Fusion Gene Transcripts Research Articles

Beyond chromosomal rearrangements: The expanding landscape of gene fusions and chimeric RNAs

BCR–ABL, the pioneering gene fusion resulting from chromosomal translocation, has marked a major milestone in understanding genetic alterations in cancer. Initially, gene fusions were linked solely to chromosomal rearrangements, serving as diagnostic markers and cancer drivers. However, advancements in high-throughput sequencing and bioinformatics have revealed additional mechanisms underlying gene fusion. The term “gene fusion” primarily refers to fusion events at the DNA level, whereas “chimeric RNA” encompasses a wide range of transcripts containing exons from different parental genes, including gene fusion transcripts. Recent developments have identified numerous chimeric RNAs in various cancer types, extending even to non-cancerous tissues. Chimeric RNAs, originating from events such as trans-splicing, read-through, and intergenic splicing, form a complex landscape with varied functions. While some chimeric RNAs have defined roles and therapeutic potential, a comprehensive understanding of their diverse functions remains a priority. Exploring the full spectrum of chimeric RNA activities is crucial for revealing their clinical and therapeutic implications. In addition, chimeric RNAs are key players in tumorigenesis, affecting cellular processes, and driving cancer progression. Understanding their intricate interactions with cellular pathways is essential for developing targeted therapies and precision medicine approaches. The dynamic nature of chimeric RNAs highlights the need for ongoing research to fully harness their diagnostic and therapeutic potential.

Mixed-phenotype (B-lymphocytic/myeloid) acute leukemia with ETV6-ABL1 expression.

Mixed-phenotype acute leukemia (MPAL) is rare in the clinic, accounting for approximately 2%-5% of acute leukemia cases. In this study the cohort included 126 patients, of which 125 cases were from re-examined published data and current patients from Shenzhen Longhua District Central Hospital, carrying an ETV6-ABL1 rearrangement from April 15, 2020 to December 19, 2020. The ETS variant transcription factor 6-Abelson proto-oncogene 1 (ETV6-ABL1) fusion gene is mainly seen in malignant hematological diseases such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myeloproliferative neoplasms (MPNs). Positivity of both MPAL and ETV6-ABL1 suggest a poor prognosis. This is the first report of B lymphocytic/myeloid mixed-phenotype acute leukemia with ETV6-ABL1 fusion gene positivity. Complete remission was achieved with chemotherapy for lymphoid and myeloid leukemia and targeted therapy with tyrosine kinase inhibitors (TKIs). The level of ETV6-ABL1/ABL decreased from 23.056% to 11.165%. After consolidation chemotherapy, the patient underwent allogeneic hematopoietic stem cell transplantation but died due to intestinal rejection. There are 126 cases of ETV6-ABL1 fusion gene transcript expression in numerous hematologic malignancies reported to date, including 48 cases of ALL, 12 of AML, and 65 of MPN. Eosinophilia is a common characteristic, especially in MPN patients. Survival analysis suggests that the survival time of ALL and MPN patients receiving TKI treatment is better than that of patients not receiving this treatment. Dasatinib or nilotinib, especially the former, is more effective than imatinib for MPN.

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 7293: Serum fusion gene transcripts to predict liver cancer

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancers and one of the most lethal malignancies for human. Early diagnosis of HCC is crucial in reducing the mortality of the disease. In this report, we analyzed a panel of 9 fusion transcripts in the serum samples from 136 individuals (61 HCC patients and 75 individuals without HCC) using TaqMan qRT-PCR. Our results showed that 7 fusion genes were frequently detected in the serum samples of HCC patients, including MAN2A1-FER (100%), SLC45A2-AMACR (62.3%), ZMPSTE24-ZMYM4 (62.3%), Pten-NOLC1 (57.4%), CCNH-C5orf30 (55.7%), STAMBPL1-FAS (26.2%) and PCMTD1-SNTG1 (16.4%). We then divided the samples into training and testing cohorts based on the sample collection time: 82 individuals with samples collected before 2016 as the training cohort while 54 individuals with sample collected after 2015 as the testing cohort. Machine learning models was constructed based on the combination of different fusion genes with various cutoffs to predict the occurrence of HCC in the training cohort using leave-one-out-cross-validation approach. One of the machine learning models called 4-fusion genes logistic regression model (MAN2A1-FER<40, CCNH-C5orf30<38, SLC45A2-AMACR<41, Pten-NOLC1<40) produced a 91.5% accuracy, with a sensitivity of 87.1% and specificity of 94.1% in the training cohort. The same model generated an accuracy of 83.3%, with a sensitivity of 73.3% and specificity of 95.8% in testing cohort. When the training and testing cohorts were combined, the model produced an accuracy of 86%. When serum α-fetal protein (AFP) level was incorporated into the machine learning model, we found a 2-fusion gene+AFP logistic regression model (MAN2A1-FER<40, CCNH-C5orf30<38, AFP) generated an accuracy of 94.8% in the training cohort with a sensitivity of 93.5% and specificity of 96.3%. The same model generated 95% accuracy in both the testing cohort and the combined cohorts in the validation. Cancer treatment reduced most of the serum fusion transcript levels, reflecting the reduction of the tumor load. As a result, serum fusion gene machine learning models may serve as important tools in screening HCC and monitoring the impact of HCC treatment. Citation Format: Jian-Hua Luo, Yan-Ping Yu, Silvia Liu, David Geller. Serum fusion gene transcripts to predict liver cancer [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 7293.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [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 7418.

The development of a custom RNA-sequencing panel for the identification of predictive and diagnostic biomarkers in glioma

PurposeVarious molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA.MethodsA total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets.ResultsAmong 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis.ConclusionsTOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

IGH::NSD2 Fusion Gene Transcript as Measurable Residual Disease Marker in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy. Approximately 15% of MM patients are affected by the t(4;14) translocation resulting in the IGH::NSD2 fusion transcript. Breakage occurs in three major breakpoint regions within the NSD2 gene (MB4-1, MB4-2, and MB4-3), where MB4-1 leads to the production of full-length protein, while truncated proteins are expressed in the other two cases. Measurable residual disease (MRD) has been conclusively established as a crucial prognostic factor in MM. The IGH::NSD2 fusion transcript can serve as a sensitive MRD marker. Using bone marrow (BM) and peripheral blood (PB) samples from 111 patients, we developed a highly sensitive quantitative real-time PCR (qPCR) and digital PCR (dPCR) system capable of detecting fusion mRNAs with a sensitivity of up to 1:100,000. PB samples exhibited sensitivity three orders of magnitude lower compared to BM samples. Patients with an MB4-2 breakpoint demonstrated significantly reduced overall survival (p = 0.003). Our novel method offers a simple and sensitive means for detecting MRD in a substantial proportion of MM patients. Monitoring may be carried out even from PB samples. The literature lacks consensus regarding survival outcomes among patients with different NSD2 breakpoints. Our data align with previous findings indicating that patients with the MB4-2 breakpoint type tend to exhibit unfavorable overall survival.

Concurrent BCR::-ABL1 and CBFB Rearrangement in De Novo Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is known for its genetic heterogeneity. Several recurrent genetic variants have been characterized including AML-defining chromosomal rearrangements involving the core binding factor (CBF), such as inv(16)/t(16;16)/ CBFB:: MYH11. 1 Less common is a subset of AML characterized by the Philadelphia chromosome (Ph+), generating a BCR::ABL1 fusion protein. 2 The Ph+ AML subtype is associated with poor prognosis and treatment resistance. 3,4 Co-occurrence of CBFB rearrangement and BCR::ABL1 fusion is rare, representing a subgroup with unique clinical and molecular features. To date, there have been less than 30 cases reported in the literature. 5-8 Case Description A 62-year-old male presented to hospital with headaches, visual changes, weakness, and a nodular rash on his back, with leukocytosis, thrombocytopenia, and 42.5% circulating blasts. A bone marrow (BM) aspirate and biopsy was diagnostic of AML with 56% myeloblasts. RT-PCR was positive for an e19a2 BCR::ABL1 fusion transcript coding for p230 kDa fusion protein (p230) and CBFB: :MYH11 fusion gene transcript. Cytogenetics identified a complex karyotype with an inv(16) and a Ph chromosome (46,XY,t(1;14)(p32;q24),t(9;22)(q34;q11.2),inv(16)(p13.1q22)[10]), consistent with concurrent BCR::ABL1 and CBFB::MYH11 rearrangements within the same clonal population of cells. Treatment consisted of hydroxyurea for cytoreduction, 7+3 induction, one cycle of low dose cytarabine consolidation, and oral azacytidine maintenance, in combination with dasatinib. He did not receive Gemtuzumab ozogamicin (GO) given patient preference and comorbidities. Post-induction course was complicated by febrile neutropenia due to Enterococcus Faceium bacteremia, pre-renal acute kidney injury, and dasatinib-related complications including rash and transaminitis. These issues resolved by holding dasatinib for 15 days. Complete morphologic remission (CR) was obtained by Day 33 post-induction and no fusion gene transcripts were detectable by nested RT-PCR from BM aspirate on day 49 post-induction, nor day 76 post-consolidation The patient was ineligible for an allogeneic stem cell transplant given his poor performance status. At present, the patient remains alive and well, continuing on maintenance with oral azacytidine and dasatinib. Discussion We present the only case, to our knowledge, of a concurrent BCR::ABL1 p230 isoform and CBFB::MYH11 with de novo AML. Based on our literature search, 26 cases have been reported. BCR::ABL1 and CBFB::MYHII rearrangements in de novo AML potentially confers an intermediate to favorable prognosis with outcomes comparable to those of CBF-AML. Studies suggest that this entity may be more akin to AML with inv(16) rather than CML-BP with inv(16), thereby retaining favorable prognosis. 5,9-11 There are no formal guidelines for management of de novo AML with concurrent BCR::ABL1 and CBFB rearrangements. Traditional chemotherapy treatment strategies for Ph+ AML are often ineffective in these cases. The presence of BCR::ABL1 fusion transcript raises the possibility of utilizing TKIs targeting the BCR::ABL1 kinase domain. In our case, the patient achieved rapid CR with induction chemotherapy and dasatinib, but experienced multiple complications, precluding further intensive chemotherapy and allogeneic stem cell transplantation. This case raises a number of questions, including the optimal timing, combination, and duration of TKI therapy in the context of dual rearrangement de novo AML. This patient subgroup may benefit from intensive chemotherapy regimens and TKIs. Prospective monitoring of these cases is warranted to assess outcomes in comparison to Ph+ AML without coinciding CBFB rearrangement. This case report underscores the importance of comprehensive genetic profiling in Ph+ AML to determine the genetic landscape and individualize therapeutic strategies.

The Efficacy and Safety of Olverembatinib Combined with Monoclonal Antibodies As Salvage Therapy for RR B ALL with ABL1 Fusion Gene Positive

Background: B cell acute Lymphoblastic Leukemia （B ALL） with ABL1 fusion gene(FG) positive（ABL1+B ALL） is classified as Ph or Ph like ALL and has a poor prognosis. With the therapy of tyrosine kinase inhibitors(TKI)CD19- or CD22-specific chimeric antigen receptor (CAR) T-cell therapy,more patients(pts)can achieve complete remission(CR) even relapsed after hematopoietic stem cell transplantation(HSCT). However, the pts relapsed/recurrence（RR）after HSCT /CART/ TKIs even ponatinib or cannot receive CART due to any reason have the worst prognosis. Recently, such pts have new hope with the wide application of a novel third-generation TKI Olverembatinib developed in China and Blinatumomab（CD19 antibody,BITE）and INOTUZUMAB OZOGAMICIN(CD22 antibody,Ino.). Aims: Observe the efficacy and safety of Olverembatinib combined with BITE or INO. therapy in RR ABL1+B ALL pts after chemotherapy combined with TKI or CART or even after transplantation. Methods: Four RR ABL1+B ALL pts received Olverembatinib with BITE or INO from June 2022 to July 2023. During treatment, the dose of Olverembatinib was adjusted and chemotherapy or veneclax may be given at the same time,and give appropriate treatment depending on the patient's condition.After treatment, efficacy was assessed by complete remission (CR) rate, minimal residual disease(MRD)neg (<0.01%) rate by MFC and complete molecular remission(CMR )(FG transcript<10-5) rate by real-time quantitative polymerase chain reaction (RT-qPCR). Results:Four pts were male, the median age was 45 (range13-72) years, 3 pts were BCR-ABL1 FG positive, 1 pts was AML1-ABL1 FG positive. Before treatment,3 pts presented with 5.5%-35.5% leukemic blasts in bone marrow（BM） and only 1 pts was MRD positive by MFC.And all pts's leukemic cell showed CD19 and CD22 expression confirmed by MFC.2 pts did not undergo HSCT: both them have T315I mutation occurred during disease progression. Pts 1 was 72 years and relapsed after chemotherapy combined with TKI( include ponatinib);Pts 2 was 61 years and relapsed after chemotherapy, TKI( include ponatinib), venetoclax ,BITE and CD19-CART CD22-CART treatments. 2 pts were eventually treated with Olverembatinib combined with INO. and chemotherapy and venetoclax. The other two pts relapsed after HSCT: neither of them had T315I mutation.Pts 3 was 29 years and had recurrence after chemotherapyTKIvenetoclax HSCT and three times CART. Pts 4 was 13 years and had reccurence after chemotherapyHSCTthree times CART and TKI( include ponatinib)venetoclax DLI. 2 pts were eventually treated with Olverembatinib combined with BITE and venetoclax.All pts survived safely and achieved CR:3 pts CMR,1 pts MRDneg by MFC and BCR::ABL1:0.0023%. Pts.2 bridged HSCT;pts.3 and 4 continued oral Olverembatinib combined with venetoclax. The longest follow-up time for 3 pts was one year nearly until today. Unfortunately, pts 1 died due to a secondary fungal infection post-COVID-19 and recurrence of ALL. Conclusions:For heavily treated RR ABL1+B ALL pts,including those after treatment with ponatinib and other TKICART and HSCT, Olverembatinib combined with monoclonal antibodies is effective and safe. In my cases, it is particularly noteworthy that one pts ,Ph like ALL with ABL1 FG, received Olverembatinib combination BITE and achieved a surprising response, so Olverembatinib may also be effective and safe for such pts. In addition, venetoclax may be used as a sensitizer for TKI or other antitumor drugs. Of course, the above needs more clinical practice to verify.

Quantum-First Trial: FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD)-Specific Measurable Residual Disease (MRD) Clearance Assessed through Induction (IND) and Consolidation (CONS) Is Associated with Improved Overall Survival (OS) in Newly Diagnosed (nd) FLT3-ITD+ AML Patients (pts)

Background Detection of MRD by flow cytometry or gene fusion transcript quantitation increasingly is used to guide treatment decisions in AML. Molecular monitoring of recurrent gene mutations for MRD detection is an alternative strategy but, other than for nucleophosmin 1 ( NPM1) gene quantitation, remains controversial. FLT3-ITD is among the most common recurrent mutations in AML and confers a worse prognosis vs FLT3-wild-type and FLT3-TKD+ AML. Routine use of FLT3-ITD detection in remission as a predictor of relapse risk or OS has been limited by the low sensitivity of both conventional PCR-based detection methods and broad NGS platforms in clinical practice settings. More recently, FLT3-ITD-specific PCR-NGS assays such as getITD (PMID: 31089248) show greater sensitivity and ease of interpretation that point to eventual routine clinical application. However, the clinical value of these measurements has not been evaluated prospectively in large scale randomized controlled trials of FLT3 inhibitors. The phase 3 QuANTUM-First study (NCT02668653) evaluated the novel, potent, and highly selective type II FLT3 inhibitor quizartinib (Quiz) in nd FLT3-ITD+ AML pts and demonstrated that Quiz added to intensive IND and CONS, ± transplant, followed by single-agent continuation (CONT) therapy (Tx) resulted in a significant improvement in OS (PMID: 37116523). We analyzed if FLT3-ITD-specific MRD in QuANTUM-First pts impacted the clinical outcome or the benefits provided by Quiz in nd FLT3-ITD+ AML pts. Methods Genomic DNA, isolated from bone marrow aspirates or peripheral blood from pts after achievement of remission after 1 or 2 courses of IND and at end of CONS (prior to transplant or CONT cycle 1 day 1 [C1D1] for transplant pts and prior to CONT C1D1 for non-transplant pts), was analyzed with a FLT3-ITD PCR-NGS assay specifically developed for this trial (PMID: 31722002). ITD mutations detected after IND were cross-validated against the ITD detected at enrollment for each patient. Using a custom bioinformatics program, ITD mutations were identified, and variant allele frequencies (VAFs) were calculated with a sensitivity of 10 −5. MRD was classified as undetectable (using the 0 cutoff) or detectable above or below a 10 −4 predefined cutoff (based on lower limit of quantification for the assay). Comparisons of complete response (CR), composite complete response (CRc=CR+CRi), and the rate of pts achieving CRc during IND with no MRD between arms were made using a stratified Cochran-Mantel-Haenszel test. Comparison of FLT3-ITD VAF during IND and during CONS between arms was made using Wilcoxon rank sum test. All P-values were not adjusted for multiplicity. Results In QuANTUM-First, 539 nd FLT3-ITD+ AML pts were randomized to Quiz (n=268) or placebo (PBO; n=271). Of the 539 randomized pts, 368 (68.3%) achieved CRc after 1 or 2 courses of IND, and MRD analysis was performed on 321 (87.2%) of these pts (162 pts in Quiz and 159 pts in PBO) using samples collected at the time of response assessment during IND, before further Tx. MRD was also assessed in 337 pts (172 pts in Quiz and 165 pts in PBO) by end of up to 4 cycles of CONS prior to CONT/maintenance Tx. Of these pts, 166 additionally received transplant (87 in Quiz, 79 in PBO). The % of pts in CRc at end of IND with FLT3-ITD MRD of <10 −4 was similar between study arms (25.4% Quiz vs 21.8% PBO, nominal P = 0.3430), however, a greater % of pts in CRc had undetectable MRD with Quiz than PBO (12.3% vs 7.0%, nominal P = 0.0403). Among pts with CRc at end of IND, the median best FLT3-ITD VAF by end of CONS was lower (0% versus 0.0017%; nominal P = 0.0006) with Quiz vs PBO (Fig 1) and, using the 0 cutoff at end of IND, a longer OS was observed with Quiz vs PBO, regardless of MRD status, with a greater effect with Quiz (HR, 0.789 in MRD− pts; HR, 0.749 in MRD+ pts; Fig 2). In MRD+ pts, the median OS was not reached with Quiz and 35.4 months with PBO (Fig 2). Performing this analysis with an MRD cutoff of 10 ‒4 yielded similar findings (HR, 0.696 in MRD− pts; HR, 0.800 in MRD+ pts). Additional data about MRD impact on pts ± transplant in this trial will be presented. Conclusions These findings demonstrate the potential prognostic utility of FLT3-ITD-specific MRD measurements in the clinical management of pts with FLT3-ITD+ AML. Our data suggest that long-term OS benefits conferred by Quiz in part derive from a deep and sustained reduction of the FLT3-ITD+ leukemia burden.

BIOM-59. FEASIBILITY AND UTILITY OF THE NOVEL CANCER COMPREHENSIVE GENOMIC PROFILING RNA-SEQ FOR THE IDENTIFICATION OF THERAPEUTIC TARGETS AND DIAGNOSTIC BIOMARKERS IN GLIOMA

Abstract BACKGROUND The detection of novel therapeutic targets is increasingly important in oncology. The Todai OncoPanel (TOP) RNA panel is a custom target RNA-seq using the junction capture method to maximize the sensitivity in detecting of known 456 fusion gene transcripts, which is approved in Japan in 2022 and covered by insurance in later 2023. Above, TOP RNA panel interrogates aberrantly spliced transcripts and analyzes the expression profiles of 1,390 genes. Primary brain tumors require various molecular profiles to be classified based on WHO2021 classification and also necessitates new therapeutic targets. In this study, we aimed to classify gliomas and identify the molecular targets by TOP RNA panel. METHODS Among 131 frozen samples of malignant gliomas, 124 samples passed quality control and were subjected to TOP RNA panel. Fusion detection and RNA expression analysis were conducted with the original pipeline. Cluster analysis and Gene Set Enrichment Analysis (GSEA) were performed to identify gene sets specific for individual subtypes. Copy numbers of tyrosine kinase genes, MDM2 and CDK4 were evaluated by digital droplet PCR (ddPCR). RESULTS Among 55 cases of glioblastoma, gene fusions were detected in 11 cases (20.0%) including novel MET fusions, and overexpression of seven tyrosine kinase genes were observed in 15 cases (27.3%). Of the 34 overexpressed genes, gene amplification was observed in 17 genes (50%) by ddPCR. In contrast to IDH-wildtype glioblastoma, IDH-mutant tumors hardly harbored fusion genes or gene overexpression. GSEA identified that the gene sets related to 1p36 and 19q were relatively enriched in astrocytoma, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. CONCLUSION TOP RNA panel detects more fusions than DNA panel and is utilized not only for the detection of molecular targets but also for the molecular classification of glioma, providing treatments optimized for the individual patients.

Deciphering the Structural Variants in B Cell Acute Lymphoblastic Leukemia By Optical Genome Mapping

Introduction Structural variants (SVs) of the genome play a key role in the leukemogenesis and development of B cell lymphoblastic leukemia (B-ALL), including gene deletion, amplification, translocation, and other forms. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection beween karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM and RNA-seq in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in B-ALL cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 70 patients with newly diagnosed or relapsed/refractory B-ALL were recruited into the study. All samples were conducted with both OGM and transcriptome sequencing (RNA-seq). Among these cases, 42 males and 28 females, 30 pediatric (< 18 years) and 40 adults (≥ 18 years) cases, ages ranged 4-60 years (median 16 years). Results Totally 50 gene fusions were reported in 48 cases, with 2 cases each carrying two fusion genes ( BCR::ABL1/ PAX5::ELK3 and P2RY8::CRLF2/ PAX5::GSE1, respectively) (Figure 1A). OGM and RNA-seq have complete concordance in reporting gene fusions, and OGM provides further insight into the genomic events that lead to gene fusion. A subtype of B-ALL characterized by UBTF microdeletion leading to UBTF::ATXN7L3 fusion and PAN3 downstream deletion-induced CDX2 aberrant expression has been reported recently, and has been accepted by the International Consensus Classification as a novel molecular subtype of UBTF/CDX2 of B-ALL. We performed the OGM investigation on 6 UBTF/CDX2 cases which were identified by RNA-seq. OGM identified both microdeletion events in all 6 cases, and further reported 1q amplification in 3 of them (Figure 1B, C). B-lymphocyte differentiation-associated transcription factor gene defects are common and important genetic abnormalities in B-ALL, often manifested as complete or partial deletion of genes. We focused on a panel including 30 genes in the OGM data and determined IKZF1 deletion in 35 patients, CDKN2A/B deletion in 32 patients, and PAX5 deletion in 25 patients (Figure 1 A). Follow-up data showed that patients with CDKN2A/B deletion had a worse prognosis than those without CDKN2A/B deletion ( P = 0.025). Conclusion This is the first B-ALL cohort to systematically compare OGM and RNA-seq in gene fusion detection, and the result showed complete consistency. RNA-seq has significant advantages in analyzing gene fusion transcripts, while OGM has technical advantages in analyzing SVs at the genome level. Combining the two technologies can accurately analyze gene fusion and other SVs in B-ALL, significantly improve the detection rate of SVs with important pathological/diagnostic classification significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Heterogeneous Gene Fusion Transcripts Found in t(7;12)(q36;p13) Acute Myeloid Leukemia but with Similar Gene Expression Profile

Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.

Philadelphia-like B-Cell Acute Lymphoblastic Leukemia in a Largely Hispanic Population: Disease Features and Outcomes in the Era of Immunotherapy a Single Institutional Study

Introduction: Philadelphia (Ph)-like B-cell acute lymphoblastic leukemia (ALL) is a high-risk subgroup of B- cell ALL with distinct genotypes, unified by a gene expression profile similar to Ph-positive ALL but lacking the BCR: ABL1 fusion. Previous studies have noted the increased prevalence of Ph-like B-cell ALL in Hispanic patients. Our study evaluates Ph-like B- cell ALL clinical/genomic features and outcomes in a predominantly Hispanic population. Methods: This is a retrospective chart review of ALL patients that underwent treatment for B-cell ALL at Norris Comprehensive Cancer Center (NCCC) between 2011 and 2023. Identification of fusions associated with Ph-like B-cell ALL was performed using cumulative results from RNA-seq, conventional cytogenetics, and fluorescence in situ hybridization (FISH) with reflex gene fusion transcript analysis. Additionally, we used the Anchored Multiplex PCR™-based multi-gene NGS assay designed to detect known and novel fusions and elevated gene expression levels using a proprietary algorithm in ALL. Cumulative incidence of relapse (CIR) was evaluated using competing risk regression (Fine-Gray method), with death as a competing outcome. Overall survival (OS) and event-free survival (EFS) were analyzed using Cox proportional hazards model. Events of interest for EFS were relapse, treatment failure, and death. Ph-negative B-cell ALL patients were set as the reference for survival analysis. Results: 253 patients were included with a median age at diagnosis of 42 years (range: 18-80). ALL subtypes included 64(25.3%) Ph-like, 77(30.4%) Ph-positive, and 112(44.3%) Ph-negative. Most were Hispanic (N = 186, 73.5%), and the median follow-up time was 27 months. The 3-year OS, EFS, and CIR were 81.3% (95% CI 75.7-87.4%), 49.1% (95% CI 42.7-56.5%), and 39.9% (95% CI 33.0-46.7%). CRLF2 related mutations constituted 44(68.8%) of Ph-like patients, with 9(14.0%) CRLF2-P2RY8, 24(37.5%) CRLF2-IGH, and 11(17.2%) CRLF2-other gene rearrangements. 19 Ph-like patients had non-CRLF2 mutations consisting of 13(20.3%) IGH, 3(4.7%) ABL1/2, 3(4.7%) JAK2, and 1(1.6%) EPOR rearrangements. Compared to Ph-negative, Ph-like patients were more likely to have a refractory disease (20.6% vs. 6.2% P=0.006) and require Blinatumomab (67.2% vs. 39.3% P<0.001), with a non-significant trend towards lower rates of CR (84.1% vs 90.9% P=0.21). Ph-like patients had significantly worse EFS (HR = 1.63; 95% CI 1.09-2.45; P=0.018), but similar OS (HR = 0.88; 95% CI 0.39-1.99; P=0.75) and CIR (HR = 1.06; 95% CI 0.65-1.70; P = 0.83). Ph-positive patients displayed improved EFS relative to Ph-negative (HR = 0.56; 95% CI 0.35-0.91; P = 0.018), similar OS (HR = 0.73; 95% CI 0.35-1.50; P = 0.39), and a trend towards improved CIR (HR = 0.67; 95% CI 0.40-1.10; P=0.11). When controlling for age, sex, and treatment type, EFS was worse in Ph-like (HR = 1.57; 95% CI 1.03-2.41; P = 0.037) and improved in Ph-positive (HR = 0.56; 95% CI 0.33-0.93; P = 0.026). Our incidences of CRLF2 mutations in Ph-like patients aged>40 compared to age ≤40 was (73.3% vs 64.7%, P=0.591). On subgroup analysis of Ph-like patients, univariate regression revealed CRLF2-related Ph-like patients had a worse CIR (HR = 5.68; 95% CI 1.91-16.9; P = 0.002) but similar OS(P=0.99) and EFS(P=0.18). PAX5 expression was also a predictor of poor OS (HR = 8.89; 95% CI 1.06-74.7; P = 0.044) and EFS (HR = 2.51; 95% CI 1.09-5.77; P = 0.031), with a trend towards increased CIR (HR = 2.30; 95% CI 0.92 -5.78; P = 0.075). Compared to non-CRLF2 mutations, CRLF2-Ph-like had worse CIR (HR = 5.68; 95% CI 1.91 -16.9; P = 0.002). Relative to Ph-like patients who achieved MRD negativity during induction, patients who remained MRD positive had worse CIR(HR = 2.92; 95% CI 1.23 -6.94; P = 0.015) and EFS (HR = 4.32; 95% CI 1.890-9.84; P < 0.001). However, after controlling for Blinatumomab administration, the effect diminished (CIR: P=0.73, EFS P=0.28). There were no differences in EFS(P=0.34), CIR(P=0.69), and OS(P=0.36) between Ph-like patients who were transplanted vs not. Conclusions: Ph-like B- Cell ALL represents a high-risk disease subtype of adult B-ALL, with poor EFS and frequent treatment failure. CRLF2 translocation confers worse CIR and EFS within Ph-like patients and requires novel treatment approaches.

Fatal intracranial haemorrhage in acute promyelocytic leukemia patients with short isoform of PML-RARα: Review of molecular and radiological data

Three major PML-RARα fusion gene transcripts (long [bcr1], variant [bcr2], and short [bcr3]) are currently used in clinical laboratories for the diagnosis and treatment monitoring of APL patients. Despite highly improved outcome, relapse and intracranial haemorrhage that may lead to early death is still an unsolved complication in APL. We reviewed APL patients confirmed by qPCR for the presence of PML-RARα transcripts (n = 27) and studied their outcome in relation to the isoform expression at diagnosis and follow-up in King Fahad Medical City. Eight in twenty-seven patients showed bcr3 and nineteen patients with bcr1 as major isoforms at diagnosis. Half of the bcr3 patients (n = 4/8) showed early mortality, prolonged qPCR positivity, 4-fold higher neutrophil/lymphocyte ratio, higher creatinine levels, and significantly reduced relapse free and overall survival time compared with bcr1 patients. Radiological findings in bcr3 patients revealed CNS involvement in the form of intracranial haemorrhage and periventricular microangiopathy and no CNS involvement in bcr1 patients. In conclusion, PML-RARα isoform expression at diagnosis in selective patients influences disease course over time and may even lead to early mortality due to haemorrhage. Thus, timely reporting of the specific PML-RARα isoform by clinical laboratories and CNS assessment by radiology can prevent complications leading to death in some APL patients.

Feasibility and utility of the custom RNA-seq for the identification of therapeutic targets and diagnostic biomarkers in glioma.

e14029 Background: The detection of novel therapeutic targets is increasingly important in oncology. The Todai OncoPanel (TOP) RNA panel is a custom target RNA-seq using the junction capture method to maximize the sensitivity in detecting of known 456 fusion gene transcripts, which is scheduled to be approved for clinical application in Japan in April 2023. Above, TOP RNA panel interrogates aberrantly spliced transcripts and analyzes the expression profiles of 1,390 genes. Malignant brain tumors, including gliomas, require various molecular profiles to be classified based on the latest WHO classification criteria, while their poor prognosis also necessitates new therapeutic targets. In this study, we aimed to classify gliomas and identify the molecular targets by TOP RNA panel. Methods: Among 131 frozen tumor samples of malignant gliomas, 124 samples passed quality control for further validation and were subjected to TOP RNA panel to classify based on the molecular profiles and to identify the molecular targets. Fusion detection and RNA expression analysis were conducted with the original pipeline. Cluster analysis and Gene Set Enrichment Analysis (GSEA) were performed to identify gene sets specific for individual subtypes. Copy numbers of tyrosine kinase genes, MDM2 and CDK4 were evaluated by digital droplet PCR (ddPCR). Results: Among 58 cases of glioblastoma, gene fusions were detected in 11 cases (19.0%) including novel MET fusions, and overexpression of seven tyrosine kinase genes were observed in 21 cases (36.2%). Of the 34 overexpressed genes, gene amplification was observed in 17 genes (50%) by ddPCR. In contrast to IDH-wildtype glioblastoma, IDH-mutant tumors including astrocytomas and oligodendrogliomas hardly harbored fusion genes or gene overexpression. GSEA to compare astrocytomas and oligodendrogliomas identified that the gene sets related to 1p36 and 19q were relatively enriched in astrocytoma, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. Conclusions: TOP RNA panel is utilized not only for the detection of molecular targets but also for the molecular classification of glioma, both of which is essential for providing treatments optimized for the individual patients.

Estimating prognostic relevant cutoff values for a multiplex PCR detecting BCR::ABL1 in chronic myeloid leukemia patients on tyrosine kinase inhibitor therapy in resource-limited settings

The prognosis of chronic myeloid leukemia (CML) on tyrosine kinase inhibitor (TKI) treatment is based on the quantification of BCR::ABL1 fusion gene transcript copy number, harmonized by an international scale (IS) based on TaqMan-based real-time quantitative PCR (qRT-PCR). In Ethiopia, as in most low- and middle-income countries (LMICs), access to standard diagnostic, follow-up, and prognostic tools is very limited, and it has been challenging to strictly follow international guidelines. This seriously compromises clinical outcome, despite the availability of TKIs through the Glivec International Patient Assistance Program (GIPAP). Multiplex PCR (mpx-PCR), conventionally regarded as a “screening tool,” offers a potential solution to this problem. A total of 219 samples from confirmed CML patients were assayed. In reference to qRT-PCR, the AUC of ROC curve for mpx-PCR was 0.983 (95% CI: 0.957 to 0.997). At the optimum cut-off value, equivalent to BCR::ABL1 (IS) transcript copy number of 0.6%, the specificity and sensitivity were 93% and 95%, respectively, with 94% accuracy. Albeit the sensitivity and accuracy of mpx-PCR decrease below the optimum cutoff of 0.6% (IS), the specificity at 0.1% (IS) was 100%, making it an attractive means to rule-out relapse and drug non-adherence at later stages of treatment, which is particularly an issue in a low income setting. We conclude that the relative simplicity and low cost of mpx-PCR and prognostic relevant cutoff values (0.1–0.6% IS) should allow its use in peripheral clinics and thus maximize the positive impact of TKIs made available through GIPAP in most LMICs.

260 Mosaic BRAF fusions are a recurrent cause of multiple congenital melanocytic naevi

Congenital melanocytic naevi (CMN) are moles present at birth, and when multiple or very extensive can involve other organ systems as well as predisposing to melanoma. Some CMN develop a highly proliferative phenotype whose progression is poorly understood. Genotypically, CMN are caused by mosaic NRAS missense mutations in 67% of cases and BRAF missense mutations in 7% of cases. Single cases of mosaic gene fusions have been previously described. To investigate the remaining 25%, skin biopsies from 19 patients shown to be double wildtype for NRAS/BRAF underwent transcriptome-wide paired-end RNA sequencing to detect gene fusion transcripts.