CD11c+ dendritic cells mediate antigen-specific suppression in extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) represents one of the most widespread and effective cell therapies for graft-versus-host disease and other T cell-mediated disorders. However, the key factors affecting the therapeutic efficacy of ECP remain unclear. We hypothesized that therapeutic effects are mediated by ECP-treated antigen-presenting dendritic cells (DC). To test this hypothesis, we used the experimental model of contact hypersensitivity (CHS). The ECP's therapeutic activity improved when the total cell dose of the ECP-treated cells was increased. We used different haptens during sensitization to demonstrate that the anti-inflammatory activity of ECP is antigen-specific. This confirmed the hypothesis that professional antigen-presenting cells are involved in the mode of action. Also, the ECP's therapeutic activity was abrogated by the depletion of CD11c+ DC, which represents fewer than 1% of all the ECP-exposed cells. Finally, we confirm the critical importance of CD11c+ DC for ECP activity by showing that only a few purified CD11c+ DC are sufficient to mediate its therapeutic effect. The finding that ECP-treated, physiological antigen-presenting DC alone mediate antigen-specific modulation of a pathological immune response may result in better-targeted interventions when treating patients.

Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

This corrects the article DOI: 10.1038/leu.2017.9.

Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

It is unknown, why only a minority of chronic myeloid leukemia (CML) patients sustains treatment free remission (TFR) after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission (MR). Here we studied, whether expression of the T-cell inhibitory receptor (CTLA-4)-ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) affects relapse risk after TKI cessation. CML patients in MR displayed significantly higher CD86+pDC frequencies than normal donors (P<0.0024), whereas TFR patients had consistently low CD86+pDC (n=12). This suggested that low CD86+pDC might be predictive of TFR. Indeed, in a prospective analysis of 122 patients discontinuing their TKI within the EURO-SKI trial, the one-year relapse-free survival (RFS) was 30.1% (95% CI 15.6-47.9) for patients with >95 CD86+pDC per 105 lymphocytes, but 70.0% (95% CI 59.3-78.3) for patients with <95 CD86+pDC (hazard ratio (HR) 3.4, 95%-CI: 1.9-6.0; P<0.0001). Moreover, only patients with <95 CD86+pDC derived a significant benefit from longer (>8 years) TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; P=0.0263). High CD86+pDC counts significantly correlated with leukemia-specific CD8+ T-cell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; P=0.0098). Our data demonstrate that CML patients with high CD86+pDC counts have a higher risk of relapse after TKI discontinuation.

NFATc1 as a therapeutic target in FLT3-ITD-positive AML.

Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Multicenter phase II trial to investigate safety and efficacy of gemcitabine combined with cetuximab as adjuvant therapy in pancreatic cancer (ATIP).

BACKGROUND: To investigate whether addition of cetuximab to standard adjuvant chemotherapy with gemcitabine improves outcome in pancreatic cancer, specifically whether the rate of disease-free survival (DFS) at 18 months (primary end point) exceeds the previously reported 35% of gemcitabine alone. PATIENTS AND METHODS: Prospective, open-label, multicenter, nonrandomized phase II study in 76 patients with R0- or R1-resected ductal adenocarcinoma of the pancreas included between October 2006 and November 2008. Gemcitabine and cetuximab were administered for 24 weeks. Secondary end points included overall survival (OS) and toxic effect. RESULTS: Seventy-three patients received cetuximab. Median DFS was 10.0 [95% confidence interval (CI) 8.9-13.6] months and the DFS rate at month 18 of 27.1% (16.7%-37.6%) was inferior to 35%. Median OS was 22.4 (18.2-27.9) months. Subgroup analyses revealed a nonsignificant increase in DFS for patients with versus without skin toxic effect ≥ grade 2 (median 14.7 versus 8.3 months, P = 0.073) and wild-type versus mutated K-Ras (median 11.5 versus 9.3 months, P = 0.57). Grade 3/4 toxic effects included neutropenia (11.0%), thrombopenia (7%), skin toxic effect (7%) and allergic reactions (7%). CONCLUSION: Addition of cetuximab to adjuvant gemcitabine does not seem to improve DFS or OS of unstratified pancreatic cancer patients. Trends for improved DFS in patients with wild-type K-Ras and skin toxic effect remain to be confirmed.

Immune regulatory plasmacytoid dendritic cells selectively accumulate in perithyroidal lymph nodes of patients with Graves disease: implications for the understanding of autoimmunity.

UNLABELLED: Endocrine orbitopathy (EO) is the most common extrathyroidal manifestation of Graves disease (GD) but the involvement of the underlying immunological dysregulations remains largely unknown. The major source for IFNa-production, plasmacytoid dendritic cells (PDC) are fundamentally involved in the integration of TH1 and TH2 immune responses but also implicated in the pathogenesis of autoimmune diseases such as lupus. AIM: To establish whether PDC may play a role in GD autoimmune reaction and in the pathogenesis of EO. MATERIAL AND METHODS: In a series of six sequential patients with GD as well as six further patients with multinodular goiter, cervical lymph nodes (LN) were sampled and preserved in the setting of thyroid resection. In parallel, peripheral blood samples were collected. The frequency of PDC from the peripheral blood and lymph nodes were determined. Mononuclear cells (MC) were enriched from lymph nodes and peripheral blood using Fiquoll-hypaque density gradient centrifugation. Mononuclear cells were stained using BDCA-2-PE and CD123-FITC monoclonal antibodies and their frequency subsequently analyzed using fluorescence activated cell sorting (FACS). Dead cells were excluded from analysis by appropriate gating strategies and propidium iodide (PI) staining. RESULTS: In all patients with GD (with or without EO), PDC frequency was significantly increased in perithyroidal LN as compared to LN of patient not suffering form autoimmune diseases, e. g. patients with multinodular goiter (p < 0.01). The number of PDC infiltrating lymph nodes (LN-PDC) was also higher when compared to peripheral blood PDC (pB-PDC) of patients with multinodular goiter (p < 0.05). Finally, LN-PDC counts in perithyroidal lymph nodes of patients with GD were substantially increased when compared to pB-PDC of the same patients (p < 0.01) indicating a migration and accumulation of PDC in the draining LN. CONCLUSIONS: We found evidence that PDC selectively accumulate in perithyoidal LN of patients with GD, but not other thyroid diseases such as multinodular goiter. Based on their central importance in the pathogenesis of autoimmune processes such as in lupus erythematoides, we suggest that the migration and accumulation of PDC in an anatomical joining point between the thyroid gland and orbita may be of critical importance for the initiation and maintenance of a chronic autoimmune stimulation. This implies a so far unknown role for PDC in GD and as putative cellular targets for new therapeutic approaches.

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter.

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.

Very mild cases of Rett syndrome with skewed X inactivation.

BACKGROUND: Rett syndrome, a common cause of mental retardation in females, is caused by mutations in the MECP2 gene. Most females with MECP2 mutations fulfil the established clinical criteria for Rett syndrome, but single cases of asymptomatic carriers have been described. It is therefore likely that there are individuals falling between these two extreme phenotypes. OBJECTIVE: To describe three patients showing only minor symptoms of Rett syndrome. FINDINGS: The patient with the best intellectual ability had predominantly psychiatric problems with episodes of uncontrolled aggression that have not been described previously in individuals with MECP2 mutations. All three patients had normal hand function, communicated well, and showed short spells of hyperventilation only under stress. Diagnosis in such individuals requires the identification of subtle signs of Rett syndrome in girls with a mild mental handicap. Analysis of the MECP2 gene revealed mutations that are often found in classical Rett syndrome. Skewed X inactivation was present in all three cases, which may explain the mild phenotype. CONCLUSIONS: Because of skewed X inactivation, the phenotype of Rett patients may be very mild and hardly recognisable.

Detection of relapse by sequential monitoring of chimerism in circulating CD34+ cells.

Relapse of the underlying malignant disease represents one of the major causes of treatment failure in patients treated with stem cell transplantation, especially in patients with acute leukemia. Detection of minimal residual disease (MRD) after allogenic stem cell transplantation (SCT) is important to schedule therapeutic intervention, e.g. donor lymphocyte infusion. We asked, whether chimerism analysis in circulating CD34+ cells might be a feasible method to monitor MRD in patients after allogeneic SCT. Eighty-seven patients undergoing allogeneic SCT were prospectively analyzed. CD34+ cells were prepared from peripheral blood samples and sorted after immunomagnetic preenrichment. Results were correlated to overall chimerism and results of nested RT-PCR for the bcr-abl rearrangement in Ph1-cases. In the 87 patients a median of 8 analyses (range 2-22) covering a median period of 295 days (range 28-1152) were performed. A hematological relapse was observed in 22/84 engrafting patients (26%). In twenty patients, the relapse was detected in the CD34+ fraction, in 14 of these patients, donor chimerism in CD34+ cells decreased 12-97 days (median 52 days) before the clinical diagnosis. In two cases, CD34+ chimerism failed to demonstrate the relapse. In patients with relapsing CML, the decrease of donor CD34+ cells was associated with reappearance of bcr-abl transcripts. Treatment (reduction of immunosuppression, DLI or STI571) was associated with an increase of donor derived CD34+ cells and clearance of bcr-abl positive cells. Sequential chimerism analysis in CD34+ cells is feasible and sensitive, allowing early detection of relapse and monitoring of therapeutic intervention. This method appears especially useful in patients with high risk leukemia lacking other markers for detection of MRD.

Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers.

Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40,000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods.

Sterols and gene expression: control of affluence.

Intracellular and extracellular cholesterol levels are tightly maintained within a narrow concentration range by an intricate transcriptional control mechanism. Excess cholesterol can be converted into oxysterols, signaling molecules, which modulate the activity of a number of transcription factors, as to limit accumulation of excess of cholesterol. Two key regulatory pathways are affected by oxysterols. The first pathway involves the uptake and de novo synthesis of cholesterol and is controlled by the family of sterol response element binding proteins, whose activity is regulated by a sterol-dependent feedback mechanism. The second pathway, which only recently has become a topic of interest, involves the activation by a feedforward mechanism of cholesterol utilization for either bile acid or steroid hormone synthesis by oxysterol-activated nuclear receptors, such as liver X receptor and steroidogenic factor-1. Furthermore, biosynthesis and enterohepatic reabsorption of bile acids are regulated by the farnesol X receptor, a receptor activated by bile acids. Both the feedback inhibition of cholesterol uptake and production and the stimulation of cholesterol utilization will ultimately result in a lowering of the intracellular cholesterol concentration and allow for a fine-tuned regulation of the cholesterol concentration.

Expression of interferon regulatory factor 4 in chronic myeloid leukemia: correlation with response to interferon alfa therapy.

PURPOSE: Mice experiments have established an important role for interferon regulatory factor (IRF) family members in hematopoiesis. We wanted to study the expression of interferon regulatory factor 4 (IRF4) in various hematologic disorders, especially chronic myeloid leukemia (CML), and its association with response to interferon alfa (IFN-alpha) treatment in CML. MATERIALS AND METHODS: Blood samples from various hematopoietic cell lines, different leukemia patients (70 CML, 29 acute myeloid leukemia [AML], 10 chronic myelomonocytic leukemia [CMMoL], 10 acute lymphoblastic leukemia, and 10 chronic lymphoid leukemia patients), and 33 healthy volunteers were monitored for IRF4 expression by reverse transcriptase polymerase chain reaction. Then, with a focus on CML, the IRF4 level was determined in sorted cell subpopulations from CML patients and healthy volunteers and in in vitro-stimulated CML cells. Furthermore, IRF4 expression was compared in the CML samples taken before IFN-alpha therapy and in 47 additional CML samples taken during IFN-alpha therapy. IRF4 expression was then correlated with cytogenetic response to IFN-alpha. RESULTS: IRF4 expression was significantly impaired in CML, AML, and CMMoL samples. The downregulation of IRF4 in CML samples was predominantly found in T cells. In CML patients during IFN-alpha therapy, a significant increase in IRF4 levels was detected, and this was also observed in sorted T cells from CML patients. The increase seen during IFN-alpha therapy was not due to different blood counts. In regard to the cytogenetic response with IFN-alpha, a good response was associated with high IRF4 expression. CONCLUSION: IRF4 expression is downregulated in T cells of CML patients, and its increase is associated with a good response to IFN-alpha therapy. These data suggest IRF4 expression as a useful marker to monitor, if not predict, response to IFN-alpha in CML.

Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions.

Acute myeloid leukemias (AML) are considered to be clonal disorders involving early hematopoietic progenitor cells. The recent advances in characterization of early stem cells give rise to the question whether it is possible to distinguish healthy progenitors from cells of the leukemic clone in leukemia patients. Differences and similarities in phenotype, genotype and biology are described for leukemic cells and normal hematological progenitors. Recent new insights into human stem cell development offer the perspective that distinction between benign and malignant progenitors might be possible in the future at a very early stage of maturation.

Detection of cytogenetic aberrations both in CD90 (Thy-1)-positive and (Thy-1)-negative stem cell (CD34) subfractions of patients with acute and chronic myeloid leukemias.

Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are thought to arise from malignant hematopoietic progenitor cells representing early and undifferentiated stem cell clones. In CML there is evidence for a progenitor cell subset free of leukemic clones, depending on the course of the disease. Additionally, it has been suggested that in AML, the early stem cell compartment (CD34+/90+) does not harbor the malignant clone. We analyzed white blood cells from leukemia patients for the presence of aberrant cells in stem cell subfractions. Sixteen patients with CML, six patients with AML, two patients with acute lymphatic leukemia (ALL) and one with chronic myelomonocytic leukemia (CMMOL), all with known cytogenetic abnormalities, were evaluated according to their CD90 (Thy-1)-positive or -negative phenotype. Subsets were sorted on to slides and further characterized by FISH and/or standard cytogenetic testing. The bcr-abl translocation or gross chromosomal abnormalities could be detected in equally high amounts of 92.2% and 89.2% in both stem cell subsets. We conclude, that in progressed AML and CML cells characterized by specific genetic aberrations implicated in the malignant state can be found in the CD34+/CD90+ and CD90- population, thus making CD90 an inappropriate marker to distinguish benign from malignant cells in these leukemias.

Stable engraftment after megadose blood stem cell transplantation across the HLA barrier: the case for natural killer cells as graft-facilitating cells.

BACKGROUND: The case of a patient with chronic myelogenous leukemia who underwent transplantation with highly purified CD34+ peripheral blood stem cells from his two-antigen-mismatched mother is reported. No graft-versus-host disease has been observed so far and stable engraftment has been documented until day 100. METHODS: Weekly analysis of chimerism in different cellular subsets was performed using a quantitative polymerase chain reaction assay for nine short tandem repeat markers in leukocytes sorted by fluorescence-activated cell sorting. RESULTS: No donor CD4+ or CD8+ T cells have been detected up to 3 months after transplantation, whereas a rapid increase of donor CD56+ natural killer (NK) cells was observed in parallel with circulating donor CD34+ progenitors and myeloid cells. CONCLUSIONS: Because the graft contained virtually no T and NK cells, we believe the rapid in vivo generation of NK cells supported stable engraftment across the HLA barrier. The differentiation of CD34+ progenitors into NK cells might be a distinct feature of megadose stem cell transplants.

Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection.

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells.

Analysis of the p53 and MDM-2 gene in acute myeloid leukemia.

The MDM-2 (murine double minute 2) gene codes for a cellular protein that can bind to the p53 tumor suppressor gene product, thereby functioning as a negative regulator of p53. In order to define the role of the MDM-2 gene in the pathogenesis of human acute myeloid leukemia, the expression and the sequence of the MDM-2 gene were examined in samples of bone marrow and/or peripheral mononuclear cells of 38 patients by using immunostaining, polymerase chain reaction (PCR), single strand conformation polymorphism, and sequencing. Immunohistochemical staining detected a weak accumulation of the MDM-2 protein in AML patients of FAB classification M4 and M5. RT-PCR analysis revealed a heterogeneous expression pattern of MDM-2 mRNA in AML samples of different FAB classification. An increased level of MDM-2 mRNA expression was observed in 17 of 38 AML patients when compared to normal controls. No structural changes in a 488 bp region extending from nucleotide 890 to 1378 of the MDM-2 cDNA were detected using RT-SSCP and sequence analysis. In addition, heterogeneous expression of p53 transcripts was found with the highest p53 mRNA levels in AML M4 and M5. Interestingly, there seems to be a correlation between the relative ratios of p53 and MDM-2 mRNA levels in AML M4 and M5: in 15 of 23 cases high p53 mRNA expression was directly associated with high levels of MDM-2 transcripts. An exclusively intranuclear p53 immunostaining pattern was found in 10 of 16 (58%) AML FAB M4 and M5, whereas the remaining AML samples tested were negative for p53 (0/10). Using RT-SSCP analysis and direct sequencing of the RT-PCR amplification products of p53 exon 5-8, we observed that only 1 of 38 AML patients showed a point mutation in the p53 gene. This missense mutation occurred in the evolutionary highly conserved region of p53 at codon 255 (Ile to Phe). These data indicated that structural alterations of the p53 gene do not play an important role in the initiation and progression of AML. However, abrogation of p53 tumor suppressor function due to MDM-2 overexpression may be an alternative molecular mechanism by which a subset of AMLs may escape from p53-regulated growth control.