Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103.

BACKGROUND AIMS: Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL). METHODS: Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. RESULTS: Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by (51)Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004). CONCLUSIONS: MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.

Modelling cytomegalovirus replication patterns in the human host: factors important for pathogenesis.

Human cytomegalovirus can cause a diverse range of diseases in different immunocompromised hosts. The pathogenic mechanisms underlying these diseases have not been fully elucidated, though the maximal viral load during infection is strongly correlated with the disease. However, concentrating on single viral load measures during infection ignores valuable information contained during the entire replication history up to the onset of disease. We use a statistical model that allows all viral load data sampled during infection to be analysed, and have applied it to four immunocompromised groups exhibiting five distinct cytomegalovirus-related diseases. The results show that for all diseases, peaks in viral load contribute less to disease progression than phases of low virus load with equal amount of viral turnover. The model accurately predicted the time of disease onset for fever, gastrointestinal disease and pneumonitis but not for hepatitis and retinitis, implying that other factors may be involved in the pathology of these diseases.

Future directions in haematology: beyond multiple myeloma.

Thalidomide acts on the microenvironment of myelodysplastic syndromes (MDS) by influencing cytokine networks, and growing evidence supports thalidomide's usefulness in the management of haematological malignancies, such as MDS. The European Collaboration Group on Myelofibrosis with Myeloid Metaplasia reviewed patients who received at least four weeks' thalidomide treatment, in doses ranging from 50 mg/day to 400 mg/day. The results showed that 30% of patients had increases in haemoglobin, and, of these, almost 40% became transfusion independent. Platelets were increased in a significant proportion of patients, and approximately 40% of patients had a reduction in their spleen size. Data on thalidomide and acute myeloblastic leukaemia (AML) are conflicting: a recently published study indicated that thalidomide does not have a role in the management of acute myeloblastic leukaemia (AML), while other studies suggest some patients may respond because of thalidomide's ability to activate natural killer cells and cytotoxic T-lymphocytes. Partial responses to thalidomide treatment have been recorded in patients with lymphoma. In a phase II study to assess the activity of thalidomide in patients with Waldenstrom's macroglobulinaemia, a partial response was seen in 25% of patients who received a starting dose of 200 mg, which was escalated in 200 mg increments every 14 days as tolerated to a maximum of 600 mg. Although further study is required, thalidomide shows promise in the treatment of a number of haematological malignancies, many of which currently have limited treatment options and poor prognosis.

Induction therapy for adults with acute lymphoblastic leukemia: results of more than 1500 patients from the international ALL trial: MRC UKALL XII/ECOG E2993.

The international acute lymphoblastic leukemia (ALL) study was designed to prospectively define the optimal therapy for adults 60 years of age or younger with newly diagnosed ALL. All patients received identical induction therapy, and 91% achieved complete remission (CR). Patients 50 years of age or younger with a compatible sibling were assigned to undergo allogeneic transplantation; the others were randomly assigned to autologous transplantation or to consolidation/maintenance therapy for 2.5 years. Patients who did not achieve CR after induction had an overall survival rate of 5% compared with 45% for patients who achieved CR. Factors at diagnosis predictive of overall survival and disease-free survival were age (P = .001), white blood cell count less than 30 x 10(9)/L for B lineage or less than 100 x 10(9)/L for T lineage (P = .001) and immunophenotype, T lineage versus B lineage (P = .001). The data demonstrate that achieving CR with induction therapy is indispensable for long-term survival in adult patients with ALL. Furthermore, with a response rate greater than 90%, the induction regimen was highly efficacious as remission-inducing therapy. This large database has validated several previously identified independent prognostic factors in ALL, such as age, white blood cell count at presentation, cytogenetics, and immunophenotype. However, the achievement of CR within 4 weeks does not appear to be an independent prognostic factor.

Clinical effects and P-glycoprotein inhibition in patients with acute myeloid leukemia treated with zosuquidar trihydrochloride, daunorubicin and cytarabine.

BACKGROUND AND OBJECTIVES: P-glycoprotein (P-gp) is a major cause of multidrug resistance (MDR) in acute myelogenous leukemia (AML) and is thought to contribute to the failure of chemotherapy. Zosuquidar trihydochloride (Z.3HCL) is a potent and selective inhibitor of P-gp which rapidly and effectively inhibits drug efflux. DESIGN AND METHODS: The aim of this study was to evaluate the clinical effects of Z.3HCL and determine its influence on P-gp activity. Sixteen AML patients were entered into a phase 1 dose ranging clinical trial of Z.3HCL, co-administered intravenously with daunorubicin and cytosine arabinoside (ARA-C). Clinical outcomes, toxicity abd adverse events were assessed. P-gp function was analyzed by flow cytometry. In vitro cytotoxicity was studied using the MTT assay. RESULTS: Eleven patients achieved a complete remission and one a partial remission with a median survival of 559 (range 38-906) days. Non-hematologic grade 3 and 4 toxicities were seen in 4 patients. Z.3HCL infusion was associated with rapid inhibition of Rh123 efflux in CD56+ cells in 16/16 patients and in CD33+ cells from 6/10 patients. The median inhibition was 95% for CD56+ cells and 85.25% for CD33+ cells was significantly elevated in 6/16 patients. The median IC50, using a MTT assay for daunorubicin, decreased significantly between Z.3HCL modulated and unmodulated cells (n=11,153 and 247 ng/mL respectively, p=0.01). INTERPRETATION AND CONCLUSIONS: The modulator Z.3HCL is a specific inhibitor of P-gp efflux and can be given safely to patients with AML in combination with induction doses of conventional cytotoxic drugs.

A strategy for treatment of Epstein-Barr virus-positive Hodgkin's disease by targeting interleukin 12 to the tumor environment using tumor antigen-specific T cells.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T cells (CTL) is effective for the prophylaxis and treatment of EBV-induced lymphoma in hematopoietic stem cell recipients. However, in EBV-positive Hodgkin's disease (HD) the efficacy of adoptively transferred EBV-specific CTL may be limited by tumor-derived immunosuppressive factors, such as T-cell growth factor (TGF) beta, interleukin (IL)13 and the chemokine TARC. Local delivery of IL12 to tumor sites by tumor-specific CTL could provide direct antitumor effects and overcome the CTL-inhibitory effects of the Th2 tumor environment while avoiding the systemic toxicity of recombinant IL12. EBV-specific CTL transduced with a retrovirus vector expressing the p40 and p35 subunits of IL12 as a single molecule (Flexi-IL12), produced IL12 following antigenic stimulation. This resulted in an elevated production of Th1 cytokines, including interferon gamma and tumor necrosis factor alpha, and a reduction in the Th2 cytokines IL4 and IL5. Flexi-IL12-transduced CTL resisted the antiproliferative and anticytotoxic effects of exogenous TGFbeta, likely by antagonizing the TGFbeta-induced downregulation of the Th1 transcriptional factor T-bet. In addition, Flexi-IL12-transduced CTL demonstrated a proliferative advantage in the presence of inhibitory supernatants from HD-derived cell lines. Tumor-specific, Flexi-IL12-transduced EBV-specific CTL should have a functional advantage over unmodified CTL, particularly in the presence of the adverse Th2 cytokine environment produced by Hodgkin tumor cells.

Fludarabine, cytosine arabinoside, granulocyte-colony stimulating factor with or without idarubicin in the treatment of high risk acute leukaemia or myelodysplastic syndromes.

The combination of fludarabine (FDR), high dose cytarabine and granulocyte colony stimulating factor (FLAG) with or without idarubicin (Ida) was used in the treatment of poor risk acute leukaemia or myelodysplastic syndrome (MDS) in a single centre experience. A total of 105 patients were treated over a 4-year period with 59% achieving a complete remission (CR); no statistical difference observed between FLAG and FLAG-Ida. For patients responding to FLAG +/- Ida, the median event-free survival (EFS) was 11 months and 23% at 5 years. Such patients proceeded either to further chemotherapy or a haematopoietic stem cell transplant (HSCT). The median EFS (13 months vs. 8 months) and projected 5-year survival (37% vs. 13%) of patients undergoing HSCT was significantly better than those who did not (P = 0.021). In all, 14 of 72 patients remain alive in continuing CR (median duration 43 months) with 10 of 31 having had a HSCT vs. four of 41 that did not (P = 0.033). Both regimens were well tolerated, with the majority of patients experiencing grade 1 or less non-haematological toxicity (mainly nausea and vomiting). The median time to neutrophil and platelet recovery was 28 and 31 d, respectively. No significant differences were seen with the addition of ida. There was a 17% incidence of treatment-related deaths, of which 39% was caused by invasive aspergillus infection. The results show that FLAG +/- Ida is an effective and well-tolerated remission induction regimen for poor risk leukaemia and MDS.

Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.

Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.

Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand.

We have studied the actions of tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super-additive apoptosis induction in approximately half of the isolates. Molecular evidence of super-additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super-additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro-apoptotic form of the BCL-2 family member BID. Our data suggest that co-administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.

The degree of matching at HLA-DPB1 predicts for acute graft-versus-host disease and disease relapse following haematopoietic stem cell transplantation.

The importance of matching for HLA-DPB1 in unrelated donor haematopoietic stem cell (HSC) transplantation is little understood. Most transplant centres do not, currently, prospectively match for DPB1, but emerging data show that DPB1 matching does play a role in determining outcome. We studied the impact of HLA-DPB1 matching on outcome in 143 recipients of T-cell depletion transplants, who matched with their respective unrelated donors (allelic level) at HLA-A, -B, -C, -DRB1 and -DQB1. Of those matched at DPB1, 47.2% (17/36) developed acute graft-versus-host disease (aGvHD) as compared to 66.3% (55/83) of those who were mismatched. This led to a 19.1% (95% CI 0.1-38.3%) increase in the chance of developing aGvHD in mismatched patients (P=0.049). Relapse of the original disease occurred in 51 recipients; 23 of 37 (62%) matched at both DPB1 alleles, 28 of 82 (34%) were mismatched at one or two DPB1 alleles. Thus, there was a significantly higher relapse rate (P=0.0011) in transplant recipients who matched at both DPB1 alleles. In conclusion, a donor/recipient DPB1 match was associated with a significantly lower incidence of aGvHD and a significantly higher incidence of disease relapse. This study provides further evidence for an immunogenic role of HLA-DPB1 in HSC transplants.

Expression of co-stimulatory molecules on acute myeloid leukaemia blasts may effect duration of first remission.

Many solid tumours have been shown to lack expression of either of the immune co-stimulatory molecules CD80 (B7.1) or CD86 (B7.2), which is thought to be one of the ways in which tumours may escape immune recognition. We have examined the surface expression of CD80, CD86, human leucocyte antigen (HLA) class I and II, CD11a, CD54, and CD58 on the blast cells from patients with acute myeloid leukaemia (AML) at presentation. CD80 was only rarely expressed on AML blasts and, in those leukaemic cells expressing CD80, the level of expression was low. In contrast, expression of CD86 was detected on the AML blasts in more than half of the samples tested and, in some cases, the level of expression was equivalent to that of mature monocytes and activated B lymphocytes. The percentage of leukaemic blasts expressing CD86 was higher in the M4 and M5 French-American-British (FAB) types, and expression of CD11a and HLA class II was higher in the M4 FAB type. There was no difference in expression of CD80, CD54, CD58, or HLA Class I between different FAB subgroups. There was no significant difference in duration of first remission with expression of CD80, CD86, CD11a, CD54 or HLA class II. However, when expression of CD80 and CD86 were considered together, a significantly longer duration of remission was found. We suggest that these molecules may play a role in immunosurveillance, resulting in prolonged remission in some patients treated for AML.

Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis.

Activation of the phosphatidylinositol 3- kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (CLL) cells. Here we report experiments that identify albumin as the major component of plasma that blocks CLL cell killing by chlorambucil or radiation. Intact plasma depleted of albumin by chromatography on Cibacron blue-Sepharose or plasma from a subject with analbuminemia failed either to activate AKT or to protect CLL cells from chlorambucil-induced apoptosis. Both functions were restored by re-addition of albumin. The protective action of albumin as well as AKT activation was compromised by the binding of lipids. Fluorescence-activated cell sorter (FACScan) analysis demonstrated the uptake of fluoresceinated albumin by CLL cells. Accumulation of albumin in intracellular vesicles was also shown by confocal microscopy. Indirect inhibition of AKT activation by the phosphatidylinositol 3-kinase inhibitor LY294002 reversed the blockade of chlorambucil-induced killing by plasma albumin. The data suggest that activation of AKT consequent to binding of albumin by CLL cells blocks chlorambucil- and radiation-induced apoptosis. Strategies designed to block albumin-induced antiapoptotic signaling may, therefore, be of value in enhancing cytotoxic drug action on CLL cells.

Comparison of outcome following allogeneic bone marrow transplantation with cyclophosphamide-total body irradiation versus busulphan-cyclophosphamide conditioning regimens for acute myelogenous leukaemia in first remission.

We evaluated transplant-related mortality (TRM), leukaemia relapse, leukaemia-free survival (LFS) and overall survival (OS) in patients receiving busulphan and cyclophosphamide (BuCy) or cyclophosphamide and total body irradiation (CyTBI) prior to allogeneic bone marrow transplantation (BMT) for acute myelogenous leukaemia (AML) in first remission. Outcomes of 381 human leucocyte antigen (HLA)-matched sibling transplants using BuCy were compared with 200 transplants using CyTBI performed between 1988 and 1996. The incidence of hepatic veno-occlusive disease was higher with BuCy (13%) than with CyTBI (6%) (P = 0.009). Risks of acute and chronic GVHD were similar. In multivariate analysis, relapse risk was higher in the BuCy group [relative risk (RR) = 1.72; 95% confidence interval (CI), 1.05-2.81; P = 0.031]. Eleven of 373 evaluable patients in the BuCy group had a central nervous system relapse in contrast to none of 194 evaluable patients in the CyTBI group (P = 0.016). There were no differences in TRM, LFS and OS. CyTBI conditioning may lower relapse risk but produces comparable TRM, LFS and OS to BuCy for HLA-matched sibling transplantation in first remission AML.

Polymerase chain reaction (PCR)- and reverse transcription PCR-based minimal residual disease detection in long-term follow-up of childhood acute lymphoblastic leukaemia.

Minimal residual disease (MRD) was investigated in 52 children with acute lymphoblastic leukaemia (ALL), using antigen receptor gene rearrangements and reverse transcription polymerase chain reaction for fusion transcripts as molecular targets. Patients [treated according to the Medical Research Council United Kingdom ALL (MRC UKALL) XI protocol or Total XI and XIII protocols] were monitored for a median period of 45 months (range, 9-110 months). Among 17 patients who relapsed, MRD persisted for longer (66.7%, 47.1%, 53.8% and 41.7% at 0-2, 3-5, 6-9, 10-24 months respectively) than patients who remained in continuous clinical and immunological remission (n = 35) (27.3%, 11.1%, 4.3%, 8.0%). Association between MRD tests and outcome was assessed and found to be significant at all time-points. The difference in survival for MRD-positive and MRD-negative patients (using the log-rank test) was statistically significant at all time intervals, as was risk of relapse for MRD-positive patients (1.89, 2.20, 2.65 and 2.16) and MRD-negative patients (0.72, 0.82, 0.65 and 0.70). Sixteen of the 52 patients had an oligoclonal pattern at presentation but oligoclonality did not have an impact on outcome. Cox regression analysis revealed that MRD assessment is an independent and prognostically significant factor during treatment and should be used for patients' stratification in future studies.

Allogeneic stem cell transplantation in the myelodysplastic syndromes: interim results of outcome following reduced-intensity conditioning compared with standard preparative regimens.

Conventional allogeneic stem cell transplantation (SCT) for myelodysplastic syndrome (MDS) is associated with excessive procedure-related mortality. The outcome following volunteer-unrelated donor (VUD) or sibling allogeneic SCT was therefore evaluated in 23 MDS patients conditioned with reduced-intensity regimens (fludarabine/busulphan/Campath-1H) because of advanced age (48 vs 37 years, P = 0.002) and/or co-morbidity (19 vs 3, P < 0.0001) which precluded conventional transplantation, and compared with 29 treated with standard protocols [busulphan/cyclophosphamide (Bu/Cy); Bu/Cy/total-body irradiation/Campath-1G]. Graft-versus-host disease (GVHD) prophylaxis comprised of cyclosporine/methotrexate. One hundred per cent donor engraftment (variable number tandem repeat analysis/cytogenetics/fluorescence in situ hybridization) was achieved in 18/19 (95%) evaluable patients receiving reduced-intensity regimens, although six (32%) have subsequently shown mixed chimaerism. Reduced-intensity conditioning was associated with significantly reduced duration of aplasia, less mucositis, fever, antibiotic, analgesia, parenteral nutrition use, less acute and chronic GVHD, and lower early procedure-related mortality [two (9%) vs nine (31%), P < 0.05]. Six patients relapsed (two standard, four reduced-intensity) and two (reduced-intensity) experienced late graft failure. The 2 year actuarial overall/disease-free survival (OS/DFS) was 48/39% in the reduced-intensity arm and 44/44% in the standard group. The 2 year non-relapse mortality was 31% and 50% respectively. In VUD recipients, OS was superior in the reduced-intensity arm (49%vs 34%). Predictors of DFS included good/intermediate-risk karyotype, low/intermediate-1 International Prognostic Scoring system score, human leucocyte antigen compatibility and attainment of complete remission. Our data demonstrates that VUD or sibling allogeneic SCT following reduced-intensity conditioning is feasible in high-risk MDS patients considered unsuitable for standard transplantation and is associated with comparable 3.5 year DFS to those receiving conventional regimens.

Thrombotic thrombocytopenic purpura after allogeneic stem cell transplantation: a survey of the European Group for Blood and Marrow Transplantation (EBMT).

A survey was carried out among the European Group for Blood and Marrow Transplantation (EBMT) centres to determine the incidence, risk factors, treatment and outcome of thrombotic thrombocytopenic purpura (TTP) following allogeneic haematopoietic stem cell transplantation. TTP was defined as the simultaneous occurrence of red cell fragmentation, laboratory findings of haemolysis, red cell transfusion requirement and de novo or persistent thrombocytopenia caused by consumption, in the absence of disseminated intravascular coagulation. Forty-five centres reported all patients (n = 406) transplanted between July and December 1996. Twenty-three patients developed TTP; the risk of developing TTP was 6.7% at 2 years (95% CI: 4.1% to 9.3%). The median time of onset was 44 d (range 13-319) post transplantation. Significant risk factors for the development of TTP were female gender (P = 0.005) and an unrelated donor (P = 0.046). To treat TTP, cyclosporin administration was discontinued in 10 cases, plasma exchanges were performed in five cases and 12 patients received plasma infusions without plasma exchange. TTP resolved in 13 of the 23 patients (57%). The only factor predictive of resolution of TTP was the absence of nephropathy. Seven patients (30%) were alive at follow-up of 38-45 months from the onset of TTP. Sixteen patients died; the causes were multiple, only three patients had TTP as a central factor. The median time to death was 41 d (range 1-762 d) from the onset of TTP. TTP is a relatively frequent complication of allogeneic stem cell transplantation and it is associated with high mortality, though death is usually caused by multiple factors.

A novel treatment approach for low grade lymphoproliferative disorders using PKC412 (CGP41251), an inhibitor of protein kinase C.

INTRODUCTION: PKC412 (formally CGP41251) selectively inhibits protein kinase C (PKC) isoforms and has been shown to be cytotoxic to malignant cells in vitro. We have undertaken a single centre, open-label, multi-dose, exploratory Phase II clinical trial of PKC412 in patients with CLL and low grade NHL. METHODS: Thirteen CLL patients and eight stage IV NHL patients were treated at three oral dose levels of 25, 150 and 225 mg/day for 14 days. RESULTS: There was a median decrease of 29.4% in the lymphocyte count in 11 out of 18 patients with circulating disease following treatment. Two NHL patients without circulating disease showed loss of immunophenotypic evidence of marrow disease and a third showed an improvement in blood counts and transfusion requirements. Adverse events were mostly gastrointestinal (16 patients) requiring little or no intervention. In nine patients there was an asymptomatic rise in serum amylase and/or transaminases. Asymptomatic hyperglycemia was also observed in eight patients. All returned to normal following cessation of treatment. In 14 out of 20 patients total PKC activity measured in peripheral blood and/or bone marrow lymphocytes was reduced during treatment to a mean of 54% of pre-treatment level. CONCLUSION: PKC412 is safe, well tolerated and reduces the tumor load in chronic B-cell malignancies. Inhibition of PKC offers a novel approach to the chemotherapy of B-cell malignancies.

Alloantigen-specific T-cell depletion in a major histocompatibility complex fully mismatched murine model provides effective graft-versus-host disease prophylaxis in the presence of lymphoid engraftment.

Graft-versus-host disease (GvHD) is a multistep immune process involving lymphocyte activation, proliferation and target cell killing. We have devised a novel method for the selective depletion of alloreactive cells from haematopoietic stem cell grafts which retains a pool of immunocompetent lymphocytes possessing antiviral activity with the potential to hasten immune reconstitution. The method is based upon the expression of the activation antigen CD69 on responding donor lymphocytes in a cytokine-modified mixed lymphocyte culture (mMLC) and depletion of these cells by paramagnetic bead sorting. We have previously demonstrated the in vitro efficacy of this system for the removal of alloreactive cells in both human leucocyte antigen-mismatched and -matched settings. Here, we describe a non-obese diabetic/severe combined immunodeficient murine model of aggressive GvHD in which we have tested its in vivo efficacy. Murine recipients of infusions of non-manipulated major histocompatibility complex class I and class II mismatched donor T cells suffered rapid onset of acute, and generally fatal, GvHD. This model is akin to aggressive clinical transfusion-related GvHD. Recipients of lymphocyte infusions depleted of CD69+ alloreactive donor cells ex vivo and monitored for 10 weeks post infusion demonstrated significantly improved survival (71.4% compared with 12.5% in the non-manipulated group) and the absence of clinical GvHD despite the presence of circulating donor lymphocytes.

Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells.

Although it has been known for more than 40 years that allogeneic immune responses cure leukaemias after bone marrow transplantation, autologous leukaemia-specific immunity remains controversial and its impact upon survival has not been established. Here we have tested 25 patients with de novo acute leukaemias, while in remission at completion of their anti-leukaemia therapy, for evidence of autologous cytolytic immunity to their leukaemic cells taken and cryopreserved at disease presentation. We have measured this degree of cell-mediated cytotoxicity in vitro and termed it "leukaemia cytolytic activity" (LCA). Patients whose disease ultimately relapsed had significantly lower LCA than those who remained in remission beyond 2 years (P < 0.001); the absence of LCA when in remission predicted subsequent relapse within 2 years with a sensitivity of 100% and specificity of 77%. LCA was mediated in vitro by CD56+/CD8alpha+/CD3- natural killer cells. We propose that it is this immune response, rather than the chemotherapy per se, which is responsible for continued remission and that measurement of LCA in patients at completion of therapy may be used as an indicator of risk of subsequent relapse. Patients lacking this response will require further treatment, either with an allogeneic donor transplant or an alternative immunotherapeutic strategy.

Mycophenolate mofetil for the treatment of refractory auto-immune haemolytic anaemia and auto-immune thrombocytopenia purpura.

The treatment of both auto-immune haemolytic anaemia (AIHA) and auto-immune thromobocytopenic purpura (AITP) remains unsatisfactory in those refractory to first-line management. Mycophenolate mofetil (MMF) is an immunosuppressive agent originally used to prevent acute rejection of solid organ transplants and used more recently in the management of auto-immune conditions. We report its use in four patients with AIHA and six patients with AITP. All four patients with AIHA and five of the six patients with AITP showed a complete or good partial response to treatment with MMF, confirming a possible role in the treatment of these conditions.