Chiara Calabrese

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.

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.

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.

Prognostic significance of The Wilms’ Tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia

Acute myeloid leukemia is a genetically heterogeneous disease characterized by the accumulation of mutations in hematopoietic progenitor cells. For its heterogeneity, prognostic markers are very useful for therapeutic choice. The most important prognostic markers are age, white blood cell count, chromosomal alterations and gene mutations. Recent works have studied the prognostic significance of WT1 polymorphisms and mutations, highlighting the role of SNP rs16754 as a positive prognostic factor in AML patients. Nevertheless, the data are still unclear. To investigate the role of WT1 rs16754 polymorphism in AML, we designed a new tool for the detection using PNA directed PCR Clamping technology. Our data were able to establish a correlation between SNP rs16754 and the clinical outcome. Our results support the hypothesis that rs16754 polymorphism is an independent positive prognostic molecular marker that could be useful for therapeutic choice. In view of this, we described a novel assay faster, more sensitive and cheaper than DNA sequencing. The assay allows evaluating WT1 rs16754 polymorphism in diagnostic routine to improve prognostic information faster and without over-costing for diagnostic laboratories.

Erythroid response during iron chelation therapy in a cohort of patients affected by hematologic malignancies and aplastic anemia with transfusion requirement and iron overload: a FISM Italian multicenter retrospective study

Emanuela Messa, Lucia Biale, Anna Castiglione, Monia Lunghi, Margherita Bonferroni, Flavia Salvi, Bernardino Allione, Dario Ferrero, Chiara Calabrese, Marco De Gobbi, Paolo Nicoli, Daniela Gioia, Alessandro Levis, Giuseppe Saglio and Daniela Cilloni Department of Internal Medicine, ASL To5-Turin, Italy; AOU Citt a della Salute e della Scienza di Torino – Presidio Ospedaliero Molinette – SC Banca del Sangue e del plasma, Turin, Italy; Unit of Cancer Epidemiology and CPO Piedmont, S. Giovanni Battista Hospital, Torino, Italy; Division of Haematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Italy, Novara, Italy; Department of Hematology, Ospedale S. Croce e Carle, Cuneo, Italy; Department of Hematology, AO SS Antonio e Biagio e C. Arrigo, Alessandria, Italy; Division of Hematology, AOU Citt a della Salute e della Scienza di Torino, Torino, Italy; Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; FISM Registry, SS Antonio e Biagio e C. Arrigo Hospital, Alessandria, Tuscany, Italy; Department of Clinical and Biological Sciences, Division of Hematology, University of Turin, Turin, Italy

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.

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.

Development of cellular and humoral response against WT1 protein vaccination in mice

To the Editor: Many anti‐cancer vaccination strategies have been tested in mice and humans in the attempt to eradicate leukemia cells 1. The vast majority of clinical trials are based on peptide vaccination which allows the induction of cellular response to specific tumor associated antigens 2. WT1(Wilms tumor‐1) gene is located on chromosome 11p13 and encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation. WT1 was originally described as a tumor suppressor gene although many evidences demonstrated that it plays an oncogenic function in the setting of leukemia. WT1 protein represents an optimal tumor antigen since it is highly expressed in acute leukemias, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms 3. By contrast, it is expressed at very low levels in normal hematopoietic progenitors. Expression of the WT1 protein is restricted to a limited set of tissues, including the gonads, uterus, kidney, and spleen.