Analytical Cellular Pathology - Volume 29, issue 4

Authors: Lanzilotta, Salvatore Giovanni | Grammatica, Luciano | Paradiso, Angelo | Simone, Gianni

Article Type: Research Article

Abstract: Novel genetic findings about papillary thyroid carcinoma identify BRAF gene as a subject of great interest. Involvement of BRAF gene in pathogenesis of PTC, diagnostic value and the putative prognostic significance of its T1799A mutation are summarized in this article. Furthermore, a particular attention is focused to the role of pre-operative detection of BRAF mutation in the FNAB specimens of thyroid nodules and to the use of this gene as target for an effective cancer therapy.

Keywords: BRAF mutation, papillary thyroid cancer, fine-needle aspiration

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 269-277, 2007

Authors: Vannini, Ivan | Bonafe, Massimiliano | Tesei, Anna | Rosetti, Marco | Fabbri, Francesco | Storci, Gianluca | Ulivi, Paola | Brigliadori, Giovanni | Amadori, Dino | Zoli, Wainer

Article Type: Research Article

Abstract: Background: Melanoma remains largely resistant to currently available chemotherapy, and new strategies have been proposed to flank standardized therapeutic protocols in an effort to improve efficacy. Such an approach requires good knowledge of the mechanisms involved in the resistance and survival of melanoma cells. In this context, the SLUG gene has recently been characterized as a major regulator of melanocytes and melanoma cell survival. Methods: We tested the hypothesis that an oligonucleotide-based short interfering RNA (siRNA) directed against the SLUG gene increases the susceptibility of melanoma cells to drugs such as cisplatin and fotemustine, which are frequently used to treat …this cancer. Results: It was found that SLUG siRNA increased cisplatin-induced cell death and rendered the drug active in vitro at half its plasmatic peak concentration. Such activity was correlated with an upregulation of the pro-apoptotic gene, PUMA. Furthermore, SLUG siRNA increased the capacity of fotemustine to elicit cell death and induced p21WAF1 upregulation, resulting in cell cycle arrest. Interestingly, this pathway did not require functional p53. Conclusion: These findings suggest that SLUG siRNA enhances the efficacy of two of the most widely used drugs to treat melanoma. Show more

Keywords: Slug gene, melanoma, short interfering RNA, cisplatin, fotemustine

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 279-287, 2007

Authors: Page, Robert E. | Klein-Szanto, Andrés J.P. | Litwin, Samuel | Nicolas, Emmanuelle | Al-Jumaily, Raid | Alexander, Peter | Godwin, Andrew K. | Ross, Eric A. | Schilder, Russell J. | Bassi, Daniel E.

Article Type: Research Article

Abstract: Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived …from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease. Show more

Keywords: Proprotein convertases, metalloproteinases, survival, ovarian cancer, tumor progression

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 289-299, 2007

Authors: Soreide, Kjetil | Buter, Tirza C.E. | Janssen, Emiel A. | Gudlaugsson, Einar | Skaland, Ivar | Körner, Hartwig | Baak, Jan P.A.

Article Type: Research Article

Abstract: Background and aims: Although adenomas may be precursors to colorectal cancers (CRC), knowledge concerning the development of metachronous CRC is scarce. We assessed whether differential expression of cell-cycle and apoptosis-regulating proteins and a monotonous population of elongated cells (MPECs) in colorectal adenomas could predict metachronous CRC. Methods: Application of immunohistochemistry on tissue microarrays in consecutive, population-based colorectal adenomas. Influence of classic features (e.g., intraepithelial neoplasia grade, histological type, size) was examined. Results: Of 171 patients with colorectal adenoma 86% (n=147) were eligible for study; 10 (7%) developed metachronous CRC. Median time to cancer was 69 months (range, 25–256). Median follow-up …was equal for the non-cancer and cancer groups. Elevated expression of cell-cycle regulators p16INK4A , p21CIP1 , and cytoplasmic/nuclear β-catenin correlated with increased CRC risk (all P<0.0001), as did elevated expression of the anti-apoptosis protein survivin (P<0.0001) and human telomerase reverse transcriptase (hTERT; P<0.001). Survivin, hTERT, and nuclear β-catenin were the most predictive molecular markers (hazard ratios [HRs]: 6.3, 9.4, and 5.8, respectively). In a combined multivariate model, MPECs had the best overall prognostic ability (HR 28.2, 95% CI: 3.6–223.0), together with survivin, and hTERT. Within adenomas containing MPECs, several molecular markers further defined high-risk patients. Conclusions: Among several markers predictive for metachronous CRC development in colorectal adenomas, MPECs, survivin and hTERT may, when validated, provide information superior to conventional histology, with relevance for the clinical management of patients with colorectal adenoma. Show more

Keywords: Colorectal, adenoma, immunohistochemistry, cell cycle, apoptosis, metachronous cancer, molecular marker, diagnosis

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 301-313, 2007

Authors: Mijatovic, Tatjana | Gailly, Philippe | Mathieu, Véronique | De Nève, Nancy | Yeaton, Paul | Kiss, Robert | Decaestecker, Christine

Article Type: Research Article

Abstract: Background: While the neurotensin (NT) roles in pancreatic cancer growth are well documented, its effects on pancreatic cancer cell migration have not been described. Methods: The NT-induced effects on the migration process of human pancreatic ductal adenocarcinoma cells (PDACs) were characterized by means of various assays including computer-assisted video-microscopy, fluorescence microscopy, ELISA-based, small GTPase pull-down and phosphorylation assays. Results: The NT-induced modifications on in vitro PDACs migration largely depended on the extra-cellular matrix environment and cell propensity to migrate collectively or individually. While NT significantly reduced the level of migration of collectively migrating PDACs on vitronectin, it significantly increased the …level of individually migrating PDACs. These effects were mainly mediated through the sortilin/NTR3 receptor. Neurotensin both induced altered expression of αV and β5 integrin subunits in PDACs cultured on vitronectin resulting in modified adhesion abilities, and caused modifications to the organization of the actin cytoskeleton through the NT-mediated activation of small Rho GTPases. While the NT effects on individually migrating PDACs were mediated at least through the EGFR/ERK signaling pathways, those on collectively migrating PDACs appeared highly dependent on the PI 3-kinase pathway. Conclusion: This study strongly suggests the involvement of neurotensin in the modulation of human PDAC migration. Show more

Keywords: Neurotensin, pancreatic cancer, cell migration, integrins, sortilin/NTR3

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 315-326, 2007

Authors: Guervós, Marta Alonso | Marcos, César Álvarez | Hermsen, Mario | Nuño, Andrés Sampedro | Suárez, Carlos | Llorente, José Luis

Article Type: Research Article

Abstract: Background: Lymph node metastasis is the mayor cause of mortality in patients with head and neck squamous cell carcinomas (45%). The genetic changes underlying metastasis are still largely unknown and genetic markers to predict lymph node positivity still need to be found. The aim of this study was to search such markers by using Multiplex Ligation-dependent Probe Amplification (MLPA), a semi-quantitative PCR technique to detect gene copy number alterations. Methods: Thirty-seven genes were analysed by MLPA in 34 larynx and 22 pharynx carcinomas. Results: Losses of CDKN2A (9p21) and MLH1 (3p22) and gains of CCND1, EMS1 (both at 11q13), RECQL4 …and PTP4A3 (both at 8q24) were the most frequent aberrations in both larynx and pharynx carcinomas. Amplifications were detected at EMS1, CCND1 and ERBB2 (17q21). A correlation between loss of N33 (8p22) and poor survival was found (p=0.02). Gain of EMS1 had the same relation with survival but not significant (p=0.08). Lymph node positive tumors presented a specific pattern of genetic alterations, with losses of N33, STK11 (19p13) and TP53 (17p13), the latter especially in larynx tumors. Conclusion: We propose that these 3 genes might play a role in the development of metastasis in larynx and pharynx squamous cell carcinomas. Show more

Keywords: Larynx and pharynx carcinoma, MLPA, lymph node metastasis, genetic alterations

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 327-334, 2007

Authors: Xiao, Qiang | Li, Lei | Xie, Yubo | Tan, Ning | Wang, Changqing | Xu, Junyi | Xia, Keyi | Gardner, Kevin | Li, Qingdi Q.

Article Type: Research Article

Abstract: The E2F family members play a critical role in cell cycle regulation and other biological processes in the cell. To better understand the involvement of E2F-1 in the development and progression of gastric tumors, we investigated the mutation and expression of E2F-1 in human gastric cancer tissues and the effect of E2F-1 overexpression on the proliferation of gastric carcinoma cells. In this study, 80 pairs of gastric cancer specimens and paratumor tissues from different patients and 40 stomach mucosa specimens from healthy individuals were examined. PCR-SSCP analysis demonstrated that mutations were not detected in any of the gastric cancer and …normal tissue specimens. In addition, the results of an immunohistochemistry assay revealed higher expression rates of E2F-1 (P<0.01) in gastric cancer tissues (72.5%) than in paratumor tissues (30.0%) of the same individuals and stomach mucosa from healthy individuals (22.5%). However, no correlation was observed between the E2F-1 levels and patients' clinical features, such as sex, age, histological types, lymph node metastasis, and clinical stages (P>0.05). Finally, the influence of E2F-1 overexpression on the growth of human gastric carcinoma MKN-45 cells in vitro was assessed by measuring colony formation, cell survival, and cell cycle progression. Our data clearly showed that cell growth and proliferation were significantly inhibited in MKN-45 tumor cells transfected with the expression vector encoding E2F-1 in comparison with nontransfected cells or cells transfected with empty vector. These findings suggest that E2F-1, a stable and conservative gene during the oncogenesis and progression of stomach cancers, may potentially serve as a biomarker for clinical diagnosis of gastric carcinomas and as a target for the development of novel therapeutic interventions to treat this disease. Show more

Keywords: E2F-1, mutation, gastric cancer, human tissues, MKN-45 cells, cell cycle, cell proliferation, cell growth, cell survival

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 335-349, 2007

Authors: Buffart, Tineke E. | Tijssen, Marianne | Krugers, Thijs | Carvalho, Beatriz | Smeets, Serge J. | Brakenhoff, Ruud H. | Grabsch, Heike | Meijer, Gerrit A. | Sadowski, Henry B. | Ylstra, Bauke

Article Type: Research Article

Abstract: Background: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. Material and Methods: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal …cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by β-globin PCR. Results: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non-amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. Conclusion: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples. Show more

Keywords: Array CGH, FFPE, DNA quality, isothermal amplification

Citation: Analytical Cellular Pathology, vol. 29, no. 4, pp. 351-359, 2007