Mutations in N-ras predominate in acute myeloid leukemia

Using synthetic oligomers we investigated fresh samples of acute myeloid leukemia (AML) for the presence of mutated ras oncogenes. Our original results showed that five of eight samples contained a mutation in codon 13 of the N-ras gene. In a subsequent study involving 37 samples, we found only one N-ras-13 mutation, and, in addition, mutations in codon 61 of the N-ras gene in four cases and a mutation in codon 12 of the Ki-ras gene in two cases. Amplification of ras genes was not observed. We conclude that in approximately 20% to 25% of AML cases, a mutated ras oncogene is present, predominantly the N-ras gene. The occurrence of mutations does not correlate with the cytological features of the leukemia.     

Clonal hematopoiesis as defined by polymorphic X-linked loci occurs infrequently in aplastic anemia

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.     

Persistent synovitis treated with radiation synovectomy using yttrium- 90: a retrospective evaluation of 83 procedures for 45 patients

The objectives were to investigate the efficacy and safety of yttrium- 90 colloid (Y-90) synovectomy in joints with persistent synovitis and to examine the effect of a second synovectomy using a double dose after an initial inadequate response. Of the 45 patients at the University Hospital Utrecht who underwent Y-90 synovectomy between July 1987 and October 1995, the effectiveness and side-effects of all yttrium procedures (n = 83) were assessed retrospectively. Glucocorticoids were administered together with the yttrium, except in 1987. Radiation synovectomy had an overall success rate of 75% (complete or almost complete remission of synovitis) initially, i.e. within 1 month of the procedure, partly due to co-administration of glucocorticoids. However, in October 1995, only 17 joints (22%) were still in remission with a mean (S.D.) duration of remission of 20.8 months (22.0), range 1-95 months (median 16 months). In 60 joints (78%), synovitis was present at that time because of an unsatisfactory initial response to the Y-90 injection (19 joints) or recurrent synovitis during follow-up (41 joints); the mean (S.D.) duration of remission of these 60 joints was 3.3 months (5.9), range 0-22 months. A second injection of a double dose of Y-90 after an initial inadequate response (n = 8) did not contribute significantly to a better result. Short-term side-effects occurred in two cases after two Y-90 injections (2%) without glucocorticoid co-administration: a post-injection flare-up of synovitis and a local skin burn lesion. Persistent synovitis can be treated by Y-90 synovectomy with an overall success rate of 75% within 1 month. However, prolonged remission of synovitis is only achieved in 29% of joints with a good initial response. It does not appear worthwhile to perform a second synovectomy with a double dose if the initial response was inadequate. Y-90 synovectomy leads to only minor short-term side-effects (2%).      

Fluorescent in situ hybridization and cytogenetic studies of trisomy 12 in chronic lymphocytic leukemia

Cytogenetic studies (CG) of 475 chronic lymphocytic leukemia (CLL) cases showed trisomy 12 in 6.1% or 26% of patients with abnormal karyotypes. Fluorescence in situ hybridization (FISH) detected trisomy 12 in 35% of 117 CLL patients. Only 34.6% of cases detected by FISH were detected by CG. Twelve patients had low levels of trisomic cells (4% to 11%) relative to clonal B cells (47.5% to 86%), suggestive of clonal evolution. Untreated patients with trisomy 12 were predominantly male (P < .05) and had an increased incidence of splenomegaly (P < .03). Patients with trisomy 12 were more likely to be previously treated and had advanced Binet stage compared with those without trisomy 12. The median survival was shorter in patients with trisomy 12 (7.8 years) and patients with other chromosomal abnormalities without trisomy 12 by FISH (5.5 years) than in patients with diploid karyotypes (14.4 years). The response to fludarabine was similar to that of patients with diploid karyotypes, but there was a trend for earlier disease progression. FISH detected residual disease in all patients with trisomy 12 in complete (n = 6) or partial remission (n = 4). As few as 1 trisomic cell in 5,000 was detected by performing FISH on fluorescence-activated cell sorter-sorted cells. Trisomy 12 was absent in T cells in patients with trisomy 12. We conclude that FISH identifies trisomy 12 approximately 2.6 times more often than CG, readily identifies minimal residual disease, and predicts for a shorter median survival.     

Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.     

Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.