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Microdissected Areas (microdissected + area)
Selected AbstractsHeterogeneity in uveal melanoma assessed by multiplex ligation-dependent probe amplification (MLPA)ACTA OPHTHALMOLOGICA, Issue 2009J DOPIERALA Purpose To study intratumour heterogeneity in primary uveal melanoma (UM) by MLPA in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Methods DNA was extracted from 2-9 areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1p, 3, 6 and 8 were tested for copy number changes using the SALSA MLPA P027.B1 assay (MRC Holland). MLPA data were displayed as dosage quotients (DQs), which were classified to 5 ranges (0.35-0.64 deleted; 0.65-0.84 equivocal deletion; 0.85-1.14 normal; 1.15-1.35 equivocal amplification; >1.35 amplified). The tumour was considered heterogeneous at a locus, if a) the difference in DQs of any two areas was higher than 0.2 (value determined by ROC analysis), and b) the DQs of the areas belonged to different ranges. Results Genetic abnormalities were detected in all 32 UMs. Monosomy 3, the most significant metastasis predictor, and gain of 8q genes MYC or DDEF1 were detected in at least 1 microdissected area of 22 (69%) and 28 (87%) of the tumours, respectively. The comparison of MLPA data obtained from different areas of UMs showed heterogeneity in 1-24 loci across chromosomes 1p, 3, 6 and 8 in 26 (81%) tumours. Interestingly, trisomy 3 was observed in 3 (9%) UMs and these tumours showed the highest degree of heterogeneity (>23 heterogeneous loci). Intratumour heterogeneity of 3p12.2 (ROBO1) and 6p21.2 (CDKN1A) were most common and present in more than 35% of the tumours. Conclusion Heterogeneity of chromosomal abnormalities of 1p, 3, 6 and 8 is present in many UM. Taking one random tumour sample for prognostic testing, therefore, may not be representative of the whole tumour. [source] Multilineage progression of genetically unstable tumor subclones in cutaneous T-cell lymphomaEXPERIMENTAL DERMATOLOGY, Issue 8 2004Albert Rübben Abstract:, Molecular analysis of solid malignant tumors has suggested multilineage progression of genetically unstable subclones during early stages of tumorigenesis as a common mechanism of tumor cell evolution. We have investigated whether multilineage progression is a feature of cutaneous T-cell lymphoma (CTCL). To identify individual tumor cell subclones, we determined the pattern of mutations within microsatellite DNA obtained from multiple histomorphologically confined tumor cell nests of mycosis fungoides (MF) and lymphomatoid papulosis (LyP) lesions. Tumor cells were isolated by laser microdissection, and allelotypes were determined at microsatellite markers D6S260, D9S162, D9S171, D10S215, TP53.PCR15, and D18S65. Nine cases of MF and one patient with anaplastic large cell lymphoma (ALCL) originating from LyP were analyzed at 277 different microdissected areas obtained from 31 individual lesions. Three specimens of cutaneous lichen planus microdissected at 26 areas served as the control tissue. Microsatellite instability in microdissected tissue [MSI(md-tissue)] was detected in tumor tissues of all CTCL patients. One hundred and fifty-seven of 469 analyzed polymerase chain reaction (PCR) amplifications contained mutated microsatellite alleles (34%). In lichen planus, MSI(md-tissue) was seen in only four of 76 PCR products (5%) (P < 0.0001). The distribution of allelotypes in tumor cells from different disease stages was consistent with multilineage progression in five MF cases, as well as in the LyP/ALCL patient. Our results suggest that CTCL may evolve by multilineage progression and that tumor subclones in MF can be detected in early disease stages by mutation analysis of microsatellite DNA obtained from multiple microdissected areas. [source] Human brain aminopeptidase A: biochemical properties and distribution in brain nucleiJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Nadia De Mota Abstract Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca2+. Kinetic studies showed that the Km (190 ,mol/L) of the human enzyme for the synthetic substrate- l -glutamyl-,-naphthylamide was close from that of the mouse enzyme (256 ,mol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension. [source] Transcriptional upregulation and unmethylation of the promoter region of p16 in invasive basal cell carcinoma cells and partial co-localization with the ,2 chain of laminin-332,THE JOURNAL OF PATHOLOGY, Issue 1 2007S Svensson Månsson Abstract Basal cell carcinoma cells show low proliferation rates at the invasive front and a concordant upregulation of the cdk-inhibitor p16, limiting proliferative capacity. Little is known about the mechanisms of p16 regulation in normal and malignant cells apart from that many transcription factors such as Ets1, Ets2, SP1, SP3, JunB and the polycomb protein Bmi1 have the potential to induce or repress p16 expression. Therefore, the aim of this study was to determine how p16 is regulated in basal cell carcinoma with special focus on its upregulation in invasive cells. By analysing various microdissected areas of basal cell carcinoma using real-time quantitative PCR we observed upregulation of p16 mRNA in invasive tumour cells compared to centrally localized tumour cells. The methylation status of the p16 promoter, analysed by methylation-specific PCR, also showed diminished methylation in tumour cells at the invasive front, supporting the hypothesis that promoter methylation can affect the transcriptional activation of p16 in vivo. There was only sporadic co-localization of Ets, or ERK1/2 phosphorylation with p16 upregulation at the invasive front, suggesting that these factors were not directly involved in the regulation of p16. Furthermore, the ,2 chain of laminin-332 has been reported to be increased at the invasive front compared to the central areas of many tumours. Interestingly, in basal cell carcinoma we observed partial co-localization between p16 and the ,2 chain of laminin-332 in tumour cells towards areas of ulceration and in the majority of clearly infiltrative tumour cells but not in p16 positive tumour cells with a more pushing invasive growth pattern. These data suggest that concurrent p16 upregulation and decreased proliferation are more general phenomena in different types of invasive growth patterns in basal cell carcinomas and that these only partially overlap with the ,2 chain of laminin-332 associated invasion patterns. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] | ||||||||||