Abstract 3127: MiR-BART9 is a prognostic biomarker associated with PD-1 expression in colon cancer patients
Authors: Mariani Marisa , Pandya Deep , Andreoli Mirko , He Shiquan , Spennato Manuela , Ferlini Cristiano 
Published: July 2016

Abstract
Background:
Epstein-Barr Virus (EBV) is widely expressed in the population in a latent stage. During cancer progression, antiviral immunity is compromised by the same mechanisms leading to suppression of anticancer immunity.
This study was aimed at developing the expression of miR-BART as biomarkers to select patients at risk of fast progression for a defect of the antiviral/anticancer immunity.
Methods:
We first analyzed the expression of EBV-miRNAs in a panel of 438 colon cancer (CC) patients of the TCGA dataset. Analysis was conducted using level 1 miRNA-seq and level 3 RNA-seq available on the same patients.
In order to confirm the results, we tested the expression of miR-BART9 in an additional set of 271 CC patients using spectral imaging and quantitative fluorescent immunohistochemistry (PD-1 and PD-L1 antigens at the protein level and miR-BART9 using a specific designed ISH probe).
In order to increase the clinical applicability of our findings, we also developed a qPCR test to analyze the expression of miR-BART9 in serum of colon cancer patients.
Results:
In the analysis of 438 CC patients of the TCGA dataset, expression of miR-BART9 was found in 78 patients (18%). Levels of miR-BART9 were prognostic of poor outcome in a multivariate Cox model including age and stage (HR = 2.8, CI 1.6-4.7, p&lt;0.0001). In patients positive for miR-BART9 we also noticed a significant higher expression of PD-1, CD3 but not PD-L1 at the gene level. These results suggest that the expression of miR-BART9 is linked to a T cell infiltrate in the cancer tissue capable to induce a potent suppression of anticancer and antiviral immunity.
In order to confirm this hypothesis, we performed spectral imaging in an additional set of 271 CC patients. Expression of miR-BART9 was found in 32 (12%) patients with a prevalent stromal pattern. Also in this case expression of miR-BART9 was associated with poor outcome in Cox-multivariate analysis including age and stage (HR = 2.5, CI 1.6-3.4, p&lt;0.001). Expression of miR-BART9 was again significantly associated with a PD-1 positive T-cell infiltrate.
Based on these results, we have designed a qPCR test to analyze the expression of miR-BART9 in serum using an EBV-infected B lymphoma cell line (Raji) as positive control. The assay was employed for monitoring miR-BART9 expression in serum from CC patients (n = 89) and healthy controls (n = 95). Human miR-486 and miR-17 were used as positive controls.MiR-BART9 expression was found in 33 (37%) of CC patients and only in 5 (5%) of the 95 healthy controls.
Conclusions: Our results reveal that expression of miR-BART9 is associated with aggressive CC. Patients featuring reactivation of latent EBV-infection have also expression of PD-1+ T cells, which will impact both anticancer and antiviral immunity. The expression of miR-BART9 can be detected also in serum and may serve to identify the right timing to treat CC patients with PD-1/PD-L1 inhibitors in order to restore an effective anticancer and antiviral immunity.
Citation Format: Marisa Mariani, Deep Pandya, Mirko Andreoli, Shiquan He, Manuela Spennato, Cristiano Ferlini. MiR-BART9 is a prognostic biomarker associated with PD-1 expression in colon cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3127.