Mette Holm

Clinical Experience with Recombinant Factor VIla in Patients with Thrombocytopenia

Platelets play a central role in primary hemostasis. The role of the coagulation mechanism during early stages of hemostasis is less clear, although increasing evidence is emerging indicating the ultimate importance of the factor VII (FVII)-tissue factor-dependent coagulation system in providing the first thrombin molecules necessary for the platelet activation to occur. Supporting this, early fibrin formation has been reported to occur within the bleeding time wound and infusion of recombinant FVIIa (rFIIa) has been shown to shorten the bleeding time in rabbits. We have investigated whether infusion of rFVIIa would enhance fibrin formation in bleeding time wounds in patients with thrombocytopenia as reflected by a shortening of the bleeding time. A reduction of the bleeding time was found in 55/105 cases (52%). The decrease was significantly more pronounced when the platelet count exceeded 20 x 10(9)/l. With the exception of an anaphylactoid reaction in 1 patient, no major adverse reactions related to the study drug were observed. Nine infusions of rFVIIa were given to 8 thrombocytopenic patients with overt bleeding. One patient received two infusions. Bleeding decreased in all patients and stopped in 6 patients.

Promoter hypermethylation of p15INK4B, HIC1, CDH1, and ER is frequent in myelodysplastic syndrome and predicts poor prognosis in early-stage patients

Abstract:  The propensity of myelodysplastic syndrome (MDS) to transform into acute myeloid leukemia (AML) suggests the existence of common pathogenic components for these malignancies. Here, four genes implicated in the development of AML were examined for promoter CpG island hypermethylation in cells from 37 patients with different stages of MDS. Aberrant methylation was detected by polymerase chain reaction amplification of bisulfite‐treated DNA followed by denaturing gradient gel electrophoresis. The highest rate of methylation was found for p15INK4B (51%), followed by HIC1 (32%), CDH1 (27%), and ER (19%). Concurrent hypermethylation of ≥3 genes was more frequent in advanced compared with early‐stage MDS (P ≤ 0.05), and hypermethylation of p15INK4B was associated with leukemic transformation in early MDS (P ≤ 0.05). The median overall survival was 17 months for cases showing hypermethylation of ≥1 genes vs. 67 months for cases without hypermethylation (P = 0.002). Specifically, promoter hypermethylation identified a subgroup of early MDS with a particularly poor prognosis (median overall survival 20 months vs. 102 months; P = 0.004). In multivariate analysis including stage and thrombocyte count, hypermethylation of ≥1 genes was an independent negative prognostic factor (P < 0.05). These data suggest that hypermethylation of p15INK4B, HIC1, CDH1, and ER contribute to the development and outcome of MDS.

Myelodysplastic Syndromes Are Propagated by Rare and Distinct Human Cancer Stem Cells In Vivo.

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.

Risk group assignment differs for children and adults 1–45 yr with acute lymphoblastic leukemia treated by the NOPHO ALL‐2008 protocol

The prognosis of acute lymphoblastic leukemia is poorer in adults than in children. Studies have indicated that young adults benefit from pediatric treatment, although no upper age limit has been defined.

Kinetics of BCR‐ABL fusion transcript levels in chronic myeloid leukemia patients treated with STI571 measured by quantitative real‐time polymerase chain reaction

Abstract: The activated tyrosine kinase, which arises as a result of the balanced t(9,22) translocation in chronic myeloid leukemia (CML), is thought to be essential for the development of the leukemic phenotype. Recently, designer drugs have been introduced which specifically inhibit such specific kinases. Among these, STI571 (GlivecTM) has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients. To evaluate the effect of STI571 at the molecular level we have employed quantitative real‐time PCR (RQ‐PCR) to measure the amount of BCR‐ABL fusion transcript in a series of 19 patients treated with STI571, either in CP(11) or in (AP)(8) of the disease for 3–9 months (median 6 months). Employing this method, which is able to detect at least one BCR‐ABL+ cell in 500 000, in serial blood and bone marrow specimens we found decreases in transcript levels in 10/11 CP patients, but only in 1/8 of the AP patients. When present such decreases were gradual and became evident only after 3 months of STI571 treatment, and their kinetics in blood closely mirrored those seen in parallel marrow samples. Moreover, decreases were between 10‐ and 100‐fold in 11/13 patients, with only two patients reaching residual disease levels below 10−2 (a 900‐fold decrease). Thus, no patient reached PCR negativity. We conclude that the RQ‐PCR method is a highly suitable tool for following the effect of STI571 in CML and that further validation of the method, performed in a prospective manner, will contribute significantly to the elucidation of the proper role of STI571 in CML.

Maintenance treatment with azacytidine for patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia following MDS in complete remission after induction chemotherapy

This prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5‐azacytidine for older patients with high‐risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS‐acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter‐methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre‐induction, in CR and 6, 12 and 24 months post CR. Twenty‐four (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13·5 months, >24 months in 17% of the patients, and 18–30·5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0·003). 5‐azacytidine treatment, at a dose of 60 mg/m2 was well tolerated. Grade III‐IV thrombocytopenia and neutropenia occurred after 9·5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5‐azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in vitro BrdUrd incorporation into normal and malignant hematopoietic cells

We have designed an assay for the simultaneous measurement of cell surface phenotype, S-phase fraction, and DNA content by single laser instrumentation for the purpose of determining the labeling index (LI), duration of S-phase (Ts), and the potential doubling time (Tpot) of leukocyte subpopulations. The procedure was optimized with regard to: mode of bromodeoxyuridine (BrdUrd) incorporation, selection of suitable leukocyte differentiation antigens (LDAs) as well as PE-conjugated monoclonal antibodies (MoAbs) against myeloid cells, overnight permeabilization and fixation (paraformaldehyde 1% and 0.05% Nonidet P40), DNase I treatment (250 Kunitz units), concentration of FITC-conjugated anti-BrdUrd MoAb (dilution 1:5), and DNA staining with 7-amino-actinomycin (7-AAD) (10 microg/ml). We validated this assay by measuring LI, Ts, and Tpot repeatedly in four leukemic cell lines and found these to be stable (coefficients of variation (CV): 0.06, 0.13, and 0.08, respectively). Finally, we employed the assay on different leukocyte preparations from normal donors (including purified CD34 + cells) and patients with malignant myeloid disorders, and we concluded that it will yield valuable data regarding the cell cycle kinetics of subsets of leukocytes in heterogeneous mixtures of hematopoietic cells.

Comparison of short term in vitro cultured human mast cells from different progenitors - Peripheral blood-derived progenitors generate highly mature and functional mast cells.

During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors. To investigate the significance of different progenitors, we have compared MC generated from CD133(+) progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD(2). The generated metachromatic (<99%) MC were 75% tryptase(+), regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcepsilonRI was comparable. The density of c-kit/CD117 receptors on CBMC was higher that of PBMC (p<0.001). The density of CD203c and FcepsilonRI was higher on PBMC (p<0.001). PBMC contained more histamine (p<0.001), expressed more FcepsilonRI (p<0.001) and released more histamine (p<0.001) and PGD(2) (p<0.001) upon ligation of FcepsilonRI, than CBMC. Culture with IL-4 increased expression of tryptase, FcepsilonRI, CD117 and CD203c, secretion of histamine and PGD(2) of PBMC, and histamine secretion of CBMC. Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used.

Normalization of Structural Cardiovascular Changes During Antihypertensive Treatment with a Regimen Based on the ACE-inhibitor Perindopril

Untreated essential hypertension is associated with left ventricular hypertrophy (LVH) and structural changes in resistance vessels. The aim of this study was to establish the effect of perindopril based antihypertensive therapy on media thickness to lumen diameter (media:lumen) ratio of peripheral resistance vessels and left ventricular mass in essential hypertension. Twenty-five patients with newly diagnosed or poorly regulated essential hypertension were treated with perindopril. Insufficient treatment response (DBP > 90 mmHg) led to addition of isradipine, and hydralazine was used as a tertiary drug if necessary. Gluteal subcutaneous biopsies were taken surgically at baseline and after 9 months of successful treatment. Two small resistance arteries were isolated and mounted in a small vessel myograph, and media:lumen ratio (%) was measured under standardized conditions. Left ventricular mass was determined by echocardiography. Mean (SD) media:lumen ratio decreased from 9.8 (2.6) % to 7.8 (1.9) % (p < 0.05), while left ventricular mass decreased from 299 (75) g to 199 (53) g (p < 0.001). Correlation was found between changes in left ventricular mass index and media:lumen ratio (r = 0.62, p < 0.01). It is concluded that a perindopril based regimen efficiently normalizes resistance artery structure and left ventricular hypertrophy in essential hypertension within one year of treatment. The impact of these findings on the excess cardiovascular morbidity and mortality in arterial hypertension remains to be investigated.

The relation between peripheral vascular structure, left ventricular hypertrophy, and ambulatory blood pressure in essential hypertension

The relations between left ventricular mass (LVM), peripheral resistance artery structure, and ambulatory BP were studied in 83 patients with previously untreated or poorly regulated essential hypertension and 20 healthy controls of similar age and sex. LVM was assessed by echocardiography. Signs of left ventricular hypertrophy (LVH) were present in 67 (81%) of the patients and in none of the controls. Peripheral resistance arteries were isolated from surgical gluteal skin biopsies and mounted in a Mulvany-Halpern isometric small vessel myograph, and their media:lumen ratio, media thickness, and media cross-sectional area were determined under standardized conditions. Mean (+/- SD) ambulatory BP was 122 +/- 9 mm Hg among patients and 96 +/- 8 mm Hg among controls (P < .001). LVM was 327 +/- 99 g among patients and 197 +/- 37 g among controls (P < .001). Media thickness of resistance arteries was 21.0 +/- 4.2 microns among hypertensives and 16.2 +/- 2.6 microns among controls (P < .001). The media:lumen ratio of arteries from patients was 10.2 +/- 2.6% v 7.9 +/- 2.0% in arteries of similar internal diameter from controls (P < .01). Both LVM index (LVMI) and media/lumen ratio correlated significantly with BP. There was significant correlation between media:lumen ratio and LVMI among hypertensive patients (r = 0.45, P < .001), but if patients were subdivided according to the presence of LVH this correlation was found only among patients with LVH (r = 0.60 P< .001) and not among patient without LVH nor controls. Multiple regression analyses of age, body surface area, media/lumen ratio, and BP on LVM or LVMI revealed independent contributions of media/lumen ratio and BP. Age had no influence in the models. Similar results were obtained when casual BP was replaced with ambulatory BP in these analyses. No correlation was found between LVMI and media cross-sectional area. A minor subset of patients with complete absence of nocturnal BP drop had particularly great LVM and media:lumen ratio. The study suggests that cardiac and arteriolar tissue undergo parallel structural remodeling in essential hypertension.