Cross-species Color Banding Research Articles

5178 Is mammographic breast density a breast cancer risk factor in women with BRCA mutations?

We have studied cytogenetic rearrangements in karyotypes of five neuroblastoma cell lines [SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, SMS-KCNR] by G-banding, cross species color banding (RxFISH), and fluorescence in situ hybridization (FISH) with chromosome painting probes. Each neuroblastoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. The number of rearranged chromosomes in SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, and SMS-KCNR was 11, 3, 7, 14 (tetraploid, 20–21), and 6, respectively. The origins of abnormal chromosomes were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The chromosomal origin of the homogeneously staining region in SH-SY5Y was identified as coamplification of chromosome bands 2p13 and 2p24 by chromosome microdissection and FISH. The non-random rearrangements of chromosomes were determined on 1p34∼p36, 6q16∼q21, 8q24, 9q34, 11q13∼q23, 16q23∼q24, 17q21, and 22q31. These results may provide useful information for further molecular characterization of neuroblastoma.

Progresses on the methods of tumor chromosome aberration analysis

Most cancers are known to be associated with chromosome aberration, and chromosome analysis is essential to understand the relationships between chromosome aberration and cancer. Here we briefly introduce several methods of chromosome aberration detection, including G-banding, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), multi-fluorescence in situ hybridization (M-FISH), cross-species color banding (Rx-FISH), comparative genomic hybridization (CGH)and Array comparative genomic hybridization (Array CGH).

Cytogenetic and FISH Study of 92 Patients with Splenic Marginal Zone B-Cell Lymphoma (SMZBCL).

Background. In the World Health Organization classification system (WHO), Splenic Marginal Zone B-cell Lymphoma (SMZBCL) is described as an indolent B-cell lymphoma, which generally presents as splenomegaly with involvement of the bone marrow and peripheral blood. The immunophenotype is usually IgM+, IgD+/−, cytoplasmic-Ig−/+, pan B antigens+, CD5−, CD10−, CD23−, CD43−/+ and cyclin D1−. A minority of cases (< 20%) express CD5 antigen. The most frequent cytogenetic aberrations are deletions at 7q22–q32, gains of chromosome 3/3q and involvement of chromosomes 1 and 8. The objective of this study is the genetic characterization of SMZBCL by conventional cytogenetics and FISH.Patients and methods. We present 92 patients with SMZBCL from a multicenter study after being diagnosed morphologically and immunologically. Conventional cytogenetic studies were carried out on lymphoid cells from 72 hour-peripheral blood and/or spleen cultures stimulated with TPA. FISH was performed with centromeric probes from chromosome 3 and 12 and locus specific probes from P53 gene and 7q31 loci. Cross-species color banding FISH (RxFISH) probe was applied in 11 patients and SKY in 4 patients to redefine/confirm G-banding karyotype.Results. A clonal chromosome abnormality was found in 62/92 patients (67%) being identified in 37 (60%) as a complex karyotype. The most frequent recurrent abnormalities were deletions of 7q (30 cases) and gains of chromosome 3 (20 cases). 6/20 cases with gains #3, were gains of 3q arm, in three of them resulting from an unbalanced translocation. Thirteen cases showed rearrangements of 3q without gain of material. The chromosomes most frequently involved were: 7q, 3q, 14q, 8q and 1q (in more than 10 cases). Four cases presented gain of chromosome 3/3q and 7q deletion in the same karyotype. Four novel cytogenetic aberrations involving 14q32 were found in this series: t(6;14)(p12;q32) in two cases, t(14;19)(q32;q13) in two cases and t(10;14)(q24;q32), t(1;14)(q21;q32) and t(9;14)(pter-p22::14q32-14q11::9q22) in one case respectively.Conclusions.-In our series, the most frequent cytogenetic aberration in SMZBCL is a deletion at 7q (48%) and could be considered as a cytogenetic marker of these entity.-Gains of chromosome 3 or partial gains affecting only 3q arm are frequently observed in SMZBCL (32%) being chromosome 3 commonly rearranged.-RxFISH ans SKY techniques help us to define new aberrations not detected by conventional cytogenetics.-We describe translocations involving 14q32 that could help us to identify novel oncogenes related to SMZBCL.

Cytogenetic and molecular genetic analyses of endometrial stromal sarcoma: nonrandom involvement of chromosome arms 6p and 7p and confirmation of JAZF1/ JJAZ1 gene fusion in t(7;17)

Endometrial stromal sarcomas (ESS) are rare neoplasms with the capacity both to invade the myometrium locally and to give rise to extrauterine metastases. Cytogenetic abnormalities have been reported in 22 cases of ESS, mostly involving rearrangements of chromosomes 6, 7, and 17. The most characteristic translocation of this tumor type, t(7;17)(p15∼p21;q12∼q21), was recently shown to generate a JAZF1/ JJAZ1 fusion gene. We report three additional cases of ESS with abnormal karyotypes, whose interpretation was based on the combined analysis by conventional cytogenetics and cross-species color banding FISH (RxFISH). The combination of G-banding and RxFISH in every case gave additional information beyond that obtained by either technique alone, determining the identity of even complex inter- as well as intrachromosomal rearrangements. In one of the three tumors, a t(7;17) was seen; molecular genetic studies identified the JAZF1/ JJAZ1 fusion gene in this case. Two tumors had aberrations that included structural changes of chromosome arms 6p and 7p. Evidently, karyotypic, and hence pathogenetic, heterogeneity exists for tumors classified as endometrial stromal sarcomas based on their phenotypic features.

A comprehensive karyotypic analysis on Korean hepatocellular carcinoma cell lines by cross-species color banding and comparative genomic hybridization

Chromosomal aberrations were investigated in hepatitis B virus integrated into the hepatocellular carcinoma (HCC) cell lines SNU-368, SNU-449, SNU-398, SNU-182, and SNU-475 using Giemsa-banding, cross species color banding, and comparative genomic hybridization (CGH). The origins of the marker chromosomes were confirmed by fluorescence in situ hybridization with constructed chromosome painting probes. Each cell line had unique modal karyotypic characteristics and showed variable numbers of numerical and structural clonal cytogenetic aberrations. The gains were commonly detected on chromosome 1, and chromosome regions 6p, 7q, 8q, 10p, 17q, and 20; the losses were often found on 4q21∼qter, 13, 18q21∼qter, and Y. In particular, the breakpoints on 1p36, 1p13∼q21, 2p13∼q11, 6q10∼q11, 7q11, 7q22, 14q10, 16q10∼q13, 17q21, 18q21, and 19p11∼q11 were involved frequently at the multiple rearranged lesions. CGH analysis further confirmed the cytogenetic data, and the nonrandom rearrangements data suggested the candidate regions for the genes to be isolated which were related to HCC.

Multicolor fluorescence in situ hybridization in clinical cytogenetic diagnostics.

Multicolor fluorescence in situ hybridization is a technology that has vastly expanded the diagnostic repertoire of the clinical cytogenetics laboratory. The limitations of conventional chromosome banding analysis can often be overcome by the high sensitivity and specificity of multicolor fluorescence in situ hybridization tests. This article reviews the latest multicolor fluorescence in situ hybridization tests (including multiplex fluorescence in situ hybridization, spectral karyotyping, cross-species color banding, and comparative genomic hybridization) that are currently limited to a few select clinical cytogenetic laboratories, but may soon have more dominant roles in clinical cytogenetic practice.

Complete cytogenetic characterization of the human breast cancer cell line MA11 combining G-banding, comparative genomic hybridization, multicolor fluorescence in situ hybridization, RxFISH, and chromosome-specific painting

The MA11 cell line was established from malignant cells isolated from the bone marrow of a breast cancer patient. It metastasizes selectively to the brain in athymic mice. Since the genomic rearrangements of only a few breast cancer cell lines have been characterized completely, we analyzed MA11 cytogenetically. Because the G-banding analysis revealed a very complex karyotype with several markers and chromosomes with additional material of unknown origin, we used multicolor fluorescence in situ hybridization (M-FISH), cross-species color banding (RxFISH), comparative genomic hybridization (CGH), and chromosome-specific probes to better characterize the chromosome abnormalities. The use of these FISH-based screening techniques allowed us to detect previously unsuspected chromosomal changes and determine the identity of chromosomal markers. Multicolor FISH was especially useful to identify the rearranged chromosomes, whereas RxFISH, G-banding, and CGH were instrumental in determining breakpoint positions, although some uncertainties were removed only after hybridization with chromosome-specific probes. The combined analysis revealed a near-triploid karyotype with no less than 20 chromosomes demonstrating structural rearrangements. The resulting imbalances included several of those known to be common in primary breast carcinomas (gain of 1q, 8q, and 20q and loss of 8p, 11q, and 13q), indicating that the MA11 cell line may serve as a good model to study breast carcinogenesis. The full cytogenetic characterization we present may guide future searches for the mechanism of organ-selective metastasis in this model system and, possibly, also in vivo.

Chromosomal aberrations in neuroblastoma cell lines identified by cross species color banding and chromosome painting

We have studied cytogenetic rearrangements in karyotypes of five neuroblastoma cell lines [SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, SMS-KCNR] by G-banding, cross species color banding (RxFISH), and fluorescence in situ hybridization (FISH) with chromosome painting probes. Each neuroblastoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. The number of rearranged chromosomes in SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, and SMS-KCNR was 11, 3, 7, 14 (tetraploid, 20–21), and 6, respectively. The origins of abnormal chromosomes were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The chromosomal origin of the homogeneously staining region in SH-SY5Y was identified as coamplification of chromosome bands 2p13 and 2p24 by chromosome microdissection and FISH. The non-random rearrangements of chromosomes were determined on 1p34∼p36, 6q16∼q21, 8q24, 9q34, 11q13∼q23, 16q23∼q24, 17q21, and 22q31. These results may provide useful information for further molecular characterization of neuroblastoma.

New t(11;12)(q12;q11) characterized by RxFISH in a patient with T-cell large granular lymphocyte leukemia

Chromosomal abnormalities in patients with large granular lymphocyte leukemia (LGLL) are rare. Herein we present a novel cytogenetic abnormality t(11;12)(q12;q11) in a patient with LGLL identified by cross-species color banding (RxFISH). The application of RxFISH allowed the rapid and easy identification of a chromosome rearrangement that was not recognized by conventional cytogenetics. Therefore, RxFISH is a suitable complement to, but not a replacement for, conventional cytogenetics.

Characterization of chromosomal aberrations in lung cancer cell lines by cross-species color banding

Using cross-species color banding (RxFISH) and chromosome painting techniques, chromosomal aberrations were investigated in six lung cancer cell lines (NCI-H524, H865, H522, H1373, H358, A549). Each cell line had a variable number of numerical and structural cytogenetic aberrations. While NCI-H524, -H865, and -H522 had near diploidy, NCI-H358, -H1373, and A549 had near triploidy. The origins of the marker chromosomes were further identified by RxFISH and chromosome painting: Nonrandom chromosomal rearrangements were seen on 1p, 3q, 5p10–p15, 6q13–q21, 7q22–q31, 9p32, 15q22–qter, 17p, 17q21–q25, and 21. These abnormal cytogenetic findings indicate that multiple genetic lesions are associated with the development of lung cancer, and thus, these might be possible candidate regions for the abnormal genes involved in lung cancer.

Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumor-specific chromosomal aberrations

Spectral karyotyping (SKY), multiple fluorescence in situ hybridization (M-FISH), cross-species color banding (Rx-FISH), multicolor chromosome banding, and other labeling techniques and strategies have been recent comprehensive technical developments in the field of molecular cytogenetics. The immediate goals of these methods are (1) to reliably characterize complex chromosomal rearrangements present in tumor karyotypes; (2) to screen for new tumor-specific chromosomal aberrations; (3) to improve genetic classification systems of different tumor types in correlation with clinical data, treatment regimens, detection of minimal residual disease, and prognosis; and (4) to identify new target regions for gene identification strategies. We present a brief overview of the different methods, including summaries of numerous published and submitted papers detailing specific cytogenetic aberrations associated with lekemias and lymphomas. To date, 640 tumor cases have been analyzed by SKY, including 410 hematologic malignancies, 146 solid tumors, and 45 mouse tumors.

Characterization of Chromosomal Breakpoints in an ALL Patient using Cross-Species Color Banding

Cross-species color-banded karyotype (Rx-FISH) results were compared with those of conventional G-banded metaphases from the same sample. Breakpoints and karyotype were confirmed as 46,XX,t(8;22)(q24;q11),der(9)t(1;9)(q21;p13) through the novel technology of cross-species color banding in an acute leukemic patient (ALL, L3); the karyotype was 46,XX,t(8;22)(q24;q11),der(9)t(1;9)(q25;p24) by conventional G-banding.

Detailed Genome-Wide Screening for Inter- and Intrachromosomal Abnormalities by Sequential G-Banding and RxFISH Color Banding of the Same Metaphase Cells

While the now-classic chromosome banding methods, such as G-banding, remain the techniques of choice for the initial screening for karyotypic abnormalities, sometimes chromosomal rearrangements involve segments too small or too similarly banded to be detected or described adequately by these techniques. The necessity to use a genome-wide, fluorescence in situ hybridization (FISH)-based screening technique as a complement to G-banding is especially obvious in cases where the information obtained by the latter analysis does not provide an adequate guide to the choice of probes for chromosome-specific FISH. Furthermore, the same metaphase cells should ideally be used for both G-banding and FISH analysis to overcome the scarcity of metaphases observed in many cases and to ensure the correct interpretation of chromosomal aberrations in cytogenetically unstable neoplasms with massive cell-to-cell karyotypic variability. We describe a protocol which enables cross-species color banding (RxFISH), a new FISH-based screening technique that simultaneously imparts specific color banding patterns on all chromosomes, of preparations that have been G-banded and mounted for up to several years, as well as a procedure allowing chromosome-specific painting of the same metaphase cells to resolve whatever doubts persist after the preceding G-banding and RxFISH analyses. This approach makes possible a detailed, genome-wide screening for inter- and intrachromosomal abnormalities including archival cases whose karyotypic rearrangements had been incompletely identified by G-banding.

Cross-species color banding in ten cases of myeloid malignancies with complex karyotypes

Cross-species color banding is a multiple-color fluorescence in situ hybridization (FISH) technique using probes developed from other animal species. Hybridization to human metaphases produces color banding patterns specific for each homologous chromosome pair. The technique has been evaluated in a complementary manner with G-banding and chromosome painting in a series of 10 myeloid malignancies with complex or unresolved karyotypes. Color banding detected the majority of chromosomal abnormalities, which had been identified by G-banding and in each case revealed chromosomal changes that G-banding had not identified. Painting was necessary to confirm these abnormalities due to the limitation of only seven colors in the color-banded karyotype. At the same time, painting fortuitously uncovered cryptic abnormalities in 6 of 10 cases that had not been detected by color banding. Insertions were visible by painting only. This study has demonstrated that in the analysis of complex karyotypes, the application of color banding revealed the involvement of the long arm of chromosome 3, indicating a poor risk, in two cases not identified by G-banding. Therefore, these techniques applied together have revealed cryptic chromosomal abnormalities with prognostic significance, which in some cases may have implications for patient management.

Establishment and characterization of chromosomal aberrations in human cholangiocarcinoma cell lines by cross-species color banding.

Cholangiocarcinoma (CC), a malignant neoplasm of the biliary epithelium, is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. Furthermore, little is known about the genetics and biology of CC. Only a few reports concerning cytogenetic studies of CC have been published, and few cell lines have been established. We recently established four CC cell lines, designated as SCK, JCK, Cho-CK, and Choi-CK, and report the first application of cross-species color banding (RxFISH) and multiple chromosome painting for the characterization of the chromosomal rearrangements of these CC cell lines. Each cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. Chromosomes 3, 6, 7, 8, 12, 14, 17, and 18 were commonly involved in structural abnormalities. Homogeneously staining regions were determined in SCK and JCK, and double minute chromosomes were found in Cho-CK. The chromosomal aberrations of the four CC cell lines were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The nonrandom rearrangements suggest candidate regions for isolation of genes related to CC.

Multiplex Fluorescence In Situ Hybridization and Cross Species Color Banding of a Case of Chronic Myeloid Leukemia in Blastic Crisis with a Complex Philadelphia Translocation

Exciting new techniques in molecular cytogenetics—namely, spectral karyotyping, multiplex fluorescence in situ hybridization (M-FISH), and cross species color banding—have been recently developed. An increasing number of reports demonstrate the success of these procedures in providing additional cytogenetic information—identifying marker chromosomes and revealing the presence of previously undetected chromosomal changes. However, these procedures have their limitations, and their absolute sensitivity in the accurate identification of subtle chromosomal abnormalities remains to be established. M-FISH and color banding have been applied to a case of chronic myeloid leukemia with a complex Philadelphia translocation involving chromosomes 9, 17, and 22, which had initially been identified from G-banded chromosome analysis. The abnormalities were confirmed by chromosome “painting” and specific probes. Although M-FISH and color banding revealed no additional cryptic chromosomal changes, this study has clearly demonstrated the success of these multiple color FISH approaches in the accurate characterization of a complex rearrangement with subtle abnormalities.

Cross-species color banding characterization of chromosomal rearrangements in leukemias with incomplete G-band karyotypes

Karyotype analysis has depended on chromosome banding techniques since their introduction in about 1970, and the information thus obtained is indispensable for the clinical management of patients with hematologic malignancies. Sometimes, however, chromosomal rearrangements involve segments too small, too similarly banded, or too complex to be described adequately or even to be detected by G-banding. Cross-species color banding is a new FISH-based screening technique that enables the generation of a specific color banding pattern for each human chromosome based on the genomic homologies between man and various species of apes. We report the first application of cross-species color banding (RxFISH) to characterize the chromosomal rearrangements of 10 leukemia samples the G-band karyotypes of which were incomplete. The combination of G-banding and RxFISH in every case yielded additional information beyond that obtained by either technique used alone, determining the identity of even the most complex, inter- as well as intrachromosomal, rearrangements. Genes Chromosomes Cancer 26:13-19, 1999.

Cross‐species color banding characterization of chromosomal rearrangements in leukemias with incomplete G‐band karyotypes

Karyotype analysis has depended on chromosome banding techniques since their introduction in about 1970, and the information thus obtained is indispensable for the clinical management of patients with hematologic malignancies. Sometimes, however, chromosomal rearrangements involve segments too small, too similarly banded, or too complex to be described adequately or even to be detected by G-banding. Cross-species color banding is a new FISH-based screening technique that enables the generation of a specific color banding pattern for each human chromosome based on the genomic homologies between man and various species of apes. We report the first application of cross-species color banding (RxFISH) to characterize the chromosomal rearrangements of 10 leukemia samples the G-band karyotypes of which were incomplete. The combination of G-banding and RxFISH in every case yielded additional information beyond that obtained by either technique used alone, determining the identity of even the most complex, inter- as well as intrachromosomal, rearrangements. Genes Chromosomes Cancer 26:13–19, 1999. © 1999 Wiley-Liss, Inc.

Combined RxFISH/G-banding allows refined karyotyping of solid tumors.

Chromosome banding analysis of solid tumors often yields incomplete karyotypes because of the complex rearrangements encountered. The addition of fluorescence in situ hybridization (FISH) methods has helped improve the accuracy of solid tumor cytogenetics, but the absence of screening qualities from standard FISH approaches has proved a severe limitation. We describe the cytogenetic analysis of ten solid tumors using G-banding followed by cross-species color banding (RxFISH), a FISH-based screening technique giving a chromosome-specific banding pattern based on the genomic homologies between humans and gibbons. The addition of RxFISH analysis in all cases led to the identification of previously unidentified intra- as well as interchromosomal rearrangements, thus giving a much more certain and detailed karyotype. In two gastric stromal sarcomas, a tumor type for which no cytogenetic data were hitherto available, numerical chromosomal aberrations dominated, but one of the tumors also carried an unbalanced 7;17-translocation with the same breakpoint in chromosome 17 as that seen in endometrial stromal sarcomas.