The size of duplication does not add to the prognostic significance of FLT3 internal tandem duplication in acute myeloid leukemia patients.
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Acute Disease↗
Biomedical subjects
A M Vannucchi
Publications and source records attributed to A M Vannucchi.
Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086.
WEB-2086 -- an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties -- also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)alpha, underwent a dose-dependent growth arrest (IC(50)=0.65+/-0.09 and 0.41+/-0.07 mM, respectively) and accumulation in G(0)-G(1) phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERalpha was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERalpha status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.
Azepines↗
A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis.
A point mutation in the Janus tyrosine kinase 2 (JAK2) gene has been described in patients with chronic myeloproliferative disorders (MPD), but the clinical significance of JAK2(V617F), which may be harbored in either the heterozygote or homozyote status, is still largely undefined. There are indirect suggestions that clinical phenotype and also some biological characteristics are dependent on the mutated allele levels. We have designed and validated in 179 MPD patients an amplification-refractory mutation sequencing PCR assay that allows the relative quantitation of mutated and normal JAK2 mRNAs using dye-labelled mutation-specific primers and capillary electrophoresis. Direct sequencing confirmed the specificity of the assay, which has a detection limit congruent with1% and allowed to identify 9% more JAK2-mutated patients as compared to conventional allele-specific PCR. The mutated mRNA ratio ranged from 5 to 51% in the JAK2(V617F) heterozygote and from 45 to 100% in the homozygote patients. Expression levels of both PRV-1 and NF-E2 gene, previously found to be overexpressed in MPD patients, were significantly correlated to the amount of mutated JAK2 mRNA. We propose that this method might complement current technologies based on genomic DNA analysis, and lead prospectively to a better clinically oriented assessment of the impact of JAK2(V617F) mutation in MPD.
Amino Acid Substitution↗
Epstein-Barr virus-associated post-transplant lymphoproliferative disease with central nervous system involvement after unrelated allogeneic hematopoietic stem cell transplantation.
Post-transplant lymphoproliferative disorders (PTLD) represent an heterogeneous group of abnormal lymphoid proliferation related to Epstein-Barr virus (EBV) reactivation that arise early after allogeneic hematopoietic stem cell transplant (HSCT). PLTD with central nervous system (CNS) involvement has been reported in few cases. We describe the case of a 31-year-old-man who developed an EBV-related PTLD with CNS involvement 2 months after an allogeneic unrelated HSCT for acute myeloid leukemia in first complete remission who was successfully treated with rituximab, cidofovir and intrathecal infusion of methotrexate and methylprednisolone.
Acute Disease↗
WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential.
PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5-1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025-0.05 microM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBT-positive NB4 cells at definitely higher levels than with 1 muM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.
Antineoplastic Agents↗
Clinical implications of the JAK2 V617F mutation in essential thrombocythemia.
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Adolescent↗
Chronic relapsing thrombotic thrombocytopenic purpura successfully treated with rituximab: case report.
Plasma therapy is a cornerstone in the treatment of idiopathic Thrombotic Thrombocytopenic Purpura (TTP); however about one-third of patients relapse. In this subset of patients different immunosuppressive approaches have been reported with variable efficacy. We describe the case of an 11-year-long chronic relapsing TTP, requiring frequent plasma exchange procedures and treated unsuccessfully with several immunosuppressive agents. On the occasion of a further relapse, the patient was treated with rituximab, and achieved normalization of hematological values and clinical status for about one year. Upon further relapse, rituximab therapy was started again successfully. A monthly administration was performed with the aim of maintaining the clinical and hematological response stable. In conclusion, rituximab is a safe and effective alternative to other immunosuppressive therapies for chronic relapsing TTP patients.
Antibodies, Monoclonal↗
Internal tandem duplications of Flt3 gene (Flt3/ITD) predicts a poor post-remission outcome in adult patients with acute non-promyelocytic leukemia.
Despite progress in AML therapy, most patients eventually relapse, even the ones with normal or favorable karyotype. Since survival is poor once relapse occurs, new genetic tools above karyotype at diagnosis are needed to predict leukemia free survival. Recently, Flt3/ITD has been reported as an independent marker for clinical outcome in most studies concerning adult AML patients. To assess the prognostic relevance of activating mutations of Flt3, pretreatment samples of 100 not-M3 AML patients, all of them subjected to an intensive chemotherapy regimen, were analyzed for Flt3/ITD; 25/100 patients had one or more Flt3-ITD. Flt3/ITD patients had higher WBC count (P = 0.005), a lower incidence of a preceding MDS (P = 0.004) and most of them had a normal karyotype. Flt3/ITD had no impact on CR achievement while karyotype remained the most powerful prognostic factor (HR 2.8 95% CI 1.2 6.3). However, post-remission outcome was significantly worsened by the presence of Flt3/ITD. Median RFS of the Flt3/ITD patients was 5 vs. 27 months compared to the patients with wild-type Flt3 (P = 0.0002); moreover, Flt3/ITD patients had a significantly poorer post-remission survival (11 vs. 38 months, P = 0.01). On multivariate analysis, the presence of Flt3-ITD significantly affected relapse free survival and post-remission survival (HR 3.1 and 2.1, respectively). Thus, post-remission outcome highly depends on Flt3 status. Flt3 mutations identify patients at high risk of relapse, who should prospectively receive, according to age, either more aggressive or alternative therapeutic approaches.
Acute Disease↗
Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).
The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.
Anemia↗
Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide.
Adoptive transfer of ex vivo-generated cytomegalovirus (CMV)-specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV-specific T lymphocytes based on the antigen-presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201-restricted pp65 nonamer peptide. CMV-specific T lymphocytes were identified following induction of interferon gamma (IFN-gamma) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV-specific T lymphocytes were selectively produced in these cultures and showed CMV-restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV-specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV-specific lymphocytes.
Adoptive Transfer↗
Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.
We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)
Animals↗
Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.
Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.
Acetylcholinesterase↗
Increased expression of the distal, but not of the proximal, Gata1 transcripts during differentiation of primary erythroid cells.
Gata1 is expressed from either one of two alternative promoters, the erythroid (proximal to the AUG) and the testis (distal to the AUG) promoter, both used by hemopoietic cells. To clarify the role of the distal and proximal Gata1 transcripts in erythroid differentiation, we determined by specific reverse transcriptase-polymerase chain reactions their relative levels of expression during the differentiation of erythroid precursors purified from the spleen of mice treated with phenylhydrazine (PHZ) or infected with the anemia-inducing strain of the Friend virus (FVA cells). PHZ cells are erythroid precursors that progress in vivo to erythroblasts in 3 days. Both PHZ and FVA cells synchronously proliferate and differentiate in vitro in the presence of erythropoietin (EPO). The levels of total and of distal, but not of proximal, Gata1 transcripts increased by five- to eightfold during in vivo and in vitro differentiation of FVA and PHZ cells. The increase in expression was temporally associated with an increase in the expression of Eklf, Scl, and Nfe2, three genes required for erythroid differentiation, and preceded by 24 h the repression of Gata2 and Myb expression. The day 1 PHZ cells that survived 18 h in the absence of EPO do not express globin genes and express detectable levels of distal but not of proximal Gata1 transcripts. These cells activate the expression of the globin genes within 2 h when exposed to EPO. Therefore, during erythroid differentiation of primary cells, increased expression of distal Gata1 transcripts underlies the increase in the expression of total Gata1 associated with the establishment of the erythroid differentiation program.
Animals↗
Detection of contaminating tumor cells by RT-PCR in breast cancer patients.
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Bone Marrow↗
An open evaluation of triple antibiotic therapy including vancomycin for febrile bone marrow transplant recipients with severe neutropenia.
Infectious complications still represent a major problem in patients submitted to bone marrow transplant (BMT); approximately 40% of febrile episodes are associated with infection and one-third of these are bacteremias. Opinions about the best appropriate empiric regimens are based on evaluation of cost, potential for adverse side-effects, development of bacterial resistance, prevalent nosocomial infections. In order to assess the clinical and microbiological effectiveness of an aggressive approach, we performed a prospective open study in 72 neutropenic febrile BMT patients, employing a triple antibiotic association including amikacin 500 mg x 8h, ceftazidime 2 g x 8 h, vancomycin 500 mg x 8 h as first-line empiric treatment. For the purpose of this study, a lasting return of temperature to normal and complete disappearance of either clinical or bacteriological signs of infection without any modification of therapy was considered as success; the persistence of fever after 72 hours or a protocol change was considered as failure. Eighty episodes were enrolled during the course of the study; bacteriological evidence of infection was obtained in 23 (28.7%) febrile episodes. Median duration of antibiotic administration and of febrile episodes were 5 and 2 days respectively. Overall response rate based on clinical responses was 87% and 91% in microbiological documented infections. Death due to sepsis nor toxicity were observed. This triple antibiotic combination appears to be a very effective regimen for the empiric treatment of febrile episodes in severely neutropenic BMT recipients.
Adolescent↗
Erythropoietin upregulates the expression of its own receptor in TF-1 cell line.
In the erythroleukemia cell line TF-1, recombinant human erythropoietin (rHEpo), but not c-kit ligand, enhanced the number of cells expressing the erythropoietin receptor (EpoR), as measured by flow-cytometric analysis of binding of the biotin-labeled Epo. Moreover, 125I-Epo binding and Scatchard analyses, indicated that TF-1 cells, maintained in standard conditions with IL-3, and those stimulated with c-kit ligand, bear a single class of EpoR. On the other hand, cells cultured in the presence of rHEpo had a higher number of receptors than IL-3 or c-kit ligand-stimulated cells, and had two binding sites with different affinities for the ligand. EpoR mRNA expression was higher in cells exposed to rHEpo than in IL-3 or c-kit-stimulated cells. This difference may have been dependent on either a higher level of transcription or an increased stability of mRNA. The observed changes of EpoR in rHEpo-stimulated TF-1 cell line could cooperate, together with the alteration of the gene (3' end deletion), in the occurrence of the erythroleukemic process. Changes induced in EpoR by rHEpo were not accompanied by an increase in the expression of glycophorin A or globin chain mRNAs. This may suggest that rHEpo is unable to induce erythroid differentiation in TF-1 cells. The results also indicate that this cell line could be a model for the investigation of the role of transcription factor(s) in the expression of EpoR, and for the study of the mechanism(s) underlying the changes in the number and affinity of the cell receptors.
Erythropoietin↗
Fatal herpesvirus 6 encephalitis after unrelated bone marrow transplant.
A complex pattern of neurological dysfunctions with generalized seizures and visual allucinations, but without focal signs, suddenly arose 20 days after an unrelated bone marrow transplant for chronic myelogenous leukemia (CML) in a 13-year-old girl, accompanied by signs of acute skin graft-versus-host disease (GVHD). Magnetic resonance imaging (MRI) revealed multiple bilateral foci of signal abnormalities, which were exclusively localized in the grey matter, sparing the white. Extensive microbiological and virological assays of cerebrospinal fluid (CSF) allowed the identification of HHV-6, variant A, DNA. Further progression of both neurological alterations and of skin and gut GVHD led to a fatal outcome 2 weeks later. A retrospective analysis of both the recipient and donor mononuclear cell suspensions supported the hypothesis that HHV-6 had been acquired from the donor with the bone marrow graft. This report suggests a pathogenetic role of HHV-6 in viral encephalitis in immunocompromised bone marrow transplant (BMT) recipients, and its possible association with GVHD.
Adolescent↗
Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias.
There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT-PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1-M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for alpha-globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo-R) and thrombopoietin receptor (c-mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB-subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.
Base Sequence↗