High-dose chemotherapy with autologous haematopoietic stem cell support for relapsed or refractory primary CNS lymphoma: a prospective multicentre trial by the German Cooperative PCNSL study group.

To investigate safety and efficacy of high-dose chemotherapy followed by autologous stem cell transplantation (HCT-ASCT) in relapsed/refractory (r/r) primary central nervous system lymphoma (PCNSL), we conducted a single-arm multicentre study for immunocompetent patients (<66 years) with PCNSL failing high-dose methotrexate)-based chemotherapy. Induction consisted of two courses of rituximab (375 mg/m2), high-dose cytarabine (2 × 3 g/m2) and thiotepa (40 mg/m2) with collection of stem cells in between. Conditioning for HCT-ASCT consisted of rituximab 375 mg/m2, carmustine 400 mg/m2 and thiotepa (4 × 5 mg/kg). Patients commenced HCT-ASCT irrespective of response after induction. Patients not achieving complete remission (CR) after HCT-ASCT received whole-brain radiotherapy. Primary end point was CR after HCT-ASCT. We enrolled 39 patients; median age and Karnofsky performance score are 57 years and 90%, respectively. About 28 patients had relapsed and 8 refractory disease. About 22 patients responded to induction and 32 patients commenced HCT-ASCT. About 22 patients (56.4%) achieved CR after HCT-ASCT. Respective 2-year progression-free survival (PFS) and overall survival (OS) rates were 46.0% (median PFS 12.4 months) and 56.4%; median OS not reached. We recorded four treatment-related deaths. Thiotepa-based HCT-ASCT is an effective treatment option in eligible patients with r/r PCNSL. Comparative studies are needed to further scrutinise the role of HCT-ASCT in the salvage setting.

Does ex vivo CD34+ positive selection influence outcome after autologous hematopoietic stem cell transplantation in systemic sclerosis patients?

This EBMT Autoimmune Disease Working Party study aimed to evaluate the influence of CD34+ positive graft selection (CD34+) on the outcome of systemic sclerosis (SSc) patients after autologous hematopoietic stem cell transplantation (AHSCT). Clinical and laboratory data from 138 SSc patients at diagnosis, before and after AHSCT were retrospectively analyzed. CD34+ selection was performed in 47.1% (n=65) patients. By multivariate analysis adjusting for all factors differing between the two groups (without or with CD34+), there was no statistically significant difference in terms of overall survival (hazard ratio (HR): 0.98, 95% confidence interval (CI) 0.40-2.39, P=0.96), PFS (HR: 1.55, 95% CI 0.83-2.88, P=0.17) and incidence of relapse or progression (HR: 1.70, 95% CI 0.85-3.38, P=0.13). We demonstrate that CD34+ does not add benefit to the outcome of SSc patient treated with AHSCT. These findings should be further confirmed by prospective randomized trials.

[Special hematological diagnostics and therapy options for ocular lymphoma taking CNS involvement into account]. / Hämatologische Spezialdiagnostik und Therapieoptionen bei okulären Lymphomen unter Berücksichtigung einer ZNS-Beteiligung.

BACKGROUND: Intraocular lymphomas are very rare and occur as either vitreoretinal or uveal tumors. Management in the clinical routine is highly variable and controversial. OBJECTIVES: To present the most important aspects of the diagnostics and therapy from the perspective of hematological oncologists and formulate management recommendations. METHODS: The English language literature was reviewed and the most important data were analyzed for presentation. RESULTS: In patients with vitreoretinal lymphoma evaluation for central nervous system (CNS) involvement should be performed due to its strong association with primary CNS lymphoma (PCNSL). The prognosis is relatively poor, particularly when the CNS is involved. Optimal therapy has not yet been established. For isolated vitreoretinal manifestations local therapy, such as intraocular methotrexate (MTX) or rituximab or radiation is recommended; however, there is a very high frequency of CNS relapse. Systemic high-dose MTX-based chemotherapy analogous to PCNSL treatment is an alternative option and is the treatment of choice in patients with simultaneous CNS and vitreoretinal lymphoma. Primary uveal lymphoma is usually an indolent lymphoma and treated by local therapy, whereas secondary uveal lymphoma predominantly occurs in aggressive systemic (non-CNS) lymphoma and is treated by systemic chemotherapy. DISCUSSION: Data on intraocular lymphoma are derived from small, usually retrospective and very heterogeneous studies with a relatively short follow-up. To gain more knowledge on this rare disease, inclusion of patients in the prospective registry, currently in progress in Germany, is desirable.

[Asymptomatic 32 year old female smoker with persistent polyclonal lymphocytosis]. / Asymptomatische 32-jährige Raucherin mit persistierender Lymphozytose.

A 32 year old female smoker (20 pack years) presented with an asymptomatic lymphocytosis of 13,000/nl and splenomegaly. The patient's blood smear showed an absolute lymphocytosis with 65% atypical lymphocytes. A total of 1% of the lymphocytes were bilobulated. Bone marrow histology and immunphenotyping of blood and bone marrow excluded leukemia and non-Hodgkin's lymphoma. IgH-CDR-3 PCR analysis revealed a polyclonal pattern. In summary, a persistent polyclonal B-cell-lymphocytosis (PPBL) was diagnosed. The exact etiology of PPBL is still unclear, however, it is associated with a polyclonal raise in the lymphocyte count of CD27+IgD+-memory-B-lymphocytes due to a defect in apoptosis signaling and leukocyte homing to secondary lymphoid tissues. An association with cigarette smoking is obvious since all patients are smokers. From all published cases, only two developed a malignancy with an uncertain association with PPBL. We have been monitoring our patient for 6.5 years without any evidence of the development of a lymphoma.

Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia.

Prognostic predictions in B-cell chronic lymphocytic leukemia (B-CLL) at early clinical stage are based on biological disease parameters, such as ZAP-70 and CD38 protein levels, genomic aberrations as well as immunoglobulin variable heavy chain gene (IgV(H)) mutation status. In the current study, ZAP-70 and CD38 expressions were examined by flow cytometry in 252 patients with B-CLL. Cytoplasmic ZAP-70 expression in more than 20% (ZAP-70(+)) and surface CD38 expression on more than 30% (CD38(+)) of B-CLL cells were associated with an unfavorable clinical course. The levels of ZAP-70 and CD38 did not change over time in the majority of patients where sequential samples were available for analysis. Combined analysis of ZAP-70 and CD38 yielded discordant results in 73 patients (29.0%), whereas 120 patients (47.6%) were concordantly negative and 59 patients (23.4%) were concordantly positive for ZAP-70 and CD38 expression. Median treatment-free survival times in patients whose leukemic cells were ZAP-70(+)CD38(+) was 30 months as compared to 130 months in patients with a ZAP-70(-)CD38(-) status. In patients with discordant ZAP-70/CD38 results, the median treatment-free survival time was 43 months. Thus, ZAP-70 and CD38 expression analyses provided complementary prognostic information identifying three patient subgroups with good, intermediate and poor prognosis. Over-representation of high-risk genomic aberrations such as 17p deletion or 11q deletion and distribution of the IgV(H) mutation status in B-CLL discordant for ZAP-70/CD38 pointed toward a distinct biologic background of the observed disease subgroups. This finding was also supported by microarray-based gene expression profiling in a subset of 35 patients. The expression of 37 genes differed significantly between the three groups defined by their expression of ZAP-70 and CD38, including genes that are involved in regulation of cell survival and chemotherapy resistance.

Lentiviral transduction of human T-lymphocytes with a RANTES intrakine inhibits human immunodeficiency virus type 1 infection.

Intrakines, modified intracellular chemokines, offer a novel strategy to prevent cellular entry of HIV-1 by blocking the surface expression of HIV-1 co-receptors. To investigate potential clinical applications of the RANTES-intrakine, we explored the use of HIV-1-based lentiviral vectors for therapeutic gene transfer into T-lymphocytes. RANTES-intrakine genes can be efficiently transduced into primary human T-lymphocytes by lentiviral vectors, especially when human T-lymphocytes were stimulated with CD3 and CD28 antibodies. The transduced T cells showed decreased surface expression of the chemokine receptor CCR-5, as well as CCR-1 and CCR-3. This lentivirus-mediated approach to intrakine gene transfer protected human T-lymphocytes from infection by a variety of R5-tropic HIV-1 strains. A quantitative real-time PCR assay, developed to monitor cells for HIV entry and persistence, revealed persistent low copy numbers of proviral HIV DNA in RANTES intrakine-transduced T-lymphocytes during 3-week culture, suggesting that viruses produced from infected untransduced cell populations were unable to infect the surrounding transduced T-lymphocytes. We conclude that targeting HIV-1 co-receptors to block virus entry with lentiviral vectors is an attractive approach to the control of HIV-1 infection.

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells.

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.

Transfection of dendritic cells (DCs) with the CIITA gene: increase in immunostimulatory activity of DCs.

Dendritic cells (DCs) are the major antigen-presenting cells. They are able to present tumor antigens to immunologic effector cells. MHC class II molecules on DC surfaces play an important role in priming effector cells against tumor cells and their antigens. The transactivator CIITA (MHC class II transactivator) is a non-DNA-binding transactivator, which regulates the expression of MHC class II, HLA-DM, and invariant chain and behaves as a master controller of constitutive and inducible MHC class II gene activation. Here, we transfected DCs with the CIITA gene using a novel transfection technique. The vector system consisted of a plasmid bound to an adenovirus via poly-L-lysine, which is covalently bound to a UV-irradiated adenovirus. After transfection, expression of MHC class II on DCs increased from 27% to 75% on day 2 after transfection. Transfected DCs were co-cultured with immunologic effector cells. Cytotoxicity of effector cells against tumor cells increased after co-culture with transfected DCs to 63% compared to 15% with effector cells co-cultured with irrelevantly transfected DCs (P=.037). This effect was dependent on the timing and period of co-culture. In conclusion, transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs. We can further conclude that DCs could be efficiently transfected with the CIITA gene. Transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs and may have a major impact on immunotherapeutic protocols for patients with cancer.

Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system.

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.