e-Poster Display Session (ID 87) Poster Display

99P - Development of a qRT-PCR-based diagnostic test to identify colorectal cancer patients with recurrent R-Spondin gene fusions (ID 245)

Presentation Number
Lecture Time
09:00 - 09:00
  • Veronica Diermayr (Singapore, Singapore)
On-Demand e-Poster Display, Virtual Meeting, Virtual Meeting, Singapore
09:00 - 20:00



In colorectal cancers (CRC) 4 different recurrent gene fusions involving R-Spondin (RSPO)2/3 and different exons of PTPRK or EIF3E are found with around 4% incidence. These fusions (Fusion A-D) occur exclusively in a sub-population of CRC with high unmet medical need that is microsatellite-stable (MSS) and mutant for KRAS, NRAS or BRAF but wild-type (WT) for APC. RSPO-fusions are known driver mutations, increasing Wnt signalling and sensitizing for treatment with upstream Wnt pathway inhibitors. Detection of the fusions is possible using Archer FusionPlex technology, but with low sensitivity. A multiplex reverse transcription, real-time PCR-based test, specific for the detection of recurrent RSPO fusions, was developed as a Clinical Trial Assay (CTA).


The CTA uses RNA extracted from FFPE or frozen tumor tissue in a qRT-PCR, simultaneously detecting the RSPO fusion, the WT fusion partner(s), and an internal control. The assays span exon-exon junctions to increase selectivity and amplify fragments of 80-140 bp in length to allow for detection in RNA extracted from FFPE tissue. A ΔCt is calculated by subtracting the Ct of the WT reaction (PTPRK or EIF3E) from the RSPO fusion Ct. Values below or equal to the set cut-off permit qualitative calling of samples positive for the fusions.


180 fresh frozen and 118 FFPE samples from CRC patients were used in the analytical validation for the 4 CTAs. All assays show high precision (100%) at the limit of detection (LoD) and high specificity in terms of residual EtOH (≤4%) or EDTA (<5 mM). Sensitivity was high with the LoD for Fusions A-D between 1.46-5.10% for frozen tissue and 2.82%-21.6% for FFPE, respectively. The assays also showed high accuracy and 100% concordance with Sanger sequencing for fusion positive samples (with a ΔCt cut-off of ≤13-15 and ≤5-7.5 for frozen and FFPE tissue, respectively).


The RSPO Fusion CTA is a quick and cost-effective test with high sensitivity (RNA input of 5 or 25 ng for frozen and FFPE tissue respectively). This CTA is currently used to pre-screen & select patients for the phase IB trial with the porcupine inhibitor ETC-1922159 (NCT02521844) ongoing in Singapore and USA.

Clinical trial identification


Legal entity responsible for the study

Experimental Drug Development Centre, A*STAR.


BMRC, NRF and NMRC Singapore.


V. Diermayr: Research grant/Funding (institution), Full/Part-time employment: A*STAR. S. Sarma, K.F.Y. Lee, B.H. Gan: Full/Part-time employment: A*STAR. M. Inoue: Licensing/Royalties, Full/Part-time employment: A*STAR. All other authors have declared no conflicts of interest.