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Twenty patients with poor prognosis B-cell chronic lymphocytic leukemia (B-CLL) underwent uniform high-dose chemoradiotherapy followed by rescue with multiple monoclonal antibody-purged autologous bone marrow (BM) (12 patients) or T-cell-depleted allogeneic BM from HLA-identical siblings (8 patients) in a pilot study to assess the feasibility of BM transplantation (BMT) in this disease. All had poor prognosis disease by either staging, BM pattern, tumor doubling time criteria, or cytogenetics. All patients achieved remission criteria (defined as < or = 2 adenopathy, absence of splenomegaly, < or = 20% of the intertrabecular space involved on BM biopsy) before BMT. Despite the use of fludarabine, a median of three treatment regimens were required to achieve BMT eligibility. After BMT, all patients achieved complete hematologic engraftment. Toxicities were not significantly different between autologous versus allogeneic BMT. Two toxic deaths were observed. Of 19 evaluable patients, 17 clinical complete clinical remissions (89%) were observed, with 2 patients (1 allogeneic and 1 autologous) exhibiting persistent BM disease. Complete clinical remissions were documented at the phenotypic and molecular level for the majority of patients in whom dual fluorescence for CD5 and CD20 (15 of 15; 100%) and Ig gene rearrangements (11 of 14; 79%) were performed. Although long-term follow-up is needed to assess any potential impact on the disease-free and overall survival of these patients, this study shows the feasibility of using high-dose chemoradiotherapy and BMT in patients with poor prognosis B-CLL.

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl-2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR-detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR-detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.

Anti-B-blocked ricin (anti-B4-bR) combines the specificity of the anti-B4 (CD19) monoclonal antibody with the protein toxin "blocked ricin." In blocked ricin, affinity ligands are attached to the ricin B-chain to attenuate its lectin binding capacity. In a phase I trial, Anti-B4-bR was administered by 7-day continuous infusion to 12 patients in complete remission after autologous bone marrow transplantation (ABMT) for relapsed B-cell non-Hodgkin's lymphoma (NHL). Patients were treated at 20, 40, and 50 micrograms/kg/d for 7 days. Potentially therapeutic serum levels could be sustained for 3 to 4 days. The maximum tolerated dose was 40 micrograms/kg/d for 7 days (total 280 micrograms/kg). The dose-limiting toxicities were reversible grade IV thrombocytopenia and elevation of hepatic transaminases. Mild capillary leak syndrome was manifested by hypoalbuminemia, peripheral edema (4 patients), and dyspnea (1 patient). Anti-immunotoxin antibodies developed in 7 patients. Eleven patients remain in complete remission between 13 and 26 months post-ABMT (median 17 months). These results show that Anti-B4-bR can be administered with tolerable, reversible toxicities to patients with B-cell NHL in complete remission following ABMT.

Using the extremely sensitive technique of polymerase chain reaction (PCR) to detect the bcl-2 translocation, only 50% of bone marrows could be purged of PCR-detectable lymphoma cells using a cocktail of three anti-B-cell monoclonal antibodies (MoAbs) and complement-mediated lysis. This observation is of clinical importance because those patients whose reinfused marrows harbored residual lymphoma cells showed a significantly increased incidence of relapse. To improve purging, we used PCR detection of the bcl-2 translocation to compare the efficiency of complement-mediated lysis with immunomagnetic bead depletion. Using either a three or a four MoAb cocktail followed by immunomagnetic bead depletion, all PCR-detectable cells were purged after three cycles of treatment. In these same patient samples, treatment with three MoAbs and complement purged only 11 of the 25 (44%) samples. The addition of a fourth MoAb followed by complement lysis purged the marrows of only an additional five patients. Immunomagnetic bead depletion was specific because there was no loss of committed myeloid progenitor cells. The above results suggest that immunomagnetic bead depletion of the harvested marrow will likely be superior to our previous method of purging and the lack of nonspecific toxicity to myeloid progenitor cells predicts that it will not impair engraftment. This methodology will now be used to determine whether the reinfusion of lymphoma free marrow affects the incidence of relapse after autologous bone marrow transplantation.

B7 is an activation antigen expressed on activated B cells and gamma-interferon-stimulated monocytes. The B7 antigen is the natural ligand for CD28 on T cells. After engagement of T-cell receptor with antigen in association with major histocompatibility complex class II, a second signal mediated through the binding of B7 to CD28 greatly upregulates the production of multiple lymphokines. We have now mapped the B7 gene to human chromosome 3 using the technique of polymerase chain reaction on a panel of hamster x human somatic cell hybrid DNAs. We have further localized the gene to 3q13.3-3q21 using in situ hybridization on human metaphase chromosomes. Trisomy of chromosome 3 is a recurrent chromosome change seen in various lymphomas and lymphoproliferative diseases, particularly diffuse, mixed, small, and large cell lymphomas, human T-cell lymphotropic virus type I-induced adult T-cell leukemia, and angioimmunoblastic lymphadenopathy. A number of chromosomal defects involving 3q21 have been described in acute myeloid leukemia and also in myelodysplastic and myeloproliferative syndromes. The mapping of B7 may permit further insight into disease states associated with aberrant lymphocyte activation and lymphokine synthesis.

Polymerase chain reaction (PCR) of bcl-2 provides an extremely sensitive method to detect minimal disease in approximately 50% of patients with non-Hodgkin's lymphomas (NHL). In an attempt to determine the clinical usefulness of this technique, we examined the bone marrow (BM) of 152 patients with advanced-stage NHL at the time of evaluation and after induction or salvage chemotherapy before autologous BM transplantation. The BM proved to be an accessible and reproducible tissue source to determine PCR positivity because all of the 102 patients examined had the same PCR-amplifiable breakpoint in their BM and lymph node. At the time of evaluation, PCR analysis in advanced-stage NHL patients added little additional information to morphologic analysis because each technique identified BM infiltration in approximately 70% of patients. PCR was significantly more useful in determining BM infiltration after induction or salvage therapy. At that time, approximately 50% of patients had morphologically normal BM, whereas PCR analysis remained positive in 100% of those with an amplifiable breakpoint. These observations were confirmed in a clinical trial attempting to induce remission in previously untreated low-grade advanced-stage NHL patients. In this series, PCR was positive in all patients after treatment although the BM was histologically uninvolved in 50% of cases, showing that conventional therapy did not eradicate bcl-2-positive cells.

Background: The use of autologous bone marrow transplantation is increasing in the management of advanced cancers. Many investigators have attempted to "purge" autologous marrow of residual tumor cells because of concern that reinfused tumor cells might contribute to relapse. The efficacy of purging remains unproved., Methods: We performed clonogenic assays in a tumor cell line in culture to determine the efficiency of immunologic purging. Amplification by the polymerase chain reaction (PCR) was used to detect residual lymphoma cells before and after purging of bone marrow from 114 patients with B-cell non-Hodgkin's lymphoma in whom a translocation (t(14;18] that could be amplified by PCR was detected at the time of their initial evaluation., Results: Immunologic purging in vitro resulted in a 3-to-6-log destruction of cells in the tumor cell line. Residual lymphoma cells were detected by PCR in the bone marrow of all patients before purging. No lymphoma cells could be detected in the marrow of 57 patients after purging. Disease-free survival was increased in these 57 patients as compared with those whose marrow contained detectable residual lymphoma (P less than 0.00001). The ability to purge residual lymphoma cells was not associated with the degree of bone marrow involvement (P = 0.4494) or the previous response to therapy (P = 0.1298)., Conclusions: The inability to purge residual lymphoma cells was the most important prognostic indicator in predicting relapse. These results provide evidence of the clinical usefulness of ex vivo purging of autologous bone marrow in the treatment of patients with lymphoma and suggest that the reinfusion of malignant cells in autologous marrow contributes to relapse

One hundred and sixty eight adult patients with B-cell non-Hodgkin's lymphoma (NHL) and other hematologic malignancies who underwent autologous or allogeneic bone marrow transplantation (BMT) were investigated for the subsequent development of hemolytic-uremic syndrome (HUS). All patients were conditioned with cyclophosphamide and total body irradiation. When examined at 3-month intervals for the first year post-BMT, all patients had uniform measurements of hematocrit (Hct) and serum creatinine. Sixteen patients who initially exhibited Hct and creatinine values that were normal range for the BMT populations developed a sudden decrease in Hct and increase in creatinine between 3 and 11 months post-BMT and fulfilled the clinical and laboratory criteria for HUS. None of these patients had known active cytomegalovirus infection, graft-versus-host disease, or cyclosporine administration. The degree of decrease in Hct and creatinine elevation ranged from solely laboratory abnormalities to a clinically significant syndrome. Twelve of the 16 patients developed acute clinical complications of congestive heart failure, hypertension (HTN), or peripheral edema. Twelve patients required red blood cell support, whereas only four patients required platelet transfusions. Both hemolytic anemia and thrombocytopenia have resolved in virtually all cases. At a mean follow up of 18 months postdiagnosis, creatinine elevations have persisted along with HTN. All patients have survived without life-threatening long-term sequelae. With the increasing use of BMT as a curative modality for patients with hematologic malignancies, it becomes important to prospectively monitor patients for the development of HUS and its potential long-term impact on renal function.

For younger patients with acute myeloid leukemia (AML), an allogeneic transplant from a matched sibling may afford the best chance of cure. In patients who are older or without a matched sibling donor, dose intensification can be achieved with an autologous bone marrow transplant (ABMT). We report here the results of a high-dose chemotherapy regime with nonpurged ABMT in 82 adult patients in first remission of AML with a median follow-up of 31 months. The median age was 40 years (range 16 to 57 years). The median interval between remission and ABMT was 5 months (range 1 to 12 months). Twenty-eight of these patients received a second course of the same high-dose chemotherapy and ABMT. The procedure related mortality rate was 6%. The projected leukemia-free survival (LFS) at 5 years is 48% for all 82 patients and 50% for the 76 patients with no known preceding myelodysplastic syndrome. For those patients with primary AML who received a double ABMT the projected LFS is 67%. The interval between remission and ABMT did not predict for either relapse or LFS. ABMT using a multidrug chemotherapy protocol is less toxic than allogeneic BMT yet results in a similar LFS.

In 4 out of 16 patients receiving recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) in phase I/II studies antibodies developed to the recombinant protein. The antibodies react with sites on the native protein backbone which are normally protected by O-linked glycosylation but which are exposed in rhGM-CSF produced in yeast and Escherichia coli. Antigenicity of recombinant human proteins due to non glycosylation may have relevance to the choice of host system for production of factors for clinical use.

Growth factors may be of value in reducing transplant-related complications. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant granulocyte colony-stimulating factor (G-CSF) are the most widely studied growth factors to date, and show promise in terms of accelerating haemopoietic recovery; however, their overall value is less clear. Non-recombinant colony-stimulating factors derived from human urine are also under investigation. Any antileukaemic or immune modulatory role for growth factors is currently speculative. The value of growth factors in allogeneic bone-marrow transplantation has not yet been adequately investigated. The use of growth factcm in combination is potentially of great value but as yet there are no human data. In addition to having proliferative effects, some colony-stimulating factors have important effects on mature phagocyte function. Large randomized studies will be needed to document the real value of growth factors in marrow transplanation.

3 patients with acute leukaemia, HLA antibodies and thrombocytopenia refractory to random donor platelet transfusions were treated with high-dose i.v. immunoglobulin. All 3 patients responded favourably with improved post-transfusion recovery of random platelets. In 1 patient, the recovery of transfused histocompatible platelets was also enhanced. Treatment was followed by reduction in the total lymphocyte count and marked changes in lymphocyte subsets in 1 patient.

This short review of autologous bone marrow transplantation (ABMT) has a distinct clinical emphasis and concentrates particularly on adult acute leukaemia and lymphoma in which the greatest amount of current clinical experience lies. In the early part of the review we discuss how escalations of dose of chemoradiotherapy might allow ablation of both marrow-derived (leukaemia) and non-marrow-derived disease (lymphoma and solid tumour) provided that haemopoiesis is reintroduced into the host in the form of autologous marrow stem cells, and how cryopreservation techniques have allowed this to proceed. Whilst discussing ABMT in acute leukaemia we describe initial results possibly suggestive of an improvement on current consolidation/maintenance chemotherapy regimens but emphasis that we are dealing only with heterogeneous registry data and not randomised controlled trials. We also suggest that there is no useful data as yet as to the value of purging autologous acute leukaemia marrow. The lymphoma data is described which may suggest a useful role of high dose therapy with ABMT in relapsed disease-timing of ABMT may need to differ profoundly in HD from NHL. Current ABMT data in lymphoma suggests that local relapse at sites of previous disease remain the major problem and emphasises the difficulties of finding satisfactory ablative regimens and timing the selection of patients at particular points in the natural history of their disease. Finally, we emphasise that although solid tumours may numerically represent the largest group of potential candidates for ABMT, the picture in this area remains essentially one of failure to be able to ablate the underlying disease despite increments in chemoradiotherapy and ABMT.

Forty-four patients with refractory Hodgkin's disease were treated with high-dose combination chemotherapy followed by autologous bone marrow rescue. Twenty-two patients (50%) entered complete remission within 6 months of the procedure and four other patients are free of disease progression. Only two patients have subsequently relapsed from complete remission (CR). Bone marrow suppression was the predictable major toxicity of this procedure, and two patients (4.5%) died of sepsis during the aplastic phase. High-dose therapy with autologous bone marrow transplantation (ABMT) appears to be an effective salvage regimen for patients with refractory Hodgkin's disease.

A recurrent theme in studies of the pathology of fatal Lassa fever in man is the lack of histological lesions to explain disordered cell function and death. Recently, we demonstrated the existence of a factor in the plasma of patients with Lassa fever which markedly inhibits the aggregation responses of normal platelets in vitro. To assess whether this factor could mediate more global cellular dysfunction, we studied the effects of Lassa plasma on the respiratory burst of neutrophils. Thirteen of 15 samples from patients in the acute phase of Lassa fever profoundly inhibited the amount of superoxide generated by normal neutrophils in response to the chemotactic peptide, f-met-leu-phe (FMLP) (mean superoxide generated = 54.7 +/- 6.1% of control). In contrast, eight of nine samples from patients who had infections other than Lassa fever enhanced the neutrophil response to the peptide. All Lassa samples which inhibited the ADP-induced aggregation responses of normal platelets inhibited the neutrophil response to FMLP. Unlike the effect on platelets, however, the inhibition of neutrophils was only apparent when the cells were stimulated within 5 min of exposure to the plasma. The inhibition of neutrophils is not due to either interference with FMLP-neutrophil binding or an effect on the NADPH-oxidase, suggesting a suppression of signal transduction. Our data suggest the inhibitory factor in Lassa plasma has global effects on cellular function, and may play a central role in the pathogenesis of this often fatal illness.

Ultrafiltration was performed in nine patients with congestive cardiac failure that was refractory to conventional medical treatment. A mean of 12 X 7 litres of fluid was removed, and there was a sustained symptomatic improvement in all patients. Weight loss continued after ultrafiltration and a sustained increase in serum sodium concentration was also noted. A transient fall in right atrial pressure was seen only at four hours after ultrafiltration. No adverse haemodynamic effects were seen four and eighteen hours after fluid removal. Intracardiac dimensions measured by echocardiography remained unchanged. Ultrafiltration can be used to relieve symptoms in patients with refractory congestive heart failure and gross oedema.

Thirty-one patients with resistant Hodgkin's disease were treated by an identical high dose chemotherapy regimen and autologous bone marrow transplantation. Twelve of these patients received recombinant human granulocyte/macrophage colony stimulating factor (rh GM-CSF) in a phase I/II study. rh GM-CSF was administered by continuous infusion into an indwelling central venous catheter for 3-21 days at doses of 100-400 micrograms/m2/day. The patients receiving rh GM-CSF did not differ significantly from those who did not receive growth factor with regard to age, previous therapy or number of bone marrow cells infused. rh GM-CSF resulted in more rapid neutrophil regeneration, the average time to achieve a neutrophil count of greater than or equal to 0.5 x 10(9)/l being 17.5 days compared to 24.9 days in the control group (p less than 0.01). Platelet recovery was very varied and not accelerated by rh GM-CSF. Patients receiving rh GM-CSF had a similar infection rate (58% vs 68% in the control group), similar number of febrile days (5.0 vs 4.7 days) and similar period of hospitalization to the control group (30.1 vs 30.2 days). Randomized controlled trials are now required to define the clinical value of rh GM-CSF in the setting of autologous bone marrow transplantation.

Autologous bone marrow transplantation (ABMT) following intensive therapy regimes is being used increasingly in relapsed lymphomas. To date no randomised studies have been performed to ascertain the value of this form of therapy. We describe here our experience of ABMT in both Hodgkin's disease and Non-Hodgkin's Lymphoma and review the situations in which we believe future randomised studies should be based.