Progressive Genomic Instability in the Nup98-HoxD13 Model of MDS Correlates with Loss of the PIG-A Gene Product

The Nup98-HoxD13 (NHD13) fusion gene was identified in a patient with therapy-related myelodysplastic syndrome (MDS). When transgenically expressed in hematopoietic cells, mice faithfully recapitulate human disease with serial progression from peripheral blood (PB) cytopenias and increased bone marrow (BM) blasts to acute leukemia. It is well accepted that genomic instability in dysplastic hematopoietic stem/progenitor cells (HSPC) drives the evolution of MDS to acute leukemia. Findings here demonstrate that reticulocytes, myeloid and lymphoid PB cells of NHD13 mice, display an increase in the age-associated loss of glycosylphosphatidylinositol-linked surface proteins versus wild type controls. These data correlate with a progressive increase in the DNA damage response as measured by γ-H2AX activity, accumulating BM blasts as the disease progresses and finally development of acute leukemia. These findings clearly demonstrate a state of progressive genomic instability that increases the likelihood of a “second hit” or complimentary mutation later in the disease to trigger development of acute leukemia and underscores the mechanistic nature of how the NUP98-HoxD13 transgene induces progression of MDS to acute leukemia. Additionally, these data support the use of the PIG-A assay as an efficient, real-time surrogate marker of the genomic instability that occurs in the MDS HSPCs.Key PointThe PIG-A assay is a sensitive, nonlethal method for the serial assessment of genomic instability in mouse models of MDS.     

Telomerase activity in myelodysplastic syndromes

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Telomeres are thought to be critical in maintaining normal hematopoiesis. In this study, we assessed telomere dynamics in order to obtain further insight into the pathogenesis of MDS. We studied telomerase activity (TA) in mononuclear cells from peripheral blood (PB) and bone marrow (BM) from patients with myelodysplastic syndrome (MDS; n=24), acute myeloid leukemia (AML; n=14), chronic myeloid leukemia (CML; n=12) and 11 normal controls using a polymerase chain reaction-based telomeric repeat amplification assay. Telomerase activities (mean+/-S.D.) were found as 0.199+/-0.09, 0.414+/-0.55, 0.253+/-0.26 and 0.181+/-0.05 pg/ml in PB mononuclear cells, respectively (P>0.05). Comparison of TA of BM mononuclear cells from 19 MDS patients versus 10 BM samples from normal controls revealed no significant difference (P=0.3). There was no correlation between the levels of TA and clinical and prognostic parameters of the patients with MDS, such as degree of anemia, platelet counts on presentation, gender, presence of organomegaly, bone marrow fibrosis and BM blast percentages. Patients who had higher TA had significantly inferior survival compared with patients who had lower TA (P=0.005). Consistent with previous data, our results suggest that in patients with MDS, telomerase activity might be insufficient to compensate for the telomere shortening. Furthermore, TA might be prognostically important in patients with MDS. Measurements of enzymatic activity in association with telomere length studies may help to understand the prognostic role of telomere dynamics in patients with myelodysplastic syndromes more reliably.     

Comparative analysis of the in vitro proliferation and expansion of hematopoietic progenitors from patients with aplastic anemia and myelodysplasia

Aplastic anemia (AA) and myelodysplasia (MDS) show great similarities in their biology. To date, however, it is still unclear to what extent hematopoietic progenitor cells (HPCs) from AA and MDS share biological properties and what the functional differences are between them. In trying to address this issue, in the present study we have analyzed, in a comparative manner, the proliferation and expansion capacities of bone marrow (BM) progenitor cells from AA and MDS in response to recombinant cytokines. BM samples from normal subjects (NBM) and patients with AA and MDS were enriched for HPC by immunomagnetic-based negative selection. Selected cells were cultured in the absence (control) or in the presence of early-acting cytokines (Mix I), or early-, intermediate- and late-acting cytokines (Mix II). Proliferation and expansion were assessed periodically. In NBM and MDS cultures apoptosis was also determined. In NBM cultures, Mix I induced a nine-fold increase in total cell numbers and a 3.6-fold increase in colony-forming cell (CFC) numbers. In Mix II-supplemented cultures, total cells were increased 643-fold, and CFC 12.4-fold. In AA cultures, no proliferation or expansion were observed in Mix I-supplemented cultures, whereas only a four-fold increase in total cell numbers was observed in the presence of Mix II. In MDS cultures, a 12-fold increase in total cells and a 2.9-fold increase in CFC were observed in the presence of Mix I; on the other hand, Mix II induced a 224-fold increase in total cells and a 5.9-fold increase in CFC. Apoptosis was reduced in cytokine-supplemented cultures from NBM. In contrast, Mix II induced a significant increase in the rate of apoptosis in MDS cultures. Our results demonstrate that, as compared to their normal counterparts, AA and MDS progenitors are deficient in their proliferation and expansion potentials. Such a deficiency is clearly more pronounced in AA cells, which seem to be unable to respond to several cytokines. MDS progenitors, on the other hand, are capable to proliferate and expand in response to cytokines; however, their rate of apoptosis is increased by intermediate- and late-acting cytokines, so that the overall proliferation and expansion are significantly lower than those of normal progenitor cells.     

Will a peripheral blood (PB) sample yield the same diagnostic and prognostic cytogenetic data as the concomitant bone marrow (BM) in myelodysplasia?

In patients with myelodysplastic syndromes (MDS), chromosome anomalies are detected by conventional cytogenetic studies (CCS) and/or interphase fluorescence in situ hybridization (FISH) of bone marrow (BM) samples and provide prognostic and diagnostic information, which can direct therapy. Whether peripheral blood (PB) can be substituted for bone marrow in these cases and can provide the same information remains unknown. Concurrent BM and PB specimens collected from 100 patients with recently diagnosed MDS were studied using both CCS and FISH. While 68% of BM samples showed an abnormal karyotype by CCS, only 31% of PB samples were abnormal by CCS. In 12% of patients, FISH and CCS were discordant due to the inability of the FISH panel to detect all possible abnormalities. However, only one case (1%) had a cryptic abnormality detected by FISH. BM and PB FISH were discordant in 3% of cases, most likely due to the smaller clone size in PB vs. BM. While PB should not be substituted for BM at diagnosis, it is a viable alternative for monitoring patients using the appropriate FISH probe(s).     

Expression of CDKN1C in the bone marrow of patients with myelodysplastic syndrome and secondary acute myeloid leukemia is associated with poor survival after conventional chemotherapy

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34⁺ cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin‐dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p < 0.001). In multivariable analysis, higher CDKN1C expression was associated with worse OS (p = 0.02). On the BM level, a total of 84 (38%) patients showed CDKN1C protein expression before start of treatment. Patient, disease and treatment characteristics did not differ between CDKN1C‐positive and ‐negative patients. Positive CDKN1C BM status was associated with shorter OS in multivariable analysis (HR 1.54, p = 0.04). There was an interaction between CDKN1C BM status and subsequent treatment with negative impact on OS being most pronounced in patients receiving conventional cytotoxic chemotherapy (n = 83, 2‐year OS 30% versus 58%, p = 0.002). In conclusion, low‐proliferative phenotype and CDKN1C expression were associated with shorter OS. CDKN1C protein expression in the BM of newly diagnosed, treatment‐naïve MDS and secondary AML patients was identified as a prognostic factor for poor survival in patients treated with antiproliferative chemotherapy.     

Having a higher blast percentage in circulation than bone marrow: clinical implications in myelodysplastic syndrome and acute lymphoid and myeloid leukemias.

Determining the percentage of peripheral blood (PB) and bone marrow (BM) blasts is important for diagnosing and classifying acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Although most patients with acute leukemia or MDS have a higher percentage of BM blasts than PB blasts, the relative proportion is reversed in some patients. We explored the clinical relevance of this phenomenon in MDS (n = 446), AML (n = 1314), and acute lymphoblastic leukemia (ALL) (n = 385). Among patients with MDS or ALL, but not AML, having a higher blast percentage in PB than in BM was associated with significantly shorter survival. In multivariate analyses, these associations were independent of other relevant predictors, including cytogenetic status. Our findings suggest that MDS and ALL patients who have a higher percentage of PB blasts than BM blasts have more aggressive disease. These data also suggest that MDS classification schemes should take into account the percentage of blasts in PB differently from the percentage of blasts in BM.     

The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.     

Re-evaluation of refractory anemia with excess blasts in transformation

The category 'refractory anemia with excess blasts in transformation (RAEBt)' consists of two sub-sets; one group is categorized based on the percentage of blasts in the marrow (> or =20%) and other is based on the percentage of blasts in the peripheral blood (> or =5%). We separated RAEBt patients based on these two criteria and compared hematologic and clinical relevance to assess the reasonable basis for the new classification. All RAEBt patients showing peripheral blood (PB) blasts of > or =5% were re-classified as RAEB by the WHO classification. This subset of RAEBt patients had lower percentages of bone marrow (BM) blasts, and notably they showed frequent complex cytogenetic abnormalities, including -5/5q- and/or -7/7q-. Moreover, the RAEBt patients of this group had shorter survivals compared to RAEBt patients with BM blasts between 20 and 30%. We next assessed hematologic and clinical relevance between refractory anemia with excess blasts (RAEB) and RAEBt patients with PB blasts of > or =5%. Except for the percentage of blasts in the PB (P=0.0037) and BM (P=0.0073), there was no significant difference in hematologic or clinical features between RAEB patients with BM blasts of > or =11% and RAEBt patients with PB blasts of > or =5%. When MDS patients with PB blasts of > or =5% (RAEBt by the FAB classification) were included as RAEB-II based on the "MDS 2000 classification', there was a high frequency of patients with complex chromosome changes, involving 5q and 7q, with significant poorer outcome compared to those with RAEB-I. Although it is still controversial whether MDS patients with BM blasts 20% or more should be considered as acute leukemia, the utilization of the 'MDS 2000 classification' might be useful to designate MDS patients diagnosed based on the percentage of blasts in the peripheral blood.     

Peripheral blood cytogenetic studies in myelofibrosis: overall yield and comparison with bone marrow cytogenetic studies

Among 59 consecutive patients with myelofibrosis (MF) in whom peripheral blood (PB) cytogenetic studies were performed, at least two analyzable metaphases (median 20, range 2-31) were obtained in 49 (81%) patients and in all 37 (100%) cases with PB myeloid progenitor cell count of 0.1 x 10(9)L(-1) or above (p=0.02). Twenty-two patients had concomitant PB and bone marrow (BM) cytogenetic studies; 6 showed similarly abnormal findings in both BM and PB. In another 2 cases, results were abnormal in BM but normal in PB; the opposite was seen in 1 case. These results suggest that PB can be considered as an alternative to BM for cytogenetic studies as currently used in MF but additional prospective studies are needed to support change in practice.     

In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.     

CD14+ peripheral blood mononuclear cells from chronic myeloid leukemia and normal donors are inhibitory to short- and long-term cultured colony-forming cells

Monocyte-induced cell-cytotoxicity has been implicated in the mechanism of suppression of normal haematopoietic progenitors in chronic myeloid leukemia (CML). We examined here the in vitro effect of CML-derived and normal peripheral blood (PB) monocytes on short- and long-term cultured haematopoietic progenitor cells. Short-term coculture (5 days) of CML or normal monocytes with CML or normal peripheral blood mononuclear cells (PBMNC)/CD34+ cells as targets resulted in a significant inhibition of colony-forming cell (CFC) growth. Coculture conditioned medium (CCM) from 5-days cocultures of normal or CML CD14+ monocytes with CD34+ cells were likewise inhibitory to CFC. In 5-week long-term cocultures of monocytes in direct contact with normal bone marrow (BM) progenitors, CML monocytes reduced the proportion of long-term cultured CFC (LTC-CFC) significantly to 52% of the controls, while normal monocytes had a less pronounced inhibitory effect (89% of the controls) on LTC-CFC. Reduction of LTC-CFC was great when CML monocytes and target cells were separated by a transwell membrane as compared to control cultures in the absence of CD14+ cells (53.5 vs. 9%). CCM from 5-week cocultures of normal or CML CD14+ monocytes with CD34+ progenitors from bone marrow (BM) cells were also inhibitory to CFC. No difference in cytokine levels for TNF-alpha, IFN-gamma, G-CSF, IL-10, IL-6 was detectable between CML CD14+ CCM and control CCM derived from short- and long-term cocultures. Our results suggest that CML monocytes may play a role in the inhibition of normal haematopoiesis through a yet not defined soluble factor supporting the expansion of the malignant clone in CML.     

The biological behavior of SDF-1/CXCR4 in patients with myelodysplastic syndrome

The purpose of this investigation is to evaluate the biological behavior of stromal cell-derived factor-l (SDF-1) in migration, adhesion, and apoptosis as well as the related signaling transduction pathways in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We chose 22 patients with MDS, 7 patients with de novo AML, and 8 patients with non-clonal cytopenia diseases. We performed flow cytometric analysis of CD34⁺ cells apoptosis using annexinV-FITC, which binds to exposed phosphatidylserine on apoptotic cells. The cell adhesion capability was detected by CCK-8 assay. The migration ability of the cell was checked by transwell assay. Furthermore, we measured SDF-1 levels in BM plasma from patients by enzyme-linked immunosorbent assay (ELISA). Our results indicated that the apoptosis of CD34⁺ cell was significantly increased in the Low-grade MDS (IPSS score ≤ 1.0) patients compared with the high-grade MDS (IPSS score ≥ 1.5) (21.33% vs. 7.27%, P < 0.001) and patients with de novo AML (21.33% vs. 7.53%, P < 0.001). SDF-1 promoted CXCR4 high expression cells adhesion to the stroma cells (MSC) and induced these cells migration. SDF-1 could trigger the occurrence of polarized morphology of the cells that expressed CXCR4 high. After addition of wortmannin or PTX, the ability of adhesion and migration of the cells that expressed CXCR4 high decreased. But in the patient’s cells that expressed CXCR4 low, there was no above-mentioned phenomenon. So we can suppose that the signaling pathway of SDF-1/CXCR4 axis is PI3K pathway, and we should do more things about this pathway and may find out the target treatment of MDS.     

Myelodysplastic syndrome: a search for minimal diagnostic criteria

We have evaluated dyshemopoietic features in bone marrow (BM) samples obtained from healthy people aged over 50 without peripheral blood (PB) cytopenia patients and compared them with MDS patients. Control group displayed BM features of dyserythropoiesis and dysgranulopoiesis in up to 15 and 27% of the considered cell elements (P90) respectively, overlapping in part with MDS patients. Interobserver agreement in dyshemopoietic features was highest for BM blast cell and pathological sideroblast counts. An algorithm based on BM blast cell and pathological sideroblast counts that has been verified on 613 patients from different Spanish centers may be of help to improve reproducibility in Myelodysplastic syndrome (MDS) diagnosis.     

Correlation of tumor necrosis factor α (TNFα) with high Caspase 3-like activity in myelodysplastic syndromes

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor α (TNFα) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNFα signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNFα in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8±13.9 at 0 h vs. 177.8±55.2 units/mg protein at 4 h, n=14, P=0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76%±0.25% vs. 3.99%±1.1%, n=14, P=0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNFα levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (ρ=0.517, n=13, P=0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (ρ=0.593, n=13, P=0.033). Additionally when TNFα-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNFα-positive marrows (189.6±66.2 vs. 25.0±14.6 units/mg protein, respectively, P=0.043). The increase in AI%, though not statistically significant, was also higher in the TNFα-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNFα-induced apoptosis were evaluated. TNFα treatment significantly increased AI% (P<0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 μM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 μM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 μM/l) as well as PTX (250 μM/l) significantly curtailed the AI% induced by TNFα The present studies thus identify the downstream effectors of TNFα-inducible apoptosis in MDS and so also the suppressors of TNFα apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.     

Peripheral blood cell‐free DNA is an alternative tumor DNA source reflecting disease status in myelodysplastic syndromes

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell‐free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma‐PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher‐risk groups than that in lower‐risk groups (P = 0.034). The concentration of plasma‐/serum‐PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160‐bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM‐DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB‐2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.     

Proliferating status of peripheral blood progenitor cells from patients with BCR/ABL-positive chronic myelogenous leukemia.

To investigate the mechanisms behind the leukemic expansion of BCR/ABL-positive chronic myelogenous leukemia (CML), we examined the cell cycle status of hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) of 37 patients with newly diagnosed BCR/ABL-positive CML. We found a high proportion of 12.51 +/- 1.19% of CD34+ peripheral blood progenitor cells (PBPC) in S/G2M phase. Comparison of PB and BM from 19 cases revealed similar proliferation rates (10.74 +/- 1.41% vs 15.97 +/- 1.95%). Furthermore, even primitive CD34+/CD38- PBPC displayed high proliferation rates (17.45 +/- 2.98%) in 10 cases examined. In contrast, PBPC from 11 patients with BCR/ABL-negative myeloproliferative disorders were almost noncycling (S/G2M 1.46 +/- 0.47%). When matched pairs of PB and BM from six patients with BCR/ABL-negative myeloproliferative disorders were examined, only 0.89 +/- 0.41% of the CD34+ PBPC, but 8.29 +/- 3.13% CD34+ cells from BM were in S/G2M phase. Consistently, as compared to 19 patients with newly diagnosed BCR/ABL-positive CML, a significantly lower PB/BM ratio of CD34+ cells in S/G2M phase was found in these six patients with BCR/ABL-negative myeloprolifrative disorders. Administration of the tyrosine kinase inhibitor STI571 to 13 patients with CML in chronic phase, accelerated phase, or blast crisis lead to an inhibition of PBPC proliferation within a few days. Interestingly, CD34+ hematopoietic progenitor cells from BM remained proliferating in five cases examined, indicating that CML PBPC are more easily inhibited by STI571 as compared to CD34+ CML hematopoietic progenitor cells from BM. These data suggest that BCR/ABL leads to an enhanced cell cycle activation of CD34+ cells, which seems to be, at least in part, independent of additional factors provided by the bone marrow microenvironment.     

Morphologic, immunophenotypic and in vitro growth characteristics of blood and bone marrow associated with stem cell mobilisation in patients with lymphoma

The proportion of CD34+ cells in the bone marrow (BM) is predictive of the size of progenitor cell mobilisation into the blood (PB). To investigate which other PB and BM parameters may be related to mobilisation, we analysed at steady state PB and BM of 23 patients with relapsed or resistant lymphoma before administering high-dose cyclophosphamide and G-CSF Cell morphology, number of CD34+ cells, and growth in clonogenic assay and in long-term cultures (LTC) were determined and then correlated with mobilisation extent (CD34+ and GM-CFC) and quality (growth of harvested cells in LTC). We found that the good mobilising patients (CD34 > 50 x 10(3)/ml, n=10) had several baseline BM characteristics (number of CD34+ MNC, GM-CFC, BFU-E, production of CFCs in LTC) similar to a group of 12 healthy controls, while patients with reduced mobilisation (CD34 < 50 x 10(3)/ml, n=13) had clearly reduced BM progenitors and LTC growth (p< 0.05). In a multivariate analysis including baseline clinical, blood and bone marrow characteristics, the most significant PB and BM factors independently associated with a higher number and/or quality of mobilised cells were a higher number of CD34+ and GM-CFC in the BM and a higher baseline haemoglobin, platelet, and CD34+ blood count. The capacity to release progenitor cells into the circulation is therefore not predicted by the distribution of morphologically distinguishable cells, marginally predicted by the BM content of highly undifferentiated cells (growth in long term culture), while it is proportional to the number of BM progenitors (CD34+, GM-CFC and BFU-E).     

Impact of transfusion dependency on survival in patients with early myelodysplastic syndrome without excess of blasts

We present a retrospective analysis of 137 patients with early MDS without excess of blasts that revealed transfusion dependency in 87% of the cases. A significant difference in overall survival was noted between patients receiving ≤2 units and those receiving >2 units of RBC transfusions/month (65.0 vs. 35.3 months, respectively, P = 0.02). Univariate statistical analysis identified the presence of disease progression to advanced MDS (χ² = 26.4, P = 0.001) and the administration of >1 or >2 units of RBC per month (χ² = 15.9 and 14.6, respectively, P = 0.001) as the most important parameters affecting survival. Nevertheless, even the administration of 1 RBC unit every 4–8 weeks had a significantly adverse impact on survival compared to non-transfused patients. Transfusion dependency itself did not affect disease progression as determined by the presence of multilineage dysplasia and adverse karyotype (expressed by the IM-1 or IM-2 score). Multivariate analysis confirmed disease progression towards leukemia as a highly significant independent variable affecting survival (P = 0.0001). None of the other evaluated parameters had a significant impact on survival in patients with progressive disease. In non-transplanted patients without MDS progression, administration of >2 units of RBC transfusions/month was the only independent variable with adverse impact on survival in patients with unilineage erythroid dysplasia (P = 0.02). In patients with multilineage dysplasia, only heavy transfusion dependency (>3 TU RBC/month) and serum ferritin >2000 μg/l adversely affected survival (P = 0.03). Modification of the WPSS by replacing transfusion dependency with initial Hb level <80 g/l retained its prognostic relevance and allowed the identification of a potential risk subset of early MDS patients with intermediate and high scores and limited survival (<40% at 5 years) as early as at the time of diagnosis. Our results confirm a significant negative impact of transfusion dependency on survival in patients with early MDS without excess of blasts. The main risk subgroup is characterized by unilineage dysplasia limited to erythropoiesis in combination with dependency on >2 TU of RBC per month. These patients usually have prolonged survival that leads to the development of heavy transfusion iron overload and they thus represent the most important target group for intensive chelation therapy.     

Acute myeloid leukemia and myelodysplastic syndrome following breast cancer: increased frequency of other cancers and of cancers in multiple family members

Adjuvant chemotherapy and radiation therapy for breast cancer are associated with therapy-related acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS), but little is known about additional risk factors. Thirty-four patients with AML (n = 26)/MDS (n = 8) following breast cancer (cases) were compared with 2029 breast cancer patients without AML/MDS (controls). Cases were older at breast cancer diagnosis (mean 60.2 years versus 54.5 years; p = 0.01) and more commonly had additional cancers (29% versus 4.9%; p < 0.0001) and ≥4 first-degree relatives with any type of cancer (OR: 5.37, CI: 1.44–19.9). Thus risk factors for AML/MDS following breast cancer include older age, other cancers and multiple first-degree relatives with cancer.