Susanne Widell

Diagnostic significance of dysplastic features of peripheral blood polymorphs in myelodysplastic syndromes.

Dysplastic features of peripheral blood granulocytes were studied to investigate the diagnostic value estimating cytoplasmatic hypogranulation and nuclear abnormalities of the pelgeroid type in myelodysplastic syndromes (MDS). Hypogranulation was measure both as the percentage of agranular neutrophils and as a score value, taking into account also cells with slight or moderate hypogranulation. We studied 62 cases of MDS (18 refractory anemia, 11 sideroblastic anemia, 26 refractory anemia with excess of blasts, three chronic myelomonocytic leukemias, four refractory anemia with excess of blasts in transformation). For comparison we studied 13 cases of myeloproliferative disorders, 18 patients with different forms of anemia and 20 normal controls. Reference values were defined as the 95% probability limit of the mean values of normal controls. In MDS 52/62 patients (84%) had increased numbers of pelgeroid cells, 40/62 (65%) had abnormal granulation scores while only 14/62 (23%) had increased percentages of agranular neutrophils. The mean granulation score (+/- S.D.) in MDS (225.0 +/- 57.4), was significantly lower (p less than 0.001) than in myeloproliferative disorders (282.7 +/- 9.0), anemias (288.8 +/- 8.0) and normal controls (281.7 +/- 12.9). Pelgeroid neutrophils were significantly (p less than 0.001) more common in MDS (11.6% +/- 7.8) compared to myeloproliferative disorders (1.1% +/- 1.0), anemias (3.1% +/- 2.0) and normal controls (1.9% +/- 1.5). There was no significant correlation between the degree of hypogranulation and the percentages of pelgeroid cells in individual patients. Hypogranulation tended to be more pronounced in the more immature forms of MDS while pelgeroid cells were equally common in the different subgroups. When the two parameters were combined peripheral blood dysplasia was recognized in 92% of the MDS cases. The results suggest that quantitative estimation of hypogranulation and of nuclear abnormalities in peripheral blood polymorphs are simple and valuable diagnostic tools in MDS.

Neutrophil dysplasia is not a specific feature of the abnormal chromosomal clone in myelodysplastic syndromes

In order to study if dysplastic cells are part of the abnormal chromosomal clone in myelodysplastic syndromes (MDS) we used fluorescence in situ hybridization in combination with standard May-Grünwald-Giemsa morphology (MGG/FISH) to investigate the penetration of chromosomal abnormalities into dysplastic neutrophil granulocytes in five MDS patients with documented monosomy 7 or trisomy 8 in the bone marrow at diagnosis. Neutrophil dysplasia was defined as neutrophil granulocytes with extreme hypogranulation or with nuclear abnormalities of the pelgeroid type. In one patient all dysplastic cells were derived from the abnormal chromosomal clone, while in the other four cases only 6-43% of the hypogranulated neutrophils and 33-40% of the pelgeroid granulocytes exhibited the chromosomal marker. The results suggest that neutrophil dysplasia is not a specific feature of the abnormal chromosomal clone in MDS. It is not clear, however, if the disomic dysplastic cells were derived from a parallel MDS clone with a normal karyotype, or represent residual non-clonal, normal hematopoietic cells.

Balloon-like platelets in myelodysplastic syndromes—a feature of dysmegakaryopoiesis?

Large bizarre platelets are a frequent finding in the peripheral blood of the myelodysplastic syndrome (MDS). In this study we describe a distinct subpopulation of platelets in MDS which by phase contrast microscopic examination seems to have a balloon-shaped bulge of the cell membrane. Increased numbers of these atypical platelets were found in 24 of 27 MDS patients (89%). Only 3 patients, all with ringsideroblastic anemia, had normal platelet morphology. The number of atypical platelets were negatively correlated (r = 0.44; p = 0.021) to the peripheral platelet counts in MDS. Among 48 patients with acute leukemia, chronic lymphoproliferative or chronic myeloproliferative disorders, 13 (27%) had atypical platelets. Here, but not in the MDS group, atypical platelets seemed to be associated with recent chemotherapy. In a group of patients with benign hematological disorders abnormal platelet morphology was seen only occasionally. The described atypical platelets most likely reflect maturation disturbances of the megakaryopoiesis. An increased value (greater than 1%) in a cytopenic patient would suggest a diagnosis of MDS, unless associated with recent cytotoxic therapy.

Inverse relationship between myeloid maturation and leukotriene C4 synthase expression in normal and leukemic myelopoiesis—consistent overexpression of the enzyme in myeloid cells from patients with chronic myeloid leukemia

OBJECTIVE Leukotriene (LT) C(4) synthase (LTC(4)S) is the key enzyme in the biosynthesis of LTC(4), which has been reported to stimulate the growth of human myeloid progenitor cells and is specifically overproduced in chronic myeloid leukemia (CML). The aim of this study was to clarify the expression of LTC(4)S during normal and leukemic myelopoiesis and to investigate the correlation between abnormal LTC(4)S expression in CML myeloid cells and the activity of the disease-specific tyrosine kinase p210 BCR-ABL. MATERIALS AND METHODS Immature and mature myeloid cell subpopulations were isolated with magnetic cell sorting from healthy volunteer bone marrow (n = 11) and CML patient peripheral blood (n = 8), respectively. The cells were subjected to analysis of LTC(4)S protein expression and activity. Expression of LTC(4)S was investigated in CD16(+) neutrophils from CML patients before and after 1 month of medication with imatinib mesylate (STI571), which is a specific inhibitor of p210 BCR-ABL. RESULTS Among normal cells, the highest enzyme activity was observed in the most immature, CD34(+) progenitor cell-enriched and CD15(+) myelocyte-enriched fractions. Subsequently, LTC(4)S activity decreased with increasing maturity, with only negligible amounts of LTC(4) produced in CD16(+) neutrophils. LTC(4)S was expressed at the protein level in the immature myeloid cell fractions but not in CD16(+) cells. In CML cells, LTC(4)S activity and expression were consistently elevated. Thus, the CML CD34(+) and CD15(+) cell fractions, as well as the CD11b(+) myelocyte/metamyelocyte-enriched fractions, produced 6 to 10 times as much LTC(4) as the corresponding normal cells. Again, enzyme expression was highest in the most immature cells, although evident LTC(4)S expression and activity remained in CML CD16(+) neutrophils. Interestingly, treatment of five CML patients with imatinib mesylate down-regulated the abnormal neutrophil LTC(4)S expression and activity. CONCLUSIONS Expression of LTC(4)S in immature myelopoid cells is in line with a role for this enzyme in myelopoiesis. In addition, consistent overexpression of LTC(4)S in CML and the correlation to p210 BCR-ABL activity suggests that LTC(4)S may be involved in leukemic pathogenesis.

Dysplastic peripheral blood polymorphs link acute myeloblastic leukaemia in elderly to the mvelodvsdastic Syndromes

Abstract: We studied dysplastic features in peripheral blood polymorphs from 80 patients with acute leukaemia. Thirty‐seven patients with de novo acute myeloblastic leukaemia (AML) were compared to 26 patients with AML that had developed after a myelodysplastic phase (MDS‐AML), and 17 cases of acute lymphoblastic leukaemia (ALL). Cytoplasmic hypogranulation in neutrophils, measured as a score value (G‐score; normal range: 255–300), and the percentage of pelgeroid polymorphs (ppp; normal range: 0–5%) were studied retrospectively by reviewing the diagnostic peripheral blood smears. The mean G‐score was decreased in MDS‐AML (178 ± 67.9), and in de novo AML (212 f 65.l), but not in ALL (275 ± 24.3). When de now AML patients were divided by age, the elderly (> 60 yr) had significantly (p = 0.0001) lower mean G‐score than the younger (<45 yr) ones; 156 ± 64.8 v 243 ± 41.4. This age‐related difference became accentuated when only patients with extreme hypogranulation (G‐score < 150) were studied. Elderly de now AML patients also had significantly (p = 0.0057) higher mean ppp. By studying the degree of polymorph dysplasia in the peripheral blood, it seems possible to identify a subset of dysplastic elderly AML patients, who might have passed a (preleukaemic) MDS phase unnoticed.

Clinical imatinib mesylate treatment induces early normalisation of aberrant neutrophil leukotriene C4 synthase expression and activity in chronic myeloid leukaemia

Clinical imatinib mesylate treatment induces early normalisation of aberrant neutrophil leukotriene C4 synthase expression and activity in chronic myeloid leukaemia

DNA image analysis in childhood acute lymphoblastic leukemia

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

DNA image cytometry in MDS bone marrow smears.

The nuclear DNA content, defined as DNA index (DI) in blasts/promyelocytes (bla/pro), were determined on Feulgen‐stained bone marrow smears from 39 patients with myelodysplastic syndromes (MDS) and eight control subjects by the use of image cytometry (ICM). The DI in patients was compared to that of corresponding normal cell types, and to cytogenetic data available in 32/39 patients. The mean DI in bla/pro of patients with MDS was significantly (P < 0.01) lower compared to corresponding cell types in control subjects. By ICM, a DNA aneuploidy in bla/pro was found in 67% of the MDS patients, and 59% expressed DNA hypodiploidy. By cytogenetics, an abnormal karyotype was found in 31%, and 6/9 MDS patients with a ‘hypodiploid’ abnormal karyotype showed DNA hypodiploidy. Of patients with a normal karyotype (69%), seven (32%) showed a normal, 12 (55%) a lower, and three (14%) a higher DI compared to controls. No difference between groups of MDS patients was found. DNA hypodiploidy is suggested to be a common feature in MDS without a relationship to cytogenetics.