T-Cell Subsets and Macrophages in the Tumor Microenvironment Are Associated with Early Outcome in Follicular Lymphoma

Introduction. Follicular lymphoma (FL) is the most frequent indolent subtype of non-Hodgkin lymphoma. Despite an indolent course, it remains an incurable disease and the overall survival (OS) is 70-80% at 8 years in the rituximab era. The event-free survival status at 24 months (EFS24) is a strong predictor of subsequent OS: FL patients that relapse within 2 years after immunochemotherapy (IC) have a poor outcome. Similarly, in FL patients observed or treated with rituximab monotherapy, EFS at 12 months (EFS12) is a strong predictor of subsequent OS. In the era of precision medicine treatment strategies, it is essential to establish strategies for risk assessment particularly that predict early disease progression. Clinical prognostic factors, including FLIPI, have a limited usefulness in predicting patients who will fail EFS12/24. The lymphoma microenvironment, including the number and location of T-cells and macrophages, potential expression of anti-tumor response, may be the key determinant of early failure. While there is variability in results reported to date for many of the microenvironment markers with prognosis, no studies have focused on early failure. In this study, we evaluated the association of immune cells in the tumor microenvironment with early event status in a subset of FL patients enrolled into University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) cohort study.

Methods. Patients with FL grade 1-3A were prospectively enrolled in the MER from 2002-2012. Tissue microarrays were assembled from available research tissue from diagnostic biopsies. Staining was performed using immunohistochemistry (IHC) markers for CD4, CD8, FOXP3, CD32b, CD14, CD68, CD70, SIRP alpha, TIM3, PD-1 and PDL-1. IHC staining was read by expert hematopathologist (ALF); stains were scored to the nearest decile as the percentages of cells with positive staining for each marker within and outside of the neoplastic follicles. Results for each stain were dichotomized (high vs low) agnostic to outcome based on median staining and/or histologic relevance. Event-free survival was defined as time from diagnosis to relapse/progression, retreatment, or death due to any cause. Early events were defined as failure to achieve EFS24 for patients treated with immunochemotherapy (IC) at diagnosis and failure to achieve EFS12 for all other therapies (including observation) at diagnosis, with EFS12/24 referring to the combined endpoint for the analysis of all patients.

Results. 166 patients were included in the analysis, of which 102 patients were initially observed or received rituximab as monotherapy, and 64 were treated with IC. In the full set of patients, 27.3% of patients with no expression of PD-1 inside the follicle failed to achieve EFS12/24 compared to 14.3% with any expression (p=0 .040), and 27.4% of patients with no CD4 expression outside of the follicle failed to achieve EFS12/24 compared to 14.6% with any expression (p=0.043). Conversely, 14.0% of patients with no FOXP3 expression outside the follicle failed to achieve EFS12/24 compared to 26.3% with any expression (p=0.046), and 16.2% of patients with <30% expression of SIRP alpha inside the follicle failed to achieve EFS12/24 compared to 33.3% with ≥30% expression (p=0 .047). None of the other markers predicted EFS12/24. In the IC subset, only two markers predicted EFS24: the expression of PD-1 inside of the follicle was associated with a lower EFS24 failure rate (18.6%) compared to no expression (47.4%; p=0 .019), and any expression of CD4 was also associated with a lower EFS24 failure rate (14.6%) compared to no expression (52.4%; p=0.0016). Unadjusted associations between these markers and early events remained consistent in multivariable models adjusted for FLIPI (all p<0.15).

Conclusion. Components of the microenvironment, including T-cell subsets and macrophages, are associated with early outcome in patients with follicular lymphoma. Replication of these results in larger cohorts of patients with follicular lymphoma is warranted