Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.     

Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers

The use of prophylactic anti-D to prevent Rh D immunization in Rh D- women and subsequent hemolytic disease in Rh D+ infants is widespread, but has led to shortages of the anti-D Ig. With the aim of substituting monoclonal anti-D for Rh D prophylaxis, we have compared the abilities of monoclonal and polyclonal anti-D to clear Rh D+ red blood cells (RBCs) infused into Rh D- male volunteers and to suppress Rh D immunization. Two human monoclonal antibodies (MoAbs), BRAD-3 (IgG3) and BRAD-5 (IgG1), produced from stable Epstein-Barr virus-transformed B-lymphoblastoid cell lines, were selected because of their proven in vitro activity in promoting RBC lysis in antibody-dependent cell- mediated cytotoxicity assays. RBC clearance was assessed by intravenous injection of 3 mL of 51chromium-labeled D+ RBCs into 27 volunteers 48 hours after intramuscular injection of monoclonal or polyclonal anti-D. Further 3-mL injections of unlabeled D+ cells were administered at 6 and 9 months to induce immunization. Blood samples were taken throughout the 12-month period of study for the serologic detection of anti-D. The mean half-life (t50%) of RBCs in 7 recipients of 300 micrograms BRAD-5 (5.9 hours) was similar to that in 8 recipients of 500 IU polyclonal anti-D (5.0 hours), whereas D+ cells were cleared more slowly in some of the 8 subjects injected with 300 micrograms BRAD- 3 (mean t50% 12.7 hours) and in 1 individual administered 100 micrograms BRAD-3 (t50% 41.0 hours). The rate of RBC clearance in both groups administered 300 micrograms monoclonal anti-D correlated with the amount of antibody bound per cell, determined by flow cytometry. There was no evidence of primary immunization having occurred in any subject after 6 months of follow-up. Five of 24 subjects produced anti- D after one or two further injections of RBCs, confirming that they were responders who had been protected by the monoclonal or polyclonal anti-D administered initially. Four of these responders were recipients of monoclonal anti-D (3 BRAD-3, 1 BRAD-5). One individual who received BRAD-5 produced accelerated clearance of D+ RBCs at the third unprotected RBC challenge but did not seroconvert. This study shows that the human MoAbs BRAD-3 and BRAD-5 can prevent Rh D immunization, and indicates that they may be suitable replacements for the polyclonal anti-D presently used in prophylaxis of Rh D hemolytic disease of the newborn.(ABSTRACT TRUNCATED AT 400 WORDS)     

Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient's age and/or the conditioning regimen, but it is not associated with an increased risk of relapse

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host- type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T- cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T- cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.     

Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype

The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.     

Does apolipoprotein(a) heterogeneity influence lipoprotein(a) effects on fibrinolysis?

High plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.     

Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells

The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.