ALBERT

Description: Interleukin-2 (IL-2) therapy may improve immune reconstitution and reduce the risk of leukemic relapse in the setting of minimal residual disease by augmenting cytotoxic effector mechanisms directed at residual malignant cells. In addition, IL-2 in vitro promotes the release of cytokines including gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF), which also possess antileukemic activity and can enhance granulocyte function. To determine if IL-2 infusion induces release of gamma-IFN and TNF in vivo in sufficient quantity to mediate these effects, we have measured serum levels of these cytokines and secretion by lymphocytes obtained from patients receiving this cytokine in a phase 1 trial. Serum gamma-IFN was undetectable pre-IL-2 and increased to 1.5 to 17 U/mL during IL-2 infusion (P less than .05). Culture of patient lymphocytes for 48 hours produced 1.2 U gamma-IFN/2 x 10(6) cells/mL pre-IL-2 rising to 50 U/2 x 10(6) cells/mL when the lymphocytes were obtained during therapy (P less than .05). Lymphocyte subset analysis showed that both CD3+ and CD16+ cells secreted gamma- IFN in response to IL-2. TNF secretion by lymphocytes also rose during IL-2 infusion from a mean of 5 U/mL to 14.4 U/mL (P less than .01) although no rise was seen in serum levels. The material secreted by IL- 2-stimulated lymphocytes is bioactive as addition of supernatants from lymphocytes obtained during IL-2 therapy to cultures of myeloid blasts significantly inhibited clonogenic growth. IL-2-induced secretion of these cytokines mediated this inhibition as it could be partially blocked by either anti-gamma-IFN or anti-TNF antibodies. Preincubation of granulocytes with the same supernatants produced enhanced oxidative metabolism, measured by chemiluminescence in response to N-formyl- methionyl-leucyl-phenylalanine (FMLP). This effect also could be partially abrogated by anti-gamma-IFN and anti-TNF antibodies. Therefore, secondary cytokine secretion may boost granulocyte function and contribute to the antileukemic effects of IL-2 infusion in patients following bone marrow transplantation or chemotherapy.

Description: Acute myelofibrosis (AM) or malignant myelosclerosis is a myeloprofilerative syndrome in which bone marrow fibrosis is associated with a proliferation of immature myeloid cells. In four patients with typical AM, investigated by the platelet-peroxidase reaction at ultrastructural level, the blast cells were found to be megakaryoblasts. One patient, treated with the drug combination DAT, achieved a complete remission of 5 mo duration. This study supports the view that megakaryoblastic leukemia is the most frequent underlying cause of AM and proposes that it should be classified as a form of acute myeloid leukemia.

Description: We studied the clinical course of 130 chronic myeloid leukemia (CML) patients (89 males and 41 females) in the European Bone Marrow Transplantation Group (EBMT) registry who received transplants before January 1, 1988 and who subsequently had evidence of recurrent leukemia. All patients had received a pretransplant conditioning regimen including total body irradiation (TBI). The first evidence of relapse was cytogenetic only in 74 (57%) patients and hematologic in 56 (43%). The overall actuarial survival from relapse was 36% at 6 years, with a significantly higher proportion of survivors among female patients (53% v 30%; P 〈 .002). In univariate analysis, the 6-year probability of survival was 52% for patients with cytogenetic relapse and 30% for patients relapsing in chronic phase (CP), while no patient who relapsed in advanced phase (AP or BC) survived more than 3.5 years from relapse (P 〈 .0001). The actuarial survival of patients relapsing before 6 months, between 6 and 12 months, and later than 12 months after transplant was 27%, 26%, and 45%, respectively (P 〈 .002). Among patients with cytogenetic relapse, partial or complete disappearance of Ph-positive cells occurred in 40% of untreated patients and in 42% of those treated with interferon (IFN). However, IFN therapy significantly delayed progression toward hematologic disease. Cytogenetic responses were observed in 25% of patients who received IFN for relapse into CP, while only one minor cytogenetic response was reported in patients on conventional chemotherapy. For patients presenting with cytogenetic relapse as well as for those in hematologic relapse, IFN therapy significantly improved the 2-year probability of survival. However, long-term survival for IFN-treated patients in either group was not different from long-term survival in comparable patients not receiving IFN therapy. Twenty-nine patients of this series underwent a second bone marrow transplant (BMT) and the projected survival at 4 years after the second transplant is 28%. In multivariate Cox regression analysis, four factors remained significantly associated with survival: disease phase at relapse (P 〈 .0001), duration of time interval from BMT to relapse (P = .0001), interferon therapy at relapse (P = .0024), and patient sex (P = .0032). This retrospective study provides evidence that some patients who relapse after BMT may benefit from treatment with IFN; a second BMT may offer the chance of cure. Data from this analysis may be useful in designing future prospective trials on posttransplant CML relapse.

Description: After bone marrow transplantation (BMT), mortality from viral infections such as cytomegalovirus (CMV) remains high. Gamma-Interferon (gamma IFN) and tumor necrosis factor (TNF) are produced constitutively after BMT and have anti-viral properties. To study the effects of these cytokines on CMV interaction with host cells, we have used patient marrow fibroblasts since marrow stroma is a target for CMV infection correlating with myelosuppression in vivo. Both gamma IFN and TNF are constitutively produced by recipient CD3+ and CD16+ lymphocytes, but not by their marrow fibroblasts. Secretion by peripheral blood mononuclear cells is increased if they are cultured with host fibroblasts infected with CMV in vitro and the levels of gamma IFN and TNF produced are within the range that protects fresh fibroblasts from CMV infection. Constitutive secretion of cytokines by lymphocytes declines by 8 weeks after BMT, a time when the risk of CMV disease increases sharply. The in vitro phenomenon that we have described needs to be evaluated in correlative studies on individual BMT recipients to determine whether such a cytokine-mediated defense mechanism against CMV may operate in vivo.

Description: Activated killer cells, unrestricted by major histocompatibility (MHC) antigens circulate in the peripheral blood of patients who have undergone autologous and allogeneic bone marrow transplant (BMT) and may contribute to the reduced risk of leukemic relapse observed after these procedures. Interleukin-2 (IL-2) in vitro augments this cytotoxicity and used therapeutically might thereby promote the eradication of minimal residual disease. In order to assess whether these effects on cytotoxicity can be reproduced in vivo, we studied changes in number, phenotype, and MHC unrestricted cytotoxicity of peripheral blood mononuclear cells obtained from patients with hematologic malignancy receiving IL-2 infusions. Patients with acute myeloid leukemia and multiple myeloma were treated after cytotoxic chemotherapy or autologous BMT. IL-2 infusions produced an initial lymphopenia, followed by a progressive recovery in mononuclear cell numbers and a rebound lymphocytosis after the termination of treatment. This affected all lymphocyte subsets; in particular CD25 (IL-2 receptor) positive cell numbers rose sevenfold. Cells with the ability to kill a natural killer (NK)-resistant, lymphokine activated killer cell (LAK)-sensitive target appeared in the circulation during 16 of 19 infusions and mean LAK activity rose from 5.9% to 15.5% during infusion (E:T ratio, 50:1; P less than .001). During IL-2 infusion, cells present in the peripheral blood inhibited the growth of myeloid leukemia blasts in agar after overnight co-culture. Depletion experiments showed that LAK activity was mediated by cells of both CD3- CD16+ (NK derived) and CD3+ CD16- (T derived) subsets. LAK precursor activity in peripheral blood also significantly increased during IL-2 infusion. Increases in major histocompatibility complex (MHC) unrestricted cytotoxicity can be produced by IL-2 infusions in vivo and may result in improved relapse-free survival following chemotherapy or BMT.

Description: Cytokine-secreting, major histocompatibility complex-unrestricted activated killer (AK) cells are toxic to a wide range of virus-infected or malignant target cells and may be generated endogenously, eg, after bone marrow transplantation, or by infusion of cytokines such as recombinant interleukin-2 (rIL-2). Although AK cells secrete cytokines such as gamma-interferon and tumor necrosis factor, which are themselves able to recruit fresh cytokine-secreting AK cells, activation in both settings is short-lived, implying the existence of homeostatic regulatory mechanisms. We now demonstrate one mechanism by which rapid homeostasis is achieved. We show that IL-4 is produced in patients with both endogenously and exogenously generated AK cells. The cytokine was detected in serum after marrow transplantation, and IL-4 transcripts appeared in circulating lymphocytes during rIL-2 infusion. Although IL-4 inhibited the induction phase of AK cell function, it had no significant inhibitory effect on the ability of AK cells from these individuals to respond to restimulation. Nonetheless, neutralization of the IL-4 induced during cell activation doubled the half-life of AK function, once activating stimuli were removed, from 18 to 44 hours and produced a 2-log increase in AK cell secretion of tumor necrosis factor and gamma-interferon. These data suggest that IL-4 induced in vivo during lymphocyte activation abbreviates AK cell responses once the triggering stimuli have been removed. Neutralization of endogenous IL-4 in vivo by appropriate monoclonal antibodies might prolong the duration of AK function.

Description: Interleukin-2 (IL-2) therapy may improve immune reconstitution and reduce the risk of leukemic relapse in the setting of minimal residual disease by augmenting cytotoxic effector mechanisms directed at residual malignant cells. In addition, IL-2 in vitro promotes the release of cytokines including gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF), which also possess antileukemic activity and can enhance granulocyte function. To determine if IL-2 infusion induces release of gamma-IFN and TNF in vivo in sufficient quantity to mediate these effects, we have measured serum levels of these cytokines and secretion by lymphocytes obtained from patients receiving this cytokine in a phase 1 trial. Serum gamma-IFN was undetectable pre-IL-2 and increased to 1.5 to 17 U/mL during IL-2 infusion (P less than .05). Culture of patient lymphocytes for 48 hours produced 1.2 U gamma-IFN/2 x 10(6) cells/mL pre-IL-2 rising to 50 U/2 x 10(6) cells/mL when the lymphocytes were obtained during therapy (P less than .05). Lymphocyte subset analysis showed that both CD3+ and CD16+ cells secreted gamma- IFN in response to IL-2. TNF secretion by lymphocytes also rose during IL-2 infusion from a mean of 5 U/mL to 14.4 U/mL (P less than .01) although no rise was seen in serum levels. The material secreted by IL- 2-stimulated lymphocytes is bioactive as addition of supernatants from lymphocytes obtained during IL-2 therapy to cultures of myeloid blasts significantly inhibited clonogenic growth. IL-2-induced secretion of these cytokines mediated this inhibition as it could be partially blocked by either anti-gamma-IFN or anti-TNF antibodies. Preincubation of granulocytes with the same supernatants produced enhanced oxidative metabolism, measured by chemiluminescence in response to N-formyl- methionyl-leucyl-phenylalanine (FMLP). This effect also could be partially abrogated by anti-gamma-IFN and anti-TNF antibodies. Therefore, secondary cytokine secretion may boost granulocyte function and contribute to the antileukemic effects of IL-2 infusion in patients following bone marrow transplantation or chemotherapy.

Description: After marrow transplantation, major histocompatibility complex (MHC)- unrestricted natural killer (NK) lymphocytes are among the first cells to appear in the circulation. After T-cell-depleted bone marrow transplantation (TD-BMT), these cells have an activated pattern of target cell killing; they also secrete lymphokines including gamma- interferon (gamma-IFN), interleukin-2 (IL-2), and tumor necrosis factor (TNF) and may have a significant role as a primary defense against viral reactivation and in the elimination of residual host malignancy. We studied 43 patients with hematologic malignancy, treated by allogeneic TD-BMT, autologous nondepleted BMT, or chemotherapy alone to investigate (a) the mechanisms underlying the generation of these activated killer cells, (b) the range of conditions under which they are produced, and (c) their surface phenotype. We showed that gamma-IFN- secreting activated killer cells with the capacity to kill MHC- nonidentical NK-resistant targets are generated 4 to 6 weeks after either allogeneic TD-BMT or autologous BMT but do not appear after treatment with chemotherapy. Production therefore is not owing to T- cell depletion per se or to host donor alloreactivity, nor is it caused by stimulation by alloantigens contained in blood product support since no significant difference exists between allograft and chemotherapy patients in the number of units of blood platelet support given in the posttreatment period. Because most patients had no evidence of stimulation from virus reactivation/infection, the phenomenon of activation therefore appears to represent posttransplant immune disregulation following repopulation of the host immune system with lymphoid subsets derived exclusively from blood and marrow. Activated killing is predominantly mediated by the CD16+ CD3- subset, but substantial activity remains in the CD16- CD3+ cell fraction. Monoclonal antibodies (MoAbs) that block interaction with class-I MHC molecules at the level of target cell (W6/32 anti-HLA class I) or effector cell (CD8) do not inhibit killing by CD16- CD3+ cells. Activated killer cells may contribute to the lower risk of relapse after marrow transplantation as compared with intensive chemotherapy.

Description: Cytomegalovirus (CMV) remains the most common single infective cause of death following allogeneic bone marrow transplantation (BMT) from major histocompatibility complex (MHC)-identical siblings, whereas Epstein- Barr virus (EBV)-related disease is infrequent. We show here that MHC- unrestricted cytotoxic effector cells in the peripheral blood of BMT recipients are highly effective at killing EBV-infected target cells, but are inactive against CMV-infected target cells. Differential cytotoxicity is associated with disparate target structure expression. Although both EBV- and CMV-infected target cells express viral antigens, it is only those infected with EBV that express the adhesion molecule lymphocyte function-associated antigen 1 (LFA1; CD11a/18). Thus, EBV-infected target cells are able to interact with the principal LFA1 ligand, intercellular adhesion molecule 1 (ICAM1; CD54), which is expressed on posttransplant peripheral blood mononuclear (PBM) effector cells. CMV-infected target cells cannot utilize this ligand. Posttransplant cytotoxicity against EBV-infected target cells is abolished by target and effector cell blockade with monoclonal antibodies (MoAbs) to LFA1 and ICAM1, respectively, demonstrating the functional relevance of this additional ligand interaction. These results provide an illustration both of the importance and of the limitations of MHC-unrestricted cytotoxicity in vivo and may explain the frequency of CMV disease and the relative rarity of EBV-related disease following allogeneic transplantation from MHC-matched siblings. The increased immunosuppression used following MHC-mismatched/matched unrelated-donor BMT may cause this MHC-unrestricted defense mechanism to fail and may contribute to the greatly increased incidence of EBV lymphoproliferative syndrome in these patients.

Description: There is substantial evidence that the volume of medical procedures in a hospital has an inverse relationship with mortality. We analyzed data for 1313 recipients of HLA-identical sibling bone marrow transplants for early leukemia (acute leukemia in first remission or chronic myelogenous leukemia in first chronic phase) to determine whether transplant outcome differed in small and large centers. Transplants were performed in 86 bone marrow transplant centers active between the years 1983 and 1988, which participated in the International Bone Marrow Transplant Registry. Twenty-one (24%) centers performed five or fewer allogeneic transplants per year during the study period; five (6%) performed more than 40 per year. After adjustment for differences in patient and disease characteristics, the relative risks of treatment- related mortality (1.53, P less than .01) and treatment failure (1.38, P less than .04) were higher among patients who received transplants at centers doing five or fewer transplants per year than among those at larger centers. Among patients receiving transplants in centers performing more than five transplants a year, there was no statistically significant correlation between number of transplants and outcome.