Jian Gu Research Articles

Chemical and Microbial Differences of Root and Rhizosphere Soil among Different Provenances of Fokienia hodginsii

Aims: Fokienia hodginsii is a threatened conifer tree species, known as the dominant nursery-grown species capable of colonizing the challenging woodland environments in southern China due to its strong root penetrating ability. The ecological phenotype of Fokienia hodginsii is not well documented during its breeding process, which limits the potential planting area and its ecological function. This study aims to understand how Fokienia hodginsii associates with microbes to conduct its key ecological function and provide a theoretical basis for further improving the forest nursery management of Fokienia hodginsii. Methods: This study explored the ecological traits of 11 main Fokienia hodginsii provenances in a homogeneous garden experiment by analyzing their nutrient utilization strategies and associated microbial features in the rhizosphere soil and roots. Results: The study found that the paramount difference in the rhizosphere soil among provenances is in Ca and Fe content. Some microbial communities, namely Crenarchaeota, Verrucomicrobiota, and Desulfobacterota, were positively correlated with the amounts of the soil nutrient elements, whereas Abditibacteriota and Dependentiae were negatively correlated. The abundance of N- and Fe-related bacteria in the Fu Jian Chang Ting (FJCT) provenance was significantly higher than that in other provenances, while the C-, P-, K-, and Mg-related fungal communities, respectively, had higher abundances in the FJCT, Fu Jian Long Yan (FJLY), Fu Jian Gu Tian (FJGT), and Fu Jian Xian You (FJXY) provenances than the others. The impacts of the Gui Zhou Li Ping (GZLP), Hu Nan Dao Xian (HNDX), Jiang Xi Shang Yao (JXSY), and Guang Dong Shi Xing (GDSX) provenances on the rhizosphere soil are similar, but the differences in nutrient utilization arise from the plant itself. Conversely, the root nutrient contents of the FJCT, Fu Jian You Xi (FJYX), Fu Jian An Xi (FJAX), FJLY, Fu Jian De Hua (FJDH), FJGT, and FJXY provenances are highly correlated with soil nutrient features. Conclusions: For the native provenances, their economic traits are better than the exotic provenances. The native provenances are more sensitive to local soil conditions, so they should benefit more from human interventions, rendering them more suitable for artificial cultivation. The growth of the exotic provenances is less affected by the soil environment, making them better suited for the ecological transformation of forest stands and soil improvement.

Abstract 3671: Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants

Abstract For in vitro Diagnostic use only. Not available in all regions including the United States. Introduction Minimally invasive blood-based liquid biopsy using cell-free total nucleic acid (cfTNA) and next generation sequencing (NGS) has substantially evolved in the field of clinical diagnostics for oncology, for detection of molecular therapeutic targets in non-small cell lung cancer (NSCLC). Here we describe analytical validation results for cfTNA using the CE-IVD Oncomine™ Dx Express Test (ODxET) assay, a qualitative in vitro diagnostic test that uses targeted NGS technology, the Ion Torrent Genexus™ Dx Integrated Sequencer to detect SNV, indel and copy number gain present in 42 genes and fusions in 7 genes from cfTNA isolated from NSCLC plasma samples. Methods Plasma samples from NSCLC patients were screened for variants of interest on several important gene loci. Screening involved isolation of cfTNA from K2-EDTA-derived plasma using the Genexus™ Cell-Free Total Nucleic Acid Purification kit on an automated purification system and sequencing was performed on the Genexus™ Dx integrated sequencer using the ODxET assay. Plasma samples having clinically relevant variants from the set of screened plasma samples were used to perform the validation studies. Results Analytical sensitivity and specificity of NSCLC plasma samples were characterized through determination of limit of detection (LOD) and limit of blank (LOB), respectively. The LOD level tested for DNA SNVs, and indels at 5 ng (minimum) input level ranged from 0.62% to 1.82% allelic frequency (AF), while 30 ng (maximum) input shows the LOD level ranging from 0.23% to 0.42% AF. The LOD for the tested RNA fusions ranged from 4.2 to 19.6 molecular counts for 5ng input and 4.9 to 8.0 molecular counts for 30ng input. Furthermore, LOB for the ODxET assay was determined by testing wild type (WT) cfTNA extracted from 30 healthy donor blood plasma samples, which were confirmed to be negative at all variant locations. The analytical accuracy study shows the false positive rate at 0.20% for SNVs, 0% for indel locations and fusion targets. In addition, the analytical reproducibility study shows 99.64% intra-run repeatability call rate for DNA variants and 98.75% for RNA variants. Conclusion In conclusion, we establish that the ODxET assay on Ion Torrent Genexus Dx Integrated Sequencer, is fast and efficient while demonstrating high sensitivity, specificity, and reproducibility for testing NSCLC liquid biopsy samples. Citation Format: Madhuri Jasti, Swasti Raut, Diarra Hassell, Nader Ezzedine, Daniela Garcia, Stephen Wunsch, Nick Siepert, Elliott Martinez, Emilia Ostrowska, Jeff Schagemann, Jian Gu, Thilanka Jayaweera, Luming He, Angie Cheng. Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3671.

Quepen() and related issues

Based on the features of the Chinese constellation Kuixiu() as seen in archaeological and documentary historical materials and the fact that it is also known as Quepen (), the reason why the pelvis was named Quepen was discussed. To verify the relationship between Jian gu (,Clavicle and shoulder blades) and Quepen on the basis of Kui in qinjian rishu corresponds to the term Jian shang () on the Divination Birth Chart() in bamboo strips of Former Han Dynasty collected byPeking University.Taking the Huangdi Neijing and other relevant documents as clues, the reason of "quepen" may refer to the jugular notch is clarifid.Taking the two acupoints of "quepen" and "Tianshu" as examples, it reveals the habit of the ancients to use the names of human body parts and transfer them to the names of acupoints.

Abstract 5601: Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation

Abstract Introduction - Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine™ Dx Express Test (ODxET) and Genexus™ Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods - Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus™ Dx Integrated Sequencer according to the user guide. Results - The Genexus™ Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusion - Here, we demonstrated that the ODxET workflow on Genexus™ Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity. For in vitro diagnostic use. Not available in all regions including the United States. Citation Format: Nicholas Siepert, Jeoffrey Schageman, Jian Gu, Thilanka Jayaweera, Madhu Jasti, Luming He, Stephen Wunch, Emilia Ostrowska, Tasha Delacour, Diarra Hassell, Thomas Bowden, Daniela Garcia, Stephanie Tong, Swasti Raut, Nader Ezzedine, Elliott Martinez, Kelli Bramlett. Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5601.

Abstract 195: A focused ultrasound blood brain barrier disruption model to test the influence of tight junction genes to treat brain tumors

Abstract A major hindrance to advances in the care of patients with malignant gliomas is the presence of the blood brain barrier (BBB) and blood-brain tumor barrier (BBTB) that greatly restricts drug access from the plasma to the tumor cells. Bubble-assisted Focused Ultrasound (BAFUS) has proven effective in opening the BBB for treatment of glial tumors in adults and pediatric cases. BAFUS has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. However, there is a lack of an in vitro preclinical model suitable for testing the genetic determinants of endothelial cell tight junction integrity and vulnerability to the physical disruption. Our BBB organ-on-chip platform will enable precision medicine of brain cancers through identifying patient-specific parameters by which to open the BBB allowing use of drugs and drug combinations otherwise unsuitable. We intend to sequence these in vitro models to verify that the genotype (alleles/SNPs) of tight junction proteins contribute to BBB structure and integrity. To initiate this effort, we report the development of an ultrasound transparent organ-on-chip model populated by iPSC-derived endothelial cells (iPSC-EC) co-cultured with astrocytes. Western blot, immunocytochemistry, permeability, and transelectrical endothelial resistance (TEER) studies all convey expression of key EC proteins and marked barrier integrity. Further benchmarking of device-ultrasound interactions, successful iPSC differentiation, tight junction formation, and fabrication of nanobubbles and their assistance in ultrasound BBB disruption will be presented. Efforts are underway to analyze nine characteristic BBB tight junction genes from WGS data to determine associations between iPSC-EC genotype and phenotype. Citation Format: Jayashree Iyer, Adam Akkad, Nanyun Tang, Sen Peng, Michael Berens, Frederic Zenhausern, Jian Gu. A focused ultrasound blood brain barrier disruption model to test the influence of tight junction genes to treat brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 195.

Abstract 78: Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus࣪ System

Abstract Introduction: Gene fusions and exon skipping events play an important oncogenic role in non-small cell lung cancer (NSCLC). Here we employed the Oncomine Precision Assay (OPA) for sequencing of 998 clinical research FFPE (Formalin-Fixed Paraffin-Embedded) lung samples using the Genexus࣪ integrated sequencing platform. The RNA assay strategy is aimed at providing a wide scope for studying known oncogenic fusions and exon skip variants, as well as a method for detection of novel fusion combinations and detection of fusions in a partner agnostic manner. We summarize the findings that include detected samples with oncogenic fusions in tyrosine kinase genes ALK, RET, ROS1 as well as MET exon14 skipping, and demonstrate the novel fusion detection capabilities of the panel. Methods: The Oncomine Precision Assay panel developed using the Ion AmpliSeq HD technology for use on both tissue and liquid biopsy samples to detect fusions with high sensitivity in low input RNA. The panel features 981 known fusion isoforms in 16 oncogenic drivers as well as assays for exon skipping and deletion in MET and EGFR. The amplicons are strategically designed around known fusion breakpoints and generate reads only if the variant is present. Results are assessed bioinformatically with a framework that includes detailed genomic annotations of the fusion breakpoint, and generates interpretable fusion calls and report in the Genxus࣪ software. In addition, the panel contains an algorithm for novel fusion detection, and 78 exon-junction amplicons for partner agnostic fusion detection in ALK, RET, NTRK1,2,3 and FGFR1,2,3 with an exon tiling expression imbalance assay. We used the Genexus࣪ instrument to sequence 998 unique lung FFPE samples and analyzed the results with the Genexus࣪ fusion analysis workflow. Results: A total of 998 unique lung FFPEs were sequenced, of which 906 (91%) resulted with >5000 aligned reads and at least 5 of the 7 RNA expression controls at the assay threshold. A total of 25 samples with ALK fusions were detected by either the targeted fusions or the expression imbalance assays. In addition, we detected 10 samples with RET fusions, 4 with FGFR3 fusions, 3 with ROS1 fusions, and additional single observations of NRG1, RSPO3 and FGFR1 fusions. Exon 14 skipping in MET was detected in 14 samples, of which we were able to detect a likely splice site variant SNV or indel at, or near the boundaries of exon 14, with the DNA assays of the panel. These variants were observed in a mutually exclusive set of 59 total samples (6.5% of the samples). Conclusions: We used the OPA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion, and demonstrated known and novel fusion detection as well exon skip variant detection capabilities in a research cohort of lung FFPE samples. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Jeoffrey Schageman, Jian Gu, Stephen Wunsch, David Chi, Paul D. williams, Scott P. Myrand, Fiona C. Hyland, Seth Sadis, Kelli S. Bramlett. Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus࣪ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 78.

Abstract 2939: Automation meets reliability: Use of Oncomine࣪precision assay on the Genexus࣪system for accurate identification of cancer biomarkers in FFPE and liquid biopsy samples

Abstract Accurate and early detection of oncogenic markers may one day be the key to fighting cancer. However, with complex workflows, long turnaround times, and numerous user touch points of most sequencing platforms does not make the process attainable. In contrast, the fully automated Genexus࣪ system provides a specimen-to-report workflow for cancer research with minimal user touchpoints and single day turnaround time. FFPE (formalin-fixed paraffin embedded) tissues and liquid biopsy are two of the main sample types used in oncology research. Here we report the use of the Oncomine࣪ Precision Assay (OPA) with the Genexus࣪ System which provides a comprehensive genetic profile across 50 key genes using DNA and RNA from FFPE tissues or cfTNA (cell free total nucleic acid) from liquid biopsy samples. To evaluate the nucleic acid (NA) extraction and sequencing performance of the Genexus࣪ system, contrived control samples with known variants and clinical research samples were used in the OPA FFPE and liquid biopsy workflows (n = 30). NA was quantified using the Genexus࣪ purification instrument’s onboard Qubit࣪ quantitation feature after purification. Output plates with extracted NA were transferred to the Genexus࣪ Integrated Sequencer for library preparation and sequencing using the OPA assay. Data were analyzed using the Ion torrent Genexus࣪ software to evaluate assay performance and variant calling.The OPA assay only requires 10ng of DNA and RNA from FFPE samples and 20ng of cfTNA from liquid biopsy samples. Genexus࣪ purification instrument onboard quantitation data showed successful extraction of NA exceeding the required yields for library preparation. Excess NA was automatically aliquoted into an archive plate and stored for future use. Sequencing results for four samples of FFPE or liquid biopsy were reported within 24 hours. Both Control and clinical research samples showed expected assay metrices including read coverage, molecular coverage, and uniformity. The results reported all expected variants at correct allele frequencies, including BRAF V600E, KRAS G12C, PIK3CA N345K, AKT1 etc. Overall, this study demonstrates that the Genexus࣪ system provides a user-friendly workflow with automated NA purification, quantitation, sample dilution, library preparation, sequencing, and data analysis with minimal hands-on time that can be performed with limited expertise to obtain results within 24 hours. Reliable identification of variants from control and clinical research samples of FFPE and liquid biopsy origin with optimal assay metrics demonstrates the successful use of the OPA assay and Genexus࣪ system that can be confidently used in clinical oncology research.(For Research Use Only. Not for use in diagnostic procedures.) Citation Format: Thilanka Jayaweera, Nicholas Siepert, Brian Hradecky, Emilia Ostrowska, Iris Casuga, kristy Lea, Priyanka Kshatriya, Jian Gu, Jeff Schageman, Ru Cao, Angie Cheng, Kelli Bramlett. Automation meets reliability: Use of Oncomine࣪precision assay on the Genexus࣪system for accurate identification of cancer biomarkers in FFPE and liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2939.

Systemic therapies for preventing or treating aromatase inhibitor-induced musculoskeletal symptoms in early breast cancer.

Systemic therapies for preventing or treating aromatase inhibitor-induced musculoskeletal symptoms in early breast cancer.

Abstract 2984: Building an in vitro blood brain barrier model to test the influence of tight junction gene alleles on disruption by focused ultrasound to treat brain tumors

Abstract Treating primary or metastatic tumors in the brain (glioblastomas, melanoma, lung cancer, breast cancer) proves challenging by virtue of the protective function of the blood brain barrier (BBB). Recently, it has been shown that low intensity focused ultrasonic (LIFU) waves stably cavitate infused microbubbles which then mechanically disrupt the tight junctions of the BBB. This leads to temporary, recoverable opening of the BBB, and passage of otherwise disqualified cancer-therapeutic drugs at precise locations targeted by the focused ultrasound. To date, potential genetic influences on the durability and vulnerability of tight junctions to LIFU have not been elucidated, nor have the determinants of tight junction repair post LIFU been thoroughly investigated. We report the development of an ultrasound transparent organ-on-chip model to test LIFU with microbubble infusion treatment on a cell-engineered BBB. The BBB is developed using brain-specific endothelial cells derived from genomically characterized immortalized pluripotent stem cells (iPSC). Furthermore, to test genetic variation effects we propose that alleles coding for the proteins involved in tight junction assembly contribute to LIFU disruption variability. Developing preclinical models of the BBB to accommodate cell sources with tight junction genes of different allele makeup will shed light on how individuals will respond to different ultrasound frequencies. The in vitro BBB device is composed of two orthogonally stacked fluidic channels formed by top and bottom polydimethylsiloxane (PDMS) membranes and a middle polyester membrane with 3 µm pores. An ultrasound system is constructed with a waveform generator, amplifier, and 1MHz ultrasound transducer. A 0.5 MHz receiving transducer and a digital storage oscilloscope are used for stable cavitation monitoring. Nanobubbles (FUS Instruments) transduce the LIFU into a mechanical vibration force to disrupt the BBB. To deliver the ultrasonic waves, the device is submerged in degassed DI water in a custom tank. Fluid flow was achieved, and subharmonic ultrasound signal is observed using the digital oscilloscope with Fast Fourier Transform (FFT). Preliminary results convey stable cavitation with LIFU and the formation of tight junctions in a brain microvascular endothelial cell monolayer in the device, eventually leading to a versatile platform to evaluate genetic-based vulnerability of the BBB. Citation Format: Jayashree Iyer, Adam Akkad, Nanyun Tang, Michael E. Berens, Frederic Zenhausern, Jian Gu. Building an in vitro blood brain barrier model to test the influence of tight junction gene alleles on disruption by focused ultrasound to treat brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2984.

Abstract 213: Demonstration of the genexus integrated sequencing system with the oncomine precision assay

Abstract Introduction: We describe the development and performance of a new sample-to-report targeted sequencing solution for testing solid tissue cancers using the Genexus Integrated Sequencing System and accompanying software. The assay is designed for research applications from either formalin fixed paraffin embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy samples. The Genexus Integrated Sequencer is a fully automated system requiring minimal touch points and hands on time allowing a novice user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types in less than two days. Methods: The Oncomine Precision Assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biological variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. With about 15 minutes of hands on time, a run is set-up using pre-filled reagent strips for a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: Reported here are the results generated from an early external test site along with development data. The Oncomine Precision Assay is designed to detect somatic variants in 50 unique genes testing all major variant types important in the oncology research. The content was selected based on published accounts of target actionability and prevalence across multiple cancer types. All major variant types are targeted including SNVs, insertions, deletions, copy number variation, fusion transcripts and alternate splice forms. Data is shared from an external test lab using the Oncomine Precision Assay on the Genexus Integrated Sequencer with control and research samples. Results from both multiplexed FFPE and liquid biopsy runs are presented. Data demonstrates use of a single assay and system to effectively call variants from both FFPE and liquid biopsy sample types with a turn-around time of less than 30 hours. Conclusion: The Oncomine Precision Assay and Genexus Integrated Sequencer enable detection of key oncology variants in 50 genes using either solid tissue or liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in less than 2 days with minimal hands-on time and touch-points from the user. Many features of the automated system increase success rates by ensuring the appropriate reagents are properly installed before a run. The user friendly, highly automated, and fully optimized sample to answer system described here has great potential for targeted oncology sequencing in the research setting. Citation Format: Jian Gu, Ru Cao, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Varun Bagai, Khalid Hanif, Jose L. Costa, Kelli Bramlett. Demonstration of the genexus integrated sequencing system with the oncomine precision assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 213.

Abstract 28: Integrating genomic and transcriptomic data to identify genetic loci associated with breast cancer risk in women of African ancestry

Abstract Background: To date, most genome-wide association studies (GWAS) of breast cancer have been conducted only among women of Asian and European ancestry. It is difficult to generalize results from those studies to women of African ancestry (AA). We conducted a large genetic association study of breast cancer in women of AA by analyzing both genetic and transcriptomic data. Methods: This collaborative study included 11,073 cases and 11,095 controls of AA who were participants in more than 15 studies conducted in the U.S. and Africa. Genotyping data were harmonized and imputed using the 1000 Genomes Project database as the reference. Imputed genotypes were used for GWAS to identify novel genetic risk loci for breast cancer. To search for susceptibility genes, we conducted a transcriptome-wide association study (TWAS), in which gene expression prediction models were built using genetic and tumor tissue RNA sequencing data from ~400 AA patients and used to impute expression levels of genes across the transcriptome for association analyses in all cases and controls included in the GWAS mentioned above. Results: We identified five loci (5p15.33, 5q31.3, 10q26.13, 18q12.1, and 19p13.11) associated with breast cancer risk at P < 5 × 10−8, including a novel locus at 5q31.3 (allelic odds ratio, OR = 1.18, 95% CI = 1.11-1.25, P = 4.65 × 10−8, nearby gene, ARHGAP26). This locus was also identified in association with estrogen receptor (ER) positive breast cancer at P < 5 × 10−8. Analyses stratified by ER status replicated known loci associated specifically with ER-positive (10q26.13) or ER-negative (2q14.2, 2p11.2, 5p15.33) breast cancer at P < 5 × 10−8. Of the 165 lead risk SNPs reported from previous breast cancer GWAS, 35 SNPs were replicated with the same association direction at P < 0.05. We constructed a polygenic risk score using these 35 replicated SNPs and the lead risk SNP at the novel locus and estimated the AUC to be 0.575. Of the 7,592 genes tested in the TWAS, we identified one gene, AC091053.1, with an association at a Bonferroni-corrected threshold of 6.64 × 10−6 (0.05/7,592). AC091053.1 is a long non-coding RNA gene at locus 11p15.4, where no risk variants have been identified in any previous breast cancer GWAS. AC091053.1 is located in the region of protein coding gene DENND2B, which acts as a regulator of MAPK1/ERK2 kinase and reduces the tumorigenic phenotype in cells. The gene AC091053.1 was associated with ER-positive breast cancer with P = 4.11 × 10−5 and ER-negative breast cancer with P = 0.032. Conclusions: Our study, the largest genetic study conducted to date in AA, identified novel breast cancer risk loci at 5q31.3 and 11p15.4 (AC091053.1) among women of AA and replicated >30 associations reported in previous studies. Studies with a larger sample size are needed to further investigate genetic variants and genes associated with breast cancer risk in AA women. Citation Format: Guochong Jia, Jie Ping, Yaohua Yang, Maureen Sanderson, Qiuyin Cai, Xingyi Guo, William J. Blot, Bingshan Li, Elisa V. Bandera, Manjeet K. Bolla, Montserrat García-Closas, Douglas F. Easton, Mary K. Fadden, Jian Gu, Dezheng Huo, Esther M. John, Kathryn L. Lunetta, Olufunmilayo I. Olopade, Xiang Shu, Melissa A. Troester, Song Yao, Breast Cancer Association Consortium, Andrew F. Olshan, Christine B. Ambrosone, Christopher A. Haiman, Jirong Long, Julie R. Palmer, Wei Zheng. Integrating genomic and transcriptomic data to identify genetic loci associated with breast cancer risk in women of African ancestry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 28.

Abstract 3118: Next generation sequencing assay for detection of gene fusions and exon deletion events in tissue and liquid biopsy samples at very low frequency

Abstract Introduction: Gene fusions caused by chromosomal rearrangements and the exon deletion events caused by aberrant RNA splicing events play a key role in oncogenesis and the progression of cancer. Next generation sequencing using molecular tagged AmpliSeq HD chemistry enables highly sensitive variant detection down to <=0.1% mutant allele fractions. Here we present the Oncomine Precision RNA Assay developed using the Ion AmpliSeq HD Technology for use on both tissue and liquid biopsy samples to detect fusion and exon deletions with very high sensitivity. The assay also includes capabilities for novel fusion detection in driver genes via exon level expression measurements. Methods: The Oncomine Precision RNA assay can detect >900 known fusion isoforms including those of ALK, ROS1, RET, NTRK1,2,3, FGFR1,2,3, and BRAF and exon deletion events in MET and EGFR genes. The assay also includes amplicons in 8 key driver genes like NTRK1,2,3 to detect novel gene fusions in a partner agnostic manner based on the expression imbalance. The amplicons to detect targeted isoforms are designed strategically around the known break-points of the targeted fusions and exon skipping events. These amplicons generate reads when the target variant is present in the sample and provide complete information of the fusion event including the precise break-point with annotations such as cosmic ID. The unique molecular tags attached on both ends of the reads are used for error correction and this enables us to detect fusions at a very low frequency with high accuracy which is critical in liquid biopsy samples . The assay also includes amplicons to measure the wild-type expression of MET and EGFR genes and the exon deletion events are detected by comparing the expression of the exon-deletion transcript to that of the wild-type transcript. Results: To test the panel, we used the GenexusTM sequencer to sequence ALK, ROS1, RET,FGFR3 and NTRK1 fusion positive cell lines and correctly identified all the targeted fusions and concordant results with expression imbalance. We sequenced cell-free total nucleic acid(cfTNA) control samples and identified the expected fusions in ALK, RET and ROS1 genes and the MET Exon 14 skipping event. We sequenced 16 FFPE samples with known truth and correctly identified all the 11 ALK and 5 ROS1 fusion isoforms and observed highly concordant results with the expression imbalance in the samples positive for ALK fusion. We sequenced the ALK, RET, NTRK1 and FGFR3 cell lines diluted to 2%, 2%, 5% and 20% respectively with the background of wild-type RNA and detected the expected fusions and observed concordant results with the expression imbalance. Conclusions: This targeted sequencing assay enables researchers to detect gene fusions and exon deletions in both FFPE and blood samples with >99% sensitivity and high specificity. For research use only. Not for use in diagnostic procedures. Citation Format: Rajesh K. Gottimukkala, Fiona C. Hyland, Amir Marcovitz, Jeoffrey Schageman, Varun Bagai, Ru Cao, Paul D. Williams, Scott P. Myrand, Jian Gu, Seth Sadis, Kelli S. Bramlett. Next generation sequencing assay for detection of gene fusions and exon deletion events in tissue and liquid biopsy samples at very low frequency [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3118.

Abstract 215: Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research

Abstract Introduction: Next generation sequencing technologies and platforms have been evolving rapidly over the last decade with improved throughput and cost-effectiveness. The application of various NGS assays using both genome wide or targeted sequencing has been widely employed in cancer research to characterize tumor biomarker profiles including SNV/indels, CNV, and fusion in various sample types. Here we will present the performance of OncomineTM Precision Assay developed at Thermo Fisher Scientific, which is compatible with both solid tumor and liquid biopsy sample types. It is the very first automated NGS assay developed with Ion Torrent Genexus system to provide a streamlined workflow directly from sample to results with minimal hands-on time. Methods: The assay employs a novel AmpliSeq™ HD library technology to provide optimal sensitivity and specificity for challenging sample types including degraded FFPE or cell-free nucleic acid from plasma. For library preparation, 10 ng DNA or RNA was used for FFPE and 20 ng cfTNA was used for plasma samples. Ion Torrent Genexus system provides an interface to guide the user to plan the run following a defined workflow according to sample type, and performs library preparation, templating and sequencing automatically. Results: The OncomineTM Precision Assay includes focused genetic content with clinical relevance including 45 hotpot genes (SNV/Indel), 14 CNVs, and 19 structural variants (fusion and exon variants). This study evaluated the assay performance using a set of controls for FFPE or cfDNA from positive cell line material or commercial sources such as Horizon discovery and SeraCare, which contain various SNV/Indels on several important gene loci (EGFR, KRAS, NRAS, BRAR, and PIK3CA), CNV (EGFR, MET amplification and pTEN deletion), and fusion variants across ALK, ROS1, RET, and NTRKs. The results showed that all the expected SNV variants were consistently detected with sensitivity at ≥95% for FFPE with 5% AF and ≥85% for cfDNA samples down to 0.1% LOD. For fusion variants and CNV targets including gain and loss, both sensitivity and PPV at ≥90% could be achieved for FFPE and cfTNA samples. Furthermore, a cohort of positive FFPE tissue was also analyzed and detected expected variants that had been previously confirmed through an orthogonal NGS test. Lastly, a set of matched FFPE and plasma samples was also evaluated using the assay and demonstrated high concordance between FFPE and plasma from each subject. Conclusion: Overall the study demonstrates that the OncomineTM Precision Assay provides an automated and sensitive NGS solution for efficient and effective mutation assessment using both FFPE and liquid biopsy samples. Citation Format: Ru Cao, Jian Gu, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Bo Ding, Khalid Hanif, Kelli Bramlett. Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 215.

Abstract B26: A new way of fabricating high-porosity parylene membranes for high-throughput capturing of viable circulating and exfoliated tumor cells from large-volume bodily fluids

Abstract Liquid biopsy is a critical component in cancer precision medicine. The ability to capture rare cancer cells from liquid samples, such as circulating tumor cells (CTCs) from peripheral blood as well as exfoliated tumor cells (ETCs) from other body fluids, has shown great promises in cancer detection and prognosis, as well as understanding of cancer biology at different stages. Different ways have been developed for rare cancer cell capture, including biomarker-based immunocapture and capture through other physical processes, such as size, mechanical or dielectric properties, hydrodynamic fluids, etc. However, the throughputs of these techniques are limited to 0.2-2 mL/min. Recently, a high-porosity parylene membrane has been shown to be able to process sample at a fluid rate of > 100 mL/min, which would greatly extend the capability of CTC and ETC-based liquid biopsy. The high porosity of the membrane can also dramatically reduce the shear stress the tumor cells encountered during capture, which has been shown to be critical to maintain cell viability. However, the current parylene membrane fabrication involves deep Si reactive ion etching, which is an expensive process and not widely available. The release of the parylene membrane can also be challenging. We report here a new method to fabricate the parylene membrane using simple photolithography and reactive ion etching techniques. The fabricated parylene membranes and their capability of processing large volume sample for viable cell capture will be presented. Citation Format: Jian Gu, Inad Rabadi, Frederic Zenhausern, David Carpentieri, Jue Wang. A new way of fabricating high-porosity parylene membranes for high-throughput capturing of viable circulating and exfoliated tumor cells from large-volume bodily fluids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B26.

Abstract B46: Development of a fully integrated sample-to-report system for a pan-cancer application

Abstract Introduction: We have developed a new sample-to-report solution for testing solid tissue cancers using the Ion Torrent System and Software. The assay is designed for research applications from either formalin-fixed, paraffin-embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy. The new sequencer is a fully automated system requiring minimal hands-on time and touch points for a user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types. Methods: The assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biologic variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. The assay is optimized for use with the Ion Torrent System. With minimal hands-on time (~15 minutes), the user prepares the system with prefilled reagents strips to start a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: The assay is designed to detect somatic variants in 50 unique genes, where 45 of these genes include substitutions, deletions, and insertions. 14 genes are targeted for copy number variation, and 19 genes are drivers of gene fusions and alternate splice forms. The content has been selected based on published accounts of target actionability and prevalence across multiple cancer types. An example of run setup is shared along with timing (~30min) to input sample information using the Torrent Suite Software. Data are shown from a multiplexed sequencing run using FFPE DNA (10 ng) and RNA (10ng) as input. The automated FFPE run on the Ion Torrent System completed in about 30 hours. Data are also presented from a sequencing run that included multiple liquid biopsy libraries utilizing cell-free total nucleic acid (cfTNA) extracted from plasma. In this run mode, variant calling is optimized for highly sensitive and specific variant calling below 1% allelic frequency for all targeted variant types assay including CNVs, fusions, and alternative splice forms and was accomplished in less than 24 hours. Conclusion: The assay and system enable detection of key oncology variants in 50 genes using both solid tissue and liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in a single 24-hour period with minimal hands-on time and touch-points from the user. This system is not yet released but is in development. Citation Format: Kelli Bramlett, Ru Cao, Jeff Schageman, Yanchun Li, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Oana Lungu, Varun Bagai, Desiree Martin, Khalid Hanif, Jian Gu. Development of a fully integrated sample-to-report system for a pan-cancer application [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B46.

Abstract 834: Epigenome-wide profiling of DNA methylation in peripheral blood leukocytes and the prognosis of prostate cancer patients in African Americans

Abstract Introduction: DNA methylation at CpG sites plays important roles in cancer development and progression. Hypermethylation of the promoter regions of tumor suppressor genes leads to gene silencing whereas global hypomethylation may affect chromosome structure and cause genomic instability. African Americans have higher incidence and mortality rates of prostate cancer than other ethnic groups. The goals of this study are to investigate the role of global DNA methylation in prostate cancer aggressiveness and identify CpG site methylations as predictors of aggressive prostate cancer in African Americans. Methods: We measured global DNA methylation level of long interspersed nucleotide elements (LINE-1) and subtelomeric repeat (D4Z4) in leukocyte DNA from ~300 African American prostate cancer patients and compared their methylation levels between different clinicopathological variables at diagnosis. We then analyzed the association of the LINE-1 and D4Z4 methylation with the risk of biochemical recurrence (BCR) using a multivariate Cox proportional hazards model. In addition, we used the Kaplan-Meier survival function and log-rank tests to assess BCR-free survival associated with D4Z4 methylation.. We also used Illumina’s HumanMethylationEPIC beadchip to profile 850K CpG site methylation in leukocytes and analzyed their associations with prostate cancer aggressiveness. Results: There was no significant differences in the methylation level of LINE-1 between clinically defined aggressive and non-aggressive PCa at diagnosis and LINE-1 methylation was not associated with BCR either. However, the methylation of subtelomeric region D4Z4 was associated with BCR. Patients with higher methylation of D4Z4 exhibited an increased risk of BCR (HR=3.47, 95% CI, 1.10 - 10.97) compared to patients in the lower methylation after adjustment of age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. In Kaplan-Meier survival analysis, patients with higher D4Z4 methylation level had a significantly shorter BCR-free survival time than those with lower methylation level (log rank P = 0.0071). When analyzing individua CpG site methylation, we identified a number of CpG site that can distinguish aggressive from non-aggressive prostate cancer and found a CpG methylation signature that can identify a subgroup of patients with aggressive prostate cancer in African Americans. Conclusions: These data suggest that methylation in the subtelomeric region D4Z4 may be able to predict worse prognosis of patients. Individual CpG site methylation may be promising biomarkers for the identification of aggressive prostate cancer in African Americans. Citation Format: Junfeng Xu, Chia-Wen Tsai, Wen-Shin Chang, Da-Tian Bau, Jian Gu. Epigenome-wide profiling of DNA methylation in peripheral blood leukocytes and the prognosis of prostate cancer patients in African Americans [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 834.

Abstract 5123: Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay

Abstract Currently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants. Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems. In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly, additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor. Overall, this study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models. Citation Format: Ru Cao, Kris Lea, Madhu Jasti, Jeff Schageman, Khalid Hanif, Yanchun Li, Jian Gu, Varun Bagai, Priyanka Kshatriya, Harriet Wikman, Sonja Loges, Kelli Bramlett. Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay [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 5123.

Abstract 3332: Genome-wide DNA methylation profiling of peripheral blood leukocytes identifies predictors of aggressive prostate cancer

Abstract Background: Prostate cancer (PCa) is the most commonly diagnosed cancer for men in the United States. Most of the PSA screening-detected PCa are slow-growing and not life-threatening. However, the majority of PSA-detected PCa receive aggressive treatment, which is often associated with significant morbidity. Clinical variables, such as Gleason Score (GS), PSA level, and tumor stage, are not sufficient to distinguish aggressive from indolent diseases at diagnosis. Biomarkers are urgently needed for more accurate prediction of individual tumor behavior and patient prognosis. DNA methylation has shown profound impact on cancer development and progression. In this study, we performed a genome-wide DNA methylation profiling of peripheral blood leukocytes to identify predictors of aggressive PCa. Methods: We performed leukocyte DNA methylation profiling in 140 GS=6 PCa patients and 147 GS ≥ 8 patients using the Illumina's Methylation 450K BeadChip. The differential methylated CpG position (DMP) was identified using the ChAMP analysis package implemented in R. Top differentially methylated CpG sites between less aggressive (GS=6) and more aggressive (GS≥ 8) patients were used to develop a signature to best cluster these two groups using semi-supervised recursively partitioned mixture modeling (SS-RPMM) algorithm. The white blood cell subtype composition was analyzed with RefbaseEWAS method. Ingenuity pathway analysis was used to find the enriched pathways that may determine aggressive prostate cancer. Results: Analyzing all the assayed CpG sites together, the mean methylation level appeared to be lower in GS ≥ 8 patients than in GS=6 patients. This global DNA hypomethylation was only significant at CpG sites located within gene body, shore, shelf, opensea and 3' UTR regions, but not in promoter and regions close to transcription start sites. There were many individual hypermethylated CpG sites in GS ≥ 8 patients that are mostly located in CpG islands within promoter and in the vicinity of transcription start sites. We then split the patients into a training (70 GS=6 and 73 GS ≥ 8 patients) and testing set (70 GS=6 and 74 GS ≥ 8 patients) to identify and validate a CpG methylation signature that best differentiates GS=6 and GS≥ 8 patients. The signature can identify a subgroup of aggressive PCa patients with higher risks of biochemical recurrence (HR=2.57, 95% Cl, 0.99-6.68). We also analyzed the white blood cell subtypes in patients and found that natural killer (NK) cell proportion is significantly decreased in patients who experienced biochemical recurrence (P=0.045), indicating weakened immune response. Conclusion: These data suggest that DNA methylation in peripheral blood leukocytes may serve as valuable biomarkers to supplement clinical variables to predict aggressive PCa at diagnosis and worse prognosis of PCa patients. Citation Format: Yuyan Han, Jia Li, Mutian Zhang, Jeri Kim, Xifeng Wu, Deqiang Sun, Jian Gu. Genome-wide DNA methylation profiling of peripheral blood leukocytes identifies predictors of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3332.

Effects of the Chinese medicine Yi Shen Jian Gu granules on aromatase inhibitor-associated musculoskeletal symptoms: A randomized, controlled clinical trial

Aromatase inhibitor-associated musculoskeletal symptoms (AIMSS) are prevalent among patients on AI therapy, which leads to a lower quality of life and poor adherence to AI treatment. We evaluated whether Yi Shen Jian Gu granules (YSJG) is effective and safe to relieve AIMSS in patients with breast cancer.Eligible participants were randomly assigned to the YSJG group or the placebo group. Both groups had a 12-week treatment period and a 12-week follow-up period. Brief Pain Inventory-Short Form (BPI-SF), Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index, and the Modified Score for the Assessment and Quantification of Chronic Rheumatoid Affections of the Hands (M-SACRAH) were obtained at baseline and at 4, 8, 12 and 24 weeks.Of 146 participants enrolled, 84 were randomly assigned, and 77 were evaluable at 12 weeks. Baseline characteristics were comparable between two groups. The primary outcome was the differences in mean BPI-SF scores at 12 weeks. The worst pain scores decreased by 3.10 points (50.2%; 95% CI, 2.50 to 3.65) for YSJG group compared with a 1.63-point decrease (26.9%; 95% CI, 3.86 to 4.97) for the placebo group (P = 0.001). Significantly improvements were also observed for the WOMAC and M-SACRAH. Possibly YSJG-related side effects were grade 1 nausea (10%) and grade 2 diarrhea (2%). Serum follicle-stimulating hormone and serum estradiol were kept in the postmenopausal range before and after YSJG treatment.Patients with AIMSS treated with YSJG granules had significant improvements in musculoskeletal symptoms. YSJG is effective, safe and well-tolerated in managing AIMSS. Trial registrationISCTN: ISRCTN06129599 (assigned 14 August 2013).

Abstract 5396: An NGS workflow to detect down to 0.1% allelic frequency in cfDNA for breast and colon cancers

Abstract Noninvasive detection of rare mutations in blood could allow tumor monitoring for research purposes. Research studies have suggested that cfDNA contains DNA from tumor cells with somatic mutations that could inform on tumor progression and therapeutic resistance. Here, we demonstrate a complete workflow from a single tube of blood through data analysis for research samples down to a 0.1% allelic frequency. The low abundance tumor mutations found in cfDNA requires sensitive and accurate mutation detection. We have developed two panels that utilize an amplification-based assay that generates tagged DNA copies, which allows detection of low abundance tumor mutations found in cfDNA. The two panels allow multiplex interrogation of primary driver and resistance mutations specific to ctDNA from breast and colon cancer. The Oncomine Colon cfDNA panel targets 236 hotspots within 14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in 10 genes. This workflow was validated from matched single blood tubes, Streck and K2EDTA. Additionally, the utility for cancer research was demonstrated with concordance studies using matched FFPE and plasma from oncology samples. To further characterize these panels we have developed an oncology control for cfDNA with nucleosome fragment sizing and minimal sonication damage. This engineered control contains SNPs and indels at 0.1% allelic frequencies, orthogonally confirmed with TaqMan based Rare Mutation assays. With this control, the Oncomine Breast cfDNA panel had over 81% sensitivity and 99.9% specificity. The Oncomine Colon cfDNA panel had over 85% sensitivity and 100% specificity. The Oncomine Breast cfDNA panel and Oncomine Colon cfDNA panel integrated into a complete workflow starting from a single tube of blood can advance oncology research with the ability to detect blood based cancer biomarkers present at 0.1%. Citation Format: Dalia Dhingra, Richard Chien, Jian Gu, Dumitru Brinza, Ruchi Chaudhary, Kunal Banjara, Yanchun Li, Efren Ballesteros-Villagrana, Kelli Bramlett. An NGS workflow to detect down to 0.1% allelic frequency in cfDNA for breast and colon cancers [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 5396. doi:10.1158/1538-7445.AM2017-5396