Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data

Abstract

Cancer gene fusions that encode a chimeric protein are often characterized by an intragenic discontinuity in the RNA\expression levels of the exons that are 5' or 3' to the fusion point in one or both of the fusion partners due to differences in the levels of activation of their respective promoters. Based on this, we developed an unbiased, genome-wide bioinformatic screen for gene fusions using Affymetrix Exon array expression data. Using a training set of 46 samples with different known gene fusions, we developed a data analysis pipeline, the "Fusion Score (FS) model", to score and rank genes for intragenic changes in expression. In a separate discovery set of 41 tumor samples with possible unknown gene fusions, the FS model generated a list of 552 candidate genes. The transcription factor gene NCOA2 was one of the candidates identified in a mesenchymal chondrosarcoma. A novel HEY1-NCOA2 fusion was identified by 5' RACE, representing an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. RT-PCR or FISH evidence of this HEY1-NCOA2 fusion was present in all additional mesenchymal chondrosarcomas tested with a definitive histologic diagnosis and adequate material for analysis (n = 9) but was absent in 15 samples of other subtypes of chondrosarcomas. We also identified a NUP107-LGR5 fusion in a dedifferentiated liposarcoma but analysis of 17 additional samples did not confirm it as a recurrent event in this sarcoma type. The novel HEY1-NCOA2 fusion appears to be the defining and diagnostic gene fusion in mesenchymal chondrosarcomas.

Copyright © 2011 Wiley-Liss, Inc.

Figures

Figure 1

Representative exon expression plots of samples with known fusions for validation of the data analysis pipeline. To visualize the exon level expression data, the normalized relative expression level of each exon (Y axis) was plotted along exons of a given gene from 5′ to 3′ (X axis). Red lines indicated possible exon clusters of the given gene, and solid black triangle indicated each corresponding probe set. Four plots on the left were FLI1 exon expression plots in four different samples with EWSR1-FLI1 fusion. Tumor cell line SK-PN-DW (upper left) and patient sample ES2 (lower left) had EWSR1-FLI1 type I fusion (fusion at FLI1 exon 6); Patient samples ES4 (upper middle) and ES6 (lower middle) had EWSR1-FLI1 type II fusion (fusion at FLI1 exon 5). Two plots on the right were WT1 exon expression plots in two different samples with EWSR1-WT1 fusion. Tumor cell line JN-DSRCT (upper right) and patient sample DS2 (lower right) both show evidence of fusion at WT1 exon 8.

Figure 2

Exon expression plot of NCOA2 in the mesenchymal chondrosarcoma sample UF5, showing the intragenic change in NCOA2 expression. The change point was mapped between exon 12 and exon 13. This exon expression pattern was not seen in any other tumor samples or in normal control tissues.

Figure 3

Identification of the novel HEY1-NCOA2 fusion in the mesenchymal chondrosarcoma case UF5. A, Upper: partial schema of HEY1 and NCOA2 gene structures in relation to the formation of HEY1-NCOA2 fusion transcript; Lower: Partial sequence of the HEY1-NCOA2 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. B, RT-PCR detection of HEY1-NCOA2 fusion. The presence of HEY1-NCOA2 fusion transcript was confirmed by RT-PCR using two independent sets of HEY1 forward and NCOA2 reverse primers, HEY1-F1/NCOA2-R3 and HEY1-F2/NCOA2-R2. Sequencing of bands a and b confirmed the HEY1-NCOA2 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA). C, Schematic diagrams of HEY1, NCOA2 and the predicted HEY1-NCOA2 chimeric proteins as well as mapping of NCOA2 breakpoints of four different fusions in protein level. Symbols and abbreviations for the diagram of HEY1: blue bar stands for the basic motif; HLH, helix-loop-helix domain. Symbols and abbreviations for the diagram of NCOA2: bHLH, basic helix-loop-helix domain; PAS, Per/ARN/Sim homologous domain; NID, nuclear receptor interaction domain; AD1/CID, transcriptional activation domain 1/CBP/p300 interaction domain; AD2, transcriptional activation domain 2; Q, glutamine rich region; red bars stand for LXXLL motifs; orange bar indicates the LLXXLXXXL motif.

Figure 3

Identification of the novel HEY1-NCOA2 fusion in the mesenchymal chondrosarcoma case UF5. A, Upper: partial schema of HEY1 and NCOA2 gene structures in relation to the formation of HEY1-NCOA2 fusion transcript; Lower: Partial sequence of the HEY1-NCOA2 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. B, RT-PCR detection of HEY1-NCOA2 fusion. The presence of HEY1-NCOA2 fusion transcript was confirmed by RT-PCR using two independent sets of HEY1 forward and NCOA2 reverse primers, HEY1-F1/NCOA2-R3 and HEY1-F2/NCOA2-R2. Sequencing of bands a and b confirmed the HEY1-NCOA2 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA). C, Schematic diagrams of HEY1, NCOA2 and the predicted HEY1-NCOA2 chimeric proteins as well as mapping of NCOA2 breakpoints of four different fusions in protein level. Symbols and abbreviations for the diagram of HEY1: blue bar stands for the basic motif; HLH, helix-loop-helix domain. Symbols and abbreviations for the diagram of NCOA2: bHLH, basic helix-loop-helix domain; PAS, Per/ARN/Sim homologous domain; NID, nuclear receptor interaction domain; AD1/CID, transcriptional activation domain 1/CBP/p300 interaction domain; AD2, transcriptional activation domain 2; Q, glutamine rich region; red bars stand for LXXLL motifs; orange bar indicates the LLXXLXXXL motif.

Figure 3

Identification of the novel HEY1-NCOA2 fusion in the mesenchymal chondrosarcoma case UF5. A, Upper: partial schema of HEY1 and NCOA2 gene structures in relation to the formation of HEY1-NCOA2 fusion transcript; Lower: Partial sequence of the HEY1-NCOA2 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. B, RT-PCR detection of HEY1-NCOA2 fusion. The presence of HEY1-NCOA2 fusion transcript was confirmed by RT-PCR using two independent sets of HEY1 forward and NCOA2 reverse primers, HEY1-F1/NCOA2-R3 and HEY1-F2/NCOA2-R2. Sequencing of bands a and b confirmed the HEY1-NCOA2 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA). C, Schematic diagrams of HEY1, NCOA2 and the predicted HEY1-NCOA2 chimeric proteins as well as mapping of NCOA2 breakpoints of four different fusions in protein level. Symbols and abbreviations for the diagram of HEY1: blue bar stands for the basic motif; HLH, helix-loop-helix domain. Symbols and abbreviations for the diagram of NCOA2: bHLH, basic helix-loop-helix domain; PAS, Per/ARN/Sim homologous domain; NID, nuclear receptor interaction domain; AD1/CID, transcriptional activation domain 1/CBP/p300 interaction domain; AD2, transcriptional activation domain 2; Q, glutamine rich region; red bars stand for LXXLL motifs; orange bar indicates the LLXXLXXXL motif.

Figure 4

RT-PCR detection of the HEY1-NCOA2 fusion in additional mesenchymal chondrosarcoma cases. Upper panel: RT-PCR performed using HEY1 forward primer HEY1-F1 and NCOA2 reverse primer NCOA2-R3. Lower panel: RT-PCR for PGK transcript. MSK_1 to MSK_10 were 10 FFPE samples and L1~L3 were frozen samples. The identical HEY1-NCOA2 fusion transcript was detected in cases MSK_3 (weak positive due to the very poor quality of RNA), MSK_5, MSK_6, L1 and L3. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA).

Figure 5

Interphase FISH detection of HEY1-NCOA2 fusion in mesenchymal chondrosarcomas. A, the schema of FISH-probe design. B, representative HEY1-NCOA2 dual-color FISH pictures. Signal pattern in normal cells was shown in the left panel. In contrast, fused signals (visible as one yellow dot, instead of green dot/red dot side by side) were seen in cells of RT-PCR positive cases MSK_3 (middle) and MSK_6 (right).

Figure 5

Interphase FISH detection of HEY1-NCOA2 fusion in mesenchymal chondrosarcomas. A, the schema of FISH-probe design. B, representative HEY1-NCOA2 dual-color FISH pictures. Signal pattern in normal cells was shown in the left panel. In contrast, fused signals (visible as one yellow dot, instead of green dot/red dot side by side) were seen in cells of RT-PCR positive cases MSK_3 (middle) and MSK_6 (right).

Figure 6

Morphology of four HEY1-NCOA2 positive mesenchymal chondrosarcoma cases showing richly cellular fields of small round or slightly spindled cells and islands of chondroid matrix.

Figure 7

Identification of the novel NUP107-LGR5 fusion in a dedifferentiated liposarcoma case (UF2). A, Exon expression plot of LGR5 in sample UF2, showing the intragenic change in LGR5 expression. The change point was mapped between exon 11 and exon 12. This exon expression pattern was not seen in any other tumor samples or in normal control tissues. B, Upper: The schema of NUP107 and LGR5 partial gene structures as well as the formation of NUP107-LGR5 fusion transcript; Lower: Partial sequence of the NUP107-LGR5 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of NUP107 exon 1 to LGR5 exon 12. C, RT-PCR detection of NUP107-LGR5 fusion. The presence of NUP107-LGR5 fusion transcript was verified by RT-PCR using NUP107 forward and LGR5 reverse primers (NUP107-E1-F, LGR5-E13-RV). Sequencing of the RT-PCR product confirmed the NUP107-LGR5 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA).

Figure 7

Identification of the novel NUP107-LGR5 fusion in a dedifferentiated liposarcoma case (UF2). A, Exon expression plot of LGR5 in sample UF2, showing the intragenic change in LGR5 expression. The change point was mapped between exon 11 and exon 12. This exon expression pattern was not seen in any other tumor samples or in normal control tissues. B, Upper: The schema of NUP107 and LGR5 partial gene structures as well as the formation of NUP107-LGR5 fusion transcript; Lower: Partial sequence of the NUP107-LGR5 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of NUP107 exon 1 to LGR5 exon 12. C, RT-PCR detection of NUP107-LGR5 fusion. The presence of NUP107-LGR5 fusion transcript was verified by RT-PCR using NUP107 forward and LGR5 reverse primers (NUP107-E1-F, LGR5-E13-RV). Sequencing of the RT-PCR product confirmed the NUP107-LGR5 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA).

Figure 7

Identification of the novel NUP107-LGR5 fusion in a dedifferentiated liposarcoma case (UF2). A, Exon expression plot of LGR5 in sample UF2, showing the intragenic change in LGR5 expression. The change point was mapped between exon 11 and exon 12. This exon expression pattern was not seen in any other tumor samples or in normal control tissues. B, Upper: The schema of NUP107 and LGR5 partial gene structures as well as the formation of NUP107-LGR5 fusion transcript; Lower: Partial sequence of the NUP107-LGR5 fusion transcript along with predicted amino acid sequence. The transcript is an in-frame fusion of NUP107 exon 1 to LGR5 exon 12. C, RT-PCR detection of NUP107-LGR5 fusion. The presence of NUP107-LGR5 fusion transcript was verified by RT-PCR using NUP107 forward and LGR5 reverse primers (NUP107-E1-F, LGR5-E13-RV). Sequencing of the RT-PCR product confirmed the NUP107-LGR5 in-frame fusion transcript. Size marker is 100bp DNA ladder (Invitrogen, Carlsbad, CA).