Anchored Multiplex PCR enables sensitive detection and internal verification of novel gene fusions

By Laura Griffin, PhD on Wed Apr 6, 2016


  • Driver fusion genes frequently detected in thyroid and lung cancers are targetable by tyrosine kinase inhibitors (TKIs) and are highly promiscuous, forming fusions with multiple partners.
  • Current methods to detect gene fusions in clinical samples, such as FISH and IHC, cannot identify fusion partners.
  • Anchored Multiple PCR (AMP™) enriches for both known and unknown gene fusions, enabling sensitive NGS-based detection of novel fusions by breakpoint identification and confirmation by expression imbalance.
  • Our data show that AMP-based NGS detected an EML4-ALK fusion in an NSCLC FFPE sample that was ambiguous by ALK break-apart FISH testing.

Gene fusions in thyroid and lung cancers

Recurrent gene fusions have been implicated as oncogenic drivers in an array of human cancers and are particularly frequent in thyroid and lung cancers (1, 2). Fusions involving ALK, RET and ROS1 are the most commonly detected genetic rearrangements in these cancers. These genes 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). However, these genes are also highly promiscuous, forming fusion genes with multiple partners (i.e. ALK has at least 22 known partners) (5-16). This diverse nature of targetable driver gene fusions in thyroid and lung cancers necessitates methods to detect fusions with both known and unknown partners in clinical sample types.

Gene fusion detection: conventional methods

Conventionally, fluorescence in situ hybridization (FISH) in conjunction with RT-PCR and Sanger sequencing has been used to detect and confirm gene fusions in clinical specimens. However, these methods cannot detect novel fusion genes. Traditional FISH requires prior knowledge of fusion partner genes, and, while break-apart FISH detects fusions with known and unknown partners, it cannot identify these partner genes. RT-PCR and Sanger sequencing also requires prior knowledge of fusion partners for opposing primer design. IHC has recently been recognized as a more sensitive alternative to FISH, however fusion events are only inferred based on resulting expression levels and therefore IHC cannot detect novel fusions (17).

Gene fusion detection: NGS-based methods

The advent of next-generation sequencing (NGS) technologies has enabled the objective identification of novel fusion genes (18). NGS-based whole transcriptome sequencing (RNA-Seq) identifies both known and unknown fusions by breakpoint identification. However, the comprehensive nature of this approach results in low sensitivity in fusion detection, a significant hurdle in detecting gene fusions from low input clinical sample types. Target enrichment strategies greatly enhance detection sensitivity, increasing the read depth over target regions of interest and enabling fusion detection from low input clinical samples. However, amplicon-based target enrichment strategies require prior knowledge of fusion partners for opposing primer design and therefore cannot be used to identify novel fusion genes. Anchored Multiple PCR (AMP™) enriches for both known and unknown fusion genes, enabling sensitive detection of novel fusions by NGS-based breakpoint identification. Archer’s FusionPlex CTL assay uses AMP to enrich for genes relevant to thyroid and lung cancers.

Imbalanced expression within rearranged genes can occur if a portion of the gene is inserted downstream of an alternate promoter. This expression imbalance is particularly frequent in fusion genes associated with thyroid and lung cancers. AMP-based target enrichment uses molecular barcodes that enable the counting of unique molecules across a region to assess differences of expression levels across target genes. Thus, expression imbalance detected using the FusionPlex CTL assay provides internal verification of gene fusions detected by breakpoint identification.

Detection of EML4-ALK by FusionPlex CTL

To test our FusionPlex CTL assay, we obtained an NSCLC FFPE specimen from a collaborator that was ambiguous by ALK break-apart FISH testing, the standard method currently used to detect ALK fusions (19-22). We extracted RNA from this sample, prepared a library using FusionPlex CTL and sequenced the library on an Illumina® MiSeq. We then used Archer Analysis v3.3 to analyze the sequencing data. As shown in the figure below, the FusionPlex CTL assay enabled NGS-based detection of an EML4-ALK fusion. Hundreds of sequencing reads originating from unique input molecules spanned the breakpoint and showed fusion of EML4 exon 12 to ALK exon 20 (top panel). This is consistent with previous reports that EML4-ALK fusions result from fusion of EML4 exons 1-13 to ALK exons 20-29 (9). Here, ALK-specific unidirectional primers used in AMP enabled sensitive and objective identification of EML4 as the fusion partner for ALK. Furthermore, expression imbalance of ALK was observed with a highly expressed region downstream of exon 20 in contrast to a lack of expression upstream of exon 20 (bottom panel). This confirms the ALK rearrangement at exon 20, supporting the fusion call by breakpoint sequence identification. Here, AMP disambiguated an inconclusive ALK break-apart FISH result by EML4-ALK breakpoint identification and ALK expression imbalance.

Detection of an EML4-ALK fusion by breakpoint sequence identification confirmed internally by expression imbalance of ALK using the Archer FusionPlex CTL Kit.
Detection of an EML4-ALK fusion by breakpoint sequence identification confirmed internally by expression imbalance of ALK using the Archer FusionPlex CTL Kit. Top: Read statistics and visualization of the fusion EML4 exon 12 and ALK exon 20 in Archer Analysis and sequencing reads spanning the breakpoint of EML4-ALK. The EML4-ALK fusion was called with 1,281 unique supporting reads. Bottom: Expression imbalance was observed with a highly expressed region of ALK downstream of Exon 20 (red) in contrast to lack of expression upstream of Exon 20 (black). Expression difference: p=0.01.


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About Laura Griffin, PhD

Laura 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.

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