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.

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.

[Avascular jaw osteonecrosis associated with bisphophonate therapy]. / Osteonecrosi avascolare del massiccio facciale in corso di terapia con bisfosfonati.

The case of an IgAk myeloma showed the following common features with more than a hundred of patients described by the literature: osteonecrosis of the jaws, prolonged administration of bisphosphonates due to neoplastic skeletal lesions, antitumoral therapies and recent dental surgery. The antiangiogenetic effects of bisphosphonates can have an important pathogenetic role and a general dental treatment should be completed before the start of bisphosphonate administration.

Survivin expression, apoptosis and proliferation in chronic myelomonocytic leukemia.

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.

Thalidomide treatment reduces apoptosis levels in bone marrow cells from patients with myelodysplastic syndromes.

We treated with thalidomide seven patients with primary MDS and observed reduction of the transfusion requirement in three cases and reduction of bone marrow blasts in one case. The apoptotic rate of bone marrow cells diminished significantly from a mean of 43.8% to a mean of 17.5%, whereas the proliferative activity did not change. Plasma TNF-alpha, bFGF, IL-1beta levels decreased variably, whereas VEGF levels tended to increase. Matrix metalloproteinases 2 and 9 expression decreased in bone marrow cells of responders. A reduction of CD4 cells and an increase of NK cells was observed in the peripheral blood. Thus, thalidomide may produce a fairly good hematological improvement in erythroid series in MDS, with complex biological mechanisms.

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.