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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.

The clinical course of peripheral T-cell lymphoma (PTCL) is usually aggressive and the prognosis unfavorable. Therefore, there is a need for improvement of treatment options. Patients with newly diagnosed (n = 27) or refractory/relapsed (n = 11) PTCL received a combination of alemtuzumab, fludarabine, cyclophosphamide, and doxorubicin. The overall response rate (ORR) was 61%, with a complete response rate of 39%. In newly diagnosed patients the ORR was 63%, the median overall survival 25.9 months, and progression-free survival 11.8 months. In relapsed/refractory patients the median OS was 6.1 months. The most frequent grade 3/4 toxicities were leukopenia (95% of patients) and thrombocytopenia (58%). Cytomegalovirus (CMV) reactivation occurred in 12 patients, but only two had CMV disease. Treatment-related deaths occurred in six newly diagnosed patients and one with relapsed/refractory disease. In conclusion, Campath-FCD is active in PTCL but is associated with significant toxicity and is, therefore, not recommended for use or further study. Further studies are warranted to investigate other approaches to combining alemtuzumab with chemotherapy for the treatment of PTCL.

Salvage therapy for patients with mantle cell lymphoma (MCL) remains a challenge. The clinical course is characterised by increasing resistance to conventional chemotherapy and a dismal long-term outcome. On the basis of studies demonstrating synergy in vitro, eight heavily pretreated patients (median age 65 years) with advanced stage MCL were individually treated with a novel combination protocol consisting of the proteasome inhibitor bortezomib (1.5 mg/m(2); Days 1 and 4), high-dose cytarabine (750-2000 mg/m(2); Days 2 and 3) and dexamethasone (40 mg daily; Days 1-4). Rituximab (375 mg/m(2)) was added in patients not refractory to prior rituximab-containing regimens. Treatment was repeated in 3-week intervals or postponed until hematologic recovery for up to four planned cycles. Toxicity consisted mainly of Grade 3/4 hematotoxicity, which occurred in all patients. Median treatment interval was 31 days. Objective responses were observed in four (50%) of eight patients, including two complete remissions. Median progression free and overall survival were 5 and 15.5 months, respectively. The combination of bortezomib and a high-dose cytarabine-containing regimen has activity in heavily pretreated patients with relapsed or refractory MCL.

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.

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.

Par-4 functions as a tumor suppressor antagonizing the transforming capacity and the resistance of malignant cells towards apoptotic stimuli. After demonstrating that par-4 promotes apoptosis by activating signaling of the intrinsic pathway of apoptosis, we hypothesized that par-4 also impacts on key molecules of the extrinsic pathway without the requirement of a receptor/ligand interaction. Here, we provide first evidence, that expression of par-4 increases cleavage of caspase-8, truncation of Bid and its translocation to the mitochondria, resulting in an augmentation of cytochrome c and AIF efflux into the cytosol, effects par-4-positive cells are able to retain to a higher extent than par-4-negative cells upon inhibition of caspase-3 activation.

In a variety of malignant cells Prostate-apoptosis-response-gene-4 (Par-4) exhibits a pro-apoptotic influence sensitizing these cells to apoptosis-inducing agents by downregulating expression of Bcl-2. Considering the crucial role of Bcl-2 in the development of chemoresistance of acute myeloid leukemia (AML) cells, we here assessed the potential of Par-4 to down-regulate Bcl-2 and to induce apoptosis in the erythroleukemic cell line HEL. Testing a potential pro-apoptotic role of Par-4 upon incubation with various conventional chemotherapeutic drugs, novel agents such as the signal transduction inhibitor STI 571 and the histone deacetylase (HDAC)- inhibitor trichostatin A (TSA), as well as with the experimental substances Fas and TRAIL, we provide evidence that in the erythroleukemic cell line HEL expression of Par-4 is not sufficient to sensitize to any of these pro-apoptotic stimuli. We further demonstrate that--in contrast to previous reports in non-AML cells--Par-4 expression in HEL cells leads to an upregulation of Bcl-2. Moreover, Par-4-positive HEL cells exhibit a decreased level of the proapoptotic protein Bax as compared to Par-4- negative cells. In addition, Par-4 increases the expression of Daxx--whose downregulation is associated with augmented chemosensitivity--as well as expression of the procaspases-8, -9 and -10, whereas the levels of the procaspases-3 and -7 remain unaltered. In conclusion we here demonstrate that in the erythroleukemic cell line HEL--in contrast to other cell types Par-4 fails to promote apoptosis and outline the underlying molecular mechanisms.

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.

CD4+ CD56+ neoplasia is a rare malignancy of unclarified origin. So far only 57 cases have been reported. We characterized in detail a case of CD4+ CD56+ malignancy with special emphasis on apoptosis induced by cytotoxic drugs and expression of sialyl Lewis X (CD15s). The disease was diagnosed in a 73-year-old female presenting with skin involvement, generalized lymphadenopathy and bone marrow infiltration. Treatment with cladribine/mitoxantrone induced a short-lasting partial response and the patient died 6 months after diagnosis. The neoplastic cells expressed CD4, CD56, HLA-DR, and CD15s. PCR for the T-cell receptor gamma chain revealed a polyclonal amplification product. In situ hybridization for Epstein-Barr Virus (EBV) was negative. Cytotoxic granule-associated proteins were not detected, consistent with the observation that the cells did not mediate cytotoxic activity against several target cells. Apoptosis of the tumor cells was inducible by anthracyclines and cladribine but not with gemcitabine. Combinations of cladribine or gemcitabine with anthracyclines however, resulted in synergistic effects on apoptosis. Expression of CD15s on the CD56+ cells was three times higher than on CD56+ cells from healthy controls. The results demonstrate that the features of the present case is in accordance with the diagnosis of CD4+ CD56+ malignancy. This is the first report demonstrating increased CD15s expression on a CD4+ CD56+ neoplasia, possibly explaining the frequent occurrence of the disease in the skin.

The pyrimidine analogue Ara-C and the purine analogues fludarabine and cladribine (2-CdA) are essential compounds in the treatment of acute myeloid leukemia (AML). Inhibition of cell proliferation and induction of apoptosis are the major mechanisms of cytotoxic agents to cause tumor cell death. Therefore, we studied whether Ara-C in combination with the purine analogues exerts synergistic or antagonistic effects on cell proliferation, phosphatidylserine exposure and disruption of mitochondrial membrane potential (MMP) in the AML cell lines HL60 and HEL. Furthermore, effects of the combination of Ara-C with bendamustine, a new bifunctional agent with alkylating activity and a purine nucleus, was investigated. Assessment by combination index analysis showed that Ara-C combined with fludarabine or bendamustine exhibited additive to antagonistic effects on inhibition of cell proliferation, induction of apoptosis as well as on disruption of mitochondrial membrane potential, independent of a simultaneous or consecutive (purine analogues before Ara-C) incubation schedule. In contrast, the combination of Ara-C with 2-CdA exclusively yielded synergistic effects. While inducing IC50 levels of apoptosis neither the antagonistic nor the synergistic drug combinations caused a specific expression pattern of apoptosis-associated proteins such as the pro- or antiapoptotic Bcl-2 family members, executioner caspases, IAPs (inhibitor of apoptosis proteins), proapoptotic Par-4, PARP, or p53. In conclusion, we here demonstrate that the in vitro efficacy of drug combinations containing Ara-C and purine analogues depends on the purine analogue applied, whereas incubation schedules or escalating dosages do not contribute to the synergistic effects.

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.

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).

Due to the emerging role of high dose chemotherapy with stem cell rescue and ex vivo purging in hematological diseases, we examined the effect of chemotherapeutic drugs on the rate of apoptosis in more mature CD34+/CD38+ and less differentiated CD34+/CD38- stem cells. CD34+ cells were obtained by cell apheresis from healthy donors (n = 25) or patients (n = 25) prepared for high dose chemotherapy and stem cell transplantation. Cells were incubated with different concentrations of doxorubicin, mitoxantrone, mafosphamide, cladribine or gemcitabine. Apoptosis was determined after 24 and 48 h. Generally, the percentage of apoptotic cells was higher in the more mature CD34+/CD38+ progenitor population than in the less differentiated CD34+/CD38- cells. By analysis of variance (ANOVA) significantly (p < 0.05) more apoptotic cells within the CD34+/CD38+ progenitors were calculated after incubation with mafosphamide, doxorubicine and cladribine. Mafosphamide induced the highest rate of apoptosis on CD34+/CD38- cells, whereas doxorubicine had nearly no effect on this immature population. Dose effect plots for mafosphamide and doxorubicin were steep, suggesting a large therapeutic index. The dose response of cladribine showed a flat course. Furthermore we found a selective induction of apoptosis by doxorubicin and cladribine on more mature CD34+/CD38+ progenitors in contrast to simultaneous protection of CD34+/CD38- progenitors. From these findings, in particular the demonstrated low stem cell toxicity, we conclude that doxorubicin and cladribine might be efficient alternatives in ex vivo purging of autologous grafts, as well as safe components in primary treatment schedules of lymphomas or prior to stem cell harvest with respect to stem cell toxicity.