Integrative analysis of DNA methylation suggests down-regulation of oncogenic pathways and reduced somatic mutation rates in survival outliers of glioblastoma

The study of survival outliers of glioblastoma can provide important clues on gliomagenesis as well as on the ways to alter clinical course of this almost uniformly lethal cancer type. However, there has been little consensus on genetic and epigenetic signatures of the long-term survival outliers of glioblastoma. Although the two classical molecular markers of glioblastoma including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation are associated with overall survival rate of glioblastoma patients, they are not specific to the survival outliers. In this study, we compared the two groups of survival outliers of glioblastoma with IDH wild-type, consisting of the glioblastoma patients who lived longer than 3 years (n = 17) and the patients who lived less than 1 year (n = 12) in terms of genome-wide DNA methylation profile. Statistical analyses were performed to identify differentially methylated sites between the two groups. Functional implication of DNA methylation patterns specific to long-term survivors of glioblastoma were investigated by comprehensive enrichment analyses with genomic and epigenomic features. We found that the genome of long-term survivors of glioblastoma is differentially methylated relative to short-term survivor patients depending on CpG density: hypermethylation near CpG islands (CGIs) and hypomethylation far from CGIs. Interestingly, these two patterns are associated with distinct oncogenic aspects in gliomagenesis. In the long-term survival glioblastoma-specific sites distant from CGI, somatic mutations of glioblastoma are enriched with higher DNA methylation, suggesting that the hypomethylation in long-term survival glioblastoma can contribute to reduce the rate of somatic mutation. On the other hand, the hypermethylation near CGIs associates with transcriptional downregulation of genes involved in cancer progression pathways. Using independent cohorts of IDH1/2- wild type glioblastoma, we also showed that these two patterns of DNA methylation can be used as molecular markers of long-term survival glioblastoma. Our results provide extended understanding of DNA methylation, especially of DNA hypomethylation, in cancer genome and reveal clinical importance of DNA methylation pattern as prognostic markers of glioblastoma.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (NRF-2018M3A9H3021707). in Korea, and the Seoul National University Hospital Research Fund (3020180010)

Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model.

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4) blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137) is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response.Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse model of glioma by a CD4+ T cell dependent mechanism and generates antigen-specific memory

Combining focal radiation therapy with anti-4-1BB and anti-CTLA-4 antibodies results in long-term survival.

<p>A) Schematic of the treatment protocol for triple therapy. B) Kaplan Meier survival curves for double immunotherapy and triple therapy (<i>n</i> = 18/group). Treatment with triple therapy was superior to double immunotherapy with anti-4-1BB and anti-CTLA-4 antibodies (<i>p</i><0.05), focal RT alone (<i>p</i><0.01) and untreated mice (<i>p</i><0.001). Furthermore, triple therapy results in long-term survival in at least 50% of treated animals, whereas immunotherapy with both 4-1BB activation and CTLA-4 blockade produced long-term survival in 3/18 mice (16.7%). Data from three repeated experiments with similar results was pooled into single Kaplan Meier curves. <i>P</i> values were calculated with the Log-Rank test.</p

The anti-tumor activity of triple therapy is CD4⁺ T cell dependent.

<p>Kaplan Meier survival curves for untreated animals, triple therapy (no depletion), triple therapy with depleted CD4⁺ T cells and triple therapy with depleted CD8⁺ T cells (<i>n</i> = 5–7 mice/group). CD4⁺ T cell depletion abolishes the survival benefit of triple therapy (<i>p</i><0.001). Depletion of CD8⁺ T cells did not interfere with the efficacy of triple therapy and resulted in long-term survival.</p

Treatment with triple therapy results in a glioma-specific memory response.

<p>A) Naïve mice (<i>n</i> = 4/group) challenged with GL261 cells in both flanks formed flank tumors in 6/8 flanks. All control animals formed GL261 flank tumors; two animals formed bilateral flank tumors and two animals formed unilateral flank tumors. B) Long-term survivors (<i>n</i> = 3/group) developed long-lasting immunity to GL261 tumors and successfully rejected flank tumor formation in 6/6 flanks. Mice were observed for 50 days. Each line represents tumor growth in one flank. C) Bioluminescent imaging data on day 21 after flank tumor implantation for an experiment where naïve mice (top row) and long-term survivors (bottom row) were inoculated subcutaneously with 10⁶ GL261-luc cells in the <i>left</i> flank and 10⁵ B16-luc cells in the <i>right</i> flank. D) Naïve mice had progressive GL261 and E) B16 flank tumor growth. Note that B16 tumors grew faster in comparison to GL261 tumors. F) Long-term survivors established a protective memory response and rejected GL261 flank tumor growth. G) Long-term survivors established a glioma-specific memory response which did not affect the formation of B16 melanoma flank tumors.</p

Triple therapy leads to increased CD4⁺ and CD8⁺ infiltrating lymphocytes in the brain.

<p>A, B) Influx of CD4⁺ and CD8⁺ tumor infiltrating lymphocytes respectively was higher in brains of animals treated with immunotherapy with 4-1BB activation and CTLA-4 blockade and triple therapy compared to brains from non-tumor bearing mice (<i>p</i><0.05). C, D) Cervical lymph nodes from naïve mice showed a higher baseline of CD4⁺ and CD8⁺ T cells which resulted in comparable densities of T cells between all groups. Error bars represent standard error of the mean (SEM) and p values were calculated with the student t-test.</p