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Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei

carpinteyrocyp

9/4/2013 11:56:00 PM


Fluorescent In Situ Hybridization on Isolated Tumor Cell NucleiAccepted 1 April 2003.Top of pageAbstractIn oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffinembedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed <a href="http://www.dtdrivingschool.co.uk/... ">tiffany and co outlet</a> 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse lowgrade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P = .001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. In contrast to other types of gliomas, oligodendroglioma is associated with generally good response to adjuvant therapy (1, 2, 3). Various clinical, radiological and histological features have been suggested as prognostic factors. However, none of these parameters predicts reliably response to adjuvant therapy (2, 4, 5, 6, 7, 8, 9, 10, 11).Recently, DNA losses on chromosomes 1p and 19q have been disclosed as common genetic alterations in oligodendroglial neoplasms and much less frequently in astrocytic neoplasms (12, 13, 14, 15, 16, 17, 18). Patients with anaplastic oligodendrogliomas harboring a 1p/19q deletion were shown to have a more favorable prognosis and significantly better response to chemotherapy as compared with patients with intact 1p/19q status (1, 17, 19, 20, 21, 22, 23). In patients with diffuse lowgrade gliomas, the 1p/19q status predicts prognosis (24) and the predictive value of 1p/19q status seems more reliable than histology (25).Therefore, it has been repeatedly suggested to amend conventional histological evaluation of anaplastic oligodendroglioma and diffuse lowgrade gliomas with molecular analysis of chromosome arms 1p and 19q (1, 3, 9, 19, 20, 21, 22, 24, 25, 26). As a consequence, 1p/19q testing is increasingly proposed for use in the diagnostic setting. Various methods are used for 1p/19q deletion analysis, in particular PCRbased investigations (microsatellite analysis, realtime PCR), comparative genomic hybridization, and fluorescent in [url=http://www.camfalconara.it/woolr...]woolrich donna[/url] situ hybridization (FISH) techniques (12, 14, 24, 27, 28, 29, 30). Consensus criteria for assessment and interpretation of analytical results in brain tumors are, however, lacking. In contrast, in peripheral neuroblastic tumors consensus criteria concerning methodology, evaluation of analytical results and data interpretation of diagnostic 1p testing are well established (31, 32).FISH is a classic technique for molecular cytogenetic testing on paraffinembedded tissues (33). FISH is not limited by age of material and does not require normal autologous or pooled normal reference tissue. Binding of probes can be controlled microscopically. However, one major limitation is nuclear truncation in tissue sections complicating evaluation and interpretation of hybridization results. To circumvent the problem of signal truncation, an improved FISH technique, that is, FISH on isolated whole tumor cell nuclei, has been developed for chromosome 1p analysis in germ cell [url=http://www.dtdrivingschool.co.uk/giusep...]giuseppe zanotti uk sale[/url] tumors. Sidebyside comparison of both methods has proven that FISH on isolated tumor cell nuclei is superior as compared with conventional FISH on tissue sections (32). FISH on isolated tumor cell nuclei has meanwhile replaced conventional FISH on tissue sections not only in germ cell tumors but also in other tumor types, most notably peripheral neuroblastic tumors (32, 34). Studies on oligodendroglial neoplasms have continued to use conventional FISH on tissue sections. Therefore, we investigated applicability of FISH on isolated tumor cells on a large series of oligodendroglial neoplasms and assessed FISH results according to consensus criteria for 1p analysis in peripheral neuroblastic tumors, as defined by the International Society of Pediatric Oncology Europe (ESIOP) Neuroblastoma Group (31).Top of pageMATERIALS AND METHODSA total of 89 paraffinembedded archival tumor specimens diagnosed as low and highgrade oligodendrogliomas and oligoastrocytomas were collected from the five major Austrian neuropathology/neurooncology units (Vienna, Graz, Innsbruck, Salzburg, and Linz). Age of the material ranged between 2 and 23 years. Control tissues comprised four cases of gliotic brain tissues (neurosurgically resected specimens of temporal lobe/Ammon's horn sclerosis) and 4 cases of diffuse lowgrade astrocytoma. Histological slides, 4 m in thickness, were deparaffinized in xylol. Hematoxylin and eosin (HE) staining and immunohistochemistry for glial fibrillary acidic protein (GFAP, polyclonal antibody Z0334; DAKO, Glostrup, Denmark) and epithelial membrane antigen (EMA, monoclonal antibody E 29, DAKO) were performed according to standard protocols (35). Immunolabeling was visualized with the ChemMate kit (DAKO) and diaminobenzidine as chromogene. The tumor specimens were reclassified according to WHO 2000 criteria (36). Accordingly, we categorized those tumors as oligodendrogliomas which displayed uniform oligodendroglial morphology, and we categorized those tumors as oligoastrocytomas that contained distinct components with oligodendroglial and astrocytic differentiation (Fig. 1). We used the antiGFAP immunostaining pattern as support for the distinction of oligodendroglioma versus oligoastrocytoma. We considered a ringlike perinuclear "gliofibrillary" immunostaining pattern as a characteristic oligodendroglial feature, and we considered immunolabeling of a fibrillary tumor matrix as a characteristic feature of an astrocytic component. We <a href="http://www.libreria-apogeo.it/monc..."&... moncler</a> used antiEMA immunostaining for initial screening of the cases to exclude immunoreactive cell surface structures, a feature that would indicate clear cell ependymal neoplasms. (A: HE, 100 ; inset a: HE; magnification, 170 ). Oligodendrogliomas are characterized by a uniform oligodendroglial, that is, clear cell morphology, whereas (B: HE, 100 ) oligoastrocytomas are composed of distinct astrocytic (inset a: HE, 170 ) and oligodendroglial (inset b: HE, 170 ) components.Full figure and legend (55K)For FISH analysis, we marked representative tumor areas on HE stained sections. Corresponding areas were identified on the paraffin blocks, and core tissue biopsies of 1.5 mm in diameter were taken from each block using a Yamshidi needle. The same procedure was used for sampling of control tissues. We prepared isolated cell nuclei of tumor and control samples as described previously (32). In detail, the paraffinembedded tissue samples and 1mmdiameter glass pellets were put into small bags of nylon gauze. The material was then deparaffinized with xylene for 1.5 hours, followed by an ethanol row with decreasing concentrations (100%, 90%, 70%, and 35% for 1 h, respectively). After transfer into distilled water, the deparaffinized tissue specimens were incubated with 0.1% protease (Sigma P8038, St. Louis, MO) in 0.1 N TrisHCl at 37 C for 3 minutes and was agitated occasionally. Afterwards, the digestion was stopped on ice. For checking of digestion, 10 L of 4'6diamidino2phenylindole2HCl (DAPI; 0.002 L/mL PBS) and 10 L of cell suspension were put on a slide, covered by coverslip and examined under the fluorescence microscope. Cytospins on glass slides were prepared by centrifugation of 50 L cell suspensions (8 min, 500 rpm) with the help of a SHANDONCytospin 2 cytocentrifuge (Pittsburgh, PA). The cytospin preparations were airdried for 2 hours and fixed with 4% buffered formaldehyde for 10 minutes. The samples were immediately processed for FISH or stored before FISH in sealed boxes at 20 C or 80 C. 1p analysis was performed with the paracentromeric probe D1Z1 (1q12) (37) and subtelomeric probe D1Z2 (1p36.3; both QBIOgene, Heidelberg, Germany) in a doublecolor FISH <a href="http://www.camfalconara.it/woolr...">woolrich donna</a> approach as described previously. For 19q analysis, probes 19pter and 19q13.3 were used (both Vysis, BergishGladbach, Germany). For visualization, the probes were either directly fluorescence labeled or biotin/digoxigeninlabeled probes were detected with fluorescencelabeled antibodies.Assessment and interpretation of FISH results were performed according to guidelines defined by the International Society of Pediatric Oncology (ESIOP Neuroblastoma Study Group) for studies of peripheral neuroblastic tumors (31). In each case, hybridization signals of 200 tumor cell nuclei were assessed. The ratios between the numbers of paracentromeric and subtelomeric regions of chromosomes 1p and 19q were calculated.Deletion: ratio of paracentromeric and subtelomeric signals = 2/1, partially in conjunction with 4/2, 3/1, 4/1 ratios. This hybridization pattern indicates the presence of an LOH.Imbalance: disproportion of the ratio of paracentromeric and subtelomeric signals, number of subtelomeric signals >1 (=3/2, 4/3, 4/2, 5/3, etc.). Such a result can indicate but does not prove an LOH. Clarification of this specific question <a href="http://www.dtdrivingschool.co.uk/giusep...">giuseppe zanotti uk sale</a> requires complementary techniques, such as PCR or Southern blot analysis.No deletion, no imbalance result: equal ratio of paracentromeric and subtelomeric signals = 2/2, 4/4.Unclear result: result is not interpretable because of material (too little tissue, calcifications, etc.) or technical reasons (enzymatic overdigestion).Statistical testing was performed using 2 test and statistic as appropriate. Twotailed P values .05 were considered as statistically significant.Top of pageRESULTSOn histological review, we classified 49 specimens as pure oligodendrogliomas and 30 specimens as oligoastrocytomas. Ten tumors were excluded from further analysis because histology revealed features of clear cell ependymoma in 3 cases, subependymoma in one case, astrocytoma in two cases, primitive neuroectodermal tumor (PNET) in one case, glioblastoma with focal oligodendroglial features in one case, and unclassifiable clear cell gliomas in two cases. The cohort of oligodendroglial neoplasms included 59 primary and 20 recurrent tumors. Fiftyseven specimens fulfilled criteria of Grade II, and 22 cases, Grade III, according to current WHO grading criteria.We performed 1p analysis in 79/79 tumor cases and 19q analysis in 76/79 tumor cases. In addition, we performed 1p/19q analysis in 8/8 controls. FISH showed intact 1p and 19q status (2:2 ratio) in 4/4 gliotic brain tissue samples and in 4/4 diffuse astrocytomas, used <a href="http://www.libreria-apogeo.it/p..."... borse 2013</a> as controls. Results in the cohort of oligodendroglial neoplasms are summarized in Table 1. FISH yielded interpretable results in 98.7% (78/79) for 1p and in 92.1% (70/76) for 19q. In the remaining cases, results were unclear, because of material (too little tissue, too low tumor cell content in tissue, calcification of the tissue) or methodology (enzymatic overdigestion). In the cohorts with interpretable results (discounting the cases with unclear results), 79.5% (62/78) had 1p deletion and 11.5% (9/78) had 1p imbalance, 80.0% (56/70) had 19q deletion, and 12.9% (9/70) had 19q [url=http://www.libreria-apogeo.it/p...]prada borse 2013[/url] imbalance. In the total group (including cases with [url=http://www.libreria-apogeo.it/monc...]spaccio moncler[/url] unclear results), 56/76 (73,7%) had 19q deletion, and 9/76 (11,8%) had 19q imbalance. Aberrations in 1p were always present in oligodendrogliomas but not in all oligoastrocytomas (100% versus 75.9%, exact 2 test: P = .001). Figure 2 shows representative FISH results. Representative hybridization results are shown in twocell nuclei (DAPI stain), respectively. A, normal result in a nonneoplastic control case with an equal 2:2 ratio of paracentromeric (green) and subtelomeric (red) signals. B, deletion status in an oligodendroglial neoplasm with a 2:1 ratio of paracentromeric (green) and subtelomeric (red) signals. C, imbalance status in an oligodendroglial neoplasm with 4:2 and 5:3 ratios of paracentromeric (green) and subtelomeric (red) [url=http://www.dtdrivingschool.co.uk/... ]tiffany and co outlet[/url] signals. (A original magnification, 3,000 )Full figure and legend (12K)There was a tight positive association of 1p and 19q alterations (Table 2): 96.9% (63/65) of the cases with 1p alteration had in addition 19q aberration. Only 5.7% (4/70) had either isolated 1p or 19q aberration. Statistical testing illustrated the high degree of association (kappa: 0.569; P



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