Magnetic affinity colloid (MAC) cell separation of leukemia cells from autologous bone marrow aspirates.

A colloidal suspension of Co2B with avidin irreversibly adsorbed to the surface has been used with biotinylated antibodies and lectins to eliminate specific cell populations from mixtures with peripheral blood or bone marrows. Using the monoclonal antibodies CF-1 and PM-81 with this magnetic affinity colloid (MAC), we can eliminate five logs of K562 cells from mixtures with peripheral blood or marrow cells as determined by a linear limiting dilution clonogenic assay. We have also used this separation to eliminate clonogenic leukemia cells from fresh samples of peripheral blood and bone marrow from relapsed acute leukemia patients. Using CF-1 alone or in combination with PM-81, we eliminated two logs of colonies and clusters of leukemia cells from the fresh samples. The same antibodies used with MAC separation of hematologically normal marrows allow recovery of greater than 30% of the hematopoietic progenitors.

T cell depletion in bone marrow transplantation.

Prior to the introduction of T cell depletion graft-versus-host disease (GVHD) was the major cause of transplant-related mortality. Why has the remarkable technical achievement of efficient T cell depletion, which has virtually eliminated severe GVHD over the last few years, failed to reduce mortality and increase disease-free survival? This review examines the value of T cell depletion based on recent clinical experience. It surveys the directions that are being pursued in an effort to solve the problems that have arisen and indicates how this is leading to a greater understanding of the mechanisms involved in GVHD and the graft-versus-leukaemia effect.

The gut mucosal barrier in bone marrow transplantation.

The damage to the small bowel mucosal barrier was studied prospectively in 26 patients undergoing bone marrow transplantation. The conditioning regimens resulted in the small bowel mucosa becoming more permeable for up to 4 weeks. Acute graft-versus-host disease also compromised barrier function. There was no difference in gut damage between conditioning with chemotherapy only and chemoradiotherapy, but damage was greater in patients 30 years of age and above.

Use of IgM enriched intravenous immunoglobulin (Pentaglobin) in bone marrow transplantation.

In a study of 63 allogeneic and autologous bone marrow transplants, patients were randomized to receive the IgM and IgA enriched intravenous immunoglobulin (IVIG) preparation (Pentaglobin). Pentaglobin has been postulated to have anti-endotoxin properties and one of the aims of the study was to measure endotoxin levels in these patients together with the clinical sequelae of infection. The anti-endotoxin effects of Pentaglobin were found to reside in the IgM fraction. Those patients who received Pentaglobin were significantly protected from dying from infection in the first 100 days after the transplant, although it was not actually possible to document bacterial infections as the cause of death in the control patients. Peak endotoxin levels were significantly reduced (p = 0.02) in those patients receiving Pentaglobin. Liver damage as assessed by liver enzyme abnormalities correlated significantly with the presence of endotoxaemia greater than 25 pg/ml and up to 70% of pyrexial episodes were associated with endotoxaemia. Our results suggest that Pentaglobin is useful in reducing hepatic toxicity and this may be related to a reduction in endotoxaemia.

Bone marrow transplantation for adults and children with poor risk acute lymphoblastic leukaemia in first complete remission.

Thirty-two patients with poor risk acute lymphoblastic leukaemia in first complete remission received bone marrow transplants (BMT) from fully matched family donors. Their ages ranged from 7 to 41 (median 23) years. Nine patients were aged 16 years or less. Patients were selected for BMT because they had risk factors for relapse with standard treatment approaches. In particular the children who were selected for BMT had presenting blast counts of greater than or equal to 90 x 10(9)/l or null immunophenotypes. The overall disease-free survival was 50% with a relapse risk of 31% at 9 years. Patients aged less than 16 years had a much lower risk of both graft-related disease and relapse than did older patients (disease-free survival 89% for those aged 7-16, 48% for those aged 17-26, 24% for those aged 26-41). We conclude that selection of BMT for young patients with poor risk features is entirely justified but that the prognosis for older patients is poor even after BMT.

Safe application of a 13-Gy split dose total body irradiation schedule prior to bone marrow transplantation.

Two conditioning regimens for bone marrow transplantation for patients with acute lymphoblastic leukaemia were compared. Both regimens (VVRAPID and VVRAPID-X) incorporated the same cytotoxic chemotherapy but differed in the radiation dose; VVRAPID employed a single fraction of 10.5 Gy and VVRAPID-X employed 10.5 Gy followed 4 days later by a further dose of 2.5 Gy. The 13-Gy split-fraction schedule was well tolerated with no significant increase in infection or requirement for blood products. An increase in gastrointestinal toxicity occurred but responded to increased oral anti-diarrhoeal agents. Overall survival and relapse rates did not differ significantly between the two groups. There was a trend for the 13-Gy schedule to reduce the graft failure rate following T cell-depleted bone marrow transplants.

Parenteral glutamine protects hepatic function during bone marrow transplantation.

Hepatic veno-occlusive disease (VOD) of the liver is a common complication following high-dose cytotoxic therapy for bone marrow transplantation (BMT). The major pathological changes are seen in centrilobular (zone 3) hepatocytes and adjacent endothelium. Glutathione (GSH) becomes depleted following chemotherapy and experimental evidence suggests reduced levels predispose to centrilobular hepatocyte and endothelial cell injury. Animal studies have shown that glutamine infusions can maintain GSH levels and protect against free radical injury. We have prospectively studied the effect of glutamine supplementation during BMT. Thirty-four patients undergoing BMT were randomised to receive either glycl-L-glutamine (n = 18) or an isonitrogenous mixture of non-essential amino acids (n = 16). Glutamine was shown to significantly preserve protein C (days +4 and +7, P < 0.05) and albumin levels (days 0 and +4, P < 0.02). Markers of thrombin and plasmin generation (thrombin-antithrombin, prothrombin fragment F1+2 and plasmin-antiplasmin levels) were not significantly changed between the two groups. These findings suggest that glutamine preserves hepatic function but does not alter thrombin or plasmin generation during BMT. Previous studies have shown reductions in protein C, albumin, factor X and factor VII levels post BMT. Falling protein C levels have been shown to be predictive of severe VOD. These data suggest a role for glutamine in the protection of hepatic function following BMT.

Glutamine and vitamin E in the treatment of hepatic veno-occlusive disease following high-dose chemotherapy.

Hepatic veno-occlusive disease (VOD) of the liver is a common complication following high-dose cytotoxic therapy for bone marrow transplantation (BMT). Liver injury is believed to occur following free radical damage to endothelial cells of the sinusoids and small hepatic veins. Glutathione the main antioxidant of the cytosol becomes depleted following chemotherapy. Animal studies have shown that glutamine infusions can maintain glutathione levels and protect against free radical injury. We present two cases of established VOD successfully treated with intravenous glutamine (as dipeptide) and oral vitamin E. Although both cases have possible confounding factors we believe that these give support to the notion that glutamine/vitamin E may have a role in the prophylaxis and treatment of VOD. Further formal trials are indicated.

Intermittent superior vena cava syndrome caused by a Hickman catheter.

A case of intermittent superior vena cava syndrome caused by a Hickman catheter is reported. The symptoms resolved on removal of the catheter. The use of anticoagulants in conjunction with indwelling venous catheters is discussed.

Polycythaemia rubra vera and myelofibrosis with spinal cord compression.

Spinal cord compression from extramedullary haemopoiesis within the spinal epidural space is a rare complication of myelofibrosis and polycythaemia rubra vera (PRV). A 69-year-old male with PRV (later transforming to myelofibrosis) who developed this complication is described here. Due to the uncertainty over its optimal treatment, previous case reports were systematically reviewed to define its presentations, treatments and outcomes. Including the present case this complication has been reported in 21 patients with myelofibrosis and PRV: 17 were male and the mean symptom duration was 7.6 months. Neurological improvement occurred in 14 patients and 12 survived. Seventy-five per cent of patients receiving combined treatment (irradiation with laminectomy or chemotherapy) showed neurological improvement and 100% survived. In contrast, 67% of those receiving single treatments exhibited improved neurology and only 33% survived. It is concluded that spinal cord compression in myelofibrosis and PRV has a high mortality, with combined treatment providing a better prognosis.

Monoclonal antibodies: the possibilities for cancer therapy.

Previous studies and current efforts to use monoclonal antibodies in human cancer therapy are reviewed. These include antibody-dependent immune reactions in vivo, conjugation of cytotoxic agents to monoclonal antibodies, and monoclonal antibody-mediated tumor cell elimination from bone marrow in vitro for use in autologous bone marrow transplantation. Our studies describe the use of magnetic immunocolloids for the removal of leukemic cells from human bone marrow. Future possibilities for the therapeutic use of monoclonal antibodies are also discussed.

Effect of IgM-enriched intravenous immunoglobulin (Pentaglobin) on endotoxaemia and anti-endotoxin antibodies in bone marrow transplantation.

Endotoxin was measured in over 1000 plasma samples from bone marrow transplant patients in a randomized trial of the IgM-enriched intravenous immunoglobulin (IVIG) Pentaglobin. Peak endotoxaemia was significantly reduced (P = 0.02) in patients receiving Pentaglobin and 70% of all pyrexias of unknown origin were associated with endotoxaemia. Gut mucosal damage, assessed by lactulose/mannitol ratios, was significantly associated (P = 0.02) with endotoxaemia. Specific IgM antibody to endotoxin core-glycolipid was significantly raised (P < 0.01) in patients receiving the IVIG, and the IgM fraction of Pentaglobin was found to contain most of the anti-endotoxin antibody activity of the IVIG. These results suggest a role for IgM-enriched IVIG as a prophylactic agent for the reduction of endotoxaemia and its consequences in bone marrow transplant patients.

Haemopoietic colony-forming cells from peripheral blood stem cell harvests: cytokine requirements and lineage potential.

We have measured the in vitro growth requirements of progenitor cells released into the blood of cancer patients following administration of chemotherapy and cytokines. In order to distinguish the direct effects of cytokines on progenitors from those activating accessory cells, we have compared clonogenic growth before and after CD34-positive selection of progenitors, in serum-free conditions. CD34 selection had little effect on the cytokine requirements of erythroid colony-forming cells and single cytokines, particularly interleukin-3, could support considerable colony growth in both mononuclear and CD34+ cell suspensions. Optimal erythroid colony growth, however, usually required the addition of a combination of stem cell factor and interleukin-3, in addition to erythropoietin, which was always required. Maximal numbers of granulocyte-monocyte progenitors in mononuclear cell cultures, could be achieved with a mixture of stem cell factor, interleukin-3 and granulocyte-monocyte colony stimulating factor. However, after CD34 selection, full myeloid colony growth was only achieved when granulocyte colony stimulating factor was added to the above mixture. This presumably reflects loss of accessory cells, during CD34 selection, which produce this cytokine. When transplanted after 8 d of culture, 16/22 myeloid colonies from erythropoietin-free cultures of peripheral blood stem cell harvests, could generate secondary erythroid colonies implying that these progenitors are multipotential. However, surface marker analysis of individual erythroid colonies revealed only the occasional presence of granulocytes and monocytes. These data demonstrate that cytokine mixtures are required for optimal colony growth, particularly after CD34 selection, and that most mobilized, blood clonogenic cells are multipotential.

Evaluation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting.

Erythroid regeneration is an important and separate element in the engraftment process in allogeneic and autologous bone marrow transplantation (alloBMT, autoBMT). Qualitative visual reticulocyte counting has proved inadequate in the evaluation of erythropoiesis after BMT but automated flow cytometry now allows the reliable quantitation of reticulocytes even to very low levels. Reticulocyte counts and highly fluorescent reticulocyte (HFR) counts (very early reticulocytes) were estimated daily in recipients of 22 autoBMT and 14 alloBMT using a Sysmex R-1000 automated reticulocyte counter. Marrow ablation caused an immediate and rapid fall in both the reticulocyte count and the HFR. Measurable numbers of reticulocytes persisted throughout the hypoplastic period, but HFR fell to zero in the majority of both the autoBMT and alloBMT. HFR rose significantly after a median time of 14 d post-autoBMT, and 12 d post-alloBMT. Attainment of 15 x 10(9)/l reticulocytes and 0.5 x 10(9)/l HFR at day 21 post-transplant was associated with ultimate engraftment in 100% cases. Inadequate engraftment was seen in the majority of patients whose responses fell below these levels. Graft-versus-host disease was associated with a transient slight reduction in reticulocyte count. Neither episodes of infection nor blood transfusions had any significant impact on trends of reticulocytes or HFR. Automated flow cytometric reticulocyte counting has been shown to provide an accessible measure of erythroid activity which may be of predictive value in the management of patients following bone marrow transplantation.

Regressing atypical histiocytosis, a regressing cutaneous phase of Ki-1-positive anaplastic large cell lymphoma. Immunocytochemical, nucleic acid, and cytogenetic studies of a new case in view of current opinion.

BACKGROUND: Regressing atypical histiocytosis is a rare multifocal cutaneous tumor characterized by large, spontaneously regressing, ulcerating skin nodules. Although initially self-remitting, the condition may progress to systemic lymphoma. METHODS: Using material from one patient, an attempt was made to clarify the nature of this condition with immunophenotyping, genotyping, and chromosome studies. RESULTS: Immunophenotyping studies indicated the condition was of T-cell lineage, although T-cell receptor gene studies showed polyclonal rearrangement. This case progressed to systemic lymphoma. CONCLUSIONS: The authors believe regressing atypical histiocytosis is a regressing phase of Ki-1-positive anaplastic large cell lymphoma of the skin.