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

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

qRT-PCR results on flash frozen chondrosarcoma tumors from the UW Sarcoma Tissue Bank relative to positive Control Cell Lines Mel375 (for NY-ESO-1 and PRAME) and JJ (For LAGE-1s).

<p>qRT-PCR results on flash frozen chondrosarcoma tumors from the UW Sarcoma Tissue Bank relative to positive Control Cell Lines Mel375 (for NY-ESO-1 and PRAME) and JJ (For LAGE-1s).</p