Investigating genes and micro-RNAs that may predict clinical benefits of anti-CTLA-4 therapy.

Abstract

3043 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, ipilimumab, has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. Methods: On an IRB-approved phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Anti-CTLA-4 treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, anti-CTLA-4 significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that anti-CTLA-4 induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. Conclusions: Anti-CTLA-4 treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.