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GENES, CHROMOSOMES & CANCER 27:418–423 (2000) 33333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333 BRIEF COMMUNICATION 333333333333333333333333333333333333333333333333333333333333 Spectral Karyotyping Combined With Locus-Specific FISH Simultaneously Defines Genes and Chromosomes Involved in Chromosomal Translocations Giovanni Tonon, Anna Roschke, Kristen Stover, Yaping Shou, W. Michael Kuehl, and Ilan R. Kirsch* Genetics Department, Medicine Branch, National Cancer Institute, Bethesda, Maryland 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. Genes Chromosomes Cancer 27:418–423, 2000. Published 2000 Wiley-Liss, Inc.† In the past three decades, cytogenetics has greatly advanced our understanding of tumor biology. Chromosomal banding techniques, the central tool of standard cytogenetics, have identified gains or losses of specific chromosomal fragments and have defined chromosomal rearrangements in metaphase cells from tumor cell lines and patient samples. In particular, the cloning of DNA proximate to the breakpoints of chromosomal translocations has led to the discovery of many genes involved in critical steps in the initiation and progression of cancer (Rowley, 1998). Cytogenetics has been particularly helpful in the identification of rearranged genes associated with hematologic tumors. This has been most easily accomplished when only one or a very few rearrangements are present. However, most solid tumors and some hematologic malignancies have complex karyotypes with many translocations (Bardi et al., 1993; Mertens et al., 1997). A great challenge is the specific definition of these breakpoints and of the genes involved in these karyotypically complex tumors, the vast majority of cancers in man. Classical cytogenetics, through G-banding, is not up to the task of accurately and reliably defining these complex karyotypes (Schrock et al., 1997). Comparative genomic hybridization (CGH) (du Manoir et al., 1995), spectral karyotyping (Garini et Published 2000 Wiley-Liss, Inc. †This article is a US Government work and, as such, is in the public domain in the United States of America. al., 1996; Schrock et al., 1996; Veldman et al., 1997; Macville et al., 1999), and fluorescence in situ hybridization (FISH) (Raap, 1998) have greatly increased the resolution of cytogenetics, introducing a new era for the study of the genetics of cancer. Thanks to these techniques, the analysis of the genome has become much more informative, comprehensive, and accurate. Spectral karyotyping and multiplex-fluorescence in situ hybridization (M-FISH) (Speicher et al., 1996) involves the simultaneous hybridization of 24 chromosome-specific painting probes labeled with different fluorochromes or fluorochrome combinations. A spectral classification then assigns a discrete pseudo-color to all pixels with the identical spectrum. Therefore, a specific color defines each chromosome. Locus-specific FISH is able to locate at which site on a specific chromosome a probe hybridizes. The introduction of fluorescent dyes for labeling DNA probes has greatly improved the spatial resolution and the sensitivity of detection of in situ hybridization (Wiegant et al., 1991). Moreover, when more than one fluorochrome is used, *Correspondence to: Ilan R. Kirsch, M.D., Genetics Department, Medicine Branch, National Cancer Institute, NNMC, 8901 Wisconsin Avenue, Bldg. 8, Room 5101, Bethesda, Maryland 20889-5105. E-mail: [email protected] Received 16 July 1999; Accepted 16 September 1999 COMBINATION OF SPECTRAL KARYOTYPING AND FISH multiple loci can be detected simultaneously in the same metaphase cell. Even with these new approaches, delineating the chromosomes and, at the same time, the genes affected by chromosomal rearrangements, is very time- and labor-intensive. Multiple rounds of FISH and chromosomal painting are often necessary, the task being additionally complex if a gene is present in multiple copies and involved in multiple translocations. To try to solve this problem, we developed a technique that combines spectral karyotyping and FISH. This approach retains the refinements of each technique, but it adds the critical feature of defining, in a single step, both the involvement of a specific gene in a translocation and the chromosomes involved. Moreover, the use of different dyes for different probes, in a single hybridization, allows the analysis of different genes in a single metaphase cell. For the preparation of the metaphase cells, we used standard cytogenetic procedures (Kirsch et al., 1982). Dr. Abraham Karpas kindly provided Karpas 620 cells. Slides of metaphase cells from peripheral normal blood lymphocytes were obtained from Vysis (Downers Grove, IL). The spectral karyotyping hybridization protocol has been described in detail (Schrock et al., 1996). Slides were hybridized simultaneously with a spectral karyotyping probe mixture, containing 24 distinctly labeled chromosome-specific probes, and FISH probes. Specific chromosomes, kindly provided by Dr. Thomas Ried, were obtained by high-resolution flow sorting, and then amplified in our laboratory by two consecutive rounds of degenerate oligo-primed (DOP)-PCR amplification. Subsequently, the probes were distinctly labeled with combinations of three fluorochromes and two haptens, to create a unique spectral definition for each chromosome. As fluorochromes, Spectrum Orange (Vysis), Rhodamine 110 (Perkin Elmer, Foster City, CA), and Texas Red (Molecular Probes, Eugene, OR) were used for the direct labeling, whereas biotin-16-dUTP and digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) were used for indirect labeling. One hundred nanograms of each chromosome-specific probe was mixed and ethanolprecipitated with variable amounts of the FISH probes, 50 µg of human Cot-1 DNA (Life Technologies, Rockville, MD), and 1 µl of salmon sperm DNA (Sigma, St. Louis, MO). The precipitate was then resuspended in 10 µl of hybridization solution (50% formamide (Fluka, Milwaukee, WI), 2⫻ standard saline citrate, 10% dextran sulfate). Metaphase 419 chromosome slides were denatured separately at 75°C for 2–3 min in 70% formamide/2⫻ SSC (volume/volume) and dehydrated in an ethanol series. The probe cocktail was applied to the slides, and hybridization was carried out for 48–72 h at 37°C. After hybridization, biotin was detected with Avidin-Cy5 (Amersham, Piscataway, NJ) and digoxigenin-11-dUTP with mouse anti-digoxin antibodies (Sigma) followed by sheep anti-mouse antibodies custom-conjugated to Cy5.5 (Amersham). The slides were counterstained with 4,6-diamidino-2phenylindole (DAPI, Sigma) and covered with antifade solution (Vector, Burlingame, CA). Spectral images were acquired with an SD200 SpectraCube system (Applied Spectral Imaging, Carlsbad, CA) mounted on a Leica DMRBE microscope (Leica, Wetzlar, Germany) through a customdesigned triple bandpass optical filter (SKY v.3; Chroma Technology, Brattleboro, VT). Spectrumbased classification of the raw spectral images was performed using SKYView 2 software (Applied Spectral Imaging). The DNA probes that we used included: the GS53N21 BAC (kindly provided by Dr. Raluca Yonescu), that maps to chromosome 7q21.11 (http://www.ncbi.nlm.nih.gov/CCAP/), a MYC plasmid that maps to 8q24.1, and a 38 IGH BAC containing sequences that hybridize to the two alpha constant/enhancer regions of the IGH locus, at 14q32.3 (Gabrea et al., 1999). For the FISH analysis, probes were labeled by nick translation according to standard procedures (Sambrook et al., 1989). We used the following dyes or haptendUTPs for the nick translation, at 1 mM concentration: Texas Red, Spectrum Orange, Rhodamine 110, Cy5 (Amersham), biotin-16-dUTP, digoxigenin11-dUTP, Cy3 (Amersham), and Alexa 594 (Molecular Probes). Nick translation yielded fragments primarily between 300 and 600 bp. The technique presented here is rapid, simple, and flexible in its applications. In both spectral karyotyping and FISH applications, probes are usually ethanol-precipitated, resuspended in a small volume, and applied to metaphase cells on a slide. We precipitated the probes for spectral karyotyping and FISH together and then hybridized the mixture of the two probes on the same slide. A strong gene-specific signal (FISH signal), localized to the expected chromosome, was evident above the background label of the specific chromosome painting probe set (spectral karyotyping signal) (Fig. 1). The amount of total DNA needed for the FISH probes could be relatively low. We tested amounts of DNA as low as 0.1 µg in the hybridization, and the signal did not decrease in intensity. Therefore, a concen- 420 TONON ET AL. Figure 1. Combination of spectral karyotyping and FISH in a normal chromosome metaphase cell, using as a FISH probe a MYC fragment inserted in a plasmid, labeled with Cy3 (arrows). Figure 3. Combination of spectral karyotyping and FISH, using two FISH probes, one for MYC, labeled with Cy3 and mapping to chromosome 8 (arrowheads) and another, BAC GS53N21, labeled with Alexa 594 and mapping to chromosome 7 (arrows). Figure 2. (a) Combination of spectral karyotyping and FISH applied to a rearranged chromosome, labeled with Spectrum Orange and Rhodamine 110. As FISH probe, a 38 IGH BAC (see text), labeled with Cy3 with a fluorescence spectrum similar to that of Spectrum Orange, maps at the breakpoint (arrows). (b) FISH image of the same derivative, from the same metaphase, using a filter specific for tetramethylrhodamine-5-isothiocyanate (TRITC). tration of probe similar to that used for routine FISH can be used here, without reduction in the signal intensity. Probes with inserts of different sizes and in different vectors have been tested. We used plasmids, cosmids, P1, and BACs, and all of them were effective in yielding a strong signal. More importantly, the size of the insert, which hybridizes to the template, can cover a wide range. COMBINATION OF SPECTRAL KARYOTYPING AND FISH We used as a FISH probe an insert as small as 7.5 kb, and we were still able to visualize easily the gene-specific probe (data not shown). Different dyes or haptens have been used for labeling of the gene-specific probes. Some of them were detected more efficiently than others. Spectrum Orange, Cy3, Alexa 594, Texas Red, and Rhodamine 110 all gave strong signals. In contrast, indirect labeling, using biotin and digoxigenindUTP as haptens, and antibodies or avidin labeled with Cy5 or Cy5.5, respectively, for detection, failed to give signals of enough intensity to be easily visualized. Cy5-dUTP also was not bright enough to be visualized. Only five chromosomes are labeled with a single dye in the standard spectral karyotyping technique that we used, chromosomes 2 (Cy5.5), 8 (Rhodamine 110), 11 (Spectrum Orange), 14 (Texas Red), and 17 (Cy5). All of the other chromosomes are defined by a combination of dyes, which give them each distinct colors. Therefore, if the probe, which is labeled with just one dye, hybridizes to a chromosome painted with more than one dye, the filter and the software easily distinguish between the signal coming from the probe and the signal coming from the chromosome. Obviously, the FISH probe may hybridize to a chromosome labeled uniquely with the same or related dye (for example, Fig. 2). Here, the signals coming from the FISH probe were located at the juxtaposition of two different chromosomes, one of which was labeled with Spectrum Orange, which has a wavelength similar to that of the Cy3-labeled FISH probe. Therefore, in this case, in some metaphase cells, it was difficult to detect the FISH probe on this rearranged chromosome (Fig. 2a). However, we could distinguish the FISH probe from the chromosome painting by analyzing the metaphase using a filter with a narrower spectrum (Fig. 2b). The FISH signals were much brighter than the chromosome painting and were easily distinguishable. Therefore, we suggest the sequential use of the filter for the spectral karyotyping and a filter with a more defined spectrum. It is possible that in certain cases confirmation of an analysis using a second independent FISH alone would be required. However, in approximately two dozen separate cases in which we have compared FISH alone versus simultaneous spectral karyotyping in combination with FISH, we have not yet encountered a situation where the independent FISH analysis gave us more or different information than the method described above using different filters. The FISH probes usually do not interfere with the spectral karyotyping analysis, 421 and the normal two ‘‘dots’’ of the FISH hybridization are easily distinguishable from the small chromosomal insertions identified by the spectral karyotyping. Therefore, it is not necessary to perform separate hybridization of the spectral karyotyping probes, without the FISH probes, to obtain reliable spectral karyotyping data. The possibility of using more than one FISH probe labeled with different dyes will be of great advantage for the analysis of complex karyotypes. For example, we hybridized two probes, one for MYC, labeled with Cy3, and a BAC, GS53N21, mapping to chromosome segment 7q21.11, labeled with Alexa 594. Signals specific for the two probes were easily detectable in the metaphase cell (Fig. 3). The major challenge in the future for cytogenetics will be the analysis of complex karyotypes, both of solid and hematopoietic tumors. To evaluate the ability of this technique to detect the involvement of genes at chromosomal breakpoints and to define the chromosomes involved in a complex karyotype, we hybridized slides of metaphase cells of a myeloma cell line, Karpas 620 (Fig. 4a and b). Gbanding-like images were obtained from DAPI staining of the slides (Fig. 5). A 38 IGH BAC was labeled with Cy3-dUTP. Dysregulation of oncogenes by translocations to the IGH locus, at 14q32, is a critical event in the pathogenesis of B-cell tumors. The multiple translocations present in the karyotype of this cell line were defined by spectral karyotyping analysis. The karyotype that we describe here is partly different from the one previously described (Nacheva et al., 1990). Eight cells were analyzed with DAPI staining, and spectral karyotyping and FISH in combination. A representative example is shown in Figures 4 and 5. The modal chromosome number was 68 (range, 60–68). In the majority of the cells, three copies of chromosomes 3, 5, 6, 17, 20, 21, and 22 and four copies of chromosomes 15, 16, 18, and 19 were present. We found two copies of chromosomes 2, 4, 7, 8, 9, 10, 12, and X, one copy of chromosome 13, and no normal chromosome 1, 11, and 14. The following rearrangements were present in the majority of the cells: a reciprocal translocation t(1;11)(q32;q13), with two copies of der(1) and one copy of der(11); one copy each of der(7)t(4;7)(q31;p15), der (7)t(7;1)(q22;p31) and der(13)t(8;13)(q23;q31); two copies in each cell of der(8)t(8;11)(q24;q13), der(11)t(11;13)(q13;q14), der(14)t(1;14)(q11;q32), and der(14)t(8;14)(q24; q32). Both conventional FISH and the combination of spectral karyotyping and FISH detected six copies 422 TONON ET AL. Figure 4. a: Combination of spectral karyotyping and FISH in Karpas 620 cell line. The FISH probe is a 38 IGH (see text), labeled with Cy3. Six separate areas of hybridization of the FISH probe were evident in all the metaphase cells analyzed. Larger arrows: der(14)t(8;14); smaller arrows: der(14)t(1;14); arrowheads: der(8)t(8;11). b: Classified image of the same metaphase cell. Figure 5. Karyotype table, showing the DAPI-stained, G-banded-like karyotype and the classified image of the Karpas 620 cell line. For a composite description of the karyotype, see text below. of the 38 IGH probe. In two chromosomes, a translocation der(14)t(8;14)(q24;q32) was evident, and the 38 IGH probe was located at the breakpoint. The 38 IGH probe also localized to the breakpoint of two other pairs of chromosome markers: two copies of der(14)t(1;14)(q11;q32) and two copies of der(8)t(8;11)(q24;q13). In the latter case, the presence of the 38 IGH probe at the breakpoint (Figs. 2, 4, and 5) suggests a small insertion of part of chromosome 14 previously undetected by conventional cytogenetic, spectral karyotyping, or more specific chromosome analysis. A conventional FISH analysis, using the 38 IGH probe and chromosome 14-specific painting had also shown the presence of 38 IGH, but not of more extensive chromosome 14 material in this derivative (not shown). Based on this analysis a composite karyotype is: 60-68, XX-X, -1,t(1;11)(q32;q13), der(1)t(1;11)(q32; q13),-2,-4,der(7)t(1;7)(p31;q22),der(7)t(4;7)(q31; p15),⫹8,der(8)t(8;11)(q24;q13)x2,-9,-10,der(11)t(11; 13)(q13;q14)x2,-12,-13,der(13)t(8;13)(q23;q31),der(14) t(1;14)(q11;q32)x2,der(14)t(8;14)(q24;q32)x2,⫹15, ⫹16,⫹18,⫹19[8].ish der(8)t(8;11)ins(14)(q32.3) (IGH)x2, der (14) t(1; 14) (IGH) x2, der (14) t (8;14) (IGH)x2. The human DNA sequence will be determined in the next 2–5 years (Collins et al., 1998). In addition, efforts are under way to establish a standard set of BAC clones spaced at 1–2 Mb intervals across the genome (C-CAP; World Web Site: http:// www.ncbi.nlm.nih.gov/CCAP/). Using these resources, the combination of spectral karyotyping and FISH will allow screening for genes and chromosome involvement in tumors in a much more straightforward manner than can be achieved by separate FISH and spectral karyotyping analyses. COMBINATION OF SPECTRAL KARYOTYPING AND FISH In summary, we describe a simple and rapid technique that allows the definition of genes and chromosomes involved in chromosomal translocations in a single step. This approach is particularly useful for the analysis of complex karyotypes and may help to test hypotheses coming from gene expression studies, like cDNA microarrays, SAGE, and CGAP (Strausberg et al., 1997; Wang and Rowley, 1998). ACKNOWLEDGMENTS We thank Dr. Q. Ying for preparing Karpas 620 slides, Dr. A. Dutra for preparing some normal metaphase cells on slides, and Dr. T. Ried for helpful discussions. REFERENCES Bardi G, Johansson B, Pandis N, Mandahl N, Bak-Jensen E, Lindstrom C, Tornqvist A, Frederiksen H, Andren-Sandberg A, Mitelman F, Heim H. 1993. Cytogenetic analysis of 52 colorectal carcinomas—non-random aberration pattern and correlation with pathologic parameters. Int J Cancer 55:422–428. Collins FS, Patrinos A, Jordan E, Chakravarti A, Gesteland R, Walters L. 1998. 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