Analytical Cellular Pathology - Volume 31, issue 5

Authors: Heideman, D.A.M. | Thunnissen, F.B. | Doeleman, M. | Kramer, D. | Verheul, H.M. | Smit, E.F. | Postmus, P.E. | Meijer, C.J.L.M. | Meijer, G.A. | Snijders, P.J.F.

Article Type: Research Article

Abstract: Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies. Methods: In this study, we describe the development of high resolution melting (HRM) technology-based assays with direct sequencing confirmation possibility for mutation screening of the EGFR gene (exons 19, 20 and 21) in routine diagnostic specimens, and compared assay findings to those of conventional nested-PCR following cycle-sequencing. Results: In reconstruction experiments, each HRM assay following sequencing demonstrated a sensitivity of ≤5% …of mutated DNA in a background of wild-type DNA. The panel of EGFR HRM assays following sequencing applied to a series of genomic DNA samples isolated from 68 FFPE NSCLC specimens correctly identified all EGFR mutations that were previously found by nested-PCR following cycle-sequencing. The HRM approach additionally scored two mutations not detected by the conventional assay. Complementary HRM following sequencing for K-ras revealed three mutations. EGFR and K-ras mutations were mutually exclusive. Conclusions: The panel of designed HRM assays with direct reflex sequencing possibility provides an effective method for mutation screening of EGFR and K-ras genes in routine diagnostic specimens, thereby allowing the selection of the treatment of choice in clinical practice. Show more

Keywords: HRM, direct cycle sequencing, K-ras, epidermal growth factor receptor, genotype, codon, (nested-) PCR, formalin-fixed paraffin-embedded, molecular diagnostics, TKI, receptor tyrosine kinase inhibitors, anti-EGFR monoclonal antibodies, EGFR-targeted therapies, personalized therapy

DOI: 10.3233/CLO-2009-0489

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 329-333, 2009

Authors: Janssen, Emiel A.M. | Øvestad, Irene T. | Skaland, Ivar | Søiland, Håvard | Gudlaugsson, Einar | Kjellevold, Kjell H. | Nysted, Arne | Søreide, Jon-Arne | Baak, Jan P.A.

Article Type: Research Article

Abstract: Background: The mitotic activity index (MAI) is a strong prognosticator in node-negative invasive breast cancer patients. Recently, a correlation between the MAI and specific chromosomal aberrations at chromosome 1p was described. Methods: Analysis of MAI, immunohistochemical staining patterns for proliferation-associated phosphohistone H3 (PPH3), phosphorylated ERK1/2, p21, cyclin E, Ki67 and cyclin D1 proteins; and prognosis in 158 adjuvant chemotherapy-treated T1-2N0M0 invasive breast cancer patients, analysis of LOH at 1p31 (including ARHI) using the dinucleotide repeats D1S207, D1S430 and D1S464 in 76 patients. Single and multivariate survival analysis was used to evaluate the importance of the various markers tested. …Results: LOH at 1p31 did not correlate with MAI nor provide prognostic information. Phosphohistone H3 was the best prognosticator for patients in all age groups with 20 year distant metastasis free survival of distant metastases 93% vs. 72% respectively (p=0.004, HR=4.5). In multivariate analysis, phosphohistone H3<13 vs. ≥13 exceeded the prognostic value of the mitotic activity index. Conclusions: LOH at 1p31 is common in breast cancer, and correlates with loss of proliferation-associated proteins, but not with MAI, PPH3 or prognosis. PPH3 is the best prognosticator in this study group of adjuvant chemotherapy-treated lymph node-negative breast cancer patients. Show more

Keywords: Breast cancer, proliferation, PPH3, loss of heterozygosity, ARHI

DOI: 10.3233/CLO-2009-0479

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 335-343, 2009

Authors: Fijneman, Remond J.A. | Bade, Lindsey K. | Peham, Johannes R. | van de Wiel, Mark A. | van Hinsbergh, Victor W.M. | Meijer, Gerrit A. | O'Sullivan, Michael G. | Cormier, Robert T.

Article Type: Research Article

Abstract: Background: The group IIA secretory phospholipase A2 gene, Pla2g2a, confers resistance to intestinal tumorigenesis in the ApcMin/+ mouse model. However, it is unclear how Pla2g2a exerts its tumor-suppressive effects and whether its mode of action depends on Apc-germline mutations. Methods: We tested whether expression of a Pla2g2a transgene provides protection against carcinogen-induced colon tumors, and examined whether the normal colon microenvironment is modulated by Pla2g2a expression. Results: Pla2g2a strongly inhibited colon tumorigenesis in mice following treatment with the DNA alkylating agent azoxymethane (AOM). Moreover, AOM-induced duodenal tumors were also attenuated by Pla2g2a expression. These tumors demonstrated …upregulation of β-catenin, indicative of involvement of the Wnt signaling pathway. Comparison of genome-wide microarray expression profiles of healthy (non-pathologic) colon tissues from Pla2g2a-transgenic to non-transgenic mice revealed 382 genes that were differentially expressed, comprising clusters of genes involved in inflammation and microbial defense, cell signaling and cell cycle, transactivation, apoptosis and mitochondrial function, DNA repair, and lipid and energy metabolism. Pathway analysis using Gene Set Enrichment Analysis (GSEA) indicated that Pla2g2a suppresses the expression of interferon-induced genes. Conclusion: Our results demonstrate that Pla2g2a attenuates colon tumorigenesis independent of Apc-germline mutations, and reveal Pla2g2a target genes and pathways in non-pathologic colon microenvironment that influence conditions for colorectal cancer development. Show more

Keywords: Colorectal cancer, Pla2g2a, microarray expression analysis, transgenic mice

DOI: 10.3233/CLO-2009-0480

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 345-356, 2009

Authors: Magnani, Ivana | Novielli, Chiara | Bellini, Melissa | Roversi, Gaia | Bello, Lorenzo | Larizza, Lidia

Article Type: Research Article

Abstract: Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma. Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and …NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines. Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines. Conclusion: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma. Show more

Keywords: Glioma, MARK4L, immunodetection, multiple subcellular localization, centrosome abnormalities

DOI: 10.3233/CLO-2009-0481

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 357-370, 2009

Authors: Baldewijns, Marcella M. | van Vlodrop, Iris J.H. | Smits, Kim M. | Vermeulen, Peter B. | Van den Eynden, Gert G. | Schot, Fiona | Roskams, Tania | van Poppel, Hein | van Engeland, Manon | de Bruïne, Adriaan P.

Article Type: Research Article

Abstract: Background: von Hippel–Lindau (VHL) inactivation is common in sporadic clear cell renal cell carcinomas (ccRCC). pVHL is part of the ubiquitin ligase complex that targets the alpha subunits of hypoxia-inducible transcription factor (HIF) for degradation under well-oxygenated conditions. In the absence of wild-type pVHL, as observed in VHL patients and most sporadic ccRCCs, constitutive upregulation of HIF results in transcriptional activation of angiogenesis-related genes, such as VEGF. Differences in angiogenic activity within the group of ccRCCs were reported and strong genotype-phenotype correlations were found in patients with VHL disease, raising a question about the importance of VHL inactivation status in …angiogenic behaviour and tumour progression. Methods: To address this question, we investigated the influence of VHL mutation (direct sequencing)/hypermethylation (methylation-specific PCR) on angiogenesis/tumour parameters (immunohistochemistry) in 150 patients with sporadic ccRCC. Results: We found no significant association between VHL mutation or methylation and angiogenesis/tumour parameters. Conclusion: These data indicate that tumour progression and angiogenesis are not directly influenced by VHL alterations and that additional genetic/epigenetic events should be considered to explain the diverse angiogenic and proliferative behaviour during tumour progression. Show more

Keywords: VHL, angiogenesis, renal cell carcinoma

DOI: 10.3233/CLO-2009-0482

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 371-382, 2009

Authors: Mannello, Ferdinando | Tonti, Gaetana A. | Medda, Virginia

Article Type: Research Article

Abstract: Background: Protein carbonyl levels are the most frequently used biomarker of protein oxidation in several human diseases, including cancer. Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium from which nipple aspirate fluid can be collected and analysed to assess tissue metabolic activity. Our aims were to perform an exploratory investigation on the protein carbonyl accumulation in breast secretions from healthy and cancer patients and its correlation with lipid peroxidation markers. Methods: Protein carbonyls were determined by ELISA in 288 Nipple Aspirate Fluids (NAF) from Control, Pre-malignant and Cancer patients. Results: Significantly higher protein carbonyl …concentration was found in NAF from breast cancer (BC) patients compared to Control subjects. Cancer patients accumulated in NAF significantly higher levels of carbonyls in post-menopausal condition. A significant inverse relationship between carbonyls and 8-F2α -isoprostanes in NAF was found in Cancer patients. NAF levels of protein carbonyls are significantly higher in women with pre-malignant conditions than in healthy subjects. Conclusion: Our results support the hypothesis that oxidative stress in breast microenvironment plays a role in breast cancer; measurement of protein and lipid oxidative products in NAF may improve the identification of women at increased breast cancer risk. Show more

Keywords: Nipple aspirate fluid, oxidative stress, protein carbonyl, breast cancer

DOI: 10.3233/CLO-2009-0483

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 383-392, 2009

Authors: Liu, Leyuan | Xie, Rui | Yang, Chaofeng | McKeehan, Wallace L.

Article Type: Research Article

Abstract: Background: Survival and evolution of aneuploid cells after an asymmetric segregation of chromosomes at mitosis may be the common initiating event and underlying cause of the genetic diversity and adaptability of cancers. We hypothesize that mechanisms exist to detect impending aneuploidy and prevent it before completion of an aberrant mitosis. Methods: The distribution of isoforms of C19ORF5, an interactive partner with mitochondria-associated LRPPRC and tumor suppressor RASSF1A, state of spindle microtubules and mitochondrial aggregation was analyzed in synchronized mitotic cells and cells stalled in mitosis after treatment with paclitaxel. Results: C19ORF5 distributed broadly across the mitotic spindle and …reversibly accumulated during reversible mitotic arrest. Prolonged stabilization of microtubules caused an accumulation of a C19ORF5 product with dual MAP and MtAP properties that caused irreversible aggregation of mitochondria and death of mitotic cells. Conclusion: Dual function microtubule-associated (MAP) and mitochondria-associated (MtAP) proteins generated by prolonged mitotic arrest trigger mitochondrial-induced mitotic cell death. This is a potential mechanism to prevent minimal survivable aneuploidy resulting from an aberrant cell division and cancers in general at their earliest common origin. Show more

Keywords: Aneuploidy, C19ORF5, genetic instability, LRPPRC, microtubule dynamics, mitochondrial dynamics, RASSF1A, paclitaxel, tumor suppression, mitochondria aggregation

DOI: 10.3233/CLO-2009-0484

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 393-405, 2009

Authors: van Houdt, I.S. | Sluijter, B.J.R. | van Leeuwen, P.A.M. | Moesbergen, L.M. | Hooijberg, E. | Meijer, C.J.L.M. | de Gruijl, T.D. | Oudejans, J.J. | Boven, E.

Article Type: Research Article

Abstract: Background: Sentinel Lymph Node (SLN) status is strongly related to clinical outcome in melanoma patients. In this study we investigated the possible association between the presence of activated and/or suppressive Tumour Infiltrating Lymphocytes (TILs) and SLN status in clinically stage I/II melanoma patients. Methods: Diagnostic primary melanoma samples from 20 patients with a sentinel lymph node metastasis were compared to melanoma samples from 20 patients with a negative sentinel lymph node, who were matched for gender, age and Breslow thickness. Presence of activated Granzyme B positive (GrB+ ) TILs, presence of suppressive (FoxP3+ ) TILs and MHC class I …antigen expression on tumour cells were analysed by immunohistochemistry. Results: FoxP3 and MHC-I expression had no direct bearing on the presence of melanoma metastases in the SLN. Whereas the presence of activated GrB+ TILs in the primary melanoma had no predictive value for SLN status either, their absence was strongly associated with the presence of metastasis in the SLN (p=0.001). While both GrB+ and FoxP3+ TILs could be detected in SLN metastases, a majority did not display MHC-I expression. Conclusion: These data support a role for cytotoxic T cells in the prevention of early metastasis of melanoma to the draining lymph nodes. Show more

Keywords: Melanoma, CTL, Tregs, FoxP3, Granzyme B, sentinel lymph node

DOI: 10.3233/CLO-2009-0485

Citation: Analytical Cellular Pathology, vol. 31, no. 5, pp. 407-413, 2009