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.

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.

Therapeutic options in relapsed or refractory peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) represents a relatively rare group of heterogeneous non-Hodgkin lymphomas with a very poor prognosis. Current therapies, based on historical regimens for aggressive B-cell lymphomas, have resulted in insufficient patient outcomes. The majority of patients relapse rapidly, and current 5-year overall survival rates are only 10-30%. It is evident that new approaches to treat patients with PTCL are required. In recent years, prospective studies in PTCL have been initiated, mainly in patients with relapsed/refractory disease. In some of these, selected histologic subtypes have been evaluated in detail. As a consequence, numerous new therapies have been developed and shown activity in PTCL, including: agents targeting the immune system (e.g. brentuximab vedotin, alemtuzumab, lenalidomide); histone deacetylase inhibitors (romidepsin, belinostat); antifolates (pralatrexate); fusion proteins (denileukin diftitox); nucleoside analogs (pentostatin, gemcitabine); and other agents (e.g. alisertib, plitidepsin, bendamustine, bortezomib). A variety of interesting novel combinations is also emerging. It is hoped that these innovative approaches, coupled with a greater understanding of the clinicopathologic features, pathogenesis, molecular biology, and natural history of PTCL will advance the field and improve outcomes in this challenging group of diseases. This review summarizes the currently available clinical evidence on the various approaches to treating relapsed/refractory PTCL, including the role of stem cell transplantation, with an emphasis on potential new drug therapies.

Long-term complete remission following radiosurgery and immunotherapy in a melanoma patient with brain metastasis: immunologic correlates.

A melanoma patient with brain metastases was treated by gamma-knife radiosurgery and immunotherapy with autologous tumor-lysate-loaded dendritic cells (DC). Ten years after the combined treatment, the patient remains in complete remission. Remarkable immunologic correlates to the clinical development were the transient induction of NY-ESO-1 antibody and the durable expansion of MAGE-A1p161-169 EADPTGHSY-specific CD8+ T cells. Although the induction of NY-ESO-1 antibody most likely resulted from gamma-knife-mediated "auto-vaccination," the persistence of circulating MAGE-A1-specific T cells, which are still detectable ex vivo in the absence of any tumor manifestation, coincides with DC-based vaccination administered monthly until today.

ABVD in older patients with early-stage Hodgkin lymphoma treated within the German Hodgkin Study Group HD10 and HD11 trials.

PURPOSE: Older patients with Hodgkin lymphoma (HL) account for approximately 20% of all HL patients. ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) chemotherapy is regarded as standard of care in these patients. However, little is known on feasibility and efficacy of ABVD in this age group. PATIENTS AND METHODS: We analyzed the feasibility and efficacy of four cycles of ABVD in older patients age 60 to 75 years with early-stage HL who were treated within the German Hodgkin Study Group (GHSG) HD10 and HD11 trials; results were compared with those of younger patients treated within these trials. RESULTS: In total, 1,299 patients received four cycles of ABVD, and 117 of those patients were older than age 60 years (median, 65 years). In 14% of older patients, treatment was not administered according to protocol, mainly because of excessive toxicity. The mean delay of treatment was twice as high in the older patients (2.2 v 1.2 weeks). Fifty-nine percent of older patients achieved a relative dose-intensity of at least 80% compared with 85% of younger patients. Major toxicity (WHO grade 3 and 4), including leucopenia, nausea, infection, and others, was documented in 68% of older patients with a treatment-related mortality of 5%. Complete response was achieved in 89% of older patients, 3% had progressive disease, and 11% relapsed. At a median observation time of 92 months, 28% of the patients had died, and the 5-year progression-free survival estimate was 75% (95% CI, 66% to 82%). CONCLUSION: In patients age ≥ 60 years with HL, four cycles of ABVD is associated with substantial dose reduction, treatment delay, toxicity, and treatment-related mortality.

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.

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.

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.

A novel regimen combining high dose cytarabine and bortezomib has activity in multiply relapsed and refractory mantle cell lymphoma - long-term results of a multicenter observation study.

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.

Epstein-Barr virus-associated B-cell lymphoma secondary to FCD-C therapy in patients with peripheral T-cell lymphoma.

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disorders occur at an increasing frequency in various hereditary and acquired states of immune dysfunction. In a few cases of T-cell lymphoma, especially in angioimmunoblastic T-cell lymphoma (AILT), EBV-associated B-cell lymphoproliferative disorders have been reported. Here, we present two cases of EBV-associated B-cell lymphoma after treatment of T-cell lymphoma (AILT and peripheral T-cell lymphoma, unspecified, PTCL-NOS) with a regimen containing alemtuzumab and fludarabine. Conventional and immunohistological tissue staining showed the typical features of highly proliferating diffuse large B-cell lymphoma in both cases. The monoclonal B-cell population displayed EBV latency type III. At the time of diagnosis the cellular immune status of both patients was severely compromised with an absolute CD4 T-cell count below <120 microl(-1). Our observation supports the notion that combination of cytotoxic drugs and immunosuppressive antibodies in patients with T-cell lymphoma may severely aggravate the already present immunodeficiency. We suggest to monitor the cellular immune status in combination with the EBV load in high risk patients for early detection-and possibly intervention-of EBV-associated lymphoma.

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.

Mitomycin C, 5-fluorouracil, leucovorin, and oxaliplatin as a salvage therapy for patients with cisplatin-resistant advanced gastric cancer: a phase I dose escalation trial.

BACKGROUND: This study aimed at evaluating the feasibility and toxicity of a salvage therapy with mitomycin C (MMC), 5-fluorouracil (5-FU), leucovorin, and oxaliplatin in patients with cisplatin-resistant advanced gastric cancer. METHODS: A 3-patient cohort dose-escalating study design was used. The patients received FLO: oxaliplatin 85 mg/m2, 5-FU 2,600 mg/m2 (24 h), leucovorin 200 mg/m2 on days 1, 15, and 29 plus MMC on day 1 (FLOM). The MMC dose was escalated from 6 to 12 mg/m2 in 2- mg/m2 steps. Cycles were repeated every 6 weeks. RESULTS: Twenty patients were enrolled in 4 treatment cohorts. The treatment was well tolerated with grade 3 or 4 nonhematological toxicities affecting less than 5% of patients. Grade 3 or 4 neutropenia, anemia, and thrombocytopenia were observed in 9 (45%), 7 (35%), and 5 (25%) of 20 patients, respectively. Mild but prolonged thrombocytopenia was dose limiting, requiring treatment discontinuation or a treatment delay >or=2 weeks in 8 (40%) of 20 patients. MMC 10 mg/m2 every 6 weeks was considered as the optimal dose in combination with FLO. Objective responses were observed in 7 (35%) of 20 patients, and 7 further patients (35%) had stable disease. Median time to progression and overall survival were 4.1 and 8 months, respectively. CONCLUSIONS: Prolonged cumulative myelotoxicity was dose limiting in the therapy with MMC, 5-FU, and oxaliplatin. This combination chemotherapy seems to overcome cisplatin resistance in patients with advanced gastric cancer.

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.

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.

In vivo drug-response in patients with leukemic non-Hodgkin's lymphomas is associated with in vitro chemosensitivity and gene expression profiling.

Only a few approaches are available to address the mechanisms of cell death in vivo which are induced by anticancer treatment in patients with malignancies. In this study in vitro chemosensitivity testing of primary peripheral blood leukemic cells of five patients suffering from different leukemic non-Hodgkin's lymphomas was combined with the analysis of the in vivo rate of apoptosis by flow-cytometry (Annexin V and depolarisation of mitochondrial membrane potential (MMP) by JC-1). Furthermore, changes in expression patterns of apoptosis related proteins during chemotherapeutic treatment were detected by Western Blot. Gene expression profiling (HG-U133A, Affymetrix, Santa Clara, CA) was employed to identify common marker genes of in vivo drug response. In vitro chemosensitivity was tested using the cytotoxic agents which the patients were scheduled to receive and was strongly correlated with effective reduction of leukemic lymphoma cells in patients resulting in complete remissions in all five cases. Due to the rapid clearance of apoptotic tumor cells in vivo neither the analysis of the in vivo rate of apoptosis and depolarisation of MMP nor the assessment of expression of regulators of apoptosis showed concordant results concerning the drug response. However, assessment of gene expression during therapy could identify a set of 30 genes to significantly discriminate between samples from patients before treatment compared to samples from the same patients after receiving cytotoxic therapy. Among these 30 genes we found a high proportion of genes associated with apoptotic cell death, cell proliferation and cell cycle signalling including complement lysis inhibitor (clusterin/CLU), beta-catenin interacting protein (ICAT), peroxisome proliferator activated receptor alpha (PPARalpha), TNF alpha converting enzyme (ADAM17/TACE), homeo box A3 (HOX1), inositol polyphosphatase 5-phosphatase type IV (PPI5PIV) and inhibitor of p53 induced apoptosis alpha (IPIA-Alpha/NM23-H6). These results indicate that in vitro chemosensitivity testing and gene expression profiling can successfully be utilised to analyse in vivo drug response in patients with leukemic NHL's and can be used to explore new pathway models of drug-induced cell death in vivo which are independent of different lymphoma subtypes and different treatment regimens.

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.

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.

Expression of Daxx sensitizes Jurkat T-cells to the apoptosis-inducing effect of chemotherapeutic agents.

BACKGROUND: 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 the extrinsic signaling of malignant lymphocytes Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. RESULTS: Assessing first the impact of Daxx expression on the rate of proliferation we demonstrate that overexpression of Daxx alone is not sufficient to alter proliferation in neoplastic lymphocytes. Nevertheless, expression of Daxx down-regulates anti-apoptotic Bcl-2 and up-regulates pro-apoptotic BID. In addition, Daxx-overexpressing Jurkat cells exhibit a decreased expression of the pro-caspase-8, -10, -9 and -3 and a concomitant increase of the inhibitors of apoptosis proteins survivin, XIAP, cIAP-1 and -2. We further demonstrate, that upon incubation with various chemotherapeutic agents these Daxx-induced molecular alterations sensitize Jurkat T-cells to the apoptosis-inducing effects of specific chemotherapeutic agents. CONCLUSIONS: We here outline the molecular changes elicited by Daxx on major components of the apoptotic cascade of malignant lymphocytes and demonstrate the capacity of Daxx to sensitize these cells to the apoptosis-inducing effect of various chemotherapeutic agents.

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.