results: Expression patterns of the basal keratinocyte marker cytokeratin …14 (CK14) were not altered by any of the viral E7 types analysed. However, expression of the early and late differentiation markers CK10 and involucrin were markedly altered in HPV 1, 4 and 38 cultures. The highest proliferation rates in basal cell layers, as judged by BrdU and Ki67 staining, were observed in HPV 1, 4 and 38 cultures. Interestingly, co-expression of cyclin E and p16INK4a within the same cell of the suprabasal cell layers was observed only in cultures generated using E7 of HPV 5 or HPV 8. Conclusion: HPV types associated with either benign or malignant lesions perturb keratinocyte proliferation and differentiation in different ways. Moreover, expression of E7 from HPV 5 or HPV 8 seem able to overcome p16INK4a induced cell cycle arrest in a subset of keratinocytes. Show more

Keywords: β-PV, human papillomavirus (HPV), human primary keratinocytes (HPK), organotypic skin cultures, p16^INK4a

DOI: 10.3233/CLO-2009-0476

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 213-226, 2009

Authors: Fransvea, Emilia | Paradiso, Angelo | Antonaci, Salvatore | Giannelli, Gianluigi

Article Type: Research Article

Abstract: Background: Hepatocellular carcinoma (HCC) poses a major challenge because of the extreme variability of the clinical outcome, which makes it difficult to properly stage the disease and thereby estimate the prognosis. There is growing evidence that this heterogeneous clinical behavior is attributable to several different biological pathways. A novel approach to mapping these differences is by investigating the epigenetics associated with certain clinical aspects. Design: Herein, the relevance of these molecular differences in combination with the biological and molecular pathways regulating the clinical outcome will be discussed. Use of a mechanistic and pathogenic approach to clarify the natural history …of HCC is not just an academic speculation but should help to develop new therapies and to tailor these therapies to each individual patient. Conclusion: New biological therapies targeting components of the tumoral or peritumoral microenvironment are crucial to the fight against HCC. However, biological redundancies and the presence of several growth factors, hormones, cytokines, etc., potentially involved in HCC tumor progression make it difficult to assess the best target. Sorafenib, a multi-tyrosine kinase inhibitor, blocks the functions of different growth factors present in the tissue microenvironment. The use of Sorafenib in patients with HCC offers a new approach to the therapy of this disease, stimulating research focusing on the development of drugs based on new molecular and pathogenic insights. Show more

Keywords: Biological therapies, HCC, molecular pathogenesis, TGF-β1, tissue microenvironment, TK-receptors, tumor progression, heterogeneity

DOI: 10.3233/CLO-2009-0473

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 227-233, 2009

Authors: Grivas, Petros D. | Tzelepi, Vassiliki | Sotiropoulou-Bonikou, Georgia | Kefalopoulou, Zinovia | Papavassiliou, Athanasios G. | Kalofonos, Haralabos

Article Type: Research Article

Abstract: Background: Estrogen receptor β (ERβ) is abundantly expressed in colorectal tissue, but its role in colorectal carcinogenesis remains elusive. ER novel co-regulator, proline-, glutamic acid- and leucine-rich protein 1 (PELP1/MNAR) has been characterized, but its expression in colorectal carcinomas has not been investigated. Methods: ERα, ERβ and PELP1/MNAR protein expression were evaluated by immunohistochemistry in colorectal normal mucosa, adenomas and adenocarcinomas from 113 patients with colorectal cancer. Results: ERα expression is extremely rare in colorectal tissue and its expression does not appear to be associated with colorectal carcinogenesis. ERβ and PELP1/MNAR were detected in the nucleus of epithelial, …endothelial, inflammatory, smooth muscle cells and myofibroblasts. When intensity of staining was taken into account, the expression of both proteins was significantly increased in epithelial cells of carcinomas compared to normal mucosa. ERβ expression in epithelial cells was correlated with decreased disease progression – free survival. PELP1/MNAR overexpression in epithelial cells was found to be an independent favorable prognostic factor. Additionally, the expression of both proteins was significantly increased in stromal myofibroblasts of carcinomas compared to adenomas and normal mucosa. Conclusion: ERβ and PELP1/MNAR appear to be involved in colorectal tumorigenesis and might have prognostic significance. Show more

Keywords: Colorectal cancer, estrogen receptor α/β, immunohistochemistry, PELP1/MNAR, tumorigenesis

DOI: 10.3233/CLO-2009-0467

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 235-247, 2009

Article Type: Other

DOI: 10.3233/CLO-2009-0475

Citation: Analytical Cellular Pathology, vol. 31, no. 3, pp. 249-250, 2009