Intensive leukapheresis as initial therapy for chronic granulocytic leukemia

Intensive leukapheresis has been used as the initial treatment of chronic granulocytic leukemia (CGL) in six patients. The number of leukaphereses ranged from 3 in 7 days to 13 in 39 days (mean, 8 in 22 days). The procedures were well tolerated, and in all patients there was improvement in hematologic values, in most cases with considerable reduction in the peripheral leukocytosis and thrombocytosis and in the proportion of immature granulocytic cells in the circulation. Splenomegaly decreased considerably in the four patients who had more than four leukaphereses. Symptoms of sweating, malaise, and pain due to splenomegaly were rapidly relieved. Problems due to hyperuricemia did not occur, but four patients required blood transfusions for correction of anemia. This method of initial treatment of CGL appears to give more rapid relief of symptoms than does conventional chemotherapy; it incurs no risk of hyperuricemia and lessens that associated with thrombocytosis. In addition, large quantities of granulocyte-rich plasma are made available for the treatment of infections in neutropenic patients. Intensive leukapheresis deserves more widespread evaluation as the initial treatment of CGL.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

c-tal, a helix-loop-helix protein, is juxtaposed to the T-cell receptor- beta chain gene by a reciprocal chromosomal translocation: t(1;7)(p32;q35)

Studies on nonrandom chromosomal translocations have been important for the identification of genes potentially involved in the malignant transformation of cells. The most widely studied translocations, involving members of the Ig supergene family, have shown juxtapositions of proto-oncogenes with the rearranging loci. Such translocations can inappropriately activate expression of the proto-oncogenes and thereby play a role in tumorigenesis. Because the cytogenetic analysis of a bone marrow sample from a child with T-cell acute lymphoblastic leukemia showed a (1;7)(p32;q35) translocation, we sought to determine if the translocation breakpoint was in the T-cell receptor (TCR)-beta gene locus on chromosome 7. Analysis of the TCR-beta gene by Southern blotting showed three rearranged bands. Nucleotide sequencing and Southern blot analysis of TCR-beta genomic clones, isolated from patient DNA, showed that one contained a normal rearrangement of the TCR-beta gene using V beta 12.2, D beta 2.1, and J beta 2.5, whereas two other clones contained DNA from derivative chromosomes 1 and 7. Chromosomal mapping showed that the (1;7) translocation breakpoint was 35 kb 3' to the c-tal gene locus. The juxtaposition of c-tal to the TCR- beta locus may enhance c-tal expression and contribute to T-cell leukemogenesis.     

High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of Hodgkin's disease occurring in Peru

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease (HD). This study was undertaken to determine whether the association of EBV with HD showed geographical variation, as in Burkitt's lymphoma. We studied 32 formalin-fixed, paraffin-embedded cases of HD occurring in Peru. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated antisense oligonucleotide complementary to the EBER1 gene of EBV. EBV immunohistochemistry was also performed, using a monoclonal antibody (MoAb) to the latent membrane protein (LMP1) of EBV. Identification of the precise cellular subset staining with EBV was accomplished via double-labeling with MoAbs directed against Reed-Sternberg cells (LeuM1/CD15) and B cells (L26/CD20). EBV RNA was identified in all or virtually all of the Reed- Sternberg cells and variants in 30 of the 32 (94%) cases of HD by in situ hybridization. LMP1 expression was identified in 83% of the EBER1- positive cases. Double-labeling studies confirmed the localization of EBV RNA to CD15-expressing Hodgkin's cells. This study found an extremely high prevalence of EBV in Peruvian HD, in contrast to the much lower percentage of EBV-associated cases of HD occurring in "Western" patients.     

Acute myeloid leukemia with T-lymphoid features: a distinct biologic and clinical entity

We studied the clinical and biologic features of 10 cases of acute leukemia that met standard French-American-British (FAB) criteria for acute myeloid leukemia (AML) but in which the blast cells also expressed the T-cell-associated CD2 surface antigen. All cases had greater than 3% myeloperoxidase and Sudan black B-positive leukemic blasts, and blasts from seven cases contained Auer rods. Reactivity of the cells with a panel of monoclonal antibodies (MAbs) indicated that leukemic cells in all cases expressed myeloid-associated (CD11b, CD13) surface antigens, further supporting the diagnosis of AML. However, blasts from every patient coexpressed the T-cell-associated surface CD2 and CD7 as well as cytoplasmic CD3 antigens. Blasts from five patients expressed surface CD25, whereas blasts from only one expressed surface CD3. Five patients had rearranged T-cell receptor beta-chain genes, whereas only three had rearranged T-cell receptor gamma-chain genes. This pattern of lineage-related gene expression appears to define a distinct subtype of AML with T-lymphoid features (CD2+ AML) and could reflect either aberrant gene expression in leukemic blasts or transformation of a pluripotent stem cell having a flexible pattern of gene expression. Clinically, these 10 patients presented at an older age with a higher leukocyte count and a higher frequency of lymphadenopathy than did children whose blast cells were characteristic of myeloid leukemia. Patients with CD2+ AML also had poorer responses to remission induction therapy (50% v 80% entered complete remission, P = .05). However, each of the five children who failed induction chemotherapy on AML protocols had a striking response to drug combinations usually reserved for lymphoid leukemia. We conclude that this leukemia with mixed lymphoid and myeloid characteristics is a distinct biologic and clinical entity.     

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.     

Improved exercise performance after exchange transfusion in subjects with sickle cell anemia

Ten patients with sickle cell anemia underwent partial exchange transfusion with hemoglobin-A-containing cells using a technique that allowed hemoglobin concentration and blood volume to remain constant. The mean fraction of hemoglobin-A in these patients increased from 9% to 55%, but the mean hemoglobin concentration increased by only 1.44 g/dl. The exchange resulted in a large improvement in submaximal exercise capacity: the mean of the anaerobic threshold (the work at which lactic acid begins to accumulate in the blood) increased from 68 to 114 W. The mean work performed at a heart rate of 170/min, an estimation of maximal work capacity, increased from 128 to 187 W. Improved exercise performance after partial exchange transfusion may result from the superior flow properties of hemoglobin-A-containing red cells. Furthermore, we believe that exercise testing in sickle cell anemia has great potential utility as a means to monitor therapy and to evaluate the benefits of exchange transfusion.     

Hepatolithiasis with biliary ascariasis – a case report

Background  

Biliary ascariasis is regarded as possible etiological factor for hepatolithiasis. Here we report one case of a patient with hepatolithiasis with biliary ascariasis who developed a liver abscess, which was treated with partial hepatectomy.     

Reduced thrombus formation in native blood of homozygous factor VII- deficient patients at high arterial wall shear rate

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII- deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P < .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P < .002 and P < .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P < .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.     

Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells

We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.