Investigating Gene Expression Phenotypes and Tumor Microenvironment Factors Associated with Response to Low Dose Radiotherapy for Follicular Lymphoma

Abstract

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) is the most effective single agent for the treatment of Follicular Lymphoma (FL) as shown in the FoRT trial, where the response to 4 Gy was 81%. However, as ∼20% do not respond and others develop rapid regrowth after RT, identifying molecular markers associated with response is imperative for personalized RT approaches. We sought to determine if gene expression phenotypes and tumor microenvironment factors are associated with response to low dose RT (4 Gy). <h3>Materials/Methods</h3> We retrospectively identified patients with grade 1-2 FL who had pre-treatment formalin-fixed paraffin embedded tumor tissue available prior to low dose RT and had not received systemic therapy within the prior 3 months. We measured baseline clinical factors and scored ‘responders' to RT as those with a complete or partial response by imaging or clinical criteria within 6 months, with all others defined as ‘non-responders'. We performed bulk RNA-sequencing with gene expression quantification using Salmon. Digital cytometry was performed using CIBERSORTx. We performed differential gene expression using DESeq2 with adjustment of all <i>P</i>-values for multiple hypothesis testing. All <i>P</i>-values were two-tailed and considered significant at <i>P</i><0.05. <h3>Results</h3> We identified 7 patients with 8 available tissue samples who met inclusion criteria. Median age at RT was 76.6 years (range, 57.1 – 79.3). Four were males and the majority (85.7%) had stage III-IV FL. One patient received 4 Gy in 1 fraction, all others received 2 Gy x 2. By treated site, 75% (n=6) exhibited a partial or complete response to low-dose RT and 2 progressed, representing non-responders. The non-responders had significantly higher expression of genes associated with immunosuppression (<i>CLEC4G, CCND1</i>) and tumorigenesis (<i>MAGED4, MAGED4B</i>) as compared to responders. Additionally, non-responders had significant under-expression of genes associated with tumor suppression (<i>XIST, BIK, AC009299.1</i>) and cell migration (<i>IGF2BP1, DCAF12</i>). We observed numerically larger cell fractions of CD4 T cells (median=10.1% v 6.4%, <i>P</i>=.26) and natural killer cells (median=2.7% vs 0.8%, <i>P</i>=.15) in responders whereas non-responders had higher relative abundances of follicular helper T-cells (median=11.7% v 8.6%, <i>P</i>=.15) and macrophages (median=8.5% v 5.6%, <i>P</i>=.47). <h3>Conclusion</h3> Based on the results of our pilot cohort, we observed that non-responders exhibited a gene expression phenotype characterized by immunosuppression and increased tumorigenesis. Furthermore, our results suggest immune cell abundances may differ in those that have a response to low dose RT. We aim to expand our investigation to a larger cohort of patients with the goal of informing precision approaches when using low dose RT for FL.