Prognostic Significance of Immune Microenvironmental Factors in Undifferentiated Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy

Abstract

EBV-associated UNPC is characterized by an abundant immune infiltrate, which may have a prognostic relevance, considering EBV-specific immune response and microenvironmental immunesuppression. Our primary aim was to retrospectively assess the significance of immunesuppression in the UNPC microenvironment as prognostic biomarker of treatment failure in a non-endemic area population. As secondary aim we investigated the immunomodulating effects of chemoradiotherapy (CRT) monitoring the variation of EBV-specific immunity before and after treatment. We reviewed 63 consecutive patients (pts) with EBV-associated stage II-IVB UNPC undergoing radical CRT. Our policy was of administering concurrent CRT in pts with stage II disease, and sequential therapy in pts with advanced stages. The number of relapsed pts was 11 (cases), while 52 pts (controls) were free from treatment failure at a minimum follow-up of 2 years. DNA and RNA were extracted from tumor (T) and adjacent normal mucosa (M) frozen biopsies at diagnosis. We verified the expression of EBV-derived Barf1 gene in T versus M samples. We investigated the expression of the immune-related genes PD-L1, CD8, FoxP3 and IFN-g in T and M tissues by quantitative RealTime PCR, and their methylation status through quantitative methylation specific PCR. Quantitative RT-PCR analyses showed significantly higher expression levels of several immune-related genes (i.e. BARF1, CD8, IFN-g, IDO, PD-L1, and PD-1) in UNPC samples respect to healthy tissues. Interestingly, CD8 expression was significantly reduced in both tumor and healthy tissues in UNPC patients who relapsed within two years (P<0.001). Similarly, quantitative methylation-specific PCR analyses identified a hypomethylated FOXP3 intron 1 exclusively in relapsed UNPC patients. Finally, we quantified the amount of EBV- and BARF1-specific T-cells in the peripheral blood of 14 patients (7 relapses) through IFN-g ELISPOT assay, and observed a significant decrease after CRT only in relapsing patients. Altogether, our data suggest that a high level of immunosuppression (low CD8, hypomethylated FoxP3) in UNPC microenvironment may predict treatment failure and early identify those patients who could benefit from the addition of an immunotherapy improving first line CRT.