PrEMeR-CG: inferring nucleotide level DNA methylation values from MethylCap-seq data.

MOTIVATION: DNA methylation is an epigenetic change occurring in genomic CpG sequences that contribute to the regulation of gene transcription both in normal and malignant cells. Next-generation sequencing has been used to characterize DNA methylation status at the genome scale, but suffers from high sequencing cost in the case of whole-genome bisulfite sequencing, or from reduced resolution (inability to precisely define which of the CpGs are methylated) with capture-based techniques. RESULTS: Here we present a computational method that computes nucleotide-resolution methylation values from capture-based data by incorporating fragment length profiles into a model of methylation analysis. We demonstrate that it compares favorably with nucleotide-resolution bisulfite sequencing and has better predictive power with respect to a reference than window-based methods, often used for enrichment data. The described method was used to produce the methylation data used in tandem with gene expression to produce a novel and clinically significant gene signature in acute myeloid leukemia. In addition, we introduce a complementary statistical method that uses this nucleotide-resolution methylation data for detection of differentially methylated features.

Clinical and pharmacodynamic activity of bortezomib and decitabine in acute myeloid leukemia.

We recently reported promising clinical activity for a 10-day regimen of decitabine in older AML patients; high miR-29b expression associated with clinical response. Subsequent preclinical studies with bortezomib in AML cells have shown drug-induced miR-29b up-regulation, resulting in loss of transcriptional activation for several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases. Thus, a phase 1 trial of bortezomib and decitabine was developed. Nineteen poor-risk AML patients (median age 70 years; range, 32-84 years) enrolled. Induction with decitabine (20 mg/m(2) intravenously on days 1-10) plus bortezomib (escalated up to the target 1.3 mg/m(2) on days 5, 8, 12, and 15) was tolerable, but bortezomib-related neuropathy developed after repetitive cycles. Of previously untreated patients (age ≥ 65 years), 5 of 10 had CR (complete remission, n = 4) or incomplete CR (CRi, n = 1); 7 of 19 overall had CR/CRi. Pharmacodynamic analysis showed FLT3 down-regulation on day 26 of cycle 1 (P = .02). Additional mechanistic studies showed that FLT3 down-regulation was due to bortezomib-induced miR-29b up-regulation; this led to SP1 down-regulation and destruction of the SP1/NF-κB complex that transactivated FLT3. This study demonstrates the feasibility and preliminary clinical activity of decitabine plus bortezomib in AML and identifies FLT3 as a novel pharmacodynamic end point for future trials.

TET2 mutations improve the new European LeukemiaNet risk classification of acute myeloid leukemia: a Cancer and Leukemia Group B study.

PURPOSE: To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS: Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays. RESULTS: TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I-risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I-risk patients and found distinct mutation-associated microRNA signatures in both ELN groups. CONCLUSION: TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.