Targeted RNA Sequencing Assay Research Articles

Detection of sarcoma fusions by a next-generation sequencing based–ligation-dependent multiplex RT-PCR assay

Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.

Detection of EWSR1 fusions in CCOC by targeted RNA-seq

To describe the application of a targeted RNA sequencing assay to detect fusion transcripts in formalin-fixed paraffin-embedded (FFPE), non-decalcified samples of clear cell odontogenic carcinoma (CCOC) and related tumors, and to add to knowledge of the genetic drivers of CCOC. Five FFPE tissues, including intraosseous CCOC (n=3), clear cell carcinoma of the salivary gland (CCC, n=1), and Ewing sarcoma (ES, n=1), were analyzed by targeted RNA-seq to detect fusions. The 3 intraosseous CCOC samples harbored EWSR1 translocations: EWSR1-ATF1 (n=2) and EWSR1-CREM (n=1); the CCC sample contained an EWSR1-ATF1 fusion; and the ES sample contained an EWSR1-FLI1 fusion detected by RNA-seq. These results demonstrate that targeted RNA-seq is a valuable tool to detect fusions in FFPE samples of rare tumors such as CCOC and CCC. The results also confirm the observations that CCOC is driven by fusions between EWSR1 and CREB family transcription factors, including ATF1 and CREM. To our knowledge, this is the second report of CCOC with an EWSR1-CREM translocation.

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

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.

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, >=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.

Detection of FGFR fusions in intrahepatic cholangiocarcinoma using targeted RNA sequencing.

e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.

Abstract 177: RNA sequencing based gene fusion detection with oncomine comprehensive assay plus

Abstract Introduction Detection of oncogenic fusions has been of great importance for understanding tumorigenesis and for precision oncology in enhancing diagnosis and selection of targeted therapies. Herein, we describe an extended Oncomine targeted RNA sequencing assay for detection of fusion transcripts and intragenic rearrangements (exon deletion/skipping). For multiple key driver genes we also supplemented the panel with a complementary transcript-based expression imbalance assay designed to identify gene fusions in a partner agnostic manner. Methods Based on evidence from Oncomine™ Knowledgebase and collaboration with leading oncology researchers, we designed an Ion AmpliSeq™ panel to target > 1,200 fusion breakpoints in > 50 driver genes, > 40 intragenic rearrangements (e.g., MET exon 14 skipping, ARv7, EGFRvIII) in 7 genes, and 5 RNA expression controls. In addition, the panel supports detection and reporting of non-targeted fusions (i.e., novel combinations of drivers and partners). We supplemented the panel with exon tiling expression imbalance assays, using amplicons tiling the exon junctions of ALK, RET, NTRK1, NTRK2 and NTRK3 to measure 3'/5' expression imbalance signatures. We developed a bioinformatic tool to call fusions from a normalized and corrected expression imbalance profile per gene (using a baseline from normal formaldehyde fixed paraffin embedded [FFPE samples]). We optimized the gene fusion algorithms and integrated them as workflows into the Ion ReporterTM Software to facilitate the summary of the results with relevant annotations, rich data visualizations and easily interpretable reports. Results We sequenced hundreds of positive and negative fusion samples including commercial reference standards, cell lines and FFPE clinical research samples on the Ion GeneStudioTM S5 sequencer. To assess the feasibility of the combined panel, we sequenced the Seraseq® FFPE tumor fusion RNA reference, 7 fusion positive cell lines with ALK, RET, ROS1, NTRK1, FGFR1, FGFR2 and FGFR3 rearrangements, and cohorts of FFPE samples using 20ng RNA as input and successfully detected the expected fusion isoforms or other RNA rearrangements in each sample. We applied the exon tiling fusion detection method for ALK, RET and NTRK1 and observed perfect concordance between the true isoform in the positive samples and the predicted breakpoint position and magnitude of 3'/5' expression imbalance indicated by the exon tiling method. Conclusions We developed an extended, multiplexed RNA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion panels and significantly expands the scope of fusion isoform detection including methods to detect gene fusions in a partner agnostic manner. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Rajesh K. Gottimukkala, Gary G. Bee, Jennifer M. Kilzer, Vinay K. Mital, Elain Wong-Ho, Chenchen Yang, Yu-Ting Tseng, Scott P. Myrand, Paul D. Williams, Seth Sadis, Fiona C. Hyland. RNA sequencing based gene fusion detection with oncomine comprehensive assay plus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 177.

Abstract 3978: Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform

Abstract Exosomes carry signals between cells and may influence cancer development, metastasis, and drug resistance. Unfortunately, current tools for exosome isolation and analysis have been limited to examining a specific class of markers, such as DNA, RNA, and proteins. We have developed a microelectrode array chip that uses a novel Alternating Current Electrokinetic method to perform rapid, affinity-free isolation of exosomes. The chip runs on our proprietary ExoVeritaTM Flex platform. Isolated exosomes can then be analyzed in situ or can be lysed to obtain DNA and RNA for use in downstream assays, such as sequencing. In this study, we used the ExoVerita Flex to isolate exosomes directly from plasma specimens obtained from cancer subjects with lung, colorectal, or pancreatic cancers as well as healthy donors. Isolated exosomes were analyzed in situ for exosome-bound proteins and nucleic acids, and then purified following isolation to examine their nucleic acid expression profiles. Using the ExoVerita Flex with plasma specimens and antibodies targeting the exosome-bound proteins CEA, PD-L1, GPC-1 and CD63, we observed differences in protein expression levels among the different cancer types (N = 35) and healthy donors (N = 20), such as increased PD-L1 expression in non-small cell lung cancer subject samples and elevated levels of CEA in colorectal cancer subject samples (N = 10). Furthermore, we co-stained samples with antibodies and DNA- or RNA-selective dyes to simultaneously measure exosome-bound proteins and circulating nucleic acid levels in the same sample at the same time. RNA sequencing analysis was performed by extracting RNA from the isolated exosomes using a proprietary buffer, and then performing RT-ddPCR and miRNA-Sequencing. RT-ddPCR analysis of the PGK-1 housekeeping gene RNA transcript confirmed the presence of viable RNA following extraction. miRNA profiles from isolated exosomes were generated using a targeted RNA sequencing assay and used for repeatability and differential expression studies. The ExoVerita Flex platform offers a novel, unbiased method for isolation and characterization of exosome-bound proteins and circulating nucleic acids while being compatible with standard downstream analysis. The ability to perform two-dimensional protein & nucleic acid analysis in the same sample run, at the same time, is both unique and unprecedented and will enable promising future clinical studies in cancer looking at diverse classes of biomarkers directly from blood. Citation Format: Jean M. Lewis, David Searson, Alfred Kinana, Heath Balcer, Debrah Thompson, David Bodkin, Juan P. Hinestrosa, Rajaram Krishnan. Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3978.

Abstract CT057: Safety and efficacy of durvalumab in combination with tremelimumab in patients with advanced gastric cancer with elevated tumor interferon-γ gene signature

Abstract Background: Anti-PD-L1 and anti-CTLA-4 agents have demonstrated clinical activity in gastric and gastroesophageal junction cancer (GC/GEJC) tumors. However, response rates are low suggesting clinical benefit in only a subset of the population. This study prospectively evaluated the ability of a tumor-based interferon (IFN)-γ gene signature to identify a subset of GC/GEJC patients more likely to respond to checkpoint inhibitor therapy. Methods: Second and third-line patients were prescreened using archival formalin-fixed paraffin-embedded tissue to assess IFN-γ gene signature (comprising IFNy, CD274, LAG3, and CXCL9) status with a custom, targeted RNA sequencing assay using Ion AmpliSeq (Thermo Fisher Scientific) sequencing technologies. The cutoff for positive was established at the upper tertile of expression from an existing data set. Patients who were positive during prescreening were eligible to screen. Enrolled patients received durvalumab 20 mg/kg and tremelimumab 1 mg/kg IV every 4 weeks (Q4W) for 4 cycles, followed by durvalumab 10 mg/kg Q2W for ≤12 months. Disease assessments were performed every 8 weeks according to Response Evaluation Criteria In Solid Tumors (RECIST) v1.1. Results: As of August 24, 2018, 176 patients were prescreened. In these patients, the IFN-γ gene signature assay had a 70% rate for providing actionable data; 37.5% of prescreened patients had high expression. The primary failure reason was insufficient tissue, resulting in insufficient nucleic acid. 19 patients were enrolled, with a median follow-up of 5.8 months. Drug-related adverse events (AEs) occurred in 8 patients (42%); 4 (21%) had grade 3/4 AEs (fatigue, elevated lipase, decreased appetite, and interstitial lung disease, the latter of which led to treatment discontinuation). There were no drug-related deaths. Objective response rate (ORR) was 15.8% (95% CI, 3.4%-39.6%), including 1 complete response and 2 partial responses; 1 patient had stable disease. Duration of response was 13.3 weeks (range, 8.1-16.3 w). Median progression-free survival (PFS) was 1.8 months (95% CI, 1.6-1.9 mo), and median overall survival (OS) was 7 months (95% CI, 2.4-7.5 mo). Conclusions: Prospective screening of patients using a novel RNA-based IFN-γ gene expression signature was feasible for patient selection with predictable assay performance. Clinical activity of durvalumab + tremelimumab was increased, with a higher ORR in enrolled patients, compared with unselected populations receiving durvalumab and/or tremelimumab (presented at ASCO 2018); however, PFS and OS remained similar. Given the added complexity of patient preselection and lack of substantial improvement in clinical outcomes, these results, while encouraging, do not support further implementation of the IFN-γ gene signature for patient selection in this indication and treatment regimen. Citation Format: Geoffrey Ku, Jeeyun Lee, Heinz-Josef Lenz, Cheng Ean Chee, Takeshi Omori, Keun-Wook Lee, Yee Chao, Daniel Catenacci, Michael Gibson, Deirdre Cohen, Zev Wainberg, Philip Z. Brohawn, Peng He, Siddharth Sheth, Judson Englert, Ronan Kelly, Yung-Jue Bang. Safety and efficacy of durvalumab in combination with tremelimumab in patients with advanced gastric cancer with elevated tumor interferon-γ gene signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT057.

Diagnosis of known sarcoma fusions and novel fusion partners by targeted RNA sequencing with identification of a recurrent ACTB-FOSB fusion in pseudomyogenic hemangioendothelioma

Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of sarcomas. We have validated and implemented a clinical molecular diagnostic assay, MSK- Fusion Solid, for detection of gene fusions in solid tumors, including sarcomas. Starting with RNA extracted from formalin-fixed paraffin-embedded tumor material, this targeted RNA sequencing assay utilizes anchored multiplex PCR to detect oncogenic fusion transcripts involving 62 genes known to be recurrently rearranged in solid tumors including sarcomas without prior knowledge of fusion partners. From 1/2016 to 1/2018, 192 bone and soft tissue tumors were submitted for MSK- Fusion Solid analysis and 96% (184/192) successfully passed all the pre-sequencing quality control parameters and sequencing steps. These sarcomas encompass 24 major tumor types, including 175 soft tissue tumors and 9 osteosarcomas. Ewing and Ewing-like sarcomas, rhabdomyosarcoma, and sarcoma-not otherwise specified were the three most common tumor types. Diagnostic in-frame fusion transcripts were detected in 43% of cases, including 3% (6/184) with novel fusion partners, specifically TRPS1-PLAG1, VCP-TFE3, MYLK-BRAF, FUS-TFCP2, and ACTB-FOSB, the latter in two cases of pseudomyogenic hemangioendothelioma, representing a novel observation in this sarcoma. Our experience shows that this targeted RNA sequencing assay performs in a robust and sensitive fashion on RNA extracted from most routine clinical specimens of sarcomas thereby facilitating precise diagnosis and providing opportunities for novel fusion partner discovery.

Abstract B036: Comprehensive transcriptomic profiling challenges the robustness of prostate cancer prognostic signatures to multifocality

Abstract Purpose: Imaging and tissue-based biomarkers are increasingly utilized in men diagnosed with low-grade prostate cancer (PCa) to guide definitive management vs. active surveillance. PCa is uniquely multifocal, with multiple clonally distinct tumor foci present in the majority of men. A single tumor focus may also be composed of both low- and high-grade components. Hence, an ideal prognostic biomarker should be robust to both undersampling of a high-grade component, as well as a not sampled multifocal high-grade tumor focus. Materials and Methods: To assess the robustness of prognostic biomarkers to multifocality, we designed a comprehensive multiplexed targeted RNA sequencing assay (mxRNAseq) capable of assessing multiple classes of transcriptional alterations and deriving available prognostic signature scores (e.g., Prolaris CCP and OncotypeDX GPS). We applied this assay to a retrospective, multi-institution cohort of over 150 formalin-fixed, paraffin-embedded tissue samples representing the range of prostate cancer progression. Single candidate biomarkers and derived prognostic signatures were compared between low-grade foci in cases with and without high-grade foci, all-low-grade foci vs. all-high-grade foci, and cases with extremes of grade differences in multifocal tumors. Frequency of clinically relevant extreme multifocality was determined by identifying patients in a single-institution consecutive prostatectomy database over 9 years who had exclusively low-grade cancer on biopsy but very-high-grade disease on prostatectomy. Results: Our mxRNAseq assay robustly detected known coding gene/lncRNA expression, gene fusions and splice variants, and expressed mutations and germline variants. Supervised hierarchical clustering of target gene expression confirmed expected transcriptional module deregulation and derived prognostic signatures across prostate cancer progression. Prognostic biomarkers (including derived signatures) showed no significant differences in expression between low-grade foci from prostates with and without high-grade multifocal tumors and were uniformly higher in high-grade foci vs. low-grade foci from the same case. In four cases of extreme multifocality (Gleason score 6 vs. >=8 foci), prognostic signatures were significantly lower in low-grade foci vs. high-grade foci. In 1,418 men with biopsy Gleason score 3+3=6 or 3+4=7, 21 (1.5%) had Gleason score ≥ 4+4=8, where the biopsy almost certainly did not sample the most clinically relevant focus. Conclusions: Using a novel comprehensive mxRNAseq assay, our results challenge the robustness of prognostic biomarkers between multifocal low- and high-grade prostate cancer foci. Tissue-based prostate cancer prognostic biomarkers should be specifically validated using an extreme multifocality design to support utility in predicting the presence of an unsampled, aggressive multifocal tumor focus. Citation Format: Simpa Salami, Daniel H. Hovelson, Jeremy B. Kaplan, Romain Mathieu, Aaron M. Udager, Nicole Curci, Matthew Lee, Lorena Lazo de la Vega, Martin Susani, Nathalie Rioux-Leclercq, Daniel E. Spratt, Todd M. Morgan, Matthew S. Davenport, Mark A. Rubin, Shariat F. Shahrokh, Scott A. Tomlins, Ganesh S. Palapattu. Comprehensive transcriptomic profiling challenges the robustness of prostate cancer prognostic signatures to multifocality [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B036.

Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications

BackgroundIntegrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported. ObjectiveTo assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. Design, setting, and participantsWe performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines. Outcome measurements and statistical analysisUnsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. Results and limitationAcross 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation. ConclusionsOur results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. Patient summaryA targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers.

MP35-10 DETECTION OF PROSTATE CANCER-ASSOCIATED TRANSCRIPTS IN URINARY EXTRACELLULAR VESICLES

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2018MP35-10 DETECTION OF PROSTATE CANCER-ASSOCIATED TRANSCRIPTS IN URINARY EXTRACELLULAR VESICLES Kathryn Pellegrini, Dattatraya Patil, Kristen Douglas, Ella Anastasiades, Kristin Larsen, Frances Kim, Almira Catic, Mersiha Torlak, Eugene Huang, Carlos Moreno, and Martin Sanda Kathryn PellegriniKathryn Pellegrini More articles by this author , Dattatraya PatilDattatraya Patil More articles by this author , Kristen DouglasKristen Douglas More articles by this author , Ella AnastasiadesElla Anastasiades More articles by this author , Kristin LarsenKristin Larsen More articles by this author , Frances KimFrances Kim More articles by this author , Almira CaticAlmira Catic More articles by this author , Mersiha TorlakMersiha Torlak More articles by this author , Eugene HuangEugene Huang More articles by this author , Carlos MorenoCarlos Moreno More articles by this author , and Martin SandaMartin Sanda More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1123AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The relatively high rate of negative prostate biopsy demonstrates the need for additional non-invasive biomarkers to improve the diagnosis and classification of prostate cancers prior to biopsy. We have previously established that post-DRE extracellular vesicles (EVs) are enriched for prostate-specific RNAs, making them an ideal target for prostate cancer biomarker discovery. In our current study, we aimed to identify genes with increased expression in the post-DRE urine EVs of patients with aggressive prostate cancer. METHODS We developed and refined a method for the targeted analysis of over 260 different RNA transcripts in post-DRE urine EVs. Genes were selected for inclusion in our assay based on their prior inclusion on tissue biomarker panels of aggressive prostate cancer, or from analysis of TCGA datasets to identify genes with both significant over-expression in aggressive prostate cancer and minimal expression in normal genitourinary tissues. RESULTS We prepared RNA from the post-DRE urine EVs of 29 men and used our targeted RNA sequencing assay to measure expression of over 260 different transcripts. We detected 92 transcripts (34%) in all 29 specimens, with only 19 transcripts (7%) being undetectable in any specimen. Comparison of patients with no evidence of disease (n = 15) and patients with Gleason Score ≥7 prostate cancer (n = 14) identified 25 genes with significantly higher expression in patients with aggressive prostate cancer, including known prostate cancer-associated genes such as PCA3, AMACR, and SPINK1, while KLK3 showed no significant difference in expression (Figure 1). CONCLUSIONS Post-DRE urine EVs contain transcripts that are indicative of prostate cancer status and represent a novel source for biomarker discovery and development. Further investigation of patient specimens with this method offers the potential to determine combinations of transcripts that could be used to non-invasively detect and monitor prostate cancer status. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e450-e451 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kathryn Pellegrini More articles by this author Dattatraya Patil More articles by this author Kristen Douglas More articles by this author Ella Anastasiades More articles by this author Kristin Larsen More articles by this author Frances Kim More articles by this author Almira Catic More articles by this author Mersiha Torlak More articles by this author Eugene Huang More articles by this author Carlos Moreno More articles by this author Martin Sanda More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Integrative molecular profiling challenges robustness of prognostic signature scores in multifocal prostate cancer.

96 Background: Tissue based biomarkers are increasingly utilized in men diagnosed with low grade prostate cancer (PCa) to guide definitive management vs. active surveillance. PCa is uniquely multifocal, suggesting ideal prognostic biomarkers should be robust to both undersampling of a high grade component of a mixed-grade tumor focus, as well as unsampled multifocal high grade tumor foci. Methods: To assess the robustness of prognostic biomarkers to multifocality, we designed a comprehensive multiplexed targeted RNA sequencing assay (mxRNAseq) capable of assessing multiple classes of transcriptional alterations and deriving available prognostic signature scores (e.g. Prolaris CCP and OncotypeDX GPS). We applied this assay to a retrospective cohort of 176 FFPE tissue samples representing the range of PCa progression. Single candidate biomarkers and derived prognostic signatures were analyzed in multifocal cases with only low-grade disease as well as those with extreme grade differences across tumor foci. Results: Our mxRNAseq assay robustly detected known coding gene/lncRNA expression, gene fusions, splice variants, and expressed somatic and germline mutations. Supervised clustering of target gene expression confirmed expected transcriptional module deregulation and derived prognostic signatures across PCa progression. Prognostic biomarkers (including derived signatures) showed no significant expression differences between low grade foci from prostates with and without high grade disease foci and were uniformly higher in high vs. low grade foci from the same case. In four cases of extreme multifocality (Gleason score 6 vs. ≥ 8 foci), prognostic signatures were significantly lower in low vs. high grade foci. In a clinical prostatectomy cohort of 1,418 men with diagnostic biopsy Gleason score 3+3 = 6 or 3+4 = 7, 21 (1.5%) had Gleason score ≥ 4+4 = 8, suggesting the initial biopsy missed or undersampled the most clinically relevant focus. Conclusions: Using a novel comprehensive mxRNAseq assay, our results challenge the robustness of prognostic biomarkers between multifocal low and high grade PCa foci, critically important in the context of un/under-sampled aggressive tumor foci.

Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors

Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan-fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

Targeted RNA Sequencing Assay Efficiently Identifies Cryptic KMT2A (MLL)-Fusions in Acute Leukemia Patients

The KMT2A gene is rearranged in pediatric and adult acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). By fusing with a number of different partner genes, the KMT2A gene generates chimeric proteins with oncogenic abilities. Due to the promiscuity of the gene, routine genetic analysis of KMT2A-rearranged leukemia is time-consuming as it often involves chromosome and FISH analyses, RT-PCR or southern blot. We have thus developed a captured-based next generation sequencing approach to detect KMT2A fusion genes, especially useful when routine methods indicate a rearrangement but cannot identify the specific fusion partner.First, we analyzed bone marrow samples from two AML cases with deletions of the distal part of the KMT2A gene and, as a control, a pre B-ALL with a known KMT2A-AFF1 fusion. The capture was carried out using custom hybridization probes (SureSelect, Agilent) encompassing the entire KMT2A gene. Sequencing was performed on the Ion Torrent PGM, a sequencing instrument well suited for routine diagnostics given its rapid turn-around time. Putative fusions were identified from sequencing reads connecting an exon within KMT2A to an exon from any other gene and the results were confirmed by RT-PCR. As comparison we performed the standard RNA-sequencing protocol using Ion Proton. In the two patients with unknown KMT2A-rearrangements, the capture approach identified 3034 and 2118 reads over a KMT2A-MLLT4 gene fusion, while only 4 and 2 reads, were detected with the standard RNA-sequencing protocol (Table 1). Similarly, RNA-sequencing and the capture approach detected in the control a KMT2A-AFF1 fusion, with a sequencing depth of 4 and 144 reads, respectively. Interestingly, with the capture approach we obtained 5032 reads encompassing the reciprocal fusion, AFF1-KMT2A, underlining the importance of the reciprocal transcript in this entity. Hence, the KMT2A capture approach resulted in a 5000-35000-fold enrichment of the KMT2A fusions compared to standard RNA-sequencing. Although the standard RNA-sequencing detected reads covering the fusion genes, it yielded far too low sequencing depth to be of diagnostic use.We then performed the KMT2A capture approach on three additional cases with distal KMT2A deletions. In one case, we detected a KMT2A-ARHGEF12 fusion, resulting from a 20Mb interstitial deletion (Table 1). This aberration is missed by standard cytogenetics and consequently likely under-diagnosed. However, in two other cases no KMT2A-fusions were identified. In these cases, the 11q-deletion was present only in a proportion of cells and in one, the deletion was absent at relapse, indicating that it was not likely the primary event in this leukemia. These results demonstrate the importance of establishing if a suspected KMT2A-rearrangement truly generates a KMT2A-fusion.In summary, the hybridization-based capture sequencing approach efficiently detects KMT2A-fusion transcripts and reliably identifies canonical and cryptic KMT2A-rearrangements, generating significantly higher number of fusion breakpoint reads, as compared to RNA-sequencing. Furthermore, sensitive RT-PCRs for follow-up (MRD) can be readily designed using the sequence information.Abstract 2406. Table 1Sequencing results using KMT2A-capture and standard RNA-sequencingPatients (diagnosis)G-bandingKMT2AFISH analysisaRNA-seq (Proton)KMT2A capture (PGM)(% cells)FusionNo of reads% of total readsFusionNo of reads% of total reads#1 (pre B-ALL)46,XX,t(4;11)(q21;q23),i(7)(q10)[18]/46,XX[2]t(4;11)(q21;q23) (94%)KMT2A-AFF145x10-6KMT2A-AFF11440,01AFF1-KMT2A263x10-5AFF1-KMT2A50320,20#2 (AML)46,XX[20]del(11)(q23q23) (80%)KMT2A-MLLT445x10-6KMT2A-MLLT430340,10#3 (AML)46,XY[20]del(11)(q23q23) (83%)KMT2A-MLLT422x10-6KMT2A-MLLT421180,07#4 (pre B-ALL)46,XX,der(9)t(3;9)(p21;q34)[9]/46,idem,der(9)t(3;9)[10]del(11)(q23q23) (87%)b--KMT2A-ARHG EF122960,02#5 (AML)46,XY[25]del(11)(q23q23) (25%)b--c--#6 (AML)47,XY,del(6)(q14q21),+8[20]del(11)(q23q23) (14%)b--c--All results are from bone marrow samples taken at time of diagnosis.aKMT2A specific FISH-analysis was performed using Vysis KMT2A break apart probe. bRNA-sequencing not performed. c No KMT2A-fusion detected. DisclosuresNo relevant conflicts of interest to declare.

NGS-based targeted RNA sequencing for expression profiling and relative quantitation of specific gene isoforms and fusions in tumor-specific panels.

11108 Background: Gene expression signatures have become a useful tool for the identification of tumor subtypes and response to specific therapies. Expression of tumor, metastatic and macrophage specific transcripts utilizing alternative promoters and transcriptional start sites can further characterize these tumors . NGS is a powerful tool for gene expression analysis, however larger sample input requirements (>100ng) and excessive sequencing depth requirements (30-40M tags/sample) to detect the expression of rare isoforms or fusions in tumor samples are prohibitive for clinical assay development. We describe the development of a targeted RNA sequencing assay for the relative quantitation of specific gene expression signatures, known splice variants and gene fusions from less than 100 ng of starting material in a single tube universal amplification format. Methods: Primers for 52 genes, isoforms and gene fusion products were designed using the universal amplification strategy. 10 ng of RNA from 5 matched tumor/adjacent normal breast cancer tumor pairs were assayed. Libraries were prepared for sequencing by emPCR and sequenced on Ion Torrent PGM. Data were aligned via TMAP. Relative expression was determined vs. housekeeping genes or wild type transcripts. Results: All gene targets were detected at significant levels in at least one tumor sample. Robust expression profiling (5 log dynamic range) was obtained from FFPE macrodissected tumor and normal samples with as little as 200K reads/sample. Immune specific transcripts demonstrated differential expression (CCL3, AIF, FCGR3A and CSF1) across patients and matched pairs as well as an upregulation of CXCL12, indicative of tumor associated macrophages. Conclusions: Targeted RNAseq demonstrates detection and quantitation of relative expression levels of not only tumor subclass specific gene expression signatures, but immune cell specific transcripts from 10ng of FFPE derived total RNA derived from macrodissected tumor samples. The lower input requirements, quicker turnaround time and incredible sensitivity of targeted RNAseq make this assay a useful tool for clinical assay development.