Comparison of 3 Tfr2-deficient murine models suggests distinct functions for Tfr2-alpha and Tfr2-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.

Deferasirox is a powerful NF-kappaB 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-kappaB 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-kappaB 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 muM deferasirox for 18h. RESULTS: Nuclear factor-kappaB 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-kappaB 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.

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.

Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.

Role of TGF-beta1 haplotypes in the occurrence of myocardial infarction in young Italian patients.

BACKGROUND: Transforming growth factor beta 1 (TGF-beta1) gene play an important role in the acute myocardial infarction (AMI), however no investigation has been conducted so far in young AMI patients. In this study, we evaluated the influence of TGF-beta1 polymorphisms/haplotypes on the onset and progression of AMI in young Italian population. METHODS: 201 cases and 201 controls were genotyped for three TGF-beta1 polymorphisms (G-800A, C-509T and Leu10Pro). The main follow-up end-points (mean follow-up, 107 +/- 49 months) were death, myocardial infarction or revascularization procedures. RESULTS: Significant risk factors were smoking (p < 10-4), family history for coronary artery disease (p < 10-4), hypercholesterolemia (p = 0.001) and hypertension (p = 0.002). The C-509T and Leu10Pro polymorphisms showed significant differences (p = 0.026 and p = 0.004) between cases and controls. The most common haplotypes revealed a possible protective effect (GCT, OR 0.75, 95% CI 0.57-0.99, p = 0.042) and an increased risk of AMI (GTC, OR 1.51, 95% CI 1.13-2.02, p = 0.005), respectively. No statistical differences were observed in genotype distribution in the follow-up study between the two groups: 61 patients with subsequent events (13 deaths) and 108 without events. CONCLUSION: Even though our results need to be further confirmed in larger studies, this is the first study reporting on a possible role of TGFbeta1 common haplotypes in the onset of AMI in young patients.

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.

A novel assay to detect calreticulin mutations in myeloproliferative neoplasms.

The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56-88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs.We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.

Liver expression of hepcidin and other iron genes in two mouse models of beta-thalassemia.

BACKGROUND AND OBJECTIVES: Homozygous beta-thalassemia patients may develop iron overload even if untransfused, due to inappropriately high intestinal iron absorption. Reduction of hepcidin synthesis has been reported both in patients and in animal models. We have measured liver hepcidin and other iron gene transcripts in two different mouse models of beta-thalassemia at different ages. DESIGN AND METHODS: Mice Hbb(th/th), characterized by spontaneous homozygous deletion of the major b1 globin gene were studied at 2 and 8 months. Mice Hbb(th/3+), characterized by the heterozygous deletion of b1 and b2 globin genes were studied at 4 and 10 months. Hematologic data were obtained and iron overload estimated by Perls' staining of the liver. Expression of liver hepcidin, Tfr2, Hjv, Fpn and Hfe RNA was assessed by real-time polymerase chain reaction. Levels of serum cytokines (interleukin-6, IL-1beta, IL-10, granulocyte-macrophage colony-stimulating factor) levels were assayed by enzyme-linked immunosorbent assay. RESULTS: Hemoglobin, hematocrit and mean corpuscular volume were significantly reduced in both beta-thalassemia models, more significantly in Hbb(th/3+), which have the greater, age-dependent, iron overload. Hepcidin RNA was not increased despite iron overload in both strains. Fpn RNA was increased and Tfr2 was decreased in older animals. Inflammatory cytokine levels were striking variable and unrelated to hepcidin levels. INTERPRETATION AND CONCLUSIONS: Although anemia is reported to inhibit hepcidin expression, normal hepcidin synthesis was maintained in both thalassemic models studied. However, hepcidin levels were inappropriate for the body iron, especially in Hbb(th/3+) 10-month-old animals. As we previously reported in wild type mice after parenteral iron overload, Tfr2 is reduced and Fpn RNA increased in thalassemic mice. Inflammatory cytokines did not play a major role in increasing hepcidin levels or in modifying iron homeostasis in this study.

Genetic abnormalities as targets for molecular therapies in myelodysplastic syndromes.

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.

The Wilms' tumor (WT1) gene expression correlates with the International Prognostic Scoring System (IPSS) score in patients with myelofibrosis and it is a marker of response to therapy.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.

Hepatic expression of hemochromatosis genes in two mouse strains after phlebotomy and iron overload.

BACKGROUND AND OBJECTIVES: Iron homeostasis is tightly regulated in mammals according to the needs of erythropoiesis and the iron stores present. This regulation is disrupted in hereditary hemochromatosis (HH), a genetic disorder characterized by increased intestinal iron absorption, leading to iron overload. The genes coding for HFE, transferrin receptor 2 (TFR2), ferroportin (SLC40A1 or FPN1), hepcidin (HEPC) and hemojuvelin (HJV or RGMC) are responsible for different types of genetic iron overload. All these genes are highly expressed in the liver and their protein products are likely components of a single hepcidin-related pathway. In order to gain insights into the molecular relationship among the HH proteins we evaluated the hepatic expression of HH genes in conditions of iron restriction or overload. DESIGN AND METHODS: Data were obtained after phlebotomy, to activate the erythroid regulators and following parenteral iron dextran loading, to activate the store regulators, in two mice strains (C57BL/6 and DBA/2). HH genes and proteins expression were analyzed by quantitative real time polymerase chain reaction and by Western blotting, respectively. RESULTS: Hepc RNA was reduced after phlebotomy and increased in iron overload. A statistically significant reduction of hepatic Fpn1 RNA expression was observed after phlebotomy; this effect was more evident in the DBA/2 strain. Fpn1 increased in C57BL/6 mice, but not in the DBA/2 ones in parenteral iron loading. Fpn1 protein did not change substantially in either condition. Hfe, Rgmc and Tfr2 expression was not influenced by phlebotomy. In parenteral iron overload, Tfr2 gene and protein expression decreased concomitant to the increase in Hepc, while Hfe RNA remained constant. INTERPRETATION AND CONCLUSIONS: Our results indicate that regulation of hepatic Fpn1 differs from that reported for duodenal Fpn1. Furthermore, taken the differences in gene expression in dietary overload (increased Hfe but not Tfr2), distinct roles are suggested for Hfe and Tfr2 in Hepc activation.

Estrogen receptor-alpha polymorphisms and angiographic outcome after coronary artery stenting.

OBJECTIVE: Because of the receptor-mediated antiproliferative effects of estradiol on vascular smooth muscle cells, our study aimed at identifying a role of PvuII and XbaI polymorphisms of the alpha-estrogen receptor (alphaER) gene in the occurrence of restenosis after coronary stent implantation (in-stent restenosis [ISR]). METHODS AND RESULTS: In 858 patients (148 women), 955 lesions were treated with stent implantation, and the PvuII C/T and XbaI G/A polymorphisms of the alphaER gene were determined. Quantitative angiography was performed before and after stenting and at 6-month follow-up. The allelic frequencies were similar between sexes (C/T allele, 0.43/0.57 and 0.44/0.56; P=0.9; G/A allele, 0.35/0.65 and 0.38/0.62; P=0.8; in women and men, respectively). A significantly higher ISR rate in women than in men homozygous for the T-allele of the PvuII polymorphism (42.6% versus 26.9%, P=0.03) or the G-allele of the XbaI polymorphism (41.2% versus 19.4%, P=0.04) was observed. At multivariate analysis, T/T genotype was the only independent predictor of ISR in women but not in men (odds ratio, 1.5; 95% CI, 1.0 to 2.1; P=0.03). XbaI polymorphism was no longer associated with ISR in both sexes. CONCLUSIONS: Women homozygous for the T-allele of the PvuII polymorphism of the alphaER gene treated with coronary stent implantation have a higher risk of ISR than men.

Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.

BACKGROUND: Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL (T315I) mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation. RESULTS: The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors. CONCLUSIONS: We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

Myelodysplastic syndromes.

Efforts made during the last few years have helped unravel the complex pathogenesis of the myelodysplastic syndromes (MDS). A large number of studies, made possible by the introduction of newer technologies, have led to major progress in understanding the heterogeneous genetic and biological abnormalities contributing to the development and progression of myelodysplasia. Better insights into these pathogenetic processes will aid the development of newer and more successful therapies for MDS patients. The identification of specific genes involved in the emergence and progression of the myelodysplastic clone has extended biological findings into the clinic. Recently, several clinical trials have used selective compounds to target and inhibit the disrupted signal transduction pathway in myelodysplastic patients. The demonstration of genetic abnormalities present not only in MDS patients but also in acute myeloid leukemia (AML) patients or in chronic myeloproliferative disorders (CMPD) has prompted extension of a number of clinical trials from AML and CMPD to MDS patients. In spite of this, the more complex and heterogeneous pathogenesis underlying the myelodysplastic process is responsible for the often different and in same cases worse clinical results obtained in MDS patients. Finally, the identification of myelodysplasia-associated antigens that may be targeted by an immunotherapeutic approach represents a future perspective in tailored therapy for MDS patients.

Association between transforming growth factor beta1 gene polymorphisms and IgA nephropathy.

BACKGROUND: Transforming growth factor beta1 (TGF-beta1) plays an important role in the modulation of cellular growth and differentiation in a wide variety of cell types and in the production/degradation of the extracellular matrix (ECM). We investigated whether G-800A, C-509T and Leu10-->Pro polymorphisms in the TGF-beta1 gene could be involved in the development and progression of immunoglobulin A nephropathy (IgAN). METHODS: DNA samples were obtained from 101 patients with biopsy proven IgA mesangial nephropathy and 118 healthy controls. The genotypes of G-800A, C-509T and Leu10-->Pro polymorphisms in the TGF-beta1 gene were determined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) with MaeIII, Eco 81I and Pst I, respectively. RESULTS: No significant differences were observed in the genotype distribution of the three TGF-beta1 polymorphisms between patients and controls. The TAC haplotype (T=Leu10, A-800 and C-509 alleles, respectively) was significantly associated with IgAN (p=0.043; odds ratio (OR) =2.334, 95 % confidence interval (95%CI) 1.01-5.41). CONCLUSION: Our study suggests that the haplotype reconstruction of TGF-beta1 gene polymorphisms could be more informative than the investigation of single nucleotide polymorphisms for defining the associated risk of developing IgAN. Further research is needed on larger cohorts to confirm TGF-beta1 involvement and test other TGF-beta1 variants with possible additive or synergistic effects.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines.

We aimed at investigating in vitro the cytotoxic activity (determined using WST-1, apoptosis and cell cycle assays) of gemcitabine, alone or in combination with mitotane, in mitotane-sensitive H295R and mitotane-insensitive SW-13 cells. Results of these experiments were compared with drug-induced modulation of RRM1 gene, the specific target of gemcitabine. In H295R cells, mitotane and gemcitabine combinations showed antagonistic effects and interfered with the gemcitabine-mediated inhibition of the S phase of the cell cycle. By contrast, in SW-13 cells, except when mitotane was sequentially administered prior to gemcitabine, the combination of the two drugs was synergistic. Such opposite effects were associated with opposite expression profiles of the target gene, with significant up-modulation in H295R but not in SW-13 under gemcitabine and mitotane combination treatment.

Aberrant activation of ROS1 represents a new molecular defect in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a clonal disorder sharing features of myelodysplastic syndromes and chronic myeloproliferative neoplasms. Although rare chromosomal aberrations and point mutations are reported in CMML, the molecular defects underlying CMML are largely unknown. ROS1 encodes a tyrosine kinase that is abnormally expressed and translocated in brain and lung cancers. In this study we show that ROS1 is abnormally activated in the CD34+ compartment of approximately 70% of CMML patients resulting in the activation of the Erk/Akt pathways through the Grb2/SOS complex thus revealing a central oncogenic role for ROS1 in CMML which might represent a molecular target.