Distinct RET Fusion Partner Genes in RET-Rearranged Spindle Cell Tumors Contribute to Varied Transformation Capacities

Distinct RET Fusion Partner Genes in RET-Rearranged Spindle Cell Tumors Contribute to Varied Transformation Capacities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Distinct RET Fusion Partner Genes in RET-Rearranged Spindle Cell Tumors Contribute to Varied Transformation Capacities Sheng Xiao, Qi Gui, Ying Zhang, Mei Yang, RONGRUI LIANG, Man Huang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6513808/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract RET -rearranged spindle cell tumors represent a group of soft tissue tumors with heterogeneous clinical presentations. While some tumors are benign, others display malignant behavior with metastatic potential. The underlying causes of this heterogeneity are unknown but are suspected to be associated with the diverse fusion partner genes involved in RET rearrangements. We describe a unique case of a spindle cell tumor with an MYH10::RET fusion that originated in the knee and metastasized to the lung. The tumor initially responded to anti- RET therapy but subsequently relapsed due to a new NTRK1 fusion. The disease has since been effectively managed with a third-generation inhibitor targeting both RET and NTRKs. This supports our hypothesis that MYH10 as a fusion partner contributes to the tumor's aggressive clinical course. We established cell lines stably expressing MYH10::RET and CCDC6::RET , and we found that MYH10::RET -expressing cells demonstrated significantly more aggressive phenotypes as compared to cells expressing CCDC6::RET . Further analysis revealed that MYH10::RET possesses more potent kinase activity than CCDC6::RET , providing a mechanistic explanation for the observed differences in tumor behavior. We conclude that distinct RET fusion partners significantly contribute to the clinical heterogeneity in RET -rearranged spindle cell tumors. Biological sciences/Cancer/Cancer therapy/Targeted therapies Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance RET Fusion Partner Genes RET-rearranged spindle cell tumors NTRK1 fusion Drug-resistant mutations Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION A group of soft tissue tumors are characterized by the activation of various tyrosine kinases, mostly through chromosome translocations, resulting in the formation of chimeric proteins, along with activation point mutations within the tyrosine kinase domains or insertions/deletions within the juxtamembrane domains. To date, 10 tyrosine kinases have been identified in this group of tumors: RET , NTRK1/2/3 , ROS1 , ALK , EGFR , MET , BRAF , and RAF1 1–3 . RET -rearranged spindle cell tumors show a wide range of histopathologic features, resembling those of lipofibromatosis-like neural tumors (LPF-NT), infantile fibrosarcomas (IF), malignant peripheral nerve sheath tumors (MPNST) and fibrosarcoma. While some of these tumors express S100 and CD34, their immunophenotype s are generally non-specific 4 . The clinical behavior of these tumors varies widely, ranging from indolent, slow-growing, and benign lesions to aggressive metastatic malignancies. For example , four of five patients with the MYH10::RET fusion were histologically sarcoma-like, and one of them developed lung metastasis. In contrast, other RET rearrangements, such as CCDC6::RET and NCOA4::RET , are associated with LPF-NT that is effectively treated with surgical resection alone, even in cases of incomplete excision 5 . The reasons why the same RET rearrangements lead to tumors with different prognoses remain to be determined. Although oncogenic receptor tyrosine kinases (RTKs) activate similar downstream signaling pathways, different fusion partners of RTKs can influence tumor cell behavior. For example, NIH3T3 cells expressing seven ALK fusions containing various 5'-fusion partners demonstrated different cell growth in soft agar cultures and varying sensitivities to ALK inhibitors. Additionally, ALK fusion proteins have different kinase activities 6 . Fusion partners can also affect cellular localization; for instance, ETV6::NTRK3 expressed in melanocytes was found in both the nucleus and cytoplasm, leading to epithelioid morphology, whereas MYO5A::NTRK3 was exclusively nuclear and resulted in spindle cell morphology 7 . Similar variability was observed for th e non-RTK fusion proteins. In acute promyelocytic leukemia (APML), patients with the classic PML::RARA fusion respond to all-trans retinoic acid (ATRA), resulting in a cure for once-deadly leukemia. Conversely, patients with non-classic RARA fusions featuring different 5' partners show varied responses to ATRA, with some being unresponsive to therapy 8 . In this study, we present a case of spindle cell sarcoma with MYH10::RET fusion showing a fibrosarcoma-like morphology and lung metastasis. To investigate why different RET fusions led to varying clinical outcomes, we established isogenic cell lines stably expressing either MYH10::RET or CCDC6::RET . We found that cells expressing MYH10::RET had increased proliferation, migration, and invasion compared to those expressing CCDC6::RET , reflecting their clinical behavior. Further analysis showed that MYH10::RET had a stronger kinase activity than CCDC6::RET . We conclude d that the activation level of RET is modulated by different fusion partners, likely contributing to the heterogeneous clinical behaviors observed in these tumors. These results may have clinical implications and may potentially influence patient management strategies. MATERIALS AND METHODS Immunohistochemistry The tumor tissue was fixed in formalin, embedded in paraffin ( FFPE , Formalin Fixed Paraffin Embedded) and cut into 5 µm sections. Immunohistochemical staining for pan-TRK (ab181560, Abcam, UK), CD34 (Kit-0004, MXB, China), and S100 (ab11428, Abcam, UK) was carried out according to the following protocol: the slides were baked for 1 h at 60 °C , deparaffinized, and rehydrated using 100% xylene, 100% ethanol, and running water, respectively. The slides were blocked with a solution containing 10% normal serum and 1% bovine serum albumin (BSA) in Tris-buffered saline and incubated with primary antibodies for 2 h. Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide, and the slides were incubated with a horseradish peroxidase-labeled polymer (DAKO). Tissue sections were developed using 3,3’-diaminobenzidine (DAKO) as the chromogen and counterstained with Mayer’s hematoxylin. Targeted DNA next-generation sequencing Genomic DNA was isolated from FFPE tumor tissue sections using a QIAamp DNA Micro Kit (Cat: 56,304; Qiagen, Germany). Three hundred nanograms DNA (300 ng) was fragmented to a size of 200–300 bp using a Bioruptor Pico (Diagenode, Denville, NJ, USA), and library preparation was performed using the Rapid Plus DNA Lib Prep Kit for Illumina (RK20208, ABclonal) according to the manufacturer's instructions. The libraries were hybridized with a pool of biotin-labeled bait oligos targeting 638 tumor-associated genes for 16 h. Targeted regions were captured with streptavidin beads, followed by PCR amplification and sequencing as paired-end 150-bp reads on an Illumina NextSeq 6000 instrument. Sequencing reads were aligned to the reference genome (hg19) using BWA-MEM. Analyses of single-nucleotide variations (SNVs), insertion/deletion (indels), copy number variations (CNVs), and structural variations (SVs) were carried out using SeqNext software (JSI, Germany) and proprietary pipelines developed by Sano Medical Laboratories (China). Fluorescence in situ hybridization (FISH) FISH analysis was performed on 5-µm FFPE tumor tissue slides with two colored split-apart probes (5’probe green and 3’ probe red) for RET and NTRK1 from Betrue, China. The slides were deparaffinized in xylene, rehydrated, and treated with 750 U/ml of pepsin digest ion solution (Cat: P6887, Sigma Aldrich, USA) for 10 min , followed by incubation in 10% buffered formalin for another 10 min. The slides and probes were separately denatured and hybridization was performed overnight at 37°C. After hybridization, the slides were washed in 0.4 × SSC/0.3% NP-40 at 73°C for 3 min and then counterstained with DAPI. Targeted RNA next-generation sequencing Total RNA from FFPE tumor tissue sections was extracted with TRIZOL reagent according to the manufacturer's instructions (Cat: 10296010, ThermoFisher, Invitrogen, USA). Reverse transcription was performed using 100 ng total RNA, and end repair and adaptor ligation were performed following standard NGS (Next-generation sequencing) p rotocols (Cat: E7771 and E6111, NEB, USA). PCR enrichment was performed using 641 gene-specific primers specific to 118 genes commonly involved in solid tumors, and the enriched PCR products were sequenced on a NovaSeq 6000 platform (Illumina, USA). Sequencing reads were analyzed using the SeqNext software (JSI, Germany). Lentivirus The CCDC6::RET and MYH10::RET fusion genes were cloned into the lentiviral plasmid vectors. These plasmids, along with empty vectors for control cells, were co transfected with PAPAX2 and PMD2.G into NIH3T3 cells. The supernatant from transfected NIH3T3 cells was collected and filtered 48–72 h post-transfection. Target cells were then infected with lentiviral particles in the presence of 10 µg/ml polybrene reagent. Following two weeks of puromycin selection, the cells were harvested for subsequent experiments. Cell counting kit-8 The cells were then transferred to 96-well plate s. After cell attachment, the cells were treated with various TKIs for 3 to 4 days, including selpercatinib (HY-114370, MCE), pralsetinib (S8716, Selleck), and cabozantinib (S1119, Selleck). The culture medium was then mixed at a 10:1 ratio with the CCK8 solution (Cat: C0039, Beyotime, China) and added to the wells in the dark. After 2 h of incubation at 37°C, the absorbance (OD) at 450 nm was measured using a microplate reader. Wound healing assay NIH3T3 cells were collected and inoculated on both sides of the scratch chamber (Culture-Insert 2 Well in µ-Dish 35 mm, Cat: 81176, IBIDI). The cells were incubated with a medium containing 10% fetal bovine serum and 1% penicillin until the cells were attached to the wall. After removing the scratch chamber, the cells were cultured in low -serum medium. The healing rates of the different cell groups were recorded at 0, 24, and 48 h. Transwell migration assay The Transwell chamber was placed in a 24-well culture plate. NIH3T3 cells were harvested and resuspended in serum-free medium. A total of 200 µL of the cell suspension was added to the upper chamber and 600 µL of medium containing 10% serum was added to the lower chamber as a chemoattractant. Following incubation at 37°C for 48 h, the chamber was gently washed twice with PBS ( Phosphate-Buffered Saline), fixed with methanol, and stained with crystal violet solution. Immunofluorescence colocalization Approximately 5 × 10⁴ cells were seeded in confocal petri dishes. After cell attachment, the cells were fixed with formaldehyde solution and incubated overnight with mouse anti-FLAG tag antibody (bsm-33346M, Bioss, 1:200). The following day, the cells were incubated with a fluorescently labeled secondary antibody (ab150115, Abcam, 1:200) for 1 h . After washing with phosphate-buffered saline (PBS), an antifade solution containing DAPI (Cat: P0131, Beyotime) was applied. Images were captured using an Olympus fluorescence microscope. Western blot analysis and Co-Immunoprecipitation (CO-IP) Cell lysates were quantified using a BCA Protein Assay Kit (Cat:23225, Thermo Fisher, Invitrogen, USA). Equal amounts of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane s (IPVH00010, Millipore). The membrane was blocked with 5% skim milk (36120ES76 ; Yeasen) for 1 h at room temperature and incubated with the corresponding antibodies overnight at 4°C in a shaker. The membrane was washed three times with TBST (Solarbio, T1082), incubated with anti-rabbit or anti-mouse antibodies for 1 h at room temperature, and detected with an enhanced chemiluminescence solution (B io-Rad). For immunoprecipitation, the protein lysates were incubated with an Anti-Flag Tag and microbeads, and the free proteins in the supernatant were removed by magnetic separation. Proteins were eluted from the beads and analyzed by western blotting. The antibodies used were as follows: Phospho-RET (Tyr905) antibody (Cat: 3221, CST), RET (E1N8X) XP® rabbit mAb (Cat: 14556, CST), Mouse Anti-Flag Tag antibody (bsm-33346M, Bioss, 1:200), and Rabbit Anti-beta-Actin (Loading Control) antibody (BS-0061R, BIOSS). Kinase Assays The activities of the two RET fusion kinases were compared using an ADP-Glo Kinase Assay Kit (Promega Corporation, Madison, WI, USA). The reagents required for the reaction were configured according to the manufacturer’s instructions. For each reaction system, CO-IP-purified RET kinase samples, IGF (1 mg/mL), and varying concentrations of ATP (from 0 to 1000 µM) were added, followed by incubation at room temperature for 60 min to allow the kinase to catalyze the substrate phosphorylation reaction. ADP-Glo™ Reagent (final concentration 10 µM) was added to each well and gently shaken to ensure mixing to terminate the kinase reaction. The wells were incubated at room temperature for 40 min to allow conversion of ADP to ATP. Kinase Detection Reagent was then added to each well, mixed with gentle shaking, and incubated at room temperature for 60 min. The luminescence signal s were measured using a chemiluminescence instrument. RESULTS Genomic profile of a RET -rearranged spindle cell tumor A 37-year-old male presented with a subcutaneous mass on the lateral side of the left knee. PET-CT revealed a well-defined, hypermetabolic soft tissue mass measuring 159 × 92 × 100 mm, containing both solid and cystic/necrotic components ( Fig. 1 A, B ). A complete surgical excision was performed. Histological examination of formalin-fixed, paraffin-embedded (FFPE) tissue showed hypercellular spindle cells arranged in fascicles with hemangiopericytoma-like vasculature and areas of necrosis ( Fig. 2 A ). Immunohistochemical staining revealed a null immunophenotype profile, with tumor cells negative for CD34, S100, and pan-TRK ( Fig. 2 B-D ) . Based on these findings, a diagnosis of malignant spindle cell tumor was established. Targeted DNA next-generation sequencing (NGS) revealed CDKN2A/B homozygous loss, 9q copy-neutral loss of heterozygosity (CN-LOH), gain of chromosomes 1p, 16, 20q, and 21, and an FGF20::RET rearrangement. The RET breakpoint is located in intron 11, which is a common site for RET rearrangements. However, due to incompatible transcription al orientation, the FGF20::RET rearrangement did not contain a reading frame ( Fig. 2 E,F ) . Fluorescence in situ hybridization (FISH) with a RET split-apart probe confirmed the RET rearrangement ( Fig. 2 G ) . Subsequently, targeted RNA NGS identified an MYH10::RET fusion with an intact reading frame and breakpoints, consistent with previously reported MYH10::RET rearrangements ( Fig. 2 H ) . The MYH10::RET fusion protein contained the myosin head domain from MYH10 and the intact kinase domain from RET. The oncogenic mechanism likely involves myosin head-mediated dimerization, leading to autophosphorylation and RET kinase activation. The discrepant results between DNA and RNA NGS are likely due to multiple RET fusions at the DNA level, including both FGF20::RET and MYH10::RET . DNA NGS detected nonfunctional FGF20::RET but missed functional MYH10::RET . This underscores the superiority of RNA NGS over DNA NGS in detecting functional fusions as it effectively eliminates all non-transcript rearrangements often observed in DNA NGS, as previously reported 9 , 10 . Ten months after the surgery, the patient developed lung metastasis ( Fig. 1 B ) , which was biopsied and confirmed to carry the same MYH10::RET fusion. The patient was enrolled in a clinical trial and treated with 400 mg of pralsetinib (BLU-667), which resulted in a significant reduction in lung metastases, nearly achieving complete remission ( Fig. 1 C ) . However, 11 months later, the patient developed a severe cough and fever. Chest computed tomography (CT) s howed severe infection in both lungs and potentially drug-associated pneumonia ( Fig. 1 D ) . Subsequently, Pralsetinib was discontinued and the patient withdrew from the clinical trial. Microbiome NGS of bronchoalveolar lavage fluid detected human herpesvirus 5 (cytomegalovirus) infection with 45.28% abundance and Pneumocystis infection. Treatment with Ganciclovir, Cotrimoxazole, and human hemoglobin effectively controlled the bilateral lung infection s . At this time, chest CT showed no evident tumor lesions ( Fig. 1 E ) , and the patient did not receive further antitumor therapy but underwent regular follow-up. Six months later, a new lesion had developed in the right lung ( Fig. 1 F ) . The patient was subsequently treated with 160 mg selpercatinib twice daily, which result ed in significant tumor shrinkage and stable disease ( Fig. 1 G ) . Nearly two years later, the patient presented with marked chest tightness and dyspnea. Chest computed tomography (CT) indicated renewed progression of the pulmonary lesion, accompanied by pericardial effusion ( Fig. 1 H ) . A lung tumor biopsy was then performed, which showed a new TPM3::NTRK1 rearrangement in addition to MYH10::RET fusion ( Fig. 3 A ) . Histologically, the tumor cells showed similar morphology to the primary knee tumor, but immunohistochemistry (IHC) showed positivity for CD34 and pan-TRK, which are negative markers in the primary tumor ( Fig. 3 B-E ) . To determine whether the same tumor cells harbored both MYH10::RET and TPM3::NTRK1 rearrangements, or if they were present in different cell populations, successive FISH was performed on the same cell group, first with a RET split-apart probe set, followed by an NTRK1 split-apart probe set. This confirmed that both rearrangements were present in the same cells ( Fig. 3 F ) , suggesting that the MYH10::RET fusion likely serves as a mechanism of resistance to the RET inhibitor selpercatinib. The patient was treated with a combination of selpercatinib (160 mg/day) and the NTRK inhibitor entrectinib (300 mg/day ). This combination significantly shrank the neoplasm in the left lung after two months of treatment ( Fig. 1 I ) , but a new lesion merged in the right lung. Over the following 6 months, however, the combination therapy became less effective, and the pulmonary lesions and pericardial effusion showed progression ( Fig. 1 J ) . Genomic profiling of a tumor biopsy specimen revealed drug-resistant mutations in the kinase domains of both kinases : RET p.G810S (variant allele frequency, VAF 1.8%) and NTRK1 p.G595R (VAF 14.2%). Consequently, the patient transitioned to a combination therapy of selpercatinib 160 mg daily and repotrectinib 160 mg daily, specifically designed to address solvent-front and gatekeeper mutations associated with 1st and 2nd generation therapies. Eleven days later, a chest CT scan revealed significant tumor shrinkage ( Fig. 1 K ) . With this combination regimen, the patient’s disease remained stable, as confirmed by the most recent follow-up ( Fig. 1 L ) . Functional evaluation of MYH10::RET and CCDC6::RET Our patient with MYH10::RET had a metastatic tumor characterized by a continuously evolving cancer genome. However, tumors with other RET rearrangements, such as CCDC6::RET , are often benign. To determine whether distinct RET fusion partners influence oncogenesis at the cellular level, we generated cell lines expressing either MYH10::RET or CCDC6::RET . NIH3T3 cells were transduced with lentiviruses expressing MYH10::RET or CCDC6::RET , and expression was confirmed by western blot ting and immunofluorescence, demonstrating cytoplasmic localization of both fusion proteins ( Fig. 4 A ). While CCDC6::RET -expressing cells showed modestly increased growth compared to control cells, MYH10::RET -expressing cells showed significantly greater proliferation than both CCDC6::RET -expressing and control cells at all time points (24, 48, and 72 h), as measured by the WST-8 assay (Cell Counting Kit-8; CCK-8) ( Fig. 4 B ). In the cell scratch assay, both MYH10::RET- and CCDC6::RET -expressing cells showed significantly faster migration than control cells (P < 0.0001), with MYH10::RET -expressing cells demonstrating markedly greater migration than CCDC6::RET -expressing cells at 24 h and 48 h post-wounding ( Fig. 4 C,D ). Similarly, in a collagen-coated transwell assay to assess invasive capacity, MYH10::RET- and CCDC6::RET -expressing cells were significantly more invasive than control cells (P < 0.0001), although MYH10::RET -expressing cells were significantly more invasive than their CCDC6::RET -expressing counterparts (P < 0.0001) ( Fig. 4 E,F ). Cells expressing CCDC6::RET and MYH10::RET fusions were treated with three tyrosine kinase inhibitors specifically targeting RET. Both cell lines were sensitive to all three inhibitors ( Fig. 4 G-I ) , although the degree of sensitivity varied. The IC50 values for MYH10::RET- expressing cells were 3.858 and 3.004 µM for cabozantinib, selpercatinib, and 2.59 µM for Pralsetinib. In contrast, the IC50 values for CCDC6::RET cells were 19.49 µM for Cabozantinib, 8.712 µM for Selpercatinib, and 3.836 µM for Pralsetinib. These results suggest that MYH10::RET -expressing cells are more sensitive to RET inhibitors than CCDC6::RET -expressing cells, likely reflecting the faster growth of MYH10::RET cells. To determine whether MYH10::RET and CCDC6::RET have different kinase activities, FLAG-tagged MYH10::RET and CCDC6::RET were immunoprecipitated from lysates of cells stably expressing these fusion proteins. The phosphorylation status of rearranged RET was assessed using a phospho-RET (Tyr905) antibody. Both MYH10::RET and CCDC6::RET are constitutively phosphorylated and activated. Notably, the phosphorylation level of MYH10::RET was significantly higher than that of CCDC6::RET. We further evaluated the phosphorylation of MAPK, a key effector of the RET signaling pathway. Both MYH10::RET- and CCDC6::RET-expressing cells had significantly higher MAPK phosphorylation than control cells. Moreover, MAPK phosphorylation was higher in MYH10::RET-expressing cells than that in CCDC6::RET-expressing cells ( Fig. 5 A ) . Finally, an in vitro kinase assay was performed to compare the kinase activities of MYH10::RET and CCDC6::RET using the IGF peptide, a known substrate of RET kinase. The results demonstrated that the kinase activity of MYH10::RET was approximately three -fold higher than that of CCDC6::RET ( Fig. 5 B,C ) . DISCUSSION We report a case of metastatic spindle cell sarcoma initially driven by an MYH10::RET rearrangement, which subsequently acquired an NTRK rearrangement as a mechanism of resistance to RET inhibitor therapy. Further genomic evolution has led to the emergence of kinase domain mutations in both RET and NTRK following treatment with combined RET and NTRK inhibitors. Ultimately, disease control was achieved using the next-generation tyrosine kinase inhibitor, repotrectinib. This case highlights the critical importance of sequential molecular profiling for monitor ing tumor evolution and guid ing precision therapy. Spindle cell tumors with RET rearrangement s present a wide clinical spectrum, ranging from benign lesions to aggressive high-grade sarcomas. The underlying mechanisms driving this variability in clinical behavior remain unclear. To date , nine RET fusion partners have been identified ( Fig. 5 D,E ) 5 . We hypothesized that differences in fusion partners would contribute to varying transformation capabilities. In this study, we established isogenic cell lines stably expressing MYH10::RET and CCDC6::RET , which are associated with metastatic tumors and benign LPF-NT s, respectively. Cellular assays revealed that MYH10::RET -expressing cells showed significantly faster growth, increased migration, and greater invasiveness than cells expressing CCDC6::RET . These observations closely mimic ked the clinical behaviors associated with these fusion proteins. Further analysis confirmed that MYH10::RET had substantially higher kinase activity than CCDC6::RET . These findings suggest that different fusion partners may contribute to distinct transformation capabilities through differential kinase activities. This study has limitations in that it did not address the endogenous expression levels of fusion proteins in tumor cells. Expression can be regulated at multiple levels, including mRNA expression, which is influenced by various promoters associated with different fusion partners. At the protein level, factors such as post-translational modifications, protein folding, and degradation pathways can affect chimeric protein stability and function. These variations may also affect the transformation activities and clinical outcomes. RET inhibitors are increasingly used for the treatment of RET-altered cancers. However, similar to many targeted therapies, resistance to RET inhibitors can develop over time, and the resistance mechanisms may include the acquisition of secondary RET mutations, activation of bypass signaling pathways (e.g., EGFR and MET), histologic transformation, pharmacokinetic factors, epigenetic alterations, and modulation of the tumor microenvironment, all of which allow tumor cells to evade targeted therapy 11 . Our patient, with the MYH10::RET rearrangement, initially responded to RET inhibitors Pralsetinib and Selpercatinib, but later relapsed due to the emergence of a new NTRK1 rearrangement. The patient then responded to combination therapy with the anti-NTRK drug entrectinib and the anti-RET drug selpercatinib, only to relapse again due to drug-resistant mutations in the kinase domains of both NTRK1 and RET 12 , 13 . When resistance mutations occur in both RET and NTRK1 kinase domains, the mutation with the strongest impact on drug binding typically plays a decisive role in determining resistance. The choice of a new targeted therapy depends on the specific mutation profiles and their impact on available inhibitors. Mutation abundance (variant allele frequency, VAF) can provide important clues for determining which resistance mutation is dominant. If RET G810C/S/R is dominant, targeted therapy can be switched to next-generation RET inhibitors, such as TPX-0046 or LOXO-260. If NTRK1 G595R/G667C is dominant, targeted therapy can be switched to second-generation TRK inhibitors, such as repotrectinib (TPX-0005), which is designed to overcome TRK resistance mutations. Our patient subsequently responded to repotrectinib and has remained stable since then. Our patient experienced a pralsetinib-related pulmonary infection, which can be easily mistaken for tumor progression on imaging. Pralsetinib (formerly BLU-667) is a potent and selective RET kinase inhibitor designed for the treatment of RET-fusion-positive malignancies, including non-small cell lung cancer (NSCLC) and thyroid cancer. Although Pralsetinib has demonstrated significant clinical efficacy in RET-driven cancers, it is associated with various adverse effects. According to data from the ARROW clinical trial, the most common adverse events include hypertension, fatigue, gastrointestinal disturbances, and hematologic al toxicities. Pulmonary toxicities were reported in 12% of patients, with most cases being of grade 1 or 2 severity, whereas severe pneumonia (grade 3 or 4) occurred in only 2% of patients, sometimes necessitating dose interruptions or discontinuation 14 . Pulmonary infections associated with pralsetinib can closely mimic tumor progression, highlighting the need for careful diagnostic differentiation. Microbiome NGS may help distinguish infection s from disease progression. In our case, the patient was diagnosed with cytomegalovirus infection, which showed improvement following appropriate treatment. The precise mechanisms underlying pralsetinib-induced pneumonia remain unclear but may involve direct pulmonary toxicity, immune-mediated reactions, or interactions with concomitant therapies and underlying lung conditions. In this patient, a key consideration was the potential off-target effects of pralsetinib, which may increase susceptibility to infections or trigger autoimmune-like reactions in the lung tissue. Conclusions In conclusion, we have characterized a rare metastatic spindle cell tumor with MYH10::RET fusion. The aggressive behavior of cells expressing MYH10::RET was confirmed using isogenic cell models. Our findings suggest that different RET fusion partners of RET contribute to the clinical heterogeneity commonly observed in this group of tumors. Furthermore, this study highlights the critical importance of continuous genomic monitoring to identify emerging drug resistance mechanisms and tailor-targeted therapies using appropriate inhibitors. Declarations Ethics Statement and Patient Consent Ethical approval was obtained from the Medical Ethics Committee of the Fourth Affiliated Hospital of the Soochow University. Consent for participation was not required. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors declare no potential conflicts of interest. Funding This study was financially supported by the Science and Technology Plan Project of Suzhou (SZM2022009), Horizontal Research Foundation of Soochow University (P142900324), and the Medical Innovation Research Project of Suzhou Industrial Park (CXYJ2024B03). Authors' contributions MT and SX contributed to the conception of the study; QG analyzed and interpreted the patient data; YZ, MY, XY, NC and XC performed the experiment; RL, MH, MW, HC and LM contributed significantly to analysis and manuscript preparation; QG, YZ and MY performed the data analyses and wrote the manuscript; MT and SX performed the analysis with constructive discussions, reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Antonescu, C. R. Emerging soft tissue tumors with kinase fusions: An overview of the recent literature with an emphasis on diagnostic criteria. Genes, chromosomes & cancer. 2020; 59: 437–444. Davis, J. 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RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2020; 15: 541–549. Griesinger, F. et al. Safety and efficacy of pralsetinib in RET fusion-positive non-small-cell lung cancer including as first-line therapy: update from the ARROW trial. Annals of oncology: official journal of the European Society for Medical Oncology. 2022; 33: 1168–1178. Additional Declarations There is NO conflict of interest to disclose. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6513808","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449705381,"identity":"88ff0e51-9bbf-48c0-8d08-319c66f4c4f1","order_by":0,"name":"Sheng Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACxgYGhgoJOfv2xsYHH4jXcsbG2IDncLPhDOItaktL3CCR3ibNQYxq+RnJjR8+sB1m3C75sEGagcFOTreBgBaDMwebJWfwHGa2nJ3YYFzAkGxsdoCQFvbGBmkeicNsDLcTG5JnMBxI3EZIi3wzY/NvHoPDPAw3DzYASSK0MBxvbJPmSUiTMLjB2NhMlBagX9osZxywMZDsSWxmnGFAhF/kZ6Q/vvHxn0R9P/vx5z8+VNjJEdSCbilpykfBKBgFo2AU4AAAhDFGlifpLA0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8852-6434","institution":"Brigham and Women's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Xiao","suffix":""},{"id":449705382,"identity":"f1b6a5a9-9845-41b9-9849-e8450093b958","order_by":1,"name":"Qi Gui","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Gui","suffix":""},{"id":449705383,"identity":"159a2ebc-7c4c-4d83-bd23-65b2122542e3","order_by":2,"name":"Ying Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":449705384,"identity":"4f838a3b-b504-463a-bd51-07a57376534b","order_by":3,"name":"Mei Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Yang","suffix":""},{"id":449705385,"identity":"e5476638-c942-4ad2-b2b9-dcf580f87942","order_by":4,"name":"RONGRUI LIANG","email":"","orcid":"","institution":"The First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"RONGRUI","middleName":"","lastName":"LIANG","suffix":""},{"id":449705386,"identity":"a78ccdab-aece-4f69-bab0-94e2339b790e","order_by":5,"name":"Man Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Man","middleName":"","lastName":"Huang","suffix":""},{"id":449705387,"identity":"942cc42a-f143-43c1-a562-4dd9a5bf8718","order_by":6,"name":"Xiaoshan Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaoshan","middleName":"","lastName":"Yang","suffix":""},{"id":449705388,"identity":"8768acc4-1155-431e-a51a-0f1b2d5cabe2","order_by":7,"name":"Nan Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Chen","suffix":""},{"id":449705389,"identity":"80303293-9eaf-4c8c-8a63-e456c9ea5f6e","order_by":8,"name":"Xiaojun Chen","email":"","orcid":"","institution":"Advanced Molecular Pathology Institute of Soochow University and SANO","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Chen","suffix":""},{"id":449705390,"identity":"580c9393-cb11-45d5-a833-92f5b58aec3a","order_by":9,"name":"Meng-Yao Wu","email":"","orcid":"","institution":"the First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Meng-Yao","middleName":"","lastName":"Wu","suffix":""},{"id":449705391,"identity":"ae2b6ae7-83b6-4dcd-b595-b33e662e1f87","order_by":10,"name":"Huafei Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huafei","middleName":"","lastName":"Chen","suffix":""},{"id":449705392,"identity":"e12a6d8b-0c71-4aae-906a-54de23bbf038","order_by":11,"name":"Lijun Meng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Meng","suffix":""},{"id":449705393,"identity":"52c61ba7-caff-49b2-8325-b83bb15fe841","order_by":12,"name":"Min Tao","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Tao","suffix":""}],"badges":[],"createdAt":"2025-04-23 14:56:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6513808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6513808/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82152077,"identity":"93b66e0d-b7ce-43df-adf3-5e6113dc654f","added_by":"auto","created_at":"2025-05-07 07:22:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1195161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisease Timeline\u003c/strong\u003e. (A) PET-CT showed a metabolically active mass on August 20, 2019. The grayscale background represented the anatomical structure. Tumor contained MYH10::RET. (B) Multiple metastatic lesions (red arrow) appeared in the left lung on June 10, 2020 (CT lung window). Lung tumor also contained MYH10::RET. (C) Significant reduction in lung metastases after Pralsetinib therapy, nearly achieving complete remission, was observed on February 8, 2021 (CT lung window). (D) Severe lung infection was evident on August 12, 2021 (CT lung window). (E) Marked improvement in lung infection was noted on September 2, 2021. (F) A new lesion in the right lung (red arrow) developed on February 15, 2022, six months after discontinuing treatment. (G) Following treatment with Selpercatinib, the lung lesion was nearly resolved by June 10, 2022. (H) Renewed progression of pulmonary lesions with pericardial effusion was observed on January 18, 2024. Biopsy revealed a TPM3::NTRK1 rearrangement in addition to the MYH10::RET fusion. (I) Significant improvement in the left pulmonary lesion and pericardial effusion was noted on April 18, 2024, with combination therapy of Entrectinib and Selpercatinib. (J) Disease progression with recurrent pulmonary lesions and pericardial effusion was observed on August 13, 2024 (CT lung window). Biopsy showed kinase domain mutations from both TPM3::NTRK1 and MYH10::RET. (K) Tumor shrinkage was significant on September 20, 2024, with combination therapy of Repotrectinib and Selpercatinib. (L) The most recent follow-up on February 20, 2025, showed continued reduction in pulmonary metastases.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/bf90cf6002c16efba16997c5.png"},{"id":82155530,"identity":"e0f249ff-cd5d-4554-b87b-ff612378da67","added_by":"auto","created_at":"2025-05-07 07:38:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3599161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic profiling of the primary spindle cell tumor.\u003c/strong\u003e (A) H\u0026amp;E staining showed spindle cells arranged in fascicles, with hemangiopericytoma-like vasculature, and areas of necrosis observed. (B-D) IHC was negative for CD34, S100, and pan-TRK. (E) Targeted DNA NGS analysis showed CNVs including \u003cem\u003eCDKN2A/B\u003c/em\u003e homozygous loss at 9p21 and gain of chromosomes 1p, 16, 20q, and 21. (F) IGV images showed a non-functional \u003cem\u003eRET \u003c/em\u003erearrangement between \u003cem\u003eFGF20 \u003c/em\u003eand \u003cem\u003eRET\u003c/em\u003e. (G) FISH showed that the 5’\u003cem\u003eRET \u003c/em\u003egreen signal separated from the 3’\u003cem\u003eRET\u003c/em\u003e red signal, confirmed \u003cem\u003eRET\u003c/em\u003e rearrangement. Note that an extra 5’\u003cem\u003eRET \u003c/em\u003esignal was present, consistent with a complex \u003cem\u003eRET \u003c/em\u003erearrangement. (H) Targeted RNA NGS identified the \u003cem\u003eMYH10::RET\u003c/em\u003e fusion, in which the first 32 exons of \u003cem\u003eMYH10\u003c/em\u003e are fused with the last 9 exons of \u003cem\u003eRET.\u003c/em\u003e The exon structure and functional domains of wild-type \u003cem\u003eMYH10, RET\u003c/em\u003e, and \u003cem\u003eMYH10::RET\u003c/em\u003efusion are illustrated in a schematic. The breakpoint is indicated by a vertical dotted line. IQ: IQ calmodulin-binding motif; CLD1-4: RET Cadherin-like domain1–4; CRD: Cysteine-rich domain; TM: transmembrane domain; TK: Tyrosine Kinase domain.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/b020ea0f2c436f06e1cf2757.png"},{"id":82154573,"identity":"4b3ac232-966d-408b-b17c-29de1f08a926","added_by":"auto","created_at":"2025-05-07 07:30:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2550795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic profiling of the metastatic lesions. \u003c/strong\u003e(A) Targeted RNA NGS identified a new \u003cem\u003eTPM3::NTRK1\u003c/em\u003e fusion, where the first 7 exons of \u003cem\u003eTPM3\u003c/em\u003e are fused to the last 8 exons of \u003cem\u003eNTRK1\u003c/em\u003e. The exon structure and functional domains of wild-type \u003cem\u003eTPM3\u003c/em\u003e, \u003cem\u003eNTRK1\u003c/em\u003e, and the \u003cem\u003eTPM3::NTRK1\u003c/em\u003e fusion are illustrated schematically, with the breakpoint marked by a vertical dotted line. C1-2: Cysteine clusters C1 and C2; LRR1-3: Leucine-rich regions (LRR) 1–3; Ig-like 1-2: Immunoglobulin-like domain 1-2; TM: transmembrane domain; TK: Tyrosine Kinase domain. (B) H\u0026amp;E staining showed that the lung tumor shared a similar morphology with the primary knee tumor. (C-E) IHC was positive for CD34 and pan-TRK, and negative for S100. (F) FISH confirmed \u003cem\u003eNTRK1\u003c/em\u003e rearrangement, with separation of 5’\u003cem\u003eNTRK1\u003c/em\u003e (green signals) from 3’\u003cem\u003eNTRK1\u003c/em\u003e (red signals). The same slide was stripped and re-hybridized with a \u003cem\u003eRET\u003c/em\u003e probe set, revealing\u003cem\u003e RET\u003c/em\u003e rearrangements in the same group of cells, indicated by the separation of 5’\u003cem\u003eRET\u003c/em\u003e (green signals) from 3’\u003cem\u003eRET\u003c/em\u003e (red signals).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/f94f0b55ec7ba9b2716d58f7.png"},{"id":82152086,"identity":"72cef3ca-9fa5-4485-9c94-4d2a02fe4004","added_by":"auto","created_at":"2025-05-07 07:22:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2765770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional evaluation of MYH10::RET and CCDC6::RET. \u003c/strong\u003e(A) Both MYH10::RET and CCDC6::RET are located in the cytoplasm of lentivirus-transduced NIH3T3 cells. (B-F) MYH10::RET-expressing cells showed increased proliferation (B), migration (C,D), and invasion (E,F) compared to CCDC6::RET-expressing cells and control cells expressing empty vectors. **p \u0026lt; 0.01, ****p \u0026lt; 0.0001.\u003cstrong\u003e \u003c/strong\u003eScale bars are 100 μm. (G-I) Both MYH10::RET-expressing cells and\u003cem\u003e \u003c/em\u003eCCDC6::RET-expressing cells were sensitive to the RET inhibitors selpercatinib (G), pralsetinib (H), and cabozantinib (I).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/8ffd80c9e2cd1ff5f66b0444.png"},{"id":82152079,"identity":"28989efd-607e-4980-9c6e-c17ac08a738d","added_by":"auto","created_at":"2025-05-07 07:22:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1149513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinase activities of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYH10::RET\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCCDC6::RET\u003c/strong\u003e\u003c/em\u003e. (A) Western blotting showed phosphorylation of both \u003cem\u003eMYH10::RET\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003eand their downstream MAPK. Signals were visualized using the LiCor Odyssey and phospho-RET、MAPK and phospho-MAPK were quantified with Image J. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. (B) \u003cem\u003eMYH10::RET\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003e were pulled down from cell lysates and kinase assay was performed by incubating with IGF peptide (1 mg/ml) and increasing concentrations of ATP. Data represent the mean ± SEM, n = 4 from two different protein preparations; ****p \u0026lt; 0.0001, two-way ANOVA Bonferroni test. (C) Catalytic efficiency constants (Kcat/KM, fold difference). (D) 9 \u003cem\u003eRET\u003c/em\u003e fusion partners have been described to date. (E) A schematic representation illustrates the breakpoints and retained structural domains of \u003cem\u003eRET\u003c/em\u003efusion variants.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/529bace62c9135752bdbd775.png"},{"id":83682487,"identity":"0702958a-3e90-4d72-b830-1e32655b4608","added_by":"auto","created_at":"2025-05-30 16:29:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11475892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6513808/v1/22cd2a25-40e8-4227-97eb-bfdbd3776b58.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Distinct RET Fusion Partner Genes in RET-Rearranged Spindle Cell Tumors Contribute to Varied Transformation Capacities","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eA group of soft tissue tumors are characterized by the activation of various tyrosine kinases, mostly through chromosome translocations, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eresulting in the formation of chimeric proteins, along with activation point mutations within the tyrosine kinase domains or insertions/deletions within the juxtamembrane domains. To date, 10 tyrosine kinases\u003c/span\u003e have been identified in this group of tumors: \u003cem\u003eRET\u003c/em\u003e, \u003cem\u003eNTRK1/2/3\u003c/em\u003e, \u003cem\u003eROS1\u003c/em\u003e, \u003cem\u003eALK\u003c/em\u003e, \u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eMET\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, and \u003cem\u003eRAF1\u003c/em\u003e\u003csup\u003e\u003cb\u003e1\u0026ndash;3\u003c/b\u003e\u003c/sup\u003e. \u003cem\u003eRET\u003c/em\u003e-rearranged spindle cell tumors show a wide range of histopathologic features, resembling those of lipofibromatosis-like neural tumors (LPF-NT), infantile fibrosarcomas (IF), malignant peripheral nerve sheath tumors (MPNST) and fibrosarcoma. While some of these tumors express S100 and CD34, their immunophenotype\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es are generally\u003c/span\u003e non-specific\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The clinical behavior of these tumors varies widely, ranging from indolent, slow-growing, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand benign lesions to aggressive metastatic malignancies. For example\u003c/span\u003e, four of five patients with the \u003cem\u003eMYH10::RET\u003c/em\u003e fusion were histologically sarcoma-like, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand one of them developed lung metastasis. In contrast, other\u003c/span\u003e \u003cem\u003eRET\u003c/em\u003e rearrangements, such as \u003cem\u003eCCDC6::RET\u003c/em\u003e and \u003cem\u003eNCOA4::RET\u003c/em\u003e, are associated with LPF-NT that is effectively treated with surgical resection alone, even in cases of incomplete excision\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The reasons why the same \u003cem\u003eRET\u003c/em\u003e rearrangements lead to tumors with different prognoses remain to be determined.\u003c/p\u003e \u003cp\u003eAlthough oncogenic receptor tyrosine kinases (RTKs) activate similar downstream signaling pathways, different fusion partners of RTKs can influence tumor cell behavior. For example, NIH3T3 cells expressing seven \u003cem\u003eALK\u003c/em\u003e fusions containing various 5'-fusion partners demonstrated different cell growth in soft agar cultures and varying sensitivities to \u003cem\u003eALK\u003c/em\u003e inhibitors. Additionally, \u003cem\u003eALK\u003c/em\u003e fusion proteins have different kinase activities\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Fusion partners can also affect cellular localization; for instance, \u003cem\u003eETV6::NTRK3\u003c/em\u003e expressed in melanocytes was found in both the nucleus and cytoplasm, leading to epithelioid morphology, whereas \u003cem\u003eMYO5A::NTRK3\u003c/em\u003e was exclusively nuclear and resulted in spindle cell morphology\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Similar variability was observed \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003efor\u003c/span\u003e th\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ee non-RTK fusion proteins. In acute promyelocytic leukemia (APML), patients with the classic\u003c/span\u003e \u003cem\u003ePML::RARA\u003c/em\u003e fusion respond to all-trans retinoic acid (ATRA), resulting in a cure for once-deadly leukemia. Conversely, patients with non-classic \u003cem\u003eRARA\u003c/em\u003e fusions featuring different 5' partners show varied responses to ATRA, with some being unresponsive to therapy\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we present a case of spindle cell sarcoma with \u003cem\u003eMYH10::RET\u003c/em\u003e fusion showing a fibrosarcoma-like morphology and lung metastasis. To investigate why different \u003cem\u003eRET\u003c/em\u003e fusions led to varying clinical outcomes, we established isogenic cell lines stably expressing either \u003cem\u003eMYH10::RET\u003c/em\u003e or \u003cem\u003eCCDC6::RET\u003c/em\u003e. We found that cells expressing \u003cem\u003eMYH10::RET\u003c/em\u003e had increased proliferation, migration, and invasion compared to those expressing \u003cem\u003eCCDC6::RET\u003c/em\u003e, reflecting their clinical behavior. Further analysis showed that \u003cem\u003eMYH10::RET\u003c/em\u003e had \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea stronger kinase activity\u003c/span\u003e than \u003cem\u003eCCDC6::RET\u003c/em\u003e. We conclude\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed that the activation level of\u003c/span\u003e \u003cem\u003eRET\u003c/em\u003e is modulated by different fusion partners, likely contributing to the heterogeneous clinical behaviors observed in these tumors. These results may have clinical implications and may potentially influence patient management strategies.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eThe tumor tissue was fixed in formalin, embedded in paraffin (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFFPE\u003c/span\u003e, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFormalin Fixed Paraffin Embedded) and cut into 5 \u0026micro;m sections. Immunohistochemical staining for pan-TRK (ab181560, Abcam, UK), CD34 (Kit-0004, MXB, China), and S100 (ab11428, Abcam, UK) was carried out according to the following protocol: the slides were baked\u003c/span\u003e for 1 h at 60\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026deg;C\u003c/span\u003e, deparaffinized, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand rehydrated using 100% xylene, 100% ethanol, and running water, respectively. The slides were blocked with a solution containing 10% normal serum and 1% bovine serum albumin (BSA) in Tris-buffered saline\u003c/span\u003e and incubated with primary antibodies for 2 h. Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand the slides were incubated with a horseradish peroxidase-labeled polymer (DAKO).\u003c/span\u003e Tissue sections were developed using 3,3\u0026rsquo;-diaminobenzidine (DAKO) as the chromogen and counterstained with Mayer\u0026rsquo;s hematoxylin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTargeted DNA next-generation sequencing\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was isolated from FFPE tumor tissue sections using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea QIAamp DNA Micro Kit\u003c/span\u003e (Cat: 56,304; Qiagen, Germany). Three hundred nanograms DNA \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(300 ng) was fragmented to a size of 200\u0026ndash;300 bp\u003c/span\u003e using a Bioruptor Pico (Diagenode, Denville, NJ, USA), and library preparation was performed using the Rapid Plus DNA Lib Prep Kit for Illumina (RK20208, ABclonal) according to the manufacturer's instructions. The libraries were hybridized with a pool of biotin-labeled bait oligos targeting 638 tumor-associated genes for 16 h. Targeted regions were captured with streptavidin beads, followed by PCR amplification and sequencing as paired-end 150-bp reads on an Illumina NextSeq 6000 instrument. Sequencing reads were aligned to the reference genome (hg19) using BWA-MEM. Analyses of single-nucleotide variations (SNVs), insertion/deletion (indels), copy number variations (CNVs), and structural variations (SVs) were carried out using SeqNext software (JSI, Germany) and proprietary pipelines developed by Sano Medical Laboratories (China).\u003c/p\u003e\n\u003ch3\u003eFluorescence in situ hybridization (FISH)\u003c/h3\u003e\n\u003cp\u003eFISH analysis was performed on 5-\u0026micro;m FFPE tumor tissue slides with two colored split-apart probes (5\u0026rsquo;probe green and 3\u0026rsquo; probe red) for \u003cem\u003eRET\u003c/em\u003e and \u003cem\u003eNTRK1\u003c/em\u003e from Betrue, China. The slides were deparaffinized in xylene, rehydrated, and treated with 750 U/ml of pepsin digest\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eion solution (Cat: P6887, Sigma Aldrich, USA) for 10 min\u003c/span\u003e, followed by incubation in 10% buffered formalin for another 10 min. The slides and probes were separately denatured and hybridization was performed overnight at 37\u0026deg;C. After hybridization, the slides were washed in 0.4 \u0026times; SSC/0.3% NP-40 at 73\u0026deg;C for 3 min and then counterstained with DAPI.\u003c/p\u003e\n\u003ch3\u003eTargeted RNA next-generation sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA from FFPE tumor tissue sections was extracted with TRIZOL reagent according to the manufacturer's instructions (Cat: 10296010, ThermoFisher, Invitrogen, USA). Reverse transcription was performed using 100 ng total RNA, and end repair and adaptor ligation were performed following standard \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eNGS\u003c/span\u003e (Next-generation sequencing) p\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003erotocols (Cat: E7771 and E6111, NEB, USA). PCR enrichment was performed using 641 gene-specific primers specific to 118 genes commonly involved in solid tumors, and the enriched PCR products were sequenced on a NovaSeq 6000 platform (Illumina, USA). Sequencing reads were analyzed using the SeqNext software (JSI, Germany).\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eLentivirus\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eCCDC6::RET\u003c/em\u003e and \u003cem\u003eMYH10::RET\u003c/em\u003e fusion genes were cloned into \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe lentiviral plasmid vectors. These plasmids, along with empty vectors for control cells, were co\u003c/span\u003etransfected with PAPAX2 and PMD2.G into NIH3T3 cells. The supernatant from transfected NIH3T3 cells was collected and filtered 48\u0026ndash;72 h post-transfection. Target cells were then infected with lentiviral particles in the presence of 10 \u0026micro;g/ml polybrene reagent. Following two weeks of puromycin selection, the cells were harvested for subsequent experiments.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell counting kit-8\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e cells were \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethen transferred to\u003c/span\u003e 96-well plate\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es. After cell attachment, the cells were treated with various TKIs for 3 to 4 days, including\u003c/span\u003e selpercatinib (HY-114370, MCE), pralsetinib (S8716, Selleck), and cabozantinib (S1119, Selleck). The culture medium was then mixed \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eat a 10:1 ratio with the CCK8 solution (Cat: C0039, Beyotime, China) and added to the wells in the dark. After 2\u003c/span\u003e h of incubation at 37\u0026deg;C, the absorbance (OD) at 450 nm was measured using a microplate reader.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eNIH3T3 cells were collected and inoculated on both sides of the scratch chamber (Culture-Insert 2 Well in \u0026micro;-Dish 35 mm, Cat: 81176, IBIDI). The cells were incubated with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea medium containing 10%\u003c/span\u003e fetal bovine serum and 1% penicillin until \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe cells were attached to the wall. After removing the scratch chamber, the cells were cultured in low\u003c/span\u003e-serum medium. The healing rates of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe different cell groups\u003c/span\u003e were recorded at 0, 24, and 48 h.\u003c/p\u003e\n\u003ch3\u003eTranswell migration assay\u003c/h3\u003e\n\u003cp\u003eThe Transwell chamber was placed in a 24-well culture plate. NIH3T3 cells were harvested and resuspended in serum-free medium. A total of 200 \u0026micro;L of the cell suspension was added to the upper chamber and 600 \u0026micro;L of medium containing 10% serum was added to the lower chamber as a chemoattractant. Following incubation at 37\u0026deg;C for 48 h, the chamber was gently washed twice with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePBS (\u003c/span\u003ePhosphate-Buffered Saline), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003efixed with methanol, and stained with crystal violet solution.\u003c/span\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence colocalization\u003c/h2\u003e \u003cp\u003eApproximately 5 \u0026times; 10⁴ cells were seeded in confocal petri dishes. After cell attachment, the \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ecells were fixed with formaldehyde solution and incubated overnight with\u003c/span\u003e mouse anti-FLAG tag antibody (bsm-33346M, Bioss, 1:200). The following day, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe cells were incubated with a fluorescently labeled secondary antibody (ab150115, Abcam, 1:200) for 1 h\u003c/span\u003e. After washing with phosphate-buffered saline (PBS), an antifade solution containing DAPI (Cat: P0131, Beyotime) was applied. Images were captured using an Olympus fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis and Co-Immunoprecipitation (CO-IP)\u003c/h2\u003e \u003cp\u003eCell lysates were quantified using a BCA Protein Assay Kit (Cat:23225, Thermo \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFisher, Invitrogen, USA). Equal amounts of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to\u003c/span\u003e polyvinylidene fluoride (PVDF) membrane\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es (IPVH00010, Millipore). The membrane was blocked with 5% skim milk (36120ES76\u003c/span\u003e; Yeasen) for 1 h at room temperature and incubated with the corresponding antibodies overnight at 4\u0026deg;C in a shaker. The membrane was washed three times with TBST (Solarbio, T1082), incubated with anti-rabbit or \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eanti-mouse antibodies for 1 h at room temperature, and detected with an enhanced chemiluminescence solution (B\u003c/span\u003eio-Rad). For immunoprecipitation, the protein lysates were incubated with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ean Anti-Flag Tag and microbeads, and the free proteins in the supernatant were removed by magnetic separation. Proteins were eluted from the beads and analyzed by\u003c/span\u003e western blotting. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e antibodies used \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewere as follows: Phospho-RET (Tyr905)\u003c/span\u003e antibody (Cat: 3221, CST), RET (E1N8X) XP\u0026reg; rabbit mAb (Cat: 14556, CST), Mouse Anti-Flag Tag antibody (bsm-33346M, Bioss, 1:200), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand Rabbit Anti-beta-Actin (Loading Control) antibody (BS-0061R, BIOSS).\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKinase Assays\u003c/h2\u003e \u003cp\u003eThe activities of the two \u003cem\u003eRET\u003c/em\u003e fusion kinases were compared using an ADP-Glo Kinase Assay Kit (Promega Corporation, Madison, WI, USA). \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e reagents required for the reaction were configured according to the manufacturer\u0026rsquo;s instructions. For each reaction system, CO-IP-purified \u003cem\u003eRET\u003c/em\u003e kinase samples, IGF (1 mg/mL), and varying concentrations of ATP (from 0 to 1000 \u0026micro;M) were added, followed by incubation at room temperature for 60 min to allow the kinase to catalyze the substrate phosphorylation reaction. ADP-Glo\u0026trade; Reagent (final concentration 10 \u0026micro;M) was added to each well and gently shaken to ensure mixing to terminate the kinase reaction. The wells were incubated at room temperature for 40 min to allow conversion of ADP to ATP. Kinase Detection Reagent was then added to each well, mixed with gentle shaking, and incubated at room temperature for 60 min. The luminescence signal\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e were measured using a chemiluminescence instrument.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eGenomic profile of a\u003c/b\u003e \u003cb\u003eRET\u003c/b\u003e\u003cb\u003e-rearranged spindle cell tumor\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA 37-year-old male presented with a subcutaneous mass on the lateral side of the left knee. PET-CT revealed a well-defined, hypermetabolic soft tissue mass measuring 159 \u0026times; 92 \u0026times; 100 mm, containing both solid and cystic/necrotic components \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e).\u003c/b\u003e A complete surgical excision was performed. Histological examination of formalin-fixed, paraffin-embedded (FFPE) tissue showed hypercellular spindle cells arranged in fascicles with hemangiopericytoma-like vasculature and areas of necrosis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Immunohistochemical staining revealed a null immunophenotype profile, with tumor cells negative for CD34, S100, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand pan-TRK\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D\u003cb\u003e)\u003c/b\u003e. Based on these findings, a diagnosis of malignant spindle cell tumor was established.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTargeted DNA next-generation sequencing (NGS) revealed \u003cem\u003eCDKN2A/B\u003c/em\u003e homozygous loss, 9q copy-neutral loss of heterozygosity (CN-LOH), gain of chromosomes 1p, 16, 20q, and 21, and an \u003cem\u003eFGF20::RET\u003c/em\u003e rearrangement. The \u003cem\u003eRET\u003c/em\u003e breakpoint is located in intron 11, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich is a common site for\u003c/span\u003e \u003cem\u003eRET\u003c/em\u003e rearrangements. However, due to incompatible transcription\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eal orientation, the\u003c/span\u003e \u003cem\u003eFGF20::RET\u003c/em\u003e rearrangement did not contain a reading frame \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F\u003cb\u003e)\u003c/b\u003e. Fluorescence in situ hybridization (FISH) with a \u003cem\u003eRET\u003c/em\u003e split-apart probe confirmed the \u003cem\u003eRET\u003c/em\u003e rearrangement \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Subsequently, targeted RNA NGS identified an \u003cem\u003eMYH10::RET\u003c/em\u003e fusion with an intact reading frame and breakpoints, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003econsistent with previously reported\u003c/span\u003e \u003cem\u003eMYH10::RET\u003c/em\u003e rearrangements \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. The MYH10::RET fusion \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eprotein contained the myosin head domain from MYH10 and the intact kinase domain from RET. The oncogenic mechanism likely involves myosin head-mediated dimerization, leading to autophosphorylation and\u003c/span\u003e RET kinase \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eactivation. The discrepant results between\u003c/span\u003e DNA and RNA NGS are likely due to multiple \u003cem\u003eRET\u003c/em\u003e fusions at the DNA level, including both \u003cem\u003eFGF20::RET\u003c/em\u003e and \u003cem\u003eMYH10::RET\u003c/em\u003e. DNA NGS detected \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003enonfunctional\u003c/span\u003e \u003cem\u003eFGF20::RET\u003c/em\u003e but missed functional \u003cem\u003eMYH10::RET\u003c/em\u003e. This underscores the superiority of RNA NGS over DNA NGS in detecting functional fusions as it effectively eliminates all non-transcript rearrangements often observed in DNA NGS, as previously reported\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTen months after \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe surgery, the patient developed lung metastasis\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, which was biopsied and confirmed to carry the same \u003cem\u003eMYH10::RET\u003c/em\u003e fusion. The patient was enrolled in a clinical trial and treated with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e400 mg of\u003c/span\u003e pralsetinib (BLU-667), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich\u003c/span\u003e resulted in a significant reduction in lung metastases, nearly achieving complete remission \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. However, 11 months later, the patient developed a severe cough and fever. Chest \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ecomputed tomography (CT) s\u003c/span\u003ehowed severe infection in both lungs and potentially drug-associated pneumonia \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Subsequently, Pralsetinib was discontinued and the patient withdrew from the clinical trial. Microbiome NGS of bronchoalveolar lavage fluid detected human herpesvirus 5 (cytomegalovirus) infection with 45.28% abundance and Pneumocystis infection. Treatment with Ganciclovir, Cotrimoxazole, and human hemoglobin effectively controlled the bilateral lung infection\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e. At this time, chest CT showed no evident tumor lesions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, and the patient did not receive further antitumor therapy but underwent regular follow-up. Six months later, a new lesion \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ehad developed in the right lung\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. The patient was subsequently treated with 160 mg \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eselpercatinib twice daily, which result\u003c/span\u003eed in significant tumor shrinkage and stable disease \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Nearly two years later, the patient presented with marked chest tightness and dyspnea. Chest \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ecomputed tomography (CT) indicated renewed progression of the pulmonary lesion, accompanied by pericardial effusion\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. A lung tumor biopsy was then performed, which showed a new \u003cem\u003eTPM3::NTRK1\u003c/em\u003e rearrangement in addition to \u003cem\u003eMYH10::RET\u003c/em\u003e fusion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Histologically, the tumor cells showed similar morphology to the primary knee tumor, but immunohistochemistry (IHC) showed positivity for CD34 and pan-TRK, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich are\u003c/span\u003e negative \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003emarkers in the primary tumor\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the same tumor cells harbored both \u003cem\u003eMYH10::RET\u003c/em\u003e and \u003cem\u003eTPM3::NTRK1\u003c/em\u003e rearrangements, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eor if they were present in different cell populations, successive FISH was performed on the same cell group, first with a\u003c/span\u003e \u003cem\u003eRET\u003c/em\u003e split-apart probe set, followed by an \u003cem\u003eNTRK1\u003c/em\u003e split-apart probe set. This confirmed that both rearrangements were present in the same cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, suggesting that the MYH10::RET fusion likely serves as a mechanism \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eof resistance to the RET inhibitor\u003c/span\u003e selpercatinib. The patient was treated with a combination of selpercatinib (160 mg/day) and the \u003cem\u003eNTRK\u003c/em\u003e inhibitor entrectinib (300 mg/day\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). This combination significantly shrank the neoplasm\u003c/span\u003e in the left lung after two months of treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e, but a new lesion merged in the right lung. Over the following 6 months, however, the combination therapy became less effective, and the pulmonary lesions and pericardial effusion showed progression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Genomic profiling of a tumor biopsy \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003especimen revealed drug-resistant mutations in the kinase domains of both kinases\u003c/span\u003e: \u003cem\u003eRET\u003c/em\u003e p.G810S (variant allele frequency, VAF 1.8%) and \u003cem\u003eNTRK1\u003c/em\u003e p.G595R (VAF 14.2%). Consequently, the patient transitioned to a combination therapy of selpercatinib 160 mg daily and repotrectinib 160 mg daily, specifically designed to address solvent-front and gatekeeper mutations associated with 1st and 2nd generation therapies. Eleven days later, a chest CT \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003escan revealed significant tumor shrinkage\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. With this combination regimen, the patient\u0026rsquo;s disease remained stable, as confirmed by the most recent follow-up \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFunctional evaluation of MYH10::RET and CCDC6::RET\u003c/h2\u003e \u003cp\u003eOur patient with \u003cem\u003eMYH10::RET\u003c/em\u003e had a metastatic tumor characterized by a continuously evolving cancer genome. However, tumors with other \u003cem\u003eRET\u003c/em\u003e rearrangements, such as \u003cem\u003eCCDC6::RET\u003c/em\u003e, are often benign. To determine whether distinct RET fusion partners influence oncogenesis at the cellular level, we generated cell lines expressing either \u003cem\u003eMYH10::RET\u003c/em\u003e or \u003cem\u003eCCDC6::RET\u003c/em\u003e. NIH3T3 cells were transduced with lentiviruses expressing \u003cem\u003eMYH10::RET\u003c/em\u003e or \u003cem\u003eCCDC6::RET\u003c/em\u003e, and expression was confirmed by western blot\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eting and immunofluorescence, demonstrating cytoplasmic localization of both fusion proteins\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e While \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing cells showed modestly increased growth compared to control cells, \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells showed significantly greater proliferation than both \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing and control cells at all time points (24, 48, and 72 h), as measured by \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe WST-8 assay (Cell Counting Kit-8; CCK-8)\u003c/span\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the cell scratch assay, both \u003cem\u003eMYH10::RET-\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing cells showed significantly faster migration than control cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells demonstrating markedly greater migration than \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing cells at 24 h and 48 h post-wounding \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D\u003cb\u003e).\u003c/b\u003e Similarly, in a collagen-coated transwell assay to assess invasive capacity, \u003cem\u003eMYH10::RET-\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing cells were significantly more invasive than control cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ealthough\u003c/span\u003e \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells were significantly more invasive than their \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing counterparts (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE,F\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells expressing \u003cem\u003eCCDC6::RET\u003c/em\u003e and \u003cem\u003eMYH10::RET\u003c/em\u003e fusions were treated with three tyrosine kinase inhibitors specifically targeting RET. Both cell lines were sensitive to all three inhibitors \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I\u003cb\u003e)\u003c/b\u003e, although the degree of sensitivity varied. The IC50 values for \u003cem\u003eMYH10::RET-\u003c/em\u003eexpressing cells were 3.858 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand 3.004 \u0026micro;M for\u003c/span\u003e cabozantinib, selpercatinib, and 2.59 \u0026micro;M for Pralsetinib. In contrast, the IC50 values for \u003cem\u003eCCDC6::RET\u003c/em\u003e cells were 19.49 \u0026micro;M for Cabozantinib, 8.712 \u0026micro;M for Selpercatinib, and 3.836 \u0026micro;M for Pralsetinib. These results suggest that \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells are more sensitive to RET inhibitors than \u003cem\u003eCCDC6::RET\u003c/em\u003e-expressing cells, likely reflecting the faster growth of \u003cem\u003eMYH10::RET\u003c/em\u003e cells.\u003c/p\u003e \u003cp\u003eTo determine whether MYH10::RET and CCDC6::RET have different kinase activities, FLAG-tagged MYH10::RET and CCDC6::RET were immunoprecipitated from lysates of cells stably expressing these fusion proteins. The phosphorylation status of rearranged RET was assessed using a phospho-RET (Tyr905) antibody. Both MYH10::RET and CCDC6::RET are constitutively phosphorylated and activated. Notably, the phosphorylation level of MYH10::RET was significantly higher than that of CCDC6::RET. We further evaluated the phosphorylation of MAPK, a key effector of the RET signaling pathway. Both MYH10::RET- and CCDC6::RET-expressing cells had significantly higher MAPK phosphorylation than control cells. Moreover, MAPK phosphorylation was higher in MYH10::RET-expressing cells than that in CCDC6::RET-expressing cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Finally, an \u003cem\u003ein vitro\u003c/em\u003e kinase assay was performed to compare the kinase activities of MYH10::RET and CCDC6::RET using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe IGF peptide, a known substrate of RET kinase. The results demonstrated that the kinase activity of MYH10::RET was approximately three\u003c/span\u003e-fold higher than that of CCDC6::RET \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe report a case of metastatic spindle cell sarcoma initially driven by an \u003cem\u003eMYH10::RET\u003c/em\u003e rearrangement, which subsequently acquired an \u003cem\u003eNTRK\u003c/em\u003e rearrangement as a mechanism \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eof resistance to RET inhibitor therapy. Further genomic evolution has led to the emergence of kinase domain mutations in both RET and NTRK following treatment with combined RET and NTRK inhibitors. Ultimately, disease control was achieved\u003c/span\u003e using the next-generation tyrosine kinase inhibitor, repotrectinib. This case highlights the critical importance of sequential molecular profiling for monitor\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eing tumor evolution and guid\u003c/span\u003eing precision therapy.\u003c/p\u003e \u003cp\u003eSpindle cell tumors with RET rearrangement\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es present a wide clinical spectrum, ranging from benign lesions to aggressive high-grade sarcomas. The underlying mechanisms driving this variability in clinical behavior remain unclear. To date\u003c/span\u003e, nine \u003cem\u003eRET\u003c/em\u003e fusion partners have been identified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. We hypothesized that differences in fusion partners \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewould contribute to varying transformation capabilities. In this study, we established isogenic cell lines stably expressing\u003c/span\u003e \u003cem\u003eMYH10::RET\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003e, which are associated with metastatic tumors and benign LPF-NT\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es, respectively. Cellular assays revealed that\u003c/span\u003e \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells showed significantly faster growth, increased migration, and greater invasiveness than cells expressing \u003cem\u003eCCDC6::RET\u003c/em\u003e. These observations closely mimic\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eked the clinical behaviors associated with these fusion proteins. Further analysis confirmed that\u003c/span\u003e \u003cem\u003eMYH10::RET\u003c/em\u003e had substantially higher kinase activity than \u003cem\u003eCCDC6::RET\u003c/em\u003e. These findings suggest that different fusion partners may contribute to distinct transformation capabilities through differential kinase activities.\u003c/p\u003e \u003cp\u003eThis study has limitations in \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethat it did not address\u003c/span\u003e the endogenous expression levels of fusion proteins in tumor cells. Expression can be regulated at multiple levels, including mRNA expression, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich is influenced by various promoters associated with different fusion partners. At the protein level, factors such as post-translational modifications, protein folding, and degradation pathways can\u003c/span\u003e affect chimeric protein stability and function. These variations may also affect \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe transformation activities and clinical outcomes.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eRET inhibitors are increasingly used for the treatment of RET-altered cancers. However, similar to many targeted therapies, resistance to RET inhibitors can develop over time, and the resistance mechanisms may include the acquisition of secondary \u003cem\u003eRET\u003c/em\u003e mutations, activation of bypass signaling pathways (e.g., EGFR and MET), histologic transformation, pharmacokinetic factors, epigenetic alterations, and modulation of the tumor microenvironment, all of which allow tumor cells to evade targeted therapy\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our patient, with the \u003cem\u003eMYH10::RET\u003c/em\u003e rearrangement, initially responded to RET inhibitors Pralsetinib and Selpercatinib, but later relapsed due to the emergence of a new \u003cem\u003eNTRK1\u003c/em\u003e rearrangement. The patient then responded to combination therapy with the anti-NTRK drug entrectinib and the anti-RET drug selpercatinib, only to relapse again due to drug-resistant mutations in the kinase domains of both NTRK1 and RET\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. When resistance mutations occur in both \u003cem\u003eRET\u003c/em\u003e and \u003cem\u003eNTRK1\u003c/em\u003e kinase domains, the mutation with the strongest impact on drug binding typically plays a decisive role in determining resistance. The choice of a new targeted therapy depends on the specific mutation profiles and their impact on available inhibitors. Mutation abundance (variant allele frequency, VAF) can provide important clues for determining which resistance mutation is dominant. If \u003cem\u003eRET\u003c/em\u003e G810C/S/R is dominant, targeted therapy can be switched to next-generation RET inhibitors, such as TPX-0046 or LOXO-260. If \u003cem\u003eNTRK1\u003c/em\u003e G595R/G667C is dominant, targeted therapy can be switched to second-generation \u003cem\u003eTRK\u003c/em\u003e inhibitors, such as repotrectinib (TPX-0005), which is designed to overcome \u003cem\u003eTRK\u003c/em\u003e resistance mutations. Our patient subsequently responded to repotrectinib and has remained stable since \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethen.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eOur patient experienced a pralsetinib-related pulmonary infection, which can be easily mistaken for tumor progression on imaging. Pralsetinib (formerly BLU-667) is a potent and selective RET kinase inhibitor designed for the treatment of RET-fusion-positive malignancies, including non-small cell lung cancer (NSCLC) and thyroid cancer. Although Pralsetinib has demonstrated significant clinical efficacy in RET-driven cancers, it is associated with various adverse effects. According to data from the ARROW clinical trial, the most common adverse events include hypertension, fatigue, gastrointestinal disturbances, and hematologic\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eal toxicities. Pulmonary toxicities were reported in 12% of patients, with most cases being of grade 1 or 2 severity, whereas severe pneumonia (grade 3 or 4)\u003c/span\u003e occurred in only 2% of patients, sometimes necessitating dose interruptions or discontinuation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Pulmonary infections associated with pralsetinib can closely mimic tumor progression, highlighting the need for careful diagnostic differentiation. Microbiome NGS may help distinguish infection\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es from disease progression. In our case, the patient was diagnosed with cytomegalovirus infection, which showed improvement following appropriate treatment. The precise mechanisms underlying\u003c/span\u003e pralsetinib-induced pneumonia remain unclear but may involve direct pulmonary toxicity, immune-mediated reactions, or interactions with concomitant therapies and underlying lung conditions. In this patient, a key consideration was the potential off-target effects of pralsetinib, which may increase susceptibility to infections or trigger autoimmune-like reactions in \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe lung tissue.\u003c/span\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we have characterized a rare metastatic spindle cell tumor with \u003cem\u003eMYH10::RET\u003c/em\u003e fusion. The aggressive behavior of cells expressing \u003cem\u003eMYH10::RET\u003c/em\u003e was confirmed using isogenic cell models. Our findings suggest that different \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eRET fusion partners of\u003c/span\u003e \u003cem\u003eRET\u003c/em\u003e contribute to the clinical heterogeneity commonly observed in this group of tumors. Furthermore, this study highlights the critical importance of continuous genomic monitoring to identify emerging drug resistance mechanisms and tailor-targeted therapies using appropriate inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement and Patient Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the Medical Ethics Committee of the Fourth Affiliated Hospital of the Soochow University. Consent for participation was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Science and Technology Plan Project of Suzhou (SZM2022009), Horizontal Research Foundation of Soochow University (P142900324), and the Medical Innovation Research Project of Suzhou Industrial Park (CXYJ2024B03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMT and SX contributed to the conception of the study; QG analyzed and interpreted the patient data; YZ, MY, XY, NC and XC performed the experiment; RL, MH, MW, HC and LM contributed significantly to analysis and manuscript preparation; QG, YZ and MY performed the data analyses and wrote the manuscript; MT and SX performed the analysis with constructive discussions, reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntonescu, C. R. Emerging soft tissue tumors with kinase fusions: An overview of the recent literature with an emphasis on diagnostic criteria. Genes, chromosomes \u0026amp; cancer. 2020; 59: 437\u0026ndash;444.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis, J. L., Al-Ibraheemi, A., Rudzinski, E. R. \u0026amp; Surrey, L. F. Mesenchymal neoplasms with NTRK and other kinase gene alterations. Histopathology. 2022; 80: 4\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKao, Y. C. \u003cem\u003eet al.\u003c/em\u003e Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis-like neural tumor pattern. Genes, chromosomes \u0026amp; cancer. 2020; 59: 575\u0026ndash;583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, B., Suurmeijer, A. J. H., Agaram, N. P. \u0026amp; Antonescu, C. R. Head and Neck Mesenchymal Tumors with Kinase Fusions: A Report of 15 Cases With Emphasis on Wide Anatomic Distribution and Diverse Histologic Appearance. The American journal of surgical pathology. 2023; 47: 248\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis, J. L. et al. Recurrent RET gene fusions in paediatric spindle mesenchymal neoplasms. Histopathology. 2020; 76: 1032\u0026ndash;1041.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChildress, M. A. et al. ALK Fusion Partners Impact Response to ALK Inhibition: Differential Effects on Sensitivity, Cellular Phenotypes, and Biochemical Properties. Molecular cancer research. 2018; 16: 1724\u0026ndash;1736.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede la Fouchardi\u0026egrave;re, A. et al. Fusion partners of NTRK3 affect subcellular localization of the fusion kinase and cytomorphology of melanocytes. Modern pathology: an official journal of the United States and Canadian Academy of Pathology. 2021; 34: 735\u0026ndash;747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeoffroy, M. C. \u0026amp; de Th\u0026eacute;, H. Classic and Variants APLs, as Viewed from a Therapy Response. Cancers (Basel). 2020; 12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Z. et al. Anchored multiplex PCR for targeted next-generation sequencing. Nature medicine. 2014; 20: 1479\u0026ndash;1484.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar-Sinha, C., Kalyana-Sundaram, S. \u0026amp; Chinnaiyan, A. M. Landscape of gene fusions in epithelial cancers: seq and ye shall find. Genome medicine. 2015; 7: 129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClifton-Bligh, R. J. Mechanisms of resistance to RET-directed therapies. Endocrine-related cancer. 2025; 32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuse, M. J. et al. Mechanisms of Resistance to NTRK Inhibitors and Therapeutic Strategies in NTRK1-Rearranged Cancers. Mol Cancer Ther. 2017; 16: 2130\u0026ndash;2143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolomon, B. J. et al. RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2020; 15: 541\u0026ndash;549.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriesinger, F. et al. Safety and efficacy of pralsetinib in RET fusion-positive non-small-cell lung cancer including as first-line therapy: update from the ARROW trial. Annals of oncology: official journal of the European Society for Medical Oncology. 2022; 33: 1168\u0026ndash;1178.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RET Fusion Partner Genes, RET-rearranged spindle cell tumors, NTRK1 fusion, Drug-resistant mutations","lastPublishedDoi":"10.21203/rs.3.rs-6513808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6513808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eRET\u003c/em\u003e-rearranged spindle cell tumors represent a group of soft tissue tumors with heterogeneous clinical presentations. While some tumors are benign, others display malignant behavior with metastatic potential. The underlying causes of this heterogeneity are unknown but are suspected to be associated with the diverse fusion partner genes involved in \u003cem\u003eRET\u003c/em\u003e rearrangements. We describe a unique case of a spindle cell tumor with an \u003cem\u003eMYH10::RET\u003c/em\u003e fusion that originated in the knee and metastasized to the lung. The tumor initially responded to anti-\u003cem\u003eRET\u003c/em\u003e therapy but subsequently relapsed due to a new \u003cem\u003eNTRK1\u003c/em\u003e fusion. The disease has since been effectively managed with a third-generation inhibitor targeting both \u003cem\u003eRET\u003c/em\u003e and \u003cem\u003eNTRKs.\u003c/em\u003e This supports our hypothesis that \u003cem\u003eMYH10\u003c/em\u003e as a fusion partner contributes to the tumor's aggressive clinical course. We established cell lines stably expressing \u003cem\u003eMYH10::RET\u003c/em\u003e and \u003cem\u003eCCDC6::RET\u003c/em\u003e, and we found that \u003cem\u003eMYH10::RET\u003c/em\u003e-expressing cells demonstrated significantly more aggressive phenotypes as compared to cells expressing \u003cem\u003eCCDC6::RET\u003c/em\u003e. Further analysis revealed that \u003cem\u003eMYH10::RET\u003c/em\u003e possesses more potent kinase activity than \u003cem\u003eCCDC6::RET\u003c/em\u003e, providing a mechanistic explanation for the observed differences in tumor behavior. We conclude that distinct \u003cem\u003eRET\u003c/em\u003e fusion partners significantly contribute to the clinical heterogeneity in \u003cem\u003eRET\u003c/em\u003e-rearranged spindle cell tumors.\u003c/p\u003e","manuscriptTitle":"Distinct RET Fusion Partner Genes in RET-Rearranged Spindle Cell Tumors Contribute to Varied Transformation Capacities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 07:22:39","doi":"10.21203/rs.3.rs-6513808/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ee13735-201f-424c-8fc5-31b526c13841","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47853349,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"},{"id":47853350,"name":"Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance"}],"tags":[],"updatedAt":"2025-05-30T16:21:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 07:22:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6513808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6513808","identity":"rs-6513808","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}