Combined lectin/monoclonal antibody purging of bone marrow for use in conjunction with autologous bone marrow transplantation in the treatment of multiple myeloma.

What was thought at the time to be a real breakthrough in myeloma therapy came in 1983 when McElwain reported that complete remission could be gained by means of a single, very high, dose of melphalan (HDM). Between 20 and 30% of patients attain complete remission with HDM (1). These observations led directly to exploration of the role of autologous bone marrow rescue following high-dose therapy, since the morbidity and mortality associated with prolonged bone marrow aplasia caused by these doses are unacceptably high (2) Most patients with myeloma are relatively old for allogeneic bone marrow transplantation, since graft-vs-host reactions tend to be poorly tolerated in people over 40. Autologous transplantation, however, carries the obvious disadvantage of potential reinfusion of malignant cells. Efforts have therefore been made to develop systems for in vitro purging to remove potentially malignant cells prior to reinfusion of the marrow (3,4). Although the nature of the initial malignant cell in myeloma is unknown, it is assumed that purging should entail at least the removal of plasma cells and B-lymphocytes.

Autologous bone marrow transplantation for myeloma patients using PNA- and CD19-purged marrow rescue.

A new method of in vitro bone marrow purging using a lectin and monoclonal antibody in combination has been used for the first time in vivo. Two patients with advanced myeloma were treated with high-dose melphalan and total body irradiation and then rescued with autologous bone marrow which had been purged in vitro to remove malignant cells by using a combination of a plasma cell-binding lectin (peanut agglutinin, PNA) and the anti-B lymphocyte monoclonal antibody anti-CD19, bound to magnetised microspheres. Both patients showed rapid engraftment of the purged bone marrow and remain well 36 and 46 months later with normal bone marrow morphology, although one patient still has a low level of circulating paraprotein. This is a promising form of therapy for what has been an invariably fatal condition.

Proliferative responses of HT29 and Caco2 human colorectal cancer cells to a panel of lectins.

BACKGROUND/AIMS: Altered lectin binding is common in malignant and premalignant epithelia, but its functional significance is unclear. This study examined the proliferative effects of four lectins on HT29 and Caco2 colon cancer cells. METHODS: Proliferation was assessed in log growth and confluent culture by thymidine incorporation and cell counts. Peanut agglutinin (PNA) binding was characterized by Scatchard analysis and electrophoresis of lectin affinity-purified cell surface-radiolabeled preparations. RESULTS: PNA, 5 micrograms/mL, increased thymidine incorporation in HT29 but had no effect on Caco2. Wheat germ agglutinin and concanavalin A stimulated proliferation slightly at 0.5-1.0 microgram/mL but were inhibitory at higher concentrations. Ulex europaeus 1 had no significant effect. Similar results were obtained when proliferation was assessed by cell counts and with confluent cell cultures. Scatchard analysis with both cell lines showed multisite best fit models with similar binding affinities. Three PNA-binding glycoproteins were identified in both cell lines, but two were of lower molecular weight in HT29 than in Caco2. Membrane preparations from a resected colorectal cancer contained a 30-kilodalton PNA-binding glycoprotein similar to that in HT29 cells. CONCLUSIONS: Lectins are plentiful in the normal diet and often escape digestion. This study suggests that altered expression of lectin receptors, particularly the upregulation of PNA receptor seen in colonic malignancy and hyperplasia, may have an important role in growth modulation.

The peanut-agglutinin (PNA)-binding surface components of malignant plasma cells.

Plasma cells within bone marrow aspirates from multiple myeloma patients have been shown to be reactive with the lectin peanut agglutinin (PNA). This has been recently exploited by using PNA for purging bone marrow of malignant cells in autotransplantation therapy of the disease. The purpose of this investigation was to isolate and characterize the PNA-binding proteins of myeloma cells. We used the malignant plasma cell-derived line Karpas-620 (K620) as a model, and showed by affinity chromatography, SDS-PAGE, and immunoprecipitation that, among several PNA-binding proteins, a major one is an incompletely sialylated form of CD44. CD44 is a well-known homing receptor protein which is rich in carbohydrate and usually completely sialylated so that it does not react with PNA. We have then examined the PNA reactivity of myeloma cells from different patients and showed a clear difference in the profile of PNA-binding proteins from case to case. Moreover, in contrast to K620 cells, some of the patient plasma cells tested did not have a PNA-binding form of CD44. In conclusion, therefore, we have shown that a number of different proteins participate in PNA binding by malignant plasma cells. Moreover, we have demonstrated a novel, incompletely sialylated form of CD44 on a myeloma cell line. It is known that the level of glycosylation of CD44 and other proteins may affect their function, but how this relates to the malignant behaviour of plasma cells remains to be determined.

A method for clinical purging of myeloma bone marrow using peanut agglutinin as an anti-plasma cell agent, in combination with CD19 monoclonal antibody.

Previous studies have shown that the lectin peanut agglutinin (PNA) binds bone marrow plasma cells in the majority of patients with myeloma and does not bind to normal haemopoietic progenitors. This lectin has been used in combination with anti-CD19 monoclonal antibody (moAb) in a system for purging myeloma bone marrow. This has now been scaled up for application to ex vivo treatment of large volumes of bone marrow suitable for autologous bone marrow transplantation. Four bone marrow harvests from patients with myeloma containing 9.5 +/- 4.9% plasma cells were depleted of erythrocytes and mature granulocytes by Ficoll separation using the Haemonetics V50 cell separator. The mononuclear fraction was then purged with magnetic beads coated with PNA and anti-CD19 moAb. The system proved highly efficient with removal of all detectable plasma cells and CD19+ cells. Average mononuclear cell recovery following purging was 71% of the concentrated marrow with 78% yield of CFU-GM. Normal progenitor recovery related to patients' weight is predicted to be adequate for haemopoietic reconstitution following ablative chemoradiotherapy. This system is therefore feasible for large-scale clinical purging.

Effective concentration of bone marrow mononuclear cells using density gradient separation within an automated cell separator.

An effective method for concentrating bone marrow is described. Concentration was achieved using an intermittent flow cell separator. Elimination of mature haemopoietic cells was enhanced by the addition of a density separation medium (Ficoll-metrizoate) which was then removed by washing. All procedures were undertaken using the cell separator, this allowed for standardization of procedure and less manipulation which is associated with enhanced mononuclear cell recovery and less risk of microbial contamination. Mature granulocytes were depleted by 86.8% and red cells by 97.7% whilst mononuclear cells showed a 49% recovery. Marrows processed in this way and subsequently purged and/or cyropreserved were shown to successfully engraft when reinfused.

Peanut agglutinin in combination with CD19 monoclonal antibody has potential as a purging agent in myeloma.

Administration of high-dose chemotherapy to patients with myeloma, followed by rescue with autologous bone marrow transplantation (ABMT), sometimes induces complete disease remission but relapse is usual. We have attempted to reduce the risk of relapse by selective in vitro removal of myeloma cells from the autologous graft. A combination of the (gal-galNac)-binding lectin peanut agglutinin (PNA), which binds all plasma cells, and the pan-B monoclonal antibody CD19 was assessed for purging marrow of myeloma cells and their putative precursors using a magnetic bead method. Preliminary experiments performed on peripheral blood mononuclear cells spiked with fluorescent-labeled PNA+ Kirk tumor cells showed that a magnetic bead: target cell ratio of 40:1 resulted in a greater than 3-log reduction in PNA+ cells. This technique was then applied to 17 samples of myeloma bone marrow and to 18 samples of normal bone marrow spiked with PNA+ Kirk cells and CD19+ hairy cell leukemia cells. In each case all detectable plasma cells and CD19+ lymphocytes were effectively removed, and normal hemopoietic progenitor cell recovery was greater than 55%. This purging system deserves further study as a means of reducing relapse rates in myeloma patients treated by a combination of high-dose chemotherapy and ABMT.

Continuous flow cell separator use for bone marrow processing.

Many techniques have been described for processing bone marrow prior to transplantation, purging or cryopreservation. Effective techniques incorporate centrifugation and, or, density separation to produce an ideal marrow concentrate. We report on the use of a continuous flow cell separator (COBE Spectra) for marrow processing. Preliminary results indicate that the improved technology incorporated in this machine together with the new algorithm control of its collection functions allows for rapid collection of an ideal marrow concentrate. The addition of an inert sedimenting agent prior to processing enhances differential mononuclear cell collection and elimination of red blood cells and granulocytes. By this technique a volume depletion of 87% was achieved with recovery of 76.4% mononuclear cells and 86.5% CFU-GM progenitor cells. Marrow processed in this manner has been successfully transplanted; patients receiving such marrow show no delay in engraftment and their grafts have been sustained.

A serum-free culture method for myeloma progenitors in vitro: proliferative and immunophenotypic characteristics.

Methods for forming multiple myeloma (MM) colonies are difficult because nonproliferative, but viable, plasma cells can survive for several weeks in culture and because MM cells tend to clump readily, forming pseudo-colonies. The present study describes a method for growing pure myeloma colonies in serum-free conditions in which genuine myeloma growth is unequivocally demonstrated. Growth was observed in 17 of 32 MM bone marrow samples. After a delay of 3-5 weeks, during which most cells died, Ig light-chain-restricted colonies emerged, expanded for approximately 3 weeks, and then showed no evidence of further proliferation. Cell doubling time was 8-10 days, and a significant number of cells in all cultures expressed Ki-67, having earlier lacked this nuclear proliferation antigen. In addition, colony formation was abrogated by irradiation, and two of eight cultures were successfully replated in 0.8% methylcellulose. Phenotypic analysis revealed a mixed population of plasma cells (RFD6+) and B-lymphocytes (CD19+, CAL-LA-), and cells were consistently Epstein-Barr virus negative. Culture of myeloma bone marrow by this serum-free method will allow appraisal of the role of various recombinant growth factors.

Peanut agglutinin shows specificity for bone marrow plasma cells.

A panel of lectins was used to study surface carbohydrate expression on myeloma cells with the aim of finding a possible agent for in vitro bone marrow purging. Peanut agglutinin (PNA, galactose beta 1,3 N-acetylgalactosamine-binding) bound to all plasma cells in 33/34 bone marrow samples from myeloma patients and to all plasma cells in 11 bone marrow from patients with monoclonal gammopathy of undetermined significance and 10 normal bone marrow samples. Bone marrow and peripheral blood monocytes reacted weakly with PNA except in one case of acute monoblastic leukaemia and two of chronic myelomonocytic leukaemia in which monocytes were strongly positive. The only case of plasma cell leukaemia studied was PNA negative. All other bone marrow mononuclear cells were negative for PNA but became positive after sialidase treatment. Peanut agglutinin may have potential as an agent to be used in myeloma for in vitro marrow purging prior to autologous transplantation in combination with high dose chemotherapy.

Empirical treatment of febrile, neutropenic patients with tobramycin and latamoxef.

One hundred and two febrile episodes in neutropenic patients were treated with intravenous tobramycin and latamoxef. After 48 h latamoxef at 6 g day-1, patients were randomized to continue this regimen or latamoxef at 3 g day-1. Infections responded to these regimens in 67% and 71% of patients, respectively. Two-thirds of the infections which failed to respond were due to coagulase-negative staphylococci in Hickman catheters, a trend which may necessitate the inclusion of additional antibiotics in future empirical regimens. Prolonged prothrombin times due to antibiotic therapy were seen in nine patients but there was only one episode of bleeding and this responded quickly to treatment with vitamin K and fresh frozen plasma. In 35 patients, coagulopathy was present before antibiotics were started, and these cases also responded to vitamin K. The study shows that the response to tobramycin and latamoxef is comparable to other broad-spectrum antibiotic regimens and that a reduction in the dose of latamoxef after 48 h treatment may safely permit cost savings.

Amodiaquine induced agranulocytosis: inhibition of colony growth in bone marrow by antimalarial agents.

Bone marrow was cultured in vitro for colonies of granulocytes and macrophages five months after a patient had recovered from amodiaquine induced agranulocytosis. The addition of amodiaquine, chloroquine, and sulfadoxine to the culture was followed by a dose dependent inhibition of colony growth in the patient's marrow but not in normal control bone marrow. Colony growth was, however, unaffected by proguanil, pyrimethamine, and quinine. These findings show that in vitro marrow culture may have important predictive value in some cases of drug induced agranulocytosis.

Disappearance of antibodies to Factor VIII in a patient with acquired haemophilia and carcinoma of the pancreas during cytotoxic therapy with fluorouracil and CCNU.

An inhibitor to clotting factor VIII (anti-VIII:C) developed in a 70 year old woman with carcinoma of the pancreas three months after palliative by-pass surgery. A life-threatening sublingual haemorrhage was controlled by infusion of human factor VIII concentrate in high dosage. With the objective of reducing pancreatic tumour size, combination cytotoxic therapy with fluorouracil and CCNU was given. Reduction in the size of the tumour was associated with disappearance of anti-VIII:C, reappearance of normal quantities of clotting factor VIII (VIII:C) in the plasma and resolution of the bleeding tendency. The anti-VIII:C was characterised as being predominantly of the IgG4 sub-class with k light chains. In vitro and in vivo studies showed the inactivation of VIII:C by anti-VIII:C was markedly non-linear. Normal quantities of factor VIII coagulant antigen (VIII:CAg) were detected in the patient's plasma when VIII:C levels were negligible.

Bone marrow endogenous colony stimulating factor(s): relation to granulopoiesis in vivo.

Granulocyte-macrophage colony stimulating factors (CSF) are produced both in peripheral tissues and within the bone marrow. Stimulators from either source might regulate granulocyte and monocyte production in vivo. The purpose of this study was to devise an assay for bone marrow endogenous CSF so that its role in regulation of granulopoiesis might be assessed. There was a significant positive correlation between the endogenous CSF level and the subsequent trend in peripheral blood neutrophil count in both normal and infected patients, suggestive of a regulatory role. In addition, granulopoietic recovery after neutropenia was associated with increased endogenous CSF levels. There are two candidate regulators of endogenous CSF production, a stimulatory humoral factor and a neutrophil derived inhibitor which inhibits endogenous CSF production in vitro. Our results suggest that the marrow neutrophil level had a much more significant inhibitory effect than the neutrophil level in the culture.