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CALL FOR ABSTRACT PER COMUNICAZIONI ORALI E POSTER Il presente modulo deve essere compilato e trasmesso via mail all’indirizzo [email protected] entro e non oltre il 31 LUGLIO 2016 Istruzioni per la compilazione: Tutti gli abstract dovranno essere redatti in lingua inglese Non saranno accettati abstract trasmessi via fax L’abstract dovrà contenere le seguenti sezioni: introduzione, metodi impiegati, risultati e conclusioni L’accettazione ufficiale dell’abstract è subordinata al giudizio del Comitato scientifico che valuterà il contributo pervenuto in base all’originalità/innovatività, metodologia ed impatto clinico della ricerca. L’abstract accettato verrà trasmesso all’Editore per gli atti congressuali, nella forma in cui sarà pervenuto Carattere “Times New Roman”, dimensione 12, Interlinea 1pt, allineamento giustificato Spazio disponibile massimo 2 pagine (figure e bibliografia incluse) Massimo 8 autori per abstract Indicare preferenza di presentazione (orale vs poster) ed area tematica per la quale si presenta l’abstract La preferenza di presentazione e l’area tematica indicata sono subordinate alla scelta insindacabile del Comitato Scientifico Indicare preferenza di presentazione: X Comunicazione orale □ E- Poster Indicare Relatore se diverso dal 1° autore: Nome____________ Cognome__________________ Mail___________________________ Cell____________________________________________ DATI 1° AUTORE PER LA CORRISPONDENZA: Nome: WALTER Cognome: ARANCIO Data di nascita 19 maggio 1977 Ente di appartenenza: Università degli studi di Palermo CF:RNCWTR77E19G273D Cell: E-mail: [email protected] 3291886211 Indicare l’area tematica di presentazione: □ Patologia feto-placentare □ Patologie apparato digerente □ Patologia pleuro-polmonare □ Patologia endocrina □ Ematopatologia □ Patologia mammaria □ Dermatopatologia □ Uropatologia □ Patologia ginecologica □ Patologia testa e collo □ Patologia molecolare □ Patologia dell’osso e tessuti molli □ Neuropatologia □ Patologia pediatrica □ Patologia cardiovascolare □ Patologia ultrastrutturale □ Patologia dei trapianti □ Paleopatologia □ Interesse Generale (biobanche, rischio clinico,statistica, linee guida, etica, tracciabilità, ecc.) Anaplastic Large T Cell Lymphoma (ALCL) is characterized by high expression of P-Selectin Glycoprotein Ligand 1 (PSGL-1) that positively correlates with CD30 expression and TCR signaling pathway. Walter Arancio1, Alessandro Gulino1, Valeria Cancila1, Pier Paolo Piccaluga2, Paolo Macor3, Arianna di Napoli4, Sabina Sangaletti5, Ada Maria florena6. 1 Laboratorio di Immunologia dei Tumori, ProSAMI, Università degli Studi di Palermo. Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, ALMA MATER STUDIORUM - Università di Bologna. 3 Dipartimento di Scienze della Vita, Università degli Studi di Trieste. 4 Dipartimento di Medicina Clinica E Molecolare, Università degli Studi di Roma "La Sapienza". 5 Struttura Complessa Immunologia Molecolare, Fondazione IRCSS Istituto Nazionale dei Tumori, Milano. 6 Scienze per la Promozione della Salute e Materno Infantile "G.D'Alessandro” Università degli Studi di Palermo. 2 INTRODUCTION: _________________________________________________________________________ P-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is one of the major ligands of selectins, it is normally expressed in microvilli of polymorphonucleated leukocytes, monocytes, activated T-lymphocytes, plasma cells and activated platelets, where it has a major role in regulating the tethering, rolling and extravasation(1). It may bind also von Willerbrand factor (vWF) and chemokines such as CCL21 and CCL19 to facilitate T-cell lymph node entry. PSGL-1 is a homodimeric disulfide-linked glycoprotein, which is highly post-translationally modified. It may bind with variable affinity to its different ligands depending on the status of its modofications. PSGL-1 cytoplasmic domain transduces signals upon activation via spleen tyrosine kinase (Syk) after clustering within lipid rafts; the signaling leads to the secretion of cytokines or the activation of membrane integrins to promote extravasation. Noteworthy, PSGL-1 is involved in core molecular programs such as Syk, PLC2, PI3K or MAPK pathways that let envisage a role for this molecule beyond cell adhesion and extravasation. The cross-linking of PSGL-1 in activated T cells is able to induce apoptosis, and PSGL-1 acts as an immune checkpoint regulator that promoting T cell exhaustion also through Programmed-cell death-protein-1 (PD-1) upregulation, and IL-2 signaling quencing (2,3). PSGL-1 expression in pathological conditions has been reported in lymphoproliferative disorders showing plasmacytic differentiation and in multiple myeloma, (MM) (4,5), where it has been suggested as a potential target for humoral immunotherapy (4). In T-cell lymphomas, the expression of PSGL-1 and its molecular correlates have been not investigated so far. Most T-cell lymphomas are classified as peripheral T-cell lymphomas (PTCL) a heterogeneous group of aggressive non-Hodgkin lymphomas (NHLs). The most common subtypes of PTCL, are PTCL not otherwise specified (PTCL-NOS), anaplastic large-cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL). Up to date, no effective therapeutic targets have been identified, except for the TNF receptor CD30, targeted by the anti-CD30 drug conjugated antibody Brentuximab-Vedotin (6). In this study we investigated the expression of PSGL-1 and its molecular co-relates in T-cell lymphomas and speculated on its suitability as functional target. METHODS:_______________________________________________________________________________ Gene Expression Profiles (GEPs) have been examined in a panel of 180 T-cell lymphomas and control samples and in a set of 498 B-cell lymphomas and control samples in order to evaluate the expression of selected genes and their correlations. Gene set enrichment and cluster analyses have been performed. PSGL-1 expression has been evaluated by Immuno-Histochemistry (IHC) on Tissue Macro Arrays (TMAs) comprising 110 ALCLs and 50 PTCLs-NOS. Immunolocalization has been performed as previously reported (4,5). CIBERSORT software has been applied on GEP data in order to infer the enrichment of specific bystander immune elements. In vitro experiments have been performed on Karpas229, SUDHL1, L82 and TS cell lines. Cellular viability was assessed by MTT assay. Inhibition of adhesion was evaluated as the ability of cells to remain adherent to endothelial Ea.hy926 cells. Capping of PSGL-1 was manually scored on confocal microscopy. RESULTS:________________________________________________________________________________ We evaluated the expression of SELPLG in 678 samples, comprising both T and B cells, and lymphomatous samples. SELPLG was first investigated in B-cell setting, being plasma cells neoplasms and naïve B-cells positive and negative controls respectively. Among B-cell lymphomas, Classical Hodgking Lymphoma (CHL) and T-cell/histiocyte-rich diffuse large B-cell Lymphoma(TCRBL) showed the highest expression of SELPLG. Notably, these histotypes are characterized by a very rich environment comprising T cells and myeloid cells. In the T cell setting consistent expression of SELPLG was detected, with ALCL showing the highest expression (Fig.1). ALCL encompasses different clinical entities that histologically share the presence of large pleomorphic T-cells expressing CD30. ALCLs can be classified according to the expression of Anaplastic Lymphoma Kinase (ALK). Surprisingly, no statistically significant difference was detected in SELPLG expression between ALK+ and ALK- ALCL specimens. Based on data from GEP analysis, we evaluated the expression of PSGL-1 in TMAs of 160 PTCL, confirming that PSGL-1 was consistently highly expressed in ALCL (Fig.2). Prompted by GEP and IHC analyses, where CD30+ ALCL and CHL resulted highly expressing SELPLG/PSGL-1, we hypothesized that a positive correlation between SELPLG and TNFRSF8 (CD30 coding gene) might occur. Our data confirmed this hypothesis. The correlation was observed both in T and B lymphoma settings, suggesting that it is not cell-type intrinsic. Network analysis by Genemania pointed to six genes potentially correlated with both SELPLG and TNFRSF8, namely MSC, SNX20, TNFRSF4, TNFRSF25, TNIP1 and TRAF1. We next evaluated the genes that showed a positive Pearson’s product-moment correlation (coefficient 0.75 to 1) with SELPLG in different settings. Strikingly, ALCL showed the highest number of genes significantly correlated with SELPLG, which do not overlap with the other settings analyzed, suggesting that SELPLG activity might be peculiar in ALCL. GSEA of such genes pointed to specific pathways and cellular functions, many of which previously associated with cancer, including RNA splicing and RNA processing functions. Subsequently, we tried to classify ALCLs according to SELPLG expression in order to identify relevant differences in the transcriptional program related with SELPLG. Almost 4000 genes differentially clustered together with high or low cases (Fig.3), TNIP1 (from Genemania) and T cell receptor (TCR) signaling genes were among them. Among TCR signaling genes, LCK, LAT, ZAP70, SYK proved to be correlated with SELPLG. These data suggest that PSGL-1 signaling correlates with TCR signaling in ALCL. In order to functionally characterize PSGL-1 in ALCL cells, we first investigated its expression on cells with two different antibodies: KPL1 (a known blocking antibody against PSGL-1) and TB5 (with non-blocking functions). Coherently, KPL1 was able to interfere with ALCL adhesion in vitro, while TB5 was not effective. As PSGL-1 required raft formation during signaling, we evaluated the capability of two antibodies to induce capping and found that only TB5 was able to do so. Consistently, since ALCL has an activated phenotype, TB5 was effective in induce apoptosis though variably in the different ALCL cell lines. Finally, we evaluated via CIBERSORT method if SELPLG expression might influence the microenvironment characterizing the enrichment of specific bystander immune elements. ALCLs result enriched in Mast cells and neutrophils in comparison with other PTCLs and normal controls, and this enrichment was particularly relevant in highlyexpressing SELPLG ALCLs. Fig. 3 Fig. 1 Fig. 2 CONCLUSIONS:___________________________________________________________________________ SELPLG expression characterize T-cell lymphoma, in particular ALCL and it correlates with CD30 expression. SELPLG expression correlates with relevant pathways such as TCR signaling. In ALCL cell lines, PSGL-1 has a functionally expression that can be used to modulate adhesion, signaling and death. 1. Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule. Evangelista V, et Al. Blood. 1999 Feb 1;93(3):876-85. 2. PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion. Tinoco R, et Al. Immunity. 2016 May 17;44(5):1190-203 3. In vivo regulation of Bcl6 and T follicular helper cell development. Poholek AC, et Al. J Immunol. 2010 Jul 1;185(1):313-26. 4. P-selectin glycoprotein ligand-1 as a potential target for humoral immunotherapy of multiple myeloma. Tripodo C, et Al. Curr Cancer Drug Targets. 2009 Aug;9(5):617-25. 5. Identification of CD162 in plasma-cell dyscrasia.. Florena AM, et Al. Lancet Oncol. 2005 Aug;6(8):632.. 6. Antibody-drug conjugates for cancer therapy. Thomas A, et Al. Lancet Oncol. 2016 Jun;17(6):e254-62.