The aims of this study were to pilot the use of low-dose decitabine in children with relapsed or refractory solid tumors and to study the pharmacokinetics, toxicity and drug exposures associated with biological and clinical response. This information will inform the design of future studies combining demethylating agents and cytotoxics. In addition, our study illustrates the challenges and logistics of pediatric patient sample collection, transport, quality control and analysis of samples for biological targets and correlates.
The MTD of decitabine, when administered for 7 days in combination with doxorubicin and cyclophosphamide, was 5 mg/m2
/d. As in adult patients [11
], neutropenia and thrombocytopenia were the dose-limiting toxicities. Non-myelosuppression-related toxicity occurs rarely in adults and includes nausea, vomiting, diarrhea, and fatigue [36
], all of which were also seen in our patients.
Similar to studies in adult patients [37
], there was wide interpatient variability in decitabine drug disposition. Drug clearance in children was greater than that reported in adults [37
]. However, direct comparisons with other trials should be made cautiously as non-compartmental methods using the trapezoidal rule tend to underestimate area under the curve during the infusion and hence lead to an overestimate of clearance.
The biologic correlates of the approach of using a demethylating agent were analyzed using several methods: caspase 8 methylation status and expression, MAGE-1
methylation and HbF expression. Observed changes after decitabine exposure included demethylation of the caspase 8 gene and an increase in mRNA expression albeit in a limited number of bone marrow samples. The limited methylation noted could be attributed to alterations in normal bone marrow cells or in the proportion of tumor cells, or deletion of one or both of the caspase-8 alleles in a subset of samples, although homozygous or heterozygous loss of the CASP8
gene is extremely rare [15
]. It is also possible that in primary tumors, as opposed to cell lines, caspase-8 expression is regulated by multiple mechanisms, only one of which may be promoter hypermethylation. This was previously noted in a study where hypermethylation-associated CD44 receptor silencing was found in neuroblastoma cell lines but not in primary tumor samples [22
methylation was examined in PBMC as a potential marker of tumor methylation. The MAGE-1
promoter is normally methylated in somatic tissues [40
]. The percentage of patients who exhibited significant demethylation of MAGE-1
in PBMCs during the first course of treatment was lower in our study than in a previous study in adults using 2 mg/m2
/d of decitabine infused continuously over 7 days (60% vs. 100%) [10
]. Others have found that a 10 mg/m2
/d decitabine dose level was required to attain plasma peak concentrations associated with biologic effects [41
], suggesting that in our study of heavily pre-treated patients optimal concentrations of drug might not have been achieved. Nonetheless, the duration of MAGE-1
demethylation in our study appears to be similar to that observed in adult studies [10
hypomethylation was retained, and in a few patients increased slightly, on day 28 as noted previously [11
]. Although this is consistent with the possibility that decitabine-induced demethylation is prolonged after exposure and that even low levels of decitabine can alter methylation to some extent, the administration of cytotoxic chemotherapy may have impacted these results. We are not aware of any published literature regarding epigenetic modifications induced by dexrazoxane.
We also examined changes in HbF expression as a biomarker of tumor demethylation. Increased expression of HbF mRNA was found in 88% of blood samples and 100% of bone marrow samples, although by HbF electrophoresis, no change in protein levels was observed. These differences likely reflect the difference in the sensitivity of the two methodologies; a recent adult study showed an increase in HbF protein levels by western blotting following decitabine [46
]. These data suggest that MAGE-1
and HbF could potentially serve as biomarkers of methylation status, especially because such an analysis is relatively easily performed on DNA (MAGE-1
) and RNA (HbF) isolated from peripheral blood cells.
Similar to other reports [35
] preliminary microarray analyses of global changes in gene expression revealed significant interpatient heterogeneity. Other studies have reported that interpatient heterogeneity can result from differences in patient age, sex, ethnicity, time of sample collection and the proportions of the various cell types in the blood and bone marrow [47
]. Since all of our patients had treatments that could alter the relative proportions of the various cell types in peripheral blood, and also because their bone marrow samples most likely would exhibit differences in the proportions of neuroblasts and stromal cells, the interpatient heterogeneity is not especially surprising. Despite this variation, sets of genes with differential expression could be identified at day 7 and 28. Only 10% of the genes whose expression was altered in response to decitabine (i.e., day 7) exhibited changes in expression on day 28, which could be due to the fact that a different set of genes responds to cytotoxic agents. In our study, increases in gene expression were more common after decitabine, while decreases were more commonly seen after treatment with cytotoxic agents. While analysis of which genes and pathways are affected by these agents is clearly more important than the number of genes with altered expression, the fact that the cellular response to these drugs differed so dramatically is nonetheless potentially interesting and may reflect differences in the mechanism of action of these agents. Our data are consistent with the proposed effect of decitabine: reactivation of epigenetically silenced genes by irreversibly binding DNMT1 and depleting intracellular DNMT1 activity. This was also noted in a study that analyzed the gene expression profile of a neuroblastoma cell line following exposure to decitabine; of 44 genes that were differentially expressed (≥ 3-fold), 27 were upregulated, among them Hsp47, and 17 were downregulated following treatment [24
]. Further studies with additional patients and quantitative analyses will be required to validate these data and eliminate false positives.
Most responses to low-dose decitabine have been observed in patients with myelodysplastic syndrome [36
] with limited evidence of activity in patients with solid tumors [11
]. In adults with hematopoietic malignancies, 15 mg/m2
administered over one hour daily, 5 days a week, for 2 weeks induced the highest response rate [14
]. Two of 9 adults with refractory solid tumors treated with continuous infusion decitabine at 2 mg/m2
/day for 7 days had stable disease [11
]. Similar results were seen in patients with ovarian cancer treated with a cytotoxic agent following decitabine [46
]. The limited clinical responses seen in our study may reflect the fact that we were not able to achieve drug exposures similar to those that have been observed in other clinical studies. It has been reported that the higher the dose of demethylating agent, the more marked the epigenetic change [50
]. Failure to achieve an adequate concentration to induce both biological and clinical response could have been due to several reasons including the inability to dose escalate due to the heavy pre-treatment of patients, the additional cytotoxicity incurred with cyclophosphamide and doxorubicin, the dosing schedule employed, the rapid plasma clearance of decitabine, and the use of single agent epigenetic therapy. Of course, we cannot exclude the possibility that decitabine may be inactive at any exposure. The use of alternative cytotoxic agents may allow further dose escalation of decitabine to achieve concentrations that would result in clinical and biologic response. In addition, targeting multiple points of the apoptotic pathway and/or the use of other agents that induce more extensive chromatin modifications may be necessary to induce response.
In summary, when administered in combination with cyclophosphamide and doxorubicin, the MTD of low-dose decitabine is 5 mg/m2/day in pediatric patients with relapsed solid tumors, with significant myelosuppression being the primary toxicity observed. At this dose, increased caspase-8 expression was found in a limited number of tumor-containing bone marrow samples. Sustained demethylation and overexpression of biomarkers MAGE-1 and HbF in peripheral blood was maintained for 4 weeks in the majority of samples tested and MAGE-1 could be used as a potential biomarker to assess demethylation status of the tumor. Since aberrant DNA methylation is often complemented by other epigenetic events that alter chromatin structure such as histone deacetylation, a combination of these two epigenetic modifications with chemotherapeutic agents is an attractive strategy for future investigation. Lastly, the methylation profiles of tumors should be explored to determine whether they correlate with response to chemotherapy.