ArcherDX combines targeted NGS library preparation kits and powerful bioinformatics software to detect gene fusions, point mutations and CNVs in clinical sample types. Learn more at AMP 2017 by attending the workshops and browsing the scientific posters. And swing by Booth 1619 for more info.
All corporate workshops are presented on Wednesday, November 15 and are open to the public.
8:00-8:50am, Room 251A
Immune repertoire (IR) characterization is a powerful approach to monitor disease, measure the adaptive immune response to disease, and develop vaccines and therapeutic interventions. IR characterization by next-generation sequencing (NGS) is often challenging and can require proprietary synthetic controls to account for differential amplification efficiency. In this workshop, Dr. Nathan Montgomery from the UNC School of Medicine and Dr. Alex Lazar from MD Anderson Cancer Center describe their ability to confidently identify clonality and characterize the IR using targeted NGS coupled with powerful, transparent bioinformatics. The speakers will be introduced by Dr. Jason Myers, co-founder and CEO of ArcherDX.
10:00-10:50am, Room 251A
Acute myeloid leukemia (AML) is a clonally heterogeneous cancer where the landscape of mutations is highly variable between children and adults, involving complex chromosomal translocations, indels and single nucleotide variants (SNVs). Traditional molecular assays rely upon multiple platforms to identify mutations but generally lack quantitation of rare clones. Advances in sequencing-based methods enable comprehensive detection of gene fusions, SNVs, gene expression and internal tandem duplications. In this workshop, Todd Druley MD, PhD from Washington University describes his use of error-corrected sequencing coupled with powerful bioinformatics to detect rare clonal variants in adult and pediatric AML. Dr. Druley will be introduced by Dr. Brian Kudlow, Vice President of R&D at ArcherDX.
2:00-2:50pm, Room 251A
Liquid biopsies are less invasive and can add value beyond tissue biopsies in advanced solid tumor patients, but circulating tumor DNA (ctDNA) is typically highly fragmented and present in low abundance. NGS-based assays must be sensitive enough to detect low-level mutations (allele fraction <2%) from low input DNA (10-100ng). In this workshop, Dr. Margaret L. Gulley from the UNC School of Medicine and Dr. A. John Iafrate from Massachusetts General Hospital describe their ability to confidently detect variants in plasma DNA using targeted NGS coupled with powerful bioinformatics. The speakers will be introduced by Dr. Jason Myers, co-founder and CEO of ArcherDX.
All posters will be presented on Saturday, November 18
Section: Solid Tumors, poster ST147
Josh Haimes1, Brian A. Kudlow1, Namitha M. Nair1, Skyler J. Mishkin1, Marc R. Bessette1, Laura M. Griffin1, Danielle A. Murphy2, Robert Shoemaker2, Joshua A. Stahl1
1ArcherDX, Boulder, CO USA; 2Ignyta, San Diego, CA USA
Introduction Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers and can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and kinase-activating point mutations. MET mutation profiling is important, as the various mutated forms of MET exhibit unique drug sensitivities. We developed targeted NGS assays based on Anchored Multiplex PCR (AMP™) to detect all types MET mutations from a single sample. Here, we demonstrate the ability of AMP-based NGS to detect these mutations, with a particular emphasis on the identification of novel MET fusions.
Methods AMP uses molecular barcoded adapters and single gene-specific primers for amplification, enabling open-ended capture of DNA and cDNA fragments. This allows for sequence identification of known and unknown mutations, including novel fusions, as well as copy number and expression analysis from DNA and RNA, respectively.
Results We demonstrate the ability of AMP-based NGS to identify novel fusion partners for MET, including HLA-DRB1, CTTNBP2, and GTF2I in RNA extracted from FFPE samples. Furthermore, AMP enables NGS-based detection of MET amplifications in DNA and overexpression in RNA. Finally, AMP-based NGS detects exon 14 skipping and driver splice site mutations, as well as kinase-activating point mutations.
Conclusions These results demonstrate that AMP-based NGS detects all types of MET mutations, including both known and novel fusions, enabling comprehensive characterization of MET deregulation from low-input clinical samples.
Section: Solid Tumors, poster ST143
Aaron Garnett, Ian Hoskins, Kaitlyn Moore, Laura Griffin, Ryan Walters
ArcherDX, Boulder, CO USA
Introduction Germline pathogenic variants in BRCA1 and BRCA2 significantly increase the lifetime risk of developing breast, ovarian and prostate cancers. As such, detecting these variants has significant prognostic implications, guiding recommendations for cancer screening, prophylactic treatments and other risk-reducing options for affected individuals and their relatives. Next-generation sequencing (NGS) has now been shown to be more sensitive and cost-effective compared to Sanger sequencing to detect BRCA1/2 variants. However, traditional amplicon-based NGS assays require large sets of opposing primer panels to capture all of the BRCA1/2 coding regions. We developed a targeted NGS assay based on Anchored Multiplex PCR (AMP) to capture all coding regions in BRCA1/2. Here, we demonstrate the ability of this assay to detect known disease-causing single nucleotide variants (SNVs) and insertions and deletions, including large multi-exon deletions.
Methods AMP is a target enrichment method for NGS that uses molecular barcoded (MBC) adapters and single, unidirectional primers for open-ended amplification of individual DNA strands. MBC adapters enable post-sequencing quantitative analysis of original unamplified template molecules, which allows for copy number assessment across all coding regions of BRCA1/2 and thus detection of partial gene deletions.
Results Here, we demonstrate that MBC adapters ligated prior to amplification enable unique molecule identification that allows for deduplication and PCR sequencing error correction. Furthermore, we show that strand-specific priming facilitates dual, independent coverage across target regions, preventing false-negative variant detection due to allele dropout. Our AMP-based NGS assay demonstrates high specificity and 100% coverage uniformity, resulting in reduced sequencing cost. We used in silico-generated sequences to validate the ability of our analysis pipeline to detect over 1,000 known pathogenic variants, including SNVs and insertions and deletions. Finally, we detected a heterozygous copy loss of BRCA2 exons 14-16 in a clinical sample, demonstrating the ability of this assay to detect homozygous and heterozygous partial gene deletions.
Conclusions These data show that AMP enables full exon sequencing coverage of BRCA1/2 for comprehensive detection pathogenic SNVs and insertions and deletions, including large, multi-exon deletions.
Section: Hematopathology, poster H23
Josh Haimes1, Skyler J. Mishkin1, Namitha M. Nair1, Thomas D Harrison1, Laura M. Griffin1, Margaret L. Gulley2, Nathan D. Montgomery2 and Brian A Kudlow1
1ArcherDX, Boulder, CO USA; 2University of North Carolina at Chapel Hill, Chapel Hill, NC USA
Introduction B-lymphocyte malignancies are characterized by monoclonal expansion of cells with related, if not identical, immunoglobulin gene sequences. Sequencing the immunoglobulin heavy chain (IGH) repertoire to define tumor-associated clonotypes has a number of potential clinical applications, including identifying specific markers for residual disease, and determining somatic hypermutation status for risk stratification in chronic lymphocytic leukemia (CLL). Compared to DNA, RNA-based clonotype sequencing has advantages, as primers can be positioned to determine isotype, and non-expressing cells do not dilute out signals from B cells. We tested an RNA-based next-generation sequencing (NGS) assay based on Anchored Multiplex PCR (AMP™) to detect dominant clonotypes, identify IGH isotypes, and characterize IGHV mutation status in a cohort of patients with multiple myeloma (MM), lymphoplasmacytic lymphoma (LPL) and chronic lymphocytic leukemia (CLL).
Methods Sequencing mRNA from MM, LPL and CLL was performed with Archer® Immunoverse™ IGH assay. Total RNA was extracted from formalin-fixed bone marrow collected from patients with MM and LPL or from fresh blood collected from patients with CLL. Libraries were sequenced on an Illumina® MiSeq® using v3 600-cycle chemistry, and data were analyzed using Archer Analysis™ v5.1 for clonotype frequencies, IGH isotypes, and IGHV mutational status.
Results In all IGH-expressing MM and LPL cases, strongly dominant clonotypes representing >90% of all clones were present. In contrast, dominant clonotypes representing ≥2% of all clones were not detectable in IGH non-expressing MM specimens nor in blood from normal donors. The assay accurately defined isotype, as results were 100% concordant with isotype defined by serum immunofixation. Similar results were observed for CLL samples, although disease-associated clonotypes were less dominant than in MM/LPL, potentially reflecting lower IGH expression in CLL. Furthermore, because reads typically covered >90% of IGHV, somatic hypermutation status was discernable and was highly concordant with mutation status inferred from ZAP70 and CD38 protein expression.
Conclusions RNA-based sequencing from both formalin-fixed tissue and fresh blood permits characterization of IGH repertoire in clinical samples. Even in formalin-fixed marrow, the targeted NGS assay robustly identified dominant clonotypes while providing IGH isotope and somatic hypermutation status. Unambiguous identification of CDR3 sequences provides a marker by which to monitor residual tumor burden after treatment.
Section: Genetics, poster G50
Matthew T. Mardison1, Kaitlyn E. Moore2, Paula G. Roberts2, Laura M. Griffin2, Ryan D. Walters2 and Brady P. Culver2
1Baby Genes, Inc., CO USA; 2ArcherDX, Inc., Boulder, CO USA
Introduction Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Carrier identification and newborn screening have significant implications in the overall prognosis of CF patients. Underlying CFTR mutations were recently shown to vary significantly across ethnic groups. However, current CFTR genotyping assays detect mutations highly prevalent in white individuals, yet fail to detect mutations that are more prevalent in nonwhite individuals. Furthermore, these assays also fail to detect large deletions, such as the CFTRdele2,3(21kb), which is prevalent in Central and Eastern European populations and confers a severe CF phenotype. Here, we present a method based on Anchored Multiplex PCR (AMP™) and next-generation sequencing (NGS) for comprehensive, pan-ethnic detection of CFTR variants, including common base substitutions and large deletions.
Methods AMP is a library preparation method for NGS that uses unidirectional gene-specific primers (GSPs) and molecular barcoded adaptors ligated to random start sites for open-ended amplification. This enables NGS-based identification of both known and unknown mutations across a panel of target regions. Furthermore, anchored GSPs amplify large genomic regions from both ends independently, permitting sequencing of both wildtype and variant alleles from the same GSPs.
Results Using a set of 150 blinded specimens from the Coriell Institute for Medical Research, we show that AMP-based NGS detects known CFTR variants with 100% accuracy. In addition, we detected the CFTRdele2,3(21kb) in a pre-validated DNA sample obtained from the Coriell Institute for Medical Research. Pan-ethnic screening of 1,585 clinical samples identified 34 unique mutations, several of which were identified in multiple individuals. 73% (25/34) of these unique mutations and 60% (74/123) of total mutations detected are not currently included in the ACMG-recommended 23-mutation panel for CF carrier screening. Furthermore, this screen revealed ethnic differences in clinically significant CFTR variants and a pan-ethnic carrier rate of approximately 7%.
Conclusions We demonstrate that AMP-based NGS enables comprehensive detection of both known and novel variants in the CFTR gene, with the ability to detect large deletions. Identification of novel variants is critical for global carrier and newborn screening, as CF driver mutations have not been fully characterized across all ethnicities. As the reagents for AMP-based NGS are lyophilized and thus do not require refrigeration, this may be a practical method for CF screening in global communities..
To address the existing bottlenecks of using NGS in translational research, we’ve created a robust platform that is purpose-built for clinical oncology research.
By combining revolutionary Anchored Multiplex PCR (AMP™) chemistry with an easy-to-use workflow and intuitive software, we are unleashing the power of translational NGS to enable accurate and scalable mutation detection.
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