Circulating hsa-miR-5096 predicts 18F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors

Martine Bocchini; Marcella Tazzari; Sara Ravaioli; Filippo Piccinini; Flavia Foca; Michela Tebaldi; Fabio Nicolini; Ilaria Grassi; Stefano Severi; Raffaele Adolfo Calogero; Maddalena Arigoni; Joerg Schrader; Massimiliano Mazza; Giovanni Paganelli

doi:10.3389/fonc.2023.1136331

Circulating hsa-miR-5096 predicts ¹⁸F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors

Front Oncol. 2023 May 23:13:1136331. doi: 10.3389/fonc.2023.1136331. eCollection 2023.

Authors

Affiliations

¹ Immunotherapy, Cell Therapy and Biobank (ITCB), IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
² Biosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
³ Scientific Directorate, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
⁴ Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy.
⁵ Unit of Biostatistics and Clinical Trials, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
⁶ Nuclear Medicine and Radiometabolic Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
⁷ Molecular Biotechnology Center, Department of Biotechnology and Health Sciences, University of Turin, Turin, Italy.
⁸ Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Abstract

Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. ¹⁸F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with ¹⁸F-FDG-PET/CT status, higher risk and lower response to PRRT.

Methods: Whole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between ¹⁸F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models.

Results: While no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with ¹⁸F-FDG-PET/CT in PanNETs (p-value:<0.005). Statistical analysis has shown that, hsa-miR-5096 can predict 6-month PFS (p-value:<0.001) and 12-month Overall Survival upon PRRT treatment (p-value:<0.05), as well as identify ¹⁸F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:<0.005). In addition, hsa-miR-5096 inversely correlated with both SSTR2 expression in PanNET tissue and with the ⁶⁸Gallium-DOTATOC captation values (p-value:<0.05), and accordingly it was able to decrease SSTR2 when ectopically expressed in PanNET cells (p-value:<0.01).

Conclusions: hsa-miR-5096 well performs as a biomarker for ¹⁸F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.

Keywords: PRRT (peptide receptor radionuclide therapy); SSTR2; functional imaging (positron-emission tomography); miRNA – microRNA; pancreatic neuroendocrine tumors.