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Empirical determination of the limit of detection. Defined oncogenic driver and background SNVs (a), oncogene and tumor suppressor indels (b), and oncogenic fusions (c) were titrated to specified VAFs, and multiple replicates were measured to determine the 95% LOD. (d) Cell lines comprising defined gene amplifications were titrated to bracket the expected LOD, and the 95% LOD was determined from statistical detection models as per the CLSI Classical Approach.

Digital Sequencing analytical performance metrics summary table.

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24–2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%–100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non–small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539–49. ©2018 AACR.

Background Circulating cell‐free tumor DNA (ctDNA)‐based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next‐generation sequencing platform in a large series of patients with non–small cell lung cancer (NSCLC). Methods Plasma‐based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co‐occurrence, and mutual exclusivity. Results Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%‐97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment‐induced resistance mutations were common in this cohort, including driver‐dependent and driver‐independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard‐of‐care, tissue‐based testing at diagnosis. A pooled data analysis on this plasma‐based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis. Conclusions Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies., Circulating cell‐free tumor DNA‐based liquid biopsy using next‐generation sequencing detects a spectrum of targetable alterations at frequencies expected in patients with advanced non–small cell lung cancer. These findings support the concept of a plasma‐first algorithm at the time of progression on targeted therapy for this population.

Background: A blood-based cancer screening test must exhibit performance metrics optimized for the cancer of interest based on associated clinical diagnostic pathways and demonstrate an ability to detect disease at an early stage when intervention has a meaningful impact on individual and net population health outcomes. We evaluated the performance of a blood-based cancer screening assay in select tumor types where we believe cancer screening can save lives. The assay interrogates cell-free DNA (cfDNA) methylation signatures for early-stage cancer (stage I/II) detection and tissue of origin identification. Methods: Whole blood samples from individuals with (N > 1,500) and without (N > 1,800) cancer were obtained from multiple cohorts. Plasma-derived cfDNA was profiled using a custom assay that enriches fragments with dense CpG methylation and further depletes uninformative background molecules. A broad genomic panel (16 Mb) targeting regions with low rates of methylation in healthy individuals was used to capture and sequence tumor-associated molecules while maintaining high sensitivity at low sequencing cost per sample. A cross-validated analysis was used to estimate out of sample performance of the predictive model. Classification thresholds corresponding to 90% and 95% specificities were established using a set of samples from individuals without a cancer diagnosis. Results: To evaluate the performance of this screening assay in cancers with guideline-directed screening protocols, colorectal and lung cancers, detection was assessed at 90% specificity. At this threshold, sensitivity for stage I/II colorectal and lung cancer was 90% and 87%, respectively. For other cancers with no current guideline-directed screening paradigms, pancreatic and bladder cancers, a specificity threshold of 95% was applied. Sensitivity was 73% and 52% for stage I/II pancreatic and bladder cancer, respectively. Tissue of origin prediction evaluated at 98% specificity had accurate identification in 99% of colorectal, 94% of lung, 88% of bladder, and 86% of pancreatic cancers. Conclusions: This multi-cancer targeted screening assay provides robust and sensitive detection of early-stage cancer at thresholds optimized for current screening paradigms with accurate tissue of origin identification. The assay is undergoing further expansion of its detection capabilities to include additional cancer types where screening can save lives. Clinical evaluation in registrational screening trials is ongoing (NCT05117840). Citation Format: Anton Valouev, Elena Zotenko, Matthew Snyder, Charbel Eid, Ngan Nguyen, Jun Min, Yupeng He, Ariel Jaimovich, Haley Axelrod, Prashanthi Natarajan, Anna Hartwig, Noam Vardi, Tam Banh, Andrew Kennedy, William Greenleaf, Stefanie Mortimer, Sven Duenwald, Darya Chudova, AmirAli Talasaz. Development of a highly-sensitive targeted cell-free DNA epigenomic assay for early-stage multi-cancer screening [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 2141.

3542 Background: Cancer screening in asymptomatic individuals who meet guideline criteria has yielded reductions in cancer death rates. However, adherence to screening guidelines remains below targets set forth by leading health-care organizations. A blood-based multi-cancer screening assay with clinically meaningful sensitivity and specificity, in cancer types where early detection and intervention can save lives, that is integrated with existing clinical pathways may increase access to and adherence with guideline recommendations, ensuring more individuals benefit from these proven interventions. We evaluated the performance of a blood-based multi-cancer screening assay that interrogates cell-free DNA (cfDNA) methylation signatures for cancer detection and tissue of origin prediction in a set of tumor types where cancer screening can save lives. Methods: Whole blood from 1,607 individuals with and 3,298 individuals without cancer was obtained from multiple unique cohorts. Plasma-derived cfDNA was profiled using a custom assay that enriches fragments with dense CpG methylation and further depletes uninformative background molecules containing unmethylated CpGs. We utilized a broad genomic panel (16 Mb) targeting regions with low rates of methylation in individuals without cancer. The panel captures tumor-associated molecules and allows for high sensitivity of detection at low sequencing costs. A cross-validated analysis was used to estimate the performance of the predictive model upon the sample set. Classification thresholds corresponding to 90%, 95%, and 98% specificities were established using samples from individuals without a cancer diagnosis. Results: At 90% specificity, overall sensitivity for lung cancer detection was 92.1% (95% CI: 80-100%; 90.2% in Stage I/II disease (N = 82) and 93.1% in Stage III/IV disease (N = 159)) and 93.1% (CI: 88-98%) for CRC detection (92% in Stage I/II disease (N = 743) and 94.5% in Stage III/IV disease (N = 623)). Tissue of origin prediction evaluated at 98% specificity yielded accurate identification in 99% of CRC and 98% of lung cancers. Lung cancer histology was known for approximately 74% of the cohort. Across Stage I – IV cancers, at 90% specificity, sensitivity was 97.3% in lung squamous cancer (N = 73) and 86.8% in lung adenocarcinoma (N = 106). At 95% and 98% specificity thresholds, overall sensitivity was 86.3% (CI: 75-98%) and 66.4% (CI: 56-77%) for lung cancer and 85.7% (CI:81-91%) and 71.6% (CI: 67-76%) for CRC, respectively. Conclusions: This blood-based multi-cancer screening assay yields clinically meaningful sensitivity and specificity for early-stage cancers. This assay is undergoing further development to expand detection capabilities to additional cancer types where screening can save lives. Clinical evaluation in registrational screening trials is ongoing (SHIELD; NCT05117840).

Summary By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 104 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020) .

High-throughput single cell RNA sequencing (scRNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional scRNA-seq captures information at the transcript level, recent technology using DNA-barcoded antibodies enable multiomic analysis, examining protein alongside mRNA. Although this approach allows analyzing a large number of protein markers, the sequencing cost can escalate quickly for large panels, particularly ones with high antigen-density markers, and workflows can be long. Therefore, panels should be optimized to gain meaningful results efficiently and cost-effectively. Here, we introduce the BD® AbSeq Immune Discovery Panel (IDP), a lyophilized panel consisting of 30 antibodies against major human immune cell-types and immune response markers for analyzing multiple immune cell subsets. The BD® AbSeq IDP can be used to uncover phenotypic changes in cell subsets such as unique cell input differences or changes in the immune cell status. To test this capability, we used the IDP alongside a mRNA analysis using targeted mRNA panel and whole transcriptome assay to compare the immune phenotypes of cells from disease and in vitro stimulated samples at the single-cell level using the BD Rhapsody™ System. We propose that the BD® AbSeq IDP can be used to identify the cell types and molecular pathways impacted by the disease at the single cell level. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Adoptively transferred antigen-specific T cells have shown great efficacy in treatment of some virus-associated diseases and malignancies. A major driver of the development of adoptive T-cell therapy has been our ability to successfully characterize the functional status and antigen specificity of T cells. However, this has been limited by inefficient detection of antigen-specific T cells possibly due to their low frequency and low binding affinities to known MHC-peptide complexes. Here, we aim to combine two powerful technologies, advanced dCODE™ Dextramer® from Immudex and single-cell multiomics analysis using the BD Rhapsody™ Single-Cell Analysis system, to detect and characterize disease-specific CD8+ T cells within thousands of PBMCs. Currently, we are able to identify over 350 mRNAs alongside a panel of over 20 BD® AbSeq cell surface protein markers which can be associated with T cell activation states. These data can be used to define T-cell phenotypes alongside antigen specificity of enriched CD8+ dextramer+ cells from a PBMC population. This study outlines our ability for high-resolution T-cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

High-throughput single cell RNA sequencing (RNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional single cell RNA-seq captures information about transcript expression, recent technological advances using oligo-conjugated antibodies enable simultaneous detection of proteins alongside mRNA in high-throughput sequencing. This multiomic technology enables high parameter protein analysis that can simultaneously discriminate 100 protein markers. However, experiment workflow time and cost can escalate quickly with high plexy assays. To address these issues we investigated a number of methods to decrease cost and assay time while maintaining data quality using the BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis sequencing system. To begin, we investigated the performance of 20 core proteins when used in a 20plex, 40plex, 60plex, 80plex, or 100plex in model cells. Results from 3 experiments showed that performance was maintained as plexy increased, however rigorous experimental approaches must be followed to minimize impact on specificity or sensitivity. After establishing performance of this 20plex we investigated pre-cocktailing of antibodies as a method to shorten the day-of-experiment workflow, and mixing of oligo-labeled and unlabeled antibodies to high-expressing proteins (“signal muting”) as a way to decrease sequencing costs. Results showed a decrease in specificity as soon as 24 hours after the cocktailing of antibodies and increasing non-specific binding with longer cocktail storage times. On the other hand, signal muting was effective at decreasing sequencing requirements by up to 70%, without impacting data for high expressors. Together these results can be used to aid in optimizing experimental design when working with high-plex AbSeq protein panels, to better harness the information gained from multiomic single cell sequencing data. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Punya Narayan, Hye-Won Song, Margaret Nakamoto, Elisabeth Walczak, Katherine Lazaruk, Stefanie Mortimer. Optimizing experimental design for high-parameter protein analysis using BD AbSeq technology [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 1348.

Advances in high-throughput single cell whole transcriptome analysis (WTA) have enabled the discovery of biomarkers and cellular pathways critical to resolving diversity in diseases. Despite these advances, challenges still remain in comprehensively profiling single cells. Here we address two challenges, limited use of frozen tissues, and inability to resolve post-transcriptional differences from cell to cell. Single-cell RNAseq often requires intact single cells to get good resolution of signal, preventing the use of archival frozen samples. To overcome this limitation, we have tested the possibility to use dissociated nuclei that can be isolated from frozen samples in the BD RhapsodyTM Single-Cell Analysis system. As a proof of concept, we compared high throughput single-nucleus WTA data to that produced from single-cell RNAseq from the same cell type. Our data showed that mRNAs from single nuclei can be captured, and amplified using the BD RhapsodyTM system. Furthermore although the number of molecules detected is lower in nucleus, probably due to lower mRNA content, their expression profiles are well correlated with Pearson's correlation coefficient over 0.9. This opens up the possibility to conduct single cell transcriptomic analysis on the large pool of archived frozen specimens, expanding the variety and number of disease samples that can be examined at the single cell level in the BD RhapsodyTM system. A second limitation of WTA analysis is that it only provides information about gene regulation at the transcript level. Given that regulation critical for cellular pathways is often found not only at the transcriptional level but also at the post-transcriptional level, quantitative analysis of proteins and mRNAs at the single cell level can provide deeper understanding of the disease cells. Here, we used DNA-barcoded antibodies BD® AbSeq to enable multiomic analysis, examining protein alongside mRNA expression and enabling simultaneous transcriptional and post-transcriptional gene profiling in single cells. Our data show that upon activation human peripheral blood mononuclear cells and isolated T cells undergo regulation of markers at the protein level that cannot be resolved at the mRNA level. Several key markers used to define activation states including CD69 and L-Selectin were found to be regulated at the protein level confirming that addition of protein analysis to WTA can generate more comprehensive cell profiling. These approaches offer flexibility and choice in experimental design and allow users to utilize archived frozen samples or to obtain mRNA and protein profiles from single cells. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Hye-Won Song, Gretchen Y. Lam, Margaret Nakamoto, Punya Narayan, Ian Taylor, Stefanie Mortimer. Advances in single cell whole transcriptome analysis: Single nucleus RNAseq and simultaneous protein and mRNA profiling using the BD RhapsodyTM Single-Cell Analysis system and BD® AbSeq [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 1345.

Obesity can weaken the body's immune system and reduce its ability to fight off infections. Current analysis on immune cells from obese mouse models suggests that the immune cells suffer from chronic inflammation. To understand the root cause of this problem, we used single cell sequencing to examine thousands of immune cells isolated from primary and secondary lymphoid organs as well as adipose tissue and compared a diet-induced obesity mouse model with control mice. Cells were stained with 30 BD® AbSeq DNA-barcoded antibodies to enable multiomic analysis, i.e. examining protein alongside mRNA expression in tandem. We also utilized DNA-barcoded universal antibodies from the BD® Single-Cell Multiplexing Kit, which allowed us to combine 8 samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost while eliminating potential batch effects. The pooled samples were loaded on the BD Rhapsody™ system to perform cell lysis and individual mRNA and cell barcoding, allowing measurement of ~400 immune-related mRNAs and 30 proteins at the single cell level. We were able to efficiently de-multiplex the pooled samples after sequencing. The targeted mRNA and AbSeq panel provided robust clustering of immune cell types and showed that genes related to critical immune responses, including inflammation and lymphocyte activation, are differentially regulated in specific immune-cell subsets in the obese mouse model. Using this multiomic analysis of genes differentially regulated in immune cells from different tissues, we propose a model to explain the immuno-phenotype we observed in obese mouse. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Hye-Won Song, Gisele Baracho, Ian Taylor, Stephanie Widmann, Stefanie Mortimer. An efficient multiplexing approach to avoid batch effects in single cell RNA- and Ab-seq studies demonstrated in a mouse model of chronic inflammation and obesity [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 2750.

High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of complex and heterogenous immune populations. New advances in scRNA-seq are expanding the molecules that can be profiled at the single cell level, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA. Although the ability to sequence the immune repertoire can provide crucial insights into understanding the complexities of the adaptive immune system and advancing discoveries in immuno-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA that are missed by conventional 3' scRNA-seq. In this study we utilized an ex vivo antigen stimulation system to measure antigen-specific T-cell activation and clonal amplification. Stimulated T cells from two donors were loaded onto the BD Rhapsody™ Single-Cell Analysis System to extract immune repertoire information in addition to gene and protein expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets, 20 BD® AbSeq protein markers associated with different status of T-cell activation, and the hypervariable region of T-cell receptors at the single cell resolution. The study is a proof of concept of combining mRNA, protein and immune repertoire analysis at the single cell level to deconvolute the heterogeneity and different activation of stimulated T cells. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Imteaz Siddique, Xiaoshan Shi, Margaret Nakamoto, Stefanie Mortimer. Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing [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 1344.

In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

High-throughput single cell RNA sequencing (scRNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional scRNA-seq captures information at transcript level, recent technology using DNA-barcoded antibodies enable multiomic analysis, examining protein alongside mRNA. Although this approach allows high parameter antibody panels, the sequencing cost can escalate quickly for high plexy panel with high antigen density markers. Therefore, thought should be put on panel design in order to gain meaningful results efficiently. Here, we have designed a panel for analyzing multiple immune responses, BD® AbSeq pan immune-response panel. Our immune response panel excludes abundant cell lineage markers which take up a huge amount of reads but provide only cell lineage information that can be easily resolved by RNA profiles only. This panel is focused on analyzing the various immune responses in multiple cell lineages. Therefore, it has a potential to be used to uncover phenotypic changes upon disease status with various immune stimuli. To test the capability of this AbSeq panel in discovery of phenotypic changes in multiple immune response, we have tested it in the various scenarios. We propose that the BD® AbSeq pan immune-response panel can be used to evaluate unpredictable disease phenotypes to identify the cell types affected from the disease as well as the responsible molecular pathways of the disease at a single cell level. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Purpose: Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.Results: Patterns and prevalence of cfDNA alterations in major driver genes for non–small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC; r = 0.90–0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10−7 for EGFR and ERBB2), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).Conclusions: Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Clin Cancer Res; 24(15); 3528–38. ©2018 AACR.

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24–2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%–100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non–small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539–49. ©2018 AACR.

A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.

Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay. Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set. Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management. Citation Format: Seung-Tae Kim, Victoria M. Raymond, Joon Oh Park, Elena Zotenko, Young Suk Park, Matthew Schultz, Won Ki Kang, Oscar Westesson, Hee-Cheol Kim, Yupeng He, Justin I. Odegaard, Stefanie A. Mortimer, William J. Greenleaf, Ariel Jaimovich, Jeeyun Lee, AmirAli Talasaz. Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC) [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 916.

High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of heterogenous immune populations. Advances in scRNA-seq, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA, are expanding the range of molecules that can be profiled at the single cell level. Although sequencing the T-cell and B-cell immune repertoire can provide crucial insights into the complexities of the adaptive immune system, thus advancing discoveries in immunology and immune-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA missed by conventional 3′ scRNA-seq. Here we demonstrate a novel approach using the BD Rhapsody™ system to extract immune-repertoire information in addition to gene-expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets and 40 protein markers as well as the CDR3 region of T- and B-cell receptors in thousands of human PBMCs. After using the mRNA and protein information to robustly annotate different lymphocyte populations, we examined the T- and B-cell repertoires for differences in their clonal diversity. We were able to identify clonally expanded T cells and B cells and examine how their gene-expression profiles differ from that of non-expanded cells. This study demonstrates the power of combining mRNA, protein, and immune repertoire analysis at the single cell level to answer complex and important biological and clinical questions. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Single cell RNA-sequencing (scRNA-seq) is a powerful tool for understanding the sample heterogeneity of individual cells. Many methods rely on whole transcriptome analysis (WTA) to get a snapshot of the entire cellular landscape. Although WTA analysis can be used to discover novel biomarkers, this technique can be expensive. To enable scaling of experiments, WTA data can be mined to design targeted gene panels. To showcase this, we used single cell sequencing to examine thousands of B cells isolated from the bone marrow and peripheral blood of chronic lymphocytic leukemia (CLL) and healthy donors. B cells that had been sorted using the BD FACSMelody™ cell sorter were multiplexed using the BD™ Human Single-Cell Multiplexing Kit and pooled before being processed on the BD Rhapsody™ system, thereby minimizing batch effects. The resulting data was mined to design a panel of differentially expressed genes between CLL and healthy B cells that could be used for subsequent CLL phenotyping. By combining this panel with the BD Rhapsody™ Immune Response Panel (together comprising ~500 mRNAs), along with 36 DNA-barcoded BD™ AbSeq antibodies, we were able to simultaneously analyze mRNA and protein targets from a new subset of CLL and healthy B cells for additional high-resolution analysis. This study showcases the power of using WTA data to design specific gene panels that can be used alone or in combination with existing targeted panels for routine and cost-effective transcriptional profiling at a single cell level. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, FACSMelody, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Obesity can weaken the body’s immune system and trigger chronic inflammation. To understand the root cause of this problem, we used single cell sequencing to examine thousands of immune cells isolated from primary and secondary lymphoid organs as well as adipose tissue and compared a diet-induced obesity mouse model with control mice. Cells were stained with 30 DNA-barcoded antibodies from BD™ AbSeq reagents to enable multiomic analysis, i.e. examining protein alongside mRNA expression in tandem. We also utilized DNA-barcoded universal antibodies from the BD™ Single-Cell Multiplexing Kit, which allowed us to combine 8 samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost while eliminating potential batch effects. The pooled samples were loaded on the BD Rhapsody™ system to perform cell lysis and individual mRNA and cell barcoding, allowing measurement of ~400 immune-related mRNAs and 30 proteins at the single cell level. We were able to de-multiplex the pooled samples with high specificity after sequencing. The targeted mRNA and AbSeq panel provided robust clustering of immune cell types and showed that genes related to critical immune responses, including inflammation and lymphocyte activation, are differentially regulated in specific immune-cell subsets in obese mouse. Using this multiomic analysis of genes differentially regulated in immune cells from different tissues, we propose a model to explain the immuno-phenotype we observed in obese mouse. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5′ end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3′ noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

Background: Translational research and enrollment in clinical trials are limited by the rarity of individual mutations and lack of sufficient tissue for comprehensive testing. To address these limitations, we developed GuardantOMNI (OMNI), a highly sensitive 500-gene cfDNA sequencing test requiring as little as 2 mL of plasma and designed for broad genomic detection of somatic single-nucleotide variants (SNVs) and small indels in 497 genes, copy number amplifications (CNAs) in 106 genes, and fusions in 21 genes. Additionally, the OMNI panel enables assessment of tumor mutational burden (TMB), and DNA damage and mismatch repair, with coverage of over 30 genes associated with the DDR pathway. Here we present the first analytical validation study of OMNI. Methods: Analytical performance was assessed as per Nex-StoCT Working Group guidelines using precharacterized cell lines and healthy normal donor-derived samples. Qualitative and quantitative orthogonal confirmation was provided by exome sequencing, microarrays, and data from published compendia. Results: Seventy-three validation and 150 development plasma samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Reportable ranges for SNVs were ≥0.04% variant allele fraction (VAF), ≥0.02% for indels, ≥2 supporting molecules for fusions, and ≥2.18 copies for CNAs. Cell line-based dilution studies demonstrated 95% limits of detection (LoD) of 0.24-0.6% VAF for SNVs (depending on known cancer association), 0.4-0.8% for non-homopolymeric indels (depending on clinical relevance), 0.1-0.2% for fusions, and 2.2-2.9 copies for 90% of CNA genes targeted. Comparison of diluted cell line and healthy donor samples to orthogonal sequencing and published genotype data demonstrated accuracies of 98.7% for SNVs, 97.2% for indels, and 100% for CNAs and fusions across the reportable range. The analytical false-positive rate per sample measured across 24 healthy donors was 0.25 for SNVs, 0.04 for indels, and 0 for CNAs and fusions, with positive predictive values (PPVs) of 97.5% for SNVs, 98% for indels, and 100% for CNAs and fusions. Quantitative correlation of allele fraction with confirmatory methods was high (r2 > 0.99). Conclusions: To our knowledge, OMNI is the largest comprehensive ctDNA cancer gene panel available. It detects alterations in genes under study in over 98% of current clinical trials with sensitivity, specificity, and accuracy similar to currently available targeted ctDNA sequencing tests. OMNI has the potential to accelerate clinical trial enrollment, research and discovery with a single, noninvasive blood sample. Citation Format: Elena Helman, Carlo Artieri, James V. Vowles, Jennifer Yen, Tracy Nance, Marcin Sikora, Joshua Gourneau, Mohit Goel, Stefanie Mortimer, Darya Chudova, Justin Odegaard, Richard B. Lanman, AmirAli Talasaz. Analytical validation of a comprehensive 500-gene ctDNA panel designed for immuno-oncology and DNA damage research [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 5603.

12044Background: ctDNA in post-op CRC pts correlates with molecular residual disease and may be useful to guide adjuvant therapy. However, initial studies employed clinically impractical assays ind...

RNA selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2'-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2'-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 degrees C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2'-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2′-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2′-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2′-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Guardant360 is a cell-free circulating tumor DNA (ctDNA) test that genotypes all guideline-recommended solid tumor somatic genomic treatment targets from a single non-invasive blood draw. The new version, v2.10, was redesigned to enhance sensitivity and specificity across 73 cancer-related genes. It detects all four major variant classes (single nucleotide variants, SNVs, in all 73 genes; indels in 23 genes; gene amplifications, CNAs, in 18 genes; and fusions in 6 genes). Analytical performance was assessed throughout the reportable range via multiple serial dilution studies of orthogonally-characterized contrived and patient samples. Analytical specificity was assessed by calculating the false positive rate in pre-characterized healthy donor sample mixtures serially diluted. Positive predictive value (PPV) was estimated as a function of allelic fraction/copy number from pre-characterized samples and prevalence-adjusted using a cohort of 2,585 consecutive clinical samples. Confirmation was performed using ddPCR. Analytical specificity was 100% for SNVs, fusions, and CNAs and 96% for indels across 25 defined samples. Relative to Guardant360v2.9, v2.10 demonstrated 20-50% increase in fusion molecule recovery. Retrospective in silico analysis of 2,585 consecutive clinical samples demonstrated a 15% increase in actionable fusion detection, a 6%-15% increase in actionable indel detection (excluding newly reportable indels), and a 3%-6% increase in actionable SNV detection. AlterationsReportable Range95% Limit of DetectionAllelic Fraction / Copy NumberAnalytical SensitivityAllelic Fraction / Copy numberPPVSNVs≥0.04%0.25%≥0.25%>99.9%≥0.25%98.7%0.05–0.25%63.8%99.9%≥0.25%98.4%0.05–0.25%67.8% Guardant360 analytical performance characteristics based on standard cfDNA input (30ng). Analytical sensitivity/limit of detection estimates are provided for clinically actionable variants and may vary by sequence context and cfDNA input. PPV is estimated across entire reportable panel space (PPV was 100% for clinically actionable variants). Conclusion: Guardant360 v2.10 comprehensively detects all adult solid tumor guideline-recommended somatic genomic variants with unparalleled sensitivity, accuracy, and specificity. Citation Format: James Vowles, Justin Odegaard, Stefanie Mortimer, Stephen Fairclough, Marcin Sikora, Diana Abdueva, Reza Mokhtari, Arthur Baca, AmirAli Talasaz. Analytical validation of Guardant360 v2.10 [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 5705. doi:10.1158/1538-7445.AM2017-5705

3522 Background: Preliminary data suggests that ctDNA can serve as a marker of minimal residual disease following colorectal cancer (CRC) tumor resection. Applicability of current ctDNA testing is limited by the requirement of sequencing known individual tumor mutations. We explored the applicability of a multi-gene panel ctDNA detection technology in CRC. Methods: Plasma was prospectively collected from CRC patients (pts) undergoing hepatic resections with curative intent between 1/2013 to 9/2016. In a blinded manner 5ml of preoperative (preop) and immediate post-operative (postop) plasma were tested using a novel 30kb ctDNA digital sequencing panel (Guardant Health) covering SNVs in 21 genes and indels in 9 genes based on the landscape of genomic alterations in ctDNA from over 10,000 advanced cancer pts with a high theoretical sensitivity (96%) for CRC. Median unique molecule coverage for this study is 9000 for cfDNA inputs ranging from 10 – 150 ng (media input preop = 27 ng, median input postop = 49 ng) with 120,000X sequencing depth on an IIlumina HiSeq2500. Results: A total of 54 pts underwent liver metastectomies with curative intent with a median follow-up of 33 months. Preop blood was a median of 49 days from last systemic chemotherapy and 3 days prior to surgery; postop blood was a median of 17 days after resection. Tumor mutations from standard of care hotspot multigene panel testing (at MDACC) were identified in 46 of 54 pts (85%). Preop ctDNA mutation detection rate was 80% (43/54) and 44% (24/54) in postop setting, with postop median allele frequency of 0.16% (range 0.01% to 20%). In pts with a minimum of 1 year follow up, sensitivity of postop ctDNA for residual disease was 58% (95%CI; 41%-74%), and specificity was 100% (66%-100%). In 43 patients who underwent successful resection of all visible disease, postop detection of ctDNA significantly correlated with RFS (P = 0.002, HR 3.1; 95% CI 1.7-9.1) with 2-year RFS of 0% vs. 47%. Recurrence was detected in ctDNA a median of 5.1 months prior to radiographic recurrence. Conclusions: The detection of postop ctDNA using an NGS panel-based approach is feasible and is associated with a very high rate of disease recurrence.

e23068 Background: Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) is a promising method for detecting and monitoring earlier stage cancers. Liquid biopsies have a large potential to detect cancer earlier upon initial recurrence and to aid in adjuvant decision making through a non-invasive and highly accurate mechanism. Methods: We developed a 30kb ctDNA capture panel based on the landscape of genomic alterations in ctDNA of over 10,000 advanced cancer patients with high theoretical clinical sensitivity for colorectal (96%) and lung (87-93%) cancers. The panel was validated with high analytical PPV (86% at MAFs < 0.025%, 96% at MAFs > 0.025%). We applied the panel to a clinical study of 63 healthy donors and also 40 early stage (II/III) CRC and lung cancer patients with both pre- and post-tumor resection blood draws. Tumor samples were also collected at the time of the surgical resection. Results: Overall, the detection rate of ctDNA in pre-op blood draws was 72% (13/18) in CRC and 67% (12/18) in lung cancer patients. In the post-op blood draws ctDNA was detectable in 11% (2/18) of CRC and 11% (2/18) of lung cancer cases. For lung cancer patients, the median MAF in pre-op was 0.06% (0.01%-8.47%, n = 51) and in post-op was 0.06% (0.02%-4.14%, n = 38). For CRC patients, the median MAF in pre-op was 0.18% (0.01%-9.26%, n = 51) and in post-op was 0.36% (0.04%-9.5%, n = 18). Sixty-three healthy donor plasma samples were screened at similar cfDNA input amounts and nine (14%) had mutations; the majority of the mutations were also detected in matched leukocyte DNA. To assess a clinically relevant biological false positive background rate in lung cancer patients, screening of cancer-free smokers is ongoing. Conclusions: We have developed a highly sensitive and specific universal assay for the detection of ctDNA in early stage CRC and lung cancer patients without the requirement of a priori knowledge of tumor mutations. This technology allows for a promising non-invasive route for molecular monitoring of residual disease post-surgery and for early detection of relapse compared to traditional methodologies.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.

Knowledge of RNA structure is critical to understanding both the important functional roles of RNA in biology and the engineering of RNA to control biological systems. This article contains a protocol for selective 2'-hydroxyl acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through a combination of structure-dependent chemical probing and next-generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure-dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high-throughput manner. Curr. Protoc. Chem. Biol. 4:275-297 © 2012 by John WileySons, Inc.

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome. © 2013 The Author(s), National Institutes of Health (NIH) [R56-AI095687 to J.H.D.C.; P50-GM102706 to J.A.D. and J.H.D.C.]; Spanish Ministry of Education through the Programa Nacional de Movilidad de Recursos Humanos del Plan Nacional de I-D+i 2008-2011 (to E.A.-P.). J.A.D. and E.N. are Howard Hughes Medical Institute Investigators. Funding for open access charge: NIH [P50-GM102706]

MicroRNAs (miRNAs) typically downregulate protein expression from target mRNAs through limited base-pairing interactions between the 5' 'seed' region of the miRNA and the mRNA 3' untranslated region (3'UTR). In contrast to this established mode of action, the liver-specific human miR-122 binds at two sites within the hepatitis C viral (HCV) 5'UTR, leading to increased production of infectious virions. We show here that two copies of miR-122 interact with the HCV 5'UTR at partially overlapping positions near the 5' end of the viral transcript to form a stable ternary complex. Both miR-122 binding sites involve extensive base pairing outside of the seed sequence; yet, they have substantially different interaction affinities. Structural probing reveals changes in the architecture of the HCV 5'UTR that occur on interaction with miR-122. In contrast to previous reports, however, results using both the recombinant cytoplasmic exonuclease Xrn1 and liver cell extracts show that miR-122-mediated protection of the HCV RNA from degradation does not correlate with stimulation of viral propagation in vivo. Thus, the miR-122:HCV ternary complex likely functions at other steps critical to the viral life cycle.

Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) allows non-invasive real-time profiling of actionable genomic alterations. Liquid biopsy provides an option for disease monitoring in early stage cancer patients post surgical resection, with a potential to aid in adjuvant decision making. However, to be applicable, tests must cover a broad enough genomic footprint to not require a priori knowledge of mutations, have high specificity, and sensitivity higher than conventional methods. NGS is necessary, since inactivating mutations are the most common alteration type in many common cancer types such as colorectal carcinomas (CRC). Here we present a highly efficient and specific NGS assay for detection of ctDNA in early stage cancer patients, capable of detecting single molecule variants across a 12 kb gene panel with an analytical sensitivity of >0.02% for single nucleotide variants (SNVs) and indels. This panel was applied to a clinical study involving 14 early stage (II/III) CRC patients with both pre- and post-op blood draws (up to 7 days post surgery). A subset (6 patients) also had tumor samples collected at the time of the surgical resection of the tumor. Overall, the detection rate of ctDNA in pre-op blood draws was 93%. In the post-op blood draws ctDNA was detectable in 43% of cases. The estimated average minor allele frequency (MAF) is 0.58% (± 0.82%) in pre-op, 0.18% (±0.21%) in post-op, and 40% (±18%) in tumor samples. When tumor tissue was available and used as a reference, the clinical sensitivity, specificity, and accuracy in pre-op blood samples were 83%, 99.995%, and 99.99%, respectively. SNVs with MAF as low as 0.04% were confirmed in tissue data. The clinical specificity of variants detected in post-op blood samples using pre-op samples as the reference is 99.996%. Cohort expansion to 50 patients and follow-up for clinical recurrence in both cohorts is ongoing. In conclusion, we have developed an assay with ultra-high accuracy and specificity, for the detection of ctDNA in early stage CRC patients that is capable of detecting alterations present in the tumor post-surgical resection. This technology allows for a promising non-invasive route for molecular monitoring of residual disease post surgery and for early detection of relapse compared to traditional methodologies. Citation Format: Stefanie A. Mortimer, Katharine Dilger, Stephen Fairclough, Diana Abdueva, Darya Chudova, Ankit Sarin, Jim Leng, Jeeyun Lee, Helmy Eltoukhy, AmirAli Talasaz. Post-surgical resection monitoring in early stage colorectal carcinoma patients using a circulating cell-free DNA assay with ultra-high accuracy and specificity. [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 506.

Analysis of cell-free circulating tumor DNA (ctDNA) enables non-invasive and serially repeatable genomic profiling of advanced cancer patients, providing options when tissue biopsy is contra-indicated or of insufficient quantity (20-25% of solid tumor patients). Liquid biopsy studies to-date have been limited to modest-sized cohorts and case studies. Here we present genomic profiling results from liquid biopsies of >10,000 advanced cancer patients in whom Guardant360TM (G360) was ordered for clinical care. G360 provides deep-coverage (15,000x average) and highly accurate sequencing of ctDNA across a 70-gene target-capture panel. It detects single nucleotide variants, indels, gene amplifications, and fusions across all NCCN-recommended solid tumor genomic alterations with analytic specificity >99.9999% and analytic sensitivity Of the >10,000 patients (>50 solid cancer types), 80% of patients had at least one somatic alteration detected in ctDNA (median = 3; mean = 4.3 alterations). The most common cancers were lung (32%), gastrointestinal (23%), and breast (14%). Mutational spectra across the 70 analyzed genes were similar to TCGA results with the notable exception of increased prevalence of resistance mutations in the liquid biopsy cohort, likely owing to ongoing/prior therapy. Canonical drivers in NSCLC patients were generally mutually exclusive, although activating EGFR or RAS mutations were observed in 12.5% of patients with fusions (5/40). Colorectal cancers showed both mutual exclusivity among KRAS, NRAS, and BRAF drivers, and convergent evolution toward downstream pathway activation under anti-EGFR therapy, with some samples showing multiple resistance mechanisms. Mutation patterns for the most frequently mutated genes were generally well correlated between ctDNA and published tissue data, including for commonly altered tumor suppressor genes (for TP53: Pearson r = 0.94, Spearman ρ = 0.80). G360 is ordered prior to initial treatment when biopsy tissue is exhausted, unobtainable or under-genotyped, but more often is ordered upon progression to identify evolving resistance mechanisms that may be targetable. Thus, relative frequencies of genomic alterations were expected to differ from published frequencies established in unselected, early-stage, treatment-naïve cohorts. Nonetheless, the specific patterns of alterations in ctDNA from this unprecedented liquid biopsy cohort largely recapitulated patterns observed in published tissue sequencing studies. Moreover, ctDNA sequencing clearly and robustly identified convergent evolution of drug resistance occurring under therapy. Citation Format: Oliver A. Zill, Kimberly C. Banks, Coyt Jackson, Stefanie Mortimer, Arthur Baca, Becky Nagy, Richard B. Lanman, Helmy Eltoukhy, AmirAli Talasaz. Comparison of over 10,000 clinical NGS circulating tumor DNA profiles to tissue-derived genomic compendia. [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 4343.

e23021Background: In non-small cell lung cancer (NSCLC), targeted treatment with first line tyrosine kinase inhibitors (TKIs) selects for the evolution of resistance at EGFR T790M in 50% of cases. ...

LBA11501 Background: Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) enables non-invasive profiling of solid tumor cancers. Liquid biopsy studies to date have been limited to modest-size cohorts and case studies. Methods: Somatic genomic profiles of over 15,000 patients with advanced-stage clinical cancer were determined by a highly accurate, deep-coverage (15,000x) ctDNA NGS test targeting 70 genes (Guardant360). Frequencies of somatic ctDNA alterations per gene were compared to those previously described in tissue sequencing projects (e.g., TCGA). Accuracy of ctDNA sequencing (PPV) was assessed by comparing with matched tissue tests for 386 patients. Results: The cohort consisted of lung (37%), breast (14%), colorectal (10%) and other cancers (38%), with ctDNA clinical sensitivity of 86%, 83%, 85%, and 78%, respectively. Cancer-type-specific frequencies and mutual exclusivity patterns among major driver alterations largely recapitulated those seen in tissue sequencing studies. Mutation frequencies per codon correlated well between ctDNA and published tissue data, both for commonly altered tumor suppressors and for oncogenes (Pearson correlations: TP53, r = 0.94; KRAS, r = 0.99; PIK3CA, r = 0.99). The overall accuracy of ctDNA sequencing in comparison with matched tissue tests was 87% (336/386). The accuracy increased to 98% when blood and tumor were collected less than six months apart. Four distinct classes of clinical outcome benefits have been observed by liquid biopsy: 1) actionable mutations in cases with tissue QNS ( ALK fusion, or EGFR or BRAF activating mutations in lung; ERBB2 amp in gastric), 2) actionable resistance mutations at time of progression ( MET amp or EGFRT790M in lung), 3) evolution of sensitivity upon progression ( ERBB2-amplified metastatic breast cancer with triple negative primary), 4) under-genotyped tumors ( BRAFV600E or ERBB2indel in lung). Conclusions: Somatic alteration patterns in ctDNA samples largely agree with tissue alteration patterns, with the exception of resistance mutations. Clinical outcome benefits have been observed for patients treated based on ctDNA findings.

e23167Identification of novel EGFR ectodomain (ECD) mutations based on a database of clinical circulating cell-free DNA sequencing testsBackground: The Cancer Genome Atlas primarily sequenced early...

11541Background: Circulating tumor DNA (ctDNA) testing must include detection of single nucleotide variants (SNVs), indels, fusions, and copy number amplifications (CNAs) to identify all NCCN-recom...

11540Background: Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) is a promising method for early detection of cancer. Applications include screening high ris...

Background: Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) enables non-invasive real-time testing. The Guardant360® (G360) ctDNA panel includes all NCCN-recommended solid tumor genomic targets and sequences complete exons of 68 genes for single nucleotide variants (SNVs), and fusions, amplifications and indels for a subset. We conducted updated validity studies for this larger panel. Methods: Analytical sensitivity was assessed via serial dilution studies of cell-free DNA (cfDNA) with known alterations spiked into healthy cfDNA background. SNVs detected with G360 were compared to whole-exome sequencing at an outside lab for analytic specificity. Tissue NGS for fusions, SNVs, indels, and/or amplifications was compared to G360 results. Consecutive G360 tests over 12 months were analyzed for assay success rate, yield, mutant allele fractions (MAF), and actionability. Results: Serial dilution studies confirmed the previously reported 0.1% lower limit of detection. The accuracy and analytic specificity studies were 98.6% concordant, with 3 putative false positives. Sanger sequencing by a 3rd lab for these discordant calls confirmed the G360 calls as true positives, verifying the previously reported >99.9999% specificity. Retrospective review of outside tumor test reports of 56 patients with varied genomic alterations found 90% concordance with G360 (77/86 variants: 4/4 fusions, 9/9 indels, 13/17 amps, 51/56 SNVs). Of 5,491 tests, 99.5% were successfully reported with somatic alterations found in 76% [median MAF 0.42% (range 0.1-92.8%)]. Lung, breast and colon cancers were most common (see Table). Overall, 10% of patients had >1 on-label matched therapy identified, 54% off-label, and 77% had clinical trial options. On-label matched therapy rates rose for lung adenocarcinoma (15%), breast (16%) and colon (16%) cancers (Table 1). Conclusion: The updated G360 test achieves single-molecule sensitivity to 0.1% MAF and analytical specificity of >99.9999%, as reported previously. Clinical concordance of plasma to tissue-based NGS across all four types of genomic alterations was high, and actionability rose modestly, especially in lung, breast and colon cancer. Clinical utility included biopsy avoidance, rescue of tissue samples with insufficient DNA and tissue-false negatives due to tumor evolution and heterogeneity. Table 1Cancer Type% of 4,888 unique patients% with ctDNA alterations identified% with on-label therapy options identified% with off-label therapy options identified% with clinical trial options identifiedMost commonly involved gene2nd most commonly involved gene3rd most commonly involved geneLung - ALL33%82%10%52%78%TP53 (55%)KRAS (18%)EGFR (18%)Lung adenocarcinoma11%82%15%56%78%TP53 (51%)EGFR (23%)KRAS (19%)Breast15%74%16%58%79%TP53 (43%)PIK3CA (28%)ERBB2/ESR1 (10%) / (9%)CRC12%82%16%72%88%TP53 (61%)APC (38%)KRAS (38%)PDAC7%70%3%57%72%TP53 (47%)KRAS (42%)PIK3CA (6%)Ovarian3%81%1%42%81%TP53 (69%)PIK3CA (15%)KRAS (11%)CUP3%85%0%64%83%TP53 (40%)KRAS (24%)PIK3CA (14%)All100%76%10%54%77%TP53 (50%)KRAS (17%)PIK3CA (14%) Citation Format: Kimberly C. Banks, Stefanie A. W. Mortimer, Oliver A. Zill, Richard B. Lanman, Helmy Eltoukhy, AmirAli Talasaz. Genomic profiling of over 5,000 consecutive cancer patients with a CLIA-certified cell-free DNA NGS test: Analytic and clinical validity and utility. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B140.