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Journal of Clinical Oncology, Vol 25, No 29 (October 10), 2007: pp. 4603-4609 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.10.8688 Phase I and Pharmacodynamic Trial of the DNA Methyltransferase Inhibitor Decitabine and Carboplatin in Solid Tumors
From the Centre for Oncology and Applied Pharmacology, Glasgow University, Cancer Research UK Beatson Laboratories, Glasgow; Cancer Research UK Section of Medicine, Institute of Cancer Research, and Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton; Cancer Research UK Drug Development Office, London; Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, United Kingdom Address reprint requests to Robert Brown, PhD, Cyclotron Building, Hammersmith Hospital Campus, Imperial College, London W12 0NN, United Kingdom. e-mail: b.brown{at}imperial.ac.uk
Purpose: The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (decitabine) induces DNA demethylation and re-expression of epigenetically silenced genes, and increases carboplatin sensitivity of tumor xenograft models. We designed a clinical study to determine the feasibility of delivering a dose of decitabine, combined with carboplatin, that would be capable of producing equivalent biologic effects in patients with solid tumors. Patients and Methods: In a two-stage design, 33 patients received escalating doses of decitabine administered as a 6-hour infusion on day 1 followed by carboplatin, area under the concentration-time curve (AUC) 5 (cohort 1) and AUC 6 (cohort 2), on day 8 of a 28-day cycle. Pharmacodynamic analyses included 5-methyl-2'-deoxycytidine levels, MAGE1A CpG island methylation, and fetal hemoglobin (HbF) expression. Results: The major toxicity was myelosuppression. Dose limiting toxicities, prolonged grade 4 neutropenia (one patient), and sepsis and grade 3 anorexia/fatigue (one patient), were seen in two of four patients treated with decitabine 135 mg/m2 and carboplatin AUC 5. Dose limiting toxicity comprising neutropenic sepsis (one patient) and grade 3 fatigue (one patient) was seen in two of 10 patients treated at decitabine 90 mg/m2 and carboplatin AUC 6. Decitabine induced dose-dependent, reversible demethylation in peripheral-blood cells (PBCs) maximally at day 10. Furthermore, decitabine 90 mg/m2 induced demethylation of the MAGE1A CpG island in PBCs, buccal cells, and tumor biopsies, as well as elevation of HbF expression. Conclusion: Decitabine can be combined safely with carboplatin at a dose and schedule that causes epigenetic changes equivalent to or greater than that observed in mice with carboplatin-sensitized xenografts. The recommended dose/schedule for phase II trials is decitabine 90 mg/m2 (day 1) followed by carboplatin AUC 6 (day 8) every 28 days.
Multiple genes become transcriptionally silenced during tumor development, which is associated causatively with aberrant DNA methylation.1 Neoplastic cells exhibit global hypomethylation with localized hypermethylation of CpG islands (CGIs) and increased levels of methyltransferases.2 Aberrant hypermethylation of CGIs is associated with transcriptional silencing of genes, which not only play a role in tumorigenesis, but may also influence response to anticancer agents.3,4 Reversal of gene methylation and epigenetic silencing has the potential to influence tumor growth, the sensitivity to anticancer agents, and ultimately clinical outcome.5 Key targets for potential DNA demethylating agents are DNA methyltransferases (DNMTs), enzymes that catalyze the addition of a methyl group to cytosine residues at CpG dinucleotides.6-8 The DNMT inhibitor decitabine (5-aza-2'-deoxycytidine) is a deoxycytidine analog that incorporates into DNA and forms irreversible covalent bonds with DNMT at cytosine sites targeted for methylation.6 Decitabine demonstrates activity against hematologic malignancy,9 and it has been hypothesized that low-dose, prolonged exposure to decitabine correlates with clinical response as a result of changes in gene expression induced by a reduction in DNA methylation (rather than cytotoxicity, which occurs with higher doses of decitabine).10-12 We have shown previously that nontoxic doses of decitabine sensitize ovarian and colon drug-resistant tumor xenografts in nude mice to a range of cytotoxic chemotherapeutic agents, including carboplatin.13 The dose and scheduling of decitabine in the mouse model resulted in reduction of GCI methylation and re-expression of previously silenced genes both in surrogate tissues, such as peripheral-blood cells (PBCs), and in tumor. Sensitization was highly schedule dependent, and was observed when decitabine was administered before the cytotoxic agent, but no effect was observed when decitabine was administered concurrently or after the cytotoxic agent. In contrast to hematologic malignancy, single-agent studies of DNMT inhibitors in solid tumors have demonstrated little activity, and previous combination studies have not been pursued.14-16 However, based on our xenograft data, we hypothesized that the schedule and dose used in earlier combination studies would not have induced DNA demethylation optimally at an appropriate time point to affect drug sensitivity.8,13 We therefore designed a phase I clinical and pharmacodynamic trial of decitabine in combination with carboplatin to determine the feasibility of administering decitabine at an equivalent dose and schedule that produced an effect on methylation in the xenograft studies. Increased methylation of a subset of CGIs in late-stage ovarian cancers significantly correlates with worse clinical outcome,17,18 suggesting that gene methylation is a valid target for resistance reversal in ovarian cancer. With a view to subsequent studies in ovarian cancer, we therefore determined to establish a recommended dose of decitabine with carboplatin area under the concentration-time curve (AUC) 6 every four weeks.
Eligibility This phase I and pharmacodynamic study was sponsored by Cancer Research United Kingdom and was approved by the local research ethics committee at each of the participating institutions. All patients entered onto this study had histologically confirmed advanced solid malignancy, for which there was no standard treatment. Eligibility criteria included life expectancy  12 weeks; Eastern Cooperative Oncology Group performance status  2; no chemotherapy, immunotherapy, or radiotherapy within 4 weeks of study entry (6 weeks for nitrosoureas and mitomycin); adequate hematologic (absolute neutrophil count [ANC] 1.5 x 109/L; platelets 100 x 109/L), hepatic (bilirubin 30 µmol/L; AST/ALT 2.5x upper limit of normal or 5x upper limit of normal in the presence of liver metastases), and renal function (EDTA clearance 50 mL/min). Patients who had received more than six cycles of carboplatin or suffered significant bone marrow suppression during prior platinum treatment were excluded.
Study Design
AUC for carboplatin was determined using the Calvert formula, in which glomerular filtration rate was determined by a radioisotope method, using chromium-51–EDTA. All patients received antiemetics according to best local practice. During the first phase of this study, the dose of decitabine was escalated in combination with carboplatin AUC 5. The starting dose, 45 mg/m2, and scheduling of decitabine were based on pharmacodynamically active dose and schedule that induced chemosensitization in xenograft studies.13 The dose of decitabine was escalated by 45 mg/m2 in subsequent cohorts. Three patients were entered at each dose level and the cohort expanded to six patients if dose-limiting toxicity (DLT) was observed. Toxicity was assessed according to National Cancer Institute Common Toxicity Criteria expanded version 2. DLT was defined as grade 4 neutropenia (febrile)
Patient Evaluation and Follow-Up
Collection of Pharmacokinetic and Pharmacodynamic Samples Ten-milliliter blood samples for methylation studies were collected in EDTA Vacutainer tubes on day 1 of decitabine treatment; days 2, 5, 8, 10, 12, and 15; and day 22 of cycle 1 (two samples collected on days 1, 8, 10, 12, and 15); and on days 1, 8, and 15 of subsequent cycles (two samples on days 1 and 8). Human genomic DNA extraction from blood was performed using the BACC2 Nucleon DNA extraction kit (GE Healthcare Life Sciences Ltd, Buckinghamshire, UK). Buccal smears were collected day 1 of decitabine treatment; days 2, 5, 8, 10, 12, and 15 of cycle 1; and days 1, 8, and 15 of subsequent cycles. DNA from buccal smears was isolated using the BuccalAmp DNA Extraction Kit (Cambio, Cambridge, UK).
Methylation-Specific Polymerase Chain Reaction
High-Performance Liquid Chromatography Detection of 5-Methyl-2-Deoxycytidine
DNA Pyrosequencing
Fetal Hemoglobin
Thirty-five patients were recruited onto this study (Table 1). Thirty-three patients received treatment with decitabine and carboplatin (two patients received only decitabine because of rapid clinical deterioration). Tumor types included carcinoma of the colon (n = 7), breast (n = 5), ovary (n = 5), melanoma (n = 4), sarcoma (n = 4), gall bladder (n = 2), pleural mesothelioma (n = 2), and others (n = 6).
In the first phase of the study, DLT (day 29 decitabine delayed by 15 days due to neutropenia) was observed in one of three patients treated with 90 mg/m2 decitabine/carboplatin AUC 5, resulting in cohort expansion to six patients with no additional DLTs. Two of four patients receiving 135 mg/m2 decitabine/carboplatin AUC 5 experienced DLT (febrile neutropenia/grade 3 anorexia, nausea and fatigue; uncomplicated prolonged neutropenia beyond day 36). The cohort receiving 90 mg/m2 decitabine and carboplatin AUC 5 was expanded (to 13) to provide additional pharmacodynamic data. One additional patient developed DLT (causing a delay in treatment of 9 days because of uncomplicated neutropenia). During the second phase of the study, the dose of decitabine was escalated in combination with carboplatin AUC 6. At decitabine 45 mg/m2, no toxicity was seen. At decitabine 90 mg/m2, one patient experienced DLT (grade 3 fatigue). This dose level was expanded to include 10 patients to assess further the toxicity and pharmacodynamic effects. One additional patient developed DLT (neutropenic sepsis). Decitabine 90 mg/m2/carboplatin AUC 6 was therefore chosen as the recommended dose (RD) for future studies.
Toxicity
Antitumor Activity Thirty patients were assessable for response; the majority had heavily pretreated disease (Table 1). Of the 10 patients treated at the RD, one patient (melanoma) had a partial response maintained for eight cycles, and one patient (mesothelioma) had stable disease maintained for six cycles. An additional two patients had stable disease at the 45 mg/m2 decitabine/AUC 6 dose level.
Demethylation of DNA Induced by Decitabine in Surrogate Tissues Global 5-methyl-2'-deoxycytidine levels in DNA of PBCs from patients were measured after decitabine treatment. Maximal mean decrease in levels of 5-methyl-2'-deoxycytidine in PBC DNA were 33%, 35%, and 48%, respectively, for 45, 90, and 135 mg/m2 decitabine. For all dose levels, this is greater than the mean demethylation of 27% observed in murine PBCs at doses that caused chemosensitization (P < .05). In this study, PBCs showed a decrease in levels of 5-methyl-2'-deoxycytidine up to day 10, which then reversed and had returned to near starting levels by day 22. This reversible nature and kinetics of demethylation induced by decitabine is consistent with previous in vitro studies of cell lines and previous clinical studies.7,13,24 There is a clear correlation between the dose of decitabine and demethylation, as shown in Figure 1 (r2 = 0.97). Data for 5-methyl-2'-deoxycytidine levels of individual patients are provided in Appendix Table A1 (online only).
CGI Demethylation at the MAGE1A Gene in PBCs and Buccal Cells The MAGE1A CGI is bi-allelically methylated in most normal tissues and transcriptionally silent.25,26 Although the assay is only semiquantitative, methylation-specific PCR for unmethylated MAGE1A CGI showed that, after 8 days, patients treated with decitabine had an increase in levels of demethylated MAGE1A CGI in peripheral blood mononuclear cells and buccal cells (Fig 2) . Demethylation is again maximal during days 8 through 12, and returns to initial levels by day 22. To evaluate more quantitatively the amount of demethylation occurring, we have determined the amounts of methylated and unmethylated cytosines at three CpG sites in the MAGE1A CGI by DNA pyrosequencing (Fig 2). There is clear evidence for dose-dependent demethylation at each of the CpG sites in the MAGE1A CGI, with higher doses of decitabine treatment showing significantly increased demethylation (P = .006).
CGI Demethylation at the MAGE1A Gene in Tumor Figure 3 shows the average levels of unmethylated CpGs at three CpG sites in the MAGE1A promoter from tumor biopsies of eight patients taken on day 10 to 12 in cycle 1 or 2 after decitabine treatment. Overall, we observed a maximal demethylation in patients of 6.8% (mean, 3.5%). The samples taken after decitabine treatment have increased methylation significantly different from the pretreatment level (P = .006). For comparison, in xenografts an 11.4% increase in cytosine demethylation in the MAGE1A gene is observed at doses of decitabine that induce chemosensitization. However, although there is evidence of demethylation after decitabine treatment, this is variable and is much less than that observed in PBCs. This may be due to the pharmacologic barriers in delivering drug to tumor. However, because decitabine needs to be incorporated into DNA during DNA replication to inactivate DNMTs, and subsequent cell proliferation is required for demethylation to occur, we reasoned that the demethylation effects in tumor may be delayed markedly compared with a more highly proliferating tissue such as PBCs.
Induction of Gene Expression in Lymphocytes The ability of decitabine to induce expression of fetal hemoglobin (HbF) has been demonstrated previously in trials of patients with sickle cell anemia.27 In the current study, levels of HbF in lymphocytes increased 8 to 12 days after treatment of patients with 90 mg/m2 decitabine and returned to approximate starting levels by day 15 in cycle 1 (Figs 4A and 4B). In subsequent cycles of treatment there was accumulation of HbF levels (Fig 4C), suggesting that repeat treatment of patients with decitabine is more effective than a single treatment in reversing gene silencing, or that increasing numbers of HbF-expressing progenitor lymphocytes are repopulating the blood.
Aberrant methylation of CGIs impacts on diverse cellular processes, including apoptosis, tumor cell growth, and expression of drug sensitivity or resistance phenotypes.3,4 Decitabine induces hypomethylation in tumor xenografts and this is associated with increased sensitivity to carboplatin.13 Here we report for the first time the results of a phase I clinical and pharmacodynamic study demonstrating the feasibility of delivering a DNMT inhibitor (decitabine) in combination with carboplatin at a dose and schedule sufficient to produce a reduction in DNA methylation equivalent to or greater than that observed at carboplatin-sensitizing doses in the xenograft model. Decitabine at 90 mg/m2 administered as a 6-hour IV infusion can be combined safely with carboplatin (AUC 5 or 6) administered on day 8 of a 28-day cycle. The toxicity we observed was predictable (mainly myelosuppression) and manageable at the recommended dose. Doses of decitabine that cause sensitization to carboplatin of xenografts in mice induce a 27% mean reduction in 5-methyl-2'-deoxycytidine levels in PBCs,13 whereas in patients, the mean decrease was 35% at the recommended dose of 90 mg/m2 decitabine. This schedule of a 6-hour infusion on day 1 produced a similar reduction in 5-methyl-2'-deoxycytidine levels in PBCs to that seen with a 7-day continuous low-dose regimen and a 1 hour infusion daily over 10 days,12,24,28 though analysis using exactly the same assays and protocols will be necessary to determine if there are subtle differences in demethylation induced by the different schedules. Less demethylation was observed in tumor biopsies than in PBCs. This is likely due to reduced uptake of decitabine and lower levels of proliferation in tumor cells versus hematopoietic cells (DNA replication is essential for demethylation, given the mechanism of action of decitabine). The level of demethylation we observed in patient tumors was up to 60% of that in xenografts at chemosensitizing doses. However, this is perhaps not a valid comparison, given that it is the level of demethylation in proliferating repopulating tumor cells that may be the crucial comparison, and these will inevitably be different in the xenografts compared with tumor biopsies. Nevertheless, there is clearly scope for improving the amount of hypomethylation induced in tumors in patients. Unfortunately, the toxicity of decitabine precludes higher doses of decitabine, but alternate delivery or scheduling approaches, as well as the development of less toxic DNMT inhibitors,8 will be fruitful future avenues to explore. In summary, the prevalence of aberrant methylation of genes in tumors makes it an attractive target for novel anticancer therapies. Decitabine produces a reduction in DNA methylation and can be combined safely with carboplatin. This study represents a rational approach to clinical trial design targeting a potential epigenetic mechanism of drug resistance. It remains to be seen if this translates into clinical benefit; a randomized phase II study in partially platinum-sensitive ovarian cancer is now underway.
The author(s) indicated no potential conflicts of interest.
Conception and design: Helen J. Mackay, Jane A. Plumb, Gordon Strathdee, Andreas Schätzlein, Chris Twelves, Stanley B. Kaye, Robert Brown Financial support: Stanley B. Kaye, Robert Brown Administrative support: Kim Appleton, Katharine Bellenger, Lynsay Mackay, Andreas Schätzlein, Robert Brown Provision of study materials or patients: Helen J. Mackay, Ian Judson, Chooi Lee, Sophie Barrett, Sarah Reade, Dalal Jadayal, Adrian Tang, Chris Twelves, Stanley B. Kaye Collection and assembly of data: Kim Appleton, Helen J. Mackay, Ian Judson, Jane A. Plumb, Carol McCormick, Chooi Lee, Sophie Barrett, Sarah Reade, Dalal Jadayal, Adrian Tang, Katharine Bellenger, Lynsay Mackay, Albert Setanoians, Robert Brown Data analysis and interpretation: Kim Appleton, Helen J. Mackay, Ian Judson, Jane A. Plumb, Carol McCormick, Gordon Strathdee, Chooi Lee, Katharine Bellenger, Lynsay Mackay, Albert Setanoians, Andreas Schätzlein, Chris Twelves, Stanley B. Kaye, Robert Brown Manuscript writing: Kim Appleton, Helen J. Mackay, Ian Judson, Stanley B. Kaye, Robert Brown Final approval of manuscript: Kim Appleton, Helen J. Mackay, Ian Judson, Jane A. Plumb, Carol McCormick, Gordon Strathdee, Chooi Lee, Sophie Barrett, Sarah Reade, Dalal Jadayal, Adrian Tang, Katharine Bellenger, Lynsay Mackay, Albert Setanoians, Andreas Schätzlein, Chris Twelves, Stanley B. Kaye, Robert Brown
Supported by Cancer Research UK Grants No. A2662 and DC0024/0201 to R.B. Presented in part at the 40th Annual Meeting of the American Society of Clinical Oncology, June 5-9, New Orleans, LA; and at the American Association for Cancer Research–National Cancer Institute–European Organisation for Research and Treatment of Cancert International Conference on Molecular Targets and Cancer Therapeutics, November 14-18, 2005, Philadelphia, PA. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Gifford G, Paul J, Vasey PA, et al: The acquisition of hMLH1 methylation in plasma DNA after chemotherapy predicts poor survival for ovarian cancer patients. Clin Cancer Res 10:4420-4426, 2004 18. Wei SH, Balch C, Paik HH, et al: Prognostic DNA methylation biomarkers in ovarian cancer. Clin Cancer Res 12:2788-2794, 2006 19. Momparler RL, Cote S, Eliopoulos N: Pharmacological approach for optimisation of the dose schedule of 5-aza-2'-deoxycitidine (Decitabine) for the therapy of leukaemia. Leukemia 11:175-180, 1997[CrossRef][Medline] 20. Herman JG, Graff JR, Myohanen S, et al: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821-9826, 1996 21. Teodoridis JM, Hall J, Marsh S, et al: CpG island methylation of DNA damage response genes in advanced ovarian cancer. Cancer Res 65:8961-8967, 2005 22. Ghent University, Faculty of Medicine and Health Sciences, Centre for Medical Genetics: methPrimer DB: DNA methylation analysis PCR primer database. http://medgen.ugent.be/methprimerdb/index.php 23. Lin KT, Momparler RL, Rivard GE: High-performance liquid chromatographic analysis of 2-deoxy-5-azacytidine. J Pharm Sci 70:1228-1232, 1981[CrossRef][Medline] 24. Samlowski WE, Leachman SA, Wade M, et al: Evaluation of a 7-day continuous intravenous infusion of decitabine: Inhibition of promoter-specific and global genomic DNA methylation. J Clin Oncol 23:3897-3905, 2005 25. De Smet C, De Backer O, Faraoni I, et al: The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci U S A 93:7149-7153, 1996 26. De Smet C, Loriot A, Boon T: Promoter-dependent mechanism leading to selective hypomethylation within the 5' region of gene MAGE-A1 in tumor cells. Mol Cell Biol 24:4781-4790, 2004 27. 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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
; Santa Cruz Biotechnology, Santa Cruz, CA). 
of 7 minutes and t1/2 β of 35 minutes (see also Appendix Table A1). (B) 5-Methylcytosine levels as percent of total cytosine over time (days). (C) Plasma levels of decitabine versus 5-methylcytosine levels. Error bars represent SEs. AAC, area above the time curve; AUC, area under the concentration-time curve.
