Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Paragangliomas of the head and neck region show complete loss of heterozygosity at 11q22-q23 in chief cells and the flow-sorted DNA aneuploid fraction.

Nonchromaffin paragangliomas of the head and neck region, also known as glomus tumors, are usually benign neoplasms consisting of clusters of chief cells surrounded by sustentacular cells arranged in so-called 'Zellballen.' Most of the patients have a familial background. In a previous study, examining all chromosome arms, we found loss of heterozygosity (LOH) predominantly at the chromosome 11q22-q23 region, where the disease causing gene PGL1 has been located by linkage analysis. However, all tumors showed only partial loss of allele signal intensities, and it was not clear whether this represented allelic imbalance or cellular heterogeneity. In the current study, we have performed LOH analysis for the 11q22-q23 region on DNA-aneuploid tumor cells, enriched by flow sorting, and on purified chief cell fractions obtained by single-cell microdissection. Complete LOH was found for two markers (D11S560 and CD3D) in the flow-sorted aneuploid fractions, whereas no LOH was found in the diploid fractions of three tumors. The microdissected chief cells from two of these tumors also showed complete LOH for both markers, indicating that the chief cells are clonal proliferated tumor cells. These results indicate that the PGL1 gene is likely to be a tumor suppressor gene, which is inactivated according to the two-hit model of Knudson. Furthermore, it shows that chief cells are a major if not the sole neoplastic component of paragangliomas.

A second case of hexasomy 8 in myelodysplastic syndrome.

Tetrasomy 8 is a rare form of acquired aneuploidy found exclusively in the myeloid leukemias. Hexasomy 8 is even rarer: only one case has been reported, thus far. We describe here the second case of hexasomy 8 as the sole abnormality in an elderly female patient with myelodysplastic syndrome (MDS).

A case of isodicentric 7p as sole abnormality in a patient with acute myeloid leukemia.

The detection of isochromosomes in the leukemias and in solid tumors has been well described in the literature, the most common being the i(17q), which is found in the blast crisis of CML and terminal stages of acute myeloid leukemia. Reports of isochromosome 7 have, however, been less well represented, particularly isochromosomes of the short arm of chromosome 7, which represent approximately 1% of all reported isochromosomes in neoplasia. We present here a case report of an elderly female patient with AML-M2 who manifested an idic(7p) in the majority of her bone marrow cells. Fluorescence in situ hybridization (FISH) studies with both centromere-7--and chromosome-7--specific DNA probes verified the diagnosis of idic(7p). To the best of our knowledge, this is the first report of this type of leukemia with an acquired idic(7p) as the sole cytogenetic abnormality.

Sublocalization of the breakpoints of a t(5;16) in myelodysplasia.

As a first step in characterizing a t(5;16)(q31;p11.2) in a patient with the diagnosis refractory anemia with ring sideroblasts, a cell fusion was carried out between bone marrow cells from the patient and the Chinese hamster cell line A3. Using PCR and FISH analysis on hybrid lines containing the human derivative 16 chromosome, the breakpoints could be mapped between the markers TCF-7 and IL-9 on chromosome 5 and OL-7 and s30A4 on chromosome 16, both regions spanning approximately 1 Mb. Since the breakpoint on 5q has occurred in a region that is frequently deleted in myeloid malignancies, the gene disrupted by this translocation could also be implicated in this aberration.

Ring chromosome 4 as the sole cytogenetic anomaly in a chondroblastoma: a case report and review of the literature.

Chromosome analysis of a chondroblastoma of the right distal femur in a 31-year-old male patient revealed a ring chromosome 4 in approximately one-third of the analyzed cells. The remaining cells had a normal karyotype. These findings were subsequently confirmed by fluorescence in situ hybridization (FISH) with a chromosome-4-specific library. FISH with cosmids pC847.351 (4p16.3) and cT171 (4q35) revealed that fewer than 300 kilobase pairs (kbp) are deleted. To our knowledge, ring chromosome 4 has never been reported in this type of neoplasm. There are, however, several reports of chondroblastoma with other chromosome abnormalities, but the relation of these anomalies to this tumor specifically is unclear. In this report, we also provide a review of the literature concerning cytogenetic studies in chondroblastoma. The possible significance of ring chromosome 4 in this type of tumor is discussed.

Characterization of a New Human B Cell Line (Bonna-12) with Trisomy 9 and Trisomy 12 Chromosomal Abnormality.

A new EBV positive human B-cell line, BONNA-12 was established from splenic cells of a patient with a hairy cell leukemia (HCL). BONNA-12 cells grew spontaneously and formed colonies in semisolid media. Although the BONNA-12 cell line was identical with the patient's spleen cells by HLA analysis and Southern blot examination of minisatellite DNA patterns, the immunoglobulin heavy and light chain rearrangement patterns differed from the original HCL. Cytogenetic analysis of the BONNA-12 cell line demonstrated in the major cell clone a 47, X, -Y, +9, +12 karyotype. Trisomy 12 is a characteristic abnormality in chronic lymphocytic leukemia that also rarely occurs in HCL. The BONNA-12 cell line is of potential value in the study of trisomy 12 in chronic B cell malignancies.

Proliferation and cytogenetic analysis of hairy cell leukemia upon stimulation via the CD40 antigen.

Using the CD40 system, in vitro proliferation of hairy cell leukemia (HCL) was examined in 43 patients. In this culture system, cells were stimulated by interleukin-4 (IL-4) and anti-CD40 monoclonal antibodies (MoAbs) that were added in soluble form or were cross-linked via their Fc part using Fc gamma RII-transfected mouse fibroblast cells. Proliferation was induced and confirmed by 3H-thymidine incorporation in 14 cases and by the presence of metaphases in 42 cases. 3H-thymidine incorporation showed a heterogeneous pattern: cross-linking of anti-CD40 gave the highest proliferation in 8 cases; in 11 cases, stimulation with anti-CD40 MoAbs alone, without cross-linking also resulted in proliferation; the addition of IL-4 further enhanced 3H-thymidine incorporation in 5 cases, but suppressed this phenomenon in 5 other cases. The CD40 system proved to be very effective in obtaining cytogenetic data. With a success rate of 42 of 43 patients tested, we found clonal abnormalities in 8 cases (19%) and nonclonal abnormalities with involvement of one or two abnormal metaphases in another 7 cases. The chromosomes most frequently involved in the abnormal karyotypes, both structurally and numerically, were chromosomes 5, 7, and 14. By fluorescence-activated cell-sorting analysis of the cultured cells, and by immunophenotypic analysis of metaphase spreads, T-cell growth could be excluded and the HCL-lineage confirmed. Stimulation via the CD40 antigen is an excellent tool for growing hairy cell leukemia cells.

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied. MGF as a single factor did not induce significant colony formation by clonogenic leukemic precursor cells. In the presence of IL-3 and/or GM-CSF, MGF weakly stimulated the colony formation by clonogenic precursor cells from patients with AML. In contrast, in the presence of IL-3 and/or GM-CSF, MGF strongly induced both size and number of leukemic colonies from patients with CML in chronic phase. Furthermore, in the presence of EPO, MGF strongly stimulated erythroid colony formation by CML precursor cells. Cytogenetic analysis of the colonies showed that all metaphases after 1 week of culture were derived from the leukemic clone. In patients with MDS, MGF strongly stimulated myeloid colony formation in the presence of IL-3 and/or GM-CSF (up to fourfold), and erythroid colony formation in the presence of EPO (up to eightfold). Not only the number, but also the size of the colonies increased. In the presence of MGF, the percentage of normal metaphases increased in three patients tested after 1 week of culture compared with the initial suspension, suggesting that the normal HPC were preferentially stimulated compared with the preleukemic precursor cells. In the absence of exogenous EPO and in the presence of 10% human AB serum, MGF in the presence of IL-3 and/or GM-CSF induced erythroid colony formation from normal bone marrow and patients with MDS or CML, illustrating that MGF greatly diminished the EPO requirement for erythroid differentiation. These results indicate that MGF may be a candidate as a hematopoietic growth factor to stimulate normal hematopoiesis in patients with acute myeloid leukemia, or with myelodysplastic syndromes.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Nonradioactive in situ hybridisation of the translocation t(1;7) in myeloid malignancies.

Bone marrow cells of four patients with t(1;7) and myelodysplasia or acute myeloid leukemia were analyzed using nonradioactive in situ hydridisation. As probes, centromeric alphoid DNA sequences of chromosomes 1 and 7, a satellite DNA probe for 1q12, and chromosome-specific libraries of chromosomes 1 and 7 were used. The breakpoints of the t(1;7)(p11;p11) as determined by banding analysis could be studied more accurately, and the recently proposed designation t(1;7)(cen;cen) was confirmed in all four cases. Colocalization of alphoid DNA sequences of chromosomes 1 and 7 by double target in situ hybridisation was demonstrated in metaphase cells and also in interphase nuclei. The in situ hybridisation method described is applicable for the screening of peripheral blood cells or archival material.

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p.

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.