Impact of Tumor Infiltrating T Cells in Patients with Follicular Lymphoma At Diagnosis

Abstract 769

Previous global gene expression profiling (GEP) studies have demonstrated non-malignant tumor-infiltrating immune cells present in the tumor at diagnosis were among the molecular features of the most important prognostic markers (Sandeep et al. NEJM 2004). We investigated the molecular mechanisms whereby tumor infiltrating T cells (TILs) are altered in the FL microenvironment and examined GEP of highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in treatment naive patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as peripheral blood (PB) (n=10) of age matched healthy donors. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH, ETV1, TNFRSF9 and NAMPT.

PMCH is not expressed in PB T cells, but its expression is highly induced when healthy PB T cells are cultured, either in cell contact or in transwell culture, with FL cells. Using well characterized tissue microarrays of diagnostic FL samples (n=172) we now show that the T-cell expressed genes of PMCH, ETV1 and NAMPT have prognostic significance for survival and time to transformation in FL. Patients with higher percentage (p=0.039, median survival 10.59yr vs 19.20yr) and number (p=0.016, median survival 8.67yr vs 19.01yr) of PMCH expressing cells in intrafollicular and higher percentage (p=0.021, median survival 8.18yr vs 16.5yr) in interfollicular area had significantly longer disease specific survival (DSS) compared with patient with low number of PMCH expressing cells. Patients with higher percentage (p=0.014, median survival 19.01yr vs 11.75) and number (p=0.032, median survival 19.01yr vs 12.41yr) of ETV1 expressing cells in intrafollicular area showed significantly shorter DSS comparing to those with lower percentage and number of ETV1 expressing cells. Furthermore, the higher total MI of NAMPT expression showed significantly longer DSS (p=0.003, median survival 7.62yr vs 18.28yr) as well.

Pathway analysis indicated disruption in multiple cellular pathways including actin-based motility/cytoskeleton formation which is in keeping with our previous studies where we have shown altered T cell expression of genes regulating actin in CLL (Gorgun et al, JCI 2005) and AML (De Lieu et al, Blood 2009). Using Time-Lapse Imaging we demonstrated both CD4 and CD8 TILs from patients with FL (n=7) have significantly impaired motility compared to those of healthy TILs from reactive tonsil (n=4) (p<0.025). We have further demonstrated that short term culture of allogeneic healthy T cells with FL cells from treatment naive patients (n=8) directly induce significantly impaired T cell motility compared to culture of these T cells with healthy allogeneic non malignant B cells (n=6)(p<0.05).

Taken together, these data indicate that TILs in patients with FL are abnormal in terms of their gene expression and function. Furthermore, we provide evidence supporting the role that FL cells play in inducing changes in immune cells in the tumor microenvironment. In addition, we have shown that changes in the protein level in tumor infiltrating T-cells have an impact on the survival and time to transformation in patients with FL. We are further characterizing the mechanisms of gene expression alteration in TILs of patients with FL and its functional consequences in the biology and of the disease. Since non-malignant infiltrating immune cells have a crucial role in the outcome of patients with FL, understanding the nature and impact of the abnormalities induced in TILs in these patients is vital before any immunotherapeutic strategies can be implemented to attempt to alter the immune microenvironment in FL.