Activity of the caspase-3/CPP32 enzyme is increased in "early stage" myelodysplastic syndromes with excessive apoptosis, but caspase inhibition does not enhance colony formation in vitro.

OBJECTIVE: Excessive apoptosis may have a role in the ineffective hematopoiesis and cytopenias observed in myelodysplastic syndromes. The goals of this study were 1) to quantify apoptosis in patients with "early stage" myelodysplasia [including patients with refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts and with less than 10% blasts (RAEB(<10))], and in patients with "late stage" myelodysplasia [including RAEB with more than 10% blasts (RAEB(>10)), RAEB in transformation (RAEB-t), and acute myeloid leukemia secondary to myelodysplasia (LAM2)]; 2) to study the activation of the caspase-3/CPP32 enzyme, a major "effector" caspase in hematopoiesis, in patients with "early stage" myelodysplasia, and 3) to evaluate the effect of caspase inhibition on the apoptotic phenotype and clonogenicity of hematopoietic progenitors in vitro in these patients. PATIENTS: Fifty-four patients with myelodysplastic syndromes, including 30 with "early stage" myelodysplasia and 24 with "late stage" myelodysplasia were studied. Study of apoptosis: TUNEL assay performed on bone marrow smears and/or quantification of annexin V positive bone marrow mononuclear cells by flow cytometric analysis. Caspacse-3/CPP32 activity: Quantitative measurement of caspase-3/CPP32 activity on total bone marrow mononuclear cells using a fluorogenic substrate. Effect of the caspase-inhibitor Z-VAD-FMK: 1) on the apoptotic phenotype of total bone marrow mononuclear cells and 2) on the clonogenicity of hematopoietic progenitor cells. RESULTS: The group of 30 patients with "early stage" myelodysplasia had statistically increased apoptosis compared to the group of 24 patients with "late stage" myelodysplasia (44.1% +/- 4.8 vs 21.8% +/- 3.6; p = 0.02) using the TDT-mediated dUTP nick-end labeling (TUNEL) assay. In the group of patients with RAEB, those with MDS(RAEB<10) had excessive apoptosis compared to those with MDS(RAEB>10) (44.0% +/- 3.5% vs 29.5% +/- 3.6%;p = 0.042) The median caspase-3 activity in 20 "early stage" myelodysplasia patients was 19,000 U (range 3,460-41,000) and significantly increased compared to normal individuals (4,256 U, range 3,200-5,200; p = 0.032) Bone marrow mononuclear cells from 12 "early stage" MDS patients (including 11 from the 20 studied for caspase-3 activity) were incubated with or without the broad-spectrum caspase inhibitor Z-VAD-FMK. In 4 of 9 evaluable patients (44.4%) with excessive apoptosis, the number of annexin V positive cells decreased in a dose-dependent manner in the presence of Z-VAD-FMK. However, in none of these patients was caspase inhibition with Z-VAD-FMK able to enhance colony formation in vitro. CONCLUSION: These results confirm that a major characteristic of patients with "early stage" myelodysplasia is increased apoptosis. The results also indicate that excessive apoptosis in these patients is accompanied by increased caspase-3/CPP32 activity. However, caspase inhibition with the broad-spectrum inhibitor Z-VAD-FMK cannot improve hematopoiesis in this group of patients, even when apoptosis is attenuated.

Preliminary evaluation of the new hematology analyzer COULTER GEN-S in a university hospital.

The COULTER GEN-S system (COULTER Corp, Miami, USA) is an automated hematology instrument that is designed to provide a complete hematological profile including white blood cells (WBC), complete blood count (CBC) differential count (diff) and the reticulocyte parameters. It was evaluated in our laboratory over a one month period. A preliminary study was performed using the GEN-S software revision 1D. The evaluation had two purposes: 1) evaluation of the GEN-S specifications; 2) comparison of its analytical performance with the hematology analyzer currently used in our laboratory. The first part of the evaluation showed that the COULTER GEN-S is reproducible, has linearity beyond the specifications given by the manufacturer and produces stable results up to 48 hours after blood collection. The evaluation of analytical performance included: 1) a comparison between the GEN-S CBC and diff numerical results and the COULTER STKS (COULTER Corp, Miami, USA). These comparisons showed that the results given by the two methods are similar and suggested that the COULTER GEN-S could replace the current hematology instrument in use in our laboratory; 2) a performance analysis, to measure the system's ability to detect morphologic abnormalities, when compared to the reference blood smear examination. This part of the evaluation was performed on both normal and abnormal samples. The results of this analysis showed that the sensitivity of the GEN-S was excellent, especially regarding blast cells, immature granulocytes, nucleated red blood cells (NRBCs) and platelet clumps when using the complete suspect flagging system of the instrument. In addition, the use of the review criteria in our laboratory allowed to detect all hematological diseases. The overall false negative rate was 0.9%. Thus we consider that the COULTER GEN-S system is suited for use in medium-to large hospital laboratories which perform more than 100 CBC/day. Overall, the instrument had excellent performance, with a throughput of more than 100 samples/h. Its user friendly workstation has complete patient data management, which is in compliance with good laboratory practices in France.

Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.

In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34(+) hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 micromol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34(+) and CD41(+)CD34(+) cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.

Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction.

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)-induced BFU-E and CFU-E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo-R in MDS marrow, we focused on Epo-induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro-apoptotic signals rather than the defect of anti-apoptotic pathways resulting from Epo-stimulated Jak2-STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.

Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia.

Thrombocytopenia is a frequent feature of myelodysplastic syndromes (MDS) that could be improved by the use of recombinant human megakaryocyte growth and development factor (rHuMGDF). Using short-term liquid cultures and progenitor assays, we have found that rHuMGDF stimulated DNA synthesis and potentiated leukemic cluster growth of bone marrow mononuclear cells in 10/38 MDS cases (26%). Cytogenetically malignant colonies were detectable in rHuMGDF-stimulated cultures (n=3) by fluorescence in situ hybridization. rHuMGDF was able to stimulate CFU-MK formation in 45% of the samples tested. Finally, rHuMGDF-induced blast cell proliferation correlated with elevated expression of c-MPL, previously identified as a bad prognosis factor in MDS.

Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes.

Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% +/- 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% +/- 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias.

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15-47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.

Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics, morphology and immunophenotype.

The t(8;21) identifies a subgroup of acute myeloid leukaemia (AML) with a relatively good prognosis which may merit different treatment. It is associated predominantly, but not exclusively, with AML M2, and corresponds to rearrangements involving the AML1 and ETO genes. AML1-ETO positive, t(8;21) negative cases are well recognized but their incidence is unknown. In order to determine optimal prospective AML1-ETO RT-PCR screening strategies, we analysed 64 unselected AML M1 and M2 cases and correlated the results with other biological parameters. Molecular screening increased the overall detection rate from 8% to 14%. AML1-ETO was found in 3% (1/32) of AML M1 and 25% (8/32) of M2, including three patients without a classic (8;21) but with chromosome 8 abnormalities. It was more common in younger patients. Correlation with morphology enabled development of a scoring system which detected all nine AML1-ETO-positive cases with a false positive rate of 7% (4/55). Although certain AML1-ETO-positive cases demonstrated characteristic immunological features (CD19 and CD34 expression, CD33 negativity), each of these markers was insufficiently specific to permit prediction in an individual case. We conclude that initial routine prospective molecular screening for AML1-ETO in all AMLs, combined with standardized morphological and immunological analysis, is desirable in order to produce improved prognostic stratification and to determine whether screening can ultimately be restricted to appropriate subgroups.

Prognostic value of c-mpl expression in myelodysplastic syndromes.

The c-mpl proto-oncogene which encodes a member of the hematopoietic cytokine receptor superfamily has been recently shown to be the receptor for thrombopoietin (TPO), which stimulates megakaryocyte progenitor expansion and differentiation. We studied c-mpl expression by Northern blot analysis, in a large series of 58 MDS. No expression was found in 14 patients with refractory anemia (RA) or with refractory anemia with ring sideroblasts (RARS). In contrast 11/26 (42%) patients with refractory anemia with excess of blasts (RAEB), or with RAEB in transformation (RAEBt), and 8/18 (44%) patients with chronic myelomonocytic leukemia (CMML) expressed c-mpl. In CMML patients, no correlation was found between c-mpl expression and any prognostic factor tested, nor with the course of the disease. In contrast, in RAEB and RAEBt, expression of c-mpl was correlated with high Bournemouth scoring (P < 0.005) and poor survival (P = 0.02) due to leukemic transformation. Forty-five per cent (5/11) of the c-mpl positive patients evolved towards AML with a mean follow-up of 10.5 months, while 13% (2/15) of the c-mpl negative patients developed a secondary leukemia, with a mean follow-up of 21.1 months. Moreover, in RAEB and RAEBt, a significant correlation was observed between c-mpl, CD34, megakaryocyte glycoprotein IIb (GPIIb) expression, and the presence of dysmegakaryopoiesis. These results indicate that patients with RAEB and RAEBt, with high expression of the c-mpl, CD34, and GPIIb genes, may identify a subgroup of patients with particularly poor prognosis, due to an increased risk of secondary leukemia. More aggressive therapy could be justified in these patients.

Detection of the chromosome 16 CBF beta-MYH11 fusion transcript in myelomonocytic leukemias.

Karyotypic detection of chromosomal 16 abnormalities classically associated with AML M4Eo can be difficult. Characterization of the two genes involved in the inv(16)(p13q22), CBF beta and MYH11, has allowed the detection of fusion transcripts by reverse-transcriptase polymerase chain reaction (RT-PCR). We have analyzed CBF beta-MYH11 fusion transcripts by RT-PCR in myelomonocytic leukemias, with or without eosinophilia, to determine whether their presence correlates with morphology. Fifty-three cases (11 AML M4Eo; 1 AML M4 with atypical abnormal eosinophils (AML M4 "Eo"); 29 AML M4; 8 AML M5; 3 CMML; and 1 AML M2 with eosinophilia) were analyzed. All 11 typical AML M4Eo were CBF beta-MYH11 positive. The single case of AML M4 with distinctive eosinophil abnormalities was negative by karyotype, RT-PCR and fluorescent in situ hybridization (FISH). Three of 29 (10%) AML M4 without abnormal eosinophils were CBF beta-MYH11 positive, 1 of which did not show any apparent chromosome 16 abnormalities by classical metaphase analysis (2 not tested). Both cases tested also showed MYH11 genomic rearrangement. None of the other leukemias were RT-PCR positive. Follow-up of three patient showed residual positivity in apparent complete remission. These data show that CBF beta-MYH11 fusion transcripts occur not only in the vast majority of typical AML M4Eo, but also in approximately 10% of AML M4 without eosinophilic abnormalities, a much higher incidence than the sporadic reports of chromosome 16 abnormalities in AML M4 would suggest. Taken together with the detection of CBF beta-MYH11 transcripts in the absence of apparent chromosome 16 abnormalities by classical banding techniques, these data show that additional screening by either RT-PCR or FISH should be performed in all AML M4, regardless of morphologic features, to allow accurate evaluation of the prognostic importance of this fusion transcript.

Expression of the Evi-1 gene in myelodysplastic syndromes.

Increased expression of the proto-oncogene Evi-1 has been shown to block the in vitro granulocytic differentiation of myeloid cells in response to granulocytic colony-stimulating factor and to interfere with the proliferation of erythroid cells in response to erythropoietin. We determined the frequency of Evi-1 expression in myelodysplastic syndromes (MDS), a disorder with altered proliferation and differentiation in the erythroid and myeloid lineages. Twenty-one patients were studied. Abnormal expression was found in 1/9 patients with refractory anemia and in 7/12 patients with refractory anemia with excess of blasts (RAEB) or in transformation (RAEBt). No correlation could be found between expression of Evi-1 and age, sex, hemoglobin level and percentage of bone marrow blasts or erythroblasts. This result suggests that the high incidence of Evi-1 expression which remains at low levels in RAEB and RAEBt is not a major determinant of ineffective erythropoiesis and myelopoiesis in MDS.

High doses of intravenous recombinant erythropoietin for the treatment of anaemia in myelodysplastic syndrome.

We studied the efficacy of high doses (100,000 IU intravenously (IV)/twice a week) of human recombinant erythropoietin (rHuEpo) in patients with transfusion dependent myelodysplastic syndromes (MDS). Rationale for such dose of IV Epo was the poor in vitro response of MDS erythroid progenitors (CFU-E) to physiological concentrations of Epo, and the usual high endogenous serum Epo levels of MDS patients. Seventeen patients (nine males, eight females) were included, five refractory anaemia (RA), six RA with blasts excess (RAEB), five RA with ringed sideroblasts (RARS). Tolerance was good, except in three patients who experienced severe flu-like syndrome after Epo injection. None of the patients showed hypertension or developed anti rHuEpo antibodies. Three patients (17.6%) with RAEB had 35-60% reduction of transfusion requirements. No progression of disease occurred. Percentage of erythroblasts, endogenous baseline Epo level and in vitro cultures of erythroid progenitors did not correlate with response to Epo treatment. This study shows that very high IV doses induce only seldom and partial improvement in the status of transfusion dependent MDS. This rate of response, not higher than described with lower dosage, probably represents the maximum expectable response to rHuEpo in this category of patients.

Use of the new Coulter MAXM for leucocyte differentials.

The Coulter MAXM is an automated instrument designed to give a complete haematological profile including cell counts and WBC differential (DIFF). The WBC DIFF is determined by tridimensional flow cytometry on the basis of cell volume, light scattering and conductivity (VCS technology). Evaluation of the MAXM DIFF was performed by comparison with microscopic examination of blood smear. A total of 467 samples were studied by the MAXM and reference techniques, according to a standard protocol employed in our laboratory to define the criteria for smear examination. Good correlation was obtained between the two methods in non-flagged samples and those flagged only with an isolated "Imm Gran" message. In a group of 407 non-haematological and 60 haematological patients, there were 12 (2.6%) false positive and 5 (1.1%) false negative reports, all in non-haematological cases and the five false negative results corresponding to no more than minor abnormalities. The Coulter MAXM was thus shown to be an excellent haematology analyser. This instrument was capable of detecting significant abnormalities of blood smears with a sensitivity of 97.9%, a specificity of 94.7% and a global efficiency of 96.3% in the samples examined in the present study.

[Prevention of AIDS transmission by syringes and needles in France and Africa]. / Prévention de la transmission par les seringues et aiguilles du V.I.H. en France et en Afrique.

A study made among drug addicts in the Northern suburbs of Paris enables establishing a stabilisation of positive H.I.V. serology prevalence among them, a fact related to the free sale of syringes and needles. In Africa, the nosocomial transmission of H.I.V. by non-sterilisable but reused needles and syringes is demonstrated, and two cases are described. Its importance is certain but must be precise. The prevention of this epidemiological modality must be done by systematic use of reusable and sterilisable injectable material.

PIP: Reuse of disposable needles and syringes plays an important role in the transmission of HIV in France as in Africa. From September 1985-March 1986, 51% of drug addicts systematically screened in the northern suburbs of Paris were found to be HIV positive. Studies in the same population through 1988 indicated that the rate of HIV infection had remained stable, as had the average age of addicts, duration of addiction, and sex ratio. Stabilization of seropositivity rates among drug addicts in the northern suburbs in believed to have been due to information programs alerting addicts to the risk of contamination through sharing of needles and to the legal sale of syringes and needles instituted in May 1987. HIV transmission through reuse of disposable needles and syringes has been demonstrated in Africa. It is attributable to the poverty of health services and of the general population. The significance of HIV transmission by reuse of disposable materials in the health services appears to be considerable but is difficult to quantify. Prevention of transmission by this means will require correct use of sterilizable needles and syringes.