Analyses of the frequency of base changes per kilobase of sequenced DNA from the AML pilot set and the control set. (A) Total sequence changes per kilobase of sequenced DNA plotted as a function of the numbers of samples with the designated frequencies. More than 98% of sequence changes detected in both sets were priority 2. (B) Total sequence changes per kilobase of sequenced DNA plotted for each pilot set AML sample and control sample arranged in rank order. (C) Total sequence changes per kilobase of sequenced DNA, plotted for each of the 12 genes analyzed. More changes were noted in the AML set for HRAS, FLT3, and c-KIT, whereas more were in the control set for c-MYC and AML-1/RUNX1 (P < 0.01). The other genes had insignificant differences between the sets. When all genes were compared there was no significant difference between the AML set and the control set.

Summary of sequencing results. (A) Data are from the genes that were resequenced in the AML pilot set. Each row represents one designated sequence change from the indicated gene. N/A, not available. Each column under the M2 and M3 headings represents one patient sample. Red indicates a sequence change that predicts altered gene function (priority 1), and green indicates that the indicated change was not found. White indicates that resequencing of a target exon was not completed for a particular sample. t(15;17) indicates the presence of the t(15;17) translocation that is found in >90% of patients with AML M3. (B) Data are from the genes that were resequenced from the 94 AML samples from the CALGB set. The color scheme is the same as for A. Purple indicates apparent homozygosity for a priority 1 sequence change.

Analysis of mutations in the FLT3, c-MYC, and PML genes. (A) Sequence tracings from FLT3 exon 20 amplicons from five AML patients. Sample identifiers and blast counts are shown on the right. These patients were from the pilot set, so the samples were not enriched on Ficoll before they were banked. The positions of the mutations are shown with red arrows. G → T substitutions are present at position 199 in four patients (2263, 2405, 2323, and 2446), creating a D835Y substitution in FLT3. The other patient (2403) has a T → G substitution at position 201, creating a D835E substitution. (B) Sequence tracings from FLT3 exon 20 amplicons derived from an AML sample and a wild-type control sample at varying ratios. The two DNA samples were premixed at the designated ratios, and then exon 20 amplicons were created by PCR and sequenced. The position of the mutation that creates the D835E mutation is shown with a red arrow. The trace from control (WT) DNA is shown at the top, and the trace from the undiluted AML sample is shown next. The 1:1 dilution is shown next, and the 1:2 dilution is shown last. The signal from the mutant allele is detectable at all dilutions, but it decreases as the proportion of control DNA increases. (C) FLT3 mutations (either ITDs in exon 14, and/or activating point mutations at amino acid position 835) are plotted against the blast count of each sample. Data from the pilot set and from the CALGB set are indicated. Means are shown as black bars, and SDs are shown as gray boxes. (D) Sequence tracings from pilot-set AML samples with the c-MYC V170I change, the NRAS G13R change, and the PML R307C change. Note that the signal intensity from the mutant allele is less than that of wild type in the designated AML samples (except for one patient with c-MYC V170I), suggesting that the mutation is not present in all of the cells of the sample. Control tracings are shown for each region as indicated.