bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletions of D13S25, D13S319 and RB‐1 mapping to 13q14.3 in T‐cell prolymphocytic leukaemia
Authors: Brito‐Babapulle V. , Baou M. , Matutes E. , Morilla R. , Atkinson S. , Catovsky D.
Published: March 2003

Deletions of 13q14.3 are well known in several malignancies and are thought to be associated with tumour suppressor function. The RB‐1 gene is a tumour suppressor gene, but other loci including D13S319 and D13S25 telomeric to this within 13q14.3 are deleted in B‐cell chronic lymphocytic leukaemia (B‐CLL), multiple myeloma and non‐Hodgkin's lymphoma, with varying clinical significance. The fluorescence in situ hybridization screening of 22 patients with T‐prolymphocytic leukaemia (T‐PLL) for deletions of 13q14.3 revealed loss of D13S25 in 17 cases (mean 40% range 13–98%), with 11 patients having at least a 20% deletion. Mapping the deletions for the RB‐1, D13S319,and D13S25 loci revealed D13S25 as the most frequently deleted marker. However, patients with only the D13S25 deletion had low percentages of cells with the deletion (12–13%), suggesting that loss of D13S25 on its own may not provide sufficient growth advantage. The use of the YAC 954c12, which maps immediately adjacent to D13S25, defined the telomeric border of the deletion in some of the cases. Inv(14)(q11q32) and t(14;14)(q11;q32) are characteristic of T‐PLL, but are also observed in premalignant T‐cell clones in patients with ataxia telangiectasia. Transition to overt leukaemia may result from loss of suppressor function. Thus, 13q14.3 deletions could contribute to the development of overt leukaemia in T‐PLL, but the involvement of more than one gene in the region cannot be excluded.
Mutations in the thrombomodulin and endothelial protein C receptor genes in women with late fetal loss
Authors: Franchi Franca , Biguzzi Eugenia , Cetin Irene , Facchetti Floriana , Radaelli Tatjana , Bozzo Maddalena , Pardi Giorgio , Faioni Elena M.
Published: March 2003

Late fetal loss can be associated with placental insufficiency and coagulation defects. Thrombomodulin (TM) and the endothelial protein C receptor (EPCR) are glycoprotein receptors expressed mainly on the endothelial surface of blood vessels and also in the placenta; they both play a key physiological role in the protein C anticoagulant pathway. Defects in these proteins might play an important role in the pathogenesis of late fetal loss. We performed a case–control study in 95 women with unexplained late fetal loss (> 20 weeks), to elucidate whether TM or EPCR gene mutations were associated with an increased risk for this complication of pregnancy. The control group comprised 236 women who gave birth to at least one healthy baby and had no history of late fetal death or obstetrical complications. The entire TM and EPCR genes, including the promoter region, were screened. In total, five mutations were identified in the TM gene in 95 patients and three in 236 control subjects, and two mutations were identified in the EPCR gene in 95 patients and one in 236 control subjects. The relative risk for late fetal loss when having a mutation in the TM or EPCR gene was estimated by an odds ratio of 4·0 (95% CI 1·1–14·9). In conclusion, identified mutations in the TM and EPCR genes of women with unexplained fetal loss are more prevalent compared with women with no obstetrical complications.
Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1
Authors: Carter Anne , Dann Eldad J. , Katz Tamar , Shechter Yael , Oliven Ana , Regev R. , Eytan Esther , Rowe Jacob M. , Eytan Gera D.
Published: March 2003

Tetramethylrosamine (TMR) is excluded from P‐glycoprotein (MDR1)‐enriched cell lines, but it stains efficiently MDR1‐poor parent lines. Application of the TMR resistance assay to cells obtained from chronic myelogenous leukaemia (CML) patients revealed, in all individuals, a significant resistance compared with healthy donors (P < 0·001). Cells from the same patients at later phases exhibited a further increase in TMR resistance. Doxorubicin was excluded from all cell samples obtained from CML patients at presentation. The resistance to TMR and doxorubicin was energy‐dependent, and was not modulated by inhibitors of MDR1 and multidrug‐resistance protein‐1 (MRP1). Transcription of mRNAs suspected as relevant to multidrug resistance was assessed using comparative reverse transcription polymerase chain reaction. All cells from the CML patients transcribed high levels of MRP3, MRP4 and MRP5 compared with healthy donors. Low levels of MDR1, MRP1, MRP2, MRP6, lung resistance‐related protein and anthracycline resistance‐associated protein were equally transcribed in cells from healthy donors and CML patients. These results indicated that neither MDR1 nor MRP1 mediate the resistance in these cells. Our results shed light on a resistance mechanism operative in CML patients, which, together with the resistance to apoptosis, is responsible for the lack of response of CML patients to induction‐type protocols used to treat acute myeloid leukaemia patients.
Mast cells express functional CD30 ligand and are the predominant CD30L‐positive cells in Hodgkin's disease
Authors: Molin Daniel , Fischer Marie , Xiang Zou , Larsson Ulrika , Harvima Ilkka , Venge Per , Nilsson Kenneth , Sundström Christer , Enblad Gunilla , Nilsson Gunnar
Published: March 2003

Hodgkin's disease (HD) tumours are characterized by the presence of few tumour cells, the Hodgkin and Reed‐Sternberg (HRS) cells, surrounded by a large amount of non‐neoplastic cells. The role of this cell infiltrate for the development of HD is not known. CD30, belonging to the tumour necrosis factor receptor superfamily, is highly expressed on HRS cells and believed to be involved in tumourigenesis and tumour progression. Tumour samples from 42 patients were immunohistochemically double‐stained for tryptase, a mast cell‐specific proteinase and CD30 ligand (CD30L). Tryptase‐positive mast cells were present in all tumours. Of these cells, 50% expressed CD30L and 66% of the CD30L‐positive cells were mast cells. CD30L mRNA in in vitro developed normal mast cells and malignant human and murine mast cell lines was detected using reverse transcription polymerase chain reaction. CD30L protein expressed on human mast cells was detected using flow cytometry. In a co‐culture assay, the human mast cell line HMC‐1 stimulated thymidine uptake in HRS cell lines, and the stimulation could be blocked using CD30L‐specific monoclonal antibodies. In conclusion, mast cells are present in HD tumours and are the predominant CD30L‐expressing cells. CD30L–CD30 interaction is a pathway by which mast cells may stimulate DNA synthesis in HRS cells.
Intracellular Fas ligand is elevated in T lymphocytes in severe aplastic anaemia
Authors: Luther‐Wyrsch Annette , Nissen Catherine , Wodnar‐Filipowicz Aleksandra
Published: March 2003

Increased expression of Fas receptor by haemopoietic progenitors in aplastic anaemia (AA) suggests that excessive apoptosis contributes to multilineage bone marrow (BM) failure. To investigate the role of Fas ligand (FasL) in triggering progenitor cell death, we examined FasL levels in T lymphocytes of patients with severe untreated AA (n = 8). Expression of FasL on the surface of CD3+cells was not detectable. However, flow cytometric analysis of saponin‐permeabilized cells demonstrated higher levels of intracellular FasL in AA than in normal T cells (P < 0·005), both prior to and following activation with phytohaemagglutinin. Confocal microscopy revealed that FasL‐specific signals overlapped with cathepsin D staining, indicating that intracellular FasL is stored in lysosomal granules. Levels of intracellular FasL in patients examined 1 month after immunosuppression with antilymphocyte globulin and cyclosporin A were lower than prior to treatment. The caspase inhibitors, DEVD and zVAD, enhanced colony formation and prolonged survival of AA BM cells in liquid cultures by about 10‐fold (P < 0·05). Taken together, these data provide further evidence that apoptosis by the Fas/FasL system plays a role in the depletion of stem cells in AA.
Induced synthesis of albumin‐like protein in damaged rat reticulocytes
Authors: Trifunović Aleksandra , Zarić Jelena , Lušić Marina , Mijatović Marija , Glišin Vladimir , Popović Zvezdana
Published: March 2003

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin‐like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation. The endogenous origin of rat RBC albumin‐like protein was demonstrated by monitoring protein synthesis in reticulocytes and by identification of reticulocyte mRNA for albumin. Haemolytic anaemia, either hereditary (b/b rat) or drug‐induced (phenylhydrazine‐treated normal rat), results in increased accumulation of the albumin‐like protein in rat RBCs as a result of its induced synthesis.
Prolonged molecular remission in advanced acute promyelocytic leukaemia after treatment with gemtuzumab ozogamicin (MylotargTM CMA‐676)
Authors: Petti Maria C. , Pinazzi Maria B. , Diverio Daniela , Romano Atelda , Petrucci Maria T. , De Santis Silvia , Meloni Giovanna , Tafuri Agostino , Mandelli Franco , Lo Coco Francesco
Published: March 2003

We report a patient with acute promyelocytic leukaemia (APL) who received two doses of gemtuzumab ozogamicin for advanced disease. Previous treatments included front‐line all‐trans retinoic acid and anthracyclines, polychemotherapy consolidation, salvage chemotherapy for the first relapse followed by autologous stem cell transplantation (ASCT), arsenic trioxide for the second relapse followed by a second ASCT and then high‐dose methotrexate for more advanced systemic disease with central nervous system involvement. The patient achieved prolonged haematological and molecular remission after monotherapy with gemtuzumab ozogamicin given at the time of the third relapse.
Analysis of expression of nuclear factor κB (NF‐κB) in multiple myeloma: downregulation of NF‐κB induces apoptosis
Authors: Ni Hongyu , Ergin Melek , Huang Qin , Qin Jian‐Zhong , Amin Hesham M. , Martinez Robert L. , Saeed Shahnaz , Barton Kevin , Alkan Serhan
Published: March 2003

Nuclear factor‐κB (NF‐κB) is an important transcription factor that regulates survival in many cells. Activated NF‐κB has been shown to protect some haematopoietic neoplastic cells from apoptosis. In the present study, we analysed NF‐κB status in 13 primary samples from patients with multiple myeloma (MM) and in four myeloma cell lines including U266, RPMI 8226, HS‐Sultan and K620. Constitutive activation of NF‐κB was evaluated by either immunohistochemistry or immunofluorescence using a monoclonal mouse anti‐human p65 (Rel A) antibody, which recognizes the unbound, active form of p65 (Rel A). Constitutively active NF‐κB was present in all MM patient samples as well as in all four myeloma cell lines. Inhibition of constitutively active NF‐κB, by either proteasome inhibitors (MG132, gliotoxin) or inhibitors of IκB phosphorylation (Bay117082, and Bay117085), induced apoptosis as demonstrated by both flow cytometric analysis and light microscopic morphological evaluation. This chemically induced apoptosis was associated with decreased DNA binding of nuclear NF‐κB as determined by the electrophoretic mobility shift assay. In addition, adenovirus vector with dominant negative IκBα (Ad5IκB) was used for inhibition of NF‐κB in the U266 cell line. Compared with wild‐type, super‐repressor‐treated cells showed an increased level of apoptosis. These results suggest that constitutive expression of NF‐κB plays an important role in plasma cell survival in MM.
Quantitative polmerase chain reaction for the rapid prenatal diagnosis of homozygous α‐thalassaemia (Hb Barts hydrops fetalis)
Authors: Chan Vivian , Yip Ben , Lam Y. H. , Tse H. Y. , Wong H. S. , Chan T. K.
Published: March 2003

A quantitative polymerase chain reaction (Q‐PCR) method based on the TaqMan technology has been devised for the prenatal diagnosis of homozygous α°−thalassaemia (south‐east Asian type deletion). Primers and TaqMan probes were designed to specifically amplify an α°‐thal chromosomal fragment or a normal α‐chromosomal fragment. Variations in input target DNA in individual sample wells were normalized by the simultaneous amplification of a β‐actin gene fragment and results expressed as a ratio to that of β‐actin. There was no overlap of the data between the homozygous α°‐thal, α°‐thal and normal subjects. Up to 5% maternal DNA (α°‐thal) contamination did not affect the specificity of the result. In 31 prenatal diagnoses, the result using Q‐PCR compared favourably with the gold standard of Southern hybridization of α‐genes.
IVIg inhibits reticuloendothelial system function and ameliorates murine passive‐immune thrombocytopenia independent of anti‐idiotype reactivity
Authors: Crow Andrew R. , Song Seng , Semple John W. , Freedman John , Lazarus Alan H.
Published: March 2003

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody‐dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region‐reactive (anti‐idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet‐specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre‐established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti‐idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody‐sensitized red blood cells. F(ab′)2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti–idiotype interactions are not required.
Cyclosporin A combined with vincristine, doxorubicin and dexamethasone (VAD) compared with VAD alone in patients with advanced refractory multiple myeloma: an EORTC–HOVON randomized phase III study (06914)
Authors: Sonneveld P. , Suciu S. , Weijermans P. , Beksac M. , Neuwirtova R. , Solbu G. , Lokhorst H. , Van Der Lelie J. , Dohner H. , Gerhartz H. , Segeren C. M. , Willemze R. , Lowenberg B.
Published: March 2003

Patients with multiple myeloma (MM) refractory to alkylating agents frequently express P‐glycoprotein (Pgp), which is associated with the multidrug resistance (MDR) phenotype. We have conducted a randomized phase II/III study of the MDR reversal agent cyclosporin A combined with VAD (vincristine, doxorubicin, dexamethasone) compared with standard VAD in patients with MM stage IIA/IIIA who were refractory to or progressive after treatment with alkylating agents. Out of 81 patients who were randomized, 75 were eligible and evaluable: 34 in the VAD + cyclosporin A arm versus 41 in the VAD arm. Toxicities of grade 2–3 were observed more often with VAD + cyclosporin A than with VAD only: nausea (30% versus 8%, P = 0·015), mucositis (18% versus 5%, P = 0·13), infection (45% versus 35%, P = 0·50). The treatment results were similar in the two arms: 53% versus 49% responded [95% CI (−18·5%, 26·9%)]. The median progression‐free survival (PFS) was 8·6 months (VAD + cyclosporin A) versus 5·8 months (VAD): [log rank P = 0·16, hazard ratio = 0·71, 95% CI (0·44, 1·15)], and median overall survival was 13 months versus 14·6 months [log rank P = 0·89, hazard ratio = 0·96, 95% CI (0·62, 1·72)]. The cause of death was progressive disease (85%), toxicity (10%) or other (5%). Bone marrow analysis performed in 23 patients showed that the response rate was 67% in Pgp‐positive versus 55% in Pgp‐negative patients. Cyclosporin A combined with VAD is relatively well tolerated. There is no effect of cyclosporin A on the overall response rate, PFS and overall survival with VAD.
In vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells
Authors: Denning‐Kendall P. A. , Evely R. , Singha S. , Chapman M. , Bradley B. A. , Hows J. M.
Published: March 2003

Summary. This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34+ cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL‐3), IL‐6 and granulocyte colony‐stimulating factor, all at 10 ng/ml in serum‐replete conditions. Transplantation of mice with fresh CB containing 3 × 104 CD34+ cells and 1–2 SCID repopulating cells (SRC) resulted in a median of 7·4% (0·4%‐76·8%) human engraftment. When mice received the expanded product of 1–2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two‐ to fourfold. Expanded SRC produced long‐term culture‐initiating cells, clonogenic cells and CD34+ cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0·8%, range 0·0–24·0%) compared with 1–2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we confirmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two‐ to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.
Identification of novel regions of amplification and deletion within mantle cell lymphoma DNA by comparative genomic hybridization
Authors: Allen Jeannette E. , Hough Rachael E. , Goepel John R. , Bottomley Sarah , Wilson Gill A. , Alcock Helen E. , Baird Margaret , Lorigan Paul C. , Vandenberghe Elisabeth A. , Hancock Barry W. , Hammond David W.
Published: March 2003

Summary. We have carried out comparative genomic hybridization (CGH) analysis on archival biopsy material from a series of 30 UK mantle cell lymphomas. The most frequent aberrations were gains of 3q (21 cases), 6p (19 cases), 7q (8 cases), 12p (8 cases), 12q (9 cases) and 17q11q21 (8 cases), and losses of 1p13p32 (10 cases), 5p13p15.3 (9 cases), 6q14q27 (11 cases), 8p (7 cases), 11q13q23 (8 cases) and 13q (18 cases). Nineteen cases (63%) had a common region of amplification at 3q28q29, which was highly amplified in three cases, suggesting the presence of a mantle cell lymphoma (MCL)‐related oncogene in this region. There was a minimal common region of deletion at 6q25q26 in nine cases (30%). No MCL‐specific locus has previously been identified on chromosome 6 and this region may contain a tumour suppressor gene specifically implicated in the development of this subtype of lymphoma. An increased number of chromosome aberrations, gain of Xq and loss of 17p were all significantly associated with a worse prognosis. A greater understanding of the genetics of mantle cell lymphoma may allow the identification of prognostic factors which will aid the identification of appropriate treatment regimens.
Differentiation of promyelocytic leukaemia: alterations in Fas (CD95/Apo‐1) and Fas Ligand (CD178) expression
Authors: Salih Helmut R. , Starling Gary C. , Brandl Stephan F. , Pelka‐Fleischer Renate , Haferlach Torsten , Hiddemann Wolfgang , Kiener Peter A. , Nuessler Volkmar
Published: March 2003

Summary.  The survival of leukaemic blasts contributes to the pathological mechanism of acute promyelocytic leukaemia (APL). While treatment of APL using retinoic acid (RA) is a model of differentiation therapy, little is known about possible effects of this treatment on the Fas/FasL system. Investigation of APL cells from patients undergoing differentiation therapy with RA and of promyelocytic HL‐60 and monoblastic U‐937 cells cultured with RA revealed a reduction of surface expression of both Fas and its ligand. Accordingly, the sensitivity of the cells to anti‐Fas‐induced apoptosis decreased proportionally and the reduced expression of FasL resulted in a decreased ability of the leukaemic cells to induce apoptosis in T cells. Our findings demonstrate that there are significant changes in Fas and FasL expression during RA treatment of APL, which probably have consequences for the interaction between host immune and leukaemia cells, and thus may be involved in the beneficial effects of differentiation therapy.
Pneumocystis carinii pneumonia in patients with malignant haematological diseases: 10 years' experience of infection in GIMEMA centres
Authors: Pagano Livio , Fianchi Luana , Mele Luca , Girmenia Corrado , Offidani Massimo , Ricci Paolo , Mitra Maria E. , Picardi Marco , Caramatti Cecilia , Piccaluga Paolo , Nosari Annamaria , Buelli Massimo , Allione Bernardino , Cortelezzi Agostino , Fabbiano Francesco , Milone Giuseppe , Invernizzi Rosangela , Martino Bruno , Masini Luciano , Todeschini Giuseppe , Cappucci Maria A. , Russo Domenico , Corvatta Laura , Martino Pietro , Del Favero Albano
Published: March 2003

Summary. A retrospective survey was conducted over a 10‐year period (1990–99) among 52 haematology divisions in order to evaluate the clinical and laboratory characteristics and outcome of patients with proven Pneumocystis carinii pneumonia (PCP) complicating haematological diseases. The study included 55 patients (18 with non‐Hodgkin's lymphoma, 10 with acute lymphoblastic leukaemia, eight with acute myeloid leukaemia, five with chronic myeloid leukaemia, four with chronic lymphocytic leukaemia, four with multiple myeloma, three with myelodysplastic syndrome, two with myelofibrosis and one with thalassemia) who developed PCP. Among these, 18 (33%) underwent stem cell transplantation; only two received an oral prophylaxis with trimethroprim/sulphamethoxazole. Twelve patients (22%) developed PCP despite protective isolation in a laminar airflow room. The most frequent symptoms were: fever (86%), dyspnoea (78%), non‐productive cough (71%), thoracic pain (14%) and chills (5%); a severe hypoxaemia was present in 39 patients (71%). Chest radiography or computerized tomography showed interstitial infiltrates in 34 patients (62%), alveolar infiltrates in 12 patients (22%), and alveolar–interstitial infiltrates in nine patients (16%). Bronchoalveolar lavage was diagnostic in 47/48 patients, induced sputum in 9/18 patients and lung biopsy in 3/8 patients. The diagnosis was made in two patients at autopsy. All patients except one started a specific treatment (52 patients trimethroprim/sulphamethoxazole, one pentamidine and one dapsone). Sixteen patients (29%) died of PCP within 30 d of diagnosis. Multivariate analysis showed that prolonged steroid treatment (P < 0·006) and a radiological picture of diffuse lung involvement (P < 0·003) were negative diagnostic factors.
Qualitative and quantitative polymerase chain reaction detection of the residual myeloma cell contamination after positive selection of CD34+ cells with small‐ and large‐scale Miltenyi cell sorting system
Authors: Voena Claudia , Locatelli Giovanni , Castellino Claudia , Omedè Paola , Ladetto Marco , Zappone Elisabetta , Milani Raffaella , Perfetti Vittorio , Boccadoro Mario , Pileri Alessandro , Lusso Paolo , Villa Chiara , Malnati Mauro , Corradini Paolo
Published: March 2003

Summary.  The purging efficacy of the Miltenyi sorting system was evaluated by qualitative and TaqMan quantitative polymerase chain reaction (PCR) in myeloma patients, using immunoglobulin genes. After small‐scale selection, qualitative PCR showed that in 6 of 12 leukaphereses myeloma cells were no longer detectable. Envisaging a possible clinical application, the leukaphereses from three patients underwent large‐scale selection. Qualitative PCR showed that myeloma cells were still detectable. Quantitative PCR, performed in two patients, showed a tumour depletion of␣1 and 2 logs respectively. Although numbers are small, the␣promising results obtained with small‐scale selection were␣not reproduced in large‐scale experiments.
Human leucocyte antigen alloimmunization after bone marrow transplantation: an association with chronic myelogenous leukaemia
Authors: Geiger Terrence L. , Woodard Paul , Tong Xin , Srivastava Deo Kumar , Johnson Robyn , Turner Victoria , Hale Gregory , Richardson Stacye
Published: March 2003

Summary. Platelet refractoriness due to human leucocyte antigen (HLA) alloimmunization is a significant risk to allogeneic bone marrow transplant recipients. To identify factors contributing to this risk, we reviewed the records of 317 consecutive, paediatric, allogeneic bone marrow transplant recipients at a single institution. The 6‐year estimated cumulative incidence of platelet refractoriness due to HLA alloimmunization was 2·6% ± 0·9%. The incidence among patients with chronic myelogenous leukaemia (CML) 12·5% ± 5·3% was significantly greater than that of other patients (1·1% ± 0·6%, P < 0·001). Graft rejection (P = 0·003) and the number of platelet transfusions during the first 90 d after bone marrow transplantation (BMT) (P = 0·0025) were also significantly associated with alloimmunization. The association with CML and with graft rejection was not seen among patients alloimmunized before transplantation. Eight patients developed alloimmunization, of whom three had mismatched grafts and four had unrelated grafts. Alloantibody specificities, identified in seven patients, were unrelated to host or graft major histocompatibility complex (MHC). Host recognition of alloantigens in transfused blood products, not graft–host recognition, therefore seems predominantly responsible for the alloimmunization. These results show that paediatric CML patients have a significantly increased risk of platelet refractoriness due to HLA alloimmunization after BMT. Identifying the mechanism for the increased alloimmunization risk may assist in the development of therapies to prevent platelet refractoriness.
Clinicopathological and prognostic characteristics of CD56‐negative multiple myeloma
Authors: Sahara Naohi , Takeshita Akihiro , Shigeno Kazuyuki , Fujisawa Shinya , Takeshita Kaori , Naito Kensuke , Ihara Michio , Ono Takaaki , Tamashima Sadahiro , Nara Kenji , Ohnishi Kazunori , Ohno Ryuzo
Published: March 2003

Summary. We analysed CD56 expression in 70 patients with multiple myeloma (MM) to determine its clinicopathological and prognostic significance. Fifty‐five (79%) patients were CD56+. CD56– patients (n = 15) had higher β2 microglobulin levels and a higher incidence of extramedullary disease, Bence Jones protein, renal insufficiency and thrombocytopenia than CD56+ patients. Their myelomas more frequently had a plasmablastic morphology. Overall survival was significantly lower in CD56– than CD56+ patients (22 vs 63 months, P = 0·0002). We conclude that CD56– MM is a discrete entity associated with more aggressive disease. The higher incidence of plasmablastic cases suggested that CD56– MM may develop from a less mature plasma cell than CD56+ MM.
Aplastic anaemia with 13q–: a benign subset of bone marrow failure responsive to immunosuppressive therapy
Authors: Ishiyama Ken , Karasawa Masamitsu , Miyawaki Shuichi , Ueda Yasunori , Noda Masaaki , Wakita Atsushi , Sawanobori Masakazu , Nagai Hirokazu , Nakao Shinji
Published: March 2003

Summary. In an attempt to determine the pathological significance of a long arm deletion of chromosome 13 (13q–) in bone marrow failure syndrome, we reviewed the clinical records of nine patients who were initially diagnosed with aplastic anaemia due to bone marrow hypoplasia without dysplasia. Six patients responded to immunosuppressive therapy and the other three improved with steroids. None of the patients developed acute leukaemia (follow up: 54–129 months) and the estimated 5‐year survival was 78%. These findings indicate that pancytopenia with 13q– represents bone marrow failure of a benign nature, similar to aplastic anaemia without karyotypic abnormalities, rather than preleukaemia.
JH probe real‐time quantitative polymerase chain reaction assay for Bcl‐2/IgH rearrangements
Authors: Jenner Michael J. , Summers Karin E. , Norton Andrew J. , Amess John A. , Arch Rachael S. , Young Bryan D. , Lister T. Andrew , Fitzgibbon Jude , Goff Lindsey K.
Published: March 2003

Summary. Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only ∼75% of the consequent Bcl‐2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region‐specific real‐time quantitative polymerase chain reaction (RQ‐PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ‐PCR assay for the quantification of Bcl‐2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ‐PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 105 cells. In addition, to generate standard curves equating to diverse Bcl‐2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl‐2/IgH breakpoints identified by long‐range or inverse PCR, one MBR+ and one mcr+) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl‐2/IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.
Mutations in the thrombomodulin gene are rare in patients with severe thrombophilia
Authors: Faioni Elena M. , Franchi Franca , Castaman Giancarlo , Biguzzi Eugenia , Rodeghiero Francesco
Published: March 2003

Summary. Because thrombomodulin plays a key role in the protein C pathway, we evaluated the contribution of thrombomodulin gene mutations to venous thrombosis. We examined 38 patients with recurrent, documented thrombotic events at a young age and a positive family history. Twelve individuals with low levels of soluble thrombomodulin in plasma were also studied. Finally, the allelic frequency of the Ala455Val polymorphism was estimated in 192 patients with at least one thrombotic event and in 369 age‐ and sex‐matched asymptomatic controls. Two mutations were identified; G/A−201, in a severely thrombophilic patient and G/T 1456, in a patient with low soluble thrombomodulin levels. The first mutation has been reported by some, but not others, to be associated with moderately reduced levels of thrombomodulin. The second was identified previously in a patient with low soluble thrombomodulin, but expression studies failed to show functional changes in the mutant. Thrombomodulin gene mutations thus appear to be rare even in highly selected thrombophilic patients, and possibly functionally irrelevant. The allelic frequency of the Ala455Val polymorphism was identical in patients and controls. Considering the lack of a phenotype and the costly screening procedure, we recommend that thrombomodulin defects be sought only for research purposes.
Molecular characterization of a new recombination of the SIL/TAL‐1 locus in a child with T‐cell acute lymphoblastic leukaemia
Authors: Carlotti Emanuela , Pettenella Francesca , Amaru Ricardo , Slater Sarah , Lister T. Andrew , Barbui Tiziano , Basso Giuseppe , Cazzaniga Giovanni , Rambaldi Alessandro , Biondi Andrea
Published: March 2003

Summary. Deletions involving the SIL‐TAL‐1 locus are seen in 15% of T‐acute lymphoblastic leukaemias (T‐ALL). To date, seven deletions have been described, spreading over 90 kb of chromosome 1, fusing SIL to the TAL‐1 gene and resulting in over expression of TAL‐1. During the diagnostic screening of the TAL‐1 deletion in 176 T‐ALL patients, we identified one case showing a new SIL rearrangement. A novel fusion transcript was identified between the SIL exon 1a and an unknown sequence (633‐cDNA). Polymerase chain reaction (PCR) screening of a human cDNA library confirmed the existence of this transcript. Using long‐distance PCR on patient DNA, we obtained a genomic fragment containing SIL exon 1b, a portion of intron 1b, an unknown sequence and the 633 sequence. Using DNA from healthy donors, a partial genomic map of 633‐DNA was found to be identical to the restriction map of the PCR fragment amplified from patient DNA. To define the chromosomal origin of 633‐DNA, a YAC human genomic library was screened. Two clones containing 633‐DNA were found, mapping to chromosomal region 1p32 and both contained SIL and TAL‐1 sequences. By searching GenBank, we identified PAC RP1‐18D14 which contains SIL, TAL‐1 and 633‐DNA, confirming this novel rearrangement as a new deletion of the SIL/TAL‐1 locus.
Both hypodiploidy and deletion of chromosome 13 independently confer poor prognosis in multiple myeloma
Authors: Fassas Athanasios B.‐T. , Spencer Tray , Sawyer Jeffrey , Zangari Maurizio , Lee Choon‐Kee , Anaissie Elias , Muwalla Firas , Morris Christopher , Barlogie Bart , Tricot Guido
Published: March 2003

Summary. Complete or partial deletion of chromosome 13 or translocations involving 13q (Δ13) by conventional cytogenetic analysis confers a poor prognosis in multiple myeloma (MM) patients, even with timely application of tandem autologous transplants. It was recently suggested that the prognostic significance of Δ13 is related to its frequent association with hypodiploidy but by itself does not have a poor prognostic significance. We therefore analysed our experience in 1475 consecutive MM patients in whom we intended treatment with tandem transplants after a melphalan‐based conditioning regimen. Patients with abnormal cytogenetic analysis were grouped into hypodiploid/hypotetraploid, pseudodiploid and hyperdiploid groups, according to their modal chromosome number. Their event‐free and overall survival were compared with those of patients with a normalkaryotype. Both hypodiploidy and Δ13 were found to independently confer poor prognosis in MM patients. Furthermore, these parameters in combination with easily obtained pretransplant levels of β‐2 microglobulin and albumin define three groups of MM patients with clearly distinct outcomes.
Exploring polycythaemia vera with fluorescence in situ hybridization: additional cryptic 9p is the most frequent abnormality detected*
Authors: Najfeld Vesna , Montella Lya , Scalise Angela , Fruchtman Steven
Published: March 2003

Summary. Between 1986 and 2001, 220 patients with polycythaemia vera (PV) were studied using conventional cytogenetics. Of 204 evaluable patients, 52 (25·4%) had clonal abnormalities. The recurrent chromosomal rearrangements were those of chromosome 9 (21·1%), del(20q) (19·2%), trisomy 8 (19·2%), rearrangements of 13q (13·4%), abnormalities of 1q (11·5%), and of chromosomes 5 and 7 (9·6%). Subsequent analysis of 32 patients, performed at follow‐up of up to 14·8 years, revealed new clonal abnormalities in five patients and the disappearance of an abnormal clone in four. Eleven patients remained normal up to 11·5 years and seven patients maintained an abnormality for over 10 years. Fifty‐three patients were studied retrospectively using interphase fluorescence in situ hybridization (I‐FISH), utilizing probes for centromere enumeration of chromosomes 8 and 9, and for 13q14 and 20q12 loci. Conventional cytogenetics demonstrated clonal chromosome abnormalities in 23% of these 53 patients. The addition of I‐FISH increased the detection of abnormalities to 29% and permitted clarification of chromosome 9 rearrangements in an additional 5·6% of patients. FISH uncovered rearrangements of chromosome 9 in 53% of patients with an abnormal FISH pattern, which represented the most frequent genomic alteration in this series.
Autologous lymphocytes as vectors to target therapeutic radiation, using indium‐114m, in patients with lymphoid cell malignancy
Authors: Cowan Richard A. , Murby Brian , Gunton Deborah , Owens Susan E. , Hoyes Kathryn P. , Sharma Harbans L. , Smith Anne‐Marie , Chang James , Van Kessel Bregje , Nuttall Pamela M. , Crowther Derek
Published: March 2003

Summary. Autologous lymphocytes provide a potential vector for the delivery of a cytotoxic agent in patients with lymphoid cell malignancy. This report describes a phase I–II study using autologous lymphocytes to target the radionuclide indium‐114m (114mIn) in patients with refractory chronic lymphocytic leukaemia or small lymphocytic non‐Hodgkin's lymphoma. Nineteen patients, the majority of whom had been heavily pretreated with conventional chemotherapy and radiotherapy, received between 69 and 211 MBq 114mIn‐labelled autologous lymphocytes. Approximately 80% of the administered activity was localized in the liver and spleen, with around 5% accumulating in the bone marrow. Ten patients (53%) responded (one complete response and nine partial responses). The median duration of response was 7 months. The median survival for the responders was 14 months and for the non‐responders was 3 months. The first notable response in every patient was a fall in peripheral lymphocyte count. The indium treatment was not associated with any subjective toxicity, although all patients suffered from myelosuppression, with thrombocytopenia being the dose‐limiting factor. This study has demonstrated a significant anti‐tumour effect in a group of patients with late‐stage highly resistant disease.
Molecular cytogenetics characterization of a novel translocation involving chromosomes 17 and 19 in a Ph+ adult acute lymphoblastic leukaemia
Authors: Specchia Giorgina , Albano Francesco , Anelli Luisa , Storlazzi Clelia Tiziana , Cimino Giuseppe , Liso Arcangelo , Zagaria Antonella , Liso Vincenzo , Rocchi Mariano
Published: March 2003

Summary. We report a case of positive Philadelphia chromosome adult acute lymphoblastic leukaemia with a novel unbalanced translocation t(17;19), leading to trisomy of 17q21‐qter. The patient did not obtain complete haematological response and died a few months after diagnosis. The significance of the 17q21‐qter trisomy, resulting from this novel translocation, and its possible role in the progression of the leukaemia is discussed.
Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease
Authors: Ghosh Kanjaksha , Kulkarni Bipin , Nair Sona , Shetty Shrimati , Mohanty Dipika
Published: March 2003

Summary. Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA‐1) system is important. Studies have shown that, in the normal population, the HPA‐1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty‐one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA‐1 to HPA‐6, using PCR–ASA (polymerase chain reaction–allele‐specific amplification), and for GPIIb‐IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA‐1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 µmol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 µmol/l ADP. Seven out of 37 patients with HPA‐1a/1a phenotype showed 1–5% fibrinogen binding and GPIIb–IIIa receptors. The two HPA‐1b/1b patients showed 34·6% and 32% fibrinogen binding and > 10% GPIIb–IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA‐1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.
Selective reduction of natural killer T cells in the bone marrow of aplastic anaemia
Authors: Zeng Weihua , Maciejewski Jaroslaw P. , Chen Guibin , Risitano Antonio M. , Kirby Martha , Kajigaya Sachiko , Young Neal S.
Published: March 2003

Summary.T cell‐mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural‐killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T‐cell receptor (TCR), consisting of Vα24JαQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T‐lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Vα24 in CD4–CD8–T cells derived from AA and healthy individuals, the CDR3 size of Vα24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Vα24 polymerase chain reaction products revealed that the NKT cell‐specific JαQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS.
Differentiation of anti‐tumour cytotoxic T lymphocytes from autologous peripheral blood lymphocytes in non‐Hodgkin's lymphomas
Authors: Chaperot Laurence , Manches Olivier , Mi Jian Qing , Moine Agnes , Jacob Marie‐Christine , Gressin Rémy , Molens Jean‐Paul , Sotto Jean‐Jacques , Leroux Dominique , Bensa Jean‐Claude , Plumas Joël
Published: March 2003

Summary. We have previously reported that specific anti‐tumour cytotoxic T cells (CTL) can be differentiated from tumour‐infiltrating lymphocytes (TIL) in non‐Hodgkin's lymphoma. We found that the combination of interleukin (IL)‐1, IL‐2 and IL‐12 was very efficient for expansion of CD8+ T‐cell receptor (TCR)αβ+ T cells and for development of their ability to specifically lyse tumour cells. In this study, we investigated whether anti‐tumour T cells could be generated from the peripheral blood of patients using the culture protocol developed for TIL. Autologous T cells and tumour B cells from five patients were included in this study. It was found that polyclonal anti‐tumour cytotoxic effector cells were generated when cultured in the presence of IL‐1β, IL‐2 and IL‐12. Interestingly, tumour cells were lysed by perforin/granzyme‐mediated cytolysis and not by CD95‐mediated apoptosis. By performing inhibition experiments, it was observed that both CD8+ and CD4+ T cells were responsible for the cytotoxic effect and that they were able to recognize malignant B cells by either a major histocompatibility complex (MHC)‐restricted or MHC‐non‐restricted mechanism. Intriguingly, in addition to interferon‐γ and tumour necrosis factor‐α, IL‐10 was secreted continuously during culture. The source of patient T cells used for the generation of anti‐tumour CTL should be based on the results obtained with peripheral blood lymphocytes and TIL.
Telomere maintenance in human B lymphocytes
Authors: Martens Uwe M. , Brass Volker , Sedlacek Lucie , Pantic Milena , Exner Carolin , Guo Yalin , Engelhardt Monika , Lansdorp Peter M. , Waller Cornelius F. , Lange Winand
Published: March 2003

Summary. Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere‐length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow‐FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B‐lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27– (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B‐cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long‐term B memory cells.
Multilineage involvement in hypereosinophilic syndrome terminating in granulocytic sarcoma and leukaemic transformation with trisomy 8
Authors: Fujii Nobuharu , Ikeda Kazuma , Takahashi Naoto , Kojima Kensuke , Kobayashi Yoshimi , Ashiba Atsuko , Takenaka Katsuto , Fukuda Shunnichi , Shinagawa Katsuji , Ishimaru Fumihiko , Niiya Kenji , Miura Ikuo , Tanimoto Mitsune , Harada Mine
Published: March 2003

Summary. We report a patient with hypereosinophilic syndrome (HES), which, 8 years later, transformed into granulocytic sarcoma in the brain and, subsequently, into acute myelocytic leukaemia. Repeated chromosome analyses showed a normal karyotype, until the time of leukaemic transformation when trisomy 8 was confirmed in cells from the bone marrow and cerebrospinal fluid. The combined techniques of May–Grunwald–Giemsa staining and fluorescence in situ hybridization identified trisomy 8 not only in blasts and eosinophils but also in neutrophils and erythroblasts. Our observation suggests that HES is a multilineage myeloproliferative disorder involving precursors of at least the eosinophil, neutrophil and erythroid lineages.