Jörg Schubert

Reduced Treatment Intensity in Patients with Early-Stage Hodgkin's Lymphoma

BACKGROUND Whether it is possible to reduce the intensity of treatment in early (stage I or II) Hodgkins lymphoma with a favorable prognosis remains unclear. We therefore conducted a multicenter, randomized trial comparing four treatment groups consisting of a combination chemotherapy regimen of two different intensities followed by involved-field radiation therapy at two different dose levels. METHODS We randomly assigned 1370 patients with newly diagnosed early-stage Hodgkins lymphoma with a favorable prognosis to one of four treatment groups: four cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by 30 Gy of radiation therapy (group 1), four cycles of ABVD followed by 20 Gy of radiation therapy (group 2), two cycles of ABVD followed by 30 Gy of radiation therapy (group 3), or two cycles of ABVD followed by 20 Gy of radiation therapy (group 4). The primary end point was freedom from treatment failure; secondary end points included efficacy and toxicity of treatment. RESULTS The two chemotherapy regimens did not differ significantly with respect to freedom from treatment failure (P=0.39) or overall survival (P=0.61). At 5 years, the rates of freedom from treatment failure were 93.0% (95% confidence interval [CI], 90.5 to 94.8) with the four-cycle ABVD regimen and 91.1% (95% CI, 88.3 to 93.2) with the two-cycle regimen. When the effects of 20-Gy and 30-Gy doses of radiation therapy were compared, there were also no significant differences in freedom from treatment failure (P=1.00) or overall survival (P=0.61). Adverse events and acute toxic effects of treatment were most common in the patients who received four cycles of ABVD and 30 Gy of radiation therapy (group 1). CONCLUSIONS In patients with early-stage Hodgkins lymphoma and a favorable prognosis, treatment with two cycles of ABVD followed by 20 Gy of involved-field radiation therapy is as effective as, and less toxic than, four cycles of ABVD followed by 30 Gy of involved-field radiation therapy. Long-term effects of these treatments have not yet been fully assessed. (Funded by the Deutsche Krebshilfe and the Swiss Federal Government; ClinicalTrials.gov number, NCT00265018.)

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.

Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes.

Abstract Membrane anchoring of cell surface proteins via glycosylphosphatidylinositol (GPI) occurs in all eukaryotic organisms. In addition, GPI-related glycophospholipids are important constituents of the glycan coat of certain protozoa. Defects in GPI biosynthesis can retard, if not abolish growth of these organisms. In humans, a defect in GPI biosynthesis can cause paroxysmal nocturnal hemoglobinuria (PNH), a severe acquired bone marrow disorder. Here, we review advances in the characterization of GPI biosynthesis in parasitic protozoa, yeast and mammalian cells. The GPI core structure as well as the major steps in its biosynthesis are conserved throughout evolution. However, there are significant biosynthetic differences between mammals and microbes. First indications are that these differences could be exploited as targets in the design of novel pharmacotherapeuticsthat selectively inhibit GPI biosynthesis in unicellular microbes.

Role of Radiotherapy to Bulky Disease in Elderly Patients With Aggressive B-Cell Lymphoma

PURPOSE R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) is standard care for aggressive B-cell lymphoma. A prospective trial was conducted to investigate the role of additive radiotherapy (RT) to bulky and extralymphatic disease. PATIENTS AND METHODS The best arm of the RICOVER-60 trial (6×R-CHOP-14+2R [R-CHOP administered once every 2 weeks plus two additional applications of rituximab] plus involved-field RT [36 Gy] to sites of initial bulky [≥ 7.5 cm] disease and extralymphatic involvement) was compared with a cohort receiving the same immunochemotherapy but without RT in an amendment to the RICOVER-60 trial (RICOVER-noRTh) in a prospective fashion. RESULTS After a median observation time of 39 months, 164 of 166 RICOVER-noRTh patients were evaluable. In a multivariable analysis of the intention-to-treat population adjusting for International Prognostic Index risk factors and age (> 70 years), event-free survival (EFS) of patients with bulky disease was inferior without additive RT (hazard ratio [HR], 2.1; 95% CI, 1.3 to 3.5; P = .005), with trends for inferior progression-free (PFS; HR, 1.8; 95% CI, 1.0 to 3.3; P = .058) and overall survival (OS; HR, 1.6; 95% CI, 0.9 to 3.1; P = .127). In a per-protocol analysis with 11 patients in RICOVER-noRTh excluded for receiving unplanned RT, multivariable analysis revealed HRs of 2.7 (95% CI, 1.3 to 5.9; P = .011) for EFS, 4.4 (95% CI, 1.8 to 10.6; P = .001) for PFS, and 4.3 (95% CI, 1.7 to 11.1; P = .002) for OS for patients not receiving RT to bulky disease. CONCLUSION Additive RT to bulky sites abrogates bulky disease as a risk factor and improves outcome of elderly patients with aggressive B-cell lymphoma. Whether RT can be spared in patients with (metabolic) complete remission after immunochemotherapy must be addressed in appropriately designed prospective trials.

Diagnosis of paroxysmal nocturnal haemoglobinuria using immunophenotyping of peripheral blood cells.

Paroxysmal nocturnal haemoglobinuria (PNH) is now generally accepted as a disease in which bone marrow derived cells are deficient in phosphatidylinositolglycan (PIG)‐anchored surface molecules. A series of new monoclonal antibodies detecting PIG‐anchored surface structures on human leucocytes (CD48, CD55, CD59) has recently been described. In the present study 12 patients with the diagnosis PNH and a positive Ham test were examined for PIG‐anchored surface antigen expression on various cell lineages using immunofluorescence. In all patients deficient cells were detected in erythrocyte, granulocyte and monocyte analysis. A deficient lymphocyte subset was also observed in all but one of these patients. Using two‐colour analysis, all lymphocyte subpopulations such as T, B and NK cells were found to be affected. In addition, peripheral blood cells of 22 patients with severe aplastic anaemia (SAA) were tested for the PIG‐anchoring defect. In five of these patients the defect was detected, and in four of the five the lack of PIG‐anchored molecules was confined to the granulocyte and monocyte lineages apparently without affecting the erythrocytes. The results of these studies demonstrate that cytofluorographic testing of peripheral blood cells provides a simple and reliable method for establishing the diagnosis of PNH. Furthermore, especially in the case of aplastic anaemia patients, the sensitivity of immunophenotyping might be superior to conventional laboratory tests.

Flares in Patients with Systemic Lupus erythematosus Are Associated with Daily Psychological Stress

Background: The aetiology of systemic lupus erythematosus (SLE) remains unclear. Clinical observations and a small number of studies performed so far suggest an association between psychological stress and self-reported symptoms of SLE patients. This longitudinal study was designed to investigate whether daily psychological stress is associated with flares in SLE patients, measured by clinical and laboratory parameters. Methods: Female SLE patients (n = 41) were followed over a period of six months. Daily stress was monitored by a hand-held PC diary programmed with 44 items based on standardized measures and clinical experience. Once every four weeks patients visited the outpatient clinic for medical evaluation. Disease activity was evaluated using the European Consensus Lupus Activity Measurement (ECLAM), laboratory parameters, and intake of steroids. Results: Classification and regression tree (CART) patient-wise analyses revealed that SLE patients with vs. without flares using complement and ECLAM as activity measures show greater negative self-ratings in mood, and social duties (p < 0.01). In addition, mixed model analysis of variance showed that daily hassles with social relationships were significantly associated with flares in SLE measured by an increase in steroid medication >5mg/d (p < 0.01). Conclusions: These results suggest that psychological stress is associated with flares in SLE. Particularly daily stress with social relationships and social duties may be factors to be related to the course of disease activity in SLE.

Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis.

The mammalian N-acetylglucosaminyl transferase for the first step in glycosylphosphatidylinositol biosynthesis has been shown to consist of at least four components: PIG-A, PIG-C, PIG-H and GPI1. Here, the enzymatic complex is further characterised. PIG-A protein, which is thought to represent the catalytic subunit of the complex, was expressed in an epitope-tagged form in the PIG-A deficient JY5 lymphoblastoid cell line. Subcellular localisation of this protein was studied using immunofluorescence and immunoelectron microscopy. The protein was localised to both perinuclear and mitochondria-associated lamellae of the endoplasmic reticulum. Using affinity chromatography, epitope-tagged PIG-A protein was partially purified. To identify regions that might be involved in the catalytic process, computer-aided comparison was performed between PIG-A and 26 distantly related glycosyl transferases. A number of residues in the membrane-proximal region of the cytoplasmic domain (230-340) were found highly conserved. Finally, a topological model of the four partners participating in the enzymatic complex is introduced to provide a working model for further structural and functional analysis.

In vitro generation of human CD86+ dendritic cells from CD34+ haematopoietic progenitors by PMA and in serum-free medium

The cytokine requirements to differentiate CD34+ progenitor cells from different origins either cord blood (CB) or peripheral blood (PB) into dendritic cells (DC) are known to be different. In addition to DC, macrophages and neutrophils are generated. On the other hand, phorbol esters such as PMA induce primary human CD34+ bone marrow (BM) progenitor cells to differentiate into functional DC and no other lineages are generated. In addition, FCS is used as culture supplement in most of the protocols described which contains additional foreign antigens potentially skewing the resulting immune response. Therefore, we evaluated the ability to differentiate CB‐ and PB‐CD34+ progenitor cells into DC with PMA and under serum‐free conditions. In this study, we delineate the maturation of cultured human blood DC by analysis of expression co‐stimulatory molecule B7–2 (CD86). Human mature DC with typical morphology and surface antigen phenotype (CD1a−, CD83+ and CD86+) were obtained from CB‐ and PB‐CD34+ progenitor cells after 1 week of culture in serum‐free medium upon stimulation with PMA alone. The same result was obtained from ex vivo‐expanded BM‐CD34+ cells. CD86+ yield was increased by PMA compared to cytokine cocktails (28·0% ± 7·0 versus 15·3% ± 5·6 for CB and 44·6% ± 7·5 versus 28·1% ± 7·5 for PB, respectively). CD86 was most up‐regulated in the presence of the calcium ionophore ionomycin. However, the number of viable cells after differentiation was decreased by PMA plus ionomycin (P < 0·05) or plus TNF‐alpha (P > 0·05) as compared with that in PMA alone. We conclude that PMA is a potent activator to differentiate human CD34+ cells into mature DC in serum‐free medium. This may be used for in vitro studies of primed or genetically modified DC against infectious and tumour‐associated antigens.

Glycosylphosphatidylinositol (GPI)-deficient Jurkat T cells as a model to study functions of GPI-anchored proteins

Many cell surface proteins attached to the membrane by GPI are involved in cell signalling. However, the role of the GPI membrane anchor itself remains poorly understood. GPI‐defective cells from patients with paroxysmal nocturnal haemoglobinuria (PNH) are relatively resistant to apoptosis induction. We developed a Jurkat T cell model for GPI deficiency by isolating a GPI‐negative mutant, which is defective in the GPI biosynthetic gene PIG‐A. Using retroviral PIG‐A gene transfer along with the transfer of a vector control, we obtained two genetically identical cell lines, distinguished only by expression of the PIG‐A gene and, thus, their ability to produce GPI. Cell proliferation and survival were not affected by this difference. Apoptotic stimuli such as serum starvation and camptothecin exposure elicited similar responses. In contrast, GPI‐defective Jurkat cells were more susceptible to Fas‐mediated apoptosis than GPI‐positive cells. These results indicate that a deficiency in GPI‐anchored proteins, as is found in PNH, does not confer resistance to apoptosis.

Paroxysmal nocturnal hemoglobinuria: Differential gene expression of EGR-1 and TAXREB107

OBJECTIVE Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells characterized by deficiency in GPI-anchored surface proteins. It is not yet known how GPI-deficient stem cells are able to expand within the bone marrow and contribute considerably to the hematopoiesis. In PNH, as well as in AA and MDS, genetic instability and increased mutation frequency have been detected. Therefore, a second event is very likely, such as additional mutations, leading to clonal expansion of GPI-deficient bone marrow stem cell in PNH. METHODS In order to elucidate the molecular basis of clonal expansion in PNH, we identified several genes differentially expressed in normal and GPI-deficient cells of PNH patients by combination of RNA fingerprinting and cDNA array hybridization. RESULTS Expression of two of these genes, EGR-1 and TAXREB107, has been further investigated. EGR-1 is upregulated in granulocytes of all PNH patients analyzed so far. In contrast, significant upregulation of TAXREB107 is present only in some of our PNH patients. Further analysis confirmed their overexpression in PNH and excluded a possible secondary event character of observed overexpression. Moreover, similar levels of expression in cases of other clonal diseases, such as MPS and MDS, has been identified. CONCLUSION Our data suggest that additional genetic alterations apart from PIG-A mutations could be present in PNH granulocytes. In addition, these genetic changes might contribute to clonal expansion of GPI-deficient cells in PNH.