Detection Of Fusion Transcripts Research Articles

Pre-transplant MRD detection of fusion transcripts is strongly prognostic in KMT2A-rearranged acute myeloid leukemia

Pre-transplant detection of KMT2Ar MRD ≥0.001% by quantitative PCR was associated with significantly inferior post-transplant survival (2-year RFS 17% vs 59%; p=0.001) and increased cumulative incidence of relapse (2-year CIR 75% vs 25%, p=0.0004).

Unusual PEComa With PRCC :: TFE3 Fusion Mimicking Sinonasal Tract Melanoma.

We report a nasal cavity unusual perivascular epithelioid cell tumor (PEComa) mimicking mucosal melanoma. Immunohistochemistry was performed using BenchMark Ultra and panel of antibodies. The Ion Torrent platform and Ion AmpliSeq cancer hotspot panel were utilized for DNA genotyping. Target-specific RNA libraries for the detection of fusion transcripts were constructed using Archer Universal RNA Reagent Kit v2 and Archer FusionPlex Solid Tumor panel and sequenced on the MiSeqDx instrument. The tumor, diagnosed in 46-year-old female, was composed of spindle cells, and lacked pigmentation. Immunohistochemically, it showed a patchy HMB-45 positivity. Other melanocytic markers (S100 protein, Melan-A, SOX10) were negative. The tumor cells were weakly positive for KIT (CD117) while negative for smooth muscle actin, pancytokeratin cocktail (AE1/AE3), and synaptophysin. Diagnosis of primary sinonasal tract mucosal melanoma was favored. Additional molecular studies detected PRCC :: TFE3 fusion as the sole genetic change, and suggested the diagnosis of unusual PEComa. Previously, TFE3 fusions were reported in a subset of PEComas but not in melanomas, while PRCC involvement has only been documented once in an ocular PEComa. Immunohistochemistry revealed strong nuclear TFE3 expression concordant with the molecular findings. This report emphasis the importance of molecular testing in the differential diagnosis between PEComa and melanoma, especially when the tumor arises in a site typical of melanoma but showing an unusual morphology and immunophenotype. The detection of TFE3 fusion transcripts suggested the diagnosis of SNT PEComa, although it cannot be excluded that this and similar tumors represent a distinct diagnostic category.

Abstract 1764: Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions

Abstract Background Acute myeloid leukemia with complex karyotype (CKAML) does frequently show del(5q), deletion of chromosome 5q. Previous work in MDS and AML showed that CKAML form clusters distinguishing del(5q) cases with commonly retained regions (CRRs) in the telomeric (≥5q34) and centromeric (≤5q14.2) ends from cases with loss of the telomeric and/or centromeric regions. Interestingly, del(5q) patients with CRRs were shown to have less genomic lesions and were associated with a better prognosis than other 5q-deleted CKAML cases. Aim: Combining the advantages of two different long read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), we aimed to more comprehensively characterize the molecular landscape of CKAML with del(5q). Our integrative workflow enabled an in-depth characterization of copy number variations (CNV), genomic rearrangements, transcriptomic and epigenomic changes. Methods: Low coverage whole genome sequencing (WGS) using ONT was performed on 209 patients with CKAML to identify del(5q) cases for further in-depth analysis. For 10 selected cases with known CRR status, genomic deep sequencing using PacBio Revio and ONT PromethION was performed, as well as PacBio Revio MAS-seq for fusion transcript detection. Results: A mean sequencing depth of 3-fold (range 0.4-17.3-fold) was obtained by low coverage WGS of the 209 CKAML cases. With a resolution of 0.1 Mbp n=134 del(5q) cases were identified in our CKAML cohort (64%) and commonly deleted regions on 5q could be resolved at 5q21.2 to 5q23.3 and 5q33.3 to 5q35 (Fisher test, FDR Benjamini-Hochberg, p<10−30). These regions separated del(5q) in n=52 cases showing CRR retention and n=82 showing partially no CRR, (either the centromeric or telomeric regions, n=74, or of both regions, n=8 involved). Subsequent co-occurrence analysis provided evidence of an association between the loss of telomeric 5q region with a 3p12-25 deletion. In-depth analysis (30-50 fold) of ten distinct del(5q) cases by PacBio and ONT revealed additional insights into the complex clonal composition of del(5q) cases. For example, one case showed two distinct subclones with 5q14.2-5q35.1 deletion and with translocation t(3;5)(p14;q14) respectively. Moreover, we could demonstrate that 5q-aberrations are far more complex than initially thought showing, e.g. inverted terminal duplications that resulted in fusion transcripts involving PDE4D and leading to its deregulated expression. Conclusion: Using our long read deep sequencing approach we could further delineate the structural complexity of del(5q) CKAML. While gene expression profiling, methylation analysis and correlation of findings with clinical outcome are ongoing and will be presented at the meeting, our results help to further pinpoint the biology underlying del(5q) and to further refine this heterogeneous CKAML subgroup. Citation Format: Felix Schick, Ekaterina Slonova, Eric Straeng, Sarah Huet, Maurizio Raso, Marlon Tilgner, Frederik Damm, Pierre Sujobert, Olga Blau, Lars Bullinger, Anna Dolnik. Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions [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 1764.

Abstract 321: Optimized RNA Seq library preparation for fusion calling from FFPE samples

Abstract Gene fusions due to chromosomal rearrangement, duplication, or deletion can be important drivers of cancer. Detection of gene fusions plays a valuable role in selecting targeted therapies. A widely used method for detecting fusion transcripts is targeted RNA sequencing, which focuses sequencing reads on known fusions but does not allow for the detection of novel fusions. Whole transcriptome analysis (WTA), where the entire transcriptome is interrogated, offers an opportunity to detect both known and novel fusions. Herein we examine the effect of sample quality, input mass, increased reverse transcriptase (RT) and insert size on fusion calling. Libraries are prepared using the Watchmaker RNA Library Preparation Kit with or without Polaris Depletion and, for target enrichment, the Twist Exome 2.0 panel prior to paired end 2 × 150 Illumina sequencing. First, control samples, specifically Seraseq Fusion RNA v4 high quality and FFPE samples, are used to determine if any of the parameters interrogated increase the number of breakpoint spanning reads both in a WTA and targeted sequencing context. Once established, the optimized workflow is applied to real FFPE samples. Targeted sequencing results in a higher number of breakpoint spanning reads, compared to WTA, when controlling for sequencing depth, resulting in a higher number of reads supporting each fusion call. Longer insert sizes, achieved by modified SPRI ratios, maintain library complexity and may enhance fusion calling. Taken together, these data demonstrate an optimized protocol for fusion calling from FFPE and demonstrate the utility of both WTA and targeted sequencing for fusion detection. Citation Format: Giulia Corbet, Josh Haimes, Travis Sanders, Martin Ranik, Thomas Harrison, Eduard Casas, Kailee Reed, Doug Wendel, Kristina Giorda, Ariele Hanek, Jen Pavlica, Brian Kudlow. Optimized RNA Seq library preparation for fusion calling from FFPE samples [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 321.

Abstract 6251: Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center

Abstract Introduction Transcriptome sequencing is a well-established diagnostic tool to characterize and quantify gene expression profiles and to detect fusion transcripts. The utilization of whole-transcriptome sequencing (WTS), in clinical workflows can tremendously help improve diagnosis, risk stratification, and therapy selection. However, there are multiple challenges to widespread clinical implementation of WTS including nucleic acid input, defining appropriate quality control (QC) criteria, tumor specific baseline controls and depth of sequencing. Here, we present a validation study to deploy routine whole transcriptome sequencing with automated analysis in a mid-size academic center. Methods Nucleic acid material was obtained through clinical residual samples in the Center for Genomics and Advanced Technology. RNA samples were extracted using the Purigen Ionic® FFPE to Pure RNA Kit. RNA input sample quantities ranged from 34-100ng. WTS libraries were prepared using the Illumina® RNA Prep with Enrichment (L) Tagmentation kit with the Illumina Exome Panel for FFPE tumor samples. The validation was performed on a set of patient samples with known clinically reported variants from the Illumina TruSight® Tumor 170 assay. A total of 84 true positives with reportable fusions, 5 splicing events, 16 negative samples, and 15 limit of detection (LOD) samples were used. Libraries were prepared in a three-plex format. Sequencing was performed on the Novaseq-6000 system using S2 and S4 flow cells with a target read count of 100 million. All data was processed through the AUGMET informatics platform for demultiplexing, quality control, fusion detection, transcript quantification and determination of alternatively spliced transcript. Results The WTS validation assay demonstrated 98% (n=49/50) sensitivity, with only one false negative, and 100% (n=68/68) specificity. A final review of our data was determined to develop stable QC standards for RNA sequenced libraries to be > 60M reads and > 130bp read length. When analyzing (LOD) for WTS, interestingly, read length was inversely proportional to input and as a result detection of the clinically relevant fusion. Based on our LOD experiments, a minimum input threshold of 20ng of RNA was determined to maintain the sensitivity and specificity. An LOD study for tumor purity was not performed due to the known lack of correlation between tumor purity and fusion transcript quantity. Discussion WTS can detect all clinically reported fusions and splicing events with comparable sensitivity to panel assays. Moreover, simultaneously available transcript quantification and variant calling provide the ability to implement several disease classifiers. This work lays the foundation for integration with our clinically available tumor exome sequencing assay to allow joint exome transcriptome analysis in a single informatics platform. Citation Format: Jillian A. Moran, Edward G. Hughes, Donald C. Green, Shrey Sukhadia, Gregory J. Tsongalis, Samantha F. Allen, Laura Tafe, Brianna Houde, Nevena B. Ognjenovic, Jennifer N. Barbuto, Ivy Riano, Parth Shah. Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center [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 6251.

Relevance of detection of RAF fusion transcripts in pan-negative melanoma in routine practice.

Pan-negative melanomas account for 30% of melanomas. In case of immunotherapy failure, therapeutic options are limited. Oncogene fusions represent a target of interest in many solid cancers. In melanoma, the frequency of oncogene fusion is not well documented and not routinely investigated. We conducted a single-center retrospective study. The objective was to determine the frequency of oncogene fusion detected by RNA sequencing, in patients with advanced or metastatic pan-negative melanoma. In parallel, an extended molecular alteration search was performed using extended targeted next-generation sequencing. We identified 59 patients with advanced pan-negative melanoma between January 2021 and January 2023. It was a cutaneous melanoma in 71.1% of the cases, a mucous melanoma in 15.2% of the cases. We identified nine patients with a RAF fusion, including seven BRAF gene fusion and two RAF1 fusion. Of the other molecular alterations, NF1 mutation was the most frequent molecular alteration identified. Among the nine patients with RAF fusions, all the patients initially received treatment with anti-PD1 ± anti-CTLA4 immunotherapy. After immunotherapy failure, five patients benefited from second-line targeted therapy (two with BRAF and MEK inhibitors combination, three MEK inhibitors alone). The response rate was 20%. In a population of pan-negative melanoma, we detected 15.2% of RAF fusion. Fusion detection allowed the introduction of a second line of targeted therapy, in the absence of a validated therapeutic option in 55.5% of cases. This study suggests the relevance of detecting RAF fusion in a selected population.

Cytogenetics in the management of hematological malignancies: An overview of alternative technologies for cytogenetic characterization

Genomic characterization is an essential part of the clinical management of hematological malignancies for diagnostic, prognostic and therapeutic purposes. Although CBA and FISH are still the gold standard in hematology for the detection of CNA and SV, some alternative technologies are intended to complement their deficiencies or even replace them in the more or less near future. In this article, we provide a technological overview of these alternatives. CMA is the historical and well established technique for the high-resolution detection of CNA. For SV detection, there are emerging techniques based on the study of chromatin conformation and more established ones such as RTMLPA for the detection of fusion transcripts and RNA-seq to reveal the molecular consequences of SV. Comprehensive techniques that detect both CNA and SV are the most interesting because they provide all the information in a single examination. Among these, OGM is a promising emerging higher-solution technique that offers a complete solution at a contained cost, at the expense of a relatively low throughput per machine. WGS remains the most adaptable solution, with long-read approaches enabling very high-resolution detection of CAs, but requiring a heavy bioinformatics installation and at a still high cost. However, the development of high-resolution genome-wide detection techniques for CAs allows for a much better description of chromoanagenesis. Therefore, we have included in this review an update on the various existing mechanisms and their consequences and implications, especially prognostic, in hematological malignancies.

A Protocol for the Detection of Fusion Transcripts Using RNA-Sequencing Data.

Fusion transcripts are formed when two genes or their mRNAs fuse to produce a novel gene or chimeric transcript. Fusion genes are well-known cancer biomarkers used for cancer diagnosis and as therapeutic targets. Gene fusions are also found in normal physiology and lead to the evolution of novel genes that contribute to better survival and adaptation for an organism. Various in vitro approaches, such as FISH, PCR, RT-PCR, and chromosome banding techniques, have been used to detect gene fusion. However, all these approaches have low resolution and throughput. Due to the development of high-throughput next-generation sequencing technologies, the detection of fusion transcript becomes feasible using whole genome sequencing, RNA-Seq data, and bioinformatics tools. This chapter will overview the general computational protocol for fusion transcript detection from RNA-sequencing datasets.

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

BackgroundGenomic 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.ResultsWe 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.ConclusionsA 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.

Somatic and Germline Cohesin Genes Alterations in Pediatric Acute Lymphoblastic Leukemia

Leukemia is a complex disease and the molecular mechanisms of malignant transformation are not fully understood yet. Cohesins are essential proteins, forming a complex which plays critical roles in various cellular processes, not only by canonical DNA binding and chromosome segregation but also in non-canonical regulation of gene expression in both proliferating and post-mitotic cells. Mutations or gene expression alterations of genes encoding the components of the cohesins' complex may affect chromosomal stability and potentially contribute to the pathogenesis of leukemia. Somatic cohesins mutations have been already reported to occur in myeloproliferative disorders and pediatric Acute Lymphoblastic Leukemia (ALL), while on the other hand, germline mutations are causative of Cohesinopathies such as Cornelia de Lange Syndrome (CdLS). Our previous first report of a patient affected by both CdLS and ALL suggested a potential involvement of cohesin germline mutations in the pathogenesis of ALL. Moreover, we demonstrated that germline mutations in STAG1 gene could play a crucial role in predisposition to pediatric hematological malignancies, including ALL. These findings suggest that both somatic and germline alterations of cohesin genes may be associated with the development of ALL. This study aims to understand the molecular mechanisms affected by Cohesin genes alterations, that could potentially pave the way for the development of targeted therapies and improved treatment strategies for leukemia patients. By employing a DNA-targeted next-generation sequencing (NGS) screening of a cohort of 120 consecutive pediatric ALL cases (enrolled in the AIEOP-BFM protocol at various Italian centers), we discovered a total of 11 germline cohesin mutations, further categorized as follows: 6 out of 11 were missense, 4 out of 11 were located in the 3' or 5' untranslated regions (UTR) and 1 out of 11 was a splice-acceptor variant. To investigate the functional implications of selected germline mutations in STAG1 gene, we took advantage of the CRISPR/Cas9 technique to generate in vitro models of two Lymphoblastoid Cell Lines (LCLs) harboring specific variants already found in two patients of our cohort. Additionally, we established control LCLs from healthy donors' samples using the same CRISPR/Cas9 approach. Subsequently, we performed RNA sequencing (RNAseq) analysis on these cell lines, and our preliminary findings revealed a total of 619 differentially expressed genes (DEGs) compared to the control group. Many of them are involved in critical intracellular pathways, like ribosomal transcription, suggesting potential disruptions of essential cellular processes caused by germline STAG1 mutations. Moreover, we have successfully developed an advanced computational analysis pipeline with the primary objective of detecting fusion transcripts from RNAseq data. This pipeline was applied to a dataset of 529 consecutive diagnose and 95 relapse samples of pediatric B/T-ALL patients and in five B-ALL cases a fusion transcript involving the STAG1 (n=1), STAG2 (n=3), and NIPBL (n=1) genes was identified. The significance of these findings was further reinforced by additional cases from both pediatric and adult cohorts through an international collaborative effort and five more fusion transcripts of STAG2 (n=2) and NIPBL (n=3) were uncovered. Investigation to clarify the underlying functional consequences of these fusion genes in pediatric ALL are ongoing. Collectively, these observations endeavor to elucidate the role of germline and somatic cohesin events in pediatric ALL. In particular, the investigation seeks to unveil the non-canonical functions of these proteins, thus paving the way for the identification and characterization of a novel and distinct subcategory of leukemia within the pediatric population.

FindDNAFusion: An Analytical Pipeline with Multiple Software Tools Improves Detection of Cancer-Associated Gene Fusions from Genomic DNA

Detection of cancer-associated gene fusions is crucial for diagnosis, prognosis, and treatment selection. Many bioinformatics tools are available for the detection of fusion transcripts by RNA sequencing, but there are fewer well-validated software tools for DNA next-generation sequencing (NGS). A 542-gene solid tumor NGS panel was designed, with exonic probes supplemented with intronic bait probes against genes commonly involved in oncogenic fusions, with a focus on lung cancer. Three software tools for the detecting gene fusions in this DNA-NGS panel were selected and evaluated. The performance of these tools was compared after a pilot study, and each was configured for optimal batch analysis and detection rate. A blacklist for filtering common tool-specific artifacts, and criteria for selecting clinically reportable fusions, were established. Visualization tools for annotating and confirming somatic fusions were applied. Subsequently, a full clinical validation was used for comparing the results to those from in situ hybridization and/or RNA sequencing. With JuLI, Factera, and GeneFuse, 94.1%, 88.2%, and 66.7% of expected fusions were detected, respectively. With a combinatorial pipeline (termed FindDNAFusion), developed by integrating fusion-calling tools with methods for fusion filtering, annotating, and flagging reportable calls, the accuracy of detection of intron-tiled genes was improved to 98.0%. FindDNAFusion is an accurate and efficient tool in detecting somatic fusions in DNA-NGS panels with intron-tiled bait probes when RNA is unavailable.

Deep and Durable Cytogenetic and Molecular Responses with Pemigatinib in Myeloid/Lymphoid Neoplasms with Fibroblast Growth Factor Receptor 1 Rearrangement: The Fight-203 Study

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1 from t[8;13][p11;q12] or BCR::FGFR1 from t[8;22][p11;q11]) and constitutive FGFR1 activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1 in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1 fusion transcripts in this same cohort. Methods: FIGHT-203 (NCT03011372) is an ongoing open-label, multicenter, phase 2 study evaluating pemigatinib in patients ≥18 years old with MLN FGFR1 and ≥1 prior therapy. The starting dose was pemigatinib 13.5 mg daily (2 weeks on, 1 week off). Following protocol amendments, patients without prior therapy were eligible to enroll, and the dosing regimen was changed to a continuous schedule. Patients were censored for cytogenetic and molecular follow-up at the time of transplantation. CCyR was defined as 0 metaphases positive for the respective reciprocal translocation in ≥20 evaluated BM metaphases on local and/or central karyotyping or 0 cells (or not exceeding the lower level of detection of the probe) with the FGFR1 rearrangement in ≥200 BM cells on local and/or central break-apart FISH assays (central FISH prioritized). As part of the translational research plan, fusion partner genes were identified with next-generation sequencing (NGS; FusionPlex, Archer, Boulder, CO, USA) and targeted PCR profiling of RNA extracted from whole blood; droplet digital PCR (ddPCR) assays were developed to monitor the frequencies of specific FGFR1 fusion transcripts in RNA extracted from whole blood samples (estimated limit of detection, 0.05 copies/μL). Results: As of June 30, 2021, 39 of 41 enrolled and treated patients were evaluable for translational analyses. Among the 39 patients with whole blood samples, FGFR1 fusion partner genes identified on translational testing were ZMYM2 (n=17), BCR (n=11), FGFR1OP (n=4), TPR (n=3), TRIM24 (n=2), and C14orf93, CCDC6, CNTRL and ETV6 (n=1 each). Two of 39 patients had 2 fusions detected ( ZMYM2 and BCR, n=1; ZMYM2 and TPR, n=1). C14orf93, CCDC6 and ETV6 have not been previously identified as FGFR1 fusion partner genes. Central FISH assessment of BM cells confirmed FGFR1-aberrantcells were reduced to undetectable levels in responding patients but rebounded in some patients during periods of treatment interruption (Figure A). Serial ddPCR of whole blood samples demonstrated marked reductions in FGFR1 fusion transcripts for the 34 patients with ≥3 measures while on treatment (Figure B). Among patients with CR, 81.0% had a >2-log reduction and 47.6% had a >3-log reduction in detectable FGFR1 fusion transcripts. Smaller reductions in FGFR1 fusion transcripts were observed in patients with stable disease. Changes in FGFR1 fusion transcripts correlated with FGFR1 FISH results (Pearson coefficient = 0.81) and reflected changes in treatment intensity. Conclusion:Several new FGFR1 fusion partner genes, including C14orf93, CCDC6, and ETV6 were identified with NGS and PCR profiling. Pemigatinib treatment resulted in marked decreases in the percentages of cells with the FGFR1 rearrangement on FISH, as well as 2-3 log reductions in FGFR1 fusion transcripts in patients with MLN FGFR1. Further investigations are ongoing to determine the relationship between pemigatinib dose intensity and the depth and durability of molecular response.

Diagnostic des sarcomes utérins et tumeurs mésenchymateuses utérines rares à potentiel de malignité. Référentiels du Groupe Sarcome Français et des Tumeurs Rares Gynécologiques

The landscape of uterine sarcomas is becoming more complex with the description of new entities associated with recurrent driver molecular alterations. Uterine sarcomas, in analogy with soft tissue sarcomas, are distinguished into complex genomic and simple genomic sarcomas. Leiomyosarcomas and undifferentiated uterine sarcomas belong to complex genomic sarcomas group. Low-grade and high-grade endometrial stromal sarcomas, other rare tumors associated with fusion transcripts (such as NTRK, PDGFB, ALK, RET ROS1) and SMARCA4-deficient uterine sarcoma are considered simple genomic sarcomas. The most common uterine sarcoma are first leiomyosarcoma and secondly endometrial stromal sarcomas. Three different histological subtypes of leiomyosarcoma (fusiform, myxoid, epithelioid) are identified, myxoid and epithelioid leiomyosarcoma being more aggressive than fusiform leiomyosarcoma. The distinction between low-grade and high-grade endometrial stromal sarcoma is primarily morphological and immunohistochemical and the detection of fusion transcripts can help the diagnosis. Uterine PEComa is a rare tumor, which is distinguished into borderline and malignant, according to a risk assessment algorithm. Embryonal rhabdomyosarcoma of the uterine cervix is more common in children but can also occur in adult women. Embryonal rhabdomyosarcoma of the uterine cervix is almost always DICER1 mutated, unlike that of the vagina which is wild-type DICER1, and adenosarcoma which can be DICER1 mutated but with less frequency. Among the emerging entities, sarcomas associated with fusion transcripts involving the NTRK, ALK, PDGFB genes benefit from targeted therapy. The integration of molecular data with histology and clinical data allows better identification of uterine sarcomas in order to better treat them.

The efficacy of the TRK inhibitor entrectinib in patients with extracranial <i>NTRK</i> fusion-positive tumors

Somatic translocations involving the NTRK genes occur in 0.34–2.2% of all malignant neoplasms in children. TRK inhibitors whose efficacy has been demonstrated in prospective clinical studies expand treatment options for patients with solid tumors harboring NTRK gene rearrangements. The aim of our study was to summarize the first Russian experience with the use of the TRK inhibitor entrectinib in patients with extracranial NTRK fusion-positive solid tumors included in the compassionate use program. This study was approved by the Independent Ethics Committee and the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included 8 patients who had been treated at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and the N.N. Blokhin National Medical Research Center of Oncology. The main criteria for inclusion in the compassionate use program were a confirmed rearrangement of either NTRK1/2/3 genes in a solid tumor in patients with unresectable disease for whom no effective standard systemic therapy was available, progressive or recurrent disease during therapy prescribed according to the established diagnosis, risk group and risk stratification criteria, and the infeasibility of non-mutilating radical surgery. The median age at diagnosis was 4.3 months (range 1.2–83.6). The male to female ratio was 1:1. The primary site distribution was as follows: head and neck (n = 6; 75%), chest wall (n = 1; 12.5%), pelvis (n = 1; 12.5%). None of the patients had regional lymph node involvement or distant metastases at diagnosis. The distribution by histology (according to histopathology reports) was as follows: infantile fibrosarcoma (n = 4; 50%), undifferentiated round cell sarcoma, low-grade (n = 1; 12.5%), undifferentiated spindle cell sarcoma, high-grade (n = 1; 12.5%), NTRK-rearranged spindle cell sarcoma, low-grade (n = 1; 12.5%), spindle cell tumor associated with an NTRK rearrangement (n = 1; 12.5%). Immunohistochemistry (IHC) with a pan-Trk monoclonal antibody was performed in 7/8 (87.5%) patients. Pan-Trk IHC was positive in 4/7 (57%) patients. Rearrangements in the NTRK1 and NTRK3 genes were confirmed in all the patients. The final methods used for the detection of fusion transcripts were as follows: reverse transcription polymerase chain reaction (n = 4; 50%) and RNA-based next-generation sequencing (n = 4; 50%). NTRK1 and NTRK3 gene translocations were detected in 3/8 (37.5%) and 5/8 (62.5%) patients, respectively. The following fusion transcripts were identified: ETV6::NTRK3 (n = 4), DIP2C::NTRK3 (n = 1), TPR::NTRK1 (n = 1), TPM3::NTRK1 (n = 1), MYH10::NTRK1 (n = 1). One (12.5%) patient received entrectinib as first-line therapy, other patients (7/8, 87.5%) received entrectinib as secondor subsequent-line therapy. Three (37.5%) patients had undergone surgery before treatment with entrectinib: 2 had R2 resection, 1 had R0/R1 resection (resection margins were not evaluated). None of the patients received radiation therapy. The median duration of entrectinib therapy at the time of analysis was 11.8 months (range 2.3–20.1). Delayed surgery was performed in 2/8 patients; according to the histopathology reports, they achieved grade IV pathomorphosis. Three patients experienced adverse events during treatment with entrectinib. The median time to adverse events was 0.23 months (range 0.2–7.96). Three patients required temporary interruption in treatment to relieve symptoms, a subsequent dose reduction by one dose level was necessary when resuming therapy in two patients. The median follow-up since diagnosis was 19.5 months (range 14.9–75.0). All the patients included in our analysis were alive, three of them had no radiologic evidence of disease. Fifty percent of the patients completed targeted therapy, another 50% of the patients continued treatment with entrectinib. Complete and very good partial response was achieved in 3/8 and 2/8 patients, respectively. Partial response, minor partial response and stable disease were observed in one patient each. These results indicate high efficacy and safety of entrectinib in pediatric patients with extracranial NTRK fusion-positive solid tumors. Further studies are needed to determine the therapeutic potential of TRK inhibitors in the treatment of different solid malignant neoplasms in children and to assess long-term treatment results.

Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements

Introduction: A variety of gene rearrangements and molecular alterations are key drivers in the pathobiology of acute leukemia and myeloid disorders; current classification systems increasingly incorporate these findings in diagnostic algorithms. Therefore, clinical laboratories require versatile tools, which can detect an increasing number and variety of molecular and cytogenetic alterations of clinical significance. Methods: We validated an RNA-based next-generation sequencing (NGS) assay that enables the detection of: (i) numerous hybrid fusion transcripts (including rare/novel gene partners), (ii) aberrantly expressed EVI1 (MECOM) and IKZF1 (Del exons 4–7) transcripts, and (iii) hotspot variants in KIT, ABL1, NPM1 (relevant in the context of gene rearrangement status). Results: For hybrid fusion transcripts, the assay showed 98–100% concordance for known positive and negative samples, with an analytical sensitivity (i.e., limit of detection) of approximately 0.8% cells. Samples with underlying EVI1 (MECOM) translocations demonstrated increased EVI1 (MECOM) expression. Aberrant IKZF1 (Del exons 4–7) transcripts detectable with the assay were also present on orthogonal reverse transcription PCR. Specific hotspot mutations in KIT, ABL1, and NPM1 detected with the assay showed 100% concordance with orthogonal testing. Lastly, several illustrative samples are included to highlight the assay’s clinically relevant contributions to patient workup. Conclusion: Through its ability to simultaneously detect various gene rearrangements, aberrantly expressed transcripts, and hotspot mutations, this RNA-based NGS assay is a valuable tool for clinical laboratories to supplement other molecular and cytogenetic methods used in the diagnostic workup and in clinical research for patients with acute leukemia and myeloid disorders.

EGFR and ERBB2 exon 20 insertion/duplication in advanced non-small cell lung cancer: genomic profiling and clinicopathologic features.

Exon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non-small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression. A total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones. Nine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767-V774 in EGFR and Y772-P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases. NSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.

Abstract LB078: pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data

Abstract Sarcomas are a broad group of soft tissue and bone cancers that can be difficult to treat leading to a high mortality rate. Sarcomas comprise two broad genomic classes: (1) simple karyotypes, where a single oncogenic structural variant (SV) clonally expands a subtype that is diagnostic and relevant to tumor burden tracking; and (2) complex karyotypes, genomic instability, where SVs continuously arise throughout tumor evolution resulting in heterogeneous cellular subtypes. Class two sarcomas are harder to characterize using genome sequencing because there may be multiple low-frequency mutations. In both genomic classes, accurate and sensitive detection of fusion transcripts is needed to interpret functional consequences, to understand tumor biology and evolution, and potentially identify new targets for therapy. Many fusions have complex structures that cannot be uniquely resolved using short reads due to a lack of exon connectivity. PacBio full-length RNA isoform sequencing resolves complex fusions, providing more accurate breakpoints, and a complete sequence readout of the associated fusion transcript. To date, long-read fusion detection software was designed for high-error sequencing. PacBio HiFi data provides both full-length transcripts and accurate base calls. Here we present a fusion detection tool, pbfusion, specifically designed for HiFi sequence data, and apply it to sarcoma patients from both classes. pbfusion converts mapped sequences (either HiFi reads or Iso-Seq isoforms) into transcript objects that are annotated with reference gene models. Annotations determine whether transcripts are discordantly mapped, overlap differing genes, strand swap, transcriptional readthrough, or contain novel exons. The discordant exonic boundaries are treated as breakpoints between two genomic locations. All breakpoints are clustered with a multi-directional chaining algorithm and annotated with exonic information, gene names, and quality information. To test our method, we applied pbfusion to twelve samples from 8 sarcoma patients from both genomic classes. We discovered the known and novel fusions, including validated driver events in the fusion-driven samples (e.g. ASPSCR1-TFE3 in alveolar soft part sarcoma and SS18-SSX2/1 fusion in synovial sarcoma). This approach demonstrates the utility of HiFi sequence data for identification of fusion transcripts in patient samples, and the use of pbfusion in quantifying and annotating these events. pbfusion provides a user-friendly interface, can process a sample in a few minutes, and is freely available to the research community on Bioconda. Citation Format: Roger Volden, Zev Kronenberg, Aaron Gillmor, Ted Verhey, Michael Monument, Donna Senger, Harsharan Dhillon, Jason Underwood, Elizabeth Tseng, Daniel Baker, Primo Baybayan, Michael A. Eberle, Jonas Korlach, Sorana Morrissy. pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB078.

Abstract 232: Fully automated comprehensive genomic profiling for detection of cancer variants, gene fusions, and complex oncology endpoints

Abstract Introduction: We present the comprehensive genomic profiling performance of the Ion Torrent Genexus system using the Oncomine Comprehensive Assay Plus (OCA Plus), a 500+ gene targeted AmpliSeq-based oncology research panel that evaluates DNA variants (including copy number alterations), RNA fusions, and key oncology research endpoints including tumor mutational burden (TMB), microsatellite instability (MSI), and homologous recombination repair deficiency (HRD) via characterization of genomic instability by the newly introduced Genomic Instability Metric (GIM). Methods: The Ion Torrent Genexus System provides comprehensive genomic profiling via automated sample-to-report workflow with next day results. The Genexus System supports oncology research panels such as OCA Plus, which is comprised of over 13,000 amplicons, and enables low input requirements of just 20ng of FFPE DNA and RNA. This study utilized cell lines, reference controls, and orthogonally tested FFPE research samples to evaluate detection of DNA variants, copy number alterations, RNA fusions, and key research endpoints, including MSI, TMB, and HRD. The OCA Plus panel was also evaluated for the ability to detect arm-level copy number changes in orthogonally validated FFPE samples. Results: Commercial reference controls and FFPE research samples were sequenced using OCA Plus on the Genexus System to an average depth of ≥24 million reads per sample, with four DNA and RNA samples supported per run. SNV and MNV calling performance was assessed using the AcroMetrix Oncology Hotspot Control which has 377 variants covered by OCA Plus and delivered SNV sensitivity and PPV >99% and MNV sensitivity of >99% and PPV >95%. MSI status was assessed using orthogonally tested FFPE samples from various tumor tissues (stomach, endometrial, colorectal) and returned status concordance of 99.4% with sensitivity and PPV >99%. The TMB endpoint was tested using commercial controls and FFPE samples with a correlation of r2 > 0.90 to orthogonal measurements. RNA Fusion reference controls showed 100% positive correlation. Copy number gain detection shows sensitivity of 99% and PPV >95%, while homologous copy loss gives 100% PPV and sensitivity of >90%. We also demonstrate high concordance to orthogonal methods in detection of HER2 amplifications, and the ability to detect arm-level copy number alterations such as 1p/19q co-deletions in IDH1 positive glioma samples. Conclusion: The Genexus System combines minimal touch points and a rapid turnaround time to enable comprehensive genomic profiling for research assays such as OCA Plus for detection of rare variants and low-level fusion transcripts. Further, by providing accurate characterization of key oncology research endpoints, the Genexus System can accelerate research in oncology. For research use only. Not for use in diagnostic procedures. Citation Format: Geoffrey Marc Lowman, Dinesh Cyanam, Emily Norris, Michelle Toro, Coleen Nemes, Tanaya Puranik, Yan Zhu, Alex Phan, Derek Wong, Portia Bernado, Anelia Kralcheva, Srinivas Nallandhighal, Loni Pickle, April Bigley, Mohit Gupta, Ying Jin, Sameh El-Difrawy, Amir Marcovitz, Fatima Zare, Charles Scafe, Yu-Ting Tseng, Jianjun Guo, Vinay Mittal, Scott Myrand, Santhoshi Bandla, Paul Williams, Eugene Ingerman, Elaine Wong-Ho, Seth Sadis, Mark Andersen, Rob Bennett. Fully automated comprehensive genomic profiling for detection of cancer variants, gene fusions, and complex oncology endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 232.

Collecting ducts carcinoma: In depth exploration and biologically driven therapy (CICERONE).

TPS754 Background: Collecting duct carcinoma (CDC) is a rare type of non-clear cell renal cell carcinoma with an aggressive course and poor prognosis. The treatment of metastatic disease remains unsatisfactory as no standard-of-care has been established yet. The gap in the clinical management is certainly linked to the poor molecular characterization. To date, a comprehensive biological characterization of CDC and the identification of predictive biomarker is still lacking. Our previous studies proved that CDC is a molecularly heterogeneous disease composed of at least two subtypes distinguished by cell signalling, metabolic and immune-related alterations and featured by a prognostic signature associating T cell immunity and angiogenesis with good clinical outcome and sensitivity to cabozantinib (Gargiuli et al, Cancers 2021 Jun 10;13(12):2903; Todoerti et al, J Clin Oncol 40, no. 16_suppl (June 01, 2022) e16507-e16507; Verzoni et al, Ann Oncol 33:S1218-S121). The aim of CICERONE trial is to change the paradigm of CDC management providing therapies in a biologically informed manner starting from our previous findings. Methods: First, we will define a molecular taxonomy of CDC through comprehensive transcriptomic profiling of 100 CDC samples retrospectively collected within 12 months from the Italian Group of Uropathology Network and the European Union Hub Center. Pathologic review by genitourinary pathologist was required. RNA-Seq will be performed. Results will be merged to that already obtained by the same methodology from the BONSAI dataset (NCT03354884) and evaluated by unsupervised clustering analysis. We will identify differentially expressed genes and pathways. The immune and angiogenic signatures will be searched validating our preliminary results to identify predictive signatures for the subsequent prospective phase II trial. RNA-seq data will be also explored to detect fusion transcripts, as potential source of novel CDC oncogenic drivers, and study immune tumor content through Cybersort deconvolution. RT-qPCR on RNA derived from tumor and normal tissue will be performed to determine tumor specific origin of the fusion transcripts. Immune infiltrate will be also characterized by IHC, together with PDL-1 scoring. Subsequently we will conduct a prospective phase II trial in order to optimize treatment choice and validate prospectively the biological data. In this phase, previously untreated patients with metastatic CDC will be treated according to baseline tumor signature. Moreover, we will assess whether a new concept of immune liquid biopsy that includes the assessment of immunosuppressive vs antitumor immune cells may represent an accessible and compliant tool to test systemic immunity and its implications in response to treatment. This study was supported by the Italian Ministry of health (RF-2019-12369602). Clinical trial information: NCT05372302 .

NovoRNABreak: local assembly for novel splice junction and fusion transcript detection from RNA-seq data

We present novoRNABreak, a unified framework for cancer specific novel splice junction and fusion transcript detection in RNA-seq data obtained from human cancer samples. novoRNABreak is based on a local assembly model, which offers a tradeoff between the alignment-based and de novo whole transcriptome assembly (WTA) methods. This approach is accurate and sensitive in assembling novel junctions that are difficult to directly align or have multiple alignments. Additionally, it is more efficient due to the strategy that focuses on junctions rather than full length transcripts. The performance of novoRNABreak is demonstrated by a comprehensive set of experiments using synthetic data generated based on genome reference, as well as real RNA-seq data from breast cancer and prostate cancer samples. The results show that our tool has a better performance by fully utilizing unmapped reads and precisely identifying the junctions where short reads or small exons have multiple alignments. novoRNABreak is a fully-fledged program available on GitHub (https:// github.com/KChen-lab/novoRNABreak).