Bendamustine plus rituximab versus fludarabine plus rituximab for patients with relapsed indolent and mantle-cell lymphomas: a multicentre, randomised, open-label, non-inferiority phase 3 trial.

BACKGROUND: Fludarabine-based chemoimmunotherapy with rituximab is frequently used in patients with indolent and mantle-cell lymphomas who relapse after alkylating chemotherapy. We aimed to compare the efficacy and safety of rituximab with bendamustine or fludarabine in patients with relapsed, indolent, non-Hodgkin lymphoma and mantle-cell lymphoma. METHODS: For this randomised, non-inferiority, open-label, phase 3 trial, we recruited patients from 55 centres in Germany, who were subsequently randomised centrally according to prespecified randomisation lists with permuted blocks of randomly variable block size to rituximab (375 mg/m(2), day 1) plus either bendamustine (90 mg/m(2), days 1 and 2) or fludarabine (25 mg/m(2), days 1-3) every 28 days for a maximum of six 28-day cycles. Patients were aged 18 years or older with a WHO performance status of 0-2 and had relapsed or refractory indolent or mantle-cell lymphoma; patients refractory to regimens that included rituximab, bendamustine, or purine analogue drugs were excluded. Patients were stratified by histological subtypes of lymphoma and by their latest previous therapies. Treatment allocation was not masked. The primary endpoint was progression-free survival and the final analysis was completed per protocol. Non-inferiority of bendamustine plus rituximab versus fludarabine plus rituximab was defined as a difference of less than 15% in 1-year progression-free survival. The protocol was amended in July, 2006, after approval of rituximab maintenance (375 mg/m(2) every 3 months for up to 2 years), which was then given to patients achieving a response to either trial treatment. This study is registered with ClinicalTrials.gov, number NCT01456351 (closed to enrolment, follow-up is ongoing). FINDINGS: Between Oct 8, 2003, and Aug 5, 2010, we randomly assigned 230 patients to treatment groups (116 bendamustine plus rituximab, 114 fludarabine plus rituximab). 11 patients were excluded for protocol violations and were not followed up further (two in the bendamustine plus rituximab group and nine in the fludarabine plus rituximab group). Thus, 219 patients were included in the per-protocol analysis (114 bendamustine plus rituximab, 105 fludarabine plus rituximab). 1-year progression-free survival with bendamustine plus rituximab was 0·76 (95% CI 0·68-0·84) and 0·48 (0·39-0·58) with fludarabine plus rituximab (non-inferiority p<0·0001). At a median follow-up of 96 months (IQR 73·2-112·9), median progression-free survival with bendamustine plus rituximab was 34·2 months (95% CI 23·5-52·7) and 11·7 months (8·0-16·1) with fludarabine plus rituximab (hazard ratio [HR] 0·54 [95% CI 0·38-0·72], log-rank test p<0·0001). Safety outcomes were similar in both groups, with 46 serious adverse events recorded (23 in the bendamustine plus rituximab group and 23 in the fludarabine plus rituximab group), most commonly myelosuppression and infections. INTERPRETATION: In combination with rituximab, bendamustine was more effective than fludarabine, suggesting that bendamustine plus rituximab may be the preferred treatment option for patients with relapsed indolent and mantle-cell lymphomas. FUNDING: Roche Pharma AG, Ribosepharm GmbH, Mundipharma GmbH, Studiengruppe indolente Lymphome (StiL).

Rituximab combined with DexaBEAM followed by high dose therapy as salvage therapy in patients with relapsed or refractory B-cell lymphoma: mature results of a phase II multicentre study.

Salvage therapy followed by high-dose therapy (HDT) remains a mainstay for patients with relapsed lymphoma, however no optimal regimen has been defined. Here we report on the results of R-DexaBEAM (rituximab, dexamethasone, carmustine, etoposide, cytarabine, melphalan) followed by HDT. Patients aged 18-65 years, Eastern Cooperative Oncology Group performance score 0-2, with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) were eligible. R-Dexa-BEAM was given for two cycles followed by stem cell mobilization and HDT. Primary endpoint of the trial was progression-free-survival (PFS). One hundred and three patients were included: aggressive NHL (aNHL): diffuse large B-cell lymphoma 55, mantle cell lymphoma 7, follicular lymphoma (FL) grade 3: 5, indolent Lymphoma (iNHL): FL grade 1-2: 29, marginal zone lymphoma 6, Immunocytoma 1. The overall response rate after salvage therapy was 62% for aNHL and 78% for iNHL patients. 66% of patients with aNHL and 86% with iNHL underwent HDT. Treatment-related mortality for HDT was 1·3%. For aNHL patients, the median PFS was 0·83 years with 44% alive at the median follow-up of 7·3 years. Corresponding figures for iNHL were: median PFS 3·7 years and 72% alive after 8 years. The combination of rituximab with DexaBEAM followed by HDT resulted in high response rates and sustained remissions in responders. R-DexaBEAM followed by HDT can be considered a valid salvage option for NHL.

Bendamustine plus rituximab versus CHOP plus rituximab as first-line treatment for patients with indolent and mantle-cell lymphomas: an open-label, multicentre, randomised, phase 3 non-inferiority trial.

BACKGROUND: Rituximab plus chemotherapy, most often CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), is the first-line standard of care for patients with advanced indolent lymphoma, and for elderly patients with mantle-cell lymphoma. Bendamustine plus rituximab is effective for relapsed or refractory disease. We compared bendamustine plus rituximab with CHOP plus rituximab (R-CHOP) as first-line treatment for patients with indolent and mantle-cell lymphomas. METHODS: We did a prospective, multicentre, randomised, open-label, non-inferiority trial at 81 centres in Germany between Sept 1, 2003, and Aug 31, 2008. Patients aged 18 years or older with a WHO performance status of 2 or less were eligible if they had newly diagnosed stage III or IV indolent or mantle-cell lymphoma. Patients were stratified by histological lymphoma subtype, then randomly assigned according to a prespecified randomisation list to receive either intravenous bendamustine (90 mg/m(2) on days 1 and 2 of a 4-week cycle) or CHOP (cycles every 3 weeks of cyclophosphamide 750 mg/m(2), doxorubicin 50 mg/m(2), and vincristine 1.4 mg/m(2) on day 1, and prednisone 100 mg/day for 5 days) for a maximum of six cycles. Patients in both groups received rituximab 375 mg/m(2) on day 1 of each cycle. Patients and treating physicians were not masked to treatment allocation. The primary endpoint was progression-free survival, with a non-inferiority margin of 10%. Analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00991211, and the Federal Institute for Drugs and Medical Devices of Germany, BfArM 4021335. FINDINGS: 274 patients were assigned to bendamustine plus rituximab (261 assessed) and 275 to R-CHOP (253 assessed). At median follow-up of 45 months (IQR 25-57), median progression-free survival was significantly longer in the bendamustine plus rituximab group than in the R-CHOP group (69.5 months [26.1 to not yet reached] vs 31.2 months [15.2-65.7]; hazard ratio 0.58, 95% CI 0.44-0.74; p<0.0001). Bendamustine plus rituximab was better tolerated than R-CHOP, with lower rates of alopecia (0 patients vs 245 (100%) of 245 patients who recieved ≥3 cycles; p<0.0001), haematological toxicity (77 [30%] vs 173 [68%]; p<0.0001), infections (96 [37%] vs 127 [50%]); p=0.0025), peripheral neuropathy (18 [7%] vs 73 [29%]; p<0.0001), and stomatitis (16 [6%] vs 47 [19%]; p<0.0001). Erythematous skin reactions were more common in patients in the bendamustine plus rituximab group than in those in the R-CHOP group (42 [16%] vs 23 [9%]; p=0.024). INTERPRETATION: In patients with previously untreated indolent lymphoma, bendamustine plus rituximab can be considered as a preferred first-line treatment approach to R-CHOP because of increased progression-free survival and fewer toxic effects. FUNDING: Roche Pharma AG, Ribosepharm/Mundipharma GmbH.

Tumor-reactive CD8+ T-cell responses after vaccination with NY-ESO-1 peptide, CpG 7909 and Montanide ISA-51: association with survival.

Peptide-based vaccines have led to the induction of antigen-specific CD8(+) T-cell responses in patients with NY-ESO-1 positive cancers. However, vaccine-induced T-cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl-deoxyguanosin oligodeoxy-nucleotides) mixed with NY-ESO-1 peptide p157-165 and incomplete Freund's adjuvants (Montanide(R) ISA-51) led to enhanced NY-ESO-1 antigen-specific CD8(+) immune responses in patients with NY-ESO-1 or LAGE-1 expressing tumors. Of 14 HLA-A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen-specific CD8(+) T-cell responses: Four had detectable CD8+ T-cells against NY-ESO-1 after only 2 vaccinations, whereas 5 patients showed a late-onset but durable induction of NY-ESO-1 p157-165 specific T-cell response during continued vaccination after 4 months. In 6 patients, vaccine-induced antigen-specific T-cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8(+) T-cells. Postvaccine T-cell clones were shown to recognize and lyse NY-ESO-1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY-ESO-1-specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.

LUD 00-009: phase 1 study of intensive course immunization with NY-ESO-1 peptides in HLA-A2 positive patients with NY-ESO-1-expressing cancer.

NY-ESO-1 is a cancer-testis antigen and an attractive target for immunotherapy in patients with different malignancies. Here we report the results of a phase I clinical study of intensive course NY-ESO-1 peptide vaccination, evaluating the safety, immunogenicity and clinical response in HLA-A2 positive patients with NY-ESO-1 expressing cancers. Of 20 patients enrolled in the trial, 14 completed at least 2 cycles of immunization and were evaluable for clinical and immunological response. Five of these evaluable patients were treated in cohort 1 (baseline seropositive) and 9 patients were treated in cohort 2 (baseline seronegative). During vaccination, NY-ESO-1-specific CD8+ T-cells were induced in 3 of 9 baseline seronegative patients. In patients with pre-existing antigen-specific CD8+ T-cells, their number increased or remained stable. In contrast to previous immunization protocols with less intensive immunization schedules, we observed a rapid induction of high magnitude NY-ESO-1 peptide-specific T-cell responses detectable already on day 15-22 of immunization. A specific immune response of high magnitude and early onset may be more effective in eliminating minimal residual disease in adjuvant treatment situations and in preventing tumor progression due to immune escape mechanisms.

Dexa-BEAM as salvage therapy in patients with primary refractory aggressive non-Hodgkin lymphoma.

Although aggressive NHL in relapse after remission can still be cured by second-line treatment followed by high-dose therapy and autologous stem cell transplantation, the long-term prognosis of patients who fail to obtain remission after first-line therapy remains extremely poor. We retrospectively evaluated a series of 29 consecutive patients with primary refractory high-grade NHL who were treated with Dexa-BEAM (DB) as uniform salvage therapy at a single institution. Twenty-nine patients with aggressive NHL primary refractory to CHOP or CHOP-like induction therapy with a median age of 47 (range, 22 - 64) years received 1 - 2 cycles of DB and were candidates for subsequent autologous stem cell (PBSC) mobilization and transplantation (PBSCT). Follow-up of all patients was updated in March 2004. Eight of 29 patients (28%) responded to one cycle of DB (1 complete/7 partial remissions); 2 of whom are alive after PBSCT (1 autologous/1 matched unrelated donor), 1 patient died after autologous PBSCT. Reasons for failure to proceed to high-dose therapy in spite of response to DB were recurrent progressive disease (n = 2), septicemia (n = 1), and allogeneic transplant-related mortality after mobilization failure to DB (n = 2). Twenty-one patients failed to respond to DB and died of progressive disease. Overall survival was 7% after 41 months. We conclude that Dexa-BEAM salvage therapy is not effective in patients with truly primary refractory high-grade NHL. The efficiency of rituximab combined with Dexa-BEAM or novel chemotherapeutic strategies needs to be established.

Intratumoral T-cell infiltrates and MHC class I expression in patients with stage IV melanoma.

The infiltration of tumors by T cells has been shown to correlate with prolonged patients' survival. However, it remains unclear why only some tumors are infiltrated with T cells. This study was designed to investigate possible correlations between intratumoral T-cell infiltrates and the expression of cancer-associated antigens and MHC class I and II molecules in patients with melanoma. Fresh frozen samples from 124 stage IV melanoma patients were analyzed by immunohistochemistry for the expression of Melan-A/MART-1, tyrosinase, gp100, NY-ESO-1, and MHC class I and II. Intratumoral T-cell and B-cell infiltrates were detected by staining with anti-CD4, anti-CD8, anti-CD3, and L26 antibodies. The NY-ESO-1 serum antibody status was assessed by Western blot analysis. Intratumoral CD8+ and CD4+ T cells were detected in 63.9% and 71.3% of patients, respectively. We observed a significant heterogeneity of the expression of the melanocyte differentiation antigens, NY-ESO-1, and MHC class I and II molecules. The only significant correlation was found between the expression of MHC class I and the presence of CD4+ and CD8+ T cells (P < 0.0001). There was a strong association between these two variables with respect to the density and distribution of infiltrating T cells and the pattern of MHC class I expression (focal versus homogenous). Intratumoral T-cell infiltration is closely correlated with the MHC class I expression but not with the expression of differentiation antigens, cancer-associated antigens, or MHC class II molecules. These results may have implications for the definition of prognostic variables and for the identification of patients who may benefit from antigen-specific cancer immunotherapy.

Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: Anaplastic large cell lymphoma (ALCL) and classical Hodgkin's lymphoma (HL) are derived from different cell types, namely T cells and B cells, respectively. However, both lymphomas share a similar cytological and immunohistochemical tumor cell phenotype with little resemblance to their cells of origin. DESIGN AND METHODS: In this study, the transcriptional profiles of ALCL cell lines, primary ALCL tumor cells from peripheral blood and HL cell lines were compared to each other and to normal B-cell subsets, B non-Hodgkin's lymphomas (NHL) and B NHL- and Epstein-Barr virus (EBV)-transformed B-cell lines in order to establish their relationship at the transcriptional level and to identify genes with possible pathobiological impact. Expression of some of the genes identified was confirmed in microdissected primary tumor cells by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: HL samples clustered separately from ALCL samples, but HL and ALCL were found to be more closely related to each other than to any normal or malignant B-cell sample in the dataset. Their relationship was determined to a large extent, but not exclusively, by lack of expression of B-cell antigens and by the over-expression of mRNA encoding activation markers and structural proteins. Apart from established differences between HL and ALCL, further genes of interest could be identified that distinguish both entities from each other and from the other samples. The differential expression of PRAME, DDR2, SOCS3 and CEBPD in HL and ALCL was confirmed in primary tumor tissue by immunohistochemistry and/or RT-PCR. INTERPRETATION AND CONCLUSIONS: At a transcriptional level HL is more closely related to Alk+ ALCL than to the B-NHL or B-cell samples investigated, although it is a B-cell derived lymphoma. The newly identified genes discriminating HL and ALCL may be pathobiologically important and may serve as possible therapeutic targets.

Daxx overexpression in T-lymphoblastic Jurkat cells enhances caspase-dependent death receptor- and drug-induced apoptosis in distinct ways.

The role of Daxx, in particular, its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in apoptosis signaling of malignant lymphocytes, Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. We thus demonstrate that ectopic expression of Daxx substantially increases the rate of apoptosis upon incubation with death receptor agonists such as Fas and TRAIL as well as upon incubation with the cytotoxic drug doxorubicin (DOX). Analysis of the molecular changes induced in the extrinsic and intrinsic apoptosis pathways reveals that augmentation of apoptosis by Daxx overexpression is conveyed by distinctly different mechanisms. Although enforced apoptosis caused by ectopic Daxx expression is caspase-dependent in both cases, major differences between Fas/TRAIL-induced apoptosis and doxorubicin-induced apoptosis are observed in expression patterns of X-linked inhibitor of apoptosis (XIAP), p53, Bid, ZIP kinase, and prostate apoptosis response gene 4 (Par-4). Moreover, we could show that addition of a CD95 blocking antibody to the clones treated with doxorubicin was able to increase apoptosis as compared to doxorubicin treatment alone and was accompanied by an enhancement of the mitochondrial branch of apoptosis. In conclusion, we here outline the major molecular mechanisms underlying the apoptosis-promoting effect of Daxx in neoplastic lymphocytes and demonstrate fundamental molecular differences elicited by the overexpression of Daxx in the extrinsic and intrinsic signaling pathways.

In lymphatic cells par-4 sensitizes to apoptosis by down-regulating bcl-2 and promoting disruption of mitochondrial membrane potential and caspase activation.

Inhibition of apoptosis is a hallmark of malignancies of the hematopoetic system. Previous studies in nonhematopoetic cells demonstrated that the prostate-apoptosis-response-gene-4 (Par-4) is up-regulated in cells undergoing programmed cell death and that Par-4 exerts its proapoptotic effect by down-regulating Bcl-2. After showing the aberrant expressional pattern of Par-4 in neoplastic lymphocytes as well as demonstrating inverse expressional patterns of Par-4 and Bcl-2 in malignant cells of patients suffering from acute lymphocytic leukemia, we assessed the functional consequences of Par-4 overexpression during apoptosis in Jurkat T lymphocytes. We show that in lymphatic cells Par-4 overexpression decreases the level of Bcl-2, whereas Bax, the proapoptotic counterpart of Bcl-2, retains unaltered levels. Moreover, Par-4 overexpression is accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). Despite these effects, overexpression of Par-4 alone is not sufficient to induce apoptosis but markedly increases the rate of apoptosis on treatment with different chemotherapeutic agents. On chemotherapeutic treatment Par-4 overexpression enhances disruption of mitochondrial membrane potential, PARP-cleaving activity, as well as activation of caspase-3. The hypothesis of caspase-dependency of Par-4-promoted apoptosis is additionally supported by demonstrating complete abrogation of programmed cell death after pretreatment with a broad spectrum caspase-inhibitor. On inhibition of caspase-3 overexpression of Par-4 enables lymphatic cells to alternatively activate caspases-9, -6, and -7 by diminishing the influence of the inhibitors of apoptosis proteins (IAPs) cIAP1 and XIAP. Our study is the first to identify Par-4 as a proapoptotic protein in lymphatic cells, outlining a model of action evaluating the role of Bcl-2/Bax, as well as demonstrating the impact of Par-4 expression on PARP cleavage, disruption of mitochondrial membrane potential, caspase activation, and interactions with inhibitors of apoptosis proteins.

Optimal use of bendamustine in hematologic disorders: Treatment recommendations from an international consensus panel - an update.

Bendamustine has achieved widespread international regulatory approval and is a standard agent for the treatment for chronic lymphocytic leukemia (CLL), indolent non-Hodgkin lymphoma and multiple myeloma. Since approval, the number of indications for bendamustine has expanded to include aggressive non-Hodgkin lymphoma and Hodgkin lymphoma and novel targeted therapies, based on new bendamustine regimens/combinations, are being developed against CLL and lymphomas. In 2010, an international panel of bendamustine experts met and published a set of recommendations on the safe and effective use of bendamustine in patients suffering from hematologic disorders. In 2014, this panel met again to update these recommendations since the clarification of issues including optimal dosing and management of bendamustine-related toxicities. The aim of this report is to communicate the latest consensus on the use of bendamustine, permitting the expansion of its safe and effective administration, particularly in new combination therapies.

Prostate apoptosis response gene-4 (par-4) abrogates the survival function of p185(BCR-ABL) in hematopoietic cells.

OBJECTIVE: Prostate apoptosis response gene-4 (par-4) is deregulated in acute and chronic lymphatic leukemia. Given its pro-apoptotic role in neoplastic lymphocytes and evidence that par-4 antagonizes oncogenic Ras in solid tumors, we hypothesized that par-4 may act as a tumor suppressor impairing transformation induced by p185(BCR-ABL). MATERIALS AND METHODS: The capacity of par-4 to interfere with factor independence induced by p185(BCR-ABL) and V12ras was evaluated by analysis of factor-independent growth of p185(BCR-ABL)/ par-4 and V12ras/par-4 transduced cells. The expression of par-4 and p185(BCR-ABL) by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185(BCR-ABL) in the absence and presence of par-4. RESULTS: Expression of p185(BCR-ABL) causes factor independence, signifying a conversion toward a transformed phenotype in hematopoietic precursors. We demonstrate that par-4 completely abolishes factor independence induced by p185(BCR-ABL) and partially abrogates factor independence caused by activated V12ras. Evaluating the underlying molecular mechanisms, we show that par-4 hinders activation of oncogenic Ras and causes concomitant disruptions of p185(BCR-ABL)-mediated signaling. CONCLUSION: We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185(BCR-ABL)-induced factor-independent proliferation in hematopoietic cells.

In vitro studies with bendamustine: enhanced activity in combination with rituximab.

Studies in vitro have shown that bendamustine, given as a monotherapy or in combination, can induce apoptosis in many cell types, including B-cell chronic lymphocytic leukemia and low-grade lymphoma cells. Evidence is accumulating to suggest that bendamustine may also have synergistic effects in combination therapies. Rituximab is a promising new agent for the treatment of hematologic malignancies and has been shown to have synergistic actions with other chemotherapeutic agents. The actions of the combination of bendamustine and rituximab on ex vivo B-cell chronic lymphocytic leukemia cells and the DOHH-2 cell line, derived from CD20-positive lymphoma cells, are discussed in this article.

In bcr-abl-positive myeloid cells resistant to conventional chemotherapeutic agents, expression of Par-4 increases sensitivity to imatinib (STI571) and histone deacetylase-inhibitors.

In a variety of malignant cells the prostate-apoptosis-response-gene-4 (Par-4) induces increased sensitivity towards chemotherapeutic agents by down-regulating anti-apoptotic B-cell lymphoma-gene 2 (Bcl-2). Hypothesizing that Par-4 also influences apoptosis in myeloid cell lines, we tested this hypothesis by stably transfecting bcr-abl transformed-K562 cells with a Par-4-expressing vector. Here we demonstrate that over-expression of Par-4 in K562 cells up-regulates expression levels of Bcl-2 and death-associated protein (Daxx). Upon treatment with different chemotherapeutic agents, Fas- or TRAIL agonistic antibodies, Par-4-positive cells did not exhibit an increased rate of apoptosis as compared to Par-4-negative control cells. However, incubation with histone deacetylase (HDAC)-inhibitors Trichostatin A (TSA) and LAQ824 or the tyrosinkinase inhibitor Imatinib (STI571) increased the rate of apoptosis in Par-4-positive K562 cells. Assessing the underlying molecular mechanisms for the Par-4-induced response to HDAC-inhibitors and STI571 we provide evidence, that these effects are associated with a down-regulation of Daxx, enforced activation of caspases and enhanced cleavage of cellular inhibitor of apoptosis (cIAP)-1 and -2.

Synergistic effects of chemotherapeutic drugs in lymphoma cells are associated with down-regulation of inhibitor of apoptosis proteins (IAPs), prostate-apoptosis-response-gene 4 (Par-4), death-associated protein (Daxx) and with enforced caspase activation.

Cytotoxic drugs mediate apoptotic tumor cell death by influencing key regulator proteins of programmed cell death. In clinical practice cytotoxic drug combinations are desired to potentiate tumor cell kill and to minimize side effects. Nevertheless, the molecular mechanisms underlying synergistic and antagonistic effects on tumor cells are still poorly understood. In order to elucidate these molecular mechanisms we established models of synergistic and antagonistic drug combinations within the same lymphoma cell lines. By combination index method we demonstrated that bendamustine in combination with either doxorubicin or mitoxantrone caused antagonistic effects on disruption of mitochondrial membrane potential as well as on the rate of apoptosis. In contrast the combination of bendamustine with cladribine acted synergistically on these parameters. By using the IC(50) (dosages causing 50% rate of apoptosis) the synergistic effect of the combination of bendamustine and cladribine was associated with an enhanced mitochondrial release of cytochrome c and Smac/DIABLO, by down-regulation of x-linked inhibitor of apoptosis (XIAP), cIAP1, Par-4 and Daxx as well as by a significantly increased activation of caspases-3, -6, -7, -8 and -9. At the same rate of apoptosis (IC(50)), the antagonistic combinations did not increase the release of cytochrome c or Smac/DIABLO, nor down-regulate the expression of XIAP, cIAP1, Par-4 and Daxx, nor increase the activation of caspases. The role of down-regulation of IAPs and of enforced caspase activation for synergism in this model was supported by the observation, that broad spectrum inhibition of caspases re-established expression of XIAP. Our study is the first to outline the molecular alterations caused by synergistic and antagonistic drug combinations within the same lymphoma cell model. The above described mechanisms were already assessable at a point where the effects of synergistic or antagonistic combinations could not yet be discriminated quantitatively by the level of apoptosis rate of the lymphoma cells.

Inhibiting effects on the induction of cytotoxic T lymphocytes by dendritic cells pulsed with lysates from acute myeloid leukemia blasts.

Dendritic cells (DCs) were established from 25 patients in complete remission of acute myeloid leukemia (AML). In patients during hematopoietic regeneration following chemotherapy the yield of DC was comparable to that of healthy donors. In patients, more than 2 months after chemotherapy, significantly less DC were generated. Comparison of the antigen-presenting capacity using tetanus toxoid of six AML patients and six healthy volunteers did not show significant differences. In six AML patients, lymphocytes stimulated with blast cell lysate pulsed DC were analyzed for cytotoxic activity against autologous blast cells. 8.4-35.6% of autologous blast cells were lysed by DC stimulated lymphocytes. In three of the six patients maximum lysis of target cells was achieved by unpulsed DC. Thus, it seems that in some patients blast cell lysates mediate inhibitory effects, which may explain to some extend immune escape mechanisms in AML.

In malignant myeloid cells expression of Daxx downregulates expression of p53 and of the inhibitors of apoptosis proteins.

The role of Daxx, in particular its ability to promote or hinder proliferation, still remains controversial. In order to elucidate the functional relevance of Daxx in malignant myelocytes, the erythroleukemia cell line HEL was stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. Assessing the molecular consequences of ectopic Daxx-expression, we present evidence that Daxx downregulates p53. Moreover, we demonstrate that Daxx overexpressing myelocytes downregulate the proapoptotic Bcl-2 family member Bax, while expression of antiapoptotic Bcl-2 is not influenced. Furthermore, expression of Daxx diminishes expression levels of the initiator-procaspase-8 and -10, and the executioner procaspase-7, whereas the procaspase-3, -6 and -9 remain unaltered. The altered protein levels of the caspases in Daxx overexpressing myelocytes are accompanied by a decrease of expression levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, -2 and survivin. Despite the described impact of Daxx expression on major molecules of the apoptotic cascade, expression of Daxx in neoplastic myelocytes does not impact on the rate of proliferation. Upon a proapoptotic stimulus such as serum withdrawal Daxx is unable to maintain its influence on expression levels of p53, Bax, IAPs and the procaspase-8, -10 and -7.

Cytotoxic activity of T- and NK-cell lymphoma cells is not dependent on a mature cytotoxic phenotype.

Cytotoxic T- and NK-cell neoplasms constitute a rare clinico-pathological entity associated with aggressive clinical behaviour and a poor prognosis. The entity comprises a heterogenous group of different diseases classified by histologic, immunologic as well as clinical features. Recently, expression patterns of "cytotoxicity-associated proteins" such as T-cell intracellular antigen (TIA), perforin and granzyme B have been applied to differentiate between an immature (TIA positive) and a mature (TIA and perforin and/or granzyme B positive) phenotype of these malignant cells. In particular, expression of perforin and granzyme B are considered to mediate cytotoxic activity. This study assesses histology/cytology, immunophenotype, expression of "cytotoxicity-associated proteins" and the actual exhibition of cytotoxic activity of lymphoma cells of 10 patients suffering from different T- and NK-cell neoplasms. As investigated by PKH67 labelling of the target cells 6 out of 10 samples exhibited cytotoxic activity. Thus, all samples of lymphoma cells with a mature phenotype exhibited cytotoxic activity. Nevertheless, the ability to induce cytotoxic cell lysis was neither restricted to mature lymphoma cells, nor to lymphoma cells expressing "cytotoxicity-associated proteins": two samples with an immature phenotype and one CD4 positive sample, completely lacking expression of "cytotoxic proteins" as well as NK cell-associated markers, destroyed target cells. Artificial activation of a mature cytotoxic phenotype by cell culture conditions or contact of lymphoma cells with target cells was excluded by demonstrating the absence of perforin expression after the incubation period in two exemplary cases. In conclusion, we demonstrate that the exhibition of cytotoxic activity is neither restricted to cells with a mature phenotype, nor does it depend on the expression of the "cytotoxicity-associated proteins" TIA, perforin or granzyme B.

Expression and function of prostate-apoptosis-response-gene-4 in lymphatic cells.

Inhibition of apoptosis contributes to the pathogenesis of lymphatic malignancies. In particular, the elevated expression of Bcl-2 is considered to be a marker of poor prognosis, since increased levels of Bcl-2 confer longevity as well as chemoresistance. After demonstrating an inverse expressional pattern of Bcl-2 and prostate-apoptosis-response-4 (Par-4) in ex vivo cells of patients suffering from acute lymphatic leukemia (ALL) as well as a deregulated expression of Par-4 in acute and chronic lymphatic neoplasias, the molecular mechanisms underlying these results were investigated. Thus, it was demonstrated that in neoplastic lymphatic cells Par-4 exerts a proapoptotic role augmenting chemosensitivity by down-regulating Bcl-2, promoting disruption of mitochondrial membrane potential and enforcing caspase-activation. Moreover, Par-4 enables cells to circumvent inhibition of the central executioner caspase-3 by alternative activation of caspases following a decrease in expression levels of inhibitors of apoptosis proteins (IAP).

Upon drug-induced apoptosis in lymphoma cells X-linked inhibitor of apoptosis (XIAP) translocates from the cytosol to the nucleus.

The X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis-1 (cIAP-1) are emerging as versatile proteins in programmed cell death with a scope of possible functions reaching far beyond their well known inhibitory effects on caspases. We previously demonstrated that the ability of drugs to modify expression and cleavage of the IAPs are crucial for the synergistic effects achieved by the combinations of different cytotoxic drugs employed to treat malignant lymphomas. In order to more clearly assess the underlying molecular mechanisms, we here evaluated the consequences of drug-induced apoptosis on the localization and aggregation of XIAP and cIAP-1. The influence of drug-induced apoptosis on localization of IAPs was investigated using immunofluorescence microscopy as well as western blot analysis. Apoptosis was induced by chemotherapeutic drugs with different modes of action (bendamustine, cladribine, fludarabine, doxorubicin and mitoxantrone) and assessed by flow-cytometry using Annexin V. We demonstrate that XIAP and cIAP-1 are downregulated and/or cleaved in a dose-dependent manner upon treatment with a variety of anti-cancer drugs. Moreover we provide evidence that in the context of drug-induced apoptosis XIAP, its BIR3-RING cleavage product and cIAP-1 undergo an extensive change of subcellular localization. Immunofluorescence microscopy reveals that XIAP, in contrast to cIAP-1, is located in discrete cytosolic protein aggregates and-upon induction of apoptosis with cytotoxic drugs--redistributes into large nuclear inclusions. This translocation of XIAP and its BIR3-RING cleavage product from the cytosol into the nucleus is confirmed by cell fractionation and western blot analyses. Of note, in this experimental setting putative interaction partners of XIAP-such as Apaf-1, caspase-3 and -7--do not co-localize with XIAP. These results imply a new unknown function of XIAP and its BIR3-RING fragment in the nucleus in the context of drug-induced apoptosis. The localization of cIAP-1 in mitochondria and its liberation from these indicate a profoundly different function of this protein despite its similar modular structure to XIAP.