It has been documented in our previous study that oncogenic fusions were significantly enriched in dMMR CRCs harboring hypermethylated MLH1 and wild-type BRAF/RAS(10). Herein, we conducted further study using integrative DNA and RNA sequencing, aimed for more accurate and comprehensive characterization of gene fusions in CRCs. We proved that RNA NGS was a valuable addition to DNA NGS for enhancing fusion detection (46–56% in MLH1me+ BRAF/RAS wild-type dMMR CRCs), as well as identifying novel or atypical fusion types. An optimizing strategy incorporating RNA NGS to screen for oncogenic fusions in CRCs was thus proposed. Next, we presented a detailed analysis of molecular genetic profile and clinicopathological features of fusion-positive dMMR CRCs. All fusions involved RTK-RAS signaling pathway, predominantly RTKs, and were mutually exclusive to other RTK-RAS driver mutations. WNT pathway alterations were also frequently detected. Fusion-positive tumors were typically diagnosed in elder patients, predominantly right-sided, preferentially occurred at hepatic-flexure and showed histologically poor-differentiated components.
Considering the distinct advantages over other techniques in gene fusion detection, the latest National Comprehensive Cancer Network guideline for non-small cell lung cancer recommended RNA-based NGS in patients with no identifiable driver oncogenes detected by broad panel DNA NGS(19). In the present study, we revealed that nearly 20% (n = 4) MLH1me+ dMMR tumors with neither oncogenic fusions nor BRAF/RAS driver mutations detected by DNA NGS were positive for gene fusions by RNA NGS. In all of these four cases, the genomic breakpoints were located at large introns or intronic repetitive elements, which were typically not sufficiently covered by large hybrid-capture based DNA NGS panel. In our cohort, fusion-positive tumors by integrative DNA and RNA NGS represented 11% of dMMR cases, 24% of MLH1me+ dMMR cases, and 56% of MLH1me+ dMMR cases with wild-type BRAF/RAS. These proportions were much higher in comparison to that reported in prior DNA-based large-scale clinical research using MSK-IMPACT assay(9), suggesting that optimizing fusion detection process by incorporating additional RNA NGS was able to achieve a considerable higher yield of gene fusions in CRCs. In addition, RNA NGS successfully identified two potentially actionable kinase fusions (SNRNP70-MET and YPEL1-MAPK1) which have not been reported in CRCs before. Therefore, we suggested the sequentially combined use of DNA NGS and RNA NGS as a highly effective strategy to uncover oncogenic gene fusions in MLH1me+ CRCs, which were suggested as markers for unfavorable prognosis and targets for personalized therapy(20). In clinical settings where BRAF/RAS PCR was applicated alternative to DNA NGS, direct RNA NGS was recommended in BRAF/RAS wild-type cases for maximized cost-efficiency.
Aberrant activation of RTK-RAS signaling pathway has been well-recognized as key molecular event in CRC tumorigenesis. Previously, among MLH1me+ dMMR CRCs, RTK-RAS activation was generally considered to be mediated by BRAF oncogenic mutation, occurring at the early stage of serrated neoplasia pathway(21). In this and our prior studies(14), we revealed that almost all gene fusions were detected in dMMR CRCs harboring hypermethylated MLH1, which presented as the only RTK-RAS driver alteration in these tumors. It is rational to suggest gene fusions as one major mechanism of RTK-RAS oncogenic activation in MLH1me+ dMMR CRCs, second only to BRAF mutation. Most of the fusion-positive cases harbored RTK fusions susceptible to tyrosine kinase inhibition therapy. In spite of the rarity, it is worth noting that a minority of fusions involved MAP3K(BRAF) and MAP1K, genes encoding key components of downstream mitogen-activated protein kinase (MAPK) cascade which were essential for intracellular RTK-RAS signal transduction. Due to the potential feedback activation of EGFR(22, 23), combination therapy consisting of both EGFR and RAS/RAF inhibitors might be required in these cases(24–26).
Despite that dMMR was typically considered as a favorable prognostic marker in CRC patients, oncogenic fusions have been shown to be associated with poorer clinical outcome(27, 28). The detected genetic fusions primarily affected RTKs, and rendered those tumors amenable to FDA approved targeted therapy that might reverse the otherwise poor prognosis. Therefore, efficient identification and detailed characterization of fusion variants is of key clinical significance. In our dMMR CRC cohort, TRK fusions, particularly NTRK1 fusions, were the most frequently detected fusion events. We observed that TPM3 was the most common fusion partner of NTRK1 in CRCs (66%), which was in consistent with previous reports(29, 30). NTRK1-LMNA and NTRK1-PLEKHA6, two other NTRK1 fusion types documented in CRCs before(29), were found to take a lesser proportion in our cases. We did not detect NTRK1 fusions with SCYL3 and TPR, which have been reported rarely before(30). In previously published reports, NTRK3 fusions were found in only a few CRCs, accounting for two out of 21 fusion events in cases assessed by MSK-IMPACT testing(9), and one out of 16 NTRK fusion events in cases screened by pan-TRK IHC testing(30). However, it has been implicated that substantial numbers of NTRK3 gene rearrangements occurred at large introns (NTRK3 intron 13 and 14), and might be omitted by DNA NGS alone(7). Also, large scale clinical researches have documented a lower sensitivity of pan-TRK IHC assay for NTRK3 fusions comparing to NTRK1/2 fusions(31, 32). In the present study, using sequentially combined DNA NGS and RNA NGS, we observed a much higher proportion of NTRK3 fusions in all detected fusion events (5/22). This finding further justified incorporating RNA NGS in clinical practice to more efficiently identify fusion-positive tumors, especially those harboring NTRK3 fusions. Although several rare NTRK3 fusion types were previously identified in CRCs, including KANK1-NTRK3, COX5A-NTRK3 and VPS18-NTRK3(11, 30), here we observed that NTRK3 exclusively formed fusion with its main partner gene ETV6 or EML4. As far as we can see, two of the gene fusions affecting RTKs presented in our cohort were not well-documented in CRCs previously. An EML4-ALK fusion was found to involve atypical ALK breakpoint within exon 19 that encoded transmembrane domain. ALK rearrangements at exon 19, instead of usual site within intron 19 or exon 20, has only been rarely described in malignant stromal sarcoma(33) and lung adenocarcinoma(34, 35) before. Except for a case demonstrating a partial response to targeted therapy(34), reports on clinical implication of this breakpoint were very limited. A MET fusion with novel partner gene SNRNP70 encoding a key component of spliceosome was identified in one case. Although MET gene copy number gain and protein overexpression were proved to drive CRC tumor malignant progression(36), MET gene fusions have not been noted in CRCs before.
Apart from RTKs, gene fusions involving the downstream MAPK cascade were also potentially actionable. Both of the two fusions affecting MAPK cascade detected in our cohort have been rarely reported before. The CUL1(e7)-BRAF(e9) fusion was previously observed in a few cases of melanoma(37) and low-grade serous carcinoma (LGSC)(38), and only once in CRC(9). Tumor cells harboring CUL1-BRAF fusion has been found to show activation of MAPK signaling pathway and sensitivity to MEK/RAF inhibition. Moreover, complete response to MEK inhibitor-based combination therapy was noted in one LGSC patient bearing CUL1-BRAF fusion(38). The YPEL1(e1)-MAPK1(e5) was a novel fusion to our limited knowledge. Typically, abnormal overactivation of MAPK1(ERK) was induced by hyperactivated upstream RTK/RAS signaling. Gain-of-function mutations in the gene itself were only seldomly documented in laboratory models or in clinical cases(39). Since only part of MAPK1 C-terminal kinase domain was involved in the detected YPEL1-MAPK1 chimeric transcript, whether this fusion gene possessed oncogenic properties awaited further investigation. Given that constitutively activated RTK fusions could concurrently induce downstream RAS and PI3K pathways, it is not surprising to find the general low frequency of PI3K pathway aberration among tumors harboring RTK fusions. However, PIK3CA and PTEN mutations were observed in these two cases with fusions involving MAPK cascade. This finding indicated that despite the well-established intimate intersection of RTK downstream pathways RAS-MAPK and PI3K-mTOR, constitutive activation of MAPK cascade by gene rearrangements might not be sufficient to cross-activate PI3K-mTOR signaling and give rise to malignant transformation events.
We observed that RNF43 was the most frequently mutated one among all genes analyzed in this study. This result strengthened our previous finding that RNF43 inactivation was directly correlated with MLH1 hypermethylation, instead of BRAF mutation status(14). Nearly 90% of the fusion-positive cases were presented with WNT pathway alterations. Additionally, four out of 12 top recurrently mutated genes (RNF43, APC, FBXW7 and ARID1A) were found to be involved in WNT signaling. It is rational to assume that synergistic cooperation of WNT pathway components might play an important role in tumorigenesis of fusion-positive CRCs. A very recent in vitro study revealed susceptibility to poly (ADP-ribose) polymerase (PARP) inhibitors in a subset of poor prognostic CRCs with DNA homologous recombination repair (HRR) pathway deficiency(40). Our data showed that one third of fusion-positive tumors harbored mutations in crucial HRR genes ATM and BRCA2, and lay a rationale for further clinical studies investigating PARP inhibitors as a potential therapeutic option for these tumors.
Based on large sample size and detailed molecular subclassification, we further conducted comparison between fusion-positive and fusion-negative tumors within MLH1me+ CRCs. Fusion-positive tumors were found to exhibit characteristic clinicopathological features, including old age, preferential hepatic flexure localization and poor differentiation. Typically, dMMR tumors were considered as a relatively homogeneous molecular entity characterized by vulnerability to immunotherapy, which have recently been approved by FDA as first-line treatment for metastatic dMMR CRCs. Our findings highlighted the delicate yet noticeable heterogeneity within dMMR CRCs, and justified more precise molecular subtyping for personalized diagnosis and therapy in CRCs. In addition, a recent study has uncovered the continuum variation of tumor molecular profile along the large intestine, and necessitated more precise classification of CRCs by tumor location(41). In this study, we not only confirmed that fusion-positive CRCs were primarily right-sided lesions, but also specified that more than half of them were localized at hepatic flexure. In clinical practice, these results implicated that CRC patients with above-mentioned clinicopathological features might be prioritized for molecular assay for gene fusions, including RNA NGS.
In summary, our study presented a practical and highly effective screening procedure for genetic fusions through integrated DNA NGS and RNA NGS in a selected subset of dMMR CRCs harboring hypermethylated MLH1. With a detailed description of fusion variants, molecular profile and clinicopathologic features, we further characterized fusion-positive CRCs as a distinctive subtype with key clinical significance.