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Ion Channels and Cancer

Department of Experimental
Oncology and Pathology
Viale GB Morgagni, 50- 50134 Florence
Tel: 055 4598206 Fax: 055 4598900

Principal Investigator: Dr. Annarosa Arcangeli

staff picture  Staff

  • Olivia Crociani, Researcher of the University of Florence
  • Alessio Masi, Post Doc
  • Elena Lastraioli, Post Doc
  • Serena Pillozzi, Ph D.
  • Emanuele Cilia, Ph.D.
  • Marika Masselli, Ph. D.
  • Emanuele De Lorenzo, Graduate student
  • Dr. Patrizia Bernini, Graduatestudent

Most of the Unit's research is performed at the Animal Facility of the University of Florence (CeSAL). This Facility contains a brand-new section fully equipped for the maintenance and manipulation of transgenic and nude mice (LIGeMA).
The Unit is involved in studies aimed at determining the role of ion channels in tumor establishment and progression. In particular, our group has demonstrated that K+ channels belonging to the hERG1 family are frequently mis/overexpressed in several human cancers and their activity contributes to the regulation of various aspects of neoplastic growth, from proliferation and apoptosis, to the acquisition of an invasive phenotype and regulation of VEGF secretion and hence of tumour angiogenesis (see list of publications).

Main Research Themes

hERG1 and cancer

  1. Expression in various types of cancer
    Recent evidence indicates that ion channels may represent novel regulators of cancer establishment and progression. In fact, the expression and activity of different types of ion channels, especially K+ channels, mark and regulate specific stages of cancer progression. We and other research groups, have demonstrated that K+ channels belonging to the hERG1 family can be included in the list of ion channels mis/overexpressed in cancer cells and whose activity regulates different aspect of neoplastic growth (reviewed in 13)
    Main achievements. During the last few years Dr.Arcangeli's group has provided evidence that hERG1 channels have an important role in establishing and regulating tumor progression in the human gastrointestinal tract, namely esophagus, stomach and colorectum.

    1. Colorectal cancer. The first studies on colorectal cancer were extended by performing immunohistochemistry (IHC) on a larger cohort of patients and including other potential molecular markers of malignancy (VEGF-A, CA IX and GLUT1), searching for correlations between the expression of hERG1, VEGF-A, CAIX and GLUT1, and clinico-pathological parameters, follow-up and response to chemotherapy. A strict correlation between hERG1 and VEGF (p<0.0001) and GLUT1 (p=0.015) expression emerged. In addition, a statistically significant correlation was also found between hERG1 expression and a better prognosis and response to chemotherapy in the group of Dukes C and N+ patients, who were treated with adjuvant therapy. Collaborators: Dr. R. Moretti, Dr. F. Di Costanzo (University Hospital of Careggi, Florence)
    2. Gastric cancer. We recently demonstrated that hERG1 channels were expressed in the majority of samples obtained from patients with Barrett's esophagus and that 89% of the patients whose lesions proceeded towards cancer expressed the hERG1 protein (4). We extended such observation to Gastric Cancer. A first immunohistochemical study on specimens of both advanced and early gastric cancers, as well as of gastric dysplasias showed that: i) hERG1 is prevalently expressed in Laurčn's intestinal type carcinomas (p =0.022); ii) hERG1 expression is correlated with VEGF-A expression (p < 0.0001); iii) a statistically significant correlation occurs between hERG1 expression in dysplasias and progression towards cancer (P=0.046). Collaborators: Prof. P. Bechi (University of Florence), Prof. G. Demanzoni (University of Verona) and GIRCG (Italian Research Group of Gastric Cancer).
    3. Acute Myeloid leukemias (AML). The quantitative expression of herg1 transcripts was analyzed by Real Time PCR (RQ-PCR)) on peripheral blood (PB) mononuclear cells from 61 patients with de novo AML. AML cases examined showed an increased level of herg1 transcript (mean value = 3.29E+01 ±1.81E+01), and an even greater increase in the level of herg1b (mean value = 5.11E+04 ± 5.18E+03). The correlation between herg1 expression and clinical features and outcome was analyzed. It emerged that: i) complete remission (CR) was obtained in 61% of hERG1+ versus 82% in hERG1- patients (p = 0.222); ii) the percentage of relapses was 79% in hERG1+ versus 21% in hERG1- patients (p = 0.035); iii) the median time to relapse was 11.9 in hERG1+ versus 25.3 months in hERG1- patients (p= 0.014). Finally, the overall survival (OS) in the same cohort of patients was analyzed: the median OS of hERG1+ AML patients was 12 months, compared with 23 months for hERG1- ( p= 0.026). This is an important clinical result emerging from our data, since herg1 expression configures as a negative prognostic factor in AML
    4. Acute Lymphoblastic leukemias (ALL). The quantitative expression of herg1 and herg1b transcripts was analysed in leukemic blasts obtained from the bone marrow (BM) of 66 pediatric patients with B-ALL. The expression in CD19+ cells from normal controls was taken as 1. While the herg1 transcript was down-regulated in all the B-ALL samples, the herg1b isoform was expressed in 80% of cases of B-ALL: among positive cases 48% showed overexpression (from 10 to 1000 fold). A higher expression of herg1b was also detected in the early-B group compared to the pro-B subtype group. The presence of herg1b overexpression was found to correlate with the presence of favorable prognostic factors at diagnosis. These results suggest that the expression of herg1b may provide prognostic information on B-ALL patient survival. Collaborators: Prof..G. Basso (University of Padova), Prof. G. Bernini and Dr. A. Lippi (University of Florence, Meyer Hospital, Florence).

    Current work and future plans: On the basis of the results obtained so far and presented above, the analysis of hERG1 expression will be continued in the four types of cancer as follows:

    1. Colorectal and gastric cancers: a wider number of cases will be examined and RQ-PCR will be performed to evaluate the expression of herg1, herg1b, vegf-a and glut1.
    2. Acute leukemias: the expression of herg1/herg1b transcripts will be evaluated by RQ-PCR in a greater number of cases and correlated with cytogenetic risk groups as well as with genetic alterations affecting outcome of cytogenetically normal leukaemia patients (NPM1 and CEBPA mutations, FLT3-ITD, MLL-PTD). In addition, the expression of the hERG1 protein(s) in normal precursor cells and in leukeimas will be studied by flow cytometry (FC), in conjunction with clinical parameters. For hERG1 detection the MoAb developed in our laboratory will be used (see below).This part of the study will be done in collaboration with Prof. G. Basso, University of Padua.

  2. Molecular mechanisms leading to overexpression
    In the past years we have provided evidence that the herg1 gene (and the corresponding protein) is constantly over-expressed in various types of tumor cell lines. In addition, we have shown that tumour cells often express the herg1 alternative transcript herg1b along with the full-length herg1 RNA (19).
    Main achievements: Another variant was identified and cloned from tumor cells in our laboratory, named herg1BUSO (Accession number AJ609614) which, having both exon 1b replacing exons 1-5 and exon USO replacing exons 9-15, is the shortest herg1 isoform discovered so far. The two USO-containing isoforms are expressed in several normal and neoplastic human tissues and cells, but do not give detectable hERG1 currents, since they are retained intracellularly. The USO-containing isoforms form heterotetramers with both hERG1 and hERG1B in vitro and in vivo, and in this way can regulate trafficking of these hERG1 proteins, thus modulating the intensity of hERG1 currents. This modulatory mechanism, although not exclusive, is peculiar of tumor cells that highly express hERG1BUSO (Guasti et al., JCS submitted). Collaborators: Prof. E. Wanke and Dr. A. Becchetti (University of Milano Bicocca, Milano).
    Current work and future plans: A relevant point in the future studies will be that of defining the genetic mechanisms leading to herg1 gene(s) overexpression in tumor cells. The herg1 promoter has been recently identified. We will further identify the herg1b promoter, as well as putative sequences responsible for cell-specific expression and gene regulation. Moreover, we will evaluate possible amplification or deletions in proximity of herg1 chromosomal location. Finally, the methylation state of the promoter region of herg1 gene will de analyzed.
  3. hERG1 signalling and control of invasiveness
    A paper from our laboratory has demonstrated that the activity of hERG1 channels regulates cell invasiveness in colorectal cancers, and that a direct correlation exists between the amount of the hERG1 protein on the plasma membrane and the invasion capacity of colorectal cancer cells (15). The regulatory role of hERG1 channels in cell invasion can conceivably be traced back to the functional and physical association between hERG1 channels and β1 integrins we have described in neoplastic cell lines (reviewed in 3). Such a complex was definitely demonstrated to occur both in transfected and in tumor cells that endogenously express hERG1 channels. Such a complex was proven to have a regulatory role on integrin-dependent signaling. In fact, we demonstrated that the Focal Adhesion Kinase (FAK) co-immunoprecipitates with hERG1 upon adhesion onto FN and that such interaction is halted by hERG1 channel blockers. This interaction turned out to be necessary for the subsequent phosphorylation of FAK on tyrosine residues (p-Tyr), and for the switching on of the activity of the small GTPase Rac1(11).
    Main achievements: More recently we showed that a similar complex exists in AML. Such a complex comprises the hERG1B isoform, the VEGF receptor 1 (FLT-1) and the β1 integrin. The FLT-1/hERG1B/β1 complex regulates FLT-1 p-Tyr and downstream signaling. In addition, we demonstrated that AML cell migration depends on both FLT-1 and integrin stimulation, and is strictly regulated by hERG1 channel activity. The same mechanism was also operative in primary leukaemia blasts and in human hemopoietic progenitors (CD34+ cells) after proper stimulation to proliferate after addition of growth factors and cytokines. Finally, the activity of the FLT-1/hERG1B/β1 complex turned out to modulate VEGF-A secretion in AML. This mechanism can apparently account for a greter malignancy of hERG1 expressing AML when injected into NOD-SCID mice (1).
    Current work and future plans: we propose to:
    1. determine the molecular architecture of the β1/hERG1complex. We will study whether a direct intermolecular contact exists between the two proteins, making use of fluorescence resonance energy transfer (FRET). We will perform FRET using fluorophore- (xFPs)-tagged β1 and hERG1 proteins, transfected into "nude" cells. The FRET technique will be combined with the TIRFM technique (Total Internal Reflection Fluorescence Microscopy). All the FRET experiments necessary for the present project will be performed in collaboration with Prof. Pavone's group at the European Laboratory for non-linear spectroscopy (LENS), Florence, Italy, to which the PI also belongs.
    2. determine the role of hERG1 channels in the regulation of integrin dependent signaling (FAK phosphorylation and Rac1 activity) and cell invasion. Experiments relative to these tasks will be performed mainly using colorectal cancer cells as a model. We will analyze whether colorectal cancer cell lines with different hERG1 expression undergo FAK phosphorylation and Rac1 activation after adhesion onto integrin-dependent substrates and whether such activation depends on hERG1 channel activity. To test this latter point we will use both drugs that specifically block hERG1 currents, and hERG1- specific siRNAs. Based on results obtained in AML cells, we will also study the activaton of the PI3K/Akt pathways.

Prof. Arcangeli is also responsible of the Unit L.I.Ge.M.A. (see Core Facilities section)

Research Grants

  • Title of project: HERG K+ channels as novel regulators of tumor progression:prognostic significance and therapeutic potential.
    source of funding: ASSOCIAZIONE ITALIANA PER LA RICERCA SUL CANCRO.
    Years: 2004-2006. Amount: € 120000
  • Title of project: STUDY OF THE ROLE OF HERG K+ CHANNELS IN TUMOUR PROGRESSION OF COLORECTAL CANCER.
    source of funding: ASSOCIATION FOR INTERNATIONAL CANCER RESEARCH.
    Years: 2006-2009. Amount: € 182000.
  • Title of project: STUDY OF HERG1 EXPRESSION IN PRIMARY PAEDIATRIC LYMPHOMA AND ACUTE LYMPHATIC LEUKEMIA.
    source of funding: ASSOCIAZIONE Noi per Voi
    Years: 2006-2009 Amount: € 75000
  • Title of project: ESTABLISHMENT OF A LABORATORY FOR PRODUCTION AND STUDY OF TRANSGENIC ANIMALS.
    source of funding: ENTE CASSA DI RISPARMIO DI FIRENZE.
    Years: 2006-2007. Amount: € 36000.
  • Title of project: STUDY OF THE DYNAMICS OF MOLECULAR INTERACTIONS BETWEEN BETA1 INTEGRIN, HERG1 POTASSIUM CHANNELS AND FOCAL ADHESION KINASE IN THE REGULATION OF INVASIVENESS IN NORMAL AND NEOPLASTIC EPITHELIAL CELLS.
    source of funding: MINISTERO DELL'UNIVERSITA' E DELLA RICERCA SCIENTIFICA E TECNOLOGICA.
    Years: 2007-2009. Amount: € 54.714
  • Title of project: HERG K+ channels as novel regulators of tumor progression: prognostic significance and therapeutic potential.
    source of funding: ASSOCIAZIONE ITALIANA PER LA RICERCA SUL CANCRO.
    Years: 2007-2009. Amount: € 120000

Publications

  1. Pillozzi, S., Brizzi, M.F., Bernabei, P.A., Bartolozzi, B., Caporale, R., Basile, V., Boddi,V.,Pegoraro, L., Becchetti, A., and Arcangeli,A. (2007). VEGFR-1 (FLT-1), β1 integrin and hERG K+ channel form a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome. Blood, 110, 1238-1250, 2007. IF 10.131
  2. Furlan F., Taccola G., Grandolfo M., Guasti L., Arcangeli A., Nistri A. and Ballerini L. (2007) ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord. J. Neurosci., 27, 919-928. IF 7.506.
  3. Arcangeli, A e Becchetti A. (2006) Complex functional interaction between integrin receptors and ion channels. Trends Cell. Biol. 16: 631-639. IF 11.791
  4. Lastraioli E., Taddei A., Messerini L., Comin C.E., Festini M., Giannelli M., Tomezzoli A., Paglierani M., Mugnai G., De Manzoni G., Bechi P. e Arcangeli A. (2006), hERG1 channels in human esophagus: evidence for their aberrant expression in the malignant progression of Barrett's esophagus. J. Cell. Physiol. 209: 398-404. IF 4.362
  5. Restano-Cassulini R., Korolkova Y.V., Diocot S., Gurrola G., Guasti L., Possani L.D., Lazdunski M., Grishin E.V., Arcangeli A. e Wanke E. (2006) Species diversity and peptide toxins blocking selectivity of ether-a-go-go-related gene subfamily K+ channels in the central nervous system. Mol. Pharmacol. 69: 1673-1683. IF 4.612
  6. Salvati A., Ciani L., Ristori S., Martini G., Masi A. e Arcangeli A. (2006) Physico-chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid. Biophys. Chem. 121: 21-29. IF 1,925
  7. Masi A., Becchetti A., Restano-Cassulini R., Polvani S., Hofmann G., Buccoliero A.M., Paglierani M., Pollo B., Taddei G.L., Gallina P., Di Lorenzo N., Franceschetti S., Wanke E. e Arcangeli A. (2005) hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines. Br. J. Cancer. 93: 781-792. IF 4.115
  8. Furlan F., Guasti L., Avossa D., Becchetti A., Cilia E., Ballerini L. e Arcangeli A. (2005) Interneurons transiently express the ERG K+ channels during development of mouse spinal networks in vitro. Neurosci.135: 1179-1192. IF 3.410
  9. Guasti L., Cilia E., Crociani, O., Hofmann G., Polvani S., Becchetti A., Wanke E., Tempia F. e Arcangeli A. (2005) Expression pattern of the ether-a-go-go-related (ERG) family proteins in the adult mouse central nervous system: evidence for coassembly of different subunits. J. Comp. Neurol. 491: 157-174. IF 3.855
  10. Biagiotti T., D'Amico M., Marzi I., Di Gennaro P., Arcangeli A., Wanke E., Olivotto M. (2005) Cell renewing in neuroblastoma: electrophysiological and immunocytochemical characterization of stem cells and derivatives. Stem cells. 24: 443-453. IF 6.094
  11. Cherubini A., Hofmann G., Pillozzi S., Guasti L., Crociani O., Cilia E, Di Stefano P., Degani S., Balzi M., Olivotto M., Wanke E., Becchetti A, Defilippi P.,Wymore R. e Arcangeli A. (2005) hERG1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signalling. Mol. Biol. Cell. 16: 2972-2983. IF 6.520
  12. Arcangeli A. e Becchetti A. (2005) "Ion channels and cell cycle" in "Cell cycle and the central nervous system" Ed. D. Janigro. Humana Press Inc., New Jersey Totowa,
  13. Arcangeli A. Expression and role of hERG channels in cancer cells (2005) The hERG cardiac potassium channel: structure, function, and long QT syndrome. Wiley, Chichester. "Novartis Foundation Symposium". 266: 225-234. IF 1.879
  14. Arcangeli A., Becchetti A., Cherubini A., Crociani O., Defilippi P., Guasti L., Hofmann G., Pillozzi S., Olivotto M. e Wanke, E. (2004). Physical and functional interaction between integrins and hERG potassium channels. Biochem.Soc.Trans. 32: 826-827. IF 3.099
  15. Lastraioli E., Guasti L., Crociani O., Polvani S., Hofmann G., Witchel H., Bencini L., Calistri M., Messerini L., Scatizzi M., Moretti R., Wanke E., Olivotto M., Mugnai G. e Arcangeli, A. (2004). herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells. Cancer Res. 64: 606-611. IF 7.616
  16. Polvani S., Masi A., Pillozzi S., Gragnani L., Crociani O., Olivotto M., Becchetti A., Wanke E. e Arcangeli, A. (2003). Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3. Gene Expr.Patterns: 3, 767-776. IF 1.794
  17. Milnes J.T., Dempsey C.E., Ridley J.M., Crociani O., Arcangeli A., Hancox J.C. e Witchel, H.J. (2003). Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin. FEBS Lett. 547, 20-26. IF 3.415
  18. Milnes J.T., Crociani O., Arcangeli A., Hancox J.C. e Witchel H. J. (2003). Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652. Br.J.Pharmacol. 139: 887-898. IF 3.410
  19. Crociani O., Guasti L., Balzi M., Becchetti A., Wanke E., Olivotto M., Wymore R.S. e Arcangeli A. (2003). Cell cycle-dependent expression of HERG1 and HERG1B isoforms in tumor cells. J.Biol.Chem. 278: 2947-2955. IF 5.854
  20. Gullo F., Ales E., Rosati B., Lecchi M., Masi A., Guasti L., Cano-Abad M.F., Arcangeli A., Lopez M.G. e Wanke E. (2003). ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death? FASEB J. 17: 330-332. IF 7.064
  21. D'amico M, Biagiotti T., Fontana L., Restano-Cassulini R., Lasagna N., Arcangeli A., Wanke E. e Olivotto M. (2003) A HERG current sustains a cardiac-type action potential in neuroblastoma S cells. Biochem Biophys Res Commun. 302: 101-8. IF 3
  22. Dabizzi S., Noci I., Borri P., Borrani E., Giachi M., Balzi M., Taddei G.L., Marchionni M., Scarselli G. e Arcangeli A. (2003) Luteinizing hormone increase human endometrial cancer cell invasiveness through activation of PKA. Canc. Res. 63: 4281-4286. IF 7.616
  23. Pillozzi S., Brizzi M. F., Balzi M., Crociani O., Cherubini A., Guasti L., Bartolozzi B., Becchetti A., Wanke E., Bernabei, P.A., Olivotto M., Pegoraro L. e Arcangeli A. (2002). HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors. Leukemia 16: 1791-1798. IF 6.612
  24. Lecchi M., Redaelli E., Rosati B., Gurrola G., Florio T., Crociani O., Curia G., Restano-Cassulini R., Masi A., Arcangeli A., Olivotto M., Schettini G., Possani L. D. e Wanke, E. (2002). Isolation of a long-lasting eag-related gene-type K+ current in MMQ lactotrophs and its accommodating role during slow firing and prolactin release. J.Neurosci. 22, 3414-3425. IF 7.506
  25. Becchetti A., De Fusco M., Crociani O., Cherubini A., Restano-Cassulini R., Lecchi M., Masi A., Arcangeli A., Casari G. e Wanke E. (2002). The functional properties of the human ether-a-go-go-like (HELK2) K+ channel. Eur.J.Neurosci. 16: 415-428. IF 3.949
  26. Cherubini A., Pillozzi S., Hofmann G., Crociani O., Guasti L., Lastraioli E., Polvani S., Masi A., Becchetti A., Wanke E., Olivotto M. e Arcangeli A. (2002) HERG K+ channels and beta1 integrins interact through the assembly of a macromolecular complex. Ann N Y Acad Sci 973: 559-61. IF 1.971
  27. Hofmann G., Bernabei P.A., Crociani O., Cherubini A., Guasti L., Pillozzi S., Lastraioli E., Polvani S., Bartolozzi B., Solazzo V., Gragnani L., Defilippi P., Rosati B., Wanke E., Olivotto M. e Arcangeli A. (2001). HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1. J.Biol.Chem. 276: 4923-4931. IF 5.854
  28. Noci I., Borri P., Bonfirraro G., Chieffi O., Arcangeli A., Cherubini A., Dabizzi S., Buccoliero A.M., Paglierani M. e Taddei G.L. (2001) Longstanding survival without cancer progression in a patient affected by endometrial carcinoma treated primarily with leuprolide. British .J. Cancer 85: 333-336. IF 4.115
  29. Fontana L., D'amico M., Crociani O., Biagiotti T., Solazzo M., Rosati B., Arcangeli A., Wanke E. e Olivotto M. (2001) Long-term modulation of herg channel gating in hypoxia. Biochem.Biophys.Res. Commun. 286: 857-862. IF 3

Collaborations

  • Prof. G. Rainaldi, Laboratory of Genic and Molecular Therapy, Institute of Clinical Physiology CNR Research Area Pisa;
  • Dr. F. Di Costanzo (ITT work group coordinator " Raccomandazioni Cliniche del Carcinoma del Colon-retto"
  • Dr. R. Moretti (I Division of General Surgery and Organ Transplant, University Hospital of Careggi, Florence);
  • Dr. F. Di Costanzo (Medical Oncology Unit, Az. University Hospital of Careggi, Florence);
  • Prof. P. Bechi (Surgical Pathology I, University of Florence);
  • Prof. G. Bernini , Dr. A.Lippi (Units of Pediatric Oncohematology and Hemotransplantation, Meyer Hospital, Florence);
  • Prof. G. De Manzoni (I Clinical Division of General Surgery, Borgo Trento Hospital, Verona, coordinator GIRCG (Italian Research Group on Gastric Cancer);
  • Prof. E. Wanke (Department of Biotechnology and Biosciences, University of Milan Bicocca, Milan);
  • Prof. A. Becchetti (Department of Biotechnology and Biosciences, University of Milan Bicocca, Milan);
  • Prof. P. De Filippi (Department of Genetics, Biology and Biochemistry, University of Turin);
  • Dr. M.F. Brizzi (Dip. Internal Medicine, University of Turin);
  • Prof. . F. Pavone (European Laboratory for non linear spectroscopy (LENS), Firenze);
  • Dr. R. Wymore (Center of Health Sciences and college of osteopathic medicine, Oklahoma State University, Tulsa, USA);
  • Dr.J. Mitcheson (Department of Cell Physiology and Pharmacology,University of Leicester, UK); Dr. M. Levin (Director, Forsyth Center for Regenerative and Developmental Biology, Boston, U.S.A.);
  • Dr.E. Gherardi MRC Centre, Cambridge, UK);
  • Dr. S. Aparicio (Dept. Pathology and Laboratory Medicine, Univ of British Columbia, Vancouver, Canada);
  • Dr. H. Duff (University of Calgary, Canada),
  • Dr. G. Basso (Laboratory of Pediatric Onohematology, University of Padua).

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