We report the first objective, durable responses in solid tumor patients using combined epigenetic therapy with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor. Objective responses to this therapy in this heavily pretreated population occurred in only 4% of the patients, but the anti-tumor responses that were observed were impressive. Several observations from this cohort of patients suggest that this therapy is quite distinct from prior experience with high-dose azacitidine, and merits additional focused investigation.
Unlike participants in older clinical trials of azacitidine or decitabine in solid tumor patients (24
), our patients received doses far below the maximally tolerated dose, permitting repetitive dosing over many months, and avoiding the cytotoxicity associated with the high-dose regimens. Low-dose azacitidine or decitabine regimens have led to successful treatment of MDS (5
) with improved survival (31
). Consistent with clinical trial observations in MDS (5
), and in contrast to typical responses to cytotoxic chemotherapy, tumor responses in NSCLC patients improved gradually and progressively over several months of treatment. Intriguingly, the clinical responses produced were sustained even after cessation of epigenetic therapy. There was no evidence of relapse of the complete responder’s original wild-type KRAS
metastatic disease at the time of her death, sixteen months after discontinuing epigenetic therapy. Similarly, at present there has been no evidence of recurrence of the partial responder’s hepatic metastases, over 2 years after stopping epigenetic treatment.
Another potential contributor to the responses seen in these patients is the methylation status of certain key genes in the tumor. Multiple studies have demonstrated the relevance of DNA methylation in lung cancer (32
). The patient with a complete response demonstrated tumor-specific promoter hypermethylation in primary tumor and mediastinal lymph nodes in a pattern prognostic of poor survival in early stage lung cancer, which may define a subset of lung cancers driven by epigenetic mechanisms (19
). The patient with partial response did not have baseline tumor available for analysis, but circulating DNA analysis from this patient confirms target gene methylation at baseline, and de-methylation with treatment. Both of these patients had methylation of 3 of 4 genes detectable in circulating DNA, with demethylation in all 3 with epigenetic therapy.
Analysis of free-circulating tumor DNA in the plasma of patients supports early de-methylation as a potential predictor of clinical benefit from this therapy, and is consistent with an on-target epigenetic mechanism of action. Evaluation of methylation changes in tumor DNA during cycle 1 of therapy as a predictor of clinical benefit should be included in future trials of epigenetically directed therapy. Other putative biomarkers of response to epigenetically targeted agents defined in recent studies could also be explored in this context (33
Two of the patients described here developed molecularly and histologically distinct second lung cancers on therapy. This has not been observed in other patients in this study. Second primary cancers are common in lung cancer patients, associated with similar carcinogenic exposure throughout the lung field (34
). While it is difficult to entirely rule out the possibility of therapy-related adverse effects, secondary malignancies have not been reported in much larger series of patients receiving similar therapies for MDS (35
A limitation of the general applicability of this therapy is the need for subcutaneous injection of azacitidine on a daily basis. This was also a common cause of low grade toxicities (e.g. injection site reactions with localized erythema) on this study. Activity and bioavailability of an oral formulation of azacitidine has been recently reported in patients with hematologic malignancies (37
). Other novel oral demethylating agents including zebularine derivatives have shown activity in experimental cancer models (38
). If bioequivalence can be demonstrated, these oral agents may have significant advantages over subcutaneous administration in terms of patient tolerance over prolonged courses of drug administration.
An important feature of this trial is combinatorial targeting of epigenetic silencing adding the HDAC inhibitor, entinostat. Preclinical data have demonstrated that while re-expression of epigenetically silenced target genes can be induced with inhibition of DNA methyltransferase alone, additional targeting of histone deacetylation results in more robust and persistent changes in gene expression (12
). Previous trials combining demethylating agents and HDAC inhibitors have used much less potent HDAC inhibitors (40
). We hypothesize that these key features contributed to the responses observed.
The median survival of 6.4 months on azacitidine and entinostat in extensively pretreated NSCLC (median number of prior therapies = 3) is similar to the median survival previously noted for patients with 1 or 2 prior therapies treated with the only FDA-approved drug for this patient population, erlotinib (median survival 6.7 months, vs. 4.7 months for placebo control) (42
). Stable changes in gene expression induced by epigenetically directed therapy could alter cancer cell sensitivity to subsequent cytotoxic therapy. Recent data from the Settleman laboratory have defined drug resistant “persisters” within clonal cancer cell populations highly sensitive to targeted therapy, and that maintenance of these drug resistant persisters is epigenetically regulated (2
). Major objective responses to immediate subsequent therapies, even among patients with primary RECIST progression on epigenetic therapy, have been observed and may have contributed to the exceptionally long survival among some patients on this study. Interestingly, a previous study also observed long-term survival in a patient following chemotherapy given after the DNA methyltransferase inhibitor, decitabine (24
). These observations suggest a testable hypothesis, that epigenetic therapy could prime cancers for response to subsequent cytotoxic therapy. Efforts to further refine characteristics of patients likely to benefit from this novel therapeutic approach, and to optimize this regimen to benefit additional cancer patients, are ongoing.