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PubMed2002

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.

Biomarkers, Tumor↗
PubMed2003

De novo 9-break-event in one chromosome 21 combined with a microdeletion in 21q22.11 in a mentally retarded boy with short stature.

We report on a moderately mentally retarded 12-year-old boy of short stature showing the most complex chromosomal rearrangement (CCR) within a single chromosome ever described. A de novo derivative chromosome 21 was recognized in GTG-banding shortly after birth. However, the nature of the rearrangement remained obscure up to the application of the chromosome 21-specific centromere-near multicolor-FISH (subcenM-FISH) probe set and of six selected locus-specific probes along chromosome 21. An unbalanced 9-break-event was uncovered with breakpoints in 21p13, 21p13-->12, 21q11.2, 21q21.1, 21q22.11, 21q22.11, 21q22.12, 21q22.22 and 21q22.3. A deletion of 21q22.11 was detected by application of the BAC probe bk249H10. The karyotype can be described as 46,XY,der(21)(:p13-->p1213::q22.3-->q22.22:: q11.2-->p1213::q11.2-->q21.1::q22.11-->q21.1::q22.12--> q22.22::p13-->p13). The clinical signs can either be due to gene inactivation in connection with structural changes at the break and fusion regions, to the building of new fusion genes within the CCR and/or to the deletion of genes in 21q22.11.

Abnormalities, Multiple↗
PubMed2005

New insights into the evolution of chromosome 1.

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.

Animals↗
PubMed2005

High-resolution physical mapping of the secalin-1 locus of rye on extended DNA fibers.

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by fluorescence in situ hybridization to extended DNA fibers of rye (Secale cereale, 2n = 14), employing DNA probes of lambda phage clones containing the omega-secalin gene. The fluorescent signals to rye extended DNA fibers revealed continuous strings of 45 microm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2-kb fragment including the entire coding region of the omega-secalin gene and a 1.0-kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. The physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of the Sec-1 locus having 15 copies of the gene. The present findings based on fiber FISH analysis support the contention that the omega-secalin genes are arranged in a head-to-tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb.

Base Sequence↗
PubMed2005

p53 as an immunotherapeutic target in head and neck cancer.

Squamous cell carcinomas of the head and neck (HNSCCs) are characterized by a high frequency of mutations in the p53 gene often leading to p53 protein accumulation. Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of ' pan' vaccines against HNSCC has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53 264-272 epitope pulsed on autologous dendritic cells to generate cytotoxic T lymphocytes (CTLs) ex vivo from circulating precursor T cells of HLA-A2.1+ patients with HNSCC. CTLs specific for the wt p53 264-272 peptide were generated from leukocytes obtained from a cohort of patients with HNSCC (group A). Paradoxically, none of those patients had tumors which adequately presented the epitope, i.e. accumulated p53. In contrast, patients who did not generate CTLs (group B) had tumors which accumulated altered p53 and potentially could present the p53 264-272 epitope. When p53 264-272-specific T cells were directly enumerated in the peripheral circulation of patients with HNSCC using tetrameric p53 264-272/HLA-A2.1 complexes by multicolor flow cytometry, group A had high and group B low percentages of tetramer+ CD3+ CD8+ T cells. These findings suggested that in vivo p53-specific CTLs in group A might play a role in the elimination of tumor cells expressing the p53 264-272 epitope ('immunoselection'), leading to the outgrowth of 'epitope loss' tumor cells. On the other hand, precursor CTLs specific for the wt p53 264-272 peptide in group B are unresponsive to the p53 antigen. Unresponsiveness of CTLs specific for the wt p53264-272 peptide detected in group B could be reversed by using more immunogenic variant peptides of the p53 264-272 epitope. In vivo, immunoselection of tumors which become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies. It calls for modified approaches, in which altered peptide variants of the wt sequence p53 264-272 epitope are used in a vaccine in order to overcome unresponsiveness of T lymphocytes to the native epitope.

Cancer Vaccines↗
PubMedDate not supplied

Heterogeneity in fetal immunocompetence during the second trimester of gestation. Implications for treatment of nonimmune genetic disorders by in utero transplantation.

OBJECTIVE: To address the role that alloreactivity may play and better define the window for histoincompatible stem cell transplantation in utero. SUBJECTS, MATERIAL AND METHODS: We studied 9 fetal blood specimens obtained by cardiocentesis during elective abortions in the second trimester by multicolor flow cytometry and in vitro stimulation. RESULTS: Lymphocytes ranged from adult levels (3/9) to >90% leukocytes. Six specimens had T cells within adult range. T cells in the other specimens were reduced, while B cells were conversely elevated. This variability did not correlate with gestational age, or leukocyte composition. Following 4 h of mitogenesis, fetal CD4+ and CD8+ T cells from 1 of 5 specimens showed a response similar to that of maternal T cells, while the other 4 specimens showed a diminished response (0.3 +/- 0.2-fold). This heterogeneity did not correlate with gestational age, or lymphocyte subset distribution. Following 18 h of in vitro mitogenesis, fetal T cells from 2 specimens showed a response similar to that of maternal T cells (0.8 +/- 0.2-fold). Despite that, one specimen gave a 3-fold greater response in a one-way mixed lymphocyte reaction vs. maternal cells compared to the other specimen. CONCLUSION: We determine that fetal immunocompetence differs greatly during the second trimester and assessment of host vs. donor reactivity prior to in utero transplantation is likely to potentiate more favorable outcomes.

Antibody Formation↗
PubMed2005

Unusual small supernumerary marker chromosome (sSMC) 9 in a Klinefelter patient.

Small supernumerary marker chromosomes (sSMC) are small additional chromosomes characterizable for their origin only by molecular cytogenetic approaches. sSMC have been reported previously in four types of syndromes associated with chromosomal imbalances: in approximately 150 cases with Turner syndrome, 26 cases with Down syndrome and only one case each with Klinefelter syndrome and "Triple-X"-syndrome. Here we report the second case with an sSMC detected in addition to a Klinefelter karyotype. Molecular cytogenetics applying centromere-specific multicolor FISH (cenM-FISH) and a specific subcentromere-specific (subcenM-FISH) probe set characterized the sSMC as a dic(9)(:p12-->q11.1::q11.1--> p11.1:). The reported patient was described with hypogonadism, gynaecomastia plus a bronchial carcinoma. The patient's clinical features are discussed in connection with other Klinefelter cases and possible consequences of presence of the sSMC(9). Furthermore, a suggestion is made for the mode of sSMC-formation in this case.

Adenocarcinoma↗
PubMed2006

A molecular cytogenetic study of chromosome evolution in chimpanzee.

We applied multitude multicolor banding (mMCB) in combination with a novel FISH DNA probe set including subcentromeric, subtelomeric and whole chromosome painting probes (subCTM) to characterize a Pan paniscus (PPA) cell line. These powerful techniques allowed us to refine the breakpoints of a pericentric inversion on chimpanzee chromosome 4, and discovered a novel cryptic pericentric inversion in chimpanzee chromosome 11. mMCB provided a starting point for mapping and high resolution analysis of breakpoints on PPA chromosome 4, which are within a long terminal repeat (LTR) and surrounded by segmental duplications, as well as the integration/expansion sites of the interstitial heterochromatin on chimpanzee chromosomes 6 and 14. Moreover, we found evidence at hand for different types of heterochromatin in the chimpanzee genome. Finally, shedding new light on the human/chimpanzee speciation, karyotypes of three members of the genus Pan were studied by mMCB and no cytogenetic differences were found although the phylogenetic distance between these subspecies is suggested to be 2.5 million years.

Animals↗
PubMed2006

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms.

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).

Cell Line, Tumor↗
PubMed2006

Imaging glomeruli in renal biopsy specimens.

Glomerular capillary loops are complex vascular filters composed of interdigitating podocytes and fenestrated endothelial cells with an intervening proteoglycan-rich extracellular matrix. This arrangement is crucial to maintaining the filtration barrier but renders the glomerulus difficult to analyze by conventional two-dimensional histochemical techniques. When pathologic lesions distort glomerular architecture, its complex morphology is even more challenging to interpret. Fortunately, recent advances in microscopes and computer software now enable glomerular enthusiasts to dissect this complex structure with finer detail. In this review we explore the application of new methodologies such as two-photon microscopy that optimize three-dimensional, multicolor imaging and single-cell segmentation of glomerular components.

Animals↗
PubMed2006

Spectral karyotyping of human, mouse, rat and ape chromosomes--applications for genetic diagnostics and research.

Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies.

Animals↗
PubMed2006

COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications.

The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.

Fluorescent Dyes↗
PubMed2006

Automated microscopy of amniotic fluid cells: detection of FISH signals using the FastFISH imaging system.

OBJECTIVE: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope fastFISH amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. RESULTS: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. CONCLUSION: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

Adolescent↗
PubMed1992

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.

Breast Neoplasms↗
PubMed1995

The development of painting probes for dual-color and multiple chromosome analysis in the mouse.

The recent development of mouse chromosome painting probes for fluorescence in situ hybridization has extended the use of this common laboratory mammal in cytogenetics. We now report the development of additional painting probes by degenerate-oligonucleotide-primed PCR on chromosomes from mouse lung fibroblast cultures, each homozygous for a single Robertsonian translocation chromosome. These probes are for Rb(1.2), Rb(1.3), Rb(4.6), and Rb(6.7). Probes were also made for the sex chromosomes by isolating shoulders from larger peaks (X) or small, clearly resolved peaks (Y) in the flow karyotype. Combinations of probes were used to paint four chromosomes simultaneously in a single color. Multicolor painting was achieved with a biotinylated Rb(1.2) probe and a digoxigenin-labeled Rb(2.8) probe. Each of the three different homologous pairs was uniquely colored by avidin-Texas Red, anti-digoxigenin-FITC, or both simultaneously. These results extend the usefulness of the mouse as a model for understanding adverse environmental exposures and genetic diseases in humans.

Animals↗
PubMed1996

Generation of mouse chromosome painting probes by DOP-PCR amplification of microdissected meiotic chromosomes.

We describe here the development of four sets of painting probes for mouse chromosomes 1 and 13, 2 and 8, 6 and 15, and X and Y by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of 10-20 copies of microdissected male meiotic chromosomes. The X,Y probe was obtained from the X-Y bivalent of diakinesis/metaphase I complements of mice with a normal karyotype, whereas the other probes were derived from tri- or quadrivalents in diakinesis/metaphase I of two reciprocal translocations, T(1;13)70H and T(2;8)2Wa, and one inversion heterozygote carrying a small deletion, In(6;15)Rb1Ald del6(15)1Wa. The specificity of these probes was established by fluorescence in situ hybridization (FISH) to meiotic and mitotic metaphase complements. The chromosomes painted by these probes could be identified by single or multicolor FISH.

Animals↗
PubMed1996

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

Chromosome Mapping↗
PubMed1997

Increased aneuploid frequency in spermatozoa from a Hodgkin's disease patient after chemotherapy and radiotherapy.

The frequency of sperm aneuploidy was investigated by fluorescence in situ hybridization (FISH) in a Hodgkin's disease patient shortly after he had received chemotherapy and radiotherapy. Sperm karyotyping of the same patient had previously shown multiple structural abnormalities in most spermatozoa immediately after radiotherapy (day 0), whereas most spermatozoa collected 5 wk later (day 38) exhibited normal metaphase divisions (Rousseaux et al., 1993). Variations in the frequency of aneuploidy could not be detected by sperm karyotyping. Multicolor FISH on interphase spermatozoa revealed an increase in the rate of disomy for chromosomes 1, 6, 11, X, and Y at day 0 as well as at day 38. The high frequency of 24,XY (nondisjunction at meiosis I) and 24,XX (nondisjunction at meiosis II) spermatozoa (8.46% and 1.64% at day 0, respectively) from the Hodgkin's disease patient suggests that both meiosis I and II are affected and that the X chromosome is frequently involved in such malsegregation events. The rate of 46,XY diploidy was also increased in the patient's sperm, up to 0.62% at day 0. While radiotherapy probably affected the postmeiotic cells (spermatids), the patient's cancer and/or chemotherapy are the two major factors that could have affected the dividing spermatogonia and/or spermatocytes, resulting in high aneuploidy rates.

Aneuploidy↗