Identification of RET fusions in a Chinese multicancer retrospective analysis by next‐generation sequencing

Abstract Fusion of RET with different partner genes has been detected in papillary thyroid, lung, colorectal, pancreatic, and breast cancer. Approval of selpercatinib for treatment of lung and thyroid cancer with RET gene mutations or fusions calls for studies to explore RET fusion partners and their eligibility for RET‐based targeted therapy. In this study, RET fusion patterns in a large group of Chinese cancer patients covering several cancer types were identified using next‑generation sequencing. A total of 44 fusion patterns were identified in the study cohort with KIF5B, CCDC6, and ERC1 being the most common RET fusion partners. Notably, 17 novel fusions were first reported in this study. Prevalence of functional RET fusions was 1.05% in lung cancer, 6.03% in thyroid cancer, 0.39% in colorectal cancer, and less than 0.1% in gastric cancer and hepatocellular carcinoma. Analysis showed a preference for fusion partners in different tumor types, with KIF5B being the common type in lung cancer, CCDC6 in thyroid cancer, and NCOA4 in colorectal cancer. Co‐occurrence of EGFR mutations and RET fusions with rare partner genes (rather than KIF5B) in lung cancer patients was correlated with epidermal growth factor receptor‐tyrosine kinase inhibitor resistance and could predict response to targeted therapies. Findings from this study provide a guide to clinicians in determining tumors with specific fusion patterns as candidates for RET targeted therapies.


| Patients and samples
Tumor samples (tissues or plasma fractions) obtained from patients between January 2017 and December 2019 were used for NGS RET fusion detection (Genetron Health).

| Papillary thyroid cancer sample sequencing
DNA and RNA extracted from PTC samples were analyzed with the FSZ-Thyroid-V1 NGS Panel using one-step multiplex PCR targeted amplicons as described previously. 45   SureSelect V5 system (Agilent) and the captured samples were subjected to Illumina HiSeq X-Ten for paired end sequencing. Sequencing reads were mapped to a human reference genome (hg19) using Hisat2-2.0.5. 46 Gene fusions were identified using FusionMap. 47

| Statistical analyses
Categorical variables were compared using Pearson's analysis and χ 2 test. Analyses and data presentation were undertaken using GraphPad Prism (8.0.1) and R (version 4.1.1).  (Table 1). In addition to lung cancer and papillary thyroid carcinoma, RET fusions have been found in 0.6%-0.7% of patients with other types of cancer, including breast, colon, esophageal, ovarian, prostate, and stomach cancers. [6][7][8][9][10][11][12][13][14][15][16] Figure 2B,C). Notably, the common partner genes were different in different cancers, implying that the hotspots of chromosome breakpoints in the partner genes are different, which might be associated with difference in sensitivity to RET inhibitors.

| Genomic breakpoints in RET of patients with different cancer types
Fusion-mediated RET activation is induced by different mechanisms, including increased kinase expression due to replacement of the 5′-upstream RET promoter with that of fusion partners, 7,49 and dimerization/oligomerization of the RET kinase domain mediated by a C-terminal domain present in the fusion partners that leads to ligand-independent kinase activation. 40,45,48,50 Breakpoints in RET and its fusion partners mainly occur in the intronic regions, therefore, the ORF is retained after mRNA splicing. A RET in intron 11, the most common breakpoint in LC patients, allowed exon 12 to be retained in the fusion product. In addition, breakpoints in introns 7, 8, 9, and 10 of RET were observed in this study ( Figure 3).
Notably, breakpoints in intron 11 were the most common types in these malignancies, and breakpoints in intron 8, 9, and 10 were also

| mRNA features of cases with novel RET fusion
Of the 17 novel fusions first reported in this study, five cases were sent for RNA sequencing to verify the breakpoint locations and fusion partners at the transcript level (   (Figures 5 and S1G,H).  The carcinogenic mechanisms of RET fused with novel and common partners could be different, which deserves more research and discussion in the future.

| Coexistence of RET fusion with other actionable variations in LC patients
Previous studies reported that driver mutations are commonly mutually exclusive. 60 (Table S3). This finding validates the function of KIF5B-RET fusion protein as a driver mutation in LC ( Figure 6). In addition, it implies that different fusion partners might have different functions during oncogenesis. low sensitivity and specificity. 13,36,37 Reverse transcription-PCR can only detect RET fusions with known fusion partners. 29,38,39 Although FISH is highly sensitive, it requires special technical expertise and it is not effective for identification of fusion partners, which could be critical for determining oncogenicity of fusion products. 38,40 The kinase domain of RET spans from exon 12 to 18. Breakpoints in RET and its fusion partners mainly occur in the intronic regions and can retain the ORF after mRNA splicing.

| D ISCUSS I ON
In this study, breakpoints of 3′-terminal RET fusion in intron 11 were the most common types, and breakpoints in RET introns 7,

D I SCLOS U R E
The authors have no conflict of interest.