RNA-seq was performed as previously described (Blevins et al., 2011). Two microgram of RNA were retrotranscribed using random nucleotides primers and RNase H. cDNAs were then sheared to an average fragment size of 200 bases. In brief, polyA-containing mRNA was purified using oligo-dT beads from 10 ug of total RNAs for each sample and fragmented into small fragments using Zn cations under elevated temperature. Cleaved RNA fragments were reverse-transcribed into first strand cDNA using random primers (Invitrogen Inc.), followed by second-strand cDNA synthesis. After end-repair processing, a single ‘A’ base was added to cDNA fragments at 3′ end. cDNAs were subsequently ligated to adapters, purified by 2%
agarose gel, and then enriched by PCR to create the final cDNA library. RNA double-end sequencing was performed using Illumina 2000 using the standard protocol. The cDNA library of each sample was loaded to a single lane of an Illumina flow cell. The data were then analyzed using dedicated tools (Bellerophontes, (Abate et al., 2012) deFuse, (McPherson et al., 2011) Chimerascan (Iyer et al., 2011) and TxFuse (Singh et al., 2012) and Pelagus (Abate F. et al. in preparation).
Mice treatment
The following drugs and routes were implemented: CEP28122 (Cephalon, 100 mg/kg body weight BID), AZD6244 (ChemieTek, 100mg/kg), SGN-35 (1mg/kg q4dx3 or 4) were administered by oral gavage, and intravenous (i.v.) injections, respectively.
Analysis of clinical data
Overall patient survival (OS) was calculated from the time of histologic transformation to the last follow-up or death of any cause. Log-rank test was used to investigate the impact on OS of categorical variables. The cumulative probability of OS was plotted according to the Kaplan-Meier method. Statistical analysis was performed with the SPSS software.
REFERENCES METHODS
Abate, F., Acquaviva, A., Paciello, G., Foti, C., Ficarra, E., Ferrarini, A., Delledonne, M., Iacobucci, I., Soverini, S., Martinelli, G., and Macii, E. (2012). Bellerophontes: an RNA-Seq data analysis framework for chimeric transcripts discovery based on accurate fusion model. Bioinformatics 28, 2114-2121.
Results
149
Blevins, T., Rajeswaran, R., Aregger, M., Borah, B. K., Schepetilnikov, M., Baerlocher, L., Farinelli, L., Meins, F., Jr., Hohn, T., and Pooggin, M. M. (2011). Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic acids research 39, 5003-5014.
Fu, L., and Medico, E. (2007). FLAME, a novel fuzzy clustering method for the analysis of DNA microarray data. BMC Bioinformatics 8, 3.
Iafrate, A. J., Feuk, L., Rivera, M. N., Listewnik, M. L., Donahoe, P. K., Qi, Y., Scherer, S.
W., and Lee, C. (2004). Detection of large-scale variation in the human genome. Nature genetics 36, 949-951.
Iyer, M. K., Chinnaiyan, A. M., and Maher, C. A. (2011). ChimeraScan: a tool for identifying chimeric transcription in sequencing data. Bioinformatics 27, 2903-2904.
Kwee, I., Rinaldi, A., De Campos, C., and Bertoni, F. (2012). Fast and Robust Segmentation of Copy Number Profiles Using Multi-Scale Edge Detection. Submitted.
Lenz, G., Wright, G. W., Emre, N. C., Kohlhammer, H., Dave, S. S., Davis, R. E., Carty, S., Lam, L. T., Shaffer, A. L., Xiao, W., et al. (2008). Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proceedings of the National Academy of Sciences of the United States of America 105, 13520-13525.
McPherson, A., Hormozdiari, F., Zayed, A., Giuliany, R., Ha, G., Sun, M. G., Griffith, M., Heravi Moussavi, A., Senz, J., Melnyk, N., et al. (2011). deFuse: an algorithm for gene fusion discovery in tumor RNA-Seq data. PLoS computational biology 7, e1001138.
Mian, M., Rinaldi, A., Mensah, A. A., Rossi, D., Ladetto, M., Forconi, F., Marasca, R., Gattei, V., Zucca, E., Cavalli, F., et al. (2012). Del(13q14.3) length matters: an integrated analysis of genomic, fluorescence in situ hybridization and clinical data in 169 chronic lymphocytic leukaemia patients with 13q deletion alone or a normal karyotype.
Hematological oncology 30, 46-49.
Piva, R., Agnelli, L., Pellegrino, E., Todoerti, K., Grosso, V., Tamagno, I., Fornari, A., Martinoglio, B., Medico, E., Zamo, A., et al. (2010). Gene expression profiling uncovers molecular classifiers for the recognition of anaplastic large-cell lymphoma within peripheral T-cell neoplasms. J Clin Oncol 28, 1583-1590.
Rinaldi, A., Mian, M., Kwee, I., Rossi, D., Deambrogi, C., Mensah, A. A., Forconi, F., Spina, V., Cencini, E., Drandi, D., et al. (2011). Genome-wide DNA profiling better defines the prognosis of chronic lymphocytic leukaemia. British journal of haematology 154, 590-599.
Results
150
Shultz, L. D., Ishikawa, F., and Greiner, D. L. (2007). Humanized mice in translational biomedical research. Nature reviews Immunology 7, 118-130.
Singh, D., Chan, J. M., Zoppoli, P., Niola, F., Sullivan, R., Castano, A., Liu, E. M., Reichel, J., Porrati, P., Pellegatta, S., et al. (2012). Transforming fusions of FGFR and TACC genes in human glioblastoma. Science 337, 1231-1235.
Affiliations and Acknowledgments
1Department of Molecular Biotechnology and Health Science and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, 10126 Italy;
2Department of Control and Computer Engineering, Politecnico di Torino, 10129, Italy;
3Department of Biomedical Informatics, Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10027 USA; 4Lymphoma and Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, 6500 Switzerland; 5Pathology & Lymphoid Malignancies Units, San Raffaele Scientific Institute, Milan, 20132 Italy; 6Molecular Imaging Center, Department of Chemistry IFM and Molecular Imaging Center, University of Torino, Torino, 10125 Italy;
7Oncology, Cephalon Inc.,41 Moores Road Frazer, PA 19355 USA; 8University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 9Institute of Hematology and Medical Oncology L. and A. Seràgnoli, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, 40138 Italy; 10Institute of Hematology, University of Perugia, Ospedale S. Maria della Misericordia, S. Andrea delle Fratte, Perugia, 06156 Italy; 11The Jackson Laboratory, Bar Harbor, ME, 04609 USA; 12Fasteris SA, Plan-les-Ouates/Geneva, 1028 Switzerland; 13IDSIA Dalle Molle Institute for Artificial Intelligence, Manno, CH-6928 Switzerland; 14SIB Swiss Institute of Bioinformatics,1015 Lausanne, Switzerland;
15Department of Pathology, and NYU Cancer Center, New York University School of Medicine, New York, NY, 10016 USA; and 16Departmentof Oncology, University of Torino and Institute for Cancer Research at Candiolo, 10060 Italy, 17Lymphoma Unit, IOSI Oncology Institute of Southern Switzerland, 6500 Bellinzona, Switzerland.
The European T-Cell Lymphoma Study Group: Italy: Cristina Abele, Luca Bessone, Antonella Barreca, Nicoletta Chiesa, Ramona Crescenzo, Antonella Fienga, Marcello Gaudiano, Filomena di Giacomo, Giorgio Inghirami, Indira Landra, Elena Lasorsa,
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Rodolfo Marchiorlatti, Barbara Martinoglio, Enzo Medico, Gian Battista Ferrero, Katia Messana, Elisabetta Mereu, Elisa Pellegrino, Roberto Piva, Irene Scafo’, Elisa Spaccarotella, Fabrizio Tabbo’, Maria Todaro, Ivana Ubezzi, Susanna Urigu (Azienda Ospedaliera Città della Salute e della Scienza di Torino, University of Torino); Francesco Abate, Elisa Ficarra, Andrea Acquaviva (Politecnico di Torino); Antonella Barreca, Domenico Novero, Annalisa Chiapella and Umberto Vitolo (ASO Molinette, and San Luigi Gonzaga Torino); Marco Chilosi and Alberto Zamó (University of Verona); Fabio Facchetti and Silvia Lonardi (University of Brescia); Anna De Chiara and Franco Fulciniti (National Cancer Institute, Napoli); Claudio Doglioni, Andrés Ferreri and Maurilio Ponzoni (San Raffaele Institute, Milano); Claudio Agostinelli, Pier Paolo Piccaluga and Stefano Pileri (University of Bologna); Brunangelo Falini and Enrico Tiacci (University of Perugia).
Belgium: Peter Van Loo, Thomas Tousseyn, and Christiane De Wolf-Peeters (University of Leuven); Germany: Eva Geissinger, Hans Konrad Muller-Hermelink and Andreas Rosenwald, (University of Wuerzburg); Spain: Miguel Angel Piris and Maria E. Rodriguez (Hospital Universitario Marqués de Valdecilla, IFIMAV, Santander and Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid)
Current address for Mangeng Cheng: In Vitro Pharmacology, Merck Research Laboratory, BMB11-138, 33 Avenue Louis Pasteur, Boston, MA 02115
GI is supported by the Italian Association for Cancer Research (AIRC) Special Program in Clinical Molecular Oncology, Milan (5x1000 No. 10007); Regione Piemonte (ONCOPROT, CIPE 25/2005); ImmOnc (Innovative approaches to boost the immune responses, Programma Operativo Regionale, Piattaforme Innovative BIO F.E.S.R.
2007/13, Asse 1 'Ricerca e innovazione' della LR 34/2004) and the Oncology Program of Compagnia di San Paolo, Torino. RR is supported by Partnership for Cure, NIH 1 P50 MH094267-01, NIH 1 U54 CA121852-05, NIH 1R01CA164152-01. FB sponsored by the Oncosuisse KLS-02403-02-2009 (Bern, Switzerland); Anna Lisa Stiftung (Ascona, Switzerland); Nelia and Amadeo Barletta Foundation (Lausanne, Switzerland); RP by Rete Oncologica del Piemonte e della Valle d’Aosta. LDS is supported by National Institutes of Health (USA) grant CA034196. M.B. is enrolled in the PhD program in Pharmaceutical Sciences, University of Geneva, Switzerland. We thank Drs. Vigliani C, Fioravanti A, and Mossino M for their technical support.
Results
revealed by global genomic analyses. Science321, 1801-1806 (2008).
3. Piva, R., et al. Gene expression profiling uncovers molecular classifiers for the recognition of anaplastic large-cell lymphoma within peripheral T-cell neoplasms. J Clin Oncol28, 1583-1590 (2010).
4. Zamo, A., et al. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene21, 1038-1047 (2002).
5. Tabbo, F., Barreca, A., Piva, R. & Inghirami, G. ALK Signaling and Target Therapy in Anaplastic Large Cell Lymphoma. Front Oncol2, 41 (2012).
6. Eckerle, S., et al. Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma. Leukemia23, 2129-2138 (2009).
7. Inghirami, G., et al. Molecular characterization of CD30+ anaplastic large-cell lymphoma: high frequency of c-myc proto-oncogene activation.
Blood83, 3581-3590 (1994).
8. d'Amore, F., Jantunen, E. & Relander, T. Hemopoietic stem cell transplantation in T-cell malignancies: who, when, and how? Curr Hematol Malig Rep4, 236-244 (2009).
9. Shultz, L.D., Brehm, M.A., Garcia-Martinez, J.V. & Greiner, D.L.
Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol12, 786-798 (2012).
10. Garber, K. From human to mouse and back: 'tumorgraft' models surge in popularity. J Natl Cancer Inst101, 6-8 (2009).
11. Macor, P., et al. An update on the xenograft and mouse models suitable for investigating new therapeutic compounds for the treatment of B-cell malignancies. Current pharmaceutical design14, 2023-2039 (2008).
12. Green, M.R., et al. Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma. Blood116, 3268-3277 (2010).
13. Boudry-Labis, E., et al. Neurofibromatosis-1 gene deletions and mutations in de novo adult acute myeloid leukemia. American journal of hematology (2013).
14. Staser, K., et al. Normal hematopoiesis and neurofibromin-deficient myeloproliferative disease require Erk. J Clin Invest123, 329-334 (2013).
15. Chang, T., et al. Sustained MEK inhibition abrogates myeloproliferative
disease in Nf1 mutant mice. J Clin Invest123, 335-339 (2013).
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