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Objectives Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma with a heterogenous genetic landscape that can require multiple assays to characterize. We reviewed a 1-step RNA-based assay to determine cell of origin (COO), detect translocations, and identify mutations and to assess the role of the assay in diagnosis. Methods Using a single custom Archer FusionPlex Lymphoma panel, we performed anchored multiplex polymerase chain reaction–based RNA sequencing on 41 cases of de novo DLBCL. Each case was subclassified by COO, and gene fusions and hotspot mutations were identified. The findings were then compared with COO classification by the Hans immunohistochemical algorithm and NanoString technology, cytogenetics, and fluorescence in situ hybridization results. Results Concordant COO classification by the FusionPlex panel and NanoString was observed in 35 of 41 cases (85.3%), with NanoString and Hans concordant in 33 of 41 cases (80.5%) and FusionPlex and Hans concordant in 33 of 41 cases (80.5%). The FusionPlex assay also detected 6 of 11 BCL6 translocations (4 cryptic), 2 of 3 BCL2 translocations, and 2 of 4 MYC translocations. Mutations were detected in lymphoma-related genes in 24 of 41 cases. Conclusion This FusionPlex assay offers a single method for COO classification, mutation detection, and identification of important translocations in DLBCL. Although not replacing traditional testing, it could offer useful data when limited tissue is available

Introduction: Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of multiple mutations for characterization. For instance, 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 low frequency mutations is also of growing importance. The ability to accurately detect variants at low allele fractions (AFs) using a single test can be used to assess treatment efficacy and potential relapse. Methods: We developed Archer® VariantPlex® myeloid assays based on Anchored Multiplex PCR (AMP™) to detect important mutations in myeloid malignancies. AMP is a target enrichment strategy that uses molecular-barcoded adapters and gene-specific primers for amplification, permitting open-ended capture of DNA fragments from a single end. This approach enables flexible and strand-specific primer design to provide better coverage of ITD-containing and GC-rich regions. We also developed a method to assess SNV sensitivity taking into account both unique coverage depth and noise for single base substitutions. This strategy enables utilization of position-specific detection thresholds and maximizes sensitivity and specificity. We tested this approach using the VariantPlex® Core Myeloid panel, by titrating reference inputs into background normal samples to examine detection of low AF variants. Results: Our assay enables calling of a 30bp FLT3-ITD down to sub-0.05% allele frequencies. Using optimized low AF conditions improves coverage depth, consistency of low AF FLT3-ITD detection, and sensitivity (98.5% of bases are powered to call a true variant at an allele frequency of 3.0% with 1M reads and 200ng of input). We show >1000X unique molecule coverage across the coding region of CEBPA and use this challenging region to visualize the minimum detectable AF (MDAF) at which a variant has a >95% probability of being detected above the noise (95MDAF). Finally, we show consistent single nucleotide variant (SNV), insertion and deletion (indel), and ITD calling at sub-0.5% allele frequencies, and demonstrate the utility of reporting variant-specific MDAFs and normal dataset P-values when analyzing low AF variants. Conclusion: AMP provides NGS-based detection of complex mutation types that are relevant in AML. We demonstrate robust calling of FLT3-ITDs and other variants at low AFs. We also demonstrate full coverage of CEBPA with high depth and low noise such that the 95MDAF predicts confident variant calling at low AFs. This approach is accurate and scalable, enabling simultaneous detection of multiple mutation types across multiple target genes in a single assay. Citation Format: Verity Johnson, Kaitlyn E. Moore, Laura M. Griffin, Aaron Berlin, Abel Licon, Ryan Walters. Accurate detection of low AF variants relevant to AML by Anchored Multiplex PCR and next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1699.

Introduction Acute myeloid leukemia (AML) oncogenesis is thought to require multiple somatic mutations in a "two-hit" process to 1) increase proliferation and 2) prevent maturation of myeloid cells. While FLT3 and KIT mutations are associated with increased proliferation, NPM1, CEBPA and several other mutations can be associated with maturation inhibition. The most common mutations in AML are internal tandem duplications (ITDs) in FLT3, which are detected in more than 20% of pediatric and adult AML cases and are associated with an aggressive phenotype. As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments. Capillary gel electrophoresis can detect ITDs but cannot be easily coupled with assays to detect other mutation types common in AML. Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types. However, detection of ITDs by NGS is particularly challenging, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference. Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that uses molecular barcoded adaptors and gene-specific primers, permitting open-ended capture of DNA fragments from a single end. We present an approach based on AMP technology and a novel bioinformatics algorithm to detect ITDs in FLT3 as well as other mutation types common in AML. Methods We developed a library preparation assay for NGS to detect FLT3-ITDs as well as other mutations relevant in AML from genomic DNA extracted from clinical samples. We designed AMP probes to cover the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. We further developed a novel de novo sequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs. Results In silico datasets enabled optimization of our sequencing data analysis algorithm, resulting in the detection of over 98% of in silico ITDs with no false positives. The AMP library preparation assay in conjunction with the optimized analysis algorithm enabled sensitive NGS-based detection of ITDs in 16 AML-positive blood samples. These results were consistent with results obtained from standard capillary gel electrophoresis. In addition, point mutations in the TKD of FLT3 and insertions in NPM1 exon 11 were detected in 2/7 and 5/7 FLT3-ITD positive blood samples. Conclusions Our data show that AMP enables accurate NGS-based detection of FLT3-ITDs from clinical DNA samples. As this approach can detect multiple mutation types from a single sample, our AMP library preparation coupled with our NGS analysis algorithm enables simultaneous detection of multiple mutations relevant in AML. Disclosures Van Deusen: ArcherDX, Inc.: Employment. Bessette:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Berlin:ArcherDX, Inc.: Employment. Banos:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Reckase:ArcherDX, Inc.: Employment. Stahl:ArcherDX, Inc.: Employment. Licon:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.

FLT3 internal tandem duplications (ITDs) are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are generally associated with a poor prognosis. 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 have developed a novel targeted assay, the Archer™ VariantPlex™ Core AML Panel, that utilizes Anchored Multiplex PCR (AMP™) with probes in multiple locations proximal to exons 13, 14, and 15 of FLT3. These exons encompass the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. This panel yields high-complexity, dual-strand coverage of known FLT3-ITD locations. Because AMP probes, unlike traditional PCR probes, function independently of each other, we are able to produce multiple overlapping “snapshots” of the region of interest, thereby enhancing the ability to confidently identify complex mutation types. The sequenced reads are assembled using a novel de novo assembly algorithm in the Archer Analysis bioinformatics pipeline, in which the resulting consensus is annotated and events that involve FLT3 exons 13 14 or 15 are marked as FLT3 abnormalities. In order to assess Archer Analysis and the VariantPlex Core AML panel, we tested it on >20 patient-derived samples with known FLT3-ITDs. In addition, we generated over two thousand in silico datasets, representing the spectrum of known ITDs, to further test our panel design and analysis algorithm. Each in silico dataset was constructed to simulate reads originating from probes in VariantPlex Core AML Panel. This methodology permitted rigorous optimization of both the VariantPlex Core AML Panel and the analysis algorithm. The VariantPlex Core AML Panel, in conjunction with our novel detection algorithm, showed both exceptional sensitivity and specificity in the detection of FLT3-ITDs. FLT3-ITDs were successfully identified in all patient samples tested, and no false positives were detected. Our in silico datasets showed similarly high sensitivity and specificity. These data indicate that AMP libraries targeting the FLT3-ITD region are ideal for the detection of this complex mutation type, largely because of the overlapping and anchored read structure. Therefore, the Archer VariantPlex Core AML panel, in conjunction with Archer Analysis represents a reliable platform for the detection of FLT3-ITDs, in addition to other mutations commonly found in AML. Citation Format: Marc Bessette, Benjamin Van Deusen, Michael Banos, Laura Johnson, Aaron Berlin, Erik Reckase, Joshua Stahl, Abel Licon, Brian Kudlow. NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3618.

Introduction: Next-generation sequencing (NGS) has become a critical diagnostic assay to identify pathologic SNVs, CNVs, and gene rearrangements. However, it has not been routinely used to assess expression levels of target genes that could indicate patient populations responsive to therapeutics. This is largely due to the absence of reliable bioinformatics tools for the assessment of expression levels within RNA assays. We have developed a novel within-sample distribution-based method that assesses the relative extremity of expression for individual genes. Our predominate focus has been on assessing the expression of NTRK1, NTRK2, NTRK3, ROS1, and ALK, however this method can be readily applied to other genes and NGS platforms. Methods: Within the kinase domain, the expression of a targeted region is represented by the number of unique deduplicated reads for NGS studies and normalized probe expression values (based on spike-in controls) for NanoString studies. A Poisson distribution is used to represent primer expression with the parameters estimated via maximum likelihood. The interquartile range (IQR) of the entirety of the sample's read counts is calculated and only those that do not exceed the third quartile bound by more than 150% of the IQR are considered during parameter estimation. A probability is then assigned to each of the primers based on their read counts. A geometric mean of the individual primer probabilities represents the probability value for the entire gene. Expression values are reported as -log10 (p-value) and cutoffs of 6 and 1 were used to call significant expression for NGS and NanoString platforms, respectively. Results: The NGS-based approach correctly identified significant expression in all gene rearrangement positive cell lines (n = 11). In cell lines not harboring gene rearrangements, the NGS and NanoString platforms showed 100% concordance in calling significant expression (n = 12). In a gene rearrangement negative FFPE cohort, concordance between NGS and NanoString platforms was 97%-99% for target genes (n = 102). ROS1 and ALK were most commonly found to be significantly expressed in FFPE samples (5% and 2%). Conclusions: We have developed a statistical-based approach to detecting expression levels for RNA-based NGS assays. This can be applied to cohort studies to not only identify clinical samples that may benefit from targeted kinase inhibitor therapeutics but also correlate with predicted outcome of disease. Citation Format: Robert Shoemaker, Aaron Berlin, Amy Diliberto, Heather Ely, Marissa Chen, Danielle Murphy, Jason Christiansen, Vince Reuter, Abel Licon. A novel, statistical-based method to determine RNA expression by next-generation sequencing in clinical FFPE samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5274.