The 2016 Association of Molecular Pathology
Annual Meeting

Posters are now available for download

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Corporate Workshops

Keeping it Real: NGS-based detection of cfDNA for liquid biopsy research

Wed, 9 Nov | 11:00-11:50am | Room 211/212B

Liquid biopsies are less invasive than tissue biopsies for advanced solid tumors, but circulating tumor DNA (ctDNA) is typically highly fragmented and present in low abundance. While NGS-based assays to detect variants in ctDNA show promise, they must be sensitive enough to detect low-frequency mutations (allele fractions <2%) from low mass inputs (10-100ng) of highly fragmented material. Here, we describe our paired ctDNA library preparation method and bioinformatic analysis software, highlighting the use of unique molecular identifiers to enable confident variant detection at low allelic frequencies.

FusionPlex: The gold standard in NGS-based translocation assays

Wed, 9 Nov | 12:00-12:50pm | Room 201-202

Gene fusions play an important role in tumorigenesis in solid tumors, sarcomas, and hematological malignancies. While RT-PCR and FISH can be used to detect fusions, the limitations of tissue availability can prevent the use of these techniques to identify low-incidence mutation types. Archer® FusionPlex® assays utilize purpose-built chemistry and bioinformatic software for sensitive NGS-based detection of known and novel fusions from RNA, as well as RNA-based variant detection and expression analysis. Join us to learn how leaders in the field are using these assays routinely, improving testing workflows and turnaround times from low-input clinical sample types.

Fusions, ITDs, and More: Advanced NGS applications to characterize hematological malignancies

Wed, 9 Nov | 3:00-3:50pm | Room 217D

The landscape of somatic mutations in hematological malignancies is complicated, frequently involving complex chromosomal translocations and single nucleotide variants, among others. While traditional testing algorithms rely upon multiple technology platforms to identify these different mutation types, advances in next-generation sequencing (NGS)-based methods enable comprehensive detection of gene fusions, SNVs, gene expression profiles, and internal tandem duplications (ITDs).

Poster Presentations
Fri, 11 Nov | 2:30pm - 3:30pm

TT20. Sensitive 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

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Introduction

Copy number variants (CNVs) are oncogenic drivers, and impact more of the cancer genome than all other types of mutations combined. Genomic analysis using next-generation sequencing (NGS) has shown promise as a method to detect CNVs from clinical sample types. However, archival storage of formalin-fixed paraffin-embedded (FFPE) specimens severely damages DNA by inducing protein-nucleic acid crosslinking, base modifications and strand cleavage, which results in poor sequencing coverage and limited 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, resulting in enhanced CNV detection sensitivity.

Methods

Based on AMP chemistry, we developed Archer® VariantPlex™ targeted DNA enrichment assays for NGS to detect both low- and high-level CNVs from FFPE specimens and other low-input clinical sample types. We used the VariantPlex Solid Tumor kit, which contains AMP primers designed to detect CNVs for 43 cancer-associated genes by NGS. To rapidly screen samples prior to library preparation and sequencing, we also developed the Archer PreSeq™ DNA QC Assay to determine the integrity of genomic DNA.

Results

We examined over 150 FFPE tumor samples for genomic DNA integrity and CNV detection using the PreSeq and VariantPlex assays for targeted NGS. Our data show that NGS-based detection sensitivity is driven primarily by the integrity of the input genomic DNA, determined by the PreSeq Assay, which further predicts the limit of CNV detection. Using optimal input amounts of genomic DNA, we detected CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity with the VariantPlex Solid Tumor kit.

Conclusions

These results demonstrate that Archer VariantPlex assays enable sensitive NGS-based detection of low-level CNVs from low-input clinical samples and in samples with low tumor cellularity. Furthermore, the PreSeq DNA QC Assay allows for the adjustment of DNA input to provide adequate genomic copies into the NGS workflow and maximize CNV detection sensitivity.


TT58. B- and T-cell immune repertoire characterization by Anchored Multiplex PCR and next-generation sequencing

Jens Eberlein, Thomas Harrison, Ian McKittrick, Megan Wemmer, Laura M. Griffin, Brady P. Culver, Laura Johnson, Brian A. Kudlow

ArcherDX, Inc., Boulder, CO, USA

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Introduction

The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. 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.

Methods

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 AMP-based B- and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples.

Results

Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%. Furthermore, 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.

Conclusions

AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire. This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples.


TT64. Comprehensive MET mutation profiling by 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, USA

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Introduction

Deregulation of the MET receptor tyrosine kinase 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, although 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 to detect all modes of MET deregulation.

Methods

AMP-based Archer® VariantPlex™ and FusionPlex® library preparation assays 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

MET amplifications and resulting overexpression were detected in FFPE samples using the VariantPlex and FusionPlex kits and confirmed by FISH. Exon 14 skipping with concomitant splice site mutations was also detected using the Archer kits and confirmed by RT-PCR in both FFPE and cells. Interestingly, we detected a novel GTF2I:MET gene fusion and a Y1253D activating point mutation in FFPE samples.

Conclusions

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.



Sat, 12 Nov | 9:45am - 10:45am

G59. Rapid comprehensive CFTR mutation detection across ethnic groups using Anchored Multiplex PCR and next-generation sequencing

Matthew T. Hardison, PhD, FACMG, Laura M. Griffin, PhD, Brady P. Culver, PhD

ArcherDX, Inc., Boulder, CO, USA

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Introduction

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by the build-up of thick mucus resulting in chronic lung infections and airway inflammation. As such, early diagnosis and treatment interventions are crucial to help prevent airway obstruction and lung infections. Therefore, carrier identification and newborn screening have significant implications in the overall prognosis of CF patients. Unfortunately, there is a selection bias in CF diagnosis of white compared to nonwhite populations. This ethnic disparity in CF diagnosis is primarily attributed to differences in underlying CFTR mutations, which were recently shown to vary significantly across ethnic groups by Iris Schrijver et al. 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 assay for comprehensive detection of CFTR mutations for pan-ethnic carrier identification and newborn screening.

Methods

BabyGenes, Inc, in partnership with ArcherDX, Inc., has developed a targeted next-generation sequencing (NGS) assay based on Anchored Multiplex PCR (AMP™) to detect all mutations currently reported in the CFTR2 database. AMP is a library preparation method for NGS that uses unidirectional gene-specific primers (GSPs) and molecular barcoded adaptors ligated to random start sites to enrich for both known and unknown mutations across a panel of target regions.

Results

The BabyGenes CFTR sequencing assay was validated on a set of reference standards purchased from the Coriell Institute for Medical Research resulting in 100% analytical sensitivity across over 100 replicates. Subsequently, we tested 150 Coriell samples, including 60 with known CFTR mutations, representing a diverse ethnic set, including Caucasians, Ashkenazi Jews, African Americans, Hispanics/Latinos and South and East Asians. In addition to the pathogenic mutations from CF-affected samples, we detected over 20 variants of unknown significance. Our results also indicate that the carrier frequency of CFTR mutations in the general pan-ethnic population is high, at about 12.5%.

Conclusions

We demonstrate that the BabyGenes, Inc. AMP-based targeted NGS assay 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.


H69. Comprehensive detection of Acute Myeloid Leukemia driver mutations including internal tandem duplications with 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

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Introduction

Acute myeloid leukemia (AML) oncogenesis is thought to require multiple somatic mutations in a “two-hit” process to 1) increase proliferation and 2) prevent maturation of myeloid cells. While FLT3 and KIT mutations are associated with increased proliferation, NPM1, CEBPA and several other mutations can be associated with maturation inhibition. The most common mutations in AML are internal tandem duplications (ITDs) in FLT3, which are detected in more than 20% of pediatric and adult AML cases 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 particularly 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 present an approach based on AMP technology and a novel bioinformatics algorithm to detect ITDs in FLT3 as well as other mutation types common in AML.

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 silicodatasets enabled optimization of the VariantPlex Core AML analysis algorithm, resulting in the detection of 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. In addition, point mutations in the TKD of FLT3 and insertions in NPM1 exon 11 were detected in 2/7 and 5/7 FLT3-ITD positive blood samples.

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.


TT15. Anchored Multiplex PCR for Targeted Sequencing of Low Molecular Weight Circulating Tumor DNA

Jerome E. Lee, Eric M. Davis, Ian McKittrick, Katelyn E. Trifilo, Laura A. Johnson, Laura M. Griffin, Brian A. Kudlow, and Brady P. Culver

ArcherDX, Inc., Boulder, CO, USA

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Introduction

Liquid biopsies are easier to collect and potentially offer a more comprehensive picture of the mutational landscape for more advanced solid tumors than tissue biopsies. However, the abundance of overall cell-free DNA (cfDNA) in these samples is generally low, ranging from less than 1ng to 30ng/mL of cfDNA in blood from healthy individuals. Unfortunately, this material is generally low abundance and cancer cell derived DNA (ctDNA) contributes a very small percentage to the overall material, except in more advanced disease states. Additionally, ctDNA is typically highly fragmented down to 100-300bp. Therefore, NGS-based assays to detect variants in ctDNA must be sensitive enough to detect low-frequency mutations (allele fractions <2%) from low mass inputs (10-100ng) of highly fragmented material.

Methods

We modified our Anchored Multiplex PCR (AMP™) method for DNA library construction to specifically enrich for low molecular weight ctDNA over larger cfDNA fragments. AMP is a target enrichment strategy for NGS that ligates molecular barcoded adapters to DNA fragments to generate random start sites for multiplex PCR. AMP is well suited to amplify small ctDNA fragments, as it only requires one intact primer-binding site within a fragment. Additionally, our method allows for capture of target regions from both strands independently. Through the use of the molecular barcoded adapters and a specific library purification step, we are able to precisely quantify the number of sequenced DNA molecules. Our ctDNA assay contains primers to enable NGS-based coverage of >3.4kb of many of the most common oncogenic driver mutations, acquired cancer drug resistance mutations, as well as full exon coverage of TP53.

Results

Here, we describe our AMP-based ctDNA library preparation method, highlighting the use and advantages of molecular barcoded adapters. Molecular barcodes are used to unambiguously identify PCR duplicates, correct for PCR or sequencer-derived sequence errors, and flag run-to-run contamination. Using a set of reference ctDNA standards, we demonstrate that these benefits of molecular barcodes enabled confident variant detection at allele frequencies ~0.5% with as little as 50ng of input DNA and less than 4M reads. Furthermore, our ctDNA profiling assay demonstrated a similar power to call variants in ctDNA derived from liquid biopsies.

Conclusions

We demonstrate that our modified AMP-based library construction method for ctDNA mutational profiling is a powerful tool for sensitive detection of variants in model ctDNA reference samples. The advantages observed with reference materials will likely be applicable to clinically derived specimens.



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