Analytical Cellular Pathology - Volume 26, issue 5-6

Authors: Meijer, Gerrit A.

Article Type: Research Article

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 271-273, 2004

Authors: Grade, Marian | Becker, Heinz | Ghadimi, B. Michael

Article Type: Other

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 275-278, 2004

Authors: Armstrong, Nicola J. | van de Wiel, Mark A.

Article Type: Research Article

Abstract: We review several commonly used methods for the design and analysis of microarray data. To begin with, some experimental design issues are addressed. Several approaches for pre‐processing the data (filtering and normalization) before the statistical analysis stage are then discussed. A common first step in this type of analysis is gene selection based on statistical testing. Two approaches, permutation and model‐based methods are explained and we emphasize the need to correct for multiple testing. Moreover, powerful approaches based on gene sets are mentioned. Clustering of either genes or samples is frequently performed when analyzing microarray data. We summarize the basics …of both supervised and unsupervised clustering (classification). The latter may be of use for creating diagnostic arrays, for example. Construction of biological networks, such as pathways, is a statistically challenging but complex task that is a relatively new development and hence mentioned only briefly. We finish with some remarks on literature and software. The emphasis in this paper is on the philosophy behind several statistical issues and on a critical interpretation of microarray related analysis methods. Show more

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 279-290, 2004

Authors: Derks, Sarah | Lentjes, Marjolein H.F.M. | Hellebrekers, Debby M.E.I. | de Bruïne, Adriaan P. | Herman, James G. | van Engeland, Manon

Article Type: Research Article

Abstract: Methylation‐specific PCR (MSP) is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1) sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2) detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical) applications.

Keywords: DNA methylation, epigenetics, methylation‐specific PCR, tumor suppressor gene silencing, tumor marker

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 291-299, 2004

Authors: Giaretti, Walter | Molinu, Silvia | Ceccarelli, Jenny | Prevosto, Claudia

Article Type: Research Article

Abstract: Whether in vivo specific gene mutations lead to chromosomal instability (CIN) and aneuploidy or viceversa is so far not proven. We hypothesized that aneuploidy among human sporadic colorectal adenomas and KRAS2 and APC mutations were not independent. Additionally, we investigated if 1p34–36 deletions by dual target FISH were associated with aneuploidy. Among 116 adenomas, 29 were DNA aneuploid by flow cytometry (25%) and 29 were KRAS2 mutated (25%). KRAS2 mutations were associated with aneuploidy (P=0.02). However, while G–C and G–T transversions were strongly associated with DNA aneuploidy (P=0.007), G–A transitions were not. Within a second series of 61 adenomas, we …found, instead, that APC mutational status and aneuploidy by flow cytometry were not associated. However, a statistically significant association was found with specific APC mutations, i.e., occurring in the mutation cluster region (MCR, codons 1200–1500) or downstream (P=0.016). Finally, the correlation of 1p34–36 deletions with flow cytometric and FISH detected aneuploidy was also significant (P=0.01). Specific KRAS2 and APC mutations and loss of genes in the 1p34–36 region appear associated with aneuploidy suggesting that these events are not independent and may cooperate in inducing human sporadic colorectal adenomas. A cause effect relationship between gene mutations and aneuploidy remains, however, to be demonstrated. Show more

Keywords: Aneuploidy, chromosomal instability, oncogenes, tumor suppressor genes

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 301-305, 2004

Authors: Weiss, Marjan M. | Kuipers, Ernst J. | Postma, Cindy | Snijders, Antoine M. | Pinkel, Daniel | Meuwissen, Stefan G.M. | Albertson, Donna | Meijer, Gerrit A.

Article Type: Research Article

Abstract: Background & aims: Pathogenesis of gastric cancer is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. In order to investigate the patterns of chromosomal aberrations in gastric carcinomas, we performed genome‐wide microarray based comparative genomic hybridisation (microarray CGH). With this recently developed technique chromosomal aberrations can be studied with high resolution and sensitivity. Methods: Array CGH was applied to a series of 35 gastric adenocarcinomas using a genome‐wide scanning array with 2275 BAC and P1 clones spotted in triplicate. Each clone contains at least one STS for linkage to the …sequence of the human genome. These arrays provide an average resolution of 1.4 Mb across the genome. DNA copy number changes were correlated with clinicopathological tumour characteristics as well as survival. Results: All thirty‐five cancers showed chromosomal aberrations and 16 of the 35 tumours showed one or more amplifications. The most frequent aberrations are gains of 8q24.2, 8q24.1, 20q13.12, 20q13.2, 7p11.2, 1q32.3, 8p23.1–p23.3, losses of 5q14.1, 18q22.1, 19p13.12–p13.3, 9p21.3–p24.3, 17p13.1–p13.3, 13q31.1, 16q22.1, 21q21.3, and amplifications of 7q21–q22, and 12q14.1–q21.1. These aberrations were correlated to clinicopathological characteristics and survival. Gain of 1q32.3 was significantly correlated with lymph node status (p=0.007). Tumours with loss of 18q22.1, as well as tumours with amplifications were associated with poor survival (p=0.02, both). Conclusions: Microarray CGH has revealed several chromosomal regions that have not been described before in gastric cancer at this frequency and resolution, such as amplification of at 7q21–q22 and 12q14.1–q21.1, as well gains at 1q32.3, 7p11.2, and losses at 13q13.1. Interestingly, gain of 1q32.3 and loss of 18q22.1 are associated with a bad prognosis indicating that these regions could harbour gene(s) that may determine aggressive tumour behaviour and poor clinical outcome. Show more

Keywords: Microarray comparative genomic hybridisation, gastric cancer, survival, lymph node, 1q gain, 18q loss, amplification, 7q21–22, 12q

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 307-317, 2004

Authors: Chamgoulov, Ravil | Lane, Pierre | MacAulay, Calum

Article Type: Research Article

Abstract: A novel optical computed‐tomographic microscope has been developed allowing quantitative three‐dimensional (3D) imaging and analysis of fixed pathological material. Rather than a conventional two‐dimensional (2D) image, the instrument produces a 3D representation of fixed absorption‐stained material, from which quantitative histopathological features can be measured more accurately. The accurate quantification of these features is critically important in disease diagnosis and the clinical classification of cancer. The system consists of two high NA objective lenses, a light source, a digital spatial light modulator (DMD, by Texas Instrument), an x–y stage, and a CCD detector. The DMD, positioned at the back pupil‐plane of …the illumination objective, is employed to illuminate the specimen with parallel rays at any desired angle. The system uses a modification of the convolution backprojection algorithm for reconstruction. In contrast to fluorescent images acquired by a confocal microscope, this instrument produces 3D images of absorption stained material. Microscopic 3D volume reconstructions of absorption‐stained cells have been demonstrated. Reconstructed 3D images of individual cells and tissue can be cut virtually with the distance between the axial slices less than 0.5 μm. Show more

Keywords: 3D imaging, computed‐tomography, histology, cancer diagnosis, optical microscopy, absorption‐stained

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 319-327, 2004

Authors: Korinth, Dirk | Donhuijsen, Konrad | Bockmühl, Ulrike | Petersen, Iver

Article Type: Research Article

Abstract: Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin‐fixed, paraffin‐embedded (FFPE) tissue. A well‐known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non‐enzymatic, PHOTOPROBE® biotin labeling for conjugation of the hapten to the DNA prior to in situ …hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE® samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE® biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material. Show more

Keywords: CGH, non‐enzymatic labeling, FFPE tissue

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 329-334, 2004

Authors: Hruska, Anett | Bollmann, Reinhard | Kovács, Rita Beáta | Bollmann, Magdolna | Bodó, Miklós | Sápi, Zoltán

Article Type: Research Article

Abstract: Background and methods: 44 peripheral nerve sheath tumors (PNST) (27 schwannomas, 9 neurofibromas and 8 malignant peripheral nerve sheath tumors (MPNST)) were analyzed to determine DNA ploidy pattern and to clarify the conflicting data in the literature concerning this topic (whether benign PNSTs are aneuploid or not). For further insight we analyzed 6 schwannomas, one atypical neurofibroma and five MPNSTs by fluorescence in situ hybridization (FISH) technique using centromeric chromosome probes (7, 17 and 18) and automatic image analysis station, Metafer 4. Results: Benign schwannomas (including the problematic variants as ancient, cellular, neuroblastoma like and multiplex schwannomas) could be …characterized by euploid‐polyploidisation and by their 4c peak height value which was usually more than 10% of total cell number measured. These characters were not found among neurofibromas and MPNST‐s. FISH analysis revealed and confirmed that the ‘normal’ euploid–polyploid cells are mainly eusomic–polysomic containing two, four, eight or sixteen signals for each chromosomes examined, but in a small proportion aneusomy was found among tumor cells of benign schwannomas (average: 2.58; range 1.33–3.44). In contrast, the atypical neurofibroma displayed marked aneusomy (18.44%) but it contained normal eusomic and polysomic cells too. Two diploid MPNSTs proved to be clearly aneusomic with trisomy of chromosome 17 and monosomy of chromosome 18. Conclusions: All these data suggest that ploidy pattern determination combined with FISH analysis may be a very useful supplementary tool for making a right diagnosis (to differentiate benign versus malignant schwannomas in problematic variants) and to understand better the malignant transformation in PNSTs. Show more

Keywords: Schwannoma, PNST, FISH, DNA cytometry, aneusomy

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 335-345, 2004

Authors: Vleugel, Marije M. | Bos, Reinhard | Buerger, Horst | van der Groep, Petra | Saramäki, Outi R. | Visakorpi, Tapio | van der Wall, Elsken | van Diest, Paul J.

Article Type: Research Article

Abstract: Objective: Hypoxia Inducible Factor‐1 (HIF‐1) is an important transcription factor that stimulates tumour growth and metastases via several pathways, including angiogenesis and altered metabolism. Activation of HIF‐1 depends on the levels of its α‐subunit, which increase during hypoxia. Recent studies showed that the HIF‐1α gene was amplified in prostate cancer, leading to overexpression of HIF‐1α at normoxia. The aim of this study was to evaluate the presence of HIF‐1α gene amplifications in invasive breast cancer as an explanation for HIF‐1α protein overexpression. Methods: Protein and gene expression of HIF‐1α were analyzed on a tissue microarray of 94 breast cancers by …immunohistochemistry and fluorescent in situ hybridization (FISH), respectively. Results: Overexpression of HIF‐1α protein was found in 58/94 (62%) of patients. No amplifications of the HIF‐1α gene were detected. Conclusion: Increased protein levels of HIF‐1α are not associated with amplification of the HIF‐1α gene in human breast cancer. Therefore, other mechanisms than gene amplification must be responsible for HIF‐α overexpression at normoxia. Show more

Keywords: HIF‐1α, gene expression, amplification, immunohistochemistry, breast cancer, FISH

Citation: Analytical Cellular Pathology, vol. 26, no. 5-6, pp. 347-351, 2004