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Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1bdeletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1bdeletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1bdeletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.

-Presence of GFI1-36N impedes homologous recombination and MGMT-mediated DNA repair selectively in AML cells.-Use of temozolomide and olaparib allows selective targeting of GFI1-36Nleukemic cells.

E1A is a multifunctional adenoviral protein expressed early after infection that interferes with numerous important regulatory processes by interaction with host cell proteins or direct transcriptional activation of target genes. Although, initially identified as the adenoviral component that can cause malignant conversion of rodent cells, remarkable tumorsuppressive effects of E1A on various types of human cancer cells were observed. Gene therapeutic approaches with E1A are currently evaluated in animal models and early clinical studies. Therapeutic applications of E1A are covered by a series of patents which include the description of small variants (mini-E1A) that can be used for tumor suppression and E1A gene transfer in combination with conventional chemotherapy. In this mini review, we provide an introduction to E1A functions, summarize relevant patents, and discuss potential clinical applications of E1A gene transfer on basis of recent results of clinical and preclinical investigations.

Hematopoietic stem cell gene transfer of the drug-resistance gene cytidine deaminase (CDD) protecting cells from the cytotoxic cytidine analogs cytarabine and gemcitabine was investigated in a murine transplant model. Following transplantation of CDD-transduced cells and cytarabine application (500 mg/kg; days 1-4; intraperitoneally) significant myeloprotection was demonstrated with nadir counts of peripheral blood granulocytes and thrombocytes of 2.9 ± 0.6/nL versus 0.7 ± 0.1/nL (P< .001) and 509 ± 147/nL versus 80 ± 9/nL (P= .008), respectively (CDDversus control). Protection also was observed from otherwise lethal gemcitabine treatment (250 mg/kg; days 1-3). Stable levels of gene-marked cells in primary and secondary recipients were demonstrated for up to 9 months, and whereas CDDoverexpression clearly reduced B- and T-lymphocyte numbers, no major toxicity was observed in the myeloid compartment. Despite the profound myeloprotective properties, however, CDD overexpression did not allow for pharmacologic enrichment of transduced hematopoiesis in our model. Thus, in summary, our data establish CDDas a drug-resistance gene highly suitable for myeloprotective purposes, which, given the lack of selection observed in our hands, might best be used in combination with selectable drugresistance genes such as MGMT (P140K)or MDR1.

Hematopoietic stem cell gene transfer of the drug-resistance gene cytidine deaminase (CDD) protecting cells from the cytotoxic cytidine analogs cytarabine and gemcitabine was investigated in a murine transplant model. Following transplantation of CDD-transduced cells and cytarabine application (500 mg/kg; days 1-4; intraperitoneally) significant myeloprotection was demonstrated with nadir counts of peripheral blood granulocytes and thrombocytes of 2.9 ± 0.6/nL versus 0.7 ± 0.1/nL (P < .001) and 509 ± 147/nL versus 80 ± 9/nL (P = .008), respectively (CDD versus control). Protection also was observed from otherwise lethal gemcitabine treatment (250 mg/kg; days 1-3). Stable levels of gene-marked cells in primary and secondary recipients were demonstrated for up to 9 months, and whereas CDD overexpression clearly reduced B- and T-lymphocyte numbers, no major toxicity was observed in the myeloid compartment. Despite the profound myeloprotective properties, however, CDD overexpression did not allow for pharmacologic enrichment of transduced hematopoiesis in our model. Thus, in summary, our data establish CDD as a drug-resistance gene highly suitable for myeloprotective purposes, which, given the lack of selection observed in our hands, might best be used in combination with selectable drugresistance genes such as MGMT (P140K) or MDR1.

The detection of residual molecular and cytogenetic disease was prospectively compared in patients with Philadelphia-chromosome (Ph1) positive first chronic phase chronic myelogenous leukemia (CML) who underwent allogeneic transplantation of unmanipulated peripheral blood stem cells (PBSCT) (n = 29) or bone marrow (BM) (n = 62) using genotypically HLA-identical sibling donors or partially HLA-matched extended family donors. A molecular relapse (MR), as defined by two consecutive positive polymerase chain reaction (PCR) assays for the detection of M-bcr-abl transcripts in a 4-week interval, was found in two of 29 (7%) patients after PBSCT compared with 20 of 62 (32%) patients after bone marrow transplantation (BMT). This corresponds to a 4-year molecular relapse estimate (± standard error) of 7% ± 5% after PBSCT and of 44% ± 8% after BMT (P < .009). With identical follow-up periods of survivors in both patient subsets between 6 and 55 months (median, 28 months), 14 of the 20 patients with MR after BMT progressed to an isolated cytogenetic (n = 10) or a hematologic (n = 4) disease recurrence, resulting in a 4-year cytogenetic relapse estimate of 47% ± 11%, while none of the patients after PBSCT has so far relapsed (P < .006). Multivariate analysis including all potential influencial factors of posttransplant disease recurrence identified the source of stem cells (P < .02) as the only independent predictor of molecular relapse. In conclusion, this prospective comparison of molecular and cytogenetic residual disease demonstrates that peripheral blood stem cell transplants have a more pronounced activity against residual CML cells than bone marrow transplants. Prospective randomized trials comparing PBSCT and BMT in patients with first chronic phase Ph1-positive CML are strictly required to further substantiate differences in the antileukemic activity of the two stem cell sources.

Two major alternative models, clonal deletion and suppression, have been proposed as possible explanations for immunological tolerance1,2. We have now isolated a functional T suppressor (Ts) cell clone to analyse the role of Tscells in the induction and maintenance of tolerance. This cloned, long-term cultured Tscell line (HF1) was derived from CBA/J mice (H–2k) in which low-zone tolerance (LZT)3had been induced by sub-immunogenic doses of bovine serum albumin (BSA). HF1 cells show BSA-specific suppressor activity in two in vitro suppressor assays. HF1 Tscells and a factor derived from them suppress imprinted, carrier-specific T helper (Th) cells in an assay measuring IgG antibody production against fluorescein-conjugated BSA (Flu-BSA). Furthermore, they block DNA synthesis in an antigen-specific in vitro T-cell proliferation assay. Their proliferation in the presence of antigen-presenting cells is antigen specific and shows restriction to the I–Akregion of the major histocompatibility complex (MHC). In addition, HF1 Tscells have a characteristic cytotoxic activity. The data reported suggest that clonal deletion and suppression models are not necessarily mutually exclusive. Clonal deletion could manifest itself by the action of Tscells.

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) a were treated in a phase I/II study with recombinant human tumor necrosis factor a to overcome IFNa resistance. Doses of 40, 80, 120 or 160 µg/m2 TNFa were given as 2-h infusions on 5 consecutive days every 3 weeks. IFNa (4 × 106 IU/m2 s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNFa cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m2 and 160 µg/m2. To examine possible pathways of TNF activity in these patients, interferon receptor status and (2'–5')-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNFa treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNFa in IFNa-resistant CML patients.

Successful transfer of yeast artificial chromosomes (YACs) into human cells has been described in only a single study. We here report on the evaluation of YAC transfer strategies into a human renal cell carcinoma cell line by yeast spheroplast fusion and cationic lipids. While the latter approach proved inefficient, significant numbers of clones containing both vector arms were obtained by spheroplast fusion. FISH analyses on such clones revealed the presence of YAC integration and the co-localization of both vector arms with insert sequences. These data demonstrate that under certain experimental conditions efficient YAC transfer into human cells by spheroplast fusion is possible and may be useful for the cloning of human disease-related genes.

Abstract: Losses of genetic material within human chromosome regions (HCR) 3p12–p14 and 3p21–p22 are observed in various neoplasias, suggesting tumor suppressor gene (TSG) loci within these regions. HCR 3p14 is particularly interesting as it contains the t(3;8) translocation breakpoint of a hereditary renal cell carcinoma, the FRA3B fragile site, and DNA markers deleted in several types of human cancer. We here report on the identification of five novel ‘expressed sequence tags’ (ESTs) within 3p14.2 which map proximal to exon 9 of the candidate TSG, FHIT. These ESTs may be valuable for elucidation of the supposed TSG content in 3p14.2.

Interferon (IFN) therapy has become widely used for the treatment of chronic myelogenous leukemia. Hematologic remissions can be induced in about 60% of patients. Moreover, in a small number of patients loss of the Philadelphia (Ph) chromosome and of the BCR-ABL rearrangement is observed. We have used genomic Southern blotting as well as a two-step polymerase chain reaction (PCR) method to score for BCR-ABL messenger RNA (mRNA) in patients with hematologic remission induced by treatment with IFNα-2b alone or in combination with IFNγ. Concomitantly, cytogenetic analysis was performed. In 11 of 115 patients reported here, a complete loss of rearranged BCR fragments was observed in Southern blots of peripheral blood (PB) and/or bone marrow (BM) cell samples. Malignant marker bands disappeared first in the PB. In six patients, this genotype remained stable, whereas in five patients, low-intensity, rearranged bands reappeared despite continuation of treatment. The reappearance of the malignant marker was not accompanied by a clinical relapse. Ph-negative metaphases were observed in PB cells of four patients and in the PB and BM cells of two of these patients. In the samples of the other patients, residual Ph-positive cells were detected. By two-step PCR, residual BCR-ABL rearranged transcripts were found in samples of 10 patients.

The common fragile site at 3p14.2 (FRA3B) has been described as the most active fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. We have previously described induction of breakage around FRA3B using aphidicolin in a somatic cell hybrid whose only human component was a single intact chromosome 3. That work led to the isolation of hybrids with breakpoints in the 3p13-p21.1 region with loss of all sequences distal to their respective breakpoints. In this report we describe the further characterization of the breakpoints in many of these cell lines using newly available molecular markers. We also report the identification of YAC clones that span the breakpoints present in many of these hybrids.

To assess the influence of pretransplant cytoreductive therapy with special reference to interferon-α (IFN-α) treatment on major endpoints of allogeneic bone marrow transplantation (BMT), we studied 133 consecutive patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in first chronic phase who received marrow grafts from HLA-identical family (n = 103) or alternative donors (n = 30) at a referral-based transplant center. Fifty of these patients (38%) were previously exposed to IFN-α for a median duration of 14 months (range, 1 to 61 months), whereas 83 patients (62%) exclusively received hydroxyurea and/or busulfan therapy between 1 and 129 months (median, 15 months) pretransplant. Using the categorized treatment duration with each pretransplant cytoreductive agent as a measure for individual patient exposure to each agent, prolonged (>12 months) IFN-α administration was identified as the sole significant pretransplant therapy-related predictor of transplant outcome by proportional hazards regression analysis. The adjusted risk ratio (RR) of transplant-related mortality (TRM) was 2.5-fold higher (95% confidence limits [95% CL], 1.4 to 4.5; P< .004) compared with other pretransplant therapy and this was mainly attributable to a 3.1-fold higher RR (95% CL, 1.4 to 6.4; P< .005) of fatal posttransplant infections after prolonged IFN-α treatment pretransplant. Marrow graft failure developed exclusively among 7 of 30 patients (23%) with donors other than HLA-identical family members and was further restricted to patients who had been previously exposed to IFN-α. The probability of graft failure was 49% ± 28% in 17 patients pretreated with IFN-α compared with 0% for the other 13 patients with mismatched family or unrelated donors (P< .008). In addition, a significant delay in neutrophil and platelet count re-constitution was observed among patients with donors other than HLA-identical family members after pretransplant IFN-α exposure. No influence of pretransplant cytoreductive therapy on either acute and chronic graft-versus-host disease or leukemic relapse was detected in this study. As a consequence of its adverse effect on TRM, prolonged pretransplant IFN-α treatment was independently associated with a 2.5-fold lower likelihood (95% CL, 1.4 to 4.5; P < .003) of 5-year overall survival and with a 2.3-fold lower likelihood (95% CL, 1.3 to 4.2; P < .004) of 5-year disease-free survival postransplant after adjustment for other significant prognostic factors in multivariate analysis. In conclusion, the present study strongly suggests that prolonged pretransplant IFN-α administration in patients with chronic-phase Ph1-positive CML may be associated with an increased risk of fatal transplant-related complications and an inferior outcome after allogeneic BMT. Future analyses on transplant results in chronic-phase CML patients should carefully evaluate the impact of treatment duration or, if applicable, of the cumulative doses of IFN-α and other cytoreductive agents administered pretransplant.

Neoplastic diseases are proliferative disorders characterized by uncoordinated cell growth. Cellular proliferation follows an orderly progression through the cell cycle, which is governed by different cyclins and cyclin dependent kinases (CDKs). Recently, CDK-inhibitors, which are a new class of small proteins involved in the negative regulation of the cell cycle, have been identified by virtue of their ability to interact physically with cyclin/CDK-complexes. As the genes encoding the CDK4-and CDK6-inhibitors (CDK4/6-inhibitors) p16INK4A/CDKN2/MTS1 and p15INK4B/MTS2 have been found to be altered in many cancer cell lines and primary neoplastic tissues, CDK-inhibitors in general and CDK4/6-inhibitors in particular are now a set of candidate tumor suppressors. The p15 and p16 genes map to a region frequently deleted in lymphoid neoplasms. Therefore, considerable efforts have been made to determine the role of CDK4/6-inhibitors in hematologic malignancies: This article will review alterations of components of the cell-cycle machinery in brief and summarize the role of the CDK4/6-inhibitors p16INK4A, p15INK4B, p18INK4C and p19INK4D in leukemias and lymphomas.

The BCR gene on chromosome 22 has received increasing attention because of its involvement in the Philadelphia (Ph' ') translocation. For most restriction enzymes, this locus has been found to be nonpolymorphic. Two alleles have only been found when Taql-digested DNA is hybridized to a 5’ bcr-specific probe. We describe another two-allele polymorphism detected by the same probe in Pvull-digested DNA. The polymorphism is characterized by an additional PvuII site in the bcr region: this causes the appearance of an additional band of about 2.3 kb or 2.5 kb besides a 4.8-kb fragment in hybridizations with the 5’ bcr or a 3’ bcr probe. The incidence of the second allele is very low. It has only been found in some patients with hematopoietic malignancies and in a group of volunteers having a leukemia patient in their families.

131 patients with Philadelphia (Ph') chromosome positive chronic myelogenous leukemia (CML) were treated with interferon (IFN) alpha or a combination of IFN alpha and IFN gamma. In study 1, 13 not pretreated and 41 pretreated patients, 48 in chronic phase disease, 6 in accelerated phase, received 4 × 106 U/m2 IFN alfa-2b initially. After achievement of hématologic remission, the individually minimum effective dose was used for maintenance. There was no response in acute phase disease. Of the 48 patients with chronic phase disease, 22 achieved a hématologic remission (HR), 13 a partial HR (PHR), and 13 did not respond (NR) to IFN. No major cytogenetic response occurred in these patients, but two patients achieved a major molecular response in Southern blots, whereas PCR tests were positive. In a further randomized study, CML patients without prior therapy were treated initially with 4 × 106 U/m2 IFN alpha alone (arm A) or in combination with 50 μg IFN gamma (arm B). For maintenance, the maximum tolerable dose of IFN alpha was given (up to 10 × 106 U/day). Thirteen patients in arm A (54%) and 14 (56%) patients in arm B achieved a HR, 7 patients (29%) in arm A and 6 patients (24%) in arm B a PHR. No response could be induced in 4 patients (17%) of arm A and 5 patients (21%) of arm B. Major cytogenetic responses were observed in 5 (20%) patients of arm A and 5 patients (20%) of arm B. Major molecular responses were observed in 3 patients of arm A and 5 patients of arm B. A single negative PCR in a sample of one patient from arm A was followed by a molecular relapse without recurrence of disease. A significant advantage of the combination could not be substantiated. The study had to be terminated because of two deaths in arm B probably due to cumulative toxicity of the different IFNs. In study III, all patients were pretreated and received a combination of IFN alpha and IFN gamma following the protocol of study II, arm B. Here, HRs occurred in 15 patients (54%), PHRs in 7 (25%), and 6 patients (21%) were NR. No complete cytogenetic response was achieved in 16 evaluable patients, but major molecular responses were demonstrated in samples of 2 patients. One patient had a negative PCR result at a single time point.

Lenz: Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy; Agios: Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding; AQUINOX: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

Introduction:Acute myeloid leukemia (AML) is a hematological malignancy characterized by poor prognosis. Interaction with bone marrow mesenchymal stromal cells (BM-MSC) supports leukemic cells to evade chemotherapy-induced apoptosis. Of particular interest, massive parallel transcriptome sequencing of MSCs from mice and patients with preleukemic syndromes identified inflammatory signaling in inducing genotoxic stress, leukemogenesis, and clinical outcome. Recently, Dexamethasone (Dex), an anti-inflammatory drug, demonstrated antileukemic activity and improved clinical outcome in AML patients. These observations intrigued us to further investigate leukemia-BM niche-associated signaling and to identify signaling cascades supporting leukemic cells and BM-MSC interaction.

Dührsen: Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding. Khandanpour:Max-Eder: Research Funding; Hospital of Essen university: Research Funding.

Dührsen: Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Khandanpour:Hospital of Essen university: Research Funding; Max-Eder: Research Funding.

Dührsen: Roche: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Göthert:Proteros: Consultancy; Ariad Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Acute myeloid leukemia (AML) develops by the acquisition of genomic alterations which initiate different pathways leading to full-blown malignancy. The type of genomic driver alteration governs AML biology and clinical disease course. Translocations involving the core-binding factor (CBF) complex such as t(8:21) and inv(16) represent distinct subgroups of AML genomic drivers. However, additional factors promoting CBF AML leukemogenesis remain largely unknown. Due to the hypoxic nature of the bone marrow the activation of the ubiquitous hypoxia-sensing pathway via HIF-1a might promote CBF leukemogenesis. Nevertheless, in different AML models HIF proteins were recently considered as oncogenic drivers and also as tumor suppressors. Therefore, we here aimed to clarify the role of HIF-1a in CBF AML.

The proper differentiation of hematopoietic stem cells (HSCs) is regulated by a concert of different so called transcription factors (TFs). A disturbed function of these TFs can be the basis of malignant diseases such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Growth Factor Independence 1b (Gfi1b) is a repressing transcription factor, with a key role in maintaining the quiescence of HSCs and the proper emergence and maturation of erythrocytes and platelets. Here we show that low expression of GFI1B in blast cells is associated with an inferior prognosis of AML and MDS patients. Using three different mice models of human AML (Nup98-HoxD13, MLL-AF9 and expression of a mutated K-Ras), we could show that reduced expression of Gfi1b accelerates AML development in mice and the latency is even more shortened when Gfi1b is conditionally deleted. Using a limiting dilution assay of transplantation of different number of Gfi1b-wildtype and Gfi1b-deficient cells, we could show that loss of Gfi1b significantly enhanced stemness of leukemic cells. Since Gfii1b is involved in epigenetic regulation of gene expression, we analyzed effect of loss of Gfi1b on an epigenetic level by analyzing the whole genome using Chip-Seq. We found that loss of Gfi1b leads to genome wide increased level of H3K9 acetylation of genes and hence expression of these genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b not only increases the levels of reactive oxygen species (ROS), but also induces gene expression changes of key AML-pathways such as the p38/ AKT pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS/AML development.

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow. The prognosis of AML is still poor with only 25% of patients living longer than 5 years. Thus, new therapeutic approaches are warranted to improve patients' survival. The growth of AML cells not only depends on cell intrinsic factors, but is also supported by stroma cells, among them mesenchymal stem cells (MSCs). We reported here that MSCs derived from human AML patients or murine models of human AML better supported the in vitrogrowth of leukemic cells than MSCs from non-leukemic patients or mice and this is dependent on cell-to-cell contact. Similarly, immortalized MSCs lines derived from AML patients supported the growth of human AML cell lines better than a MSC line originating from a healthy donor. In addition, the frequency of MSCsin the BM of leukemic mice was increased. On a molecular level, the polarization of MSCs towards an AML-supporting state depended on the expression of the transcription factor Growth factor independence 1 (Gfi1). Loss of Gfi1 abrogated the tumor-supporting state of AML-associated MSCs in vitroand in vivo. In summary, MSCs from AML patients support the growth of AML cells in vitroin a Gfi1-dependent manner which could open the path to new therapeutic approaches.

No relevant conflicts of interest to declare.

Dührsen: Celgene: Honoraria, Research Funding.

The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML.

Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

The proper differentiation of hematopoietic stem cells is regulated by a concert of different so called transcription factors. Growth Factor Independence 1b (Gfi1b) is a repressing transcription factor, which is pivotal for the proper emergence and maturation of erythrocytes and platelets. Furthermore, Gfi1b controls quiescence as well as cell cycle progression of hematopoietic stem cells and early progenitor cells. It has been shown for other transcription factors that a disturbed function of these transcription factors can be the basis of malignant diseases such acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). MDS is characterized by disturbed differentiation of one or several hematopoietic lineages. The accumulation of malignant blast cells, which are arrested in their development, is a key feature of AML.

The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes.

The differentiation of hematopoietic stem cells to mature cells is essential for the function of the hematopoietic system. Disturbance of this process can lead to the emergence of Acute Myeloid Leukemia (AML). AML is characterized by an accumulation of immature, malignant blasts, which disturb the function of the “normal” hematopoietic cells. The differentiation to myeloid cells is regulated among others by transcription factors. Growth factor independence 1 (GFI1) is such a hematopoietic transcription factor regulating the differentiation of myeloid cells.

May: InSCREENeX GmbH: Employment, Equity Ownership, Patents & Royalties.

Abstract 1491

No relevant conflicts of interest to declare.

Abstract 1047

No relevant conflicts of interest to declare.

Abstract 2261

Purpose. Cytomegalovirus (CMV) reactivation after transplant occurs frequently and is generally thought to be associated with an increased transplant-associated mortality. We aimed first to evaluate the cytotoxic effects of CMV on AML in vitro, and further, the clinical outcome of patients with AML with a documented CMV reactivation after transplant. We hypothesized that CMV directly affects AML cells thereby reducing the risk for leukemic relapse in AML-patients after transplant. Patients and Methods. We retrospectively evaluated 236 patients with AML in two cohorts (108 patients in the 1st cohort transplanted from an HLA-identical sibling donor, and 127 patients in a 2nd cohort transplanted from an HLA-identical unrelated donor). All patients were transplanted in Essen. Endpoints of the study were probabilities of relapse and overall survival (OS). For the definition of a CMV reactivation treatment with ganciclovir and the detection of pp65 positive cells in peripheral mononuclear cells at minimum at two different occasions were required. For in vitro studies we used the cell lines Kasumi-1 (AML-M2), SD-1 (ALL) and K562 (CML in blast crises) and infected them with the AD169 CMV strain. 14 days after infection we evaluated the apoptosis rate by measuring annexin V by FACS, the proliferation rate by BRDU assay, the expression of disease-specific marker (AML1/ETO, BCR-ABL), pro-and antiapoptosis marker p21, c-myc by real-time PCR in infected cells and controls. Results. Infection of CMV in Kasumi-1 cells (AML) induced in 99.8% of cells apoptosis. Apoptosis was induced also in SD-1 cells (ALL) in 31.3% of cells after CMV infection, whereas no difference in the apoptosis rate was seen for K562 cells (CML) after CMV infection (6.4%) compared to uninfected controls (9.0%). These results were in concordance with the clinical observation of a CMV reactivation after transplant in AML, ALL, and CML. In 25.9% of AML- patients of cohort 1 and in 29.9% of AML-patients of cohort 2 we detected a CMV reactivation. AML-Patients with a documented CMV reactivation had in both cohorts a markedly reduced risk for leukemic relapse (probability of risk for relapse at 5-year 9.2% versus 52.6% in cohort 1 (p<0.0005) and 12.2% versus 47.2% in cohort 2 (p<0.013), which resulted also in a significant improved outcome after transplant (cohort 1 OS 73.6% versus 42.5% (p<0.04) and 48.0% versus 33.5% in cohort 2 (p<0.04). There were no significant differences in the characteristics of patients with or without CMV reactivation as age, disease stage, donor type or conditioning regimens. Patients with CMV reactivation had a higher incidence of acute GVHD grade 2 – 4 (82% versus 38%, p <0.0001), but this was also seen in patients with ALL and CML who did not have a reduced risk for leukemic relapse after a CMV reactivation. When stratified according to the occurrence of acute GVHD grade 2–4, the probability for risk of relapse rate remained significant lower in patients with a CMV reactivation compared to their counterparts (p<0.003). In multivariate analyses, age of patients, acute GVHD grade2–4, chronic GVHD, disease stage, HLA-constellation between donor and recipient, donor type (unrelated versus sibling), sex-constellation between donor and recipient were tested in each cohort as well as combined in both cohorts together. The only significant factors impacting the outcome and relapse rate were disease stage, chronic GVHD and CMV reactivation. Conclusions. We provide strong evidence that the occurrence of a CMV reactivation after HSCT is associated with a reduced risk for leukemic relapse in patients with AML. Our data indicate an unrecognized role of CMV reactivation after HSCT for AML, which is probably mediated by CMV induced apoptosis.

Purpose. Cytomegalovirus (CMV) reactivation after transplant occurs frequently and is generally thought to be associated with an increased transplant-associated mortality. We aimed first to evaluate the cytotoxic effects of CMV on AML in vitro, and further, the clinical outcome of patients with AML with a documented CMV reactivation after transplant. We hypothesized that CMV directly affects AML cells thereby reducing the risk for leukemic relapse in AML-patients after transplant.

In vitro gene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control). Protection from araC was investigated in three experiments. Gene transfer efficiency was analysed by EGFP expression in the peripheral blood after hematopoietic reconstitution. In these experiments 42±4%, 31±1% and 69±2% EGFP+ granulocytes and 9±1%, 10±1% and 36±3% EGFP+ lymphocytes were detected following SF91-CDD-IRES-EGFP transduction (n=5–6 for each experiment). After araC application (500mg/kg; 1xdaily; d1-4; i.p.) substantial protection of granulocytes and thrombocytes was observed. Following the first treatment cycle nadir counts [x103/μl] were 2.9±0.6 vs. 0.7±0.1, 1.1±0.2 vs. 0.5±0.1 and 2.3±0.3 vs. 0.7±0.2 for granulocytes and 510±148 vs. 80±9, 165±48 vs. 70±42 and 728±133 vs. 85±22 for thrombocytes, respectively (SF91-CDD-IRES-EGFP vs. control; all differences significant at p<0.01 for granulocytes and p<0.05 for thrombocytes). Similar protection was noted during repetitive araC treatment cycles. However, no significant enrichment of CDD transduced cells following araC application was seen and lack of selection was confirmed in two additional experiments utilizing low efficiency gene transfer (<5% and <12% EGFP+ granulocytes or lymphocytes throughout the experiment). No major toxicity of CDD-expression in hematopoietic cells was noted and steady levels of gene marked cells were seen for up to six months in primary and at 5 weeks in secondary recipients. Substantial protection of hematopoiesis by CDD overexpression also was observed for gemcitabine. Mice receiving CDD transduced cells and exposed to 250mg/kg gemcitabine (1xdaily i.p., d1-2), showed granulocyte and thrombocyte nadirs [x103/μl] of 2.4±0.6 vs. 1.4±0.2 and 416±62 vs. 277±97 (SF91-CDD-IRES-EGFP vs. control). When a third day of gemcitabine treatment was added, all mice in the control group died within 4 days while animals receiving CDD-transduced bone marrow survived with nadir counts [x103/μl] of 0.8±0.1 granulocytes and 1130±292 thrombocytes [n=3]. Again, no enrichment of transduced cells was observed. In summary, our data demonstrate efficient protection from araC and gemcitabine hematotoxicity by CDD overexpression. This protective effect was on progenitor rather than stem cells, as no selection of CDD-expressing cells was demonstrated. Thus, a promising approach to utilize CDD clinically may be the combination with a selectable drug resistance gene such as MGMT or MDR-1 to allow enrichment of transduced cells while at the same time protecting against additional drugs.

This study evaluates cellular and humoral immune parameters in myeloma patients focusing on the effect of treatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyeloma immunoglobulins (Ig) were analyzed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibited significantly reduced CD4+/45RO+, CD19+, CD3+/HLA-DR+, and natural killer (NK) cells as well as nonmyeloma IgA, IgG, and IgM. Conventional-dose chemotherapy (conv-CTX) resulted in significantly reduced CD4+ and even further decline of CD4+/CD45RO+ and CD19+ cells, most notably in relapsed patients. Patients treated with conv-CTX in combination with thalidomide showed significantly increased counts of monocytes as well as serum levels of nonmyeloma IgA and IgM as compared to patients on conv-CTX without additional thalidomide. Following high-dose chemotherapy (HD-CTX) prolonged immunosuppression was observed. While CD8+, NK, CD19+, and CD4+/CD45RO+ cells recovered to normal values within 60, 90, 360, and 720 days, respectively, CD4+ counts remaining reduced even thereafter. Nine opportunistic infections were observed including 5 cytomegalovirus (CMV) diseases, 1 pneumocystis carinii pneumonia (PCP), and 3 varicella zoster virus (VZV) infections with CMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reduced CD4+ as well as CD4+/CD45RO+, and CD19+ counts. Thus, myeloma patients display cellular and humoral immunodeficiencies, which increase following conv- as well as HD-CTX and constitute an important predisposing factor for opportunistic infections.

In vitrogene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control).

This study evaluates cellular and humoral immune parameters in myeloma patients focusing on the effect of treatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyeloma immunoglobulins (Ig) were analyzed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibited significantly reduced CD4+/45RO+, CD19+, CD3+/HLA-DR+, and natural killer (NK) cells as well as nonmyeloma IgA, IgG, and IgM. Conventional-dose chemotherapy (conv-CTX) resulted in significantly reduced CD4+ and even further decline of CD4+/CD45RO+ and CD19+ cells, most notably in relapsed patients. Patients treated with conv-CTX in combination with thalidomide showed significantly increased counts of monocytes as well as serum levels of nonmyeloma IgA and IgM as compared to patients on conv-CTX without additional thalidomide. Following high-dose chemotherapy (HD-CTX) prolonged immunosuppression was observed. While CD8+, NK, CD19+, and CD4+/CD45RO+ cells recovered to normal values within 60, 90, 360, and 720 days, respectively, CD4+ counts remaining reduced even thereafter. Nine opportunistic infections were observed including 5 cytomegalovirus (CMV) diseases, 1 pneumocystis carinii pneumonia (PCP), and 3 varicella zoster virus (VZV) infections with CMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reduced CD4+ as well as CD4+/CD45RO+, and CD19+ counts. Thus, myeloma patients display cellular and humoral immunodeficiencies, which increase following conv- as well as HD-CTX and constitute an important predisposing factor for opportunistic infections.