Germline mutations predisposing to diffuse large B-cell lymphoma.

Genetic studies of diffuse large B-cell lymphomas (DLBCLs) in humans have revealed numerous targets of somatic mutations and an increasing number of potentially relevant germline alterations. The latter often affect genes involved in DNA repair and/or immune function. In general, defects in these genes also predispose to other conditions. Knowledge of these mutations can lead to disease-preventing measures in the patient and relatives thereof. Conceivably, these germline mutations will be taken into account in future therapy of the lymphoma. In other hematological malignancies, mutations originally found as somatic aberrations have also been shown to confer predisposition to these diseases, when occurring in the germline. Further interrogations of the genome in DLBCL patients are therefore expected to reveal additional hereditary predisposition genes. Our review shows that germline mutations have already been described in over one-third of the genes that are somatically mutated in DLBCL. Whether such germline mutations predispose carriers to DLBCL is an open question. Symptoms of the inherited syndromes associated with these genes range from anatomical malformations to intellectual disability, immunodeficiencies and malignancies other than DLBCL. Inherited or de novo alterations in protein-coding and non-coding genes are envisioned to underlie this lymphoma.

[A man with FIP1L1/PDGFRA-positive chronic eosinophilic leukemia]. / Een man met FIP1L1-PDGFRA-positieve chronische eosinofiele leukemie.

A 39-year-old man was referred from Surinam to the Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands, for a right ventricular tumour, hypereosinophilia and mild thrombocytopenia. He appeared to have chronic eosinophilic leukaemia that was positive for the 'FIP1-like-1-platelet-derived growth factor receptor alpha' (FIP1L1-PDGFRA) gene. In addition, he had signs of a right ventricular thrombus that had existed for at least 6 months. The patient was treated with oral anticoagulants and the tyrosine kinase inhibitor imatinib. The latter therapy resulted in normalisation of leukocyte count and differential values. After 3 months of therapy, the FIP1L1-PDGFRA fusion transcript was no longer detectable in peripheral blood. After 1 year of follow up, the patient was in complete haematological and molecular remission for chronic eosinophilic leukaemia. The cardiac mass remained unchanged, but caused no haemodynamic problems.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Reactive hemophagocytic syndrome as a presenting feature of Hodgkin's disease.

We report the case of a 60-year-old man with fever of unknown origin, severe pancytopenia, and rapidly developing splenomegaly due to reactive hemophagocytic syndrome and Hodgkin's disease. Reactive hemophagocytic syndrome is often rapidly fatal and, once this diagnosis is considered, an underlying infection or malignancy should be treated promptly. An extensive search of the literature revealed only two other cases of reactive hemophagocytic syndrome and Hodgkin's disease. This is the only reported patient who survived after being diagnosed as having reactive hemophagocytic syndrome and Hodgkin's disease.

BCR-ABL directed immunotherapy: a virtual reality?

Nearly ten years of research on the feasibility of specific immunotherapy targeting the junctional regions of BCR-ABL has considerably increased our knowledge of which MHC alleles might present BCR-ABL peptides, yet has failed to provide us with definite proof of appropriate processing of the hybrid oncoprotein into such antigenic peptides. This paper intends to provide an overview of the current state of affairs as well as to delineate limitations and future directions of this line of research.

A BCR-ABL oncoprotein p210b2a2 fusion region sequence is recognized by HLA-DR2a restricted cytotoxic T lymphocytes and presented by HLA-DR matched cells transfected with an Ii(b2a2) construct.

Peptides corresponding to the fusion site in 210 kD BCR-ABL protein b3a2 (p210b3a2) were previously shown to bind to several HLA class I and II alleles. We have found that b3a2 peptide-specific CD4-positive T-helper cells were able to recognize p210b3a2-positive chronic myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until now, there were no reports of b2a2 breakpoint-specific human T-cell responses. Here we show that repetitive stimulation of T lymphocytes with a 17mer peptide covering the fusion region in p210b2a2 also leads to specific T-cell responses. CD4 and CD4/CD8 double-positive clones obtained from a b2a2 peptide-specific cell line were cytotoxic and proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous Epstein-Barr virus (EBV) transformed cells, expressing BCR-ABL(b2a2) on transfection, and allogeneic HLA-DR matched p210b2a2-positive cells from CML patients were, however, not lysed. BCR-ABL peptide-specific T-cell clones did respond to autologous EBV cells transfected with invariant chain (li) cDNA in which the HLA class II-associated invariant chain peptide (CLIP) was replaced by a BCR-ABL b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABL(b2a2) ligand.

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation.

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.

Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide.

In chronic myeloid leukemia (CML) the classical 9;22 translocation results in a BCR-ABL fusion gene, which encodes chimeric BCR-ABL fusion 210 kD oncoproteins (p210BCR-ABL). The two main p210BCR-ABL fusion variants in CML, b2a2 and b3a2 are examples of well characterized antigens expressed by malignant cells. The possibility of an immunotherapeutic approach involving the fusion part of p210BCR-ABL in CML has previously been illustrated by observed peptide binding to major histocompatibility complex (MHC) class I alleles and by demonstrating the immunogenicity of p210BCR-ABL breakpoint peptides. In this report we show that in vitro immunization of human T cells with a 17 amino acid (aa) peptide representing the p210BCR-ABL fusion region resulted in peptide specific CD4+ T-cell lines designated P4, P6, and P7. HLA DR4 (DRB1*0401) restricted T-cell line P4 and several subsequently derived clones recognized HLA-DRB1*0401 and p210b3a2-mRNA expressing blasts from an allogeneic patient with CML in blast crisis. Recognition appeared DR expression-dependent. No responses were observed with DR4 positive p210BCR-ABL negative cells or with p210b3a2 leukemic cells with absent or insufficient expression of DR4. These observations indicate that oncoprotein p210b3a2 can be degraded and processed for presentation by MHC class II molecules at the surface of leukemic cells. The BCR-ABL fusion region is in all likelihood presented as peptides by HLA DR and thus capable to act as a distinctive tumor antigen to peptide specific CD4+ T cells.

Recognition of peptides corresponding to the joining region of p210BCR-ABL protein by human T cells.

In chronic myeloid leukemia (CML) the proto-oncogene c-abl from chromosome 9 q34 is translocated to the breakpoint cluster region (bcr) gene on chromosome 22 q11. This translocation results in a BCR-ABL fusion gene, which encodes chimeric fusion oncoproteins p210BCR-ABL. Here we demonstrate that a peptide with joining region sequence ATGFKQSSKALQRPVAS (eight amino acids (aa) encoded by BCR exon 3; one novel lysine, encoded by the fusion codon; eight aa encoded by ABL exon 2) is immunogenic to human T cells. Primary immune response induction with this peptide resulted in a HLA DR2(DRB1*1501) restricted CD4+ BCR-ABL peptide specific T cell line P1. Responses of P1 were negatively affected by individual aa replacement by alanine at eight aa positions within the 17mer peptide (F4, K5, Q6, K9, L11, Q12, R13, P14). These findings were supported by experiments with a panel of overlapping 11mer b3a2 peptides. Only two of these peptides with an aa sequence encompassing all residues which could not be replaced by alanine induced P1 proliferation. Since presentation of cytosolic oncoproteins as peptides by DR molecules has been observed, the present findings provide a possible explanation for post interferon-alpha persisting remissions in spite of the presence of BCR-ABL PCR positive progenitors.

The role of splenectomy in the treatment of relapsing thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a serious disorder of unknown etiology. Clinical findings are the result of vascular occlusions by platelet aggregates. Treatment with plasma exchange, often used in combination with corticosteroids, vincristine, aspirin, and dipyridamole, has reduced mortality to 20%. Relapses may occur even after long disease-free intervals. In this report we describe our experience with splenectomy in patients with relapsing TTP. Between July 1978 and March 1994, 16 patients with TTP were treated in our hospital. Five of the 13 patients surviving the first episode of TTP had relapses. Most relapses were treated as the first episode of TTP with plasma exchange with fresh-frozen plasma, followed by plasma infusions, corticosteroids, and vincristine. Sometimes aspirin and dipyridamole were added. Splenectomy was performed after five relapses in the first two patients and after two and three relapses in the other patients. Before splenectomy the disease-free interval varied from 3 weeks to 27 months and the incidence rate of relapses was 1.5 relapse/patient/year. None of the patients had relapses after splenectomy. The mean follow-up after splenectomy is 39 months with a range of 9-62 months. We conclude that patients with relapsing TTP can benefit from splenectomy, since it seems to increase disease-free intervals. Further investigation is necessary to understand the role of the spleen in the pathogenesis of TTP.

[Primary treatment of acute promyelocytic leukemia using all-trans-retinoic acid]. / Primaire behandeling van acute promyelocytenleukemie met all-trans-retinoïnezuur.

We describe the medical history of a 68-year-old woman with acute promyelocytic leukaemia. As primary treatment she received all-trans retinoic acid. For the major part this treatment could be given on an outpatient basis. After 60 days there was a complete remission of the leukaemia with disappearance of the characteristic cytogenetic abnormality t(15;17)(q22;q21).

Effectiveness of low dose cyclosporine in acquired aplastic anaemia with severe neutropenia.

Six patients with acquired aplastic anaemia were treated with cyclosporine (5 mg/kg/day) either alone or in combination with corticosteroids. A favourable response was observed in 4, including 2 patients presenting with an absolute granulocyte count of less than 0.2 x 10(9)/l. The 6th patient showed no effect after 6 wk of therapy and was thereafter successfully treated with anti-thymocyte globulin (ATG). Side effects of cyclosporine therapy were minimal (maximum follow-up 20 months). Temporary discontinuation of the drug in 1 patient resulted in a relapse which responded to reinstitution of therapy. Our results indicate that cyclosporine may be an effective, well-tolerated agent in acquired aplastic anaemia even in previously untreated patients with severe neutropenia.

Alloantigenic composition of the endothelial vitronectin receptor.

Endothelial cells synthesize a heterodimeric adhesion molecule, the vitronectin receptor (VnR), which is similar to the platelet glycoprotein (GP)IIb/IIIa complex. The subunits of the endothelial VnR (VnR alpha and GPIIIa) have been studied for their ability to express alloantigens associated with platelet GPIIb and IIIa. We previously showed that endothelial GPIIIa can express the platelet alloantigen Zwa or PIA1, which is associated with GPIIIa. We studied the relationship between the expression of Zwa on platelets and endothelial cells in neonates (n = 13). Using immunoprecipitation and immunofluorescence techniques, we showed that the Zwa antigen is either expressed or absent from both platelets and endothelial cells of the same individual. This finding indicates that in both cell types the same gene is expressed. We also showed that Zwa-negative endothelial cells express Zwb (PIA2), in analogy to Zwa-negative platelets. Moreover, our results strongly suggest expression on endothelial cells of Yukb, a recently described platelet alloantigen, also located on GPIIIa. However, we could not demonstrate expression on the endothelial VnR alpha subunit of Baka, an alloantigen located on platelet GPIIb. These findings are in agreement with the concept that the endothelial GPIIIa subunit is more closely related to its platelet counterpart than to the endothelial VnR alpha subunit.

The platelet alloantigen Zwa or PlA1 is expressed by cultured endothelial cells.

Recently, the synthesis by cultured human endothelial cells of a membrane protein complex immunologically related to platelet glycoprotein (GP) IIb/IIIa complex was demonstrated. Since platelet GP IIIa is known to carry the platelet alloantigen Zwa or PlA1, studies were performed to establish whether this antigen is also expressed on endothelial cells. The present report describes the results of these studies, which provide evidence for the presence of the Zwa or PlA1 antigen on the surface of cultured human endothelial cells. This evidence is based on the following observations: (1) cultured endothelial cells react with anti-Zwa (PlA1) antibodies as shown by indirect immunofluorescence; (2) two proteins are precipitated by anti-Zwa (PlA1) antibodies from lysates of 125I-labelled endothelial cells with an electrophoretic mobility corresponding with that of GP IIb and IIIa; (3) anti-Zwa (PlA1) reacts specifically, as shown by immunoblotting of sodium-dodecylsulphate polyacrylamide gels of solubilized endothelial cells, with a protein with a mobility similar to that of platelet GP IIIa.

Normal synthesis and expression of endothelial IIb/IIIa in Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an "endotheliopathy."

Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation.

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A-peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.

Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex.

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S-methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.