Antibody detection of translocations in Ewing sarcoma

Abstract

The detection of chromosomal translocations has important implications in the diagnosis, prognosis and treatment of patients with cancer. Current approaches to translocation detection have significant shortcomings, including limited sensitivity and/or specificity, and difficulty in application to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach called antibody detection of translocations (ADOT) that avoids the shortcomings of current techniques. ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor-quality unprocessed total ribonucleic acid (RNA). Furthermore, the technique is readily generalizable to detect any potential fusion transcript, including previously undescribed fusions. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumour xenografts and FFPE primary tumours. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers.

Figure 1. Schematic representation of EWS/FLI isoforms and mechanism of ADOT

1. Ten different EWS/FLI isoforms are represented by fusions between different EWS and FLI exons.

2. The ADOT approach detects hybrids between RNA transcripts from samples and DNA oligonucleotides on the microarray. These RNA–DNA duplexes are recognized by antibody S9.6 which is then detected by PE-labelled secondary antibody.

Figure 2. Proof of principle of ADOT

1. ADOT detects translocation transcripts in RNA from 293 cells overexpressing the EWS/FLI 7/6 fusion. The central heatmap shows signal intensity of fusion probes, while the left and bottom heatmaps show those of EWSR1 and FLI exon (Ex) and splice (Spl) probes, respectively. Signal intensities are shown in colour scale. In this case, high signal intensity at row 7, column 6, indicates a fusion between EWSR1 exon 7 and FLI1 exon 6.

2. Optimization of probe length and signal-to-noise ratio for ADOT. Fusion probes of increasing length as indicated were printed on microarray and hybridized to total RNA from EWS/FLI 7/6 overexpressing 293 cells. Signal-to-noise ratio was then calculated and plotted as a function of probe length.

Figure 3. ADOT detects known (A) and unknown (B) translocations at endogenous levels in patient-derived Ewing sarcoma cells

1. Heatmaps indicate that A673 cells contain EWS/FLI 7/6 translocation (left); RDES cells harbour EWS/FLI 7/5 translocation (middle); TC466 cells contain EWS/ERG 7/8 translocation (right).

2. ST97-894 cells were identified by ADOT to contain an EWS/FLI 10/8 translocation.

3. RT-PCR and sequencing analysis confirming the translocation detected in ST97-894 cells.

Figure 4. Sensitivity of ADOT

Heatmaps were generated by using decreasing amounts (5, 0.5, 0.2 and 0.05 µg) of total RNA from A673 cells to hybridize with microarray.

Figure 5. ADOT detects translocations in frozen (A) or FFPE (C and D) tumour samples

1. A. Heatmaps showing two frozen tumours that contains an EWS/FLI 7/5 and an EWS/ERG 7/8 translocation, respectively.

2. B. RT-PCR and sequencing analysis confirming translocation types in frozen tumours tested in A.

3. C, D. Heatmaps showing EWS/FLI 7/6 translocation in FFPE xenograft (C) and patient tumour (D).