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American Journal of Pathology, 152, 1107- 1123
1998

DNA Copy Number Amplifications In Human Neoplasms

S. Knuutila, A.-M. Björkqvist, K. Autio, M. Tarkkanen, et al, M. Wolf

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.

Proc. Natl. Acad. Sci. USA, 95, 167- 171
1998

Creation of monosomic derivatives of human cultured cell lines

D.J. Clarke, J.F. Giménez-Abián, H. Tönnies, H. Neitzel, K. Sperling, C.S. Downes, R.T. Johnson

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.

Genes Chromosomes Cancer, 21, 1- 4
1998

Minimal Sizes of Deletions Detected by Comperative Genomic Hybridization

M. Bentz, A. Plesch, S. Stigenbauer, H. Döhner, P. Lichter

<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>

Genes, Chromsomes & Cancer, 23, 141- 152
1998

Genetic Heterogeneity of Neuroblastoma Studied by Comparative Genomic Hybridization

J. Vandesompele, N. Van Roy, et al, M. Van Gele

<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>

J Neuropathol Exp Neurol, 56, 743- 748
1997

Detection of Malignant Cells in Cerebrospinal Fluid Using Fluorescence In Situ Hybridization

R.J. Van Oostenbrugg, A.H.N. Hopman, M.H. Lenders, P. Van Heerde, J.-W. Arends, F.C.S. Ramaekers, A. Twijnstra

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.

Cancer Genet Cytogenet, 97, 135- 142
1997

Comparative Genomic Hybridization Analysis of Human Neuroblastomas: Detection of Distal 1p Deletions and Further Molecular Genetic Characterization of Neuroblastoma Cell Lines

N. Van Roy, A. Jauch, M. Van Gele, G. Laureys, R. Versteeg, A. De Paepe, T. Cremer, F. Speleman

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.

Cytogenetics and Cell Genetics, 78, 96- 102
1997

Monosomy 6 in human cultured fibroblast-like cells after long-term stimulation with acidic fibroblast growth factor (FGF1)

W. Krone, H. Kehrer-Sawatzki, A. Siegel, H. Röck, H. Götz

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.

Genes Chromosomes Cancer, 16, 31- 34
1996

Ring chromosomes in parosteal osteosarcoma contain sequences from 12q13-15. A combined cytogenetic and comparative genomic hybridization study

J. Szymanska, N. Mandahl, F. Mertens, M. Tarkkanen, E. Karaharju, S. Knuutila

<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>

American Journal of Pathology, 2, 463- 468
1996

Translocation t(2/5) is not a primary event in Hodgkin's disease. Simultaneous immunophenotyping and interphase cytogenetics

K. Weber-Matthiesen, J. Deerberg-Wittram, A. Rosenwald, M. Poetsch, W. Grote, B. Schlegelberger

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.

Mutation Research, 372, 259- 268
1996

Spontaneous rates of sex chromosomal aneuploidies in sperm and offspring of mice: a validation of the detection of aneuploid sperm by fluorescence in situ hybridization

I.-D. Adler, J. Bishop, X. Lowe, T.E. Schmid, G. Schriever-Schwemm, W. Xu, A.J. Wyrobek

<p>This study was designed to evaluate the frequency of aneuploid sperm in young adult mice of the genotype (102/E1 x C3H/E1)F1 determined by the fluorescence in situ hybridization (FISH) procedure and to evaluate the frequencies of aneuploid sperm observed by FISH compared with the frequencies of aneuploid offspring. Three-chromosome FISH was applied to determine the fractions of hyperhaploid and diploid sperm with DNA probes specific for chromosomes X, Y and 8. The animals were treated with three common solvents. Sperm smears were prepared for FISH by two similar protocols and were scored by different persons and in two different laboratories. There were no significant differences between scorers or laboratories. The frequencies of the sex chromosome aneuploidies in sperm (Y-Y and X-Y) were compared to the frequencies of mice carrying sex chromosome aneuploidy among controls of the heritable translocation assay in studies conducted from 1975-1995. To identify aneuploid individuals, untreated males and females of the genotype (102/E1 x C3H/E1)F1 were mated to assess their fertility by observing three consecutive litters. Semisterile and sterile animals were further analysed by meiotic cytogenetics and by karyotyping to determine the incidence of reciprocal translocations and sex chromosome aneuploidies (XXY and XYY). Based on the analysis of 175247 sperm and 9840 progeny, the frequency of Y-Y sperm was 0.01% while 0.03% of the offspring were XYY. The frequency of X-Y sperm was 0.005% while 0.02% of the offspring were XXY. The frequencies of aneuploid sex chromosomes were not significantly different between sperm and offspring. This allows two conclusions. First, there was no detectable prenatal selection against these sex-chromosomal aneuploid offspring, and second, germ cell aneuploidy can be reliably determined in mice by sperm FISH analyses.</p>

British Journal of Haematology, 92, 673- 680
1996

Secondary acute leukaemias with 11q23 rearrangement: clinical, cytogenetic, FISH and FICTION studies

Y. Zhang, M. Poetsch, K. Weber-Matthiesen, K. Rhode, M. Winkemann, T. Haferlach, W. Gassmann, W.-D. Ludwig, W. Grote, H. Löffler, B. Schlegelberger

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.

Blood, 87, 2459- 2463
1996

The Abnormal Eosinophils Are Part of the Leukemic Cell Population in Acute Myelomonocytic Leukemia With Abnormal Eosinophils (AML M4Eo) and Carry the Pericentric Inversion 16: A Combination of May-Grünwald-Giemsa Staining and FISH

T. Haferlach, M. Winkemann, H. Löffler, R. Schoch, W. Gassmann, C. Fonatsch, C. Schoch, M. Poetsch, K. Weber-Matthiesen, B. Schlegelberger

The French-American-British subtype acute myelomonocytic leukemia with abnormal eosinophils (FAB AML M4Eo) with pericentric inversion of chromosome 16 is cytomorphologically defined by a myelomonoblastic blast population and abnormal eosinophils. Until now, it remained an open question whether these abnormal eosinophils are part of the malignant clone or an epiphenomenon. We analyzed five cases of AML M4Eo with inv(16) and combined May-Grünwald-Giemsa staining with fluorescence in situ hybridization using yeast artificial chromosome clone 854E2, which spans the inv(16) breakpoint on 16p. In the case of inv(16), three instead of the normal two hybridization signals can be observed both on metaphase spreads and in interphase cells. With this approach, we were able to show inversion 16 in abnormal eosinophils and, therefore, identified them as a part of the leukemic cell population.

Cancer Genet Cytogenet, 89, 7- 13
1996

Gains and losses of DNA sequences in malignant mesothelioma by comparative genomic hybridisation

P. Kivipensas, A.-M. Björkqvist, R. Karhu, K. Pelin, K. Linnainmaa, L. Tammilehto, K. Mattson, O.-P. Kallioniemi, S. Knuutila

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.

Blood, 87, 5269- 5278
1996

DNA copy number changes in diffuse large B-cell lymphoma - Comparative genomic hybridization study

O. Monni, H. Joensuu, K. Franssila, S. Knuutila

We studied DNA copy number changes in diffuse large B-cell lymphoma using comparative genomic hybridization analysis on 20 primary tumors and on 12 recurrent tumors excised after chemotherapy or radiotherapy. Twenty-nine (91%) of the cases showed abnormal copy number karyotypes. Chromosomal regions at X (41%), 1q (38%), 7 (31%), 3 (24%), 6p (21%), 11 (21%), 12 (21%), and 18 (21%) were most frequently gained, and the most common losses involved 6q (38%), X (21%), 1p (14%), and 8p (10%). High-level amplifications were observed at 6p23-ter, 10p12-14, 17p1l.2, 18q21-ter, and Xq22-ter, all but 18q appearing only in the recurrent tumors. Gains (median, 2; range, 0 to 10) were more frequent than losses (median, 1; range, 0 to 7; P = .0004). The median number of aberrations found in the recurrent tumors (6.5) was greater than that in the primary tumors (2; P = .01). The copy number changes found in the recurrent tumors were more random than those found in the primary tumors, which were mainly located in the most frequently affected regions. Our findings are in line with those observed using conventional cytogenetic analysis, but especially novel high-level amplifications were detected. Southern blot analysis showed BCL2 amplification, but not translocation t(14;18)(q32;q21), in cases in which a gain at 18q was detected by comparative genomic hybridization, which strongly suggests that, in addition to translocation, gene amplification is another mechanism for the overexpression of the BCL2 protein.

Cancer Research, 55, 1334- 1338
1995

Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization

M. Tarkkanen, R. Karhu, A. Kallioniemi, I. Elomaa, A.H. Kivioja, J. Nevalainen, T. Böhling, E. Karaharju, E. Hyytinen, S. Knuutila, O.P. Kallioniemi

Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.

Blood, 87, 2459- 2463
1995

The abnormal eosinophils are part of the leukemia cell population in Acute Myelomonocytic Leukemia with abnormal eosinophils (AML M4Eo) and carry the pericentric inversion 16: A combination of May- Grünwald-Giemsa staining and FISH

T. Haferlach, M. Winkemann, H. Löffler, R. Schoch, W. Gassmann, C. Fonatsch, M. Poetsch, K. Weber-Matthiesen, B. Schlegelberger

<p>The French-American-British subtype acute myelomonocytic leukemia with abnormal eosinophils (FAB AML M4Eo) with pericentric inversion of chromosome 16 is cytomorphologically defined by a myelomonoblastic blast population and abnormal eosinophils. Until now, it remained an open question whether these abnormal eosinophils are part of the malignant clone or an epiphenomenon. We analyzed five cases of AML M4Eo with inv(16) and combined May-Grünwald-Giemsa staining with fluorescence in situ hybridization using yeast artificial chromosome clone 854E2, which spans the inv(16) breakpoint on 16p. In the case of inv(16), three instead of the normal two hybridization signals can be observed both on metaphase spreads and in interphase cells. With this approach, we were able to show inversion 16 in abnormal eosinophils and, therefore, identified them as a part of the leukemic cell population.</p>