ArcherDX at the 2016 ESMO Congress

Copenhangen, Denmark
7-11 October, 2016


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Featured Posters

Copy number variant detection by Anchored Multiplex PCR and next-generation sequencing

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, USA


Background

Copy number variants (CNVs) are common oncogenic drivers, impacting more of the cancer genome than all other types of mutations combined. Next generation sequencing (NGS) of cancer genomes is a highly sensitive method to detect CNVs from clinical sample types. However, routine formalin-fixed paraffin-embedded (FFPE) storage of clinical specimens severely damages DNA by inducing cross-linking of proteins to nucleic acids, base modifications and strand cleavage. This results in poor sequencing coverage and limits CNV detection sensitivity. Therefore, NGS-based detection of low-level CNVs (<3-fold) and CNVs in samples with low tumor cellularity remains challenging. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that enables digital read counting in low-quality samples, potentially enhancing CNV detection sensitivity.

Methods

Based on AMP technology, we developed 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. Specifically, we used the VariantPlex Solid Tumor kit, which contains AMP primers designed to enable NGS-based detection of CNVs for 43 cancer-associated genes. We also developed the PreSeq™ DNA QC Assay to determine the integrity of genomic DNA prior to library preparation.

Results

We examined over 150 FFPE tumor samples for genomic DNA integrity and CNV detection using the PreSeq DNA QC Assay and VariantPlex Solid Tumor kit 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 kit enabled detection of CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity.

Conclusions

These results demonstrate that 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.


Comprehensive profiling of thyroid and lung cancers by Anchored Multiplex PCR and next-generation sequencing

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, USA

Background

Thyroid and lung cancer tumorigenesis can be driven by many mutation types occurring across a large set of genes. These include single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs) and fusions. As such, a comprehensive assay for multiple classes of genomic aberrations targeting a spectrum of relevant genes has significant implications for the characterization of thyroid and lung tumors. Anchored Multiplex PCR (AMP™) is a target enrichment strategy engineered to preserve the complexity of degraded samples by ligating molecular barcodes with a universal primer binding site for amplification. This enables next generation sequencing (NGS)-based detection of known and novel fusions from RNA, as well as RNA-based variant detection and expression analysis. In addition, high complexity DNA-based libraries allow for high-confidence SNV/indel and CNV detection.

Methods

Sixty two non-small cell lung carcinoma samples were subjected to AMP-based NGS using Archer® VariantPlex™ and FusionPlex® CTL parallel assays. Analysis of all samples was carried out with Archer Analysis.

Results

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.

Conclusion

These results demonstrate that AMP-based libraries support simultaneous NGS-based detection of multiples types of genomic aberrations across many genes in lung and thyroid cancer. Furthermore, combined use of RNA- and DNA-based libraries offer cross-validation of findings within a sample.


Internal tandem duplications in FLT3 detected by Anchored Multiplex PCR and next-generation sequencing

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

ArcherDX, Inc., Boulder, CO, USA


Background

Internal tandem duplications (ITDs) in FLT3 are detected in more than 20% of pediatric and adult acute myeloid leukemia (AML) and are associated with an aggressive phenotype. As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments. Capillary gel electrophoresis can detect ITDs but cannot be easily coupled with assays to detect other mutation types common in AML. Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types. However, detection of ITDs by NGS is challenging, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that uses molecular barcoded adaptors and gene-specific primers, permitting open-ended capture of DNA fragments from a single end. We tested whether AMP-based NGS is suitable for FLT3-ITD detection.

Methods

We developed the Archer® VariantPlex™ Core AML library preparation assay for NGS to detect FLT3-ITDs from genomic DNA extracted from clinical samples. We designed AMP probes to cover the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. We further developed a novel de novo sequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs.

Results

In silico datasets enabled optimization of the VariantPlex Core AML analysis algorithm, resulting in the detection over 98% of in silico ITDs with no false positives. The VariantPlex Core AML library preparation assay in conjunction with the optimized analysis algorithm enabled sensitive NGS-based detection of ITDs in 16 AML-positive blood samples. These results were consistent with results obtained from standard capillary gel electrophoresis.

Conclusion

Our data show that 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 kit enables simultaneous detection of multiple mutations relevant in AML.


Genetic aberrations driving MET deregulation detected with Anchored Multiplex PCR and next-generation sequencing

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

ArcherDX, Inc., Boulder, CO

Background

Deregulation of the receptor tyrosine kinase, MET, is associated with aggressive phenotypes in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. 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. Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes. However, low detection sensitivity, high input requirement and high costs render these approaches impractical for routine detection of mutations from low-input clinical sample types. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that, by its scalable and quantitative nature, is well suited for detection of each of the modes deregulation of MET.

Methods

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.

Results

VariantPlex and FusionPlex kits enabled detection of MET amplifications, confirmed by FISH, and the resulting overexpression in FFPE samples. Exon 14 skipping was also detected and 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.

Conclusions

These results show that AMP enables comprehensive and sensitive NGS-based detection of multiple mutation types from low-input clinical sample types.


Exhibition Dates

Friday, 7 October 2016, 13:00 – 17:00

Saturday, 8 October 2016, 10:00 – 17:00

Sunday, 9 October 2016, 10:00 – 17:00

Monday, 10 October 2016, 10:00 – 16:30


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.