Abstract

The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #9421 (n=144) was 36%. We previously reported a subgroup of patients (n=68) with AML and NK that could be divided into 2 groups whose clinical outcomes correlated with abnormalities of FLT3 [internal tandem duplications (ITD) or activating loop mutations]. EFS estimates were 13% for patients with mutant FLT3 and 61% for children with wild-type FLT3 (P=0.01). We hypothesized that gene expression profiling would identify signatures that are linked to clinical outcome and can be used for risk determination. Cytogenetic testing was carried out in clinical laboratories at the institutions in which AML was diagnosed and then centrally reviewed. We analyzed bone marrow from 45 patients with NK on 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. FLT3 status (mutant or wild type) was determined by RT-PCR analysis of RNA from these 45 samples (exon 11 for ITDs, exon 17 for point mutations): 18 expressed mutant FLT3, 27 expressed wild-type FLT3. Using prediction analysis for microarrays (PAM) to find the minimum number of genes that identified samples associated with and unassociated with events (relapse or death), we identified a 128 gene cluster that differentiated patients with NK on the basis of clinical outcome (classification error rates were 15% for samples associated with events and 12.5% for event-unassociated samples). Significance analysis of microarrays (SAM) identified, with a false-discovery rate of 1.25%, 82 genes in the cluster whose expression differed significantly between the event-associated samples and the event-unassociated samples. Hierarchical clustering based on these 82 genes yielded 2 signatures: one event-associated and one event-unassociated.

FLT3 Status Event-Associated Signature Event-Unassociated Signature 
Wild-type EFS=44% (n=15) EFS=90% (n=12) 
Mutant EFS=7% (n=13) EFS=60% (n=5) 
FLT3 Status Event-Associated Signature Event-Unassociated Signature 
Wild-type EFS=44% (n=15) EFS=90% (n=12) 
Mutant EFS=7% (n=13) EFS=60% (n=5) 

The median WBC counts at the time of diagnosis were 68 x 109/L in the event-associated group and 61 x 109/L in the event-unassociated group (P=0.27). The gene list and d-scores from SAM analysis were analyzed using Ingenuity Pathway Analysis software (Ingenuity™ Systems, Mountain View, CA). Canonical pathways associated with poor outcome included apoptosis signaling (↑BCL2A1, ↓BAK1), ERK/MAP signaling (↑RAC2), cell cycle (↓ABL1), SAPK/JNK signaling (↑RAC2, ↓CDC42), integrin signaling (↑RAC2, ↓BCAR3, ↓ABL1, ↓CDC42), and IL6 signaling (↑IL6R). We conclude that risk assignment for patients with NK can be more precisely determined by combining FLT3 analysis and gene expression profiling. Such an approach identified 4 distinct groups with different outcomes. We will validate these findings by analyzing additional diagnostic samples with normal karyotype. Prospective validation of this strategy in clinical trials may be warranted.

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