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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.

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.

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.

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.

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

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.

Background: Genomic alterations of metastatic cancers may be discordant with alterations in primary tumors due to continuous evolution of disease and tumor heterogeneity. Current NGS approaches based on tissue biopsies can fail to capture a complete picture of the real-time cancer profile. Biopsy-free genomic profiling of circulating tumor DNA (ctDNA) can provide deep insights into the disease when biopsies are not an option. Methods: We developed Guardant360 for comprehensive profiling of ctDNA with high sensitivity (detection in 90%+ of advanced cancer pts) and ultra-high specificity (>99.9999%). Guardant360 has been validated to analyze a 69-oncogene panel covering over 120,000 bases and detects SNVs, gene amplifications, indels and genomic rearrangements, including all NCCN-guidelines recommended biomarkers. We previously reported high concordance of ctDNA profiles with concurrent matched biopsies. Results: In our first 1,000 clinical tests (77% across breast, colorectal and lung and 23% distributed over many other indications), we successfully processed 99.8% of samples. In 3 cases, we canceled the test as we detected secondary genome carry-over (confirmed to be pregnancy or liver transplant cases). Despite the wider range of clinical stages in commercial samples, we detected ctDNA in 78%. Additionally, we identified actionable mutations with associated treatments in 74% of those cases. Clinical benefit of the test was shown in many patients. Examples: A refractory IBC patient with Her2 status change, showed strong response to Her2 TKI. Patients with quantity-not-sufficient biopsy sequencing, showed strong actionable alterations (ERBB2 amplification, BRAF mutation, EGFR activating and resistance mutations, etc) Conclusion: Comprehensive sequencing of a patient's cancer in real -time through a simple blood test can empower oncologists to make better informed treatment decisions, especially when repeat tissue biopsy is not a desirable or viable option. Citation Format: Eric Collisson, Stefanie Mortimer, Dragan Sebisanovic, Reza Mokhtari, Somayeh Bakhtiari, Rene Lopez, Devi M. Gadde, Maria M. Vidamo, Heena Patel, Bahram G. Kermani, Helmy Eltoukhy, Richard B. Lanman, AmirAli Talasaz. Biopsy-free comprehensive tumor profiling of 1,000+ consecutive cancer patients using CLIA-certified commercial test and its clinical utility. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2015-2403

Background: Analysis of circulating tumor nucleic acids by massively parallel DNA sequencing has created an exciting new tool for capturing an accurate, more complete and real-time picture of a patient's tumor profile throughout the body and the course of disease. However, the widespread use of this biomarker has been limited by the high-quality false positives and non-systematic distortion present in current NGS assays, especially where the tumor-derived fraction is low ( Methods: Our differentiated sequencing assay, Digital Sequencing Technology, has enabled robust, ultra-sensitive and ultra-specific detection of rare genomic abnormalities in cell-free circulating DNA. Digital Sequencing relies on enabling single-molecule sequencing using current next-gen sequencers. Our assay is able to eliminate most of the errors and distortion introduced by these sequencers and produce near-perfect representations of all rare variants. Results: We have shown that in sequencing 0.1% cancer cell line titrated samples across a broad 80kbp cancer panel, standard Illumina sequencing generates several high-quality false positives with allele frequencies of 0.05-5%, while our novel Digital Sequencing Technology results in high sensitivity and completely error-free detection of rare variants. Furthermore, we have applied our technology to analyze rare tumor-derived cell-free DNA from more than 300 cancer patient plasma samples collected at multiple sites (various US and international locations) and across different cancer types. We investigated the concordance of tumor mutation profiles derived from circulating cell-free DNA with those derived from matched tumor biopsy samples in colorectal, breast, NSCLC, prostate and melanoma cancers across patients (Stage II-IV). We also studied the presence of de novo mutations (SNV and CNV) in these cancer patient plasma samples. In general, the range of tumor DNA in plasma was determined to be between 0.03%-85.9% in these studies. This comprehensive dataset illustrates the broad power of cell-free DNA analysis as a diagnostic biomarker in cancer patients. Conclusion: The present work shows the strong potential clinical impact of Digital Sequencing in analysis of circulating cell-free tumor-derived nucleic acids, thereby allowing researchers and clinicians to comprehensively and non-invasively monitor the genetic dimension of cancer throughout the body. Citation Format: Stefanie A. Mortimer, Dragan Sebisanovic, Gangwu Mei, Benjamin Schiller, Lai Mun Siew, Aubrey Zapanta, Helmy Eltoukhy, AmirAli Talasaz. Comprehensive non-invasive tumor sequencing: High fidelity sequencing of tumor-derived circulating cell-free DNA across 300 cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2014-3573