ArcherDX and GenomOncology team up at AGBT-Precision Health 2016

Scottsdale, Arizona
September 22-24, 2016
Booth #5

Stop by to see how combining Archer® NGS assays with GenomOncology GO Clinical Workbench™ reporting software can enable your precision oncology research.

Want to meet up? Request a meeting.

ArcherDX Poster Presentations

Anchored Multiplex PCR enables sensitive NGS-based detection of copy number variants from genomic DNA

Josh D. Haimes, James Covino, Namitha Manoj, Elina Baravik, Laura Johnson, Laura M. Griffin, Joshua Stahl, Brady P. Culver, Brian Kudlow

ArcherDX, Inc., Boulder, CO


Copy number variations (CNVs) impact more of the cancer genome than all other mutation types combined. Current methods to detect CNVs, such as fluorescence in situ hybridization (FISH), are not amenable to multiplexing and cannot be easily coupled with assays to detect other mutation types common in cancer. Next-generation sequencing (NGS) is a scalable alternative to FISH, detecting CNVs as well as other mutation types across the entire genome. However, routine formalin-fixed paraffin-embedded (FFPE) storage of clinical specimens severely damages DNA, limiting detection sensitivity. Therefore, NGS-based detection of low-level CNVs and CNVs in samples with low tumor cellularity remains challenging.

Anchored Multiplex PCR (AMP™) is a target enrichment strategy that ligates molecular barcoded adapters to DNA fragments prior to PCR, amplifying fragments as small as 50bp. AMP therefore increases read depth and coverage of target regions and enhances CNV detection sensitivity by NGS. We developed AMP-based Archer® VariantPlex™ targeted DNA enrichment assays for NGS to detect both low- and high-level CNVs from low-input clinical sample types, such as FFPE specimens. We also developed the PreSeq™ DNA QC Assay to determine the integrity of genomic DNA prior to library preparation.

We examined over 150 FFPE tumor samples for genomic DNA integrity and CNV detection using the PreSeq DNA QC Assay and VariantPlex Solid Tumor assay for NGS. Our data show that NGS-based detection sensitivity is primarily driven by the integrity of the input genomic DNA, which further predicts the limit of CNV detection. Using optimal input amounts of genomic DNA, the VariantPlex Solid Tumor assay enabled detection of CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity. Therefore, AMP-based target enrichment enables sensitive NGS-based detection of low-level CNVs from low-input clinical samples and in samples with low tumor cellularity.


Anchored multiplex PCR enables NGS-based detection of FLT3-ITDs from genomic DNA

Benjamin Van Deusen, Marc Bessette, Laura Johnson, Aaron Berlin, Michael Banos, Laura Griffin, Erik Reckase, Joshua Stahl, Abel Licon, Brian A. Kudlow

ArcherDX, Inc., Boulder, CO

Internal tandem duplications (ITDs) in FLT3 are associated with poor prognosis of acute myeloid leukemia (AML) and are detected in more than 20% of pediatric and adult AML cases. As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments. ITDs are routinely detected by capillary gel electrophoresis, however this assay cannot be easily coupled with assays to detect other mutations common in AML. Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types. However, detection of ITDs by NGS is challenging, as many variant callers fail to identify these highly variable repeated sequences.

Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS whereby molecular barcoded adaptors and unidirectional gene-specific primers are used for amplification of target sequences. We developed the AMP-based Archer® VariantPlex™ Core AML assay with a set of primers designed to amplify commonly mutated regions of FLT3 from genomic DNA. We also developed a novel de novosequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs.

Using our optimized analysis algorithm, we detected over 98% of in silico ITDs with no false positives. The VariantPlex Core AML assay for NGS in conjunction with this analysis algorithm enabled detection of 7/7 ITD-containing and 9/9 wild-type FLT3 genes in AML-positive blood samples, concordant with capillary gel electrophoresis. Therefore, AMP enables accurate NGS-based detection of FLT3-ITDs from clinical DNA samples. As this approach can detect multiple mutation types from a single sample, our VariantPlex Core AML assay enables simultaneous detection of multiple mutations relevant in AML.


Anchored Multiplex PCR enables sensitive and comprehensive profiling of thyroid and lung cancers by NGS-based methods

Joshua A. Stahl, Josh Haimes, Laura Johnson, James Covino, Namitha Manoj, Marc Bessette, Elina Baravik, Abel Licon, Ryan D. Walters, Laura M. Griffin, Brady P. Culver, Brian A. Kudlow

ArcherDX, Inc., Boulder, CO


Oncogenic drivers of thyroid and lung cancers are complex, involving multiple mutation types occurring across multiple genes. These include single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs) and fusions. Therefore, characterization of thyroid and lung cancers requires comprehensive detection of multiple classes of genomic aberrations across a spectrum of relevant genes. However, current methods to detect each of these mutation types are not scalable and cannot be easily coupled. Therefore, characterization of a single sample requires multiple tests, posing significant challenges for low-input sample types, such as formalin-fixed paraffin-embedded (FFPE) specimens.

Next-generation sequencing (NGS) enables comprehensive detection of multiple mutation types in a single assay. However, high cost and low detection sensitivity associated with NGS-based methods have rendered this approach impractical for routine use. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that increases read depth and coverage of target regions, thus enhancing detection sensitivity. As AMP amplifies target regions using unidirectional gene-specific primers, AMP enables unbiased NGS-based detection of all mutation types across enriched target regions.

We developed the AMP-based Archer® VariantPlex™ and FusionPlex® library preparation assays for NGS that enable detection of mutations in genomic DNA and RNA, respectively. Specifically, comprehensive thyroid and lung (CTL) assays were designed with AMP primers to amplify relevant genes in thyroid and lung cancers.

Our data show that parallel interrogation of DNA and RNA using VariantPlex and FusionPlex CTL assays, respectively, enables simultaneous detection of SNVs, indels, CNVs and fusions from low-input clinical sample types. Furthermore, we show that characterization of gene expression, including detection of splice variants, expression imbalances and whole gene expression levels provides orthogonal verification of detected mutations. Therefore, AMP-based NGS enables simultaneous detection and orthogonal confirmation of multiples types of genomic aberrations across many genes in a single sample.


All types of mutations driving MET deregulation detected by Anchored Multiplex PCR-based NGS

Brian Kudlow, Josh Haimes, Marc Bessette, Namitha Manoj, Danielle Murphy, Robert Shoemaker, Laura M. Griffin, Josh Stahl

ArcherDX, Inc., Boulder, CO

Deregulation of the receptor tyrosine kinase, MET, is associated with aggressive phenotypes in a variety of human cancers. Several types of genetic aberrations can drive MET deregulation, including gene amplification, overexpression, single nucleotide variants (SNVs), exon 14 skipping and fusions. MET is a target of intensive drug development efforts, however the various mutated forms of MET exhibit unique drug sensitivities. Therefore, detection of these mutations has the potential to guide treatments for cancers driven by MET deregulation.

Current methods to detect mutations driving MET deregulation are specific to the mutation type under interrogation. Consequently, multiple tests are required to characterize MET deregulation, which poses a significant challenge for low-input clinical sample types. Next-generation sequencing (NGS) is a powerful alternative to these methods, enabling comprehensive detection of all mutation types from a single sample. However, low detection sensitivity, high input requirements and high costs render these approaches impractical for routine use.

Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that increases read depth and coverage of target sequences, thereby enhancing the detection sensitivity of all mutation types by NGS. We developed AMP-based VariantPlex™ and FusionPlex® library preparation assays for NGS to detect mutations from DNA and RNA, respectively. We designed AMP probes covering the MET gene to detect copy numbers and SNVs from DNA, and fusions, exon skipping and expression levels from RNA.

Here, we show that VariantPlex and FusionPlex assays enable detection of MET amplifications, confirmed by FISH, and resulting overexpression in FFPE samples. We also detected exon 14 skipping, confirmed by RT-PCR, in FFPE and in cells, with concomitant splice site mutations. Lastly, a GTF2I:MET gene fusion and a Y1253D activating point mutation were detected. Therefore, AMP enables comprehensive and sensitive NGS-based detection of multiple mutation types from low-input clinical sample types.


GenomOncology Poster Presentation

Enhanced biomarker-based clinical trials matching

James T. Cole, Ian T. Maurer, Baiju Parikh, Matthew R. Stachowiak, Manuel J. Glynias

GenomOncology, Cleveland, OH


Identifying candidates for enrollment in clinical trials with biomarker-based eligibility criteria is a complex and data analysis-intensive task. Surprisingly, it has been estimated that ~60-95% of eligible patients go unidentified; for patients this means a missed opportunity at a potential cure, and for the drug approval system, a huge loss of time and resources. To ensure accurate trial matching, not only does each patient’s cancer genome need to be properly sequenced and analyzed for potentially matching biomarkers, but the subtleties of clinical trial eligibility requirements need be fully understood at the biomarker-level.

The creation of a large-scale database of biomarker based clinical trials that enables users to identify best-match trials for a single patient presents several technical challenges, including:

  • Cancer diagnosis subtypes (e.g. Patients with Lung Adenocarcinoma could enroll in a NSCLC trial, but not patients with Small Cell Neuroendocrine Carcinoma)
  • Region-based mutations (e.g. EGFR Exon 19 Deletions)
  • Karyotype mutation types and their implicit mutations (e.g. Monosomy 17, i(17)(q10) or del(17)(p10) could both imply TP53 Loss)
  • Complex “boolean-logic” requirements (e.g. must have either a CBFB-MYH11 Fusion, CBFB-MYH11 Inversion, or del(5)(q10), and not a PML-RARA Fusion)
  • Location of a trial relative to a patient

To solve this problem, GenomOncology has developed a custom clinical trial curation tool used by the team at My Cancer Genome (MCG) to curate biomarker-based and disease-type eligibility criteria, which is part of our Knowledge Management System (KMS). To date, over 2500 clinical trials have been imported from ClinicalTrials.gov and NCI PDQ and curated by the MCG team. The result of this process is a database where each clinical trial exists as a set of content based assertions, that interacts with the GO Clinical Workbench™, in which patients with particular alterations and disease combinations are matched to the appropriate clinical trials in the most accurate manner possible.


About ArcherDX

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.

How to contact us

Address

2477 55th Street, Suite 202

Boulder, CO 80301

Phone

Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2017 ArcherDX, Inc.

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.