Ewing tumors that do not overexpress BMI-1 are a distinct molecular subclass with variant biology: a report from the Children's Oncology Group

Abstract

Purpose: Ewing sarcoma family tumors (ESFT) are aggressive tumors of putative stem cell origin for which prognostic biomarkers and novel treatments are needed. In several human cancers, high expression of the polycomb protein BMI-1 is associated with poor outcome. We have assessed the potential clinical significance of BMI-1 expression level in ESFT.

Experimental design: BMI-1 expression was assessed in 130 tumors by immunostaining and associations with clinical features and outcome determined. The molecular signatures of BMI-1-low and BMI-1-high tumors were compared using microarrays and differentially activated canonical pathways identified by gene-specific enrichment analysis. Automated quantitative analysis of phosphoproteins was used to assess relative levels of pathway activation. Sensitivity to IGF1-R inhibition was determined using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays.

Results: BMI-1 is overexpressed by the vast majority of ESFTs. However, in 20% of cases, BMI-1 levels are low to undetectable. Significantly, although clinical presentation and outcome were similar between BMI-1-high and BMI-1-low tumors, whole genome expression array analysis showed marked differences in their respective gene expression profiles. Gene-specific enrichment analysis identified that several cancer-associated canonical biological pathways, including IGF1, mTOR, and WNT, are significantly downregulated in BMI-1-low compared with BMI-1-high tumors. Consistent with these in vivo data, the response to IGF1-R inhibition in vitro was diminished in BMI-1-low compared with BMI-1-high ESFT cells.

Conclusion: ESFT that do not overexpress BMI-1 represent a novel subclass with a distinct molecular profile and altered activation of and dependence on cancer-associated biological pathways.

Figure 1

BMI-1 expression in ESFT. Most tumors robustly and diffusely express BMI-1 (A & B: BMI-1-high). In contrast, in nearly 20% of tumors BMI-1 staining is absent or minimal (C & D: BMI-1-low). BMI-1 negative cells in (B) are non-tumor stromal cells (see corresponding H&E section).

Figure 2

Kaplan-Meier survival analysis of 72 patients with localized disease shows no significant association of BMI-1 expression with outcome. A. Event free survival (EFS) and B. Overall survival (OS).

Figure 3

Gene expression profiling studies demonstrate distinct molecular signatures of BMI-1-low tumors. A. BMI-1 raw signal intensities from 5 BMI-1-low and 5 BMI-1-high primary tumors (arbitrary units). B. Principal components analysis of 10 tumors using all core transcripts segregates BMI-1-low and BMI-1-high tumors into two distinct clusters. Cluster ellipses encompass 2 standard deviations. C. BMI-1-low tumors display down-regulation of genes in the IGF1 pathway relative to BMI-1-high tumors. The fold change in median signal intensity for each gene was calculated for BMI-1-high and BMI-1-low tumors relative to median expression in all 10 tumors. Error bars represent SEM for 5 tumors. D. AQUA analysis reveals variable expression of BMI-1 protein in an independent cohort of primary ESFT. Comparison between the lowest BMI-1 expressors (9 tumors, bottom 15% of 58 cases) and the highest BMI-1 expressors (29 tumors, top 50%) demonstrates that phospho-IGF1R, phospho-mTOR and total mTOR levels are significantly reduced in BMI-1-low tumors.

Figure 4

A. Hierarchical clustering of 19 ESFT cell lines and 10 tumors identifies TC-248 as a BMI-1-low cell line. B. Western blot confirms low expression of BMI-1 protein in TC-248 compared to 2 BMI-1-high cell lines. C. RT-PCR confirms expression of a type 1 EWS-FLI1 fusion in TC-248 cells. D. Treatment with an IGF1-R-specific small molecule inhibitor picropodophyllin (PPP) results in growth inhibition of BMI-1-high cells after 72 hours. In contrast, TC-248 cells are significantly less sensitive to PPP (p<0.0001 compared to each of 4 BMI-1-high cell lines at 500 nM dose). E. Growth of TC-248 cells is not significantly inhibited by exposure to the anti-IGF1-R-targeted antibody IMC-A12.