Detection of a novel RET gene fusion in a non-small cell lung cancer patient using AMP chemistry

Abstract

In lung cancer, several driver Receptor Tyrosine Kinases (RTKs) have been identified in gene fusions, including ALK, RET, ROS1, NTRK1, NRG1 and FGFR1/2/3. These oncogenic gene fusions have become either direct targets or biomarkers for molecular therapies for patients with lung cancer. Screening patients for the presence of these gene fusions would be critical for oncologists to select appropriate therapeutic intervention or for drug developers to design novel therapeutics and conduct clinical trials. However, detection of fusions, especially unknown fusions, from FFPE samples is challenging for many diagnostic techniques. Archer™ FusionPlex™ Lung Thyroid Panel is built on Anchored Multiplex PCR (AMP™) Chemistry, allowing detection of both novel and known fusions. Here we report the results from our study using the Archer Lung-Thyroid Panel. We identified and verified the presence of a novel fusion between Myosin Phosphatase Rho Interacting Protein (MPRIP) and RET oncogene, in an FFPE sample from a non-small cell lung cancer (NSCLC) patient. FFPE samples from 20 NSCLC patients were analyzed in the study. An RNA sample containing an ALK and ROS1 fusion was used as the positive control, and FFPE processed GM12878 was used as the negative control. In addition, FFPE controls containing TMP3-NTRK1 and CCDC6-RET were also used to evaluate the performance of the panel to detect fusions. Total nucleic acid (TNA) was extracted from the FFPE sections using the Agencourt™ FORMAPURE™ kit. For each sample, 200 ng of TNA was used for library preparation. The barcoded libraries from 14 samples were pooled and sequenced on an Illumina MiSeq using a v2 flow cell. The FASTA files from the MiSeq were then analyzed by the ArcherDX data analysis pipeline to determine the presence of gene fusions with driver genes including ALK, FGFR3, MET, NTRK1, NTRK3, NTRK3, PPARG, RET, and ROS1. The detected fusions in clinical samples were further confirmed by Sanger sequencing and FISH. The known fusions, including EM4-ALK, SLC34A2-ROS1, TMP3-NTRK1, and CCDC6-RET were successfully detected in the positive RNA and FFPE control samples. For all of the clinical FFPE samples, one 10 micron section was sufficient to yield more than the required 200 ng TNA for library preparation. Although three of the twenty (15%) FFPE samples failed the initial sample quality metrics, libraries were prepared for all 20 NCSLC FFPE samples and sequenced on MiSeq. Among the three samples that did not pass the initial sample QC, two of them also failed the QC metrics for fusion detection. From the seventeen samples that did pass the initial QC, one sample failed the fusion QC. Overall, the success rate for the clinical samples tested with this panel was 85%. Among the 20 NSCLC samples, one sample was found to harbor a novel, in-frame fusion between exon 19 of MPRIP and exon 12 of RET. This type of fusion would retain an intact kinase domain (exons 12-18) of RET, and would presumably be oncogenic. Using the RNA portion of the TNA extracted, cDNA was synthesized using an independent method, and sequenced by Sanger. The results confirmed the presence of an RNA transcript spanning exon 19 of MPRIP and exon 12 of RET. Furthermore, FISH was performed with the RET (10q11.2) break-apart probe, and confirmed the rearrangement involving the RET region of chromosome 10. These results show the Archer™ FusionPlex™ Lung Thyroid Panel can accurately detect gene fusions with both known and unknown partner and driver genes. Further investigation is needed to better understand the clinical significance and prevalence of the novel MPRIP and RET fusion.