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Results for “Spectral Karyotyping”

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PubMed2002

Multicolor spectral karyotyping of serous ovarian adenocarcinoma.

We applied multicolor spectral karyotyping (SKY) to decipher the chromosomal complexity of a panel of seven cell lines and four primary tumors derived from patients with high-grade serous adenocarcinoma of the ovary. By this method we identified a total of 188 unbalanced translocations, nine reciprocal translocations [t(2;15)(q13;q23), t(7;17) (q32;q21), t(8;22)(p11;q11), t(8;22) (q24;q13), t(10;19) (q24;q13.2), t(11;19) (q13;p11), t(12;21)(q13;q22),t(18;20) (q?11;q?11), t(18;22)(q?11;q?13)], 6 isochromosomes [i(1q), i(7q), i(8q), i(9p), i(17q), i(21q)], and 23 deletions. By detailed mapping of rearrangement breakpoints, it was possible to identify several recurring breakpoint clusters at chromosomal bands 1p36, 2p11, 2p23, 3p21, 3q21, 4p11, 6q11, 8p11, 9q34, 10p11, 11p11, 11q13, 12p13, 12q13, 17q21, 18p11, 18q11, 20q11, and 21q22. Recurrent interstitial deletion of chromosomal bands 8p11, 11p11, and 12q13 and a recurrent unbalanced translocation--der(6)t(6;8)(q11;q11)-were also identified. In addition, a homogeneously staining region localized in one cell line to 11q13 was found using SKY to be derived from genetic material originating from chromosome 12. Subsequent comparative genomic hybridization (CGH) studies on this tumor revealed the amplification of DNA sequences derived from the short arm of chromosome 12 at the 12p11.2 region. These studies demonstrate the power of SKY, CGH, and G-banding to resolve the full spectrum of chromosomal rearrangements in serous ovarian adenocarcinoma.

Adenocarcinoma, Papillary↗

PubMed2000

Sensitivity of multiple color spectral karyotyping in detecting small interchromosomal rearrangements.

Multiple color spectral karyotyping (SKY) has been proven to be a very useful tool for characterization of the complex rearrangements in cancer cells and the de novo constitutional structural abnormalities. The sensitivity of SKY in detecting interchromosomal alterations was assessed with 10 constitutional translocations involving subtelomeric regions. Among the 13 small segments tested, 9 were clearly visualized and 8 were unambiguously identified by SKY. Fluorescence in situ hybridizations (FISH) with subtelomeric probes confirmed the reciprocity in three of the four translocations in which a small segment was not detectable by SKY. On the basis of resolution level of G-banding and the information obtained from the FISH analysis, the minimum alteration that SKY can detect is estimated to be 1,000-2,000 kbp in size with the currently available probes. This study has demonstrated the power, but also the limitations, of SKY in detecting small interchromosomal alterations, particularly those in subtelomeric regions.

Adult↗

PubMed2000

Spectral karyotyping (SKY) refinement of a complex karyotype with t(20;21) in a Ph-positive CML patient submitted to peripheral blood stem cell transplantation.

A patient with a Ph-positive chronic myeloid leukaemia (CML) was submitted to allogeneic peripheral blood stem cell transplantation from an HLA-haploidentical related donor 7 years after the diagnosis. Six months later, he showed a disease relapse while cytogenetic analysis displayed a complex karyotype. To characterise the chromosomal rearrangements spectral karyotype (SKY) analysis was used. This redefined all chromosome rearrangements and revealed a t(20;21)(q11;q22). FISH analysis with a specific probe for the AML1 gene disclosed disruption of this gene which was partially translocated on to the long arm of chromosome 20. It is likely that this rearrangement, unusual for CML, was implicated in the disease evolution towards blastic crisis (BC).

Adult↗

PubMed2003

Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids.

Mouse-human somatic cell hybrids have been extensively used in the molecular genetic dissection of human disease-related chromosome rearrangements because of their ability to selectively and randomly eliminate human chromosomes. This technology allows the isolation of structural chromosome abnormalities, which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions and translocations, down to the nucleotides involved. The main confounding problem with the analysis of somatic cell hybrids is determining the exact chromosome complement unequivocally and quickly. Spectral karyotyping can identify each of the individual human chromosomes in a normal metaphase spread, as well as structural chromosome rearrangements-although, because of potential cross-hybridization between the human probe and mouse DNA sequences during the hybridization reaction, it has not been determined whether the same analysis will selectively identify human chromosomes on a mouse background. We show (to our knowledge, for the first time) that, under modified conditions of chromosomal in situ suppression hybridization, the standard spectral karyotyping probe does not cross-react with mouse chromosomes and can be used to identify subtle structurally rearranged chromosomes in hybrid cells. This analysis allows for the rapid and unequivocal identification of the human chromosome complement in these hybrids, as well as structural chromosome rearrangements that occur between mouse and human chromosomes that might otherwise confound the analysis.

3T3 Cells↗

PubMed2002

The utility of spectral karyotyping in the cytogenetic analysis of newly diagnosed pediatric acute lymphoblastic leukemia.

We applied multicolor spectral karyotyping (SKY) to a panel of 29 newly diagnosed pediatric pre B-cell ALLs with normal and abnormal G-banded karyotypes to identify cryptic translocations and define complex chromosomal rearrangements. By this method, it was possible to define all add chromosomes in six cases, a cryptic t(12;21)(p13;q11) translocation in six cases, marker chromosomes in two cases and refine the misidentified aberrations by G-banding in two cases. In addition, we identified five novel non-recurrent translocations - t(2;9)(p11.2;p13), t(2;22) (p11.2;q11.2), t(6;8)(p12;p11), t(12;14)(p13;q32) and t(X;8)(p22.3;q?). Of these translocations, t(2;9), t(2;22) and t(12;14) were identified by G-banding analysis and confirmed by SKY. We characterized a t(12;14)( p13;q32) translocation by FISH, and identified a fusion of TEL with IGH for the first time in ALL. We identified a rearrangement of PAX5 locus in a case with t(2;9)(p11.2;p13) by FISH and defined the breakpoint telomeric to PAX5 in der(9)t(3;9)(?;p13). These studies demonstrate the utility of using SKY in combination with G-banding and FISH to augment the precision with which chromosomal aberrations may be identified in tumor cells.

Acute Disease↗

PubMed2001

Characterization of chromosomal abnormalities in uroepithelial carcinomas by G-banding, spectral karyotyping and FISH analysis.

Chromosome analysis by G-banding, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) was performed on 24 short-term cultured transitional cell bladder carcinomas and 5 cell lines established from bladder carcinomas. Except for one tumor with an apparently normal chromosomal constitution, clonal chromosome abnormalities were detected in all examined cases by the combined approach. The application of SKY and FISH techniques improved the karyotypic descriptions, originally based on G-banding only, by identifying 32 additional numerical changes, by establishing the chromosomal origin of 27 markers and 2 ring chromosomes, by redefining 53 aberrations and by detecting 15 hidden chromosomal rearrangements. No recurrent translocation, however, was detected. The most prominent karyotypic feature was thus the occurrence of deletions and losses of whole chromosome copies indicating the importance of tumor suppressor genes in transitional cell carcinoma pathogenesis. Invasive carcinomas were karyotypically more complex than were low grade superficial tumors. Specific losses of material from chromosome 9 and from chromosome arms 11p and 8p, and gains of 8q and 1q seem to be early changes appearing in superficial tumors, whereas losses from 4p and 17p and the formation of an isochromosome for 5p were associated with more aggressive tumor phenotypes.

Aged↗

PubMed2001

Translocation (14;19)(q32;q13) detected by spectral karyotyping and lack of BCL3 rearrangement in CD5-positive B-cell lymphoma associated with hemophagocytic syndrome.

It has been shown that some cases of B-cell non-Hodgkin lymphoma associated with a hemophagocytic syndrome (B-LAHS) have chromosomal abnormalities at 14q32 or 19q13. We report here a 64-year-old woman with B-LAHS and a complex karyotype including add(14)(q32). We applied spectral karyotyping and revealed that the add(14)(q32) was derived from a der(14)t(14;19)(q32;q13). However, rearrangement of the BCL3 gene at 19q13 could not be detected by Southern blot analysis. Our results indicate that the translocation involving 19q13 may be one of the recurrent aberrations in B-LAHS and that the molecular mechanism of t(14;19)(q32;q13) in B-LAHS appear to be different from that observed in chronic lymphocytic leukemia.

B-Cell Lymphoma 3 Protein↗

PubMed2000

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.

Chromosome Aberrations↗

PubMedDate not supplied

Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements.

Spectral karyotyping (SKY) is a new fluorescence in situ hybridisation (FISH) technique that refers to the molecular cytogenetic analysis of metaphase preparations by means of spectral microscopy. For SKY of human metaphase chromosomes, 24 chromosome-specific painting probes are used in just one FISH experiment. The probes are labelled by degenerate oligonucleotide-primed PCR using three fluorochromes and two haptens. Each probe is differentially labelled with one, two, three or four fluorescent dyes, resulting in a unique spectral signature for every chromosome. After in situ hybridisation and immunodetection, a spectral image is acquired using a conventional fluorescence light microscope equipped with a custom-designed triple-bandpass filter and the SpectraCube, which is able to retrieve spectral information for every pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the unique emission spectra of the chromosomes. Together with chromosome banding information from an inverted DAPI or a G-banded metaphase, a comprehensive overview of chromosomal aberrations is presented.

Color↗

PubMed1999

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band-like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q- and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q- and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336-345, 1999.

Adult↗

PubMed2004

Identification of 14 rare marker chromosomes and derivatives by spectral karyotyping in prenatal and postnatal diagnosis.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.

Chromosome Aberrations↗

PubMed2005

Chromosome abnormalities in 10 lung cancer cell lines of the NCI-H series analyzed with spectral karyotyping.

The karyotypes of 10 lung cancer cell lines of the NCI-H series were analyzed with spectral karyotyping (SKY): 7 non-small lung cancer (NSCLC) lines and 3 small cell lung cancer (SCLC) lines. Modal chromosome number ranged from 42 (NCI-H2171) to 72 (NCI-H2126). All lines showed at least six structural abnormalities, and most had amplifications visible as double minutes or homogeneously staining regions (HSRs). Four reciprocal translocations were found: t(1;17)(p10;p10) in NCI-H82, t(3;6)(q24;q21) and t(12;17)(p10;p10) in NCI-H2009, and a complex t(2;6) in NCI-H1437. NCI-H1770 had a striking HSR containing many copies of the NMYC region. Karyotypes showed a wide range of relationship between numerical and structural change. Two of the lines showed little numerical change but many structural rearrangements (NCI-H209 with mode 46, but 12 rearrangements, and NCI-H2009 with mode 48 but 27 rearrangements). A second group had karyotypes that appeared to have evolved by unbalanced translocation leading to proportionate loss of chromosomes, with or without endoreduplication. In other lines, notably NCI-H2122, the structurally abnormal chromosomes appeared to have been added to a near-diploid karyotype. The karyotypes contribute to a full genomic characterization of these lines, almost all of which have matching normal lymphoblastoid cell lines.

Adenocarcinoma↗

PubMed2003

Spectral karyotype (SKY) analysis of human prostate carcinoma cell lines.

BACKGROUND: Well-characterized in vitro model systems provide an invaluable tool for studying prostate cancer in the laboratory. Detailed karyotypes have been reported using modern techniques such as multiplex fluorescence in situ hybridization (M-FISH) and spectral karyotyping (SKY) for LNCaP, DU 145, NCI-H660, and PC-3 cell lines. However, karyotypic data for more recently established prostate carcinoma cell lines are still limited. METHODS: Classical (G-banding) and SKY analyses were performed on ten prostate carcinoma cell lines: 22Rv1, CWR-R1, DuCaP, LAPC-4, MDA PCa 1, MDA PCa 2a, MDA PCa 2b, PC-346C, PSK-1, and VCaP. RESULTS: Chromosomal abnormalities were identified in all cell lines, although the number and complexity varied greatly among them. PC-346C, established from a primary tumor, exhibited the lowest number (3) of clonal structural abnormalities, while DuCaP, established from a metastasis from a hormone-refractory patient, exhibited both the highest number (31) and largest complexity of structural abnormalities. In various subsets of these models, breakpoints were identified in chromosomal regions previously described as being involved in prostate cancer (e.g., 8p, 10q, 13q, and 16q). CONCLUSIONS: The present study provides a comprehensive karyotypic analysis of a large number of prostate carcinoma cell lines, and offers a valuable resource for future investigations.

Carcinoma↗

PubMed2005

Identification of a ring chromosome with spectral karyotyping in a pleural synovial sarcoma.

The origin of a ring chromosome in a monophasic synovial sarcoma of the diaphragmatic pleura of an 18-year-old man was investigated using spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Conventional cytogenetic analysis revealed the following clonal karyotypic abnormalities: 47,Y,t(X;18)(p11.2;q11.2),t(11;19)(q12;q13.4),+12,-13,+r[6]. The SYT-SSX1 fusion transcript was detected with reverse transcriptase-polymerase chain reaction analysis. SKY analysis suggested that the ring chromosome was composed of material from chromosome 8. Subsequent FISH analysis with a whole-chromosome 8 paint probe confirmed the SKY results. This study demonstrates the usefulness of SKY as an adjunct for determining the chromosomal composition of ring chromosomes.

Adolescent↗

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↗

PubMed2001

Molecular cytogenetic analysis of non-small cell lung carcinoma by spectral karyotyping and comparative genomic hybridization.

The overall pattern of chromosomal changes detected by spectral karyotype (SKY) analysis of two cell lines of each major histological subtype of NSCLC, namely squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), indicated a greater degree of chromosomal rearrangement, than was present or predicted by either comparative genomic hybridization (CGH) or G-banding analysis alone. To investigate these observations, CGH was used to screen DNA derived from 8 primary tumors and 15 cell lines. The results indicated that the most frequently gained chromosome arms were 5p (70%), 8q (65%), 15q (52%), 20q (48%), 1q (43%), 19q (39%), 3q (35%), and 11q (35%). Chromosomal losses were less frequently observed, and included 18q (39%), 9 (35%), 6q (30%), 13q (21%), 5q12-q32 (17%), and 19p (17%). Amplifications were found on 2p23-p24, 3q24-q27, 5p, 6cen-p21.1, 6q26, 7p21, 7q31, 8q, 11q13-qter, 20q12-q13.2. Comparison between CGH findings of the two major histological subtypes showed that gains at 1q22-q32.2, 15q, 20q, and losses at 6q, 13q, and 18q was common in ADCs, whereas SQCCs exhibited gains/amplifications at 3q. Distal 8q was gained by CGH in 65% of tumors of both subtypes. Low level MYCC amplification was confirmed by direct fluorescence in situ hybridization (FISH) analysis. The pattern of overall chromosomal changes detected using combinations of molecular cytogenetic analytical methods suggests that it will be easier to detect recurrent subtype-dependent aberrations in NSCLC.

Adenocarcinoma↗

PubMed1998

Baboon/human homologies examined by spectral karyotyping (SKY): a visual comparison.

Baboon (Papio hamadryas) metaphase chromosomes were analyzed using spectral karyotyping (SKY), a technique combining fluorescence microscopy, CCD-imaging, and Fourier spectroscopy. Results from a comparison of SKY analyses using probes derived from human chromosomes on baboon metaphases were consistent with the majority of comparative gene mapping data between the two species. These data were also compatible with earlier studies comparing macaque and human chromosomes. Human (HSA) chromosome 2 was homologous to baboon (PHA) chromosomes 12 (HSA 2q) and 13 (HSA 2p), whereas three baboon chromosomes corresponded to two different human chromosomes: PHA 3 to HSA 7 and HSA 21, PHA 7 to HSA 14 and HSA 15, and PHA 10 to HSA 20 and HSA 22. These results support the retained synteny between the Hominidae and Cercopithecidae genomes.

Animals↗

PubMed2006

Characterization of chromosomal aberrations in diffuse large B-cell lymphoma (DLBL) by G-banding and spectral karyotyping (SKY).

Cytogenetic chromosome analysis by classical G-banding was supplemented by spectral karyotyping (SKY) in 12 cases of diffuse large B-cell lymphoma (DLBL). SKY is a fluorescence in-situ-based, genome-wide screening technique allowing identification of genetic material even in highly condensed metaphase chromosomes of poor morphology. By simultaneous hybridization of whole chromosome painting probes onto tumor chromosome spreads genetic rearrangements are visualized permitting the clarification of even complex karyotype alterations and the identification of genetic material of previously unknown origin, so-called marker chromosomes. Taking the SKY results into account, we reevaluated the G-banding karyotypes initially carried out, thus generating a more precise karyotype in ten of twelve (83%) cases investigated. In particular, thirteen chromosomal rearrangements not correctly recognized by classical cytogenetics were identified, the genetic origin of seven marker chromosomes was elucidated and three structural genetic rearrangements were redefined. We found SKY to be a valuable technique to establish a definite karyotype in addition to classical cytogenetics.

Chromosome Aberrations↗