Gundula Thiel

Karyotypes in 90 human gliomas

Cytogenetic studies were performed on 90 human gliomas including 26 astrocytomas, 12 oligodendrogliomas, three oligo-astrocytomas, seven ependymomas, eight pilocytic astrocytomas, and 33 malignant gliomas (anaplastic astrocytomas and glioblastomas). The most common abnormalities were trisomy 7 in 23 cases, monosomy 22 in 15 cases, losses of the Y chromosome in 19 of 50 male cases, and losses of the X chromosome in 10 of 39 female cases. There are evident differences between the particular subgroups of gliomas. Monosomy 10 and double minutes are typical for malignant gliomas. The 58 determined chromosomal breakpoints were located on 45 different sites. Chromosomes 1, 9, 6, 3, 10, and 17 were predominantly involved.

Distinct methylation profiles of glioma subtypes

Gliomas are tumors of the central nervous system with a wide spectrum of different tumor types. They range from pilocytic astrocytoma, with a generally good prognosis, to the extremely aggressive malignant glioblastoma. In addition to these 2 types of contrasting neoplasms, several other subtypes can be distinguished, each characterized by specific phenotypic, as well as genotypic features. Recently, the epigenotype, as evident from differentially methylated DNA loci, has been proposed to be useful as a further criterion to distinguish between tumor types. In our study, we screened 139 tissue samples, including 33 pilocytic astrocytomas, 46 astrocytomas of different grades, 7 oligoastrocytomas, 10 oligodendrogliomas, 10 glioblastoma multiforme samples and 33 control tissues, for methylation at CpG islands of 15 different gene loci. We used the semiquantitative high throughput method MethyLight to analyze a gene panel comprising ARF, CDKN2B, RB1, APC, CDH1, ESR1, GSTP1, TGFBR2, THBS1, TIMP3, PTGS2, CTNNB1, CALCA, MYOD1 and HIC1. Seven of these loci showed tumor specific methylation changes. We found tissue as well as grade specific methylation profiles. Interestingly, pilocytic astrocytomas showed no evidence of CpG island hypermethylation, but were significantly hypomethylated, relative to control tissues, at MYOD1. Our results show that glioma subtypes have characteristic methylation profiles and, with the exception of pilocytic astrocytomas, show both locus specific hyper‐ as well as hypomethylation.

Molecular Characterization and Gene Content of Breakpoint Boundaries in Patients with Neurofibromatosis Type 1 with 17q11.2 Microdeletions

Homologous recombination between poorly characterized regions flanking the NF1 locus causes the constitutional loss of approximately 1.5 Mb from 17q11.2 covering > or =11 genes in 5%-20% of patients with neurofibromatosis type 1 (NF1). To elucidate the extent of microheterogeneity at the deletion boundaries, we used single-copy DNA fragments from the extreme ends of the deleted segment to perform FISH on metaphase chromosomes from eight patients with NF1 who had large deletions. In six patients, these probes were deleted, suggesting that breakage and fusions occurred within the adjacent highly homologous sequences. Reexamination of the deleted region revealed two novel functional genes FLJ12735 (AK022797) and KIAA0653-related (WI-12393 and AJ314647), the latter of which is located closest to the distal boundary and is partially duplicated. We defined the complete reading frames for these genes and two expressed-sequence tag (EST) clusters that were reported elsewhere and are associated with the markers SHGC-2390 and WI-9521. Hybrid cell lines carrying only the deleted chromosome 17 were generated from two patients and used to identify the fusion sequences by junction-specific PCRs. The proximal breakpoints were found between positions 125279 and 125479 in one patient and within 4 kb of position 143000 on BAC R-271K11 (AC005562) in three patients, and the distal breakpoints were found at the precise homologous position on R-640N20 (AC023278). The interstitial 17q11.2 microdeletion arises from unequal crossover between two highly homologous WI-12393-derived 60-kb duplicons separated by approximately 1.5 Mb. Since patients with the NF1 large-deletion syndrome have a significantly increased risk of neurofibroma development and mental retardation, hemizygosity for genes from the deleted region around the neurofibromin locus (CYTOR4, FLJ12735, FLJ22729, HSA272195 (centaurin-alpha2), NF1, OMGP, EVI2A, EVI2B, WI-9521, HSA272196, HCA66, KIAA0160, and WI-12393) may contribute to the severe phenotype of these patients.

Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts

Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.

Recurrent gain of chromosome arm 7q in low‐grade astrocytic tumors studied by comparative genomic hybridization

Consistent tumor‐specific chromosomal aberrations have not been described in low‐grade astrocytic tumors. The most frequent genetic alterations are mutations of the TP53 tumor suppressor gene and/or loss of heterozygosity (LOH) on 17p that occur in about 30% of the cases in adult patients but that are uncommon in childhood tumors. We used comparative genomic hybridization (CGH) to map DNA copy number alterations in 18 primary low‐grade astrocytic tumors (ten adult patients and eight children). A gain of chromosome arm 7q was the most frequent event detected in five of ten astrocytomas (50%) from adult patients, followed by DNA amplification on chromosome arm 8q and gain on 12p (two cases). Loss of chromosomal regions on 1p, 4q, and the X chromosome was observed in two of ten cases each [including one patient afflicted with Turner syndrome (45,X)]. In contrast, no consistent changes were observed in low‐grade astrocytomas in children. A loss of the X chromosome was the sole aberration detected in two of eight cases using DNA extracted from normal brain tissue. The findings suggest that a gain of 7q is an early event in the initiation of astrocytomas in adult patients. The discrepant findings in low‐grade astrocytic tumors in adults compared to tumors in children support the hypothesis that there might be different mechanisms responsible for tumor development. Genes Chromosom Cancer 15:199–205 (1996).

Involvement of chromosome 22 in ependymomas

We have karyotyped a total of twelve ependymomas using GTG-banding including seven for which preliminary results have already been published. One case showing hyperdiploid main line with two marker chromosomes was further analyzed by nonisotopic chromosome in situ suppression hybridization. It was shown that the marker chromosomes consisted of 1q, 14q and 1q, and 22q. The possible role of chromosome 22 in ependymomas and the usefulness of fluorescence in situ hybridization for cytogenetic analysis in tumor investigation are discussed.

Numerical aberrations of chromosomes 1, 2, and 7 in astrocytomas studied by interphase cytogenetics.

For both juvenile astrocytomas and astrocytomas of adults, numerical and structural aberrations of chromosomes 1 and 7 have been described. To study the frequency of those aberrations in more detail and to exclude in vitro artifacts, we investigated directly prepared material from 18 juvenile and 26 astrocytomas of adults by fluorescence in situ hybridization with DNA probes specific for chromosome regions 1p36, 1q12, 2cen, and 7cen. Chromosome 2 was used as control in the hybridization with chromosome 7. To exclude tissue‐specific alterations, we tested cerebral and cerebellar paraffin‐embedded material from persons who had died from other diseases. In 13 of the juvenile astrocytomas, we found a loss of 1p36 in relation to 1q12. In 16 astrocytomas of adults, a gain of signals from 1p36 or both probes for chromosome 1 was seen. Gain of chromosome 7 was found in 25 cases. Unexpectedly, gain of chromosome 2 occurred in 32 cases. For both probes, there was no difference between astrocytomas of children and those of adults. Our data suggest that loss of 1p is an early event in the development of juvenile astrocytomas and that trisomy 7 is frequent in malignant tumors and tumors containing a potential of growing malignantly. Genes Chromosom. Cancer 19:6–13, 1997.

Age-related nonrandom chromosomal abnormalities in human low-grade astrocytomas

We report a cytogenetic investigation of 55 low-grade astrocytomas in 52 patients, 15 children and 37 adults. In addition to numerical aberrations such as trisomy 7 and gonosomal losses, we found structural and/or numerical aberrations of chromosome 1 in eight astrocytomas. There was a striking difference between the rearranged chromosomes in pediatric and adult patients. Whereas the pediatric tumors revealed monosomies 1p with accompanying trisomy 1q, the astrocytomas in adults showed partial or complete monosomies 1q.

Cytogenetic studies in acute leukemia patients relapsing after allogeneic stem cell transplantation.

We analyzed karyotype stability in 22 patients with acute leukemia at relapse or disease progression after allogeneic stem cell transplantation (allo-SCT). Karyotypes before and at relapse after allo-SCT were different in 15 patients (68%), the most frequent type being clonal evolution either alone or combined with clonal devolution (13 patients). Patients with and without a karyotype change did not differ significantly in overall survival (OS) (median, 399 vs. 452 days; P = 0.889) and survival after relapse (median, 120 vs. 370 days; P = 0.923). However, acquisition of additional structural chromosome 1 abnormalities at relapse after allo-SCT occurred more frequently than expected and was associated with reduced OS (median, 125 vs. 478 days; P = 0.008) and shorter survival after relapse (median, 37 vs. 370 days; P = 0.002). We identified a previously undescribed clonal evolution involving t(15;17) without PML-RARA rearrangement in an AML patient. We conclude that a karyotype change is common at relapse after allo-SCT in acute leukemia patients. Moreover, our data suggest that additional structural chromosome 1 abnormalities are overrepresented at relapse after allo-SCT in these patients and, in contrast to a karyotype change per se, are associated with reduced OS and shorter survival after relapse.

De novo t(12;17)(p13.3;q21.3) translocation with a breakpoint near the 5′ end of the HOXB gene cluster in a patient with developmental delay and skeletal malformations

A boy with severe mental retardation, funnel chest, bell-shaped thorax, and hexadactyly of both feet was found to have a balanced de novo t(12;17)(p13.3;q21.3) translocation. FISH with BAC clones and long-range PCR products assessed in the human genome sequence localized the breakpoint on chromosome 17q21.3 to a 21-kb segment that lies <30 kb upstream of the HOXB gene cluster and immediately adjacent to the 3′ end of the TTLL6 gene. The breakpoint on chromosome 12 occurred within telomeric hexamer repeats and, therefore, is not likely to affect gene function directly. We propose that juxtaposition of the HOXB cluster to a repetitive DNA domain and/or separation from required cis-regulatory elements gave rise to a position effect.