Imbalances of chromosome 17 in medulloblastomas determined by comparative genomic hybridisation and fluorescence in situ hybridisation.

AIMS: To investigate the status of chromosome 17 in a series of medulloblastomas using comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). METHODS: Frozen tissue and formalin fixed, paraffin was embedded tissue from 27 medulloblastomas were analysed by CGH and FISH, respectively. CGH ratio profiles for chromosome 17 were compared with the results of FISH, for which loss or gain of 17p or 17q was assessed in two distinct ways using a combination of differentially labelled subtelomeric and centromeric probes and analysing 200 nuclei in each tumour. RESULTS: CGH revealed imbalances consistent with isochromosome 17q in eight of 27 tumours. Either loss of 17p or gain of 17q was identified in a further nine tumours, whereas 10 tumours were apparently balanced. Using control results from preparations of paraffin wax embedded tonsils, thresholds for the detection of abnormalities by FISH were established, either by determining the dominant pattern of signals in each case, or the mean ratio of subtelomeric to centromeric signals. Results by CGH and FISH were concordant in 21 of 27 tumours. In the remainder, most discrepancies related to methodological differences. CONCLUSIONS: CGH has a role in disclosing common, genome wide chromosomal gains or losses in tumours, the clinical relevance of which can then be studied in large archival series of paraffin wax embedded tumours using FISH.     

Near-haploidy and subsequent polyploidization characterize the progression of peripheral chondrosarcoma

Chondrosarcomas are malignant cartilaginous tumors arising centrally in bone (central chondrosarcoma), or secondarily within the cartilaginous cap of osteochondroma (peripheral chondrosarcoma). We previously used DNA flow cytometry to demonstrate that near-haploidy is relatively frequent in peripheral chondrosarcomas. We performed fluorescence in situ hybridization (FISH) to interphase nuclei using centromeric probes, a genome wide loss of heterozygosity (LOH) analysis, and comparative genomic hybridization on five peripheral chondrosarcomas. We demonstrated near-haploidy in two low-grade tumors with only one copy and LOH of most chromosomes. Few chromosomes are disomic, with retention of heterozygosity and overrepresentation at comparative genomic hybridization. One tumor contains both a near-haploid clone with chromosomes in monosomic and disomic state, and an exactly duplicated clone. Two high-grade tumors clearly demonstrate polyploidization because most chromosomes show LOH and two copies at FISH, whereas few chromosomes have four copies with retention of heterozygosity. Using DNA from a relative, we demonstrate that chromosome loss is random regardless of parental origin. Using FISH on paraffin slides, we exclude near-haploidy to result from meiosis-like division in binucleated cells, characteristic for chondrosarcoma. In conclusion, our results indicate that near-haploidy characterizes the progression from osteochondroma toward low-grade chondrosarcoma. Moreover, further progression toward high-grade chondrosarcoma is characterized by polyploidization.     

Unusual chromosome patterns of renal cell carcinomas common to two brothers

In this study, we describe two renal cell carcinomas (RCC) that occurred at the same time in two brothers, yielding information on the carcinogenic process. We used flow cytometry (FCM) to evaluate nuclear DNA content, and performed cytogenetic analysis. We also carried out fluorescence in situ hybridization (FISH) with a panel of centromeric probes for chromosomes 3, 7, 8, 9, 12, 17, 20, and Y in interphase cells. Flow cytometry analysis revealed diploid histograms in the tumor and "nonmalignant" samples of patient 1, while an aneuploid cell subpopulation was found in the tumor and "nonmalignant" samples of patient 2. Tumor samples from the two brothers were studied by FISH, and had common numerical chromosome aberrations: trisomy of chromosomes 3 and 7, and monosomy and trisomy of chromosomes 9 and 17. Moreover, in normal samples from both brothers, we found monosomy 9, and in a normal sample from patient 1, monosomy 17. Cytogenetic analysis revealed trisomy 3 in some cells grown from normal kidney tissue of each brother. The identification of the same chromosome alterations in both brothers appears to provide evidence of an unusual process of carcinogenesis, probably due to a common genetic basis.     

Satellite DNA hypomethylation in karyotyped Wilms tumors

Previously, a high percentage of Wilms tumors was found to be hypomethylated in the unusually long region of pericentromeric satellite DNA on chromosome 1. We now show that these pediatric cancers are also frequently hypomethylated in centromeric satellite DNA throughout the genome and compare satellite DNA hypomethylation with chromosome rearrangements. Relative to normal somatic tissues, 83% of the tumors were hypomethylated in centromeric satellite alpha DNA. This was assessed by blot hybridization under low-stringency conditions after digestion with CpG methylation-sensitive restriction endonucleases. Similar results were obtained with different enzymes, indicating generalized hypomethylation of centromeric DNA. Hypomethylation of another heterochromatic sequence, juxtacentromeric satellite 2 DNA of chromosome 1, was observed in 51% of the tumors. By cytogenetic analysis, rearrangements in the centromeric or juxtacentromeric heterochromatin of chromosome 1 were the most frequent structural aberration and were seen in 14% of the tumors. Tumors with such rearrangements had hypomethylation of satellite DNA in the pericentromeric region. These results show a high degree of targeting of DNA hypomethylation to centromeric and juxtacentromeric satellite DNA sequences in cancer and are consistent with satellite DNA hypomethylation contributing to, but not sufficing for, karyotypic instability in cancer and possibly playing other roles in carcinogenesis.     

DNA measurement by image analysis of paraffin-embedded breast carcinoma tissue. A comparative investigation.

Measurement of cellular DNA content may provide useful prognostic information in several human neoplasms. The DNA content by image analysis of fresh tissue has been compared with flow cytometry with good correlation, but the use of paraffin-embedded tissue for image analysis has not been studied widely. This study reports the DNA content of 54 breast carcinomas and compares the results of image analysis of paraffin-embedded tissue and results of image analysis and flow cytometry of corresponding fresh tumors. Image analysis of paraffin blocks and fresh tumors showed comparable results in 51 tumors (94%), whereas 47 tumors (87%) were similar by all three methods. The DNA indices from image analysis of fresh and paraffin-embedded tumors showed significant correlation by linear regression analysis (r = 0.96; P less than 0.001). Discordances between image analysis of fresh and paraffin-embedded tumors are the result of technical problems, such as staining and tissue preservation. Discordances between image analysis and flow cytometry reflect the advantages and pitfalls of the two techniques. These data suggest that image analysis of paraffin-embedded tissue is a viable technique that will permit the performance of retrospective studies with long-term follow-up data for the evaluation of the prognostic significance of DNA content.     

Flow-cytometric DNA analysis of intracranial tumors in children

The objective of this study was to investigate flow-cytometric DNA values of pediatric intracranial tumors, and to establish DNA analysis as a potential prognostic parameter. Twenty-nine brain tumor specimens from 26 pediatric patients were cryo-preserved within a 3-year period. The DNA content was measured by flow cytometry. Six of the tumor specimens had aneuploid DNA patterns. The median of the proliferation index was lower in the survivor group compared with the non-survivor group (36.4% and 47.5%, respectively). Ten of the 26 patients are still alive, eight were lost to follow up, and eight died. Flow-cytometric DNA analysis may be a helpful tool for examining brain tumors in children. The small size of this study could not establish flow cytometry as a definite prognostic factor, but further prospective multicenter studies will evaluate the prognostic significance of flow-cytometric DNA analysis.     

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe

The DNA content distributions of micronuclei induced in mouse3T3 cells by ionizing radiation and chemicals was measured byflow cytometry. For a quantitative understanding of these distributions,micronuclei with increasing DNA contents were sorted and analysedfor the presence of centromeric signals using fluorescent insitu hybridization (FISH) with a mouse centromeric gamma satelliteprobe. Radiation-induced micronuclei were found to be producedmainly by chromosome fragments, whereas micronuclei inducedby the tear gas chlorobenzylidene malonitrile (CS) were foundto be produced mainly by whole chromatids. In contrast, micronucleiinduced by vinblastine (VBL) were, according to the shape oftheir DNA content distributions, produced mainly by whole chromosomesand by combinations of two or more whole chromosomes. With increasingDNA content, micronuclei induced by ionizing radiation alsocontained one or more whole chromosomes, whereas micronucleiinduced by CS or VBL were found to contain several whole chromatidsor chromosomes respectively. Computerized random breakage ofchromosomes and random combination of chromosome fragments,whole chromatids and whole chromosomes were used according tothe FISH results to simulate the measured DNA content distributionsof micronuclei. A good agreement was obtained between measuredand simulated distributions of micronuclei as well as betweenresults of the measured frequency of micronuclei showing centromericsignals as a function of their DNA content and those predictedby the simulations. These results demonstrate the usefulnessof flow cytometry and sorting combined with the FISH techniqueand computer simulations for producing a more detailed analysisof mechanisms of micronucleus induction. ⁵To whom correspondence should be addressed     

Detection of chromosomal abnormalities of chromosome 12 in uterine leiomyoma using fluorescencein situ hybridization

Summary Fifty uterine leiomyomas were examined using conventional cytogenetic method and fluorescencein situ hybridization (FISH) for detection of chromosomal, abnormalities of chromosome 12. Of the 50 tumors, nine were examined using FISH on the non-cultured samples. Two (4.0%) of 50 tumor samples examined showed chromosomal abnormalities of chromosome 12 by the conventional cytogenetic analysis. For FISH, the whole-chromosome painting probe and D12Z3 probe specific for the centromeric region were used. Of the 50 cultured samples, 10 showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the nine non-cultured samples, four showed structural abnormalities of chromosome 12, all of which also showed structural abnormalities of chromosome 12 on the cultured samples. These results indicate that chromosomal abnormalities of chromosome 12 are important in the biology of at least some types of uterine leiomyoma, and that FISH is a useful complement to the conventional cytogenetic analysis in the study of solid tumors.     

DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluorescence in situ DNA hybridization

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.     

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.     

Detection and analysis of origin of i(12p), a diagnostic marker of human male germ cell tumors, by fluorescence in situ hybridization.

The i(12p) chromosome marker has been shown to be a diagnostic and prognostic marker of human male germ cell tumors (GCTs). An analysis of the i(12p) and chromosome 12 aneuploidy was performed in five primary cell cultures and three established cell lines derived from human male GCTs by fluorescence in situ hybridization (FISH) with a chromosome 12 centromere-specific alpha-satellite DNA probe. Distinct differences in the centromeric signals originating from the i(12p) and normal chromosome 12 were detected, which were found to be useful for unambiguous distinction between the i(12p) and normal chromosomes 12 at interphase as well as at metaphase in these cultures. This method can be used for rapid screening of large numbers of interphase cells, eliminating the main limitation of conventional karyotypic analysis, namely, frequent inability to obtain sufficient numbers of dividing cells in direct preparations or in short-term culture of fresh biopsies. Our analysis of chromosome 12 centromeric signal size along with karyotypic data and results of analysis of restriction fragment length polymorphisms (RFLPs) on 12q in four GCTs suggested that the i(12p)s are formed by nonreciprocal centromeric interchanges between nonsister chromatids of homologous chromosomes.     

Sensitive detection of chromosome copy number aberrations in prostate cancer by fluorescence in situ hybridization.

The pattern of chromosomal aberrations and their significance in prostate cancer are poorly understood. We studied 23 prostate cancer and 10 benign prostatic hyperplasia (BPH) specimens by fluorescence in situ hybridization (FISH) using pericentromeric repeat-specific probes for 10 different chromosomes. The aims of the study were: 1) to compare the sensitivity of FISH and DNA flow cytometry in aneuploidy detection, 2) to determine which chromosome copy number changes are most common, and 3) which probe combinations would be most effective in aneuploidy diagnosis. Disaggregated tumor cells from formalin-fixed, paraffin-embedded tissues were pretreated with our newly developed method based on hot glycerol solution to improve probe penetration. All BPH specimens were diploid by DNA flow cytometry and showed no numerical chromosome aberrations by FISH. In prostate cancer, flow cytometry showed abnormal DNA content in 35% of cases, whereas 74% were abnormal by FISH. Aberrant copy number of chromosomes 8 (48% of cases), X (43% of cases), and 7 (39% of cases) were most common. Ninety-four percent of all aneuploid cases would have been detected with these three probes alone. Simple chromosome losses were uncommon but in DNA tetraploid tumors relative losses (trisomy or disomy) of several chromosomes were often found, suggesting progression of prostate cancer through tetraploidization followed by losses of selected chromosomes. In conclusion, our results indicate that FISH using three selected chromosome-specific probes is two to three times more sensitive than flow cytometric DNA content analysis in aneuploidy detection.     

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non‐DNA binding genotoxins (e.g., aneugens), unlike DNA‐binding genotoxins, are theoretically expected to show thresholded concentration‐effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose‐response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non‐linear dose‐dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg^?1, respectively. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.     

Chromosomal translocations in human soft tissue sarcomas by interphase fluorescence in situ hybridization

In soft tissue sarcomes, clonal rearrangement of chromosomes has been shown by cytogenetic analysls to be unique and specific for tumor types. The development of fluorescence in situ hybridbation (FISH) has allowed detection of chromosomal rearrangements In the interphase nuclel isolated from paraffin-embedded tissues. Three kinds of trans-locations in the interphase nuclel that were isolated from 47 cases of soft tissue sarcomas ware examined by FISH with chromosome-speclfic DNA probes of centromeric and total probes. of 47 soft tissue sarcomas 42 (89.4%) revealed tumor-specitic transiocations by retrospective cytogenetic analysis. Transiocation t(X;18) was detected In 25/28 synovial sarcomas; translocation t(11;22) In 5/6 Ewlng's sarcomas and primitive neuroectodermal tumors (PNET); and translocation t(12;16) was found in 12/13 liposarcomas, including 10 myxold and two round cell lypes as clonal chromosomal aberrations specific for both subtypes. Based on the cytogenetic analysis, Ewing's sarcoma is related closely with PNET as shown by MIC2–protein reactivity. Other cytogenetic findings of translocation t(12;16) Indicate that round cell liposarcomas share chromosomal changes with myxoid lipo sarcomas, and further suggest that both tumor subtypes of liposarcoma may possess common precursor cells. FISH is a useful aid in determining the tumor type of soft tissue sarcomas with regard to histogenetic origin.     

Complex genomic organization of Indian muntjac centromeric DNA

A 69-kb Indian muntjac bacterial artificial chromosome (BAC) clone that screened positive for Cervid satellites I and IV was selected for complete sequence analysis and further characterization. The sequences of this BAC clone were found in the centromeres and in some interstitial sites of Indian muntjac chromosomes. Sequence analyses showed that the BAC clone contained a 14.5 kb Cervid satellite I-like DNA element and a 9 kb Cervid satellite IV-like DNA element. In addition, it contained 51 regions each organized in a complex fashion, with sequences homology to intersperse repetitive sequences such as LINEs, SINEs, LTRs, other published DNA elements, and unassigned sequences. The FISH patterns of seven non-satellite sequence elements generated from the BAC clone showed mainly specific to centromeres of the Indian muntjac representing novel centromeric DNAs of the species. Furthermore, FISH signals and Southern blot patterns of these elements suggest the existence of a not yet identified repetitive sequence with giant repeated monomers. Positive FISH signals of these elements were also detected in the centromeric regions of Formosan muntjac. This suggests that these newly identified non-Cervid satellite DNA sequences have been conserved in the centromere of the Formosan muntjac.     

DNA flow cytometric evaluation of serous and mucinous cystic neoplasms of the pancreas

There have been few reports of flow cytometric studies of pancreatic neoplasms, whether endocrine or exocrine. In this report, the DNA content of three mucinous and seven serous cystic tumors of the pancreas was evaluated by flow cytometry on formaldehyde solution-fixed, paraffin-embedded tissue. Two serous cystadenomas were aneuploid, with the remaining five serous and all three mucinous being diploid. All cases had a low S phase (ranging from 0.1% to 3.1%), indicating neoplasms with a low turnover. From these results, it appeared that DNA flow cytometry as an independent factor did not discriminate between the benign pancreatic serous cystadenomas and the more aggressive mucinous cystic neoplasms.     

Flow cytometric analysis of genetic damage, effect on cell cycle progression, and apoptosis by thiophanate-methyl in human lymphocytes

Flow cytometric technique was used to study the effects of the fungicide Thiophanate-methyl on cell proliferation, micronucleus induction, and apoptosis in human peripheral blood lymphocytes treated in vitro. In particular, a combined approach of flow cytometry and fluorescence in situ hybridization (FISH) with a pancentromeric alpha-satellite probe was used to evaluate the mechanism of micronucleus induction by Thiophanate-methyl. Flow sorted micronuclei (MN) induced in human lymphocytes by Thiophanate-methyl were analyzed by FISH and the results were compared with results from FISH analysis on MN in binucleated cells. It could be shown that most MN induced by Thiophanate-methyl did not reveal any centromeric signal, thus demonstrating clastogenic action of this fungicide. Moreover, it was found that as a function of the concentration of Thiophanate-methyl, cellular proliferation was delayed and the frequency of apoptotic cells was increased.     

Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis

The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry-sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.