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The role of plerixafor in optimizing peripheral blood stem cell mobilization for autologous stem cell transplantation

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte- macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)- induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.

We evaluated inflammatory markers in febrile neutropenic lymphoma patients undergoing high-dose chemotherapy with autologous stem cell support. Based on MASCC scores, our patients had a low risk of serious complications and a perspective of a benign initial clinical course of the febrile neutropenia. We also studied the impact of tobramycin given once versus three times daily on these immune markers. Sixty-one patients participating in a Norwegian multicentre prospective randomized clinical trial, comparing tobramycin once daily versus three times daily, given with penicillin G to febrile neutropenic patients, constituted a clinically homogenous group. Four patients had bacteraemia, all isolates being Gram-positive. Thirty-two patients received tobramycin once daily, and 29 patients received tobramycin three times daily. Blood samples were taken at the onset of febrile neutropenia and 1-2 days later. All samples were frozen at -70 °C and analysed at the end of the clinical trial for C-reactive protein (CRP), procalcitonin (PCT), complement activation products, mannose-binding lectin (MBL) and 17 cytokines. We found a mild proinflammatory response in this series of patients. CRP was non-specifically elevated. Ten patients with decreased MBL levels showed the same mild clinical and proinflammatory response. Patients receiving tobramycin once daily showed a more pronounced proinflammatory response compared with patients receiving tobramycin three times daily. Overall, febrile neutropenic cancer patients with a benign clinical course show a mild proinflammatory immune response.

J. Crown, B. Coiffier, H. Cortes-Funes, Th. Guillaume, L. Kanz, G. Kvalheim, M. Marty & M. Symann 'St. Vincent's Hospital, Dublin, Ireland; Centre Hospilalier Lvon-Sud, Lyon, France; Hospital Universitario 12 de Octubre, Madrid, Spain; Catholic University of Louvain, Brussels, Belgium; Universitat Tubingen, Tubingen, Germany; The Norwegian Radium Hospital, Oslo, Norway; Hopital Saint-Louis, Paris, France

PURPOSE/EXPERIMENTAL DESIGN: The importance of detection of disseminated isolated tumor cells (ITCs) in bone marrow (BM) is still not settled. BM aspirates from 920 patients with primary breast cancer were analyzed for tumor cells by standardized direct immunocytochemical analysis (ICC) of 2 x 10(6) mononuclear cells (MNCs) using anticytokeratin monoclonal antibody (AE1/AE3). Samples (637) were analyzed by negative immunomagnetic enrichment (IMS) followed by ICC (10 x 10(6) MNCs). Analyses of the primary tumor specimens have been performed, including histomorphology, tumor-node-metastasis (TNM) staging, grading, and immunohistochemical analyses.Of the patients with infiltrating carcinoma, 63% were node negative (N0) and 33%, node positive (N+). The results show the presence of tumor cells in 13.4% of the evaluable patients after direct ICC analysis. The presence of tumor cells correlated to the nodal- and tumor stage, showing BM positivity in 9.9% of the N0 cases and 20.6% in the N+ group (P0.0005), 11.2% of the stage T(1) were positive, and 15.0% and 22.6% were positive in the T(2) and T(3/4) groups, respectively (P = 0.013). No correlation between detection of ITC and detection of p53 and cathepsin D expression was found. Vascular invasion and c-erbB2 expression were associated with ITCs in BM (P = 0.045 and P = 0.024, respectively). Node-negative patients with estrogen receptor (ER)+ and/or progesterone receptor (PgR)+ tumors had lower frequency of ITCs than ER-/PgR- (P = 0.004). The use of negative IMS increased the frequency of positive BM by 63% (P0.0005).The direct ICC detection of ITCs in BM correlated with primary tumor stage, nodal stage, vascular invasion, c-erbB2 expression, and ER/PgR status. Analysis of larger BM samples by negative IMS resulted in increased number of ITC-positive patients.

In this study we explored whether a standard chemotherapy regimen consisting of mitoguazone, ifosfamide, methotrexate and etoposide (MIME) combined with 5 micrograms/kg or 10 micrograms/kg G-CSF was capable of mobilizing peripheral blood progenitor cells (PBPC) in lymphoma patients. Thirty-three patients with Hodgkin's disease (HD) and 108 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3-40) of chemotherapy prior to mobilization. Eight of 141 patients failed to mobilize PBPC and bone marrow was harvested. In addition, 10 patients obtained a harvest of2.0 x 10(6) CD34+ cells/kg. More than 2.0 x 10(6) CD34+ cells/kg were achieved in all HD patients and in 83% of the NHL patients. Fifty-eight per cent of the patients harvestedor = 5 x 10(6) CD34+ cells/kg. Eleven per cent of the patients developed neutropenic fever during the mobilization and 3% had nadir platelet values below 20 x 10(9)/L. An inverse correlation was observed in high-grade NHL (H-NHL) patients between the number of chemotherapy cycles given before mobilization and yield of CD34+ cells. Such an association was not seen among patients with HD, transformed and low-grade NHL. When G-CSF 10 micrograms/kg was used in combination with MIME, this correlation was no longer seen in patients with H-NHL. There was also association between CD34+ cell yield and prior radiotherapy in patients with HD or transformed NHL or low-grade NHL. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF, can be successfully used to mobilize PBPC. Although no significant effect of increased dose of G-CSF was found, our data suggest that MIME/G-CSF 10 micrograms/kg should preferentially be used to mobilize PBPC in H-NHL patients pre-treated withor = 12 cycles of chemotherapy, in irradiated HD patients and in all low-grade and transformed lymphomas.

The incidence of breast cancer is increasing dramatically in the developed countries. Although attention is being paid to diagnose breast cancer early through screening programs, still a significant number of patients classified to have a localised disease at diagnosis, later experience a systemic recurrence. Therefore, more sensitive and reliable methods to detect the early spread of the disease, permitting the early use of additional systemic therapy, seem justified. New treatment strategies, such as immunotherapy employing monoclonal antibodies against breast cancer cells, is promising. Since such a treatment is most efficient on patients with limited disease, sensitive methods to monitor the therapeutic effect are needed. Immunocytochemistry using tumour associated monoclonal antibodies and reverse transcription polymerase chain reaction assays (RT-PCR), that screen for carcinoma-specific expression of mRNA in bone marrow and blood, have been developed. Many standardisation problems with the detection methods currently in use are unsolved. Despite this, we discuss here recent data showing that the presence of occult tumour cells in the bone marrow is a prognostic factor in patients with breast cancer.

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination.

The feasibility of mobilizing peripheral blood stem cells (PBSC) using anthracycline containing polychemotherapy and G-CSF on the first 50 patients randomized to the high-dose arm in the adjuvant SBG 9401 is investigated. The patients were treated with standard FEC (5-fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2) for two courses followed by a modified third FEC course with a C dose of 1200 mg/m2 supported with subcutaneous G-CSF (filgrastim) at 5 mg/kg followed by harvest around day 11. The mean yield of CD34+ cells per patient was 10.6x10(6)/kg (range 2.6-29.1). The side effects after the third course were low and only one patient developed an uncomplicated granulopenic fever. Our data indicated a correlation between number of transfused CD34+ cells and days to neutrophil and platelet recovery. In conclusion, the modified FEC regimen followed by G-CSF is a feasible method for PBSC mobilization in the adjuvant setting.

Since 1994, 17 breast cancer patients and 16 lymphoma patients have been treated at the Norwegian Radium Hospital with high-dose therapy supported by autologous peripheral progenitor cells. All the patients were given granulocyte colony stimulating factor in the recovery phase after cytotoxic treatment in order to mobilize and harvest peripheral progenitor cells. Aphareses were successful in all patients and the mean number of CD34 cells reinfused per kilo body weight was 7.05 x 10(6) for the lymphoma patients and 11.1 x 10(6) for the breast cancer patients. The mean time to recoveror = 0.5 x 10(9)/l granulocytes andor = 20 x 10(9)/l platelets after reinfusion of stem cells was 10 days and 11.7 days respectively for the lymphoma patients, while the breast cancer patients engrafted at day 8.6 and day 9.3. No severe treatment-related complications were observed.

In this article we report our initial clinical experiences in connection with immunomagnetic isolated CD34-positive cells from peripheral blood progenitor cells. Six patients, five with breast cancer and one with non-Hodgkin's lymphoma, were mobilized by chemotherapy and G-CSF (5ug/kg). CD34-positive cells were isolated by means of immunomagnetic beads (Dynalbeads) and Isolex 300 Cell Separator (Baxter, USA). Mean purity of isolated CD34-positive cells was 97% (94.7-99.7) and mean yield was 54% (35-68). Three patients were treated with high dose therapy followed by reinfusion of CD34-positive cells as stem cell support. Recovery of neutrophils (0.5 x 10(9) leucocytes/liter) occurred at day 8, 11 and 13 and of platelets (20 x 10(9) platelets/litre) at day 9,14 and 32. It is concluded that immunomagnetic isolated CD34-positive cells give high purity and yield. Although use of CD34-positive cells reduces the content of contaminating tumour cells in the graft, breast cancer cells were still detectable in two out of five CD34-positive cell products.

Residual leukemia was evaluated in autologous bone marrow grafts harvested in first (n = 11) or second (n = 3) complete remission from 14 patients with BCR-ABL-positive acute lymphoblastic leukemia after treatment according to the German multicenter ALL protocols. The intervals from diagnosis to BM harvest were median 159 (range 78-463) and from preceding chemotherapy to BM harvest median 39 (range 26-69) days, respectively. A limiting log(10)-dilution RT-PCR was used to semiquantify BCR-ABL-positive cells. All autografts appeared to be significantly contaminated with residual leukemic cells. The BCR-ABL-specific titers ranged from 1:10(3) to 1:10(6) (median 1:10(4)) above the limit of detection. This was the rationale to purge the grafts using two cycles of IgM anti-CD10, CD19, and AB4 MoAbs-coated immunomagnetic beads (IMB). Purging depleted median 3 (range 2-4) logs of residual leukemia, resulting in a median 1:10(1) (range 1:10(0) to 1:10(3)) postpurge BCR-ABL-specific titer. The second purging cycle accounted for 1 log of depletion. The mean +/- s.e.m. post-purge recoveries of MNC and CFU-GM were 59 +/- 4%, and 61 +/- 9%, respectively. We conclude that all BCR-ABL-positive ALL patients achieving CR by cytological criteria have critically high levels of residual leukemia in their bone marrow, which can be reduced by median 3 log using immunomagnetic bead purging.

Immunocytochemistry using tumour-associated monoclonal antibodies has led to improvements in the ability to detect occult breast cancer cells in bone marrow aspirates and peripheral blood. Nevertheless, the immunocytochemistry method needs to be further developed before it can be used routinely in the clinic. Reverse transcription polymerase chain reaction assays (RT-PCR) that screen for carcinoma-specific expression of mRNA in bone marrow and blood have been developed. However, it is not yet clear whether the most frequently employed RT-PCR assay for cytokeratin 19 has the specificity required to be safely used in the clinic. In spite of many unsolved standardization problems with micrometastatic detection methods, recent data show that the presence of occult tumour cells in the bone marrow at diagnosis and in the reinfused autograft after high-dose therapy appears to increase the rate of recurrence in the patients.

A variety of techniques has been used for the purpose of removing normal T cells and tumor cells from bone marrow. Here, the various methods devised and the problems associated with each procedure will be briefly discussed.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

Between 1986 and 1991, 29 patients with malignant lymphomas were treated with total body irradiation and high-dose chemotherapy followed by autologous bone marrow transplantation at the Norwegian Radium Hospital. Owing to treatment-induced bone marrow toxicity, the leucocyte count in peripheral blood rapidly fell to zero. The aplastic phase lasted for 25 days (median). All patients experienced fever in the posttransplant period and needed broad-spectrum antibiotics. 12 patients had documented bacteremia, mostly with Gram-positive isolates. There were seven cases of documented focal infections, and antibiotic-associated colitis was seen in two cases. Other complications included bleedings and skin rashes. Four patients died in the post-transplant period from complications related to the cytotoxic treatment. We discuss guidelines for antimicrobial treatment of suspected and confirmed infections in neutropenic patients.

An immunomagnetic method was developed to purge human bone marrow of malignant T cells, for use in conjunction with autologous bone marrow transplantation in patients with acute lymphoblastic leukemia and lymphoma. Three monoclonal antibodies anti CD2 (BH1), CD5 (BB8) and CD7 (BF12) were used. In model experiments employing MOLT4 cells it was found that with 50-fold excess of immunobeads relative to antigen-positive cells, the use of each antibody alone resulted in a 3.3-3.6 log tumor cell depletion, as assessed in a sensitive and reproducible clonogenic soft agar assay. When all three antibodies were used in a mixture, a purging efficacy of 5 logs was achieved. Two treatment cycles improved these figures to about 4 logs and more than 5 logs. When MOLT4 cells were mixed 1:10 with fresh bone marrow cells the antibody mixture yielded 3.1 and more than 5 log tumor cell depletion with one and two treatment cycles, respectively. This procedure resulted in only an insignificant reduction of the number of CFU-GM and CFU-GEMM progenitor cells. In two patients autotransplanted with purged marrow, the loss of CFU-GM was 37% and 48%, and no tumor cells could be detected by immunocytochemistry after purging. Rapid and sustained engraftment was achieved and both patients remain in complete remission after more than 20 months.

In acute lymphoblastic leukemia (ALL) the expected duration of a second complete remission (CR) following conventional aggressive multi-drug treatment is rather short, and long-term survival is rarely seen. Myeloablative consolidation treatment with ABMT in second CR is thought to benefit "high risk" patients thus allowing the escalation of cytoreductive treatment into a lethal range. In three patients with relapsed ALL bone marrow was collected in the very early second remission phase. The autografts were incubated with a panel of monoclonal antibodies bound to magnetobeads (DYNABEADS M450). The pretransplant regimen consisted of total body irradiation (TBI) (14.4 Gy) and cyclophosphamide (CY) (50 mg/kg b.w. x4). Following autologous transplantation of those immunomagnetobead purged marrow grafts, complete hematologic reconstitution was seen in all three patients. They are alive and disease-free for +26, +24 and +17 months post transplant, which is also proven by the absence of detectable gene rearrangements after transplantation. In two of them disease-free survival exceeds the duration of the preceding remission y 20 and 12 months respectively, which is a definitive parameter for therapy effectiveness. Therefore, "high dose" consolidation treatment in very early second CR followed by autologous transplantation of purged bone marrow seems to allow long-term disease-free survival. The technique of ex vivo immunomagnetic bead purging, i.e. the "direct" and "indirect" method of binding monoclonal antibodies to magnetobeads is described.

Autologous bone marrow transplantation permits the use of greatly intensified cytoreductive therapy for cancer. Since 1983 seven children with disseminated neuroblastoma (stage IV) were treated by this method. Five were treated in first, and one in second complete remission; one child was in partial remission. Tumor cell purging of the marrow inoculum was performed in five cases. All children had engraftment and were discharged from hospital free of disease. Relapse was observed in three children within two years. Four children remain healthy at follow-up 5-77 months after autotransplantation. We describe and discuss indications, methods, side effects and results.

The principle of activated swelling has made available polymer particles in the range of 1–100 μm, highly uniform in size, and with great variations in morphology and chemical composition. A short description of the method is given. On the basis of monosized porous macroreticular particles, monosized magnetic polymer particles (MMP) are prepared which are uniform in size as well as in content of magnetic oxides. These particles have found applications in a number of selective cell separation procedures. Special types of MMP have been developed for isolation of organelles and viruses. The use of MMP as contrast agent in magnetic resonance imaging is shortly described.

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.

The efficacy of a novel monosized and magnetizable polymer bead, denoted M-280, in immunomagnetic removal of Rael B-lymphoma cells was compared with that of M-450 beads, previously used in bone marrow purging. The M-280 beads which are smaller (diameter 2.8 microns) and contain less iron than the M-450 beads were coated with polyclonal IgG sheep antimouse (SAM) antibody. The two types of immunobeads were equally efficacious when used together with the mouse monoclonal IgG antibodies HH1 or FN1, giving tumor cell depletions of about 3 logs in one cycle of operation. However, when used together with the primary IgM monoclonal antibodies (MoAbs) AB1 or HH2, the new immunobeads were significantly more efficacious than the M-450 immunobeads. To elucidate the underlying mechanism flow cytometric studies and measurements of the binding of the labeled primary MoAbs to the cellular antigens as well as to the immunobeads were carried out. Competition experiments showed that in the case of IgG MoAbs, the SAM beads bind predominantly to the Fc portion, whereas in the case of the IgM MoAbs, the Fab part plays a relatively greater role in the binding. The results indicate that if M-280 immunobeads are used, IgM MoAbs may profitably be included in antibody cocktails together with IgG antibodies in immunomagnetic purging of B-lymphoma cells. They suggest that in the case of cell bound MoAbs, the epitopes on IgG are more accessible to SAM beads than those of surface bound IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

A Phase I study was carried out with ricin, a plant toxin acting by inhibiting protein synthesis, on 54 cancer patients with advanced disease. Ricin was given as i.v. bolus injections every two weeks at dose levels ranging from 4.5 to 23 micrograms/sq m of estimated body surface area. Ricin was well tolerated at doses up to 18 to 20 micrograms/sq m. At these levels and at higher levels, flu-like symptoms with fatigue and muscular pain appeared and, in some patients, nausea and vomiting occurred also. No myelo-suppression was seen. Antibodies to ricin were detected in serum after two to three ricin injections. Ricin was eliminated from blood according to first order kinetics. At each dose level, the plasma concentrations, as well as the side effects, showed only minor differences between patients. The highest dose given, 23 micrograms/sq m, gave plasma concentrations twice those found previously to be therapeutically effective in tumor-bearing mice. Of 38 evaluable patients, one patient with lymphoma had a partial response. Stable disease was observed in four patients with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A dose of 23 micrograms/sq m is recommended for Phase II trials of ricin.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.