Wilfried Schroyens

Induction of complete and molecular remissions in acute myeloid leukemia by Wilms’ tumor 1 antigen-targeted dendritic cell vaccination

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms’ tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8+ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-γ–producing CD8+ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

Platelet-mediated erythromelalgic, cerebral, ocular and coronary microvascular ischemic and thrombotic manifestations in patients with essential thrombocythemia and polycythemia vera: A distinct aspirin-responsive and coumadin-resistant arterial thrombophilia

Microvascular circulation disturbances including erythromelalgia, its microvascular ischemic complications, and migraine-like atypical or typical transient ischemic cerebral, ocular, and coronary ischemic attacks are specific clinical manifestations in patients with essential thrombocythemia (ET), and polycythemia vera (PV) associated with thrombocythemia. Thrombocythemia (ET and PV) patients with microvascular disturbances have shortened platelet survival, increased β-thromboglobulin (β-tg), platelet factor 4 (PF4), and thrombomoduline (TM) levels, and increased urinary thromboxane B2 (TxB2) excretion indicating platelet-mediated processes in vivo. Inhibition of platelet cyclooxygenase (COX 1) by aspirin is followed by relief of microvascular disturbances, correction of shortened platelet survival, and return of plasma levels of β-tg, PF4, TM levels and TxB2 excretion to normal. The transient ischemic attacks and thrombotic complications in thrombocythemia are very likely caused by hypersensitive platelets produced by spontaneously proliferating enlarged megakaryocytes in the bone marrow of ET and PV patients. In contrast to normal platelets in healthy individuals the circulating hypersensitive thrombocythemic platelets spontaneously activate and secrete their products, thus forming aggregates that transiently plug the microcirculation, or result in occlusive platelet thrombi in arterioles or small arteries. Clear evidence is presented that the microvascular transient ischemic and occlusive thrombotic complications in thrombocythemia patients are relieved by treatment with aspirin and by reduction of platelet counts to normal (<400 × 109/l), but not by coumadin. In patients with thrombocythemia associated with PV, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular ischemic and thrombotic syndrome of thrombocythemia to major arterial and venous thrombotic complications. Correction of hematocrit and blood viscosity by phlebotomy significantly reduces the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in PV patients are obtained by treatment with low-dose aspirin on top of phlebotomy or by treatment with the platelet lowering agents, anagrelide, interferon or hydroxyurea.

Acquired von Willebrand syndrome type 1 in hypothyroidism: reversal after treatment with thyroxine.

In 16 cases, acquired von Willebrand syndrome (AvWS) and hypothyroidism have been described that occur with each other: 15 women and one man, at a mean age of 32 years, range, 13 to 82 years of age. Activated partial thromboplastin time (APTT) was normal in six patients, and five patients had factor VIII concentration (factor VIIIc) levels in excess of 60%. The bleeding time was prolonged in nine of 13 evaluable patients. Activated partial thromboplastin time was prolonged in seven patients, and five of these had factor VIIIc levels between 18 and 45%, with two patients having levels in excess of 60%. A deficiency of other coagulation factors, including factor VII, V. IX, and X, caused by a generalized diminution in protein synthesis in hypothyroidism, may have contributed to the prolongation of the APTT. The AvWS was very likely type 1 in all cases because of a normal von Willebrand factor antigen/ristocetin cofactor (vWF Ag/RCF) ratio. Acquired von Willebrand syndrome was documented via cross immunoelectrophoresis in three patients and via multimeric analysis of vWF in six patients. A definite diagnosis of AvWS type I has to be confirmed by a normal response to 1-desamino-8-D-arginine vasopressin (DDAVP). Treatment of hypothyroidism with thyroxine was associated with the disappearance of the AvWS and the bleeding diathesis. Decreased factor VIIIc, vWF Ag ,and vWF RCF levels (50%, 33%, and 36% respectively) before thyroxine treatment increased to normal values (97%, 93%, and 107% respectively) after treatment. The absence of bleeding, or mild bleeding, symptoms, in relation to those more commonly recognized with hypothyroidism, has led to the complication of acquired vWF deficiency being under-diagnosed. Acquired von Willebrand syndrome type I should be considered whenever hypothyroidism is diagnosed and thyroid biopsy or surgery is contemplated. The complete relief of AvWS via treatment of hypothyroidism with thyroxine is the final proof of this association and causal relationship.

Decreasing antibiotic resistance of Enterobacteriaceae by introducing a new antibiotic combination therapy for neutropenic fever patients.

Prompt empiric antibiotic therapy is of critical importance for patients with neutropenic fever. However, a major concern with important clinical consequences is the emergence of bacterial resistance to antibiotics. After using ceftazidime with a glycopeptide as initial empiric therapy for neutropenic fever, we were confronted with a 75% reduced susceptibility rate to ceftazidime of inducible Enterobacteriaceae collected in 1994. The initial empiric therapy was therefore replaced in May 1995 by a combination of cefepime with amikacin, with addition of a glycopeptide after 48 h if necessary. After this change, we observed a significant decrease in reduced susceptibility of inducible Enterobacteriaceae, not only to ceftazidime, but also to amikacin, cotrimoxazole and ciprofloxacin. There was also a decrease in reduced susceptibility of non-inducible Enterobacteriaceae, such as Klebsiella spp, to ceftazidime. The reduction of resistance may be related at least in part to the combined use of cefepime together with an aminoglycoside. This study shows that it is possible to reverse bacterial resistance by modifying the antibiotic regimen used.

Philadelphia (Ph) chromosome-positive thrombocythemia without features of chronic myeloid leukemia in peripheral blood: natural history and diagnostic differentiation from Ph-negative essential thrombocythemia

We have evaluated the clinical symptoms, hematological features, and natural history of 3 cases and 20 reported cases described as Philadelphia chromosome-positive (Ph+) essential thrombocythemia (ET). The presence of increased small mononuclear megakaryocytes in bone marrow smears and biopsy material in patients with pronounced thrombocytosis and no evidence of chronic myeloid leukemia (CML) in peripheral blood appeared to be a diagnostic clue to the diagnosis of Ph+ (essential) thrombocythemia. As compared to cases of reactive thrombocytosis, the megakaryocytes in Ph+ thrombocythemia are smaller than normal ones and typically have hypolobulated round nuclei. This contrasts with the finding of clustered mature and enlarged megakaryocytes in Ph-negative true ET. Patients diagnosed as Ph+ ET may progress to CML and show a high tendency to myelofibrosis and blastic transformation. These observations indicate that both Ph+ ET and Ph+ thrombocythemia associated with CML can be regarded as early manifestations of the chronic stable phase of CML.

Classification and Characterization of Hereditary Types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (Unclassifiable) von Willebrand Disease

All variants of type 2 von Willebrand disease (VWD) patients, except 2N, show a defective von Willebrand factor (VWF) protein (on cross immunoelectrophoresis or multimeric analysis), decreased ratios for VWF:RCo/Ag and VWF:CB/Ag and prolonged bleeding time. The bleeding time is normal and FVIII:C levels are clearly lower than VWF:Ag in type 2N VWD. High resolution multimeric analysis of VWF in plasma demonstrates that proteolysis of VWF is increased in type 2A and 2B VWD with increased triplet structure of each visuable band (not present in types 2M and 2U), and that proteolysis of VWF is minimal in type 2C, 2D, and 2E variants that show aberrant multimeric structure of individual oligomers. VWD 2B differs from 2A by normal VWF in platelets, and increased ristocetine-induced platelet aggregation (RIPA). RIPA, which very likely reflects the VWF content of platelets, is normal in mild, decreased in moderate, and absent in severe type 2A VWD. RIPA is decreased or absent in 2M, 2U, 2C, and 2D, variable in 2E, and normal in 2N. VWD 2M is usually mild and characterized by decreased VWF:RCo and RIPA, a normal or near normal VWF multimeric pattern in a low resolution agarose gel. VWD 2A-like or unclassifiable (2U) is distinct from 2A and 2B and typically featured by low VWF:RCo and RIPA with the relative lack of high large VWF multimers. VWD type 2C is recessive and shows a characteristic multimeric pattern with a lack of high molecular weight multimers, the presence of one single-banded multimers instead of triplets caused by homozygosity or double hereozygosity for a mutation in the multimerization part of VWF gene. Autosomal dominant type 2D is rare and characterized by the lack of high molecular weight multimers and the presence of a characteristic intervening subband between individual oligimers due to mutation in the dimerization part of the VWF gene. In VWD type 2E, the large VWF multimers are missing and the pattern of the individual multimers shows only one clearly identifiable band, and there is no intervening band and no marked increase in the smallest oligomer. 2E appears to be less well defined, is usually autosomal dominant, and accounts for about one third of patients with 2A in a large cohort of VWD patients.

Bleeding prophylaxis for major surgery in patients with type 2 von Willebrand disease with an intermediate purity factor VIII-von Willebrand factor concentrate (Haemate-P)

The parameters to diagnose von Willebrand disease (vWD) include factor VIII coagulant activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), von Willebrand factor ristocetin cofactor activity (vWF:RCo), and von Willebrand factor collagen binding activity (vWF:CB). Type 2 vWD is associated with a moderate bleeding diathesis due to low levels of vWF:RCo and vWF:CB as compared with near normal or normal values for FVIII:C and vWF:Ag. As the factor VIII/von Willebrand factor (vWF) concentrate, Haemate-P, is featured by a vWF:RCo/FVIII:C ratio of about 2.2, the recommended loading dose of 50 U/kg FVIII:C followed by 25 U/kg FVIII:C every 12 h for several days for bleeding prophylaxis in type 2 vWD patients undergoing major surgery demonstrated a predicted significant over-treatment reaching vWF:RCo levels above 2 U/ml. Therefore, we restricted Haemate-P substitution for major surgery to one loading dose of 40–50 U/kg FVIII:C (88–110 U/kg vWF:RCo) followed by 15–20 U/kg FVIII:C (33–44 U/kg vWF:RCo) every 12 h for several days and evaluated this strategy in a prospective pharmacokinetic and efficacy study for bleeding prophylaxis in five type 2 vWD patients. Pre-treatment and peak levels (1 h after Haemate-P loading dose) rose from 0.43–0.66 to 1.5–2.5 U/ml for FVIII:C, from 0.23–0.45 to 1.5–2.5 U/ml for vWF:Ag, from 0.10–< 0.20 to 1.5–2.5 U/ml for vWF:RCo, and from < 0.05–0.10 to 1.0–2.0 U/ml for vWF:CB. Mean in vivo recoveries per transfused IU FVIII:C/kg body weight were 3.2% for FVIII:C, 3.9% for vWF:RCo, and 2.8% for vWF:CB. Mean in vivo recoveries per transfused IU vWF:RCo/kg were 1.45% for FVIII:C, 1.7% for vWF:RCo and 1.25% for vWF:CB. The biological half-life times after transfused Haemate-P were about 12 h for both vWF:RCo and vWF:CB. Based on these pharmacokinetic data, we propose to adapt the loading dose factor VIII/vWF concentrate (Haemate-P) to 60–80 U/kg vWF:RCo followed by 30–40 U/kg vWF:RCo every 12 h for no longer than several days (less than 1 week) for bleeding prophylaxis during major surgery or trauma, and to one loading dose of 40–60 U/kg vWF:RCo for minor surgery, trauma or mucotaneous bleedings in patients with type 2 vWD unresponsive to DDAVP.

Intravenous DDAVP and Factor VIII-von Willebrand Factor Concentrate for the Treatment and Prophylaxis of Bleedings in Patients With von Willebrand Disease Type 1, 2 and 3

The current standard set of von Willebrand factor (VWF) parameters used to differentiate type 1 from type 2 VWD include bleeding times (BTs), factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetine cofactor activity (VWF:RCo), VWF collagen binding activity (VWF:CB), ristocetine induced platelet aggregation (RIPA), and analysis of VWF multimers in low and high resolution agarose gels and the response to DDAVP. The BTs and RIPA are normal in asymptomatic carriers of a mutant VWF allele, in dominant type 1, and in recessive type 2N VWD, and this category has a normal response of VWF parameters to DDAVP. The response of FVIII:C is compromised in type 2N VWD. The BTs and RIPA are usually normal in type Vicenza and mild type 2A VWD, and these two VWD variants show a transiently good response of BT and VWF parameters followed by short in vivo half life times of VWF parameters. The BTS are strongly prolonged and RIPA typically absent in recessive severe type 1 and 3 VWD, in dominant type 2A and in recessive type 2C (very likely also 2D) VWD and consequently associated with low or absent platelet VWF, and no or poor response of VWF parameters to DDAVP. The BTs are prolonged and RIPA increased in dominant type 2B VWD, that is featured by normal platelet VWF and a poor response of BT and functional VWF to DDAVP. The BTs are prolonged and RIPA decreased in dominant type 2A and 2U, that all have low VWF platelet, very low VWF:RCo values as compared to VWF:Ag, and a poor response of functional VWF to DDAVP. VWD type 2M is featured by the presence of all VWF multimers in a low resolution agarose gel, normal or slightly prolonged BT, decreased RIPA, a poor response of VWF:RCo and a good response of FVIII and VWF:CB to DDAVP and therefore clearly in between dominant type 1 and 2U. The existing recommendations for prophylaxis and treatment of bleedings in type 2 VWD patients with FVIII/VWF concentrates are mainly derived from pharmocokinetic studies in type 3 VWD patients. FVIII/VWF concentrates should be characterised by labelling with FVIII:C, VWF:RCo, VWF:CB and VWF multimeric pattern to determine their safety and efficacy in prospective management studies. As the bleeding tendency is moderate in type 2 and severe in type 3 VWD and the FVIII:C levels are near normal in type 2 and very low in type 3 VWD patients. Proper recommendations of FVIII/VWF concentrates using VWF:RCo unit dosing for the prophylaxis and treatment of bleeding episodes are proposed and has to be stratified for the severity of bleeding, the type of surgery either minor or major and for type 2 and type 3 VWD as well.

Changing concepts of diagnostic criteria of myeloproliferative disorders and the molecular etiology and classification of myeloproliferative neoplasms: From Dameshek 1950 to Vainchenker 2005 and beyond

The Polycythemia Vera Study Group (PVSG) and WHO classifications distinguished the Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia from the Ph1-negative myeloproliferative neoplasms (MPN) essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (MF) or primary megakaryocytic granulocytic myeloproliferation (PMGM). Half of PVSG/WHO-defined ET patients show low serum erythropoietin levels and carry the JAK2V617F mutation, indicating prodromal PV. The positive predictive value of a JAK2V617F PCR test is 95% for the diagnosis of PV, and about 50% for ET and MF. The WHO-defined JAK2V617F-positive ET comprises three ET phenotypes at clinical and bone marrow level when the integrated WHO and European Clinical, Molecular and Pathological (ECMP) criteria are applied: normocellular ET (WHO-ET), hypercellular ET due to increased erythropoiesis (prodromal PV) and hypercellular ET associated with megakaryocytic granulocytic myeloproliferation (EMGM). Four main molecular types of clonal MPN can be distinguished: JAK2V617F-positive ET and PV; JAK2 wild-type ET carrying the MPL515; mutations in the calreticulin (CALR) gene in JAK2/MPL wild-type ET and MF, and a small proportion of JAK2/MPL/CALR wild-type ET and MF patients. The JAK2V617F mutation load is low in heterozygous normocellular WHO-ET. The JAK2V617F mutation load in hetero-/homozygous PV and EMGM is clearly related to MPN disease burden in terms of splenomegaly, constitutional symptoms and fibrosis. The JAK2 wild-type ET carrying the MPL515 mutation is featured by clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow (WHO-ET), and lacks features of PV. JAK2/MPL wild-type, CALR mutated hypercellular ET associated with PMGM is featured by dense clustered large immature dysmorphic megakaryocytes and bulky (cloud-like) hyperchromatic nuclei, which are never seen in WHO-ECMP-defined JAK2V617F mutated ET, EMGM and PV, and neither in JAK2 wild-type ET carrying the MPL515 mutation. Two thirds of JAK2/MPL wild-type ET and MF patients carry one of the CALR mutations as the cause of the third distinct MPN entity. WHO-ECMP criteria are recommended to diagnose, classify and stage the broad spectrum of MPN of various molecular etiologies.

Relationship between clinical and BK virological response in patients with late hemorrhagic cystitis treated with cidofovir : a retrospective study from the European Group for Blood and Marrow Transplantation

To investigate the relationship between clinical response and modification of BK viremia, we assessed retrospectively 32 cases of hemorrhagic cystitis (HC) after allogeneic hematopoietic SCT that were treated with i.v. cidofovir (CDV). They were 22 men (69%) and 10 women (31%) with a median age of 24 years, range 3–62. The median number of CDV doses was 3, range 1–8, and the treatment lasted for a median of 3 weeks, range 1–10. Clinical improvement of HC was observed in 27 patients (84%). In 12 of 32 episodes (37.5%), BK viremia was determined before every CDV administration and a complete clinical response was observed in 10 of 12 patients (83%), the reduction of BK viremia load being ⩾1 log by 2 weeks after starting CDV. Nephrotoxicity related to CDV was observed in nine patients. Among 26 patients with 100-day follow-up, 4 of 4 patients who had a complete clinical response by 30 days were alive vs 16 of 22 (73%) who did not have the resolution of HC in this time frame. We conclude that in patients with HC, the response to CDV treatment is usually associated with a significant reduction of BK viremia load.