Laurence Pearce

Outcomes of alemtuzumab-based reduced intensity conditioning stem cell transplantation using unrelated donors for myelodysplastic syndromes

This prospective study evaluated the outcomes of 75 successive patients receiving a FBC (fludarabine, busulphan, alemtuzumab) reduced‐intensity conditioning (RIC) regimen for myelodysplastic syndromes (MDS) using volunteer unrelated donors(VUD). The prognostic significance of a variety of clinical variables including the recently described haematopoietic cell transplantation co‐morbidity index (HCT‐CI) was assessed. The median age of the cohort was 52·0 years (range: 19–68 years) with a median follow‐up of 1038·5 d. Forty‐nine patients (65%) had an International Prognostic Scoring System stage of ≥Intermediate‐2, 35 (46%) had intermediate or poor risk cytogenetics, and 23 patients(31%) were human leucocyte antigen‐mismatched. The actuarial 3‐year overall survival (OS) and disease‐free survival (DFS) was 43% [95% confidence interval (CI): 37–49] and 41% (95%CI: 35–47) respectively, and the cumulative incidence of extensive chronic graft‐versus‐host disease was 22%. On multivariate analysis, presence of either one class II mismatch or a two‐antigen mismatch adversely influenced transplant‐related mortality, DFS and OS. In addition, disease status at transplantation and the haematopoietic cell transplantation‐specific comorbidity index were independent variables for overall survival. In contrast, both advanced age and pre‐transplant cytogenetic status did not significantly affect overall outcomes. RIC regimens using VUD was associated with durable long‐term survival even in older patients with MDS, and the use of a pre‐transplant comorbidity index may help to improve patient selection for transplantation.

Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells.

Interactions in the tumour microenvironment can promote chronic lymphocytic leukaemia (CLL) cell survival, proliferation and drug resistance. A detailed comparison of three co‐culture systems designed to mimic the CLL lymph node and vascular microenvironments were performed; two were mouse fibroblast cell lines transfected with human CD40LG or CD31 and the third was a human microvascular endothelial cell line, HMEC‐1. All three co‐culture systems markedly enhanced CLL cell survival and induced a consistent change in CLL cell phenotype, characterized by increased expression of CD38, CD69, CD44 and ITGA4 (CD49d); this phenotype was absent following co‐culture on untransfected mouse fibroblasts. In contrast to HMEC‐1 cells, the CD40LG and CD31‐expressing fibroblasts also induced ZAP70 expression and marked CLL cell proliferation as evidenced by carboxyfluorescein succinimidyl ester labelling and increased Ki‐67 expression. Taken together, our data show that co‐culture on different stroma induced a remarkably similar activation phenotype in CLL cells but only the CD40LG and CD31‐expressing fibroblasts increased ZAP70 expression and CLL cell proliferation, indicating that ZAP70 may play a critical role in this process. This comparative study reveals a number of striking similarities between the co‐culture systems tested but also highlights important differences that should be considered when selecting which system to use for in‐vitro investigations.

Delayed attainment of full donor chimaerism following alemtuzumab-based reduced-intensity conditioning haematopoeitic stem cell transplantation for acute myeloid leukaemia and myelodysplastic syndromes is associated with improved outcomes

This prospective study evaluated the kinetics of lymphoid (CD3) engraftment in 110 patients with acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) after allogeneic transplantation and conditioning with fludarabine, busulphan and alemtuzumab, using ciclosporin for post‐transplant immunosuppression. Declining donor CD3 chimaerism beyond day+100 was treated with pre‐emptive donor lymphocyte infusion (pDLI). The median age of patients was 53·0 years (range: 19–72 years), and the median follow‐up was 690 d (range:168–1470 d). Patients achieving full CD3 donor chimaerism (FDC, n = 46) by day+100 had a significantly inferior disease‐free survival (DFS) and overall survival (OS) compared to patients with mixed donor chimaerism (MDC, n = 59). Twenty patients had stable MDC and did not require pDLI. Patients attaining early FDC had a higher transplant‐related mortality compared to those who maintained stable levels of MDC (P = 0·02), with no difference between the FDC and pDLI groups (P = 0·07). There was no difference in relapse between all three groups (P = 0·21). On multivariate analysis, only CD3 chimaerism status at day+100 and disease status at transplantation had a significant effect on DFS and OS. In patients with AML/MDS undergoing alemetuzumab based‐RIC HSCT, prolonged MDC beyond day+100 is associated with an improved OS. Future studies need to be directed towards establishing the underlying factors that dictate T‐cell engraftment, expansion and homing post‐transplantation.

Expression of AML1-ETO in human myelomonocytic cells selectively inhibits granulocytic differentiation and promotes their self-renewal

The t(8;21) translocation is one of the most frequent translocations in acute myeloid leukaemia (AML), giving rise to the AML1-ETO fusion protein (or RUNX1-CBF2T1). This abnormality is associated with myelocytic leukaemia with dysplastic granulopoiesis. Here, we demonstrate that when expressed in a normal human (CD34+) progenitor population, AML1-ETO selectively inhibits granulocyte colony formation but not monocyte colony formation. In bulk liquid culture, we found that though AML1-ETO transiently inhibited the proliferation of CD34+ cells, it promoted long-term growth of myeloid cells for more than 80 days, suggesting that differentiation was inhibited. In support of this, cultures expressing AML1-ETO demonstrated enhanced retention of colony-forming capacity. Phenotypic examination of AML1-ETO cultures revealed a defect in granulocytic differentiation in terms of retention of CD34+ cells within the culture and delayed CD11b upregulation. Morphologically, granulocyte terminal differentiation in AML1-ETO-expressing cells was inhibited by 83±5%, giving rise to a build-up of early to intermediate granulocytes that exhibited a number of morphological features associated with t(8;21) leukaemias. In contrast, AML1-ETO had little or no effect on monocytic differentiation. Taken together, these results suggest that expression of AML1-ETO selectively inhibits the differentiation of granulocytic cells and promoted extensive self-renewal, supporting a causal role for t(8;21) translocations in leukaemogenesis.

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Graft-versus-host disease (GVHD) remains the downside of the graft-versus-leukemia effect following allogeneic stem cell transplantation. This investigation of 25 patients with myeloid malignancies treated with reduced intensity conditioning and with GVHD prophylaxis, has focused on cell populations and their correlation with outcome. Interesting imbalances of effector and regulatory CD4+ T cells were detected, which, if confirmed in larger cohorts, should provide insight into how the immunological storms accompanying allogeneic stem cell transplantation can be harnessed and weathered. Background A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care. Design and Methods Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease. Results Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO+ CD27−) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25high Foxp3+) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease. Conclusions Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.

Two novel aspirin analogues show selective cytotoxicity in primary chronic lymphocytic leukaemia cells that is associated with dual inhibition of Rel A and COX-2

Objectives:  Non‐steroidal anti‐inflammatory drugs have been shown to induce apoptosis in primary B‐cell chronic lymphocytic leukaemia (CLL) cells, but the molecular mechanisms that underpin this observation have not been fully elucidated. Here, we have analysed the effect two novel aspirin analogues, 2‐hydroxy benzoate zinc (2HBZ) and 4‐hydroxy benzoate zinc (4HBZ), on primary CLL samples.

Chimerism does not predict for outcome after alemtuzumab-based conditioning: lineage-specific analysis of chimerism of specific diseases may be more informative.

Chimerism does not predict for outcome after alemtuzumab-based conditioning: lineage-specific analysis of chimerism of specific diseases may be more informative

Rapid recovery of lymphocyte subsets is not associated with protection from relapse of myelodysplastic syndromes and acute myeloid leukaemia after haematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab.

Graft‐versus‐leukaemia (GvL) and graft‐versus‐host disease (GvHD) are both caused by alloreactive lymphocytes. We previously reported that GvHD correlated with higher numbers of effector CD4 T cells and Natural Killer cells early after allogeneic transplantation using a regimen comprising fludarabine, busulphan and alemtuzumab. Here, we assessed immune cell subset recovery in these patients in the context of early myeloid malignant disease relapse. Despite the close relationship between the GvL and GvHD immune responses, rapid recovery of lymphocyte subsets was not associated with protection from disease relapse. These results indicated that GvL may be weak in this treatment setting for patients with myelodysplastic syndromes and acute myeloid leukaemia. Consistent with low GvL activity, we previously reported that mixed T cell chimaerism had no detrimental effect on relapse in this treatment setting and instead correlated with better outcome because of reduced GvHD incidence. We now report that patients with significantly higher lymphocyte numbers prior to transplantation subsequently maintained the mixed T cell chimaeric state. This pre‐transplant profile, together with absence of the early post‐transplant signature indicative of GvHD predisposition, could potentially be used to identify patients suitable for early withdrawal of immunosuppression and prophylactic donor leucocyte infusion to boost GvL activity.

Mixed donor chimaerism in recipient fingernails following reduced-intensity conditioning haematopoietic SCT

Mixed donor chimaerism in recipient fingernails following reduced-intensity conditioning haematopoietic SCT

Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%±8.5% and the mean cell viability 74%±17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.