Purpose Patients with high grade gliomas (HGG) routinely receive radiation, temozolomide, and glucocorticoids. As each of these is immunosuppressive, we conducted a prospective, multicenter study to follow CD4 counts over time and determine if low CD4 counts were associated with adverse outcomes. Experimental design Patients with newly diagnosed HGG had CD4 counts drawn before initiating standard therapy and monthly thereafter for one year. Information on hospitalizations, infections, glucocorticoid use, survival, and cause of death were also collected. Results Ninety-six evaluable patients were accrued (85% glioblastoma, median age of 57, median KPS 90). The median CD4 count before radiation and temozolomide was 664 cells/mm3. The CD4 count nadir occurred 2 months after initiating therapy when 73% of patients had CD4 counts <300 cells/mm3 and 40% had <200 cells/mm3. CD4 counts remained low throughout the year of follow-up. Patients with CD4 counts <200 cells/mm3 at 2 months had shorter survival than those with higher counts (median 13.1 versus 19.7 months, p=0.002). Median survival was related to CD4 toxicity grades (I=23.8 months, II=19.7 months, III–IV=13.1 months, p=0.009). The adjusted hazard ratio for death attributable to 2-month CD4 count below 200 was 1.66 (p=0.03). Eighty-eight percent of deaths resulted from disease progression while only 2.5% were due to infection. Conclusions Severe reductions in CD4 counts in patients with newly diagnosed HGG treated with radiation and temozolomide are common, treatment-related, long-lasting, and associated with early death from tumor progression.

Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials. (Cancer Res 2006; 66(12): 6361-9)

Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially contributing to pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDSs), are responsive to DNA methyltransferase inhibitors. To determine the extent of promoter hypermethylation in such tumors, we compared the distribution of DNA methylation of 14 000 promoters in MDS and secondary acute myeloid leukemia (AML) patients enrolled in a phase 1 trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34 ؉ bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34 ؉ bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alupoor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. This trial was registered at www.clinicaltrials.gov as J0443.

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