Ilaria Defilippi

Early prediction of treatment outcome in acute myeloid leukemia by measurement of WT1 transcript levels in peripheral blood samples collected after chemotherapy.

The Wilms’ tumor gene WT1 is a reliable marker for minimal residual disease assessment in acute leukemia patients. The study was designed to demonstrate the potential use of WT1 to establish quality of remission in acute leukemia patients for early identification of patients at high risk of relapse. A prospective study based on a quantitative Real–Time PCR (TaqMan) assay in 562 peripheral blood samples collected from 82 acute leukemia patients at diagnosis and during follow-up was established. The evaluation of WT1 in peripheral blood samples after induction chemotherapy can distinguish the continuous complete remission patients from those who obtain only an “apparent” complete remission and who could relapse within a few months. WT1 helps identify patients at high risk of relapse soon after induction chemotherapy allowing post-induction therapy in high risk patients to be intensified.

Deferasirox is a powerful NF-κB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging

Background Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-κB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes. Design and Methods We evaluated deferasirox activity on nuclear factor-κB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 μM deferasirox for 18h. Results Nuclear factor-κB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate. Conclusions Nuclear factor-κB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

Comparison of three Tfr2 -deficient murine models suggests distinct functions for TFR2 alpha and beta isoforms in different tissues

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.

Sensitivity to imatinib therapy may be predicted by testing Wilms tumor gene expression and colony growth after a short in vitro incubation

The objective of the current study was to verify the ability to predict response to imatinib therapy using in vitro assays to evaluate the inhibition of Wilms tumor gene (WT1) expression and colony growth after samples obtained from patients with chronic myelogenous leukemia (CML) before the start of treatment were subjected to short‐term incubation with imatinib.

Valproate enhances imatinib‐induced growth arrest and apoptosis in chronic myeloid leukemia cells

The objective of this study was to evaluate the ability of the clinically available histone deacetylase (HDAC) inhibitor valproate to enhance the cytotoxicity of the Bcr‐Abl inhibitor imatinib in imatinib‐resistant cell lines.

WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia

Idiopathic hypereosinophilic syndromes (HES) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can be present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The hybrid gene FIP1L1-PDGRFα was identified in a subset of patients presenting with HES or chronic eosinophilic leukemia (CEL). In spite of this, the majority of HES patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. In this study we explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on WT1 transcript amount. For this purpose, 312 patients with hypereosinophilia were characterized at the molecular and cytogenetic level and analyzed for WT1 expression at diagnosis and during follow-up. This study clearly demonstrates that WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD

Background Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD. Methods Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD. Results In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A+ cell numbers were higher than old control non-smokers (p<0.05). Angiogenin+, B2R+ and B1R+ cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin+ cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R+ cells was positively related to the numbers of B1R+ (rs=0.43), angiogenin+ (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin+ cell numbers were negatively related to forced expiratory volume in 1 s (rs=−0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10−6M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD. Conclusions Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Genetic abnormalities as targets for molecular therapies in myelodysplastic syndromes.

Abstract:  Recent advances in molecular genetics have increased knowledge regarding the mechanisms leading to myelodysplastic syndrome (MDS), secondary acute myeloid leukemia (AML), and therapy‐induced MDS. Many genetic defects underlying MDS and AML have been identified thereby allowing the development of new molecular‐targeted therapies. Several new classes of drugs have shown promise in early clinical trials and may probably alter the standard of care of these patients in the near future. Among these new drugs are farnesyltransferase inhibitors and receptor tyrosine kinase inhibitors including FLT3 and VEGF inhibitors. These agents have been tested in patients with solid tumors and hematologic malignancies such as AML and MDS. Most of the studies in MDS are still in early stages of development. The DNA hypomethylating compounds azacytidine and decitabine may reduce hypermethylation and induce re‐expression of key tumor suppressor genes in MDS. Biochemical compounds with histone deacetylase inhibitory activity, such as valproic acid (VPA), have been tested as antineoplastic agents. Finally, new vaccination strategies are developing in MDS patients based on the identification of MDS‐associated antigens. Future therapies will attempt to resolve cytopenias in MDS, eliminate malignant clones, and allow differentiation by attacking specific mechanisms of the disease.

WT1 overexpression: A clinically useful marker in acute and chronic myeloid leukemias

Monitoring of acute leukemia patients during and after treatment for the presence of remaining leukemic cells minimal residual disease (MRD) have been shown to give major insight into the effectiveness of treatment. However, so far applicability of this strategy has been limited to those leukaemia subsets characterized by genetic markers amenable to sensitive detection by PCR. Although PCR for immunoglobulin and T-cell receptor gene rearrangement represents the gold standard for MRD detection in most cases of acute lymphoblastic leukemias (ALL) lacking the availability of fusion gene transcripts as molecular markers, the situation in AML is more complicated because, at present, more than 50% of them lack any sort of clonality markers suitable for MRD monitoring. Thus, a number of studies have been performed in the attempt to identify cytogenetic and molecular abnormalities associated with leukemic transformation. The Wilms Tumor Gene (WT1) represents a molecular marker for the detection of the leukemic clone useful for monitoring the presence of leukemic cells in all the patients affected by acute and chronic leukemias as well as myelodysplastic syndromes. The WT1 gene, cloned in 1990 by Call et al. [1] encodes for a protein with the characteristics of a zinc finger transcription factor. WT1 expression is restricted to a small number of tissues [2] including testis, ovaries, myometrium, stromal cells of the uterus, heart, lung, intestine, liver and in the supportive stroma and splenic capsule of the spleen [2]. In contrast, several other tissues and cell lines were negative for WT1 expression. Although the role of the WT1 gene in the development of malignancies in the kidney appears quite well defined, currently its potential function in human hematopoiesis still needs to be clarified. The role of WT1 in the leukemogenesis process appears

Detection of humoral immune responses against WT1 antigen in patients affected by different hematological malignancies.

(MM) patients suggests an evident clinical benefit, although the WT1 gene is not overexpressed in their cells. Furthermore, it was clearly demonstrated that MM cells are highly sensitive to WT1-specific cytotoxic T lymphocytes [15] . In vitro and in vivo evidence in many hematological disorders suggests the possibility that the humoral immune responses towards WT1 protein could be elicited, too. To verify this hypothesis, we have analyzed the presence of WT1 antibodies in a large cohort of untreated patients affected by different types of hematological malignancies. Furthermore, in order to establish whether WT1 expression could predict the immune response and to calculate the level of correlation between WT1 mRNA and antibodies, we tested patients with high, low or absent levels of WT1 transcript. Finally, we tested a group of healthy subjects since few data are available regarding their WT1 antibody level [14] . To address this point, serum samples were collected at diagnosis from 139 patients affected by the following hematological disorders: 25 AML, 40 MDS [12 refractory The Wilms tumor gene (WT1) is highly expressed in many types of hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML) and Ph-negative myeloproliferative disorders such as idiopathic myelofibrosis (IMF), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndromes or chronic neutrophilic leukemia [1–5] . WT1 overexpression prompted many researchers to develop vaccine strategies using WT1 as immunological target [6–10] . The clinical results, although still preliminary, are encouraging in terms of feasibility, safety and efficacy although this latter point requires strategy improvements being at a very early phase of development [6–13] . One important step could the adequate selection of patients who are candidates for vaccine protocols. Few years ago, the presence of humoral immune responses against the WT1 product was reported in a cohort of patients affected by AML, MDS and CML [14] . The presence of WT1 antibodies in the serum of leukemia patients is relevant in view of the development of vaccination approaches since it demonstrates that WT1 protein can be recognized by the immune system. Interestingly, WT1 peptide vaccination in multiple myeloma Received: January 15, 2008 Accepted after revision: June 25, 2008 Published online: September 30, 2008