Combined Results of Two Cross-Sectional Surveys on the Participation in Clinical Trials and the e-Consent Procedure in the Landscape of Haematology.

Despite the motivation of oncology patients to take part in clinical trials, only a minority of them are enrolled in clinical trials. Implementation of new practical procedures can become a barrier that withholds patients from participating in clinical trials. Treating physicians are crucial in augmenting trial accrual. The drivers that promote physicians to allocate patients for clinical trials need further assessment. We conducted two separate cross-sectional surveys, addressing patients with a haematological disease in one survey and haematologists in another survey. The patient survey was filled out by 420 patients. Significant relationships between the willingness to participate in a trial and trial knowledge (p < 0.001) and between doctor-patient relationship and participation willingness (p = 0.007) were noted. Patients above 60 years were less willing to use an electronic consent procedure vs. patients younger than 60 (p < 0.001). The physician questionnaire was completed by 42 participants of whom most (83%) were active in and (94%) motivated for clinical trials. Apart from the patient benefit and scientific interest, prestige was an equal motivator closely followed by financial remunerations. First goal was not to harm the patient. Our study confirms the high willingness of patients for trial participation and the need to rethink the structure of trial organisation. The e-consent procedure is not the method preferred by most patients above 60 years old.

Disease Perception Is Correlated with Health-Related Quality of Life in Patients Suffering from Myelodysplastic Syndromes: Results of the Belgian Be-QUALMS Study.

Patients with myelodysplastic syndromes suffer from an impaired quality of life that is only partially explained by physical symptoms. In an observational study, we aimed to investigate the impact of current MDS treatments and the influence of disease perception on quality of life. Serial measurement of health-related quality of life was performed by 'the QUALMS', a validated MDS-specific patient reported outcome tool. Disease perception was evaluated by means of the Brief Illness Perception Questionnaire (B-IPQ). We prospectively collected data on 75 patients that started on a new treatment and could not demonstrate a significant change in QUALMS score or B-IPQ score during treatment. Six out of eight items evaluated in the B-IPQ correlated significantly with QUALMS score. In this small sample, no significant difference in QUALMS score was found between lower vs. higher risk MDS patients or other studied variables, e.g., targeted hemoglobin showed no correlation with QUALMS score. In daily practice attention must be paid to initial formation of disease perception as it correlates independently with health-related quality of life and does not change during treatment (clinicaltrials.gov identifier: NCT04053933).

Comparison of the Power of Procalcitonin and C-Reactive Protein to Discriminate between Different Aetiologies of Fever in Prolonged Profound Neutropenia: A Single-Centre Prospective Observational Study.

Management of fever in prolonged, profound neutropenia remains challenging with many possible infectious and non-infectious causes. We investigated whether procalcitonin (PCT) is superior to C-reactive protein (CRP) in discriminating between different aetiologies of fever in this setting. CRP and PCT were tested daily during 93 neutropenic episodes in 66 patients. During this study period, 121 febrile episodes occurred and were classified into four categories based on clinical and microbiological findings: microbiologically documented infection (MDI); clinically documented infection (CDI); proven or probable invasive fungal disease (IFD); fever of unknown origin (FUO). Values of PCT and CRP at fever onset as well as two days later were considered for analysis of their performance in distinguishing aetiologies of fever. At fever onset, no significant difference in PCT values was observed between different aetiologies of fever, whereas median CRP values were significantly higher in case of IFD (median 98.8 mg/L vs 28.8 mg/L, p=0.027). Both PCT and CRP reached their peak at a median of 2 days after fever onset. Median PCT values on day 2 showed no significant difference between the aetiologies of fever. Median CRP values on day 2 were significantly higher in IFD (median 172 mg/L versus 78.4 mg/L, p=0.002). In MDI median CRP values rose > 100 mg/L, whereas they did not in CDI or FUO. PCT has no added value over CRP for clinical management of fever in prolonged, profound neutropenia. When performing reassessment 2 days after fever onset, CRP has better discriminatory power between aetiologies of fever.

A predictive model for risk of early grade ≥ 3 infection in patients with multiple myeloma not eligible for transplant: analysis of the FIRST trial.

Infections are a major cause of death in patients with multiple myeloma. A post hoc analysis of the phase 3 FIRST trial was conducted to characterize treatment-emergent (TE) infections and study risk factors for TE grade ≥ 3 infection. The number of TE infections/month was highest during the first 4 months of treatment (defined as early infection). Of 1613 treated patients, 340 (21.1%) experienced TE grade ≥ 3 infections in the first 18 months and 56.2% of these patients experienced their first grade ≥ 3 infection in the first 4 months. Risk of early infection was similar regardless of treatment. Based on the analyses of data in 1378 patients through multivariate logistic regression, a predictive model of first TE grade ≥ 3 infection in the first 4 months retained Eastern Cooperative Oncology Group performance status and serum ß2-microglobulin, lactate dehydrogenase, and hemoglobin levels to define high- and low-risk groups showing significantly different rates of infection (24.0% vs. 7.0%, respectively; P < 0.0001). The predictive model was validated with data from three clinical trials. This predictive model of early TE grade ≥ 3 infection may be applied in the clinical setting to guide infection monitoring and strategies for infection prevention.

A Belgian consensus protocol for autologous hematopoietic stem cell transplantation in multiple sclerosis.

Multiple sclerosis is considered to be an immune mediated inflammatory disorder of the central nervous system. It mainly affects young, socioeconomic active patients. Although our armamentarium for this disease has significantly evolved in recent years some patients remain refractory to conventional therapies. In these cases, autologous hematopoietic stem cell transplantation can be considered as a therapeutic option. Decreasing morbidity, mortality, and increasing patient awareness have led to rising inquiry by our patients about this treatment option. With the aim of a standardized protocol and data registration, a Belgian working party on stem cell therapy in multiple sclerosis was established. In this paper, we report the consensus protocol of this working party on autologous hematopoietic stem cell transplantation in multiple sclerosis.

Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia.

Relapse is a major problem in acute myeloid leukemia (AML) and adversely affects survival. In this phase 2 study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms' tumor 1 (WT1) messenger RNA (mRNA) as postremission treatment in 30 patients with AML at very high risk of relapse. There was a demonstrable antileukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which were sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in nonresponders (53.8% vs 25.0%; P = .01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25%, and 5-year relapse-free survival was higher in responders than in nonresponders (50% vs 7.7%; P < .0001). In patients age ≤65 and >65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared with 51.7% and 18% in the Swedish Acute Leukemia Registry. Long-term clinical response was correlated with increased circulating frequencies of polyepitope WT1-specific CD8+ T cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed-type hypersensitivity-infiltrating CD8+ T lymphocytes. In conclusion, vaccination of patients with AML with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224.

Adult acute precursor B-cell lymphoblastic leukemia presenting as hypercalcemia and osteolytic bone lesions.

BACKGROUND: Osteolytic bone lesions and hypercalcemia without peripheral blasts B-cell acute lymphoblastic leukemia (B-ALL) is reported in children but rarely seen in adults. CASE PRESENTATION: We describe the case of a 34-year old man presenting with hypercalcemia and symptomatic osteolytic bone lesions of vertebrae and ribs who was initially suspected as having a solid malignancy. Diagnostic work-up including peripheral blood examination, radiographic and nuclear studies could, however, not detect a primary tumor. Because of a mild thrombocytopenia and the lack of a primary focus, a bone marrow biopsy was performed leading to the diagnosis of Philadelphia chromosome positive precursor B-ALL. After correction of the hypercalcemia with intravenous fluid administration, corticoids and bisphosphonates, the patient was treated according to the HOVON 100 protocol achieving complete molecular remission after the first cycle of induction chemotherapy. CONCLUSION: Hypercalcemia and osteolytic bone lesions are rare complications of adult B-ALL and can occur in the absence of peripheral blastosis. With this case report we would like to emphasize the importance of clinical awareness. Immediate treatment of hypercalcemia and initiation of antileukemic treatment is mandatory as a delay of diagnosis might pose a real and possible life-threatening risk in these patients.

Induction of cytomegalovirus-specific T cell responses in healthy volunteers and allogeneic stem cell recipients using vaccination with messenger RNA-transfected dendritic cells.

BACKGROUND: Infection with human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in solid organ and hematopoietic stem cell transplant (HSCT) recipients. METHODS: The present study explored the safety, feasibility, and immunogenicity of CMV pp65 messenger RNA-loaded autologous monocyte-derived dendritic cells (DC) as a cellular vaccine for active immunization in healthy volunteers and allogeneic HSCT recipients. Four CMV-seronegative healthy volunteers and three allogeneic HSCT recipients were included in the study. Four clinical-grade autologous monocyte-derived DC vaccines were prepared after a single leukapheresis procedure and administered intradermally at a weekly interval. RESULTS: De novo induction of CMV-specific T-cell responses was detected in three of four healthy volunteers without serious adverse events. Of the HSCT recipients, none developed CMV disease and one of two patients displayed a remarkable threefold increase in CMV pp65-specific T cells on completion of the DC vaccination trial. CONCLUSION: In conclusion, our DC vaccination strategy induced or expanded a CMV-specific cellular response in four of six efficacy-evaluable study subjects, providing a base for its further exploration in larger cohorts.

Clinical and microbiological impact of discontinuation of fluoroquinolone prophylaxis in patients with prolonged profound neutropenia.

BACKGROUND: Infections remain a leading cause of morbidity and mortality in patients with reduced immunity caused by haematological disease and chemotherapy-induced neutropenia. We evaluated the clinical and microbiological impact of discontinuing fluoroquinolone prophylaxis in these patients. METHODS: We analysed 154 admissions in three sequential periods of 8 months: long-standing use, discontinuation of prophylaxis and reintroduction of prophylaxis. Clinical endpoints were occurrence of febrile neutropenia, bacteraemia, severe sepsis, septic shock, response to antibiotic therapy, total antibiotic consumption and duration of hospital stay. Microbiological analysis included bacterial isolates from stool and blood cultures and their resistance pattern. RESULTS: No significant increase in serious infectious complications was seen with the discontinuation of prophylaxis. The overall incidence of bacteraemia did not change, but a higher proportion of bacterial isolates were Gram-negative (22.2% vs. 5.9% & 8.6%; P = 0.030), more often multisusceptible (50% vs. 0%) and less fluoroquinolone resistant (10% vs. 100%). Screening of stools showed a higher prevalence of organisms in the discontinuation period (86.7% vs. 37.3% & 55.2%; P ≤ 0.001), but they were more frequently multisusceptible (53.8% vs. 10.5% & 6.3%; P ≤ 0.001). After discontinuation of prophylaxis, fluoroquinolone resistance decreased rapidly from 73.7 to 7.7%, in association with a significant decrease in extended spectrum beta-lactamase (ESBL)-producing isolates from 42.1 to 10.3%. Resistance figures immediately returned to prediscontinuation values after reinstitution of prophylaxis. CONCLUSIONS: No clinically relevant short-term drawbacks were observed with the discontinuation of fluoroquinolone prophylaxis in patients with chemotherapy-induced prolonged profound neutropenia, which led to a significant decrease in fluoroquinolone resistance as well as occurrence of ESBL-producing isolates.

Adequate iron chelation therapy for at least six months improves survival in transfusion-dependent patients with lower risk myelodysplastic syndromes.

BACKGROUND: Most patients with myelodysplastic syndromes (MDS) require transfusions at the risk of iron overload and associated organ damage, and death. Emerging evidence indicates that iron chelation therapy (ICT) could reduce mortality and improve survival in transfusion-dependent MDS patients, especially those classified as International Prognostic Scoring System (IPSS) Low or Intermediate-1 (Low/Int-1). METHODS: Follow-up of a retrospective study. Sample included 127 Low/Int-1 MDS patients from 28 centers in Belgium. Statistical analysis stratified by duration (≥6 versus <6 months) and quality of chelation (adequate versus weak). RESULTS: Crude chelation rate was 63% but 88% among patients with serum ferritin ≥1000 µg/L. Of the 80 chelated patients, 70% were chelated adequately mainly with deferasirox (26%) or deferasirox following deferoxamine (39%). Mortality was 70% among non-chelated, 40% among chelated, 32% among patients chelated ≥6 m, and 30% among patients chelated adequately; with a trend toward reduced cardiac mortality in chelated patients. Overall, median overall survival (OS) was 10.2 years for chelated and 3.1 years for non-chelated patients (p<0.001). For patients chelated ≥6 m or patients classified as adequately chelated, median OS was 10.5 years. Mortality increased as a function of average monthly transfusion intensity (HR=1.08, p=0.04) but was lower in patients receiving adequate chelation or chelation ≥6 m (HR=0.24, p<0.001). CONCLUSION: Six or more months of adequate ICT is associated with markedly better overall survival. This suggests a possible survival benefit of ICT in transfusion-dependent patients with lower-risk MDS.

Determining factors predictive of CD34+ cell collection efficiency in an effort to avoid extended and repeated apheresis sessions.

INTRODUCTION: Collection efficiency (CE) is a reflection of the proportion of cells passing through a cell separator that is harvested. The aim of our study was to evaluate which factors influence CE independently in order to find ways to improve CE and therefore minimize the costs and risks of leukapheresis and graft processing. MATERIALS AND METHODS: A total of 206 consecutive apheresis procedures performed on 128 donors/patients were studied retrospectively. We explored the association between CE and the following factors: age, sex, weight, mobilization (granulocyte-colony-stimulating factor with or without chemotherapy), collection type (autologous versus allogeneic), venous access (peripheral versus central), total processed blood volume (TPV), hematocrit, white blood cell (WBC) count, thrombocyte count, and peripheral blood CD34+ cell concentration (PBCD34+). RESULTS: Stepwise multiple regression analysis showed WBC count to be the single best predictor of CE, accompanied by TPV. When performing subgroup analysis for autologous apheresis procedures, the inverse correlation of WBC count and TPV with CE becomes stronger (r = -0.563 with P < 0.001 and r = -0.198 with P = 0.020 respectively), whereas those correlations disappear when analyzing only allogeneic apheresis procedures. CONCLUSION: The negative correlation between TPV and CE present only in autologous collection procedures can be explained by the limited intra-apheresis recruitment of CD34+ cells into the blood which is negatively influenced by extensive pre-treatment. As a result of this study we decided to limit TPV to a maximum of three times the patient's blood volume in autologous apheresis procedures at our center.

mRNA-based dendritic cell vaccination induces potent antiviral T-cell responses in HIV-1-infected patients.

BACKGROUND: In an effort to raise protective antiviral immunity, dendritic cell immunotherapy was evaluated in six adults infected with human immunodeficiency virus (HIV)-1 and stable under highly active antiretroviral therapy (HAART). DESIGN AND METHODS: Autologous monocyte-derived dendritic cells electroporated with mRNA encoding Gag and a chimeric Tat-Rev-Nef protein were administered, whereas patients remained on HAART. Feasibility, safety, immunogenicity and antiviral responses were investigated. RESULTS: Dendritic cell vaccine preparation and administration were successful in all patients and only mild adverse events were seen. There was a significant increase post-dendritic cell as compared to pre-dendritic cell vaccination in magnitude and breadth of HIV-1-specific interferon (IFN)-γ response, in particular to Gag, and in T-cell proliferation. Breadth of IFN-γ response and T-cell proliferation were both correlated with CD4(+) and CD8(+) polyfunctional T-cell responses. Importantly, dendritic cell vaccination induced or increased the capacity of autologous CD8(+) T cells to inhibit superinfection of CD4(+) T cells with the vaccine-related IIIB virus and some but not all other HIV-1 strains tested. This HIV-1-inhibitory activity, indicative of improved antiviral response, was correlated with magnitude and breadth of Gag-specific IFN-γ response. CONCLUSIONS: Therapeutic immunization with dendritic cells was safe and successful in raising antiviral cellular immune responses, including effector CD8(+) T cells with virus inhibitory activity. The stimulation of those potent immunological and antiviral effects, which have been associated with control of HIV-1, underscores the potential of dendritic cell vaccination in the treatment of HIV-1. The incomplete nature of the response in some patients helped to identify potential targets for future improvement, that is increasing antigenic spectrum and enhancing T-cell response.

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-gamma-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.

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial.

BACKGROUND AIMS: RNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients. METHODS: CD14(+) cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients. RESULTS: In a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14(+) cells (94.5+/-3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25+/-10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients. CONCLUSIONS: Our method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.

Immunotherapy of hematological malignancies using dendritic cells.

The arsenal of therapeutic weapons against hematological malignancies is constantly growing. Unravelling the secrets of tumor immunobiology has allowed researchers to manipulate the immune system in order to stimulate tumor immunity or to bypass tumor-induced immunosuppression. An area of great interest is active specific immunotherapy where dendritic cell (DC)-based therapeutic vaccines for cancer have definitely grabbed the spotlight. DC are intensively investigated as cellular adjuvants to harness the immune system to fight off cancer by augmenting the number and effector functions of tumor-specific CD8+ cytotoxic T lymphocytes. In the present review we present a comprehensive synopsis and an update of the use of DC in hematological malignancies. In the future, more basic research as well as more clinical trials are warranted to fully establish the value of DC vaccination as an adjuvant therapy for modern hematological oncology.

Characterization of recessive severe type 1 and 3 von Willebrand Disease (VWD), asymptomatic heterozygous carriers versus bloodgroup O-related von Willebrand factor deficiency, and dominant type 1 VWD.

Recessive type 3 von Willebrand disease (VWD) is caused by homozygosity or double heterozygosity for two non-sense mutations (null alleles). Type 3 VWD is easy to diagnose by the combination of a strongly prolonged bleeding time (BT), absence of ristocetine-induced platelet aggregation (RIPA), absence of von Willebrand factor (VWF) protein, and prolonged activated partial thromboplastin time (aPTT) due to factor VIII:coagulant (FVIII:C) deficiency. VWD type 3 is associated with a pronounced tendency to mucocutaneous and musculoskeletal bleedings since early childhood. Carriers of one null allele are usually asymptomatic at VWF levels of 50% of normal. Recessive severe type 1 VWD is caused by homozygosity or double heterozygosity for a missense mutation. Recessive type 1 VWD differs from type 3 VWD by the presence of detectable von Willebrand factor: antigen VWF:Ag and FVIII:C levels between 0.09 and 0.40 U/mL. Patients with recessive type 1 VWD show an abnormal VWF multimeric pattern in plasma and/or platelets consistent with severe type 2 VWD. Carriers of a missense mutation may have mild bleeding and mild VWF deficiency and can be diagnosed by a double VWF peak on cross immunoelectrophoresis (CIE). There will be cases of mild and moderate recessive type 1 VWD due to double heterozygosity of two missense mutations, or with the combination of one missense mutation with a non-sense or bloodgroup O. Mild deficiency of VWF in the range of 0.20 to 0.60 U/mL, with normal ratios of von Willebrand factor: ristocetine cofactor/antigen VWF:RCo/Ag and VWF:collagen binding/antigen (VWF:CB/Ag), normal VWF multimers, and a completely normal response to desmopressin acetate (DDAVP) with VWF level rising from below to above 1.00 U/mL are very likely cases of so-called pseudo-VWF deficiency in individuals with normal VWF protein and gene. Autosomal dominant type 1 VWD variants are in fact type 2 variants caused by a heterozygous missense mutation in the VWF gene that produces a mutant VWF protein that has a dominant effect on normal VWF protein produced by the normal VWF allele with regard to the synthesis, processing, storage, secretion, and/or proteolysis of VWF in endothelial cells. A DDAVP challenge test clearly differentiates between dominant type 1 VWD phenotype and dominant type 2 M VWD.

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.

Characterization, classification, and treatment of von Willebrand diseases: a critical appraisal of the literature and personal experiences.

Recessive type 3 von Willebrand disease (vWD) is a severe hemophilia-like bleeding disorder caused by homozygosity or double heterozygosity for two nonsense mutations (null alleles) and characterized by a strongly prolonged bleeding time (BT), absence of ristocetin-induced platelet aggregation (RIPA), absence of von Willebrand factor (vWF) protein, and prolonged activated partial thromboplastin time (APTT) due to factor VIII (FVIIIC): deficiency. Recessive severe type 1 vWD is caused by homozygosity or double heterozygosity for a missense mutation and differs from type 3 vWD by the detectable presence vWF:antigen (Ag) and FVIII:C levels between 0.09 and 0.40 U/mL. Carriers of one null allele or missense mutations are usually asymptomatic at vWF levels of 50% of normal. Mild recessive type 1 vWD may be due to a missense mutations, or one missense mutation plus blood group O. The so-called dominant type 1 vWD secretion defect and type 1 Vicenza are caused by a heterozygous missense mutation in the vWF gene that produces a mutant vWF protein having a dominant effect on the normal vWF protein produced by the normal vWF allele with regard to the defective processing, storage secretion, and/or proteolysis of vWF in endothelial cells and clearing from plasma consistent with a type 2 phenotype of vWD. Typical type 2 vWD patients, except 2N, show a defective vWF protein, decreased ratios for vWF:ristocetin cofactor [vWF:RCo]/vWF:Ag and vWF:collagen binding factor [vWF:CB]/vWF:Ag and prolonged BT. The BT is normal and FVIII:C levels clearly are lower than vWF:Ag in type 2N vWD. 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 band (not present in types 2M and 2U). 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 RIPA. RIPA 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 and dominant type 1. vWD 2M is usually mild and features decreased vWF:RCo and RIPA, and 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 features low vWF:RCo and RIPA with the relative lack of large vWF multimers. vWD type 2C is recessive; the dominant type 2D is rare. The response to desmopressin acetate (DDAVP) of vWF parameters is normal in pseudo-vWD and mild type 1. The responses to DDAVP of FVIII:C and vWF parameters in vWD 2M, Vincenza, 2E, and mild 2A, 2U, and 2N are transiently good for a variable number of hours to arrest mucocutaneous bleeding episodes or to prevent bleeding during minor surgery or trauma. However, the responses are not good enough to treat major bleedings or to prevent bleeding during major surgery or trauma. The response to DDAVP of vWF parameters is poor in recessive type 3, 1 and 2C, and dominant 2A, 2B, and 2U. Proper recommendations of FVIII/vWF concentrates using FVIII:C and vWF:RCo unit dosing for the prophylaxis and treatment of bleeding episodes in type 2 disease that is nonresponsive to DDAVP and in type 3 vWD are proposed.