Conference Posters

Featuring Archer technologies



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

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

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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.

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

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

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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, 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.

Comprehensive detection of Acute Myeloid Leukemia driver mutations including internal tandem duplications with Anchored Multiplex PCR and next-generation sequencing

The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016

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

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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 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.

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

The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016

Jens Eberlein, Thomas Harrison, Jennifer Sims, Ian McKitrick, Megan Wemmer, Brady P. Culver, Laura Johnson, Brian A. Kudlow

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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, called Archer® Immunoverse™ TCR/BCR, to analyze the IR using a minimal set of gene-specific primers.

Comprehensive MET mutation profiling by Anchored Multiplex PCR and next-generation sequencing

The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016

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

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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.

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

The Association for Molecular Pathology (AMP) | Charlotte, North Carolina 2016

Matthew T. Hardison, Kaitlyn E. Moore, Laura M. Griffin, Brady P. Culver

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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.

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

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

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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.

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

European Society for Medical Oncology (ESMO) | Copenhagen, Denmark 2016

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

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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.

Development of an NGS-based CDx assay for multiple drug targets across solid tumor malignancies

Next Generation Dx Summit | Washington, D.C. 2016

Jill Stefanelli, Josh Stahl, Brian Kudlow, Ryan Walters, Laura Griffin, Josh Haimes

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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.

NGS based CNV detection sensitivity is dependent upon nucleic acid input quality

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

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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.

Detection of Novel t(12;17)(p12;p13) in Treatment-Refractory/Relapsed Acute Myeloid Leukemia by Anchored Multiplex PCR(AMP™)-based Next Generation Sequencing

American Association for Cancer Research (AACR)| New Orleans, Louisiana 2016

Laura Johnson, Katelyn Trifilo, Helen Wang, Brian Kudlow, Eric Padron, Peter R. Papenhausen, Mohammad Hussaini.

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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).

NGS based detection of FLT3-ITDs with Anchored Multiplex PCR

American Association for Cancer Research (AACR)| New Orleans, Louisiana 2016

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

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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.

Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of Anchored Multiplex PCR-based next-generation sequencing

American Association for Cancer Research (AACR)| New Orleans, Louisiana 2016

Laura Johnson, Helen Wang, Katelyn Trifilo, Brian Kudlow, Peter R. Pappenhausen, Mohammad Hussaini

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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.

Detection of copy number variants by next generation sequencing is driven by genomic DNA integrity

Cancer Genomics Consortium (CGC)| Denver, Colorado 2016

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

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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.

Anchored Multiplex PCR enables comprehensive profiling of thyroid and lung cancer mutations by next generation sequencing

Cancer Genomics Consortium (CGC)| Denver, Colorado 2016

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

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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.

Anchored Multiplex PCR enables NGS-based detection of FLT3-ITDs

Cancer Genomics Consortium (CGC)| Denver, Colorado 2016

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

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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.

Validation of the Archer FusionPlex Solid Tumor Panel in the JAX Cancer Treatment Profile

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

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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.

Characterization of Fusion Transcripts of Soft tissue and Bone Tumors Using Next Generation Sequencing

American College of Medical Genetics and Genomics (ACMG) | Tampa, Florida 2016

A. Laubenstein, X. Li,, and M Warren

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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.

Targeted detection of copy number variants and fusion transcripts greatly expands the ability to detect oncogenic drivers in NSCLC

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.

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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.

Analytical Validation of a NGS-Based Diagnostic Test to Detect Common Fusions in Solid Tumors from Formalin-Fixed Paraffin-Embedded Tissues

The Association for Molecular Pathology (AMP) | Austin, Texas 2015

Milhan Telatar PhD, Maria Teresa Cuellar, Carrie Louie PhD, Michelle Afkhami MD, Raju Pillai MD, Dongqing Gu, Peiguo Chu MD and Patricia Aoun MD

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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.

Detection of chromosome translocations associated with myeloid malignancies using targeted RNA next generation sequencing panel

The Association for Molecular Pathology (AMP) | Austin, Texas 2015

Thomas Schneider MD, Geoffrey Smith MD, Charlie Hill MD/PhD, LinshengZhang MD/PhD

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Background

  • Recurrent chromosome translocations are frequently associated with myeloid malignancies; some being identified as disease defining abnormalities.
  • Most clinical laboratories rely on fluorescence in situ hybridization (FISH) or conventional karyotyping to detect these translocations.
  • Next generation sequencing (NGS) provides the opportunity to inquire multiple fusions at the same time with greater analytic sensitivity. The nucleotide sequences around the fusion can also be acquired.
  • In this study, we examine the performance of a commercially available RNA based NGS method designed to detect translocations in hematologic malignancies (The ArcherDxFusionPlexHemepanel, ArcherDx, MA).

Clinical Utility of the ArcherDx FusionPlex Sarcoma Panel in Formalin Fixed Tissue: A Proof of Principle Study

The Association for Molecular Pathology (AMP) | Austin, Texas 2015

Thomas M. Schneider MD, Michael Clay MD, Geoffrey Smith MD, Charlie Hill MD/PhD

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Background

  • Soft tissue tumors are increasingly defined by unique recurrent molecular alterations.
  • Unfortunately, due to RNA degradation in formalin fixed paraffin-embedded (FFPE) tissue, there have been relatively w next generation sequencing (NGS) library preparation methods for these specimens.
  • Routine molecular studies in soft tissue tumors are heavily reliant upon fluorescent in-situ hybridization (FISH). This can be problematic as some cases require multiple FISH studies before a diagnosis can be reached, and even then subsequent PCR may be needed to identify prognosticallysignificant translocation partners (as is the case in alveolar abdomyosarcoma).
  • ArcherDX has recently released an NGS Sarcoma Panel (ArcherDxFusionPlexSarcoma Panel, ArcherDx, MA) specifically signed for FFPE samples in an effort to alleviate these problems.
  • This study sought to validate its use.

Optmization of Nested PCR Conditions to Improve Target Coverage Uniformity in Archer VariantPlex BRCA Panel

Advances in Genome Biology and Technology (AGBT) | Marco Island, Florida 2015

Laura Johnson and Brady Culver

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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:

  • the ability to identify unique starting molecules and consolidate sequencing information from PCR duplicates
  • low input DNA requirements
  • applicability to both Illumina® and Ion Torrent™ sequencing platforms

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.

Development and Validation of a Directed, Rapid and Highly Multiplexed Assay to Detect Copy Number Variations in Clinical Samples

Advances in Genome Biology and Technology (AGBT) | Marco Island, Florida 2015

Josh D. Haimes, Namitha Manoj, Abel Licon, Joshua A. Stahl, Brian A. Kudlow, Maria Cuellar, Milhan Telatar

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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.

Archer™ FusionPlex™ NGS Data Analysis, Algorithms and Methods: Hypothesis-Free Gene Fusion Detection at analysis.archerdx.com

Advances in Genome Biology and Technology (AGBT) | Marco Island, Florida 2015

Aaron Berlin, Erik Reckase, Joseph Heimiller, Doug Wendel, Michael Banos, Jeremy Widmann, Thon DeBoer, Brian Kudlow, Brady Culver, Jason Myers, Josh Stahl and Abel Licon

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Abstract

Anchored Multiplex PCR for Targeted Next Generation Sequencing.

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

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