High-dose cyclophosphamide, carmustine (BCNU), and etoposide (VP16-213) with or without cisplatin (CBV +/- P) and autologous transplantation for patients with Hodgkin's disease who fail to enter a complete remission after combination chemotherapy

Patients with Hodgkin's disease (HD) who fail to enter a complete remission after an initial course of combination chemotherapy are usually considered to have an induction failure (IF); this subset of patients has an extremely poor outcome with further conventional therapy. Since 1985, we have entered 30 IF patients into protocols using conditioning with high-dose cyclophosphamide, carmustine (BCNU), and etoposide (VP16-213) with or without cisplatin (CBV +/- P) followed by autologous stem cell transplantation (ASCT) with bone marrow (19 patients), peripheral blood stem cells (PBSCs; 8 patients), or both (3 patients). All except 2 patients had previously received chemotherapy regimens for HD that contained at least 7 drugs, and 9 had received prior radiotherapy (RT). After documentation of IF, the majority of patients received some cytoreductive therapy as specified by protocol (local RT in 9, two cycles of conventional chemotherapy in 2, both modalities in 2, or high-dose cyclophosphamide to enhance PBSC collection in 11) before CBV +/- P. Five treatment-related deaths occurred, all before day 150 posttransplant. Eleven patients have had progressive HD at a median of 6 months (range, 0.1 to 45 months) after ASCT. The actuarial progression-free survival (PFS) at a median follow- up of 3.6 years (range, 0.2 to 8.2 years) is 42% (95% confidence intervals, 21% to 61%). The statistical analysis identified only prior clinical bleomycin lung toxicity as an adverse risk factor for PFS, mainly because of the increased nonrelapse mortality seen in these patients. CBV +/- P and ASCT can produce durable remission in a substantial proportion of IF HD patients who otherwise have a poor survival, and we believed ASCT approaches represent the best therapy currently available for these patients. Additional measures are needed to reduce the primary problem of disease progression despite high-dose chemotherapy and stem cell transplantation.     

Heterozygous Insulin Receptor (INSR) Mutation Associated with Neonatal Hyperinsulinemic Hypoglycaemia and Familial Diabetes Mellitus: Case Series

Mutations in the insulin receptor (INSR) gene are associated with insulin resistance and hyperglycaemia. Various autosomal dominant heterozygous INSR mutations leading to hyperinsulinemic hypoglycaemia (HH) have been described in adults and children (more than 3 years of age) but not in the neonatal period. Family 1: A small for gestational age (SGA) child born to a mother with gestational diabetes presented with persistent hypoglycaemia, was diagnosed with HH and responded well to diazoxide treatment. Diazoxide was gradually weaned and discontinued by 8 months of age. Later, the younger sibling had a similar course of illness. On genetic analysis a heterozygous INSR missense variant p.(Met1180Lys) was found in the siblings, mother and grandfather but not in the father. Family 2: A twin preterm and SGA baby presented with persistent hypoglycaemia, which was confirmed as HH. He responded to diazoxide, which was subsequently discontinued by 10 weeks of life. Genetic analysis revealed a novel heterozygous INSR missense variant p.(Arg1119Gln) in the affected twin and the mother. Family 3: An SGA child presented with diazoxide responsive HH. Diazoxide was gradually weaned and discontinued by 9 weeks of age. Genetic analysis revealed a novel heterozygous INSR p.(Arg1191Gln) variant in the proband and her father. We report, for the first time, an association of INSR mutation with neonatal HH responsive to diazoxide therapy that resolved subsequently. Our case series emphasizes the need for genetic analysis and long-term follow up of these patients.     

Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression

Extensive immunologic marker analysis was performed to characterize the various leukemic cell populations in eight patients with inv(16)(p13q22) in association with acute myeloid leukemia with abnormal bone marrow eosinophilia (AML-M4Eo). The eight AML cases consisted of heterogeneous cell populations; mainly due to the presence of multiple subpopulations, which varied in size between the patients. However, the immunophenotype of these subpopulations was comparable, independent of their relative sizes. Virtually all AML-M4Eo cells were positive for the pan-myeloid marker CD13. In addition, the AML were partly positive for CD2, CD11b, CD11c, CD14, CD33, CD34, CD36, CDw65, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Double immunofluorescence stainings demonstrated coexpression of the CD2 antigen and myeloid markers and allowed the recognition of multiple AML subpopulations. The CD2 antigen was expressed by immature AML cells (CD34+, CD14-) and more mature monocytic AML cells (CD34-, CD14+), whereas TdT expression was exclusively found in the CD34+, CD14- cell population. The eight AML-M4Eo cases not only expressed the CD2 antigen, but also its ligand CD58 (leukocyte function antigen-3). Culturing of AML-M4Eo cell samples showed a high spontaneous proliferation in all three patients tested. Addition of a mixture of CD2 antibodies against the T11.1, T11.2, and T11.3 epitopes diminished cell proliferation in two patients with high CD2 expression, but no inhibitory effects were found in the third patient with low frequency and low density of CD2 expression. These results suggest that high expression of the CD2 molecule in AML-M4Eo stimulates proliferation of the leukemic cells, which might explain the high white blood cell count often found in this type of AML.     

Methotrexate, cyclosporine, or both to prevent graft-versus-host disease after HLA-identical sibling bone marrow transplants for early leukemia?

Optimal prophylaxis of graft-versus-host disease (GVHD) is controversial. We compared efficacy of three posttransplant immune suppressive regimens in 2,286 recipients of HLA-identical sibling bone marrow transplants for acute lymphoblastic leukemia (ALL) in first remission, acute myelogenous leukemia (AML) in first remission, or chronic myelogenous leukemia (CML) in first chronic phase. Six hundred forty received methotrexate, 977 received cyclosporine, and 669 received combined cyclosporine and methotrexate. In children, the three regimens resulted in similar outcomes. In adults, cyclosporine and methotrexate had comparable risks of acute and chronic GVHD. Compared with methotrexate, cyclosporine was associated with less interstitial pneumonia (relative risk [RR] = 0.6; P < .001), less treatment-related mortality (RR = 0.6; P < .001), more relapses (RR = 1.6; P < .05), and less treatment failure (relapse or death from any cause; RR = 0.7; P < .001). Different effects were observed in different leukemias. In ALL, the rate of leukemia relapse was increased with cyclosporine versus methotrexate, with no effect on other outcomes. In AML and CML, interstitial pneumonia, treatment-related mortality, and treatment failures were decreased with cyclosporine, with no increase in relapse. Similar analyses comparing cyclosporine plus methotrexate with cyclosporine alone showed that adults receiving the combination had less acute GVHD (RR = 0.5; P < .001), less chronic GVHD (RR = 0.7; P < .01), and less interstitial pneumonia (RR = 0.7; P < .001). Treatment failure (RR = 0.8; P < .05) was marginally reduced. Separate analyses in ALL and AML showed less acute GVHD with combined therapy, but no significant effect on other outcomes. In CML, acute GVHD, interstitial pneumonia, treatment-related mortality, and treatment failure were decreased with combined therapy.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

Prevention of transfusion-induced graft-versus-host disease in dogs by ultraviolet irradiation

Ten dogs were given 9.2 Gy of total body irradiation and autologous bone marrow infusion followed by ten daily transfusions of leukocytes for a total of 11.5 to 36.2 (median, 18.8) x 10(8)/kg obtained via leukapheresis from histoincompatible unrelated donors. Four dogs were given unirradiated leukocytes, and all developed graft-versus-host disease (GVHD). In contrast, only two of three dogs given leukocytes irradiated with 20 mJ/cm2 of ultraviolet (UV) light (200 to 300 nm), and none of three dogs given leukocytes irradiated with 1,000 mJ/cm2 developed GVHD. These data indicate that UV irradiation abrogates the alloreactive potential of transfused leukocytes, and suggest that UV irradiation can be used to prevent the development of transfusion- induced GVHD.     

Monoclonal and oligoclonal gammopathy after bone marrow transplantation

Serial serum protein electrophoreses were performed on 60 patients undergoing allogeneic and syngeneic bone marrow transplantation (BMT). More than 50% of patients (31 of 60) developed transient oligoclonal and monoclonal gammopathies that appeared an average of 84 days posttransplantation (range 27 to 336 days) and persisted an average of 175 days (range 14 to 652 days). Immunofixation analysis revealed 82% of the M components to be of the immunoglobulin G (IgG) type and 18% to be IgM; 56% were kappa and 44% were lambda. A strong correlation between development of graft versus host disease (GVHD) and appearance of M components was observed (73% incidence in GVHD patients v 27% in non-GVHD patients, P = .0003). Two of the three syngeneic graft recipients also developed monoclonal gammopathies. Evidence of oligoclonal circulating B-cell populations was found in 68% of patients posttransplantation by flow cytometric B-cell clonal excess assay. No correlation of recovery of particular B- or T-lymphocyte subsets and development of M components was seen. The development of transient oligoclonal and monoclonal gammopathies after transplantation may be a ubiquitous finding reflecting recapitulation of early B-cell ontogeny.     

Human vascular endothelial cells express a membrane protein complex immunochemically indistinguishable from the platelet VLA-2 (glycoprotein Ia-IIa) complex

Endothelial cells express surface molecules that are involved in cell- matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets. The molecule recognized by CLB-10G11 from endothelial cells was characterized as follows. (1) The monoclonal antibody precipitated two proteins from surface-labeled endothelial cells that corresponded to the platelet VLA-2 subunits (glycoprotein Ia and IIa) as judged by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional nonreduced/reduced SDS- PAGE. (2) Preclearing of endothelial cells with monoclonal antibody A- 1A5, an antibody that is directed against the common VLA beta subunit, removed all the CLB-10G11-binding material. (3) Crossed immunoelectrophoresis revealed that CLB-10G11 recognizes a single precipitation arc from either platelets or endothelial cells. Analysis of these two cell types in one gel again revealed one precipitation arc. The antigen of either cell type, recognized by CLB-10G11 could be precipitated by either polyclonal antiplatelet or polyclonal antiendothelial cell antiserum. Hence, it appears that endothelial cells express at least three different surface molecules (the vitronectin receptor, the fibronectin receptor and a collagen receptor), which may play an important role in controlling the anchorage of endothelial cells to the extracellular matrix.     

Improvement of platelet storage conditions by using new polyolefin containers

BACKGROUND : Storage of pooled platelet concentrates (PCs) with yields above 3.0 × 10¹¹ platelets per unit in a 1-L PL-732 polyolefin container for 5 days often results in a drop in pH to below 6.0. Recently, new oxygen-permeable platelet containers (1-L PL-2410, 1-L and 1.5-L Compoflex) have been developed. The maximal platelet storage capacities of the new containers and the PL-732 were compared. STUDY DESIGN AND METHODS : Large platelet pools (n = 27) with platelet concentrations between 1.2 and 1.4 × 10¹¹ per L were made from 3 to 5 PCs prepared from buffy coats. The pools were divided in equal volumes among the PL-732 and the three new platelet containers. Platelet counts in the PCs ranged from 1.0 to 5.0 × 10¹¹ per unit. All PCs were stored on a flatbed shaker at 22 ± 2°C and evaluated on Days 1, 3, 5, and 7 by measuring platelet count, pH, pO₂, pCO₂, HCO₃-, glucose, lactate, platelet swirling, and soluble p-selectin. RESULTS : Day 7 storage of PCs (n = 6) with yields between 3.0 and 4.0 × 10¹¹ platelets in PL-732 showed mean ± SD pH values of 5.93 ± 0.05 and lactate values of 32.3 ± 7.9 mmol per L; in 4 of these 6 PCs, pH was below 6.0. In contrast, storage of these PCs in 1-L PL-2410 and 1.5-L Compoflex containers and of 2 of these 6 PCs in 1-L Compoflex containers showed pH values above 6.8. Lactate values were 15.5 ± 1.3, 15.3 ± 1.8, and 19.5 ± 4.7 mmol per L, respectively (p < 0.001 vs. PL-732). The platelet storage capacity of the new containers with platelet yields between 4.0 and 5.0 × 10¹¹ per unit (n = 6) was evaluated. Day 7 storage of these PCs in the 1.5-L Compoflex showed an average pH value of 6.74 ± 0.20; in 2 of 6 PCs, pH was below 6.8. The average pH value in the PL-2410 was 6.38 ± 0.31, and in all PCs, pH was below 6.8. Average lactate values were 17.8 ± 5.7 and 25.8 ± 5.6 mmol per L (p < 0.05), respectively. Soluble p-selectin values on Day 7 of storage increased approximately twofold in all PCs. CONCLUSION : The new oxygen-permeable containers showed platelet quality comparable to that with the PL-732 and for longer storage periods and at higher platelet counts.     

Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.