Conventional Dose Hypomethylating Agents Induce CG Antigen Genes In Vivo

Cancer testis or cancer germline (CG) antigens are a group of genes whose usual expression is limited to embryonic or germ cell tissues and which can also be aberrantly expressed in a variety of malignancies (Fratta E et al. Mol Oncol. 2011;5:164). The CG antigen genes MAGE-A1, XAGE-1, NY-ESO-1, and PRAME (among others) are epigenetically regulated. Most adult non-germline tissues demonstrate dense CpG island promoter methylation, with no gene expression. Endogenous expression of CG genes in epithelial malignancy can be immunogenic and a number of vaccine strategies are currently underway, however studies in hematologic malignancy have shown limited baseline expression, and thus there has been limited interest in them as a target in AML. Recently, several groups have shown that treatment with the hypomethylating agents, azacitidine(Aza) and decitabine(Dac) are sufficient to re-induce gene expression in myeloid cell lines, prompting interest in these genes as a therapeutic target in myeloid malignancy (Almstedt M et al. Leuk Res 2010;34:899). Re-expression of these genes has also been observed in MDS/AML patients treated with Dac, albeit at higher doses (15–270mg/m2) than those currently in standard use (Sigalotti L et al. Blood 2003;101:4644).

We hypothesized that conventional therapy with Aza(75mg/m2) and Dac(20mg/m2) induces CG promoter hypomethylation and gene expression, and that this may be sufficient to induce anti-CG antigen directed antibody responses.

We obtained samples every 2–4 days from 2 AML patients during the first cycle of Aza or Dac treatment, and analyzed baseline and post-treatment samples for methylation (by pyrosequencing) and CG gene expression (by RT-PCR) of the above genes. We further tested serum from 10 patients for the presence of baseline and post treatment (following 1–10 cycles of Aza or Dac) anti-CG antibodies using an ELISA-based method.

Both AML patients treated with hypomethylating agents demonstrated a time dependent decrease in global methylation (by LINE-1 pyrosequencing), and CG antigen promoter methylation which nadired at day 6 post treatment. Baseline methylation of MAGE-A1 (90%), XAGE-1 (85%), NY-ESO-1 (92%) and PRAME (50–60%) was similar and high in both patients pre-treatment. Both Dac and Aza induced hypomethylation of all the CG genes studies, and this hypomethylation correlated with changes in LINE-1 methylation. Low level expression of MAGE-A1, XAGE-1, and PRAME were induced in a time dependent fashion following treatment with both Aza and Dac, however only limited re-expression of NY-ESO-1 was observed. ELISA testing for antibodies against a panel of 25 CG antigens (including MAGE-A1, XAGE-1 and NY-ESO-1) was performed on 10 patients with either MDS or AML. Following hypomethylating agents there was evidence for increased antibody levels, most notably against MAGE proteins, compared to baseline.

Conventional hypomethylating agent therapy can induce CG gene expression, which may be sufficient to induce anti-CG antigen antibody responses. Augmentation of anti-tumor immune responses may be possible in myeloid malignancies using a combination of hypomethylating agents and CG antigen-directed vaccines.

Odunsi:Ludwig Institute for Cancer Reserach: Membership on an entity's Board of Directors or advisory committees, Research Funding.