Klaus Lechner

Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group

Diagnosis and management of immune thrombocytopenic purpura (ITP) remain largely dependent on clinical expertise and observations more than on evidence derived from clinical trials of high scientific quality. One major obstacle to the implementation of such studies and in producing reliable meta-analyses of existing data is a lack of consensus on standardized critical definitions, outcome criteria, and terminology. Moreover, the demand for comparative clinical trials has dramatically increased since the introduction of new classes of therapeutic agents, such as thrombopoietin receptor agonists, and innovative treatment modalities, such as anti-CD 20 antibodies. To overcome the present heterogeneity, an International Working Group of recognized expert clinicians convened a 2-day structured meeting (the Vicenza Consensus Conference) to define standard terminology and definitions for primary ITP and its different phases and criteria for the grading of severity, and clinically meaningful outcomes and response. These consensus criteria and definitions could be used by investigational clinical trials or cohort studies. Adoption of these recommendations would serve to improve communication among investigators, to enhance comparability among clinical trials, to facilitate meta-analyses and development of therapeutic guidelines, and to provide a standardized framework for regulatory agencies.

HIGH PLASMA LEVELS OF FACTOR VIII AND THE RISK OF RECURRENT VENOUS THROMBOEMBOLISM

BACKGROUND A high plasma level of factor VIII is a risk factor for venous thromboembolism. We evaluated the risk of a recurrence of thrombosis after an initial episode of spontaneous venous thromboembolism among patients with high plasma levels of factor VIII. METHODS We studied 360 patients for an average follow-up period of 30 months after a first episode of venous thromboembolism and discontinuation of oral anticoagulants. Patients who had recurrent or secondary venous thromboembolism, a congenital deficiency of an anticoagulant, the lupus anticoagulant, hyperhomocysteinemia, cancer, or a requirement for long-term treatment with antithrombotic drugs or who were pregnant were excluded. The end point was objectively documented, symptomatic recurrent venous thromboembolism. RESULTS Recurrent venous thromboembolism developed in 38 of the 360 patients (10.6 percent). Patients with recurrence had higher mean (+/-SD) plasma levels of factor VIII than those without recurrence (182+/-66 vs. 157+/-54 IU per deciliter, P=0.009). The relative risk of recurrent venous thrombosis was 1.08 (95 percent confidence interval, 1.04 to 1.12; P<0.001) for each increase of 10 IU per deciliter in the plasma level of factor VIII. Among patients with a factor VIII level above the 90th percentile of the values in the study population, the likelihood of recurrence at two years was 37 percent, as compared with a 5 percent likelihood among patients with lower levels (P<0.001). Among patients with plasma factor VIII levels above the 90th percentile, as compared with those with lower levels, the overall relative risk of recurrence was 6.7 (95 percent confidence interval, 3.0 to 14.8) after adjustment for age, sex, the presence or absence of factor V Leiden or the G20210A prothrombin mutation, and the duration of oral anticoagulation. CONCLUSIONS Patients with a high plasma level of factor VIII have an increased risk of recurrent venous thromboembolism.

1,25-Dihydroxyvitamin D3 inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.

How I treat autoimmune hemolytic anemias in adults

Autoimmune hemolytic anemia is a heterogeneous disease with respect to the type of the antibody involved and the absence or presence of an underlying condition. Treatment decisions should be based on careful diagnostic evaluation. Primary warm antibody autoimmune hemolytic anemias respond well to steroids, but most patients remain steroid-dependent, and many require second-line treatment. Currently, splenectomy can be regarded as the most effective and best-evaluated second-line therapy, but there are still only limited data on long-term efficacy and adverse effects. The monoclonal anti-CD20 antibody rituximab is another second-line therapy with documented short-term efficacy, but there is limited information on long-term efficacy and side effects. The efficacy of immunosuppressants is poorly evaluated. Primary cold antibody autoimmune hemolytic anemias respond well to rituximab but are resistant to steroids and splenectomy. The most common causes of secondary autoimmune hemolytic anemias are malignancies, immune diseases, or drugs. They may be treated in a way similar to primary autoimmune hemolytic anemias, by immunosuppressants or by treatment of the underlying disease.

Diagnosis and treatment of systemic mastocytosis: state of the art

Mast cells are tissue-fixed cells originating from uncommitted and mast cell-committed haematopoietic progenitors (Kitamura et al, 1981; Kirshenbaum et al, 1992; Agis et al, 1993; Rottem et al, 1994; Kempuraj et al, 1999). Mast cellcommitted progenitors co-express CD13 and KIT with CD34 (Kirshenbaum et al, 1999) and are detectable in the bone marrow as well as in the peripheral blood (Valent et al, 1992; Rottem et al, 1994; Valent, 1994). Homing, differentiation and maturation of mast cell progenitor cells are regulated by a complex network of growth factors, receptors and other antigens (Galli, 1990; Valent, 1994). The most important growth factor for human mast cells appears to be stem cell factor (SCF) (Irani et al, 1992; Kirshenbaum et al, 1992; Valent et al, 1992; Mitsui et al, 1993). This cytokine is a natural ligand for the c-kit proto-oncogene product, KIT, a tyrosine kinase receptor expressed on the surface of precommitted myelopoietic progenitor cells, mast cell-committed progenitor cells as well as mature mast cells (Galli et al, 1993; Simmons et al, 1994; Valent, 1994). Based on their unique phenotype and distinct functional properties, mast cells represent a distinct myeloid cell lineage within lympho-haematopoietic tissues. Likewise, mast cells express a unique composition of CD antigens and granular mediators when compared with other myeloid cells (Schwartz, 1985; Valent et al, 1989; Valent & Bettelheim, 1992; Agis et al, 1996) (Table I). Moreover, in contrast to blood basophils and other myeloid cells, mast cells exhibit an extremely long life span in vivo ranging from several months to years (Galli, 1990; Födinger et al, 1994). In contrast to other haematopoietic cells, mast cells produce substantial amounts of histamine and heparin and express the high-affinity IgE receptor on their surface (Ishizaka & Ishizaka, 1984; Schwartz, 1985; Galli, 1990). The concept that mast cells represent a unique myeloid lineage is in line with the notion that systemic mastocytosis (SM) is a distinct haematopoietic (myeloid) neoplasm with unique pathogenetic and clinical features (Lennert & Parwaresch, 1979; Parwaresch et al, 1985; Metcalfe, 1991a; Valent, 1996). The clonal nature of the disease has been reinforced by the association with the somatic c-kit mutation Asp-816-Val (Nagata et al, 1995; Longley et al, 1996, 1999; Buttner et al, 1998). This transforming mutation is detectable in the bone marrow (mast cells) in a majority of patients with SM but usually is not found in other myeloid neoplasms (Fritsche-Polanz et al, 2001). Remarkably, in a group of patients with (advanced) SM, the c-kit mutation Asp-816-Val is detectable not only in mast cells but also in other haematopoietic lineages, including blood monocytes (Akin et al, 2000a; Sotlar et al, 2000; Yavuz et al, 2002). Based on this notion and several clinical observations, SM can be regarded as a myeloproliferative disorder. In line with this concept, patients with SM are at a certain risk of acquiring a secondary myeloid leukaemia (Travis et al, 1988a,b; Horny et al, 1990a; Lawrence et al, 1991; Sperr et al, 2000). In the management of patients with SM, two major problems have to be faced. The first is mediator release from mast cells with respective clinical symptoms that can be observed frequently in these patients (Horan & Austen, 1991; Metcalfe, 1991a; Austen, 1992; Valent, 1996). In fact, mast cells store (in their granules) or generate a number of vasoactive mediators [histamine, tumour necrosis factor-a (TNFa), vascular endothelial growth factor (VEGF), leukotrienes, prostaglandin D2 (PGD2)] and other biologically active molecules (interleukins, proteases, heparin) (Roberts et al, 1980; Lewis & Austen, 1981; Serafin & Austen, 1987; Burd et al, 1989; Plaut et al, 1989; WodnarFilipowicz et al, 1989; Gordon et al, 1990; Gordon & Galli, 1990) (Table II). In response to activating stimuli, mast cells can generate and ⁄ or release their mediator substances (Lewis & Austen, 1981; Ishizaka & Ishizaka, 1984; Schwartz, 1985; Burd et al, 1989; Plaut et al, 1989; Wodnar-Filipowicz et al, 1989; Gordon et al, 1990). Resulting clinical symptoms include headache, flushing, pruritus, diarrhoea, vascular instability, hypotension and shock (Austen, 1992) (Table II). Such symptoms may be grave and life threatening, especially in patients with SM who also have a co-existing disease predisposing for mediator secretion (allergies). The second management problem in SM results from the uncontrolled (aggressive) growth and infiltration of mast cells in diverse organs with consecutive organopathy (Lennert & Parwaresch, 1979; Parwaresch et al, 1985; Metcalfe, 1991a; Valent, 1996). Such organopathies are seen in patients with aggressive systemic mastocytosis (ASM), mast cell leukaemia (MCL) and in a group of patients with an associated clonal haematological nonmast cell lineage disease (SM-AHNMD), but not in those with indolent systemic mastocytosis (ISM). The organ systems most frequently affected in patients with aggressive Correspondence: Peter Valent, Department of Internal Medicine I, Division of Haematology & Haemostaseology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria. E-mail: peter.valent@akh-wien.ac.at British Journal of Haematology, 2003, 122, 695–717

Clinical Studies and Thrombin Generation in Patients Homozygous or Heterozygous for the G20210A Mutation in the Prothrombin Gene

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.

Clinical significance of lupus anticoagulants in children

OBJECTIVES To determine the spectrum of associated clinical manifestations and time course of lupus anticoagulants (LA) in children. STUDY DESIGN Retrospective study of 95 consecutive children (46 boys and 47 girls), with a median age of 5.3 years (range, 1.7 to 17.1 years), diagnosed with presence of LA at a hemostasis referral center; 83 were followed up over a median of 2.9 years (range, 6 weeks to 21.6 years). RESULTS At diagnosis, 80 of 95 (84%) children were free of symptoms, and presence of LA was found incidentally. Nine children (10%) had bleeding symptoms, 5 (5%) had thrombotic events, and 1 had systemic lupus erythematosus. Among the patients with bleeding, 5 had transient severe hypoprothrombinemia after adenovirus infections, and 3 had thrombocytopenia. None of the children who were initially free of symptoms had bleeding, thrombotic complications, or autoimmune disease subsequently. At follow-up, 48 of 83 (58%) patients had normal activated partial thromboplastin time values after 1.9 years (5 weeks to 19.1 years). Thirty-two (38%) still had activated partial thromboplastin time elevations but did not fulfill all criteria for presence of LA after 3.2 years (7.4 months to 9.3 years). Three (4%) patients, who had presented with thrombosis, had persistent positive LA, anti-cardiolipin, and antinuclear antibodies after 1.4, 2.8, and 7.5 years, respectively. One of these had recurrent thrombosis. CONCLUSIONS In most children the presence of LA did not lead to clinical complications and was transient. Bleeding occurred with additional hypoprothrombinemia or thrombocytopenia. Thrombosis was rare and strongly associated with persistently positive LA.

Prognostic significance of WT1 gene expression at diagnosis in adult de novo acute myeloid leukemia

We examined the presence of WT1-specific mRNA in bone marrow samples of 125 patients with de novo acute myeloid leukemia at diagnosis by two-step RT-PCR. The sensitivity of the assay was 1:100 (first step) and 1:10 000 (second step), respectively. WT1-specific mRNA was detected in 73% of patients. No correlation was found between WT1 gene expression and age, FAB type, LDH and karyotype at diagnosis. All patients were treated with standard induction chemotherapy. There was no difference in the CR rate between WT1-positive and -negative patients. Using Kaplan and Meier plot analysis we found no difference in disease-free survival (DFS) and overall survival (OS) between patients displaying the WT1 transcript and WT1-negative patients. Furthermore, no significant interactions between WT1 PCR results and age, FAB type, LDH and karyotype on DFS and OS were demonstrable using Cox regression analysis. Eight patients who were WT1 PCR positive at diagnosis and achieved complete hematological remission following chemotherapy were monitored during the course of the disease. Based on our limited data demonstrating a heterogenity of WT1 PCR results in CR we cannot draw any conclusions regarding the usefulness of WT1 PCR analysis for the early detection of relapse. We conclude that WT1 gene expression at diagnosis is not associated with specific characteristics of AML blast cells and is not a prognostic factor for CR, remission duration and overall survival in acute myeloid leukemia.

Response to therapy with interferon alpha-2b and prednisolone in aggressive systemic mastocytosis: report of five cases and review of the literature

Aggressive systemic mastocytosis (ASM) is a hematopoietic neoplasm characterized by infiltration of visceral organs by neoplastic mast cells (MCs) with consecutive organopathy and respective clinical and laboratory findings (so called C-Findings). Whereas, it is generally appreciated that patients with ASM are candidates for pharmacological intervention, no ideal drug or drug combination have been identified yet. One drug proposed to work in ASM is interferon alpha-2b (IFN-alpha2b). However, little is known so far about the quality of responses to IFN-alpha2b and actual response rates. We here report on five ASM patients treated with either a combination of IFN-alpha2b (3x3 million units per week) and prednisolone (n=4), or IFN-alpha2b alone (n=1). During therapy, two of the five patients showed a major response defined by complete resolution of C-Finding(s), one a partial response (partial regression of C-Findings), and one a stable disease (no changes in C-Findings). In one patient, progression to mast cell leukemia was seen after 3 months. In contrast to the other patients, this patient exhibited >10% MCs in his bone marrow (bm) smear at first presentation. In summary, our data confirm beneficial effects of IFN-alpha2b (plus prednisolone) for a group of patients with ASM, whereas patients with mast cell leukemia may require more aggressive therapy. Prospective trials with more patients are now required to further document these drug effects and to better define subgroups of patients with ASM who show good and long-lasting responses to IFN-alpha2b.

Clinical and biologic diversity of leukemias occurring in patients with mastocytosis.

Patients with systemic mast cell (MC) disease, but not those with cutaneous mastocytosis, are at a high risk (10–30%) to develop life-threatening myelogenous malignancies. In a significant proportion of cases, myeloid leukemias occur. Using conventional criteria, such leukemias resemble acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or myelomonocytic leukemia (CMML). Mast cell leukemia (MCL) may also occur. Myeloid leukemias (AML, CML, CMML) can develop in indolent or aggressive mastocytosis (skin lesions present or absent) with a variable prephase of MC disease. By contrast, MCL (typically without skin lesions) often develops on a “de novo” basis, and, if at all recognized, a prephase resembling (malignant) mastocytosis, is short. MCL differs from myeloid leukemias (AML, CML, CMML) by morphologic and phenotypic cellular characteristics. In fact, MCL are strongly tryptase-positive, c-kit-positive, myeloperoxidase (MPO) -negative neoplasms with variable metachromasia and chloroacetate esterase expression, whereas an MPO-positive, tryptase-negative phenotype supports the diagnosis of a myeloid non-MC lineage disease. Thus, MCL, but also myeloid non-MC lineage leukemias can develop in patients with (systemic) mastocytosis. Little is known, however, about the pathophysiologic basis of co-evolution. In the present article, the concomitant occurrence of mastocytosis and leukemia is discussed in the light of the literature and of concepts proposed to explain the biologic basis of this phenomenon.