Fusion Gene Detection Research Articles

Cryptic TCF3 fusions in childhood leukemia: Detection by RNA sequencing.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5%-11% of ALL patients. In < 1%, the TCF3 alteration in ALL leads to a TCF3-HLF fusion gene. Even though this is a very rare event, the detection of a TCF3-HLF fusion gene is associated with a very poor prognosis with incurable relapses in almost all patients. The frequent TCF3-PBX1 fusion gene, which is detectable in 5%-10% of childhood B-cell precursor ALLs and ~3.8% of adult B-cell precursor ALLs, is associated with a rather good prognosis, that is, an observed event-free 5-year survival of approximately 85%. Thus, the distinction of the different partner genes fused to TCF3 is essential for risk assessment. To verify RNA sequencing as a tool for detection of known and unknown fusion genes, we screened 200 cases of pediatric B-cell precursor ALL with "targeted" RNA sequencing in a pilot project in comparison to classical cytogenetic analyses (chromosome R-banding analysis), fluorescence in situ hybridization, and PCR. We observed a TCF3 fusion gene in 6.5% (13/200) of the patients. Ten (5%) patients displayed a TCF3-PBX1 fusion gene, two (1%) patients a TCF3-FLI1 fusion gene, and one (0.5%) patient a TCF3-HLF fusion gene. For the TCF3 fusions, we obtained discrepant results with the different methods, which are described in the article. Taken together, translocations leading to TCF3 fusion genes might appear cryptic and may remain undetected by a single method.

1269P Identification of rare gene fusions in non-small cell lung cancer with next-generation sequencing

Given the limited efficacy of conventional chemotherapeutic agents in non-small lung cancer (NSCLC), targeted therapies are being pursued as potential treatment alternatives. For driver gene-negative NSCLC, the detection of rare gene fusions can improve the patient's chance with medication.

Integrative Analysis of Gene Expression Data by RNA Sequencing for Differential Diagnosis of Acute Leukemia: Potential Application of Machine Learning.

BCR-ABL1–positive acute leukemia can be classified into three disease categories: B-lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), and mixed-phenotype acute leukemia (MPAL). We conducted an integrative analysis of RNA sequencing (RNA-seq) data obtained from 12 BCR-ABL1–positive B-ALL, AML, and MPAL samples to evaluate its diagnostic utility. RNA-seq facilitated the identification of all p190 BCR-ABL1 with accurate splicing sites and a new gene fusion involving MAP2K2. Most of the clinically significant mutations were also identified including single-nucleotide variations, insertions, and deletions. In addition, RNA-seq yielded differential gene expression profile according to the disease category. Therefore, we selected 368 genes differentially expressed between AML and B-ALL and developed two differential diagnosis models based on the gene expression data using 1) scoring algorithm and 2) machine learning. Both models showed an excellent diagnostic accuracy not only for our 12 BCR-ABL1–positive cases but also for 427 public gene expression datasets from acute leukemias regardless of specific genetic aberration. This is the first trial to develop models of differential diagnosis using RNA-seq, especially to evaluate the potential role of machine learning in identifying the disease category of acute leukemia. The integrative analysis of gene expression data by RNA-seq facilitates the accurate differential diagnosis of acute leukemia with successful detection of significant gene fusion and/or mutations, which warrants further investigation.

Diagnostic Utility of Gene Fusion Panel to Detect Gene Fusions in Fresh and Formalin-Fixed, Paraffin-Embedded Cancer Specimens

Somatic gene fusions are common in leukemias/lymphomas and solid tumors. The detection of gene fusions is crucial for diagnosis. NanoString fusion technology is a multiplexed hybridization method that interrogates hundreds of gene fusions in a single reaction. This study's objective was to determine the performance characteristics and diagnostic utility of NanoString fusion assays in a clinical diagnostics laboratory. Validation using 100 positive specimens and 15 negative specimens by a combined reference standard of fluorescence in situ hybridization (FISH)/RT-PCR/next-generation sequencing (NGS) assays achieved 100% sensitivity in leukemias/lymphomas and 95.0% sensitivity and 100% specificity in solid tumors. Subsequently, 214 consecutive clinical cases, including 73 leukemia/lymphoma specimens and 141 formalin-fixed, paraffin-embedded solid tumor specimens, were analyzed by gene fusion panels across 638 unique gene fusion transcripts. A variety of comparator tests, including FISH panels, conventional karyotyping, a DNA-based targeted NGS assay, and custom RT-PCR testing, were performed in parallel. The gene fusion assay detected 31 gene fusions, including 16 in leukemia/lymphoma specimens and 15 in solid tumor specimens. The overall sensitivity, specificity, and accuracy of gene fusions detected by the gene fusion panel in all 329 specimens (validation and consecutive clinical specimens) tested in this study were 94.8%, 100%, and 97.9%, respectively, compared with FISH/RT-PCR/NGS assays. The gene fusion panel is a reliable approach that maximizes molecular detection of fusions among both fresh and formalin-fixed, paraffin-embedded cancer specimens.

Expression of androgen receptor splice variant, AR-V7, in high-grade serous ovarian cancer

<h3>Objectives:</h3> Androgen receptors (AR) are expressed in up to 55% of ERα-negative breast cancers overall and up to 35% of those classified as TNBC. Similarly, AR is expressed in approximately 40% of high grade serous ovarian cancer (HGSOC). The development of AR splice variants (AR-SV), particularly AR-V7, has been shown to be a poor prognostic feature, and mechanism of androgen resistance in breast and prostate cancers. We sought to determine if AR-V7 was present in HGSOC and if there was a correlation with platinum sensitivity and/or treatment outcomes. <h3>Methods:</h3> Pair-matched chemo-naïve and platinum-treated HGSOC specimens were identified from our tissue repository. Formalin-fixed paraffin-embedded tumor (FFPE) blocks were collected and tested for expression of AR-V7. Gene fusion detection and variant detection were performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required. <h3>Results:</h3> Twelve chemo-naïve specimens collected at time of diagnosis by initial biopsy (n=8) or primary debulking surgery (n=4) were pair-matched with the patients' subsequent platinum-treated specimens from either interval debulking surgery (n=6) or biopsy of recurrence (n=6). Following penultimate platinum, 50% were platinum resistant and 50% were platinum sensitive at time of second pathology. Only 19 of the 24 specimens submitted had sufficient RNA for testing, 8 chemo-naïve and 11 platinum-treated. AR-V7 variant transcript was not detected in any of the specimens submitted. <h3>Conclusions:</h3> AR-V7 was not expressed in any specimen before or after platinum based chemotherapy suggesting it may not be a useful prognostic marker in the management of HGSOC. Further investigation into gynecologic malignancies with high AR expression and treated with androgen deprivation therapy may elucidate different results.

Methods for actionable gene fusion detection in lung cancer: now and in the future.

Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.

Identification of Tissue of Origin and Guided Therapeutic Applications in Cancers of Unknown Primary Using Deep Learning and RNA Sequencing (TransCUPtomics)

Cancers of unknown primary (CUP) are metastatic cancers for which the primary tumor is not found despite thorough diagnostic investigations. Multiple molecular assays have been proposed to identify the tissue of origin (TOO) and inform clinical care; however, none has been able to combine accuracy, interpretability, and easy access for routine use. We developed a classifier tool based on the training of a variational autoencoder to predict tissue of origin based on RNA-sequencing data. We used as training data 20,918 samples corresponding to 94 different categories, including 39 cancer types and 55 normal tissues. The TransCUPtomics classifier was applied to a retrospective cohort of 37 CUP patients and 11 prospective patients. TransCUPtomics exhibited an overall accuracy of 96% on reference data for TOO prediction. The TOO could be identified in 38 (79%) of 48 CUP patients. Eight of 11 prospective CUP patients (73%) could receive first-line therapy guided by TransCUPtomics prediction, with responses observed in most patients. The variational autoencoder added further utility by enabling prediction interpretability, and diagnostic predictions could be matched to detection of gene fusions and expressed variants. TransCUPtomics confidently predicted TOO for CUP and enabled tailored treatments leading to significant clinical responses. The interpretability of our approach is a powerful addition to improve the management of CUP patients.

Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms.

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

Mutational profiling in acute lymphoblastic leukemia by RNA sequencing and chromosomal genomic array testing.

BackgroundComprehensive molecular and cytogenetic profiling of acute lymphoblastic leukemia (ALL) is important and critical to the current standard of care for patients with B‐acute lymphoblastic leukemia (B‐ALL). Here we propose a rapid process for detecting gene fusions whereby FusionPlex RNA next‐generation sequencing (NGS) and DNA chromosome genomic array testing (CGAT) are combined for a more efficient approach in the management of patients with B‐ALL.MethodsWe performed RNA NGS and CGAT on 28 B‐ALL samples and, in four patients, compared fixed cell pellets to paired cryo‐preserved samples as a starting material to further assess the utility of cytogenetic fixed pellets for gene expression analysis.ResultsAmong the fixed specimens, when using alternative techniques as references, including karyotype, fluorescence in situ hybridization, CGAT, and RT‐qPCR, fusions were detected by RNA NGS with 100% sensitivity and specificity. In the four paired fixed versus fresh cryopreserved samples, fusions were also 100% concordant. Four of the 28 patients showed mutations that were detected by RNA sequencing and three of four of these mutations had well‐known drug resistance implications.ConclusionsWe conclude that FusionPlex is a robust and reliable anchored multiplex RNA sequencing platform for use in the detection of fusions in both fresh cryopreserved and cytogenetic fixed pellets. Gene expression data could only be obtained from fresh samples and although limited variant data are available, critical hotspot variants can be determined in conjunction with the fusions.

Clinicopathological features and molecular genetic changes of lung salivary gland-type clear cell carcinoma

Objective: To investigate the clinicopathological features, immunophenotype, differential diagnosis, molecular genetic changes and prognosis of salivary gland-type clear cell carcinoma (CCC) of the lung. Methods: Eight cases of salivary gland-type CCC of the lung diagnosed at Fudan University Shanghai Cancer Center and Shanghai Pulmonary Hospital, China from March 2017 to December 2020 were retrieved and analyzed. The pathological sections of these cases were studied using immunohistochemical staining, fluorescence in situ hybridization (FISH), and RNA-seq fusion gene detection based on next generation sequencing technique. The patients were followed up and the relevant literature was reviewed. Results: The 8 patients included 3 males and 5 females, with age ranging from 43 to 64 years (average, 58 years). All patients underwent radical lobectomy and lymph node dissection, while only one had lymph node metastases. The eight patients were followed up for 6 to 45 months, and were all recurrence-free. Histopathologically, the tumor was mainly composed of eosinophilic and clear cells arranged in trabecular, ribbon and nest patterns. Hyalinization was often observed in the stroma around the nest. Immunohistochemical staining showed that 8/8 cases were positive for EMA and CK7; 5/8 cases were positive for p63 and p40; 4/8 cases were positive for SOX10; and the cases were all negative for S-100, SMA and calponin. EWSR1 gene fusion was detected in all cases by FISH. RNA-seq fusion gene was detected in 6 cases based on next generation sequencing. The EWSR1-ATF1 gene fusion was detected in 5 cases, among which one case also had the ATF1-SPTLC2 gene fusion. All 5 cases with EWSR1-ATF1 gene fusion showed that EWSR1 exon 12/13 fused with ATF1 exon 3. And EWSR1-CREM gene fusion was detected in one case. Conclusions: Salivary gland-type CCC of the lung is an extremely rare primary lung tumor arising from the bronchial mucosa. The diagnosis and differential diagnosis of this tumor depend on classic histomorphology, especially the auxiliary detection of EWSR1 fusion gene. The primary treatment choice of this tumor is complete surgical resection. Lymph node metastases may occur, but the overall prognosis is good.

Targeted RNA sequencing in the routine clinical detection of fusion genes in salivary gland tumors.

Salivary gland tumors represent a diverse group of neoplasms that occasionally pose a diagnostic challenge for pathologists, particularly with limited sampling. Gene fusions, which may reflect genetic drivers, are increasingly recognized in a subset of these neoplasms, and can be leveraged for diagnostic purposes. We performed a retrospective analysis on a cohort of 80 benign and malignant salivary gland tumors, enriched for subtypes known to harbor recurrent fusion events, to validate the diagnostic use of a targeted RNA sequencing assay to detect fusion transcripts. Testing identified fusion genes in 71% (24/34) of pleomorphic adenoma and carcinoma-ex-pleomorphic adenoma, with 56% of cases showing rearrangement of PLAG1 and 15% HMGA2. In addition to confirming known partners for these genes, novel PLAG1 fusion partners were identified, including DSTN, NTF3, and MEG3; CNOT2 was identified as a novel fusion partner for HMGA2. In adenoid cystic carcinoma, 95% of cases (19/20) were positive for a fusion event. MYB was rearranged in 60% (12/20), MYBL1 in 30% (6/20), and NFIB in 5% (1/20); two tumors exhibited novel fusion products, including NFIB-TBPL1 and MYBL1-VCPIP1. Fusion genes were identified in 64% (9/14) of cases of mucoepidermoid carcinoma; MAML2 was confirmed to partner with either CRTC1 (43%) or CRTC3 (21%). One salivary duct carcinoma was found to harbor a novel RAPGEF6-ACSL6 fusion gene. Finally, as anticipated, gene fusions were not detected in any of the five acinic cell carcinomas included in the cohort. In summary, targeted RNA sequencing represents a diagnostically useful ancillary technique for identifying a variety of existing, and novel, fusion transcripts in the classification of salivary gland neoplasms.

Molecular Testing on Cytology for Gene Fusion Detection.

Cytology samples are suitable for the study of genotypic and phenotypic changes observed in different tumors. Being a minimally invasive technique, cytology sampling has been used as an acceptable alternative to track the alterations associated with tumor progression. Although the detection of gene mutations is well-established on cytology, in the last few years, gene fusion detections are becoming mandatory, especially in some tumor types such as lung cancer. Different technologies are available such as immunocytochemistry, fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and massive parallel sequencing approaches. Considering that many new drugs targeted fusion proteins, cytological samples can be of use to detect gene fusions in solid and lymphoproliferative tumor patients. In this article, we revised the use of several techniques utilized to check gene fusions in cytological material.

Pursuit of Gene Fusions in Daily Practice: Evidence from Real-World Data in Wild-Type and Microsatellite Instable Patients.

Simple SummaryThe main aim of our study is to provide real-world data on the enrichment of gene fusions in patients affected by colorectal carcinomas, melanomas, and lung adenocarcinomas characterized by the absence of mutations in the main driver genes and in colorectal tumours with microsatellite instability, using a comprehensive method. By demonstrating this enrichment in a “real-world” cohort, we confirm the feasibility of this approach, suggesting a workflow applicable in diagnostic practice. A second aim points towards a thorough investigation of NTRK gene fusions detected in the study by applying different techniques. We therefore provide comparative data across in situ methods and in vitro nucleic acid-based assays to document effective NTRK gene fusion detection.Agnostic biomarkers such as gene fusions allow to address cancer patients to targeted therapies; however, the low prevalence of these alterations across common malignancies poses challenges and needs a feasible and sensitive diagnostic process. RNA-based targeted next generation sequencing was performed on 125 samples of patients affected either by colorectal carcinoma, melanoma, or lung adenocarcinoma lacking genetic alterations in canonical driver genes, or by a colorectal carcinoma with microsatellite instability. Gene fusion rates were compared with in silico data from MSKCC datasets. For NTRK gene fusion detection we also employed a multitarget qRT-PCR and pan-TRK immunohistochemistry. Gene fusions were detected in 7/55 microsatellite instable colorectal carcinomas (12.73%), and in 4/70 of the “gene driver free” population (5.71%: 3/28 melanomas, 10.7%, and 1/12 lung adenocarcinomas, 8.3%). Fusion rates were significantly higher compared with the microsatellite stable and “gene driver positive” MSKCC cohorts. Pan-TRK immunohistochemistry showed 100% sensitivity, 91.7% specificity, and the occurrence of heterogeneous and/or subtle staining patterns. The enrichment of gene fusions in this “real-world” cohort highlights the feasibility of a workflow applicable in clinical practice. The heterogeneous expression in NTRK fusion positive tumours unveils challenging patterns to recognize and raises questions on the effective translation of the chimeric protein.

Abstract 465: Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples

Abstract Background RNA sequencing (RNA-Seq) has been widely used for gene expression and fusion detection in fresh frozen samples for research use. In clinical studies, RNA was more commonly derived from FFPE samples, which may degrade or yield low due to preparation methods, storage conditions and time. We evaluate the feasibility, quality, and analytical performance of a targeted region RNA sequencing assay (we called as Master RNA Panel) on clinical FFPE tumor samples for gene fusion detection and expression quantification. Methods Total RNA was extracted from FFPE tumor samples. cDNA synthesis and library preparation were used to prepare next-generation sequencing (NGS) libraries that were captured by a customized panel contains 31 gene fusion/splicing regions and 1813 gene expression regions. NGS libraries were then sequenced on Illumina NexSeq CN 500 or NovaSeq 6000 instrument. Sequencing data were analyzed and annotated with an in-house developed pipeline. A set of experimental and data quality control parameters were set up. Results In both fresh tissue and FFPE samples, all known fusion sites (100%, 7/7) can be detected stably with the RNA input volume as low as 5 ng and 1 Gbp data, the gene expression of 5 ng RNA input volume shows a very high correlation with 100ng even under 1Gb data (R2≥ 0.95). Master RNA Panel can detect as low as 100 copies of the fusion gene with 50 ng RNA and the specificity was 100% based on a test of 10 fusion negative samples. The consistency of fusion gene detection by Master RNA Panel was 96.2% (51/53) compared with RNA-Seq in 44 clinical samples. To validate the accuracy of gene expression, we used both qPCR and RNA-Seq as comparison methods. Gene expression of 12 tumor-related genes (BAG1, BIRC5, CD68, ERBB2, ESR1, GRB7, GSTM1, MKI67, MMP11, MYBL2, PGR, SCUBE2) in 5 samples were profiled with qPCR assay and the correlation for each gene is greater than 0.8. 27 clinical samples were profiled with both Master RNA panel and RNA-Seq, the total correlation of gene expression between the two assay was also greater than 0.8. The precision verification results show that under the inspection of different operation dates, reagent batches and operators, the gene expression correlation between 5 replicates of 2 FFPE samples is greater than 0.99. The consistency of the fusion test for 5 replicates of 2 FFPE samples and 3 standards was 95.3% (81/85, &amp;gt;=100 copies), indicating that the operation date, reagent batch and operator have no significant influence on the test results, and the test method has good precision. Conclusions Master RNA Panel can help to identify both gene fusions and gene quantification in cancer patients with extremely low RNA input and high limit of detection (LoD), which is a good supplement for DNA based NGS assay, especially for gene expression quantification and fusion detection. Citation Format: Jingbo Zhao, Shuang Yang, Hua Dong. Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 465.

Abstract 2288: Identification of gene fusions in soft tissue sarcoma improved by integrative DNA and RNA sequencing

Abstract Background: More than 50 different histologic subtypes of soft tissue sarcoma (STS) with mesenchymal origin have been recognized, and their overlapping morphology and histology make clinical diagnosis challenging. Gene fusion has been described as an important molecular biological feature of STS, which can assist in its diagnosis and treatment. Conventional DNAseq methods have limitations for gene fusion detection, while the RNAseq method might circumvent them. In this study, we evaluated the clinical value of integrated DNA and RNA sequencing in the detection of STS gene fusions. Methods: Formalin-fixed and paraffin embedded (FFPE) tumor samples and matched blood of 142 Chinese STS patients were collected and tested by the next-generation sequencing (NGS)-based 825-gene DNA panel and 395-gene RNA panel at Genetronhealth, a laboratory accredited by College of American Pathologists and Clinical Laboratory Improvement Amendments. Results: The genetic landscape of 75 male and 67 female Chinese patients with STS was analyzed. The median age of enrolled patients was 39 years (range, 1-82 years). Multiple histological subtypes were collected, including rhabdomyosarcoma (n = 15), liposarcoma (n = 15), myofibroblastic sarcoma (n = 11) and other types which were all less than 10 cases. Overall, 83 different fusions were detected in 60 patients. Of these, 25 fusions could be detected both by DNA and RNA panels in 18 patients, five fusions were detected only by the DNA panel in four patients, and 53 fusions only by the RNA panel in 39 patients. Compared to DNA-NGS individual testing, integrative panels increased the detection rate of fusion in patients with STS by 177% (53/30). The five gene fusions detected only by the DNA panel, including FOSL1-RELA, intergenic-RUNX2, ADAMTS16-TERT, NCR3LG1-KMT2A and YAP1-MAMLD1, remain clinical significance uncertain in STS. Fusion genes, such as PAX3-FOXO1, ASPSCR1-TFE3, COL1A1-PDGFB, EWSR1-ERG, were detected separately by the RNA panel, which could assist the diagnosis or targeted therapy. Additionally, unreported DIP2B-EWSR1 and AMLD1-SSX1 gene fusions were detected in a patient with clear cell sarcoma and a patient with synovial sarcoma respectively, which might be helpful in clinical diagnosis. Conclusions: Our results showed that the integrated DNA and RNA sequencing can effectively improve the detection rate of STS gene fusions, which might benefit more STS patients. Citation Format: Jiayong Liu, Shu Li, Tian Gao, Chunyang Wang, Qifan He, Xiaojuan Wang, Tonghui Ma. Identification of gene fusions in soft tissue sarcoma improved by integrative DNA and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2288.

Abstract 2154: Expression of androgen receptor splice variant, AR-V7, in high grade serous ovarian cancer

Abstract Objective: Androgen receptors (AR) are expressed in up to 55% of ERα-negative breast cancers overall and up to 35% of those classified as TNBC. Similarly, AR is expressed in approximately 40% of high grade serous ovarian cancer (HGSOC). The development of AR splice variants (AR-SV), particularly AR-V7, has been shown to be a poor prognostic feature, and mechanism of androgen resistance in breast and prostate cancers. We sought to determine if AR-V7 was present in HGSOC and if there was a correlation with platinum sensitivity and/or treatment outcomes. Methods: Pair-matched chemo-naïve and platinum-treated HGSOC specimens were identified from our tissue repository. Formalin-fixed paraffin-embedded tumor (FFPE) blocks were collected and tested for expression of AR-V7. Gene fusion detection and variant detection were performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required. Results: Twelve chemo-naïve specimens collected at time of diagnosis by initial biopsy (n=8) or primary debulking surgery (n=4) were pair-matched with the patients' subsequent platinum-treated specimens from either interval debulking surgery (n=6) or biopsy of recurrence (n=6). Following penultimate platinum, 50% were platinum resistant and 50% were platinum sensitive at time of second pathology. Only 19 of the 24 specimens submitted had sufficient RNA for testing, 8 chemo-naïve and 11 platinum-treated. AR-V7 variant transcript was not detected in any of the specimens submitted. Conclusions: AR-V7 was not expressed in any specimen before or after platinum based chemotherapy suggesting it may not be a useful prognostic marker in the management of HGSOC. Further investigation into gynecologic malignancies with high AR expression and treated with androgen deprivation therapy may elucidate different results. Citation Format: Benjamin Wilson, Joanne Xiu, Michael Ulm, Tiffany Redfern, Ramesh Narayanan, Adam ElNaggar. Expression of androgen receptor splice variant, AR-V7, in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2154.

Abstract 2261: A multimodal single-cell workflow to interrogate cellular responses to cancer therapy

Abstract Understanding the genomic landscape of cancer in single cells can be valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using microfluidics paired with next generation sequencing. Our procedure determines targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts and DNA amplicons. We employ oligo-conjugated antibody panels to probe cell surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with the antibody panel then loaded onto the Mission Bio Tapestri for generation of a droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA/mRNA. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced yielding a multimodal readout. We applied this technique to several dynamic immunology and oncology in vitro models. As fusion gene detection has long been a focus of cancer research, we demonstrate this multi-modal workflow on cell lines containing variants of BCR-ABL transcripts with high sensitivities and specificities. We further explored the relationship between genotype-to-phenotype in imatinib impact on retinoblastoma and BCR-ABL driven leukemic cells. Our pilot studies demonstrate the utility of multimodal resolution in further illuminating cellular states and biological responses in cancer cells. Citation Format: Dalia Dhingra, Aik Ooi, Alex Li, Adam Sciambi, Shu Wang, Saurabh Gulati, David Ruff. A multimodal single-cell workflow to interrogate cellular responses to cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2261.

Abstract 449: A standard operating procedure for the curation of gene fusions

Abstract Despite the well-established role of recurrent gene fusions as oncogenic drivers, current practices for characterizing and interpreting gene fusion events in clinical testing and in biomedical literature are inconsistent. From the conceptual definition of gene fusions to the salient elements that characterize these alterations, a lack of community-driven standards for the curation of gene fusions has resulted in a disparate landscape of fusion representations and supporting tools. Consequently, the evidence-based clinical evaluation of gene fusions requires extensive expert review for accurate interpretation of observed gene fusions with respect to putative evidence from biomedical literature. Furthermore, the lack of these standards inhibits the interoperability of tools, resources, and pipelines - impeding data sharing and downstream utility.To address these challenges, a cross-consortia initiative between the Variant Interpretation for Cancer Consortium and ClinGen was formed to develop a standard operating procedure (SOP) for the curation of gene fusions. The SOP is under development by an international and diverse set of experts in the representation, detection, and clinical interpretation of gene fusions. Participating stakeholders across academic, government, and industry sectors showcased challenges and solutions, and participated in community surveys and discussions to define and develop the SOP for this diverse class of alterations.An initial result of this effort was the precise molecular definition of genomic events and features constituting gene fusions. We distinguish these from similar but distinct classes of structural alterations through clinically-relevant examples. Next, we discuss our findings on community practices around the description and evaluation of gene fusions. We provide our recommendations for characterization and representation of gene fusions from these practices, and compare these recommendations to existing variant representation standards and formats (e.g. HGVS variant nomenclature). We also discuss the concurrent application of formats for standardized human- and machine-readable representations of gene fusion events.We conclude with discussion of the salient elements to enable rapid, scalable, and consistent evaluation of fusions curated from the biomedical literature. Recommendations are provided for the standardized capture of these elements to enable both intuitive and precise characterization of this diverse class of alterations in clinical reporting and literature. In summary, we provide a clinical-practice driven framework and nomenclature for gene fusions, including recommendations for human readability, computational precision, and data integrity within the SOP. This work is a substantial advancement towards standardized communication, investigation, and sharing of gene fusion data across clinical and research domains and specialties. Citation Format: Alex H. Wagner, Ioannis S. Vlachos, Dmitriy Sonkin, Panieh Terraf, Chimene Kesserwan, Andrea Sboner, Thomas Coard, Christian Reich, Deborah I. Ritter, Peter Horak, Ying S. Zou, Anna Tanska, Aaron M. Berlin, Anna Lu, Daniel Cameron, Heather E. Williams, Wan-Hsin Lin, Gokce Toruner, Arpad Danos, Jason Saliba, Huiling Xu, Xinjie Xu, Georgina Ryland, Michele Ceccarelli, Liying Zhang, Sarah Rapisardo, Catherine Rehder, Xuelu Liu, Aparna Pallavajjala, Nicole Park, Laveniya Satgunaseelan, Kristy Lee, Jie Liu, Obi Griffith, Robert R. Freimuth, Albrecht Stenzinger, Linda B. Baughn, Michael Baudis, Jennifer Lee, Marilyn Li, Angshumoy Roy, Gordana Raca. A standard operating procedure for the curation of gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 449.

Abstract 642: Genomes for Kids: Comprehensive DNA and RNA sequencing defining the scope of actionable mutations in pediatric cancer

Abstract Clinical genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. In the Genomes for Kids study (NCT02530658) we used a three-platform sequencing approach, including whole genome (WGS), whole exome (WES) and RNA sequencing, to examine tumor and paired germline genomes from prospectively identified children with cancer. The goal of the study was to assess the potential of comprehensive next generation sequencing to elucidate the molecular mechanisms underlying tumor formation and investigate the potential of this information to influence clinical decision-making.The cohort, with a median age of 6 yrs, range 0 - 26 yrs, included 301 patients with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type or stage. Patients with hematologic malignancies accounted for 41% of cases, 31% had CNS tumors, and 28% had other non-CNS solid tumors. This cohort also included 18 patients with very rare tumor types, defined here as occurring in less than 2 cases per million person per year.Two hundred fifty three patients (84%) had sufficient tumor for three-platform sequencing and all 301 had adequate paired germline samples. Following analysis, 86% of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer predisposing (18%) variants. The inclusion of WGS enabled detection of oncogenic gene fusions, as well as 22 cases in which oncogenes were activated through enhancer hijacking, a particularly frequent occurrence in hematologic malignancies. In addition, WGS effectively detected clinically relevant small intragenic deletions (15% of tumors) and a variety of mutational signatures, which were not detectable through analysis of whole exome data. Evaluation of 56 pathogenic germline variants in the context of paired tumor sequence data helped establish the disease relevance of several genes that are not typically associated with the cancer type in question, providing critical insights on a case-by-case basis. Examples include a pathogenic germline variant in MUTYH in a patient with retinoblastoma whose tumor exhibited a mutation signature associated with reactive oxygen species indicative of loss of MUTYH function; and conversely, a likely pathogenic variant in PMS2 in a rare brain cancer, which did not exhibit a mutation signature associated with microsatellite instability. This study successfully demonstrated the power of this three-platform approach to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. As a result of these findings, we have incorporated this three-platform approach into our routine real-time clinical service at St. Jude Children's Hospital. Citation Format: David A. Wheeler, Scott Newman, Joy Nakitandwe, Chimene A. Kesserwan, Elizabeth M. Azzato, Michael C. Rusch, Sheila Shurtleff, Armita Bahrami, Brent Orr, Jeffery M. Klco, Dale J. Hedges, Kayla V. Hamilton, Scott G. Foy, Michael N. Edmonson, Andrew Thrasher, Jiali Gu, Lynn W. Harrison, Lu Wang, Roya Mostafavi, Manish Kubal, Jamie Maciaszek, Michael Clay, Annastasia Ouma, Antonina Silkov, Yanling Liu, Zhaojie Zhang, Yu Liu, Samuel W. Brady, Xin Zhou, Mark Wilkinson, Delaram Rahbarinia, Jay Knight, Jian Wang, Charles G. Mullighan, Rose B. McGee, Emily A. Quinn, Elsie L. Gerhardt, Leslie M. Taylor, Regina Nuccio, Jessica M. Valdez, Stacy J. Hines-Dowell, Alberto Pappo, Giles Robinson, Liza-Marie Johnson, Ching-Hon Pui, David W. Ellison, James R. Downing, Jinghui Zhang, Kim E. Nichols. Genomes for Kids: Comprehensive DNA and RNA sequencing defining the scope of actionable mutations in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 642.

Abstract 290: Comparison of clinically relevant fusions detection using two multiplexing RNA based platforms: nCounter and GeneReader

Abstract Background: Fusions involving the tyrosine kinase receptor genes ALK, ROS1, RET, NTRK or MET exon 14 skipping variant (METex14) are present in a significant percentage of advanced solid tumors and their accurate identification is critical to guide targeted therapies. While FISH has traditionally been considered the gold standard for fusion analysis, it is costly and shows biases. GeneReader Next Generation Sequencing (NGS) (Qiagen) and nCounter (Nanostring) are two technologies allowing simultaneous detection of fusion transcripts and splicing variants. In this work we compared the performance of both platforms for fusion and splicing variant detection in advanced solid tumor patients. Methods: RNAs from 40 selected solid tumors were purified using High Pure FFPET RNA Isolation Kit (Hoffman-La Roche) and prospectively analyzed by GeneReader and nCounter. The custom nCounter codeset used targets fusions involving ALK, ROS1, RET, NTRK1-3, NRG1 and MET exon 14 skipping variant based on a dual strategy: detection of specific fusion transcripts and imbalances between the 3' and 5' mRNA regions, enabling the recognition of even those fusions not identified with the specific primers. Reporter counts from nCounter were collected with the nSolver Analysis software (Nanostring) and analyzed using an algorithm developed in the laboratory. The design of QIAact Lung Fusion Custom GeneReader panel contains specific junction probes for the detection of fusions in ALK, ROS1, RET, FGFR1- 3, NRG1, NTRK1- 3, EGFR, BRAF, and MET exon 14 skipping variant. GeneReader analysis and interpretation were performed with the QCI-Analyze and QCI-Interpret software's (Qiagen). Results: Valid results were obtained for 40/40 (100%) of samples tested. Paired analysis showed a 97.5% concordance (39/40 cases) between the results obtained by nCounter and GeneReader NGS, corresponding to a Cohen's kappa of 0.935 [CI=0.809-1.0]. Overall, 8 samples tested positive for fusion transcripts, namely EML4-ALK (n=4), CCDC6-RET (n=1), KIF5B-RET (n=1), EZR-ROS1 (n=1) and ETV6-NTRK3 (n=1). In addition, MET exon 14 skipping variant was detected in two samples. The discordant case between nCounter and NGS corresponded to a rare RET fusion only detected by 3'-5' imbalance using nCounter, while the remaining 29 patients were pan-negative. In one of them, an uncommon HLA-DRB1-MET fusion not included in the nCounter codeset, was found by the GeneReader custom panel. Conclusions: RNA-based NGS and nCounter show excellent concordance for detection of gene fusions and MET splicing variant in advanced solid tumors. Citation Format: Mónica Garzón Ibañez, Ana Gimenez-Capitán, Marta Vives Usano, Ruth Román Lladó, Sonia Rodriguez, Erika Aldeguer, Beatriz García Pelaez, Nuria Jordana Ariza, Cristina Aguado, Santiago Viteri, Andrés Aguilar, Irene Moya, Carlos Cabrera, Maria Jose Catalán, Maria Gonzalez cao, Silvia Garcia-Roman, Jordi Bertran Alamillo, Florencia Garcia Casabal, Rafael Rosell, Miguel Angel Molina-Vila, Clara De La Caridad Mayo De Las Casas. Comparison of clinically relevant fusions detection using two multiplexing RNA based platforms: nCounter and GeneReader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 290.