IFDlong: an isoform and fusion detector for accurate annotation and quantification of long-read RNA-seq data.

Advancements in long-read transcriptome sequencing (long-RNA-seq) technology have revolutionized the study of isoform diversity. These full-length transcripts enhance the detection of various transcriptome structural variations, including novel isoforms, alternative splicing events, and fusion transcripts. By shifting the open reading frame or altering gene expressions, studies have proved that these transcript alterations can serve as crucial biomarkers for disease diagnosis and therapeutic targets. In this project, we proposed IFDlong, a bioinformatics and biostatistics tool to detect isoform and fusion transcripts using bulk or single-cell long-RNA-seq data. Specifically, the software performed gene and isoform annotation for each long-read, defined novel isoforms, quantified isoform expression by a novel expectation-maximization algorithm, and profiled the fusion transcripts. For evaluation, IFDlong pipeline achieved overall the best performance when compared with several existing tools in large-scale simulation studies. In both isoform and fusion transcript quantification, IFDlong is able to reach more than 0.8 Spearman's correlation with the truth, and more than 0.9 cosine similarity when distinguishing multiple alternative splicing events. In novel isoform simulation, IFDlong can successfully balance the sensitivity (higher than 90%) and specificity (higher than 90%). Furthermore, IFDlong has proved its accuracy and robustness in diverse in-house and public datasets on healthy tissues, cell lines and multiple types of diseases. Besides bulk long-RNA-seq, IFDlong pipeline has proved its compatibility to single-cell long-RNA-seq data. This new software may hold promise for significant impact on long-read transcriptome analysis. The IFDlong software is available at https://github.com/wenjiaking/IFDlong.

FusionVAC22_01: a phase I clinical trial evaluating a DNAJB1-PRKACA fusion transcript-based peptide vaccine combined with immune checkpoint inhibition for fibrolamellar hepatocellular carcinoma and other tumor entities carrying the oncogenic driver fusion.

The DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in fibrolamellar hepatocellular carcinoma (FL-HCC), also known as fibrolamellar carcinoma (FLC), as well as in other tumor entities, thus representing a broad target for novel treatment in multiple cancer entities. FL-HCC is a rare primary liver tumor with a 5-year survival rate of only 45%, which typically affects young patients with no underlying primary liver disease. Surgical resection is the only curative treatment option if no metastases are present at diagnosis. There is no standard of care for systemic therapy. Peptide-based vaccines represent a low side-effect approach relying on specific immune recognition of tumor-associated human leucocyte antigen (HLA) presented peptides. The induction (priming) of tumor-specific T-cell responses against neoepitopes derived from gene fusion transcripts by peptide-vaccination combined with expansion of the immune response and optimization of immune function within the tumor microenvironment achieved by immune-checkpoint-inhibition (ICI) has the potential to improve response rates and durability of responses in malignant diseases. The phase I clinical trial FusionVAC22_01 will enroll patients with FL-HCC or other cancer entities carrying the DNAJB1-PRKACA fusion transcript that are locally advanced or metastatic. Two doses of the DNAJB1-PRKACA fusion-based neoepitope vaccine Fusion-VAC-XS15 will be applied subcutaneously (s.c.) with a 4-week interval in combination with the anti-programmed cell death-ligand 1 (PD-L1) antibody atezolizumab starting at day 15 after the first vaccination. Anti-PD-L1 will be applied every 4 weeks until end of the 54-week treatment phase or until disease progression or other reason for study termination. Thereafter, patients will enter a 6 months follow-up period. The clinical trial reported here was approved by the Ethics Committee II of the University of Heidelberg (Medical faculty of Mannheim) and the Paul-Ehrlich-Institute (P-00540). Clinical trial results will be published in peer-reviewed journals. Trial registration numbers: EU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

ALK-Rearranged Epithelioid and Spindle Cell Neoplasm of the Sinonasal Tract.

Anaplastic lymphoma kinase (ALK)-rearranged mesenchymal neoplasms (non-inflammatory myofibroblastic tumor and non-epithelioid fibrous histiocytoma) have been recently described which tend to occur in the superficial and deep soft tissues. Occurrence as a primary sinonasal neoplasm has not been reported thus far. Herein, we describe the first case of sinonasal ALK-rearranged mesenchymal tumor that harbored remarkable epithelioid and spindle cell morphology. The tumor affected a 40-year-old man who presented with flu-like symptoms and was thought to have influenza A. However, computed tomography demonstrated a nasal polypoid lesion causing curvature of the nasal septum. Histological examination revealed a heterogeneous tumor composed of round to epithelioid cells with foci of spindle cells. The tumor cells exhibited moderate pleomorphism and mitotic activity. By immunohistochemistry, they showed diffuse staining of CD34, S100, ALK (D5F3) and CD30. Fluorescence in situ hybridization analysis demonstrated ALK rearrangement. Subsequent next-generation sequencing (RNA-seq) identified a rare PLEKHH2exon6::ALKexon20 fusion. This study further demonstrates the importance of molecular profiling in identifying kinase fusion-positive soft tissue tumors, particularly for those that arise at unusual sites and display atypical cytomorphology.

Imaging characterization of paediatric tumours with the neurotrophic tyrosine receptor kinase fusion transcript.

OBJECTIVES: The neurotrophic tyrosine receptor kinase (NTRK) fusion transcript (FT) is a major genetic landmark of infantile fibrosarcoma (IFS) and cellular congenital mesoblastic nephroma (cCMN) but is also described in other tumours. The recent availability of NTRK-targeted drugs enhances the need for better identification. We aimed to describe the anatomic locations and imaging features of tumours with NTRK-FT in children. CASE SERIES: Imaging characteristics of NTRK-FT tumours of 41 children (median age: 4 months; 63% <1 year old; range: 0-188) managed between 2001 and 2019 were retrospectively analysed. The tumours were located in the soft tissues (n = 24, including 19 IFS), kidneys (n = 9, including 8 cCMN), central nervous system (CNS) (n = 5), lung (n = 2), and bone (n = 1). The tumours were frequently deep-located (93%) and heterogeneous (71%) with necrotic (53%) or haemorrhagic components (29%). Although inconstant, enlarged intratumoural vessels were a recurrent finding (70%) with an irregular distribution (63%) in the most frequent anatomical locations. CONCLUSION: Paediatric NTRK-FT tumours mainly occur in infants with very variable histotypes and locations. Rich and irregular intra-tumoural vascularization are recurrent findings. ADVANCES IN KNOWLEDGE: Apart from IFS of soft tissues and cCMN of the kidneys, others NTRK-FT tumours locations have to be known, as CNS tumours. Better knowledge of the imaging characteristics may help guide the pathological and biological identification.

Novel MFSD7-ATP5I fusion promotes migration and invasion of human sarcoma.

Fusion genes have been implicated in the development and progression of several types of sarcomas, serving as valuable diagnostic and prognostic markers, as well as potential therapeutic targets. We discovered a novel major facilitator superfamily domain-containing 7 (MFSD7) and adenosine triphosphate 5I (ATP5I) gene fusion from sarcomas. In this study, the MFSD7-ATP5I fusion transcript was screened using RNA sequencing in 55 sarcoma samples and sixteen normal samples. The MFSD7-ATP5I fusion transcript was detected in 58% of sarcoma samples. The correlation between the expression of MFSD7-ATP5I fusion transcript and clinicopathological information was analyzed, and MFSD7-ATP5I expression is associated with marked pleomorphism and lower tumor necrosis. Cell migration and invasion was significantly reduced by knock-down of MFSD7-ATP5I. Cell migration and invasion was increased by overexpression of MFSD7-ATP5I. A phosphokinase assay demonstrated that MFSD7-ATP5I is involved in the GSK-3 pathway. The current study found that MFSD7-ATP5I is associated with increasing pleomorphism and decreasing necrosis of tumors. And our gain and loss of function experiments prove that MFSD7-ATP5I promotes the invasiveness of tumor cells.

Improved detection of clinically relevant fusion transcripts in cancer by machine learning classification.

BACKGROUND: Genomic rearrangements in cancer cells can create fusion genes that encode chimeric proteins or alter the expression of coding and non-coding RNAs. In some cancer types, fusions involving specific kinases are used as targets for therapy. Fusion genes can be detected by whole genome sequencing (WGS) and targeted fusion panels, but RNA sequencing (RNA-Seq) has the advantageous capability of broadly detecting expressed fusion transcripts. RESULTS: We developed a pipeline for validation of fusion transcripts identified in RNA-Seq data using matched WGS data from The Cancer Genome Atlas (TCGA) and applied it to 910 tumors from 11 different cancer types. This resulted in 4237 validated gene fusions, 3049 of them with at least one identified genomic breakpoint. Utilizing validated fusions as true positive events, we trained a machine learning classifier to predict true and false positive fusion transcripts from RNA-Seq data. The final precision and recall metrics of the classifier were 0.74 and 0.71, respectively, in an independent dataset of 249 breast tumors. Application of this classifier to all samples with RNA-Seq data from these cancer types vastly extended the number of likely true positive fusion transcripts and identified many potentially targetable kinase fusions. Further analysis of the validated gene fusions suggested that many are created by intrachromosomal amplification events with microhomology-mediated non-homologous end-joining. CONCLUSIONS: A classifier trained on validated fusion events increased the accuracy of fusion transcript identification in samples without WGS data. This allowed the analysis to be extended to all samples with RNA-Seq data, facilitating studies of tumor biology and increasing the number of detected kinase fusions. Machine learning could thus be used in identification of clinically relevant fusion events for targeted therapy. The large dataset of validated gene fusions generated here presents a useful resource for development and evaluation of fusion transcript detection algorithms.

Fast and sensitive validation of fusion transcripts in whole-genome sequencing data.

BACKGROUND: In cancer, genomic rearrangements can create fusion genes that either combine protein-coding sequences from two different partner genes or place one gene under the control of the promoter of another gene. These fusion genes can act as oncogenic drivers in tumor development and several fusions involving kinases have been successfully exploited as drug targets. Expressed fusions can be identified in RNA sequencing (RNA-Seq) data, but fusion prediction software often has a high fraction of false positive fusion transcript predictions. This is problematic for both research and clinical applications. RESULTS: We describe a method for validation of fusion transcripts detected by RNA-Seq in matched whole-genome sequencing (WGS) data. Our pipeline uses discordant read pairs to identify supported fusion events and analyzes soft-clipped read alignments to determine genomic breakpoints. We have tested it on matched RNA-Seq and WGS data for both tumors and cancer cell lines and show that it can be used to validate both new predicted gene fusions and experimentally validated fusion events. It was considerably faster and more sensitive than using BreakDancer and Manta, software that is instead designed to detect many different types of structural variants on a genome-wide scale. CONCLUSIONS: We have developed a fast and very sensitive pipeline for validation of gene fusions detected by RNA-Seq in matched WGS data. It can be used to identify high-quality gene fusions for further bioinformatic and experimental studies, including validation of genomic breakpoints and studies of the mechanisms that generate fusions. In a clinical setting, it could help find expressed gene fusions for personalized therapy.

Neurotrophic tropomyosin receptor kinase (NTRK) fusion positive tumors: a historical cohort analysis.

BACKGROUND: NTRK gene fusions have been identified in various tumors; some requiring aggressive therapy and sometimes new TRK inhibitors (TRKi). We aimed to describe a national, unselected, retrospective, multicenter cohort. RESEARCH DESIGN AND METHODS: Patients were identified through the French sarcoma diagnostic laboratory at Institut Curie through samples analyzed by RT-qPCR or whole-transcriptome sequencing. RESULTS: From 2001 to 2019, 65 NTRK fusion tumors were identified within 2120 analyses (3.1%): 58 by RNA sequencing (including 20 after RT-qPCR analysis) and 7 exclusively by RT-qPCR. Of the 61 patients identified, 37 patients had infantile soft tissue or kidney fibrosarcomas (IFS), 15 other mesenchymal (Other-MT) and nine central nervous system (CNS) tumors. They encompassed 14 different tumor types with variable behaviors. Overall, 53 patients had surgery (3 mutilating), 38 chemotherapy (20 alkylating agents/anthracycline), 11 radiotherapy, two 'observation strategy' and 13 received TRKi. After a median follow-up of 61.0 months [range, 2.5-226.0], 10 patients died. Five-year overall survival is, respectively, 91.9% [95%CI, 83.5-100.0], 61.1% [95%CI, 34.2-100.0] and 64.8% [95%CI, 39.3-100.0] for IFS, Other-MT, and CNS groups. CONCLUSIONS: NTRK-fusion positive tumors are rare but detection is improved through RNA sequencing. TRKi could be considered at diagnosis for CNS NTRK-fusion positive tumors, some IFS, and Other-MT. TRIAL REGISTRATION: Not adapted.

Fusion transcriptome profiling defines the monoclonal origin of multifocal epithelioid haemangioma of bone.

AIMS: Epithelioid haemangioma (EH) of bone remains a highly controversial entity. Indeed, the WHO classifies EHs of soft tissues as benign tumours, whereas bone EHs are considered intermediate-locally aggressive tumours due to common multifocal presentation and local destructive growth. To gain insights into the clinical behaviour and biology of EH of bone we retrospectively analysed 42 patients treated in a single institution from 1978 to 2021. METHODS AND RESULTS: Multifocal presentation was detected in 17 of 42 patients (40%) primarily as synchronous lesions. Patients were treated with curettage (57%), resection (29%) or biopsy, followed by radiotherapy or embolisation (14%). Follow-up (minimum 24 months) was available for 38 patients, with only five local recurrences (13%) and no death of disease. To clarify whether the synchronous bone lesions in multifocal EH represent multicentric disease or clonal dissemination, four cases were profiled by RNA-sequencing. Separate lesions from the same patient, which showed a similar transcriptional profile, expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints. CONCLUSIONS: These results indicate that, in EH of bone, multifocal lesions are clonally related and therefore represent the spread of a same neoplastic clone rather than simultaneous independent tumours. This finding is in apparent contradiction with the benign clinical course of the disease, and suggests that tumour dissemination in bone EH probably reflects a phenomenon of passive spreading, with tumour cells colonising distal sites while maintaining their benign biological nature.

Disruption of regulatory domains and novel transcripts as disease-causing mechanisms.

Deletions, duplications, insertions, inversions, and translocations, collectively called structural variations (SVs), affect more base pairs of the genome than any other sequence variant. The recent technological advancements in genome sequencing have enabled the discovery of tens of thousands of SVs per human genome. These SVs primarily affect non-coding DNA sequences, but the difficulties in interpreting their impact limit our understanding of human disease etiology. The functional annotation of non-coding DNA sequences and methodologies to characterize their three-dimensional (3D) organization in the nucleus have greatly expanded our understanding of the basic mechanisms underlying gene regulation, thereby improving the interpretation of SVs for their pathogenic impact. Here, we discuss the various mechanisms by which SVs can result in altered gene regulation and how these mechanisms can result in rare genetic disorders. Beyond changing gene expression, SVs can produce novel gene-intergenic fusion transcripts at the SV breakpoints.

Clinical characteristics and outcomes for children, adolescents and young adults with "CIC-fused" or "BCOR-rearranged" soft tissue sarcomas: A multi-institutional European retrospective analysis.

BACKGROUND: In certain rare undifferentiated small round cell sarcomas new specific molecular CIC-DUX4/other partner, BCOR-CCNB3/other partner, YWHAE fusions, or BCOR-ITD (internal tandem duplication) were identified. These new "CIC fused" (CIC-fused/ATXN1::NUTM1) and "BCOR rearranged" (BCOR fused/ITD/ YWHAE) soft tissue sarcomas (STS) are not well described. METHODS: Multi-institutional European retrospective analysis of young patients (0-24 years) with CIC-fused and BCOR rearranged STS. RESULTS: Overall, out of the 60 patients selected, the fusion status was CIC-fused (n = 29), ATXN1::NUTM1 (n = 2), BCOR::CCNB3 (n = 18), BCOR-ITD (n = 7), and YWHAE (n = 3), MAML::BCOR STS (n = 1). The main primaries were abdomen-pelvic (n = 23) and limbs (n = 18). Median age was 14 years (0.9-23.8) and 0.9 (0.1-19.1) for CIC-fused and BCOR-rearranged groups, respectively (n = 29; p < 0.001). IRS stages were I (n = 3), II (n = 7), III (n = 35), and IV (n = 15). Overall, 42 patients had large tumors (>5 cm) but only six had lymph node involvement. Patients received mainly chemotherapy (n = 57), local surgery (n = 50), and/or radiotherapy (n = 34). After a median follow-up of 47.1 months (range, 3.4-230), 33 (52%) patients had an event and 23 patients died. Three-year event-free survivals were 44.0% (95% CI 28.7-67.5) and 41.2% (95% CI 25.4-67.0) for CIC and BCOR groups (p = 0.97), respectively. Three-year overall survivals were 46.3% (95% CI 29.6-72.4) and 67.1% (95% CI 50.4-89.3; p = 0.24), respectively. CONCLUSIONS: Pediatric patients often present with large tumors and metastatic disease, especially CIC sarcomas. Overall outcome is dismal. New treatment options are needed.

Performance Characteristics of Oncomine Focus Assay for Theranostic Analysis of Solid Tumors, A (21-Months) Real-Life Study.

Next generation sequencing analysis is crucial for therapeutic decision in various solid tumor contexts. The sequencing method must remain accurate and robust throughout the instrument lifespan allowing the biological validation of patients' results. This study aims to evaluate the long-term sequencing performances of the Oncomine Focus assay kit allowing theranostic DNA and RNA variants detection on the Ion S5XL instrument. We evaluated the sequencing performances of 73 consecutive chips over a 21-month period and detailed the sequencing data obtained from both quality controls and clinical samples. The metrics describing sequencing quality remained stable throughout the study. We showed that an average of 11 × 106 (±0.3 × 106) reads were obtained using a 520 chip leading to an average of 6.0 × 105 (±2.6 × 105) mapped reads per sample. Of 400 consecutive samples, 95.8 ± 16% of amplicons reached the depth threshold of 500X. Slight modifications of the bioinformatics workflow improved DNA analytical sensitivity and allowed the systematic detection of expected SNV, indel, CNV, and RNA alterations in quality controls samples. The low inter-run variability of DNA and RNA-even at low variant allelic fraction, amplification factor, or reads counts-indicated that our method was adapted to clinical practice. The analysis of 429 clinical DNA samples indicated that the modified bioinformatics workflow allowed detection of 353 DNA variants and 88 gene amplifications. RNA analysis of 55 clinical samples revealed 7 alterations. This is the first study showing the long-term robustness of the Oncomine Focus assay in clinical routine practice.

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Integrated Analysis Identifies Novel Fusion Transcripts in Laterally Spreading Tumors Suggestive of Distinct Etiology Than Colorectal Cancers.

BACKGROUND: Laterally spreading tumors (LSTs) of the colon and rectum are a class of abnormality which spreads laterally and appears ulcerated. They are a subclass of colorectal cancer (CRCs) with higher invasive potential than CRCs. Moreover, the etiology of LST still remains obscure. METHODS: This study aimed to identify unique fusion transcript(s) in LSTs and evaluate their role in LST development and progression. RNA-Seq data for LST samples from the EMBL-EBI database were used to identify fusion transcripts. An integrated approach using Gene Ontology, pathway analysis, hub gene, and co-expression network analysis functionally characterized fusion transcripts to shed light upon the etiology of LSTs. RESULT: We identified 48 unique fusion genes in LSTs. GO terms were enriched in mRNA metabolic (p ≤ 2.06E-06), mRNA stabilization (p ≤ 1.60E-05), in cytosol (1.20E-05), RBP (p ≤ 2.30E-04), and RNA binding activity (p ≤ 3.51E-08) processes. Pathway analysis revealed an inflammatory phenotype of LSTs suggesting a distinct etiology than CRCs as pathways were enriched in salmonella infection (p ≤ 4.41 e-03), proteoglycans in cancer (p ≤ 1.18 e-02), and insulin signaling (p ≤ 2.13 e-02). Our exclusion and inclusion criteria and hub gene analysis finally identified 9 hub genes. Co-expression analysis of hub genes identified the most significant transcription factors (NELFE, MYC, TAF1, MAX) and kinases (MAPK14, CSNK2A1, CDK1, MAPK1) which were implicated in various cancer pathways. Furthermore, an overall survival analysis of hub genes was performed. Our predefined criterion resulted in the enrichment of NPM1-PTMA (NPM1: p ≤ 0.005) and HIST1H2BO-YBX1 (YBX1: p ≤ 0.02) fusion transcripts, significantly associated with the patient's overall survival. CONCLUSION: Our systematic analysis resulted in novel fusion genes in LSTs suggesting a different etiology than CRCs. Fusion transcripts were observed more frequently in non-granular LSTs suggestive of genetically more unstable than granular LST. We hypothesize that NPM1-PTMA and HIST1H2BO-YBX1 could be implicated in LST development and progression and may also serve as a prognostic or diagnostic biomarker in future for better management of LSTs.

Expanding the molecular spectrum of tenosynovial giant cell tumors.

Background: While great advances in clinical and pathological description of tenosynovial giant cell tumors (TGCT) have been made, TGCT molecular heterogeneity represents an ongoing challenge. The canonical oncogenic fusion CSF1::COL6A3 is not systematically observed, suggesting that other oncogenic mechanisms are involved in tumorigenesis. This study aims to explore by RNA sequencing a retrospective series of tumors diagnosed as TGCT, in order to provide a better description of their molecular landscape and to correlate molecular features with clinical data. Methods: We analyzed clinicopathological data and performed whole-exome RNA sequencing on 41 TGCT samples. Results: RNAseq analysis showed significant higher CSF1 and CSF1-R expression than a control panel of 2642 solid tumors. RNA sequencing revealed fusion transcripts in 14 patients including 6 not involving CSF1 and some previously unreported fusions. Unsupervised clustering on the expression profiles issued from this series suggested two distinct subgroups: one composed of various molecular subtypes including CSF1 and FN1 rearranged samples and one composed of four tumors harboring an HMGA2::NCOR2 fusion, suggesting distinct tumor entities. Overall, 15 patients received at least one systemic anti-CSF1R treatment and clinical improvement was observed in 11 patients, including patients from both clusters. Discussion: This study reported molecular heterogeneity in TGCT, contrasting with the clinical and pathological homogeneity and the ubiquitous high CSF1 and CSF1R expression levels. Whether molecular diversity may impact the efficacy of systemic treatments needs to be further investigated.

mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

Neoplasia-associated Chromosome Translocations Resulting in Gene Truncation.

Chromosomal translocations in cancer as well as benign neoplasias typically lead to the formation of fusion genes. Such genes may encode chimeric proteins when two protein-coding regions fuse in-frame, or they may result in deregulation of genes via promoter swapping or translocation of the gene into the vicinity of a highly active regulatory element. A less studied consequence of chromosomal translocations is the fusion of two breakpoint genes resulting in an out-of-frame chimera. The breaks then occur in one or both protein-coding regions forming a stop codon in the chimeric transcript shortly after the fusion point. Though the latter genetic events and mechanisms at first awoke little research interest, careful investigations have established them as neither rare nor inconsequential. In the present work, we review and discuss the truncation of genes in neoplastic cells resulting from chromosomal rearrangements, especially from seemingly balanced translocations.

Fusion of the HMGA2 and BNC2 Genes in Uterine Leiomyoma With t(9;12)(p22;q14).

BACKGROUND/AIM: The translocation t(9;12) (p22;q14~15) has been reported in lipomas, pleomorphic adenomas, a myolipoma, two chondroid hamartomas, and two uterine leiomyomas. In lipomas and pleomorphic adenomas, the translocation fuses HMGA2 (12q14) with the NFIB gene from 9p22; in myolipoma, it fuses HMGA2 with C9orf92 from 9p22; and in chondroid hamartomas, fluorescence in situ hybridization (FISH) investigations showed the chromosomal aberration to cause intragenic rearrangement of HMGA2. The translocation's molecular consequence in a uterine leiomyoma is described here. MATERIALS AND METHODS: A typical leiomyoma was investigated using banding cytogenetics, FISH, RNA sequencing, reverse transcription polymerase chain reaction and Sanger sequencing. RESULTS: A single translocation, t(9;12)(p22;q14) leading to an HMGA2::BNC2 chimera, was found in tumor cells. A sequence of the untranslated part of exon 5 of HMGA2 (nucleotide 1035 in the NCBI reference sequence NM_003483.4) had fused with a sequence from the untranslated part of exon 7 of BNC2 from 9p22 (nucleotide 9284 in reference sequence NM_017637.6). CONCLUSION: At the molecular level, the t(9;12)(p22;q14~15) found in several benign tumors appears to be heterogeneous fusing HMGA2 with either BNC2, C9orf92 or NFIB which all three map close to one another within a 3 Mbp region in 9p22. Because the fusion point in HMGA2 in the present tumor lays downstream from the first Let-7 miRNA consensus binding site, we conclude that deletion of the first Let-7 miRNA binding site is not important for the transcriptional upregulation of HMGA2 caused by the genomic rearrangement.