Friday, 23 June | 12:45 to 14:15 | Heinrich-Lades-Halle, Rathausplatz 1, 91052 Erlangen
Address: Heinrich-Lades-Halle, Rathausplatz 1, 91052 Erlangen
Volker Endris, Ph.D.
Center for Molecular Pathology
University Hospital Heidelberg
Florian Haller, Ph.D.
Head of Biobank of the Comprehensive Cancer Center
University Hospital Erlangen
Matthew Callan, Ph.D.
"Applications of Anchored Multiplex PCR for the detection of genomic rearrangements"
"Detection of novel gene fusions in rare sarcoma subtypes using next-generation sequencing"
Moderator, Questions and answers
Posters shown at the following times:
Matthew T. Hardison, PhD, FACMG, Laura M. Griffin, PhD, Brady P. Culver, PhDRead the abstract
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. We present a rapid, cost-effective NGS assay based on Anchored Multiplex PCR (AMP™) for comprehensive detection of CFTR mutations across ethnic groups.
AMP is a library preparation method for NGS that uses unidirectional gene-specific primers and molecular barcoded adaptors ligated to random start sites to enrich for both known and unknown mutations across a panel of target regions. Following analytical and clinical validation of the panel, a total of 1,585 clinical samples representing a diverse ethnic set were analyzed for clinically significant CFTR variants.
We demonstrate 100% accuracy of CFTR variant detection, and identified 34 unique mutations in a blinded screen of 1,585 U.S. clinical samples. This revealed ethnic-specific CFTR variants, 73% of which are not included in current ACMG guidelines for CF carrier screening in the U.S. Furthermore, we revealed a pan-ethnic carrier rate of ~7%, higher than previously predicted.
AMP-based NGS enables rapid, highly sensitive, and comprehensive detection of both known and novel mutations in the CFTR gene. This is critical for global carrier and newborn screening, as CF driver mutations have not been fully characterized across all ethnicities. As this entire assay can be performed in under 96 hours and the reagents do not require refrigeration, AMP is a practical and economical method for global communities.
Jerome E. Lee1, Namitha Manoj1, Josh D. Haimes1, Skyler J. Mishkin1, Sandra Elmore2, Brian A. Kudlow1, Margaret L. Gulley2, Brady P. Culver1
1 ArcherDX, Boulder, CO USA; 2 University of North Carolina School of Medicine, Chapel Hill, NCRead the abstract
Liquid biopsies are a promising, minimally invasive alternative to tissue biopsies that have potential cost, time and safety benefits, as well as a greater ability to interrogate heterogeneous tumors. However, except in advanced disease states, cell free DNA (cfDNA) is typically of low abundance and only a small portion of cfDNA originates from tumor cells as circulating tumor DNA (ctDNA), which tends to be highly fragmented (100-300bp). Therefore, NGS-based assays to detect variants in ctDNA must be sensitive enough to detect mutations at allele frequencies (AF) <2% from <100ng of highly fragmented DNA.
We developed the Archer® Reveal ctDNA™ 28 assay based on Anchored Multiplex PCR (AMP™), a target enrichment method for NGS that uses unidirectional gene-specific primers and molecular barcoded (MBC) adapters for amplification. AMP is well suited to amplify small cfDNA fragments, as it only requires one intact primer-binding site within a fragment. MBC adapters ligated prior to amplification permit post-sequencing error correction, reducing background noise and increasing analytical sensitivity. Finally, variant filtering in the Archer Analysis pipeline further increases the specificity of variant calls.
AMP enabled interrogation of more than 65% of the input molecules from 50ng starting material. As a result, we show 100% detection sensitivity for 1% AF variants using 10ng DNA input and 71.9% detection sensitivity for 0.1% AF variants using 50ng DNA input. MBC-enabled post-sequencing error correction and variant filtering reduced the number of false positives by 98%, resulting in 91.7% specificity. Finally, mutations detected from liquid biopsy-derived ctDNA showed cancer type-dependent concordance with tissue biopsy findings, and revealed additional oncogenic driver mutations.
These results indicate that the Archer Reveal ctDNA 28 assay is a powerful tool for sensitive and specific NGS-based detection of variants in liquid biopsies, showing cancer type-dependent concordance of tissue and plasma mutation profiles, as well as identification of additional oncogenic driver mutations in ctDNA.
Laura Johnson, Marc Bessette, Aaron Berlin, Josh Haimes, Katelyn Trifilo, Namitha Manoj, Helen Wang, Brian Kudlow
ArcherDX, Inc., Boulder, CO, USARead the abstract
Hematologic malignancies can be driven by a diversity of mutation types, including single nucleotide variants (SNVs), copy number variants (CNVs), gene fusions, insertions and deletions (indels) and changes in gene expression profiles. However, comprehensive detection of these mutation types from a single clinical sample is challenging, as specific assays are required to detect each mutation type. We developed targeted next-generation sequencing (NGS) assays based on Anchored Multiplex PCR (AMP™) to simultaneously detect multiple mutation types, including novel gene fusions and ITDs, as well as relative gene expression levels relevant in hematologic malignancies.
AMP is a library preparation method for NGS that uses molecular barcoded (MBC) adapters and single gene-specific primers (GSPs) for amplification. AMP-based Archer® VariantPlex™ and FusionPlex® assays enable NGS-based detection of mutations from DNA and RNA, respectively. Open-ended amplification permits identification of both known and novel gene fusions with FusionPlex assays. Furthermore, our novel bioinformatics algorithm enables ITD detection with VariantPlex assays. Finally, MBC adapters ligated to RNA fragments prior to amplification permit determination of relative gene expression levels.
We detected a KMT2A-MLLT3 fusion through breakpoint identification, with reads extending 6 exons into MLLT3. AMP also enabled NGS-based detection of a novel RUNX1 fusion, RUNX1-G6PD, in a case of acute unclassifiable leukemia. Furthermore, we show that single, unidirectional GSPs provide bidirectional coverage of a BCR-ABL1 fusion, which was detected with reads originating from ABL1 as well as BCR GSPs. Using our optimized bioinformatics algorithm and the VariantPlex assay, we accurately and reliably detected ITDs of varying sizes and insertion points, with simultaneous point mutation detection in AML-positive blood samples. Finally, we show NGS-based expression profile analysis with the FusionPlex assay, resulting in identification of Diffuse Large B-Cell Lymphoma subtypes in a small cohort of samples.
Our results demonstrate that AMP-based NGS enables comprehensive detection of multiple mutation types, including novel fusions and ITDs, as well as gene expression levels relevant in hematologic malignancies.
Jens Eberlein, Thomas Harrison, Ian McKittrick, Megan Wemmer, Laura M. Griffin, Brady P. Culver, Laura Johnson, Brian A. Kudlow
ArcherDX, Inc., Boulder, CO, USARead the abstract
Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Here, we describe an Anchored Multiplex PCR (AMP™)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias.
AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs.
We validated the quantitative reproducibility and sensitivity of the AMP-based IGH assay using mRNA isolated from peripheral blood leukocytes of healthy and B-cell chronic lymphocytic leukemia (B-CLL) donors. Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%, with the ability to determine IGHV mutational status. We also validated the quantitative reproducibility and sensitivity of the AMP-based T-cell receptor (TCR) assay using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples. Our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality.
AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
Brian A. Kudlow1, Josh Haimes1, Marc Bessette1, Namitha Manoj1, Laura M. Griffin1, Danielle Murphy2, Robert Shoemaker2, Joshua Stahl1
1 ArcherDX, Inc., Boulder, CO; 2 Ignyta, San Diego, CARead the abstract
Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. MET deregulation can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and single nucleotide variants (SNVs), such as kinase-activating point mutations. MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities. We developed a targeted NGS assay based on Anchored Multiplex PCR (AMP™) to detect all types of mutations driving MET deregulation from a single sample.
AMP only requires a single gene-specific primer for amplification, enabling open-ended capture of DNA and cDNA fragments for NGS-based detection of known and unknown mutations. We developed AMP-based Archer® VariantPlex™ and FusionPlex® library preparation assays to detect mutations from DNA and RNA, respectively. AMP probes were designed to cover the MET gene for detection of copy numbers variants (CNVs) and SNVs from DNA, and known and novel fusions, exon skipping and expression levels from RNA.
We show that the VariantPlex assay enables NGS-based detection of MET amplifications from DNA in concordance with FISH results. Further NGS analysis of RNA from the same sample using the FusionPlex assay revealed the resulting overexpression of MET. We also demonstrate that AMP-enabled open-ended capture of cDNA fragments allows for reliable detection of exon 14 skipping in FFPE samples and in cells, consistent with RT-PCR results. Parallel analysis of DNA from the cell samples revealed splice site mutations that have been previously reported to drive exon 14 skipping. Furthermore, this open-ended capture also permitted identification of a novel GTF2I:MET gene fusion in a patient-derived xenograft model. Finally, we detected an kinase-activating point mutation in MET, p.Y1253D, by analysis of genomic DNA with the VariantPlex NGS assay.
These results show that AMP-based VariantPlex and FusionPlex Assays enable comprehensive detection of multiple mutation types from low-input clinical sample types, such as FFPE specimens. As MET deregulation can be driven by many different genetic aberrations, this allows for NGS-based characterization of MET deregulation from a single sample.
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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