Jeoffrey Schageman

Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with < 20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.

The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers.

Exosomes are tiny vesicles (30–150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.

Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease

BACKGROUND Bronchial airway expression profiling has identified inflammatory subphenotypes of asthma, but the invasiveness of this technique has limited its application to childhood asthma. OBJECTIVES We sought to determine whether the nasal transcriptome can proxy expression changes in the lung airway transcriptome in asthmatic patients. We also sought to determine whether the nasal transcriptome can distinguish subphenotypes of asthma. METHODS Whole-transcriptome RNA sequencing was performed on nasal airway brushings from 10 control subjects and 10 asthmatic subjects, which were compared with established bronchial and small-airway transcriptomes. Targeted RNA sequencing nasal expression analysis was used to profile 105 genes in 50 asthmatic subjects and 50 control subjects for differential expression and clustering analyses. RESULTS We found 90.2% overlap in expressed genes and strong correlation in gene expression (ρ = .87) between the nasal and bronchial transcriptomes. Previously observed asthmatic bronchial differential expression was strongly correlated with asthmatic nasal differential expression (ρ = 0.77, P = 5.6 × 10(-9)). Clustering analysis identified TH2-high and TH2-low subjects differentiated by expression of 70 genes, including IL13, IL5, periostin (POSTN), calcium-activated chloride channel regulator 1 (CLCA1), and serpin peptidase inhibitor, clade B (SERPINB2). TH2-high subjects were more likely to have atopy (odds ratio, 10.3; P = 3.5 × 10(-6)), atopic asthma (odds ratio, 32.6; P = 6.9 × 10(-7)), high blood eosinophil counts (odds ratio, 9.1; P = 2.6 × 10(-6)), and rhinitis (odds ratio, 8.3; P = 4.1 × 10(-6)) compared with TH2-low subjects. Nasal IL13 expression levels were 3.9-fold higher in asthmatic participants who experienced an asthma exacerbation in the past year (P = .01). Several differentially expressed nasal genes were specific to asthma and independent of atopic status. CONCLUSION Nasal airway gene expression profiles largely recapitulate expression profiles in the lung airways. Nasal expression profiling can be used to identify subjects with IL13-driven asthma and a TH2-skewed systemic immune response.

Methods for the extraction and RNA profiling of exosomes

AIM To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques.

Evaluation of NGS and RT-PCR methods for ALK rearrangement in European NSCLC patients: Results from the ETOP Lungscape Project

Introduction: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse‐transcriptase polymerase chain reaction (RT‐PCR) has also been advocated, and most recently, the advent of targeted next‐generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. Methods: A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT‐PCR, and NGS. An H‐score higher than 120 defines IHC positivity. RNA was extracted from the same formalin‐fixed, paraffin‐embedded tissues. For RT‐PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen &kgr; coefficient (two‐sided &agr; ≤ 5%). Results: NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT‐PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT‐PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT‐PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. Conclusion: NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT‐PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

Abstract 3575: The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel

Chromosomal translocations and corresponding gene fusions play an important role in the initiation of tumorigenesis and these processes have been strongly associated with distinct tumor subtypes. The recent association of ALK, ROS and RET fusion transcripts as lung tumor therapy predictive biomarkers has increased the need of a technology that could detect these biomarkers starting from limited amount of material. Life Technologies and OncoNetwork consortium collaborated for the development of a lung fusion panel based on Ion AmpliSeq™ RNA chemistry. The OncoNetwork consortium is comprised of twelve -translational cancer research institutes with many years of experience in adopting the latest molecular techniques for lung therapy research. Material and Methods: Consortia9s requirements for the panel development : 1) detect all variants of ALK, ROS1, or RET fusion transcripts described in COSMIC in a single reaction using 10 ng of total RNA 2) Include 5′ and 3′ ALK, ROS1, RET gene expression assays as an indicator of a translocation at this gene. 3) Include endogenous RNA assay controls to determine if the quality of the results could be affected by RNA quality. 4) Provide similar results on archived FFPE samples tested by FISH. Human lung adenocarcinoma cell lines H2228 (EML4-ALK positive), HCC78 (SLC4A2-ROS1 positive ) ,LC-2/ad (CCDC6-RET positive) and Ambion® FirstChoice® Human Brain Reference (HBR) RNA was used as positive and negative control for the study. Formalin fixed paraffin embedded (FFPE) tissue was isolated using different extraction methods. After amplification using Ion AmpliSeq™ RNA chemistry samples were sequenced on the Ion Torrent PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit. The presence of the fusions was confirmed by custom TaqMan® gene expression assays when possible. Preliminary results of the panel on ALK or ROS1 or RET positive cell lines and FFPE archived cancer research samples gave good concordance with FISH results. Expected negative samples were confirmed negative. We found a KIF5B-RET fusion positive sample in a sample not previously tested for RET fusions. Two of the expected positive samples by FISH were found negative due to limited amount to tumor cells present in the sample. Cell line RNA dilutions were performed to determine the panel9s limit of detection. We demonstrate a limit of detection of 1 % tumor RNA in the presence of 99 % normal RNA using the panel with 10 ng of RNA extracted by cell lines. Gene expression controls work well as an indicator of the RNA quality and of the translocation presence. The Ion AmpliSeq™ RNA lung cancer fusion panel workflow is easier and faster to perform in comparison to the FISH method. The results obtained to date are highly encouraging for panel to be used in the clinical research setting. More data needs to be analyzed before a final conclusion is made. Citation Format: Susan M. Magdaleno, Angie Cheng, Rosella Petraroli, Orla Sheils, Bastiaan Tops, Delphine Le Corre, Henriette Kurth, Helene Blons, Eliana Amato, Andrea Mafficini, Anna Maria Rachiglio, Anne Reimann, Christoph Noppen, Chrysanthi Ainali, Jin Katayama, Renato Franco, Harriet Feilotter, Jeoffrey Schageman, Ian Cree, Andrew Felton, Jose Luis Costa, Alain Rico, Aldo Scarpa, Jose Carlos Machado, Kazuto Nishio, Nicola Normanno, Marjolijn Ligtenberg, Cecily P. Vaughn, Ludovic Lacroix, Pierre Laurent-Puig. The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2014-3575

Profiling of glycosylation gene expression in CHO fed-batch cultures in response to glycosylation-enhancing medium components

Characterization of the glycosylation profile of a recombinant protein product is an important part of defining product quality in the bioproduction industry. Development of a protein with desired characteristics would require the capacity to modify and target specific glycosylation patterns as well as an understanding of the implications of changes to these glycosylation profiles. Previous cell culture studies have demonstrated the ability to modulate glycan profiles without negative impact to culture growth and product titer through the addition of glycosylation-enhancing medium components. With new methods, including increased measurement sensitivity and new capabilities in RNA-Seq technology, it is possible to develop a glycosylation gene expression profile for CHO cells. Specific glycosylation genes can then be tracked to ensure that the addition of these compounds will not negatively impact gene expression. Analyses comparing growth and titer, glycan distribution, and transcriptome differences can present us with potential insight into what changes are taking place on a genetic level in the cell in response to changes in medium and culture conditions.

Abstract 1386: Clinical research results for a NGS-based kit for targeted detection of clinically relevant gene rearrangements in lung tumor samples

In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumor subtypes may inform diagnosis and allow the clinician to tailor a specific therapeutic approach in the future. Here, we present the testing results of one such NGS based kit used to detect specific chromosomal translocations in retrospective non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts. The included panel tested consists of a single primer pool containing amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumor research samples and achieved high-sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5’ and 3’ ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends. Testing was carried out at three external clinical research laboratories. In addition to positive and negative control samples, each site contributed FFPE lung tumor research samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq™ workflow. For site-specific samples (n = 144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively. Citation Format: Jeoffrey J. Schageman, Jose Luis Costa, Orla Sheils, John E. Glassco, David Chi, Jon Sherlock, John Bishop, Rosella P. Petraroli, Kelli S. Bramlett. Clinical research results for a NGS-based kit for targeted detection of clinically relevant gene rearrangements in lung tumor samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1386.

Abstract 5267: Gene fusion database to create custom panels: Enabling detection of fusion transcripts and gene expression assays

Gene fusions play an important role in tumorigenesis and are increasingly recognized as important entities for the diagnosis and treatment of hematological malignancies and solid tumors. Fusion events generate a hybrid mRNA transcript comprising sequence from multiple otherwise distinct genes. Oncogenic fusion events often involve tyrosine kinases or transcription factors, leading to aberrant growth signaling, making these events potentially attractive drug targets. For instance, targeted therapies such as known tyrosine kinase inhibitors are currently approved to treat ALK fusion positive Non-Small Cell Lung Carcinoma (NSCLC) patients. Detection of known gene fusion events is an important part of genomic characterization which can inform patient diagnosis. Current methods for fusion detection include chromosome banding analysis (CBA), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). New developments in next-generation sequencing (NGS) enable the efficient and simultaneous assessment of multiple gene fusion targets with high sensitivity. To enable researchers to design their own custom panels and assess a set of gene fusions of interest, we developed a comprehensive RNA gene fusion database. Oncology researchers now have the capability to create custom panels from this comprehensive database which includes over 1,000 well annotated and optimized gene fusion assays and over 20,000 gene expressions assays. To build this comprehensive gene fusion database, we identified breakpoint information for 1,178 well annotated fusions described in publications and in the COSMIC and NCBI databases. We prepared a target RNA sequence for each breakpoint using transcript sequences from the Ensembl database. We used a proprietary primer designer to generate candidates for each fusion target amenable to the AmpliSeq™ product line requirements. Quality control was performed throughout the design process to identify the best primer set for each target, to avoid primers overlapping common germline SNPs, potential primer/primer or primer/amplicon interactions, or off-target or wild-type amplifications. With this comprehensive database we provide a complete range of solutions available on ampliseq.com. Making use of the AmpliSeq™ technology, researchers now have the capability to create their own custom fusion panel and place the order within an hour. These custom panels are used with AmpliSeq™ Library reagents and Ion Torrent™ sequencing platforms for targeting next-generation sequencing. The analysis solution is provided through the Ion Reporter™ (IR) software package. Custom fusion panel workflows in IR are used to analyze sequencing data coming from the custom panels, which includes visualization of fusion transcripts and gene expression levels in a heat map feature. Citation Format: Efren Ballesteros-Villagrana, Jeoffrey Schageman, Kelli Bramlett, Paul Williams, Scott Myrand, Guoying Liu, Fiona Hyland, Seth Sadis. Gene fusion database to create custom panels: Enabling detection of fusion transcripts and gene expression assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5267.