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[Analysis of micronuclei induced under hyperthermic conditions in human lymphocyte culture by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) methods].

The spectral karyotyping (SKY) method is a novel molecular cytogenetic technique which simultaneously discerns entire chromosomes. In order to elucidate the origins of micronuclei induced under hyperthermic conditions in human lymphocyte culture, peripheral blood cells were cultured at 40 degrees C or 42 degrees C for 3-24 h, using the cytokinesis-block method with cytochalasin B. The induced micronuclei were identified by the fluorescence in situ hybridization (FISH) and SKY methods. At 42 degrees C for more than 6 h, the frequency of occurrence of micronuclei in binucleated cells rose with increasing incubation time. By the FISH method, 83.3% of micronuclei induced in 24 h culture at 42 degrees C were shown to be positive for the human centromeric probes. By the SKY method, each micronucleus induced under the hyperthermic conditions was identified unequivocally and shown to contain a specific chromosome. These results suggest that the micronuclei induced under the hyperthermic conditions in human lymphocyte culture contain chromosomes which do not migrate to the poles at the anaphase of the cell cycle because of the breakdown of the spindle apparatus.

Cells, Cultured↗

Spectral karyotyping and fluorescence in situ hybridization analysis of de novo partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter).

An 8-year-old boy presenting with hypotonia, moderate mental retardation, developmental delay, and psychomotor retardation is reported. Magnetic resonance imaging of the brain at age 3 years revealed a Dandy-Walker variant. Cytogenetic analysis of the peripheral blood revealed a derivative chromosome 12 with unknown additional material attached to the distal region of the long arm of chromosome 12. The parental karyotypes were normal. Spectral karyotyping (SKY) using the 24-color SKY probes and fluorescence in situ hybridization (FISH) using the specific 7p, 7q, 12p, and 12q telomeric probes confirmed a duplication of distal 7p and a deletion of terminal 12q. The karyotype of the proband was designated as 46,XY.ish der(12)t(7;12) (p21.2;q24. 33)(SKY+, 7pTEL+, 12qTEL-). The present case provides evidence for the association of partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter) with a cerebellar malformation and the usefulness of SKY and FISH in the identification of a de novo aberrant chromosome resulting from an unbalanced translocation.

Child↗

Increased karyotype precision using fluorescence in situ hybridization and spectral karyotyping in patients with myeloid malignancies.

We studied seven patients with various malignant hematologic disorders using fluorescence in situ hybridization (FISH) and one of these patients with spectral karyotyping (SKY). With appropriate probes, the t(8;21) and inv(16) were confirmed in two patients and the karyotypic precision was increased in five others using FISH and SKY. Two of three patients with 12p rearrangements had a deletion of one TEL allele. Thus, these newer techniques are an important adjunct to accurate chromosome analysis in malignancy.

Adolescent↗

A comprehensive karyotypic analysis on a newly developed hepatocellular carcinoma cell line, HKCI-1, by spectral karyotyping and comparative genomic hybridization.

A continuously growing human hepatocellular carcinoma (HCC) cell line was established from a Chinese male, carrier of the hepatitis B virus (HBV). This cell line, designated HKCI-1, grows as an adhering monolayer of polygonal epithelial cells that embody one or more nuclei. HKCI-1 secretes alpha-fetoprotein but shows no evidence of HBV carriage. Conventional banding analysis of the short-term cultured primary tumor and the propagated HKCI-1 revealed a chromosome modal number of near-triploidy. It was, however, impossible to derive their complete karyotype due to the complex nature of chromosomal rearrangements and many marker chromosomes of uncertain origin. Spectral karyotyping (SKY) is a newly developed molecular cytogenetic technique that allows the unprecedented discernment of chromosomal abnormalities. Spectral karyotyping analysis on HKCI-1 and the primary tumor elucidated all aberrant chromosomes and revealed complex karyograms. Recurring aberrations detected in both primary tumor and HKCI-1 included der(X)t(X;11)(q10;p10), der(1)t(1;10)(q10;?pq), der(4)t(4;16)(p10;q10), i(5p), del(5)(q13), der(7)t(7;21)(q32q10::q10), der(8)t(8;17)(q10;p10), and der(9)t(9;22)(q34;?pq). Comparative genomic hybridization (CGH) was employed to monitor the culture evolution in vitro. Genomic imbalances in HKCI-1 involved chromosomal losses on 4q, 5q13-qter, 8p, 9pter-q33, 10q, 11q, 13q, 16q, 17q12-qter, and 22, and low-level gains on 6pter-q22, 7p, 8q, 9q34, 10p, 11p, 12, 17pter-q11.2, 18, 19, 20, 21, and Y. High-level amplifications were also detected on 5pter-q12, 7q11.2-qter, and Xq. The corresponding CGH finding on the primary tumor indicated similar imbalances. TP53 mutational analysis showed that both HKCI-1 and the primary tumor had the aflatoxin-associated mutation in codon 249 and an additional TP53 polymorphism in codon 72. Our present study demonstrates the value of combined SKY and CGH study in defining complex rearrangements and identifying cryptic translocations, and provides a comprehensive analysis on the chromosomal abnormalities in HKCI-1.

Adult↗

Analysis of marker or complex chromosomal rearrangements present in pre- and post-natal karyotypes utilizing a combination of G-banding, spectral karyotyping and fluorescence in situ hybridization.

The significance of complex chromosomal rearrangements presents a diagnostic dilemma. In the past, the use of G-banding coupled with fluorescence in situ hybridization (FISH) has been the standard approach. The recent development of spectral karyotyping (SKY) and multicolor FISH (M-FISH) has resulted in an increased accuracy of identification of marker or other complex chromosomal rearrangements. However, owing to the additional cost and time associated with SKY or M-FISH, and the restricted availability of such imaging facilities in many centers, it is not feasible to perform these procedures routinely on every sample. In addition, the identification of an aberration by SKY or M-FISH will often require confirmation by FISH. A practical approach is needed to take advantage of the complementary strengths of each method. In our center we utilize an algorithm that dictates the use of routine G-banding for the initial preliminary evaluation of a patient, followed by SKY characterization if marker chromosomes or complex translocations are detected by the G-banding analysis. According to this algorithm, FISH is used to verify the results once the origin of the abnormal chromosome has been determined by SKY. To demonstrate the effectiveness of this algorithm, we have analyzed both amniocyte and lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirm that an algorithm which selectively uses SKY or M-FISH will provide an efficient and improved method for pre- and post-natal chromosomal analysis.

Algorithms↗

[Study on the mechanism of oocyte aneuploidy by spectral karyotyping].

OBJECTIVE: To establish the method of detecting oocyte aneuploidy by spectral karyotyping (SKY). METHODS: The unfertilized oocytes were fixed 1-2 days after oocyte retrieval. Spectral karyotyping was performed according to the protocol. RESULTS: 64% of oocytes were normal, 36% of oocytes were aneuploidy, of which 22% were due to nondisjunction and 14% unbalanced predivision. CONCLUSION: SKY is an effective method for detecting oocyte aneuploidy. Both nondisjunction and unbalanced predivision are involved in oocyte aneuploidy formation.

Aneuploidy↗

Spectral karyotyping analysis of head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: The genetic content of head and neck squamous cell carcinomas is ill defined. Spectral karyotyping (SKY) is a new technique that allows the simultaneous detection of all chromosomal translocations by labeling each individual chromosome with different fluorescent agents. In the current study we used SKY to analyze cell lines and a primary tumor derived from head and neck squamous cell carcinomas (HNSCC) to delineate recurrent translocations and breakpoints. STUDY DESIGN: Spectral karyotyping analysis of head and neck cancer. METHODS: Two cell lines (MDA886 and MSK922) and one primary tumor in short-term culture were subjected to metaphase growth arrest with colcemide in their exponential growth phase and fixed onto glass slides. Painting probes for each of the autosomes and the sex chromosomes were generated from flow-sorted human chromosomes using sequence-independent DNA amplification. The probes were labeled using a polymerase chain reaction-based reaction and hybridized to metaphase preparations for 2 days at 37 degrees C. Biotinylated probes were detected using avidin Cy5 and digoxigenin-labeled probes with an anti-mouse digoxigenin antibody followed by goat anti-mouse antibody conjugated to Cy5.5. Chromosomes were counterstained with 4,6-diamino-2-phenyliodole (DAPI), and a minimum of five metaphases were captured and analyzed for each case. Breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding. RESULTS: Spectral karyotyping analysis revealed a complex pattern of chromosomal abnormalities. A total of 66 translocations were identified in the three cases, with one new recurrent translocation at (der(4)t(4;20)(q35;?)). Nine complex translocations, involving three or more chromosomes, were identified in these cases. Overall, 96 breakpoints were assigned to metaphase chromosomes and another 74 breakpoints could not be assigned. Breakpoints most commonly involved chromosomes in genetic rearrangements were 1, 3, 5, 8, 13, 16, and 17. CONCLUSIONS: Spectral karyotyping analysis reveals the true complexity of chromosomal aberrations in cell lines derived from head and neck squamous cell carcinomas. The use of SKY, in combination with other techniques, may allow for a more complete assessment of the genetic abnormalities of head and neck cancers and serve as a starting point for gene identification.

Carcinoma, Squamous Cell↗

Karyotypic evolution pathways in medulloblastoma/primitive neuroectodermal tumor determined with a combination of spectral karyotyping, G-banding, and fluorescence in situ hybridization.

Medulloblastomas (MBs) or primitive neuroectodermal tumors (PNETs) represent 15%-30% of pediatric brain tumors and are the most common brain tumors in children; they are rare in adults. Classification of these tumors is based on tissue morphology and is often controversial and problematic. Karyotypic analysis of these tumors using conventional cytogenetic methods is often a difficult process that may be hindered by a limited number of metaphase cells and poor chromosome morphology, often leading to only partial characterization of the chromosomal abnormalities. We investigated three primary human tumors and four cell lines (CHO-707, DAOY, D-341, and PFSK) utilizing a combination of conventional G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH) techniques. A high level of intratumoral heterogeneity was seen, with multiple numerical and structural chromosomal aberrations. The chromosomes most frequently involved in structural aberrations were chromosomes 1 (14 rearrangements), 7 (9 rearrangements), and 21 (9 rearrangements). The chromosomes most frequently involved in numerical aberrations were chromosomes 1, 12, and 13 (four cases) and chromosomes 14, 17, 19, 21, 22, and X (three cases). Numerous aberrant chromosomes were characterized only with the SKY analysis, and based on these findings multiple clones were identified, facilitating analysis of karyotypic evolution. The most frequent evolution mechanism was via polyploidization, followed by acquisition of additional numerical or structural aberrations (or both); however, the results showed that the karyotypic evolution process in these tumors is typically divergent and complex.

Adult↗

Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.

Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.

Animals↗

Chromosome 11 rearrangements and specific MLL amplification revealed by spectral karyotyping in a patient with refractory anaemia with excess of blasts (RAEB).

A patient with refractory anaemia with excess of blasts (RAEB) had a complex karyotype with multiple markers. Spectral karyotyping (SKY) showed rearrangements including three different der(11), containing a very high number of MLL gene copies, shown by fluorescence in situ hybridization (FISH) analysis. Fibre-FISH experiments disclosed the presence of chromatin fibres with multiple MLL copies with a head-to-tail pattern. Apparently, no other region flanking the MLL site was present in the three der(11). MLL amplification was confirmed by the reverse transcription polymerase chain reaction (RT-PCR). The patient died 6 months after diagnosis, supporting the severe prognosis of sole MLL amplification.

Aged↗

Detailed characterization of a complex karyotype in a patient with primary plasma cell leukaemia using multicolour spectral karyotyping and micro-FISH.

INTRODUCTION: Plasma cell leukaemia is a rare disorder that usually carries an aggressive course with a rapidly fatal outcome. A variety of chromosomal abnormalities have been reported in plasma cell leukaemia but the clinical significance of an abnormal karyotype is still unclear. MATERIALS AND METHODS: We have applied the molecular cytogenetic techniques multicolour spectral karyotyping and microdissection in combination with fluorescence in situ hybridization on metaphases from a patient with primary plasma cell leukaemia and a fatal outcome. RESULTS AND CONCLUSION: The chromosome analysis showed severe hypodiploidy and 12 marker chromosomes. Identification of the structural rearrangements was not possible using routine cytogenetic methods. Utilizing the methods above, all marker chromosomes could be identified in detail and the karyotype was shown to be very complex. Forty-three breakpoints were found, and 25 could be identified at the band level, among others 14q32 where the immunoglobulin heavy chain locus is situated. Thus, these techniques provide the opportunity to resolve very complex chromosomal changes in a way that has not been previously possible and will consequently be of great importance in the search for hot spots that may harbour new cancer genes.

Adult↗

Patterns of aneuploidy in stage IV clear cell renal cell carcinoma revealed by comparative genomic hybridization and spectral karyotyping.

We report the use of spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to describe the numerous genomic imbalances characteristic of stage IV clear cell renal cell carcinoma (CCRCC). SKY and CGH were performed on 10 cell lines established from nephrectomy specimens, and CGH on uncultured material from five of the primary renal tumors. The mutational status of VHL (3p25) and MET (7q31), genes implicated in renal carcinogenesis, were determined for each case. Each case showed marked aneuploidy, with an average number of copy alterations of 14.6 (+/-2.7) in the primary tumors and 19.3 (+/-4.6) in the cell lines. Both whole-chromosome and chromosome-segment imbalances were noted by CGH: consistent losses or gains included +5q23-->ter (100%), -3p14-->ter (80%), and +7 (70%). All VHL mutations and 83% of the genomic imbalances found in the primary tumors were also found in the cell lines derived from them. SKY showed many complex structural rearrangements that were undetected by conventional banding analysis in these solid tumors. All cases with VHL inactivation had 3p loss and 5q gain related primarily to unbalanced translocations between 3p and 5q. In contrast, gains of chromosome 7 resulted primarily from whole-chromosome gains and were not associated with mutations of MET. SKY and CGH demonstrated that genomic imbalances in advanced RCC were the result of either segregation errors [i.e., whole chromosomal gains and losses (7.8/case)] or chromosomal rearrangements (10.7/case), of which the majority were unbalanced translocations.

Adenocarcinoma, Clear Cell↗

Interphase fluorescence in situ hybridization and spectral karyotyping reveals hidden genetic aberrations in children with acute lymphoblastic leukaemia and a normal banded karyotype.

Twenty-two cases of childhood acute lymphoblastic leukaemia (ALL) with normal G- or Q-banded karyotypes were studied by interphase fluorescence in situ hybridization (FISH) and spectral karyotyping. Probes detecting MLL, BCR/ABL and TEL/AML1 rearrangements were used for the interphase studies, along with centromere-specific probes from chromosomes 17 and X. In 10 patients (45%), previously undetected aberrations were demonstrable. Specific gene rearrangements and structural changes were found in six cases and numerical changes in five. Five of these aberrations have previously been reported to have an impact on prognosis. Three cases were massively hyperdiploid and, in one, the prognostically important BCR/ABL fusion was detected. In addition, a near-haploid karyotype with 27 chromosomes was found in one patient and TEL/AML1 rearrangements were detected in two cases. This study indicates that about half of childhood ALL cases with apparently normal karyotypes harbour genetic aberrations that may be detected using interphase FISH and spectral karyotyping.

Adolescent↗

Non-random chromosomal rearrangements in pancreatic cancer cell lines identified by spectral karyotyping.

The molecular events involved in pancreatic cancer are becoming increasingly well characterized, with mutations in the dominant oncogene KRAS and the tumour suppressor genes TP53, CDKN2A and MADH4 being typically observed. However, other genetic abnormalities remain to be identified and molecular cytogenetics may be useful to detect chromosomal loci involved in recurrent rearrangements. We have used spectral karyotyping to characterize cytogenetic aberrations in a panel of 20 human pancreatic carcinoma cell lines and confirmed their identities by dual and triple color fluorescence in situ hybridization. The most common partial or whole-arm gains involved 5p, 7q, 12p, 1q, 7p, 5q, 9p, 9q and 11p. The most common partial or whole-arm losses affected 9p, 11q, 18q, 3p, 2q and 1p, as well as the short arms of the acrocentric chromosomes. Spectral karyotyping allowed us to identify a number of recurrent structural aberrations, all of them unbalanced: most frequently i(5)(p10), del(11)(q23), i(12)(p10), i(1)(q10), del(7)(q22) and del(10)(p11). Spectral karyotyping mapped the complex aberrations occurring in pancreatic cancer cell lines and identified non-random patterns of chromosomal rearrangement. This comprehensive characterization should be useful to direct future investigation. The observation that loss at 11q and gains at 5p with i(5)(p10) and 12p with i(12)(p10) are more frequent changes than previously reported would justify more intensive investigation of these chromosomal regions.

Chromosome Aberrations↗

Spectral karyotyping (SKY) of mouse meiotic chromosomes.

The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.

Animals↗

Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas.

Conventional cytogenetic studies have shown that osteosarcomas (OSs) are often highly aneuploid, with a large number of both structural and numerical chromosomal alterations. To investigate the complexity of OS karyotypes in detail, we applied spectral karyotyping (SKY) to a series of 14 primary OS tumors and four established OS cell lines. A total of 531 rearrangements were identified by SKY, of which 300 breakpoints could be assigned to a specific chromosome band. There was an average of 38.5 breakpoints identified by SKY per primary tumor. Chromosome 20 was involved in a disproportionately high number of structural rearrangements, with 38 different aberrations being detected. Chromosomal rearrangements between chromosomes 20 and 8 were evident in four tumors. FISH analysis using a 20q13 subtelomeric probe identified frequent involvement of 20q in complex structural rearrangements of OS cell lines. Characterization of the structural aberrations of chromosomes 8 and 17 by use of SKY demonstrated frequent duplication or partial gains of chromosome bands 8q23-24 and 17p11-13. Other chromosomes frequently involved in structural alteration were chromosomes 1 (47 rearrangements) and 6 (38 rearrangements). Centromeric rearrangements often involving chromosomes 1, 6, 13, 14, 17, and 20 were present. Four of the 14 primary OS tumors were characterized by nonclonal changes that included both structural and numerical alterations. In summary, OS tumors have a very high frequency of structural and numerical alterations, compounded by gross changes in ploidy. This intrinsic karyotype instability leads to a diversity of rearrangements and the acquisition of composite chromosomal rearrangements, with the highest frequency of alteration leading to gain of 8q23-24 and 17p11-13 and rearrangement of 20q. These findings suggest that specific sequences mapping to these chromosomal regions will likely have a role in the development and progression of OS.

Adolescent↗

Genetic alterations in doxorubicin-resistant hepatocellular carcinoma cells: a combined study of spectral karyotyping, positional expression profiling and candidate genes.

Despite a prolongation of patient survival, the overall response of doxorubicin (DX) treatment on patients with hepatocellular carcinoma (HCC) remains modest. This is largely attributed to the development of tumor drug resistance either at the onset or during the course of treatment. To investigate the genetic changes associated with DX chemo-resistance, we examined the cytotoxic effect of DX on a panel of 9 HCC cell lines (HepG2, Hep3B, PLC/PRF/5, and six in-house established, HKCI-1, 2, 3 and 4, C1 and C2). The karyotypic abnormalities were examined by spectral karyotyping (SKY) and the chromosome loci defined were investigated for underlying deregulated genes by positional expression profiling. Quantitative RT-PCR was employed to verify the profiling findings, and also used to examine a number of drug resistance-related candidate genes (MDR1, MRP1, MGMT, PTEN, BCL2, BAX, TP53 and P21). Our results indicated that the cytotoxic effect of DX in cell lines exhibited IC50 values that ranged from sensitive to resistant (0.07 to 3.55 microM). While the overall chromosome aneuploidy did not correlate with DX resistance, aberrations on chromosome 10 demonstrated significant correlation with increasing IC50 (p=0.007). Positional profiling further suggested the consistent down-regulation of CGI-18 and ECHS1 on chromosome 10q. The array findings were substantiated by quantitative RT-PCR, which further pointed to a repressed ECHS1 expression in correlation with DX resistance (p=0.021). Among the candidate genes studied, an inverse relationship of P21 (p=0.034) and BAX (p=0.002) expression with DX resistance was also indicated. Our present study highlights the usefulness of multimodality approaches in identifying genetic markers, and further describes the novel finding of ECHS1 down-regulation in the DX chemo-resistance of HCC.

Antibiotics, Antineoplastic↗

Recurrent chromosomal rearrangements at bands 8q24 and 11q13 in gastric cancer as detected by multicolor spectral karyotyping.

AIM: To identify chromosomal translocations specific to gastric cancer (GC), spectral karyotyping (SKY) analysis was performed on established cell lines and cancerous ascitic fluids. METHODS: SKY analysis of 10 established cell lines and seven cancerous ascitic fluid samples identified recurrent chromosomal breakpoints and translocations in GC, several of which involved chromosomal loci of oncogenes or tumor suppressor genes. RESULTS: A total of 630 chromosomal breaks were identified. Chromosome no.8 was the most frequently involved in rearrangements (65 breaks), followed by chromosomes no.11 (53), no. 1 (49), no. 7 (46), no. 13 (37), no. 3 (36), no. 17 (33), and no. 20 (29). Frequent breakpoints were detected in 8q24.1 (30 breaks), 11q13 (29), 13q14 (16), 20q11.2 (14), 7q32 (13), 17q11.2 (13), 18q21 (12), 17q23 (9), 18q11.2 (9). SKY analysis identified a total of 242 chromosomal rearrangements including 190 reciprocal and non-reciprocal translocations. The recurrent combinations of chromosomal bands involved in translocations were 8q24.1 and 13q14 (3 cases), 8q24.1 and 11q13 (3), 11q13 and 17q11.2 (2), and 18q11.2 and 20q11.2 (2). Our study validated the ability of SKY to characterize in detail the chromosomal rearrangements in solid tumors and derived cell lines. Moreover, fluorescence in situ hybridization helped to identify the insertions, translocations, and homogeneously staining regions of MYC and CCND1 gene loci. CONCLUSION: The non-random co-localization of certain cytogenetic bands suggests the importance of chromosomal translocations in gastric carcinogenesis, by serving as landmarks for the cloning of GC causing genes.

Cell Line, Tumor↗