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Accurate Detection of FLT3-ITDs and CEBPA Variants in Acute Myeloid Leukemia by Anchored Multiplex PCR and Next Generation Sequencing

Association for Molecular Pathology (AMP) | San Antonio, USA 2018

Verity Johnson, Kaitlyn E. Moore, William M. Castor, Laura M. Griffin, Aaron Berlin, Abel Licon, Ryan D. Walters. ArcherDX, Boulder, CO. USA.

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Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of mutations across multiple genes for characterization. FLT3-ITDs and CEBPA mutations represent important markers in AML, however they are difficult to detect by NGS due to the highly variable nature of ITDs, the high GC content of CEBPA, and the difficulty in mapping repeated sequences to a wild-type reference. Tracking of very low frequency mutations is also of growing interest with regards to AML. The ability to accurately detect variants at very low allele fractions (AFs) can be used to assess treatment efficacy and potential relapse.

Anchored Multiplex PCR Enables Sensitive NGS-Based Mutation Detection in the Context of Large Primer Panels

Association for Molecular Pathology (AMP) | San Antonio, USA 2018

Verity Johnson, Kaitlyn E. Moore, Laura Griffin, Abel Licon, Aaron Berlin, Ryan Walters. ArcherDX, Boulder, CO. USA.

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The mutational landscape of pediatric and adult cancers is changing at a rapid pace. Unfortunately, some targeted high-throughput sequencing assays lack the flexibility required to quickly incorporate new discoveries. Adding new targets to existing panels often requires re-optimization, and may risk reductions in variant detection sensitivity. Here, we describe a target enrichment method, Anchored Multiplex PCR (AMP™) that performs consistently regardless of panel size and content, which enables modularity in panel design and confidence in assay adaptability as the field advances.

FLT3-ITD and CEBPA Variant Detection by Anchored Multiplex PCR and Next Generation Sequencing

Association for Molecular Pathology (AMP) Europe | Rotterdam, The Netherlands 2018

Verity Johnson, Marc Bessete, Benjamin Van Deusen, Laura Johnson, Namitha M. Nair, Josh D. Haimes, Kaitlyn E. Moore, Laura M. Griffin, Abel Licon, Ryan D. Walters, Brian Kudlow. ArcherDX, Boulder, CO. USA.

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Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of mutations across multiple genes for characterization. Internal tandem duplications (ITDs) in FLT3, detected in about 25% of AML cases, cause aberrant cell growth leading to tumorigenesis and are associated with poor prognosis. CEBPA has an important role in myeloid differentiation, and mutations in the gene are the most common mutations detected in cytogenetically normal AML. Consequently, the WHO recommends characterization of CEBPA mutations for hematopoietic and lymphoid tumor classification. While FLT3-ITDs and CEBPA mutations represent important markers in AML, they are difficult to detect by NGS due to the highly variable nature of ITDs, the high GC content of CEBPA, and the difficulty in mapping repeated sequences to a wild-type reference.

Use of Highly Multiplexed Reference Materials to Facilitate Validation of a Clinical NGS Tumor Fusion RNA Assay

American Assocation for Cancer Research (AACR) | Chicago, Illinois 2018

Catherine Huang, Subit Barua, Deepika Philkana, Russell Garlick, Bharathi Anekella, and Helen Fernandes

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Next-generation sequencing (NGS) assays for detection of tumor RNA fusions must undergo rigorous validation before clinical implementation. Validations include assessment of the assay’s accuracy, precision, reproducibility, and limits of detection (LOD) or reportable range. Obtaining samples with all needed variants is difficult and time consuming. When found, they are often in limited quantities such that repeated testing for precision and reproducibility studies is not possible. Sample heterogeneity or lack of characterization further complicates interpretation of results. This study demonstrates how highly characterized, uniformly manufactured reference materials can facilitate clinical NGS assay validation.

Donor-Derived Circulating Cell-Free DNA (ccfDNA) Reference Materials for Concordance Studies

American Assocation for Cancer Research (AACR) | Chicago, Illinois 2018

Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Katherine Bianco, Karl G. Sylvester, Bharathi Anekella, Russell K. Garlick

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The limited quantities of ccfDNA in plasma and the limited amount of ccfDNA that can therefore be obtained from a single donor can make it difficult to assess the sensitivities and specificities of circulating tumor DNA (ctDNA) assays because there is often insufficient material to confirm the presence or absence of particular mutations by orthogonal assays. Additionally, there have been reports of discordance between different ctDNA assays with patient samples at lower variant allele frequencies (VAFs). The limited and ephemeral nature of patient samples has been a bottleneck to investigating and resolving the causes of discordance. To overcome this, we developed a method to amplify nanograms of ccfDNA from donors to microgram quantities.

In order to evaluate the suitability of amplified ccfDNA for ctDNA concordance and other studies, we amplified ccfDNA from pregnant female donors and assessed reported variants and compatibility with non-invasive prenatal tests (NIPT) and various NGS-based tumor assays. In such samples, the presence of a fetus is expected to contribute some paternal SNPs at lower – but similar – frequencies that can be used to simulate somatic mutations. Additionally, the donors are expected to be free from cancer, so variants present at frequencies below those of the paternal SNPs are either from the sample (e.g., due to clonal hematopoiesis or the amplification method) or introduced by the assay.

In order to assess the impact of the amplification method on the apparent variants in a sample, we split ccfDNA samples into two parts – one for analysis in their native state, and the other for amplification and analysis thereafter.

Development of Well Characterized Breast, Lung, and Brain Cancer Copy Number Variation Reference Materials

American Assocation for Cancer Research (AACR) | Chicago, Illinois 2018

Dana Ruminski Lowe, Jessica Dickens, Catherine Huang, Yves Konigshofer, and Bharathi Anekella

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Copy number variation (CNV) can be a key driver of oncogenesis. Pathogenic CNVs are often associated with unfavorable prognosis and drug resistance. Therefore, detection and characterization of these types of genetic changes are important for personalized treatment. Well-characterized reference materials for CNVs are not widely available, which makes it difficult to assess accuracy and sensitivity of CNV calls made using next-generation sequencing or other methods. With cell lines, the CNV content can be variable and may change with passages, the genomic background may not be diploid, and germline and somatic variants may not be well-characterized. Remnant patient samples generally lack characterization and the volumes needed for large validation studies.

Therefore, we have developed and evaluated Seraseq® CNV reference materials for accuracy, precision, and limit-of-detection (LOD) studies for tumor profiling assay development and validation. By providing a highly consistent source of purified DNA with stable CNV content, these reference materials allow for repeatable assessment of assays targeting CNVs.

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All content © 2019 ArcherDX, Inc.

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