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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.

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

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

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

List of potentially actionable alterations per patient

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.

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

3627 Background: Blood-based colorectal cancer (CRC) screening tests can improve adherence to screening guidelines. Yet, current commercially available options have poor sensitivity and specificity inhibiting incorporation into routine clinical care. Here we report the validation of a blood-based test for the detection of colorectal cancer and advanced neoplasia. Methods: This blood-based test aims to detect colorectal neoplasia by identifying tumor-associated biomarkers including genomic or epigenomic (methylation and fragmentomics) signatures in cell-free DNA (cfDNA). cfDNA is partitioned based on methylation level, enriched for informative genomic regions, and sequenced. This novel workflow enables high-fidelity analysis of multi-modal information in majority of extracted cfDNA molecules. Results are integrated into a binary “detected” versus “not-detected” result using a proprietary bioinformatic pipeline (Guardant Health, USA). The assay was trained on samples obtained from >6,000 unique individuals (2,685 cancer-free and 1,698 with advanced colorectal neoplasia (ACN) for training, 1,072 cancer-free and 551 with ACN for threshold setting). The thresholds were frozen prior to validation targeting a specificity of > 91.5%. Each aspect of the validation study followed Nex-StoCT CLIA working group and CLSI guidelines. Results: Limit of detection (LoD) was established across six dilutions. Even for low cfDNA mass inputs of less than 4ng, the 95% LoD was determined to be less than 1 tumor-derived genomic equivalent (0.5), indicating over at least 10-fold increase in assay sensitivity compared to best-in-class assays for somatic mutation detection. Precision studies in 60 positive and negative replicates from clinical samples yielded >90% average positive and negative percent agreement both within and between batches. Endogenous interference studies yielded > 90% positive and negative percent agreement between reference control and common endogenous substances, including albumin, bilirubin, hemoglobin, triglycerides, and genomic DNA, in clinical positive and negative samples and minimally manipulated samples. The clinical validation of the test was conducted in > 300 cases (biobanked pre-operative cohort for CRC cases and screening cohort for advanced adenoma and negative cases). Conclusions: Here we present the validation of a multi-modal blood-based test for the detection of colorectal cancer. This test is currently being evaluated in a registrational study (ECLIPSE: NCT04136002).

Purpose Hotspot blood cell-free DNA (cfDNA) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma. We investigated the utility of a sensitive 54–cancer gene digital next-generation sequencing approach targeting blood cfDNA single nucleotide variants (SNVs) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis (DOM). Patients and Methods A total of 142 blood samples were evaluated by digital next-generation sequencing across two patient cohorts. Cohort 1 contained 44 patients with stage II, III, or IV disease with matched tumor DNA at the time of surgery or DOM. Cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to DOM. Results In cohort 1, cfDNA SNVs were detected in 75% of patients. Tumor-cfDNA somatic SNV concordance was 85% at a variant allele fraction of ≥ 0.5%. An SNV load (number of unique SNVs detected) of greater than two SNVs and an SNV burden (total cumulative SNV VAF) of > 0.5% were significantly associated with worse overall survival ( P < .05) in stage IV patients. In cohort 2, 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage III patients were analyzed before complete lymph node dissection and throughout disease progression. cfDNA SNV levels correlated with tumor burden ( P = .019), enabled earlier detection of recurrence compared with radiologic imaging ( P < .01), captured tumor heterogeneity, and identified increasing SNVs levels before recurrence. Conclusion This study demonstrates significant utility for cfDNA profiling in patients with melanoma with regional and/or distant metastasis for earlier detection of recurrence and progression and in capturing tumor evolution and heterogeneity, thus impacting how patients with melanoma are monitored.

This article reports unique aspects of the management of hepatocellular carcinoma. The study aimed to determine if next‐generation sequencing of blood‐derived circulating tumor DNA from patients with hepatocellular carcinoma could identify actionable somatic molecular alterations. Illustrative examples of treated patients and of in silico molecular dynamic simulation to reveal genomic variant function are included., Background. Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood‐derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC. Materials and Methods. We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild‐type allele fraction was calculated. Results. All patients (100%) had somatic alterations (median = 3 alterations/patient [range, 1–8]); median mutant allele fraction, 0.29% (range, 0.1%–37.77%). Mutations were identified in several genes: TP53 (57% of patients), CTNNB1 (29%), PTEN (7%), CDKN2A (7%), ARID1A (7%), and MET (7%); amplifications, in CDK6 (14%), EGFR (14%), MYC (14%), BRAF (7%), RAF1 (7%), FGFR1 (7%), CCNE1 (7%), PIK3CA (7%), and ERBB2/HER2 (7%). Eleven patients (79%) had ≥1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a CDKN2A‐inactivating and a CTNNB1‐activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX‐2/Wnt inhibitor); des‐gamma‐carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ≤7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a PTEN‐inactivating and a MET‐activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0–15 ng/mL]). Conclusion. ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC. Implications for Practice. This study reports that blood‐derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy.

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.

3045 Background: Detection of minimal residual disease (MRD) by circulating tumor DNA (ctDNA) post-curative intent treatment is predictive of recurrence in many solid tumors. Due to biological challenges with low ctDNA shed in early-stage disease and potential to detect non-tumor derived alterations in plasma (e.g. CHIP), most ctDNA MRD assays are dependent on a priori knowledge of genomic alterations from tumor tissue to achieve high sensitivity and specificity. Tissue-dependent methods limit the clinical application of a MRD assay, especially in cancer types where tissue biopsy is challenging, neoadjuvant therapy (NAT) is standard of care, and/or rapid turnaround times are needed for clinical decision making. We previously validated an assay (Guardant Reveal) that combines somatic and epigenomic analysis to detect ctDNA from early-stage colorectal tumors without tumor tissue or peripheral blood cells. Here we describe the expansion of this assay to detect MRD across multiple tumor types. Methods: Cell-free DNA (cfDNA) fragments are extracted from patient plasma, partitioned based on methylation fraction, enriched using a panel to target informative genomic and epigenomic regions, barcoded, and pooled for sequencing. Methylation status is determined non-destructively and with minimal loss of molecules, allowing sensitive genomic and epigenomic analysis of the same cfDNA fragments. A single assay with a total panel size of 5.3 Mb was developed for MRD analysis in multiple cancer types, including CRC, Lung and Bladder. A “ctDNA detected” result is defined by the de novo identification of tumor-derived somatic variants and/or the observation of a tumor-specific methylation profile exceeding predefined thresholds. Results: The assay performance was tested using 163 pre-treatment clinical samples from patients with early-stage non small cell lung cancer (NSCLC) and bladder cancer and 133 self-declared healthy donors. Sensitivity for pre-treatment detection in NSCLC was 68.9% at 95% specificity (20/42 Stage I: 47.6%, 25/30 Stage II: 83.3%, 19/21 Stage III: 90.5%). Sensitivity for bladder cancer was 44.2% at 95% specificity (13/43 NMIBC: 30.2%, 18/27 MIBC: 66.7%). Additional development from larger cohorts and other tumor types is ongoing and data will be presented as available. Conclusions: Using cancer-specific genomic and epigenomic signals combined with learning-based classifiers, we developed a highly specific method for detecting the presence of ctDNA in early-stage NSCLC and bladder cancer patients from plasma without the need for tumor tissue but with comparable sensitivities to tissue-dependent approaches. A plasma-only MRD assay will allow for greater clinical impact by overcoming the challenges of tissue procurement, particularly following NAT, and enabling faster time to results.

3536 Background: To improve average risk CRC screening compliance, additional options are needed, especially options that address patient and provider reported barriers such as time and convenience. LUNAR-2 is a multimodal blood-based colorectal neoplasia detection assay incorporating ctDNA assessment of somatic mutations and tumor derived methylation and fragmentomic patterns, aimed to maximize sensitivity for early stage CRC detection. We evaluated this test in a large patient cohort with newly diagnosed CRC. Methods: Individuals diagnosed with CRC between 2013-2016 consented to provide blood samples prior to surgical resection. Those treated with neoadjuvant chemotherapy were excluded. Isolated plasma samples (median 3mL from EDTA) from 434 individuals were analyzed with LUNAR-2 (Guardant Health, USA) and included in the analysis. Median age at CRC diagnosis was 63 years (range 28 - 89) and 41% were female. Control samples were from 271 age-matched cancer free individuals. “ctDNA detected” and “ctDNA not detected” results were generated by a model trained on a separate sample set (N=614) from both cancer free individuals and those with CRC. Calling threshold was determined based on this held-out set to target 90% specificity. ctDNA results and clinical characteristics were correlated. Results: Overall CRC sensitivity was 91% (393/434), with high sensitivity across all stages; 88% Stage I/II, 93% Stage III (Table). Specificity was 94% (255/271). There was no difference in sensitivity when excluding those with early (84 years) onset CRC (90% sensitivity; 388/429; p=0.95; 88% Stage I/II, 93% Stage III). There were no differences in sensitivity for asymptomatic CRC (88%) compared to symptomatic CRC (91%; p=0.4; Table). However, higher cell-free DNA tumor fractions were observed in the symptomatic cohort. Sensitivity for detection of right-sided and left-sided CRC was similar (93% vs. 90%; p=0.5; Table). Conclusions: In this large early-stage CRC cohort, multimodal ctDNA assessment has high sensitivity for CRC detection with high specificity. Equivalent sensitivity in the asymptomatic cohort suggests this test will have clinically meaningful performance in an average risk screening population. A prospective registrational study is ongoing to evaluate the test in an average risk CRC screening cohort.[Table: see text]

3572 Background: Serial ctDNA can measure dynamic changes in disease burden over time, however utility of serial profiling to detect changes in actionable alterations remains unclear. Methods: We evaluated 501 patients with ≥3 serial Guardant360 assays performed between 09/2016 and 11/2020 and compared MSI, fusion, amplification and single nucleotide variant (SNV) detection over time. This comprised 2147 assays with a median of 4 assays per patient (min 3, max 18) occurring an average of 163 days apart (+/- SD of 147 days). Maximum detected variant allele frequency in samples (maxVAF) was assessed for relation to changes in detected alterations as a surrogate for tumor volume. Results: Among 406 patients with assays assessable for MSI-status, 17 (4.2%) had MSI detected. New MSI detection on a subsequent assay always occurred with a rising maxVAF (3/3) that was also ≥0.7%, while loss of detectable MSI between assays always associated with falling maxVAFs (7/7) with 6/7 occurring when maxVAF fell below 0.4%. Fusions were noted in 9/501 (2%) patients. Among 3 patients who lost a detectable fusion, maxVAF decreased in 1 patient and changed ≤0.2% between assays in 2, while 2/3 patients with new fusions had rising maxVAFs and 1 patient had a falling maxVAF. Amplifications were detected in 242/501 patients (48%). While most genes had highly variable amplification detection between assays (9% serially detected), ERBB2 amplifications were more consistent and serially detected in 39% of detected cases (P < 0.0001). New detection of amplifications occurred more commonly in cases with rising maxVAF (OR 11.70, 95% CI 7.61-18.00, P < 0.0001) and loss of detectable amplifications occurred more between samples with falling maxVAF (OR 12.37, 95% CI 8.35-18.66, P < 0.0001). Change in maxVAF correlated with change in number of detected amplifications (r = 0.62, P < 0.0001), but only partially explained changes seen (R2= 0.39). Between serial assays, SNVs changed a median of 0 variants (IQR -1 to 1), however some patients had significant changes (max gain 21/max loss 18). Among 1646 serial time points, 454 (28%) had no change in SNVs, 674 (41%) gained SNVs, and 518 (31%) lost SNVs on subsequent assays. Gains were more common in samples with rising maxVAF (OR 7.76, 95% CI 6.18-9.73, P < 0.0001) while losses were more common when maxVAF fell (OR 6.90, 95% CI 5.47-8.66, P < 0.0001). The correlation between maxVAF change and SNV change was significant (r = 0.29, P < 0.0001), but minimally explained SNV changes (R2= 0.086) and was a much weaker association than noted for amplification changes. Conclusions: We noted significant differences in detection of actionable alterations across serial ctDNA assays. Increased ctDNA volume (higher maxVAF) due to tumor progression may explain some variation over time, but variability also occurs outside these changes, likely reflecting clonal evolution following therapy.