W. Heit

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.

Ex vivo T‐cell depletion with the monoclonal antibody Campath‐1 plus human complement effectively prevents acute graft‐versus‐host disease in allogeneic bone marrow transplantation

Summary. We have developed a rapid and simple procedure for the elimination of mature T‐cells from the donor marrow using a single incubation with the monoclonal antibody Campath‐1 and donor complement. This resulted in a reduction of T‐cell contamination to a mean of 1%. This regimen reduced the incidence of acute graft‐versus‐host disease significantly in 21 consecutive bone marrow grafts in 18 patients with leukaemia and non‐Hodgkins lymphoma. Purging was responsible for an increased incidence of graft rejection in HLA‐identical transplants (13%).

Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.

Reproductive and endocrine gonadal functions in adults following multidrug chemotherapy for acute lymphoblastic or undifferentiated leukemia.

The impact of aggressive chemotherapy on reproductive and endocrine gonadal function was studied in ten women and ten men with acute lymphoblastic leukemia (ALL) or acute undifferentiated leukemia (AUL) in complete remission. Hormone determinations, sperm analyses, measurements of basal body temperature, and interviews with a standardized questionnaire were used for diagnostic evaluation. Elevated serum follicle-stimulating hormone (FSH) levels and azoospermia were seen in all male patients after completion of induction and consolidation therapy as a result of germ cell and stem cell loss. Recovery of spermatogenesis, as indicated by normalization of serum FSH values and sperm density, occurred in the second year of maintenance therapy in all men. Serum testosterone and luteinizing hormone (LH) values remained within normal limits indicating resistance of Leydig cells to chemotherapy. All female patients showed normal serum levels of gonadal steroids and gonadotropins, as well as an adequate increase in basal body temperature after intensified chemotherapy, indicating intact follicle function and ovulation. Most patients reported normal sexual functions after induction and consolidation therapy. These results demonstrate that multidrug chemotherapy induced significant impairment of reproductive function in all male patients with early and complete recovery. In contrast, endocrine gonadal function was unaffected in men treated with ALL/AUL. In female patients, neither reproductive nor endocrine functions were influenced by aggressive chemotherapy.

Granulocytic progenitor cells in aplastic anaemia.

The characteristics and the concentration of granulopoietic colony forming cells (CFC) were examined in 22 patients with aplastic anaemia at different stages of their disease. Additionally the ability of the patients’peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (CSA) was studied. The ability of the patients’cells to generate CSA was shown to be unaffected. However, the incidence of CFC within the marrow and peripheral blood suspensions was significantly reduced in all patients. The results suggest a reduced compartment size of CFC even in those patients who have recovered from aplastic anaemia. This may indicate that the disturbances in the preceding compartments of the haemopoietic cell renewal system still persist after recovery from the acute bone marrow failure.

THE IN VITRO DIFFERENTIATION OF DENSITY SUB‐POPULATIONS OF COLONY‐FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY‐STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU‐c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub‐populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony‐stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post‐endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU‐c was dependent on the type of CSF. Functional heterogeneity was found among CFU‐c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro‐phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU‐c.

Patterns of growth in human septal cartilage: a review of new approaches

Recent results of the growth activities of the septal cartilage in children, adolescents, and adults are reviewed. Cartilaginous biopsies were obtained during septoplasty . Matrix synthesis was measured by in vitro incorporation of labeled sulphate; DNA synthesis was measured by in vitro incorporation of labeled thymidine. The cell density of the septal cartilage was determined by cell counting, and chondrocytes were grown in culture in an assay to determine the number of chondrocytes capable of proliferation. With data from these 4 types of experiments 5 different areas could be distinguished in human septal cartilage: (1) anterior free end, (2) suprapremaxillary area, (3) central area, (4) posterior area, and (5) caudal prolongation. Metabolic activities, degree of cell replication, and proliferative capacity are highest in childhood in all areas; they decline with age, but remain surprisingly high in the central area and in the anterior free end, even in adults. These results help to explain some clinical observations and may help the rhinosurgeon in his decision to resect the septal cartilage, especially in children.

Aggressive chemotherapy combined with G-CSF and maintenance therapy with interleukin-2 for patients with advanced myelodysplastic syndrome, subacute or secondary acute myeloid leukemia--initial results

SummaryAggressive chemotherapy of advanced myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) evolving from MDS, subacute AML and secondary AML has usually been associated with low complete remission (CR) rates, a high incidence of early death, and low disease-free survival. We therefore have initiated a phase-III trial of aggressive chemotherapy consisting of idarubicin, cytosine arabinoside, and VP-16 to improve the CR rate. Each chemotherapy cycle is followed by G-CSF to accelerate neutrophil recovery and to reduce the incidence of infections. Until now, 19 patients with high-risk AML have been entered. The CR rate is 47%, with only one death during induction. Patients achieving CR are randomized to receive either high-dose or low-dose interleukin-2 to eliminate residual leukemic cells and to prolong the duration of remission.

Human leukemic cells: Receptor binding and biological effects of insulin and insulin-like growth factors

Receptor binding and biological effects of insulin and insulin-like growth factors I and II (IGF I/II) were assessed in three human malignant cell lines: a Burkitt-type ALL-cell line, a ANLL-cell line and a Hodgkins disease-cell line. Insulin receptor binding could be demonstrated in Burkitt-type ALL cells and ANLL cells, whereas no insulin receptor binding was detectable in Hodgkin cells. IGF I and IGF II binding could be demonstrated in all leukemic cells. Insulin stimulated glycogen synthesis in the insulin receptor bearing cell lines. DNA synthesis was stimulated by insulin, IGF I and II. IGF I was more active in stimulating DNA synthesis than IGF II.