Heterogeneity Of The Antigen-Expression Pattern In Primary And Recurrent Glioblastoma Patients.

Abstract Introduction: Immunotherapy leads to promising results in the treatment of glioblastoma (GBM) patients. Recent clinical trials revealed a survival benefit of patients treated with monospecific CAR-T-cells. However, antigen escape mechanisms due to heterogeneous surface expression of target antigens still constitute a critical hurdle in the immunotherapeutic treatment of GBM and are a major reason for non-response or relapse. Therefore, we aim to characterize GBM's heterogeneity, the intratumoral distribution and changes of surface antigens in relapse. Methods: We performed immunohistochemical staining of the ten in vitro and in vivo most targeted antigens in CAR-T cell therapy (HER2, EGFRvIII, CD70, B7H3, Il13Rα2, NY-ESO1, GD2, CD133, CSPG4 and EphA2). We analyzed 35 patients, who underwent surgery for primary (PT) as well as recurrent GBM (RL). To address intrapersonal heterogeneity, we examined five fields of view per slide. As controls, we stained cerebral, cerebellar and brain stem slides of three healthy specimens. We quantified the percentage of low, medium and high antigen expressing cells on each slide by applying a specialized macro in ImageJ. We further weighted this expression score by multiplication with 1 (low), 2 (medium) or 3 (high expression). Finally, we calculated the median expression values of all tumors combined for each antigen. Summary: Median values of EGFRvIII (PT: 0.14; RL: 0.73), HER2 (PT: 0.67; RL: 4.00) and NY-ESO1 (PT: 0.00; RL: 0.16) expression were very low. Antigen expression of CSPG4 (PT: 4.67; RL: 2.78), Il13Rα2 (PT: 3.84; RL: 2.78), GD2 (PT: 7.50; RL: 27.03) and B7H3 (PT: 8.81; RL: 16.62) was mild. CD133 (PT: 52.16; RL: 56.13), CD70 (PT: 19.18; RL: 25.02) and EphA2 (PT: 60.65; RL: 55.46) showed moderate antigen expression. None of the evaluated antigens displayed high expression. Median values of HER2 (p=0.005, Cohen's r=0.48), EGFRvIII (p=0.042, Cohen's r=0.344), and GD2 (p=0.013, Cohen's r=0.42) were significantly increased in RL compared to PT. We found significant intrapersonal expression differences for each antigen. However, none of the healthy brain tissue specimens expressed any of the ten antigens. Conclusions: Inter- and intrapersonal heterogeneity constitutes a major obstacle for the implementation of GBM immune therapies which avoid antigen escape mechanisms. Citation Format: Vera Dufner, Moritz Meyer-Hofmann, Jonas Feldheim, Katja Maurus, Camelia Monoranu, Thomas Nerreter, Michael Hudecek, Carsten Hagemann, Ralf-Ingo Ernestus, Mario Löhr. Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3146.