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Extramedullary (EM) colonization is a rare complication of acute myeloid leukemia (AML), occurring in about 10% of patients, but the processes underlying tissue invasion are not entirely characterized. Through the application of RNAseq technology, we examined the transcriptome profile of 13 AMLs, 9 of whom presented an EM localization. Our analysis revealed significant deregulation within the extracellular matrix (ECM)-receptor interaction and focal-adhesion pathways, specifically in the EM sites. The transcription factor TWIST1, which is known to impact on cancer invasion by dysregulating epithelial–mesenchymal-transition (EMT) processes, was significantly upregulated in EM-AML. To test the functional impact of TWIST1overexpression, we treated OCI-AML3s with TWIST1-siRNA or metformin, a drug known to inhibit tumor progression in cancer models. After 48 h, we showed downregulation of TWIST1, and of the EMT-related genes FN1and SNAI2. This was associated with significant impairment of migration and invasion processes by Boyden chamber assays. Our study shed light on the molecular mechanisms associated with EM tissue invasion in AML, and on the ability of metformin to interfere with key players of this process. TWIST1may configure as candidate marker of EM-AML progression, and inhibition of EMT-pathways may represent an innovative therapeutic intervention to prevent or treat this complication.

In the current World Health Organization (WHO)-classification, therapy-related myelodysplastic syndromes (t-MDS) are categorized together with therapy-related acute myeloid leukemia (AML) and t-myelodysplastic/myeloproliferative neoplasms into one subgroup independent of morphologic or prognostic features. Analyzing data of 2087 t-MDS patients from different international MDS groups to evaluate classification and prognostication tools we found that applying the WHO classification for p-MDS successfully predicts time to transformation and survival (both p< 0.001). The results regarding carefully reviewed cytogenetic data, classifications, and prognostic scores confirmed that t-MDS are similarly heterogeneous as p-MDS and therefore deserve the same careful differentiation regarding risk. As reference, these results were compared with 4593 primary MDS (p-MDS) patients represented in the International Working Group for Prognosis in MDS database (IWG-PM). Although a less favorable clinical outcome occurred in each t-MDS subset compared with p-MDS subgroups, FAB and WHO-classification, IPSS-R, and WPSS-R separated t-MDS patients into differing risk groups effectively, indicating that all established risk factors for p-MDS maintained relevance in t-MDS, with cytogenetic features having enhanced predictive power. These data strongly argue to classify t-MDS as a separate entity distinct from other WHO-classified t-myeloid neoplasms, which would enhance treatment decisions and facilitate the inclusion of t-MDS patients into clinical studies.

We evaluated the impact of genomic polymorphisms in folate-metabolizing, DNA synthesis and DNA repair enzymes on the clinical outcome of 108 patients with myelodysplastic syndromes (MDS) receiving best supportive care (BSC) or azacitidine. A statistically significant association between methylenetetrahydrofolate reductase (MTHFR) 677T/T, thymidylate synthase (TS) 5′-untranslated region (UTR) 3RG, TS 3′-UTR −6 bp/−6 bp, XRCC1 399G/G genotypes and short survival was found in patients receiving BSC by multivariate analysis (P<0.001; P=0.026; P=0.058; P=0.024). MTHFR 677T/T, TS 3′-UTR −6 bp/−6 bp and XRCC1 399G/G genotypes were associated with short survival in patients receiving azacitidine by multivariate analysis (P<0.001; P=0.004; P=0.002). We then performed an exploratory analysis to evaluate the effect of the simultaneous presence of multiple adverse variant genotypes. Interestingly, patients with ⩾1 adverse genetic variants had a short survival, independently from their International Prognostic Scoring System (IPSS) and therapy received. To our knowledge, this is the first study showing that polymorphisms in folate-metabolizing pathway, DNA synthesis and DNA repair genes could influence survival of MDS patients.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low–intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia–retinoic acid receptor-a (PML–RARa) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3–internal tandem duplication (FLT3–ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML–RARa transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA–ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML–RARa transcripts was detected in the ATRA–ATO as compared with the ATRA–CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3–ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA–ATO, whereas a trend for inferior EFS was observed in FLT3–ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA–ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA–CHT in low–intermediaterisk APL. The data also suggest that ATRA–ATO may abrogate the negative prognostic impact of FLT3–ITD.

Monoclonal antibodies are a new class of agents targeted at specific receptors on cancer cells. In addition to direct cellular effects, antibodies can carry substances to the targeted cells, such as radioactive isotopes, toxins, and antineoplastic agents. At present monoclonal antibodies, directed against both lymphoid antigens (CD 20 and CD 52) and a myeloid antigen (CD33) are available for clinical use. In ALL, rituximab, a humanised anti CD20 antibody, has been combined to chemotherapy mainly in mature B-ALL and Burkitt's lymphoma and preliminary results are promising. Alemtuzumab is an anti-CD52 humanised antibody, which showed anti-tumour activity in CLL; clinical effects were observed in some patients with relapsed adult ALL. Monoclonal antibodies against myeloid antigens have been prevalently used in acute myeloid leukaemias (AML), where the most utilised immunological target is CD33. The CD33 molecule is expressed by approximately 90% of AMLs but not on CD34+ bone marrow-resident hematopoietic stem cells. The humanised anti-CD33 monoclonal antibody HuM195 has only modest activity against overt AML, but it can eliminate minimal residual disease. Radioimmunotherapy with β-particle-emitting isotopes targeting CD33 shows a major efficacy. Targeted chemotherapy with the anti-CD33-calicheamicin construct gemtuzumab ozogamicin (GO) has produced remissions as a single agent in patients with relapsed AML and appears promising when used in combination with standard chemotherapy. GO is composed of a humanised immunoglobulin G4 (IgG4) monoclonal antibody (mAb), targeting the CD33 antigen and linked to a calicheamicin derivative, a cytotoxic anthracycline antibiotic. GO has been approved by FDA as second-line therapy in older patients with AML. The most common adverse effects of monoclonal antibodies are myelosuppression, infusion-related reactions, and hypersensitivity reactions. Rituximab may cause tumour lysis syndrome. Alemtuzumab causes immunosuppression, increasing the risk of infection. GO may cause hepatotoxicity.

Abstract We report on the efficacy and toxicity of a sequential high-dose therapy with peripheral blood stem cell (PBSC) support in 107 patients with high-risk stage II/III breast cancer. There were 90 patients with more than 9 tumour-positive axillary lymph nodes. An induction therapy of two cycles of ifosfamide (total dose, 7,500 mg/m²) and epirubicin (120 mg/m²) was given, and PBSC were harvested during granulocyte colony-stimulating factor (G-CSF)-supported leukocyte recovery following the second cycle. The PBSC-supported high-dose chemotherapy consisted of two cycles of ifosfamide (total dose 12,000 mg/m²), carboplatin (900 mg/m²) and epirubicin (180 mg/m²). Patients were autografted with a median number of 4.1 × 10⁶ CD34+ cells/kg (range 1.9-26.5 × 10⁶), resulting in haematological reconstitution within approximately 2 weeks following high-dose therapy. The toxicity was moderate in general, and there was no treatment-related toxic death. Twenty-nine patients (27.1% of all patients) relapsed between 3 and 46 months following the last cycle of high-dose therapy (median 15 months). The probability of disease-free and overall survival at 3 years was 56% and 83%, respectively. A multivariate analysis showed that patients with stage II disease had a significantly better probability of disease-free survival (71%) in comparison with patients with stage III disease (30%). The probability of disease-free survival was also significantly better for patients with oestrogen receptor-positive tumours (62%) compared with patients with receptor-negative ones (40%). In conclusion, sequential high-dose chemotherapy with PBSC support can be safely administered to patients with high-risk stage II/III breast cancer. Further intensification of the therapy including the addition of non-cross-resistant drugs or immunological approaches may be envisaged for patients with stage III disease and hormone receptor-negative tumours.

Transcription factors have been shown to play a role as "master switch" factors in the programming of hematopoietic cell commitment and differentiation. PU.1 is a hematopoietic-specific member of the Ets family of transcription factors. In human bone marrow CD34-enriched progenitor cells, PU.1 expression was upregulated during the early phases of granulocytic/monocytic differentiation, preceding expression of its target genes encoding CD11b and the macrophage-colony-stimulating factor receptor, whereas PU.1 was expressed at stable levels throughout erythroid differentiation. To study PU.1 function, we synthesized double-stranded phosphorothioate oligonucleotides containing a characterized PU.1 site and demonstrated their ability to specifically compete for PU.1 DNA binding. When added to CD34+ cells in vitro, wild-type PU.1-binding oligonucleotides significantly blocked hematopoietic colony formation, whereas mutated PU.1 oligonucleotides which no longer bind PU.1 had no specific inhibitory effect. These results demonstrate that PU.1 is developmentally upregulated during normal human myelopoiesis and that the function of PU.1 is critical for the development of in vitro hematopoiesis.

Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.

In order to study the effect of high‐dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)‐derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6–27 months after transplantation. Following PBSCT, the proportion of CD34+cells was significantly smaller than before mobilization (BM 1.99±0.24 versus 0.8±0.09, P<0.001), and no change was observed at several follow‐up visits thereafter. The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34+/DR−and CD34+/Thy‐1+cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples. No relationship was found between the decrease in the proportion of CD34+cells and any particular patient characteristics, kind of high‐dose therapy or the CD34+cell content in the autograft. In conclusion, high‐dose therapy with PBSC transplantation is associated with a long‐term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post‐transplantation were normal in the majority of patients.

Background: The growth of primary low-grade B-cell gastric mucosa-associated lymphoid tissue (MALT) lymphoma is an antigen-dependent process. Case: We were able to document the influence of Helicobacter pylori on the natural history of primary low-grade B-cell gastric MALT lymphoma in a case investigated by means of polymerase chain reaction for IgH rearrangement. In this case the presence or absence of the bacterium appeared to affect both clinical manifestations and histologic features of the neoplasia, with an acceleration or a slowing down of the neoplastic expansion, respectively. Conclusions: We identified H. pylori as an antigenic stimulus that supports the growth of MALT-type lymphoma. This finding enables us to make some considerations about the management of the disease.