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Tumor marker levels and cfDNA mutant allele fractions across 19 time intervals for eight patients that were serially monitored via blood draws.

Complete patient-level and mutation-level concordance information, mutation identities, and monitoring status for each patient in study.

Comparisons of CA 19-9 concentration and cfDNA mutant allele fractions in plasma. (A-E) cfDNA mutant allele fractions ("cfDNA percentage") versus CA 19-9 units per milliliter (U/mL) were determined at similar times for five patients with three or more serial blood draws. cfDNA percentage represents the mutant allele fraction of the most abundant cfDNA mutation, as determined at time zero, for each patient. (F) cfDNA mutant allele fractions and CA 19-9 marker measurements for patient 56 over the course of four time points while on therapy (gemcitabine nab-paclitaxel). Note that draw number 1 occurred 17 days after diagnosis.

Suppementary Table S1: Gene lists and exon coverage; Supplementary Table S2: Patient Demographics; Supplementary Table S3. Mutational landscape in cfDNA; Supplementary Figure S1: Schema for identification of unique patient cases for analysis; Supplementary Figure S2. Impact of the number of mechanisms of resistance to anti-EGFR therapy on clonality and relative mutant allele frequency; Supplementary Figure S3: Clonal dynamics during anti-EGFR therapy in patient #18.

List of genes and exons captured in the Guardant360 cfDNA sequencing test.

Characteristics of the 26 patients in study.

Supplemental figure 3. Mutation frequencies in pre-and on-treatment plasma samples as determined by next-generation sequencing of cfDNA using the Guardant360 assay.

Purpose:Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies.Experimental Design:We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA).Results:A total of 153/206 (74.3%) were men; median age, 62 years (range, 18–91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1–13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%–55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20–38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04).Conclusions:This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

70 gene panel (ctDNA) (N = 120 patients)

Supplementary Figures

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.

Genes on the Guardant360 panel.

68 gene panel (ctDNA) comprising amplifications in 16 genes as well as some fusions and indels.

Noninvasive genomic profiling of tumors may be possible with next-generation sequencing (NGS) of blood-derived circulating tumor DNA (ctDNA), but proof of concept in a large cohort of patients with diverse cancers has yet to be reported. Here we report the results of an analysis of plasma-derived ctDNA from 670 patients with diverse cancers. The tumors represented in the patient cohort were mainly gastrointestinal (31.8%), brain (22.7%), or lung (20.7%). ctDNA obtained from most patients [N = 423 (63%)] displayed at least one alteration. The most frequent alterations seen, as characterized mutations or variants of unknown significance, occurred in TP53 (32.5% of patients), EGFR (13%), KRAS (12.5%), and PIK3CA (9.1%); for characterized alterations, 30.7% (TP53), 7.6% (EGFR), 12.2% (KRAS), and 7.7% (PIK3CA). We found that 32% of brain tumors had at least one ctDNA alteration. Head and neck tumors were independently associated with a higher number of alterations in a multivariable analysis (P = 0.019). Notably, 320/670 (48%) of patients displayed potentially actionable alterations, with 241 patients possible candidates for on-label or off-label treatment with an FDA-approved drug. Several illustrations of the clinical utility of the information obtained for improving treatment of specific patients is provided. Our findings demonstrate the feasibility and impact of genomic profiling of tumors by ctDNA NGS, greatly encouraging broader investigations of the application of this technology for precision medicine in cancer management. Cancer Res; 77(19); 5419–27. ©2017 AACR.

Supplemental Methods, Supplemental References, Supplemental Figure Legends, Figure S1-13, Table S1-5 and S14

Digital Sequencing analytical performance metrics summary table.

cfDNA variants associated with resistance to on-label targeted therapies across six cancer types

Supplementary Figure 2

Purpose:Detection of persistent circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who will ultimately recur. Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. We evaluated a plasma-only ctDNA assay integrating genomic and epigenomic cancer signatures to enable tumor-uninformed MRD detection.Experimental Design:A total of 252 prospective serial plasma specimens from 103 patients with colorectal cancer undergoing curative-intent surgery were analyzed and correlated with recurrence.Results:Of 103 patients, 84 [stage I (9.5%), II (23.8%), III (47.6%), IV (19%)] had evaluable plasma drawn after completion of definitive therapy, defined as surgery only (n = 39) or completion of adjuvant therapy (n = 45). In “landmark” plasma drawn 1-month (median, 31.5 days) after definitive therapy and >1 year follow-up, 15 patients had detectable ctDNA, and all 15 recurred [positive predictive value (PPV), 100%; HR, 11.28 (P < 0.0001)]. Of 49 patients without detectable ctDNA at the landmark timepoint, 12 (24.5%) recurred. Landmark recurrence sensitivity and specificity were 55.6% and 100%. Incorporating serial longitudinal and surveillance (drawn within 4 months of recurrence) samples, sensitivity improved to 69% and 91%. Integrating epigenomic signatures increased sensitivity by 25%–36% versus genomic alterations alone. Notably, standard serum carcinoembryonic antigen levels did not predict recurrence [HR, 1.84 (P = 0.18); PPV = 53.9%].Conclusions:Plasma-only MRD detection demonstrated favorable sensitivity and specificity for recurrence, comparable with tumor-informed approaches. Integrating analysis of epigenomic and genomic alterations enhanced sensitivity. These findings support the potential clinical utility of plasma-only ctDNA MRD detection.See related commentary by Bent and Kopetz, p. 5449

Supplemental figure 1. Abdominal CT scans from a patient who achieved minor response following 2 cycles of treatment. Representative sections from baseline (left) and on-treatment (right) scans are shown.

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.

Supplemental table 1

Driver alteration prevalence in the cfDNA cohort versus in TCGA

Quantitative precision of Digital Sequencing results. (a) SNV VAFs in 375 consecutive SNV control runs. (b) Indel and fusion VAFs (left y-axis) and CNA copy number (right y-axis) in consecutive CFI control runs. (c) Variation distribution of DS results in 375 consecutive analyses of a single SNV control lot (blue) and multiple consecutive lots of indel (red), fusion (grey), and CNA (orange) controls.

Detailed results of Digital Sequencing and clinical ddPCR comparison. Results shown in individual 2x2 tables for each biomarker tested. *Note, all discordances were below DFCI ddPCR's reportable range. Detailed clinical information for each is presented in Supplemental Table 3.

Purpose: On the basis of preclinical evidence of synergistic activity between MEK and EGFR inhibitors in pancreatic ductal adenocarcinoma (PDAC), we evaluated the safety and efficacy of selumetinib, a MEK1/2 inhibitor, plus erlotinib in patients with previously treated advanced PDAC.Experimental Design: In this single-arm phase II trial, eligible patients received the combination of erlotinib 100 mg plus selumetinib 100 mg daily in 3-week cycles. Study assessments included measurement of clinical outcomes, with a primary endpoint of overall survival, and exploration of potential molecular predictors of treatment benefit.Results: Forty-six patients were enrolled and received a median of two cycles (range, 1–7). Although no objective responses were observed, 19 patients (41%) showed evidence of stable disease for ≥6 weeks, and 13 of 34 patients (38%) had a CA19-9 decline ≥50%. Median progression-free survival was 1.9 months [95% confidence interval (CI), 1.4–3.3 months], with a median overall survival of 7.3 months (95% CI, 5.2–8.0 months). Common adverse events included rash, diarrhea, and nausea/vomiting. Patients with tumors exhibiting an epithelial phenotype (demonstrated by a high level of E-cadherin expression) were more likely to be sensitive to study treatment. Tumor-derived DNA was detectable in plasma from the majority of patients using next-generation digital DNA sequencing, and its relative abundance correlated with tumor burden.Conclusions: A therapeutic strategy of dual targeted inhibition of the MEK and EGFR pathways shows modest antitumor activity in pancreatic cancer. Specific molecular subtypes may derive greatest benefit from this combination. Further exploration, both with more potent MEK inhibitors and in molecularly enriched patient subsets, is warranted. Clin Cancer Res; 22(1); 61–68. ©2015 AACR.

Purpose: Genomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with non–small cell lung adenocarcinoma (NSCLC) were ascertained and correlated with clinical characteristics and therapeutic outcomes.Experimental Design: Comprehensive plasma ctDNA testing was performed in 88 consecutive patients; 34 also had tissue next-generation sequencing; 29, other forms of genotyping; and 25 (28.4%) had no tissue molecular tests because of inadequate tissue or biopsy contraindications.Results: Seventy-two patients (82%) had ≥1 ctDNA alteration(s); among these, 75% carried alteration(s) potentially actionable by FDA-approved (61.1%) or experimental drug(s) in clinical trials (additional 13.9%). The most frequent alterations were in the TP53 (44.3% of patients), EGFR (27.3%), MET (14.8%), KRAS (13.6%), and ALK (6.8%) genes. The concordance rate for EGFR alterations was 80.8% (100% vs. 61.5%; ≤1 vs. >1 month between ctDNA and tissue tests; P = 0.04) for patients with any detectable ctDNA alterations. Twenty-five patients (28.4%) received therapy matching ≥1 ctDNA alteration(s); 72.3% (N = 16/22) of the evaluable matched patients achieved stable disease ≥6 months (SD) or partial response (PR). Five patients with ctDNA-detected EGFR T790M were subsequently treated with a third generation EGFR inhibitor; all five achieved SD ≥ 6 months/PR. Patients with ≥1 alteration with ≥5% variant allele fraction (vs. < 5%) had a significantly shorter median survival (P = 0.012).Conclusions: ctDNA analysis detected alterations in the majority of patients, with potentially targetable aberrations found at expected frequencies. Therapy matched to ctDNA alterations demonstrated appreciable therapeutic efficacy, suggesting clinical utility that warrants future prospective studies. Clin Cancer Res; 23(17); 5101–11. ©2017 AACR.

54 gene panel (ctDNA) identifies potential tumor-related genomic alterations within 54 cancer-related genes

Discordant results between Digital Sequencing and clinical ddPCR. Details of the cases discordant between DS and Dana-Farber Clinical Laboratory's ddPCR results with orthogonal follow-up data. *Note, while approved specifically for T790M+ NSCLC, osimertinib has been reported to have clinical activity in T790-wild type patients as well; however, osimertinib efficacy in T790M-wild type patients resistant to 1st generation EGFR TKIs is unclear.

Supplemental figure 2. Distribution of proportion of E-cadherin expressing tumor cells across all cases.

Shows the 3 ctDNA panels, the list of alterations identified, the concordance analysis for EGFR, and a diagram on response evaluation

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.

Mutual exclusivity statistics for cfDNA alterations in lung adenocarcinoma, breast cancer, and colorectal cancer

Per-sample mutation burden distribution by tumor type. Per-sample adjusted mutation count relative to other samples within clinical indication. Green bars indicate the 95th percentile.

List of potentially actionable alterations per patient

Quantitative accuracy of Digital Sequencing. Quantitative correlation between expected and observed VAFs for SNVs (a) and CNAs (b) in the analytical validation studies. (c) DS copy number calls compared to those reported in the Cancer Cell Line Encyclopedia (CCLE).

Supplementary Tables

Supplementary Figure 1

Background: Inactivating HRR gene mutations can lead to HRR deficiency (HRD) and predict response to PARPi therapy in patients with breast cancer. Copy number loss and large genomic rearrangements (LGR) can result in HRD but are challenging to detect in ctDNA. Here, we present the analytical validation of homozygous deletions, loss of heterozygosity (LoH) and LGR detection on the Guardant360 (G360) liquid biopsy panel, previously validated for detection of small variants, copy number amplifications, and fusions. We present real-world outcomes of BRCA1/2-mutant PARPi-treated patients to demonstrate the clinical validity of the detected variants. Methods: Analytical validation was performed using the G360 83-gene ctDNA panel. Cell line DNA and clinical patient cfDNA were titrated into matched normal cell line DNA or healthy donor cfDNA to establish the limit of detection (LoD) and precision for copy number loss and LGRs, respectively. Accuracy results were compared to those from an orthogonal, externally validated tissue and ctDNA panel. De-identified, longitudinal, claims data were linked to the cancer genomic profiles in Guardant INFORM, a clinical-genomics database. Advanced PARPi treated breast cancer patients with an inactivating or reversion BRCA1/2 mutation detected by G360 were assessed. Results: The analytical sensitivity (95% LoD) for detecting homozygous and LoH deletions for deletion sizes >10MB was established at tumor fractions (TF) of 12.5% and 25%, respectively. The 95% LoD for LGRs was 0.2% variant allele fraction (VAF). The per-sample false positive rate for copy number loss and LGRs was 1000 advanced breast cancer patients was 1.8%, 16.6% and 0.25% respectively, compared to 2.4%, 56.7% and 0.3% in TCGA. To verify the clinical impact of cfDNA-detected HRR alterations, overall survival was determined for PARPi-treated patients with >1 BRCA1/2 germline or somatic SNV, indel or LGR reversion mutation to be 23.2 months [16.4, 30, CI, n=75] compared to 54.4 [28, NA, CI, n=14] months for BRCA1/2-mutant patients without a reversion (p-value=0.049). Conclusion:. This analytical validation demonstrates that G360 detection of inactivating mutations, including copy number loss and LGRs, is highly sensitive, reliable and robust. Real-world evidence analysis confirmed worse survival outcomes in PARPi treated patients harboring a BRCA1/2 reversion compared to BRCA1/2-mutant patients with no reversion. This data further supports ctDNA as a compelling non-invasive means to identify potential PARPi sensitizing and resistance mutations in patients with advanced breast cancer. Citation Format: Jennifer Yen, Leylah Drusbosky, Caroline Weipert, Nicole Zhang, David Hanna, Catalin Barbacioru, Hao Wang, Alex Artyomenko, Arielle Yablonovitch, Yu Fu, Aaron Hardin, Nagesh Alla, Robert Foley, Max Maligska, Bhargavi Panchangam, Phil Yen, Jane Meisel, Neelima Vidula, Massimo Cristofanilli, Jeremy Force, Michael Dorschner, Martina Lefterova, Elena Helman, Becky Nagy, Darya Chudova, AmirAli Talasaz. Analytical and clinical validation of a ctDNA assay for detecting copy number loss and structural rearrangement variants contributing to homologous recombination and repair (HRR) deficiency [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-29.

Purpose: Detection of persistent circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who will ultimately recur. Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. We evaluated a plasma-only ctDNA assay integrating genomic and epigenomic cancer signatures to enable tumor-uninformed MRD detection. Experimental Design: A total of 252 prospective serial plasma specimens from 103 patients with colorectal cancer undergoing curative-intent surgery were analyzed and correlated with recurrence. Results: Of 103 patients, 84 [stage I (9.5%), II (23.8%), III (47.6%), IV (19%)] had evaluable plasma drawn after completion of definitive therapy, defined as surgery only (n = 39) or completion of adjuvant therapy (n = 45). In “landmark” plasma drawn 1-month (median, 31.5 days) after definitive therapy and >1 year follow-up, 15 patients had detectable ctDNA, and all 15 recurred [positive predictive value (PPV), 100%; HR, 11.28 (P < 0.0001)]. Of 49 patients without detectable ctDNA at the landmark timepoint, 12 (24.5%) recurred. Landmark recurrence sensitivity and specificity were 55.6% and 100%. Incorporating serial longitudinal and surveillance (drawn within 4 months of recurrence) samples, sensitivity improved to 69% and 91%. Integrating epigenomic signatures increased sensitivity by 25%–36% versus genomic alterations alone. Notably, standard serum carcinoembryonic antigen levels did not predict recurrence [HR, 1.84 (P = 0.18); PPV = 53.9%]. Conclusions: Plasma-only MRD detection demonstrated favorable sensitivity and specificity for recurrence, comparable with tumor-informed approaches. Integrating analysis of epigenomic and genomic alterations enhanced sensitivity. These findings support the potential clinical utility of plasma-only ctDNA MRD detection. See related commentary by Bent and Kopetz, p. 5449

The VIKTORY (targeted agent eValuation In gastric cancer basket KORea) trial was designed to classify patients with metastatic gastric cancer based on clinical sequencing and focused on eight different biomarker groups (RAS aberration, TP53 mutation, PIK3CA mutation/amplification, MET amplification, MET overexpression, all negative, TSC2 deficient, or RICTOR amplification) to assign patients to one of the 10 associated clinical trials in second-line (2L) treatment. Capivasertib (AKT inhibitor), savolitinib (MET inhibitor), selumetinib (MEK inhibitor), adavosertib (WEE1 inhibitor), and vistusertib (TORC inhibitor) were tested with or without chemotherapy. Seven hundred seventy-two patients with gastric cancer were enrolled, and sequencing was successfully achieved in 715 patients (92.6%). When molecular screening was linked to seamless immediate access to parallel matched trials, 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional 2L chemotherapy. Circulating tumor (ctDNA) analysis demonstrated good correlation between high MET copy number by ctDNA and response to savolitinib. Significance: Prospective clinical sequencing revealed that baseline heterogeneity between tumor samples from different patients affected response to biomarker-selected therapies. VIKTORY is the first and largest platform study in gastric cancer and supports both the feasibility of tumor profiling and its clinical utility. This article is highlighted in the In This Issue feature, p. 1325

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.

Background: Several computational methods have been developed to identify copy number alterations (CNA) leading to or associated with cancer development and shown in recent studies to precede cancer diagnosis by many years. Current methods involving cell-free DNA (cfDNA) targeted sequencing data are based on the depth of coverage of on-target or off-target regions, whereas computational methods incorporating germline SNP information for making inferences on copy number alterations and tumor fraction remain underdeveloped. Methods: Using sequencing data from a large database of more than 100k clinical cell-free DNA (cfDNA) patient samples (Guardant Health, CA), we developed a probabilistic model to simultaneously normalize molecular coverage, segment the genome, predict copy number alterations, and estimate the tumor content in cfDNA samples, while accounting for mixtures of cell populations. The model is using off-target and on-target coverage. Copy number status, including loss of heterozygosity (LoH), is inferred in order to predict gene level somatic CNAs or genome wide instability/LoH. Results: We demonstrated the improvement from the off-target incorporation in three aspects. First, to estimate sensitivity improvement in detections of CNAs, we simulated deletions and amplifications of regions exceeding 40 Mb, using coverage and MAF variability observed in existing data. Combining coverage of on-target and off-target regions is expected to improve the LoD for detection of CNAs by 20%, when compared to CNA detection from on-target coverage. Next, we obtained samples from 15,618 cancer patients of different cancer types processed on GuardantOMNI® RUO and determined human leukocyte antigen (HLA) allele-specific copy number using this off-target assisted method. We observed a high prevalence (more than 15%) of LoH in HLA in bladder cancer, prostate cancer, NSCLC and HNSC, consistent with previous studies that HLA LOH is a common feature of several cancer types and diminishes immunotherapy efficacy. Finally, tumor fraction (TF) estimate was validated by comparing the TF against the maximum variant allele fraction of known oncogenic driver mutations in 6,000 cancer cases of various types. High concordance was observed in CRC samples (R2=0.75), gastric cancer (R2=0.63) and bladder cancer (R2=0.6), which suggest the use of this metric to better estimate tumor shedding levels in cfDNA in cases when driver mutations are not represented on a targeting panel. Conclusion: Our results show that probabilistic modeling of coverage data generated from both on-target and off-target cfDNA sequencing can detect gene specific or whole genome level somatic copy number alterations and LoH. This method may enable improvements in CNA detection accuracy, sensitivity, and specificity in plasma and provides more precise interrogation of LoH status and tumor fraction. Citation Format: Catalin Barbacioru, Han-Yu Chuang, Rebecca Nagy, Darya Chudova, AmirAli Talasaz. Detection of somatic copy number alterations from on-target and off-target sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 571.

Background: NTRK rearrangements, though rare in common cancers, are clinically actionable targets with two FDA-approved drugs for pan-cancer indications, larotrectinib and entrectinib. Fusion detection from ctDNA provides an opportunity to facilitate screening for this biomarker, though technical challenges such as heterogeneity in fusion partners remain. To address this, we developed an assembly-based de novo fusion algorithm that does not rely on a fixed set of partner genes and applied it to >18,000 clinical samples to detect NTRK1 fusions. Methods: A cohort of 18,867 patients across multiple cancer types (lung adenocarcinoma, breast cancer, and colorectal adenocarcinoma), plus 276 healthy control samples were previously tested with Guardant360(R), a CLIA-validated 74-gene cfDNA NGS-based assay. The median unique molecule coverage was approximately 3,000 molecules sequenced to 15,000x read depth. Samples were reanalyzed in silico using the de novo fusion algorithm: in brief, reads aligned to candidate fusion breakpoints were assembled into de Bruijn graphs. Resulting contigs were aligned to the reference and analytical filters were applied to achieve high specificity. Results: NTRK1 fusions were detected in 0.13% of patients, a similar prevalence to what has been previously described in tissue (Rosen et al., 2020). No fusions were identified in 276 healthy control samples. When examining intergenic fusions, 53% of fusions included previously characterized partner genes (TPM3, TPR, LMNA, and TP53), while the remaining fusions contained novel partners.The majority of NTRK1 fusion partners (76%) were detected only once, consistent with previous studies. KRAS G12D and BRAF V600E were detected in 17% and 13% of NTRK1 fusion-positive patients, respectively, and occurred predominantly in the colorectal cohort; most variants (60%) were subclonal to the NTRK1 fusion, and have been previously shown to be associated with resistance to TRK inhibitors through downstream activation of the MAPK pathway. The on-target resistance variant NTRK1 G595R was detected in 8% of patients positive for NTRK1 fusions, all of which were subclonal to the fusion. EGFR L858R was detected in 13% of NTRK1 fusion-positive patients, all of which were clonal to the fusion and in the lung cohort, in accordance with previous studies showing NTRK1 fusions as a resistance mechanism to TKIs in EGFR-positive NSCLC (Xia et al., 2020). Conclusions: NTRK1 fusions were detected in cfDNA at a similar prevalence to tissue NGS, demonstrating high sensitivity of plasma-based assays to detect these fusions. NTRK1 fusion partners were diverse, with the majority of partner genes observed only once across the cohort. Both clonal and subclonal NTRK1 rearrangements were detected, affirming that this biomarker can emerge as an oncogenic driver or as a mechanism of resistance. Citation Format: Arielle Yablonovitch, Sante Gnerre, Jennifer Yen, Scott Shell, Elena Helman, Stephen Fairclough, Rebecca J. Nagy, Justin Odegaard, Darya Chudova, AmirAli Talasaz. NTRK1 fusion detection from clinical cfDNA NGS using a de novo fusion caller [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 537.

Background: Molecular response (MR) estimated as a change in circulating tumor (ctDNA) load between an early on treatment sample (usually 2-9 weeks post treatment start) and pre-treatment baseline has been shown to predict patient response and outcomes across solid tumors and therapy types in many retrospective studies. There is no consensus, however, regarding the best method for assessing molecular response. Therefore, we aimed to assess several molecular response calculations and determine the optimal method for predicting outcomes in individual advanced cancer patients. Method: Aggregate results of > 4,000 patient sample pairs (3-10 weeks apart), > 1000 patient sample technical replicates, > 100 contrived sample dilutions, and in silico simulations were analyzed with Guardant360TM or GuardantOMNITM. Baseline and on-treatment paired patient samples were collected from advanced cancer patients with over 12 tumor types, including lung, colon, and breast. MR calculations included variant allele fractions (VAFs) of somatic SNVs, indels and fusions. Methods including Ratio treatment/baseline (R) of Maximum VAF (maxVAF), Ratio of Mean VAF (mVAF), and Mean of VAF Ratios were compared, with consideration of VAF precision. Analytical accuracy, reproducibility and limit of detection (LoD) were assessed. Results: Comparison of methods for calculating net change in ctDNA load on > 1500 sample pairs showed high correlation (ρ ranged from 0.93 to 0.98) and categorical agreement split by the median (93%). Therefore selecting an optimal method based on outcome prediction would require prohibitively large patient cohorts. Analytical evaluation and in silico simulations can predict the behavior of each method. Simulations of changes in tumor fraction of real pre-treatment samples found that RmVAF or RmaxVAF are more accurate than mVAFR, which can be skewed by low VAF ratios. Almost 25% of sample pairs have a tumor driver or resistance mutation that is not the maxVAF, suggesting tumor dynamics are better captured by mVAF than maxVAF. Newly-detected on-treatment variants can be an important signal of rising ctDNA levels, impacting MR in approximately 2% of sample pairs. Importantly, MR accuracy for all methods decreases as maxVAF approaches or falls below the variant LoD, due to both stochastic detection and higher CV of variants at low VAF. Thus the assay variant LoD is a key determinant of the fraction of patients who can receive MR evaluation. Technical replicates identified the variant criteria at which a 50% change in tumor fraction differs significantly from technical variation, and could define analytical reporting limits. Conclusions: Comparison of MR methods in a large set of patient samples and simulations supports RmVAF with inclusion of newly-detected mutations. Clinical validation of these methods will support patient-specific MR prediction of outcomes. Citation Format: Allysia J. Mak, Katie J. Quinn, Carin Espenschied, Kyle Chang, Han-Yu Chuang, Elena Helman, Darya Chudova, Justin Odegaard, Becky Nagy, AmirAli Talasaz. Comparison of molecular response calculations for prediction of patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 401.

Introduction: A novel non-invasive blood-based cell-free DNA (cfDNA) analysis incorporating genomic and epigenomic assessment has demonstrated high sensitivity and specificity in patients with newly diagnosed colorectal cancer (CRC) (Kim, et al. CCR, 2019). Using an improved epigenomic analysis, we tested a new cohort of individuals with either a negative colonoscopy for advanced neoplasia (CRC or advanced adenoma) or newly diagnosed early-stage CRC. Methods: Whole blood samples were collected from 162 patients with a known diagnosis of CRC (pre-operative; 20 Stage I; 98 Stage II; 39 Stage II; 5 Stage IV), 38 self-declared cancer-free donors, and 205 individuals who were screen-negative for advanced neoplasia by colonoscopy. 5-8mL of plasma was isolated and total cfDNA was extracted and partitioned based on methylation level. Sequencing libraries were prepared and enriched using an integrated genomic and epigenomic cfDNA panel (Guardant Health, Redwood City, CA, USA). Sequencing results were used to analyze genomic, methylation, and fragmentomic signals. By using a thermodynamic model to approximate the physical binding process of methylation partitioning, modeling cfDNA fragmentation with increased resolution, and training on colonoscopy screen-negative samples, we improved the performance of the cfDNA epigenomic analysis. A training cohort of 117 colonoscopy screen-negative controls, 38 self-declared cancer-free controls, and 49 CRC patients (10 Stage I; 21 Stage II; 13 Stage III; 5 Stage IV) was used to train a linear model to combine these multimodal signals. The same cohort was used to establish the classification threshold prior to generating results for any of the validation samples. Results: The assay performance was tested on a blinded held-out cohort of 113 CRC patients (10 Stage I; 77 Stage II; 26 Stage III) and 88 colonoscopy screen-negative controls. Median age was 66 years (39-86) for CRC patients (52% female) and 57 years (20-84) in the colonoscopy screen-negative cohort (73% female). Overall sensitivity for CRC detection was 90.3% (90% Stage I; 88% Stage II; 96% Stage III) and specificity was 96.6%. We define a per sample ‘signal-to-noise-ratio' (SNR) score as the number of standard deviations (SD) from the mean model score observed in the colonoscopy screen-negative cohort. In these terms, our cfDNA detection threshold is 3.8 SD above the mean. The median SNR observed per stage was 6.8 SD (1.2 - 8.6) for Stage I, 6.9 SD (-0.18 - 8.6) for Stage II, and 7.3 SD (2.4 - 8.6) for Stage III. Conclusion: These results provide further support that an integrated genomic and epigenomic cfDNA assay consistently provides sufficient sensitivity and specificity for clinical detection of early-stage CRC. A prospective CRC screening study in a larger cohort of participants is needed to further validate assay performance. Citation Format: Oscar Westesson, Haley Axelrod, Jason Dean, Yupeng He, Paul Sample, Elena Zotenko, Ruth McCole, Mohit Goel, Charbel Eid, Jessica Kurata, Yu Kong, Anna Hartwig, Matthew Snyder, Will Greenleaf, Victoria M. Raymond, Darya Chudova, Ariel Jaimovich, AmirAli Talasaz. Integrated genomic and epigenomic cell-free DNA (cfDNA) analysis for the detection of early-stage colorectal cancer [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 2316.

Background: PARP inhibition can cause synthetic lethality and increased therapeutic sensitivity in patients with HRR deficiency (HRD), which can be detected through the molecular profiling of HRR genes. Prostate cancer has a high prevalence of HRD (20-25%, Athie 2019). High failure rates for tissue biopsy in metastatic prostate cancer patients (25-75%) (Ross 2005, Spritzer 2013) pose challenges for HRD profiling, underscoring the need for a non-invasive, ctDNA alternative. Copy number loss, a frequent cause of HRD, is further difficult to call due to signal dilution by cell-free leukocytic DNA. We developed a pipeline that detects loss-of-function SNV/Indels, structural rearrangements, and gene deletions to identify HRD on GuardantOMNITM, a 500-gene liquid biopsy panel. We present its performance across >620 prostate cancer GuardantOMNITM samples. Methods: Samples from 627 prostate cancer patients were processed on GuardantOMNITM RUO, with median unique coverage of 4982 molecules sequenced to 20,000x read depth. Somatic and germline SNVs and small indels were called using the Guardant bioinformatics pipeline (Helman 2018). A novel HRD module was developed to annotate pathogenic SNV/Indels and identify structural rearrangements, gene-level homozygous deletions, loss-of-heterozygosity (LOH) and genome-wide LOH, comprising of a novel CNV (Barbacioru 2019) and de-novo fusion caller (Gnerre, submitted). Loss-of-function variants were analyzed in 24 HRD genes. Results: Pathogenic alterations in HRD genes were called in 260/620 (42%) prostate cancer samples with ctDNA detected: 28% of all samples had a pathogenic somatic or germline SNV/Indel, 20% had a homozygous deletion, 3.4% had a rearrangement involving an HRD gene. The majority of SNV/Indels occurred in BRCA2 (31% of all 158 SNV/Indels) and ATM (22%), similar to tissue (Dahwan 2016), but mutations also occurred across an additional 21 genes, including CDK12 (8%), CHEK2 (5%) and NBN (3.8%). Of prostate patients with a germline BRCA1/2 SNV/Indel and sufficient tumor shedding for LOH detection (max MAF>10%), 10/19 (52%) also had LOH, compared to 86% in tissue (Jonsson 2019). Homozygous deletions were enriched in BRCA2 (6.9% of all samples), ATM (4.4%) and CHEK1 (2.3%). Rearrangements, including fusions and multi-exonic deletions, accounted for 6.5% of inactivating HRD mutations detected. In total, 24% of prostate samples had a biallelic inactivation involving an SNV, Indel or deletion. Conclusion: We demonstrate in a prostate cancer cohort that GuardantOMNITM ctDNA profiling calls all classes of mutations contributing to HRD, with relative prevalence of alterations consistent with those in tissue. CfDNA presents a potential alternative for identifying patients who may benefit from PARP or cisplatin/platinum therapies, expanding the prevalence from 28% using small variants to 42% with the complete HRD biomarker set. Citation Format: Jennifer Yen, Sante Gnerre, Catalin Barbacioru, Elena Helman, Ohad Manor, Arielle Yablonovitch, Ravi Vijaya Satya, Leo Liu, Jennifer Saam, Stephen Fairclough, Becky Nagy, Richard Lanman, Darya Chudova, AmirAli Talasaz. Landscape of homologous recombination repair (HRR) mutations in prostate cancer profiled by ctDNA next-generation 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 729.

Background: In addition to BRCA1/2 germline and somatic inactivating mutations, loss of heterozygosity (LOH) and biallellic somatic copy number loss are associated with BRCA1/2 loss of function. Patients with cancers harboring these somatic features may benefit from treatment with PARP inhibitors. ctDNA analysis has proven a viable alternative to tumor tissue genotyping, especially in tissue- or time-limited clinical scenarios. However, ctDNA assessment of LOH and biallelic copy number loss is challenging given that ctDNA is diluted by cell-free leukocytic DNA making it difficult to confidently call these genomic events. We developed a method to identify somatic biallelic copy number loss in ctDNA using targeted sequencing of cell-free (cfDNA) across a wide range of cancer types. Methods: A novel statistical model was developed using coverage profiles and single nucleotide polymorphism (SNP) allelic frequencies estimated from plasma samples to determine the presence of LOH and biallellic copy number loss in BRCA1 or BRCA2. For each gene of interest, the model uses observed coverage and fragment size distribution, together with allele frequency of germline SNPs. The model was applied to plasma samples from 28,000 patients with advanced solid tumors sequenced using a 73-gene next generation sequencing ctDNA panel (Guardant360®, Guardant Health, Redwood City, CA). Results: The model was analytically validated using in-silico simulations in order to assess both the limit of blank and limit of detection. This method shows 95% sensitivity in detecting LOH and bi-allellic copy number loss for samples with a maximum somatic variant allele frequency as low as 9%. Sensitivity is mainly driven by the panel size and the depth of coverage of the gene of interest. The model was then applied to the 28,199 patient cohort. BRCA1 and BRCA2 LOH was observed in 2.4% (134/5568) and 7.4% (415/5568) of classic homologous recombination deficient (HRD) cancers including breast, ovarian, prostate, and pancreas. BRCA1 and BRCA2biallelic copy number loss was observed in 0.3% (19/5568) and 0.6% (31/5568) of this same group of HRD cancers. Discussion: In this cohort of 5,568 patients with classic HRD associated cancers, somatic LOH and biallelic copy number loss was detected in BRCA1 in 2.7% of samples and in BRCA2 in 8.0% of samples, which is aligned with previously reported tissue prevalence. BRCA1/2 somatic LOH and biallelic copy number loss can accurately be detected in ctDNA utilizing likelihood-based scores based on coverage and germline SNPs allele frequencies. The ability to identify this therapeutically targetable genomic alteration through a non-invasive ctDNA assessment has significant clinical implications. Citation Format: Catalin Barbacioru, Victoria M. Raymond, Marcin Sikora, Elena Helman, Sante Gnerre, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes [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 435.

Background: Clonal hematopoiesis (CH) is the acquisition of mutations in hematopoietic progenitor cells that can lead to clonal expansion. Recent studies suggest that CH-derived mutations can confound interpretation of cell-free DNA (cfDNA) sequencing results. To better understand the contribution of CH to cfDNA analysis in the metastatic cancer setting, we characterized CH-associated alterations observed in the cfDNA of late-stage cancer patients. Methods: We analyzed somatic variants from cfDNA profiles of over 62,000 patients in four late-stage cohorts: lung (>35,000), gastro-intestinal (>14,000), urogenital (>4,700), breast (>8,600). Matched white blood cell (WBC) and cfDNA was obtained for a subset of patient samples. Plasma cfDNA was processed using a 73-gene (150Kb, Guardant360TM) or 500-gene (2Mb, GuardantOMNITM) panel and sequenced to average depth of ~5000 molecules, with a 95% limit of detection (LOD) of 0.3-0.4% and 0.15-0.6% variant allele fraction (VAF) for SNVs, and 0.2%-0.7% and 0.4-0.8% for indels (for G360 and OMNI, respectively). Results: In samples processed on the 500-gene panel, DNMT3A, TET2, PPM1D, ASXL1 and SF3B1 were the most frequently mutated CH-associated genes (75% of samples). While the majority (73%) of these mutations were at low variant allele fractions (VAF) of 60,000 samples, JAK2 V617F, a known CH mutation, was observed in 927 samples with VAFs between 0.29% and 98%. Across the combined cohort, we found that in samples with a known CH variant (n=7,717), half of samples had a max CH VAF less than 50% of max tumor VAF. Interestingly, in >25% of samples, the max CH VAF was higher than the max tumor VAF. Initial comparisons of matched plasma and WBC DNA show that 100% (16/16) of clinically relevant and 92% (84/91) of variants of unknown significance (VUSs) across the 73-gene panel were found exclusively in plasma DNA and not in WBC DNA testing. Conclusions: We characterize the distribution of CH mutations across a large number of late-stage plasma samples and show that within highly pre-treated metastatic patients, the VAFs of CH mutations often differ from the tumor and can surpass the level of tumor shedding in circulation. Further investigation of these variants will enable improved understanding of CH in metastatic disease and its differentiation from the circulating tumor DNA. Citation Format: Jennifer Yen, Katie Quinn, Elena Helman, Andrey Chursov, Tracy Nance, Ariel Jaimovich, Kimberly Banks, Aleksandra Franovic, Kristin Gleitsman, John Latham, Arielle Yablonovitch, Marcin Sikora, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Analysis of clonal hematopoiesis-associated mutations in the cell-free DNA of advanced cancer patients [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 2509.

Background: Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitor (ICI) therapy. Current panel-based TMB algorithms aggregate signal from certain types of somatic variants (e.g. non-synonymous coding SNVs); however, delineating the contributions of these and other types of mutations may refine TMB calculation from gene panels. Moreover, early studies suggest other possible genomic correlates of patient outcome to ICI which may be complementary to TMB. Here, we explore the landscape of mutations comprising TMB and other genomic features correlating with TMB on a subset of several thousand late-stage plasma samples run on GuardantOMNITM (OMNI), a highly sensitive 500-gene cfDNA sequencing platform. Methods: We developed a cfDNA-based TMB algorithm which is robust to variable tumor shedding levels and presence of clonal hematopoiesis. We assessed cfDNA-based TMB in over 1,000 plasma samples across six tumor types, including lung and prostate. We examine the contribution of nonsynonymous, synonymous, intronic SNVs, and indels to TMB score. We investigate correlations between TMB and additional genomic features, including chromosomal instability, loss of HLA-bearing chromosome 6p, microsatellite instability (MSI), and common oncogenic and resistance mutations. Results: We found that the distribution of WES-calibrated TMB scores across this cohort of samples is consistent with TCGA, with median 10 mutations/Mb and upper-tertile of 14 mutations/Mb across tumor types. The number of non-synonymous coding SNVs per sample correlated highly with synonymous coding SNV and intronic SNV counts (Pearson’s r > 0.7 for each). Including this additional signal in TMB calculation improves clinical sensitivity by up to 5%. In MSS samples, indels were highly correlated with SNVs, indicating that both likely arise from a similar underlying mechanism. We found no clear correlation between high TMB and chromosomal instability, with high TMB samples exemplifying a range of tumor ploidies. TMB association with oncogenic drivers is consistent with existing literature, with lower median TMB in EGFR-driven lung tumors (p < 0.01), but little to no correlation between TMB and KRAS or PIK3CA driver status, or STK11 loss of function (p > 0.05), suggesting these latter events could be independent clinical biomarkers to TMB. Conclusions: Panel-based TMB scores can leverage synonymous and non-coding mutations to strengthen the signal of exome-wide mutation load. As more patient outcome data becomes available, TMB algorithms and orthogonal biomarkers of tumor genome immunogenicity will evolve further for improved guidance of patient response to immunotherapy. Sequencing panels with high sensitivity for TMB, via large panel space, and the ability to detect copy-number variations and MSI-status, will be important for biomarker development and clinical applications. Citation Format: Katie Quinn, Elena Helman, Tracy Nance, Jennifer Yen, John Latham, Kristin Gleitsman, Ravi Vijaya-Satya, Carlo Artieri, Alex Artyomenko, Marcin Sikora, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Landscape and genomic correlates of ctDNA-based tumor mutational burden across six solid tumor types [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 3404.