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.     

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.     

Clustering of chromosomal breakpoints in neoplasia

It is becoming increasingly difficult to assimilate in a meaningful fashion the wealth of new information regarding chromosomal changes in human cancer. Compilation and analysis of this data is essential if a clear picture of the incidence and specificity of cancer associated chromosome abnormalities is to be seen. This report provides an overview of our most recent and comprehensive analyses on chromosomal changes in 5345 neoplasms, providing further evidence for a clustering of chromosomal breakpoints in human neoplasia.     

A new chromosome banding technique,spectral color banding (SCAN), for full characterization of chromosomal abnormalities

We have developed spectral color banding (SCAN) as a new chromosome banding technique based on spectral analysis of differentially labeled chromosome band-specific painting probes. In this study, we succeeded in displaying a multicolor banding pattern for chromosome 3, which was almost identical to the pattern obtained with the corresponding G-banding. We applied this method to metaphase cells from different normal male donors with various levels of G-banding resolution, ranging from 250 to 850 bands per haploid set. The same multicolor banding pattern was observed in all samples regardless of the length of the chromosomes or the quality of the G-banding. We then used SCAN in a diffuse large B-cell lymphoma case for a complete analysis of the intrachromosomal change for chromosome 3, which could not be fully characterized by G-banding or even by spectral karyotyping (SKY). SCAN could detect the duplicated segment and identify the origin of the chromosome band on the basis of the specific spectral color of each band. This study demonstrates that SCAN is a useful tool for full characterization of chromosomal abnormalities not identified by SKY.     

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.     

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 approximately qter, 13, 18q21 approximately qter, and Y. In particular, the breakpoints on 1p36, 1p13 approximately q21, 2p13 approximately q11, 6q10 approximately q11, 7q11, 7q22, 14q10, 16q10 approximately q13, 17q21, 18q21, and 19p11 approximately 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.     

Improved structural characterization of chromosomal breakpoints using high resolution custom array‐CGH

Lindstrand A, Schoumans J, Gustavsson P, Hanemaaijer N, Malmgren H, Blennow E. Improved structural characterization of chromosomal breakpoints using high resolution custom array‐CGH. Array‐CGH is a powerful tool for the rapid detection of genomic imbalances. By customizing the array it is possible to increase the resolution in a targeted genomic region of interest and determine the structure of the breakpoints with high accuracy, as well as to detect very small imbalances. We have used targeted custom arrays to zoom in on 38 chromosomal breakpoints from 12 different patients carrying both balanced and unbalanced rearrangements. We show that it is possible to characterize unbalanced breakpoints within 17–20,000 bp, depending on the structure of the genome. All of the deletion and duplication breakpoints were further refined and potential underlying molecular mechanisms of formation are discussed. In one of seven carriers of apparently balanced reciprocal translocations we detected a small deletion of 200 bp within the previously FISH‐defined breakpoint, and in another patient, a large deletion of 11 Mb was identified on a chromosome not involved in the translocation. Targeted custom oligonucleotide arrays make it possible to perform fine mapping of breakpoints with a resolution within the breakpoint region much higher compared to commercially available array platforms. In addition, identification of small deletions or duplications in apparently balanced rearrangements may contribute to the identification of new disease causing genes.     

Human chromosomal banding by in situ hybridization of isochores

Further to the classical methods that involve different chromosome treatments followed by staining, in situ hybridization of isochores represents a novel approach to chromosomal banding. Isochores are long compositionally homogeneous DNA segments that, in the human genome, belong to five families, two GC-poor families (L1 and L2) representing 30% and 33% of the genome, respectively, and three GC-rich families (H1, H2 and H3) representing 24%, 7.5% and 4-5- of the genome, respectively. Gene concentration increases with increasing GC levels, reaching an up to 20-fold higher level in H3 compared to L1 isochores. In situ hybridization of DNA from different isochore families on metaphase chromosomes allow to distinguish different sets of Giemsa and Reverse bands. In addition, it also provides information on the chromosomal distribution of genes.     

Cloning of the breakpoints of a submicroscopic deletion in an Angelman syndrome patient

The majority of cases of the two distinct disorders Prader–Willisyndrome (PWS) and Angelman syndrome (AS) result from cytogeneticdeletions of chromosome 15q11–q13. These deletions areexclusively of maternal origin in AS but of paternal originin PWS indicating that the 15q11–q13 region is subjectto genomic imprinting. Transmission of a submicroscopic deletionin one three generation family resulted in AS only upon maternaltransmission of the deletion with no clinical phenotype associatedwith paternal transmission (1, 2). The breakpoint of this submicroscopicdeletion has been cloned and sequenced. This is the first deletionjunction from the AS/PWS region which has been so characterized.The nucleotide sequence of the deletion junction revealed a19 bp insertion of unknown origin with no evidence of repetitiveelements. A probe from the proximal deletion breakpoint, PB11,lies within the currently defined minimum region of deletionoverlap in PWS, which contains the SNRPN and D15S63 locl. Ourresults suggest that the imprinted gene(s) responsible for thePWS phenotype are proximal of pB11 in this deletion overlapregion.     

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma

Chromosomal rearrangements of the MYC locus, which often involve the IG loci, are recurrent events in multiple myeloma (MM) and plasma cell leukemia (PCL). We used dual-color fluorescence in situ hybridization (FISH) to characterize the breakpoint locations of chromosomal translocations/rearrangements involving the MYC locus at 8q24 found in a panel of 14 MM cell lines and 70 primary tumors (66 MM and 4 PCL). MYC locus alterations were observed in 21 cases: MYC/IG (mainly IGH@) fusions in 11 cell lines and three patients (2 MM and 1 PCL), and extra signals and/or abnormal MYC localizations in seven patients (5 MM and 2 PCL). Fourteen of these cases were investigated by FISH analyses by use of a panel of BAC clones covering about 6 Mb encompassing the MYC locus. The breakpoints were localized in a region 100-250 kb centromeric to MYC in four cases, a region 500-800 kb telomeric to the gene in four cases, and regions > or = 2 Mb centromeric or telomeric to MYC in five cases. Two different breakpoints were detected in the KMS-18 cell line, whereas the insertion of a MYC allele was found in a complex t(16;22) chromosomal translocation in the RPMI8226 cell line. Our data document a relatively high dispersion of 8q24 breakpoints in MM.     

Molecular cytogenetic mapping of recurrent chromosomal breakpoints in tenosynovial giant cell tumors

Tenosynovial giant cell tumor (TGCT) is the most common benign tumor of synovium and tendon sheath. Cytogenetic data indicate that 1p11-13 is the region most frequently involved in structural rearrangements. With the aim of eventually identifying the genes associated with TGCT development, we have investigated 1p11-13 breakpoints using fluorescence in situ hybridization (FISH) analysis, with a panel of yeast artificial chromosome (YAC) probes covering 1p11-21. Twenty-six tumors were analyzed by G-banding, and 24 of these showed a breakpoint in 1p11-13. The cytogenetic findings add to previous observations that, among a variety of translocations involving 1p11-13, chromosome 2 is the most common translocation partner, with a breakpoint in 2q35-37. This aberration was found in eight cases. Other recurrent translocation partners, found in two or three cases, were 5q22-31, 11q11-12, and 8q21-22. Material from 21 tumors was available for FISH analysis, which revealed that the breakpoints clustered to one region spanned by two YAC probes, 914F6 and 885F12 located in 1p13.2, in 18 cases. Bacterial artificial chromosome probes were used to map the recurrent breakpoint on chromosome 2. In four of seven cases there was a breakpoint within the sequence covered by probe 260J21, where the RDC1 gene is located, a gene reported to fuse with HMGIC in lipomas with a 2;12 translocation.     

Characterization of chromosomal rearrangements in hematological diseases using spectral karyotyping.

Since chromosomal changes are used both as independent prognostic factors and for therapy design in hematological disorders, it is necessary to elucidate chromosomal changes as accurately as possible. We used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) to further characterize chromosomal abnormalities in 35 patients with hematological disorders. SKY confirmed 149 aberrations, refined 117, and detected 11 hidden changes. Eighteen abnormalities were detected only by G-banding. Ten monosomies and two deletions described by G-banding were shown to be involved in translocations or ring chromosomes. These results demonstrate that SKY increases the accuracy of karyotype interpretation, which is important for proper diagnosis and management of hematological malignancies.     

New chromosomal breakpoints in non-Hodgkin's lymphomas revealed by spectral karyotyping and G-banding

Chromosomal rearrangements in short term cultures from nine cases of non-Hodgkin's lymphomas (NHL) were characterized by G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). Eight of the nine cases showed complex karyotypes with chromosomal aberrations which, in most cases, could not be fully characterized by traditional G-banding analysis alone. Karyotypic abnormalities of special interest were marker chromosomes and chromosomes with added unidentified chromosomal material, as previously non-identified chromosomal translocations were hidden behind these aberrations. SKY and FISH analysis, as a complement to banding analysis, significantly improved the karyotypes in seven of the nine cases and unveiled 21 previously unidentified rearrangements with novel translocation breakpoints. Traditional G-banding alone revealed seven new rearrangements, which were all confirmed by SKY. None of these new aberrations occurred as single clonal rearrangements but as parts of complex karyotypes. Nevertheless, the chromosomal break-point regions identified should be considered as potential hot spots for genes involved in the tumorigenesis of the malignancy.     

Characterization of chromosomal aberrations in human gastric carcinoma cell lines using chromosome painting

Using chromosome painting, a study of chromosomal abnormalities was performed in six gastric carcinoma cell lines (SNU-484, 601, 620, 638, 668, 719) from Korean patients. Each carcinoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. SNU-484, SNU-620, and SNU-668 had near-triploidy; SNU-601, SNU-638, and SNU-719 had near-diploidy. The origins of the marker chromosomes of these cell lines were identified by fluorescence in situ hybridization with constructed painting probes. In all of six cell lines, rearrangement of chromosome 17 resulting in partial deletion of 17p (and/or partial duplication of 17q) was found. The most frequent marker was a partial gain of chromosome 7 with the breakpoints on 7q22 and 7q31. The nonrandom rearrangements of chromosomes were also determined on 1q32, 5q11-q22, 8q, 14q22, 14q34, and 15q15; suggesting that they may be the candidate regions for the isolation of the genes related to gastric cancer.     

BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints

The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genomewide scale, offering a resolution as high as >30,000 "bands" (as defined by the number of BACs within the currently highest-density BAC array) [Ishkanian et al., 2004]. We have tested the possibility of further increasing this resolution using PCR fragments generated from individual BAC clones. Using this approach, we have efficiently defined the proximal and distal breakpoints in two cytogenetic cases, one duplication and one deletion, to within 5-20 kb. The results support the potential use of BAC-based PCR fragments to further improve the resolution of the microarray-CGH strategy by an order of magnitude.     

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.     

Linkage studies exclude the AT-V gene(s) from the translocation breakpoints in an AT-V patient

An 8-year-old girl with severe microcephaly of prenatal onset, borderline intelligence, defects of skin pigmentation, deficiency of both humoral and cellular immunity, a normal serum α-fetoprotein level and hypersensitivity to ionizing irradiation is described. Spontaneous chromosomal breakage in lymphocytes together with the clinical presentation led to the diagnosis of ataxia telangiectasia variant (AT-V). In addition, the patient carried a constitutional translocation of paternal origin: 46,XX,t(3;7)(q12;q31.3) pat. In subsequent linkage and haplotype studies in 12 AT-V families with microsatellite markers from each of the translocation breakpoint regions, we could clearly exclude the localization of an AT-V gene to these regions.     

Characterization of the 5q- breakpoint in an acute nonlymphocytic leukemia patient using pulsed-field gel electrophoresis

Multiple genes of hematopoietic importance have been localized to the long arm of chromosome 5 including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukins (IL) 3, 4 and 5 to 5q23-31, colony stimulating factor 1 (CSF1) to 5q33.1 and its receptor (c-fms ) to 5q33.3. The genes coding for platelet-derived growth factor receptor (PDGFR) and acidic fibroblast growth factor (FGFA) have been localized to 5q31-32 and 5q31.3-33.2, respectively. These genes fall in the region of chromosome 5 which is deleted in the 5q- refractory anemia syndrome (5q- RA) and acute nonlymphocytic leukemia (ANLL). We have characterized this region in a 5q- patient with therapy-related ANLL (t-ANLL) by pulsed-field gel electrophoresis and Southern blotting analysis utilizing DNA probes for PDGFR, c-fms , and FGFA. A single 300 kbp MluI restriction fragment was detected in the patient using a PDGFR probe as compared to a 200 kbp fragment in normal controls. BssHII digestions also showed restriction fragment length difference. Similar data for both MluI and BssHII digestions were also obtained when c-fms was used as a probe. Southern blotting analysis of EcoRI-digested DNA showed that each of the PDGFR, c-fms , and FGFA alleles were deleted. These results suggested that one chromosome 5 has a large deletion involving PDGFR, c-fms and FGFA, which is consistent with the cytogenetic analysis of the patient. In contrast, the other chromosome 5, which appeared normal cytogenetically, may have a smaller deletion (or alteration) in proximity to but not involving any of these 3 genes.     

Sequence analysis of amplified t(14;18) chromosomal breakpoints in B-cell lymphomas

We have explored different strategies for sequencing of major breakpoint (mbr) junctional regions in t(14;18) chromosomal translocations--the most frequent chromosomal abnormality observed in B-cell lymphomas. We demonstrate that coupling of the preparation of single-stranded DNA by asymmetric polymerase chain reaction (PCR) and direct sequencing is the method of choice for the rapid and precise determination of clone-specific bcl-2/JH fusion gene sequences. The rapidity, relative ease, and accuracy of the technique, described for the nucleotide sequence analysis of mbr t(14;18) breakpoints, permits the analysis of a relatively large number of samples and should be considered as part of the clinical evaluation of lymphoma patients. Furthermore, by providing sequence information of clone-specific DNA regions, the procedure should reduce the risk of false-positive results from PCR.