Lutz Uharek

Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial

The purpose of this prospective multicenter phase 2 trial was to investigate the long-term outcome of reduced-intensity conditioning allogeneic stem cell transplantation (alloSCT) in patients with poor-risk chronic lymphocytic leukemia. Conditioning was fludarabine/ cyclophosphamide-based. Longitudinal quantitative monitoring of minimal residual disease (MRD) was performed centrally by MRD-flow or real-time quantitative polymerase chain reaction. One hundred eligible patients were enrolled, and 90 patients proceeded to alloSCT. With a median follow-up of 46 months (7-102 months), 4-year nonrelapse mortality, event-free survival (EFS) and overall survival (OS) were 23%, 42%, and 65%, respectively. Of 52 patients with MRD monitoring available, 27 (52%) were alive and MRD negative at 12 months after transplant. Four-year EFS of this subset was 89% with all event-free patients except for 2 being MRD negative at the most recent assessment. EFS was similar for all genetic subsets, including 17p deletion (17p-). In multivariate analyses, uncontrolled disease at alloSCT and in vivo T-cell depletion with alemtuzumab, but not 17p-, previous purine analogue refractoriness, or donor source (human leukocyte antigen-identical siblings or unrelated donors) had an adverse impact on EFS and OS. In conclusion, alloSCT for poor-risk chronic lymphocytic leukemia can result in long-term MRD-negative survival in up to one-half of the patients independent of the underlying genomic risk profile. This trial is registered at http://clinicaltrials.gov as NCT00281983.

A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS

This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation.

From 2002 to 2007, 103 patients with primary myelofibrosis or postessential thrombocythemia and polycythemia vera myelofibrosis and a median age of 55 years (range, 32-68 years) were included in a prospective multicenter phase 2 trial to determine efficacy of a busulfan (10 mg/kg)/fludarabine (180 mg/m(2))-based reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation from related (n = 33) or unrelated donors (n = 70). All but 2 patients (2%) showed leukocyte and platelet engraftment after a median of 18 and 22 days, respectively. Acute graft-versus-host disease grade 2 to 4 occurred in 27% and chronic graft-versus-host disease in 43% of the patients. Cumulative incidence of nonrelapse mortality at 1 year was 16% (95% confidence interval, 9%-23%) and significantly lower for patients with a completely matched donor (12% vs 38%; P = .003). The cumulative incidence of relapse at 3 years was 22% (95% confidence interval, 13%-31%) and was influenced by Lille risk profile (low, 14%; intermediate, 22%; and high, 34%; P = .02). The estimated 5-year event-free and overall survival was 51% and 67%, respectively. In a multivariate analysis, age older than 55 years (hazard ratio = 2.70; P = .02) and human leukocyte antigen-mismatched donor (hazard ratio = 3.04; P = .006) remained significant factors for survival. The study was registered at www.clinicaltrials.gov as #NCT 00599547.

MyD88/TLR9 mediated immunopathology and gut microbiota dynamics in a novel murine model of intestinal graft-versus-host disease

Background The bacterial microflora aggravates graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation, but the underlying mechanisms of manifestations of intestinal GvHD (iGvHD) in the gut remain poorly understood. Aim To analyse the gut flora composition and the impact of bacterial sensing via Toll-like receptors (TLRs) in iGvHD. Methods By mimicking clinical low-intensity conditioning regimens used in humans, a novel irradiation independent, treosulfan and cyclophosphamide-based murine allogeneic transplantation model was established. A global survey of the intestinal microflora by cultural and molecular methods was performed, the intestinal immunopathology in TLR-deficient recipient mice with iGvHD investigated and finally, the impact of anti-TLR9 treatment on iGvHD development assessed. Results The inflammatory responses in iGvHD were accompanied by gut flora shifts towards enterobacteria, enterococci and Bacteroides/Prevotella spp. Analysis of iGvHD in MyD88-/-, TRIF-/-, TLR2/4-/-, and TLR9-/- recipient mice showed that bacterial sensing via TLRs was essential for iGvHD development. Acute iGvHD was characterised by increasing numbers of apoptotic cells, proliferating cells, T cells and neutrophils within the colon. These responses were significantly reduced in MyD88-/-, TLR2/4-/-, TRIF-/- and TLR9-/- mice, as compared with wild-type controls. However, TRIF-/- and TLR2/4-/- mice were not protected from mortality, whereas TLR9-/- mice displayed increased survival rates. The important role of TLR9-mediated immunopathology was independently confirmed by significantly reduced macroscopic disease symptoms and colonic apoptosis as well as by reduced T-cell and neutrophil numbers within the colon after treatment with a synthetic inhibitory oligonucleotide. Conclusions These results emphasise the critical role of gut microbiota, innate immunity and TLR9 in iGvHD and highlight anti-TLR9 strategies as novel therapeutic options.

Use of natural killer cells in hematopoetic stem cell transplantation

Summary:Adoptive immunotherapy using natural killer (NK) cells may prove useful, especially in situations where infusion of T cells is impractical such as in recipients of haploidentical stem cell transplantation (HSCT) from haploidentical donors. NK cells may induce potent antileukemic and possibly antirejection activity and may even mitigate graft versus host disease (GvHD). Whether such effects are clinically important and whether they are mediated mainly or exclusively by KIR–HLA class I interactions remains to be determined. Recent advances in graft engineering provide for methods to isolate large numbers of purified NK cells. Several groups have shown that clinical grade NK cells up to a dose of 107/kg may be collected and purified for the purpose of infusion to patients. Early results, in a limited number of patients, show that these cell doses may be administered without adverse events and without inducing GvHD. Whether such infusions will be useful in preventing graft rejection, or exerting graft versus leukemia effects and hastening immune recovery requires further study.

TP53, SF3B1, and NOTCH1 mutations and outcome of allotransplantation for chronic lymphocytic leukemia: six-year follow-up of the GCLLSG CLL3X trial

The purpose of this analysis was to provide 6-year follow-up of the CLL3X trial, which studied reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) in patients with poor-risk chronic lymphocytic leukemia (CLL), and to investigate the effect of TP53, SF3B1, and NOTCH1 mutations on HSCT outcome. For 90 allografted patients, 6-year overall survival (OS) was 58% and 6-year event-free survival (EFS) was 38%. TP53, SF3B1, and NOTCH1 mutations were found in 30%, 26%, and 14% of the trial population, respectively. By univariate and multivariate analyses, the mutational status of the TP53, SF3B1, and NOTCH1 genes had no significant effect on OS and EFS. Studies of minimal residual disease confirmed durability of CLL eradication in mutated patients. We conclude that HSCT can provide long-term disease control in patients with poor-risk CLL independent of the presence of TP53, SF3B1, and NOTCH1 mutations. The trial has been registered at the US National Cancer Institute as #EU-20554, NCT00281983.

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation.

Human cytomegalovirus (CMV) is a major cause of death after transplantation. The frequency of pp65-specific T cells was examined in 38 HLA-A2+ stem cell recipients during the first year after transplantation. Patients were divided into four groups based on donor/recipient serostatus: d+/r+ (n=17), d+/r− (n=7), d−/r+ (n=9) and d−/r− (n=5). Peripheral blood mononuclear cells were stimulated with the CMVpp65 peptide NLVPMVATV, and the specific T-cell frequency was assessed by interferon gamma (IFN-γ) ELISPOT assay. Responding T cells were characterized by flow cytometry revealing a terminal differentiated effector phenotype. Surveillance of CMV infection was carried out by real-time polymerase chain reaction (n=26) or immunofluorescence (n=12). Infection was present in 7/9 d−/r+ high-risk patients, and CMV disease occurred exclusively in this group with delayed or absent virus-specific T-cell recovery. In contrast, 16/24 intermediate-risk patients showed CMV-specific T cells. Our data suggest that CMV infection and disease rates are elevated in high-risk patients with delayed CMV-specific T-cell immune reconstitution and lower in those with early recovery of T-cell immunity. We recommend preferring CMV seropositive donors for CMV seropositive recipients, as this should lead to durable CMV-specific T-cell responses soon after transplantation with consecutive protection from CMV disease.

Efficacy of the interleukin-2 receptor antagonist basiliximab in steroid-refractory acute graft-versus-host disease

Acute graft‐versus‐host disease (aGVHD) occurs in up to 80% of patients who undergo allogeneic stem cell transplantation (SCT) and contributes significantly to transplant‐related mortality (TRM). We conducted a prospective phase II trial to assess the efficacy and feasibility of treating steroid‐refractory aGVHD with basiliximab, a chimaeric monoclonal antibody directed against the alpha chain of the interleukin‐2 (IL‐2) receptor. Basiliximab was administered intravenously at a dose of 20 mg on days 1 and 4. Twenty‐three patients were enrolled between October 1999 and July 2004. We found a primary overall response rate of 82·5% with four patients (17·5%) showing a complete response and 15 patients (65%) a partial response. Six patients were again treated successfully with an IL‐2 receptor antagonist because of recurrence of aGVHD. The rates of infections, chronic GVHD, malignancy recurrence and 1‐year TRM following immunosuppression with basiliximab were comparable with those found with other treatment modalities for aGVHD. We conclude that basiliximab is efficient and feasible for steroid‐refractory aGVHD and merits further evaluation.

Treatment of Acute Myeloid Leukemia or Myelodysplastic Syndrome Relapse after Allogeneic Stem Cell Transplantation with Azacitidine and Donor Lymphocyte Infusions—A Retrospective Multicenter Analysis from the German Cooperative Transplant Study Group

To expand the current knowledge about azacitidine (Aza) and donor lymphocyte infusions (DLI) as salvage therapy for relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify predictors for response and survival, we retrospectively analyzed data of 154 patients with acute myeloid leukemia (AML, n = 124), myelodysplastic (MDS, n = 28), or myeloproliferative syndrome (n = 2). All patients received a median number of 4 courses of Aza (range, 4 to 14) and DLI were administered to 105 patients (68%; median number of DLI, 2; range, 1 to 7). Complete and partial remission rates were 27% and 6%, respectively, resulting in an overall response rate of 33%. Multivariate analysis identified molecular-only relapse (hazard ratio [HR], 9.4; 95% confidence interval [CI], 2.0 to 43.5; P = .004) and diagnosis of MDS (HR, 4.1; 95% CI, 1.4 to 12.2; P = .011) as predictors for complete remission. Overall survival (OS) at 2 years was 29% ± 4%. Molecular-only relapse (HR, .14; 95% CI, .03 to .59; P = .007), diagnosis of MDS (HR, .33; 95% CI, .16 to .67; P = .002), and bone marrow blasts <13% (HR, .54; 95% CI, .32 to .91; P = .021) were associated with better OS. Accordingly, 2-year OS rate was higher in MDS patients (66% ± 10%, P = .001) and correlated with disease burden in patients with AML. In summary, Aza and DLI is an effective and well-tolerated treatment option for patients with relapse after allo-HSCT, in particular those with MDS or AML and low disease burden. The latter finding emphasizes the importance of stringent disease monitoring and early intervention.

Angiotensin-(1–7) stimulates hematopoietic progenitor cells in vitro and in vivo

This study demonstrates that the angiotensin II metabolite Ang-(1-7) stimulates the proliferation of hematopoietic progenitor cells and promotes their engraftment in a xenograft model, suggesting that the renin-angiotensin system is a regulator of blood cell formation. See related perspective article on page 745. Effects of angiotensin (Ang)-(1–7), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1–7) to stimulate proliferation of human CD34+ and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1–7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I+ and CD34+ cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(1–7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.