© 2007 European Society for Medical Oncology
miscellaneous tumors |
A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors
1 Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas (IIB), Universidad Nacional Autonóma de Mexico (UNAM), Instituto Nacional de Cancerología, Mexico City
2 Division de Investigación Clinica, Instituto Nacional de Cancerologia, Mexico City
3 División de Medicina, Instituto Nacional de Cancerologia, Mexico City, Mexico
* Correspondence to: Dr A. Dueñas-González, Dirección de Investigación, Instituto Nacional de Cancerología, San Fernando 22, Tlalpan, 14080 México, D.F., México. Tel: +52-55-56280486; Fax: +52-55-55734662; E-mail: alfonso_duenasg{at}yahoo.com
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Abstract |
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Background: Epigenetic aberrations lead to chemotherapy resistance; hence, their reversal by inhibitors of DNA methylation and histone deacetylases may overcome it.
Patients and methods: Phase II, single-arm study of hydralazine and magnesium valproate added to the same schedule of chemotherapy on which patients were progressing. Schedules comprised cisplatin, carboplatin, paclitaxel, vinorelbine, gemcitabine, pemetrexed, topotecan, doxorubicin, cyclophosphamide, and anastrozole. Patients received hydralazine at 182 mg for rapid, or 83 mg for slow, acetylators, and magnesium valproate at 40 mg/kg, beginning a week before chemotherapy. Response, toxicity, DNA methylation, histone deacetylase activity, plasma valproic acid, and hydralazine levels were evaluated.
Results: Seventeen patients were evaluable for toxicity and 15 for response. Primary sites included cervix (3), breast (3), lung (1), testis (1), and ovarian (7) carcinomas. A clinical benefit was observed in 12 (80%) patients: four PR, and eight SD. The most significant toxicity was hematologic. Reduction in global DNA methylation, histone deacetylase activity, and promoter demethylation were observed.
Conclusions: The clinical benefit noted with the epigenetic agents hydralazine and valproate in this selected patient population progressing to chemotherapy’ and re-challenged with the same chemotherapy schedule after initiating hydralazine and valproate’ lends support to the epigenetic-driven tumor-cell chemoresistance hypothesis (ClinicalTrials.gov Identifier: NCT00404508 [ClinicalTrials.gov] ).
Key words: epigenetic therapy, hydralazine, magnesium valproate, phase II, refractory, solid tumors
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
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Chemotherapy resistance, either innate or acquired, requires expression changes in a large number of genes for its development; thus, it has been hypothesized that epigenetic-mediated changes could be the driving force responsible for chemotherapy resistance [1]; hence, it could be expected that agents targeting DNA methylation and histone deacetylation would, by reverting the epigenetic marker, overcome chemotherapy resistance [2].
Hydralazine, one of the first oral antihypertensives developed, is a weak non-nucleoside DNA methylation inhibitor [3–5] whose mechanism of action depends on its ability to inhibit dnmt1 activity [6, 7] and/or by decreasing dnmt1 and –3a expression in a similar manner to PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor [7, 8]. Hydralazine's demethylating and gene-reactivating activity in tumors was demonstrated in cervical cancer in an oral dose range of 50–150 mg/day [9], which is below that used for hypertension (50–200 mg/day) or heart failure (200–300 mg/day). Pharmacokinetic characterization in healthy volunteers of the slow-release hydralazine formulation employed in this study (investigator brochure) demonstrated area-under-curve (AUC) concentrations of 6034 + 1899 ng/h/ml and 2751 + 954 ng/h/ml for slow and rapid acetylators, respectively, utilizing 182 mg regardless of acetylator status; hence, we chose for further testing doses of 182 mg/day and 83 mg/day for rapid and slow acetylators, respectively. This dose adjustment was adequate, as no statistically significant differences were found in hydralazine plasmatic levels between both types of acetylators (means, 246 and 249 ng/ml, respectively) in our previous study in patients with breast cancer [10].
The anti-epileptic valproic acid, an 8-carbon, branched-chained fatty acid, causes hyperacetylation of the N-terminal tails of histones H3 and H4 in vitro and in vivo and inhibits HDAC activity, most probably by binding to the catalytic center and thereby blocking substrate access [11, 12]. Its ability to inhibit deacetylase activity in solid tumors has recently been demonstrated in patients with cervical cancer and other malignancies at doses between 20 and 60 mg/kg [13–15]. At doses of 30 mg/kg daily, we found histone deacetylase (HDAC) inhibition in the breast cancer study at a mean plasma concentration of 87.5 µg/ml [10].
Hydralazine and valproic acid can significantly reduce cancer cell survival in a dose-dependent manner and can induce re-expression of the tumor suppressor gene p16 [16]. In combination, these show inhibitory growth effect in vitro and in vivo, chemosensitization, a synergistic effect upon global gene expression, and up-regulation of human leukocyte antigen (HLA) class-I expression and antigen-specific cytotoxic T lymphocyte (CTL) response in cervical cancer cells [17, 18]. In vivo studies have demonstrated that both drugs plus chemotherapy exhibit a higher anti-tumor effect when administered daily as compared with their administration only during the 5 days previous to and following chemotherapy administration (manuscript in preparation).
We recently completed a proof-of-principle study of epigenetic therapy with hydralazine and magnesium valproate added to neoadjuvant doxorubicin cyclophosphamide for locally advanced breast cancer, in which we demonstrate global DNA hypomethylation and HDAC activity inhibition in the peripheral blood of patients, as well as gene up-regulation of >1000 genes in primary breast tumors. Interestingly, the therapy was safe and tumor responses appeared higher as compared with historical controls [10]. These data demonstrate the ability of these epigenetic drugs to modulate global gene expression in tumors; thus, we performed a phase II study to test whether hydralazine and valproate could overcome chemotherapy resistance in patients with advanced and refractory solid tumors.
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patients and methods |
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patients
Eligible patients were 18 years of age and older with histologically proven malignant solid tumors who were receiving their 2nd, 3rd, or 4th line of palliative chemotherapy and who showed—at the second or third chemotherapy course—progressive disease as their maximum response according to Response Evaluation Criteria in Solid Tumors (RECIST), or Gynecologic Cancer Intergroup (GICG) CA125 criteria in the case of patients with ovarian cancer. In addition, patients were required to have measurable disease as defined by one of the following criteria: any unidimensionally measurable lesion
10 mm by standard magnetic resonance imaging (MRI) or computed tomography (CT) scan for solid tumors, or at least one non-measurable lesion evaluable by nuclear medicine, tumor markers, or other reliable measurements. Additional eligibility requirements included the following: Eastern Cooperative Oncology Group (ECOG) performance status 
2; absolute leukocyte count 4000/mm3; platelets 100 000/mm3; hemoglobin 9.0 g/dl; total bilirubin, aspartate amino transferase, and alanine amino transferase (<1.5 the normal upper limit); creatinine 1.2 mg/dl; life expectancy of >3 months, and written informed consent. Patients were excluded from the study if they referred a history of allergy to hydralazine or valproate, past or present condition of rheumatic disease, central nervous system disease, heart failure from aortic stenosis and postural hypotension as diagnosed by a physician, and previous use of the experimental drugs, as well as if patients were pregnant or breast-feeding. The protocol was approved by the Institutional Review Boards of the Instituto Nacional de Cancerología and carried out in accordance with the Declaration of Helsinki, good clinical practices, and local ethical and legal requirements.
study design
This was a MinExpSize 2-stage, phase II, open-label, single-arm study. A sample size of 15 patients was established, estimating a 10% probability of clinical benefit (complete response [CR], partial response [PR], and stable disease [SD]) if patients who showed progressive disease were to continue with the chemotherapy regimen, and a 40% probability of clinical benefit when hydralazine and magnesium valproate were added to chemotherapy. With these parameters the true type-I error rate, alpha is 0.043 and power of 0.818%. In the first stage of the study, four patients were evaluated; it was required that at least one of these obtain clinical benefit to continue to the second stage. The null hypothesis would be rejected if four or more patients presented complete response, partial response, or stable disease (clinical benefit). Sample size was calculated with the ExpDesign Studio for Windows version 1.5.0 from CtriSoft.
treatment plan
Patients received a single oral dose of 500 mg of sulphamethazine early in the morning, and urine was collected within the ensuing 6 h for phenotyping of acetylator status as reported [19]. Afterward, patients began treatment (day –7) with a daily dose of a slow-release formulation of hydralazine tablets containing either 182 mg for rapid, or 83 mg for slow, acetylators. Magnesium valproate tablets of 700 mg were also administered as a slow-release formulation at a dose of 40 mg/kg t.i.d. Both were administered until the last day of the final chemotherapy cycle.
chemotherapy
Chemotherapy was initiated at day 1 with the same pre-study protocol regimen. Drugs and schedules are shown in Table 1.
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therapy duration and dose modifications
Drugs were discontinued if the patient was unable to tolerate these, developed concurrent illness, or underwent changes in medical condition that rendered them unacceptable for further treatment. Toxicities were graded according to National Cancer Institute Common Toxicity Criteria (NCICTC) version 2.0. For hematologic toxicity, chemotherapy was withheld until recovery of absolute neutrophil count (ANC) to
1500/mm3 and platelet count to 75 000/mm3. Non-hematologic toxicity of grade 3 that was considered as possibly related with hydralazine and magnesium valproate required a delay in hydralazine and magnesium valproate treatment until the toxicity recovered returned to grade 2 or to pre-hydralazine and -magnesium valproate baseline.
pre-treatment and assessment of response and safety
Radiologic and clinical evaluation including spiral/helical computed tomography (CT), magnetic resonance imaging (MRI) scans, or bone scintigraphy were performed at baseline. CA125 levels were obtained several times in order to meet GCIG criteria for evaluation of response and progression [20]. Response evaluation was performed after the 1st or 2nd chemotherapy course and repeated 4 weeks later to confirm tumor response. Patients with stable disease (SD), complete response (CR), or partial response (PR) continued on protocol treatment and underwent repeated tumor assessment at 6-week intervals until progressive disease (PD). To be evaluable for response, a patient must have received at least one cycle of treatment (for 21 or 28 days) and must have completed at least one follow-up tumor assessment for response for the following month. RECIST was employed to determine best overall response in tumors other than ovarian. CR was defined as complete disappearance of all measurable (target) and non-measurable (non-target) disease, no new lesions, and no disease-related symptoms. PR was defined as an at least 30% decrease in the sum of the longest diameters (LDs) of measurable target lesions, using as baseline the sum of the LDs of measurable lesions as reference. PD was defined as an at least 20% increase in the sum of the LDs of target lesions, compared with the smallest sum of the LDs (nadir) recorded since baseline, or the appearance of new lesions. SD was defined as neither sufficient tumor reduction to qualify for PR nor sufficient increase to qualify for PD. The 50% response definition for ovarian cancer comprised a 50% decrease in serum CA125 level from two initially elevated samples; a sample showing a 50% decrease must be confirmed by a fourth sample (i.e. four samples are required) that had been analyzed at least 28 days after the previous (third) sample. Patients with ovarian cancer were defined as progressing in order to be eligible when CA125 doubled the nadir value on two occasions during the last line of treatment immediately prior to entering the protocol and with the same criteria during the study protocol. SD was considered when the patient did not meet criteria for response or progression. Safety analyses were performed on all patients who received hydralazine and magnesium valproate plus chemotherapy.
nucleic acid extraction
Genomic DNA from peripheral mononuclear blood cells was obtained with the standard method of proteinase-K digestion and phenol-chloroform extraction. Serum DNA was extracted from 10-ml blood samples in vacutainer SST Gel tubes. After centrifugation at 2500 rpm x 10 min at room temperature, the serum was filtered (0.45-µm membrane Millipore) and then stored at –70°C. DNA was purified from 1 ml of serum using the QIAamp Blood Mini kit (Qiagen, Hilden, Germany) according to manufacturer instructions.
global DNA methylation
Quantification of genomic 5Methylcytosine from the peripheral blood DNA was carried out by capillary electrophoresis as previously described [21]. The relative methylation of each DNA sample was taken as the percentage of mC in total cytosine: mC peak area x 100/(C peak area + mC peak area). Samples were taken at baseline (day 0) and on day 8 after initiation of hydralazine and valproate (before the 1st chemotherapy cycle).
Gene promoter methylation in serum DNA
Promoter methylation of hMLH, RARß, and DAPK genes was assessed in serum DNA from samples taken at day 8 (before the first course of chemotherapy and after 7 days of being on hydralazine and valproate) by methylation-specific polymerase chain reaction (PCR). Briefly, 250 ng of DNA in a volume of 50 µl of each sample was denaturated with freshly prepared NaOH at a final concentration of 0.2 M for 20 min at 42°C. One µg of salmon sperm DNA (Sigma) was added as a carrier before modification. Denaturated DNA was modified with 30 µl of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (pH 5) for 16 h at 50°C. Bisulfite-modified DNA was purified using Wizard DNA Clean Up System (Promega, Madison, WI) and eluted into 50 µl of water. Modification was completed by NAOH (final concentration, 0.3 M) treatment for 15 min at 37°C, followed by neutralization with 5 µl of ammonium acetate (10 M) and precipitated with 1 µl of glycogen and 200 µl of ethanol. After precipitation, the DNA was washed twice with 70% ethanol and dried under vacuum. DNA was resuspended in water and used immediately or stored at –20°C. The PCR mixture contained 1x PCR buffer, 0.5 U of Taq Gold polymerase, dNTPs (each 1.25 mM), 300 ng of primers, and bisulfite-modified DNA (100 ng) or unmodified DNA (50 ng) in a final volume of 20 µl. The sequence and annealing temperature of primers used for methylation-specific PCR are shown in Table 2. Products were visualized in a 2% agarose gel under ultraviolet (UV) light.
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histone deacetylase assay
Assays were performed using the colorimetric HDAC activity assay from BioVision (BioVision Research Products, Mountain View, CA) according to manufacturer instructions as described [13]. Blood samples were taken at baseline (day 0) and on day 8 after initiation of hydralazine and valproate (before the first cycle of chemotherapy).
valproic acid plasma levels
Valproic acid was measured in plasma using fluorescence polarization immunoassay (FPIA) technology as previously described [13]. Samples were analyzed at day 8 after the start of hydralazine and valproate (before the first cycle of chemotherapy).
hydralazine plasma levels
Hydralazine was determined in plasma by high-performance liquid chromatographic assay (HPLC) as previously described [22].
statistical analysis
Survival was calculated according to Kaplan–Meier method. Progression-free survival (PFS) and overall survival were registered from date of the patient's entry into the protocol until date of progression, death, or last visit. Comparisons between biological measurements were evaluated by t test. A P value of <0.05 was considered significant. Statistical analysis was done on SPSS-10 software.
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results |
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patients
The flow diagram of the trial is shown in Figure 1. A total of 27 patients signed informed consent. Three of these were ineligible and did not initiate hydralazine and valproate, seven patients abandoned the study during the first week of hydralazine and valproate prior to the first application of chemotherapy due to clinical deterioration, and two patients completed two chemotherapy cycles in the study protocol but were unevaluable because progression before their inclusion in the protocol could not be confirmed. Thus, 17 patients were evaluable for toxicity, and 15 for response. Primary sites were cervix (3), breast (3), lung (1), testis (1), and ovarian (7) carcinomas. The two patients who were not analyzed for response had testicular and breast carcinoma. Table 1 shows diagnosis and lines of treatment of evaluable patients. The majority of patients were heavily treated, and had received two and three previous lines of treatment. After entering the protocol, patients re-started the schedule employing the same dose they received previously.
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clinical response
Clinical benefit was observed in 12 (80%) of the 15 patients; 4 PR and 8 SD. Among partial responses, 3 were in ovarian cancer according to IGCG CA125 criteria, and a PR was observed in one patient with cervical cancer by MRI of a supraclavicular lymph node disease. Regarding patients with SD, 4 occurred in patients with ovarian cancer and 1 each in patients with cervix, lung, testis, and breast (Table 3). The majority of patients who exhibited response or stabilization also demonstrated improvement in symptoms such as dyspnea, cough, and pain. Regarding evaluation of response according to total number of evaluable disease sites, there were 16 evaluable lesions in the 8 cases excluding ovarian malignancies. Of these, 1 (6.6%) complete and 3 (18.75%) partial responses were achieved, whereas 9 (56.2%) had disease stabilization (Table 4).
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toxicity
Treatment was well-tolerated despite the fact that the study population was heavily pre-treated. Median number of weeks that patients received protocol therapy was 9.5 (range, 6–17 weeks). The most significant toxicity was hematologic, as shown in Table 5. Two patients required hospitalization and growth factor support due to neutropenic fever, and one patient needed platelet transfusion. Likewise, hematologic toxicity led to a decrease in dose intensity in 9 (60%) patients. Comparison between dose intensity before and during protocol therapy is shown in Table 6. Regarding non-hematologic toxicity, the only grade 3 toxicities that could be attributed to valproate were drowsiness in 3 and delirium in 1 patient, respectively. There were no grade 3 or 4 toxicities attributed to hydralazine. No toxicity-related deaths were observed.
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follow-up
Median progression-free survival was 3.3 months (range, 2.4– 5.7 months) and median survival was 6.1 months (range, 3.8–12.8 months). At the time of this writing, eight patients are alive.
DNA methylation
Analysis of methylation could only be performed in 8 patients and results are based on single measurements because of limitations in DNA amount. Results showed a reduction in 5mC content in 6 cases. Mean 5mC in the 8 pre-treatment samples was 5.5% (standard deviation [SD], 1.69), which decreased to 3.95% (SD, 0.53), (P = 0.0484) (Figure 2a). This change corresponds to a 21.2% mean decrease taking the pre-treatment 5mC content value as 100% in each sample (Figure 2b).
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histone deacetylase activity
As shown in Figure 2c, there was a reduction in enzymatic activity in all patients as determined by absolute values of optical density (OD) units. For comparison, reductions were of the same magnitude as those from the positive control of the HeLa cell kit. Means were 0.35 (SD, 0.080) and 0.237 (SD, 0.046), respectively (P = 0.0158). This change corresponds to a 29.3% mean decrease taking the pre-treatment OD in each sample as 100% (Figure 2d).
valproic acid levels
Mean concentration of valproic acid in plasma from 39 measurements was 86.3 µg/ml (SD, 32.5).
hydralazine plasma levels
Mean plasma level of hydralazine was 108.0 ng/ml (SD, 85.9) for slow acetylators (7 patients), whereas mean value was 288.4 (SD, 278.5) for rapid acetylators (3 patients), this difference statistically significant (P = 0.0151.)
gene promoter demethylation
In 12 (of 27) pre- and post-treatment MSP reactions, the genes were found unmethylated (unmethylated band pre- and post-treatment) and therefore considered non-informative. Promoter demethylation was observed in patient 5 (ovarian cancer) in the RARß gene, patient 9 (ovarian cancer) in DAPK, patient 10 (ovarian cancer) in both of these genes, and in the patient with lung cancer, at the RARß gene (patient 15). Overall, of 15 informative MSP reactions five (33.3%) showed promoter demethylation. As for patients, 8 of 9 were informative and 4 (50%) had demethylation of at least one gene (Figure 3). Table 3 summarizes clinical and biological findings.
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
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This phase II study suggests that DNA methylation and HDAC inhibitors overcome resistance to chemotherapy because clinical benefit (PR + SD) was observed in 80% of patients. This study design is unique in that the majority of patients were actively progressing to chemotherapy; this is best illustrated by the fact that seven patients deteriorated clinically during the week of hydralazine and valproate administration and prior to the first chemotherapy cycle. Therefore, the achievement of response and disease stabilization can only be attributed to the use of the epigenetic agents because this was the only difference between pre-study and protocol regimens. Further, chemotherapy-regimen dose intensity during hydralazine and valproate administration was actually lower, which strengthens the results.
Current data suggest that epigenetic changes are not solely a consequence of cancer cell-resistant phenotype, but that indeed these contribute to its development. It has been demonstrated that chemotherapy induces DNA hypermethylation in cultured cells [23–25]. Likewise, MCF-7 cells develop DNA hypermethylation when they acquire the multidrug-resistant phenotype, and both hypermethylation and doxorubicin resistance are reversed by hydralazine or by antisense treatment against DNA methyltransferases [18]. Resistance to cisplatin is also accompanied by over-expression of DNA methyltransferases [26] and the resistance itself is induced by over-expression of DNA methyltransferase genes [27]. The role of histone deacetylation in creating the resistant phenotype has not yet been demonstrated; however, a number of malignant cell lines and tumors over-express HDAC 1, 2, 4, 5, and 7A [28]. Whether exposure to chemotherapy agents leads to transcriptional or post-transductional changes upon HDAC is not known; nonetheless, cooperation between gene hypermethylation and histone deacetylation in silencing gene transcription is a well-established phenomenon [29].
Both DNA methylation and HDAC inhibitors up- and down-regulate a number of genes ranging from 0.5 to 2% of the whole genome [17, 30, 31]. In breast cancer [10], we found that hydralazine and magnesium valproate up- and down-regulated 1091 and 89 genes, respectively, by at least 3-fold (Gene Expression Omnibus [GEO] accession number GSE6304). Among these were p53, p21, IFN-response pathway genes, and HLA-class I genes. Nevertheless, the present study does not address the question of whether responses and disease stabilizations were the consequence of major transcriptome changes in accordance with the epigenetic-mediated resistance concept [1, 2, 32], or whether they were the result of transcription changes in specific genes involved in the resistance of particular drugs [33–35]. Notwithstanding this, a strong indication that responses obtained in our patients were due to the epigenetic effects of hydralazine and valproate is supported by the fact that reduction in global DNA methylation and inhibition of HDAC activity were observed. Regarding DNA methylation, hydralazine—although it is a weak DNA inhibitor as compared with nucleoside analogs [5]—led to a statistically significant reduction in the percentage of 5mC content in peripheral mononuclear cells after 7 days of treatment. This degree of reduction (1.5%) is similar to the 0.9% achieved by 5-aza-2-deoxycytidine at day 14 when evaluated by a related quantitative HPLC-based assay after 7 days of continuous infusion in patients with solid tumors [36].
Mean valproic acid plasma levels achieved (86.3 µg/ml) were similar to those found in our previous studies [10, 13]; nevertheless, while hydralazine levels in rapid acetylators were also similar to those of our study in patients with breast cancer [10] these levels were significantly lower in slow acetylators. Because dose adjustment for acetylation status yielded similar levels in patients with breast cancer (slow 246 vs. rapid 299 ng/ml; P = 0.2445), a likely explanation could be poor compliance. Ongoing studies will clarifiy this issue.
Further, as another surrogate marker we assessed methylation at the promoter of certain genes found to be methylated in serum DNA with cancer, with the rationale that these genes are always unmethylated in the serum of normal individuals; thus, if these are methylated they must derive from the primary tumor [37, 38]. Interestingly, of 15 informative MSP reactions, 5 (33.3%) showed promoter demethylation, whereas 8 (53.3%) remained methylated; in addition, of 8 informative patients, 4 (50%) had demethylation of at least one gene. The frequency of gene promoter demethylation of 33% is grossly similar to that obtained in our phase I study of hydralazine in which MSP was employed in primary tumors at baseline and after 10 days of hydralazine treatment, which ranged from 32 to 52% [9]. This is the first time that methylated sequences have been detected in serum DNA after epigenetic therapy; however, interpretation of these results must be taken with caution as there are many unknowns regarding the factors that determine DNA shedding to the circulation, as well as the exact proportion of serum DNA that derives from tumor and non-tumor cells. Nevertheless, assessment of methylated promoters in serum DNA holds promise as a simple and non-invasive means to evaluate the effect of epigenetic drugs in a clinical scenario.
It should be noted that pharmacodynamic evaluation was done at a single time point: day 8. This was specifically aimed to assess the pure effect of hydralazine and valproate as a starting point for future clinical studies in which we will measure these parameters during chemotherapy administration in order to evaluate the overall changes in these parameters exerted by epigenetic drugs and cytotoxic agents.
The only toxicity that could be attributed to the experimental therapy
specifically, to valproatewas drowsiness in the majority of patients. Regarding hydralazine, we observed only edema, headache, dizziness, and ischemia transitory attacks grades 1 and 2, which comprise hydralazine's cardiovascular effects. The lack of minor side effects observed can be at least partly due to the slow-release formulation that avoids hydralazine plasma peaks. Hematologic toxicity was clearly increased. Valproic acid is known to induce tri-lineage hematologic toxicity [39] and to increase the hematologic toxicity of fotemustine and cisplatin [40].
The study was a difficult test for hydralazine and valproate in that patients were re-challenged with the same chemotherapy schedules under which they were actively progressing. Thus, it could be expected that if patients were switched to different chemotherapy drugs and schedules responses and response duration could possibly have been better. We found no suggestions that hydralazine and valproate may work better in reverting resistance for a particular drug schedule or tumor type. It is noteworthy that the 7 patients with ovarian cancer achieved partial response (3) and disease stabilization (4). In this regard, some authors have suggested that response criteria by CA125 may overestimate the response [41], although others have found that GCIG CA125 response criteria comprise a better prognostic tool than RECIST in second-line treatment in patients with ovarian carcinoma [42]. In our study, 4 of 7 patients received the protocol therapy as third line of treatment, and in that setting CA125 criteria have not been validated. Nevertheless, the overall results are consistent if we solely analyze the seven patients with tumor types other than ovarian cancer; the clinical benefit in these was 62.5% (1 partial, 4 stabilizations, and 3 progressions).
In conclusion, the response and disease stabilization rates observed suggest that hydralazine and valproate overcome epigenetic changes, mediating chemotherapy resistance regardless of chemotherapy drug and tumor type. In addition, despite the fact that this epigenetic therapy increased the myelotoxicity of chemotherapy the toxicity is manageable. Nevertheless, this is a single-arm study; therefore, the possibility that patients would have responded to or stabilized with subsequent cycles of chemotherapy cannot be ruled out. To confirm the benefit of epigenetic therapy, placebo-controlled randomized phase III trials of hydralazine and valproate added to chemotherapy are ongoing as first-line therapy in patients with metastatic, persistent, or recurrent cervical cancer and in cisplatin-resistant ovarian cancer.
Received for publication February 28, 2007. Revision received April 18, 2007. Accepted for publication April 19, 2007.
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