Fusion Gene Detection Research Articles

Rhabdomyosarcoma in children and young adults.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood, accounting for 3% of all pediatric malignancies and 50% of all pediatric soft tissue sarcomas. In adolescents and young adults (AYA) however, RMS comprises only 6.5% of all soft tissue sarcomas. Historically, diagnosis and treatment of RMS was based on histologic recognition of the alveolar subtype, which was associated with a worse prognosis. Within the past 20 years, the biologic characteristics of RMS have become clearer, with canonical fusion drivers, PAX3/7::FOXO1, characterizing the alveolar subtype (ARMS) and in turn associated with poor outcome, while chromosomal gains/losses in addition to RAS pathway alterations characterize the embryonal subtype (ERMS). Accordingly, detection of a FOXO1 gene fusion has become a commonplace diagnostic and prognostic tool allowing tumors to be treated based on presence or absence of a FOXO1 gene fusion. However, these cytogenetic and molecular alterations represent only a portion of the molecular landscape found in RMS, and other alterations are found with increasing frequency in various subsets of RMS. Clinical trials basing risk stratification on the presence or absence of the canonical PAX3/7::FOXO1 fusions have had success in identifying the poor responders. Due to poor outcomes, the presence of MYOD1 and TP53 alterations which are common in spindle cell sclerosing RMS (SSRMS) and RMS with anaplasia have also been integrated into trial risk stratification. Therefore, complete histologic and immunophenotypic characterization remain important to better recognize and study these rare subsets of RMS. This article will discuss the challenges of RMS classification including how to combine morphologic, immunophenotypic and molecular data to arrive at an integrated diagnosis. The use of newer techniques such as liquid biopsy and methylation profiling, will also continue to shape the classification of RMS and may further refine risk stratification and prognosis.

Genetic screening of α-thalassemia fusion gene using routine flow-through hybridization.

The fusion gene is a rare form of α-thalassemia. Patients carrying the fusion gene could be misdiagnosed as normal or -α4.2deletion by the conventional thalassemia detection methods. The aim of this study was to present the detection of fusion genes using routine flow-through hybridization, as well as to analyze hematological and molecular characteristics. Samples were collected at our hospital from January 2019 to January 2024. Common thalassemia mutations in the Chinese population were conducted by flow-through hybridization. Samples showing faint coloration at the -α4.2 mutation site on hybridization membrane were considered suspicious. Samples detected as suspicious for -α4.2deletion were rechecked by conventional Gap-PCR. Those samples suspected of having -α4.2deletions were finally confirmed with specific primers for Gap-PCR and Sanger sequencing. Of the 32,083 samples, 25 samples (0.08%) were detected as suspected of having -α4.2 deletion by flow-through hybridization. However, upon reevaluation wtih conventional Gap-PCR reagents capable of detecting -α4.2 deletion, all were found to be negative for the deletion. Specific primers for Gap-PCR were designed, and fusion gene fragments were amplified. DNA sequencing of the HBA gene showed a 7-base mutation corresponding to the α-thalassemia fusion gene. Among the 25 samples, 22 were heterozygous carriers. Three samples were combined: one with Hb QS, one with β-thalassemia, and one with Hb G-Honolulu.Most hematological indices and capillary electrophoresis results were in the normal reference range. The fusion gene was present in 0.08% of the population in the Guangzhou region of Guangdong province, southern China. Conventional genetic methods tend to misdiagnose the fusion gene but can be effectively screened with flow-through hybridization.

SARCP, a Clinical Next-Generation Sequencing Assay for the Detection of Gene Fusions in Sarcomas: A Description of the First 652 Cases

An amplicon-based targeted next generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 of 652 (36.5%) of cases, including 83 distinct fusions in the 238 fusion positive cases, 10 of which had not been previously described. Among the 238 fusion positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR ITDs were identified in 6 of 485 (1.2%) patients. Among the 138 genes in the panel, 66 were involved in one or more fusions and 72 were not involved in any fusions. There was little overlap between the genes involved as 5’-partners (31 different genes) and 3’-partners (37 different genes). This study demonstrates the clinical utility of a next generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas.

Transcriptomic profiles of myxofibrosarcoma and undifferentiated pleomorphic sarcoma correlate with clinical and genomic features.

Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) are two common and aggressive subtypes of soft tissue sarcoma. The aim of this study was to assess potential transcriptomic differences between MFS and UPS tumours and to evaluate the extent to which differences in gene expression profiles were associated with genomic and clinical features. The study included 162 patients with tumours diagnosed as MFS (N = 62) or UPS (N = 100). The patients had been diagnosed and treated at two Swedish sarcoma centres during a 30-year period. For gene expression profiling and gene fusion detection all tumours were analysed using RNA-sequencing and could be compared with data on clinical outcome (N = 155), global copy number profiles (N = 145), and gene mutations (N = 128). Gene expression profiling revealed three transcriptomic clusters (TCs) without any clear separation of MFS and UPS. One TC was associated with longer metastasis-free survival. These tumours had lower tumour mutation burden (TMB), were enriched for a copy number signature representative of focal LOH and chromosomal instability on a diploid background, and were relatively immune-depleted. MFS and UPS showed extensive genomic overlap, with whole genome doubling occurring more frequently among the latter. The results support the idea that MFS and UPS tumours have largely overlapping genomic and transcriptomic features, with UPS tumours showing more aggressive behaviour and more complex genomes. Independently of the tumour type, clinically relevant subgroups were revealed by gene expression analysis, and the finding of multiple genomic subgroups strongly suggest the existence of subgroups of relevance to treatment stratification. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Deep Diving Into the Fusion Across Cancer Types in the Indian Population From Formalin-Fixed Paraffin-Embedded RNA-Exome Data: A Road to Discovering Novel Rearrangements With Clinical Relevance.

Gene fusions are critical oncogenic mutations that drive cancer development and serve as diagnostic and prognostic biomarkers. Despite the increasing cancer burden in India, large-scale analyses of molecular landscapes, particularly gene fusions, have been relatively scarce. This retrospective study used RNA-exome data from 1,392 Indian patients with cancer across 15 major cancer types to explore gene fusions. The study used a comprehensive framework that integrated open-source and proprietary tools to detect gene fusions from formalin-fixed paraffin-embedded tumor samples. The process involved RNA extraction, RNA-exome library preparation, and analysis using tools such as FastQC, DRAGEN RNA Pipeline, STAR-Fusion, and FusionInspector. We validated and filtered potential false-positive fusion calls using AGFusion and FusionAnnotator to annotate fusion breakpoints and their functional impact through various in silico tools. The study found a notable prevalence of FGFR fusions across cancer types, especially FGFR3, with FGFR3::TACC3 as the most recurrent. Kinase fusions were prevalent in the cohort accounting for 37% of incidence in the patients. We also identified 91 novel potential driver fusions, including those involving FGFR2, MET, ESR1, and PDGFRA. This study underscores the critical role of gene fusions as biomarkers in cancer, extending beyond fusion-driven malignancies to encompass all cancer types. Gene fusions serve as both diagnostic markers and tumor-agnostic therapeutic targets within the current cancer treatment paradigm. Our insights into the prevalence of oncogenic drivers and novel targets expand the understanding of gene fusions, shedding new light on their mechanisms and clinical implications.

Transcriptome amplification and fusion gene detection using ultra-trace cells derived from cytology specimens

Tumours are complex and despite scientific breakthroughs, there is much to be elucidated about cancer and its mechanisms. By personalising treatment, outcomes can be improved and genetic analysis is key to this as it allows genetic alterations to be identified and treatment to be precisely targeted. Dr Kenji Amemiya is a cytotechnologist and Chief Clinical Laboratory Technician at the Yamanashi Prefectural Central Hospital Genome Analysis Center (GAC). He is working to better understand the complex inner landscape of cancer and therefore improve cancer detection, diagnosis and treatment. Formalin‐fixed paraffin‐embedded (FFPE) specimens are typically used for genetic testing but there are drawbacks to this, including the FFPE DNA becoming damaged during the fixation process with formalin, resulting in low quality and quantity of DNA. Amemiya is collaborating closely with Chairman of the GAC, Professor Emeritus Masao Omata. They are interested in the use of cytology samples instead of tissue samples in genome analysis, as a less invasive method. Survival rates are increased when genomic analysis is utilised in non-small cell lung cancer (NSCLC). In one project, Amemiya, Omata and the team explored the potential of performing NGS analysis with archived cytological specimens (ACSs). They utilised an integrated NGS platform called the Oncomine Dx Target Test Multi-CDx system to compare the quantity and quality of extracted nucleic acids, the sequencing quality control (QC) and the detected variants between ACSs and FFPE tissues. They found that the RNA integrity was higher in ACSs and there was no difference in sequencing results, compared with FFPE tissues.

A Genomic Language Model for Chimera Artifact Detection in Nanopore Direct RNA Sequencing.

Chimera artifacts in nanopore direct RNA sequencing (dRNA-seq) can significantly distort transcriptome analyses, yet their detection and removal remain challenging due to limitations in existing basecalling models. We present DeepChopper, a genomic language model that precisely identifies and removes adapter sequences from base-called dRNA-seq long reads at single-base resolution, operating independently of raw signal or alignment information to effectively eliminate chimeric read artifacts. By removing these artifacts, DeepChopper substantially improves the accuracy of critical downstream analyses, such as transcript annotation and gene fusion detection, thereby enhancing the reliability and utility of nanopore dRNA-seq for transcriptomics research.

Comparative Analysis of MYB Expression by Immunohistochemistry and RNA Sequencing in Clinical Gene Fusion Detection in Adenoid Cystic Carcinoma.

MYB has been shown to play a central role in oncogenesis in a majority of adenoid cystic carcinomas (ACC). Testing for MYB expression via immunohistochemistry (IHC) or testing for the MYB gene fusion by next-generation sequencing(NGS) have become useful tools for the diagnosis of ACC. In addition, detection of MYB expression may have implications for patient management. A cohort of 35 ACC cases was identified from the archival pathology files of the Massachusetts General Hospital. Cases were tested for MYB expression using a panel of 4 different commercially available MYB antibodies and scored using a modified Allred system. RNA-based NGS for MYB gene fusion detection was also performed. Among 4 different MYB antibodies, the sensitivity for MYB detection ranged from 26 to 97%. When a 30% threshold for determination of MYB immunohistochemical positivity was used, the AB_10900735 IHC clone showed the maximum sensitivity (97%). RNA sequencing revealed 19 (54%) cases positive for MYB fusions, and expression analysis derived from the sequencing data confirmed a significant association between MYB expression and fusion status (p = 0.036). Although less sensitive, the AB_778878 MYB clone showed a significant positive association between IHC staining and MYB RNA expression (R2 = 0.15, p = 0.023). The detection of MYB expression using immunohistochemistry varies significantly depending on the antibody used. Comparison with MYB fusion and transcription levels, as determined by NGS, reveals that MYB has a complex relationship between genetic alterations, transcript levels, and protein abundance.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Detection of rare and novel gene fusions in patients with diffuse glioma: An institutional retrospective study.

Detection of rare and novel gene fusions in patients with diffuse glioma: An institutional retrospective study.

All-in-one bimodal DNA and RNA next-generation sequencing panel for integrative diagnosis of glioma

Previously, we constructed a DNA-based next-generation sequencing (NGS) panel for an integrated diagnosis of gliomas according to the 2021 World Health Organization classification system. The aim of the current study was to evaluate the feasibility of a modified panel to include fusion gene detection via RNA-based analysis. Using this bimodal DNA/RNA panel, we analyzed 210 cases of gliomas and others to identify fusion genes in addition to gene alterations, including TERT promoter (TERTp) mutation and 1p/19q co-deletion, in formalin-fixed paraffin-embedded tissues. Of the 210 patients, fusion genes were detected in tumors of 35 patients. Eighteen of 112 glioblastomas (GBs) harbored fusion genes, including EGFR and FGFR3 fusions. In IDH-mutant astrocytoma, 6 of 30 cases showed fusion genes such as MET and NTRK2 fusions. Eleven molecular GBs and 20 not-elsewhere-classified cases harbored no gene fusions. Other 11 tumors including ependymoma, pilocytic astrocytoma, diffuse hemispheric glioma, infant-type hemispheric glioma, and solitary fibrous tumors exhibited diagnostic fusion genes. Overall, our results suggest that the all-in-one bimodal DNA/RNA panel is reliable for detecting diagnostic gene alterations in accordance with the latest WHO classification. The integrative pathological and molecular strategy could be valuable in confirmation of diagnosis and selection of treatment options for brain tumors.

Fusion partner agnostic approaches improve detection of targetable gene fusions in thyroid cancers

Fusion partner agnostic approaches improve detection of targetable gene fusions in thyroid cancers

Evaluation of Hi-C Sequencing for Detection of Gene Fusions in Hematologic and Solid Tumor Pediatric Cancer Samples.

Hi-C sequencing is a DNA-based next-generation sequencing method that preserves the 3D genome conformation and has shown promise in detecting genomic rearrangements in translational research studies. To evaluate Hi-C as a potential clinical diagnostic platform, analytical concordance with routine laboratory testing was assessed using primary pediatric leukemia and sarcoma specimens. Archived viable and non-viable frozen leukemic cells and formalin-fixed paraffin-embedded (FFPE) tumor specimens were analyzed. Pediatric acute myeloid leukemia (AML) and alveolar rhabdomyosarcoma (A-RMS) specimens with known genomic rearrangements were subjected to Hi-C to assess analytical concordance. Subsequently, a discovery cohort consisting of AML and acute lymphoblastic leukemia (ALL) cases without known genomic rearrangements based on prior clinical diagnostic testing was evaluated to determine whether Hi-C could detect rearrangements. Using a standard sequencing depth of 50 million raw read-pairs per sample, or approximately 5X raw genomic coverage, we observed 100% concordance between Hi-C and previous clinical cytogenetic and molecular testing. In the discovery cohort, a clinically relevant gene fusion was detected in 45% of leukemia cases (5/11). This study provides an institutional proof of principle evaluation of Hi-C sequencing to medical diagnostic testing as it identified several clinically relevant rearrangements, including those that were missed by current clinical testing workflows.

Research progress on fusion genes in tumours

AbstractBackgroundThe concept of gene fusion describes the process of fusing two genes into one, which is closely linked to tumour occurrence and development and may even be the direct cause of some tumours. Due to their tumour‐specific expression and ability to drive tumour occurrence and development, there is great potential for fusion genes to be used as diagnostic markers and targets for specific types of tumours.Main bodyAlthough many fusion genes have been detected so far, they mainly focus on a small number of highly recurrent fusion genes detected in patients' tumours. There are few studies on the functional mechanism and clinical relevance of rare gene fusions. Our review discusses the generation mechanisms, detection methods, biological functions, and mechanisms of action of fusion genes. Additionally, we discuss the clinical significance of fusion gene detection in some tumour types.ConclusionThe function mechanism research of rare gene fusion is very necessary, and more functions of fusion genes independent of unfused/normal genes can be explored in future studies. There is still a long way to go in implementing precision tumour therapy targeting gene fusion.

TLE3 Is a Novel Fusion Partner of JAK2 in Myeloid/Lymphoid Neoplasm With Eosinophilia Responding to JAK2 Inhibition.

Chromosomal rearrangements involving Janus kinase 2 (JAK2) are rare but recurrent findings in lymphoid or myeloid neoplasia. Detection of JAK2 fusion genes is important as patients with aberrantly activated JAK2 may benefit from treatment with tyrosine kinase inhibitors such as ruxolitinib. Here, we report a novel fusion gene between the transcriptional co-repressor-encoding gene transducin-like enhancer of split 3 (TLE3) and JAK2 in a patient initially diagnosed with chronic eosinophilic leukemia with additional mutations in PTPN11 and NRAS. The patient was successfully treated with the JAK2 inhibitor ruxolitinib for 8 months before additional somatic mutations were acquired and the disease progressed into an acute lymphoblastic T-cell leukemia/lymphoma. The present case shows similarities to previously reported cases with PCM1::JAK2 and BCR::JAK2 with regard to disease phenotype and response to ruxolitinib, and importantly, provides an example that also patients harboring other JAK2 fusion genes may benefit from treatment with JAK2 inhibitors.

Performances of the Idylla GeneFusion Assay: contribution to a rapid diagnosis of targetable gene fusions in tumour samples

AimsWe aimed to evaluate the performances of the Idylla GeneFusion Assay (IGFA) designed to detect, in a single, rapid and fully automated assay, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3...

NanoTrans: an integrated computational framework for comprehensive transcriptome analysis with nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) provides the direct access to native RNA strands with full-length information, shedding light on rich qualitative and quantitative properties of gene expression profiles. Here with NanoTrans, we present an integrated computational framework that comprehensively covers all major DRS-based application scopes, including isoform clustering and quantification, poly(A) tail length estimation, RNA modification profiling, and fusion gene detection. In addition to its merit in providing such a streamlined one-stop solution, NanoTrans also shines in its workflow-orientated modular design, batch processing capability, all-in-one tabular and graphic report output, as well as automatic installation and configuration supports. Finally, by applying NanoTrans to real DRS datasets of yeast, Arabidopsis, as well as human embryonic kidney and cancer cell lines, we further demonstrate its utility, effectiveness, and efficacy across a wide range of DRS-based application settings.

Biphenotypic sinonasal sarcoma diagnosed by detection of PAX3-MAML3 fusion gene using integrated whole-genome and transcriptome sequencing.

Biphenotypic sinonasal sarcoma (BSNS) is a double-phenotype sarcoma that shows differentiation in both the nervous and muscular systems. To date, whole-genome and transcriptome sequencing (WGTS) has not been used to analyze BSNS. We report a patient with BSNS who was diagnosed based on PAX3 rearrangement using WGTS. A 71-year-old Japanese male without remarkable symptoms showed a nasal tumor when undergoing computed tomography. Although pathological examination revealed a non-characteristic spindle cell tumor, a definitive diagnosis could not be made based on this examination. Endoscopic sinus surgery was performed for subsequent diagnosis, treatment, and WGTS. WGTS revealed a t(2; 4)(q35; q31.1) reciprocal translocation, resulting in a PAX3-MAML3 fusion gene, leading to a definitive diagnosis of BSNS. We also detected upregulation of the expression of PAX3, MAML3, and 11 known genes involved in neural and myogenic differentiation relevant to the BSNS phenotype. Hence, using WGTS in combination with conventional pathological diagnosis can contribute to a definitive diagnosis of rare cancers, including BSNS, by detecting chromosomal rearrangements or diagnostic markers.

HGG-06. MOLECULAR PATTERNS OF PEDIATRIC HIGH-GRADE GLIOMAS IN SOUTHEAST ASIA

Abstract BACKGROUND Molecular characteristics of pediatric high-grade gliomas are largely based on cohorts derived from North America and Europe. In this study, we compare the molecular findings of a Southeast Asian cohort of pediatric high-grade gliomas with published data. METHODS We collated clinical, histopathological, and molecular data from pediatric high-grade gliomas diagnosed in our laboratory over the past 5 years. Molecular testing methods used included Illumina’s Childhood Cancer Panel (26 cases), FoundationOne’s CDx panel (2 cases), and Archer FUSIONPlex Pan Solid Tumour v2 panel (2 cases). 7 cases had no molecular data. 11 cases underwent additional DNA methylation analysis using Illumina’s Infinium MethylationEPIC v1.0 kit. RESULTS 37 cases of pediatric high-grade gliomas from 34 patients were identified. Patient age ranged from 2 to 26 years (median 14 years). The male-female ratio was 1.1:1. Diffuse midline gliomas, H3K27-altered (DMG, H3K27-altered) constituted 51% of the cases (19/37), followed by diffuse pediatric-type high-grade gliomas, H3-wildtype and IDH-wildtype (32%; 12/37) and diffuse hemispheric gliomas H3G34-mutant (16%; 6/37). The commonest genes with pathogenic single nucleotide variants were TP53 (79%;22/28), followed by H3F3A (54%;15/28). The commonest amplified genes were MYCN and EGFR (both 4/28; 14%). Three gene fusions were detected, none of which arose from infants – 2 were found in diffuse pediatric-type high-grade gliomas, H3-wildtype and IDH-wildtype (CSTF3::ALK and PRCC::NTRK1 fusions), and one occurred in a DMG, H3K27-altered (ERC1::RET fusion). The most common methylation groups identified were DMG, H3K27-altered and diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype, MYCN subtype (both 3/11; 27%). CONCLUSIONS Although small, our Southeast Asian cohort comprised a higher percentage of TP53-mutant high-grade gliomas amongst all pediatric high-grade gliomas as a group. The commonest amplified genes were MYCN and EGFR. We detected gene fusions in non-infant gliomas. These findings suggest geographical differences in the biology of pediatric high-grade gliomas.

Detection of GRM1 gene rearrangements in chondromyxoidfibroma: a comparison of fluorescence in-situ hybridisation,RNA sequencing and immunohistochemical analysis.

Chondromyxoid fibroma (CMF) is a rare, benign bone tumour which arises primarily in young adults and is occasionally diagnostically challenging. Glutamate metabotropic receptor 1 (GRM1) gene encodes a metabotropic glutamate receptor and was recently shown to be up-regulated in chondromyxoid fibroma through gene fusion and promoter swapping. The aim of this study was to interrogate cases of CMF for the presence of GRM1 gene rearrangements, gene fusions and GRM1 protein overexpression. Selected cases were subjected to testing by fluorescent in-situ hybridisation (FISH) with a GRM1 break-apart probe, a targeted RNA sequencing method and immunohistochemical study with an antibody to GRM1 protein. Two cases were subjected to whole transcriptomic sequencing. In 13 of 13 cases, GRM1 protein overexpression was detected by immunohistochemistry using the GRM1 antibody. Of the 12 cases successfully tested by FISH, nine of 12 showed GRM1 rearrangements by break-apart probe assay. Targeted RNA sequencing analysis did not detect gene fusions in any of the eight cases tested, but there was an increase in GRM1 mRNA expression in all eight cases. Two cases subjected to whole transcriptomic sequencing (WTS) showed elevated GRM1 expression and no gene fusions. GRM1 gene rearrangements can be detected using FISH break-apart probes in approximately 75% of cases, and immunohistochemical detection of GRM1 protein over-expression is a sensitive diagnostic method. The gene fusion was not detected by targeted RNA sequencing, due most probably to the complexity of fusion mechanism, and is not yet a reliable method for confirming a diagnosis of CMF in the clinical setting.