Epidemiological, genetic, and clinical characterization by age of newly diagnosed acute myeloid leukemia based on an academic population-based registry study (AMLSG BiO).

We describe genetic and clinical characteristics of acute myeloid leukemia (AML) patients according to age from an academic population-based registry. Adult patients with newly diagnosed AML at 63 centers in Germany and Austria were followed within the AMLSG BiO registry (NCT01252485). Between January 1, 2012, and December 31, 2014, data of 3525 patients with AML (45% women) were collected. The median age was 65 years (range 18-94). The comparison of age-specific AML incidence rates with epidemiological cancer registries revealed excellent coverage in patients < 70 years old and good coverage up to the age of 80. The distribution according to the European LeukemiaNet (ELN) risk categorization from 2010 was 20% favorable, 31% intermediate-1, 28% intermediate-2, and 21% adverse. With increasing age, the relative but not the absolute prevalence of patients with ELN favorable and intermediate-1 risk (p < 0.001), with activating FLT3 mutations (p < 0.001), with ECOG performance status < 2 (p < 0.001), and with HCT-CI comorbidity index < 3 (p < 0.001) decreased. Regarding treatment, obesity and favorable risk were associated with an intensive treatment, whereas adverse risk, higher age, and comorbidity index > 0 were associated with non-intensive treatment or best supportive care. The AMLSG BiO registry provides reliable population-based distributions of genetic, clinical, and treatment characteristics according to age.

Outcome and prognostic factors in patients with mantle-cell lymphoma relapsing after autologous stem-cell transplantation: a retrospective study of the European Group for Blood and Marrow Transplantation (EBMT).

BACKGROUND: Autologous stem-cell transplantation (autoSCT) is considered a standard treatment of non-frail patients with mantle cell lymphoma (MCL), but little is known about outcome of MCL patients relapsing after autoSCT. We therefore sought to analyse the outcome after autoSCT failure and the efficacy of a rescue stem-cell transplantation (SCT) in this setting. PATIENTS AND METHODS: Patients with MCL were eligible if they had relapsed after autoSCT performed between 2000 and 2009. A total of 1054 patients could be identified in the EBMT registry. By contacting the transplant centres, a full dataset could be retrieved for 360 patients. RESULTS: Median overall survival (OS) after relapse of the whole study group was 19 months. A long (>12 months) interval between autoSCT and relapse [P < 0.001, hazard ratio (HR) 0.62], primary refractory disease (P < 0.02, HR 1.92), prior high-dose ARA-C treatment (P = 0.04, HR 1.43), and the year of relapse (P = 0.02, HR 0.92) significantly influenced OS from relapse in multivariate analysis. Eighty patients (22%) received a rescue allogeneic SCT (alloSCT). Relapse incidence, non-relapse mortality, and OS 2 years after alloSCT was 33% [confidence interval (95% CI 21% to 45%)], 30% (95% CI 19% to 42%), and 46% (95% CI 33% to 59%), respectively. Remission duration after autoSCT was the only variable significantly affecting the outcome of salvage alloSCT. In contrast, rescue autoSCT was not associated with long-term disease control. However, individual patients survived long term even without salvage transplantation. CONCLUSIONS: MCL recurrence within 1 year after autoSCT has an extremely dismal outcome, while the prognosis of patients with longer remission durations after autoSCT is significantly better. AlloSCT may offer the possibility of durable survival when performed for patients with a remission duration of more than 12 months after first autoSCT, but the favourable effect of a salvage alloSCT in this setting needs further validation.

Long-term follow-up of the AML97 study for patients aged 60 years and above with acute myeloid leukaemia: a study of the East German Haematology and Oncology Study Group (OSHO).

INTRODUCTION: Treatment of patients (pts) with acute myelogenous leukaemia (AML) above 60 years remains a challenge. We report long-term follow-up of the AML97 study, where pts were registered at diagnosis and received treatment dependent on their comorbidities: dose-intense cytarabine (AraC) and anthracycline in the curative arm, and low-dose chemotherapy in the palliative arm or best supportive care. MATERIALS AND METHODS: A total of 618 pts were enrolled in this protocol (curative 471, palliative 115 and supportive 32). In the curative arm, complete remission (CR) was obtained in 66.8 % of pts and the estimated probability of being alive at 2 years was 0.30 (±0.02 SE). In multivariate analysis, gender (p = 0.005), performance status (p = 0.04) and cytogenetics (p = 0.002) were significant factors for CR. With a median follow-up of 10 (range 0.1-11.8) years, the estimated probability of being event-free after 2 and 5 years according to cytogenetics was 0.48 ± 0.11 and 0.48 ± 0.11 for favourable, 0.20 ± 0.03 and 0.09 ± 0.03 for normal, 0.18 ± 0.06 and 0.10 ± 0.05 for other standard risk and 0.10 ± 0.03 and 0.05 ± 0.02 for unfavourable karyotypes, respectively. The median survival time for pts treated with palliative chemotherapy was 54 and 11 days with best supportive care only. CONCLUSION: In conclusion, treatment of older AML pts with dose-intense AraC is feasible in the majority of pts and induces high rates of CR. Nevertheless, except for favourable karyotype, OS and event-free survival remain low. These results need to be viewed in relation to the new modalities including stem cell transplantation following non-myeloablative conditioning, epigenetic and molecular therapies.

Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.

Very early detection of response to imatinib mesylate therapy of gastrointestinal stromal tumours using 18fluoro-deoxyglucose-positron emission tomography.

AIM: To investigate whether 18F-FDG-PET allows early assessment of response to imatinib mesylate in GIST patients. PATIENTS AND METHODS: Five gastrointestinal stromal tumour (GIST) patients and one patient with a KIT-positive small cell cancer were studied. Treatment consisted of 400 mg imatinib mesylate daily. 18F-deoxyglucose-positron emission tomography (18F-FDG-PET) scans were done before and one week after starting imatinib mesylate therapy. RESULTS: Metabolic responses were detected after only one week of therapy in all GIST patients (four partial and one complete response). The mean decrease of the standardised uptake value was 60% (range 43 to 77%). In contrast, the tumour of the non-GIST patient was metabolically stable. Four of the 5 GIST patients achieved a partial response on CT after a mean duration of 23 weeks (range 6 to 48 weeks). CONCLUSION: 18F-FDG-PET is a valuable method for the detection of response to imatinib mesylate in patients with KIT-positive tumours as early as one week after starting therapy.

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and primary lymphoma cells can be efficiently transfected using an adenoviral vector system. By adding cationic liposomes, the efficiency of adenoviral gene transfer to primary tumor cells could be further improved. This protocol may have an impact on the use of lymphoma cells in cancer gene therapy.

High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses.

Preliminary evidence suggests that the multikinase inhibitor sorafenib has clinical activity in FLT3-ITD-positive (FLT3-ITD) acute myeloid leukemia (AML). However, the quality and sustainability of achievable remissions and clinical variables that influence the outcome of sorafenib monotherapy are largely undefined. To address these questions, we evaluated sorafenib monotherapy in 65 FLT3-ITD AML patients treated at 23 centers. All but two patients had relapsed or were chemotherapy-refractory after a median of three prior chemotherapy cycles. Twenty-nine patients (45%) had undergone prior allogeneic stem cell transplantation (allo-SCT). The documented best responses were: hematological remission in 24 patients (37%), bone marrow remission in 5 patients (8%), complete remission (with and without normalization of peripheral blood counts) in 15 patients (23%) and molecular remission with undetectable FLT3-ITD mRNA in 10 patients (15%), respectively. Seventeen of the patients without prior allo-SCT (47%) developed sorafenib resistance after a median treatment duration of 136 days (range, 56-270 days). In contrast, allo-SCT patients developed sorafenib resistance less frequently (38%) and significantly later (197 days, range 38-225 days; P=0.03). Sustained remissions were seen exclusively in the allo-SCT cohort. Thus, sorafenib monotherapy has significant activity in FLT3-ITD AML and may synergize with allogeneic immune effects to induce durable remissions.

Anti-tumor immunity is involved in the thymidine kinase-mediated killing of tumors induced by activated Ki-ras(G12V).

We have established a syngeneic mouse tumor model to test the efficacy of the drug-sensitizing enzyme thymidine kinase from herpes simplex virus (HSVtk) in vivo. Activated mutant Ki-ras(G12V) is frequently found in human colon cancer and adenocarcinomas of the lung and pancreas. We have transformed BALB/c-3T3 cells by stable transfection of a plasmid directing the expression of the mutant Ki-ras cDNA. To transfer the HSVtk gene into tumor cells we used a Moloney murine leukemia virus (MoMLV)-based retroviral vector that carries the HSVtk gene. In this study we show that the activity of HSV-TK inhibits tumor growth in immune-compromised nude mice following GCV treatment for up to 50 days but is not sufficient to completely eliminate all tumor cells in these mice as evidenced by the occurrence of tumors between 40 and 50 days after tumor cell implantation. By contrast, immune-competent BALB/c mice develop a long-lasting antitumor immunity in response to HSVtk transduction and GCV treatment, indicating that the immune system is important for the long-term tumor suppression in vivo. In the presence of GCV co-culturing of tumor cells with HSVtk transfected cells leads to the efficient killing of HSVtk negative tumor cells. While this retroviral vector independent HSV-TK/GCV-mediated bystander effect is not sufficient to inhibit tumor formation in athymic animals it is very efficient in immune-competent syngeneic mice. Taken together the data indicate that the antitumor activity of HSV-TK is enhanced by an intact immune system.

Inotropic response to beta-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts.

1. The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti-adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS-/-) mice. 2.eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT-PCR analysis. 3. In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS-/- hearts were not significantly different from those of WT hearts (LVP: 97 +/- 11 mmHg WT vs. 111 +/- 11 mmHg eNOS-/-; dP/dtmax: 3700 +/- 712 mmHg s(-1) WT vs. 4493 +/- 320 mmHg s)-1) eNOS-/-). 4. The dobutamine (10-300 nM)-induced increase in LVP was enhanced in eNOS-/- hearts. In contrast, L-type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS-/- hearts showed no differences after beta-adrenergic stimulation. Dibutyryl-cGMP (50 microM) reduced basal ICa,L in WT cells to 72 +/- 12 % while eNOS-/- ICa,L was insensitive to the drug. The pre-stimulated ICa,L (30 nM isoproterenol) was attenuated by dibutyryl-cGMP in WT and eNOS-/- cells to the same extent. 5. The Ca2+ (1.5-4.5 mM)-induced increase in inotropy was not different between the two experimental groups and beta-adrenergic receptor density was increased by 50% in eNOS-/- hearts. 6. The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti-adrenergic effect of both compounds was identical in WT and eNOS-/- hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7. These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to beta-adrenergic stimulation and with elevated -adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS-/- cardiac myocytes to beta-adrenergic stimulation suggests that endothelium-derived NO is important in mediating the whole-organ effects and (3) eNOS is unimportant for the anti-adrenergic effect of ACh and adenosine.