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ABSTRACT: Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8 (+) T-cell clones reacting to Flu matrix protein (Flu-M) and 6 CD4 (+) T-cell clones reacting to Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies.
Human vaccines & immunotherapeutics. 02/2013; 9(6).
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Carol Stocking,
Manuel Grez, Boris Fehse,
Dorothee von Laer,
Katsuhiko Itoh,
Vladimir Prassolov,
Joachim Nowock,
Klaus Kühlcke,
Ursula Just,
Timm Schröder,
Elke Grassman,
Johann Meyer,
Zhixiong Li,
Axel Schambach,
Ute Modlich,
Olga Kustikova,
Melanie Galla,
Jürgen Bode,
Axel Zander,
Christopher Baum
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ABSTRACT: With dismay and sorrow we learned that Wolfram Ostertag, a great mentor and conceptual driver of research in our field, and former editor of Human Gene Therapy, passed away at the age of 72, only 7 years after his retirement. His broad vision, his enthusiasm in addressing the big questions, and his never-ending commitment to create long-lasting, international connections and true friendships between basic researchers, biotechnologists, and translationally ori-ented physicians will remain unforgotten and continue to stimulate us. To illustrate the scope of his work: Would you agree that a deeper understanding of cell and virus ge-netics forms the basis of gene therapy? That viral gene vectors can be adapted to host cells by evolutionary mechanisms? That the safe and effi-cient genetic modification of stem cells is hindered by innate control mechanisms such as cellular blocks to vector entry and posttransduction silencing of gene expression? That the definition of appropriate retroviral pseudotypes and of cis elements that support long-term expression is of special importance for the genetic modification of hematopoietic stem cells? That antiretroviral therapies can be developed on the basis of pharmacological and genetic principles? That insertional mutagenesis by semirandomly integrating gene vectors is both a great challenge to their therapeutic use and an intriguing tool with which to discover pathways that regulate self-renewal and differentiation? And that basic principles of stem cell biology are regulated through the interplay of cell-intrinsic, stochastic mechanisms with di-rective signals originating from cytokines and their recep-tors? If your answer to at least one of these questions is yes, then you will certainly have dealt with his work. If your answer is yes to more than one of those questions, then be welcome in our club of the Ostertag disciples. Work in his laboratory was always stimulated by Wol-fram's eagerness to see and discuss our results, fresh from or even at the bench (while still pipetting). No matter the day or hour, his demand for more experiments, often several in parallel, coupled with never-ending philosophical discus-sions about science or whatever the topic of the day was, had an unforgettable impact on all of us. This intense scientific atmosphere and enduring enthusiasm turned out to be dif-ficult to find elsewhere. Of course Wolfram, like all of us, de-pended on and enjoyed being stimulated by discoveries made in the larger international research community. But to shed some light on his approach to the above questions, here is a short list of his and his laboratory's key achievements. Wolfram studied biology, chemistry, physics, and anthropology in Mainz, Ger-many, and Bloomington, Indiana; worked with Nobel laureate H.J. Muller to obtain his Ph.D. in Drosophila genetics, and continued his career as a postdoctoral researcher at the Max Planck Institute in Gö ttingen, Germany to study approaches to mutagenesis in cul-tured cells. After 3 years as a guest scientist at Johns Hopkins University, Baltimore, Maryland, he returned to Gö ttingen in 1968 to investigate mechanisms of transformation in erythroid cells before being recruited to the Beatson Institute, Glasgow, Scotland in 1979, where he started his work to develop retroviral vectors for genetic modification of hematopoietic and embryonic cells. From 1980 until his retirement in 2002, he was head of the Department of Experimental Virology and Immunology at the Heinrich Pette Institute in Hamburg, Germany. Inspired by the fascinating interplay of retroviruses and hematopoi-etic cells, he chose the name ''Department of Cell and Virus Genetics'' for his growing empire, a striking programmatic summary of his work. In his collaboration and interaction with other key inves-tigators in the institute, including Rudolf Jaenisch, Klaus Harbers, Wolfgang Deppert, Hans-Georg Krä usslich, Hans Will, and Fritz Lehmann-Grube, and many other great col-leagues from Europe, the United States, and Japan, Wolfram's approach to retroviral mutational adaptation to host cells became an early paradigm for combining principles of evolution and intelligent design in vector development, and culminated in the development of the murine embryonic stem cell virus (MESV) (Franz et al., 1986; Grez et al., 1990). Basic components of MESV were subsequently used to create the murine stem cell virus (MSCV), the predominant vector for the genetic modification of hematopoietic and embryonic cells before the advent of lentiviral vectors. Related and widely used vectors with strong LTR-derived promoters based on spleen focus-forming virus (SFFV) or the myeloproliferative sarcoma virus (MPSV) also originated in his laboratory and have been used Dr. Wolfram Ostertag
Human Gene Therapy 02/2013; 21(November):1501-1503. · 4.22 Impact Factor
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ABSTRACT: BACKGROUND: Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. METHODS: We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. RESULTS: We demonstrated the successful use of this approach for the screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. CONCLUSIONS: The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.
AIDS Research and Therapy 01/2013; 10(1):1. · 2.54 Impact Factor
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Nicolaus Kröger,
Anita Badbaran,
Tatjana Zabelina,
Francis Ayuk,
Christine Wolschke,
Haefaa Alchalby,
Evgeny Klyuchnikov,
Djordje Atanackovic,
Georgia Schilling,
Timon Hansen,
Sabine Schwarz,
Marion Heinzelmann,
Silke Zeschke,
Ulrike Bacher,
Thomas Stübig, Boris Fehse,
Axel R Zander
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ABSTRACT: Within a prospective protocol the incidence and impact of achievement of molecular remission (mCR)and high risk cytogenetics was investigated in 73 patients with multiple myeloma (MM) after auto-allo tandem transplantation. After induction chemotherapy patients received melphalan 200 mg/m(2)and autologous stem cell transplantation (SCT) and after 3 months melphalan 140 mg/m(2) and fludarabine 180 mg/m(2) followed by allogeneic SCT. 16 patients had high risk cytogenetic features defined by positive FISH for del(17p13) and/or t(4;14).Overall, 66% achieved complete or near complete remission and 41% molecular remission, which was sustained negative (at least four consecutive samples negative) in 15 patients (21%), and did not differ between high risk cytogenetics and others (p=0.7).After a median follow-up of 6 years the 5 year progressive-free survival was 29 % without difference between del 17p13/t(4;14) harbouring patients and others (24% vs. 30%, p=0.7). The 5 y PFS differs substantially according to the achieved remission and was for PR, CR, mCR, and sustained mCR 17 %, 41 %, 57 %, and 85 %, respectively. These results suggested that auto-allo tandem transplantation may overcome negative prognostic effect of del(17p13) and/or t(4;14) and achievement of molecular remission resulted in long-term freedom from disease. (registration: NCT 00781170).
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 10/2012; · 3.15 Impact Factor
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ABSTRACT: SOCS3 is a feedback regulator of cytokine signaling that affects T-cell polarization. Human tuberculosis is accompanied by increased SOCS3 expression in T cells and this may influence susceptibility against Mycobacterium tuberculosis. Since the role of SOCS3 in human T-cell function is not well defined, we characterized cytokine expression and proliferation of human T cells with differential SOCS3 expression in the present study. We established a flow cytometry-based method for SOCS3 protein quantification and detected higher SOCS3 levels upon Mycobacterium tuberculosis specific T-cell activation and a transient decrease of SOCS3 expression in the presence of mycobacteria-infected macrophages. Notably increased SOCS3 expression was detected in Interleukin-17 expressing T-cell clones and in CD161(+) T helper type 17 cells ex vivo. Ectopic SOCS3 expression in primary CD4(+) T cells by lentiviral transduction induced increased Interleukin-17 production but diminished proliferation and viability. Recombinant Interleukin-7 inhibited SOCS3 expression and reduced Interleukin-17 expressing T-cell proportions. We concluded that higher SOCS3 expression in human T cells favors T helper type 17 cells. Therefore increased SOCS3 expression in human tuberculosis may reflect polarization towards Interleukin-17 expressing T cells, as well as T-cell exhaustion marked by reduced proliferation.
Blood 10/2012; · 9.90 Impact Factor
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Kerstin Cornils,
Cynthia C Bartholomae,
Lars Thielecke,
Claudia Lange,
Anne Arens,
Ingmar Glauche,
Ulrike Mock,
Kristoffer Riecken,
Sebastian Gerdes,
Christof von Kalle,
Manfred Schmidt,
Ingo Roeder, Boris Fehse
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ABSTRACT: Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus-long terminal repeat-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of linear amplification-mediated PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for linear amplification-mediated PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral long terminal repeat vectors.
Experimental hematology 09/2012; · 3.11 Impact Factor
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ABSTRACT: Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3-4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1-12 h). The initial efficiency of RGB marking can be assessed after 2-4 d by flow cytometry and/or fluorescence microscopy.
Nature Protocol 01/2012; 7(5):839-49. · 8.36 Impact Factor
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Jochen Schulze,
Kristoffer Weber,
Anke Baranowsky,
Thomas Streichert,
Tobias Lange,
Alexander Simon Spiro,
Joachim Albers,
Sebastian Seitz,
Josef Zustin,
Michael Amling, Boris Fehse,
Thorsten Schinke
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ABSTRACT: Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.
Cancer letters 11/2011; 317(1):106-13. · 4.86 Impact Factor
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ABSTRACT: Several cases of T-cell leukemia caused by gammaretroviral insertional mutagenesis in children treated for x-linked severe combined immunodeficiency (SCID) by transplantation of autologous gene-modified stem cells were reported. In a comparative analysis, we recently showed that mature T cells, on the contrary, are highly resistant to transformation by gammaretroviral gene transfer. In the present study, we observed immortalization of a single T-cell clone in vitro after gammaretroviral transduction of the T-cell protooncogene LMO2. This clone was CD4/CD8 double-negative, but expressed a single rearranged T-cell receptor. The clone was able to overgrow nonmanipulated competitor T-cell populations in vitro, but no tumor formation was observed after transplantation into Rag-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptors were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. This study demonstrates that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results show that signaling of the IL-2 receptor and the protooncogene LMO2 can act synergistically in maligniant transformation of mature T lymphocytes.
Molecular Medicine 07/2011; 17(11-12):1223-32. · 3.76 Impact Factor
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ABSTRACT: Suicide gene therapy is a promising concept in oncology. We have recently introduced a novel suicide gene, TK.007, which was shown to excel established herpes simplex virus thymidine kinase (HSVtk) variants when used for donor-lymphocyte modification in adoptive immunotherapy models. Here, the potential of TK.007 in killing cancer cells was studied. Initially, we transduced tumour cell lines derived from different neoplasias (glioblastoma, melanoma, lung cancer, colon cancer) with lentiviral LeGO vectors encoding TK.007 or the splice-corrected (sc)HSVtk together with an eGFP/Neo-marker. Based on direct in vitro comparison, we found that TK.007 facilitates more efficient tumour cell killing at significantly lower ganciclovir doses in all tumour cell lines tested. Also, using different readout systems, we found a significantly stronger bystander effect of TK.007 as compared to scHSVtk. Importantly, in vitro data were confirmed in vivo using a subcutaneous G62 glioblastoma model in NOD/SCID mice. In mice transplanted with scHSVtk-positive tumours, treatment with low (10 mg/kg) or standard (50 mg/kg) ganciclovir doses resulted only in short-term growth inhibition or transient tumour remission, respectively. In striking contrast, in the TK.007 group, all animals achieved continuous complete remission after both standard and low-dose ganciclovir. Finally, a substantial bystander effect for TK.007 was also confirmed with the G62 model in vivo, where significantly prolonged survival for mice bearing tumours containing only 10% or 50% TK.007-expressing cells was observed. In summary, our data indicate strongly improved anti-tumour activity of TK.007 as compared to conventional HSVtk. We therefore suppose that TK.007 is an excellent candidate for cancer suicide gene therapy.
Journal of Molecular Medicine 06/2011; 89(11):1113-24. · 4.67 Impact Factor
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Kristoffer Weber,
Michael Thomaschewski,
Michael Warlich,
Tassilo Volz,
Kerstin Cornils,
Birte Niebuhr,
Maike Täger,
Marc Lütgehetmann,
Jörg-Matthias Pollok,
Carol Stocking,
Maura Dandri,
Daniel Benten, Boris Fehse
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ABSTRACT: We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.
Nature medicine 03/2011; 17(4):504-9. · 27.14 Impact Factor
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Evgeny Klyuchnikov,
Andreas Sputtek,
Olga Slesarchuk,
Michael Lioznov,
Thomas Stübig,
Ulrike Bacher,
Gitta Amtsfeld,
Edeltraut Merle,
Marie-Luise Reckhaus, Boris Fehse,
Christine Wolschke,
Raissa Adjallé,
Francis Ayuk,
Axel Zander,
Nicolaus Kröger
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ABSTRACT: Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 03/2011; 17(3):374-83. · 3.15 Impact Factor
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Joachim Albers,
Jochen Schulze,
F Timo Beil,
Matthias Gebauer,
Anke Baranowsky,
Johannes Keller,
Robert P Marshall,
Kristofer Wintges,
Felix W Friedrich,
Matthias Priemel, [......], Boris Fehse,
Thomas Streichert,
Guido Sauter,
Franz Jakob,
Karl L Insogna,
Barbara Pober,
Klaus-Peter Knobeloch,
Uta Francke,
Michael Amling,
Thorsten Schinke
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ABSTRACT: Although Wnt signaling in osteoblasts is of critical importance for the regulation of bone remodeling, it is not yet known which specific Wnt receptors of the Frizzled family are functionally relevant in this process. In this paper, we show that Fzd9 is induced upon osteoblast differentiation and that Fzd9(-/-) mice display low bone mass caused by impaired bone formation. Our analysis of Fzd9(-/-) primary osteoblasts demonstrated defects in matrix mineralization in spite of normal expression of established differentiation markers. In contrast, we observed a reduced expression of chemokines and interferon-regulated genes in Fzd9(-/-) osteoblasts. We also identified the ubiquitin-like modifier Isg15 as one potential downstream mediator of Fzd9 in these cells. Importantly, our molecular analysis further revealed that canonical Wnt signaling is not impaired in the absence of Fzd9, thus explaining the absence of a bone resorption phenotype. Collectively, our results reveal a previously unknown function of Fzd9 in osteoblasts, a finding that may have therapeutic implications for bone loss disorders.
The Journal of Cell Biology 03/2011; 192(6):1057-72. · 10.26 Impact Factor
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ABSTRACT: In acute myeloid leukemia (AML), the selection of poor-risk patients for allogeneic hematopoietic stem cell transplantation (HSCT) is associated with rather high post-transplant relapse rates. As immunotherapeutic intervention is considered to be more effective before the cytomorphologic manifestation of relapse, post-transplant monitoring gains increasing attention in stem cell recipients with a previous diagnosis of AML. Different methods for detection of chimerism (e.g., microsatellite analysis or quantitative real-time PCR) are available to quantify the ratio of donor and recipient cells in the post-transplant period. Various studies demonstrated the potential use of mixed chimerism kinetics to predict relapse of the AML. CD34+-specific chimerism is associated with a higher specificity of chimerism analysis. Nevertheless, a decrease of donor cells can have other causes as well. Therefore, efforts continue to introduce minimal residual disease (MRD) monitoring based on molecular mutations in the post-transplant period. The NPM1 (nucleophosmin) mutations can be monitored by sensitive quantitative real-time PCR in subsets of stem cell recipients with AML, but for approximately 20% of patients, suitable molecular mutations for post-transplant MRD monitoring are not available so far. This emphasizes the need for an expansion of the panel of MRD markers in the transplant setting.
TheScientificWorldJOURNAL 01/2011; 11:310-9. · 1.66 Impact Factor
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Florian Rothweiler,
Martin Michaelis,
Peter Brauer,
Jürgen Otte,
Kristoffer Weber, Boris Fehse,
Hans Wilhelm Doerr,
Michael Wiese,
Jörg Kreuter,
Yousef Al-Abed,
Ferdinando Nicoletti,
Jindrich Cinatl
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ABSTRACT: The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors.
Neoplasia (New York, N.Y.) 12/2010; 12(12):1023-30. · 5.48 Impact Factor
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Bettina Wiedemann,
Evgeny Klyuchnikov,
Nicolaus Kröger,
Tatjana Zabelina,
Tanja Stahl,
Silke Zeschke,
Anita Badbaran,
Francis Ayuk,
Haefaa Alchalby,
Christine Wolschke,
Carsten Bokemeyer, Boris Fehse,
Axel Rolf Zander,
Ulrike Bacher
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ABSTRACT: Chimerism is well-established for surveillance of acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HSCT), but interpretation of the results and techniques is not standardized.
We correlated chimerism in 75 AML patients (38 male, 37 female) who underwent myeloablative (n = 36)/reduced (n = 39) allo-HSCT with the risk of relapse and survival. Chimerism was evaluated by quantitative real-time polymerase chain reaction for donor/recipient specific polymorphisms/Y-specific sequences.
After HSCT, 40 patients (53%) achieved stable complete donor chimerism (≥ 99.0% of donor alleles), while 35 (47%) failed to achieve stable donor chimerism. Thirty-one patients (41%) showed decreasing donor alleles after having first achieved complete donor chimerism. To investigate the kinetics of mixed chimerism, patients were separated whether they showed subsequent increasing or decreasing donor alleles. Subsequent decrease of donor alleles was associated with relapses in 17 of 18 cases (94%), while no patient with subsequent increasing donor alleles relapsed (p < 0.001). Patients with mixed chimerism and increasing donor alleles had better 2-year disease-free survival (85%) than those with decreasing donor alleles (0%; p < 0.001).
The kinetics of mixed chimerism as assessed by quantitative real-time polymerase chain reaction is an important prognostic predictor in the post-transplantation period of AML patients.
Experimental hematology 12/2010; 38(12):1261-71. · 3.11 Impact Factor
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Haefaa Alchalby,
Anita Badbaran,
Tatjana Zabelina,
Guido Kobbe,
Joachim Hahn,
Daniel Wolff,
Martin Bornhäuser,
Christian Thiede,
Herrad Baurmann,
Wolfgang Bethge,
York Hildebrandt,
Ulrike Bacher, Boris Fehse,
Axel R Zander,
Nicolaus Kröger
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ABSTRACT: Allogeneic stem cell transplantation (ASCT) after reduced-intensity conditioning has become a reasonable treatment option for patients with advanced myelofibrosis. The role of characteristic molecular genetic abnormalities, such as JAK2V617F on outcome of ASCT, is not yet elucidated. In 139 of 162 myelofibrosis patients with known JAK2V617F mutation status who received ASCT after reduced-intensity conditioning, the impact of JAK2 genotype, JAK2V617F allele burden, and clearance of mutation after ASCT was evaluated. Overall survival was significantly reduced in multivariate analysis in patients harboring JAK2 wild-type (hazard ratio = 2.14, P = .01) compared with JAK2 mutated patients. No significant influence on outcome was noted for the mutated allele burden analyzed either as continuous variable or after dividing into quartiles. Achievement of JAK2V617F negativity after ASCT was significantly associated with a decreased incidence of relapse (hazard ratio = 0.22, P = .04). In a landmark analysis, patients who cleared JAK2 mutation level in peripheral blood 6 months after ASCT had a significant lower risk of relapse (5% vs 35%, P = .03). We conclude that JAK2V617F-mutated status, but not allele frequency, resulted in an improved survival and rapid clearance after allografting reduces the risk of relapse.
Blood 11/2010; 116(18):3572-81. · 9.90 Impact Factor
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Carol Stocking,
Manuel Grez, Boris Fehse,
Dorothee von Laer,
Katsuhiko Itoh,
Vladimir Prassolov,
Joachim Nowock,
Klaus Kühlcke,
Ursula Just,
Timm Schröder, [......],
Elke Grassman,
Johann Meyer,
Zhixiong Li,
Axel Schambach,
Ute Modlich,
Olga Kustikova,
Melanie Galla,
Jürgen Bode,
Axel Zander,
Christopher Baum
Human gene therapy 11/2010; 21(11):1501-3. · 4.20 Impact Factor
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Ute Brassat,
Stefan Balabanov,
Daniel Bali,
Judith Dierlamm,
Melanie Braig,
Ulrike Hartmann,
Hüseyin Sirma,
Cagatay Günes,
Henning Wege, Boris Fehse,
Artur Gontarewicz,
Ekkehard Dikomey,
Kerstin Borgmann,
Tim H Brümmendorf
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ABSTRACT: In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy.
To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells.
BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis.
These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.
Experimental hematology 10/2010; 39(1):66-76.e1-2. · 3.11 Impact Factor
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Tanja Stahl,
Anita Badbaran,
Nicolaus Kröger,
Evgeny Klyuchnikov,
Tatjana Zabelina,
Silke Zeschke,
Philippe Schafhausen,
Waltraud Schultz,
Svetlana Asenova,
Anna Smirnova,
Christine Wolschke,
Francis Ayuk,
Axel R Zander, Boris Fehse,
Ulrike Bacher
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ABSTRACT: Considering the high relapse rates of AML after allogeneic hematopoietic stem cell transplant, research aims to improve post-transplant surveillance. To determine the value of peripheral blood (PB) for post-transplant minimal residual disease monitoring, we compared 38 PB and bone marrow (BM) sample pairs in 25 stem cell recipients with NPM1-mutated AML (12 males, 13 females, ages 21-73 years). NPM1A mutation levels and chimerism ratios were determined in non-separated BM/PB. We observed congruent results in 28/38 (74%). In 14/38 sample pairs (37%), BM and PB were negative for the NPM1A mutation. Fourteen sample pairs were positive in BM and PB, albeit at higher mutation levels in the BM in 11 cases (4- to 278-fold). Results were discordant in 10 cases (26%), with weakly positive mutation levels in the BM but negative levels in the PB. Cases with ≥0.2% NPM1A mutation level in BM were always positive in PB. Chimerism was concordant in BM and PB in 21/34 (62%) of sample pairs. In conclusion, MRD monitoring with qPCR for the NPM1 mutation and chimerism from non-separated PB contributes to surveillance in patients with AML in the post-transplant period, but even with highly sensitive qPCR there is a risk of failure to detect the mutation in PB.
Leukemia & lymphoma 10/2010; 51(10):1837-43. · 2.40 Impact Factor
