<p>Characterisation of chromosome rearrangements using conventional banding techniques often fails to determine the localisation of breakpoints precisely. In order to improve the definition of chromosomal breakpoints, the high-resolution multicolour banding (MCB) technique was applied to identify human chromosome 5 breakpoints from 40 clinical cases previously assessed by conventional banding techniques. In 30 cases (75%), at least one breakpoint was redefined, indicating that MCB markedly improves chromosomal breakpoint localisation. The MCB pattern is highly reproducible and, in contrast to conventional banding pattern, is consistent in both short and elongated chromosomes. This might be of fundamental interest for the detection of chromosomal abnormalities, especially in tumour cells. Moreover, MCB even allows the detection of abnormalities that remain cryptic in GTG-banding analysis.</p>

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T-cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

The comparative genomic hybridization technique (CGH) was used to identify common chromosomal imbalances in osteosarcomas (OS), which frequently display complex karyotypic changes. We analyzed 13 high-grade primary tumors, 5 corresponding cell lines, 2 primary tumors grade 2, and 1 recurrent tumor from a total of 16 patients. Some of the CGH results have been verified by fluorescence in situ hybridization (FISH) studies. Gains of chromosomal material were more frequent than losses. Most common gains were observed at 8q (11 cases), 4q (9 cases), 7q (8 cases), 5p (7 cases), and 1p (8 cases). The smallest regions of overlap have been narrowed down to 8q23 (10 cases), 4q12-13 (8 cases), 5p13-14 (7 cases), 7q31-32 (7 cases), 8q21 (7 cases), and 4q28-31 (5 cases). These data demonstrate that a number of chromosomal regions and even two distinct loci on 4q and 8q are involved in the pathogenesis of OS, with gain of 4q12-13 chromosomal material representing a newly identified locus. Seven of 16 cases displayed, besides gain of 8q23 sequences, gain of MYC copies in CGH and FISH. Previous CGH reports confined gain of 8q material to 8cen-q13, 8q21.3-8q22, and 8q23-qter, whereas our data suggest that the loci 8q21 and 8q23-24 are affected in the development of OS. In contrast to recent reports, copy number increases at 8q and 1q21 did not have an unfavorable impact on prognosis in the present series. Genes Chromosomes Cancer 28:329-336, 2000.

We have investigated the requirements for NDJ1 in meiotic telomere redistribution and clustering in synchronized cultures of Saccharomyces cerevisiae. On induction of wild-type meiosis, telomeres disperse from premeiotic aggregates over the nuclear periphery, and then cluster near the spindle pole body (bouquet arrangement) before dispersing again. In ndj1Δ meiocytes, telomeres are scattered throughout the nucleus and fail to form perinuclear meiosis-specific distribution patterns, suggesting that Ndj1p may function to tether meiotic telomeres to the nuclear periphery. Since ndj1Δ meiocytes fail to cluster their telomeres at any prophase stage, Ndj1p is the first protein shown to be required for bouquet formation in a synaptic organism. Analysis of homologue pairing by two-color fluorescence in situ hybridization with cosmid probes to regions on III, IX, and XI revealed that disruption of bouquet formation is associated with a significant delay (>2 h) of homologue pairing. An increased and persistent fraction of ndj1Δ meiocytes with Zip1p polycomplexes suggests that chromosome polarization is important for synapsis progression. Thus, our observations support the hypothesis that meiotic telomere clustering contributes to efficient homologue alignment and synaptic pairing. Under naturally occurring conditions, bouquet formation may allow for rapid sporulation and confer a selective advantage.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

Acquired ring chromosomes have been found in most types of human neoplasia, with a frequency approaching 10% in malignant mesenchymal tumours. In this study, the composition and dynamics of ring chromosomes were analysed in eight cases of acute myelogenous leukaemia, 17 solid tumours, and five cases with constitutional rings. Chromosomal banding and fluorescence in situ hybridisation were performed to determine the content and the structural heterogeneity of the rings. Telomeric repeats were detected using peptide nucleic acid probes or primed in situ labelling, whereas centromeric activity was evaluated by detection of kinetochore proteins. Mitotic instability was assessed by the frequency of anaphase bridges. The results suggest that human ring chromosomes can be structurally and functionally divided into two categories. In the first of these, size variation is minimal and rearrangement at cell division is uncommon. The majority of such rings contain subtelomeric sequences. Constitutional ring chromosomes and most rings in leukaemias belong to this group, whereas only a few mesenchymal tumours exhibit rings of this type. The second category consists of rings with amplified sequences, primarily from chromosome 12, characteristically occurring in atypical lipomatous tumours and other subtypes of low or borderline malignant mesenchymal neoplasms. Variation in size and number is extensive, and breakage-fusion-bridge events occur at a high frequency. Abnormalities in pericentromeric sequences are common and, in some cases, kinetochores assemble in the absence of alphoid DNA. We conclude that it is not only the ring structure per se or the neoplastic nature of the host cell that determines ring instability, but probably also the functional role of the genes carried in the ring.

<p>We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, "large cell variant" of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffin-embedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested.</p>

<p>Comparative genomic hybridization (CGH) analysis was performed on 36 neuroblastomas of both low and high stage of disease. This study significantly increases the number of neuroblastoma tumors studied by CGH. Analysis of larger series of tumors is particularly important in view of the different clinical subgroups that are recognized for this tumor. The present data and a comparison with all published CGH data on neuroblastoma provide further insights into the genetic heterogeneity of neuroblastoma. Stage 1, 2, and 4S tumors showed predominantly whole chromosome gains and losses. A similar pattern of whole chromosome imbalances, although less frequent, was observed in stage 3 and 4 tumors, in addition to partial chromosome gains and losses. An increase in chromosome 17 or 17q copy number was observed in 81% of tumors. The most frequent losses, either through partial or whole chromosome underrepresentation, were observed for 1p (25%), 3p (25%), 4p (14%), 9p (19%), 11q (28%), and 14q (31%). The presence of 3p, 11q or 14q deletions defines a genetic subset of neuroblastomas and contributes to the further genetic characterization of stage 3 and 4 tumors without MYCN amplification (MNA) and 1p deletion. The present study also provides additional evidence for a possible role of genes at 11q13 in neuroblastoma. In a few cases, 1p deletion or MNA detected by FISH or Southern blotting was not found by CGH, indicating that the use of a second, independent technique for evaluation of these genetic parameters is recommended.</p>

<p>Comparative genomic hybridization (CGH) has been used widely for the molecular cytogenetic analysis of tumors. Until now, the spatial resolution of this technique for diagnosing deletions of chromosomal sequences has not been assessed in detail. In the present study, we performed CGH analyses on five DNA samples derived from B-cell leukemias with 11q deletions, the sizes of which ranged from 3 Mbp to 14-18 Mbp. CGH experiments were evaluated by two established commercial analysis systems. Deletions down to a size of 10-12 Mbp were diagnosed based on a diagnostic threshold value of 0.8, if the vast majority of cells carried the deletion. For cases with smaller deletions, the ratio profiles were shifted toward underrepresentation at the respective chromosomal bands; however, the diagnostic threshold value was not reached. In all five cases, there was complete agreement between the two image analysis systems.</p>

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.

Long-term exposure to fibroblast growth factor type 1 (FGF1) of fibroblast-like cells derived from neurofibromas of patients with neurofibromatosis type 1, from angiofibromas of patients with tuberous sclerosis, and from foreskin of unaffected donors resulted in the outgrowth of monosomy 6 in 7 out of 14 cell lines examined. After their initial detection by cytogenetic analysis, the proportion of cells which had lost one chromosome 6 was monitored by FISH using a-satellite probes specific for chromosome 6 and 7, and by PCR analysis of polymorphic microsatellite markers. Monosomy 6 exceeding baseline levels developed only in cultures exposed to FGF 1, and the emergence of monosomic cells could not be correlated with a given donor's genotype. During serial culture, the proportion of monosomic cells increased to over 90% in 5 of the 7 affected strains. A conspicuous change of cellular morphology from spindle-shaped to more epithelial-type cells was noted in monosomic cultures, even though none of them converted to a permanent cell line during the observation period. We conclude that long-term exposure of human fibroblast-like cell strains to FGF1 results in the emergence of monosomy 6 in 50% of the cultures so treated. A selective advantage for such monosomic cells is the most likely explanation for their steady increase during serial culture.

Cytologic examination of cerebrospinal fluid (CSF) is the diagnostic gold standard for leptomeningeal metastasis (LMM). However, this technique is only moderately sensitive when routine staining procedures are applied. The use of fluorescence in situ hybridization (FISH) to identify malignant cells may have an additional value in diagnosing LMM, since numerical chromosomal aberrations (NCA) can be detected at the single cell level. We tested the feasibility of FISH to detect tumor cells in CSF by analyzing 22 samples with proven LMM with a probe for chromosome 1 (1q12) to detect NCA in the cells. A control group consisted of samples from 10 patients with inflammatory neurologic disease. In 7 LMM samples no cells or only lysed cells were found, due to time delay before fixation. Of the other 15 LMM samples analyzed, 13 showed NCA (87%), while no NCA were found in the control group. Our results indicate that FISH may be a useful additional diagnostic tool to the cytodiagnosis of CSF in cases of LMM. We expect that FISH can become an additional marker for malignancy at the single cell level in patients with LMM, which may also be of use to determine the effect of therapy for LMM.

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

<p>Seven parosteal osteosarcoma (POS) samples, six of which were cytogenetically characterized, were studied by using comparative genomic hybridization (CGH). All samples showed DNA sequence copy number changes (mean, six aberrations/tumor; range, 1-13); gains were more frequent than losses. Gain of 12q13-15 sequences was found in every tumor and correlated with the presence of ring chromosomes. High-level amplification, which was detected in four tumors, was seen only in chromosome 12, with 12q13-14 as the minimal common region. By using chromosome painting, one of the rings of one case was shown to be composed entirely of chromosome 12 material. Together with previous data, our findings show that gain of 12q13-15 sequences is a characteristic feature of POS and that these sequences are contained within the ring chromosomes.</p>

A number of neoplastic disorders are characterized by recurrent chromosome aberrations. One of these is the translocation t(2;5), which is found in a considerable percentage of large-cell anaplastic lymphomas. This translocation results in the fusion of two genes, alk and npm. The recent discovery of alk/npm mRNA in 11 of 13 cases of Hodgkin's disease has caused a controversial discussion concerning the question of whether t(2;5) is also present in Hodgkin and Reed-Sternberg cells. We tackled this problem on the molecular cytogenetic level by combined CD30 immunophenotyping and interphase cytogenetics. Using a pair of DNA probes flanking both sides of the npm gene breakpoint at 5q35 we were able to prove, at least in 12 of 13 cases of Hodgkin's disease, that all CD30-positive Hodgkin and Reed-Sternberg cells lacked the translocation t(2;5). Fifteen to forty-five Hodgkin/Reed-Sternberg cells were analyzed per case (mean, 27). Our findings indicate that this translocation is not a primary event in the development of Hodgkin's disease.

Three patients with secondary acute leukaemia after treatment with topoisomerase II inhibitor agents are described. Two patients had acute myeloid leukaemia (AML). FAB M5a, one had pro-B-acute lymphoblastic leukaemia (ALL). The interval between initiation of chemotherapy and the onset of secondary acute leukaemia was 19-20 months. 11q23 rearrangements were detected in all cases. They were due to translocations t(11;19) (q23;p13.3), t(11;16)(q23;p13) and t(4;11)(q21;q23), respectively. Fluorescence in situ hybridization (FISH) with Yeast Artificial Chromosome (YAC) probe 13HH4 spanning the ALL-1 gene on 11q23 confirmed that in each case the ALL-1 gene had been disrupted by the translocations. The study underlined the relationship between the development of secondary acute leukaemias with 11q23 rearrangement and previous chemotherapy with topisomerase II inhibitor agents. So far, however, only six adult patients with secondary ALL with t(4;11) after treatment with topoisomerase II inhibitor agents have been reported. All with t(4;11) mostly occurs in infants or young children. Our patient received epirubicin continuously for >19 months. This indicates that both myeloid and lymphoid leukaemias with involvement of the ALL-1 gene can be induced by exogenous agents, especially topoisomerase II inhibitors. Thus they may have a common biological background. This hypothesis was substantiated by means of combined immunophenotyping and FISH (FICTION). In the case of AML M5a with t(11;19), the tumour cells with ALL-1 rearrangement expressed CD34. Moreover, the pro-B-ALL with t(4;11) was CD34 positive. These findings suggest that the cell of origin of secondary AML and ALL with 11q23 rearrangement is an immature haemopoietic progenitor cell.