Aiming at the Achilles’ heel of cancer: multiplexed lung cancer fusion detection without FISH or NGS

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Lung cancer is the most commonly diagnosed cancer worldwide, and the leading cause of cancer deaths in the United States. It is generally divided into two types, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), based on the size of the affected cells when viewed under a microscope. NSCLC accounts for 85 percent of lung cancer, while SCLC accounts for the remaining 15 percent. Treatment for these patients has historically consisted of systemic cytotoxic chemotherapy.

Since the early 2000s there has been great progress in the identification of mutational driver genes in lung cancer. Driver mutations within a gene confer a selective growth advantage, resulting in cancer initiation and/or progression. The good news is that they are also the “Achilles’ heel” of tumors:  inhibiting the driver gene could suppress cancer progression. These discoveries have led to a greater understanding of the molecular circuitries driving cancer cells’ uncontrolled replication and to the development of drugs that target pivotal pathways, with little or no damage to normal cells — an idea that only a few years ago was considered the holy grail of cancer treatment. This also means that accurate and timely identification of these markers has now become paramount.

Classically, in NSCLC the detection of anaplastic lymphoma kinase (ALK) gene fusion events are detected by FISH at the DNA level or by IHC at the protein level, but these two methods are time consuming and may still produce discordant results as FISH is often subject to interpretation.

In the case of detecting ALK fusions, for example, IHC is more accurate than FISH since the wild type ALK protein is rarely expressed while the fusion protein is. However, protein detection alone may not be enough for diagnosis or for targeted therapy because of the high percentage of equivocal IHC results and the lack of quantitative scoring.  In addition, protein detection does not allow for differentiation between fusion variants because detection with IHC does not measure activity.

There are at least 15 different well-characterized variants of the ALK-EML4 gene fusion, but there is emerging evidence that expression of particular variants directly impacts the response of patients to ALK inhibitors; some inhibitors are more effective towards a particular ALK fusion. Therefore, it is becoming essential to develop assays that easily and specifically detect multiple gene fusions in a short amount of time and at a low cost, requirements that are fulfilled only in part by NGS.

In this regard, Dr. Leon Van Kempen at the University Medical Center Groningen in The Netherlands recently spoke with us about his work to develop a lung cancer fusion assay that delivers results faster and at a lower cost per target than FISH or NGS. Dr. Van Kempen is a clinical specialist in laboratory medicine who is currently studying novel tumor suppressor genes to identify therapeutic targets applicable to a wide variety of cancers. He set out to develop a sensitive, low cost and accurate lung fusion assay using the NanoString nCounter Vantage 3D Lung Fusion Panel.

This panel allows for multiplexed identification of aberrant transcripts using a patented Junction Probe Design which enables the highly specific detection of fusion junction sequences in a background of abundant normal tissue. The Lung Fusion Panel also makes use of a 3’/5’ imbalance probe strategy that uses the ratio of multiple probes detecting driver gene expression upstream and downstream of a purported fusion junction to detect fusions with unknown partners.

Samples that contain the wild-type gene are expected to have a ratio of 3’ to 5’ probes close to 1, whereas samples that contain a fusion are often expected to have a ratio different from 1 as the 3’ end of the driver gene may be fused with the 5’ end of a partner gene. This strategy can be used to confirm fusions detected with probes for specific junctions as well as to detect fusions with unknown partners.  nCounter Vantage 3D Fusion panels can be customized with additional spike-in probes (either using the junction probe design or an imbalanced probe design) to detect fusions not included in the panel.

Dr. Van Kempen and his team designed and spiked in their own fusion probes to detect the ALK, ROS1, RET, TRK, and NRG fusion variants plus the MET-14 exon skipping fusion gene. Their efforts paid off and they were able to deliver a fully customizable, single tube, PCR-independent multiplexed assay for FFPE samples requiring a minimal percentage of tumor cells. Watch Dr. Van Kempen’s webinar to learn more about their design strategy, challenges encountered, and solutions adopted.

We hope you enjoy this highly informative webinar as much as we did.

You can find Dr. Leon Van Kempen’s webinar here.


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Sarah R. Sabir et al, Cancers (Basel). 2017 Sep; 9(9): 118

By Laura Tabellini Pierre
For research use only. Not for use in diagnostic procedures.