Justin Hasenkamp

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.

Rituximab after lymphoma-directed conditioning and allogeneic stem-cell transplantation for relapsed and refractory aggressive non-Hodgkin lymphoma (DSHNHL R3): an open-label, randomised, phase 2 trial

BACKGROUND Allogeneic stem-cell transplantation has had limited success for patients with refractory and relapsed aggressive B-cell or T-cell lymphoma. We investigated the effect of adding rituximab to standard prophylaxis for graft-versus-host disease after transplantation and estimated overall survival when using a lymphoma-directed myeloablative conditioning regimen. METHODS We did this randomised, open-label, phase 2 study at seven German transplantation centres. We enrolled patients with aggressive B-cell or T-cell lymphoma and primary refractory disease, early relapse (<12 months after first-line treatment), or relapse after autologous transplantation. Conditioning with fludarabine (125 mg/m(2)), busulfan (12 mg/kg oral or 9·6 mg/kg intravenous), and cyclophosphamide (120 mg/kg) was followed by allogeneic stem-cell transplantation. Patients were randomly assigned (1:1) to receive rituximab (375 mg/m(2) on days 21, 28, 35, 42, 175, 182, 189, and 196) or not. Allocation was done with a centralised computer-generated procedure; patients were stratified by histological subtype (B-cell vs T-cell lymphoma) and donor match (HLA-identical vs non-identical). Neither investigators nor patients were masked to allocation. The primary endpoints were the incidence of acute graft-versus-host disease grade 2-4 in each treatment group and overall survival at 1 year in both groups combined. All analyses were done for the intention-to-treat population. The study is registered with ClinicalTrials.gov, number NCT00785330. FINDINGS Between June 16, 2004, and March 24, 2009, we screened 86 patients and enrolled 84; 42 were randomly assigned to each group. The cumulative incidence of grade 2-4 acute graft-versus-host disease was 46% (95% CI 32-62) in the rituximab group and 42% (95% CI 29-59) in the no rituximab group (hazard ratio [HR] 0·91, 95% CI 0·52-1·60; p=0·74). Overall survival at 1 year for the whole study population was 52% (95% CI 41-62). Grade 4 haematological toxic effects and grade 3 alopecia occurred in all patients. The most common non-haematological grade 5 toxic effects were pneumonia (nine in the no rituximab group vs ten in the rituximab group) and other infections (seven vs four). INTERPRETATION The lymphoma-directed myeloablative conditioning regimen developed here is promising for patients with refractory and relapsed aggressive B-cell and T-cell lymphomas. However, the addition of rituximab did not affect the incidence of graft-versus-host disease or overall survival. FUNDING Hoffmann-La Roche, Amgen, Astellas Pharma.

Reduced intensity conditioning and allogeneic stem cell transplantation after salvage therapy integrating alemtuzumab for patients with relapsed peripheral T-cell non-Hodgkin's lymphoma

Reduced intensity conditioning and allogeneic stem cell transplantation after salvage therapy integrating alemtuzumab for patients with relapsed peripheral T-cell non-Hodgkins lymphoma

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression

OBJECTIVE Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells. MATERIALS AND METHODS A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)-defined NK cells from human leukocyte antigen (HLA)-typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay. RESULTS HLA, but not CD20 expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications. CONCLUSION KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA.

Reduced-intensity conditioning prior to allogeneic transplantation of hematopoietic stem cells: the need for T cells early after transplantation to induce a graft-versus-lymphoma effect

Summary:In patients with poor-risk relapse of aggressive lymphoma, reduced-intensity conditioning followed by allogeneic PBSCT may have its limitations because of rapid regrowth of the tumor. We tried to address this problem by intermediate-intensity conditioning followed by allogeneic SCT. A total of 21 patients received fludarabine, busulfan and cyclophosphamide prior to allogeneic SCT. In the first 10 patients, GVHD prophylaxis by CD34+ selection of the grafts was employed (group I). The next 11 patients received nonmanipulated grafts and mycophenolat mofetil plus cyclosporinA (group II). In group I, no GVHD was observed. In contrast, patients in group II had a significant risk of acute GVHD (aGVHD) (six patients with grade II–IV acute GVHD). However, in group I, all surviving patients progressed within 9 months. In contrast, eight of nine surviving patients of group II remain in remission after a median observation time of 10.5 months (range 4–22 months). Survival differed significantly between the groups (P=0.004). Multivariate analysis identified intensive GVHD prophylaxis as important risk factor for survival. These results support the existence of a clinically relevant GVL effect in aggressive lymphoma. T-cell depletion (or CD34 selection) of grafts is not recommended in patients with poor-risk aggressive NHL.

A molecular perspective on rituximab: A monoclonal antibody for B cell non Hodgkin lymphoma and other affections

Rituximab (a chimeric anti-CD20 monoclonal antibody) is the first Food and Drug Administration approved anti-tumor antibody. Immunotherapy by rituximab, especially in combination-therapy, is a mainstay for a vast variety of B-cell malignancies therapy. Its therapeutic value is unquestionable, yet the mechanisms of action responsible for anti-tumor activity of rituximab and rituximab resistance mechanisms are not completely understood. Investigation of the mechanisms of action that contribute to the rituximab activity have eventually directed to a suite of novel combinations and novel treatment schedules, and also have resulted new generations of antibodies with more desired effects. Although, further investigations are needed to define the mechanisms of rituximab resistance and prominent effector activity of the altered next generation anti-CD20 to improve their efficacies and develop new anti-CD20 monoclonal antibodies in NHL treatment. This article focuses on the properties of CD20 which led scientists to select it as an effective therapeutic target and the molecular details of mechanisms of rituximab action and resistance. We also discuss about the impact of rituximab in monotherapy and in combination with chemotherapy regimens. Finally, we comparatively summarize the next generations of anti CD20 monoclonal antibodies to highlight their advantages relative to their ancestor: Rituximab.

Resistance Against Natural Killer Cell Cytotoxicity: Analysis of Mechanisms

Target cell resistance against natural killer (NK) cell‐mediated cytotoxicity obstructs NK cell‐based immunotherapy of leukaemia. Several mechanisms of resistance have been described. Because of lack of simple assays for analysing these mechanisms, their relative impact on a given effector–target pair is mostly unknown. We here analysed the combination of the Granzyme B (GrB) enzyme‐linked immunospot assay (ELISPOT) for the assessment of NK cell reactivity and cytotoxicity assays to estimate target cell escape mechanisms. Target cell recognition failure leads to negative GrB ELISPOT results, whereas target cell resistance shows positive GrB ELISPOT results in the absence of cytotoxicity. We confronted NK cells with the sensitive target cell line K562, and with the resistant cell lines ML2, SupB15 and Raji. ML2 cells sufficiently activated GrB‐release whilst being resistant against cytotoxic granules of NK cells. Partial resistance of Raji results from the interaction of HLA class I with inhibitory killer immunglobulin‐like receptors (KIR) on the NK cells. Failure of target recognition by HLA class I–KIR interaction, lacking ligands to stimulatory NK cell receptors and partial resistance to cytotoxic granules all contributed to resistance of SupB15. In conclusion, revealing the mechanisms of resistance against NK cell‐mediated cytotoxicity may allow improving the results of NK‐based immunotherapy.

Self-tolerance of Human Natural Killer Cells Lacking Self-HLA-specific Inhibitory Receptors

Natural killer (NK) cells identify cells with altered human leucocyte antigen (HLA) expression as targets through lacking engagement of self‐HLA‐specific inhibitory receptors (e.g. killer cell immunoglobulin‐like receptor, KIR). Thus, they eliminate cells with ‘missing self’ because of viral or malignant transformation. We performed analysis of HLA, KIR genotypes and KIR receptor expression patterns at single cell level in NK cells in 17 donors. The function of NK cell subsets is determined by degranulation assays using target cells expressing self, cognate, control or no HLA class I. Donors could be grouped into three groups: their NK cells possess potential for alloreactivity, autoreactivity based on the presence of NK cells expressing particular KIR only (mono‐KIR) in the absence of its ligand or lack alloreactivity. All donors possess NK cells lacking all detectable inhibitory receptors. Both potential autoreactive subpopulations did not respond to HLA class I‐positive target cells. They retain partial reactivity against HLA class I‐negative tumour target cells. Mono‐KIR NK cells without the corresponding ligands in the individuals and NK cells lacking all inhibitory receptors behave self‐tolerant. Our results suggest alternative mechanisms than HLA‐specific inhibitory receptors to control NK cell activity. But HLA seems to be involved in shaping effector function of the NK cell repertoire.

Granulocytic Sarcoma by AML M4eo (inv16) after Allogeneic Stem Cell Transplantation without Bone Marrow Involvement

Granulocytic sarcoma (GS) represents a rare type of extramedullar manifestation from the acute myeloid leukaemia (AML). We report the case of a patient with recurrences of AML M4eo leukaemia in the uterus and the small intestine at 3 and 5 years, respectively, after matched related peripheral blood stem cell transplantation (PBSCT). The patient underwent the withdrawal of immunosuppression, hysterectomy, and local irradiation at first relapse, as well as systemic chemotherapy and donor lymphocyte infusions at second recurrence, inducing a second and third complete remission, respectively. At year six after transplantation, the patient experienced disease progression by meningeosis leukaemia to which she succumbed despite intrathecal chemotherapy. Following allogeneic stem cell transplantation, awareness for atypical manifestations of granulocytic sarcoma appears prudent, the cellular immunotherapy should aim at immunological disease control.

Progression of a CD4+/CD56+ blastic plasmacytoid DC neoplasm after initiation of extracorporeal photopheresis in an allogeneic transplant recipient

Progression of a CD4+/CD56+ blastic plasmacytoid DC neoplasm after initiation of extracorporeal photopheresis in an allogeneic transplant recipient