Continuing our theme this Sarcoma Awareness month, this article explores the challenges of detecting unknown fusions in cancers such as Sarcoma.

Gene fusions in cancer

Cancer progression is characterised by the gradual breakdown of cellular DNA repair mechanisms. On one hand, this leads to the accumulation of small-scale DNA errors that disrupt the function of individual genes. On a larger scale, however, cancerous cells are also more likely to experience chromosomal breakage. Worse still, they are prone to repairing these breaks incorrectly, re-connecting the wrong chromosome ends together.

Gene fusions are created in this way when a gene spans a chromosome breakpoint. If the break is repaired incorrectly, a gene may lose or gain new exons or regulatory sequences. If expressed, the resulting fusion gene may produce a novel protein with altered or entirely new functions, or it may be expressed at different times or levels compared with the original gene. In the context of oncology, gene fusion events can drive cancer progression through the activity of the fusion protein or through the absence of the wildtype protein.

Measuring and understanding these fusion events is important for cancer research and also has practical benefits to patients in the clinic. An increasing number of targeted cancer therapies or drugs are becoming available that are effective against cancers with specific fusions. A significant challenge, however, is matching the right therapy or drug to the right patient, avoiding the use of expensive treatments in patients who lack the pertinent gene fusion and are therefore unlikely to benefit from treatment. Therefore, an important first step is the development of tests to detect these gene fusions.

Challenges of fusion detection

Despite the importance of gene fusions for cancer care, testing for these remains a significant technical challenge. Of the ~20,000 genes spread across the 3.2 billion base pairs of the human genome, any two regions may fuse together at random. The only approach capable of detecting all theoretically possible fusion events is next generation whole-genome/transcriptome sequencing. However, these techniques are expensive, time-consuming and may be difficult to implement in clinical or research settings. Equally, detecting every possible fusion may be unnecessary or overwhelming if the majority are of unknown clinical significance. A more practical option is to develop targeted assays that focus only clinically relevant gene fusions.

Some gene fusions are easier to detect than others. The easiest to detect are “known” fusions, characterised by having two conserved partner genes either side of the mis-repaired breakpoint, in a “one-to-one” relationship. As the sequence of both partners is known, it is easy to design Fluorescence In Situ Hybridisation (FISH) probes or PCR primers for both sides of the breakpoint. Whilst this approach is effective for such fusions, it is not suitable for all situations.

Unknown fusions

Many gene fusions only have one conserved fusion partner, meaning that the identity and sequence of the other partner are not known in advance. A conserved fusion partner may participate in “one-to-many” or “one-to-any” fusion events, and in many cases, all of these possibilities are clinically relevant.

For these unknown fusions, FISH or traditional two-primer PCR is no longer effective. One solution is to use hybridisation-based next generations sequencing, where a single hybridisation probe is anchored within the conserved fusion partner only, enabling the capture of the entire fusion molecule. This is an effective though often slow step, with most commercial assays taking several days to complete due to the 16-hour hybridisation step.

Introducing XCeloSeq Fusion kits

The XCeloSeq range of fusion detection kits are designed to address this detection challenge. They are fast, relying only on PCR polymerase-driven detection of known and unknown fusions in a single working day, without any hybridisation.

Rapid fusion detection is achieved using GeneFirst’s novel NGS library preparation technology, ATOM-Seq. Here, the fusion cDNA molecule is extended using a DNA polymerase to introduce a universal priming site. This creates a DNA molecule with the conserved fusion partner at one end and a universal priming site at the other, each flanking the central unknown fusion partner. PCR-based target enrichment between the two ends of the molecule then allows for detection of unknown fusion partners as part of a NGS sequencing panel. Click here to learn more about how this works.

Several cancer-specific XCeloSeq fusion detection panels are available, including sarcoma or lung cancer. We also offer comprehensive pan-cancer, solid cancer or actionable-target panels. For researchers and organisations seeking to develop new fusion detection tests or to expand existing testing capabilities, GeneFirst offers a bespoke assay development service, working closely with partners to deliver tailored solutions for research and clinical requirements.