NYESO-1/LAGE-1s and PRAME Are Targets for Antigen Specific T Cells in Chondrosarcoma following Treatment with 5-Aza-2-Deoxycitabine

Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157-165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist

Genome biology of the paleotetraploid perennial biomass crop Miscanthus

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. x giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy

Background Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1–specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression.Method We performed a phase I clinical trial evaluating the safety of NY-ESO-1–specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15.Results Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1–specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor–based products at other centers.Conclusions ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1–specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression

General Surgery Considerations in the Era of Mechanical Circulatory Assist Devices

Much of the success of left ventricular assist devices (LVAD) can be attributed to the second-generation HeartMate II (Thoratec, Pleasanton, CA, USA), which is the most commonly used device to date. The latest generation of LVADs is currently undergoing clinical trials worldwide. Developers have focused on improving the limitations of the second generation with emphasis on enhancing efficiency further, decreasing complications, and increasing ease of implantability. Clinical management of a patient with an LVAD is also an excellent example of the multidisciplinary approach of care that is undoubtedly the future of medicine

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BackgroundSynovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression.MethodWe performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15.ResultsFour patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers.ConclusionsETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression

qRT-PCR primers used for these experiments.

<p>qRT-PCR primers used for these experiments.</p

qRT-PCR expression in the chondrosarcoma cell line FS, JJ, 105KC and SW1353 with and without treatment using 5-Aza-dC for 48 hours.

<p>Treated cells rested 48 hours prior to RNA extraction. Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032165#pone-0032165-t001" target="_blank">Table 1</a>. Data is presented relative to a reference sample: either Mel375 for NY-ESO-1 and PRAME or JJ for LAGE-1s normalized to GAPDH. Asterisk indicates statistical significance of p<0.05. Note that all CT antigens were significantly increased following 5-Aza-dC in all chondrosarcoma lines with the exception of LAGE-1s expression in the 105KC and JJ. These cell lines both strongly expresses LAGE-1s without treatment and were not changed significantly by 5-Aza-dC. The JJ cell line is not displayed in 1C as it is used for a reference value for LAGE-1s. Error bars describe variation between multiple values within a single experiment. Experiments were repeated at least three times to ensure reproducibility.</p

Chromium Release using two A*0201 expressing chondrosarcoma cell lines: FS and JJ.

<p>Asterisks indicate statistical significance of p<0.05. Each cell line had significantly increased lysis using antigen specific effectors following treatment with 5-Aza-dC with the exception of JJ in combination with NY-ESO-1/LAGE-1s effectors at high E∶T ratios. JJ is highly sensitive to NY-ESO-1/LAGE-1 specific effectors without 5-Aza-dC treatment and the fact that increased lysis was not seen at high E∶T ratios probably represents a maximum lysis that can be detected for the JJ cell line with these effectors in the given conditions. To control against non-specific cell lysis, targets were tested with a MART-1 specific CTL clone used at an E∶T ratio of 25∶1. Minimal killing was seen testing the control effectors against either treated or untreated targets. No cell line had increased lysis following 5-Aza-dC treatment using the control MART-1 effectors (p>0.1). To be sure the MART-1 specific cells were effective, we also targeted the MART-1 expressing cell line Mel526 during each experiment resulting in over 40% lysis (not shown). Error bars describe variation between multiple values within a single experiment. Experiments were repeated at least three times to ensure reproducibility.</p