Multicolor Fluorescence In Situ Hybridization Research Articles

Les anomalies moléculaires dans les lymphomes

Numerous molecular abnormalities have been described in lymphomas. They are of diagnostic and prognostic value and are taken into account for the WHO classification of these tumors. They also shed some light on the underlying molecular mechanisms involved in lymphomas. Overall, four types of molecular abnormalities are involved: mutations, translocations, amplifications and deletions of tumor suppressor genes. Several techniques are available to detect these molecular anomalies: conventional cytogenetic analysis, multicolor FISH, CGH array or gene expression profiling using DNA microarrays. In some lymphomas, genetic abnormalities are responsible for the expression of an abnormal protein (e.g. tyrosine-kinase, transcription factor) detectable by immunohistochemistry. In the present review, molecular abnormalities observed in the most frequent B, T or NK cell lymphomas are discussed. In the broad spectrum of diffuse large B-cell lymphomas microarray analysis shows mostly two subgroups of tumors, one with gene expression signature corresponding to germinal center B-cell-like (GCB: CD10+, BCL6 [B-Cell Lymphoma 6]+, centerine+, MUM1–) and a subgroup expressing an activated B-cell-like signature (ABC: CD10–, BCL6–, centerine–, MUM1+). Among other B-cell lymphomas with well characterized molecular abnormalies are follicular lymphoma (BCL2 deregulation), MALT lymphoma (Mucosa Associated Lymphoid Tissue) [API2-MALT1 (mucosa-associated-lymphoid-tissue-lymphoma-translocation-gene1) fusion protein or deregulation BCL10, MALT1, FOXP1. MALT1 transcription factors], mantle cell lymphoma (cycline D1 [CCND1] overexpression) and Burkitt lymphoma (c-Myc expression). Except for ALK (anaplastic lymphoma kinase)-positive anaplastic large cell lymphoma, well characterized molecular anomalies are rare in lymphomas developed from T or NK cells. Peripheral T cell lymphomas not otherwise specified are a heterogeneous group of tumors with frequent but not recurrent molecular abnormalities. Gene profiling analysis shows that the expression of several genes is deregulated including PDGFRA (platelet-derived growth factor receptor) gene, encoding a receptor with tyrosine kinase activity. In angio-immunoblastic T-cell lymphomas molecular abnormalities are found in follicular helper T-cell (TFH) that express some distinctive markers such as CD10, PD-1, CXCR5 and the CXCL13 chemokine. ALK-positive anaplastic large cell lymphoma is a paradigme of T-cell lymphoma since it is associated with an X-ALK oncogenic fusion protein due to a translocation involving ALK gene at 2p23. ALK tyrosine kinase activates downstream pathways (Stat3/5b, Src kinases, PLCγ, PI3 kinase) implicated in lymphomagenesis, proliferation and protection against apoptosis. Specific ALK inhibitors are currently in clinical evaluation. Lastly several lymphomas are associated with infectious agents that play a direct (EB virus, HTLV1) or indirect role (e.g. Helicobacter pylori in MALT lymphoma) in lymphomagenesis.

Multicolor FISH (UroVysion) Facilitates Follow-up of Patients With High-Grade Urothelial Carcinoma of the Bladder

The aim of the present prospective study was to assess the diagnostic benefit of UroVysion (Vysis-Abbott Laboratories, Downers Grove, IL) in the follow-up of patients with a history of high-grade non-muscle-invasive urothelial carcinoma of the bladder (NMIBC). An unselected cohort of 25 patients with a history of high-grade NMIBC was prospectively followed up by office-based cystoscopy, cytology, and UroVysion in 210 events. The sensitivity and specificity for standard combined cystoscopy and cytology were 78% and 83%, respectively. UroVysion yielded a considerably higher detection rate with 94% and 93%, respectively. In 89% of the follow-up events of patients with a history of previous carcinoma in situ (CIS) and negative cystoscopy but a positive UroVysion finding, CIS recurrence was noticed within 5 months. UroVysion is a worthwhile approach in patients with previous CIS, a high risk for the development of CIS, or previous unequivocal cytology suggestive of CIS, especially during or shortly after instillation therapy.

Complex chromosomal rearrangements in infertile males: complexity of rearrangement affects spermatogenesis

In this report, we describe 10 male cases of complex chromosome rearrangements (CCRs) with fertility problems: seven of them showed impairment of spermatogenesis, oligoasthenoteratozoospermia or azoospermia; in the other three cases, recurrent abortions were observed. The CCRs were characterized by conventional fluorescence in situ hybridization (FISH) and multicolor FISH methods as well as by the routine G-banding technique. CCRs found in three cases with recurrent abortions were double two-way exchanges, which were the simplest forms of CCRs; three oligoastenoteratozoospermic cases were double two-way exchanges or three-way exchanges. However, the CCRs in four azoospermic cases were much more complicated forms of CCRs. From our results and a review of the literature, we conclude that the complexity of CCRs might affect the severity of spermatogenetic impairment rather than the number of chromosomes involved or the location of breakpoints.

Characterization of 23 small supernumerary marker chromosomes detected at pre-natal diagnosis: The value of fluorescence in situ hybridization

Small supernumerary marker chromosomes (sSMCs) cannot be identified or characterized unambiguously by conventional cytogenetic banding techniques. Until recently, the large variety of marker chromosomes, as well as the limitations in their identification, have presented a diagnostic problem. In order to determine the origin of sSMCs, we used a variety of fluorescence in situ hybridization (FISH) methods, including centromere-specific multicolor FISH, acrocentric specific multicolor FISH, subcentromere-specific multicolor FISH and multicolor FISH with whole chromosome paint probes. Moreover, uniparental disomy testing was in all cases attempted. From a total of 28,000 pre-natal samples from four diagnostic genetics laboratories in Greece, 23 (0.082%) supernumerary marker chromosomes were detected. The mean maternal age was 36.2 years (range 27-43) and the mean gestational age at which amniocentesis was performed was 18.5 weeks (range 16-23). Eighteen markers were denovo and 5 markers were inherited. Molecular cytogenetic methods were applied to determine the chromosomal origin and composition of the sSMC. In total, 17 markers were derived from acrocentric chromosomes (14, 15, 21 and 22) and 6 markers were non-acrocentric, derived from chromosomes 9, 16, 18, 20 and Y. Uniparental disomy was not detected in any of the cases studied. With regard to pregnancy outcome, 13 pregnancies resulted in normal healthy neonates, while 10 pregnancies were terminated due to ultrasound abnormalities. A total of 23 marker chromosomes from 28,000 pre-natal samples (0.082%) were identified. Molecular cytogenetic techniques provided valuable information on the chromosomal origin and composition of all the sSMCs. Especially in cases with normal ultrasound, the FISH results rendered genetic counseling possible in a category of cases previously considered a diagnostic problem. Abnormal outcome was observed in 10 cases (43,5%), 7 of which showed abnormal ultrasound findings. New technologies, such as array-comparative genomic hybridization, should be used in future genotype-phenotype correlation studies, although the high mosaicism rate poses a problem.

Leukemia: Methods and Protocols

One of the newest editions to the Humana Press series Methods in Molecular Biology is Leukemia: Methods and Protocols, which provides a detailed, stepwise approach to up-to-date laboratory methods and protocols in the vast world of leukemia. In the preface, the editor clearly states that this book serves as a useful reference guide for clinical and research scientists and investigators who are interested in the following techniques: prenatal backtracking of leukemic clones, molecular diagnosis, detection of genome-wide genetic abnormalities and profiling, identification of unknown fusion genes, monitoring of minimal residual diseases, disease modeling using murine and human primary hematopoietic cells, studying of normal and malignant hematopoiesis findings, identification of interacting partners with leukemia-associated oncoproteins, and global characterization of genome-wide epigenetic changes in leukemic cells.With 21 chapters, the book begins by introducing key developments and the latest technologies in acute leukemia and then prepares the reader with an outline of the edition's contents. The following chapter begins with the methods of detecting early genetic events from the use of archived neonatal blood spots. Chapter 3 describes leukemic analysis using fluorescence in situ hybridization (FISH), whereas Chapter 4 further details the use of FISH with fluorescence immunophenotyping and multifluor or multicolor FISH (M-FISH). The next 2 chapters discuss the MLL gene using long-distance inverse polymerase chain reaction (PCR) and panhandle PCR. Chapter 7 moves in a different direction toward evaluating minimal residual disease in patients with acute lymphoblastic leukemia using immunoglobulin and T-cell receptor genes in real-time quantitative PCR. This chapter is filled with useful tables and figures with important and relevant background information. The next 2 chapters advance to 2 relevant techniques of array-based comparative genomic hybridization and single-nucleotide polymorphism microarray. In array-based comparative genomic hybridization, the 2 basic categories discussed include constructs from large-insert DNA clones (BAC array-based comparative genomic hybridization) and another with shorter oligonucleotide sequences (oligo array-based comparative genomic hybridization). Chapter 10 uses retroviral and lentiviral transduction and translocation to find patients with acute leukemia and nonrandom chromosomal translocations with overexpression of proto-oncogenes or novel fusion genes.The next 6 chapters cover different aspects of leukemic stem cells, hematopoietic stem cells and progenitor cells, embryonic stem cells, and the hematopoietic stem cell niche. Chapter 12 uses the xenotransplantation model of immunodeficient mice for identification and characterization of human leukemic stem cells. Chapter 13 describes the methodology of isolating human CD34+ cells and examining the effects of leukemia protein expression by retroviral transduction. Chapter 14 discusses an ex vivo assay with bone marrow stromal coculture system of CD34+ cells from patients with acute myeloid leukemia to study self-renewal and long-term expansion. Chapter 15 explains the use of murine bone marrow to identify and characterize hematopoietic stem and progenitor cells with multiparameter flow cytometry. Chapter 16 covers murine embryonic stem cells with protocols and methods for the culture of embryonic stem cells, their hematopoietic development, and quantitative analyses by gene expression. Last in the realm of stem cells, Chapter 17 details the steps taken to isolate and analyze hematopoietic stem cells from murine midgestation placentas.Chapter 18 covers protein-protein interaction using the yeast 2-hybrid system. The following chapter discusses the expression of the HOX gene network by real-time PCR. Chapter 20 moves into the topic of epigenetics with genome-wide determination of DNA methylation by a microarray-based technique called the HpaII tiny fragment enrichment by ligation-mediated PCR assay. The final chapter explains chromatin immunoprecipitation to analyze histone modifications and chromatin-binding regulatory proteins.Overall, the book covers relevant advanced methods and protocols in leukemia. Each topic is comprehensive and thorough, with excellent and valuable notes at the end of each chapter. The diagrams, figures, and tables are instrumental in supporting the text and helping the reader understand the concepts and procedures. For example, even the simple diagram on page 40, on the method of streaking agar plates with a sterile inoculating loop, portrays the fundamental technique needed for accurate results. Colored plates and pictures are provided at the end of Chapters 9 and 16, which are essential for color distinction, especially for FISH analysis.Geared toward clinical researchers and scientific investigators, this book serves as a practical resource in different aspects of leukemia research. In summary, Leukemia: Methods and Protocols, serves as a precise cookbook, with many carefully formulated ingredients gathered together to form the perfect recipe for leukemia research.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Meiotic analysis of XX/XY and neo-XX/XY sex chromosomes in Phyllostomidae by cross-species chromosome painting revealing a common chromosome 15-XY rearrangement in Stenodermatinae

We analyzed the meiotic behavior of the sex chromosomes of the bats Glossophaga soricina (XX/XY), Artibeus cinereus and Uroderma magnirostrum (neo-XX/XY) using multicolor FISH. The X chromosome and pair 15 autosome probes are from Phyllostomus hastatus and the Y from Carollia brevicauda. On both species with the neo-XX/XY system, the autosome translocated to the sex chromosomes is the pair 15 in P. hastatus, a synapomorphy. The analysis of meiosis confirms that the X and Y have a pseudo-autosomal region, with a typical end-to-end pairing. The autosomal regions of the neo-XX/XY shows different levels of condensation when compared to the original XX/XY. The compound system presented a characteristic shape, as if it was a closed ring with a tail. The ring represents the non-paired segments of the X and Y and the small pairing region of the original sex chromosomes. The tail corresponds to the pairing of the 15 P. hastatus autosomal bivalent, which are translocated to the sex chromosomes. Probably the non-pairing is responsible for the meiotic silencing of these segments.

FISH Applications for Genomics and Plant Breeding Strategies in Tomato and Other Solanaceous Crops

This paper describes the use of advanced fluorescence in situ hybridization (FISH) technologies for genomics and breeding of tomato and related Solanum species. The first part deals with the major determinants of FISH technology: (1) spatial resolution, which depends on the diffraction limit of the microscope and the type of chromosome, chromatin or isolated DNA fibres as target for the hybridisation; (2) the detection sensitivity, which is limited by the sensitivity and dynamic range of the CCD camera and the quality of the microscope, and the amplification system of the weak signals from tiny probe molecules; (3) simultaneous detection of multiple probes labelled directly or indirectly with up to 5 different fluorophores, whether or not in different combinations and/or mixed at different ratios. The power and usability of such multicolour FISH is indispensable when large numbers of bacterial artificial chromosomes (BACs) or other vectors with genomic DNA are available. Mapping of multiple BACs on chromosomes are powerful instruments confirming their assumed genetic position, whereas pooled BACs for a given chromosome arm will reveal the gaps between the BACs or derived contigs of their physical maps. Tandem and dispersed repeats, which are abundant in the genomes of most species, can be analysed in repeat bar coding FISH, showing the major blocks of repeats in heterochromatin and euchromatin areas. Repeat-rich areas of the chromosomes can also be demonstrated by hybridisation of probed Cot fractions of sheared genomic DNA; a powerful method to elucidate the heterochromatin domains for genomic studies. In addition, unlabelled Cot DNA, as blocking agent in BAC-FISH painting, suppresses repetitive sequences from the BACs to hybridise on the chromosomes. Cross-species BAC-FISH painting with labelled probes from tomato and potato BACs and hybridised on the chromosomes of related species, under appropriate conditions, is a powerful instrument to demonstrate chromosomal rearrangements, including inversions and translocations. The technology not only supports phylogenetic studies between the taxa under study but can also be helpful in breeding programs with crops containing introgressed regions from related species when linkage drag or meiotic pairing disturbances between the homoeologues are assumed. In the next steps in comparative genomics, we now can detect smaller chromosomal and DNA rearrangements, diminutions and amplifications of repeats and changes of the epigenetic status of introgressed regions.

Integration of Genetic, Physical, and Cytogenetic Maps for Brassica rapa Chromosome A7

Bacterial artificial chromosome (BAC) contigs have been genetically mapped to the 10 linkage groups of Brassica rapa by BAC end sequences (BES). To integrate the genetic, physical, and cytogenetic maps, fluorescence in situ hybridization (FISH) was used to anchor the assembly of BAC contigs onto Brassica chromosomes using representative BACs. This BAC-FISH approach can be used to identify chromosome arms on separate mitotic metaphase chromosomes or to map multiple BACs to single long pachytene chromosomes. As part of an international consortium that is sequencing the B. rapa genome, we integrated the linkage and physical maps with the B. rapa cytogenetic map for chromosome A7 by hybridizing BACs to mitotic chromosomes and along the length of pachytene chromosome spreads. A total of 31 BACs that were putatively located on A7 were used as probes for FISH analyses; however, only 19 BACs mapped unambiguously to A7 while the remaining BACs either mapped to other chromosomes or hybridized to multiple locations. We then created a multicolor FISH cocktail of 16 BAC probes to simultaneously hybridize the entire length of the A7 chromosome. We successfully applied the 16 A7 BAC probe mix to B. rapa, B. oleracea, and domesticated and resynthesized genotypes of B. napus to demonstrate that this approach can facilitate studies of genome evolution by integrating the genetic, physical, and cytogenetic maps among closely related species of Brassica.

PHYSICAL MAPPING OF 5S AND 45S RDNAS BY FLUORESCENT IN SITU HYBRIDIZATION IN MANGO (MANGIFERA INDICA L.)

Mango (Mangifera indica L.) is thought to be diploid despite the many numbers of chromosomes (2n=2x=40). Although the allopolyploid nature of M. indica is reported by Mukherjee (1950), no chromosome studies are available to elucidate genome composition of mango until now. We have applied fluorescent in situ hybridization (FISH) technique using 5S and 45S ribosomal DNAs (rDNAs) as probes on the somatic metaphase chromosome of mango for the first step to elucidate genome composition of mango. The digoxigenin (DIG)-labeled 5S or 45S probe was hybridized onto the chromosomes separately, and both of them were visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA were observed on two chromosomes, and these two seemed to be homologous chromosomes due to their size and identical chromosomal location of 5S rDNA loci. On the other hand, six chromosomes were demonstrated to carry 45S rDNA loci. Among these six chromosomes, two chromosomes seemed to be homologous from their size and the location of rDNA loci in addition to signal intensity. But, another four chromosomes could not be identified whether or not these four form two pairs of chromosomes, since no clear images of these chromosomes were observed. In addition, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of mango. We could distinguish eight chromosomes among forty chromosomes of mango in this study, but no evidence was obtained for a possibility of heterozygotic karyotype in mango not like in sweet orange.

Abstract 3037: Integrated Genomic, MicroRNA (miRNA) and Proteomic Profiling of Ovarian Carcinoma for Biomarker Discovery

Abstract Ovarian cancer (OCa) is the fifth leading cause of cancer-related deaths in North American women and the first due to a gynecologic malignancy. The long-term effectiveness of standard therapy is generally poor and is accompanied by serious side effects. Thus there is a need for developing markers not only for diagnosis and prognosis, but also for predicting therapeutic response. Tumour progression and resistance to therapy is a consequence of the complexity of DNA, RNA and proteins. The search for effective and specific biomarkers should integrate aspects of all these factors. We have previously demonstrated that KLK6 is a promising biomarker for OCa and its observed over-expression is linked to copy-number gains of the 19q13.3/4 locus. Here, we demonstrate by multi-colour FISH analyses that the KLK locus in 81 OCas is subject to high-level of genomic instability (p<0.001); and such instability is significantly co-related to grade (p<0.001). KLK6-specific immunohistochemistry (IHC) showed no strong corelation with KLK6 copy-number, suggesting that other mechanisms, together with copy-number, drive its over-expression. Because 19q contains the highest number of annotated microRNAs (miRNAs) and copy-number instability may affect expression of these miRNAs, we investigated the role of miRNAs in OCa, not only for regulating KLK6, but as biomarkers for OCa. miRNA profiling of OCa cell lines and primary tumours by RT2-PCR showed the differential expression of miRNAs, consistent with other published studies in OCa. Since miRNAs can potentially affect the protein expression of hundreds of genes, the identification of such differentially expressed proteins not only provides putative biomarkers, but may also elucidate pathways for therapeutic intervention. Using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry for the OVCAR-3 cell line, cultures were labeled separately in light-Arg/Lys and heavy-Arg/Lys. In this control experiment, over 2,800 proteins were identified with 2,465 quantified. Over 94% of these quantified proteins showed a heavy:light ratio between 0.8 and 1.2, making this a robust system for quantitatively distinguishing differentially expressed proteins in the presence of miRNA precursors or inhibitors. Our profiling, as well as others, has shown the loss of expression of let-7 family members and hsa-125a-5p in OCas, and both miRNAs show decreased expression in OVCAR-3. Interestingly, these miRNAs are also predicted to target KLK6. Thus, we hypothesize that these miRNAs will not only affect the expression of KLK6, but also the expression of other target genes. In the future, using SILAC, we will identify the differentially expressed proteins affected upon re-introduction of these miRNAs through the differential labeling of miRNA-transfected OVCAR-3 vs. non-transfected OVCAR-3; thus revealing novel biomarkers for OCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3037.

Multi-colour FISH on preoperative renal tumour biopsies to confirm the diagnosis of uncertain renal masses

In some cases with uncertain renal tumour lesions, it would be helpful to perform biopsies for the preoperative differential diagnosis. In our study, we evaluated the benefit of multi-colour interphase fluorescence in situ hybridization (M-FISH) on fine-needle core biopsies in uncertain renal masses. We prospectively performed three ultrasound-guided percutaneous biopsies in 25 patients with indeterminate renal masses preoperatively. Histopathology was performed on two remaining cores samples. M-FISH was performed on one core for chromosomes 1, 2, 6, 9, 7, 17, the loci 3p24pter, and 3p13p14. After interphase FISH evaluation, we classified tumours and compared the results with histopathological findings. 16 were classified as renal malignancies: 14 (56%) clear cell renal cell carcinomas (RCCs), 1 papillary RCCs (4%), and 1 "adenocarcinoma" (4%). Seven patients (28%) had a benign tumour, i.e. 6 (24%) were oncocytomas and 1 was classified as leiomyoma (4%). In two cases (8%), no renal neoplasms were found. In 19 out of 21 cases (90.5%), the preoperative diagnostic fine-needle biopsy matched the final histological findings. The combination of histopathological examination and M-FISH leads to a higher (95.5 vs. 90.5%) diagnostic fidelity as histology alone. Ultrasound-guided percutaneous renal tumour biopsy is an accurate and safety method for the histopathologic evaluation of uncertain renal masses. The M-FISH represents a new highly sensitive and specific method to confirm histopathological classification in less than 24 h which can be used in routine laboratory diagnosis.

Classifying ambiguous melanocytic lesions with FISH and correlation with clinical long-term follow up

Recently, initial studies describing the use of multicolor fluorescence in situ hybridization (FISH) for classifying melanocytic skin lesions have been published demonstrating a high sensitivity and specificity in discriminating melanomas from nevi. However, the majority of these studies included neither histologically ambiguous lesions nor a clinical long-term follow up. This study was undertaken to validate a special multicolor FISH test in histologically ambiguous melanocytic skin lesions with known clinical long-term follow up. FISH was scored by three independent pathologists in a series of 22 melanocytic skin lesions, including 12 ambiguous cases using four probes targeting chromosome 6p25, centromere 6, 6q23, and 11q13. The FISH results were compared with array comparative genomic hybridization data and correlated to the clinical long-term follow up (mean: 65 months). Pair-wise comparison between the interpretations of the observers showed a moderate to substantial agreement (κ 0.47–0.61). Comparing the FISH results with the clinical behavior reached an overall sensitivity of 60% and a specificity of 50% (χ2=0.25; P=0.61) for later development of metastases. Comparison of array comparative genomic hybridization data with FISH analyses did not yield significant results but array comparative genomic hybridization data demonstrated that melanocytic skin lesions with the development of metastases showed significantly more chromosomal aberrations (P<0.01) compared with melanocytic skin lesions without the development of metastases. The FISH technique with its present composition of locus-specific probes for RREB1/MYB and CCND1 did not achieve a clinically useful sensitivity and specificity. However, a reassessment of the probes and better standardization of the method may lead to a valuable diagnostic tool.

Human interphase chromosomes: a review of available molecular cytogenetic technologies.

Human karyotype is usually studied by classical cytogenetic (banding) techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB). Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions) being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations). Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB) allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.

Chromosome composition of an F2 Triticum aestivum×T. turgidum spp. durum cross analysed by DArT markers and MCFISH

This study has employed multicolour fluorescence in situ hybridisation (MCFISH) and Diversity Arrays Technology (DArT) markers to determine the segregation of parental A, B and D genome material into the progeny of a cross between a hexaploid bread wheat (Triticum aestivum L. var. 2-49) and a tetraploid durum wheat [T. turgidum L. spp. durum (Desf.) var. Bellaroi]. In the F2 progeny from a 2-49/Bellaroi cross, 82 out of 83 F2 plants investigated with DArT analysis carried some D genome material, principally as entire chromosomes, while 40 plants included at least one complete copy of all seven D genome chromosomes. Twelve plants containing partial D chromosomes were identified. MCFISH analysis of 26 additional F2 plants of the same cross showed that all 26 plants contained varying amounts of D genome material of which three carried single A-D translocations. In addition two telocentric D genome chromosomes were detected. The D genome content of each line and the breakpoint positions of the three A-D translocations were confirmed with DArT marker analysis. Overall results indicate a random recombination of A and B genome loci from the hexaploid female parent and the tetraploid male parent in this F2 population and a significant retention of the maternal D genome material. This study illustrates that the combined application of the MCFISH and DArT techniques provides a powerful approach for the analysis of crosses between cereal genotypes of different ploidy.

Recurrent involvement of heterochromatic regions in multiple myeloma—A multicolor FISH study

Chromosome aberrations are important prognostic markers in multiple myeloma (MM), but their identification may be hampered by complexity of the karyotypes. Using multicolor fluorescence in situ hybridization (mFISH), we found cryptic aberrations in 7 of 10 patients with a complex karyotype. Moreover, in addition to typical aberrations involving 1q, 13q, 14q and 17p and structural aberrations in chromosomes 1, 6, 9 and 19, (iso)dicentric chromosomes and whole-arm translocations were detected. These chromosome aberrations were generated by breaks in heterochromatic regions indicating an increased breakage of these regions, which may predispose to the generation of chromosome aberrations in multiple myeloma.

Clonally Expanded T Lymphocytes from Atomic Bomb SurvivorsIn VitroShow No Evidence of Cytogenetic Instability

Abstract Genomic instability has been suggested as a mechanism by which exposure to ionizing radiation can lead to cancer in exposed humans. However, the data from human cells needed to support or refute this idea are limited. In our previous study on clonal lymphocyte populations carrying stable-type aberrations derived from A-bomb survivors, we found no increase in the frequency of sporadic additional aberrations among the clonal cell populations compared with the spontaneous frequency in vivo. That work has been extended by using multicolor FISH (mFISH) to quantify the various kinds of chromosome aberrations known to be indicative of genomic instability in cloned T lymphocytes after they were expanded in culture for 25 population doublings. The blood T cells used were obtained from each of two high-dose-exposed survivors (>1 Gy) and two control subjects, and a total of 66 clonal populations (36 from exposed and 30 from control individuals) were established. For each clone, 100 metaphases were examined. In the case of exposed lymphocytes, a total of 39 additional de novo stable, exchange-type aberrations [translocation (t) + derivative chromosome (der)] were found among 3600 cells (1.1%); the corresponding value in the control group was 0.6% (17/3000). Although the ratio (39/3600) obtained from the exposed cases was greater than that of the controls (17/3000), the difference was not statistically significant (P = 0.101). A similar lack of statistical difference was found for the total of all structural chromosome alterations including t, der, dicentrics, duplications, deletions and fragments (P = 0.142). Thus there was no clear evidence suggesting the presence of chromosome instabilities among the clonally expanded lymphocytes in vitro from A-bomb survivors.

Spontaneous aneuploidy level in blood cells of fertile females

In this study we present the results of molecular-cytogenetic investigation of blood cells of fertile women of reproductive age. We used cultivated and uncultivated cells and two sets of FISH probes with direct and indirect labeling. The mean aneuploidy levels of 4 chromosomes were determined and statistical limits were scored. Aneuploidy levels determined in cultivated lymphocytes by multicolor FISH varied from 0.1 to 1.3%. The mean aneuploidy level for all chromosomes (13, 18, 21 and X) was 1.39%. The limit mutation detection was 3.4%. We found differences in results obtained with the use of direct and indirect labeling. It was shown that cultivating process influenced the aneuploidy level. Absolute error of FISH technique was 0.13%, relative error - 4.08%.

Gene amplification in myeloid leukemias elucidated by fluorescence in situ hybridization

Gene amplification in hematologic malignancies is uncommon. When karyotyping leukemia cells, gene amplification is generally seen as double-minute (dmin) chromosomes and homogeneously staining regions (hsr). One of the more commonly amplified regions is MYC at 8q24.21, but amplification of MLL at 11q23 and regions on 9p, 19q, and elsewhere on 11q have been reported. Increased copy number of these genes has been associated with poor prognosis. Over an 11-year period, we identified 31 cases of possible gene amplification, 27 of which had enough sample material for further investigations. A total of 17 cases had dmin only, 13 cases had hsr only, and 1 case had both dmin and hsr in the karyotype. Fluorescence in situ hybridization (FISH) analysis identified amplification of MYC in 12 cases, all on dmin, and amplification of MLL in eight cases, all on hsr. Regions other than MYC and MLL were amplified in eight cases and, using multicolor FISH and multicolor banding, we identified a number of novel regions of amplification: 13q11∼q12.1, 15q26.1∼q26.3, and 17q12. We also identified one case where two different chromosomal regions were simultaneously amplified in the same cell line.

Hemangiopericytoma with chromosomal aberrations on chromosomes 8 and 10: Evidence for a distict brain tumor entity

Hemangiopericytoma is not universally recognized as a tumor entity, and there is a trend to reassign it piecemeal to other tumor categories. However, it has been included in the new WHO classification of brain tumors and has been distinguished from both meningioma and fibrous tumor. Since there are few genetic studies in existence, we have performed a comprehensive cytogenetic analysis of an infratentorial hemangiopericytoma in a 55 y‐old female. It was originally classified as a grade II tumor but recurred as a grade III tumor with a proliferation index of 20 %. By use of trypsin‐Giemsa staining (GTG‐banding) and multicolor fluorescence in situ hybridization (M‐FISH) we could confirm the loss of chromosomal material 10q that has been previously described in hemangiopericytoma, and furthermore we identified de novo chromosomal aberrations on chromosomes 8. Applying high‐density single nucleotide polymorphism array (SNP‐A) karyotyping, we detected several regions suggestive of partial uniparental disomy and clonal deletions as well as segments with loss or gain. These findings together with the results of immunohistochemical investigations seem to justify the nosological separation of hemangiopericytoma as a biologically different entity and should be verified in larger patient series.