Martine Guesnu

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 t(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.

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

Abstract 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 systems 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.

Deciphering leukemic B-cell chronic lymphoproliferative disorders

Diagnosis of leukemic B-cell chronic lymphoproliferative disorders (B-CLPD) is a frequent challenge in hematology. In this multicentric study, we prospectively studied 165 new consecutive leukemic patients with B-CLPD selected on the basis of Royal Marsden Hospital scoring system ≤3. The primary aim of the study was to try to decipher the atypical cases and identify homogenous subgroups. Overall, morphological examination contributed to diagnosis in only 20% cases, all of them CD5 negative. Thirty additional cases were CD5 negative suggestive of leukemic marginal zone lymphoma in most cases. The significantly poorer survival of the 26 cyclin D1 positive cases justifies recommending its systematic determination among atypical B-CLPD. CD20 expression segregated clearly two subgroups among CD5 positive cyclin D1 negative B-CLPD. The 17 patients with the CD20 dim profile represent a homogeneous subgroup very close to typical B-cell chronic lymphocytic leukemia (B-CLL) on morphological, phenotypical and cytogenetical criteria. In contrast, the subgroup of 51 patients with a CD20 bright profile is heterogeneous. Their significantly lower p27 expression level suggest the presence of a proliferative component, underlying a more aggressive disease. Further genomic studies are warranted to establish their precise nature. These cases should not be included in the same therapeutic trials as B-CLL.

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.

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.