A phase II study of alemtuzumab, fludarabine, cyclophosphamide, and doxorubicin (Campath-FCD) in peripheral T-cell lymphomas.

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.

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.

Down-regulation of wt1 expression in leukemia cell lines as part of apoptotic effect in arsenic treatment using two compounds.

Arsenic trioxide (As2O3) induces remission in patients with acute promyelocytic leukemia (APL). To better understand molecular mechanisms of arsenic actions, this study investigated the effect of two different arsenic compounds on gene expression of apoptosis and cellular proliferation related genes. The Wilms' tumor gene (wt1) is up-regulated in acute myeloid leukemia (AML) and a variety of leukemia cell lines. The expression of wt1 in these cells is proposed to have an anti-apoptotic effect. HL-60 and K562 were treated with arsenic trioxide (As2O3) and sodium arsenite (NaAsO2) at concentrations between 0 - 10 microM for up to 48 h. The induction of apoptosis was accompanied by down-regulation of hTERT and wt1 mRNA and protein expression but up-regulation of par-4. Low concentrations of 0.1 microM arsenic induced expression of the anti-apoptotic bcl-2 gene in both cell lines HL-60 and K562. There were no major differences encountered between compounds. After arsenic treatment of the leukemia cell lines HL-60 and K562 the up-regulation of par-4 may contribute to the induction of apoptosis rather than down-regulation of bcl-2. The therapeutic effect of arsenic is the induction of apoptosis by modulating the gene expression profile of pro- and anti-apoptotic genes including the wt1 gene.

Bendamustine plus rituximab is effective and has a favorable toxicity profile in the treatment of mantle cell and low-grade non-Hodgkin's lymphoma.

PURPOSE: The aim of this multicenter-study was to evaluate the progression-free survival, response rate and toxicity of the combination of bendamustine and rituximab (BR) in patients with mantle cell or low-grade lymphomas in first to third relapse or refractory to previous treatment. PATIENTS AND METHODS: A total of 245 courses (median, four courses per patient) were administered to 63 patients. Bendamustine was given at a dose of 90 mg/m2 as a 30-minute infusion on days 1 and 2, combined with 375 mg/m2 rituximab on day 1, for a maximum of four cycles every 4 weeks. Histologies were 24 follicular, 16 mantle cell, 17 lymphoplasmacytoid, and six marginal zone lymphoma. RESULTS: Fifty-seven of 63 patients responded to BR, corresponding to an overall response rate of 90% (95% CI, 80% to 96%) with a complete remission rate (CR) of 60% (95% CI, 47% to 72%). The median time of progression-free survival was 24 months (range, 5 to 44+ months), and the median duration of overall survival has not yet been reached. In mantle cell lymphomas, BR showed a considerable activity, achieving a response rate of 75% (95% CI, 48% to 93%) with a CR rate of 50%. Myelosuppression was the major toxicity, with 16% grade 3 and 4 leukocytopenia. Thrombocytopenia was rare, with only 3% grade 3 and 4. CONCLUSION: These results demonstrate that the BR combination is a highly active regimen in the treatment of low-grade lymphomas and mantle cell lymphomas.

In vitro chemosensitivity to gemcitabine, oxaliplatin and zoledronic acid predicts treatment response in metastatic gastric cancer.

Individual response of disseminated cancer to chemotherapy is unpredictable. In vitro chemotherapy-induced apoptosis can be measured and might be a method to evaluate in vivo activity of tested drugs. In this report, tumor cells of a patient with signet cell carcinoma of the stomach and diffuse bone marrow infiltration were cultured and tested for in vitro chemosensitivity. The drugs gemcitabine, oxaliplatin and zoledronic acid were found to induce in vitro tumor cell apoptosis synergistically, and subsequently were used as combination chemotherapy regimen. An initially existing disseminated intravascular coagulopathy quickly resolved and after 6 months of treatment on ongoing complete response was induced, thus confirming the results of in vitro chemosensitivity testing.

Prostate apoptosis response gene-4 sensitizes neoplastic lymphocytes to CD95-induced apoptosis.

Evaluating the functional consequences of prostate apoptosis response gene-4 (par-4) expression in CD95-induced apoptosis of neoplastic lymphocytes, we demonstrate that par-4 increases apoptosis by upregulating the CD95 receptor on the cell surface and--with a concomitant decrease of the FLICE-like inhibitory protein (FLIP)--by promoting cleavage of the initiator caspases-8 and -10. This results in an enforced activation of the executioner caspases-6, -7, and -3 as well as in an activation of the mitochondrial pathway. Upon inhibition of caspase-8, overexpression of par-4 enables Jurkat cells to maintain a higher sensitivity to CD95-induced apoptosis by downregulating cIAP-2 and XIAP and by enforcing activation of the initiator caspase-10 as well as of the executioner caspases-6, -7, and -3.

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 AML cell lines Ara-C combined with purine analogues is able to exert synergistic as well as antagonistic effects on proliferation, apoptosis and disruption of mitochondrial membrane potential.

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.

CD4+ CD56+ neoplasia: clinical and biological features with emphasis on cytotoxic drug-induced apoptosis and expression of sialyl Lewis X.

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.

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.

Induction of apoptosis by cladribine (2-CdA), gemcitabine and other chemotherapeutic drugs on CD34+/CD38+ and CD34+/CD38- hematopoietic progenitor cells: selective effects of doxorubicin and 2-CdA with protection of immature cells.

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.

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.

Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases.

BACKGROUND AND OBJECTIVES: Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine). DESIGN AND METHODS: DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined. RESULTS: We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis. INTERPRETATION AND CONCLUSIONS: We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.

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).

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.