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In this paper a microplate method is described for diagnosing acute leukemia and for investigating the reactivity of monoclonal antibodies (MoAbs) against membrane antigens in combination with rabbit or murine antibodies to nuclear terminal transferase (TdT). The speed of this method facilitates the investigation of fresh leukemic cells from individual patients and assesses the cytolytic efficacy of the relevant MoAbs in the presence of complement (C'). Lymphoblasts (TdT+) are mixed in equal proportions with known numbers of "inert" cells, eg, RBC or nonleukemic bone marrow (BM). Following incubation with MoAbs and C' the ratio of residual TdT+ cells and inert cells is determined on cytospin preparations. Initially, percentages of TdT+ cells are counted in a unit volume of 5,000 inert cells, followed by the scanning of greater than 2 X 10(4) inert cells on entire slides. With this method more than 4 log cytoreduction of TdT + cells is detected. The method is also applicable for studying the cytolysis of malignant B cells by using mostly monoclonal lg expression rather than TdT for the identification of residual B cells. Ten representative patients selected from a group of greater than 100 are reported. In some cases cytoreduction of greater than 4 log with no identifiable residual TdT + cells is achieved by a single C'-fixing MoAb: anti-CD10 (RFAL3) in common acute lymphoid leukemia (ALL) and anti-CD7 (RFT2) in T cell ALL (T-ALL). Other cases require cocktails of anti-CD10, anti-CD19, and anti-CD24 in common ALL or anti-CD7 and anti-CD8 in T-ALL. In T-ALL a few TdT + cells remain that exhibit the features of normal TdT + BM cells (CD7-, HLA-DR+). This is particularly noticeable when patients are studied in partial remission or if nonleukemic BM is used as a source of inert cells. The methods described here contribute to establishing a range of MoAbs (ie, of IgM class) and techniques for efficient purging and to comparing the efficacy of "clean-up," in remission, of common ALL, T-ALL, and B cell malignancies.
In order to test the numerous variable biological parameters which may contribute to the efficiency of the immunomagnetic depletion method, experiments have been carried out with different Burkitt lymphoma cell lines. The Hoechst dye method, a sensitive assay able to detect 3 log elimination, and a liquid culture assay suitable for identifying an elimination greater than 4 logs, were used to measure malignant cell elimination. These assays showed that the separation of bone marrow mononuclear cells on Ficoll-hypaque, the use of beads in excess, and a double treatment of the samples are needed to obtain optimal depletion of malignant cells. Under these conditions, the immunomagnetic procedure is capable of depleting 1% tumour cell infiltration at the lower limit of detection of a very sensitive culture assay (i.e. greater than 4 log elimination).
The effectiveness of an antibody-enzyme immunotoxin (eIT) was investigated on human T cells. This enzyme immunotoxin contained glucose oxidase (GO) and lactoperoxidase (LPO) chemically coupled to the pan-leukocyte-specific mouse monoclonal antibody (MoAb) 097 (097-GO and 097-LPO). Human peripheral blood mononuclear cells or tumor cells were suspended in a mixture of 097-GO and 097-LPO for 30 min, and then for 2 h with glucose and NaI. The effectiveness of this eIT system was indicated by the almost complete reduction of T cell viability, as estimated by a phytohemagglutinin induced proliferation assay (99.4% +/- 0.31 depletion, mean +/- SEM of 15 experiments). The specificity of the cytotoxicity reaction was indicated by the lack of cytotoxicity of control irrelevant MoAb conjugates to T cells (1.9% +/- 4.17 of T cell depletion, eight experiments). The growth of human bone marrow myeloid progenitors (CFU-GM) was not affected by the conjugates even by increasing 100-fold the optimal cytotoxic dose. T cells were susceptible to the conjugates in the presence of up to 90% of erythrocytes. This eIT system may thus represent a new alternative immunospecific procedure for allograft and/or autograft purging, and appears to effectively replace complement-mediated methods of T cell depletion.
From August 1987 to March 1995, 25 patients with high-grade B cell non-Hodgkin's lymphoma (NHL) were treated with high-dose therapy (HDT) followed by bone marrow purged with immunomagnetic beads. At the time of transplantation, 20 patients were in sensitive relapse and five in first complete or partial remission. Ten patients had secondary high-grade NHL transformed from low-grade NHL. The HDT consisted of TBI followed by high-dose cyclophosphamide. All patients engrafted, except for two patients with early treatment-related death. Eleven patients relapsed, of whom nine died of lymphoma, and two are alive in new CR. The estimated event-free and overall survivals at 5 years were 40% and 48%, respectively, with a median follow-up of 48 months (range 1-123). Eight of the tumours contained the translocation t(14;18) at the major breakpoint region (MBR) of BCL-2. In these patients the presence of tumour cells in the bone marrow graft before and after purging were assessed by PCR. Four of five patients infused with non-detectable minimal residual disease in their autografts are in complete remission, while two of three patients reinfused with t(14;18) positive cells after purging, experienced a fast and aggressive relapse. As found by others, our data suggest that reinfusion of tumour-free autografts obtained by efficient in vivo purging using chemotherapy before harvesting, and/or by in vitropurging of the stem cell products, influence the patients remission status after HDT.
Complete and sustained hemopoietic function following myeloablative therapy can be successfully achieved by autologous transfusion of blood derived hemopoietic stem cells. It was the purpose of this study to compare autologous blood stem cell transplantation (ABSCT) in 20 patients with autologous transplantation of a mafosfamide purged marrow (pABMT) in 23 patients; all were transplanted in first complete remission (CR) of acute myelogenous leukemia (AML) using the same pretransplant regimen (14.4 Gy total body irradiation and 200 mg/kg cyclophosphamide). The autografts, mostly differing in source of hemopoietic stem cells, cell composition and CFU-GM number, were evaluated for their ability to reconstitute hemopoiesis and induce long-term disease-free survival (DFS). Prior to harvest, hemopoietic stem cells were mobilized by inducing transient myelosuppression (ara-C 100 mg/m2 every 12 h s.c. days 1-5 and daunorubicin 45 mg/m2, days 3 and 4) followed by an overshooting of peripheral stem cell concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
This paper differs markedly from the others that are being presented at this symposium, in that it does not describe a particular technique, or have a single unifying theme. Its aim is to draw to the attention of the purging community a list of variables that we all have tended to regard as only secondarily important to our primary goal of target cell depletion, or collection. It is often difficult to remember that ex vivo purging treatments are composed of multiple steps, and that, in turn, purging is a single component of a complex treatment protocol. In order to cope with this diversity, the natural tendency has been to focus on manageable segments. While this can speed development, we need to constantly be aware that there are multiple factors, both within and outside of our own particular segment, that can impact on the final result. Variables that we often regard as unimportant, or techniques that are thought of as routine, may ultimately be influencing clinical outcome. It would be naive to suggest that we can ever have control over all of the variables in any procedure that is part of a clinical treatment. One purpose of this paper is simply to draw the existence of these factors to the attention of investigators, and suggest that their potential impact may not always be fully appreciated. Secondly, the selective separation of cells from bone marrow is sufficiently well established that it should now be possible to evaluate the technology, reagents and variables in a systematic and collaborative fashion. While we may not emerge from such an exercise with unity of option, we may at least lay the foundations for the further development of this type of therapy, and provide the framework for addressing future questions.
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Bone marrow purging is being performed increasingly in an effort to deplete residual tumor cells from the graft prior to reinfusion. Several studies have suggested that the removal of tumor cells is an important clinical goal. In this review the utility of the polymerase chain reaction (PCR) for the detection of small numbers of tumor cells in bone marrow and peripheral blood is discussed. Using sensitive assays such as PCR, it is expected that the efficacy of bone marrow purging strategies will be improved and this will hopefully result in decreased relapse rates following autologous bone marrow transplantation.
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The prognosis for adults with B lineage ALL who have relapsed after an initial remission is poor. High-dose chemoradiotherapy followed by autologous BMT can induce prolonged clinical remissions in some children with recurrent ALL. In this study, we evaluated the efficacy of autologous BMT in adults. Autologous marrow was treated in vitro with J5 and J2 monoclonal antibodies (CD10/CD9) plus rabbit complement to purge residual ALL cells. Twenty-two adults with B lineage ALL were treated with high-dose chemoradiotherapy followed by infusion of J2/J5 purged autologous BM. The median age was 28 years (range 18-54 years). Twenty-one of 22 patients had experienced at least one relapse prior to BMT. All patients achieved complete hematologic engraftment. Disease-free survival (DFS) in this cohort of patients was 20%, with all survivors alive and free of disease between 2.5 and 7.5 years post-BMT. Age at the time of BMT was an important prognostic factor, with patients < 28 years old faring much better than older individuals (DFS, 45% vs 0%, p = 0.01). Our experience suggests that high-dose chemoradiotherapy followed by infusion of J2/J5 purged autologous marrow is as efficacious in young adults as it is in children and is a reasonable alternative for patients who lack HLA-matched donors. Results in older adults are poor, however, and demonstrate the need for more effective transplant strategies in these individuals.
Hematologic engraftment and immune reconstitution were examined in patients who received cyclophosphamide and total body irradiation therapy followed by infusion of autologous bone marrow purged with anti-B1 monoclonal antibody (MoAb) and complement as therapy for non-Hodgkin's lymphoma. Hematologic engraftment was prompt with return of greater than or equal to 0.5 X 10(3)/microL granulocytes and greater than or equal to 2 X 10(4)/microL platelets at a median of 26 and 29 days posttransplant, respectively. Immunologic reconstitution, in contrast, was prolonged. Normal numbers of circulating B cells were consistently noted by five months posttransplant, whereas return of normal immunoglobulin levels in some patients did not occur for one year. Normal numbers of T cells were evident within the first month posttransplant, but a reversed T4:T8 ratio persisted in some patients up to three years. In vitro responses of either B cells to triggers of activation or of T cells to mitogens and antigens were not normal for at least three months posttransplant. Natural killer (NK) cells predominated early after transplant and may demonstrate cytotoxicity against tumor cells. Our studies demonstrate that transplantation with anti-B1 purged autologous bone marrow results in complete hematologic and delayed immunologic engraftment. No significant acute or chronic clinical toxicities have been observed.
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