NGS Assays by disease
NGS Assays by mutation
Reveal ctDNA kit
Controls and input QC
FusionPlex RNA kits
VariantPlex DNA kits
For Research Use Only. Not for use in diagnostic procedures.
The Association for Molecular Pathology (AMP) | Salt Lake City, Utah 2017
Nathan D. Montgomery, Claire H. Edgerly, Renee Betancourt, Jen Crimmins, A. Michelle Tanner, Jose P. Zevallos, Angela L. Mazul, N. Lynn Ferguson, J. Todd Auman, Sandra Elmore, Margaret L Gulley
Olga Sala-Torra, Rhonda E Ries, Soheil Meshinchi, Ryan D Cassaday, Derek L Stirewalt, Jerald P Radich, Cecilia C Yeung
Identification of gene fusions in hematologic malignancies is part of the initial patient workup as it contributes to confirm diagnosis, prognosis, and often helps patient management.
Cytogenetics, FISH, and PCR are the current gold standard methods to identify translocations and gene fusions. Limitations to these techniques are the inability to detect rare variant translocations, cryptic fusions, and alternative breakpoints. Additionally these techniques require knowledge of both partner genes in a fusion to design assays.
Next Generation Sequencing can detect fusions, mutations, and insertions or deletions in leukemias with normal karyotype.
We tested the diagnostic utility of three Archer FusionPlex panels in different hematologic malignancies including a series of leukemias with novel fusions.
Carmela Paolillo, Amanda Oran, Jason N. Rosenbaum, Jennifer JD Morrissette, Robyn Sussman, Kojo Elenitoba-Johnson
The treatment of non-small cell lung cancer (NSCLC) patients has been revolutionized over the last decade by the discovery of targetable genomic alterations in the tyrosine kinase (TK) pathway. Hepatocyte growth factor (HGF) receptor (MET) is a TK receptor. It plays a fundamental role in regulating development and cell growth. Activating mutations and amplification in MET are well-characterized drivers of oncogenesis and potential targets. However, the results have been disappointing . Instead, actionable MET exon 14 skipping alterations (METex14) occur in up to 7%  of NSCLCs, and have been recently described as promising targets for small-molecule kinase inhibitors and monoclonal antibody therapies. The most common alterations are shown in Fig 1. The exon 14-spliced protein is missing the c-CBL E3 ubiquitin ligase binding domain, resulting in a decreased degradation (Fig 2).
From data published in the last decade, 221 distinct METex14 events have been identified from 126 unique genomic sequence alterations (both intronic and exonic). The Cancer Genome Atlas also detected some METex14 alterations at the DNA level with an incomplete METex14 skipping (80%). Such evidence highlights the need for comprehensive next-generation sequencing (NGS)-based genomic profiling detection of such variants. However, most clinical NGS targeted oncology panels are DNA based, and either cover hotspots or exons, rendering them inadequate at detecting the diverse composition of variants that lead to METex14.
Robyn Sussman, Angela Viaene, Priya Velu, Sydney Schaffer, Jason Rosenbaum, Jennifer Morrissette, and MacLean Nasrallah
As the prognostic and therapeutic importance of genomictesting increase for tumors of the central nervous system(CNS), turnaround time (TAT) and the ability to maximizetesting from limited samples becomes ever more important. Next-generation sequencing (NGS) can provide data on manygenomic changes in a single assay; however, for solid tumors,testing from formalin-fixed, paraffin embedded tissue (FFPE)imposes a minimum of 2-4 days for processing and histologicreview, and degrades the quality of the extracted nucleic acids. Moreover, the available material often has to serve multiplepurposes, including histologic examination,immunohistochemistry (IHC) and other ancillary tests,sometimes leaving limited material for NGS. To improve TAT,nucleic acid quality, and ease the burden on limited FFPEspecimens, we investigated the use of intraoperativespecimens for NGS testing of CNS tumors.
Skyler J. Mishkin, Josh D. Haimes, Namitha M. Nair, Thomas D. Harrison, Laura M. Griffin, Margaret L.
Gulley, Nathan D. Montgomery, Brian A. Kudlow
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-associatedclonotypes 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 MultiplexPCR (AMP™) to detect dominant clonotypes, identify IGH isotypes, and characterize IGHV mutation status in a cohort of patientswith multiple myeloma (MM), lymphoplasmacytic lymphoma (LPL) and chronic lymphocytic leukemia (CLL).
Methods: Sequencing mRNA from MM, LPL and CLL was performed with ArcherDX Immunoverse™ IGH/K/L assay. Total RNA wasextracted from formalin-fixed (FFPE) 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 were present. In contrast, dominant clonotypeswere not detectable in IGH non-expressing MM specimens nor in blood from normal donors. The assay accurately definedisotype, 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 expressionin CLL. Furthermore, because reads covered 100% of IGHV, somatic hypermutation status was discernable and was highly concordant with mutation status called by an orthogonal clinical-grade NGS assay.
Conclusions: RNA-based sequencing from both formalin-fixed tissue and fresh blood permits characterization of IGH repertoire inclinical 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 and treatment.
Aaron Garnett, Ian Hoskins, Kaitlyn Moore, Verity Johnson, Thomas D. Harrison, Abel Licon, Paula G. Roberts, Aaron Berlin, Laura Griffin, Kiri R. Burrow, Darius Fugere, Joshua Stahl, Ryan Walters
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 (InDels), including large multi-exon deletions.
Matthew T. Hardison, Josh Haimes, Laura M. Griffin, Brady P. Culver
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. 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.
The Connective Tissue Oncology Symposium (CTOS) | Maui, Hawaii 2017
Laura M. Griffin, Alexander J. Lazar, Wei-Lien Wang, Angshumoy Roy, Xia Li, Mika Warren, Darius Fugere
An estimated 20-30% of sarcomas are driven by expressed gene fusions, with specific sarcoma subtypes associated with unique gene fusions. Thus gene fusions represent important biomarkers for sarcoma subtype classification and potentially minimal residual disease (MRD) detection. Targeted next-generation sequencing (NGS) provides a valuable tool to detect fusion genes in low-input clinical samples, however, conventional target-enrichment methods require prior knowledge of partner genes and do not detect novel fusions. We developed and deployed with partners a targeted NGS assay based on Anchored Multiplex PCR (AMP™) that permits amplification of fusion transcripts from a single end, enabling identification of novel fusion partners. Here, we present a compilation of novel fusion variants recently identified in sarcoma with this assay, providing an updated view of the molecular landscape of translocation-associated (TA) sarcomas.
IASLC World Lung Conference | Yokohama, Japan 2017
Kurtis D. Davies, Dara L. Aisner, Anh T. Le, Jamie Sheren, Hala Nijmeh, Marileila Varella-Garcia, Robert C. Doebele
ROS1 gene fusions are well characterized oncogenic drivers of lung cancer and other cancersTargeting ROS1 fusion proteins in positive cases is now considered standard of care for lung cancer patients with advanced disease. Accurate clinical detection of ROS1 rearrangements/fusions is critical in ensuring optimal therapy selection for lung cancer patients. Here we compare the performance of 3 different methodologies for clinical detection of ROS1 rearrangement/fusion: break apart fluorescence in situ hybridization (FISH), RNA-based next generation sequencing (NGS) (ArcherDx FusionPlex), and DNA-based NGS (custom assay design using Roche SeqCap hybrid capture target enrichment system).
The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016
Josh D. Haimes, James Covino, Namitha Manoj, Elina Baravik, Laura Johnson, Laura M. Griffin, Joshua Stahl, Brady P. Culver, Brian Kudlow
Introduction 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. We developed Archer® VariantPlex™ library preparation assays for NGS based on Anchored Multiplex PCR (AMP™), a target enrichment technique that enables digital read counting in low-quality samples, potentially enhancing CNV detection sensitivity. We also developed the PreSeq™ DNA QC Assay to determine the integrity of genomic DNA prior to library preparation. Here, we 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 assay enabled detection of CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity. 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.
The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016
Jerome E. Lee, Namitha Manoj, Josh D. Haimes, Skyler J. Mishkin, Paula G. Roberts, Eric M. Davis, Ian McKittrick, Sandra Elmore, Laura M. Griffin, Ryan D. Walters, Brian A. Kudlow, Margaret L. Gulley, Brady P. Culver
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, 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. Here, we describe Archer® Reveal ctDNA™, a library preparation method for NGS based on Anchored Multiplex PCR (AMP™) that enables amplification of low-input, fragmented material for ctDNA mutation profiling.
Benjamin Van Deusen, Marc Bessette, Laura Johnson, Aaron Berlin, Michael Banos, Laura M. Griffin, Erik Reckase, Joshua Stahl, Abel Licon, Brian A. Kudlow
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 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. 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) enables comprehensive detection of multiple mutation types, but NGS-based detection of FLT3-ITDs is challenging because we developed an approach using Anchored Multiplex PCR (AMP™) and bioinformatic analysis tools to amplify, detect and size FLT3-ITDs as well as other mutation types from clinical samples. Using 16 AML-positive blood samples, we demonstrate concordance of this assay with standard methods to detect FLT3-ITDs. We also detected an NPM1 insertion and a point mutation in the tyrosine kinase domain of FLT3 in FLT3-ITD-positive samples, demonstrating the ability of AMP to simultaneously identify multiple mutation types in a single sample.
Jens Eberlein, Thomas Harrison, Jennifer Sims, Ian McKitrick, Megan Wemmer, Brady P. Culver, Laura Johnson, Brian A. Kudlow
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, called Archer® Immunoverse™ TCR/BCR, to analyze the IR using a minimal set of gene-specific primers.
Brian Kudlow, Josh D. Haimes, Marc Bessette, Namitha Manoj, Laura M. Griffin, Danielle Murphy, Robert Shoemaker, Joshua A. Stahl
Introduction 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. Each aberrant form of MET exhibits unique drug sensitivities, however current detection methods are specific to the mutation type. As a consequence, multiple tests are required to characterize MET deregulation, which poses a significant challenge for low-input clinical sample types. While next-generation sequencing (NGS) can detect all mutation types from a single sample, 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. Here, we show that AMP probes covering the MET gene detect copy numbers and SNVs from DNA, and fusions, exon skipping and expression levels from RNA. These results demonstrate that AMP-based NGS detect all modes of MET deregulation from low-input clinical sample types.
Matthew T. Hardison, Kaitlyn E. Moore, Laura M. Griffin, Brady P. Culver
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.
European Society for Medical Oncology (ESMO) | Copenhagen, Denmark 2016
Joshua A. Stahl, Josh D. 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
Introduction 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 cancers. Next generation sequencing (NGS) of target-enriched libraries is a highly sensitive and scalable method to detect these mutations. Anchored Multiplex PCR (AMP™) is a target enrichment strategy that uses unidirectional gene-specific primers and molecular barcoded adapters ligated to DNA ends for amplification. This enables amplification of both known and unknown mutations within target regions and increases coverage of target regions. Here, we demonstrate that parallel interrogation of DNA and RNA using with AMP-based NGS enables simultaneous detection of known and novel SNVs, indels, CNVs and fusions from low-input clinical sample types.
Next Generation Dx Summit | Washington, D.C. 2016
Jill Stefanelli, Josh Stahl, Brian Kudlow, Ryan Walters, Laura Griffin, Josh Haimes
Abstract Recurrent genetic mutations as well as rearrangements involving numerous tyrosine kinases including genes like EGFR, ALK, ROS1 and NTRK1/2/3 have the potential to be oncogenic drivers. Clinical utility has been demonstrated in non-small cell lung cancer (NSCLC) for EGFR exon 19 deletions and exon 21 L858R substitution mutation as well as gene fusions with ALK or ROS1. In addition to these, additional genes are currently targeted in various phases of clinical development and are often present in small proportions of a variety of tumor types, including NSCLC, colorectal cancer, breast cancer, melanoma cancer, salivary gland cancer, papillary thyroid cancer, melanoma and sarcoma making them challenging to detect for potential patient enrollment. We are developing a universal CDx assay based on the ArcherDX AMP technology to be used as a “one test, multiple drug” approach to solve some of the current challenges including sufficient sample material, development and regulatory costs. This assay can interrogate DNA and RNA simultaneously, and has great potential to address many challenges with single gene based approaches. In this approach, multiple pharmaceutical companies can align to develop a product which life cycle manages current therapeutics with demonstrated clinical utility as well as future drugs and utility. As the industry moves to rare target specific compounds, close interaction between pharmaceutical and diagnostic developers will be critical to identify, select, and enroll enough patients for a successful trial outcome and drug approval.
American Association for Cancer Research (AACR)| New Orleans, Louisiana 2016
Josh Haimes, James Covino, Namitha Manoj, Elina Baravik, Laura Johnson, Laura Griffin, Joshua Stahl, Brady P. Culver, Brian Kudlow
Introduction Copy number variations (CNVs) impact more of the cancer genome than all other mutation types combined. Arising from large-scale genomic amplifications and deletions, CNVs alter expression profiles due to altered gene dosages. Next-generation sequencing (NGS) is an invaluable tool to simultaneously detect multiple CNVs from genomic DNA. However, routine formalin-fixed paraffin-embedded (FFPE) storage of clinical specimens severely damages DNA, inducing DNA fragmentation, cross-linking of proteins to nucleic acids, strand cleavage, and base modifications. This poor integrity of genomic DNA can negatively impact sequencing coverage and can limit the sensitivity of NGS-based CNV detection from FFPE samples. Anchored Multiplex PCR (AMP™) is a target enrichment strategy that increases read depth of target sequences, thus enhancing detection sensitivity. Here, we demonstrate that NGS-based CNV detection sensitivity is primarily driven by the integrity of the input genomic DNA, as measured by our PreSeq™ DNA QC Assay. Higher integrity inputs yield greater read depth and base coverage, and detection sensitivity in damaged DNA samples is partially mitigated by increasing input quantity. Furthermore, AMP-based NGS enabled detection of CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity. These results demonstrate that AMP enables detection of low-level CNVs from low-input clinical samples and in samples with low tumor cellularity.
Laura Johnson, Katelyn Trifilo, Helen Wang, Brian Kudlow, Eric Padron, Peter R. Papenhausen, Mohammad Hussaini.
Introduction Our current understanding of leukemogenesis posits the sequential acquisition of key “driver” mutation and genetic alterations resulting in the pathogenic phenotype. In some cases, the association with genetic aberrations and disease phenotype is strong enough to be diagnostic. Most of these genetic aberrations are rearrangements that deregulate the target gene(s) resulting in leukemic transformation. Gene fusions have been implicated as oncogenic drivers in several cancers. Current methods to detect gene fusions, such as FISH and IHC, cannot identify novel fusion partners and cannot simultaneously interrogate multiple genes. Next generation sequencing (NGS) of expressed RNA (RNA-Seq) enables discovery of novel fusion genes across the entire transcriptome, however lacks the sensitivity to detect mutations from low input clinical sample types. Anchored Multiplex PCR (AMPTM) is a target enrichment strategy that enhances NGS-based detection sensitivity. Unidirectional primers used in AMP enable detection of novel fusion genes. Therefore, AMP-based NGS can be used to detect novel fusion genes with high sensitivity across a panel of relevant gene targets. Here, AMP-based NGS was used to discover a novel t(12;17)(p13;p13) translocation resulting the production of an HIC1-ETV6 fusion in a 56-year-old man diagnosed with AML (2013).
Marc Bessette, Benjamin Van Deusen, Laura Johnson, Aaron Berlin, Erik Reckase, Laura Griffin, Joshua Stahl, Abel Licon, Brian A. Kudlow
Introduction FLT3 encodes a tyrosine kinase that is involved in p53 activation and has roles in cell growth arrest and apoptosis. Internal tandem duplications (ITDs) in the juxtamembrane domain result in constitutive activation of FLT3, causing aberrant cell growth leading to tumorigenesis. FLT3-ITDs are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are generally associated with a poor prognosis. The presence of a FLT3-ITD in AML renders chemotherapy ineffective, however these cancers are sensitive to tyrosine kinase inhibitors. FLT3-ITDs are therefore important biomarkers to guide AML treatment. Standard methods to detect FLT3-ITDs include Sanger sequencing and capillary gel electrophoresis. These methods, however, cannot detect other mutations commonly associated with AML. Next-generation sequencing (NGS) enables comprehensive profiling of multiple mutation types. Nevertheless, detection of FLT3-ITDs presents a challenge to NGS-based approaches, as many variant callers fail to identify the highly variable repeated sequences associated with FLT3-ITDs. We developed an approach using Anchored Multiplex PCR (AMP™) and bioinformatic analysis tools to amplify, detect and size FLT3-ITDs from clinical sample types. Here, we demonstrate our bioinformatic approach using 2000 in silico data sets and show concordance of our assay with standard methods to detect FLT3-ITDs in 16 AML-positive samples.
Laura Johnson, Helen Wang, Katelyn Trifilo, Brian Kudlow, Peter R. Pappenhausen, Mohammad Hussaini
Introduction Development of leukemia involves the sequential acquisition of a circumscribed number of driver mutations which result in a block in differentiation and proliferation of the leukemic cells. The WHO classification of hematopoietic and lymphoid tumors designates an entire category as acute myeloid leukemia (AML) with recurrent genetic abnormalities. Most of these include specific chromosome rearrangements such as AML with inv(16)(p13.1q22). In other situations, the presence of a certain translocation can have therapeutic implications such as the presence of t(15:17)(q22:q12): PML-RARA fusion which would warrant treatment with ATRA rather than standard 7+3 induction therapy. For this reason, cytogenetic analysis is routinely performed in cases of AML at diagnosis for their diagnostic, prognostic, and therapeutic implications. Anchored Multiplex PCR (AMPTM) is a target enrichment strategy for next-generation sequencing (NGS) with the sensitivity to detect mutations from low input clinical sample types. Unidirectional primers used in AMP enable identification of novel fusion genes. Therefore, AMP-based NGS can be used to detect novel fusion genes with high sensitivity across a panel of relevant gene targets. Here, AMP-based NGS was used to discover a novel t(X;21)(q28;q22) translocation resulting the production of an RUNX1-G6PD fusion and detected a FLT3-ITD in a 78-year-old man diagnosed with AML.
Cancer Genomics Consortium (CGC)| Denver, Colorado 2016
Introduction Copy number variations (CNVs) impact more of the cancer genome than all other mutation types combined. Next-generation sequencing (NGS) is an invaluable tool to simultaneously detect multiple CNVs from genomic DNA, but routine formalin-fixed paraffin-embedded (FFPE) storage of clinical specimens severely damages DNA. This poor integrity of genomic DNA negatively impacts sequencing coverage and limits the sensitivity of NGS-based CNV detection from FFPE samples. Anchored Multiplex PCR (AMP™) is a target enrichment strategy that increases read depth of target sequences, thus enhancing detection sensitivity. Here, we demonstrate that CNV detection sensitivity by AMP-based NGS is primarily driven by the integrity of the input genomic DNA, as measured by our PreSeq™ DNA QC Assay. Higher integrity inputs yield greater read depth and base coverage, and detection sensitivity in damaged DNA samples is partially mitigated by increasing input quantity. Furthermore, AMP-based NGS enabled detection of CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity. These results demonstrate that AMP enables detection of low-level CNVs from low-input clinical samples and in samples with low tumor cellularity.
Josh Haimes, Laura Johnson, Namitha Manoj, Helen Wang, Marc Bessette, Skyler Mishkin, Abel Licon, Ryan D. Walters, Laura M. Griffin, Brady P. Culver, Joshua A. Stahl, Brian A. Kudlow
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 cancers. We developed a parallel set of targeted sequencing assays, VariantPlex and FusionPlex® Comprehensive Thyroid and Lung (CTL), to detect all mutation types in DNA and RNA, respectively, across genes relevant in thyroid and lung cancer. These assays are based on Anchored Multiplex PCR (AMP™), a target enrichment strategy for next-generation sequencing (NGS) that amplifies both known and novel mutations within target regions. Here, we demonstrate that parallel interrogation of DNA and RNA using VariantPlex and FusionPlex CTL assays, enables simultaneous detection of known and novel SNVs, indels, CNVs and fusions from low-input clinical sample types.
Marc Bessette, Benjamin Van Deusen, Laura Johnson, Aaron Berlin, Michael Banos, Laura Griffin, Erik Reckase, Joshua Stahl, Abel Licon, Brian A. Kudlow
Introduction FLT3 encodes a tyrosine kinase that is involved in p53 activation and has roles in cell growth arrest and apoptosis. Internal tandem duplications (ITDs) in the juxtamembrane domain result in constitutive activation of FLT3, causing aberrant cell growth leading to tumorigenesis. FLT3-ITDs are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are associated with chemotherapy resistance. As FLT3-ITDs are sensitive to tyrosine kinase inhibitors, they are important biomarkers to guide AML treatment. Standard methods to detect FLT3-ITDs include Sanger sequencing and capillary gel electrophoresis (CGE). These methods, however, cannot detect other mutations commonly associated with AML. Next-generation sequencing (NGS) enables comprehensive profiling of multiple mutation types, but NGS-based detection of FLT3-ITDs is challenging because many variant callers fail to identify the highly variable repeated sequences characteristic of ITDs. We developed an approach using Anchored Multiplex PCR (AMP™) and bioinformatic analysis tools to amplify, detect and size FLT3-ITDs from clinical sample types. Here, we demonstrate our bioinformatic approach using 2000 in silico data sets and show concordance of our assay with standard methods to detect FLT3-ITDs in 16 AML-positive samples.
American Association for Cancer Research (AACR) | New Orleans, Louisiana 2016
Samantha Helm, Aleksandra Ras, Vanessa Spotlow, Kevin Kelly, Susan Mockus, Cara Statz, Guruprasad Ananda, Joan Malcolm, Gregory J. Tsongalis
A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the analytical validity of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment ProfileTM (JAX-CTP) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay.
American College of Medical Genetics and Genomics (ACMG) | Tampa, Florida 2016
A. Laubenstein, X. Li,, and M Warren
Introduction Gene fusions resulted from chromosome translocations are seen in 20-30% of soft tissue and bone tumors in children. Because ealy-stage sarcomas are often missed for diagnosis until substantial metastasis has occurred, sarcomas bear a particularly high mortality rate and represent an important challenge for cancer research. Conventional methods to detect chromosomal rearrangements and gene fusions include G-banding, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). However, each method has its own limitation. We hereby employ the next generation sequencing (NGS) technology for detecting a broad spectrum of fusion transcripts from soft tissue sarcomas. This technology permits simultaneous detection of 26 genes with its fusion partners characteristic for soft tissue sarcomas. Among 30 Formalin fixed paraffin embedded (FFPE) specimens examined, 20 were positive for fusion by NGS. These results demonstrated that the NGS is a robust and powerful tool for detection of gene fusions, which reinforces the complementary roles of molecular and cytogenetic testing in diagnosis of soft tissue and bone tumors.
The Association for Molecular Pathology (AMP) | Austin, Texas 2015
Josh Haimes, Laura Johnson, James Covino, Namitha Manoj, Marc Bessette, Elina Baravik, Abel Licon, Ryan D. Walters, Brady P. Culver, Joshua A. Stahl and Brian A. Kudlow.
Introduction The genetic mutations of non-small cell lung cancer (NSCLC) include single nucleotide variations (SNVs), insertions and deletions (indels), copy number variations (CNVs) and rearrangements. A tumor is typically driven by a single class of mutations, and the proto-oncogenes in each class are diverse. Thus, to comprehensively and economically understand a given NSCLC sample, an assay needs to assess disparate mutation types multiplexed across many gene targets. We present results from a NSCLC screen using a pair of targeted NGS panels that, when combined, permit simultaneous detection of SNVs, indels, CNVs and rearrangements across oncogenes and tumor suppressors relevant to solid tumors. We found driver mutations harbored in many genes across each class of mutation, demonstrating the benefit of a targeted sequencing approach with comprehensive detection ability. In addition, we show the complementarity of two panels when used in combination to interrogate individual samples.
Milhan Telatar PhD, Maria Teresa Cuellar, Carrie Louie PhD, Michelle Afkhami MD, Raju Pillai MD, Dongqing Gu, Peiguo Chu MD and Patricia Aoun MD
Introduction A next generation sequencing (NGS) based Fusion Panel that simultaneously detects and characterizes fusions of 7 genes associated with solid tumors including non-small cell lung cancer (NSCLC) and thyroid cancer was designed and analytically validated. The Fusion Panel detects targeted regions of interest, including any known or novel fusion partners. Table 1 lists some, but not all of the possible fusion genes detectable by this assay.
Thomas Schneider MD, Geoffrey Smith MD, Charlie Hill MD/PhD, LinshengZhang MD/PhD
Thomas M. Schneider MD, Michael Clay MD, Geoffrey Smith MD, Charlie Hill MD/PhD
Advances in Genome Biology and Technology (AGBT) | Marco Island, Florida 2015
Laura Johnson and Brady Culver
Abstract Anchored Multiplex PCR (AMP™) chemistry for NGS applications is a robust tool to detect both known and unknown fusions. However, characteristics associated with AMP transcend RNA assays and make it a valuable tool for DNA sequencing as well. These characteristics include the following:
Additionally, the enzymatic DNA fragmentation method used in Archer™ VariantPlex™ assays generates a broad sequenceable fragment size distribution ranging from less than 100bp to more than 1 kilobase in length. The presence of longer fragments facilitates read mapping over repetitive or conserved regions in the genome.
The first Archer DNA assay for widespread release is the Archer VariantPlex BRCA1/2 Panel. Inherited harmful BRCA1/2 mutations substantially increase the lifetime risk of developing cancer, especially breast and ovarian cancer. The VariantPlex BRCA1/2 Panel design covers all coding and non-coding exons in RefSeq annotated transcript models for these genes. In an effort to further improve panel performance, we investigated alterations to PCR cycling conditions that enhanceed target coverage uniformity.
Our data shows that a balance must be struck between primer performance uniformity and the portion of total reads that are on-target. Despite losses in on-target reads, fewer reads were required to cover all targeted bases than conditions with higher on-target percent. In addition, coverage and sensitivity were universally improved by utilizing a new polymerase. These changes are being implemented across all Archer NGS kits. Finally, we demonstrate the performance of this panel and our variant calling software by correctly identifying the known BRCA1 or BRCA2 mutations present in 10 different samples from Coriell Institute.
Josh D. Haimes, Namitha Manoj, Abel Licon, Joshua A. Stahl, Brian A. Kudlow, Maria Cuellar, Milhan Telatar
Abstract Copy number variation (CNV) results from oncogene amplification or tumor suppressor gene deletion and is a common mode of gene deregulation in cancer (1). Several techniques have been employed to determine copy number, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR); however, none of these methods are amenable to high-throughput, directed CNV detection. We developed a directed next-generation sequencing (NGS)-based method to rapidly and quantitatively measure the copy number of tens and potentially hundreds of genes simultaneously. This complete workflow, found in the Archer™ Universal DNA Kit , is powered by Anchored Multiplex PCR (AMP™) chemistry and processes dozens of samples in about 6 hours. By ligating a molecular barcode to randomly fragmented input DNA and then using AMP to simultaneously enrich for several regions of each target gene, we can accurately measure the relative copy number of each target gene in test samples by counting unique molecular barcodes associated with each target region.
We validated our methodology with a 25-gene panel on a subset of NCI-60 cell lines by comparing our copy number measurements to those determined by both aCGH and qPCR. Results from both orthogonal methods strongly correlated with data from our NGS-based method. We multiplexed hundreds of samples on a single MiSeq® run and detected CNVs, both amplifications and deletions, of 2X magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples. Furthermore, we demonstrate that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin-fixed, paraffin embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications.
Aaron Berlin, Erik Reckase, Joseph Heimiller, Doug Wendel, Michael Banos, Jeremy Widmann, Thon DeBoer, Brian Kudlow, Brady Culver, Jason Myers, Josh Stahl and Abel
Advances in Genome Biology and Technology (AGBT) | Marco Island, Florida 2013
Zongli Zheng, Boryana Zhelyazkova, Divya Panditi, Hayley E Robinson, A. John Iafrate, Long Le
Abstract Current clinical genotyping based on next generation sequencing is mostly driven by targeted gene panels. Particularly in the field of cancer genotyping, the need for high sequencing depth to achieve both complete gene coverage and the analytical sensitivity required for detecting low frequency variants in heterogeneous specimens renders whole genome sequencing and whole exome sequencing impractical. In addition, there is an unmet demand for a rapid, focused, and economical
variant confirmation sequencing method with the ability to detect single nucleotide variants, insertions/deletions, copy number changes, and rearrangements. We have developed a novel multiplex polymerase chain reaction assay termed anchored multiplex PCR (AMP) which can detect these four types of mutations. The assay may be performed with low amounts of RNA or DNA in a one- or twotube format using commerically available reagents, custom primers, and standard library preparation
instrumentation in one working day and then sequenced by Ion Torrent or Illumina sequencing. Targeting double-stranded cDNA generated from total RNA, we have developed a one-tube lung cancer panel to detect druggable rearrangements in ALK, ROS1, and RET for clinical testing without prior knowledge of the heterologous fusion partners. Targeting genomic DNA, we have designed a 96 loci assay with miminal optimization which showed 100% 100X and >99.9% 500X minimum fold coverage at the targeted bases. A similar genomic DNA based assay targeting 370 exons (52.3 kilobases) with 626 total amplicons in a two-tube format showed >97% 100X and >93% 500X minimum fold coverage at the targeted bases in an initial run without any optimization. With efficient primer design solutions and higher oligo synthesis capacity, our techinque may scale many fold higher for rapid, facile target enrichment in clinical, discovery, and confirmation sequencing applications.
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