Charles Craddock

Guidelines on the management of acute myeloid leukaemia in adults

1 Bone marrow aspirate and trephine biopsy unless the peripheral blast count is high. 2 Immunophenotyping [CD3, CD7, CD13, CD14, CD33, CD34, CD64, CD117 and cytoplasmic myeloperoxidase (MPO)]. 3 Cytochemistry (MPO or Sudan Black, combined esterase). Can be omitted if four-colour flow cytometry is available. 4 Cytogenetics [with reverse-transcription polymerase chain reaction (RT-PCR) for AML 1-ETO and CBFBMYH11 in non-acute promyelocytic leukaemia (APL) and promyelocytic leukaemia (PML) and retinoic acid receptor-alpha (RARA) in suspected APL; fluorescent in situ hybridisation (FISH) in selected cases].

Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia.

Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5-azacitidine and sodium valproate (VAL–AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL–AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

Rituximab is effective in the management of refractory autoimmune cytopenias occurring after allogeneic stem cell transplantation

Summary:Autoimmune haemolytic anaemia (AIHA), immune thrombocytopenia (ITP) and autoimmune neutropenia (AIN) are well-recognised complications of allogeneic stem cell transplantation (SCT), but have previously only been reported in the context of myeloablative conditioning regimens. Management of AIHA, ITP or AIN occurring after allogeneic SCT is unsatisfactory since they frequently prove refractory to conventional therapies including splenectomy. As a consequence, autoimmune cytopenias occurring after allogeneic SCT are associated with substantial morbidity and mortality. We report four patients who developed AIHA or ITP after allogeneic transplantation – three of which occurred after a reduced-intensity conditioning (RIC) regimen. All patients demonstrated a complete response to Rituximab, having failed to respond to conventional treatment including high-dose prednisolone and intravenous immunoglobulin (IVIg). We conclude that Rituximab can be a valuable agent in the management of autoimmune cytopenias occurring after allogeneic SCT and that autoimmune cytopenias may be a hitherto unrecognised complication of RIC regimens.

Factors predicting long-term survival after T-cell depleted reduced intensity allogeneic stem cell transplantation for acute myeloid leukemia

Background Reduced intensity conditioning regimens permit the delivery of a potentially curative graft-versus-leukemia effect in older patients with acute myeloid leukemia. Although T-cell depletion is increasingly used to reduce the risk of graft-versus-host disease its impact on the graft-versus-leukemia effect and long-term outcome post-transplant is unknown. Design and Methods We have characterized pre- and post-transplant factors determining overall survival in 168 patients with acute myeloid leukemia transplanted using an alemtuzumab based reduced intensity conditioning regimen with a median duration of follow-up of 37 months. Results The 3-year overall survival for patients transplanted in CR1 or CR2/CR3 was 50% (95% CI, 38% to 62%) and 44% (95% CI, 31% to 56%), respectively compared to 15% (95% CI, 2% to 36%) for patients with relapsed/refractory disease. Multivariate analysis demonstrated that both survival and disease relapse were influenced by status at transplant (P=0.008) and presentation cytogenetics (P=0.01). Increased exposure to cyclosporine A (CsA) in the first 21 days post-transplant was associated with an increased relapse risk (P<0.0001) and decreased overall survival (P<0.0001). Conclusions Disease stage, presentation karyotype and post-transplant CsA exposure are important predictors of outcome in patients undergoing a T-cell depleted reduced intensity conditioning allograft for acute myeloid leukemia. These data confirm the presence of a potent graft-versus-leukemia effect after a T-cell depleted reduced intensity conditioning allograft in acute myeloid leukemia and identify CsA exposure as a manipulable determinant of outcome in this setting.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Optimizing the use of cyclosporin in allogeneic stem cell transplantation

Cyclosporin remains the most widely used immunosuppressive agent in patients undergoing allogeneic stem cell transplantation (SCT). The increased awareness of the impact of the intensity of post-transplant immunosuppression on determining outcome after allogeneic SCT has resulted in a re-examination of whether cyclosporin is currently being optimally used in this population of patients. Recent studies in solid organ transplantation have questioned whether the use of trough levels provides the most accurate reflection of the immunosuppressive actions of cyclosporin and alternative strategies to monitor cyclosporin dosage after liver and kidney transplantation are increasingly being used. As a result there is now interest in examining whether there is scope for translating these advances into the arena of haematopoietic transplantation. In this paper, we will review the rationale underlying the current schedules for dosing and monitoring cyclosporin after allogeneic SCT and identify specific areas in which the use of cyclosporin requires re-evaluation. These include evaluation of whether patient outcome would be improved by using peak cyclosporin levels to determine dosing schedules, analysis of optimal cyclosporin dosing schedules in patients undergoing reduced intensity allografts and investigation of surrogate markers of cyclosporins immunosuppressive activity.

Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage.

Quek and colleagues identify human leukemic stem cells (LSCs) present in CD34− AML. In-depth characterization of the functional and clonal aspects of CD34− LSCs indicates that most are similar to myeloid precursors.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event.

Allogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

Prediction of intravenous cyclosporine area under the concentration-time curve after allogeneic stem cell transplantation

Currently, routine monitoring of cyclosporine in patients undergoing allogeneic stem cell transplantation is based on analysis of trough, or C0, predose concentrations. However, recent studies in solid organ transplant recipients have demonstrated that monitoring cyclosporine exposure by analyzing 2-hour postdose concentrations (C2) or area under the concentration-time curve (AUC) may improve clinical outcome. This study investigated the ability of single samples to predict exposure to intravenous cyclosporine in eight patients undergoing allogeneic stem cell transplantation. Patients received cyclosporine at a starting dose of 2.5 mg/kg 12-hourly by intravenous infusion over 4 hours. Blood samples were taken at 0, 1, 2, 3, 4, 4.17, 4.33, 4.67, 5, 6, 8, and 12 hours after the start of the infusion. Linear regression was undertaken to investigate the relationship between AUC and concentrations measured at individual time points; bias and precision were also examined. Cyclosporine doses ranged from 250 mg to 430 mg/day, AUC from 3.85 to 8.39 mg·h/L, clearance from 19.1 to 48.1 L/h, and elimination half-life from 3.7 to 15.5 hours. Although Cmax and the concentration measured at 3 hours (C3) provided the best prediction of AUC (r2 = 0.90 and r2 = 0.87, respectively), the infusion protocol made the time of Cmax difficult to predict. Concentrations measured at the end of the infusion (Cend) and 12 hours postdose (C12) gave similar results (r2 = 0.87 and 0.77, respectively). These data suggest that C12 concentrations provide an acceptable marker of total exposure to intravenous cyclosporine in patients undergoing allogeneic stem cell transplantation.

High-hyperdiploidy in Philadelphia positive adult acute lymphoblastic leukaemia: case-series and review of literature.

Summary:Patients with Philadelphia positive (Ph+) adult acute lymphoblastic leukaemia (ALL) have a poor prognosis. Stem cell transplantation (SCT) is increasingly being recognised as the treatment of choice in eligible patients with Ph+ALL, but disease-relapse remains a problem in a proportion of patients prior to and after SCT. Genetic abnormalities in addition to the Ph chromosome may influence the biology and clinical course of ALL, but there are not many studies on the potential genetic heterogeneity of adult Ph+ALL and clinical outcomes. Here, we report on five patients with ALL who were double Ph+ and also had a high-hyperdiploid karyotype (Ph+/hyperdiploid) at diagnosis. In contrast to the presence of the Ph+ chromosome, high-hyperdiploidy (>50 chromosomes) as the sole karyotypic abnormality in ALL is associated with a favourable clinical outcome. In our series, four patients with a Ph+/hyperdiploid karyotype achieved a cytogenetic remission after induction chemotherapy and proceeded to stem cell transplantation (SCT). The fifth had five subclones including Ph+/hyperdiploid, as well as those that were only Ph+; the latter emerged as the dominant clone after induction therapy and the patient died of disease-relapse. Of the patients who underwent SCT, only one relapsed, but achieved a durable remission with donor lymphocyte infusions. Thus, it is conceivable that the presence of high-hyperdiploidy as an additional karyotypic abnormality may confer a better prognosis to Ph+ALL, presumably by altering the kinetics of Ph+ neoplastic cells. We have discussed these results in the context of recent studies on the significance of high-hyperdiploidy in Ph+ adult ALL.