Expression profiling reveals fundamental biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics

Abstract

Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Normal cytogenetics (CN) constitutes the single largest group, while trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How trisomy contributes to tumorigenesis is unknown. We used oligonucleotide-based DNA microarrays to study global gene expression in AML+8 patients with +8 as the sole chromosomal abnormality and AML-CN patients. CD34(+) cells purified from normal bone marrow (BM) were also analyzed as a representative heterogeneous population of stem and progenitor cells. Expression patterns of AML patients were clearly distinct from those of CD34(+) cells of normal individuals. We show that AML+8 blasts overexpress genes on chromosome 8, estimated at 32% on average, suggesting gene-dosage effects underlying AML+8. Systematic analysis by cellular function indicated up-regulation of genes involved in cell adhesion in both groups of AML compared with CD34(+) blasts from normal individuals. Perhaps most interestingly, apoptosis-regulating genes were significantly down-regulated in AML+8 compared with AML-CN. We conclude that the clinical and cytogenetic heterogeneity of AML is due to fundamental biological differences.

Figure 1

(A) Scatter plots of the log-intensity values for the 6,606 unique genes assayed with the HuGeneFL array. The intensity values were scaled and averaged over each of three groups: AML+8, AML-CN, and CD34⁺ cells. (B) Dendrogram from two-way hierarchical cluster analysis of 1,959 genes passing a variation filter generated by using the programs cluster and TreeView (19). See also Fig. 5, which is published as supplemental data on the PNAS web site, www.pnas.org.

Figure 2

(A) Genes distinguishing AML and CD34⁺ samples. Expression profiles for the 30 most up-regulated (left, AML↑, from highest to lower) and down-regulated (right, AML↓, from lowest to higher) genes between AML and CD34⁺ samples. Normalized intensities are presented for each gene as standard deviations of log intensity above the mean (red) and below the mean (blue) across samples. Similarly generated data for genes distinguishing AML+8 and AML-CN are shown in Fig. 6, which is published as supplemental data on the PNAS web site at www.pnas.org. (B) Coordinate plot obtained by averaging the unit normal deviates for genes in A that were down-regulated (coordinate 1) and up-regulated (coordinate 2) in AML vs. CD34⁺. Cross-validation revealed perfect class prediction based on this simple rule. (C) The corresponding analyses for the most significantly dysregulated genes in AML+8 vs. AML-CN. Genes are identified by their GenBank accession number and symbol. Preliminary symbols are indicated in parentheses. Genes on chromosome 8 are highlighted (red).

Figure 3

(Left) AML+8 samples exhibit higher expression for genes on chromosome 8 than do AML-CN samples. The average expression levels for genes by chromosome in AML+8 relative to AML-CN are shown. (Right) The increased expression of genes on chromosome 8 as a function of chromosomal location. Of the 169 genes with a specific localization on the integrated radiation hybrid map of chromosome 8, 42 were expressed in a majority of AML samples. For these, the intensities in the AML+8 group are expressed as a percentage of the intensities in the AML-CN group. For comparison, the results for chromosome 1, which shows no gene dosage effect, are presented in the Inset.

Figure 4

Systematic functional analysis of AML samples based on SWISS-PROT database functional annotations for expressed genes. z statistics were calculated for each gene to describe the expression difference in that gene across sample groups. The plots show the cumulative distribution for the ranks of the genes in the functional category relative to all expressed genes. The tick marks at the top of the plots show the ranks of the individual genes; only those showing significant dysregulation (z scores below −1 or above +1) are indicated. (A) Twenty-three expressed genes involved in cell adhesion showed a shift to the right indicating up-regulation of genes in the AML compared with CD34⁺ samples (P = 0.010). (B) Twenty-three genes involved in apoptosis showed a shift to the left indicating down-regulation in AML+8 compared with AML-CN (P < 0.001).