bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3618: NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR
Authors: Bessette Marc , Van Deusen Benjamin , Banos Michael , Johnson Laura , Berlin Aaron , Reckase Erik , Stahl Joshua , Licon Abel , Kudlow Brian 
Published: July 2016

Abstract
FLT3 internal tandem duplications (ITDs) are found in &gt; 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 &gt;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.
Abstract 3623: Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay
Authors: Choi Jonathan 
Published: July 2016

Abstract
Circulating tumor DNA can be found in the plasma of cancer patients, and can be utilized for non-invasive cancer detection and genotyping through Next Generation Sequencing. However, unique characteristics of ctDNA pose technical challenges that limit technical sensitivities and robustness. ctDNA are fragmented, short, rare in abundance, and found in high background of normal DNA from leukocytes. Cancer variants range as low as 0.1-1% minor allele fractions, within the noise of NGS methods. Furthermore, clinical interest includes large number of genes and mutation types including single nucleotide variants, copy number amplification, and fusions. We compared two popular target enrichment categories, digital PCR and a hybridization-based capture panel for target enrichment. We assessed the assay performance and detection limitations, using a diverse sample set to test the range of input material that reflects ctDNA found in plasma. We titrated important cancer mutations (BRAF, EGFR, KRAS, PI3KCA, etc.) testing minor allele fractions ranging from 33% down to 0.1%, MET amplification ranges of 14x to 5x, and CCDC6-RET fusions of 50% to 2%. We also assessed input DNA ranges of 1ng and 10ng, well below manufacturer recommend input of library prep methods. Testing the performance of a 29kb amplicon panel and a 114kb hybridization-probes panel, we found superior on-target rates with digital PCR compared to the hybridization capture method across all samples. However, despite higher depths of coverage with droplet PCR, hybridization based methods had superior read mapping, uniformity (1.4x better), error rate (0.16% vs 0.02%), and greater linearity of SNV variants. Hybridization methods had greater accuracy of variant detection and were able to accurately identify the RET fusion. We demonstrated the feasibility of utilizing target enrichment methods for next generation sequencing for ctDNA variant detection.
Citation Format: Jonathan Choi. Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay. [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 3623.
Abstract 3628: Improving sequencing quality of libraries prepared from FFPE DNA
Authors: liu pingfang , Chen Lixin , Ettwiller Laurence , Sumner Christine , Stewart Fiona J. , Dimalanta Eileen T. , Davis Theodore B. , Thomas Evans C. 
Published: July 2016

Abstract
Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples is currently the main source for NGS-based cancer profiling in clinical settings. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. Artifacts associated with FFPE DNA have limited the mutation detection sensitivity to ≥ 5% mutant allele frequency (Frampton et al, Nature Biotechnology 2013), which would unfortunately leave many low-abundance genetic variants of clinical significance undetected. For example, clinical resistance-causing KIT and EGFR mutations can be present in tumors at levels &lt;&lt; 1% (Milbury et al, Clin. Chem. 2012).
In this study, we investigated the effects of DNA repair and different sample handling workflows on sequencing quality of libraries prepared from FFPE samples. Careful analysis of sequencing data showed that base calling qualities for all 4 bases are improved, and aberrant G:C to A:T mutations were significantly reduced upon DNA repair. Because the large majority of mutations encountered in human tumors are G:C to A:T mutations (Greenman, C. Nature 2007), we expect that lowering the damage induced background noise of FFPE DNA would allow more reliable detection of clinically important, actionable mutations at lower abundance. In addition, we observed specific sequencing artifacts associated with the method of handling FFPE samples and have since identified effective measurements to avoid such artifacts. We expect that these improvements in sequencing quality of FFPE samples would ultimately enable more sensitive and robust detection of many low level genetic variations in clinically and biologically relevant cancer genes.
Citation Format: pingfang liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona J. Stewart, Eileen T. Dimalanta, Theodore B. Davis, Evans C. Thomas. Improving sequencing quality of libraries prepared from FFPE DNA. [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 3628.
Abstract 3629: Gene fusion detection in sarcoma using TRAC technology
Authors: Mattinen Laura , Rautio Jari , Salmivaara Jani , Halleen jussi , Sirkiä Marko 
Published: July 2016

Abstract
Genomic rearrangements are a defining feature of cancer, and can lead to gene fusions and chimeric transcripts with respective fusion proteins that have oncogenic properties. The fusion transcripts can have diagnostic, prognostic and therapeutic implications. However, the fusion partners and the exact breakpoints vary from patient to patient and from cancer type to another. This can result in significant sequence variation, and together with the large number of potential genes able to participate in fusion events makes reliable detection of fusion events challenging for many traditional methods. The TRAC (Transcript Analysis with aid of Affinity Capture) method solves a number of these issues by a combination of specific capture and detection probes. By capturing all known 5’ fusion partners and detecting potential 3’ partners for a given cancer type, only a very limited set of positive signals are detected from an individual sample, enabling high-level of multiplexing. In practice, dozens of different fusion events can be detected per sample by using an easily modifiable pool of capture/detection probes. This effectively enables detection of all known fusion types in sarcomas from one sample without the prerequisite of knowing the exact cancer type. TRAC is a direct hybridization based assay that enables rapid quantification of multiple mRNA targets simultaneously from large number of samples. Transcript levels can be measured directly from cell or tissue lysates without the need for RNA extraction, cDNA conversion or PCR-amplification. The TRAC protocol is straightforward and enables sample analysis in a high-throughput format with little hands-on time. In this analysis, TRAC was used to detect the fusion transcripts COL1A1-PDGFB, FUS-DDIT3, and SS18-SSX1/2 from three types of sarcoma tissue biopsies. All fusion types were detected for the respective targets. In addition, twenty Ewing sarcoma cell lines were analyzed for the EWSR1-FLI1 fusion, identifying 12 cell lines positive for the chimeric transcript. Sarcoma tissue lysate was found applicable for direct TRAC analysis. These results demonstrate that the TRAC Fusion Transcript Assay is functional and capable of discriminating sarcomas based on fusion transcript expression. The assay is easy to set up and it can be run using standard laboratory equipment with little hands-on time.
Citation Format: Laura Mattinen, Jari Rautio, Jani Salmivaara, jussi Halleen, Marko Sirkiä. Gene fusion detection in sarcoma using TRAC technology. [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 3629.
Abstract 3633: OncoGxOneTM - an advanced genomic test for precision cancer treatment
Authors: Han Yang , Yu Pengfei , Song Qingxuan , Wei Min , Hu Guanghui 
Published: July 2016

Abstract
The fundamental cause of cancer is the aberration of genes that control cell proliferation and apoptosis. With the rapid advancement of genomic technologies and increased arsenal of targeted therapies, genomic profiling has been largely accepted by physicians to assist cancer diagnosis and treatment. Developed by Admera Health, OncoGxOneTM is a CLIA certified and CAP accredited genomic profiling panel detecting all actionable mutations in 64 cancer genes. All the FDA approved targeted therapies and registered clinical trials are covered by this carefully designed panel. Utilizing cutting edge Next Generation Sequencing technology combined with accurate analysis and data interpretation, OncoGxOneTM detects all 4 types of genomic alterations including single nucleotide mutation, insertion/deletion, copy number variation and gene rearrangement. Using fresh frozen tissue or routine FFPE samples, OncoGxOne TM offers both high sensitivity and specificity greater than 99% from samples with as low as 10% tumor cell content.
Citation Format: Yang Han, Pengfei Yu, Qingxuan Song, Min Wei, Guanghui Hu. OncoGxOneTM - an advanced genomic test for precision cancer treatment. [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 3633.
Abstract 3636: Improved clinical variant calling and HLA genotyping with GRCh38
Authors: Chapman Brad , Meynert Alison , Church Deanna , Johnson Justin , Hofmann Oliver 
Published: July 2016

Abstract
The Genome Reference Consortium released human genome build 38 (hg38, GRCh38) two years ago and it offers major improvements over the six year old previous build, 37 (hg19, GRCh37). The new build reflects our increased understanding of the heterogeneity within human sub-populations, contains a large number of alternative genomic loci that better capture our knowledge of genome structure, and includes highly diverse human leukocyte antigen (HLA) regions enabling HLA typing.
To help with the migration of clinical sequencing workflows we provide support for variant calling on build 38, validate methods using truth sets from the Genome in a Bottle consortium and Illumina's Platinum Genome and show that feature projection approaches are a viable intermediate approach to port existing annotation resources. We show how the refined genomic representation improves mapping and variant calling by reducing the number of false positive SNP calls and improving InDel detection, and how post-processing of sequence reads mapping to HLA regions enables accurate HLA genotyping from targeted panels, exome- and WGS data. Using Omixon HLA validation data, integrated OptiType HLA calling achieved 100% accuracy on 8 targeted panels and better than 90% accuracy on 1000 genome exome samples.
This work provides a freely available, validated, ready to run pipeline making use of the improved heterogeneity representation in build 38 and demonstrating the value of moving towards a more accurate, graph-based representation of human genomes.
Citation Format: Brad Chapman, Alison Meynert, Deanna Church, Justin Johnson, Oliver Hofmann. Improved clinical variant calling and HLA genotyping with GRCh38. [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 3636.
Abstract 3644: Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification
Authors: Thorner Aaron R. , Ziaugra Liuda , Ducar Matthew D. , Lin Ling , Laing Angelica , Coleman Haley A. , McShane Suzanne R. , Clapp Andrea , Paquette Rachel R. , Wollison Bruce M. , Hoeftberger Johann , Patel Neil A. , Hunter Samuel S. , Manam Monica D. , MacConaill Laura E. , Hahn William C. , Meyerson Matthew L. , van Hummelen. Paul 
Published: July 2016

Abstract
Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis.
Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged.
Citation Format: Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification. [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 3644.
Abstract 3645: Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples
Authors: Gerstner Arpad , Schreiber Edgar , Jackson Stephen , Varma Kamini 
Published: July 2016

Abstract
Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging.
To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes implicated in tumorigenesis (e.g. TP53, KRAS and NRAS). We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations in Sanger sequencing studies. To demonstrate the workflow of these panels with MVF, we analyzed DNA from lung cancer FFPE samples. We initially determined variants of TP53 and KRAS in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing (NGS). We confirmed the identity and variant allele frequency of these variants by Sanger sequencing coupled with MVF. Furthermore, we were able to confirm these results in 1 ng, 0.5 ng or 0.1 ng of DNA from these samples. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD). We show that this workflow can detect as little as 3% of a minor variant in an FFPE sample.
Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available.
For Research Use only - Not for use in diagnostic procedures.
Citation Format: Arpad Gerstner, Edgar Schreiber, Stephen Jackson, Kamini Varma. Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (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 3645.
Abstract 3646: Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing
Authors: Kujawa Steve , Sethuraman Anand , Eng Kevin , Baybayan Primo , Heyrman Lien , Del Favero Jurgen 
Published: July 2016

Abstract
Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust but also capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT®) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (&gt;QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Diagnostics, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay.
First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. We also sequenced a second sample containing a series of mixes, each with known mutations, at levels below 10% and down to 0.01%. PCR-amplified targets were sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.
Citation Format: Steve Kujawa, Anand Sethuraman, Kevin Eng, Primo Baybayan, Lien Heyrman, Jurgen Del Favero. Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing. [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 3646.