CHARACTERIZATION OF IMMUNE-RELATED GENE EXPRESSION PROFILES IN HIV+ PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA

INTRODUCTION AND OBJECTIVES: diffuse large B cell lymphoma (DLBCL) is one of the most common cancers in HIV+ subjects. Early age at diagnosis, worse overall survival and gene expression signatures are specific features of DLBCL in the HIV+ context. However, little is known about its immunological pathways. Our aim is to characterize the expression profile of immune-related genes in HIV+ DLBCL cases. MATERIAL AND METHODS: this project received ethical committee approval (CAAE: 93856918.0.0000.5274). We included 104 Formalin-Fixed Paraffin-Embedded (FFPE) samples, 32 were from HIV+ patients with DLBCL (DLBCL-H) and 72 were from HIV- subjects with DLBCL (DLBCL-I). Currently, 58 FFPE blocks were used for gene expression analysis. RNA was extracted with AllPrep DNA/RNA FFPE Kit (Qiagen) and reverse transcribed into first-strand cDNA using SuperScript III (Invitrogen). We performed Real Time PCR assessment of immune checkpoint genes (PD-1, PD-L1 and PD-L2) using the ViiA 7 System (Applied Biosystems). Duplex reactions were prepared in triplicates with GAPDH as the housekeeping gene. Samples that showed amplification curves for both GAPDH and target gene were considered positive. Statistical analysis were performed on GraphPad Prism 6.0. RESULTS AND CONCLUSION: The study groups did not differ significantly on age at diagnostic, gender and DLBCL subtypes, confirming pairing efficiency. Median age was 49.46 and 48.38 for DLBCL-I and DLBCL-H respectively (p = 0.759). Both groups were composed of 75% men and 25% women. Among DLBCL-I subjects, 62.9% samples were from Germinal Center B Cell-like (GCB) subtype, while 61.1% were GCB in DLBCL-H patients (p = 0.901). The most frequent tumor site in DLBCL-I was head and neck (40.3%), followed by gastrointestinal tract (17%). Among DLBCL-H, the most frequent site was gastrointestinal tract (24.1%), followed by head and neck (20.7%) and central nervous system (20.7%). The average RNA concentration (N = 58) was 223.21 ng/μL. Among all samples, 6.89% did not show amplification curves neither for GAPDH nor for the target genes and were excluded from further analysis. 20.37% of all samples were positive for PD-L1. Stratifying for HIV status, 14.3% and 22.5% were positive in DLBCL-H and DLBCL-I groups respectively (p = 0.431). For PD-L2, 50% of all samples were positive. While 57.5% of DLBCL-I patients were PD-L2+, only 28.57% expressed PD-L2 in the DLBCL-H group (p = 0.062). For PD-1, one DLBCL-I sample (1.85%) was positive. The 95% confidence intervals were 9.62-31.11%; 36.66-63.33% and 0-5.44% for PD-L1, PD-L2 and PD-1, respectively. The non-overlapping intervals indicate statistical significance, suggesting that PD-L2 was more frequently expressed among DLBCL samples than PD-L1 and PD-1. Those results indicate a potential relevance of PD-L2 as an immunotherapy target for DLBCL patients since it was expressed more often than other immune checkpoint genes tested.