- Driver fusion genes in thyroid and lung cancers are targetable by tyrosine kinase inhibitors (TKIs) and are highly promiscuous, forming fusions with multiple partners.
- Expression imbalance can be used to infer the presence of a gene fusion without prior knowledge of the fusion partner.
- However, Carol Beadling and colleagues showed that expression imbalance is less accurate than breakpoint identification to detect gene fusions by amplicon-based NGS.
- Anchored Multiplex PCR (AMP™) enables NGS-based detection of fusion breakpoints and expression imbalance to detect both known and novel gene fusions.
- Here, our data show that breakpoint identification is more sensitive than expression imbalance to detect gene fusions by AMP-based NGS.
Gene fusions in lung and thyroid cancers
Recurrent gene fusions that drive oncogenesis are frequent in thyroid and lung cancers, with RET fusions detected in ~20% of papillary thyroid cancers and ALK, RET and ROS1 fusions detected in ~9% of non-small cell lung cancers (NSCLCs) (1, 2). These proto-oncogenes encode receptor tyrosine kinases and are targetable by tyrosine kinase inhibitors (TKIs) (i.e. crizotinib is clinically approved for ALK fusions in NSCLC) (3, 4). Gene rearrangements involving the receptor tyrosine kinase, ALK, are the most frequently detected rearrangements in NSCLC. ALK rearrangements typically result in fusion to EML4, however other fusion partners have been identified and several different EML4-ALK fusions have been identified to date (5-12). Furthermore, RET and ROS1 genes are highly promiscuous, forming fusion genes with multiple partners (13-15). This diverse nature of targetable gene fusions in thyroid and lung cancers necessitates methods to detect fusions with both known and unknown partners in clinical sample types.
Expression imbalance of fusion genes
Imbalanced expression within rearranged genes can occur due to insertion of a portion of the gene downstream of a different promoter. This expression imbalance is particularly frequent in fusion genes associated with thyroid and lung cancers. In these cancers, fusion genes often exhibit induced expressed of the portion of the gene encoding the tyrosine kinase domain. Expression imbalance can therefore indicate the presence of an expressed gene fusion and can be detected by hybrid capture-based methods or sequencing across transcripts. Since only one gene is interrogated for expression imbalance, these methods can detect gene fusions without prior knowledge of fusion partners, but cannot identify fusion partner genes. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that enriches for both known and unknown fusion genes. Molecular barcodes used in AMP enable the counting of unique molecules across a region to assess differences in expression levels across target genes. Importantly, AMP also enables NGS-based detection of fusion breakpoint sequences and identification of novel fusion genes.
Breakpoint identification vs expression imbalance to detect gene fusions
Data recently obtained from amplicon-based NGS indicate that identification of breakpoint sequences is much more accurate than expression imbalance to detect gene fusions (16). In a recent publication, Carol Beadling and colleagues demonstrated that expression imbalances detected by amplicon-based sequencing exhibited gene-specific variability in sensitivity and specificity (16). In one observation, expression imbalance was not sensitive enough to detect BRAF and ROS1 fusions in known fusion-positive samples. It was also reported that ALK and RET expression imbalances were detected in 11% of known fusion-negative samples. Due to this lack of sensitivity and specificity, the authors of this study concluded that fusions detected solely based on expression imbalance require confirmatory testing.
To determine the relative sensitivities of AMP-enabled expression imbalance and breakpoint identification to detect fusion genes, we extracted RNA from KM12 cells, which are known to express a TPM3-NTRK1 fusion. We then serially diluted the KM12 RNA into RNA extracted from thyroid cells, which do not express NTRK1. Diluting TPM3-NTRK1 positive RNA into RNA that is completely void of endogenous NTRK1 enables the highest possible sensitivity of expression imbalance detection. Next, we prepared libraries from these dilutions with the FusionPlex CTL assay, which uses AMP to enrich for multiple target genes relevant in thyroid and lung cancers including NTRK1. The figure below shows that the sequencing data obtained from these libraries reliably detected the TPM3-NTRK1 fusion down to 1.56% KM12 RNA and detected it in 2 out of 3 replicates in 0.39% KM12 RNA. However, NTRK1 expression imbalance was only detected down to 25% KM12 RNA. These results suggest that AMP-enabled breakpoint identification is more sensitive than AMP-enabled detection of expression imbalance to detect gene fusions. Unlike hybrid capture or amplicon-based methods, AMP-based NGS can detect novel gene fusions by breakpoint identification, with expression imbalance serving as an internal confirmation.
Breakpoint sequence identification enabled by FusionPlex CTL is more sensitive than expression imbalance to detect NTRK1 gene fusions. RNA from KM12 cells was serially diluted into thyroid RNA in triplicate. Libraries were prepared using FusionPlex CTL and 100ng total input was used for sequencing. Top: Raw data from individual replicates of each dilution. Fusions are called based on sequencing reads spanning the fusion breakpoint. Expression imbalance p-values < 0.05 indicate a statistically significant difference in expression levels between the 5’ and 3’ ends of NTRK1 transcripts and suggest the presence of an NTRK1 gene fusion. Bottom: Graphical representation of raw data, showing the average expression imbalance p-value for each dilution and standard error of the mean (SEM). Limits of fusion detection by expression imbalance and breakpoint identification are indicated with red lines.
- Espinosa, A., J. Gilbert, J. Fagin. 2014. RET Fusions in Thyroid Cancer. My Cancer Genome https://www.mycancergenome.org/content/disease/thyroid-cancer/ret/127/ (Updated October 20).
- Lovly, C., L. Horn, W. Pao. 2016. Molecular Profiling of Lung Cancer. My Cancer Genome https://www.mycancergenome.org/content/disease/lung-cancer/ (Updated March 28).
- G. R. Oxnard, A. Binder, P. A. Jänne, New targetable oncogenes in non-small-cell lung cancer. J. Clin. Oncol. 31, 1097–1104 (2013).
- T. Kohno et al., RET fusion gene: translation to personalized lung cancer therapy. Cancer Sci. 104, 1396–1400 (2013).
- K. Rikova et al., Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 131, 1190–1203 (2007).
- Y. L. Choi et al., Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res. 68, 4971–4976 (2008).
- L. Horn, W. Pao, EML4-ALK: honing in on a new target in non-small-cell lung cancer. J. Clin. Oncol. 27, 4232–4235 (2009).
- J. P. Koivunen et al., EML4-ALK Fusion Gene and Efficacy of an ALK Kinase Inhibitor in Lung Cancer. Clin Cancer Res. 14, 4275–4283 (2008).
- M. Soda et al., Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature. 448, 561–566 (2007).
- K. Takeuchi et al., Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 14, 6618–6624 (2008).
- K. Takeuchi et al., KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res. 15, 3143–3149 (2009).
- D. W.-S. Wong et al., The EML4-ALKfusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFRand KRAS. Cancer. 115, 1723–1733 (2009).
- T. Nakaoku et al., Druggable oncogene fusions in invasive mucinous lung adenocarcinoma. Clin Cancer Res. 20, 3087–3093 (2014).
- A. Drilon et al., Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discovery. 3, 630–635 (2013).
- M. E. Lira et al., A single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer. J Mol Diagn. 16, 229–243 (2014).
- C. Beadling et al., A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing. J Mol Diagn. 18, 165–175 (2016).
About Laura Griffin, PhDLaura Griffin earned her PhD in Microbiology from the University of Colorado Denver, Anschutz Medical Campus. Her research focused on cancer virology, dissecting virus-host interactions during Human Papillomavirus infection. Laura is passionate about cancer research as well as effective education and communication of scientific ideas. Laura joined the ArcherDX team as Scientific Editor in January, 2016. In her spare time, Laura is a group fitness instructor and enjoys cycling, hiking, skiing, and snowshoeing in the Rocky Mountains of Colorado.