bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of platelet FcγRIIA polymorphism in refractory idiopathic (immune) thrombocytopenic purpura
Authors: Williams Yvonne , Lynch Sara , McCann Shaun , Smith Owen , Feighery Conleth , Whelan Alex
Published: March 2003

FcγRIIA, a low affinity receptor for IgG, is a polymorphic molecule: FcγRIIA‐HH131, FcγRIIA‐HR131 and FcγRIIA‐RR131. This polymorphism influences the efficiency of the receptor to bind with IgG2. Recent reports on altered distribution amongst individuals with heparin‐induced thrombocytopenia (HIT) prompted us to examine the FcγRIIA polymorphism in a cohort of patients with refractory idiopathic (immune) thrombocytopenic purpura (ITP), in whom severe disease had required them to undergo splenectomy. 29 post splenectomy ITP individuals and 61 normal controls were investigated. Polymerase chain reaction (PCR) and a Southern blotting technique were used to determine the FcγRII polymorphism. Although the distribution of the allotypes of FcγRIIA in the control population was similar to that found in other European studies of Caucasian populations (15 (25%) HH131; 35 (57%) HR131; 11(18%) RR131), the patient group was skewed towards the RR131 allotype which has least efficiency for IgG2 binding (3 (10%) HH131; 12 (42%) HR131; 14 (48%) RR131; P < 0.005). These findings suggest that FcγRIIA polymorphisms may be implicated in the pathophysiology of ITP or may be responsible for modulating the immune response in this heterogenous autoimmune disease.
TAL1 expression does not occur in the majority of T‐ALL blasts
Authors: Delabesse Eric , Bernard Muriel , Meyer ValÉrie , Smit Linda , Pulford Karen , Cayuela Jean‐Michel , Ritz Jermoe , Bourquelot Priscille , Strominger Jack L. , Valensi FranÇoise , Macintyre Elizabeth A.
Published: March 2003

The TAL1 gene is disrupted by translocation or deletion (tald) in up to 30% of T‐cell acute lymphoblastic leukaemia (T‐ALL), leading to aberrant transcriptional activation, as a SIL‐TAL1 fused transcript in tald. It has been suggested that TAL1 transcription occurs in approximately 50% of a T‐ALLs without apparent rearrangement. SIL‐TAL1 was positive in 15/60 (25%) of T‐ALL, whereas wild‐type TAL1 transcripts were detected in all 13 SIL‐TAL1 and in 19/43 (44%) T‐ALL without SIL‐TAL1. To investigate the cellular origin of TAL1 we exploited the fact that GATA1 and TAL1 are co‐ordinately expressed in non‐lymphoid haemopoietic cells, whereas only the latter is found in T‐ALL. GATA1 was detected in 10/23 (43%) TAL1‐negative T‐ALLs but in 17/19 (89%) ‘unexplained’TAL1‐positive cases, suggesting a common non‐lymphoid cellular origin. Immunocytochemical analysis with a TAL1‐specific monoclonal antibody showed nuclear expression in the blasts of 10/34 (29%) cases, including 8/10 SIL‐TAL1+ and two RT‐PCR TAL1+, SIL‐TAL1− cases. In the remaining cases TAL1 expression was restricted to a minor population (< 5%) of larger, strongly TAL1‐positive cells which comprised erythroid cells, CD34+CD3− precursors and an unidentified TAL1+CD45− population which morphologically resembled monocytes/macrophages. We therefore suggest that appropriate diagnostic evaluation of T‐ALL should include molecular detection of SIL‐TAL1 transcripts and in situ immunocytochemical detection of TAL1 protein expression by leukaemic blasts. This approach will enable accurate analysis of the prognostic significance of TAL1 deregulation in T‐ALL.
Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients
Authors: Ambo Hironobu , Kamata Tetsuji , Handa Makoto , Kawai Yohko , Oda Atsushi , Murata Mitsuru , Takada Yoshikazu , Ikeda Yasuo
Published: March 2003

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele‐specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)‐289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)‐324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)‐747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild‐type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild‐type β3 cDNA. Flow cytometric analysis using an anti‐αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild‐type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.
Factor binding to the human γ‐globin gene distal CCAAT site: candidates for repression of the normal gene or activation of HPFH mutants
Authors: Partington  , Patient
Published: March 2003

We have examined factor binding to the distal human γ‐globin CCAAT site and three naturally occurring hereditary persistence of fetal haemoglobin (HPFH) mutations of this site. Factor binding was examined using nuclear extracts from the erythroleukaemic cell lines K562 and MEL, and from A4 cells, a non‐transformed mouse bone marrow stem cell line, using the electrophoretic mobility shift assay. Under standard binding conditions, in addition to the previously reported binding by a CCAAT factor (CP1) and GATA‐1, the wild‐type (wt) sequence bound high mobility factors which appeared to be GATA‐2 isoforms. However, when the non‐specific competitor conditions were varied, the binding profile with K562, but not MEL nuclear extract, was substantially altered. CP1 and GATA‐1 were absent, and two new factors were detected, one of which bound preferentially to the Greek and Japanese non‐deletion HPFH mutants. However, binding by the GATA‐2 isoforms to the wt sequence was maintained with both cell types, as it was using the A4 cell line. With modified binding conditions, in A4 cells the two non‐deletion and the Black deletion HPFH mutants each had a different protein binding profile which was lost on erythroid induction of the cells. We discuss the possiblity that the GATA‐2 isoforms bound to the wt sequence may function to suppress wt γ gene expression in the bone marrow. Additionally, those factors which bind preferentially either to the deletion or non‐deletion HPFH mutants may play positive roles in establishing an active chromatin structure.
Senescent human neutrophil binding to thrombospondin (TSP): evidence for a TSP‐independent pathway of phagocytosis by macrophages
Authors: Murphy Joseph F. , Mcgregor John L. , Leung Lawrence L. K.
Published: March 2003

This study investigated the interaction of apoptotic polymorphonuclear neutrophils (PMN) with thrombospondin (TSP), an important event mediating the clearance of apoptotic neutrophils by macrophages. We developed an in vitro assay to examine this interaction. Based on this assay, we found that apoptotic but not fresh PMN bound specifically to surface‐immobilized TSP (33 ± 0.03 × 103 cells/well) compared to fibrinogen, fibronectin or laminin (8.0 ± 0.3 × 103 cells/well). Moreover, the binding was specific for surface bound but not soluble TSP and appeared to be divalent cation dependent, was not significantly inhibited by heparin and was sensitive to cycloheximide (CHX) treatment of senescent PMN (>90% inhibition at 10 μM CHX). In contrast to the binding studies, phagocytosis of senescent PMN by macrophages was not affected by EDTA or cycloheximide. Phosphatidyl‐
L‐serine liposomes, phospho‐
L‐serine, glucosamine, galactosamine, and the acetylated sugars had no effect on phagocytosis. We conclude that: (i) there was specific binding of senescent human PMN to immobilized TSP, which is divalent cation dependent and requires new protein synthesis in the PMN during senescence; (ii) in addition to the recently defined TSP‐dependent pathway, there is a TSP‐independent pathway mediating phagocytosis of senescent PMN by macrophages. The identity of this pathway remains to be defined.
A comparison of linear and orthogonal regression analysis for local INR determination in ECAA coagulometer studies
Authors: Poller  , Van Den Besselaar  , Jespersen  , Tripodi  , Houghton
Published: March 2003

International sensitivity index calibrations based on the W.H.O. recommended method depend on orthogonal regression analysis. As this is not readily available in statistical packages, comparison has been made with simple linear regression analysis in a study of coagulometer effects on the International Normalized Ratio (INR) at 155 European centres. Sets of seven lyophilized normal and 20 lyophilized artificially depleted abnormal plasmas were provided with five coumarin test plasmas and two European Concerted Action on Anticoagulation reference thromboplastins (low International Sensitivity Index (ISI) human and high ISI rabbit). Local ISI based on the artificially depleted lyophilized plasmas using conventional orthogonal regression gave good correction for local coagulometer effects on the human reagent and minimal correction with the rabbit reagent INR. Results were considerably worse after attempts at correction using calibration based on linear regression analysis with both reagents. The results indicate that calibration of coagulometer prothrombin time systems using simple linear regression is not appropriate.
Association and clonal distribution of trisomy 12 and 13q14 deletions in chronic lymphocytic leukaemia
Authors: Navarro Blanca , GarcÍA‐Marco JosÉ A. , Jones David , Price Cathy M. , Catovsky Daniel
Published: March 2003

The coexistence of trisomy 12 and deletions of chromosome 13 (13q12‐q32) has rarely been observed in chronic lymphocytic leukaemia (CLL). Fluorescence in situ hybridization (FISH) performed on 600 consecutive CLL patients revealed the association of trisomy 12 and 13q14 deletion, of at least one of the three markers analysed (RB1, D13S319 and D13S25), in 55 cases (9% of 600 and 46% of 120 trisomy 12 cases). Trisomy 12 and isolated RB1 deletion were seen in 14/120 cases, trisomy 12 and D13S319/D13S25 deletion with diploid RB1 in 19/118, and trisomy 12 and deletion encompassing the three 13q markers studied in 22/118 cases. The heterogenous distribution of trisomy 12 and 13q deletions within the neoplastic B cells suggests that they are secondary rather than primary events in CLL leukaemogenesis.
Chromosomal breakage analysis in dyskeratosis congenita peripheral blood lymphocytes
Authors: Coulthard Stephanie , Chase Aandrew , Pickard Julie , Goldman John , Dokal Inderjeet
Published: March 2003

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by reticulate skin pigmentation, nail dystrophy and mucosal leucoplakia. Bone marrow failure occurs in the majority of cases and there is a predisposition to malignancy. Following conflicting reports of increased spontaneous and induced chromosomal breakage in DC lymphocytes, we examined chromosomal breakage with and without clastogen treatment in 10 DC patients from six different families. Peripheral blood cultures were stimulated with phytohaemagglutinin and treated with three clastogenic agents and γ‐irradiation. There was no significant difference in the chromosomal breakage in DC lymphocytes with or without exposure to bleomycin, DEB, MMC or γ‐irradiation. DC can therefore be distinguished from Fanconi's anaemia in which lymphocytes show increased spontaneous and clastogen‐induced chromosomal breakage.
BCR‐ABL‐positive progenitors in chronic myeloid leukaemia patients in complete cytogenetic remission after treatment with interferon‐α
Authors: Reiter Andreas , Marley Stephen B. , Hochhaus Andreas , Sohal Jastinder , Raanani Pia , Hehlmann RÜdiger , Gordon Myrtle Y. , Goldman John M. , Cross Nicholas C. P.
Published: March 2003

To determine the source of residual disease detected in patients with chronic myeloid leukaemia (CML) in complete cytogenetic remission (n = 8) after treatment with interferon‐α (IFN‐α), we have tested CFU‐GM colonies grown from bone marrow mononuclear cells or from plastic‐adherent (PΔ) cells for BCR‐ABL mRNA using a nested multiplex RT‐PCR. We compared our results with those obtained by analysis of colonies from newly diagnosed patients (n = 4) and patients achieving no cytogenetic response (n = 1) or incomplete cytogenetic response to treatment with IFN‐α (n = 5). A total of 1239 informative colonies were analysed. A small proportion of BCR‐ABL‐positive colonies was detected in all eight patients in complete cytogenetic remission, suggesting the persistence of leukaemia that could potentially lead to relapse. The overall proportion of BCR‐ABL‐positive colonies in patients achieving a cytogenetic response to IFN‐α correlated with the levels of BCR‐ABL transcripts detected in the peripheral blood by competitive RT‐PCR (P = 0.004). We conclude that residual disease detected in the peripheral blood of complete cytogenetic responders to IFN‐α is at least partly derived from clonogenic myeloid cells. It is probable that the leukaemia clone in CML is only very rarely or never entirely eradicated by treatment with IFN‐α.
Cytogenetic and molecular study of 32 Down syndrome families: potential leukaemia predisposing role of the most proximal segment of chromosome 21q
Authors: Cavani Simona , Perfumo Chiara , Argusti Alessandra , Pierluigi Mauro , Perroni Lucia , Schmiegelow Kjeld , Petersen Michael B. , Cotter Finbarr E. , Strigini Paolo , Dagna‐Bricarelli Franca , NižetiĆ Dean
Published: March 2003

Down syndrome (DS) children have a 10–20‐fold increased risk of developing ALL or AML compared to non‐DS children. An increased disomic homozygosity of the polymorphic DNA markers in the pericentromeric region of chromosome 21q (21q11) has repeatedly been found in DS patients with ANLL‐M7 and DS‐specific transient abnormal myelopoiesis (TAM), compared to the majority of DS subjects without leukaemia. Analysis of cytogenetic heteromorphisms and 26 polymorphic DNA markers from chromosome 21q showed an increased number of pericentromeric crossovers between the non‐disjoined chromosomes in DS‐ANLL cases (3/11), compared to DS‐ALL (0/9) and DS‐non‐leukaemic cases (0/12). These findings are compatible with the model of disomic homozygosity of the predisposing allele of a putative pericentromeric gene, as an explanation for the high prevalence of ANLL in DS.
Patterns of cytokine expression in AIDS‐related non‐Hodgkin's lymphoma
Authors: Pastore Cristina , Gaidano Gianluca , Ghia Paolo , Fassone Lucia , Cilia Anna Maria , Gloghini Annunziata , Capello Daniela , Buonaiuto Daniela , Gonella Silvana , Roncella Silvio , Carbone Antonino , Saglio Giuseppe
Published: March 2003

The pathogenesis of AIDS‐related non‐Hodgkin's lymphomas (AIDS‐NHL) involves accumulation of genetic lesions, stimulation and selection by antigen, as well as infection by viruses. Deregulation of cytokine loops has also been proposed to contribute to AIDS‐NHL development, although data are available only for a limited number of cytokines. In this study we have utilized a panel of AIDS‐NHL cell lines to investigate in detail the pattern of tumour expression and production of a wide spectrum of cytokines. The cytokines investigated included interleukin (IL)‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, IL‐13, TNFα, TNFβ, IFNγ, TGFβ2, G‐CSF, GM‐CSF and SCF. The AIDS‐NHL cell lines utilized were representative of both AIDS‐related Burkitt lymphoma (AIDS‐BL) and AIDS‐related body cavity‐based lymphoma (AIDS‐BCBL). Overall, AIDS‐NHL were found to produce IL‐6, IL‐10 and TNFβ, although with different patterns depending upon the biological features of the tumour. Production of high levels of IL10 preferentially associated with Epstein‐Barr virus (EBV) positive AIDS‐BL and AIDS‐BCBL, although lower levels of the cytokine were also detectable among EBV‐negative AIDS‐BL. Production of IL‐6 was restricted to EBV‐positive AIDS‐BL and AIDS‐BCBL, whereas it was absent among EBV‐negative AIDS‐BL. Production of TNFβ clustered with AIDS‐BL, whereas this was absent among AIDS‐BCBL. These results define that the pattern of cytokine expression of AIDS‐NHL depends upon the biological features of the tumour and may have implications for the pathogenesis of these disorders.
Expression and functional role of urokinase‐type plasminogen activator receptor in normal and acute leukaemic cells
Authors: Lanza  , Castoldi  , Castagnar  , Todd  , Moretti  , Spisani  , Latorraca  , Focarile  , Roberti  , Traniello
Published: March 2003

Urokinase‐type plasminogen activator receptor (UPA‐R‐CD87) is a GPI‐anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA‐R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA‐R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34+ cells were UPA‐R negative. We also attempted to clarify whether UPA‐R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA‐R was neutralized by CD87 McAb. In contrast, the anti‐UPA‐R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA‐R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA‐R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA‐R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples; in addition, since co‐expression of UPA‐R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA‐receptors >12 × 103 ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA‐R content positively correlates with the invasive potential of AML cells. The combination of higher UPA‐R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.
Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May‐Grünwald‐Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)
Authors: Haferlach Torsten , Löffler Helmut , Nickenig Christina , Ramm‐Petersen Lotte , Meeder Marlies , Schoch Robert , Schlegelberger Brigitte , Schnittger Susanne , Schoch Claudia , Hiddemann Wolfgang
Published: March 2003

Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May‐Grünwald‐Giemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B‐ and T‐lymphocytes could be identified with CD19 and CD3. In addition, HLA‐DR‐positive cells were studied for the presence of t(15;17).

Morphologically identified myeloblasts were relocated on the smear after FISH and were found to be PML/RARA positive in 91%, abnormal promyelocytes in 97%. In contrast, a positive signal was obtained in only 18% of PMN. Erythroblasts, lymphocytes and plasma cells did not show a PML/RARA rearrangement. Accordingly, all cells expressing CD3 or CD19 were PML/RARA negative. CD34 positive precursor cells identified by FICTION were PML/RARA positive in 97%. HLA‐DR‐positive cells contained a PML/RARA rearrangement in 24% of cells in one case and were negative in the two other cases investigated. These data indicate that APL appears to originate from a level of haemopoietic precursor cells but is restricted to the myeloid lineage. The low proportion of PML/RARA‐positive PMN points to the impairment of blast cell differentiation beyond the promyelocyte stage but also emphasizes the existence of normal residual haemopoietic stem cells from which PML/RARA‐negative PMN must be derived.
Exposure to myelotoxic agents and myelodysplasia: case–control study and correlation with clinicobiological findings
Authors: Rigolin Gian Matteo , Cuneo Antonio , Roberti Maria Grazia , Bardi Antonella , Bigoni Renato , Piva Nadia , Minotto Claudia , Agostini Paola , De Angeli Cristiano , Del Senno Laura , Spanedda Romedio , Castoldi Gianluigi
Published: March 2003

To better define the role of exposure to myelotoxic agents in the genesis of myelodysplastic syndrome (MDS), we carried out (a) a case–control study for the determination of the relative risk (RR) of developing MDS, including 178 consecutive patients and 178 sex‐ and age‐matched controls; (b) a study of clinicobiological features in MDS arising after occupational exposure to myelotoxic agents and in MDS in ‘non‐exposed’ patients. The definition of the ‘exposure’ status was based on a predetermined questionnaire, with calculation of an ‘exposure’ index (hours/day × days/year × years). Cumulative exposure to pesticides or to organic solvents, for >2400 h, was recorded in 48 and 25 MDS patients, respectively, compared to 27 and four controls (P < 0.00001; RR 3.74; 95% confidence interval 2.02–5.37). Older age and an excess of refractory anaemia with ringed sideroblasts and refractory anaemia with excess of blasts was noted among ‘exposed’ MDS‐patients (group 1), compared to non‐exposed MDS‐patients (group 2). 68.3% patients in group 1 had clonal chromosome changes, compared with 43.2% patients in group 2. Complex karyotypes, −7/7q−, −5/5q−, +8, 7p and 17p aberrations were seen more frequently in group 1, whereas a normal karyotype, isolated 5q− or 20q− occurred more frequently in group 2. The association of exposure to myelotoxic agents with older age at presentation and with unfavourable chromosome changes accounted for the shorter survival observed in ‘exposed’ patients. These data show that occupational exposure to pesticides and organic solvents in our region resulted in an increased RR of developing MDS and that a distinct cytogenetic profile was associated with MDS in ‘exposed’ patients. These findings provide strong indirect evidence that these agents may play a role in the pathogenesis of MDS, preferentially targeting some of the chromosome regions which are frequently involved in therapy‐related myeloid neoplasias.
Cryptic deletions and inversions of chromosome 21 in a phenotypically normal infant with transient abnormal myelopoiesis: a molecular cytogenetic study
Authors: Kempski Helena M , Craze Janet L , CHESSElls Judith M , Reeves Brian R
Published: March 2003

A case of transient abnormal myelopoiesis in a normal newborn without features of Down syndrome is described. The majority of bone marrow cells analysed belonged to a chromosomally abnormal clone with trisomy for chromosomes 18 and 21. Complex intrachromosomal rearrangements of one chromosome 21, demonstrated by fluorescence in situ hybridization using locus‐specific probes, were found in a minor population of the clonal cells. These rearrangements involved loci previously shown to be rearranged in the leukaemic cells from patients with Down syndrome and leukaemia. However, the child's myeloproliferation resolved rapidly, with disappearance of the abnormal clone, and 3.5 years later she remains well.
Paclitaxel plus high‐dose cyclosphosphamide with G‐CSF support in patients with relapsed and refractory aggressive non‐Hodgkin's lymphoma
Authors: Younes Anas , Romaguera Jorge , Mesina Ofelia , Hagemeister Frederick , Sarris Andreas H. , Rodriguez Maria A. , Mclaughlin Peter , Preti Hector A. , Bachier Carlos , Cabanillas Fernando
Published: March 2003

Based on the single‐agent activity of both paclitaxel and cyclophosphamide in the treatment of non‐Hodgkin's lymphoma (NHL), we conducted a phase II study to evaluate the efficacy of the combination of the two drugs in patients with refractory and relapsed aggressive NHL. All patients received 900 mg/m2 bolus of cyclophosphamide intravenously daily for 3 consecutive days with a concurrent infusion of 150 mg/m2 of paclitaxel over 72 h (50 mg/m2/d). 24 h after the completion of chemotherapy, patients received subcutaneous injections of 5 μg/kg of granulocyte‐colony stimulating factor (G‐CSF) daily until white cell count recovery. Treatment was repeated every 3 weeks. Patients who had at least a partial response (PR) after two courses continued to receive a maximum of four courses. Patients with responding disease were allowed to undergo high‐dose chemotherapy followed by stem‐cell/bone marrow transplantation if they were eligible. Of the 77 patients who were eligible for the study, 74 (96%) were evaluable for toxicity and treatment response. The overall response rate was 45% (95% CI 33–57%). Patients who received treatment after their disease relapsed from a complete response (CR) had an 81% response rate (38% CRs), whereas those with primary refractory disease had a 22% response rate. Toxicities of >grade 2 included alopecia (100%) and stomatitis (25%). Neutropenic fever of grade >2 occurred after 18% of the courses, and platelet count of 20 × 109/l developed after 20% of the courses. Thus, the combination of paclitaxel plus high‐dose cyclophosphamide is an effective new regimen in the treatment of refractory and relapsed NHL.
Activation of the human delta‐globin gene promoter in primary adult erythroid cells
Authors: Tang Delia C. , Rodgers Griffin P.
Published: March 2003

Restoration of the CCAAT box or insertion of an erythroid Krüppel‐like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell (hAEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at −70 bp, or insertion of an EKLF binding site at −85 bp or −95 bp in the promoter significantly increased delta globin gene expression in hAEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in δ‐globin contribute to its low level of expression in the hAEC model as well.
‘Low‐risk’ myelodysplastic syndrome is associated with excessive apoptosis and an increased ratio of pro‐ versus anti‐apoptotic bcl‐2‐related proteins
Authors: Parker  , Fishlock  , Mijovic  , Czepulkowski  , Pagliuca  , Mufti
Published: March 2003

We performed flow cytometric analysis of CD34+ cell apoptosis in 59 patients with myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) secondary to MDS (MDS‐AML) using annexin V‐FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in FAB subtypes RA, RARS and RAEB (<10% blasts) (56.5% (15.1–86.5%)) compared to normal controls (18.5% (3.4–33.4%), P < 0.0001) and RAEB‐t/MDS‐AML (16% (2.1–43.2%), P < 0.0001). There was no correlation between % apoptosis, Full blood count or cytogenetics in any disease category. Two‐colour cytometric analysis of permeabilized CD34+ cells stained with antibodies to Bcl‐2, Bcl‐X (anti‐apoptotic), Bax and Bad (pro‐apoptotic), demonstrated significantly higher ratios of pro‐ v anti‐apoptotic proteins in early MDS (2.47 (1.19–9.42) compared to advanced disease (1.14 (0.06–3.32), P = 0.0001). Moreover, using repeated measures of variants (ANOVA), we found that variations between individual Bcl‐2‐related proteins differed significantly according to disease subtype (P < 0.0005). Our results confirm that CD34+ cell apoptosis was significantly increased in MDS subtypes RA and RARS and fell with disease progression. Early MDS was also associated with a significantly higher CD34+ cell pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratio than advanced disease. Furthermore, patterns of expression of individual Bcl‐2 related proteins differed significantly between different disease categories. However, no correlation between pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratios and the degree of apoptosis was observed.
VH gene analysis of Burkitt's lymphoma in children from north‐western Iran
Authors: Chapman Caroline J. , Wright Dennis , Feizi H. Pour , Davis Zadie , Stevenson Freda K.
Published: March 2003

Cases of Burkitt's lymphoma (BL) from north‐western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional VH genes were amplified from 6/12 of the tumour masses and all of these were derived from the VH3 family, with 4/6 being derived from the most commonly used VH3 family member, V3‐23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated VH sequences showed a significant clustering of replacement mutations in the complementarity‐determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in VH sequences of EBV+ve endemic BL (eBL) and EBV−ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, VH gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.
Course of the International Normalized Ratio in response to oral vitamin K1 in patients overanticoagulated with phenprocoumon
Authors: Penning‐van Beest F. J. A. , Rosendaal F. R. , Grobbee D. E. , Van Meegen E. , Stricker B. H. Ch.
Published: March 2003

Oral vitamin K1 is used for the treatment of excessive anticoagulation. Detailed information on changes in the International Normalized Ratio (INR) in response to vitamin K1 is not available. We therefore measured the INR for the first 7 d following the oral intake of 1–5 mg of vitamin K1 in 24 patients routinely treated with phenprocoumon who had an INR 6.0 at presentation. On the first 2 d after administration of vitamin K1, the mean INR decreased by 40% and 23% respectively. After day 2, the day‐to‐day proportional change in the mean INR depended on the dose of vitamin K1 and varied from a decrease of 12% to an increase of 21%. On day 7 the mean INR was higher than on day 2 in three out of five treatment groups. Between day 2 and day 7, in general, 32% of the patients had an INR value within the target zone, 25% had an INR value 6.0 and 8% had an INR value <2.0. These findings suggest that our routine treatment of overanticoagulation in patients on phenprocoumon should be intensified to improve its efficacy.
Haemostatic and fibrinolytic responses to bone marrow transplantation
Authors: BROWN S. A. , DAVIES S. V. , FEGAN C. , WEST R. , GIDDINGS J. , WHITTAKER J. , BURNETT A. K.
Published: March 2003

Thrombin and plasmin activation markers were serially measured in 80 patients undergoing bone marrow transplantation (BMT). There were prothrombotic and fibrinolytic responses observed during autograft and allograft BMTs. Thrombin–antithrombin and prothrombin fragment F1 + 2 levels increased from day −7 to −3 (P < 0.0001) from 3.7 to 7 ng/ml and 1.2 to 1.63 nmol/l, respectively. A rise in plasmin–antiplasmin levels occurred between days 4 and 14 (P < 0.0004), from 393 ng/ml on day −7 to a peak of 795 ng/ml on day 11. No correlation between reduced protein C levels post‐BMT and a prothrombotic state was observed.
Platelet‐reactive IgG antibodies cloned by phage display and panning with IVIG from three patients with autoimmune thrombocytopenia
Authors: Fischer Peter , Jendreyko Nina , Hoffmann Melanie , Lerch Heike , Uttenreuther‐Fischer Martina M. , Chen Pojen P. , Gaedicke Gerhard
Published: March 2003

Autoimmune thrombocytopenic purpura (AITP) is a severe disease in children with a still unknown aetiology. It is not known why AITP can either be transient and self limiting or become chronic. The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of AITP patients has been proven. It is, however, not clear how IVIG functions. To analyse patient‐derived monoclonal IgG platelet autoantibodies that interact with IVIG in an anti‐idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients a large number of clones specifically reacting with IVIG molecules were derived. Many of these IVIG binders also reacted strongly with platelets in ELISA and FACS, in contrast to IVIG binders derived from a healthy individual. The heavy and light chain variable regions were sequenced and compared with each other and with databases. In all three AITP patients clones with a striking complementarity‐determining region (CDR) sequence homology to each other and to many of the known anti‐platelet antibodies were observed. Selected Fab‐phages representing the characteristic variable regions that occurred in the investigated patients with AITP may now be used to clone potentially regulatory anti‐idiotypes from healthy donors by phage display.
Tumour lysis syndrome complicating high‐dose treatment in patients with multiple myeloma
Authors: Fassas Athanasios B.‐T. , Desikan K. R. , Siegel David , Golper Thomas A. , Munshi Nikhil C. , Barlogie Bart , Tricot Guido
Published: March 2003

Tumour lysis syndrome (TLS), because of its low proliferative activity, is thought to only rarely complicate the treatment of patients with multiple myeloma. However, as more aggressive therapeutic approaches are increasingly used in the management of this disease, it is conceivable that clinicians will encounter this complication more frequently.

A retrospective analysis of > 800 patients with multiple myeloma treated at the University of Arkansas identified nine patients who developed a marked tumour lysis syndrome following intermediate‐ or high‐dose chemotherapy. Evaluation of disease characteristics revealed association with high tumour mass, high proliferative activity, increased lactic dehydrogenase levels, plasmablastic morphology, and unfavourable cytogenetic abnormalities.

Recognition of multiple myeloma patients at high risk for the development of tumour lysis syndrome and prompt intervention are required especially in the presence of abnormal baseline renal function frequently complicating the clinical course of these patients.
Complement‐induced procoagulant alteration of red blood cell membranes with microvesicle formation in paroxysmal nocturnal haemoglobinuria (PNH): implication for thrombogenesis in PNH
Authors: Ninomiya Haruhiko , Kawashima Yasuko , Hasegawa Yuichi , Nagasawa Toshiro
Published: March 2003

Complement‐induced procoagulant alteration of red blood cell (RBC) membranes in paroxysmal nocturnal haemoglobinuria (PNH) was examined. Microvesicles, deficient in acetylcholinesterase, were generated and released from PNH RBC upon complement activation. The microvesicles generated from complement‐activated PNH RBC accelerated factor Xa‐dependent plasma coagulation more than those generated from RBC by the treatment with ionophore A23187. When assessed by factor Xa‐catalysed prothrombin activation, complement activation enhanced procoagulant properties of both normal and PNH RBC similarly, although PNH RBC were lysed but normal RBC were not. This enhancement of factor Xa‐dependent prothrombinase activity of complement‐activated RBC was inhibited by the treatment of the RBC with annexin V, a protein with binding affinity for anionic phospholipids especially for phosphatidylserine (PS). Neither the enhanced procoagulant properties of RBC nor apparent RBC population with annexin V‐binding affinity were demonstrated before complement activation in any of the four PNH patients studied. PS‐externalized PNH RBC and microvesicles may contribute to the removal of PNH RBC from the circulation. We conclude that although PNH RBC do not constantly exhibit enhanced procoagulant properties in vivo, complement activation induces a procoagulant alteration of RBC membranes with microvesicle formation, potentially contributing to the thrombogenesis in PNH.
SDF‐1 suppresses cytokine‐induced adhesion of human haemopoietic progenitor cells to immobilized fibronectin
Authors: Gotoh Akihiko , Reid Suzanna , Miyazawa Keisuke , Broxmeyer Hal E.
Published: March 2003

The process of haemopoietic cell homing to the microenviroment includes migration and adhesion. SDF‐1 is a C‐X‐C chemokine that acts as a chemoattractant for haemopoietic progenitors. Adhesion of haemopoietic progenitors to immobilized fibronectin is up‐regulated by stimulation with cytokines. We assessed the effects of SDF‐1 on cytokine‐induced adhesion of progenitor cells to fibronectin. In factor‐dependent human MO7e cells and primary CD34+ cord blood cells, treatment with SDF‐1 dose‐dependently suppressed cytokine‐induced adhesion. Pretreatment with pertussis toxin reversed adhesion‐inhibition, suggesting that activation of G‐coupled proteins are involved in the intracellular signalling of this process. These data suggest that SDF‐1 not only acts as a chemoattractant but also participates in modulating adhesiveness of haemopoietic progenitors to extracellular matrix components.
Monoclonal Epstein‐Barr virus‐related lymphoproliferative disorder following adult acute lymphoblastic leukaemia
Authors: Foran James M. , Slater Sarah E. , Norton Andrew J. , Wilkes Susanna J. , Hart Ian J. , Rohatiner Ama Z. S.
Published: March 2003

A 31‐year‐old patient in remission of acute lymphoblastic leukaemia (ALL), receiving oral maintenance chemotherapy (6‐mercaptopurine, methotrexate (MTX), cyclophosphamide), developed a monoclonal, Epstein‐Barr virus (EBV)‐related lymphoproliferative disorder (LPD). Treatment consisted of excisional biopsy and the discontinuation of maintenance chemotherapy. To our knowledge, this is the first such report in an adult. The histological similarity to previous reports of ‘lymphomatoid granulomatosis’ following paediatric ALL suggests that they are the same disease. MTX may play a central role in the development of LPD in this setting. Although it is a rare complication of ALL, EBV‐related LPD should be considered in patients who develop lymphadenopathy.
High levels of lung resistance related protein mRNA in leukaemic cells from patients with acute myelogenous leukaemia are associated with inferior response to chemotherapy and prior treatment with mitoxantrone
Authors: Xu Dawei , Areström Irène , Virtala Robert , Pisa Pavel , Peterson Curt , Gruber Astrid
Published: March 2003

Expression of the mdr1 (multidrug resistance), mrp (multidrug resistance associated protein), and lrp (lung resistance related protein) genes is associated with transport related MDR (multidrug resistance). We quantified mRNA levels of these genes using competitive reverse transcription polymerase chain reaction (RT‐PCR) in 128 samples of leukaemic cells from 92 patients with acute myelogenous leukaemia (AML).

There was a wide variation between the samples in mRNA levels of all three genes. The mean mdr1 mRNA level was 1.3 transcripts per cell (range undetectable to 15.8), the mean mrp level was 7.9 (range 0.1–36.2) and mean lrp 3.9 (range 0.1–29).

Lrp mRNA levels were higher in samples drawn at diagnosis from the 15 patients with resistant disease than from the 37 with chemosensitive disease (4.9 SD 3.1 v 2.9 SD 2.3, P = 0.016). Neither mdr1 nor mrp mRNA levels were predictive for response to chemotherapy. In samples from patients who had received chemotherapy, those that had received mitoxantrone (n = 24) had higher lrp mRNA levels (mean 4.8, SD 2.5) than those that had not (n = 20, mean 2.8, SD 2.4, P = 0.012).

In conclusion, the results indicate that lrp expression is associated with inferior response to chemotherapy in AML and that lrp expression increases after exposure to mitoxantrone.
The effect of p53 dysfunction on purine analogue cytotoxicity in chronic lymphocytic leukaemia
Authors: Pettitt  , Sherrington  , Cawley
Published: March 2003

To clarify the role of p53 in the killing of chronic lymphocytic leukaemia (CLL) cells by purine analogues, we examined the cytotoxic effects of chlorodeoxyadenosine and fludarabine on CLL cells that had been characterized according to their p53 functional status.

Cases of CLL with p53 dysfunction (n = 7) displayed slight, but significant, resistance to nucleoside‐induced cell killing when compared with cases with functionally intact p53 (n = 12). The small difference between the two groups indicated that p53 plays a minor role in such killing.

These findings suggest that the poor therapeutic response to purine analogues observed in patients with p53 defects is likely to be caused by the emergence, on a background of genomic instability, of CLL‐cell clones that are resistant to nucleoside‐induced killing for reasons unrelated to p53.
Molecular features of primary mediastinal B‐cell lymphoma: involvement of p16INK4A, p53 and c‐myc
Authors: Scarpa Aldo , Moore Patrick S. , Rigaud Gildas , Inghirami Giorgio , Montresor Marina , Menegazzi Marta , Todeschini Giuseppe , Menestrina Fabio
Published: March 2003

Primary mediastinal B‐cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c‐myc, p53, N‐ras, bcl‐1, bcl‐2, bcl‐6 and for Epstein‐Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c‐myc rearrangements. Three additional cases showed sequence variations in the c‐myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl‐6 rearrangement. In conclusion, our study shows that p16INK4, c‐myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl‐1, bcl‐2 and N‐ras. Thus, none of the common genetic aberrations seen in other types of non‐Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.
Mcl‐1 and Bcl‐xL are co‐regulated by IL‐6 in human myeloma cells
Authors: Puthier Denis , Derenne Sophie , Barillé Sophie , Moreau Phillipe , Harousseau Jean‐Luc , Bataille Régis , Amiot Martine
Published: March 2003

Multiple myeloma (MM) is a slowly proliferative malignancy in which malignant plasma cells accumulate within the bone marrow. The expression of several anti‐apoptotic proteins was evaluated by immunoblotting in human myeloma cell lines and in highly purified native myeloma cells. Expression of Bcl‐xL, Mcl‐1 and Bcl‐2 was found in most of the samples; expression of Bcl‐xL and Mcl‐1 seemed to be related on myeloma cells. In a system of apoptosis by growth factor deprivation on myeloma cells, we showed that the effect of Bcl‐2 seemed minimal whereas Mcl‐1 and Bcl‐xL were tightly regulated by interleukin (IL)‐6. These findings underline the important role of Mcl‐1 and Bcl‐xL instead of Bcl‐2 in IL‐6‐induced survival of myeloma cells.
Cord blood progenitor cells have greater transendothelial migratory activity and increased responses to SDF‐1 and MIP‐3β compared with mobilized adult progenitor cells
Authors: Yong K. , Fahey A. , Reeve L. , Nicholls C. , Thomas N. S. B. , Pizzey A. , Ings S. J. , Watts M. J. , Linch D. C.
Published: March 2003

When cord blood is used as a source of haemopoietic stem cells for transplantation, fewer cells are required per kg of recipient. This greater engraftment efficiency of cord blood cells may relate to an increased ability to traverse sinusoidal endothelium, a crucial step in the homing of stem cells. We report that freshly isolated cord blood progenitors migrated more efficiently than mobilized adult cells. Cord blood progenitors responded rapidly to growth factor stimulation with an increase in migratory ability within 24 h whereas mobilized adult cells responded only after 72 h (P < 0.01). Cord blood cells also exited G0/G1 rapidly; after 24 h of growth factor exposure, 20.2 ± 1.2% of cord blood CD34+ cells were in S + G2/M compared to 6.9 ± 1.2% of adult CD34+ cells (P < 0.01). Proliferating CFC migrated more efficiently (13.3 ± 3.4% for GM‐CFC) than non‐proliferating CFC (1.4 ± 0.5%, P < 0.01) as determined using a 3H‐thymidine suicide assay. Cord blood progenitor cells also demonstrated a greater transmigratory response to chemokine stimulation compared with adult cells; this was manifested as a differential response of freshly isolated cells to SDF‐1, and of growth factor activated cells to MIP‐3β. Finally, cord blood CD34+ cells express higher levels of the chemokine receptor for SDF‐1, CXCR4, when compared with mobilized adult CD34+ cells (P < 0.05).
Circulating clonal cells in multiple myeloma do not express CD34 mRNA, as measured by single‐cell and real‐time RT‐PCR assays
Authors: Rasmussen Thomas , Jensen Linda , Honoré Lone , Andersen Heidi , Johnsen Hans Erik
Published: March 2003

The peripheral blood (PB) mononuclear cells in patients with multiple myeloma (MM) have been reported to include CD34‐expressing cells that are clonally related to the myeloma cells. To determine whether there were elevated levels of CD34 mRNA or whether CD34+ cells in the PB include myeloma‐related cells, we developed a quantitative real‐time and a competitive CD34 RT‐PCR assay working on single flow‐sorted cells. Myeloma‐specific cells were detected with allele‐specific oligonucleotides (ASO) IgH PCR. PBSC products and mononuclear cell fractions in blood from normal donors, untreated and treated myeloma patients were analysed.

When measured by flow cytometry, the numbers of CD34+/CD19+ cells were consistently < 0.1% of the mononuclear cells. In addition, no significant difference was found in the levels of CD34 mRNA between normal subjects and untreated MM patients (P = 0.935). In the treated group of MM patients the CD34 mRNA levels were significantly reduced (P = 0.052) because of the stem cell toxicity of melphalan. Further, no cells clonally related to the MM clone were found within the CD34 compartment, defined by a sort‐gate that included all cells expressing CD34 mRNA, as no cells outside the used CD34 sort‐gate had a detectable level of CD34 mRNA. We conclude that in myeloma patients, the myeloma clone is not found within the CD34 compartment.