Immune profiling of tumor-infiltrating lymphocytes in patients with primary and metastatic soft tissue sarcomas.

Abstract

e23544 Background: Soft tissue sarcomas (STSs) are rare and heterogeneous cancers with limited responsiveness to immune checkpoint inhibitors (ICIs), with most STSs exhibiting low expression of neoantigens and immune response genes. While tumor-infiltrating lymphocytes (TILs) can be readily isolated from many STSs, resistance of TILs to ICIs remains poorly understood. To investigate TIL phenotypes and function, we performed comprehensive profiling by flow cytometry of TILs obtained from STS patients undergoing routine surgical resections. Methods: Patients consented to an IRB-approved tumor banking protocol to collect clinical data and specimens. TILs were extracted/expanded from fragments of fresh STS surgical specimens by culture in AIMV media containing human IL-2 for 10-15 days. Cryopreserved TILs along with matched peripheral blood mononuclear cells (PBMCs) were analyzed using multiplex flow cytometry to assess TIL phenotypes, memory/activation states, and checkpoint protein expression. A subset of TIL samples were stimulated with PMA/ionomycin followed by flow cytometry/ELISA to assess cytokine production. Clinical and demographic data were correlated with TIL and PBMC immune variables using linear models with empirical Bayesian methods, adjusted for multiple comparisons, and followed by principal components analysis. Results: 23 patients were profiled, with 21 patients having evaluable paired samples, with most patients having liposarcoma, leiomyosarcoma or GIST. 43.5% of patients had no prior systemic therapy. TILs demonstrated a significantly higher CD8:CD4 ratio (median 1.05 [IQR 0.56–2.65] vs PBMCs 0.25 [0.14–0.43], p<0.0001). Tumor-resident memory cells (CD8⁺CD103⁺CD39⁺) were enriched in TILs (4.3% [1.6–10.5] vs PBMCs 0.09% [0.02–0.16], p<0.0001). T regulatory cells were overall rare but more prevalent in older patients and males. Most TILs were effector memory phenotype (CD8 61.2% [41.5-70.6] and CD4 81.6% [62.8-89.1]), whereas PBMCs showed higher central memory cells. Most CD3- TILs and PBMCs were CD56+ NK cells. Expression of PD1 was higher in TILs vs. PBMCs (mean MFI 7057 ± SEM 831.8 vs. PBMCs 1924 ± SEM 150.4, p=<0.0001), with trends towards higher expression of TIM3 and LAG-3 in TILs. No correlations were observed between STS types or prior treatments with quantity or phenotypes of TILs. Full analysis of clinical correlations and functional activity will be presented at the meeting. Conclusions: STS TILs display features of antigen experience with exhaustion, including higher CD8:CD4 ratio, greater EM populations, and higher checkpoint expression relative to matched PBMCs. While limited by small sample size and patient heterogeneity, our data will help inform future clinical trials of combination ICI therapies to increase TIL infiltration, lessen exhaustion, and overcome resistance, to improve clinical outcomes.