Inducible Expression of Cancer Testis Antigens in Myelodysplastic Syndrome (MDS) Patients Following Treatment with an Oral 5-Azacytidine

The identification of appropriate target antigens is critical to the success of cancer immunotherapies. Cancer-testis antigens (CTAs) are ectopically expressed immunoprivileged antigens transcriptionally controlled by DNA methylation which have utility as immunotherapeutic targets. 5-azacytidine (AZA), an analogue of cytidine, has been shown to induce expression of CTAs in cancer. We analyzed matched samples from 42 myelodysplastic syndrome patients receiving an oral AZA to determine if an anti-CTA immune response was generated.

Immunoglobulin G (IgG) responses to CTAs were analyzed by High Throughput Immunoblot assay (HTI), 29 CTAs were bound in a concise pattern to nitrocellulose filters and developed in the following methodology. Patient sera were incubated with the HTI filters and any CTA specific antibodies bound to respective antigens and reactive antibodies were detected by secondary anti-human antibodies in a colorimetric assay. Filters were read by 5 blinded individuals, spots were scored positive or negative against control antigens. These results were compared to the paired post-therapy sample, a score of 5 was maximal change of pre to post. Those CTAs with strong antibody presence post-therapy were then confirmed via ELISA resulting in an antibody titer reflected as ug/mL. A standardization of human IgG, as well as a healthy donor control was used to determine the validity of identified responses.

When HTI was examined thoroughly several CTAs, MAGEs, SSX2, NY-ESO1 and MAD-CTs were of particular interest. All patients regardless of pre- or post- therapy exhibited SSX2 expression at high levels this was further confirmed with ELISA exhibiting little to no change from pre- to post-therapy. HTI examination of MAD-CT antigens revealed that pre-treatment, MAD-CT-1 and MAD-CT-2 antibody presence, responses were nearly identical, and thirty-four had the exact same response levels. PRAME, a CTA absent from our HTI filter, was evaluated only through ELISA. We determined that eighteen patients presented with an increased anti-PRAME antibody titers at post-treatment when compared to pre-treatment. Of the cancer testis antigens analyzed patients had the highest average changes in antibody titer to PRAME at post-treatment, with a 0.128 ug/mL average increases.

Our findings suggest that CTAs could be further pursued as an immunotherapeutic target in myelodysplastic syndrome and that treatment with AZA could be exploited to modulate antigen expression in combination with immunotherapeutic approaches. Although responses were variable throughout the patients in total, some patients had robust responses at post-treatment. The CTAs, MAGEs, SSX2, NY-ESO1 and PRAME, are of particular interest, due to prior and new findings, as well as, on-going clinical trials. Five patients had a robust antibody response according to HTI to at least six antigens. Eight patients were of further interest according to ELISA examination. Further data on the relation with clinical response and possible prognostic factors of response to therapy will be presented.

Skikne:Celgene: Employment, Equity Ownership. MacBeth:Celgene: Employment.