J. Abels

Dutch β°‐thalassaemia: a 10 kilobase DNA deletion associated with significant γ‐chain production

Summary. A unique β°‐thalassaemia in a Dutch family results in fetal haemoglobin expression comparable to that of δ°β°‐thalassaemia. Haemoglobin analysis and restriction endonuclease mapping studies of DNA suggest that the β‐globin gene is entirely deleted, but that the δ‐globin gene is intact. The 5’break point of the deletion is 3–4 kilobases 3’to the δ‐globin gene, while the 3’break point is 6–7 kilobases 3’to the β‐globin gene (relative to the normal DNA restriction map). The result is a ˜ 10 kilobase deletion of DNA whose 3’end point may lie very close to that for one δ°β°‐thalassaemia, within a cluster of Kpn I‐family repetitive sequences. The Dutch β°‐thalassaemia deletion is thus the shortest one which, in the absence of additional chromosomal rearrangements, results in enhancement of γ‐chain synthesis above that seen for haemoglobin Lepore. These data support the hypothesis that the region of DNA 3’to the β‐globin gene may be important to the developmental regulation of fetal γ versus adult β chain production.

Pseudothrombocytopenia: a cold autoantibody against platelet glycoprotein GP IIb.

Summary. Increased binding of IgM to donor platelets was detected in serum of a patient demonstrating the phenomenon of pseudothrombocytopenia. The elevated IgM binding was dependent on EDTA concentration and temperature, and could not be demonstrated when platelets of patients with Glanzmanns disease were used. Immunoblotting and crossed immunoelectrophoresis showed that the IgM antibody reacted with platelet glycoprotein GP IIb. Evidence is presented that the antigenic binding site on GP IIb is made accessible to the antibody by means of the effect of EDTA on the Ca2+ dependent GP IIb/IIIa complex.

Antibodies against glycosphingolipids in sera of patients with idiopathic thrombocytopenic purpura

Summary. Evidence is presented for the existence of antibodies against platelet glycosphingolipids in sera of patients with chronic idiopathic thrombocytopenic purpura and antiplatelet antibodies. Increased binding of IgG/IgM to neutral glycosphingolipids or gangliosides extracted from donor platelets was measured by an ELISA in 17 sera of 30 patients. Thirteen sera, five with anticardiolipin antibodies, demonstrated an increased binding to sulphatides confirmed by immuno HPTLC. Four sera showed reactivity with the platelet minor gangliosides established by immuno‐HPTLC.

Clinicopathological diagnosis and treatment of malignant histiocytosis

Summary. The diagnostic findings of malignant histiocytosis (MH) were analysed in 12 consecutive patients in a single institution. Most patients presented with systemic symptoms and lymphadenopathy (92%), splenomegaly (100%) and hepatomegaly (67%). Neurologic symptoms were present in three patients, while involvement of other organs was present in five patients. The incidence of severe thrombocytopenia was 92%, of anaemia 92% and of leucocytopenia 67%. Serum angiotensin converting enzyme, α1‐antitrypsin and lysozyme were independently increased in 6/9, 3/10 and 1/9 patients respectively. High serum levels of tumour necrosis factor (TNF) were present in 3/10 patients, while serum levels of interleukin‐1 were normal in 10/10 patients. Histologic evidence of MH was obtained in all patients by repeated biopsies of involved tissues. Four patients died prior to treatment. Seven patients were treated with combination chemotherapy, consisting of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or MOPP (chloromethine, vincristine, procarbazine, prednisone), in some cases followed by non‐cross‐resistant second line chemotherapy, if no complete response was attained. The response rate of treated patients was 5 7%, and progression was observed in two patients. The median duration of response was 38 months. Three patients are alive without evidence of disease and off therapy (30+, 83+, 85+ months).

TdT positive B-cell acute lymphoblastic leukaemia (B-ALL) without Burkitt characteristics.

Summary The leukaemic cells in a 23‐year‐old man were small to medium‐sized lymphoblasts with no cytoplasmic vacuoles and negative with PAS as well with peroxidase and acid phosphatase staining. Cytogenetic analysis showed ‐6,+12,‐22,+mar(6p::22q), resulting in a trisomy 12 and monosomy of the long arm of chromosome 6. Immunological marker analysis revealed that the majority of the blasts was positive for terminal deoxynucleotidyl transferase (TdT) as well as surface membrane immunoglobulin (SmIg, ü, λ), although B‐ALL are supposed to be negative for TdT. The blasts were also positive for HLA‐DR, CD9 (BA‐2), CD10 (VIL‐A1) and CD24 (BA‐1), but negative for the B‐cell markers CD20 (B1) and Y29/55. Double immunofluorescence staining confirmed that almost all TdT+ cells were also positive for Smμ, Smλ, HLA‐DR and CD10. We thus made a diagnosis of TdT+ B‐ALL without Burkitt characteristics. Since we could not detect SmIg+/TdT+ cells in bone marrow samples from adult healthy volunteers and from 10 children with ALL in complete remission, we conclude that TdT+ B‐ALL cells may not have a normal counterpart in bone marrow or represent a malignant counterpart of a very rare cell in an intermediate differentiation stage between the pre‐B‐cell and the early B lymphocyte.

Nitrous oxide reduces growth of experimental rat leukemia

The ability of nitrous oxide to inhibit the in vivo growth of hematological neoplasms was investigated in a rat model for acute myeloid leukemia (BNML). Nitrous oxide, administered in a concentration of 67% with 33% oxygen, resulted in a reduction of spleen and liver weights of approx. 30%, as compared with leukemic rats kept in ambient air. Peripheral white cell counts were also considerably lower in the treated rats. Plasma levels of vitamin B12 were found to be elevated in untreated leukemia, but fell to about normal levels after nitrous oxide exposure. On the contrary, folic acid levels were low in untreated leukemic rats, and significantly higher in animals exposed to nitrous oxide. The observed effects of nitrous oxide appeared to be dose-dependent. The deoxyuridine suppression test performed with leukemic cells became abnormal after nitrous oxide inhalation, in accordance with the effect on normal bone marrow. These results indicate that the interference of nitrous oxide with vitamin B12-related metabolism, which leads to impairment of de novo thymidine synthesis, has the potency to reduce leukemic proliferation in vivo.

Analysis of the breakpoints in translocation (15;17) Observed in 4 patients with acute promyelocytic leukemia

SummaryFour patients with acute promyelocytic leukemia (APL) and the chromosomal translocation t(15;17) are described in detail. One of the patients presented with the microgranular variant form of APL and the standard translocation. Another patient is the third reported case with isochromosome formation of the 17q- derivative. Use of high resolution culture technique with methotrexate treatment allowed us to define the break-points at 15q2200 and 17q12.

Effect of nitrous oxide and methotrexate on folate coenzyme pools of blast cells from leukemia patients

The effects of methotrexate (inhibiting dihydrofolate reductase) and nitrous oxide (inactivating methionine synthase) on intracellular folate coenzyme levels of leukemic cells were studied. Blast cells from 10 cases of acute myeloid leukemia (AML) and 5 cases of acute lymphoid leukemia (ALL) were incubated with 5 x 10(-8) M [3H] 5-formyltetrahydrofolate (5-formylTHF) for 18 h to label intracellular folate pools, which were subsequently quantitated by high performance liquid chromatography (HPLC). In AML, 5-methylTHF made up 53% of the total folate pool followed by 10-formylTHF (26%), 5-formylTHF (10%), THF (9%) and DHF (1%). Cells from ALL differed from AML (p less than 0.05) with respect to 10-formylTHF (17%) and DHF (10%). Exposure to nitrous oxide (8 h) caused an equal decrease of 10-formylTHF and 5-formylTHF in both AML (30%) and ALL (45%), whereas 5-methylTHF increased (130%). Methotrexate (4 h, 10(-6) M) caused an accumulation of DHF and a decrease of 5-methylTHF in both AML (32%) and ALL (12%). A specific reduction of the 10-formylTHF (50%) and 5-formylTHF (25%) pools was noticed in ALL. Exposure to nitrous oxide prior to methotrexate treatment aggravated the reduction of 10-formylTHF and 5-formylTHF presumably by impaired replenishment from the 5-methylTHF pool. In conclusion, this study demonstrates a significant difference in folate coenzyme distribution between cells from AML and ALL. Moreover it is shown that nitrous oxide and methotrexate treatment of leukemic cells cause an accumulation of 5-methylTHF and DHF respectively at the expense of other folate forms. The presence of substantial amounts of DHF in cells from ALL together with the specific reduction of 10-formylTHF (necessary for purine synthesis) during MTX treatment may in part explain the efficacy of methotrexate in the treatment of ALL.

Secondary hematologic neoplasm after intravesical chemotherapy for superficial bladder carcinoma

Two cases are reported of patients who developed a hematologic malignancy several years after intravesical chemotherapy of superficial bladder cancer with etoglucid, doxorubicin, and mitomycin C. In one patient, karyotypic abnormalities (−5, 7q‐) typical of a therapy induced malignancy were associated with rapid progression of a refractory anemia with excess blasts in transformation to an acute non‐lymphocytic leukemia. Intravesical chemotherapy may be associated with a risk of secondary malignancy.

Nitrous oxide selectively reduces the proliferation of the malignant cells in experimental rat leukemia

A considerable reduction of hepatosplenomegaly and leucocytosis in leukemic rats of the Brown Norway Myeloid Leukemia (BNML) can be achieved by exposure to 50% nitrous oxide/50% oxygen. In this study the differential antiproliferative effect of nitrous oxide, inactivating vitamin B12, on normal and leukemic hemopoiesis was investigated in this rat model. Rats injected with leukemic cells and exposed to nitrous oxide for 10 days showed 30% reduction of hepatosplenomegaly and 50% reduction of leukocytosis. Similarly treated healthy rats showed no signs of impaired hemopoiesis as measured by peripheral blood parameters. Clonogenic assays of erythroid and myeloid progenitors from both healthy and leukemic rats revealed that exposure to nitrous oxide did not suppress normal bone marrow functioning. On the contrary, the reduction of leukemic proliferation by nitrous oxide retarded the leukemic infiltration of the bone marrow compartment.