PDGFRA Activity Is Mediated by An Autocrine Loop in Peripheral T-Cell Lymphoma/Not Otherwise Specified (PTCL/NOS).
Authors: Rossi Maura , Alviano Francesco , Laginestra Antonella , Tazzari Pierluigi , Ricci Francesca , Esposito Aurora , Agostinelli Claudio , Sista Maria Teresa , Gazzola Anna , Mannu Claudia , Righi Simona , Sapienza Maria Rosaria , Bagnara Gian Paolo , Hartmann Sylvia , Hansmann Martin Leo , Zinzani Pier Luigi , Pileri Stefano Aldo , Piccaluga Pier Paolo 
Published: October 2019

Abstract
Abstract 4631

Background
Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of tumors representing approximately 12% of lymphoid neoplasms, basically subdivided into specified and not specified (NOS) forms. PTCL/NOS, corresponding to about 60%–70% of PTCLs, cannot be further classified on the basis of morphology, phenotype, or conventional molecular studies. Clinically, PTCLs/NOS are highly aggressive lymphomas, with a poor response to therapy, and dismal overall survival (20-30%). Their pathobiology is poorly known, though recent gene expression profiling (GEP) studies have provided some hints for better understanding their pathogenesis. In particular, GEP and immunohistochemical studies on tissue-microarrays (TMAs) demonstrated PDGFRA to be systematically activated in almost all PTCLs/NOS, by nominating it as potential therapeutic target.

Aims
In this study, we aimed to identify the determinants of PDGFRA activation in PTCL/NOS. Specifically, we studied PDGFRA locus in order to identify possible mutations, translocations, or copy number variations and we explored the possible existence of an autocrine/paracrine loop sustaining an otherwise integer kinase.

Methods
The PDGFRA locus (4q1.1-4q1.3) was studied by FISH and wide-genome SNPs analysis (Affymetrix 500K Array). Direct sequencing of all PDGFRA exons and introns as well as of the promoter region was also performed in 90 cases. IHC and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells as well as in a FIP1L1-PDGFRApos chronic eosinophilic leukemia cell line (EOL-1) before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&D System), given at various concentrations (20-40-60-80 ug/mL). Vitality assessments, proliferation/cell cycle assay (by In Situ Cell Proliferation kit, FLUOS – Roche) and evaluation of PDGFRA and p-PDGFRA were performed at 24, 48, 96 hours. A human PDGF peptide (R&D Sytems) was added to cultured cells for 6 hours to evaluate whether PDGFRA de-phosphorilation was really due to PDGF ligand remotion.

Results
First, FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. Thus we tested the hypothesis of an autocrine/paracrine stimulation. PDGF-A, PDGF-B and PDGF-C were found to be expressed by neoplastic cells at IHC in 93-95% of cases. In addition, PDGF-AA was found to be secreted by cultured neoplastic cells by ELISA. Notheworthy, PTCL cells secreted much more ligand than any other cell taken as control. We then tested whether PDGFRA phosphorylation was actually due to the presence of a PDGF ligand. Indeed, PTCL cells treated with anti-PDGF ligand neutralizing antibody at various concentrations showed PDGFRA dephosphorilation ranging from 30% up to 90% in a time dependent manner. Notably, the effect was specific as in EOL-1 PDGFRA phosphorylation was not modified at all. In addition, PTCL cells treated with a minimum of 20ug/mL of anti-PDGF ligand neutralizing antibody for 48h showed a 70% blockade of proliferation in comparison to untreated cells (BrdU assay). A further addition of 20 ug/ml of inhibitory antibody at 48 hours, increased the proliferation arrest up to 80% at 96 hours. Finally, the addition of a natural human PDGF peptide to cells previously treated with the anti-PDGF antibody, could restore PDGFRA phosphorylation confirming that PDGFRA de-phosphorilation was due to ligand remotion.

Conclusions
Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS. In fact, though, in vivo, a possible additive paracrine effects mediated by reactive components cannot be excluded, we provide evidence that the phenomenon is largely due to neoplastic cells. Importantly, as PDGFRA signaling abrogation was associated to proliferation arrest, PDGFRA was confirmed as potential therapeutic target.
Acknowledgments: this work was supported by Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006, and Vanini-Cavagnino grant.

Disclosures:
No relevant conflicts of interest to declare.