Walter Fiedler

Retinoic acid and arsenic trioxide for acute promyelocytic leukemia.

BACKGROUND All-trans retinoic acid (ATRA) with chemotherapy is the standard of care for acute promyelocytic leukemia (APL), resulting in cure rates exceeding 80%. Pilot studies of treatment with arsenic trioxide with or without ATRA have shown high efficacy and reduced hematologic toxicity. METHODS We conducted a phase 3, multicenter trial comparing ATRA plus chemotherapy with ATRA plus arsenic trioxide in patients with APL classified as low-to-intermediate risk (white-cell count, ≤10×10(9) per liter). Patients were randomly assigned to receive either ATRA plus arsenic trioxide for induction and consolidation therapy or standard ATRA-idarubicin induction therapy followed by three cycles of consolidation therapy with ATRA plus chemotherapy and maintenance therapy with low-dose chemotherapy and ATRA. The study was designed as a noninferiority trial to show that the difference between the rates of event-free survival at 2 years in the two groups was not greater than 5%. RESULTS Complete remission was achieved in all 77 patients in the ATRA-arsenic trioxide group who could be evaluated (100%) and in 75 of 79 patients in the ATRA-chemotherapy group (95%) (P=0.12). The median follow-up was 34.4 months. Two-year event-free survival rates were 97% in the ATRA-arsenic trioxide group and 86% in the ATRA-chemotherapy group (95% confidence interval for the difference, 2 to 22 percentage points; P<0.001 for noninferiority and P=0.02 for superiority of ATRA-arsenic trioxide). Overall survival was also better with ATRA-arsenic trioxide (P=0.02). As compared with ATRA-chemotherapy, ATRA-arsenic trioxide was associated with less hematologic toxicity and fewer infections but with more hepatic toxicity. CONCLUSIONS ATRA plus arsenic trioxide is at least not inferior and may be superior to ATRA plus chemotherapy in the treatment of patients with low-to-intermediate-risk APL. (Funded by Associazione Italiana contro le Leucemie and others; ClinicalTrials.gov number, NCT00482833.).

Incidence and Prognostic Influence of DNMT3A Mutations in Acute Myeloid Leukemia

PURPOSE To study the incidence and prognostic impact of mutations in DNA methyltransferase 3A (DNMT3A) in patients with acute myeloid leukemia. PATIENTS AND METHODS A total of 489 patients with AML were examined for mutations in DNMT3A by direct sequencing. The prognostic impact of DNMT3A mutations was evaluated in the context of other clinical prognostic markers and genetic risk factors (cytogenetic risk group; mutations in NPM1, FLT3, CEBPA, IDH1, IDH2, MLL1, NRAS, WT1, and WT1 SNPrs16754; expression levels of BAALC, ERG, EVI1, MLL5, MN1, and WT1). RESULTS DNMT3A mutations were found in 87 (17.8%) of 489 patients with AML who were younger than 60 years of age. Patients with DNMT3A mutations were older, had higher WBC and platelet counts, more often had a normal karyotype and mutations in NPM1, FLT3, and IDH1 genes, and had higher MLL5 expression levels as compared with patients with wild-type DNMT3A. Mutations in DNMT3A independently predicted a shorter overall survival (OS; hazard ratio [HR], 1.59; 95% CI, 1.15 to 2.21; P = .005) by multivariate analysis, but were not associated with relapse-free survival (RFS) or complete remission (CR) rate when the entire patient cohort was considered. In cytogenetically normal (CN) AML, 27.2% harbored DNMT3A mutations that independently predicted shorter OS (HR = 2.46; 95% CI, 1.58 to 3.83; P < .001) and lower CR rate (OR, 0.42; 95% CI, 0.21 to 0.84; P = .015), but not RFS (P = .32). Within patients with CN-AML, DNMT3A mutations had an unfavorable effect on OS, RFS, and CR rate in NPM1/FLT3-ITD high-risk but not in low-risk patients. CONCLUSION DNMT3A mutations are frequent in younger patients with AML and are associated with an unfavorable prognosis.

Impact of IDH1 R132 Mutations and an IDH1 Single Nucleotide Polymorphism in Cytogenetically Normal Acute Myeloid Leukemia: SNP rs11554137 Is an Adverse Prognostic Factor

PURPOSE We assessed the prognostic impact of IDH1 R132 mutations and a known single nucleotide polymorphism (SNP) located in the same exon of the IDH1 gene in patients with cytogenetically normal acute myeloid leukemia (CN-AML) in the context of other prognostic markers. PATIENTS AND METHODS IDH1 exon four was directly sequenced in 275 CN-AML patients from two subsequent AML multicenter treatment trials and 120 healthy volunteers. Moreover, mutations in NPM1, FLT3, CEBPA, and WT1 were analyzed, and mRNA expression of IDH1 was quantified. RESULTS IDH1 R132 mutations were found in 10.9% of CN-AML patients. IDH1 SNP rs11554137 was found in 12% of CN-AML patients and 11.7% of healthy volunteers. IDH1 R132 mutations had no impact on prognosis. In contrast, IDH1 SNP rs11554137 was an adverse prognostic factor for overall survival in univariate and multivariate analysis. Other significant factors were age, NPM1/FLT3 mutational status, WT1 SNP rs16754, and platelet count. The impact of IDH1 SNP rs11554137 was most pronounced in the NPM1/FLT3 high-risk patients (either NPM1 wild-type or FLT3-internal tandem duplication positive). Patients with IDH1 SNP rs11554137 had a higher expression of IDH1 mRNA than patients with two wild-type alleles. CONCLUSION IDH1 SNP rs11554137 but not IDH1 R132 mutations are associated with an inferior outcome in CN-AML.

Prognostic impact of IDH2 mutations in cytogenetically normal acute myeloid leukemia

Mutations in the nicotinamide adenine dinucleotide phosphate(+)-dependent isocitrate dehydrogenase gene 2 (IDH2) have recently been found in patients with acute myeloid leukemia (AML) as well as in patients with leukemic transformation of myeloproliferative neoplasms. We analyzed 272 adult patients with cytogenetically normal AML (CN-AML) for the presence of IDH2 mutations in codons R140 and R172. IDH2 mutations of amino acid 140 or 172 could be identified in 12.1% of CN-AML patients, with the majority of mutations (90%) occurring at position R140. The incidence of IDH2 mutations in AML patients with aberrant karyotypes (n = 130) was significantly lower (3.8%, P = .006). IDH2 mutations were mutually exclusive with mutations in IDH1. IDH2 mutation status alone or in combination with IDH1 mutations had no impact on response to therapy, overall survival, and relapse-free survival in patients with CN-AML. In conclusion, IDH2 mutations are frequently found in CN-AML, but in our analysis these mutations did not influence treatment outcome. This study was registered at www.clinicaltrials.gov as #NCT00209833.

Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against imatinib-resistant BCR-ABL mutations including T315I

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL-positive and -negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34(+) cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.

VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN NORMAL HUMAN TESTICULAR TISSUE

Expression of vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), and its receptors Flt-1 and KDR (Flk-1 in mouse) and their localization in the human testis were analyzed by means of reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. VEGF mRNA was detected in the human testicular tissue and in fragments of seminiferous tubules by means of RT-PCR, while fragments of blood vessels isolated from testes were negative. Western blotting procedure using a specific VEGF antibody, revealed two protein bands corresponding to 24 and 49 kDa in the extracts prepared from the whole testis and in the seminiferous tubules while no such bands were found in isolated fragments of human testicular blood vessels. Also immunohistochemically, human testicular blood vessels show no VEGF immunoreactivity, while Leydig cells and Sertoli cells were positive. The mRNA of the VEGF receptor Flt-1 was found to be expressed in human testicular tissue, in isolated fragments of testicular blood vessels and in seminiferous tubules as determined by RT-PCR procedure. In accordance with these results, the Flt-1 protein was immunohistochemically localized in Leydig, Sertoli and perivascular cells. Endothelial cells of certain segments of human testicular microvasculature also stained positive for Flt-1. Expression of VEGF receptor, KDR, could be demonstrated in human testicular tissue, in isolated seminiferous tubules and in isolated fragments of human testicular blood vessels by means of RT-PCR. Immunohistochemically, the KDR protein was localized in endothelial cells and perivascular cells of capillaries within the lamina propria of seminiferous tubules. Leydig cells and Sertoli cells show KDR immunoreactivity, too. Thus we demonstrate the presence of both types of VEGF receptors Flt-1 and KDR on Leydig as well as on Sertoli cells which are normal non-endothelial cells, suggesting hitherto unrecognized and novel functions for such receptors. The results obtained permit us to suggest VEGF as a paracrine mitogenic and angiogenic factor, responsible for modulating the capillarization of the human testicular tissue and maintaining the functions of testicular microvasculature. VEGF may also influence the permeability of capillaries passing through the groups of Leydig cells and those localized within the lamina propria of human seminiferous tubules. The differences in the expression pattern of the VEGF receptors in the human testicular tissue probably reflect different VEGF effects in different compartments of human testis.

Randomized, phase 2 trial of low-dose cytarabine with or without volasertib in AML patients not suitable for induction therapy

Treatment outcomes for older patients with acute myeloid leukemia (AML) have remained dismal. This randomized, phase 2 trial in AML patients not considered suitable for intensive induction therapy compared low-dose cytarabine (LDAC) with or without volasertib, a highly potent and selective inhibitor of polo-like kinases. Eighty-seven patients (median age 75 years) received LDAC 20 mg twice daily subcutaneously days 1-10 or LDAC + volasertib 350 mg IV days 1 + 15 every 4 weeks. Response rate (complete remission and complete remission with incomplete blood count recovery) was higher for LDAC + volasertib vs LDAC (31.0% vs 13.3%; odds ratio, 2.91; P = .052). Responses in the LDAC + volasertib arm were observed across all genetic groups, including 5 of 14 patients with adverse cytogenetics. Median event-free survival was significantly prolonged by LDAC + volasertib compared with LDAC (5.6 vs 2.3 months; hazard ratio, 0.57; 95% confidence interval, 0.35-0.92; P = .021); median overall survival was 8.0 vs 5.2 months, respectively (hazard ratio, 0.63; 95% confidence interval, 0.40-1.00; P = .047). LDAC + volasertib led to an increased frequency of adverse events that was most pronounced for neutropenic fever/infections and gastrointestinal events; there was no increase in the death rate at days 60 + 90. This study was registered at www.clinicaltrials.gov as #NCT00804856.

Analysis of Concerted Expression of Angiogenic Growth Factors in Acute Myeloid Leukemia: Expression of Angiopoietin-2 Represents an Independent Prognostic Factor for Overall Survival

PURPOSE Bone marrow neoangiogenesis plays an important pathogenetic and possible prognostic role in acute myeloid leukemia (AML). Members of the vascular endothelial growth factor (VEGF) and angiopoietin family represent the most specific inducers of angiogenesis secreted by AML blasts. We therefore correlated expression of angiogenic factors with clinical variables. PATIENTS AND METHODS We investigated the expression of VEGF-A, VEGF-C, angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the receptor Tie2 by quantitative polymerase chain reaction in a cohort of 90 patients younger than 61 years with de novo AML entered into the German AML Süddeutsche Hämoblastose Gruppe Hannover 95 trial. Uni- and multivariate analyses were performed using clinical and gene expression variables. RESULTS Univariate analysis of overall survival indicated the following variables as prognostic factors: good response on a day-15 bone marrow examination after initiation of induction chemotherapy, karyotype, and high Ang2 expression. In multivariate analysis, only bad response and log Ang2 expression remained of statistical significance, with a hazard ratio of 3.51 (95% CI, 1.91 to 6.47) and 0.75 (95% CI, 0.61 to 0.91), respectively. Subgroup analysis suggested that the prognostic impact of Ang2 expression was especially evident in cohorts with low VEGF-C and Ang1 mRNA levels. CONCLUSION These results show that expression of Ang2 represents an independent prognostic factor in AML. Additional research into interactions of angiogenic cytokines in the pathogenesis of bone marrow angiogenesis in AML is warranted.

Expression of a mutated form of the p85α regulatory subunit of phosphatidylinositol 3-kinase in a Hodgkin's lymphoma-derived cell line (CO)

Phosphatidylinositol (PI) 3-kinase plays an important role in a variety of biological processes, including proliferation and apoptosis. PI3-kinase is a heterodimer consisting of an 85 kDa adapter protein (p85) containing one SH3 domain and two SH2 domains and a 110 kDa catalytic subunit (p110). Recently an oncogenic form of p85 named p65-PI3K lacking the C-terminal SH2 domain has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65-PI3K in T lymphocytes develop a lymphoproliferative disorder. Here we describe the cloning of a C-terminal truncated form of p85 expressed in a human lymphoma cell line (CO) with a T cell phenotype derived from a patient with Hodgkins disease. As a result of a frame-shift mutation at amino acid 636, p76 is lacking most of the C-terminal SH2 domain, but contains the inter-SH2 domain and is associated with an active form of PI3-kinase. A PI3-kinase-dependent constitutive activation of Akt was detected in CO cells which was only partially reduced after serum starvation. Treatment of CO cells with the PI3-kinase inhibitor wortmannin resulted in a concentration-dependent inhibition of cell proliferation associated with an increased number of apoptotic cells. This is the first detection of a mutated form of the p85 subunit of PI3-kinase in human hematopoietic cells further underlining a potential role of PI3-kinase/Akt signaling in human leukemogenesis.

Identification of the Adult Human Hemangioblast

Recent studies show that human CD133(+) (previously known as AC133(+)) cells from mobilized peripheral blood consist of stem cells with either hematopoietic or endothelial potential. To test whether this population also contains individual precursors with both capacities, the defining characteristics of the elusive adult hemangioblast, we developed a culture system that allows single-cell analyses of differentiation. In the presence of vascular endothelial growth factor (VEGF), stem cell growth factor (SCGF), and FLT-3 ligand, CD133(+)-enriched cells were first expanded and the amplified cells were transduced with a vector encoding an enhanced green fluorescent protein (EGFP) marker gene. Single EGFP(+) cells were then cocultured with corresponding non-transduced cells from the same experiment, yielding 50-100 marked cells in 8% of the wells after 2 weeks. The resultant cells were divided and differentiated with either granulocyte colony-stimulating factor (G-CSF) or with SCGF and VEGF. These culture conditions resulted in the formation of neutrophil or endothelial cells, respectively, as identified morphologically and by phenotypic staining. Dual differentiation of EGFP(+) cells could be observed in one-quarter of clones from single-seeded cells, suggesting that 2% of EGFP(+) cells were in fact human hemangioblasts. These cells could be expanded for at least 28 days without losing this dual capacity. Hence, this culture system may be of clinical relevance in the development of cellular therapies for disorders involving hematopoiesis and the vascular system. In addition, our results provide important information related to the development of the vasculature and the potential role of hemangioblasts in vasculogenesis in adult humans.