Driver Fusion Research Articles

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.

NanoString nCounter-Based Assay for Detection of Fusion-Associated Salivary Gland Tumors.

Salivary gland tumors include numerous subtypes that vary from benign to highly aggressive, with many showing overlapping histopathological features that can make diagnosis challenging. Most subtypes express driver fusion genes that are tumor specific, and detection of such fusions is useful for differentiating amongst specific diagnoses, determining appropriate tumor grading, and guiding effective treatment. Currently, fusions can be detected by FISH, RT-PCR or through next-generation sequencing approaches, all of which are highly effective methodologies but can be costly or time consuming. We developed a rapid NanoString nCounter platform-based assay to detect salivary gland tumor fusions using a combination of fusion junction-specific probes and an approach through differential exon expression analysis. The assay includes 68 junction-specific probes and analysis of exon expression across 9 fusion-associated genes in a single multiplex assay. Out of 55 retrospective and 171 prospective cases assayed, we accurately detected the majority of cases of pleomorphic adenoma, adenoid cystic carcinoma, cribriform adenocarcinoma, clear cell carcinoma, secretory carcinoma and NUT-rearranged carcinoma, including cases of these tumor types arising in non-salivary gland sites, with the major drawback being an inability to detect MAML2-containing mucoepidermoid samples. With mucoepidermoid carcinoma excluded, the assay shows an overall sensitivity of 96.1% and specificity of 100%. We show that the majority of salivary gland tumor fusions can be effectively detected with a single rapid NanoString based assay, which can serve as a useful adjunctive tool for routine diagnostic head and neck pathology. The assay is low cost with a rapid turnaround time (30h total assay time per sample batch, with minimal technician input required) compared to alternate detection methods.

Integrating the Idylla™ System Alongside a Real-Time Polymerase Chain Reaction and Next-Generation Sequencing for Investigating Gene Fusions in Pleural Effusions from Non-Small-Cell Lung Cancer Patients: A Pilot Study.

Malignant pleural effusion (MPE) from patients with advanced non-small-cell lung cancer (NSCLC) has been proven valuable for molecular analysis; however, simultaneous detection of driver fusions in MPE is still challenging. In this study, we investigated the Idylla™ GeneFusion Panel, a stand-alone test in tissue samples, in the evaluation of ALK, ROS1, RET and MET ex14 skipping mutations in MPE and compared its performance with routine reference methods (Real-time-based and Next-generation Sequencing-NGS). The inclusion criteria for sample selection were as follows: advanced NSCLC harboring ALK, ROS1, RET fusions or MET exon-skipping alterations and the availability of MPE collected at diagnosis or disease progression. Molecular alterations have been investigated on tissue by fluorescence in situ hybridization (FISH) or Real-time PCR or NGS. For molecular profiling with the Idylla™ GeneFusion, 200 µL of MPE supernatants combined with 50 µL of RNA Later solution were loaded into the Idylla™ cartridge without cfRNA extraction. The Idylla™ GeneFusion Assay performed on MPEs was able to confirm molecular profile, previously diagnosed with conventional methods, in all cases. Our data confirm that MPE are suitable material for investigating fusion alterations. The Idylla™ GeneFusion, although indicated for investigation of tissue samples, offers the possibility of performing a molecular characterization of supernatants without undertaking the entire cfRNA extraction procedure providing a rapid and reliable strategy for the detection of actionable genetic alterations.

Fusion Challenges in Solid Tumors: Shaping the Landscape of Cancer Care in Precision Medicine.

Targeting actionable fusions has emerged as a promising approach to cancer treatment. Next-generation sequencing (NGS)-based techniques have unveiled the landscape of actionable fusions in cancer. However, these approaches remain insufficient to provide optimal treatment options for patients with cancer. This article provides a comprehensive overview of the actionability and clinical development of targeted agents aimed at driver fusions. It also highlights the challenges associated with fusion testing, including the evaluation of patients with cancer who could potentially benefit from testing and devising an effective strategy. The implementation of DNA NGS for all tumor types, combined with RNA sequencing, has the potential to maximize detection while considering cost effectiveness. Herein, we also present a fusion testing strategy aimed at improving outcomes in patients with cancer.

Novel gene fusions in human oropharyngeal carcinoma

Few reports have analyzed the fusion genes involved in carcinogenesis in the oropharynx, where the incidence of human papillomavirus-associated tumors is relatively low. The aim of this study was to identify novel driver fusion genes in patients with oropharyngeal cancer. The study enrolled fifty-seven patients who were diagnosed with oropharyngeal carcinoma. RNA sequencing data from fresh-frozen specimens were used to identify candidate fusion genes via the JAFFA, arriba, and STAR-Fusion pipelines. Candidate fusion genes were confirmed by direct sequencing. The expression level of a candidate fusion gene was compared to that of tumors without fusion genes. Finally, filtering was performed for driver genes using the annoFuse pipeline. In addition, the VIRTUS pipeline was used to analyze the presence of human papillomavirus in the tumors. We identified 5 (8.8 %) novel potential driver in-frame fusion genes, MKNK2::MOB3A, ICMT::RPS6KA3, ATP1B3::GRK7, CSNK2A1::KIF16B, and FGFR3::MAEA, and 1 (1.8 %) known in-frame fusion gene, FGFR3::TACC3, in 57 patients with pharyngeal carcinoma. Our results suggest that sporadic fusion genes may contribute to tumorigenesis in oropharyngeal carcinomas.

Cancer fusion transcripts with human non-coding RNAs.

Cancer chimeric, or fusion, transcripts are thought to most frequently appear due to chromosomal aberrations that combine moieties of unrelated normal genes. When being expressed, this results in chimeric RNAs having upstream and downstream parts relatively to the breakpoint position for the 5'- and 3'-fusion components, respectively. As many other types of cancer mutations, fusion genes can be of either driver or passenger type. The driver fusions may have pivotal roles in malignisation by regulating survival, growth, and proliferation of tumor cells, whereas the passenger fusions most likely have no specific function in cancer. The majority of research on fusion gene formation events is concentrated on identifying fusion proteins through chimeric transcripts. However, contemporary studies evidence that fusion events involving non-coding RNA (ncRNA) genes may also have strong oncogenic potential. In this review we highlight most frequent classes of ncRNAs fusions and summarize current understanding of their functional roles. In many cases, cancer ncRNA fusion can result in altered concentration of the non-coding RNA itself, or it can promote protein expression from the protein-coding fusion moiety. Differential splicing, in turn, can enrich the repertoire of cancer chimeric transcripts, e.g. as observed for the fusions of circular RNAs and long non-coding RNAs. These and other ncRNA fusions are being increasingly recognized as cancer biomarkers and even potential therapeutic targets. Finally, we discuss the use of ncRNA fusion genes in the context of cancer detection and therapy.

Optimization of fusion detection in non-small cell lung cancer by amplicon-based RNA sequencing.

e20042 Background: In non-small cell lung cancer (NSCLC), more and more driver fusions are suitable for targeted therapy. Sequential or parallel DNA and RNA sequencing is an option, but it is costly and requires sufficiently high quality biopsy material. The purpose of this study was to confirm the consistency of fusion genes detected by DNA-based NGS and RNA-based NGS in the same sample, and to validate the performance of FFPE samples for RNA-based NGS. Methods: In this study, we evaluated the benefits of capture-based DNA sequencing and amplicon-based RNA sequencing in identifying gene fusion and exon-skipping events in 98 NSCLC patients. All cases were FFPE samples stored for 1-5 years. In addition, we investigated the limiting performance of RNA-based NGS for RNA initiation. Results: In the cases of positive DNA sequencing fusion samples, the fusion consistency detection rate of RNA sequencing was 95% (38/40). Two cases were not detected by RNA-based NGS, one was a five-year FFPE sample and the other was due to a probe not covered problem. In patients with negative DNA fusion genes, RNA sequencing could detect 12.07% more fusion-positive cases involving ALK (n = 1), ROS (n = 3), MET 14 exon skipping (n = 1), RET (n = 1), and BRAF (n = 1). Moreover, the FFPE samples stored for 3-5 years inevitably degrade the RNA, but could still be used for RNA-based sequencing. Among 66 FFPE samples stored for 3-5 years, the detection rate of fusion consistency between DNA sequencing fusion samples and RNA sequencing was 96.77%(30/31). In patients with negative DNA fusion genes, RNA sequencing was able to detect 14.29% (5/35) more fusion-positive cases. The performance verification of the other 8 samples with different RNA input amounts found that 87.5% (7/8) of the samples could meet the quality control of RNA sequencing and accurately detect fusion mutations as low as 1 ng. Conclusions: Molecular profiling based on mixed DNA and RNA sequencing improves the effectiveness of detection of fusion-driven NSCLC. The method was feasible on small tissue samples as well as FFPE samples stored for 3-5 years, and could greatly reduce the complexity and cost of molecular examination.

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.

TPS2702 Background: The DNAJB1-PRKACA fusion transcript is the driving force behind tumor development in fibrolamellar hepatocellular carcinoma (FL-HCC) and other tumor types, such as oncocytic neoplasms of the pancreas and bile ducts. Consequently, it serves as a versatile target for innovative cancer therapies. Recently, the DNAJB1-PRKACA fusion protein was recognized as a source of HLA-presented neoepitopes targetable by to T cell-based immunotherapy (1). The FusionVAC-22 neoepitope, derived from DNAJB1-PRKACA fusion, is computationally predicted to bind to 1,290 different HLA class II alleles. Furthermore, it includes embedded HLA class I ligands for 13 out of the 20 most common HLA class I alleles, covering 93.8% of the global population This broad coverage enables the widespread application of FusionVAC-22-based immunotherapies. The initial use of FusionVAC-22-based peptide vaccines, adjuvanted with the TLR1/2 agonist XS15 emulsified in Montanide ISA 51 VG, in two FL-HCC patients was well-tolerated and demonstrated the induction of robust and long-lasting T cell responses. Notably, T cell responses coincided with 32 months and 13 months of progression-free survival for the respective patients. Methods: Building on these promising outcomes, we have initiated a Phase I open-label, multicentric clinical trial to assess the immunogenicity, safety, toxicity, and initial signs of efficacy of the FusionVAC-22-based peptide vaccine combined with the immune checkpoint inhibitor (ICI) atezolizumab. The trial includes 20 patients with locally advanced or metastatic FL-HCC or other malignancies carrying the DNAJB1-PRKACA fusion transcript without available standard therapy. Key eligibility criteria involve the absence of autoimmune phenomena due to prior immunotherapy agents (≥ grade 3) and no history of tissue or organ allografts. The FusionVAC-22-based peptide vaccine is administered twice with a 4-week interval, with an option for a booster vaccination after 11 months. ICI administration begins on day 15 after the first vaccination and continues every 4 weeks for 1 year, followed by a 6-month follow-up. Primary trial objectives include assessing immunogenicity in terms of peptide-specific T cell responses up to 28 days after the second vaccination, as well as evaluating the safety and toxicity of the peptide vaccine in combination with ICI. Safety assessment is based on the frequency of adverse events according to CTCAE v5.0. Clinical efficacy is determined through iRECIST assessment on imaging. Additionally, disease control rate, quality of life, and overall and progression-free survival will be evaluated. So far, 3 out of 20 planned patients have been enrolled. 1. Bauer et al., Nat. Commun, 2022. Clinical trial information: NCT05937295 .

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.

EU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

Abstract 562: Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor that predominantly occurs in children, adolescents, and young adults. The treatment for OS that combines surgery with chemotherapy, which consists of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, was established in 1970s, and it is still used as a standard therapy. Oncogenic driver mutations and fusion genes common to OS have not been identified, which is one of the reasons for the lack of success in drug development for OS. Herein, to explore the characteristic vulnerability in OS, molecular targeted drugs were screened against OS cell lines and patient-derived cells; obatoclax, a pan-BH3 mimetic, and a concomitant drug that enhances obatoclax-induced apoptosis, OSI906, a dual inhibitor of IGF1R and IR, were found. Fluorescence in situ hybridization (FISH) analysis revealed that copy number gains of MCL1, an antiapoptotic Bcl-2 family protein, were observed in 46.3% of formalin-fixed paraffin-embedded (FFPE) specimens from patients with OS (N = 41) and in 45.5% of OS cell lines (N = 11), thereby causing vulnerability to BH3 mimetics. IHC staining also revealed high expression of Mcl-1 protein in specimens with MCL1-amplification in FISH analysis. Pharmacological or genetic inhibition of Mcl-1 induced potent apoptosis in MCL1-amplified OS cells, which was enhanced by the co-inhibition with Bcl-xL. Moreover, chromosome 1q21.2-3, where MCL1 is located, contains several IGF1R/PI3K pathway-related genes, including PIP5K1A, TARS2, OUTD7B, and ENSA, whose copy numbers were also increased in MCL1-amplified OS cells, suggesting that 1q21.2-3-amplified OS cells readily activate downstream pathways via IGF1R. IGF signaling activation in 1q21.2-3-amplified OS cells negated the obatoclax-induced apoptosis, which was overcome by OSI906 treatment. Combined obatoclax and OSI906 administration remarkably suppressed MCL1-amplified OS tumor growth in the xenograft mouse model and resulted in tumor regression; however, none of the treatments reduced tumor growth of OS without MCL1 amplification. These results suggest that combining obatoclax with OSI906 could be a potential therapeutic intervention for 1q21.2-3-amplified OS, and that prior stratification of OS based on the 1q21.2-3 amplification status would be important for the success of this combination therapy. Thus, we elucidated the mechanism of action of drugs and their potential as a novel therapeutic strategy for approximately 50% of patients with OS. Moreover, a FISH analysis that can detect copy number gains of MCL1 in FFPE specimens from patients with OS was established to stratify patients eligible for this therapy. Citation Format: Satoshi Takagi, Ryohei Katayama. Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 562.

Abstract 3365: Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort

Abstract Background Focal high-level oncogene amplifications (FH-amp) (e.g., MYC, EGFR) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular non-chromosomal DNA. FH-amp on ecDNA promote intra-tumoral heterogeneity, resistance to therapies, and poor prognosis. Recently, a Phase 1 clinical trial exploring ecDNA-directed treatments has begun. However, to date, bioinformatic detection of ecDNA has relied on whole-genome sequencing data, and no standard method exists to detect ecDNA on targeted NGS. We thus sought to explore the feasibility of detecting ecDNA in a large cohort of patients with solid tumors sequenced with the targeted NGS panel, MSK-IMPACT, and describe the pan-cancer prevalence of oncogenes amplified on ecDNA. Methods We utilized a novel bioinformatic ecDNA-diagnostic algorithm, ECHO, to determine the presence of oncogene FH-amp on ecDNA (including HSR) vs. chromosomal in clinical-grade NGS data. 72,651 tumor samples profiled by MSK-IMPACT were queried for high level oncogene FH-amp (AR, CCNE1, CDK4/6, EGFR, FGFR1-4, KRAS, MDM2, MDM4, MET, MYC, MYCL, and MYCN) above a fold-change of three, and subsequently annotated with ECHO. Patients with concomitant oncogenic driver mutations or fusions (e.g., EGFR L858R, EML4-ALK) were excluded based on the hypothesis that they might be mutually exclusive with oncogene FH-amp. Results FH-amp of any above listed oncogene were found in 3065 (4.2%) samples. Cancer types with frequent FH-amp without concomitant driver alterations were liposarcoma (LPS) (61% FH-amp; 43% were ecDNA+), glioblastoma (GBM) (22%; 72%), gastroesophageal adenocarcinoma (GE AD) (10%; 57%) squamous cell lung cancer (7%; 18%), and breast cancer (BRCA) (7%; 23%). Among tumors with oncogene FH-amp, GBM and GE AD had high frequencies of ecDNA+ FH-amp of EGFR (39% and 15% of all EGFR ecDNA+). LPS had the highest frequency of ecDNA+ FH-amp of CDK4 (64%), followed by GBM (15%). The majority of ecDNA+ FGFR1 and FGFR2 FH-amp were in BRCA (75% and 47%). EGFR in GBM and EGFR or FGFR2 in GE AD were ecDNA+ in more than 90% of patients, while CDK4 in LPS and FGFR1 in BRCA were ecDNA+ in 45% and 39% respectively.Evaluation the relationship between ecDNA+ by ECHO and amp FC range showed higher ecDNA+ in samples with higher level amp (FC > 5; 58% ecDNA+) compared to lower level amp (FC 4~5; 12%, FC 3~4; 6% ecDNA+). Conclusions A novel ecDNA clinical diagnostic tool, ECHO, was successfully applied to a large clinical cohort of patients enriched for actionable oncogene amplifications sequenced with MSK-IMPACT. The results demonstrate that detection of ecDNA using clinical-grade NGS assays is feasible and that key oncogene FH-amp on ecDNA (e.g., EGFR, FGFR2) are common in select tumor types. ECHO may be developed as a clinical trial device to prospectively identify patients with oncogene ecDNA+ FH-amp for clinical testing of ecDNA-directed therapies. Citation Format: Matteo Repetto, Klaus Wagner, Shalabh Suman, Peter Krein, Pavitra Rao, Maria Arcila, Christian Hassig, Mark Donoghue, Alexander Drilon, Michael Berger. Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3365.

Abstract 6: Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy

Abstract Sarcomas are a heterogenous group of mesenchymal cell malignancies that are difficult to treat and often respond poorly to current immunotherapies. Approximately 30% of sarcomas are associated with oncogenic driver fusion proteins. Desmoplastic small round cell tumor (DSRCT) is a prototypical fusion-driven sarcoma defined by a pathognomonic EWSR1-WT1 fusion event. The resultant EWSR1-WT1 fusion protein contains a shared junctional amino acid sequence that is clonally conserved and significantly divergent from normal human proteins. Consequently, the EWSR1-WT1 fusion might yield a highly immunogenic shared, or public, neoantigen (NeoAg) that may serve as a target for novel immunotherapeutic approaches.Using a functional HLA-immunoprecipitation/mass spectrometry (HLA-IP/MS) screen, we identified a 9-amino acid peptide sequence derived from the junction of the most common EWSR1-WT1 fusion protein that is presented in the context of the prevalent HLA-A*03 and -A*11 alleles. We confirmed that HLA-A*03+ DSRCT cells physiologically present the same peptide sequence. Using fluorophore-conjugated HLA-multimers loaded with the HLA-IP/MS-derived fusion NeoAg, we identified a rare population of circulating NeoAg-specific CD8+ T cells in HLA+ DSRCT patients. Through antigen-directed clonal expansion, we discovered n=3 HLA-A*03-restricted and n=1 HLA-A*11-restricted fusion NeoAg-specific T cell clones and retrieved the paired TCRαβ gene sequences of their T cell receptors (TCRs) using 10x single-cell sequencing. Polyclonal CD8+ T cells transduced with candidate TCRαβ gene sequences robustly upregulated TNFα after coculture with HLA+/Fusion+ target cells but not HLA−/Fusion+ or HLA+/Fusion− control cells. Further, T cells expressing candidate TCRs, but not viral-protein-specific TCRs, lysed HLA+ DSRCT cells in vitro and controlled established DSRCT tumors in vivo. Finally, we discovered that one TCR was capable of cross HLA allele fusion NeoAg recognition and could lyse both HLA-A*03+ and -A*11+ DSRCT cells. These findings imply that a single TCR therapeutic could cover >36% of North American DSRCT patients.Collectively, our data establish for the first time that DSRCT cells present a junction-spanning fusion-derived neoantigen in the context of two prevalent HLA alleles that may be therapeutically targeted using TCR gene therapy. These data lay the foundation for first-in-human clinical translation of a T-cell based therapy targeting EWSR1-WT1. More broadly, these findings establish proof-of-principle that recurrent fusion proteins are an actionable source of immunogenic public NeoAgs, providing a framework for developing targeted immunotherapies for other fusion-driven malignancies. Citation Format: Lauren Banks, Hannah Arkin, Smita Chandran, Emily Slotkin, Madelyn Espinosa-Cotton, Neerav Shukla, Luc Morris, Marc Ladanyi, Andrew Kung, Martin Klatt, Christopher Klebanoff. Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6.

Abstract 1081: A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma

Abstract Background: Tumor necrosis factor receptor superfamily member 10b (TNFRSF10b, also known as DR5) agonists are attractive targeted agents for cancer cells because these agents activate cellular apoptosis. Ewing sarcoma (ES) is a bone and soft-tissue sarcoma occurring in adolescence defined by a t(11;22)(q24;q12) balanced chromosomal translocation that results in chimeric transcription factor and oncogenic driver protein, EWS-FLI1. Independent studies and unbiased cell line screens have reported ES to be sensitive to DR5 targeting. INBRX-109 is a tetravalent, DR5-targeted antibody. Here, we analyzed the sensitivity of ES models to DR5 targeting by INBRX-109 and explored how the EWS-FLI1 transcriptional program impacts the continuum of sensitivity-resistance to this targeting. Methods: In a panel of 11 ES lines, in vitro sensitivity was assessed to both single agent and INBRX-109 combinations. A screen of 1,363 FDA approved compounds identified cooperative combinations that were prioritized mechanistically. Differential sensitivity for single agent and in combination with irinotecan was confirmed in vivo in xenograft models. Resistant clones were isolated from in vivo experiments and evaluated for DR5 expression and sensitivity. Cellular factors associated with sensitivity and resistance were determined by siRNA knockdown and Cleavage Under Targets and Tagmentation assay (CUT and Tag). Results: ES models showed variable sensitivity to INBRX-109, with a GI90 as low as 10 picomolar in some models. All models showed robust expression of DR5, but absolute expression of DR5 did not correlate with sensitivity. In vitro sensitivity was replicated in vivo, with all 7 mice bearing RD-ES xenografts demonstrating initial complete tumor regression and 3 of 7 mice demonstrating no recurrence beyond 90 days. Importantly, cells isolated from recurrent tumors showed a novel mechanism of resistance: direct repression of DR5 by EWS-FLI1. INBRX-109 was ineffective as a single agent at 10 nM in resistant ES models. However, there was striking synergy in vitro with SN38, the active metabolite of irinotecan, and this effect translated into tumor regressions of a resistant TC32 xenograft in vivo. The FDA compound screen captured novel synergy with a panel of agents. Conclusion: INBRX-109 is an exciting candidate for ES as a single agent in subsets of tumors or in combination with irinotecan, which is currently being evaluated in the clinic. Investigation of the exact relationship between EWS-FLI1 and sensitivity to DR5 targeting is ongoing. Initial findings suggest EWS-FLI1, combined with other disease modifying mutations, is a key mediator of this sensitivity. Further, EWS-FLI1 plays a direct role repressing DR5 in the setting of therapeutic challenge in one model in vivo. These findings highlight the potential for INBRX-109 in combination with irinotecan or novel candidates as therapies for ES patients. Citation Format: Curtis C. Parker, Lauren M. Gaetano, Elissa A. Boguslawski, Seneca Kinn-Gurzo, Rebecca Kaufman, Matthew C. Stout, Chase Deveraux, Patrick J. Grohar. A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1081.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 141.

Anchored-fusion enables targeted fusion search in bulk and single-cell RNA sequencing data

Here, we present Anchored-fusion, a highly sensitive fusion gene detection tool. It anchors a gene of interest, which often involves driver fusion events, and recovers non-unique matches of short-read sequences that are typically filtered out by conventional algorithms. In addition, Anchored-fusion contains a module based on a deep learning hierarchical structure that incorporates self-distillation learning (hierarchical view learning and distillation [HVLD]), which effectively filters out false positive chimeric fragments generated during sequencing while maintaining true fusion genes. Anchored-fusion enables highly sensitive detection of fusion genes, thus allowing for application in cases with low sequencing depths. We benchmark Anchored-fusion under various conditions and found it outperformed other tools in detecting fusion events in simulated data, bulk RNA sequencing (bRNA-seq) data, and single-cell RNA sequencing (scRNA-seq) data. Our results demonstrate that Anchored-fusion can be a useful tool for fusion detection tasks in clinically relevant RNA-seq data and can be applied to investigate intratumor heterogeneity in scRNA-seq data.

Real-World Clinical Performance of a DNA-Based Comprehensive Genomic Profiling Assay for Detecting Targetable Fusions in Nonsquamous NSCLC.

Genomic fusions are potent oncogenic drivers across cancer types and many are targetable. We demonstrate the clinical performance of DNA-based comprehensive genomic profiling (CGP) for detecting targetable fusions. We analyzed targetable fusion genes in >450 000 tissue specimens profiled using DNA CGP (FoundationOne CDx, FoundationOne). Using a de-identified nationwide (US-based) non-small cell lung cancer (NSCLC) clinico-genomic database, we assessed outcomes in patients with nonsquamous NSCLC (NonSqNSCLC) who received matched therapy based on a fusion identified using DNA CGP. Lastly, we modeled the added value of RNA CGP for fusion detection in NonSqNSCLC. We observed a broad diversity of fusion partners detected with DNA CGP in conjunction with targetable fusion genes (ALK, BRAF, FGFR2, FGFR3, NTRK1/2/3, RET, and ROS1). In NonSqNSCLC with oncogenic ALK, NTRK, RET, and ROS1 fusions detected by DNA CGP, patients treated with a matched tyrosine kinase inhibitor had better real-world progression-free survival than those receiving alternative treatment regimens and benefit was observed regardless of the results of orthogonal fusion testing. An estimated 1.3% of patients with NonSqNSCLC were predicted to have an oncogenic driver fusion identified by RNA, but not DNA CGP, according to a model that accounts for multiple real-world factors. A well-designed DNA CGP assay is capable of robust fusion detection and these fusion calls are reliable for informing clinical decision-making. While DNA CGP detects most driver fusions, the clinical impact of fusion detection is substantial for individual patients and exhaustive efforts, inclusive of additional RNA-based testing, should be considered when an oncogenic driver is not clearly identified.

Epithelioid hemangioendothelioma (EHE) with WWTR1::TFE3 gene fusion, a novel fusion variant.

Epithelioid hemangioendothelioma (EHE) is a rare endothelial sarcoma associated with a high incidence of metastases and for which there are no standard treatment options. Based on disease-defining mutations, most EHEs are classified into two subtypes: WWTR1::CAMTA1-fused EHE or YAP1::TFE3-fused EHE. However, rare non-canonical fusions have been identified in clinical samples of EHE cases and are challenging to classify. In this study, we report the identification of a novel WWTR1::TFE3 fusion variant in an EHE patient using targeted RNA sequencing. Histologically, the tumor exhibited hybrid morphological characteristics between WWTR1::CAMTA1-fused EHE and YAP1::TFE3-fused EHE. In addition to the driver fusion, there were six additional secondary mutations identified, including a loss-of-function FANCA mutation. Furthermore, in vitro studies were conducted to investigate the tumorigenic function of the WWTR1::TFE3 fusion protein in NIH3T3 cells and demonstrated that WWTR1::TFE3 promotes colony formation in soft agar. Finally, as the wild-type WWTR1 protein relies on binding the TEAD family of transcription factors to affect gene transcription, mutation of the WWTR1 domain of the fusion protein to inhibit such binding abrogates the transformative effect of WWTR1::TFE3. Overall, we describe a novel gene fusion in EHE with a hybrid histological appearance between the two major genetic subtypes of EHE. Further cases of this very rare subtype of EHE will need to be identified to fully elucidate the clinical and pathological characteristics of this unusual subtype of EHE.

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.

TPS580 Background: The DNAJB1-PRKACA fusion transcript is the driver of tumor pathogenesis in fibrolamellar hepatocellular carcinoma (FL-HCC) as well as in other tumor entities, e.g. oncocytic neoplasms of the pancreas and bile duct, thus representing a broad target for novel therapies in cancer. Recently, the DNAJB1-PRKACA fusion protein was identified as a source for HLA-presented neoepitopes that can be targeted by T-cell-based immunotherapy (Bauer et al. Nat. Commun., 2022). The DNAJB1-PRKACA fusion-derived neoepitope FusionVAC-22 is in silico predicted to bind to 1,290 HLA class II alleles and contains HLA class I ligands from 13 of the 20 most frequent HLA class I alleles that cover 93.8% of the world population with at least one HLA allotype, enabling the broad application of FusionVAC-22-based immunotherapies. The first application of FusionVAC-22-based peptide vaccines adjuvanted with the TLR1/2 agonist XS15 emulsified in Montanide ISA 51 VG in two FL-HCC patients was well tolerated and showed the induction of profound and long-lasting T-cell responses. Of note, T-cell responses were accompanied by progression-free survival of both patients for so far 32 months and 13 months, respectively. Methods: Based on these encouraging results, we established a Phase I open-label, multicentric clinical trial to evaluate the immunogenicity along with safety and toxicity, as well as first signs of efficacy of the FusionVAC-22 based peptide vaccine, combined with the immune checkpoint inhibitor (ICI) atezolizumab, in 20 patients with locally advanced or metastatic FL-HCC or other malignant diseases that carry the DNAJB1-PRKACA fusion transcript. Further, key eligibility criteria include the absence of autoimmune phenomena due to prior immunotherapy agents (≥ grade 3) as well as tissue or organ allografts. The FusionVAC-22-based peptide vaccine is applied twice in a 4-weekly interval with the opportunity of a booster vaccination after 11 months. Atezolizumab application starts on day 15 after first vaccination and is continued 4-weekly for 1 year, followed by a 6-month follow-up. The primary objectives of the trial are immunogenicity, in terms of induction of peptide specific T-cell responses until 28 days after second vaccination, as well as safety and toxicity of the peptide vaccine in combination with ICI. Safety assessment is based on the frequency of adverse events according to CTCAE v5.0. Clinical efficacy is determined by iRECIST assessment on imaging. Furthermore, disease control rate, quality of life as well as overall and progression free survival will be assessed. Clinical trial information: NCT05937295 .

Strategic Approach to Heterogeneity Analysis of Cutaneous Adnexal Carcinomas Using Computational Pathology and Genomics

Cutaneous adnexal tumors are neoplasms that arise from skin appendages. Their morphologic diversity and phenotypic variability with rare progression to malignancy make them difficult to diagnose and classify, and there is currently no established treatment strategy. To overcome these difficulties, this study investigated the transcription factor SOX9 expression, morphology, and genetics of skin adnexal tumors for understanding their biology, especially their histogenesis. We showed that cutaneous adnexal tumors and their nontumor counterparts of skin and appendages exhibit expression patterns similar to that of SOX9. Its expression intensity and pattern, as well as histopathologic evaluation of tumors, were analyzed using digital images of 69 normal skin adnexal 9-type organs and 185 skin adnexal 29-type tumors as references. It was possible to distinguish basal cell carcinoma from squamous cell carcinoma, sebaceous carcinoma, and pilomatrixoma with significant differences, along with porocarcinoma from squamous cell carcinoma. Furthermore, unsupervised machine learning "computational pathology" was used to derive a multiregion whole-exome sequencing fusion method termed "genocomputed pathology." The genocomputed pathology of three representable adnexal carcinomas (porocarcinoma, hidradenocarcinoma, and spiradenocarcinoma) was evaluated for total nine cases. We showed that there was more heterogeneity than expected within the tumors as well as the coexistence of components lacking driver fusion genes. The presence or absence of potential driver genes, such as PIK3CA, YAP1, and PTEN, in each region was identified, highlighting a therapeutic strategy for cutaneous adnexal carcinoma encompassing heterogeneous tumors.

Targeting the Clear Cell Sarcoma Oncogenic Driver Fusion Gene EWSR1::ATF1 by HDAC Inhibition.

This study reveals the epigenetic and transcriptional suppression mechanism of the fusion oncogene EWSR1::ATF1 in clear cell sarcoma by histone deacetylase inhibitor treatment as well as identifying SOX10 as a transcription factor that regulates EWSR1::ATF1 expression.