Analytical Cellular Pathology - Volume 33, issue 3-4

Authors: Tommasi, Stefania | Mangia, Anita | Iannelli, Giuseppina | Chiarappa, Patrizia | Rossi, Elena | Ottini, Laura | Mottolese, Marcella | Zoli, Wainer | Zuffardi, Orsetta | Paradiso, Angelo

Article Type: Research Article

Abstract: Background: Male breast cancer (MBC) is a rare disease and little is known about its etiopathogenesis. Array comparative genomic hybridization (aCGH) provides a method to quantitatively measure the changes of DNA copy number and to map them directly onto the complete linear genome sequences. The aim of this study was to investigate DNA imbalances by aCGH and compare them with a female breast cancer dataset. Methods: We used Agilent Human Genome CGH Microarray Kit 44B and 44K to compare genomic alterations in 25 male breast cancer tissues studied at NCC of Bari and 16 female breast cancer deposited with …the Gene Expression Omnibus (GSE12659). Data analysis was performed with Nexus Copy Number 5.0 software. Results: All the 25 male and 16 female breast cancer samples displayed some chromosomal instability (110.93 alterations per patient in female, 69 in male). However, male samples presented a lower frequency of genetic alterations both in terms of loss and gains. Conclusion: aCGH is an effective tool for analysis of cytogenetic aberrations in MBC, which involves different biological processes than female. Male most significant altered regions contained genes involved in cell communication, cell division and immunological response, while female cell–cell junction maintenance, regulation of transcription and neuron development. Show more

Keywords: aCGH, male breast cancer, familiarity

DOI: 10.3233/ACP-CLO-2010-0544

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 113-119, 2010

Authors: Nyhan, Michelle J. | El Mashad, Shereen M. | O'Donovan, Tracey R. | Ahmad, Sarfraz | Collins, Chris | Sweeney, Paul | Rogers, Eamonn | O'Sullivan, Gerald C. | McKenna, Sharon L.

Article Type: Research Article

Abstract: Background: von Hippel–Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-α (HIF-α), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. Methods: The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by real-time RT-PCR. …Expression of pVHL's protein targets were determined by Western blotting in 17 paired patient samples. Results: VHL genetic alterations were identified in 43.5% (10/23) of CCRCCs. HIF-1α, HIF-2α and CAIX were up-regulated in 88.2% (15/17), 100% (17/17) and 88.2% (15/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. Conclusion: As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), these data suggests that other pVHL targets may be more crucial in renal carcinogenesis. Show more

Keywords: Clear cell renal cell carcinoma (CCRCC), Hypoxia Inducible Factor (HIF), mutation, putative targets, von Hippel–Lindau (VHL)

DOI: 10.3233/ACP-CLO-2010-0541

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 121-132, 2010

Authors: Suijkerbuijk, Karijn P.M. | Pan, Xiaojuan | van der Wall, Elsken | van Diest, Paul J. | Vooijs, Marc

Article Type: Research Article

Abstract: Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance. Methods: We used Methylation-Specific PCR (MSP), Quantitative Multiplex MSP (QM-MSP) and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) to compare methylation of CCND2, SCGB3A1, RARB and RASSF1 on DNA from 40 breast carcinomas. Results: A comparison between MSP and QM-MSP on the same samples showed …a high discrepancy: 20% of tumors that showed no methylation in MSP gave >10% methylation in QM-MSP. In contrast, QM-MSP correlated strongly with MS-MLPA when targeting the same sequence in DNA from paraffin embedded as well as fresh frozen tissue. This correlation declined when target sequences were non-overlapping. In titration experiments, MSP and MS-MLPA performed robust with 10 ng of DNA, while QM-MSP was at least ten-fold more sensitive. Conclusion: Despite the difference in molecular basis, QM-MSP and MS-MLPA showed moderate to strong correlations. In contrast, there was a poor concordance between either of these techniques and non-quantitative MSP. For biological samples with scarce DNA, QM-MSP is the method of choice. Show more

Keywords: Breast cancer, DNA methylation, methodology, MS-MLPA, MSP, QM-MSP

DOI: 10.3233/ACP-CLO-2010-0542

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 133-141, 2010

Authors: Geddert, Helene | zur Hausen, Axel | Gabbert, Helmut E. | Sarbia, Mario

Article Type: Research Article

Abstract: Background: Epstein–Barr virus (EBV)-associated gastric carcinomas (GC) constitute a distinct clinicopathological entity of gastric cancer. In order to determine underlying distinct aberrant promoter methylation we tested cardiac and non-cardiac GC with regard to the presence of EBV. Methods: One hundred GC were tested by RNA-in situ hybridization for the presence of EBV by EBV-encoded small RNA (EBER). Aberrant promoter methylation was investigated by methylation-specific real-time PCR for p16, p14, APC and hMLH1. P16 protein expression was assessed by immunohistochemistry. Results: In our selected study cohort, EBER-transcripts were detected in 19.6% (18/92) of GC. EBV-positive GC revealed significantly more …often gene hypermethylation of p16, p14 and APC (p<0.0001, p<0.0001 and p=0.02, respectively) than EBV-negative GC. The majority of GC with p16 hypermethylation showed a p16 protein loss (22/28). In contrast, no correlation between the presence of EBV and hMLH1 hypermethylation was found (p=0.7). EBV-positive GC showed a trend towards non-cardiac location (p=0.06) and lower stages (I/II) according to the WHO (p=0.05). Conclusions: Hypermethylation of tumor suppressor genes is significantly more frequent in EBV-associated GC compared to EBV-negative GC. Our data add new insights to the role of EBV in gastric carcinogenesis and underline that EBV-associated GC comprise a distinct molecular-pathologic as well as a distinct clinicopathological entity of GC. Show more

Keywords: Gastric, cancer, cardia, EBV, methylation

DOI: 10.3233/ACP-CLO-2010-0540

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 143-149, 2010

Authors: Grimm, M. | Lazariotou, M. | Kircher, S. | Höfelmayr, A. | Germer, C.T. | von Rahden, B.H.A. | Waaga-Gasser, A.M. | Gasser, M.

Article Type: Research Article

Abstract: Introduction: The progressive growth of malignancies is accompanied by a decline in the immune response through mechanisms which are poorly understood. Apoptosis and induction of inflammation by tumor released cytokines as tumor escape mechanisms have been proposed to play an important role in colorectal carcinogenesis. Methods: Expression of Tumor necrosis factor-alpha (TNF-α) was analyzed in colorectal cancer specimen and the cancer cell line HT-29 by immunohistochemistry and RT-PCR. TNF-α expression on protein and mRNA level were correlated with clinical characteristics and impact on survival. TNFR-1 was co-labelled with TNF-α and CD8+ cytotoxic T cells in immunofluorescence double staining experiments. …Results: 94% (n=98/104) of the patients with CRC expressed TNF-α. High TNF-α expression was significantly associated with positive lymph node stage and recurrence of the tumor. Multivariate analysis revealed high TNF-α expression as an independent prognostic factor. Immunohistochemistry was correlated with RT-PCR results (τ=0.794). Immunofluorescence double staining experiments revealed increased TNFR-1 expression by CD8+ cells. Conclusions: TNF-α expression by tumor cells may be an efficient immunological escape mechanism by inflammation-enhanced metastases and probably by induction of apoptosis in tumor-infiltrating CD8+ immune cells resulting in a down regulation of the tumoral immune response. Our data support the role of tumor-derived TNF-α expression as an important promoter of tumoral immune escape mechanisms and malignant progression, and suggest that analysis on either protein (immunohistochemistry) or RNA level (RT-PCR) can be used effectively in this respect. Targeting TNF-α may be a promising option, especially in cases with high TNF-α expression and positive lymph node metastases. Show more

Keywords: Tumor escape mechanism, death receptor signalling, apoptosis, inflammation, colorectal carcinoma

DOI: 10.3233/ACP-CLO-2010-0539

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 151-163, 2010

Authors: Moelans, Cathy B. | de Weger, Roel A. | Monsuur, Hanneke N. | Maes, Anoek H.J. | van Diest, Paul J.

Article Type: Research Article

Abstract: Ductal carcinoma in situ (DCIS) accounts for approximately 20% of mammographically detected breast cancers. Although DCIS is generally highly curable, some women with DCIS will develop life-threatening invasive breast cancer, but the determinants of progression to infiltrating ductal cancer (IDC) are largely unknown. In the current study, we used multiplex ligation-dependent probe amplification (MLPA), a multiplex PCR-based test, to compare copy numbers of 21 breast cancer related genes between laser-microdissected DCIS and adjacent IDC lesions in 39 patients. Genes included in this study were ESR1, EGFR, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, …TOP2A, MAPT, BIRC5, CCNE1 and AURKA. There were no significant differences in copy number for the 21 genes between DCIS and adjacent IDC. Low/intermediate-grade DCIS showed on average 6 gains/amplifications versus 8 in high-grade DCIS (p=0.158). Furthermore, alterations of AURKA and CCNE1 were exclusively found in high-grade DCIS, and HER2, PRDM14 and EMSY amplification was more frequent in high-grade DCIS than in low/intermediate-grade DCIS. In contrast, the average number of alterations in low/intermediate and high-grade IDC was similar, and although EGFR alterations were exclusively found in high-grade IDC compared to low/intermediate-grade IDC, there were generally fewer differences between low/intermediate-grade and high-grade IDC than between low/intermediate-grade and high-grade DCIS. In conclusion, there were no significant differences in copy number for 21 breast cancer related genes between DCIS and adjacent IDC, indicating that DCIS is genetically as advanced as its invasive counterpart. However, high-grade DCIS showed more copy number changes than low/intermediate-grade DCIS with specifically involved genes, supporting a model in which different histological grades of DCIS are associated with distinct genomic changes that progress to IDC in different routes. These high-grade DCIS specific genes may be potential targets for treatment and/or predict progression. Show more

Keywords: DCIS, IDC, MLPA, laser microdissection, breast cancer

DOI: 10.3233/ACP-CLO-2010-0546

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 165-173, 2010

Authors: van Diest, Paul J. | Suijkerbuijk, Karijn P.M. | Koop, Esther A. | de Weger, Roel A. | van der Wall, Elsken

Article Type: Other

DOI: 10.3233/ACP-CLO-2010-0543

Citation: Analytical Cellular Pathology, vol. 33, no. 3-4, pp. 175-176, 2010