Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

In patients with thrombotic thrombocytopenic purpura (TTP), excessive intravascular platelet aggregation has been associated with appearance in plasma of unusually large von Willebrand factor (vWF ) multimers. These extremely adhesive vWF multimers may arise due to deficiency of a “depolymerase” cleaving vWF to smaller molecular forms, either by reducing the interdimeric disulfide bridges or by proteolytic degradation. We studied the activity of a recently described vWF-cleaving protease in four patients with chronic relapsing TTP. Diluted plasma samples of TTP patients were incubated with purified normal human vWF in the presence of a serine protease inhibitor, at low ionic strength, and in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. Four patients, that included two brothers, with chronic relapsing TTP displayed either substantially reduced levels or a complete absence of vWF-cleaving protease activity. In none of these patient plasmas was an inhibitor of or an antibody against the vWF-cleaving protease established. Our data suggest that the unusually large vWF multimers found in TTP patients may be caused by deficient vWF-cleaving protease activity. Deficiency of this protease may be inherited in an autosomal recessive manner and seems to predispose to chronic relapsing TTP. The assay of the vWF-cleaving protease activity may be used as a sensitive diagnostic tool for identification of subjects with a latent TTP tendency.

Purpose: The recognition of a number of leukemiaspecific cytogenetic abnormalities and their role as independent prognostic factors have provided considerable insights into leukemia pathogenesis and have paved the way to adopt risk-adapted treatment. However, f 50% of newly diagnosed acute myeloid leukemia (AML) have a normal karyotype. There has therefore been much interest in identifying molecular markers that could help to improve the prognostic stratification of patients with normal-karyotype AML.Experimental Design: Consecutive untreated AML patients (n = 67) from a single institution all with normal karyotype were analyzed for the presence of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/ enhancer binding protein-A), for internal tandem duplications (ITD) of the tyrosine kinase receptor gene FLT3 (for fms-like tyrosine kinase 3), and for expression of the BAALC gene (for brain and acute leukemia, cytoplasmic).Results: 17.9% of normal-karyotype AML had mutations in the CEBPA gene, and 28.4% had FLT3-ITD; 65.7% (44 of 67) had high BAALC expression and 34.3% (23 of 67) had low BAALC expression. Patients with CEBPA mutations had a very favorable course of their disease. Median diseasefree survival (DFS) and overall survival (OS) were 33.5 and 45.5 months, respectively, compared with 10 (e.g., 12 months in patients without CEBPA mutations; P = 0.0017; P = 0.0007). AML patients with FLT3-ITD had significantly shorter median DFS (P = 0.0328) and OS (P = 0.0148) than patients without FLT3-ITD. High BAALC expression predicted for a shorter DFS (P = 0.0152) and OS (P = 0.0210) compared with AML with low BAALC expression; 53.7% of normal-karyotype AML had neither FLT3-ITD nor CEBPA mutations. We found that high BAALC expression in normalkaryotype AML with neither FLT3-ITD nor CEBPA mutations (18 of 67) indicates adverse prognosis for both DFS and OS (P = 0.0001; e.g., P = 0.0001) compared with the group with low BAALC expression and absent FLT3-ITD and CEBPA mutations (18 of 67). Thus, BAALC expression represents a novel prognostic marker particularly for normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations.Conclusions: Assessment of CEBPA mutations, FLT3-ITD, and BAALC expression permits to split normalkaryotype AML into clinically distinct subgroups.

Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease with a characteristic chronic phase (cp) of several years before progression to blast crisis (bc). The immune system may contribute to disease control in CML. We analyzed leukemia-specific immune responses in cpCML and bcCML in a retroviral-induced murine CML model. In the presence of cpCML and bcCML expressing the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen, leukemia-specific cytotoxic T lymphocytes (CTLs) became exhausted. They maintained only limited cytotoxic activity, and did not produce interferon-␥ or tumor necrosis factor-␣ or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking the PD-1/PD-L1 interaction by generating bcCML in PD-1-deficient mice or by repetitive administration of ␣PD-L1 antibody prolonged survival. In addition, we found that PD-1 is up-regulated on CD8 ؉ T cells from CML patients. Taken IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. 1 The disease is bi-or triphasic, comprising a chronic, an accelerated, and a terminal blast phase in which the patients develop an acute leukemia of either myeloid (AML) or, less often, lymphoid (ALL) cell type. More than 90% of all CML cases are associated with the presence of the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22 forming the breakpoint cluster region/ Abelson protein tyrosine kinase (BCR/ABL) fusion protein, a constitutively activated tyrosine kinase. 2,3 Depending on the precise breakpoints in the BCR gene, different forms of BCR/ABL fusion protein with different molecular weights can be generated (p190BCR/ABL, p210BCR/ABL, and p230BCR/ABL). CML patients predominantly express p210BCR/ABL. 1 Currently, BCR/ABL-selective tyrosine kinase inhibitors are the standard treatment for CML. However, resistant clones often develop during treatment. At present, the only curative treatment for CML is allogeneic hematopoietic stem cell transplantation. 4 Several earlier studies suggested that the immune system plays an important role in the control of CML. CML cells are susceptible to lysis by CD8 ϩ T cells 5 and natural killer (NK) cells in vitro. 6 For unknown reasons, CML is the most graft-versus-leukemiasensitive leukemia. 7 In addition, cytotoxic T lymphocytes (CTLs) directed against leukemia antigens are found in CML patients without hematopoietic stem cell transplantation, including CTLs specific for BCR/ABL, overexpressed self-proteins such as proteinase-3, and Wilms tumor 1 protein. 5,8 However, the physiologic relevance of these leukemia-specific CTL responses in the control of CML is unknown. The presence of CTL escape mechanisms during CML disease progression to blast crisis suggests that CTLs are involved in the control of the chronic phase of the disease. ...

Plasma of patients with thrombotic thrombocytopenic purpura (TTP) has been shown to contain unusually large von Willebrand factor (vWF) multimers that may cause platelet agglutination in vivo. Fresh frozen plasma infusions and plasma exchange represent the most efficient therapy of acute TTP. A specific protease responsible for cleavage of vWF multimers has been recently isolated from normal human plasma and was found to be deficient in four patients with chronic relapsing TTP. We examined the activity of the vWF-cleaving protease in plasma samples collected over a period of 400 days from a further patient with recurrent episodes of TTP who was treated by plasma exchange, plasma infusion, vincristine, corticosteroid therapy, and splenectomy. Complete deficiency of the vWF-cleaving protease was established during the first episode of TTP. The ensuing normalization of the platelet count was associated with the appearance of the protease activity. Three months after remission from the initial TTP event, the vWF-cleaving protease again disappeared and the platelet count gradually decreased. Relapses of severe thrombocytopenia occurred 7 and 11 months after the first acute episode of TTP. Deficient protease activity was associated with the presence in the patient plasma of an inhibitor that was found to be an IgG. Plasma exchange/infusion was followed by a temporary increase in the antibody titer, whereas treatment with vincristine led to a recovery of the platelet count without affecting the inhibitor concentration. Splenectomy and corticosteroid treatment resulted in disappearance of the autoantibody and normalization of the protease activity and of the platelet count. Our data suggest that the thrombocytopenia in this patient with TTP was associated with a lack of the vWF-cleaving protease activity depleted by an autoimmune mechanism. This case, together with our previously reported patients, leads us to conclude that acquired as well as constitutional deficiency of the vWF-cleaving protease may predispose to TTP.