Background: Recurrent glioblastoma multiforme (GBM) is a highly aggressive primary malignant brain tumor that is resistant to existing treatments. Recently, we reported that activated autologous natural killer (NK) cell therapeutics induced a marked increase in survival of some patients with recurrent GBM. Methods: To identify biomarkers that predict responsiveness to NK therapeutics, we examined immune profiles in tumor tissues using NanoString nCounter analysis and compared the profiles between 5 responders and 7 non-responders. Through a three-step data analysis, we identified three candidate biomarkers (TNFRSF18, TNFSF4, and IL12RB2) and performed validation with qRT-PCR. We also performed immunohistochemistry and an NK cell migration assay to assess the function of these genes. Results: Responders had higher expression of many immune-signaling genes compared with non-responders, which suggests an immune-active tumor microenvironment in responders. The random forest model that identified TNFRSF18, TNFSF4, and IL12RB2 showed a 100% accuracy (95% CI: 73.5% - 100%) for predicting the response to NK therapeutics. The expression levels of these three genes by qRT-PCR were highly correlated with the NanoString levels, with high Pearsons correlation coefficients (0.419 (TNFRSF18), 0.700 (TNFSF4), and 0.502 (IL12RB2)); their prediction performance also showed 100% accuracy (95% CI: 73.54% - 100%) by logistic regression modeling. We also demonstrated that these genes were related to cytotoxic T cell infiltration and NK cell migration in the tumor microenvironment. Conclusions: We identified TNFRSF18, TNFSF4, and IL12RB2 as biomarkers that predict response to NK cell therapeutics in recurrent GBM, which might provide a new treatment strategy for this highly aggressive tumor.

The authors have declared no competing interest.

This work was funded by a grant (HI16C1559) from the Korea Health Technology R&D Project through The Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. NRF-2019R1A6A1A03032888).

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This study was approved by the Institutional Review Board of Bundang CHA Medical Center (#2012-12-172, #2021-01-024). Informed consent was obtained from each patient before the clinical trial.

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All expression data are available in the GEO Database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE207753.