Rediscovering biomarkers in for the diagnosis and early treatment response in NEN. Reborn Study

Introduction: Neuroendocrine neoplasms (NENs) are heterogeneous in terms of primary site, behavior, and response to treatment. The possibility to rely on diagnostic and prognostic circulating biomarkers is an unmet need in NENs. Despite promising, the clinical role of circulating angiogenic markers remains unclear. In addition, liquid biopsy is currently receiving growing attention in oncology, but data in NENs are available only for circulating nucleic acids and tumor cells while the potential role of circRNA sequencing from tumor educated platelets (TEPs) has never been explored. The aim of this study was to evaluate the role of angiogenic markers and circRNA sequencing from TEPs in NENs diagnosis and prognosis. Materials and Methods: We performed a prospective observational study including 46 consecutive patients with proven NENs of pulmonary and gastro-entero-pancreatic (GEP) origin and 29 controls. Circulating pro-angiogenic factors were measured by ELISA assay, and ANG2 tissue expression was evaluated by immunohistochemistry. A limited subgroup of patients, affected by well-differentiated GEP NET, grade G1 or G2, naïve to any medical treatment, was also included in a proof-of-concept pilot study, for analyzing the expression profile of circRNA derived from TEPs, both at baseline and early follow-up (after 3 months of treatment). Results: The study demonstrated a significantly higher level of ANG2, ANG1, sTIE2, and PROK2 in patients affected by NENs compared to controls. In the subgroup of patients with NENs, ANG2 levels were higher in poorly differentiated NENs (4.9, 2.8–7.4) than in well-differentiated (3.2, 1.7–6.4) ng/ml, p = 0.046 and in tumor stage 3–4 compared to stage 1–2: 4.2 (2.7–8.7) vs 2.7 (1.5–5.7), p = 0.044. ANG2 and PROK2 were significantly higher in patents with progressive disease compared to stable disease at the moment of sampling: ANG2 = 6.3 (4.0–11.0) vs 2.7 (1.7–4.7) pg/ml, p = 0.001; PROK2 = 29.2 (28.4–32.3) vs 28.4 (28.1–28.9) pg/ml, p = 0.035. ANG2 was also higher in patients who developed progression (or died) during the follow-up (one year after the enrollment) than in patients with stable disease (2.3 (1.5-3.8) ng/ml vs 6.3 (4.2-10.1) ng/ml, p<0.001). Immunohistochemistry confirmed ANG2 and PROK2 expression in tumor specimens. We identified a large number of circRNA in this study (98,735), of which 63,562 were not previously annotated and 35,173 annotated. To investigate the potential role of circRNAs expression profile from TEPs as diagnostic and prognostic biomarkers, a bioinformatic analysis is ongoing to evaluate differently expressed circRNA from TEPs between patients and controls and in the same patients before and after treatment. Conclusions: We demonstrated higher levels of angiogenic markers in NENs, with a correlation between ANG2 serum levels and NENs morphology and staging. In both GEP and lung NENs, ANG2 and PROK2 are higher in case of tumor progression, suggesting a potential role as prognostic markers in NENs patients. The study also demonstrated that TEPs are a good source of circRNA in patients affected by NENs. The bioinformatics analyses are currently ongoing and could be the base for the development of novel markers for the diagnosis and follow-up of patients affected by NENs.