An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples

<div><p>Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent <i>in situ</i> hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.</p></div

EIF4E3-FOXP1 fusion transcript.

<p>(A) Structure of the fusion transcript based on UCSC genome browser tracks. Red arrow indicates the in frame fusion of EIF4E3 exon7 to FOXP1 exon 4. The Sanger sequencing trace below indicates the sequence of the fusion point, while the blue arrow over the sanger sequence trace indicates the 3’ end of the targeting probe. (B) RT-PCR result using PCR primers indicated described in S2 for detecting this fusion transcript. Lane 1: RT-PCR product showing the correct 209bp size, Lane 2: 50bp ladder.</p

Description of the Ovation Target Enrichment System.

<p>(A) Experimental steps of the assay and time required for each step. Adaptors (green) are ligated on to generated double stranded cDNA (ds-cDNA). Probes (shown in red and yellow) are hybridized to target cDNA and extended with a polymerase (dashed grey lines). All probes have common tail sequences (blue), which are used as priming sites along with adaptor sequences in subsequent library amplification PCR steps. (B) Example of probe positioning across different exons in a full length double stranded cDNA. Each exon (demarked by blue vertical lines) will have probes (green arrows; arrow points in the 3’ direction) designed to hybridize near the predicted exon-exon junctions. Exons larger than 300 nucleotides (nt) may have additional probes tiled along the length of the exon to obtain more complete sequence coverage. Probes are designed against both strands of the cDNA to enable identification of gene fusions when only one of the pair of genes is targeted. Translation start sequence (ATG) and poly A tail are labeled.</p

HCC1937 Breast ductal carcinoma RNA.

<p>(A) FFPE RNA Bioanalyzer trace. (B) Bioanalyzer trace of sequencing library derived from 100 ng of RNA input shown in A. (C) Sequencing metrics for targeted RNA show that FFPE RNA is efficiently targeted.</p

Ovation Fusion Panel Target Enrichment System identifies known and novel gene fusion events in Universal Human Reference RNA.

<p>The number of sequencing reads determined to be derived from gene fusions in two different targeted sequencing libraries (blue and green) compared to the events identified in a standard, untargeted RNA-Seq library. The untargeted library (red) consists of 125 million total sequencing reads while the targeted libraries consist of 1.6 and 8.7 million sequencing reads.</p

Decreased cardiomyocyte proliferation in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts.

<p>Microtome sections of paraffin-embedded hearts were prepared from <i>Nkx2.5-Cre/Foxm1^fl/fl</i> and control mice at E14.5 (A–D, J–K), E17.5 (E–F) and P11 (G–H) and stained for proliferation markers phospho-histone 3 (PH3) (A–B, E–H, J–K) and Ki-67 (C–D). The percent of PH3-positive cardiomyocytes was significantly lower in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts compared to control at E14.5 and E17.5, though proliferation was unaltered in the lung and in endocardial cells (I). Proliferating cardiomyocytes were more prevalent in control (J) than in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts (K) as evidenced by colocalization of PH3-positive nuclei with α-actinin. Consistent with decreased proliferation in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts, there was decreased mRNA expression of the cell cycle regulators Cdc25B, Cyclin B₁, Plk-1 and nMyc as determined by qRT-PCR (M). There was no change in the expression of Cyclin D₁ or cMyc. Also consistent with decreased proliferation, there was increased mRNA expression of the cell cycle inhibitor p21^cip1. Gene expression was determined using whole heart RNA and normalized to β-actin mRNA. Decreased gene expression of Foxm1, Cyclin B₁, and increased p21^cip1 expression translated to changes in protein levels as demonstrated by Western blot analysis (L). β-actin was used as a loading control. Significant differences (p<0.05) were indicated by asterisk. “N” values were represented by boxes inside bars. Scale bars represent 50 µm in A–H and J–K and 10 µm in insets E–H.</p

Cardiac morphologic parameters.

<p>Hearts from <i>Nkx2.5-Cre/Foxm1^fl/fl</i> embryos had significantly decreased myocardial thickness compared to littermate control mice at E14.5 and E17.5. Five sections from 3 <i>Foxm1^fl/fl</i> and <i>Nkx2.5-Cre/Foxm1^fl/fl</i> embryos were measured to calculate mean±SEM. Significant differences (p<0.05) shown with asterisks. Abbreviations: RV, right ventricle; LV, left ventricle; IVS, interventricular septum.</p

Breeding table Nkx2.5-Cre/Foxm1^fl/+ X Foxm1^fl/fl.

<p>Cumulative breeding data between <i>Nkx2.5-Cre/Foxm1^fl/+</i> and <i>Foxm1^fl/fl</i> mice shows significant lethality in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> embryos by day 17.5 (E17.5). 47% of <i>Nkx2.5-Cre/Foxm1^fl/fl</i> embryos died by E17.5 and 94% lethality was observed in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> embryos at birth.</p

Generation of <i>Nkx2.5-Cre/Foxm1^fl/fl</i> mice and efficiency of Foxm1 deletion.

<p>The Cre-LoxP system was utilized to generate a mouse line in which Foxm1 was selectively deleted from myocardial cells (A). <i>Foxm1^fl/fl</i> mice were crossed with mice expressing <i>Nkx2.5-Cre</i> to generate a mouse line in which Foxm1 is truncated in cardiomyocytes early in embryonic development. Microtome sections of paraffin-embedded hearts were prepared from <i>Nkx2.5-Cre/Foxm1^fl/fl</i> and control (<i>Foxm1^fl/fl</i>) mice at E14.5 (B–C, J–O), E17.5 (D–E) and P11 (F–G) and stained with Foxm1 antibodies. Foxm1 was observed in myocardial (arrows) and endocardial (arrowheads) cells. Quantification of the percentage of Foxm1-positive nuclei showed that cardiomyocytes positive for Foxm1 were significantly decreased in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts compared to control at E14.5 and E17.5 (H). Similarly, Foxm1 mRNA was decreased in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts at E14.5 and E17.5 as determined by qRT-PCR (I). Foxm1 expression was normalized to β-actin mRNA. Foxm1 was more readily detectable in cardiomyocytes from control (J–K, N) than <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts (L–M, O) as evidenced by colocalization of Foxm1-positive nuclei with α-actinin (N–O). However, Foxm1-positive endocardial cells could be detected in control (K) and <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts (M). Significant differences (p<0.05) were indicated by asterisk. “N” values were represented by boxes inside bars. Scale bars represent 200 µm in B–G and 50 µm in J–O.</p

Decreased capillary density and cardiac fibrosis in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> heart.

<p>Microtome sections of paraffin-embedded hearts were prepared from either <i>Nkx2.5-Cre/Foxm1^fl/fl</i> or littermate control mice at P11 and stained for PECAM-1 (A–B), α-smooth muscle actin (αSM) (C–D) or Masson's Trichrome (E–H). Capillary density was decreased in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts at P11 (A–B, I), while coronary vessel formation was not altered (C–D). Numbers of capillaries were counted in PECAM-stained hearts using 10 random sections and mean±SEM was determined to confirm decreased capillary density (I). Significant fibrosis was detected in the IVS and ventricular walls of <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts (F, H) while none was detected in control hearts (E, G). Significant differences (p<0.05) were indicated by asterisk. Scale bars represent 100 µm in A–F and 50 µm in G–H.</p