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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

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: 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.

Purpose Genomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with colorectal cancers were correlated with clinical outcomes. Patients and Methods Next-generation sequencing of ctDNA (54- to 73-gene panel) was performed in 94 patients with colorectal cancer. Results Most patients (96%) had metastatic or recurrent disease at the time of blood draw. The median number of nonsynonymous alterations per patient was three (range, zero to 30). The most frequently aberrant genes were TP53 (52.1% of patients), KRAS (34%), and APC (28.7%). Concordance between tissue and blood next-generation sequencing ranged from 63.2% ( APC) to 85.5% ( BRAF). Altogether, 74 patients (79%) had one or more nonsynonymous alterations, 69 (73%) had one or more potentially actionable alterations, and 61 (65%) had an alteration actionable by a drug approved by the US Food and Drug Administration (on or off label). Lung metastases correlated with improved survival from diagnosis in univariable analysis. ctDNA of 5% or more from blood tests as well as EGFR and ERBB2 (HER2) nonsynonymous alterations correlated with worse survival (but only ERBB2 remained significant in multivariable analysis). No two patients had identical molecular portfolios. Overall, 65% versus 31% of patients treated with matched (n = 17) versus unmatched therapy (n = 18) after ctDNA testing achieved stable disease for 6 months or more, partial response, or complete response ( P = .045); progression-free survival, 6.1 versus 2.3 months ( P = .08); and survival not reached versus 9.4 months ( P = .146; all by multivariable analysis). Conclusion Patients with colorectal cancer have heterogeneous ctDNA profiles, and most harbor potentially actionable ctDNA alterations. Matched therapy yielded higher rates of stable disease for 6 months or more, partial response, or complete response. ctDNA assessment may have clinical utility and merits further investigation.

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.

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.

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.

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.

Background Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or recurrence prediction in early stage lung cancer patients. Methods We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing assay incorporating somatic and epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants. Table: 111P . Cell type Stage Somatic mutation Epigenetic pattern Total number Recurrence+, n (%) Site of recurrence Adenocarcinoma stage 1 ctDNA- methylation- 9 1 (11) Lung n = 17 methylation+ 6 2 (33.3) Stump, bone ctDNA+ 2 1 (50) lung stage 2 ctDNA- methylation- 0 0 (0) n = 2 methylation+ 0 0(0) ctDNA+ 1 1 (100) multiple stage 3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 2 0 (0) ctDNA+ 2 2 (100) brain, multiple Sqaumous cell carcinoma stage1 ctDNA- methylation- 0 0 (0) n = 7 methylation+ 3 0 (0) ctDNA+ 4 1 (25) multiple stage2 ctDNA- methylation- 0 0 (0) n = 9 methylation+ 0 0 (0) ctDNA+ 9 2 (22.2) multiple, lung stage3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 1 0 (0) ctDNA+ 3 1 (33.3) Mediastinal LNs Results Somatic mutation analysis alone detected ctDNA in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 67% (37/55). ctDNA detection rate varied by pathological subtypes; using combined approach, ctDNA was detected in all squamous cell carcinoma patients, while only 55% (12/22) in adenocarcinoma (ADC) (p=0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, within 2 years of follow-up, pre-operative ctDNA status was correlated with tumor recurrence after resection; among 17 stage I ADC patients, three of eight (38%) ctDNA-positive cases showed recurrence, while only one of nine (11%) ctDNA-negative cased did (p=0.29). Interestingly, patients with somatic mutation in their ctDNA have shown higher recurrence rate. Conclusion Utilizing a plasma-only sequencing assay incorporating somatic genomic and epigenomic analysis, ctDNA detection rate in early stage lung cancer (stage I-III) can far outperform the detection rate of somatic sequence variant detection alone. And, the presence of pre-operative ctDNA in patients with early stage lung adenocarcinoma may identify those who are more likely to have disease recurrence. Legal entity responsible for the study: Guradant Health, Inc. Funding Guardant Health, Redwood City, CA, USA. Disclosure I. Kim: Full / Part-time employment, Officer / Board of Directors: Guardant Health. M. Shultz: Officer / Board of Directors: Guardant Health. A. Jaimovich: Officer / Board of Directors: Guardant Health. J. Odegaard: Officer / Board of Directors: Guardant health, Inc. S. Olsen: Officer / Board of Directors: Guardant Health, Inc. A. Talasaz: Officer / Board of Directors: Guardant health. J. Kim: Research grant / Funding (self): Guardant health, Inc. All other authors have declared no conflicts of interest.

Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay. Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set. Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management. Citation Format: Seung-Tae Kim, Victoria M. Raymond, Joon Oh Park, Elena Zotenko, Young Suk Park, Matthew Schultz, Won Ki Kang, Oscar Westesson, Hee-Cheol Kim, Yupeng He, Justin I. Odegaard, Stefanie A. Mortimer, William J. Greenleaf, Ariel Jaimovich, Jeeyun Lee, AmirAli Talasaz. Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 916.

3602 Background: ctDNA identifies patients (pts) at high risk for disease recurrence post CRC resection (post-op). Current ctDNA residual disease detection approaches assess only genomic alterations (alts) and rely on tissue sequencing to identify tumor-derived alts. We evaluated a plasma-only ctDNA assay to identify high risk pts. Methods: 72 CRC pts (surgery only = 42; adjuvant therapy (adj) = 30) had post-op and/or post-adj plasma samples (3-4mL). Extracted cfDNA (median 27 ng) was analyzed using a single-sample NGS test validated in early stage CRC that integrates assessment of genomic alts with epigenomic cancer signature (Guardant Health, CA). A variant classifier was applied to differentiate tumor-derived from non-tumor derived alts in a tumor tissue-uninformed approach. Results: In the surgery cohort, samples were collected a median of 31 days (d) post-op. 7/8 pts with post-op ctDNA detected (ctDNA+) recurred (PPV 88%; median time to recurrence (mTTR) 248d). The recurrence-free pt has < 180d follow-up. 7/34 pts without ctDNA detected (ctDNA-) recurred (NPV 79%; mTTR 333d). 1/1 Stage 0-II ctDNA+ pt recurred (PPV 100%; TTR 440d) while 1/20 ctDNA- recurred (NPV 95%; TTR 440d). 27 pts in the adj cohort had samples collected a median of 37d post-adj. 6/6 ctDNA+ pts recurred (PPV 100%, mTTR 239d). 4/21 ctDNA- pts recurred (NPV 81%, mTTR 466d). 2/2 ctDNA+ and 0/11 ctDNA- Stage III pts recurred (PPV, NPV 100%, mTTR 420d). All 3 post-op ctDNA+/post-adj ctDNA+ (ctDNA persistence) pts recurred. 1/2 post-op ctDNA+/post-adj ctDNA- (ctDNA clearance) pts is recurrence free (306d). 2 post-op ctDNA-/post-adj ctDNA+ pts recurred. In the entire cohort, ctDNA+ after standard therapy completion had a recurrence PPV 93%, NPV 80%, HR 11.29 (p < 0.0001). Conclusions: In post-op CRC, ctDNA detection utilizing a tumor-uninformed integrated genomic and epigenomic assay has high recurrence PPV and NPV following standard therapy completion. ctDNA identifies pts who may benefit from post-op adj therapy or additional/modified post-adj therapy. These findings demonstrate that ctDNA detection from a single post-op/post-adj plasma sample stratifies high/low risk pts and informs therapy decision making.

e14557 Background: Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or predicting tumor recurrence in early stage lung cancer patients. Methods: We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing to detect somatic mutations and differential epigenomics patterns, including methylation signatures. Results: Using somatic mutation analysis alone, ctDNA was detected in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 71%. ctDNA detection rate also varied markedly between lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC);using combined analysis of somatic mutations and epigenomic patterns, ctDNA was detected in all SCC patients, while only 55% of ADC (12/22) were ctDNA-positive (p= 0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, pre-operative ctDNA status was correlated with tumor recurrence post-resection; three of eight (38%) ctDNA-positive stage I ADC patients recurred within 2 years of resection, while only one of nine (11%) ctDNA-negative stage I ADC patients recurred (p= 0.29). Conclusions: Taken together, we show that the combination of somatic mutation detection and epigenomic analysis outperforms each individual biomarker in the detection of ctDNA in early stage lung cancer. Importantly, we also demonstrate that pre-operative ctDNA detection may identify a high-risk population of early stage lung cancer patients that may benefit from (neo)adjuvant therapy.

Background Inflammatory Breast Cancer (IBC) is a clinicopathologic diagnosis characterized by rapid progression, resistance to treatment and poor prognosis. It is often an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Monitoring disease by performing multiple biopsies may not be feasible and puts the patient at risk with each invasive procedure. Circulating DNA fragments carrying tumor-specific sequence alterations (ctDNA) are found in blood and offer the possibility of longitudinal non-invasive molecular monitoring of the disease by detecting actionable mutations. Methods This is an observational analysis of 35 IBC patients who failed standard therapies and had plasma analyzed for ctDNA detection. Selection criteria included progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of genomic abnormalities already demonstrated in tissue analysis. Guardant Health performed the plasma analysis (Guardant360™); first ctDNA was isolated from plasma, then a panel of 54 gene mutations associated with solid tumors as reported in the COSMIC database were sequenced to concurrently analyze somatic mutations and gene amplification using single-molecule digital sequencing technology. Results All patients had IBC and 80% had metastatic disease; 37% of patients were ER+/HER2-, 14% ER+/HER2+, 23% ER-/HER2+, and 26% ER-/HER2-. 94% of patients with stage III or IV tumors had ctDNA alterations detected. The most common mutations were TP53 (49%), PIK3CA (20%), ERBB2 (17%), NOTCH1 (17%), and ALK (11%). Twelve patients also had next generation sequencing (NGS) analysis of tissue biopsy and 75% of these patients demonstrated at least one concordant mutation. The genomic information obtained from ctDNA, NGS or both was used to select treatments in 11 cases (31%). HER2 targeted therapy was continued in four patients with HER2+ disease after ctDNA confirmed ERBB2 alteration or amplification. A patient with ER+/HER2+ disease who progressed on HER2 targeted therapies, demonstrated ERBB2 and PIK3CA mutations on ctDNA; she was changed to exemestane/everolimus with objective response for several months. Repeat ctDNA at time of progression showed ERBB2 mutation only; she was changed to Trastuzumab/everolimus/vinorelbine. Moreover, a combination of lapatinib and capecitabine was initiated on a patient with a triple negative, chemo-refractory tumor that on ctDNA revealed EGFR and ERBB2 mutations; a repeated ctDNA after 5 months of therapy EGFR and ERBB2 mutations were not detected and she remains without evidence of progression. Conclusions Evaluation and longitudinal monitoring of IBC patients using ctDNA allows for identification of genomic abnormalities in all patients with advanced disease and to perform real-time molecular monitoring. The discovery of actionable genomic abnormalities is driving the management of this aggressive and treatment refractory form of breast cancer with potential future impact on outcome. Citation Format: Laura Austin, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, Benjamin J Schiller, Gangwu Mei, Helmy Eltoukhy, AmirAli Talasaz, Massimo Cristofanilli. Circulating tumor DNA (ctDNA) provides molecular monitoring for inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-03.

Analysis of cell-free DNA using next-generation sequencing (NGS) is a powerful tool for the detection/monitoring of alterations present in circulating tumor DNA (ctDNA). Plasma extracted from 171 patients with a variety of cancers was analyzed for ctDNA (54 genes and copy number variants (CNVs) in three genes (EGFR, ERBB2 and MET)). The most represented cancers were lung (23%), breast (23%), and glioblastoma (19%). Ninety-nine patients (58%) had at least one detectable alteration. The most frequent alterations were TP53 (29.8%), followed by EGFR (17.5%), MET (10.5%), PIK3CA (7%), and NOTCH1 (5.8%). In contrast, of 222 healthy volunteers, only one had an aberration (TP53). Ninety patients with non-brain tumors had a discernible aberration (65% of 138 patients; in 70% of non-brain tumor patients with an alteration, the anomaly was potentially actionable). Interestingly, nine of 33 patients (27%) with glioblastoma had an alteration (6/33 (18%) potentially actionable). Overall, sixty-nine patients had potentially actionable alterations (40% of total; 69.7% of patients (69/99) with alterations); 68 patients (40% of total; 69% of patients with alterations), by a Food and Drug Administration (FDA) approved drug. In summary, 65% of diverse cancers (as well as 27% of glioblastomas) had detectable ctDNA aberration(s), with the majority theoretically actionable by an approved agent.

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

Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in 9 patients (35%). In the remaining 17, 90.3% (95% confidence interval, 73.1%–97.5%) of mutations detected in tumor biopsies were also detected in cfDNA. The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across five informative genes. Changes in cfDNA correlated well with tumor marker dynamics in serial sampling (r = 0.93). We demonstrate that cfDNA sequencing is feasible, accurate, and sensitive in identifying tumor-derived mutations without prior knowledge of tumor genotype or the abundance of circulating tumor DNA. cfDNA sequencing should be considered in pancreatobiliary cancer trials where tissue sampling is unsafe, infeasible, or otherwise unsuccessful. Significance: Precision medicine efforts in biliary and pancreatic cancers have been frustrated by difficulties in obtaining adequate tumor tissue for next-generation sequencing. cfDNA sequencing reliably and accurately detects tumor-derived mutations, paving the way for precision oncology approaches in these deadly diseases. Cancer Discov; 5(10); 1040–8. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 1005

Background: FC-7 is a phase Ib study hypothesizing that dual ERBB blockade (neratinib and cetuximab) could overcome an acquired resistance mechanism to anti-EGFR tx in KRAS/NRAS/BRAF/PI3KCA wild type (wt) mCRC patients (pts). Previously we demonstrated a 36% rate of HER2 amp in post- anti-EGFR biopsies (bxs). Assay of plasma cfDNA is minimally invasive and may provide comprehensive genomic profiling. We tested the accuracy of a cfDNA assay for HER2 amp, comparing results from matched bxs to evaluate molecular changes in HER2 copy number. Methods: 23 pts with mCRC resistant to anti-EGFR tx were enrolled and treated with cetuximab and neratinib. 17 paired bxs (6 post-tx bxs had insufficient tumor) were assessed for HER2 amp using chromogenic in situ hybridization (CISH) (Dako PharmDxTM). HER2 was considered amplified when HER2/CEP17 ratio was ≥2. cfDNA was extracted from plasma of 11 pts after 1st line anti-EGFR tx but before addition of cetuximab and neratinib. 5-30 ng of DNA was sequenced across 512 exons in 54 genes with HiSeq (Illumina). Copy number variants (CNV) were quantified using a customized pipeline (Guardant 360). One plasma sample failed to yield sufficient cfDNA. 10 pts had comparison of cfDNA with pre- or post- anti-EGFR bx. Results: 4 of 9 plasma samples (1 non evaluable) had HER2 amp, all concordant with pre- or post- anti-EGFR tx tissue. 3 pts converted to HER2 amp after anti-EGFR tx (pts 3, 5, 7), and for 2 of these conversions (pts 5, 7), the amplification was evident in cfDNA. PtPre- anti-EGFR CISH RatioPost- anti-EGFR CISH RatioPost- anti-EGFR cfDNA CNVConcordance of cfDNA with pre- or post-anti-EGFR Tissue Samples1Neg (1.0)Neg (1.3)Non evaluableNA2Pos (2.5)Neg (1.5)Pos (2.66)Pre-Tx3Neg (1.0)Pos (3.7)NegPre-Tx4Neg (1.0)Neg (1.8)NegBoth5Neg (1.2)Pos (2.7)Pos (2.48)Post-Tx6Pos (>10)QNSPos (21.86)Pre-Tx7Neg (1.1)Pos (2.0)Pos (2.20)Post-Tx8Neg (1.3)Neg (1.1)NegBoth9Neg (1.0)Neg (1.3)NegBoth10Neg (1.0)Neg (1.5)NegBoth Conclusion: In this sample set, HER2 amp detected in plasma-derived cfDNA from mCRC pts resistant to anti-EGFR tx is comparable to amp assessed in primary or metastatic tissue. Given advantages of liquid bx relative to repeated tissue bxs, measurement of HER2 amp in cfDNA may be of utility in monitoring pts for resistance to anti-EGFR tx. This will need to be confirmed in a trial incorporating both plasma and tissue samples. Support: Puma Biotechnology, Inc. Citation Format: S. Rim Kim, Ashok Srinivasan, Carmen J. Allegra, Samuel Jacobs, Rebecca J. Nagy, Richard B. Lanman, AmirAli Talasaz, Patrick Gavin, Rekha Pal, Peter C. Lucas, Kay Pogue-Geile. NSABP FC-7 correlative study: HER2 amplification (amp) in circulating cell-free DNA (cfDNA) in metastatic colorectal cancer (mCRC) resistant to anti-EGFR therapy (tx) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5684. doi:10.1158/1538-7445.AM2017-5684

4534 Background: Cell-free ctDNA may be potentially actionable, may have prognostic/predictive role and evolve after therapy. We updated our analysis of our retrospective study to shed light on UC/BC biology. Methods: Patients (pts) with UC/BC with ctDNA analysis for potentially actionable alterations using Guardant360 were identified. A 70-gene ctDNA next generation sequencing panel from a CLIA-licensed, CAP-accredited laboratory (Guardant Health, Inc.) offers complete exon sequencing for 29 cancer genes, critical exons in 39 genes and amplifications (16 genes), fusions (6 genes) and indels (3 genes) harvested from 10 mL of peripheral blood. Descriptive statistics were used. Results: There were 246 pts with 276 samples. At least 1 alteration was detected in 249 (90%) samples. Median age at time of ctDNA collection was 67 years (39-85), 78% men, median number of alterations per sample was 3.5 (1-35) most pts had MIBC. In MIBC pts, the most common alterations at the 1st ctDNA sample were in TP53 (52%), PI3KCA (18%) ARID1A (17%), FGFR2 (15%), MET & NF1 (14%), EGFR (13%), BRAF (12%), FGFR3 (11%), RAF1 (10%), BRCA1 & CCNE1 (9%). In MIBC pts, the most common genes with increased copy number were RAF1 & CCNE1 (8%), ERBB2 & PI3KCA (7%), EGFR, BRAF, FGFR1, MYC (each 5%), MET (4%), KRAS (3%). Most common altered pathways included TP53 signaling (56%), RAS/RAF/MEK/ERK (51%), RTK (48%), cell cycle (38%), FGFR family (34%), DNA damage response (25%), PI3KCA/AKT/mTOR (23%) and chromatin remodeling (17%). Interestingly, FGFR3 and RAS alterations were mutually exclusive in most cases, but each may co-occur with TP53alterations. 54 serial ctDNA samples from 24 pts (18 pts with 2 samples; 6 pts with 3 samples) revealed persistent, lost and new gene alterations. Conclusions: ctDNA was detected in 90% of pts and alterations were similar to those previously seen in UC tumor tissue. Tumor heterogeneity, interim therapy, genomic instability and clonal evolution can explain differences in serial samples. Correlation assessment with prior therapies and outcomes is being pursued to inform trial designs. Prospective validation, assessment of ctDNA concordance with tumor tissue DNA, and evaluation of clinical utility is warranted.

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

334 Background: Cell-free ctDNA may be potentially actionable, prognostic for outcomes and evolve after therapy. We conducted a retrospective study to evaluate these issues and shed light on UC biology. Methods: Patients (pts) with advanced UC who underwent ctDNA analysis for potentially actionable alterations using Guardant360 were identified. Data were requested for prognostic factors, current and prior therapies, TTF (time to treatment failure) and overall survival (OS). A 70-gene ctDNA next generation sequencing panel from a CLIA-licensed, CAP-accredited laboratory (Guardant Health, Inc.) offers complete exon sequencing for 29 cancer genes, critical exons in 39 genes and amplifications (16 genes), fusions (6 genes) and indels (3 genes) harvested from 10 mL of peripheral blood. Alterations were reported and association of non-synonymous potentially functional alterations with outcomes and prior therapy was examined. Results: There were 217 pts with 238 samples. ctDNA was detectable in 212 (89%) samples. Median age was 62 (range 39-91). The most common recurrent somatic mutations were in TP53 (n = 122, 57%) ARID1A (n = 47, 22%), NF1 (n = 35, 16%), FGFR2, FGFR3, and BRCA1 (n = 30, 14% each), MET (n = 27, 13%), ERBB2 (n = 25, 12%), PIK3CA (n = 24, 11%), and EGFR (n = 22, 10%). Most common genes with increased copy numbers were ERBB2 (n = 19, 9%) and RAF1 (n = 18, 8%). Clinical data were available for 64 pts, of whom 38 (59%) had prior chemotherapy. FGFR1 alterations noted in 3 of those pts (5%, 2 functional, 1 amplification) were associated with shorter OS (HR 2.95, p = 0.05). Pts with prior chemotherapy showed trend towards frequent alterations in DNA repair genes (45% vs. 23%, p = 0.11). Serial ctDNA profiling of 21 pts receiving therapy revealed the clonal evolution of mutations in BRCA2, NF1 and GATA3. Conclusions: ctDNA was very frequently detected in pts with advanced UC, and alterations were similar to those previously seen in muscle-invasive UC tumor tissue. FGFR1 alterations predicted for unfavorable outcome and DNA repair gene alterations evolved commonly after chemotherapy. Drugs exploiting these targets such as FGFR1 and PARP inhibitors may warrant exploration as salvage therapy in selected pts.

149 Background: Cell-free ctDNA may be prognostic and evolve following therapy. We report ctDNA profiling of patients (pts) with mCRPC and their association with clinical outcomes and evolution with therapy. Methods: Pts with mCRPC that underwent baseline ctDNA analysis for potentially actionable alterations using Guardant360 before new systemic therapy were identified. Data were requested for clinical factors, current and prior therapy, TTF (time to failure) and survival. A 70-gene cfDNA next generation sequencing panel from a CLIA-licensed, CAP-accredited laboratory (Guardant Health, Inc.) offers complete exon sequencing for 29 genes, critical exons in 39 genes and amplifications (16 genes), fusions (6 genes) and indels (3 genes) harvested from 10 mL of peripheral blood. Alterations were reported and association of alterations with outcomes and prior therapy was examined. Results: Of 514 with confirmed mCRPC, 482 (94%) had ≥ 1 ctDNA alteration. The median age was 70 years (range 39-91). The most common recurrent somatic mutations were in TP53 (36% of patients), AR (22%), APC (10%), NF1 (9%), EGFR, CTNNB1 and ARID1A (6% each) and BRCA1, BRCA2 and PIK3CA (5% each) The most common genes with increased copy numbers were AR (30%), MYC (20%) and BRAF (18%). Clinical outcomes were available for 163 pts, of whom 46 (28.8%) were untreated for mCRPC. A higher number of ctDNA alterations was associated with shorter TTF (HR: 1.05, p = 0.026). AR alterations had a trend for shorter TTF (HR: 1.42, p = 0.053) and survival (HR: 2.51, p = 0.09). Pts who received prior therapy had new alterations in AR (56% vs. 37%, p = 0.028) compared to untreated pts. Serial ctDNA profiling of 64 pts revealed the evolution of alterations in AR, BRCA1 and BRCA2 following therapy. Conclusions: ctDNA was frequently detected in patients with mCRPC, and alterations appear similar to tumor tissue alterations. A higher number of overall gene alterations and AR alterations appeared associated with poor clinical outcomes and new AR and BRCA alterations appeared following therapy. These data suggest that developing salvage agents targeting AR alterations and PARP inhibitors hold promise.

47 Background: Esophageal (EC), gastroesophageal junction (GEJ) and gastric cancer (GC), together GEC, have a poor prognosis with few targeted therapeutic options. As genomic profiling is becoming increasingly useful, we queried whether comprehensive genomic profiling of ctDNA would reveal relevant genomic alterations leading to targeted therapies. Methods: Patients (pts) with advanced GEC undergoing next-generation sequencing (NGS) of ctDNA in a CLIA certified lab were identified. ctDNA was analyzed using Guardant360, a digital NGS assay to identify single nucleotide variants, indels, amplifications and fusions in 54-70 genes. Results: 546 pts with GEC had ctDNA testing and 54 pts (10%) had more than one ctDNA test (range 2-6). Pts were similar across the 3 groups, with the exception of an increased male:female ratio (5:1) in the GEJ and EC cohorts. Mean age was 61.5 yrs (range 24-91). ctDNA alterations were detectable in 455 pts (83.3%). Recurrent alterations and therapeutic options for each cohort are shown in Table 1. Serial monitoring of ctDNA correlated with tumor markers, imaging and clinical response. Multiple antitumor responses to targeted therapy will be presented. Conclusions: ctDNA was detected in 83.3% of pts with advanced GEC with a frequency similar to TCGA. The diverse genomic landscape of GEC, obtained noninvasively, along with matched therapies have potential to improve outcomes for this aggressive disease. [Table: see text]

Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) allows non-invasive real-time profiling of actionable genomic alterations. Liquid biopsy provides an option for disease monitoring in early stage cancer patients post surgical resection, with a potential to aid in adjuvant decision making. However, to be applicable, tests must cover a broad enough genomic footprint to not require a priori knowledge of mutations, have high specificity, and sensitivity higher than conventional methods. NGS is necessary, since inactivating mutations are the most common alteration type in many common cancer types such as colorectal carcinomas (CRC). Here we present a highly efficient and specific NGS assay for detection of ctDNA in early stage cancer patients, capable of detecting single molecule variants across a 12 kb gene panel with an analytical sensitivity of >0.02% for single nucleotide variants (SNVs) and indels. This panel was applied to a clinical study involving 14 early stage (II/III) CRC patients with both pre- and post-op blood draws (up to 7 days post surgery). A subset (6 patients) also had tumor samples collected at the time of the surgical resection of the tumor. Overall, the detection rate of ctDNA in pre-op blood draws was 93%. In the post-op blood draws ctDNA was detectable in 43% of cases. The estimated average minor allele frequency (MAF) is 0.58% (± 0.82%) in pre-op, 0.18% (±0.21%) in post-op, and 40% (±18%) in tumor samples. When tumor tissue was available and used as a reference, the clinical sensitivity, specificity, and accuracy in pre-op blood samples were 83%, 99.995%, and 99.99%, respectively. SNVs with MAF as low as 0.04% were confirmed in tissue data. The clinical specificity of variants detected in post-op blood samples using pre-op samples as the reference is 99.996%. Cohort expansion to 50 patients and follow-up for clinical recurrence in both cohorts is ongoing. In conclusion, we have developed an assay with ultra-high accuracy and specificity, for the detection of ctDNA in early stage CRC patients that is capable of detecting alterations present in the tumor post-surgical resection. This technology allows for a promising non-invasive route for molecular monitoring of residual disease post surgery and for early detection of relapse compared to traditional methodologies. Citation Format: Stefanie A. Mortimer, Katharine Dilger, Stephen Fairclough, Diana Abdueva, Darya Chudova, Ankit Sarin, Jim Leng, Jeeyun Lee, Helmy Eltoukhy, AmirAli Talasaz. Post-surgical resection monitoring in early stage colorectal carcinoma patients using a circulating cell-free DNA assay with ultra-high accuracy and specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 506.

Background: Mutational analysis has become standard of care in lung cancer. However, many patients do not have tissue available due to lack of tissue, or more commonly due to tissue exhaustion by previous tissue analysis. Next generation sequencing of circulating cell-free DNA (cfDNA) provides a non-invasive alternative. EGFR point mutations and indels are well reported in this manner, however gene copy number amplification is more challenging. Here we present a cases where MET amplification has been detected and guided highly efficient therapy in advanced lung cancer patient. Methods: Guardant360TM is a targeted cfDNA NGS panel using hybrid capture and complete exon sequencing exons for single nucleotide variant detection in 68 genes, copy number amplifications (CNA) in 16 genes, and selected indels and fusions. CNA has been validated against cell lines with known amplifications. Results: A 70 y old former light smoker (15 PY) with pulmonary fibrosis and moderate pulmonary hypertension was diagnosed with a 30 mm RMB lung adenocarcinoma that was treated by stereotactic beam radiotherapy due to poor pulmonary reserve. After 5 months, mediastinal, liver and multiple bone metastases were diagnosed. Genomic analysis of the primary tissue biopsy yielded a rare EGFR mutation (I744F) and afatinib therapy was started to cover uncommon EGFR mutations. However, 2 months later, a significant progression has occurred and the patient had severe local progression with respiratory near-failure. The patient was not a candidate for chemotherapy and there was no further tissue available for molecular testing. Therefore cfDNA testing was performed finding no residual EGFR mutation in the blood but a MET amplification reported above the 90th percentile for the Guardant Health laboratory with copy number of 53.6 in the blood. Upon this result, crizotinib 250 mg BID was started with immediate clinical improvement, followed with a dramatic imaging response on CT/PET scans. Currently, the patient is in PS-0 and free of any symptoms, 3 months since treatment has been started. Conclusion: cfDNA detection of MET amplification as a key resistance mechanism after EGFR TKI therapy is feasible in patients where tissue is not accessible or was undergenotyped and may be accompanied by a fast and dramatic clinical improvement. Citation Format: Nir Peled, Anna Belilovski, Lior Soussan-Gutman, Richard B. Lanman, AmirAli Talasaz. Salvage MET amplification detection and therapy through cell-free DNA NGS in a progressing lung cancer patient. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 491.

Background: Metastatic breast cancer (MBC) is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tumor genomic evaluation using next generation sequencing (NGS). Furthermore, analysis of circulating tumor DNA (ctDNA) in patients with advanced disease offers the possibility of non-invasive molecular monitoring. Methods: A patient with MBC was tested at each progression with a ctDNA NGS panel (Guardant360™) that includes all NCCN-recommended somatic genomic variants for solid tumors and sequences complete exons of >50 genes to report single nucleotide variants (SNVs), fusions, amplifications, and indels with high sensitivity and ultra-high specificity (>99.9999%). The patient was diagnosed with invasive breast cancer at age 44 and treated with surgery and hormonal therapy. At age 61, she had axillary adenopathy and liver metastases. Treatment details are in Table 1. Results: ctDNA analysis was performed at the time of metastatic diagnosis and at 5 additional time points over the course of treatment. All samples revealed an ERBB2 exon 19 indel (p.Leu755_Glu757delinsSer), and multiple SNVs and gene amplifications. ERBB2 amplification was seen in 4 of 6 samples. Mutant allele fractions (Table 1) correlated with clinical response to treatment and progression. Conclusions: Analysis of ctDNA in this patient identified an ERBB2 exon 19 indel, which are present in 2-4% of non-small cell lung cancers but 1-2% in breast cancer. Treatment with anti-HER2 monoclonal antibody or dual anti-EGFR/ERBB2 tyrosine kinase inhibitor therapies may show clinical benefit. ctDNA analysis can detect emergence of actionable resistance mutations with the advantage of serial evaluation, allowing capture of inter- and intra-tumor heterogeneity and illustration of molecular progression and response. Table 1.Blood DrawDisease status at time of blood drawMutant allele fraction of ERBB2 indel (* = ERBB2 amplification)Treatment1Initial diagnosis9%trastuzumab, pertuzumab and docetaxel (TPD), cycle 12Stable disease1%*TPD cycle 23Progressing10%*TPD cycle 34Progressing62%*trastuzumab, emtansine (TDM1) 3 cycles5Progressing70%*vinorelbine, trastuzumab, everolimus6Partial response1%flourouracil, epirubicin, cyclophoasphamide Citation Format: Laura Austin, Rebecca Nagy, Oliver Zill, Richard B. Lanman, AmirAli Talasaz, Massimo Cristofanilli. Managing metastatic breast cancer via serial monitoring with circulating cell-free tumor DNA next generation sequencing testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 172.

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

8553Background: Outcomes for curative-intent chemoradiotherapy of unresectable stage III non-small cell lung cancer (NSCLC) remains poor. Heterogeneity in tumors is correlated with therapeutic resi...

4528Background: Urothelial carcinoma of the bladder (UCB) exhibits one of the highest somatic mutation burdens. Circulating cell-free DNA (cfDNA) is obtained non-invasively from peripheral blood an...

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

Background: Several molecular alterations drive acquired resistance to anti-EGFR therapies in pts with KRAS WT mCRC. We designed a clinical trial to identify and treat MET amplification (amp), a well described driver of acquired EGFR resistance. Methods: Pts with KRAS WT mCRC were enrolled in 3 cohorts: 1) C+P Dose Finding (Dose Find): 3+3 design to determine the maximum tolerated dose (MTD) and recommended phase II dose (RPTD) of C+P; 2) C+P Expansion (Exp): to define the safety, tolerability and activity of C+P; and 3) MET amplified monotherapy (MET Mono): to determine the response rate of C alone in pts with prospectively identified MET amp/ EGFR refractory mCRC. Response assessment occurred every 2 months with computed tomography (CT) using RECIST criteria, version 1.1. cfDNA was collected at baseline and each restaging until progression. In the C+P Dose Find and Exp cohorts, peripheral blood was retrospectively sequenced for 54 gene mutations (mut) and focal amplifications, including MET (Guardant Health, Inc.). MET expressing circulating tumor cells (CTCs) were captured utilizing nanomagnetic particles conjugated to an antibody targeting extracellular MET (Janssen R&D, LLC). Genomic amp was tested in tumor tissue using next generation sequencing (NGS) or FISH. These data allowed subgroup and individual pt profiling. Results: There were no dose limiting toxicity (DLT) events at the RPTD (C 60mg PO daily and P 6 mg/kg IV Q2 weeks) in the Dose Find cohort (n = 6). Assessment of treatment response in C+P cohorts: As of 7/21/2015, 13 pts were treated at the RPTD. 3 pts had not yet received response assessment; 10 pts (6 Dose Find; 4 Exp) were evaluable for response. 8/10 evaluable pts had failed prior anti-EGFR therapy. 6/10 had reduction in RECIST lesions (2/2 EGFR naïve; 4/8 EGFR refractory). Median change was -10% (range -40% to +18%). 2/2 evaluable pts with MET amp cfDNA had a reduction in target lesions (both EGFR refractory). 7/10 had a decrease in tumor marker (CEA). Median CEA change was -39% (range -86% to +103%). Baseline cfDNA profiling in C+P and MET Mono cohorts: 13 pts with EGFR refractory mCRC (6 Dose Find; 3 Exp; 4 MET mono) had baseline cfDNA profiling. 4/13 BRAF mut (2/4 subclonal), 3/13 KRAS mut (2/3 subclonal), 1/13 NRAS mut (0/1 subclonal), 3/13 HER2 amp (3/3 confirmed in tissue), 4/13 MET amp (0/2 in tissue; 2 not tested). Pt examples: One subject (EGFR refractory) had a lymph node biopsy 3 months prior to C+P that revealed no MET amp by NGS (Foundation Medicine). Pre-treatment cfDNA revealed MET amp. Treatment with C+P resulted in a partial response (-40% in RECIST lesions). MET amp was no longer detected in cfDNA at first restaging. A second subject had MET amp in pre-treatment cfDNA. MET-expressing CTCs were captured from peripheral blood. Single cell FISH on CTCs-but not archival tissue-confirmed MET amp. Conclusions: This ongoing phase I/II study demonstrates the feasibility of: 1) using cfDNA to detect MET amp in pts with EGFR refractory mCRC, even when archival tissue is negative; and 2) capturing MET-expressing CTCs to confirm MET amp. Preliminary clinical results support prospective cfDNA screening to identify and treat pts with EGFR refractory/ MET amp mCRC. Citation Format: John H. Strickler, Tian Zhang, Andrew J. Armstrong, Donna Niedzwiecki, Hope E. Uronis, Michael A. Morse, S. Yousuf Zafar, Shiao-Wen D. Hsu, Christy C. Arrowood, Rebecca Nagy, AmirAli Talasaz, Richard Lanman, Sherri Haley, Herbert I. Hurwitz. Cell free DNA (cfDNA) to monitor clonal evolution in patients (pts) with KRAS wild-type (WT) metastatic colorectal cancer (mCRC): Preliminary results of a phase I/II clinical trial of the anti-MET multi-kinase inhibitor cabozantinib (C) plus the anti-EGFR monoclonal antibody panitumumab (P). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A52.

Background MBC is an incurable disease characterized by genomic abnormalities including somatic mutations, amplifications and gene rearrangements. It is believed that treatment resistance and disease progression is related to genomic instability making “molecular monitoring” and consequent therapeutic management a dynamic and potentially more effective approach. The detection of circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients is the ideal diagnostic tool for non-invasive molecular monitoring. Methods This is a retrospective evaluation of 75 patients with advanced or MBC who both completed primary therapy and were NED (stage 1-3), or failed standard therapies and had baseline plasma analyzed for ctDN before starting new therapy (stage 4). For patients with progression, selection criteria included: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves ctDNA isolation from plasma using a Qiagen circulating nucleic acid kit, then a panel of 54 gene mutations associated with solid tumors as reported in the COSMIC database sequenced using single-molecule digital sequencing technology. Results Most of the patients have metastatic disease (95%), but four patients had stage III disease and are now NED after neo-adjuvant therapy and surgery. According to subtype, 41% ER+/HER2-, 17% ER+/HER2+, 11% ER-/HER2+, and 31% TNBC. ctDNA was detected in 84% (63) of all patients; however, only 20% of patients who are now NED had ctDNA detected. Of patients with detected mutations, 52% had an actionable mutation that was incorporated into treatment planning. The most common mutations were TP53 (65%), PIK3CA (38%), ALK (21%) and NOTCH1 (17%). One patient with ER+/HER2- disease had ctDNA drawn at progression demonstrating a PIK3CA mutation, started on clinical trial with Letrozole/Palbociclib with clinical response and no ctDNA detected on two serial draws. Another patient with ER+/HER2- disease with 11 alterations noted in her blood, including PIK3CA and ATM, was started on targeted therapy with Trastuzumab based on HER2+ CTCs with clinical response but upon progression, ctDNA showed a dramatic increase in the resistant ATM clone, increasing from 1.1% to 27.8%. Conclusions ctDNA can be detected in the majority of patients with MBC irrespective of disease subtype. Actionable mutations can be identified and used for selection of targeted therapies. Longitudinal monitoring can accurately predict treatment response but also detect the onset of new mutations likely related to resistant clones. The real-time molecular monitoring and therapeutic implications hold the promise to significantly change our approach to the management of MBC with survival benefit. Citation Format: Laura K. Austin, Rebecca Jaslow, Tiffany Avery, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Clinical utility of circulating tumor DNA (ctDNA) in advanced and metastatic breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4928. doi:10.1158/1538-7445.AM2015-4928

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

Background MBC is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tissue genomic evaluation using next-genomic sequencing (NGS). Furthermore, circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients with advanced disease offer the possibility of non-invasive molecular monitoring. ctDNA detects actionable mutations with the advantage of serial evaluation and allowing capture of inter- and intra-tumor heterogeneity. Methods This is a retrospective evaluation of 28 patients with MBC who failed standard therapies and had baseline plasma analyzed for ctDNA and tissue analysis (NGS) before starting new therapy. We were interested in the performance of the ctDNA test and the concordance rate of genomic alterations detected in the two tests. Selection criteria: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves comprehensive sequencing of a panel of 54 gene associated with solid tumors using single-molecule digital sequencing technology. FoundationOne®(Foundation Medicine) performed the NGS on tissue evaluates the entire coding sequence of 315 cancer-related genes. Results All patients had biopsy-proved metastatic disease, and had ctDNA and NGS performed. 93% of patients had ctDNA alterations detected (with 0.1%-27.8% circulating tumor fraction), and 9 patients had serial ctDNA. Overall, for the patients we detected mutations in ctDNA and tissue, 89% of patients had a specific alteration on ctDNA that matched the NGS analysis. Among all mutations detected in tumors which are in overlapped genes, 71% of alterations were common (83% excluding gene amplifications). Interestingly, for patients who had both tests done within 8 weeks, 70% of had additional alterations in the ctDNA that were not found on NGS, such as ERBB2 mutations. Conclusions Genomic analysis using ctDNA and NGS detects genomic abnormalities in all patients with MBC with high concordance. However, each method was able to detect alterations that the other did not, suggesting the two methods can be complementary in detecting actionable mutations and expanding therapeutic options. Intriguingly, this occurred more frequently with the ctDNA, demonstrating its utility as an adjunct to tissue sampling which permits capture of the inter- and intra-tumor heterogeneity of the disease, and warrants further investigation prospectively. Citation Format: Laura K. Austin, Tiffany Avery, Rebecca Jaslow, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4918. doi:10.1158/1538-7445.AM2015-4918

Background: Monitoring molecular events in response to treatment with targeted agents is particularly challenging in pancreatic cancer due to its poor accessibility for biopsies and high stroma content. Recent advances in digital sequencing of rare DNA species allows for detection of circulating tumor-derived free DNA (ctDNA) in plasma of patients with cancer. Here, we explored the potential of gaining information on the genomic composition of pancreatic cancers in patients treated with a combination of the MEK inhibitor selumetinib (AZD6244) and the EGFR kinase inhibitor, erlotinib. Methods: 46 patients were enrolled into this study at two study centers. Treatment consisted of erlotinib 100 mg + AZD6244 100 mg daily in 3-week cycles, with tumor evaluation by CT scan every 2 cycles. Primary objective was overall survival (OS). Correlative endpoints included assessment of expression of EMT-related proteins, and next-generation sequencing analysis of ctDNA. Plasma “on treatment” samples were uniformly obtained at the 6 week time point. Results: Our preclinical studies had demonstrated that inhibition of MEK leads to enhanced signaling through EGFR with hyperactivation of a parallel RAS effector pathway (PI3K), supporting a therapeutic strategy of combined target inhibition in PDAC to overcome this negative feedback loop. Disease control rate was 58% (0 PR; 19 with stable disease (SD) > 6 weeks, 12 with SD > 12 weeks, including 12 (26%) minor responses). 13/34 patients (38%) demonstrated CA19-9 decline > 50%. Assessment of E-cadherin expression in pre-treatment FFPE biopsy material revealed that high expression of the protein was associated with greater likelihood of CA19-9 decline. Paired pre- and on-treatment plasma samples were available for digital sequencing of ctDNA using the Guardant360 assay for 32 patients. Sequence variants likely originating from the tumor (based on allele frequency and absence in the above-mentioned germ-line variants) were found in 27 (84%) pre-treatment and 25 (78%) on-treatment samples. Most frequently mutated gene in pre-therapeutic plasma samples, in which circulating tumor fraction is > 0.4%, was KRAS (85%), followed by TP53 (60%), ATM (30%), and CDKN2A (15%). Relative change in allele frequency for those alleles that were present in pre- and on-treatment biopsies were positively correlated with differences in RECIST measurements. Conclusions: In summary, dual targeting of EGFR/MEK signaling shows antitumor activity in PDAC in a subset of patients, in particular those with high levels of E-cadherin expression, which is in agreement with our preclinical findings. Digital sequencing of ctDNA provides information about genomic alterations in the majority of patients and holds the potential of providing early information on response to therapy. Supported by CTEP and NIH R21 CA149939. Citation Format: Andrew H. Ko, Tanios Bekaii-Saab, Ryan Courtin, Olga K. Mirzoeva, Sharvina Ziyeh, Robin K. Kelley, Elizabeth Ditto, Anna Ong, Regina Linetskaya, Margaret Tempero, Alan P. Venook, Amirali Talasaz, Wolfgang Michael Korn. Next-generation sequence analysis of cell-free DNA in patients with chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) treated with selumetinib (AZD6244) and erlotinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2015-5244

Background:Recent finding have suggested that discordance between primary and metastatic colorectal tumors is influenced by time, intervening therapy, and sites of metastatic disease. CfDNA allows a non-invasive assessment of the present molecular features of the tumor, but the degree of information provided and the clinical utility of the testing remains to be defined.Methods: Patients were prospectively consented for collection of cfDNA from plasma samples for sequencing on a 54-gene platform optimized for very low allele frequencies (Guardant360), a CLIA certified lab, and concurrent sequencing of clinically available and FFPE tissue following institutional standard of care (Ion-Torrent panel). Results were returned to patients and physicians within 12-19days, and used for evaluating clinical trial options. Surveys of treating physicians were provided to assess clinical utility of the additional ctDNA testing.Results: 105 patients were enrolled after a median of two prior lines of therapy for metastatic disease. Plasma testing was successful in all patients, and tissue sequencing was only able to be performed in 90% of patients, due to limited tissue availability. In the plasma testing, 85% of samples had a detectable genomic alterations (including 21% with detectable amplifications in EGFR, MET, or ERBB2), with an average of 4.6 mutations detected per patient. In the cases with tissue sequencing results, 38% of the cases had mutations detected in the plasma that were not present in the historic tissue. Only 8% of these were the previously described ‘acquired’ mutations in KRAS, NRAS, and EGFR with anti-EGFR monoclonal inhibition. Mutation in TP53 and SMAD4 genes were the most common newly detected in plasma. In many cases, it appeared that these represented minor clones not represented in the sequenced FFPE tumor. In support of this, the median allele quantification for these newly detected mutations was only 4% (IQR = 1.7% to 31%) of the dominant clone, in contrast to concordant mutations that were present at 87% (IQR = 54% to 100%) of the allele burden of the dominant clone (P Citation Format: Maria Pia Morelli, Michael Overman, Eduardo Vilar, Van Morris, David Fogelman, Imad Shureiqi, Chris Garret, Raghav Kanwal, Cathy Eng, Brian Kee, Shanequa Manuel, Robert Wolff, Dragon Sebisanovic, LaiMun Sew, Aubey Zapanta, Ben Shiller, Gangwu Mei, Helmy Eltoukhy, AmirAli Talasaz, Scott Kopetz. Prospective clinical application of circulating cell-free DNA sequencing in metastatic colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5236. doi:10.1158/1538-7445.AM2015-5236

107 Background: Pancreatobiliary carcinomas (PC) carry a poor prognosis but have not yet benefitted from the revolution in precision oncology, in part because biopsy tissue is often inadequate for ...

3604 Background: Concordance of somatic mutations between primary and metastatic colorectal tumors is high in colorectal cancer (CRC), circulating cell-free DNA (cfDNA) accurately reflects the mutation status of metastatic disease. Intervening treatment and metastatic dissemination may result in clonal evolution from pre-existing intratumoral heterogeneity, with the resulting discordance in minor clones.Methods: Patients (pts) were prospectively consented for collection of cfDNA from plasma samples for sequencing on a 54-gene platform optimized for very low allele frequencies (Guardant360), with concurrent sequencing of clinically available and macrodissected historic FFPE tissue (50-gene Ion-Torrent panel), both performed in CLIA-compliant labs. Eligible cases had plasma and tissue sequencing where the modal mutation was present with ≥ 1% and ≥ 5% allele frequencies, respectively. Minor clones were defined as those with an allele frequency < 10% of the highest detected allele frequency in the sample.Resu...

572 Background: MET inhibition is emerging as a potent therapeutic strategy and MET gene amplification has shown predictive significance. MET amplification rate in mCRC, as previously reported in literature, varies from 9% in primary to 18% in metastases but intermixes increased copy number from chromosomal level aberrations with focal gene amplification. Validation of MET amplification rate in mCRC is needed. Methods: We performed analyses of MET amplification in mCRC patients (pts) (n = 636) across multiple cohorts. Cohort 1 (n = 103) included tissue microarray from liver metastases analysed using fluorescence in situ hybridization (FISH) [cMET and CEP7 probes, MET/CEP7 ratio > 2]. Cohort 2 (n = 205) included pts referred for phase I trials who had MET amplification testing using FISH. Cohort 3 (n = 279) included cases sequenced with HiSeq (Illumina) with full exome coverage for 202 genes including MET (average depth 800) with focal gene amplification (≥ 4 copies) identified by an in-house algorithm. Cohort 4 (n = 49) included pts refractory to EGFR monoclonal antibodies enrolled in the ATTACC (a prospective molecular screening) program for mCRC, in whom plasma circulating-free DNA (cfDNA) was analyzed by Guardant sequencing technology. Results: In tissue based analyses, focal MET amplification rate was 1.7% and was higher in primary tumors compared to metastases [3.1% (9/291) vs. 0.4% (1/288), p = 0.02] [Table]. In cohorts 2 & 3 MET amplification was found in 4 [MET/CEP7: 2.1 – 7.7; primary (4/130), metastases (0/75)] and 6 [copy number: 4.0 – 6.7; primary (5/161), metastases (1/110)] cases, respectively. MET amplification rate in pts who had progressed on anti-EGFR therapy was 14.3% (Table). Conclusions: Contrary to prior reports, in this large cohort, MET amplification was a rare event in mCRC pts and the rate was not higher in metastatic sites. However, MET amplification occurred in a sizable subset of pts refractory to anti-EGFR therapy as identified by cfDNA analysis. MET amplification appears to play a minor role in de novo colorectal carcinogenesis but may play an important role in acquired resistance to anti-EGFR therapy. [Table: see text]

e22041 Background: Analysis of genomic alterations in advanced malignant disease is quickly becoming the standard of care in oncology. Biopsies are risky, often costly and may not capture genetic c...