Comparable incidence and severity of cytomegalovirus infections following T cell-depleted allogeneic stem cell transplantation preceded by reduced intensity or myeloablative conditioning.

Reports on infectious complications following reduced intensity conditioning (RIC) before allogeneic stem cell transplantation (allo-SCT) are equivocal. This prospective follow-up study compared the impact of cytomegalovirus (CMV) infections following RIC with fludarabine, ATG and busulphan or conventional myeloablative conditioning (MAC). Forty-eight RIC and 59 MAC patients were enrolled. The occurrence and severity of CMV infections within 100 days following allo-SCT were assessed, using plasma CMV DNA load kinetics. CMV DNAemia was observed in 21 RIC (60%) and in 19 MAC (44%) patients at risk for CMV. The mean CMV DNAemia free survival time was comparable following RIC and MAC: 70 days (95% (confidence interval) CI: 59-80 days) and 77 days (95% CI: 68-86 days), respectively (P=0.24). Parameters indicative for the level of CMV reactivation, including the area under the curve of CMV DNA load over time as well as the onset, the peak values and duration of CMV infection episodes, the numbers and duration of CMV treatment episodes and recurrent infections, were not different in both groups. During follow-up, none of the patients developed CMV disease. RIC with fludarabine, ATG and busulphan demonstrated safety comparable to conventional MAC with regard to frequency and severity of CMV infections within 100 days following T cell-depleted allo-SCT.

Liver chimerism after allogeneic blood stem cell transplantation.

UNLABELLED: Blood stem cells can mature into elements of many different lineages. We investigated the presence and nature of donor-derived (chimeric) cells within the liver after allogeneic stem cell transplantation. METHODS: Liver biopsy autopsy specimens were examined from nine female patients who had undergone allogeneic bone marrow (n = 6) or peripheral stem cell (n = 3) transplantation from a male donor. To identify the male origin of cells within the liver, in-situ hybridization for Y-chromosomes was performed in conjunction with CD45 staining to identify leucocytes. RESULTS: Hematopoietic stem cell engraftment was confirmed in all nine recipients. Histologic examination of the liver tissue sections revealed 5.6-fold more Y-chromosome-positive than CD45-positive staining cells (P < .02), indicative of considerable nonleucocytic chimerism. This was particularly observed in patients who had developed graft-versus-host disease. CONCLUSIONS: Donor-derived cells can be found in liver tissue specimens after allogeneic stem cell transplantation. A considerable fraction of chimeric (donor-derived) cells appeared to be of nonlymphohematopoietic origin. This finding supports the theory of blood stem cells developing into liver cells of mesenchymal origin.

Oral valganciclovir as pre-emptive therapy has similar efficacy on cytomegalovirus DNA load reduction as intravenous ganciclovir in allogeneic stem cell transplantation recipients.

The efficacy and safety of oral valganciclovir was compared to ganciclovir i.v. in pre-emptive treatment of cytomegalovirus (CMV) in T-cell-depleted allogeneic stem cell transplant (allo-SCT) recipients. A therapeutic guideline was developed to allow the safe application of valganciclovir in allo-SCT recipients requiring CMV therapy. In total, 107 consecutive transplant recipients were evaluated. Cytomegalovirus DNA load in plasma was monitored longitudinally; details on antiviral therapy and treatment responses were analyzed retrospectively. Fifty-seven CMV treatment episodes were recorded in 34 patients: 20 with valganciclovir (900 mg twice-daily) and 37 with ganciclovir (5 mg/kg twice-daily). Median CMV DNA load reduction was 0.079 and 0.069 log10 copies/ml/day in the ganciclovir and valganciclovir group, respectively. Good response on CMV DNA load (reduction below 3.0 log10 copies/ml) was observed in 75.7% of ganciclovir and 80.0% of valganciclovir treatment episodes. Severe adverse effects were not observed and CMV-related disease did not occur. However, the percentage of patients receiving erythrocyte transfusion was higher in the group of patients receiving ganciclovir as compared to valganciclovir (41 versus 20%, P=0.116). In conclusion, pre-emptive treatment with valganciclovir and ganciclovir, led to similar reduction of CMV DNA load. Oral valganciclovir is an attractive and safe alternative for pre-emptive CMV treatment in T-cell-depleted allo-SCT recipients.

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.

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.

8-Bromo-cAMP induces a proliferative response in an IL-3 dependent leukemic cell line and activates Erk 1,2 via a Shc-independent pathway.

In a number of cell types, elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells, which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response.

Treatment of T-cell prolymphocytic leukemia with human CD52 antibody.

PURPOSE: T-prolymphocytic leukemia (T-PLL) is an aggressive malignancy of mature T cells refractory to conventional chemotherapy, with a median survival duration of 7.5 months. We report here promising results with the use of a genetically reshaped human CD52 antibody, CAMPATH-1H. PATIENTS AND METHODS: Fifteen patients with T-PLL, most of whom had received the purine analog deoxycoformycin (DCF), were treated with CAMPATH-1H. Results were compared with those of 25 patients treated with DCF. RESULTS: Major responses occurred in 11 patients (73%) treated with CAMPATH-1H compared with 40% with DCF. Complete remissions (CRs) were documented in nine (60%) of the CAMPATH-1H cases and only three (12%) were obtained with DCF. CRs with CAMPATH-1H were durable, and re-treatment with the antibody resulted in second CRs in three relapsed patients. Two of them were successfully autografted with peripheral-blood and bone marrow stem cells collected during the first CR. Apart from first-dose reactions, infusions of CAMPATH-1H were well tolerated. However, two responding patients developed severe bone marrow aplasia that was fatal in one; the second remained moderately pancytopenic 21 weeks after stopping CAMPATH-1H therapy. The cause of this adverse effect is unknown. CONCLUSION: CAMPATH-1H is an effective agent in T-PLL and represents a significant improvement over other types of therapy. However, CAMPATH-1H alone is not sufficient for long-term remissions, and the role of autologous stem-cell transplantation needs further investigation.

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.

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.

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells.

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.

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.

Unconventional rapid Erk1,2 activation is indispensable for proliferation of the growth factor-independent myeloid leukemic cell line KG1.

While normal hematopoietic progenitor cells are dependent on colony-stimulating factors for in vitro proliferation, myeloid leukemic cells are frequently factor-independent. In this study we investigated several signalling intermediates of the Ras-Er1,2 pathway which may be involved in the development of growth factor independence. In the growth factor independent cell line KG1, an extremely short activation pattern of Erk1,2 with a maximum at 30 s was observed in response to FBS. In contrast, stimulation of the IL-3 receptor in AML193 cells resulted in a transient Erk activation peaking at 5 min and returning to base levels after 15 min. Although the Erk activation in KG1 cells is short-lived, using the MEK inhibitor PD98059, we demonstrated that Erk phosphorylation is essential for proliferation of these cell lines. We also detected major differences in Shc phosphorylation between factor-dependent and -independent cells. These data suggest that Erk activation is essential for proliferation of growth factor-dependent and -independent leukemic cells. The minimal Erk activation observed in KG1 cells in response to serum is sufficient for mitogenesis of these cells.

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.

Differential involvement of caspases in apoptosis of myeloid leukemic cells induced by chemotherapy versus growth factor withdrawal.

To assess the potential involvement of the caspase family in the IL-3-dependent murine myeloid leukemic cell line 32D, we studied the effect of bcl-2, crmA and three synthetic caspase inhibitors on apoptosis induced by chemotherapy or IL-3 withdrawal. Apoptosis induced by IL-3 deprivation or by ActD appears to be mediated by a crmA-insensitive pathway. Cell death by IL-3 withdrawal is inhibited by the caspase-inhibitor ZVAD-fmk, but not DEVD-fmk or YVAD-cmk. In contrast, DEVD-fmk as well as ZVAD-fmk protect 32D cells from ActD-induced apoptosis. These results indicate that different caspases are involved in apoptosis induced by growth factor withdrawal and by chemotherapy.

Comparison of allogeneic T cell-depleted peripheral blood stem cell and bone marrow transplantation: effect of stem cell source on short- and long-term outcome.

We report the results of a retrospective single-center study comparing engraftment, acute and chronic GVHD, relapse and survival in patients with malignant hematological disorders transplanted with allogeneic peripheral blood stem cells (alloPBSCT, n = 40) or bone marrow cells (alloBMT, n = 42). All transplants were T cell depleted by in vitro incubation with the Campath-1 monoclonal antibody. Primary graft failure occurred in none of the patients receiving an alloPBSCT compared with 3/42 of the recipients of an alloBMT. In addition, two patients in the alloBMT group showed no platelet engraftment. Recipients of PBSC had a more rapid recovery of neutrophils (median 14 days) compared to BM transplant recipients (median 32 days). Platelet recovery was also accelerated in PBSC recipients compared to BM recipients (11 vs 38 days). There was an increase in the incidence of grade II acute GVHD and chronic GVHD in patients after alloPBSCT (18% and 23%, respectively) compared to patients receiving alloBMT (5% and 8%, respectively). The 2-year cumulative incidence of relapse was similar in both groups (47%). At 6 months after transplantation, transplant-related mortality (TRM) was lower in PBSCT recipients than in BMT recipients. However, at a follow-up of 3 years TRM was similar in both groups. The disease-free survival rate at 3 years after transplantation did not differ between the groups (42% for PBSCT and 41% for BMT recipients). Our results indicate that T cell-depleted alloPBSCT compared to alloBMT is associated with a more rapid hematopoietic reconstitution and a decreased TRM at 6 months follow-up after transplantation. However, at a follow-up of 3 years, no sustained survival benefits were observed.

CD52 antibodies for prevention of graft-versus-host disease and graft rejection following transplantation of allogeneic peripheral blood stem cells.

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic bone marrow transplantation, but can be avoided by removing T lymphocytes from the donor bone marrow. However, T cell depletion increases the risk of graft rejection. In this study, two strategies are used to overcome rejection: (1) use of high doses of stem cells obtained from peripheral blood (PBSC), (2) admixture with a CD52 monoclonal antibody in order to deplete both donor and residual recipient lymphocytes. Two antibodies are compared: CAMPATH-1G (rat IgG2b) and its humanized equivalent CAMPATH-1H (human IgG1). A total of 187 consecutive patients at six centers received PBSC transplants from HLA-matched siblings between 1997 and 1999. A wide spectrum of diseases, both malignant and non-malignant, was included. The recovery of CD34+ cells after antibody treatment was close to 100%. The risk of acute GVHD (grade 2 to 4) was 11% in the CAMPATH-1G group and 4% in the CAMPATH-1H group (P = NS). The risk of chronic GVHD (any grade) was 11% in the CAMPATH-1G group and 24% in the CAMPATH-1H group (P = 0.03) but the risk of extensive chronic GVHD was only 2%. The overall risk of graft failure/rejection was 2%, not significantly different between the two antibodies. Antibody treatment was equally effective at concentrations between 10 microg/ml and 120 microg/ml and it made no significant difference to the outcome whether the patients received post-transplant immunosuppression or not (87% did not). Transplant-related mortality in this heterogenous group of patients (including high-risk and advanced disease) was 22% at 12 months. It is proposed that treatment of peripheral blood stem cells with CAMPATH-1H is a simple and effective method for depleting T cells which may be applicable to both autologous and allogeneic transplants from related or unrelated donors. Special advantages of this approach are the simultaneous depletion of donor B cells (which reduces the risk of EBV-associated lymphoproliferative disease) and the concomitant infusion of CAMPATH-1H to deplete residual recipient T cells and thus prevent graft rejection.

Biological action of pancreatic amylin: relationship with glucose metabolism, diabetes, obesity and calcium metabolism.

Amylin, also called islet amyloid polypeptide (IAPP), or diabetes-associated peptide (DAP) is a recently discovered 37 amino acid polypeptide which has been shown to be co-secreted with insulin from the pancreatic beta-cell. The peptide turned out to be the major constituent of pancreatic amyloid deposits which are frequently found in the pancreas of type II diabetic patients. Therefore, a role for amylin in the aetiology of type II diabetes was hypothesized. To investigate this possibility, several studies have been performed to elucidate whether amylin is able to impair insulin secretion and action, two characteristic features of type II diabetes mellitus. These studies suggest that it is unlikely that amylin has a direct inhibitory effect on insulin secretion. Amyloid deposits, however, which are derived from the in situ polymerization and precipitation of amylin, may impair beta-cell function during type II diabetes by damaging and covering beta-cells. Furthermore, it has been shown that amylin has the potential to antagonize the action of insulin on glucose metabolism by increasing hepatic glucose production and by decreasing muscle, but not adipocyte glucose uptake. For these reasons, it has been suggested that amylin might be involved in the pathophysiology of type II diabetes and obesity, disease states which are characterized by abnormal beta-cell function and insulin resistance. In addition, amylin was shown to induce hypocalcaemia by inhibiting osteoclast-mediated bone resorption in a calcitonin-like manner. Therefore, amylin is likely to be involved in both the modulation of glucose and calcium metabolism.

[Mycetoma of the foot; a disease from the tropics]. / Mycetoom van de voet; een ziekte uit de tropen.

Two patients, a Surinamese man aged 50 and a Surinamese woman aged 56 exhibited a mycetoma of the foot, 30 and 28 years, respectively, after a local injury. Pathological examination revealed an aspecific chronic granulomatous inflammation. As causative agents a Fusarium species and a Cladosporium normodendrum, respectively, were cultured. The treatment consisted of curettage of fistulous ducts and administration of itraconazole.

[A patient with chronic mucormycosis]. / Een patiënt met chronische mucormycosis.

Rhinocerebral mucormycosis was diagnosed in a 75-year-old woman with a history of type II diabetes mellitus. This rare opportunistic infection is caused by fungi belonging to the order of Mucorales. The patient had a severe osteomyelitis of the base of the skull, resulting in complaints of headache and diplopia. She was treated with intravenous colloidal amphotericin B, surgical excision, and later with liposomal amphotericin B. She died of respiratory failure. Mucormycosis is usually a rapidly fulminant infection. This patient showed a remarkably chronic course.