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Supplementary Figures 1-5 from Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system

Supplementary Tables 1-4 from Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system

Supplementary Methods, Figure Legends 1-5, Table Legends 1-4 from Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system

The regulation of the necrotic death and its relevance in anticancer therapy are largely unknown. Here, we have investigated the proapoptotic and pronecrotic activities of two ubiquitin-proteasome system inhibitors: bortezomib and G5. The present study points out that the glioblastoma cell lines U87MG and T98G are useful models to study the susceptibility to apoptosis and necrosis in response to ubiquitin-proteasome system inhibitors. U87MG cells show resistance to apoptosis induced by bortezomib and G5, but they are more susceptible to necrosis induced by G5. Conversely, T98G cells are more susceptible to apoptosis induced by both inhibitors but show some resistance to G5-induced necrosis. No overt differences in the induction of Noxa and Mcl-1 or in the expression levels of other components of the apoptotic machinery were observed between U87MG and T98G cells. Instead, by comparing the transcriptional profiles of the two cell lines, we have found that the resistance to G5-induced necrosis could arise from differences in glutathione synthesis/utilization and in the microenvironment. In particular, collagen IV, which is highly expressed in T98G cells, and fibronectin, whose adhesive function is counteracted by tenascin-C in U87MG cells, can restrain the necrotic response to G5. Collectively, our results provide an initial characterization of the molecular signals governing cell death by necrosis in glioblastoma cell lines. [Mol Cancer Ther 2009;8(11):3140–50]

Genetic vaccination using naked plasmid DNA is an immunization strategy both against infectious diseases and cancer.In order to improve efficacy of DNA vaccines, particularly in large animals and humans, different strategies have been pursued. These vaccination strategies are based on different application routes, schedules and coexpression of immunomodulatory molecules as adjuvants. Our mouse tumor model offers the possibility to investigate Her2/neu DNA vaccines in different settings, that is, intramuscular or intradermal application with or without coexpression of adjuvants. The immunogenicity of predicted peptides for Her2/neu specific memory T cells were screened and confirmed after intramuscular and intradermal application. Protection from tumor growth in tumor challenge experiments and both T cell and humoral immune responses against Her2/neu peptides are used as surrogate parameters for vaccine efficacy.

(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires—patients’ peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model—to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients’ own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor’s TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.

BackgroundAdoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC–peptide presentation.MethodsHere we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas.ResultsGenerated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity.ConclusionTaken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.

Adoptive T cell therapy (ATT) has revolutionized the treatment of cancer patients. A sufficient number of functional T cells are indispensable for ATT efficacy; however, several ATT dropouts have been reported due to T cell expansion failure or lack of T cell persistence in vivo. With the aim of providing ATT also to those patients experiencing insufficient T cell manufacturing via standard protocol, we evaluated if minimally manipulative prolongation of in vitro expansion (long-term [LT] >3 weeks with IL-7 and IL-15 cytokines) could result in enhanced T cell yield with preserved T cell functionality. The extended expansion resulted in a 39-fold increase of murine CD8(+) T central memory cells (Tcm). LT expanded CD8(+) and CD4(+) Tcm cells retained a gene expression profile related to Tcm and T memory stem cells (Tscm). In vivo transfer of LT expanded Tcm revealed persistence and antitumor capacity. We confirmed our in vitro findings on human T cells, on healthy donors and diffuse large B cell lymphoma patients, undergoing salvage therapy. Our study demonstrates the feasibility of an extended T cell expansion as a practicable alternative for patients with insufficient numbers of T cells after the standard manufacturing process thereby increasing ATT accessibility.

Aims: It is largely unknown whether cancer patients seen in routine care show ventricular arrhythmias in 24 h electrocardiograms (ECGs), and whether when they are detected they carry prognostic relevance. Methods and Results: We included 261 consecutive cancer patients that were referred to the department of cardiology for 24 h ECG examination and 35 healthy controls of similar age and sex in the analysis. To reduce selection bias, cancer patients with known left ventricular ejection fraction

Simple Summary It is largely unknown how frequently cancer patients seen in routine care show ventricular arrhythmias during 24-h electrocardiograms. We have found that non–sustained ventricular tachycardia episodes of ≥3 and ≥4 beats duration were more frequent in cancer patients than controls. Non–sustained ventricular tachycardia with ≥4 beats and ≥20 premature ventricular contractions/day seen in routine 24-h electrocardiograms of patients with cancer carry prognostic relevance. Abstract Aims: It is largely unknown whether cancer patients seen in routine care show ventricular arrhythmias in 24 h electrocardiograms (ECGs), and whether when they are detected they carry prognostic relevance. Methods and Results: We included 261 consecutive cancer patients that were referred to the department of cardiology for 24 h ECG examination and 35 healthy controls of similar age and sex in the analysis. To reduce selection bias, cancer patients with known left ventricular ejection fraction

Graft-versus-host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High-resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E-stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD.

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.

Adoptive transfer of engineered T cells has shown remarkable success in hematopoietic malignancies. However, the current most common strategy of targeting lineage-specific antigens often leads to undesirable side effects and a high relapse rate. Therefore, novel treatment approaches are still needed. Oncogenic somatic mutations represent ideal targets because of tumor specificity: such (neo)antigens can be recognized by T cell receptors (TCR) in the context of MHC-peptide presentation. Here we have generated T cell lines from multiple healthy donors targeting one of the most common driver mutations found in B-cell lymphomas; a missense mutation on adaptor protein MyD88 changing leucine at position 265 to proline (L265P). T cell lines generated by autologous in vitro priming were reactive selectively against the predicted mutant epitope restricted to HLA-B7, but not against the corresponding wild-type peptide. Cloned TCRs from these lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with mutation-specific TCR (TCR-T cells) recognized and killed cell lines of diffuse large B-cell lymphoma characterized by intrinsic MyD88 L265P. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models, while initial safety screening did not indicate any sign of cross- or allo-reactivity risk. Taken together, our data suggest that mutation-specific TCRs can be used to target MyD88 L265P mutation, and hold promise for precision therapy for a significant subgroup of B-cell malignancies. Disclosures Keller: Bristol Myers Squibb: Honoraria, Other: Travel support, Speakers Bureau. Busse:Daiichi Sankyo: Other: Travel Support; Hexal: Honoraria, Research Funding; Roche: Honoraria; BMS: Honoraria; Novartis: Research Funding.

Korrektur zu 10.1038/s41375-020-0826-9

Oncogenic MYD88 mutations, most notably the Leu 265 Pro (L265P) mutation, were recently identified as potential driver mutations in various B-cell non-Hodgkin Lymphomas (NHLs). The L265P mutation is now thought to be common to virtually all NHLs and occurs in between 4 and 90% of cases, depending on the entity. Since it is tumor-specific, the mutation, and the pathways it regulates, might serve as advantageous therapeutic targets for both conventional chemotherapeutic intervention, as well as immunotherapeutic strategies. Here, we review recent progress on elucidating the molecular and cellular processes affected by the L265P mutation of MYD88, describe a new in vivo model for MyD88 L265P-mediated oncogenesis, and summarize how these findings could be exploited therapeutically by specific targeting of signaling pathways. In addition, we summarize current and explore future possibilities for conceivable immunotherapeutic approaches, such as L265P-derived peptide vaccination, adoptive transfer of L265P-restricted T cells, and use of T-cell receptor-engineered T cells. With clinical trials regarding their efficacy rapidly expanding to NHLs, we also discuss potential combinations of immune checkpoint inhibitors with the described targeted chemotherapies of L265P signaling networks, and/or with the above immunological approaches as potential ways of targeting MYD88-mutated lymphomas in the future.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

Transformation of follicular lymphoma (FL) into aggressive disease and relapse of de novo diffuse large B cell lymphoma (DLBCL) are considered highly unfavourable events. However, most published data were acquired when rituximab was not routinely used. We retrospectively analysed 50 patients with transformed FL (tFL) in a multicenter study and compared them to 50 individuals with relapsed DLBCL (rDLBCL) who all obtained rituximab for the treatment of their disease. Our goal was to identify factors that predict a more favourable prognosis. After a median follow-up of 5.4 years from diagnosis, there was no significant difference in median overall survival (OS) from the date of transformation (tFL) or date of the first relapse (rDLBCL) (1.9 versus 3.9 years, P = .542). Of note, 5-year OS of patients with tFL was 46 %. Follicular lymphoma patients, treatment naïve prior to transformation, fared significantly better than pretreated patients (median not reached versus 1.4 years, P = .014). Regarding rDLBCL, female gender (13.9 versus 1.8 years, P = .019) and absence of rituximab prior to the first relapse (14.0 versus 1.8 years, P = .035) were favourable prognostic factors in a uni- and multivariate analysis. Only a proportion of patients received high-dose chemotherapy with autologous stem cell transplantation (HDT-ASCT), i.e. 38 and 52 % of patients with tFL and rDLBCL, respectively. Our data indicate that a favourable prognosis is conferred by treatment naivety in tFL and by rituximab naivety in rDLBCL. In contrast, we did not find a prognostic impact of HDT-ASCT in our series.

Tumor-induced immunosuppression remains a major challenge for immunotherapy of cancer patients. To further elucidate why an allogeneic gene-modified [interleukin-7 (IL-7)/CD80-cotransfected] renal cell cancer (RCC) vaccine failed to induce clinically relevant TH-1-polarized immune responses, peripheral blood mononuclear cells from enrolled study patients were analyzed by gene expression profiling (GEP) both prior and after vaccination. At baseline before vaccination, a profound downregulation of gene signatures associated with antigen presentation, immune response/T cells, cytokines/chemokines and signaling/transcription factors was observed in RCC patients as compared to healthy controls. Vaccination led to a partial reversion of preexisting immunosuppression, however, GEP indicated that an appropriate TH-1 polarization could not be achieved. Most interestingly, our results suggest that the nuclear factor-kappa B signaling pathway might be involved in the impairment of immunological responsiveness and the observed TH-2 deviation. In summary, our data suggest that GEP might be a powerful tool for the prediction of immunosuppression and the monitoring of immune responses within immunotherapy trials.

OBJECTIVES: Chemoimmunotherapy with cyclophosphamide, doxorubicin, vincristine, prednisolone and rituximab (R-CHOP) is the standard of care for patients with diffuse large B-cell lymphoma (DLBCL). However, management of elderly patients is challenging as critical comorbidities often account for increased number of treatment related complications. PATIENTS AND METHODS: In the past 8 years, we have treated elderly patients with a full-dose R-CHOP regimen by splitting the administration of cyclophosphamide and doxorubicin over two days (R-split-CHOP) to reduce peak plasma level. Here, we retrospectively analyzed the results of 30 patients with newly diagnosed DLBCL. RESULTS: The overall response rate was found to be 87%, the overall survival probability after 3 years was 60.6% (95% CI, 42.1%-79.0%), the progression-free survival probability 49.7% (95% CI, 30.4%-68.9%), respectively. Grade 3/4 infectious complications were reported in 30% of patients, yet no treatment-related deaths occurred. CONCLUSION: We suggest that R-split-CHOP could be a valuable option to safely administer full-dose intensity R-CHOP to elderly patients at risk of treatment-related complications.

BACKGROUND: Marginal zone lymphoma (MZL) is a non-Hodgkin lymphoma that occurs as extra nodal, nodal, or splenic. While MZL is generally considered an indolent disease, a substantial percentage of patients follow an unfavorable course. The objective of this retrospective analysis was to identify predictors for a reduced overall survival (OS), or conversely an increased OS. PATIENTS AND METHODS: One hundred and ninety-seven MZL patients were analyzed. Apart from assessing previously published risk factors, concomitant morbidity at diagnosis, transformation into aggressive lymphoma, and occurrence of additional malignancies were evaluated. RESULTS: Next to the known risk factors, i.e. above 60 years of age and elevated serum lactate dehydrogenase (LDH), we demonstrate that transformation into aggressive lymphoma, as well as additional malignancies, are important independent risk factors for a shortened OS in a multivariate analysis, irrespective of the MZL localization. Impressively, in the group of patients lacking LDH elevation, transformation, and/or additional malignancies, only 1 of 63 patients died during follow-up compared with 37 of 87 patients in the high-risk group (HR = 22.8; 95% confidence interval 3.1-167.0; P = 0.002). CONCLUSIONS: Our analysis proposes novel risk factors and warrants for a continuous follow-up to detect the occurrence of transformation and additional malignancies early on.

Angioimmunoblastic T cell lymphoma (AITL) belongs to the subgroup of mature T cell lymphomas according to the World Health Organization and is one of the common T cell lymphomas in Western countries. Particularly in cases in which histological confirmation cannot be easily achieved, immunophenotyping of peripheral blood can give important information for the differential diagnosis of AITL. sCD3− CD4+ T cells are a typical feature of AILT in flow cytometry of peripheral blood. In this retrospective study, the diagnostic value of flow cytometry for the diagnosis ‘AITL’ was assessed by comparing the frequency of sCD3− CD4+ T cells in leukemic AITL patients and in patients with other leukemic CD4+ T cell lymphomas. Immunophenotyping of peripheral blood by flow cytometry was performed in a lymphocyte gate using fluorochrome-labelled antibodies against CD3, CD2, CD4, CD5, CD7, CD8, CD10, CD14, CD16, CD19, CD56, CD57 and T cell receptor. In 17/17 leukemic AITL patients, a small but distinct population of sCD3− CD4+ T cells was detected (mean percentage of sCD3− CD4+ T cells in the lymphocyte gate: 11.9 ± 15.4%, range 0.1–51.8%). In contrast, sCD3− CD4+ T cells were found in only 1/40 patients with other leukemic CD4+ T cell lymphomas (one patient with mycosis fungoides). sCD3− CD4+ T cells have a high positive predictive value (94%) for the diagnosis ‘AITL’. Flow cytometry is particularly useful in the differential diagnosis of AITL, even if the aberrant T cell population has a very low frequency. Further biological characterization of this subfraction of lymphoma cells is warranted. Copyright © 2013 John Wiley & Sons, Ltd.

Leukemia-associated antigens such as proteinase-3 (PR3) and Wilms' tumor protein-1 (WT-1) are potential targets of T-cell responses, which can be monitored by T-cell assays within vaccination trials and after allogeneic stem cell transplantation (SCT). In chronic myeloid leukemia (CML) an aberrant cytokine profile of antigen-specific T-cells with predominant TNF-α secretion has previously been described. The aim of this study was to investigate whether these TNF-α(+)/IFN-γ(-) CD8(+) T-cells can also be observed in AML patients after SCT. Eight HLA-A2(+) AML patients at different time points after SCT were evaluated for HLA-A2-restricted CD8(+) T-cell responses against PR3, WT-1 and influenza-A using pentamer staining and different cytokine-based T-cell assays. Antigen-specific T-cell immune responses against influenza-A and PR3 were observed in 4/8 patients, WT-1-specific T-cells could be detected in 3/8 patients. Interestingly, four different cytokine secretion profiles of antigen-specific T-cells were detected: (1) IFN-γ(+)/TNF-α(+), (2) IFN-γ(+)/TNF-α(-), (3) TNF-α(+)/IFN-γ(-) and (4) IFN-γ(-)/TNF-α(-). TNF-α(+)/IFN-γ(-) CD8(+) T-cells are an interesting biological phenomenon which can obviously be observed also in AML patients. This finding has important implications for both T-cell biology and monitoring within immunotherapy trials. The functional characterization of these TNF-α(+)/IFN-γ(-) CD8(+) T-cells needs further investigations.

Using genetic engineering a humanized Fab fragment with specificity for CD19 was fused to a disulfide-stabilized single-chain antibody (dsFv) recognizing CD5. This format should show reduced immunogenicity and improved tissue penetration. The specificity of bsAb FabCD19xdsFvCD5 binding to target cells was verified by flow cytometry on B and T lymphoma cell lines. Binding affinities of both arms were compared with the bivalent parental antibodies against CD19 and CD5 by binding competition assay. Redirected lysis of B lymphoma cells by preactivated PBMC from healthy donors was demonstrated in a chromium-release assay. A clear dose-response relationship could be established in the range from 1 ng/mL to 10 g/mL bsAb. To evaluate the in vivo efficacy of bsAb FabCD19xdsFvCD5, NOD/SCID mice were intravenously injected with luciferase transfected Raji lymphoma cells together with pre-activated PBMC. Mice received five injections of therapeutic bsAb or control antibodies. While in the control groups all mice died within 40 to 50 days, 40% of bsAb treated animals survived longer than 60 days.

DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu+ syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumo...

In Chronic Myeloid Leukemia (CML), standard treatment consists of modern tyrosine-kinase inhibitors (TKI). Nevertheless, there is evidence that immune responses against leukemia-associated antigens (LAA) may play an important role in disease control. Dendritic cell (DC)- based immunotherapy is able to induce T cell responses against LAA and might therefore pose an interesting therapeutic option in CML, especially in the setting of minimal residual disease (MRD). GMP production of DC for clinical vaccination remains a time- and cost- intensive procedure and standardized DC generation is warranted. We asked whether maturation-induction with IFN-{gamma} and IFN-{alpha} has an influence on functional properties of DC derived from peripheral blood mononuclear cells (PBMC) in CML patients. Monocyte-derived DC from healthy donors and from patients with CML were analyzed after maturation-induction with our TNF-{alpha}-containing standard cytokine cocktail with or without addition of IFN-{alpha} and/or IFN-{gamma}. Our results confirm that the addition of IFN-{gamma} leads to enhanced IL-12 secretion in healthy donors. In contrast, in CML patients, IFN-{gamma} was not able to increase IL-12 secretion, possibly due to a higher degree of cell adherence and lower cell yield during the cell culture. Our data suggest, that- in contrast to healthy donors-, additional interferons are not beneficial for maturation induction during large-scale DC production in patients with CML.

Purpose In this phase II study (NCT00942747), temsirolimus was tested in patients with relapsed or refractory primary CNS lymphoma (PCNSL). Patients and Methods Immunocompetent adults with histologically confirmed PCNSL after experiencing high-dose methotrexate-based chemotherapy failure who were not eligible for or had experienced high-dose chemotherapy with autologous stem-cell transplant failure were included. The first cohort (n = 6) received 25 mg temsirolimus intravenously once per week. All consecutive patients received 75 mg intravenously once per week. Results Thirty-seven eligible patients (median age, 70 years) were included whose median time since their last treatment was 3.9 months (range, 0.1 to 14.6 months). Complete response was seen in five patients (13.5%), complete response unconfirmed in three (8%), and partial response in 12 (32.4%) for an overall response rate of 54%. Median progression-free survival was 2.1 months (95% CI, 1.1 to 3.0 months). The most frequent Common Toxicity Criteria ≥ 3° adverse event was hyperglycemia in 11 (29.7%) patients, thrombocytopenia in eight (21.6%), infection in seven (19%), anemia in four (10.8%), and rash in three (8.1%). Fourteen blood/CSF pairs were collected in nine patients (10 pairs in five patients in the 25-mg cohort and four pairs in four patients in the 75-mg cohort). The mean maximum blood concentration was 292 ng/mL for temsirolimus and 37.2 ng/mL for its metabolite sirolimus in the 25-mg cohort and 484 ng/mL and 91.1 ng/mL, respectively, in the 75-mg cohort. Temsirolimus CSF concentration was 2 ng/mL in one patient in the 75-mg cohort; in all others, no drug was found in their CSF. Conclusion Single-agent temsirolimus at a weekly dose of 75 mg was found to be active in relapsed/refractory patients with PCNSL; however, responses were usually short lived.

Chemokines are key regulators of both innate and adaptive immune responses. CCL4 (macrophage inflammatory protein-1β, MIP-1β) is a CC chemokine that has a broad spectrum of target cells including immature dendritic cells, which express the cognate receptor CCR5. We asked whether a plasmid encoding CCL4 is able to improve tumor protection and immune responses in a Her2/neu+ mouse tumor model. Balb/c mice were immunized twice intramuscularly with plasmid DNA on days 1 and 15. On day 25, a tumor challenge was performed with 2 × 10(5) syngeneic Her2/neu+ D2F2/E2 tumor cells. Different groups of mice were vaccinated with pDNA(Her2/neu) plus pDNA(CCL4), pDNA(Her2/neu), pDNA(CCL4) or mock vector alone. Our results show that CCL4 is able to (i) improve tumor protection and (ii) augment a TH1-polarized immune response against Her2/neu. Although Her2/neu-specific humoral and T-cell immune responses were comparable with that induced in previous studies using CCL19 or CCL21 as adjuvants, tumor protection conferred by CCL4 was inferior. Whether this is due to a different spectrum of (innate) immune cells, remains to be clarified. However, combination of CCL19/21 with CCL4 might be a reasonable approach in the future, particularly for DNA vaccination in Her2/neu+ breast cancer in the situation of minimal residual disease.

Radioimmunotherapy in relapsed/refractory mantle cell lymphoma patients: final results of a European MCL Network Phase II Trial

Depression and anxiety are the core disorders causing emotional distress in patients (pts) with metastatic breast cancer. The aim of our study was to screen metastatic breast cancer outpatients for anxiety and depression, and to investigate the influence of age, Karnofsky Performance Status (KPS), cancer activity, and inflammation as represented by IL-6 levels on these two mood disorders. Pts treated with chemotherapy for metastatic breast cancer (n = 70) were assessed using the Hospital Anxiety and Depression Scale (HADS) for symptoms (scores 0–21) and caseness (score ≥11) of clinical depression and anxiety. Blood samples for IL-6 concentrations were collected at 10:00 a.m. A total of 22 (31.4 %) pts were diagnosed with caseness of clinical depression and 23 (32.9 %) pts with clinical anxiety, while 12 pts were diagnosed positive for both mood disorders. Depression and anxiety were positively but moderately correlated (Spearman’s r 2 = 0.24, p < 0.001). IL-6 was significantly correlated with symptoms of depression (r 2 = 0.42, p < 0.001) and to a lesser extent to symptoms of anxiety (r 2 = 0.16, p = 0.001). In addition, IL-6 was positively associated with tumor progression (p < 0.001). Multiple linear regression analysis showed that tumor progression (standardized b = 0.226, p = 0.047), symptoms of anxiety (b = 0.292, p = 0.016), and IL-6 (b = 0.314, p = 0.007) were independently associated with clinical depression, whereas anxiety was linked to tumor progression (b = 0.238, p = 0.030), symptoms of depression (b = 0.407, p < 0.001) and age (b = −0.381, p < 0.001), but not to IL-6 (b = 0.168, p = 0.134). Even though a positive correlation between depression and anxiety exists, clinical parameters like age, cancer activity, KPS, and IL-6 do influence depression and anxiety differently. Unlike clinical depression, anxiety is not associated with increased IL-6 levels, however, shows a reciprocal correlation with age.

The aim of this study was to evaluate the efficacy of the chemokine CCL19 (ELC) as an adjuvant for intradermal gene gun delivery of Her2/neu DNA and to investigate the role of B cells in CCL19-mediated enhancement of immune responses. Balb/c mice were immunized intramuscularly (i.m.) on days 1 and 15 with plasmid DNA (pDNA) (100 μg DNA) or intradermally (i.d.) by gene gun delivery (1-2 μg DNA). Administration of pDNA encoding Her2/neu (pDNA(Her2/neu) was compared with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(CCL19), mock vector or uncoated gold particles/phosphate-buffered saline (PBS). Tumor challenge was performed subcutaneously on day 25 with syngeneic Her2/neu(+) tumor cells (D2F2/E2). Intradermal immunization by gene gun led to an enhancement of tumor protection by the DNA vaccine as compared with i.m. immunization. The protective effect of the vaccine was further enhanced by coadministration of pDNA(CCL19) both after i.m. and i.d. immunization. Tumor protection was associated with Her2/neu-specific T cell and humoral immune responses. Experiments in B-cell-deficient μMT mice showed that B cells are crucial for CCL19-mediated enhancement of tumor rejection, most likely as antigen-presenting B cells. DNA vaccines against Her2/neu may play a future role in the treatment of Her2/neu-positive breast cancer patients in a clinical situation of minimal residual disease.

The measurement of tumor-associated proteins is of high diagnostic value in the follow-up of cancer patients. Most tests ignore that various forms of the protein can exist; especially in epithelial cancers and the soluble receptors they produce. We choose EpCAM as model-antigen to analyze whether tests recognizing different domains of the protein give different results in patients' sera. EpCAM-reactive autoantibodies are present in the sera of patients with colorectal carcinoma, however little is known about the existence and possible relevance of circulating soluble EpCAM protein. Most monoclonal EpCAM-antibodies recognize the first EGF-like repeat and fail to detect N-terminal trimmed protein. We developed a novel ELISA to determine the concentration of serum EpCAM with mAbs recognizing the second EGF-like repeat. In 59 healthy controls, EpCAM concentrations ranged from 232 to 8893ng/ml (mean 1525ng/ml). Levels of EpCAM in 412 patients with adenocarcinoma were somewhat higher with concentrations ranging from 176 to 36,259ng/ml (mean 1971ng/ml). In direct comparison, the untrimmed protein specific ELISA detected lower levels and frequencies as compared to the EGFII-specific ELISA. Only sera with less than 1μg/ml circulating EGFII-EpCAM (66% of the sera) contained EpCAM-specific IgG antibodies. The absence of IgG antibodies in the sera with more than 1μg/ml circulating EpCAM was not due to immune complex formation. Anti-EpCAM IgA and IgM antibodies did not show such a correlation. It will be important to assess whether the presence of high levels of circulating EGFII-EpCAM is associated with side effects in patients given immunotherapy.

Background DNA vaccination is an attractive approach for tumor vaccination because plasmid DNA (pDNA) can be used as a ‘general vaccine’ across major histocompatibility complex barriers. Coexpression of immunomodulatory molecules can help to amplify the immunogenicity of DNA vaccines. CCL19 (ELC) is a CC chemokine with immunoregulatory properties, binding to the chemokine receptor CCR7 that is expressed on dendritic cells (DCs) and T cells. In vivo, CCL19 is a key regulator for the interactions between DCs and T cells in regional lymph nodes. Methods pDNA encoding Her2/neu and CCL19 was used as an intramuscular vaccine. Vaccination was performed in BALB/c mice, which were subsequently challenged with syngeneic Her2/neu+ tumor cells. Groups of mice were immunized with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(Her2/neu) plus pDNA(CCL19) plus pDNA(GM-CSF), pDNA(Her2/neu) plus pDNA(GM-CSF), pDNA(Her2/neu), pDNA(CCL19), pDNA(GM-CSF) or mock vector. Tumor protection by the vaccine and immune responses were monitored. Results Coadministration of pDNA(Her2/neu) and pDNA(CCL19) led to substantial improvement of tumor protection by the vaccine and induced a TH1-polarized, Her2/neu-specific immune response. Forty-seven days after the tumor challenge, 58% of the mice coinjected with pDNA(Her2/neu) and pDNA(CCL19) remained tumor-free compared to 22% after vaccination with pDNA(Her2/neu) alone. Additional administration of pDNA(GM-CSF) led to further improvement of tumor protection and an amplification of Her2/neu-specific immune responses. Conclusions CCL19 is able to induce a TH-1 polarization of the anti-Her2/neu immune response, which can be further amplified by granulocyte macrophage-colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu-CCL19 ± GM-CSF) vaccine might be promising in Her2/neu + breast cancer in the clinical situation of minimal residual disease. Copyright © 2012 John Wiley & Sons, Ltd.

Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.

[§ 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.

Preclinical studies showed that the allogeneic tumor cell line RCC-26 displayed natural immunogenic potential that was enhanced through expression of CD80 costimulatory molecules and secretion of interleukin-2. Here we report the study of RCC-26/CD80/IL-2 cells in a phase 1 vaccine trial of renal cell carcinoma patients with metastatic disease (mRCC). Fifteen patients of the HLA-A*0201 allotype, with at least one metastatic lesion, were included. Irradiated vaccine cells were applied in increasing doses of 2.5, 10, and 40 x 10(6) cells over 22 weeks. Primary study parameters included safety and toxicity. Sequential blood samples were analyzed by interferon-gamma enzyme-linked immunospot assays to detect tumor antigen-associated (TAA) effector cells. The vaccine was well tolerated and the designated vaccination course was completed in 9 of 15 patients. Neither vaccine-induced autoimmunity nor systemic side effects were observed. Delayed-type hypersensitivity skin reactions were detected in 11 of 12 evaluated patients and were particularly strong in patients with prolonged survival. In parallel, vaccine-induced immune responses against vaccine or overexpressed TAA were detected in 9 of 12 evaluated patients. No tumor regressions occurred according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria; however, median time to progression was 5.3 months and median survival was 15.6 months, indicating substantial disease stabilization. We conclude that vaccine use was safe and feasible in mRCC. Clinical benefits were limited in these patients with advanced disease; however, immune monitoring revealed vaccine-induced responses against multiple TAAs in the majority of study participants. These results suggest that this vaccine could be useful in combination therapies and/or minimal residual disease.

Background Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400mg/day (n=400) could be optimized by doubling the dose (n=420), adding IFN (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). Methods From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. The impact of patients' and disease factors on survival was prospectively analyzed. At the time of evaluation, at least 62% of patients still received imatinib, 26.2% were switched to 2nd generation tyrosine kinase inhibitors. Results After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival 80% and 10-year relative survival 92%. In spite of a faster response with IM800mg, the survival difference between IM400mg and IM800mg was only 3% at 5 years. In a multivariate analysis, the influence on survival of risk-group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs. other) was significant in contrast to any form of initial treatment optimization. Patients that reached the response milestones 3, 6 and 12 months, had a significant survival advantage of about 6% after 10 years regardless of therapy. The progression probability to blast crisis was 5.8%. Blast crisis was proceeded by high-risk additional chromosomal aberrations. Conclusions For responders, monotherapy with IM400mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease and blast crisis, more life-time can currently be gained by carefully addressing non-CML determinants of survival. Disclosures Hehlmann: Novartis: Research Funding; BMS: Consultancy. Saussele: Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Pfirrmann: BMS: Honoraria; Novartis: Honoraria. Krause: Novartis: Honoraria. Baerlocher: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Bruemmendorf: Novartis: Research Funding. Müller: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Jeromin: MLL Munich Leukemia Laboratory: Employment. Hänel: Roche: Honoraria; Novartis: Honoraria. Burchert: BMS: Honoraria. Waller: Mylan: Consultancy, Honoraria. Mayer: Eisai: Research Funding; Novartis: Research Funding. Link: Novartis: Honoraria. Scheid: Novartis: Honoraria. Schafhausen: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Hochhaus: Incyte: Research Funding; MSD: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Research Funding.

Even in the era of multitargeted therapies, cytokines remain at least one of different treatment options in renal cell cancer (RCC), particularly for patients belonging to the good prognostic risk category according to Memorial Sloan Kettering Cancer Center criteria. Granulocyte macrophage-colony stimulating factor plays a central role in the differentiation and activation of antigen presenting cells. This clinical phase 1/2 chemoimmunotherapy trial in metastatic RCC used sequential application of alpha-interferon /5-fluorouracil followed by granulocyte macrophage-colony stimulating factor/interleukin-2. The study was performed before multikinase inhibitors were available for routine use. Twenty patients with metastatic RCC were enrolled into this phase 1/2 protocol. Sequential chemoimmunotherapy was feasible and safe on an outpatient basis. The regimen had only modest antitumor activity with 4 mixed responses and 4 stable diseases being documented after 4 treatment cycles. Enhanced proliferative and stimulatory capacity of peripheral blood mononuclear cells was only observed in patients with mixed responses/stable diseases whereas patients with progressive disease did not show any change. Most interestingly, there was a significant increase of T cells expressing the costimulatory molecules CD80/86 in patients with progressive disease. This finding is reported here for the first time under chemoimmunotherapy of RCC. In conclusion, clinical response rates of this cytokine-based regimen do not justify further clinical evaluation. However, the study suggests that CD80/86+ T cells might have negative regulatory function under cytokine treatment and are possibly useful as a negative predictive marker for clinical response.

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.