ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Advances in therapy, in particular stratification of patients to appropriate treatment according to risk factors improved long term survival attaining cure rates of up to 80 %. Despite the efforts achieved by the stratification strategies the majority of relapse patients are recruited from the low risk groups emphasizing the need for additional independent risk factors. In a recent study we transplanted primary leukemia samples obtained from pediatric patients with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample transplanted from date of transplant to date of leukemia manifestation in the recipients. We demonstrated that patients whose leukemia cells engrafted rapidly leading to manifestation of the disease within 10 weeks (TTLshort) showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth (TTLlong). Interestingly, the same distinct difference in relapse free survival was observed in the low risk groups only. Multivariate analysis showed an almost 45- fold increased risk for relapse in TTLshort patients. In order to further characterize the biological properties of the leukemia cells in the two groups, gene expression profiles of samples with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0). Here, we used quantitative traits analysis (QTA) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks). 14 different xenograft samples (TTLshort n= 7, mean TTL 8.14 weeks; TTLlong n= 7, mean TTL 19.71 weeks; T-test P = .03) isolated from leukemia bearing mice were investigated. All 14 patients included were stratified in low (standard or intermediate) risk groups. All patients were negative for TEL/AML1- fusion, BCR/ABL- fusion or MLL rearrangement. By QTA we identified 5 genes that were significantly correlated (Spearman correlation, for all 5 genes P 〈 .0001) to time from transplant to leukemia manifestation in the recipient mice (TTL). Analysis of the 14 xenograft samples using the 5 genes identified showed clustering of all but one sample in one group. The 5 genes were than explored for their power to predict relapse in an independent cohort of pediatric ALL patients. For these patients expression profiles were analyzed in leukemia samples obtained at diagnosis. All patients in this cohort were also stratified in low risk groups and negative for the presence of TEL/AML1-, BCR/ABL- or MLL- fusion transcripts. 8 of the 38 patients encountered relapse, 5 at early and 3 at late time points (〈 or 〉 24 months after diagnosis). Clustering according to the 5 genes identified by QTA lead to separation of the patients into two groups. Interestingly, all relapsed patients except one clustered in the group associated with the signature characteristic for TTLshort. Most importantly, all 5 patients with early relapse gathered in this cluster. Taken together, we used a novel approach directly correlating gene expression values to the continuous variable time to leukemia (TTL) which might be reflected more accurately by this strategy than comparing two groups. We applied this gene signature characteristic for TTLshort, thereby identifying poor overall survival in a set of independent pediatric ALL patients and found clustering of all early relapse patients in one group. This new independent risk factor might identify patients with high risk for early relapse avoiding xenografting in the mouse model.

Description: Background: The EXtended CLinical prophylaxis in Acutely Ill Medical patients (EXCLAIM) trial was a randomized, double-blind, placebo-controlled, multicenter, international study that demonstrated a 38% relative risk reduction (RRR) for venous thromboembolism (VTE) with extended-duration enoxaparin prophylaxis compared with placebo (2.5% vs 4.0%; absolute difference [AD], −1.5% 95.8% CI −2.5 to −0.5%; P=0.002). Major bleeding occurred in 0.7% (20/2975) and 0.2% (7/2988) of patients who received enoxaparin and placebo, respectively (AD, 0.4% [CI 0.1% to 0.8%]; P=0.012). As age is a known independent risk factor for VTE, we conducted a pre-specified sub-analysis of the EXCLAIM trial to compare the efficacy and safety of extended-duration enoxaparin prophylaxis in patients 〉75 years old with patients ≤75 years old. Methods: EXCLAIM eligibility required a recent (≤3 days) reduction in mobility due to acute medical illness, an anticipated level 1 (total bed rest/sedentary) or level 2 (level 1 with bathroom privileges) decreased mobility, and age ≥40 years. During the latter part of the study, a protocol amendment required patients with level 2 mobility to have ≥1 of 3 additional pre-specified risk factors (i.e., active or prior cancer, history of VTE, age 〉75 years). Of the 7500 patients enrolled, 7415 received open-label enoxaparin 40 mg SC od for 10±4 days. Of these, 6085 were randomized to double-blind therapy (enoxaparin 40mg SC od or placebo) of 28±4 additional days duration. The incidence of VTE, the primary efficacy end point, was defined as the composite of symptomatic or asymptomatic proximal deep vein thrombosis (DVT), symptomatic pulmonary embolism (PE), or fatal PE during the double-blind treatment period. Patients were screened for DVT with bilateral proximal lower extremity compression ultrasound at the end of randomized therapy. The incidence of major bleeding, the primary safety end point, was assessed through 48 hours after the last dose of study treatment. Results: Of the 5963 randomized patients that received at least one dose of double-blind therapy, 29.9% (1781) were 〉75 years of age (mean age 81.5 years) and 70.1% (4182) were ≤75 years old (mean age 61.8 years). In patients 〉75 years old, the incidence of VTE was 2.5% (18/725) in the enoxaparin group compared with 6.7% (50/743) in the placebo group (AD −4.2% [95.8% CI −6.5 to −2.0%]; P75 years old (0.6% vs 0.2%; AD 0.3%, 95% CI −0.2 to 0.9%; P=0.282) and significantly higher in patients ≤75 years old (0.7% vs 0.2%; AD 0.5%, 95%CI 0.1 to 0.9%; P=0.041). Though the older group had a higher death rate compared to the younger group (3.2% vs 1.6%), the survival between the treatment groups was similar within the two age groups. Without extended prophylaxis (i.e., placebo group), patients 〉75 years old had a significantly higher risk of VTE than those 75 years of age.

Description: Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50〉1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50

Description: Venous thromboembolism (VTE) is a major therapeutic issue in cancer. Advances in this field and heterogeneities in clinical practices prompted us to establish guidelines related to VTE treatment and to central venous catheter thrombosis (CVCT) management. in cancer patients according to the SOR Standards, Options: Recommendations (SOR) methodology for the development of evidence-based Clinical Practice Guidelines (CPG) as endorsed by the French National Cancer Institute. Methods: After reviewing the published studies on the topics between 1999 and 2007, a first version of the guidelines was based on the levels of evidence derived from analysis of the 38 out of 418 selected studies for VTE treatment and the 40 out of 175 selected studies for the CVCT management. The recommendations were classified as Standards or Options and then peer-reviewed by 65 independent experts. Detailed methodology is available at www.sor-cancer.fr Standards in cancer patients: The treatment of VTE should be based on Low Molecular Weight Heparins (LMWH) at curative doses for at least 3 months. During the initial treatment (up to 10 days), there are no specific requirements and all drugs approved (including LMWH, Unfractionnated Heparin (UFH), fondaparinux and danaparoid) may be used. Beyond the first 10 days, VTE treatment should be based on LMWH at curative doses for at least 3 and optimally for 6 months, as validated with the following drugs and dosage regimens: dalteparin 200 IU/kg once daily for one month, then 150 IU/kg once daily; enoxaparin 150 IU/kg once daily; and tinzaparin 175 IU/kg once daily. In case of: severe renal impairment, UFH should be used rapidly followed by Vitamins K Antaogonist (VKA) for at least 3 months; severe Pulmonary Embolism (hemodynamic failure), the indications and usages of thrombolytic drugs are the same as in non-cancer patients; absolute contra-indication to anticoagulation or VTE recurrence despite optimal anticoagulation, vena cava filters (VCF) should be considered; intracranial malignancies, VTE treatment is the same as in cancer patients with non-intracranial tumors. CVCT treatment relies on long term use of LMWH. In case of severe renal failure, UFH with early AVK must be used. Treatment is to be continued as long as the catheter is maintained. This can only be achieved if the catheter is functional, well positioned, not infected and if adapted anticoagulation has resumed the CVCT. If catheter withdrawal is necessary, there is no standard concerning the anticoagulation management. CVCT prophylaxis relies on positioning the catheter distal extremity at the “superior vena cava - right atrium” junction. Systematic CVCT anticoagulant prophylaxis is not recommended. Options: Treatment of VTE: If LMWH administration for 3 months is impossible, short-term use of LWMH followed by VKA for at least 3 months may be proposed. It is recommended to administer LMWH for 3 to 6 months; LMWH should be used according to the same curative dosage regimen as during the first 3 months. Beyond the first 6 months, the anticoagulant treatment should be continued as long as the cancer is active or treated. In the event of a first VTE episode secondary to a transient risk factor and if the cancer is not active nor treated, anticoagulation may be discontinued after 6 months. The choice between LMWH and VKA depends on their benefit-risk ratio (influenced by drug interactions, chemotherapy, invasive procedures, and general health status) and acceptability. If a VCF is considered, a retrievable VCF may be discussed. CVCT treatment: If another catheter has to be inserted, prior evaluation of the venous circulation by scanner or ultrasound examination is recommended. If prolonged use of LMWH is impossible, VKA can be proposed. In case of severe superior vena cava syndrome, fibrinolytics can be used in the absence of contra-indications. Treatment by LMWH can be stopped 6 weeks after catheter withdrawal in non active cancer or after 3 to 6 months of LMWH followed by VKA in the other cases. CVCT prophylaxis: Right side catheter insertion and vein localisation by ultrasonography are preferred. Conclusion: The French recommendations further support the 2006 Italian and the 2007 North American guidelines on VTE treatment in cancer patients and were extended to the use of VCF and treatment of patients with intracranial malignancies. In addition, we provide recommendations on CVCT treatment in cancer patients.

Description: ROR1 is an oncofetal protein expressed in chronic lymphocytic leukemia (CLL). We generated a monoclonal antibody (mAb) specific for ROR1 (4A5) and used this to stain blood or marrow cells from patients with CLL and other adults. We found that 4A5 reacted with the CLL cells of all patients examined and also with some cells that had an immunophenotype of B lymphocyte precursors (BLP) (CD10+, CD19+, CD20-variable, dim CD45+, surface immunoglobulin-negative). 4A5 showed no reactivity with other blood or marrow cells. We speculated that this mAb could be used to detect CLL cells in blood or marrow of patients after therapy, providing a means with which to assess minimal residual disease (MRD). For this purpose, we evaluated a four-color combination of mAbs using CD10-FITC, CD19-PE, CD5-PerCP-Cy5.5, and 4A5-Alexa-647. MRD detection limits were established through reconstitution studies in which we made serial dilutions of CLL cells into normal blood or marrow samples. MRD was measured by whole blood lysis and acquisition of 100,000 events using a FACSCalibur and a gating strategy designed to exclude CD5-negative, CD10-positive, and 4A5-negative B lymphocytes (CD19+). CLL cells stained with 4A5 had a mean fluorescence intensity (MFI) of 31 (N=100) and a MFI ratio (MFIR) relative to that of isotype control stained CLL cells of more than 10:1. On average, 94% of the CLL cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 88–98%). Marrow samples (N=9) from adults with diagnoses other than CLL and that had 1–10% BLP had CD5− negative, CD10+, CD19+ cells that reacted with the 4A5-Alexa 647. These cells in such samples had a MFI of 19 and a MFIR relative to that of isotype-control-stained BLP of 6.3. On average, 48% of such cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 18–79%). Four color flow cytometric analysis with CD10, CD19, CD5, and 4A5 detected CLL cells present at less than 0.1% in reconstituted blood or marrow samples, including marrow with 3–5% BLP. Background was between 0.01% and 0.1% with whole blood (marrow) lysis when we acquired a total of 100,000 flow-cytometric events. These data indicate that anti-ROR1 mAbs can be used with mAbs specific for CD10, CD19, and CD5 for sensitive detection of MRD in CLL.

Description: In a two centre study (laboratories in Diagnostica Stago and Biomnis) we compared the in vitro effect on thrombin generation (TG) of Dabigatran and Bivalirudin (reversible direct anti-IIa inhibitors) with that of Lepirudin (an irreversible direct anti-IIa inhibitor) spiked into normal pool plasma. The effect of Lepirudin, Bivalirudin and Dabigatran were evaluated in both centres using the CAT (Diagnostica Stago, France) TG method in a concentration ranges up to 5, 20 and 1 μg/mL respectively. Testing was done in triplicate and repeated over 2 days. To reduce assay variability both centres used the same reagents lots and the same normal pool plasma (George King, USA). The range of each drug tested extended well above the therapeutic range concentrations normally found in patient plasma (0.5 to 1.0 μg/mL, 5 to 10 μg/mL and 0.1 to 0.3 μg/mL respectively for Lepirudin, Bivalirudin and Dabigatran). To see the effect of increasing activation forces, TG was performed at 3 different final concentrations of Tissue Factor (TF) - 1, 5 and 20 pM. All reagents were used as recommended by the manufacturer (Thrombinoscope, The Netherlands). A prolongation in the lag time (LT) is observed with all 3 drugs with all 3 concentrations of TF, but this is more marked for Lepirudin and Bivalirudin than it is for Dabigatran. In the therapeutic range Dabigatran (at 5pM TF) shows both an increase in LT and a decrease in peak thrombin and the ETP. At low concentration of Bivalirudin or Lepirudin, there is a paradoxical increase in peak height, which is even more pronounced at low TF concentration. At 1pM TF, this paradoxical peak increase is also observed with Dabigatran. Results obtained in both laboratories are similar and complement our previous results and those reported elsewhere (1–4). The effect of Lepirudin and Bivalirudin on TG is different from that of Dabigatran. We also note that at lower TF concentration the anticoagulant effect on TG initiation is more intense but the test becomes less reproducible.

Description: To improve the results of allogeneic SCT for high-risk AML and MDS, the FLAMSA-RIC conditioning regimen for allogeneic SCT combines cytoreductive chemotherapy (fludarabine, HD AraC, amsacrine), followed three days later by reduced intensity conditioning (4Gy TBI/EDX). Since in particular patients with an unfavorable karyotype seemed to benefit from this approach (Schmid et al., JCO, 2005), we analysed the outcome of 172 patients with poor risk cytogenetics according to NCCN criteria, who had been allografted following FLAMSA-RIC conditioning in 11 European centres between 1999 and 2008. Median time from diagnosis to transplantation was 5 months. Donors were matched siblings, matched unrelated, or mismatched unrelated donors in 34%, 47%, and 19%. Patients suffered from progressive MDS (10%), de novo AML (47.5%), or secondary AML (43.5%). SCT was performed upfront, after primary induction failure, in CR1 and in relapsed disease in 17%, 33%, 22% and 28% of patients, respectively. Median patient age was 53 (18–71) years. 95 patients (56%) had a complex aberrant karyotype, 55 and 65 had abnormalities of chromosome 5 (−5/5q-) and 7 (−7/7q-), respectively. After a median follow up of 20 months, overall survival (OS) at 2 and 4 years was 46.4% and 40.5%, the respective leukemia-free survival was 37.7% and 32.0%. Causes of death were leukemia in 30%, and non-relapse mortality in 21%. Encouraging results were observed in patients with chromosome 7 aberrations or with a complex karyotype leukemia (4y OS=49.3% and 40.3%). In contrast, results were inferior in patients with chromosome 5 aberrations (4y OS=30%), mainly due to an increased rate of leukemia-associated death (p=.008). Patiens with MDS, who received allogeneic SCT as first line treatment, achieved a 4y OS of 80% despite unfavorable cytogenetics. Unlike, patients with secondary AML after MDS had an inferior outcome (4y OS=28%, p=.018). In a Cox regression model, a stage of remission at transplantation, a 8/8 or 10/10 matched family or unrelated donor, and lack of monosomy 5 or deletion 5q were associated with superior OS (p=.025, .05, and .05). In conclusion, allogeneic SCT following the FLAMSA-RIC regimen is a highly effective treatment for MDS and AML with unfavorable karyotype, comparing favourably with published data. In MDS, SCT should be performed before transformation into sAML. Long term remission is achieved in a substantial percentage of patients with complex karyotype disease and aberrations of chromosome 7. Aberrations of chromosome 5 may require alternative or additive strategies.

Description: Background: The anti-VEGF drug, bevacizumab (Bev), has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Prevailing opinion on the molecular mechanism behind Bev-associated bleeding and thrombosis is that tissue factor driven coagulation, secondary to vascular endothelial cell dysfunction, may cause thrombosis due to VEGF suppression by Bev. Bev forms immune complexes (IC) with VEGF (vascular endothelial growth factor), a heparin-binding protein. In our previous in vitro studies we showed that, in the presence of heparin, Bev+VEGF immune complexes activate platelets via the IgG receptor FcγRIIa —a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia (HIT). Objectives: First, we investigated whether Bev-associated thrombosis might be replicated in mice. Because mouse platelets do not carry FcγRIIa, we used mice transgenic for this human IgG receptor (hFcR mice) in order to enable the signaling pathway identified above. Second, using human platelets in vitro, we studied the functional roles of heparin and platelet surface localization of IC in Bev-induced FcγRIIa activation. Methods: Bev+VEGF IC were preformed using VEGF165 or VEGF121 (similar to VEGF165 but lacking the heparin-binding domain). Platelet dense granule release and aggregation were measured by the serotonin release assay (SRA) and Chrono- Log aggregometers, respectively. Platelet surface localization was assessed by flow cytometry (50,000 events/test condition) and fluorescence microscopy using Alexa488- labeled Bev (Bev488). For in vivo studies, Bev+VEGF+Heparin IC (60–500 nM) or control reagents were injected intravenously into wild-type (WT) or hFcR mice. Platelet counts were measured 10–60 minutes following IC injection after obtaining blood (0.45 ml) by cardiac puncture. Immediately afterward, lungs were processed for hematoxylin and eosin staining and analyzed microscopically for evidence of thrombosis. Results: IC consisting of Bev+VEGF165+Heparin (0.2U/ml) caused thrombotic thrombocytopenia in hFcR but not WT mice, showing a requirement for FcγRIIa. Injection of Bev+VEGF121+Heparin (0.2U/ml) into hFcR mice did not cause thrombocytopenia, suggesting a requirement for the VEGF165 heparin binding domain. Bev+VEGF165 was without effect in the absence of heparin or in the presence of excess (200 U/ml) heparin demonstrating that a limited range of heparin concentrations enable Bev-induced thrombocytopenia and thrombosis. This mechanism is similar to that observed in HIT and our in vivo results were consistent with SRA and aggregation in vitro studies. By flow cytometry, maximal Fab-dependent Bev488 platelet surface binding occured only with VEGF165+0.2U/ml heparin. Saturating IV.3 (anti-FcγRIIa antibody) concentrations, present in all samples, excluded Bev-Fc binding to FcγRIIa. Furthermore, binding of Bev488+VEGF121+0.2 U/ml heparin was not detected, suggesting the VEGF heparin binding domain is required for heparin-enhanced surface binding. Conclusions: In the presence of heparin, Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of FcγRIIa receptor. This mechanism may be relevant to the thromboembolic complications observed in patients receiving Bev therapy.

Description: Donor lymphocyte infusions (DLI) are increasingly used to treat minimal residual disease or mixed hematopoietic donor-recipient chimerism in T-cell depleted allogeneic stem cell transplantation (SCT). In addition, several clinical trials currently investigate the prophylactic application of DLI to promote donor T-cell reconstitution after transplantation. However, DLI carry a substantial risk of inducing graft-versus-host disease (GVHD). We investigate DLI heavily depleted of CD8 T cells using a clinical grade immunomagnetic in vitro procedure in an ongoing clinical study [Meyer et al., Blood2007, 109:374]. These DLI are administered in a prophylactic setting to patients with hematological malignancies who are off immunosuppressive treatment and free of GVHD early after allogeneic SCT. The reduced-intensity conditioning regimen consists of fludarabine and melphalan and in vivo T-cell depletion (TCD) by the anti-CD52 antibody Alemtuzumab. Up to now, 24 patients have been treated with 1 to 4 increasing doses of CD8-depleted DLI starting with 1x10^6 CD4+ T cells/kg bodyweight. The median time between SCT and first DLI was 119 days (range, 60–194). Seven of 24 patients (29%) developed acute GVHD of grade 2 to 4 or extensive chronic GVHD following CD8-depleted DLI. We did a longitudinal analysis of lineage-specific T-cell chimerism in 20 patients who received CD8-depleted DLI in comparison to 17 patients who did not qualify for DLI due to spontaneously occurring acute GVHD (n=14) or unavailable donors (n=3). The patients’ characteristics in both groups were comparable with a median age of 55 (range, 35–64) years in the DLI group and of 57 (range, 29–67) years in the non-DLI group. The donor types were matched sibling (DLI: n=6; non-DLI: n=2), matched unrelated (DLI: n=8; non-DLI: n=8), and unrelated with 1 HLA-mismatch (DLI: n=6; non-DLI: n=7), respectively. Twelve of the 20 patients in the DLI group and 6 of 17 patients in the non-DLI group showed a secondary decrease of donor T-cell chimerism to a median of 52% (range, 10–90%) between 7 and 35 weeks after transplantation (median, 12 weeks). Only one of the latter spontaneously reconverted to a full donor T-cell chimera. Of the remaining 5 non-DLI patients, 4 patients subsequently relapsed with their underlying disease and one patient still had a mixed T-cell chimerism of 50% two years after transplantation. In contrast, in patients receiving CD8-depleted DLI the proportion of donor T cells significantly increased and 11 of 12 patients converted to durable full donor chimerism. Three patients of the DLI group subsequently developed disease relapse. By monitoring CD52-expression on reconstituting T cells by flow cytometry, we were able to demonstrate the impact of CD8-depleted DLI on post-transplant T-cell reconstitution: In the non-DLI group, the majority of CD4 T cells remained CD52-negative 9 months after transplantation. Simultaneously, the proportion of CD52-expressing CD4 T cells was significantly higher in the DLI group (mean: 42% versus 86%; t-test, p