Broad differentiation capacity has been described for mesenchymal stem cells (MSC) from human bone marrow. We sought to identify genes associated with the immature state and pluripotency of this cell type. To prove the pluripotent state of the MSC, differentiation into osteocytes, adipocytes, and chondrocytes was performed in vitro. In contrast, normal skin cells did not harbor these differentiation abilities. We compared the expression profile of human bone marrow MSC with cDNA from one primary human skin cell line as control, using a cDNA chip providing 9600 genes. The identity of all relevant genes was confirmed by direct sequencing. Data of gene array expression were corroborated employing quantitative PCR analysis. About 80 genes were differently expressed more than threefold in MSC compared to mature skin fibroblasts. Interestingly, primary human MSC were found to upregulate a number of genes important for embryogenesis such as distal-less homeo box 5, Eyes absent homolog 2, inhibitor of DNA binding 3, and LIM protein. In contrast, mesenchymal lineage genes were downregulated in MSC in comparison to skin cells. We also detected expression of some genes involved in neural development, indicating the broad differentiation capabilities of MSC. We conclude that human mesenchymal stem cells harbor an expression profile distinct from mature skin fibroblast, and genes associated with developmental processes and stem cell function are highly expressed in adult mesenchymal stem cells.

In patients with multiple myeloma, irradiation of bone marrow prior to mobilization of autologous peripheral blood progenitor cells (PBPCs) may lead to a reduced yield of CD34+ cells. Quantitative effects have not been sufficiently assessed. We retrospectively performed a multivariate analysis in 114 patients (67 men, 47 women) with multiple myeloma, of whom 53 (47%) patients had been irradiated prior to mobilization chemotherapy. High-dose cyclophosphamide followed by granulocyte colony-stimulating factor was used for mobilization in 84% of patients. In addition to previous chemotherapy, we quantitatively evaluated the dose and fractionation of prior irradiation, the volume of the irradiated bone marrow, and the time interval between radiation therapy and mobilization of PBPCs. The median volume of irradiated bone marrow was 9% (range 1–30%) of the estimated total hematopoietic bone marrow. The irradiated bone marrow volume and the number of CD34+ cells per kilogram of body weight in the first leukapheresis product showed no correlation. However, the time between irradiation and mobilization seemed to influence the yield of CD34+ cells. A comparison of irradiated patients with nonirradiated patients revealed no differences with respect to the CD34+ cell counts. We did not find a significant influence of the extent or the total dose of irradiation on the yield of CD34+ cells in the first leukapheresis product in patients with multiple myeloma. However, there may be an inverse correlation between the time elapsed since the last irradiation and the number of mobilized CD34+ cells.

BACKGROUND:Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induced in vitro differentiation of primary acute myeloid leukemia (AML) blasts, an effect enhanced by all-trans retinoic acid (ATRA). Clinical responses to VPA were recently observed in patients with myelodysplastic syndrome (MDS). Herein, the authors have described results of a clinical trial with VPA plus ATRA in 26 patients with poor-risk AML. METHODS:VPA (5-10 mg/kg starting dose) and ATRA (45 mg/m(2)) were administered orally. Low-dose AraC or hydroxyurea were permitted to control leukocytosis. Biologic activity of VPA was confirmed by serial analysis of HDAC2 protein levels in peripheral blood (PB) mononuclear cells. RESULTS:Nineteen of 26 patients completed at least 4 weeks of VPA/ATRA treatment; 7 patients were withdrawn prematurely because of rapidly progressive disease (n = 3) or unacceptable neurologic and cardiovascular toxicity (n = 4). Additional cytoreductive treatment was required in 58% of patients enrolled. Median treatment duration was 3 months. No patient achieved complete remission, one with de novo AML had a minor response, and two patients with secondary AML arising from myeloproliferative disorder (MPD) achieved a partial remission and clearance of PB blasts, respectively. The latter responses were accompanied by profound granulocytosis and erythrocytosis in both patients, reminiscent of the response pattern known from ATRA treatment of acute promyelocytic leukemia. However, cytogenetic analysis of isolated CD34(+) cells and granulocytes did not reveal terminal differentiation of leukemic blasts. CONCLUSIONS:Treatment with VPA/ATRA results in transient disease control in a subset of patients with AML that has evolved from a myeloproliferative disorder but not in patients with a primary or MDS-related AML.

A 19-year-old girl presented with a septical condition with fever of 40 degrees C, swelling of the right sternocleidomastoid region and abdominal pain. Except for a sore throat with pain strictly localized to the right side of her neck and fever over the last week there were no other clues in her past medical history. An abdominal ultrasound and MRT scan as well as a chest X-ray only showed non-specific findings. The diagnosis of Lemierre's syndrome was established by ultrasonographical detection of right jugular venous thrombosis and perivascular inflammation together with blood cultures positive for Fusobacteria. The patient recovered within days after treatment was initiated with metronidazol according to the antibiogram. Lemierre's syndrome is a life-threatening disease especially in an age group which is less frequently affected by septicaemia. A history of sore throat, Fusobacterium positive blood cultures and ultrasonographical detection of jugular venous thrombophlebitis together with the knowledge of the "forgotten disease" will lead the way to the diagnosis.

1. Klinik für Innere Medizin mit SP Hämatologie, Onkologie und Immunologie, PhilippsUniversität Marburg, Baldinger Strasse, 35043 Marburg, Germany 2. Institut für Medizinische Biometrie und Epidemiologie; Philipps-Universität, Bunsenstraße 3, 35037 Marburg, Germany 3. Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-MannkopffStr. 2, 35037 Marburg, Germany 4. Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus, Fetscherstr. 74, 01307 Dresden, Germany 5. Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-5222, USA

Reports on the prognostic impact of mutations in the RAS proto-oncogenes in patients with acute myeloid leukemia (AML) are conflicting. A peptide nucleic acid (PNA)-based technique was used on 232 AML samples to detect point mutations of the hotspots in N-RAS and K-RAS. No significant correlations between RAS mutations and clinical features, karyotype or FLT3 were found.

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with −7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI , a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with −7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RAR α signaling in AML. Ski was co-immunoprecipitated and colocalized with RAR α . We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RAR α signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RAR α signaling.

BCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFN gamma-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors.

Constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (TKD), has been described in patients with acute myelogenous leukemia (AML). We analyzed the prevalence and the potential prognostic impact of FLT3 mutations in 979 AML patients. Results were correlated with cytogenetic data and the clinical response. FLT3-ITD mutations were found in 20.4% and FLT3-TKD mutations in 7.7% of the patients. Each mutation was associated with similar clinical characteristics and was more prevalent in patients with normal karyotype. Significantly more FLT3 aberrations were found in patients with FAB M5, and fewer were found in patients with FAB M2 and M6. Although less frequent in patients with cytogenetic aberrations, FLT3-ITDs were found in 13 of 42 patients with t(15;17) and in 9 of 10 patients with t(6;9). The prevalence of the ITD allele on the DNA level was heterogeneous, ranging from faint mutant bands in some patients to predominant mutant bands in others. Based on quantitative analysis, the mutant-wild-type (wt) ratio ranged from 0.03 to 32.56 (median, 0.78). Patients with a high mutant/wt ratio (ie, greater than 0.78) had significantly shorter overall and disease-free survival, whereas survival in patients with ratios below 0.78 did not differ from those without FLT3 aberrations. Multivariate analysis confirmed a high mutant/wt ratio to be a strong independent prognostic factor. Taken together, these data confirm that FLT mutations represent a common alteration in adult AML. Constitutive activation may be associated with monocytoid differentiation. A high mutant/wt ratio in ITD-positive patients appears to have a major impact on the prognostic relevance.

Mutations in ras genes have been found to be the most frequent genetic aberrations in adult myeloid leukaemia (AML). Some reports have shown an improved outcome of ras-mutated AML. In order to understand the biology of ras mutation in AML, we studied a cohort of patients treated in a prospective multicentre trial for ras mutational status and resistance gene expression. Blast samples from 162 adult patients with de novo or secondary AML were examined for resistance gene expression (mdr1, mrp1 and lrp) and ras mutations using reverse transcription-polymerase chain reaction and protein nucleic acid-competitive polymerase chain reaction strategies respectively. Ras mutations were confirmed using DNA sequencing. Ras mutations leading to an exchange of amino acids were found in 40 (25%) patients. Thirty AML patients had N-ras mutations and nine patients had K-ras mutations. One patient showed both N-ras and K-ras mutations. Resistance gene expression was positive for mdr1 in 30%, for mrp1 in 43% and for lrp in 62% of patients. There was a strong inverse correlation between the presence of ras mutation and mdr1 expression (P = 0.005). However, no significant difference was seen between patients with or without ras mutations and mrp1 or lrp expression. Whereas mdr1 expression was associated with a lower complete remission rate (P < 0.04), ras mutations had no significant influence on remission status. Neither ras mutation nor mdr1 expression had a significant impact on overall or disease-free survival to date. For the first time, there is evidence that activated ras genes are associated with lower mdr1 expression in AML.

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.

The normal human stomach is devoid of any organized lymphatic tissue. Acquisition of mucosa-associated lymphatoid tissue (MALT) in the stomach is considered to be a direct consequence of chronic infection with Helicobacter pylori. Thus, MALT appears to be part of the host defense against the pathogen H. pylori. Consequently, lymphomas arising from gastric MALT may be seen as an end point of a clonal evolution starting from the infection. Cumulative data from several studies show that eradication of H. pylori induces complete histologic remissions in about 70%–80% of the patients. Here we present data of an extended analysis of an ongoing multicenter trial. Eightyfour patients with low-grade gastric MALT lymphoma in stage EI were treated using a dual regimen to eradicate H. pylori. Complete remission was observed in 68 (81%) patients; a partial remission was found in seven (8%) patients. In contrast, nine (11%) patients revealed “no change” and were referred for alternative treatment strategies. The majority of these cases were found to harbor high-grade lymphomas in deeper mucosal areas. Polymerase chain reaction (PCR) performed on the VDJ rearrangements of the immunoglobulin heavy chain yielded monoclonal bands in 50 of 65 analyzed patients (77%) at diagnosis. Interestingly, in patients analyzed during follow up after achieving complete histologic remission, ongoing PCR monoclonality was found in 19 of 39 eligible patients (49%). Several patients who developed local relapse of the lymphoma were found in the group with ongoing PCR monoclonality. Together with data from the literature, these results suggest that the majority of low-grade gastric MALT lymphomas in stage EI respond to eradication of H. pylori. Longer follow-up investigations are necessary to determine whether remissions really indicate a cure from the disease and to elucidate whether PCR monoclonality after complete histological remission is predictive of increased relapse rate.

We investigated the ability of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts to differentiate into dendritic cells (DC) in vitro. Cytokine-supplemented suspension cultures of leukemic blasts in 98 patients with AML and five patients with ALL (normal karyotype, n = 2; BCR/ABL, n = 3) were performed. Mononuclear cells out of peripheral blood or bone marrow containing between 60% and 90% leukemic blasts were cultured for eight days using different growth factor combinations. The highest yield of CD1a(+)/CD14(-) cells could be obtained with stem cell factor, transforming growth factor-beta, tumor necrosis factor-alpha, GM-CSF, and FLT-3-ligand. In the AML samples the median content of CD1a(+)/CD14(-) cells after eight days of culture was 3.5% (r = 0%-82%). In five informed patients CD1a(+)/CD14(-) cells were sorted by fluorescence-activated cell sorting or immunomagnetic separation. Cytogenetic and polymerase chain reaction analyses showed known primary chromosomal aberrations (monosomy 7 and inversion 16) in the sorted fractions, respectively. Dendritic cells (DC) could be generated out of leukemic blasts in 68% of AML patients. Leukemic DC showed no phagocytosis of latex beads, but stimulated allogeneic naive cord blood-derived T cells more efficiently than did uncultured blasts. In ALL patients the median percentage of CD1a(+)/CD14(-) cells was 1.2% (r = 0.7%-3.8%) after culture. The sorted CD1(+)/CD14(-) fractions were BCR/ABL-negative when analyzed with fluorescence in situ hybridization, indicating their nonleukemic origin. Leukemic DC can be generated out of leukemic progenitors in patients with AML. These cells might become relevant for autologous and allogeneic immunotherapy in selected patients. BCR/ABL-positive lymphoblasts could not be transformed into cells with an early dendritic phenotype with the cytokines used in our experiments.

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.