Chiara Benatti

Biological and clinical relevance of matrix metalloproteinases 2 and 9 in acute myeloid leukaemias and myelodysplastic syndromes

We analysed by immunocytochemistry metalloproteinase (MMP)‐2 and MMP‐9 expression in bone marrow cells from 54 acute myeloid leukaemia (AML) patients, 153 myelodysplastic syndrome (MDS) patients, and 52 non‐haemopathic subjects, in order to evaluate whether MMP expression abnormalities were associated with relevant laboratory or clinical findings. In normal samples MMP‐2 was detected in rare myeloid cells, MMP‐9 in most maturing myeloid cells. In MDS MMP‐2 myeloid levels were higher than in controls (P < 0.0001); MMP‐2 and MMP‐9 were often co‐expressed. Also many erythroblasts expressed MMP‐2. There was a positive correlation between MMP‐2 erythroblast expression and erythroid dysplasia (P = 0.002) and an inverse correlation between MMP‐2 or MMP‐9 myeloid expression and blast cell percentage (P = 0.05 and P = 0.04 respectively). High MMP levels in myeloid cells were associated with longer overall survival (P = 0.03) and evolution‐free survival (P = 0.04). In AML MMP‐2 levels were lower than in MDS (P < 0.0001) and MMP‐9 levels lower than in MDS and controls (P < 0.0001). MMP levels did not predict response to therapy. The release of active MMPs was detected by colorimetric analysis in cell cultures from representative MDS and AML cases. In conclusion, we have demonstrated an abnormal MMP expression in AML as well as in MDS. The production and release of these enzymes may influence haematopoietic cell behaviour. In MDS, the detection of MMP deregulated expression may be important also from the clinical point of view: it may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.

Survivin expression, apoptosis and proliferation in chronic myelomonocytic leukemia.

Abstract:  We analyzed the expression of the inhibitor of apoptosis survivin by immunocytochemistry in bone marrow cells from patients with chronic myelomonocytic leukemia (CMML) to evaluate possible abnormalities in comparison with other myelodysplastic (MDS) and myeloproliferative syndromes, and to investigate a possible correlation between survivin expression and altered apoptosis or proliferation, or relevant laboratory and clinical findings. Thirty‐four patients with CMML [18 MDS‐CMML and 16 myeloproliferative disorder (MPD)‐CMML], 90 with MDS, 41 with acute myeloid leukemia (AML), 19 with chronic MPD and 25 control subjects were studied. In normal samples survivin was never detectable. In CMML survivin levels higher than in MDS and AML (P < 0.0001), but similar to those found in MPD were observed. In CMML and MDS apoptosis was significantly higher compared to normal controls and all other subtypes of leukemias (P < 0.0001). Proliferation did not differ significantly in normal controls, MDS and CMML; the lowest levels were observed in AML and MPD (P < 0.0001). In CMML there was no correlation between survivin expression and blast cell percentage, apoptosis or proliferation, FAB or WHO subgroup. Proliferation was higher in MDS‐CMML and tended to correlate with overall survival. CMML‐2 cases with higher survivin expression showed higher evolution rate and shorter survival. In conclusion, CMML is characterized by high proliferation and apoptosis. Survivin overexpression, by disrupting the balance between cell proliferation/differentiation and apoptosis, may play an important role in its pathophysiology. The detection of survivin‐deregulated expression may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.

Overexpression of the Doppel protein in acute myeloid leukaemias and myelodysplastic syndromes

We investigated the expression patterns and distribution of Doppel (Dpl), the product of the prion‐like gene PRND, in the leukaemic cell lines HL‐60 and K562 and in bone marrow cells from 44 patients with acute myeloid leukaemia (AML) and 63 patients with myelodysplastic syndrome (MDS). Whereas normal samples were negative or showed very weak expression that was restricted to CD34+ cells, Dpl was detected in both cell lines and in most AML and MDS cases by immunocytochemistry and Western blotting. Quantitative reverse transcription polymerase chain reaction revealed variable mRNA levels in almost all AML and MDS cases, but barely detectable levels in normal bone marrow. These differences were confirmed by in situ hybridization. PRND expression was higher in advanced compared with early MDS (P = 0·01), but Dpl levels did not predict disease progression. In AML there was no correlation between Dpl levels and clinical or laboratory findings. In conclusion, this is the first time that the expression of PRND has been demonstrated in human bone marrow. The molecular mechanism of the observed overexpression is unknown; however, the differential Dpl distribution in AML and MDS versus healthy subjects makes it a possible leukaemia‐associated antigen that could be useful for diagnostic and therapeutic purposes.

Biological Effects of Pegfilgrastim on Circulating Neutrophils in Breast Cancer Patients Undergoing Dose-Dense Chemotherapy

Pegfilgrastim is a covalent conjugate of filgrastim and polyethylene glycol that has proved to be effective in supporting myelopoiesis during chemotherapy. Since very limited information is available on the biological effects of pegfilgrastim on neutrophils exposed to chemotherapy, we analyzed the following parameters in neutrophils of patients undergoing dose-dense chemotherapy for breast cancer: apoptosis, by a TUNEL technique; actin polymerization, using FITC-labeled phalloidin, and alkaline phosphatase activity by cytochemistry. Peripheral blood buffy coat smears were obtained before starting treatment and immediately before each chemotherapy course. After pegfilgrastim stimulation we observed the following: (1) stability of the absolute neutrophil count for the whole duration of treatment and no infectious events; (2) a reduction in the neutrophil constitutive apoptosis rate in comparison with that observed in control patients treated with standard chemotherapy courses with no growth factor support; (3) persistent abnormalities of actin assembly in neutrophils, indicative of changes in cytoskeletal organization, and (4) a significant increase in the activity of leukocyte alkaline phosphatase, a sensitive marker of the later stages of neutrophil maturation. In conclusion, these results suggest that pegfilgrastim improves the neutrophil functions in patients exposed to chemotherapy by inhibition of constitutive apoptosis, thereby prolonging the survival of these cells.

A complex chromosome 3 rearrangement not affecting RPN1, EVI1/MDS1 genes in a patient with an atypical refractory cytopenia with multilineage dysplasia

Dear Editor, Defects of band 3q21q26 are revealed in 7–10% of myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cytotypes and rarely in the blast crisis of chronic myeloid leukemia (CML) [7]. These abnormalities target the same regions of bands 3q21 and 3q26 [13] and cause the overexpression of the EVI1 gene through several distinct mechanisms [8]. However, this event not only occurs in 12.5% of AML cases with 3q26 rearrangements [1] but also in 7–20% of de novo AML, 27–55% of secondary AML, 29–58% of MDS without any 3q26 rearrangement and no clinical features typical of 3q aberrations [14]. Thus, it has been suggested that EVI1 expression is neither necessary nor sufficient to evoke the clinical characteristics of all these patients, and a molecular heterogeneity in MDS/AML with 3q21q26 rearrangements has been proposed [4]. Morphologically, most 3q aberrations present trilineage dysplasia, dysplastic megakaryocytes, and normal/increased platelets. Clinically, 3q defects are more often revealed in young patients and, when affecting EVI1 expression, predict an unfavorable prognosis [1, 2]. Herein, we report on a 67-year-old man submitted to surgery for aortic and mitral valve replacement. He presented with fatigue, weight loss, and a history of mild anemia lasting for a few years. There was no previous exposure to environmental mutagens or cancer treatments. Physical examination showed pallor and slight hepatomegaly. Hematologic data were: Hb 8.9 g/dl, MCV 108 fl, reticulocytes 38×10/l, WBC 1.53×10/l with a normal differential count and platelet count 54×10/l. A peripheral blood smear showed remarkable red cell anisopoikilocytosis and platelet anisocytosis with many giant platelets. Bone marrow aspirate displayed hypercellular marrow. Erythroid precursors were less than 50% of bone marrow cells and showed dysplastic features. Myeloid lineage was hyperplastic with very few dysplastic neutrophils and a predominance of intermediate precursors containingmany azurophilic granules in their cytoplasm and showing bilobed or abnormally shaped nuclei. Blast cells were 4%. There were also many abnormal megakaryocytes. Perls reaction showed some 30% ring sideroblasts. A diagnosis of atypical refractory cytopenia with multilineage dysplasia and ring sideroblasts was made, and the patient underwent supportive therapy only. Up to now, the patient has been followed up for 32 months, and the disease is stable with an unchanged transfusional need. The 20 metaphases from marrow cells revealed a complex chromosome 3q rearrangement, and the karyotype was tentatively described as: 45,XY, 3,der(9)t(3;9)(9pter→ 9q33::3q21→3qter),22,+der(22)[12]/46,XY,idem,+r [8]. FISH with a Whole Chromosome 3 Painting probe revealed that chromosome 3 was rearranged not only with the long arm of chromosome 9 but also with the short arm of chromosome 22, and the ring chromosome was entirely derived from number 3 (Fig. 1a). In addition, a 3p painting probe (Q-Biogene) showed that the short arm of chromosome 3 was translocated onto der(22) and participated in the generation of the ring chromosome (Fig. 1b). To check whether EVI1 and MDS1 were targeted by the translocation, the rp11-94J18, rp11-13M6, and rp11-3K16 BAC probes (Welcome Trust Sanger Institute, Cambridge, UK), Ann Hematol (2008) 87:147–150 DOI 10.1007/s00277-007-0398-z