Paolo Nicoli

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.

Increase sensitivity to chemotherapeutical agents and cytoplasmatic interaction between NPM leukemic mutant and NF-κB in AML carrying NPM1 mutations

Mutations in nucleophosmin (NPM) exon 12 and the resulting delocalization of NPM into the cytoplasm are the most specific and frequent cellular events in acute myeloid leukemia patients (AML) with normal karyotype. Cytoplasmatic NPM (NPMc+) is associated with responsiveness to chemotherapy and better prognosis. The activation of nuclear factor-κB (NF-κB) has been demonstrated to occur in a subset of AML patients and is thought to induce resistance to many chemotherapeutical agents. In this study, we demonstrate the increased in vitro sensitivity of NPMc+ cells to chemotherapeutical agents and their reduced NF-κB activity. Furthermore, we provide evidence of the interaction between NPMc+ and NF-κB in the cytoplasm, resulting in the sequestration and inactivation of NF-κB. The cytosolic localization and consequent inactivation of NF-κB justifies the reduced NF-κB DNA-binding activity observed in NPMc+ patients. These data, taken together, may provide a possible explanation for the increased rate of chemosensitivity observed among the NPMc+ patients.

HHV8-positive, HIV-negative multicentric Castleman’s disease: early and sustained complete remission with rituximab therapy without reactivation of Kaposi sarcoma

Multicentric Castleman’s disease (MCD) is a rare lymphoproliferative disorder with systemic symptoms and poor prognosis and is characterized by an abnormal proliferation of polyclonal plasmablasts in the mantle zone of B-cell follicles. The disease is found primarily in chronic HIV carriers and is usually strictly associated with human herpes virus type 8 (HHV-8) coinfection, which is believed to play a key role in the pathogenesis of MCD. The disease is also diagnosed in HIV-negative patients, who are usually elderly or immunosuppressed; however, in about half of these cases, no evidence of HHV8 infection is found. The anti-CD20 monoclonal antibody rituximab is now the preferred treatment for HIV-positive MCD. However, it is not clear whether rituximab is effective in HIV-negative patients with MCD, particularly in the HHV8-positive subset. We report here the clinical and biologic courses of two HIV-negative, HHV8-positive patients with MCD who were treated with rituximab. In both cases, a significant clinical improvement was observed after the first two infusions, which was shortly followed by a drop in HHV8 viremia to undetectable levels. Both patients underwent complete clinical remission, which persisted without relapse at 30 and 9 months of follow-up, respectively. No reactivation of the Kaposi sarcoma found in a lymph node of one of the patients was observed. Our report, along with additional data present in the literature, suggests that rituximab may be an appropriate and safe first-line therapy for HIV-negative, HHV8-positive MCD.

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.

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.

Clinical significance of TFR2 and EPOR expression in bone marrow cells in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are heterogeneous haematopoietic disorders characterized by bone marrow failure, cytopenias and a tendency to transform into acute myeloid leukaemia (AML) (Tefferi & Vardiman, 2009; Vardiman et al, 2009). Scoring systems are used to predict the prognosis of MDS (Greenberg et al, 2012). Percentage of blasts (>10%) and unfavourable cytogenetic abnormalities are the strongest predictors for poor outcome and are associated with high risk or disease progression to AML. Chronic anaemia is the main clinical problem in lowerrisk MDS but responds in 30–50% of cases to erythropoiesisstimulating agents (ESAs). Some prognostic factors of response to ESAs have been identified, with better response rates in patients with no or limited red blood cell transfusion requirement, low baseline serum erythropoietin (EPO) level and no-aberrant myeloid blasts by flow cytometry (Park et al, 2008). Nevertheless, the clinical care of MDS patients is still challenging due to lack of well-established markers that effectively monitor MDS natural history. Therefore, predicting, at diagnosis, those patients with risk of treatment failure is pivotal to personalize treatments in order to improve quality of life and prolong survival. Transferrin receptor 2 (TFR2), homologous to TFR1 (also termed TFRC), is a protein that is mutated in haemochromatosis-type-3 and contributes to the regulation of hepcidin in the liver (Ramos et al, 2011). It is also expressed in erythroid cells and in malignant myeloid disorders (Kawabata et al, 2001). TFR2 associates with erythropoietin receptor (EPOR) (Forejtnikova et al, 2010) and is required for efficient erythropoiesis (Nai et al, 2015). The TFR2 gene gives rise to two isoforms: the full-length TFR2a and the shorter TFR2b (Kawabata et al, 1999). We retrospectively investigated whether TFR2 isoforms and EPOR are differentially expressed in MDS patients and whether the expression is associated with patients’ clinical outcomes. RNA was obtained from the total bone marrow (BM) cells of individuals with non-malignant hematological disorders and in 46 treatment-naive patients at the diagnosis of MDS. After informed consent and ethical approval, TFR2a, TFR2b and EPOR expression was quantified by real-time polymerase chain reaction. Statistical analyses were performed using GraphPad-Prism software (GraphPad Software Inc., La Jolla, CA, USA). Comparison between groups was performed by Mann–Whitney U-test (nonparametric analysis). P values < 0 05 indicated a significant difference. In MDS patients, TFR2a and TFR2b showed higher variability in expression (TFR2a 6 92 5 45; TFR2b 3 16 1 61) than in non-malignant BM cells (TFR2a 8 23 2 97; TFR2b 3 68 0 47). We evaluated TFR2a and TFR2b expression in the different World Health Organization subgroups (Fig 1A–B). TFR2a and TFR2b expression was significantly lower in refractory anaemia with excess, blasts type 2 (RAEB2) (TFR2a 4 44 2 11; TFR2b 2 15 0 59; P < 0 05 and P < 0 01, respectively). A similar expression level was also seen in refractory anaemia with ringed siderobalsts. TFR2a and TFR2b expression was not correlated with total white blood cell, neutrophil and platelet counts, age at diagnosis and no significant differences were observed between sexes (data not shown). We next compared TFR2 expression with that of EPOR. Similarly to TFR2, EPOR expression varied more widely in MDS patients (18 45 18 34) than in non-malignant individuals (17 31 3 97) and was statistically lower in RAEB2 (8 18 2 62, P < 0 005) (Fig 1C). In addition, we found that TFR2a and TFR2b expression was positively correlated with that of EPOR (Fig 1D–E). To assess the clinical implication of this, we analysed the erythroid response in the patients that underwent EPO treatment (Table I). Patients with an increase in haemoglobin of ≥15 g/l after 12 weeks of treatment had TFR2 and/or EPOR levels comparable to normal controls (Fig 1F–G). In contrast, all non-responders had either very high (n = 2) or low (n = 10) TFR2 and EPOR levels. Statistical analysis performed on the 10 TFR2/EPOR low expressing patients showed that the TFR2 and EPOR products were significantly lower compared to EPO responding or non-malignant individuals (Table I). We then tested the effects of TFR2a, TFR2b and EPOR expression on survival in RAEB1-2 and refractory cytopenia with multilineage dysplasia (Fig 1H-J). In the first year of follow-up, patients with very low/low TFR2a or TFR2b expression levels had a significantly worse overall survival than those with normal/high TFR2 expression. Patients in the very low/low EPOR expression group also showed a tendency for poorer survival. Ever since it was demonstrated that TFR2 is a component of the EPOR complex (Forejtnikova et al, 2010), there has been interest in understanding its extra-hepatic function. TFR2 erythroid function has been recently described in normal erythropoiesis in mouse models lacking systemic or Correspondence

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

BackgroundMutations 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-ABLT315I 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.ResultsThe 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.ConclusionsWe 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.

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

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.

Design and application of a novel PNA probe for the detection at single cell level of JAK2V617F mutation in Myeloproliferative Neoplasms

BackgroundMutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative Myeloproliferative Neoplasms (MPN) patients and its detection is now a cornerstone in the diagnostic algorithm.MethodsWe developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence.ResultsUsing this assay we found a percentage of mutated CD34+ cells ranging from 40% to 100% in Polycythemia Vera patients, from 15% to 80% in Essential Thrombocythemia and from 25% to 100% in Primary Myelofibrosis. This method allows to distinguish, with a high degree of specificity, at single cell level, between CD34+ progenitor stem cells harbouring the mutated or the wild type form of JAK2 in NPM patients.ConclusionsThis method allows to identify multiple gene abnormalities which will be of paramount relevance to understand the pathophysiology and the evolution of any type of cancer.