Aleksandra Ras

332 The Jackson Laboratory for Genomic Medicine ActionSeq Plus Offering Provides a Comprehensive Analysis of Cancer Diagnoses

technologists tasked with running the assay. The benefits and value of building a custom LIMS is no doubt appealing, but the challenges and risks faced while accomplishing that goal proved to be a difficult endeavor. Our clinical genomics laboratory was granted the opportunity to construct a custom LIMS to be utilized for whole exome sequencing workflows. With careful consideration of the cost and time required to develop a custom software tool that needs to mimic our current procedures, our team quickly set forth to develop a project plan. Unlike many “out of the box” systems, our development would require specific insight and technical configurations. Our team arranged workshops with both laboratory technicians and software developers to gather user requirements. We vigilantly managed time as to not disrupt ongoing clinical work. Ultimately, despite setbacks and pivots, we accomplished our mission. Due to intense project planning, multi-team communication, a clear vision, and, most importantly, user input, we created and implemented a robust automated LIMS without disrupting our clinical workflow. At the conclusion of this project, much was learned. It is critical to avoid “scope-creep” (the tendency for project goals to grow beyond the original vision). For the sake of time, implement a minimal viable product, test it, release it, and then iterate on later versions. Budget for both cost and personnel bandwidth. Understand risks, and be resourceful enough to adapt to changing goals.

Abstract 757: Development and validation of the ActionSeqTMtest system

Introduction In the constantly changing field of oncology precision medicine, it is exceedingly important to keep diagnostic and therapeutic assays clinically relevant. Next generation sequencing (NGS) panels in oncology are greatly impacted by new findings in clinical actionability. In order to ensure that cancer panels continue to provide the most beneficial results to patients, they must be regularly updated. In keeping with this idea, JAX has launched a new 212 oncology gene panel which focuses on genes and variants with documented actionability, referred to as ActionSeq. Methods Development of ActionSeq included the optimization of a new targeted capture assay. This process included running multiple batches of samples through the assay to determine appropriate DNA input, ligation times, PCR cycles, and pooling conditions. The fully optimized assay was then validated using 24 uncharacterized FFPE samples. The validation was executed in 5 phases: (1) confirm that assay optimizations yielded sufficient wet lab results; (2) LOD & sensitivity (3) inter-assay concordance; (4) intra-assay concordance; (5) specificity and accuracy. Results During development, the standard protocol was optimized using a 200ng input, 30 minute ligation period, 5 cycles of pre-PCR, and the pooling of 4 samples per hybridization reaction. Wet lab processing results of the first validation batch can be seen in Table 1. The inter- and intra-assay concordances were found to be ≥ 96% for variants and 100% for CNVs. The sensitivity was calculated to be 98.92% at a LOD of 3% for SNVs, 100% at a LOD of 8% for INDELs, 100% at a LOD of 6 copies for CNV amplifications, and 100% at a LOD of 0 copies for CNV deletions. The specificity and accuracy were found to be 100% for all mutation types. Conclusion Based on the success of this validation ActionSeq has been incorporated into the JAX clinical test menu. This addition accomplished the goal of providing a more clinically relevant (actionable) somatic tumor profiling assay to patients and clinicians. Citation Format: Samantha Helm, Vanessa Spotlow, Aleksandra Ras, Kevin Kelly, Guruprasad Ananda, Sara Patterson, Honey V. Reddi. Development and validation of the ActionSeq TM test system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2017-757

Simultaneous extraction of DNA and total RNA from varying specimen types to enhance tissue utilization for molecular analysis.

e23209Background: The isolation of both genomic DNA (gDNA) and total RNA (RNA) from tissues of various types is critical for comprehensive molecular profiling, especially as submitted specimens bec...

Abstract 3634: Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method: Table 1.

Introduction: The purification of genomic DNA (gDNA) and total RNA (RNA) from various specimen types using a single sample is currently in high demand in genomic settings due to the limitation of available biological material. There is an increased need for simultaneous isolation of nucleic acids in cancer genetics for use in downstream applications such as genotyping, microarrays or next generation sequencing. The Qiagen AllPrep® technique provides a comprehensive solution for extraction of gDNA and RNA from a single specimen. In this study, we tested the AllPrep® kits for concurrent isolation of gDNA and RNA from five different sample types. Materials and Methods: Genomic DNA and RNA were extracted simultaneously according to Qiagen AllPrep® manufacturer9s instructions from five specimen types: bone marrow mononuclear cells (BMMC), cryopreserved cultured cell lines, whole blood, fresh frozen and FFPE samples. Quantity and quality of both nucleic acids were assessed by the NanoDrop 2000 and Qubit fluorometer 2.0. Purified gDNA from BMMC, fresh frozen, whole blood and FFPE samples was further evaluated by library preparation, targeted hybridization capture and sequencing on an Illumina NextSeq500. FastQ files were submitted to a Clinical Genome Analytics (CGA) pipeline for quality metrics analysis. Results: The AllPrep® technique resulted in effective isolation of high quality gDNA and RNA (Table1). All set wet lab and sequencing QC metrics were met for each sample type. Conclusion: We have successfully verified the AllPrep® method can be used for simultaneous extraction of both gDNA and RNA using the same input material without compromising the yield or purity of both final products. This simple procedure based on spin column technology not only gave the advantage of generating high quality results using limited specimen resources, but also decreased the hands on time by 3.5 hours and cost when compared to extraction done with two separate kits. Citation Format: Aleksandra Ras, Samantha Helm, Kevin Kelly, Vanessa Spotlow, Guruprasad Ananda, Gregory Tsongalis. Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method. [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 3634.

Abstract 3630: Validation of the Archer FusionPlex solid tumor panel in the JAX cancer treatment profileTM

Introduction: A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the clinical utility of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment Profile TM (JAX-CTP TM ) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay. Methods: AFPSTP was validated using 24 samples: 5 JAX Patient Derived Xenograft (PDX) cases, 4 translocation positive controls, 2 FFPE cancer samples, 1 normal tissue sample, and 12 cell lines. Nine of the cell lines were previously identified as positive for fusion transcripts and 3 lacked detectable fusion events. The validation was executed in 5 phases: (1) confirm that AFPSTP was able to detect known fusion or lack of fusion events in characterized specimens; (2) determine inter-assay concordance; (3) determine intra-assay concordance; (4) LOD and (5) sensitivity. Results: The fusion detection results for this validation are listed in Table 1. All but one of these fusion events was previously identified. The one novel fusion was confirmed using TaqMan RT-PCR. In addition to the expected fusions, 4 false positive events were detected, 2 due to mispriming and 2 determined to be WT read through transcripts. The fusion detection inter and intra-assay concordance was found to be 100% and the sensitivity was calculated to be 91.67% at a LOD of 5%. Conclusion: This analysis outlines the clinical validation of the incorporation of AFPSTP into the JAX-CTP TM test system. Once incorporated, the AFPSTP assay will accomplish the goal of making JAX-CTP TM a more comprehensive somatic tumor profiling assay without affecting the current acceptable turnaround time or required input material. Citation Format: Samantha Helm, Aleksandra Ras, Vanessa Spotlow, Kevin Kelly, Susan Mockus, Cara Statz, Guruprasad Ananda, Joan Malcolm, Gregory J. Tsongalis. Validation of the Archer FusionPlex solid tumor panel in the JAX cancer treatment profile TM . [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 3630.