Involvement of caspase-8 in chemotherapy-induced apoptosis of patient derived leukemia cell lines independent of the death receptor pathway and downstream from mitochondria.

Resistance of leukemic cells to chemotherapy frequently occurs in patients with acute leukemia, which may be caused by alterations in common apoptotic pathways. Controversy exists whether cytostatic agents induce the mitochondrial or death receptor pathway of apoptosis. In the mitochondrial pathway cytochrome C release and caspase-9 activation play a central role in the induction of apoptosis, while formation of a Death Inducing Signaling Complex (DISC) and caspase-8 activation have been reported to be essential in death receptor-induced apoptosis. Here, we show in human derived myeloid and lymphoblastic leukemia cell lines that caspase-8 plays a more important role than previously expected in apoptosis mediated via the mitochondrial pathway. We demonstrated in these malignant cells chemotherapy-induced apoptosis independent of the death receptor pathway, since blocking this pathway using a retroviral construct encoding Flice inhibitory protein (FLIP) did not inhibit drug-induced apoptosis or caspase-8 activation, while overexpression of Bcl-2 completely inhibited both events. Furthermore, we showed that activation of caspase-8 by cytostatic agents occurred downstream from mitochondria. Since caspase-8 plays a central role in both death receptor- and chemotherapy-induced apoptosis of malignant cells from patients with acute leukemia, therapeutic strategies focusing at modulation and activation of caspase-8 may be successful in the treatment of drug-resistant malignancies.

Long-term follow-up of myeloablative allogeneic stem cell transplantation using Campath "in the bag" as T-cell depletion: the Leiden experience.

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT) but can be prevented by removing T-lymphocytes from the graft. Campath (anti-CD52) antibodies have been widely used in vivo for T-cell depletion following conventional and reduced intensity conditioning regimens. The use of Campath in vivo was associated with a significant reduction in GVHD but at the cost of impaired immune reconstitution. We evaluated the long-term outcome of 73 myeloablative allogeneic stem cell transplants with HLA-identical sibling donors using Campath "in the bag" as method of in vitro T-cell depletion. All patients engrafted and hematopoietic recovery was uneventful, resulting in a median of 99% donor chimerism at 3 months after alloSCT. Cytomegalovirus (CMV) reactivation occurred in 53% of the patients. No CMV disease was observed probably as a result of pre-emptive (val)ganciclovir treatment. The incidence of aGVHD was low (22% grade II). No grades III-IV aGVHD was observed and extensive chronic GVHD (cGVHD) occurred in 19% of the patients. The low incidence of GVHD and successful pre-emptive antiviral therapy resulted in low TRM of 8%. Sixteen patients died due to disease relapse after alloSCT, resulting in an overall survival of 48% at 5-years after alloSCT.

Clinical relevance of quantitative varicella-zoster virus (VZV) DNA detection in plasma after stem cell transplantation.

Detection of Varicella-Zoster virus (VZV) DNA in plasma can facilitate the early recognition of complicated VZV-infection in immunocompromised hosts. The correlation of VZV-DNA in plasma with clinical presentations of VZV-infection and subsequent aciclovir treatment in allogeneic stem cell transplant (allo-SCT) recipients was studied. In 81 consecutive VZV-IgG positive allo-SCT recipients, VZV-DNA was measured at regular time points (1, 2 and 4 months) following allo-SCT and patient records were screened for VZV-related symptoms and aciclovir treatment. Subsequently, possible VZV-cases were studied in detail for the course of VZV-DNA and treatment effects. During the initial screening, VZV-DNA was detectable in seven patients. The survey of VZV-related symptoms revealed five additional possible VZV-cases. In cases where suitable plasma samples were available (10 out of 12), VZV-DNA was present almost simultaneously with the first clinical manifestations. No evidence of a preceding phase detectable by VZV-DNA only could be observed. Treatment with aciclovir was associated with a prompt reduction of VZV-DNA load. Detection of VZV-DNA in plasma in allo-SCT recipients accurately reflected the clinical presentation of VZV-infection and treatment with aciclovir. VZV-DNA detection in plasma of allo-SCT recipients appears clinically relevant as this may support early recognition and therapeutic management of VZV-infections following allo-SCT.

Efficacy and toxicity of gemtuzumab ozogamicin in patients with acute myeloid leukemia.

Gemtuzumab ozogamicin (GO) is a recently developed antibody-targeted chemotherapeutic agent and has been expected to be less toxic than conventional chemotherapy. We retrospectively evaluated the use of GO in 38 patients. Patients with acute myeloid leukemia (AML) at diagnosis and relapsed AML were treated with 6 and 9 mg/m(2) GO. Efficacy and toxicity of GO were analyzed, as well as several prognostic factors. A complete response (CR) was observed in 12 of 38 patients, including five patients with CR plus incomplete regeneration of platelets. In one patient a partial remission was observed. Twenty-five patients showed no change or progressive disease. The overall response (OR) in patients with AML at diagnosis was 47%, with the best response in patients with primary AML (OR 60%, compared with 21% OR in non-primary AML, P = 0.045). The OR in patients with relapsed AML was 22%. Median white blood cell (WBC) before treatment, CD33 expression on leukemic blasts, and kinetics of response were analyzed as prognostic factors. Median WBC was significantly lower in patients who responded to GO, compared with non-responders (2.1 x 10(9)/L vs. 6.8 x 10(9)/L, P = 0.036). CD33 expression and kinetics of response was not correlated to clinical outcome. Median days to reach 500 x 10(6)/L neutrophils and 100 x 10(9)/L platelets were 36 and 39 d, respectively. Infections and bleedings occurred in 45% and 12%, respectively. This report shows that GO has potent clinical activity and that the OR rate was by far the best in untreated primary AML patients.

How to improve the search for an unrelated haematopoietic stem cell donor. Faster is better than more!

Many patients do not reach haematopoietic stem cell transplantation. Shortage of unrelated donors (UDs) is still seen as the main cause. However, with a worldwide UD pool containing more than 8 million donors, it is possible that other impediments are becoming more important. We analysed 549 UD searches for Dutch patients, performed between 1987 and 2000, in order to find the reasons for failure or success to reach transplantation. Between 1996 and 2000, 59% of the patients of Northwest European origin received a graft from an UD with a median time span of 4.4 months from the start of the search. In all, 11% of the patients lacked a compatible donor, while 30% became medically unfit for transplantation. This is in contrast to the patients of non-Northwest European origin for whom UD shortage is still the most important impediment; only 32% were transplanted while 50% lacked a compatible donor. We conclude that the shortage of donors is no longer the biggest constraint in unrelated stem cell transplantation for patients of Northwest European origin. It may be more effective to optimize the chance on transplantation by making the search process more efficient.

The importance of identifying a back-up donor for unrelated stem cell transplantation.

The importance of identifying a back-up donor, once a primary suitable unrelated stem cell donor has been found, is often underestimated. Transplant centres erroneously count on the unrelated volunteer donors to be willing, available and medically fit for actual donation. According to our data, which includes 502 unrelated donor work-up procedures performed for 425 Dutch patients between 1987 and 2002, one of 11 work-ups ended in the primary requested donor failing to donate. Of all donor-related cancellations (N=46), 78% of the procedures were deferred due to medical reasons and 22% due to nonmedical reasons. Most of the donors deferred for medical reasons were female (P=0.005). In 50% of the cases for which a back-up donor was already identified, the patients were transplanted with a delay of less then 2 weeks; when no back-up donor was available, the median delay increased to 18 weeks. We strongly encourage implementing a search for at least one back-up donor in the primary search. Identifying a back-up donor can save precious time and complicated logistic rescheduling.

Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Internalization and cell cycle-dependent killing of leukemic cells by Gemtuzumab Ozogamicin: rationale for efficacy in CD33-negative malignancies with endocytic capacity.

Multicenter phase II trials with Gemtuzumab Ozogamicin (GO/Mylotarg), consisting of a CD33 antibody linked to the cytotoxic drug calicheamicin, have shown a 30% overall response rate in relapsed acute myeloid leukemia patients. However, no clear correlation was observed between CD33 expression on leukemic blasts and response to GO therapy. We analyzed the CD33 specificity of GO-induced cell death and the effect of GO on CD33-negative malignancies. We demonstrate that lysis induced by clinically relevant GO concentrations is partially CD33 mediated, and that efficient non-CD33-mediated GO uptake can occur via endocytosis. In agreement with these results, we observed GO-mediated death of human CD33-negative acute lymphoblastic leukemia cells both in vitro and in vivo in an NOD/SCID mouse model. Finally, sensitivity to GO-induced cell death was at least partially determined by the activation status of leukemic cells, with cells in activated phases of the cell cycle being most effective in both CD33-specific GO internalization, renewed expression of CD33 molecules, and non-CD33-mediated GO uptake via endocytosis. In conclusion, these data provide mechanistic insight into the efficacy of GO in CD33-positive as well as in CD33-negative malignancies with endocytic capacity, and provide a rationale for the use of GO in the treatment of malignancies with endocytic capacity.

High susceptibility of human leukemic cells to Fas-induced apoptosis is restricted to G1 phase of the cell cycle and can be increased by interferon treatment.

In this study, we analyzed the influence of cell cycle status manipulations of leukemic cells on Fas-mediated apoptosis using the GM-CSF-dependent human myeloid leukemia cell line AML-193 as a model. GM-CSF and long-term treatment with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha) were used to manipulate the cell cycle status. Control cells were GM-CSF deprived, nonproliferating cells. IFN-gamma or IFN-alpha treatment did not induce proliferation in control cells, but resulted in recruitment of cells from resting G(0) phase into activated G(1) phase. Using agonistic anti-Fas antibodies (FAS18), we demonstrated that this shift from G(0) to G(1) was accompanied by a 2.5-fold increase in Fas sensitivity. A similar increase in sensitivity to FAS18 could be obtained by induction of proliferation with GM-CSF. Quantitative FACS analysis of surviving cells after FAS18-induced apoptosis showed deletion of the G(1) compartment, but complete protection of resting G(0) cells. Cells in S or G(2)/M phase were relatively protected against Fas induction. In conclusion, sensitivity to Fas-mediated apoptosis was restricted to cells in G(1) phase of the cell cycle, and can be increased by treatment of cells with interferons. By this mechanism, interferon treatment may render leukemic cells more susceptible to lysis by T cells during immunotherapeutic interventions.