The Role of RASSF1A in Uveal Melanoma

1, 5, 6 PURPOSE. RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. We investigated the effect of RASSF1A re-expression on the UM phenotype in vivo and in vitro. METHODS. The phenotypic effect of methylation-induced inactivation of RASSF1A in UM was explored using a stable RASSF1A-expressing UM-15 clone. RASSF1A expression was assessed using QRT-PCR. Proliferation was evaluated in vitro using MTT assays. Additionally, athymic NOD/SCID mice were injected subcutaneously or intraocularly with RASSF1A-expressing and -non-expressing UM-15 clones, and euthanized when tumors reached a volume of 1500 mm 3 , or at 56 or 46 days, respectively. Tumor tissues, eyes, and livers were analyzed histologically. RESULTS. In vitro analysis confirmed the lack of RASSF1A expression and full methylation of the RASSF1A promoter region in the UM-15 cell line, which was reversible following treatment with 5-Aza-2-deoxycytidine. Cells expressing exogenous RASSF1A showed slower proliferation than controls and regained sensitivity to cisplatin. Compared to mice injected with control cells, mice treated with UM-15 cells expressing exogenous RASSF1A did not acquire intraocular tumors, and their subcutaneous tumors were relatively delayed and small. Neither group had liver metastases. CONCLUSIONS. UM cells reduced tumorigenicity in the presence of activated RASSF1A. RASSF1A apparently has an important role in the development of UM, and its reactivation might be applied in the development of new treatments. (Invest Ophthalmol Vis Sci. 2012;53:2611-2619) DOI:10.1167/ iovs.11-7730 U veal melanoma (UM) is the most common form of primary eye cancer in adults, with an annual incidence of 6-7 cases per million per year. 1 It accounts for 80% of all noncutaneous melanomas and 13% of all deaths caused by melanoma. The tumor carries up to 50% 5-year mortality from metastasis. 3,4 However, their biological and clinical behaviors differ. 2 Additionally, although alterations of chromosomes 1 and 6 are common to both tumors, aberrations, such as monosomy of chromosome 3 and gain of 8q, in addition to other aberrations, typically are found only in UM. 9 Studies revealed that a mutation in the alpha subunit of the heterotrimeric G gene (GNAQ) was present in almost half of all UMs examined, 9-15 and that UM metastatic spread was related to mutations in the BRCA associated protein 1 (BAP1) gene on chromosome 3. 9 Aberrant promoter hypermethylation of CpG islands is thought to have an important role in the inactivation of tumor suppressor genes (TSGs) in cancer. 21 From th

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT