Akos Czibere

Sorafenib (Nexavar®) induces molecular remission and regression of extramedullary disease in a patient with FLT3-ITD+ acute myeloid leukemia

The fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) can be found in about one quarter of patients with acute myeloid leukemia (AML) [Small D. FLT3 mutations: biology and treatment. Hematology Am Soc Hematology. Educ. Program 2006;178-84 [Review]]. Patients who carry this mutation have a high risk of relapse even after allogeneic stem cell transplantation [Sheikhha MH, Awan A, Tobal K, Liu Yin JA. Prognostic significance of FLT3 ITD and D835 mutations in AML patients. Hematol J 2003;4:41-6; Meshinchi S, Arceci RJ, Sanders JE, Smith FO, Woods WB, Radich JP, et al. Role of allogeneic stem cell transplantation in FLT3/ITD-positive AML. Blood 2006;108(1):400-1]. Recent reports show that Sorafenib, a multikinase inhibitor has significant activity against FLT3-ITD(+) blasts in vitro [Auclair D, Miller D, Yatsula V, Pickett W, Carter C, Chang Y, et al. Antitumor activity of sorafenib in FLT3-driven leukemic cells. Leukemia 2007;21(3):439-45]. We here report the first clinical case of molecular remission induced by Sorafenib in a patient with FLT3-ITD(+) AML and extramedullary disease after allogenic stem cell transplantation.

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Dexamethasone/ascorbic acid/glycerolphosphate (DAG) and bone morphogenic protein (BMP)‐2 are potent agents in cell proliferation and differentiation pathways. This study investigates the in vitro interactions between dexamethasone and BMP‐2 for an osteoblastic differentiation of mesenchymal stem cells (MSCs). Bone marrow‐derived human MSCs were cultured with DAG (group A), BMP‐2 + DAG (group B), and DAG + BMP‐2 combined with a porous collagen I/III scaffold (group C). RT‐PCR, ELISA, immuncytochemical stainings and flow cytometry analysis served to evaluate the osteogenic‐promoting potency of each of the above conditions in terms of cell morphology/viability, antigen presentation, and gene expression. DAG induced collagen I secretion from MSCs, which was further increased by the combination of DAG + BMP‐2. In comparison, the collagen scaffold and the control samples showed no significant influence on collagen I secretion of MSCs. DAG stimulation of MSCs led also to a steady but not significant increase of BMP‐2 level. A DAG and more, a DAG + BMP‐2, stimulation increased the number of mesenchymal cells (CD105+/CD73+). All samples showed mRNA of ALP, osteopontin, Runx2, Twist 1 and 2, Notch‐1/2, osteonectin, osteocalcin, BSP, and collagen‐A1 after 28 days of in vitro culture. Culture media of all samples showed a decrease in Ca2+ and PO  42− concentration, whereas a collagen‐I‐peak only occurred at day 28 in DAG‐ and DAG + BMP‐2‐stimulated bone marrow cells. In conclusion, BMP‐2 enhances DAG‐induced osteogenic differentiation in mesenchymal bone marrow cells. Both agents interact in various ways and can modify osteoblastic bone formation.

Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease

Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS.

Treatment of Acute Myeloid Leukemia or Myelodysplastic Syndrome Relapse after Allogeneic Stem Cell Transplantation with Azacitidine and Donor Lymphocyte Infusions—A Retrospective Multicenter Analysis from the German Cooperative Transplant Study Group

To expand the current knowledge about azacitidine (Aza) and donor lymphocyte infusions (DLI) as salvage therapy for relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify predictors for response and survival, we retrospectively analyzed data of 154 patients with acute myeloid leukemia (AML, n = 124), myelodysplastic (MDS, n = 28), or myeloproliferative syndrome (n = 2). All patients received a median number of 4 courses of Aza (range, 4 to 14) and DLI were administered to 105 patients (68%; median number of DLI, 2; range, 1 to 7). Complete and partial remission rates were 27% and 6%, respectively, resulting in an overall response rate of 33%. Multivariate analysis identified molecular-only relapse (hazard ratio [HR], 9.4; 95% confidence interval [CI], 2.0 to 43.5; P = .004) and diagnosis of MDS (HR, 4.1; 95% CI, 1.4 to 12.2; P = .011) as predictors for complete remission. Overall survival (OS) at 2 years was 29% ± 4%. Molecular-only relapse (HR, .14; 95% CI, .03 to .59; P = .007), diagnosis of MDS (HR, .33; 95% CI, .16 to .67; P = .002), and bone marrow blasts <13% (HR, .54; 95% CI, .32 to .91; P = .021) were associated with better OS. Accordingly, 2-year OS rate was higher in MDS patients (66% ± 10%, P = .001) and correlated with disease burden in patients with AML. In summary, Aza and DLI is an effective and well-tolerated treatment option for patients with relapse after allo-HSCT, in particular those with MDS or AML and low disease burden. The latter finding emphasizes the importance of stringent disease monitoring and early intervention.

Monocyte derived dendritic cells generated by IFN-α acquire mature dendritic and natural killer cell properties as shown by gene expression analysis

BackgroundDendritic cell (DC) vaccines can induce antitumor immune responses in patients with malignant diseases, while the most suitable DC culture conditions have not been established yet. In this study we compared monocyte derived human DC from conventional cultures containing GM-CSF and IL-4/TNF-α (IL-4/TNF-DC) with DC generated by the novel protocol using GM-CSF and IFN-α (IFN-DC).MethodsTo characterise the molecular differences of both DC preparations, gene expression profiling was performed using Affymetrix microarrays. The data were conformed on a protein level by immunophenotyping, and functional tests for T cell stimulation, migration and cytolytic activity were performed.ResultsBoth methods resulted in CD11c+ CD86+ HLA-DR+ cells with a typical DC morphology that could efficiently stimulate T cells. But gene expression profiling revealed two distinct DC populations.Whereas IL-4/TNF-DC showed a higher expression of genes envolved in phagocytosis IFN-DC had higher RNA levels for markers of DC maturity and migration to the lymph nodes like DCLAMP, CCR7 and CD49d. This different orientation of both DC populations was confined by a 2.3 fold greater migration in transwell experiments (p = 0.01).Most interestingly, IFN-DC also showed higher RNA levels for markers of NK cells such as TRAIL, granzymes, KLRs and other NK cell receptors. On a protein level, intracytoplasmatic TRAIL and granzyme B were observed in 90% of IFN-DC. This translated into a cytolytic activity against K562 cells with a median specific lysis of 26% at high effector cell numbers as determined by propidium iodide uptake, whereas IL-4/TNF-DC did not induce any tumor cell lysis (p = 0.006). Thus, IFN-DC combined characteristics of mature DC and natural killer cells.ConclusionOur results suggest that IFN-DC not only stimulate adaptive but also mediate innate antitumor immune responses. Therefore, IFN-DC should be evaluated in clinical vaccination trials. In particular, this could be relevant for patients with diseases responsive to a treatment with IFN-α such as Non-Hodgkin lymphoma or chronic myeloid leukemia.

A novel pathway involving melanoma differentiation associated gene-7/interleukin-24 mediates nonsteroidal anti-inflammatory drug-induced apoptosis and growth arrest of cancer cells.

Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.

Combinatorial hexapeptide ligand libraries (ProteoMiner™): An innovative fractionation tool for differential quantitative clinical proteomics

Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner™). Here, we demonstrate that ProteoMiner™ can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 μg to 300 μg) of whole E. coli lysate, processed it with ProteoMiner™ and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner™ treatment. Therefore, we conclude that the ProteoMiner™ technology can be used for quantitative analysis of low abundant proteins in complex biological samples.

Escalation therapy with bortezomib, dexamethasone and bendamustine for patients with relapsed or refractory multiple myeloma

In order to improve remission rates without causing undue toxicity, we treated 50 patients with relapsed/refractory multiple myeloma according to an institutional sequential treatment algorithm. Bortezomib was given as monotherapy (1.3 mg/m2 on day 1 + 4 + 8 + 11) followed by the addition of dexamethasone in a first (40 mg on day 1 + 4 + 8 + 11) and bendamustine (50 – 100 mg/m2 on day 1 + 8) in a second escalation step for patients with less than a minor response. Bortezomib monotherapy was sufficient in 23 (46%) patients, treatment escalation with dexamethasone was necessary in 20 (40%) patients and 7 (14%) patients needed triple combination therapy. Overall response rate was 84% while toxicity was manageable. Median time to progression and overall survival were 8 and 20 months, respectively. In conclusion, this treatment algorithm resulted in responses in the majority of heavily pre-treated patients while at the same time restricting the toxicity of triple combination therapy to only 14% of non-responding patients.

Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.

Abstract Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2–4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.

Treatment of Chronic Myelogenous Leukemia by Blocking Cytokine Alterations Found in Normal Stem and Progenitor Cells

Leukemic cells disrupt normal patterns of blood cell formation, but little is understood about the mechanism. We investigated whether leukemic cells alter functions of normal hematopoietic stem and progenitor cells. Exposure to chronic myelogenous leukemia (CML) caused normal mouse hematopoietic progenitor cells to divide more readily, altered their differentiation, and reduced their reconstitution and self-renewal potential. Interestingly, the normal bystander cells acquired gene expression patterns resembling their malignant counterparts. Therefore, much of the leukemia signature is mediated by extrinsic factors. Indeed, IL-6 was responsible for most of these changes. Compatible results were obtained when human CML were cultured with normal human hematopoietic progenitor cells. Furthermore, neutralization of IL-6 prevented these changes and treated the disease.