Deletion of 11q23 is a highly specific nonrandom secondary genetic abnormality of ETV6/RUNX1-rearranged childhood acute lymphoblastic leukemia.
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Adolescent↗
Biomedical subjects
S Strehl
Publications and source records attributed to S Strehl.
The MLL recombinome of acute leukemias.
Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.
Acute Disease↗
RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.
The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.
Acetyltransferases↗
PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13).
Recurrent chromosomal abnormalities present in malignant cells often define subentities with unique biological and clinical features. The molecular identification of genes involved in genetic alterations has led to the characterization of fusion genes with neoplastic properties. However, for many nonrandom translocations including the dic(9;12)(p11-13;p11-12), the molecular equivalent has not as yet been identified. The dicentric translocation dic(9;12) is a recurrent chromosome abnormality that accounts for close to 1% of childhood acute lymphoblastic leukemia (ALL). This specific alteration occurs almost exclusively in B-progenitor ALL, and unlike many other nonrandom translocations, is associated with an excellent prognosis. In this work, we provide strong evidence that the PAX5/ETV6 fusion transcript defines the clinical and biological entity that is associated with the presence of a dic(9;12) chromosome. As the PAX5 and ETV6 genes are localized at 9p13 and 12p13, respectively, the cytogenetic description of the dic(9;12)-PAX5/ETV6 rearrangement should be refined to dic(9;12)(p13;p13).
Adolescent↗
Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.
Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.
Adult↗
Multiplex reverse transcriptase-polymerase chain reaction screening in childhood acute myeloblastic leukemia.
To determine the incidence of leukemia-specific rearrangements, 60 cases of childhood acute myeloblastic leukemia and transient myeloproliferative disorder were screened with a novel multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and the results were correlated with the cytogenetic findings. The RT-PCR assay detects 28 different fusion genes and more than 80 different fusion transcript variants. RNA was isolated from methanol/acetic acid-fixed cells that had been routinely prepared for cytogenetic analysis. Nine different fusion transcripts were found in 40% of the cases, whereas 78.3% of the cases had abnormal karyotypes. Two cases with a t(6;11) and an MLL/AF6 gene fusion were missed cytogenetically. Conversely, cytogenetic analysis revealed 10 other well-defined chromosome rearrangements. Although cytogenetic analysis reveals a much broader range of abnormalities, multiplex RT-PCR serves as quality control and provides the essential information for minimal residual disease studies. Moreover, discrepant findings lead to the detection of new rearrangements on the molecular genetic level.
Adolescent↗
Replication timing of CD4 and CD8 in single-positive peripheral blood lymphocytes.
The regulatory events leading to the mutually exclusive expression of CD4 and CD8 on peripheral lymphocytes are not fully understood. In particular, the association between DNA replication timing and transcriptional activity of these genes has not been previously investigated. Here, the replication kinetics of the CD4 and CD8 loci in mature single-positive T-cell populations have been examined using a novel approach to the separation of CD4(+) or CD8(+) lymphocytes into discrete cell cycle fractions and a competitive PCR replication timing assay. While the timing of replication of each of these loci is independent of their expression in mature CD4 or CD8 single positive T-cells, the replication of CD8, but not of CD4, shifts to a later time in S phase in transcriptionally silent HS68 fibroblast cells. These findings suggest that changes in DNA replication timing are associated with the developmentally regulated but not with the tissue-specific expression of CD4 and CD8.
CD4 Antigens↗
Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors.
Ewing's sarcoma, peripheral primitive neuroectodermal tumors, and Askin tumors are referred to as Ewing tumors (ETs), and are characterized by high MIC2 expression and a t(11;22)(q24;q12) or other rearrangements involving 22q12. In addition to these constant aberrations, facultative numerical and structural aberrations have been reported: gains of chromosomes 8 and 12, the unbalanced translocation t(1;16), and deletions at the short arm of chromosome 1. To evaluate the frequency and to study the biological impact of these facultative aberrations, we analyzed tumor specimens from 58 ET patients by classical cytogenetics and/or in situ hybridization techniques and compared these data with clinical parameters. Gains of chromosomes 8 and 12 were detected in 55% (32/58) and 24% (14/58) of the cases, respectively. Loss of chromosome 16 or der (16)t(1;16) chromosomes were found in 20% (10/51); deletions at 1p36 were observed in 18% (9/51) of the cases evaluated. The presence of these aberrations did not correlate with age and sex of the patients, with the location of the primary tumor or with the extent of disease at diagnosis by chi-square analysis and Fisher's exact test. Patients with tumors harboring gains of chromosome 8 showed a slightly better clinical outcome (n = 14/30, P = 0.17), whereas gains of chromosome 12 did not influence the clinical outcome (n = 7/30, P = 0.63). However, Kaplan and Meier analysis revealed that deletions at the short arm of chromosome 1 were associated with an unfavorable outcome in patients with localized disease (n = 6/22; P = 0.004).
Adolescent↗
Characterization of two novel protocadherins (PCDH8 and PCDH9) localized on human chromosome 13 and mouse chromosome 14.
The protocadherins are a subfamily of the calcium-dependent cell-cell adhesion and recognition proteins of the cadherin superfamily. In this study we describe the isolation and characterization of two novel protocadherins, PCDH8 and PCDH9, that constitute a new linkage group on human chromosome 13 and mouse chromosome 14. Like other protocadherins both genes are predominantly expressed in brain, but PCDH9 is also expressed in a broader variety of tissues, and the expression patterns appear to be developmentally regulated. We have determined the genomic organization of PCDH8, which differs significantly from that of the other cadherin subfamilies. In contrast to the classical and desmosomal cadherins, which in general consist of 15-17 exons and share a remarkable degree of conservation in intron position, PCDH8 consists of only three exons and lacks introns in the extracellular domain. The first exon encodes the extracellular domain, the transmembrane region, and part of the cytoplasmic tail. The second exon encodes the remainder of the cytoplasmic region and is partially untranslated. The differences in the genomic structure of cadherin subfamilies will be discussed in the context of the evolution of the cadherin superfamily.
Amino Acid Sequence↗
Molecular-cytogenetic characterization of the Vicia faba genome--heterochromatin differentiation, replication patterns and sequence localization.
A comprehensive survey of the molecular-cytogenetic features of the Vicia faba chromosome complement (2n = 12) is given. It includes previous as well as new original data. Various Giemsa, restriction endonuclease and fluorochrome banding patterns, azacytidine-mediated segment extension, replication patterns, lateral A/T asymmetry and sequence localization data for tandemly arranged simple sequence repeats, dispersed repeats and coding sequences as well as histone acetylation patterns are considered. This allows not only to distinguish and characterize telomeres, subtelomeres, centromeres and the NOR, but also the structure of the 5S rRNA gene loci and two main types of interstitial heterochromatin. Additionally, it offers physical landmarks within euchromatic areas. Thus, the field bean genome, exemplified by the reconstructed karyotype ACB, belongs to the cytogenetically best investigated plant genomes.
Chromosome Banding↗
Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance?
Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance.
Cell Division↗
High-resolution analysis of DNA replication domain organization across an R/G-band boundary.
Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.
Cell Cycle↗
Isochromosome 12p and maternal loss of 1p36 in a pediatric testicular germ cell tumor.
Analysis of a pediatric germ cell tumor by conventional cytogenetic investigation and fluorescence in situ hybridization showed consistently the presence of two isochromosomes 12p, loss of the maternal band 1p36, and other numerical and structural chromosome changes. The rearrangements observed resulted mainly from breaks occurring at paracentromeric regions. This report represents the first description of i(12)(p10) in a pediatric testicular embryonal carcinoma.
Adolescent↗
Demonstration of the translocation der(16)t(1;16)(q12;q11.2) in interphase nuclei of Ewing tumors.
The der(16)t(1;16) has been detected cytogenetically in a number of malignancies including Ewing tumors (ETs). To enable fast and reliable analysis of der(16) chromosomes, we established an interphase cytogenetic approach. By using two DNA probes hybridizing to the heterochromatic portions on the long arms of chromosomes 1 and 16, this technique allows the detection of this chromosomal aberration in nonproliferating cells. Formation of the der(16) leads to partial excess of 1q material and partial loss of the long arm of chromosome 16. Double-target fluorescence in situ hybridization (FISH) experiments were performed on cytospin slides of 13 ETs, near-triploid tumor cells and normal cells to assess whether the FISH technique used permits the discrimination of nuclei harboring this aberration from nuclei without a der(16) chromosome. In five ETs, we found evidence for the presence of one or two der(16)t(1;16) chromosomes both by FISH and by conventional cytogenetics. Tumor cells displayed two signals for intact chromosomes 1, one or two additional fused signals for the der(16) chromosomes, and one signal for the intact chromosome 16. In one case without fused signals, the presence of a der(16) was demonstrated by hybridizing a painting probe for chromosome 16 simultaneously with the paracentromeric probe for chromosome 1. Our results suggest that double-target FISH on interphase nuclei offers an ideal tool for analyzing tumors prospectively and retrospectively to assess the biological role and the possible prognostic impact of the der(16) in ETs and in other solid tumors.
Adolescent↗
High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA.
Here we report a high-resolution fluorescence in situ hybridization (FISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chromosomes, decondensed interphase nuclear chromatin, and linearly extended chromatin fibers. We analyzed the EBV DNA integrated into the human genome in the well-characterized Burkitt's lymphoma cell line Namalwa, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromosome band 1p35. Using the biotinylated BamHIW fragment of the viral DNA, we observed distinct pairs of signals or small nuclear RNA "tracks" within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and; therefore, enables analysis of the structural organization of DNA. Application of this methodology to linearly extended chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length measurements of 30 images showed a good correlation between the mean physical length of hybridized EBV DNA of 52.8 microns (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of integration processes.
Burkitt Lymphoma↗
Regression and progression in neuroblastoma. Does genetics predict tumour behaviour?
Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation.
Adolescent↗
Screening for neuroblastoma.
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Abdominal Neoplasms↗
Interphase cytogenetic study of childhood acute lymphoblastic leukemia.
We used the fluorescence in situ hybridization (FISH) technique and centromere-specific probes for chromosomes 1, 6, 8, 10, 12, 17, 18, X, and Y to investigate the presence and number of the respective chromosomes in interphase nuclei of 14 cases of childhood acute lymphoblastic leukemia (ALL) which were shown to be hyperdiploid by DNA flow cytometry irrespective of their cytogenetic pattern. Numerical anomalies for one or more chromosomes were detected in all 14 cases. The FISH results were compared with those obtained by conventional cytogenetic analysis. A hyperdiploid karyotype was evident in 5 cases, the others were either normal or lacking cytogenetic results because of technical failure. In the 5 cytogenetically hyperdiploid cases, 14 numerical abnormalities were observed with both techniques, whereas 4 numerical deviations were found only with FISH. In 9 other cases which had a DNA content indicating hyperdiploidy, 34 trisomies and 2 tetrasomies were detected by FISH analysis. Furthermore, in 1 case duplication of the Y chromosome and in 3 male cases duplication of the X chromosome were evident. Double-target FISH experiments in 2 patients allowed the correlation of numerical aberrations of 2 chromosomes in one and the same cell. By such analyses, detection of subpopulations of tumor cells was found to be relatively easy. Our results indicate that the FISH technique with chromosome-specific repetitive centromeric probes is a rapid, simple to use, and easy to interpret technique for the evaluation of numerical chromosomal aberrations in interphase nuclei of leukemias.
Aneuploidy↗