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IntroductionThe protective effect of previous infection versus vaccination is poorly studied. Among a clinical laboratory that has been conducting routine workforce screening since the beginning of the pandemic, we aimed to assess the relative risk of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection among individuals who were SARS-CoV-2 naïve, previously infected, or vaccinated.MethodsUsing an electronic laboratory information system, employees were divided into three groups: (1) SARS-CoV-2 naïve and unvaccinated, (2) previous SARS-CoV-2 infection, and (3) vaccinated. Person-days were measured from the date of the employee first test and truncated at the end of the observation period. SARS-CoV-2 infection was defined as two positive SARS-CoV-2 PCR tests in a 30-day period. Individuals with fewer than 14 days of follow up were excluded. Incidence estimates and the 95% confidence intervals were calculated using the Poisson Exact equation. The incidence rate ratio (IRR) was used as a measure of association between groups. Analyses were performed on StataSE (StataCorp, College Station, TX).ResultsWe identified 4313, 254 and 739 employee records for groups 1, 2, and 3, respectively. The median age of employees was 29.0 years (interquartile range: 23.6, 39.9). During the observation period, 254, 0, and 4 infections were identified among groups 1, 2, and 3, respectively. Group 1 had an incidence of 25.9 per 100 person-years (95% CI: 22.8-29.3). Group 2 had an incidence of 0 per 100 person-years (95% CI: 0-5.0). Group 3 had an incidence of 1.6 per 100 person-years (95% CI: 0.04-4.2). The IRR of reinfection among those with previous infection compared to SARS-CoV-2 naïve was 0 (95% CI: 0-0.19). The IRR of those vaccinated compared to SARS-CoV-2 naïve was 0.06 (95% CI: 0.02-0.16). The IRR of those vaccinated compared to prior SARS-CoV-2 was 0 (95% CI: 0-4.98).ConclusionPrevious SARS-CoV-2 infection and vaccination for SARS-CoV-2 were associated with decreased risk for infection or re-infection with SARS-CoV-2 in a routinely screened workforce. The was no difference in the infection incidence between vaccinated individuals and individuals with previous infection. Further research is needed to determine whether our results are consistent with the emergence of new SARS-CoV-2 variants.

e15549 Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.

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.

3057 Background: ctDNA sequencing has been rapidly adopted for the identification of targetable somatic alterations (alts) in patients with advanced cancers. However, early stage disease detection has been hindered by low levels of ctDNA in circulation and the presence of confounding non-tumor-related somatic alts. We developed and validated a ctDNA assay that combines somatic and epigenomic signals to detect early stage tumors without tumor tissue or white blood cells (WBC). Methods: Using a single input sample, our assay integrates the sensitive detection of genomic alts with quantification of epigenomic signals associated with cancer. Non-tumor alts (e.g., clonal hematopoiesis of indeterminate potential; CHIP) are excluded using a newly developed bioinformatic classifier. To assess analytical sensitivity, specificity, and positive and negative reproducibility, we tested 337 clinical and contrived samples. Results: Clinical specificity was determined using 80 plasma samples from 50-75 year old presumptive cancer-free donors, and resulted in a single false positive (99% specificity). Analytical sensitivity (limit of detection) was established using a dilution series of 4 different late stage CRC pts tested in triplicate at a clinically relevant DNA input (30 ng) across multiple batches. 100% sensitivity was maintained even at the lowest tested level (estimated 0.1% tumor level). Positive/negative reproducibility was assessed by testing triplicates of diluted late stage samples and age-matched healthy donors, respectively, across different cfDNA inputs, and multiple reagent lots. Both positive and negative calls were 100% concordant across all replicates. Independent estimation of tumor levels from epigenomic or genomic signals produced highly concordant results (correlation r-value: 0.82, p-value: 3e-16). Conclusions: We designed and validated a highly specific ctDNA assay that integrates both genomic and epigenomic signals to allow for accurate and quantitative tumor level detection in early stages of the disease without requiring tumor tissue or WBC.

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

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.

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.