Mark Wade

Evaluation of a 7-Day Continuous Intravenous Infusion of Decitabine: Inhibition of Promoter-Specific and Global Genomic DNA Methylation

PURPOSEnThe nucleoside analog 5-aza-2-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients.nnnMETHODSnTen patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m(2)/d [DOSAGE ERROR CORRECTED] via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment.nnnRESULTSnTransient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient.nnnCONCLUSIONnA 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation-mediated gene silencing.

Use of Oral N-Acetylcysteine for Protection of Melanocytic Nevi against UV-Induced Oxidative Stress: Towards a Novel Paradigm for Melanoma Chemoprevention

Purpose: Induction of oxidative stress has been implicated in UV-induced melanoma. We sought to determine whether the antioxidant N-acetylcysteine (NAC) could be safely administered to protect melanocytic nevi from the oxidative stress resulting from acute UV exposure. Experimental Design: Patients at increased risk for melanoma were recruited from a screening clinic. Induction and detection of oxidative stress (reactive oxygen species and glutathione depletion) was optimized in nevi following ex vivo UV irradiation. Nevi were removed from patients before, and following, oral ingestion of a single (1,200 mg) dose of NAC, and then these nevi were UV irradiated (4,000 J/m2). Results: Oxidative stress was induced in nevi 24 to 48 hours following ex vivo UV irradiation. A single oral dose of NAC was well tolerated in all patients (n = 72). Basal levels of reduced glutathione and the NAC metabolite cysteine were well correlated between similar-appearing nevi from the same patient and were significantly increased in nevi removed 3 hours after NAC ingestion compared with nevi removed before drug ingestion. In approximately half (9 of 19) of patients tested, UV-induced glutathione depletion was attenuated in the postdrug (compared with predrug) nevus. Conclusions: NAC can be safely administered to patients for the purpose of modulating UV-induced oxidative stress in nevi. This study suggests the feasibility of patients taking NAC prophylactically before acute UV exposure, to prevent pro-oncogenic oxidative stress in nevi and ultimately reduce long-term melanoma risk. (Clin Cancer Res 2009;15(23):7434–40)

A Phase III study of radiation therapy (RT) and O6-benzylguanine + BCNU versus RT and BCNU alone and methylation status in newly diagnosed glioblastoma and gliosarcoma: Southwest Oncology Group (SWOG) study S0001

AimsTo determine the efficacy of methylguanine methyltransferase (MGMT) depletion + BCNU [1,3-bis(2-chloroethyl)-1- nitrosourea: carmustine] therapy and the impact of methylation status in adults with glioblastoma multiforme (GBM) and gliosarcoma.MethodsMethylation analysis was performed on GBM patients with adequate tissue samples. Patients with newly diagnosed GBM or gliosarcoma were eligible for this Phase III open-label clinical trial. At registration, patients were randomized to Arm 1, which consisted of therapy with O6-benzylguanine (O6-BG)xa0+xa0BCNU 40xa0mg/m2 (reduced dose) + radiation therapy (RT) (O6BGxa0+xa0BCNU arm), or Arm 2, which consisted of therapy with BCNU 200xa0mg/m2 + RT (BCNU arm).ResultsA total of 183 patients with newly diagnosed GBM or gliosarcoma from 42 U.S. institutions were enrolled in this study. Of these, 90 eligible patients received O6-BGxa0+xa0BCNU + RT and 89 received BCNU + RT. The trial was halted at the first interim analysis in accordance with the guidelines for stopping the study due to futility (<40xa0% improvement among patients on the O6BGxa0+xa0BCNU arm). Following adjustment for stratification factors, there was no significant difference in overall survival (OS) or progression-free survival (PFS) between the two groups (one sided pxa0=xa00.94 and pxa0=xa00.88, respectively). Median OS was 11 [95xa0% confidence interval (CI) 8–13]xa0months for patients in the O6BGxa0+xa0BCNU arm and 10 (95xa0% CI 8–12)xa0months for those in the BCNU arm. PFS was 4xa0months for patients in each arm. Adverse events were reported in both arms, with significantly more grade 4 and 5 events in the experimental arm.ConclusionsThe addition of O6-BG to the standard regimen of radiation and BCNU for the treatment patients with newly diagnosed GBM and gliosarcoma did not provide added benefit and in fact caused additional toxicity.

Retrieval yield of total and messenger RNA in mesenchymal tissue ex vivo.

Mesenchymal neoplasms are a heterogeneous group of tumors comprising more than 200 benign entities and approximately 100 sarcomas. Large intraobserver and interobserver variability mandates improvements in diagnostic criteria. Gene expression microarrays are one tool in an evolving field of technology that permits the screening of tissue for massive amounts of information regarding its genetic composition. Such information may aid clinicians to diagnose and treat sarcomas. Complementary deoxyribonucleic acid microarrays, although very promising, are limited by the fact that messenger ribonucleic, the genetic messenger that permits deoxyribonucleic acid to encode for proteins and is the element retrieved from tumor samples ex vivo, is highly unstable, degrading quite readily. We found that even with optimal retrieval times and processing, total ribonucleic acid extraction from tumor tissue ex vivo is retrieved in adequate amounts to avoid amplification in 23% to 55% (mean 36%) of specimens. The percentage of high-grade tumors that yielded sufficient total ribonucleic acid was significantly higher than low grade and benign tumors. When adequate retrieval is achieved, the quantity and quality of messenger ribonucleic acid is robust. Surgeons, pathologists, and clinical intermediaries must be aware of issues surrounding messenger ribonucleic acid retrieval from surgical specimens to optimize collection.

Fiberoptic Resonance Raman Spectroscopy to Measure Carotenoid Oxidative Breakdown in Live Tissues

Based on compelling epidemiologic and corroboratory in vitro studies, carotenoids are thought to have great potential as dietary prevention against cancer. Yet, carotenoid-based chemopreventive trials have found very contradictory results. Definitive conclusions from these trials are hampered by an inability to accurately and safely measure carotenoids in specific tissues at risk of cancer development. Raman spectroscopy has been proposed as an optical technology with which to analyze various molecules in live tissues. One major obstacle that impedes the clinical use of this powerful technology is the lack of a fiberoptic Raman probe suitable for endoscopic tissue evaluation. A single-fiber resonance Raman Spectroscope capable of noninvasive “optical biopsies” to measure carotenoid concentrations in live tissues has been developed. The accuracy of this Raman instrument was confirmed by comparison with more standard methods of spectrophotometry and high-pressure liquid chromatography using solubilized β-carotene (BC) and BC-loaded cells before use in a small patient cohort. This Raman instrument detected intact BC as well as BC oxidative breakdown as a decrement of its Raman signal in cells. Use of the Raman instrument in our small cohort study showed its feasibility for measuring human tissues and raised some potentially intriguing possibilities about BC tissue pharmacokinetics and oxidative biology. Based on these results, our newly developed single fiberoptic resonance Raman instrument may provide a very useful method of measuring carotenoids and their oxidative breakdown within live tissue during future carotenoid chemopreventive trials. This proof-of-concept study provides the foundation to justify future validation of our Raman prototype. Cancer Prev Res; 3(4); 529–38. ©2010 AACR.

Discriminate gene lists derived from cDNA microarray profiles of limited samples permit distinguishing mesenchymal neoplasia ex vivo

Background Mesenchymal neoplasia comprises a heterogeneous group of tumors with over 200 benign neoplasms and 100 sarcomas. Currently, tumors are classified using histologic and immunocytologic characteristics, with diagnostic error rates reported as high as 40% of cases. As a feasibility study, our goal was to generate a preliminary discriminatory gene list for selected mesenchymal tumors, including sarcomas. This technique may enable an eventual molecular classification schema based on expression profiles that can complement current clinical and pathologic diagnostic procedures in mesenchymal tumors.Methods cDNA microarray analyses were preformed on connective tissue tumors obtained at time of surgical resection or biopsy. Messenger RNA (mRNA) from four general tumor classes was competitively hybridized against a human dermal fibroblast cell line comparator and the resulting gene expression profiles processed by ANOVA and linear discriminate analysis.Results The tissue classification involved 18 patients with malignant peripheral nerve sheath tumors, giant cell containing tumors, benign spindle cell lesions, or Ewing’s family of tumors. Lymph nodes from two patients served comparative purposes. Twenty-five differentially regulated genes considered most variable among the five tissue classes were identified. The tissues were segregated into five classes by linear discriminate analysis.Conclusions Linear discriminate analysis of cDNA gene expression profiles partitioned mesenchymal tumor classes, even when constrained by limited sample sizes.

Evaluating 2-chlorodeoxycytidine for its novel mechanism as a DNA methylation inhibitor

3125 Background: Many tumor suppressor genes are silenced by epigenetic alterations of CpG islands in the promoter regions. A clinical example of this is seen in colorectal cancer. Although the adenomatous polyposis coli gene is frequently somatically mutated in colon carcinoma, methylation of the adenomatous polyposis coli gene is seen in early colon adenomas. Thus, epigenetic silencing of tumor suppressor genes may play a role in the progression of colon polyps to carcinomas. Further, reactivation of critical tumor suppressor genes in carcinomas may decrease chemotherapy resistance. 5-aza-deoxycytidine is a DNA methyltransferase inhibitor. This drug has problems of DNA mutagenesis. We are evaluating 2-chlorodeoxycytidine for its inhibitory properties of s-adenosylhomocysteine hydrolase. Since s-adenosylmethionine is the only source of methyl donor for all mammalian methyltransferases, we can indirectly inhibit DNA methylation by depleting s-adenosylmethionine.nnnMETHODSnA growth inhibition experiment is done comparing HT-29 cells treated with 2-chlorodeoxycytidine and cells treated with vehicle. This time point is then used for future experiments. Genomic DNA is isolated and digested with a methylation sensitive restriction enzyme. Methylation sensitive endonuclease digested DNA will be amplified by real time polymerase chain reaction to assess for promoter methylation. We will next evaluate global methylation level of the genome using HPLC. We have also performed microarray on vehicle and treated samples. The effect of 2-chlorodeoxycytidine compared to 5-aza-deoxycytidine on gene expression is evaluated.nnnRESULTSn300 nM 2-chlorodeoxycytidine inhibits cell proliferation within 48 hours. Many known genes silenced by methylation, including genes in the interferon pathway, were upregulated by at least 2 fold after 48 hours of 2-chlorodeoxycytidine.nnnCONCLUSIONn2-chlorodeoxycytidine, a known adenosine deaminase inhibitor, also may indirectly inhibit DNA methylation, reactivating critical tumor suppressor genes in various malignancies. [Table: see text].