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XLSX file - 31K, Antigen expression of several melanoma metastases by RT-PCR.

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. Cancer Immunol Res; 2(5); 404–9. ©2014 AACR.

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BACKGROUND: Based on the encouraging activity and manageable safety profile observed in a phase 1 study, the ECHELON-2 trial was initiated to compare the efficacy and safety of brentuximab vedotin, cyclophosphamide, doxorubicin, and prednisone (A+CHP) versus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) for the treatment of CD30-positive peripheral T-cell lymphomas.METHODS: ECHELON-2 is a double-blind, double-dummy, randomised, placebo-controlled, active-comparator phase 3 study. Eligible adults from 132 sites in 17 countries with previously untreated CD30-positive peripheral T-cell lymphomas (targeting 75% with systemic anaplastic large cell lymphoma) were randomly assigned 1:1 to receive either A+CHP or CHOP for six or eight 21-day cycles. Randomisation was stratified by histological subtype according to local pathology assessment and by international prognostic index score. All patients received cyclophosphamide 750 mg/m2 and doxorubicin 50 mg/m2 on day 1 of each cycle intravenously and prednisone 100 mg once daily on days 1 to 5 of each cycle orally, followed by either brentuximab vedotin 1·8 mg/kg and a placebo form of vincristine intravenously (A+CHP group) or vincristine 1·4 mg/m2 and a placebo form of brentuximab vedotin intravenously (CHOP group) on day 1 of each cycle. The primary endpoint, progression-free survival according to blinded independent central review, was analysed by intent-to-treat. This trial is registered with ClinicalTrials.gov, number NCT01777152.FINDINGS: Between Jan 24, 2013, and Nov 7, 2016, 601 patients assessed for eligibility, of whom 452 patients were enrolled and 226 were randomly assigned to both the A+CHP group and the CHOP group. Median progression-free survival was 48·2 months (95% CI 35·2-not evaluable) in the A+CHP group and 20·8 months (12·7-47·6) in the CHOP group (hazard ratio 0·71 [95% CI 0·54-0·93], p=0·0110). Adverse events, including incidence and severity of febrile neutropenia (41 [18%] patients in the A+CHP group and 33 [15%] in the CHOP group) and peripheral neuropathy (117 [52%] in the A+CHP group and 124 [55%] in the CHOP group), were similar between groups. Fatal adverse events occurred in seven (3%) patients in the A+CHP group and nine (4%) in the CHOP group.INTERPRETATION: Front-line treatment with A+CHP is superior to CHOP for patients with CD30-positive peripheral T-cell lymphomas as shown by a significant improvement in progression-free survival and overall survival with a manageable safety profile.FUNDING: Seattle Genetics Inc, Millennium Pharmaceuticals Inc, a wholly owned subsidiary of Takeda Pharmacuetical Company Limited, and National Institutes of Health National Cancer Institute Cancer Center.

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.

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. Cancer Immunol Res; 2(5); 404–9. ©2014 AACR.

TPS2651 Background: The two new strata of the INSIGHT trial evaluate feasibility and safety of s.c. injections of IMP321 (eftilagimod alpha) in combination with either SOC first/second-line drug therapy (Stratum C) or in combination with an PD-L1 inhibitor (avelumab; Stratum D) in advanced stage solid tumors as well as to generate first efficacy data. This proof-of-concept data could build the basis for further clinical studies exploring the therapeutic potential of combinations of active immunotherapy using IMP321 with SOC drug therapies or immunotherapies targeting the PD-1/PD-L1 axis in various solid tumor entities. IMP321 is a MHC class II agonist that activates antigen-presenting cells (primary target cells) and then CD8 T cells (secondary target cells). Activation of the dendritic cell network and subsequent T cell recruitment at the tumor site with IMP321 may lead to enhanced anti-tumor CD8 T cell responses. Thus, especially combinations with PD-1/PD-L1 inhibitors might display interesting effects by activating immune cells and disabling immune inhibitory mechanisms at the same time. Methods: This is a prospective investigator initiated phase I trial consisting of four strata. New stratum C: Patients with solid tumors treated with SOC chemo- or targeted therapy in first or second line receive concomitant s.c. IMP321 injections. This combination is aimed to enhance the immune response against tumor cells compared to chemo-/targeted SOC therapy alone. New stratum D: Patients will receive avelumab i.v. q2w along with s.c. IMP321 injections. This combination is aimed to enhance efficacy by combining IMP321’s activating effects on immune cells with the release of immune inhibitory effects caused by interruption of the PD-1/PD-L1 axis. It is planned to enroll 20 patients in Stratum C and 12 patients in stratum D. Main efficacy endpoint is the overall response rate (RECIST 1.1). Overall recruitment has started; currently (Feb 2019) 14 patients have been enrolled. EudraCT: 2016-002309-20. Clinical trial information: NCT03252938.

[§ share last authorship] Background: In 2000-2010, the first large prospective trials in peripheral T-cell lymphoma (PTCL) showed outcomes burdened by high failure rates during induction. Concurrently, trials with the anti-CD52 monoclonal antibody alemtuzumab (ALZ) yielded promising responses in PTCL while demonstrating the feasibility of combining ALZ with CHOP. Hence, the Nordic Lymphoma Group initiated the randomized ACT-1 trial to test, in younger patients (pts) (18-65yrs), the addition of ALZ to CHOP + autologous stem cell transplant (ASCT). Primary endpoint was the 3 years event-free survival (EFS). Here, we present the final analysis of the ACT-1 trial (ClinicalTrials.gov: NCT00646854). Patients and Methods: Overall, 136 pts were randomized (43% of planned sample size due to slow accrual), five did not receive study treatment, and 131 were analyzed (ALZ-CHOP: 65; CHOP: 66). Due to lack of tumoral CD52 expression, anaplastic large cell lymphomas (ALCL) were not included in the ACT-1 trial. An amendment tapering ALZ dose from 360 mg (30 mg on days 1+2 of each CHOP course) to 120 mg (30 mg on day 1 of CHOP courses 1-4) was introduced early on due to systemic fungal infections in 2 pts. Of the 65 pts treated with ALZ-CHOP, 4 received the pre- and 61 (94%) the post-amendment dose. Monitoring for CMV- and EBV-DNA and antimicrobial prophylaxis were mandatory. Results: The median observation time for the Full Analysis Set was 66 months and the median age 51 yrs. The ALZ-CHOP and CHOP cohorts were well balanced with regard to classical prognostic factors and histological subtypes (PTCL-NOS 58% vs 54%, AILT 21% vs 25%, other 21% vs 21%). Feasibility: Neither CHOP nor ALZ-CHOP pts experienced substantial treatment delay. ALZ exposure did not affect stem cell harvest nor hematopoietic recovery. Grade 4 leucopenia was more frequent in ALZ-CHOP pts (73% vs 35%; p=0.001), whereas the occurrence of grade 3-4 anemia and thrombocytopenia did not differ significantly. After ALZ dose amendment, the frequency of bacterial and fungal infections of grade ≥3 was similar in both treatment arms. ALZ treated pts had more viral events (22/57=42% vs 4/23=17%), mainly due to asymptomatic CMV reactivations. The ratio of serious adverse events per ALZ-CHOP treated patient dropped markedly (from 3.25 to 0.86, comparable with 0.46 for CHOP) after dose amendment. Additional toxicity was mild and similar in both arms. Treatment related mortality was 4% (5% vs 3%). Efficacy: Complete remission (CR) was 52% in ALZ-CHOP vs 42% in CHOP. Primary refractory disease occurred for ALZ-CHOP and CHOP in 23% and 38% of pts, respectively. Overall, females had a significantly better outcome than males (p=0.004), also after adjustment for classical prognostic factors. Analyzing time-related endpoints without knowledge of CD52 expression, 3-years EFS, progression-free, and overall survival (PFS, OS) did not differ significantly between ALZ-CHOP and CHOP (EFS 35% vs 26%, PFS 37% vs 26%, OS 52% vs 50%). Fig.1A shows EFS by treatment arm, by gender, and by gender and treatment arm. Although not significantly different, EFS, PFS and OS values of ALZ-CHOP treated females in the ACT-1 trial were consistently higher than those of non-ALZ treated females or of males regardless of treatment group. RNA sequencing from evaluable pre-therapeutic tumor biopsies defined a signature of differentially expressed genes to be predictive of clinical outcome in ALZ-CHOP but not CHOP treated pts (n=33). Tumor microenvironment genes were prominent in determining response to ALZ. Tumors rich in B-cell milieu showed good responses, while the opposite was observed in tumors with signatures enriched with high endothelial cell genes (p Disclosures Leppä: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Bayer: Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Silva:Gilead Sciences: Research Funding; Abbvie, Gilead Sciences, Janssen, BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche, Janssen, Celgene: Other: Travel Support; Roche, Janssen: Other: Institution's payment for consultancy. Hagberg:Roche: Honoraria. Lugtenburg:takeda: Consultancy, Research Funding; servier: Consultancy, Research Funding; roche: Consultancy; BMS: Consultancy; Celgene: Consultancy; Sandoz: Consultancy; GenMab: Research Funding. Walewski:Roche, GSK/Novartis, Takeda, and Janssen-Cilag: Research Funding; Roche, Celgene, Takeda, Janssen-Cilag, and Servier: Honoraria; Roche, Celegene, Takeda, Janssen-Cilag, and Servier: Membership on an entity's Board of Directors or advisory committees. Hopfinger:Janssen: Honoraria; Gilead: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Novartis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Jantunen:Amgen: Honoraria; Genzyme/Sanofi: Honoraria; Takeda: Honoraria. Steidl:Seattle Genetics: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Roche: Consultancy. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding.

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.

Background Rituximab plus combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is widely recommended for the treatment of aggressive B-cell lymphomas. However, there is very little information regarding the management of elderly patients. Patients and methods We initiated a phase II study of first-line treatment with rituximab and bendamustine in elderly patients (≥80 years) with aggressive B-cell lymphomas who were not eligible for R-CHOP or who did not agree to aggressive treatment. The treatment decision on eligibility for R-CHOP was left to discretion of the physicians. Results Fourteen patients with a median age of 85 years (range 80–95 years) were included. The age-adjusted international prognostic index was zero in five patients, one in three patients, and two in six patients. Thirteen patients were assessable for response. Seven patients (54%) had a complete response, two (15%) a partial response, and four (31%) progressive disease. The median overall survival was 7.7 months, and the median progression-free survival 7.7 months; however, six patients (43%) were alive without disease at 20–72 months from the start of treatment. Major toxicity was neutropenia (17% grade 3 and 6% grade 4). All other grade 3 and 4 hematotoxicities and non-hematological toxic effects ranged between 2% and 11% Conclusions Because of its efficacy and low toxicity, bendamustine in combination with rituximab may be an alternative treatment for aggressive lymphomas in old patients not eligible for R-CHOP. These results, however, need to be confirmed in larger studies.

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.

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 below120 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.

Infection with the SARS (Severe Acute Respiratory Syndrome)-associated coronavirus results in respiratory failure probably by immunological mechanisms in 10% of patients. Laboratory markers that predict subsequent respiratory failure would therefore be useful in patient management. We describe the clinical course, hematologic parameters, lymphocyte subpopulations and markers of inflammation in two patients with SARS, i.e., one man with diabetes mellitus and one pregnant woman, infected by the same viral isolate. The patient with underlying diabetes mellitus developed respiratory failure after admission in the second week of the illness while the second patient developed only a mild disease without respiratory failure. Subsequent respiratory dysfunction was associated with low numbers of Natural Killer (NK) cells at presentation and elevated CRP levels during the illness. NK cells and CRP levels at the end of the first week of the disease might be related to subsequent respiratory dysfunction and may link underlying conditions to disease severity.

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.

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.

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 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.

Background Bendamustine, an alkylating agent with a nitrogen mustard group and a purine-like benzimidazol group, has been shown to be effective in several solid tumors and indolent non-Hodgkin’s lymphomas, but has not yet been studied for efficacy in aggressive lymphomas. Patients and methods We conducted a phase II study in patients with relapsed or refractory high-grade non-Hodgkin’s lymphomas, using bendamustine at a dose of 120 mg/m2 on days 1 and 2, every 3 weeks for up to six cycles. Twenty-one patients were enrolled; 18 were evaluable for response and toxicity, 10 of whom were refractory to previous chemotherapy. Results With three patients achieving a complete response (at 6, ≥8 and ≥22 months) and five a partial response (three at 2 months, one at 3 months and one at 10 months), the total response rate of the evaluable patients was 44% (eight out of 18; 38% of all patients). Two complete and two partial responders were refractory to prior treatment. In 10 patients, treatment had to be stopped after one to three cycles due to progressive disease or hematological toxicity (n = 2). Non-hematological side effects were mild. Eight (13%) WHO grade 3 and no grade 4 events were observed in 60 evaluable treatment cycles. Hematologic toxicity was moderate (grade 3 and 4): anemia in five cycles (8%), leukopenia in seven (12%) and thrombocytopenia in eight (13%). Conclusions Bendamustine as a single agent is effective against aggressive lymphoma, even in cases of refractory disease. Further studies are warranted to determine the significance of bendamustine in the treatment of aggressive lymphomas.

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.

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.

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 (p0.05) 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.

7501 Background: This multicenter, randomized, phase III study compared B-R and CHOP-R as first-line treatment in patients (pts) with indolent lymphomas or mantle cell lymphoma and was first published in The Lancet in 2013. The final analysis demonstrated a significantly prolonged progression-free survival (PFS) in the B-R group compared to the CHOP-R group, with a median PFS of 69.5 vs. 31.2 months, respectively. In the current analysis, we present updated results for overall survival (OS), time-to-next-treatment (TTNT), and secondary malignancies (sNPL) with a median follow-up of 113 months for patients with indolent lymphomas (excluding MCL). Methods: 447 pts with indolent lymphomas were randomized to receive B-R or CHOP-R for a maximum of 6 cycles. The primary endpoint was PFS; secondary endpoints included OS, TTNT, and sNPL. Results: Patient characteristics were well balanced between arms; median age was 64 years. The difference in OS between the two treatment arms was not statistically significant, with 60 deaths in the B-R group vs 68 deaths with CHOP-R (HR 0.82, 95% CI 0.58 – 1.15, p = 0.249). The estimated 10-year survival rates were 71% for B-R and 66% for CHOP-R. TTNT was significantly prolonged with B-R compared with CHOP-R (HR 0.52, 95% CI 0.38 – 0.69, p < 0.001). Median TTNT was not yet reached in the B-R group (95% CI 124.9 – n.y.r) vs. 56 months in the CHOP-R group (95% CI 39.1 – 82.0). Patients treated initially with B-R needed fewer second-line treatments due to disease progression compared to CHOP-R treated pts: 73 pts (34%) in the B-R group received salvage treatment compared with 106 pts (52%) in the CHOP-R group. For B-R pts, CHOP-R was used as second-line therapy 26 times (36%), whereas B-R was used for pts initially treated with CHOP-R 49 times (46%). 36 pts with sNPL were observed in the B-R group compared with 39 in the CHOP-R group, with 7 hematological malignancies in both groups to date. Conclusions: In pts with previously untreated indolent lymphomas, B-R demonstrates a PFS and TTNT benefit over CHOP-R. Clinical trial information: NCT00991211.

Summary 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.

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. peerReviewed

AC133 is an antigen expressed on CD34+ hematopoietic progenitor cells. In acute myeloid leukemia (AML) it is expressed on leukemic blasts of most FAB subtypes. However, few data are available regarding coexpression of other surface antigens. We measured AC133 expression on AML blasts from 28 consecutive patients at initial diagnosis (n=26) or at diagnosis of first relapse (n=2) and on 26 leukapheresis products from 14 patients. In AML AC133 correlated with CD34 expression (Spearman r=0.4711, P=0.0114) and even stronger with combined CD34/CD33 expression (Spearman r=0.5083, P=0.0068). In leukapheresis products AC133 expression correlated with CD34 expression (Spearman r=0.7495, P=0.002) and the yield of the obtained amount of CD34+ cells (Spearman r=0.6484, P=0.0121). In conclusion AC133 expression is closely related to CD34 expression in AML. In leukapheresis products AC133 provides an additional marker for selection of PBPC autografts in AC133- AML.

The Wilms' tumor gene (wt1) encodes a transcription factor involved in urogenital development, in particular in renal differentiation, and in hematopoietic differentiation. Differentiation of a number of solid tumor and leukemic cells lines can be mediated by 1,25-dihydroxyvitamin D(3). This is predominantly mediated by the nuclear receptor for 1,25-dihydroxyvitamin D(3), the vitamin D receptor (VDR). In initial experiments addressing a possible link between WT1 and VDR, we observed a correlated expression of WT1 and VDR mRNA in samples from renal tissues. HT29 colon carcinoma cells, stably transfected to express WT1, exhibited elevated endogenous VDR levels compared with control cells transfected with a control construct. Elevated VDR expression was found in wt1-transfected human embryonic kidney 293 cells, as well. In transient cotransfection experiments, we observed an activation of a vdr promoter reporter by WT1 through a WT1 recognition element, indicating transcriptional regulation of the vdr gene expression by WT1. The responsive sequence element was specifically bound by wild-type, but not by mutated WT1, in electrophoretic mobility shift assays. HT29 colon carcinoma cells, which respond to 1,25-dihydroxyvitamin D(3) with slow induction of growth arrest, were investigated for the influence of WT1 on 1,25-dihydroxyvitamin D(3)-mediated growth suppression. Although HT29 cells transfected with a control construct responded moderately to 1,25-dihydroxyvitamin D(3), the response of HT29 cells expressing WT1 was strikingly enhanced. Stimulation with dihydroxyvitamin D(3) caused an up to 3-fold reduction in the growth rate of different HT29 clones expressing WT1 as compared with control cells lacking WT1 expression. Thus, induction of VDR by WT1 leads to an enhanced response to 1,25-dihydroxyvitamin D(3). We conclude that the vitamin D receptor gene is a target for transcriptional activation by WT1, suggesting a possible physiological role of this regulatory pathway.

Induction of apoptosis in vitro using gemcitabine (dFdC) in combination with cladribine (2-CdA) and other cytotoxic drugs on malignant mononuclear cells (MNCs) of patients with acute myeloid leukemia (AML, n=20) and chronic lymphocytic leukemia (CLL, n =20) in myeloid (HL60, HEL) and lymphatic cell lines (HUT78, JURKAT) was investigated using different incubation conditions (simultaneous and consecutive). Furthermore, the influence of dFdC on the level of intracellular metabolites of 2-CdA was studied using high-performance liquid chromatography (HPLC). Apoptosis was evaluated using flow cytometry with 7-aminoactinomycin D. In MNCs of patients with CLL, dFdC + 2-CdA showed an antagonistic effect when applied simultaneously. This antagonism was reduced by consecutive application. The combination of dFdC with doxorubicin was synergistic, independent of incubation schedule. In blasts from newly diagnosed patients with de novo AML, all drug combinations (dFdC+2-CdA, doxorubicin, or cytosine arabinoside) were antagonistic by simultaneous incubation. Reduced antagonism or even synergism was shown (P0.001) by consecutive incubation. The simultaneous combination of dFdC with 2-CdA in all tested cell lines resulted in a competitive inhibition on the rate of apoptosis. By changing the incubation period to a consecutive schedule, the antagonism was diminished or synergism of apoptosis was measured (P0.001). Using similar incubation conditions, these experiments were supported by HPLC measurement of intracellular metabolites of 2-CdA influenced by dFdC application. In conclusion, we demonstrated that the efficacy of dFdC in vitro in combination with other cytotoxic drugs depends on the incubation condition and on the origin of neoplastic cells (lymphatic vs myeloid). The data suggest that simultaneous combination therapy with purine and pyrimidine analogues may not improve the clinical efficacy of one or the other drug administered alone.

Peripheral T cell lymphomas are a heterogeneous group of post-thymic, mature lymphoid malignancies, accounting for approximately 10-15% of all non-Hodgkin's lymphomas. A rare entity within this group is represented by hepatosplenic gammadelta T cell lymphoma, which is characterized by primary extranodal disease with typical sinusoidal or sinusal infiltration of the liver and the spleen, respectively, by expression of the T cell receptor gammadelta chain, and by a number of other frequent clinicopathological features including aggressive course of disease. In contrast to these common attributes some biologic characteristics such as expression of cytotoxic proteins and cytotoxic activity have been controversial. In this review, clinicopathological, immunophenotypical, molecular biological, cytogenetical and biological findings, and diagnostic and therapeutic difficulties in hepatosplenic gammadelta T cell lymphoma are discussed.

Although the kidney is frequently involved in malignant monoclonal gammopathy, the clinical outcome of the patients varies considerably. We retrospectively assessed the clinical course in seventeen patients with acute and chronic renal failure suffering from multiple myeloma, and analyzed their case history focusing on the therapeutic management, the possible clinical improvement as well as the patients' outcome. Treatment included chemotherapy (n = 17), forced diuresis (n = 3), hemodialysis (n = 11, 7 chronic, 4 intermittent) and plasmapheresis (n = 3). Renal function improved in five patients, and was stabilized compensated in four. Seven patients developed end-stage renal disease, one refused further treatment and was lost for follow up. In addition to renal failure, the most frequent complications included local bone destruction (all), anemia (n = 12), low platelet count (n = 11), and bacterial infections (n = 9). One year survival rate after admission to the nephrology department was 76 percent. Chemotherapy in combination with renal replacement therapy may improve the clinical course even in MM patients with serum creatinine levels above 3.0 mg/dL or end-stage renal disease.

The association between CD4 cell count and duration of virus load suppression was investigated in 558 patients in the Frankfurt HIV Clinic Cohort who had begun highly active antiretroviral therapy and who had virus load declines to/=500 copies/mL. The Kaplan-Meier method estimated viral rebound in 42.5% of patients by 24 weeks and in 64.3% by 84 weeks. Risk of viral rebound was independently associated with baseline and changes from baseline CD4 cell count. The achieved CD4 cell count was the most important factor for prediction of viral rebound (relative hazard, 5.4 for an updated CD4 cell count of20 vs.500 copies/mL; P=.0001). Baseline virus load was not associated with virus load rebound. Lower baseline CD4 cell counts were associated with increased risk of viral rebound; however, this risk was significantly reduced in persons with low baseline CD4 cell counts who experienced substantial increases in CD4 cell counts during follow-up.

New antiretroviral strategies aim to reduce plasma HIV RNA (viral load) to below the limits of detectability of assays with the objective of reducing viral replication in order to stop or reverse the pathogenic process and prevent development of drug resistance. First use of a protease inhibitor might offer the most realistic chance of achieving this aim. Our objective was to study the virological response to protease inhibitors in patients taking them for the first time.A total of 901 patients from a large outpatient clinic were followed a mean of 15 months from the time of starting a protease inhibitor until 1 May 1998. Viral load and CD4 cell count measurements were made on average every 34 days.Overall there was a 79% [95% confidence interval (CI), 76-82] probability of the patients achieving a viral load500 copies/ml by 24 weeks after starting the protease inhibitor. In a multiple Cox regression model, those with lower initial viral load [relative hazard (RH), 0.72; P0.0001], higher CD4 cell count (RH, 1.07; P = 0.002), those starting other new drugs at same time as the protease inhibitor (RH, 1.46 for two versus none; P = 0.003), those who were antiretroviral-naive, and those using indinavir or nelfinavir were more likely to achieve such levels. In those 651 patients achieving viral load500 copies/ml within 24 weeks, there was an estimated 53% (95% CI, 51-55) probability of rebound of viral load to500 copies/ml by 52 weeks from the first undetectable value. Again, those who had started other new drugs at the same time as the protease inhibitor (RH, 0.57; P = 0.003 for starting two versus none) tended to experience a lower probability of viral load rebound, as did those with higher initial CD4 cell count (RH, 0.87 per 100 x 10(6)/l higher; P = 0.0007). Those who took saquinavir achieved less durable virological responses than those who took other protease inhibitors.Starting protease inhibitor therapy with two other new antiretroviral drugs simultaneously with protease inhibitor therapy offers a better best chance of achieving sustained viral load500 copies/ml than starting fewer new drugs.

OBJECTIVE Recent reports suggest that complex interactions exist between the neuroendocrine and immune systems. It has been shown for example that cytokines are able to stimulate the hypothalamo-pituitary-adrenal axis. In addition, some studies present evidence that endothelin is able to modulate the activity of several hypothalamic-pituitary axes, e.g. by inducing the ACTH production. DESIGN We investigated the effects of interleukin-2 on endothelin levels and the hypothalamo-pituitary-adrenal axis. We determined the interleukin-6, big-endothelin, endothelin-1, ACTH, cortisol and AVP responses to intravenously and subcutaneously administered interleukin-2 in 8 cancer patients in a randomized placebo controlled trial. PATIENTS 8 Patients (2 female and 6 male), age 44 ± 4.8 years, were enrolled. All patients had a World Health Organization performance status of 1 or less and a Karnofsky Index of at least 80%. MEASURMENTS Blood-samples were taken before and 15, 30, 45, 60, 120, 180, 240, 300 and 360 min after interleukin-2 injection. Cytokine serum levels and the plasma levels of big-endothelin, endothelin, ACTH and AVP were analysed using radioimmunoassays. Cortisol was assayed by an enzyme-linked immunosorbent assay. RESULTS Interleukin-2 treatment significantly increased plasma big-endothelin levels (P

Summary Background: The bcl-2 oncoprotein is suggested to be directly involved in the emergence of drug resistance by disrupting or delaying the apoptotic program and promoting tumor survival. Patients and methods: In order to define the clinical relevance of the bcl-2 mRNA expression in acute myeloid leukemia (AML) and its correlation to therapy outcome and prognosis, we analyzed 219 AML bone marrow (BM) samples, including 119 patients with de novo AML at presentation, 37 with AML following myelodysplastic syndrome (MDS), as well as 42 BM samples of AML in relapse and 21 in complete remission (CR) using RT-PCR. For performing quantitative measurements of bcl-2 mRNA, we developed a quantitative RT-PCR. Results: Bcl-2 mRNA was detectable in 133 of 156 (84%) patients at diagnosis and 40 of 42 (95%) at relapse. AML patients with high bcl-2 mRNA expression achieved lower CR rates than those with no or low expression. Concerning the long-term outcome, the overall (OS) and disease-free survival (DFS) was significantly worse in AML patients with high expression levels of bcl-2 mRNA. The three-year OS for all newly diagnosed AML patients was 49% and 10% (P = 0.028), respectively, and 71% and 15% (P = 0.0004) for patients 60 years, the bcl-2 expression was not associated with remission rate or survival. Conclusions: The expression oibcl-2 mRNA may serve as a prognostic factor predicting remission outcome and long-term prognosis in AML.

The tumor suppressor gene wt1 (Wilms' tumor gene) encodes for a zinc finger DNA-binding protein with predominantly transcription repressing properties. Because wt1 has been shown to be expressed in the vast majority of patients with acute myeloid leukemias (AML), we investigated the relevance of wt-1 mRNA expression regarding prognosis and possible prediction of relapse during follow-up. Totally bone marrow-derived blasts of 139 AML patients (129 newly diagnosed AML patients, 22 AML patients again in first relapse, and 10 AML patients analyzed primarily in first relapse) were studied for wt1 mRNA expression. Seventy-seven patients were analyzed for wt1 mRNA expression during follow-up. wt1-specific reverse transcription-polymerase chain reaction (RT-PCR) was performed and the amplification product was visually classified as not, weakly, moderately, or strongly amplified, as described previously. PCR products were quantitated by competitive PCR using a shortened homologous wt1 construct standard in representative cases. The expression of wt1 transcripts was correlated to age, French-American-British (FAB) subtype, phenotype, karyotype, and long-term survival. wt1 mRNA was detectable in 124 of 161 (77%) samples at diagnosis and in first relapse. wt1 expression was independent from age, antecedent myelodysplastic syndrome or FAB subtype, with the exception of a significant difference in M5 leukemias showing wt1 transcripts in only 40% (P = .0025). There was no correlation between the level of wt1 mRNA and response to treatment or the prognostic groups defined by the karyotype. Concerning long-term survival, patients with high levels of wt1 had a significantly worse overall survival (OS) than those with not detectable or low levels. The 3-year OS for all newly diagnosed AMLs was 13% and 38% (P = .038), respectively, and 12% and 43% (P = .014) for de novo AMLs. The difference was more distinct in patients less than 60 years of age. During follow-up, all patients achieving complete remission became wt1 negative. Reoccurrence of wt1 transcripts predicted relapse. The data indicate that high expression of wt1 mRNA is associated with a worse long-term prognosis.

The Wilms' tumor gene product (WT-1) is suggested to act as a tumor suppressor in childhood malignancies of the kidney and as a transcription factor with regulating activity on a number of growth and differentiation factors. Wt-1 has been shown to be expressed in blast cells of the vast majority of patients with acute myeloid and lymphoblastic leukemias (AL) by a number of workers. High levels of wt-1 mRNA expression in blast cells of newly diagnosed AML patients predict worse prognosis when compared to patients with no or low wt-1 mRNA expression. Patients achieving complete responses after chemotherapy usually lose detectable signals of wt-1. In relapse of the disease reoccurrence of wt-1 mRNA can be determined in almost all patients with initially detectable wt-1 mRNA. Using sensitive techniques such as reverse transcription polymerase chain reaction (RT-PCR) relapses are preceded by wt-1 expression in some cases. Although a subpopulation of normal hematopoietic precursor cells has also been shown to express message for wt-1, detectable levels of wt-1 during follow-ups in AML patients have been shown to be useful as a marker for residual blast populations or even to predict relapse of AML. Whether the high level of wt- expression is a non-specific phenomenon resulting from malignant transformation or whether it has an impact on the pathophysiology of AML or the uncontrolled growth of AML blasts is still controversial. However, there are indicators for an involvement of wt-1 in malignant events of AML blasts such as the downregulation of wt-1 in chemically induced differentiation of AML blast cell lines or the interactions of wt-1 with the protooncogene bcl-2 and the tumor suppressor gene p53. In conclusion, its possible relevance as an AML marker and its role in pathophysiological mechanisms in AML will still have to be defined in the future.

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.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 4021335.274 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; p0.0001). 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; p0.0001), haematological toxicity (77 [30%] vs 173 [68%]; p0.0001), infections (96 [37%] vs 127 [50%]); p=0.0025), peripheral neuropathy (18 [7%] vs 73 [29%]; p0.0001), and stomatitis (16 [6%] vs 47 [19%]; p0.0001). 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).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.Roche Pharma AG, Ribosepharm/Mundipharma GmbH.

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of 51Cr and/or spontaneously release high amounts of 51Cr. Recently, we established a culture system which promotes the outgrowth of cytotoxic T lymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T-cell lines and clones, we modified a previously described cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20:1). However, a minority of AML blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.

We have previously shown that human squamous cell carcinomas (SCC) express the interleukin 2 receptor (IL2R)-alpha and -beta chains, and that the ligand, IL2, directly inhibits growth of the tumor in vitro and in vivo in the tumor xenograft-nude mice model. We now show that the alpha and beta chains of IL2R are expressed on a variety of human carcinoma cell lines and on normal human keratinocytes in early-stage cultures. While all carcinoma cells in a population expressed IL2R-alpha and -beta proteins, in keratinocytes obtained from different normal donors, variable proportions of cells were positive, as measured by flow cytometry. The carcinoma lines and 2/5 keratinocyte lines studied were also found to contain transcripts for the IL2R-gamma chain detectable by combined reverse transcription-PCR (RT-PCR) and hybridization with the specific cDNA probe. Incubation of the gastric (HR) or renal cell carcinoma (RCC) cell lines, but not of other IL2R+ carcinoma cell lines or normal keratinocytes, in the presence of IL2 resulted in dose-dependent inhibition of tumor cell growth. Monoclonal antibodies (MAbs) specific for IL2R-beta chain completely reversed this growth inhibitory effect of IL2. The ligand, IL2, also down-regulated surface expression of its own receptor and of intercellular adhesion molecule-I (ICAM-I) or class I major histocompatibility complex (MHC) antigens on IL2R+ tumor cells. All carcinoma cells studied incubated in the presence of IL2 exhibited significantly increased sensitivity to growth-inhibitory effects of other cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta. IL2 inhibited growth of the HR cells by arresting a significant proportion of tumor cells in the G0/G1 phase of the cell cycle. Thus, IL2 can have direct effects on IL2R+ carcinoma cells, leading to changes in growth or to increases in sensitivity of tumor cells to cytostatic activities of other cytokines.

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.

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.

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable beta regions (V beta). To perform the TCR-V beta analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5' primers specifically recognizing the sequences of 20 V beta gene families and a 3' primer annealing to the constant region of the beta chain. The TCR-alpha constant region (C alpha) gene was co-amplified as a standard for the calculation of the percentage of each TCR-V beta gene expressed. The frequency of individual V beta regions expressed on TIL was computed from the ratio of cpm V beta to cpm C alpha for each V beta region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of V beta genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of V beta-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the V beta-genes usually expressed in normal PBL were not expressed in fresh TIL in melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of V beta genes occurred. Although in 4/5 TIL cultures this selection involved the V beta 7 gene, no relationship could be established between V beta gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3+CD8+ T-cell lines with V beta-gene expression restricted to 1 or 2 V beta families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.

Background: Regarding standardization of treatment, classification, and pathophysiology of peripheral T- and NK-cell neoplasias the current knowledge is markedly behind that of B-cell lymphomas, which are approximately 10 times more frequent. In May 2000, the study group ‘Peripheral T- and NK-cell Neoplasias’ was founded in Frankfurt/M. This group decided on a clinical protocol and a scientific program for research on the pathophysiology of these entities. Rationales for the therapeutic regimen are the efficacy of cyclophosphamide and doxorubicine as shown in protocols for treatment of high grade lymphoma, the synergism of cyclophosphamide and fludarabine, and reports demonstrating the efficacy of fludarabine in T-cell neoplasias. Patients and Methods: Patients will be treated with a combination of fludarabine (30 mg/m2 days 1–3), cyclophosphamide (1000 mg/m2 day 1) doxorubicine (25 mg/m2 day 2+3) (FCD). For patients ≧65 years a dose-reduced FCD regimen will be administered. In patients included in the treatment study and additionally in patients with indolent disease not requiring therapy, scientific projects on the biology of peripheral T- and NK-cell neoplasias will be performed. Conclusions: Expected conclusions of the projected study are the establishment of treatment and diagnostic standards, and improvement of classification of these entities by clinical, morphologic and biologic parameters.

We describe a case of cytotoxic T-large granular lymphocyte leukaemia showing typical morphological features, expressing antigens characteristic for cytotoxic T cells and exhibiting marked natural killer-like cytotoxicity towards different target cells. Moreover, characterization of the T-cell receptors revealed simultaneous expression of two different types of β-chains as well as α-chains by the malignant cell clone. The patient had an 8 year history of indolent disease, before progressing to an aggressive clinical course hardly responsive to chemotherapeutic treatment. This is the first description of a peripheral T-cell neoplasm expressing four distinct types of T-cell receptor molecules.

Abstract 2395 While already tested in T-PLL, the impact of chemo-immunotherapy with alemtuzumab in combination with bi-weekly CHOP on autologous stem cell harvest (ASCH) has not yet been analysed in the context of first line treatment of primary systemic peripheral T-cell lymphoma (PTCL). We therefore evaluated the feasibility of ASCH in the first 23 patients of the ongoing international multicenter ACT-1 study, the first, and so far only, randomized trial in primary systemic PTCL. The ACT trials (ACT-1 + ACT-2) test the addition of alemtuzumab to CHOP followed, in younger patients (ACT-1), by high-dose therapy with autologous stem cell transplant (HDT+ASCT). The aims of the analysis were: (i) feasibility of ASCH in alemtuzumab + CHOP (A-CHOP) treated patients as compared to patients not receiving the antibody, but otherwise treated and managed in the same way, and (ii) comparison of ASCH counts in the two treatment arms. By July 2010, 20 patients, 11 in the standard treatment (arm A) and 9 in the experimental treatment (arm B) cohort, had undergone induction therapy and had been primed for subsequent ASCH according to local guidelines. Histological subtype distribution in the two treatment groups showed: PTCL not otherwise specified N=4 (arm A) and N=5 (arm B), angioimmunoblastic N=5 (arm A) and N=4 (arm B), extranodal NK/T-cell, nasal type N=1 (arm A), hepatosplenic N=1 (arm A). Pre-therapeutic evidence of bone marrow involvement was present in 4 (arm A) and 2 (arm B) patients, respectively. Of the original 23 patients, three did not undergo stem-cell harvest due to progressive disease (1 pt), patient's decision (1 pt), and pre-therapeutic CNS involvement (1 pt). Among the 20 harvested patients, ASCH failure was experienced in three patients (standard arm N=1 and experimental arm N=2; p=0.57). In two patients (one in each treatment cohort) a suboptimal stem cell yield could be optimized by the use of plerixafor according to local guidelines. A comparison of stem cell counts (CD34+ cells × 106/kg body weight) from the two treatment cohorts showed a trend towards moderately lower stem cell yields in alemtuzumab-treated patients (3.34 CD34+ cells × 106/kg body weight in arm B vs 6.54 CD34+ cells × 106/kg body weight in arm A, p=0.03). In conclusion, the addition of alemtuzumab to bi-weekly CHOP in the setting of first-line therapy of primary systemic PTCL does not significantly impair ASCH prior to upfront autologous stem cell transplant. Disclosures: No relevant conflicts of interest to declare.

Krebserkrankungen mit unbekanntem Primartumor sind durch fehlenden Nachweis einer Primarmanifetstion bei histologisch gesichertem, metastasiertem Malignom nach Durchfuhrung der konventionellen Tumordiagnostik und gegebenenfalls weiterfuhrenden Untersuchungen definiert. Diese Gruppe von Erkrankungen wird synonym auch als »cancer of unknown origin« (CUO) oder als »unknown primary tumor« (UPT) bezeichnet, am gebrauchlichsten und in der internationalen Literatur am meisten etabliert ist jedoch der Begriff »cancer of unknown primary « (CUP bzw. CUP-Syndrom). Aufgrund des nicht bekannten Primartumors kann die Einteilung dieser Erkrankungen nicht organbezogen erfolgen, sondern muss nach histologischen Kriterien festgelegt werden. Unterschieden werden vier grosere Gruppen, die Adenokarzinome (ACUP), die undifferenzierten Karzinome (UCUP), die Plattenepithelkarzinome (SqCUP) und die neuroendokrinen Karzinome (NeCUP). Andere Histologien sind mit einer Frequenz von 1–3% selten.

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.

After a 5-day period of continuous intravenous infusion of recombinant interleukin 2 (rIL-2) in seven patients with malignant melanoma or gastric or pancreatic cancer, different lymphocyte subsets were separated from patients' blood and tested ex vivo for cytotoxic activity against various tumour cell lines. Lytic activity was mediated by CD3+CD56+, CD3−CD56+, CD3−CD2+ and CD8+CD56+ lymphocytes. No cytotoxic activity could be observed within the CD3+CD56−, CD3+CD2+ or CD4+ T cell subsets. To characterize CD56+ cytotoxic cells further, the expression of other antigens on this population was analysed before and after IL-2 therapy. CD3, CD4, CD16 and CD57 antigens were weakly expressed, and the IL-2 receptor (CD25) was not detectable on these cells either before and after treatment with IL-2. In contrast, increased expression of CD2, CD8 and HLA-DR antigens occurred following therapy. The divergence of CD3 and CD8 antigen expression after IL-2 therapy was caused by an increase in CD3−CD8+ cells, detectable as a low-density CD8+ subset. This study shows that cytotoxic activity of in vivo IL-2-activated killer cells is predominantly, but not exclusively, mediated by CD3−CD56+ lymphocytes, partially coexpressing the CD8 antigen and lacking the expression of CD 16 antigens.