Plasma Next-generation Sequencing Research Articles

Use of Plasma Metagenomic Next-generation Sequencing for Pathogen Identification in Pediatric Endocarditis.

Pediatric infective endocarditis incurs significant morbidity and generally occurs among children with underlying heart disease. Identification of a pathogen is critical in determining appropriate therapy. However, standard diagnostic testing has limited sensitivity. We describe a case series of children with infective endocarditis in whom plasma next-generation sequencing (Karius, Redwood, CA) identified an organism in 8 of 10 cases.

Circulating Tumor DNA as a Biomarker in Oligometastatic Non-small Cell Lung Cancer

Selected patients with oligometastatic non-small cell lung cancer (NSCLC) benefit from aggressive local consolidative therapy. We explore the clinical utility of liquid biopsy for circulating tumor DNA (ctDNA) as a biomarker for patients with oligometastatic NSCLC. Patients with metastatic lung cancer underwent liquid biopsy with plasma next-generation sequencing of ctDNA at diagnosis or disease progression using a validated ctDNA assay targeting 23 genes. Oligometastatic state was defined < 5 sites of metastatic disease at initial diagnostic imaging or progression of < 5 sites of disease. Driver alterations were defined by the National Comprehensive Cancer Network guidelines to include EGFR, ALK, ROS1, KRAS, BRAF, MET, RET, HER2, or NTRK. ctDNA detection rates were compared based on clinical and treatment characteristics using Fisher’s exact test. Survival was evaluated using Kaplan-Meier analysis from the time of local consolidative radiation therapy. Among 820 patients with advanced NSCLC, 109 patients (13%) had oligometastatic disease and 711 patients (87%) had polymetastatic disease. Among the oligometastatic cohort, 73 patients (67%) were oligometastatic at initial diagnosis whereas 36 patients (33%) were oligoprogressive. Oligometastatic disease was associated with a lower rate of ctDNA detection compared to polymetastatic disease (52/109, 48% vs 478/711, 67%; p < 0.001). The rate of ctDNA detection was higher among oligometastatic patients with extrathoracic metastases compared to patients with disease confined to lungs, pleura, and/or thoracic lymph nodes (43/79, 54% vs 9/30, 30%, p = 0.031). ctDNA detection among oligometastatic patients was not associated with the presence of brain metastases (p = 0.2), liver metastases (p = 0.6), or prior smoking history (p = 0.7). Among patients with oligometastatic disease, 47% (51/109) underwent consolidative radiation therapy including stereotactic body ablative therapy and conventionally fractionated radiation therapy. Detection of a driver alteration in ctDNA was associated with decreased progression-free survival (HR 2.62, 95% CI 1.27 – 5.43, p = 0.009) following consolidative radiation therapy. In the setting of advanced NSCLC cancer, the rate of ctDNA detection varies according to the number of disease sites and anatomic distribution of disease. ctDNA shows potential as a biomarker for identification of oligometastatic NSCLC patients likely to benefit from local consolidative therapy.

Turnaround Time of Plasma Next-Generation Sequencing in Thoracic Oncology Patients: A Quality Improvement Analysis.

Genomic analysis of plasma cell-free DNA has become a widespread tool for advanced non-small-cell lung cancer care. Whereas accuracy has been reported on widely, its usefulness is also tied tightly to its turnaround time (TAT), which is not well studied. We studied the TAT of commercial plasma next-generation sequencing (NGS; Guardant360) for 533 results from 461 patients at our center between August 2016 and October 2019. The study received institutional review board approval as a quality improvement study; therefore, the results of the test and clinical setting were not analyzed. TAT from blood draw to result (median of 9 days) was slightly longer than the TAT from laboratory receipt to result, a median of 7 days. Testing volume at our center increased three-fold over the time of the study. During this period, clinical TAT decreased from an initial median of 12 days to a median of 8 days in 2018, but more recently the median increased slightly to 9 days. During the most recent 12 months, 231 (95%) of 247 cases resulted within 14 days from blood draw, with delayed results usually because of billing, whereas 44 cases (18%) resulted within 7 days of blood draw. Studying 92 cases drawn in the most recent 3-month period, the median time of result receipt was 4:01 pm Eastern Time/1:01 pm Pacific Time; 39 results (43%) were returned after 5:00 pm Eastern Time. In a large single-institution experience, we find that plasma NGS results can routinely be expected within 2 weeks, but uncommonly result within 1 week, supporting the need for new strategies to incorporate plasma NGS into the initial genotyping of advanced non-small-cell lung cancer.

1939P Potential role of longitudinal plasma next generation sequencing (NGS) in advanced non-small cell lung cancer (aNSCLC) patients (pts) experiencing hyperprogression (HPD) and early death (ED) during treatment with immune checkpoint inhibitors (ICIs)

The introduction of ICIs in clinical practice has revolutionized aNSCLC treatment but a proportion of pts does not benefit from ICIs and could even derive detrimental effects. No predictive markers are currently available for their early detection. Aim of the study was to characterize pts experiencing HPD and ED with plasma NGS to analyze their modification at an early time-point during treatment.

When should we order a next generation sequencing test in a patient with cancer?

Technical advances in genome sequencing and the implementation of next-generation sequencing (NGS) in clinical oncology have paved the way for individualizing cancer patient therapy based on molecular profiles. When and how to use NGS testing in the clinic is at present an unsolved issue, although new research results provide evidence favoring this approach in some types of advanced cancer. Clinical research is evolving rapidly, from basket and umbrella trials to adaptative design precision oncology clinical studies, and genomic and molecular data often displace the classical clinical validation procedures of biomarkers. In this context, physicians must be aware of the clinical evidence behind these new biomarkers and NGS tests available, in order to use them in the right moment, and with a critical point of view. This review will present the status of currently available targeted drugs that can be effective based on actionable molecular alterations, and the NGS tests that are currently available, offering a practical guide for the application of Clinical Precision Oncology in the real world routine practice.

Abstract A28: SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC

Abstract Introduction: Precision therapy for cancer drug resistance requires detection of resistance mutations and treatment with appropriate targeted therapies. This paradigm is well established in EGFR-mutant NSCLC, yet our understanding of drug resistance in ALK-positive NSCLC is limited. Next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA) now permits noninvasive interrogation of drug resistance. To facilitate improved understanding of ALK drug resistance and the effectiveness of treatment strategies, we launched this remote participation study. Methods: The SPACEWALK study offers plasma NGS to patients with advanced NSCLC with systemic progression on a next-generation ALK TKI to assess for mechanisms of resistance. Patients consent, enroll, and participate remotely, permitting a larger cohort of ALK positive patients to be studied. Remote phlebotomists can be dispatched to the patient’s home when necessary to ease the burden of study participation and to ensure study compliance. Blood is sent to Resolution Biosciences for ctDNA extraction and hybrid capture NGS, with results returned to the study team and the patient’s physician. Patients are followed for 2 years and blood is collected again after starting a new treatment or at progression. This study is supported by the Addario Lung Cancer Medical Institute (ALCMI). Results: Of 143 patients contacting us via the study website, 61 were offered consent and 53 signed consent. 46 enrolled from 23 different US states and 38 different medical centers. Plasma NGS results were returned a median of 13 days after blood draw (range 8-21). Of the 46 patients, the known ALK fusion was detected in 20 (43%) with a median ALK fusion AF of 3.5% (range 0.3%-37%). 10 patients also underwent plasma NGS with Guardant360, with a median time between the two plasma NGS draws of 1.5 weeks (range 0-22.6). In 4 cases, Resolution Bioscience identified an ALK fusion (AF 0.2-37%) not detected on Guardant360. Among the 20 patients with ALK fusions detected, 12 (60%) were found to carry ALK resistance mutations, 5 with G1202R; 2 patients had 2 or more ALK mutations detected. In addition, 6 participants (30%) had a MET amplification detected with a median of 8 copies (range 3-22) and 1 had a KRAS G12V mutation detected (10% AF). One patient with high MET amplification received combination therapy with alectinib and crizotinib and had dramatic radiographic response. Conclusions: Plasma NGS permits the detection of targetable resistance mechanisms in patients with ALK-positive NSCLC and drug resistance. Sensitivity of different plasma NGS assays for ALK fusions varies. This assay may help guide oncologists across the country to select best treatment options after resistance. Such remote-participation studies may offer a new mechanism for characterizing resistance to emerging targeted therapies in rare cancer populations. Citation Format: Marissa N. Lawrence, Rubii M. Tamen, Alicia Sable-Hunt, Seyed Ali Hosseini, George R. Simon, Jonathan W. Riess, Geoffrey R. Oxnard. SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A28.

Selected Articles from This Issue

Paradoxically, patients with melanoma and lung cancer harboring BRAF V600E mutations respond to combined BRAF/MEK inhibition, although patients with colorectal cancer (CRC) harboring the same mutation do not. Two transcriptional subtypes of BRAF V600E-mutant CRC (BM1 and BM2) have been described, with BM1 associated with poor prognosis. Middleton and colleagues sought to determine the impact of BM subtyp. on response to combination dabrafenib, trametinib, and panitumumab (D+T+P) in patients with CRC. Patients with the BM1 subtype of CRC responded more favorably to this regimen and showed evidence of immunologic enrichment in their tumors compared with patients with BM2 CRC. These data suggest that further subtyping may be necessary to derive maximum benefit from targeted cancer therapies.Patients with metastatic renal cell carcinoma (mRCC) generally have a poor prognosis and frequently experience treatment-associated toxicity. To better understand the impact of treatment on health-related quality of life (HRQOL) in patients with mRCC, Atkins and colleagues assessed patient-reported outcomes (PROs) from the phase III IMmotion151 trial. Patients were previously untreated and received either atezolizumab plus bevacizumab or sunitinib, one of the current standards of care for this disease. Patients who received atezolizumab plus bevacizumab reported a reduced treatment burden, including better overall daily functioning and HRQOL and better tolerated treatment-related side effects than those who received sunitinib. Furthermore, more favorable PROs correlated favorably with progression-free survival. These results further support the clinical benefit of atezolizumab plus bevacizumab for patients with mRCC.Patients with ALK-positive non-small cell lung cancer, despite initially responding, ultimately develop resistance to this treatment strategy. Furthermore, resistance to the third-generation ALK inhibitor, lorlatinib, is often due to ALK-independent mechanisms. Dagogo-Jack and colleagues identified MET amplification in 15% of tumor biopsies from patients who relapsed following treatment with next-generation ALK inhibitors and detected novel ST7-MET rearrangements in two cases. MET amplification and/or fusion of MET with ST7 decreased sensitivity of ALK-positive cells to ALK inhibitors, and combined blockade of ALK and MET overcame this resistance. Consistent with these in vitro findings, two patients with ALK-positive lung cancer and acquired MET alterations responded to combined inhibition of ALK and MET. This study justifies the further study of ALK/MET combination therap. in patients developing resistance to ALK inhibition.Tyrosine kinase inhibitors (TKIs) are expected to become a standard treatment option for patients with non-small cell lung cancer harboring mutations in MET exon 14. However, acquired resistance to these agents hinders their ultimate efficacy, and the molecular mechanisms of resistance remain poorly understood. Recondo and colleagues assessed genomic alterations occurring at the time of progression on MET TKIs via next-generation sequencing of plasma and/or tissue. Genomic alterations known or suspected to be mechanisms of resistance were detected in most resistant patients, including on-target mutations in MET and off-target alterations in KRAS, EGFR, HER3, and BRAF. Two patients who developed on-target resistance mutations demonstrated second partial responses after switching between type I and type II MET TKIs. These results provide rationale for monitoring on- and off-target alterations in patients treated with MEK inhibitors.

Rapid, Noninvasive Diagnosis of Balamuthia mandrillaris Encephalitis by a Plasma-Based Next-Generation Sequencing Test.

Granulomatous amoebic encephalitis (GAE) caused by Balamuthia mandrillaris is a rare subacute infection with exceptionally high mortality. Diagnosis is typically made by brain biopsy or at autopsy. Detection of Balamuthia mandrillaris cell-free DNA by next-generation sequencing of plasma enabled rapid, noninvasive diagnosis in a case of amoebic encephalitis.

Real-world data on liquid biopsies utilization in lung cancer patients in the community setting: The Mount Sinai experience.

e21502 Background: Liquid biopsy describes the extraction of circulating tumor DNA (ctDNA) from peripheral blood via next-generation-sequencing (NGS). The yield of adding plasma-based NGS to tissue NGS for the detection of Actionable Mutations (AM) have been reported scarcely and additional “real world” data is needed. Methods: In this retrospective data review, we included patients with non-small cell lung cancer (NSCLC) who had plasma NGS genomic testing ordered between Nov 2014 and July 2019, as part of routine management. Plasma testing was performed using Guardant360 commercial platform and was analyzed for therapeutically AM in: EGFR, ALK, MET, BRCA1, BRCA2, KRAS, ROS1, RET, ERBB2, BRAF, and NTRK2. Results: Over a period of 5 years, 337 samples taken from 279 patients were sent for ctDNA NGS testing. The median age at diagnosis was 73 years [range 36-93, SD:10.4], 51% (N=141) were males and 49% (N=138) were females. Excluding synonymous mutations and variants of unknown significance, 239 samples (71%) and 201 patients (72%) had 705 somatic mutations. Of them, 36% (N=254) were therapeutically actionable and were detected in 139 samples (58%), taken from 106 patients (53%). Commonly detected AM were EGFR (N=127; 50%), KRAS (N=61; 24%), BRAF (N=24; 9.5%), MET (N=23; 9%), RET (N=5; 1.9%), BRCA1 (N=5; 1.9%), BRCA2 (N=4; 1.6%), ERBB2 (N=4; 1.6%) and ALK (N=1, 0.4%). None of the samples had mutations in ROS1, NTRK2. One patient had Microsatellite instability (MSI). Common co-occurring mutations were EGFR and BRAF (N=14), EGFR and MET (N=13) and MET and BRAF (N=10) (p&lt;0.001). Common mutually exclusive mutations were KRAS and EGFR (p&lt;0.001). Tissue NGS analysis was performed in 62/106 patients (58%), with AM found in 45 patients (72%). Concordance agreement in AM between plasma and tissue NGS modalities was detected in 39/45 patients (87%) and was demonstrated in EGFR (N=25), KRAS (N=11), BRAF (N=2) and MET (N=1). In the reminder 6 patients (13%), tissue NGS was discordant from their plasma NGS results. In 44/106 patients, for whom tissue sampling was not performed, utilizing plasma NGS increased AM detection yield by 41%. Conclusions: Integration of plasma NGS testing into the routine management of NSCLC patients demonstrated substantial yield of therapeutically targetable mutations detection rate, when tissue NGS was not available.

Plasma cell free PD-L1 RNA expression correlated with tissue PD-L1 immunohistochemical staining and tumor mutation burden in non-small cell lung cancer.

e15049 Background: Programmed death ligand-1 (PD-L1) protein expression by immunohistochemical staining (IHC) correlates with response to immune checkpoint inhibitor (ICI) therapies in non-small cell lung cancer (NSCLC). Tumor mutational burden (TMB) is another immune biomarker of ICI response in NSCLC. Clinical studies indicate tissue PD-L1 protein expression and TMB are independent yet complementary. Both are limited by tissue acquisition and potential heterogeneity. Plasma cell free PD-L1 RNA (cfRNA) levels and tissue protein PD-L1 expression and TMB was analyzed and correlated in patients with advanced NSCLC. Methods: Patients with advanced NSCLC underwent complementary plasma and tissue next generation sequencing (NGS) with immune biomarkers prior to treatment. Plasma PD-L1 cfRNA was assessed by the Circulogene proprietary direct-on-specimen enrichment technology NGS platform. Correlating tissue testing was performed by the Caris Molecular Intelligence platform with the anti-PD-L1 22C3 IHC antibody and TMB measuring the total number of non-synonymous somatic mutations per megabase. Results: 107 patients with advanced NSCLC were evaluated with simultaneous plasma and tissue NGS testing. 17% (95% confidence interval [CI] 10-24%) patients plasma PD-L1 positive. 48.5% (CI 38-57%) tissue IHC PD-L1 positive. 7 of 18 plasma PD-L1 positive patients were tissue PD-L1 negative. 48% (CI 39-58%) total patients TMB ≥ 10 mutations per megabase (TMB high). Correlating TMB high in 72% (CI 50-94%) of plasma PD-L1 positive and 56% (CI 42-69%) of tissue PD-L1 positive patients. TMB high in 49% (CI 37-59%) plasma PD-L1 negative and 50% (CI 35-65%) tissue PD-L1 negative patients. Conclusions: Although less frequent than tissue PD-L1 protein expression, plasma PD-L1 cfRNA expression correlated with a higher association of tissue TMB high findings than tissue PD-L1 positive patients. There was not any TMB high difference between plasma PD-L1 negative and tissue PD-L1 negative patients. Over one-third of plasma PD-L1 positive patients were tissue PD-L1 negative. Clinical correlation with immune checkpoint inhibitor therapies is ongoing.

Plasma next-generation sequencing (NGS) in advanced non-small cell lung cancer (aNSCLC) patients (pts) treated with immune checkpoint inhibitors (ICIs): Impact of STK11 and TP53 mutations on outcome.

3046 Background: ICIs revolutionized aNSCLC treatment. The next challenge lays on the search for predictive markers. Detection of multiple tumor-related genetic alterations through NGS in cell free DNA is a promising tool, provided the limited availability of tumor tissue in most cases. Methods: Between January 2017 and October 2019, aNSCLC pts consecutively referring to our Institution were prospectively screened with plasma NGS while included in two clinical trials: VISION (NCT02864992) and MAGIC trial, an observational study. In VISION trial NGS was performed in plasma (Guardant360 test) and tissue (Oncomine Focus Assay). In MAGIC Myriapod NGS-IL 56G Assay was used. Aim of the study was to evaluate the impact of STK11, KRAS and TP53 mutations (muts) on outcome of ICI-treated pts, with overall survival (OS) as primary endpoint. A control group of pts not receiving ICIs was also analyzed. Results: A total of 235 NSCLC pts were enrolled and received ICIs. 93 pts were analyzed in plasma at the time of beginning ICIs: median OS was 18.9 m (95% CI: 13.7-24.1) and median immune-related progression free disease (irPFS) 3.8 m (95% CI: 2.5-5.1). 49 (52.7%), 22 (23.7%) and 8 (8.6%) pts carried TP53, KRAS and STK11 pathogenic alterations, respectively. STK11 mutated pts showed a trend for worse OS compared with wildtype counterpart (14.9 m, 95% CI: 6.5-23.3, versus 20.3, 95% CI: 13.4-27.2, p = 0.192) KRAS muts had no impact on outcome. Pts with TP53 or STK11/KRAS co-mut (n = 3) had worse OS (12.3 m, 95% CI: 9.2-15.4; HR = 3, 95% CI: 1.6-5.8, p = 0.001 and 5.9 m, 95% CI: 1.4-7.6; HR = 2.9, 95% CI: 1.4-6.3, p = 0.007) and worse irPFS (2.8 m, 95% CI: 1.7-3.9, HR = 1.8 95% CI: 1.1-3.1, p = 0.03 and 1.2 m, 95% CI: 0.9-1.5, HR = 2.2 95% CI: 1.2-4.1, p = 0.01). Number of muts negatively impacts pts’ OS (HR = 1.2, 95% CI: 1.1-1.3, p = 0.02) and was higher among TP53 mutated pts (p &lt; 0.001, Mann-Whitney test). In multivariate analysis, TP53 and STK11/KRAS retained significance. A control group of pts not receiving ICIs was analyzed (n = 101): median OS was 16.8 m (95% CI: 13-20.6). Nor STK11 (n = 10), nor STK11/KRAS (n = 6) had impact on OS (HR = 1.8, 95% CI: 0.7-4.7, p = 0.267 and 1.4, 95% CI: 0.7-3.0, p = 0.293) while the presence of TP53 muts (n = 41) was associated with shorter OS (11.4 m, 95% CI: 7.3-15.5; HR = 2.2, 95% CI: 1.2-4.2, p = 0.009). Conclusions: NGS performed in plasma might be used to detect predictive markers. TP53 muts in plasma at baseline had prognostic value, while STK11/KRAS muts were associated with worse outcome to ICIs.

RELAY study of erlotinib (ERL) + ramucirumab (RAM) or placebo (PL) in EGFR-mutated metastatic non-small cell lung cancer (NSCLC): Biomarker analysis using circulating tumor DNA (ctDNA) in Japanese patients (pts).

9527 Background: The phase III RELAY study (NCT02411448) showed significantly improved progression-free survival (PFS) for RAM+ERL vs PL+ERL in 449 pts with previously untreated EGFR mutation-positive metastatic NSCLC (median PFS 19.4 vs 12.4 mo, HR 0.591 [95% CI 0.461–0.760], p&lt;.0001). To understand baseline genetic mutations and treatment-emergent (TE) resistance mechanisms, this exploratory liquid biopsy substudy examined biomarkers in ctDNA from participating Japanese pts by next-generation sequencing (NGS) and droplet digital PCR (ddPCR). Methods: Plasma samples were collected at baseline, during treatment (Cycle 4, 13, and every 6 cycles to Cycle 53) and post-study treatment discontinuation (30-day follow-up [30d FU]). Mutations were assessed at baseline and 30d FU by NGS (Ion AmpliSeq Colon and Lung Cancer panel). EGFR mutations and MET and ERBB2 copy number (CN) were assessed at all time points by ddPCR. Baseline markers were analyzed in pts with any detectable baseline mutation (to confirm ctDNA presence; NGS N=84, ddPCR N=74). TE mutations were analyzed in pts with any detectable mutation at baseline and 30d FU (NGS N=26, ddPCR N=28). Among these pts, 81% and 57% for NGS and ddPCR, respectively, had progressed by 30d FU. Results: By plasma NGS, baseline EGFR activating mutations (exon 19 deletion or exon 21 [L858R] mutation) were detected in 83.3% of pts. Common co-occurring baseline mutations were TP53 (42.9%), PTEN (7.1%) and KRAS (6.0%). Baseline TP53 mutation rate was higher in men vs women (p=.02). No difference in PFS was detected by baseline TP53 status (interaction predictive p=.45, prognostic p=.33). TE mutations were detected in EGFR (including T790M), FGFR3, KRAS and TP53. Slightly higher rates of TE KRAS (p=.03) and TP53 (p=.07) mutations were detected in RAM+ERL than in PL+ERL. TE total EGFR mutations (p=.65) or TE T790M (p=.69) did not differ by arm. By ddPCR, baseline EGFR activating mutations were detected in all pts. T790M was detected at baseline in 2/37 pts/arm (5.4%) and was TE in 6/11 (55%) RAM+ERL pts and 7/17 (41%) PL+ERL pts. There was a trend (p=.054) for greater ERBB2 CN in RAM+ERL vs PL+ERL at Cycle 4. MET CN decreased slightly at Cycle 4 in both arms (significant in PL+ERL, p=.003). Biomarker levels by ddPCR across all time points will be presented. Conclusions: Though limited by sample size and likely inconsistent tumor shedding, this exploratory study identified potential differences in TP53, KRAS, ERBB2 and MET by demographics, treatment and/or time. Clinical trial information: NCT02411448 .

Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p &lt; 0.0001) and sequencing performance (p &lt; 0.0001). In addition, 0.1% limit of detection could be achieved even when &lt; 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.

Detection of Circulating Tumor DNA in Patients with Pancreatic Cancer Using Digital Next-Generation Sequencing

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normal-range digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.

Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance

Discordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored. To measure these variables, we tested four next-generation sequencing (NGS) gene panel assays for mutations in circulating tumor DNA (ctDNA) using replicate sets of 24 plasma samples and compared the results with matched tumor-normal tissue pairs. Our orthogonal approach identified false-negative (FN) and false-positive (FP) variants with high confidence and revealed substantial variability among the ctDNA assays, with a range of sensitivity (38% to 89%) and positive predictive value (36% to 80%). Most discordance in our cross-vendor study was observed below 1% variant allele frequency. FP variants displayed mutational biases and tended to be novel variants not found in somatic databases. Of the 56 unique variants called by all four ctDNA assays, 41 (68%) resulted from technical discordance. These findings suggest that most NGS assay discordance is a result of technical variations and, to a lesser extent, biologic factors such as clonal hematopoiesis of indeterminate potential and tumor heterogeneity.

PATH-49. CLINICAL UTILITY OF PLASMA CELL-FREE DNA IN ADULT PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA – A PILOT PROSPECTIVE STUDY

Abstract BACKGROUND Liquid biopsy has been not been widely utilized in patients with glioblastoma (GBM) compared to other solid tumors. However, the clinical utility of plasma cell-free DNA (cfDNA) in GBM has not been assessed prospectively or at the time of initial diagnosis. METHODS We conducted a prospective cohort study of patients with newly diagnosed GBM. Whole blood was collected in Streck® tubes at baseline prior to initial surgical resection and longitudinally during the course of adjuvant chemoradiotherapy. Plasma cfDNA concentration (ng/mL) was quantified by qPCR for a 115 bp amplicon of the human ALU repeat element, correlated with radiographic tumor burden by volumetry at multiple time points using Spearman rank correlation, and assessed for its impact on progression-free (PFS) and overall survival (OS) by Cox regression. RESULTS Prior to initial resection, GBM patients (N=42) had higher plasma cfDNA concentration compared to age-matched healthy controls (N=42) (mean 13.43 vs. 6.70 ng/mL, p&lt; 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on subjects’ first post-radiation MRI scan (r=0.77, p=0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Pre-operative plasma cfDNA concentration above the mean (&gt;13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, p=0.038) and OS (median 8.9 vs. 14.8 months, p=0.078). The impact on PFS persisted after adjusting for age, extent of resection, performance status, MGMT promoter methylation, and IDH1/2 mutational status (HR 2.48, 95% CI 1.1–6.1, p=0.046). CONCLUSIONS Plasma cfDNA may be an effective prognostic tool and noninvasive surrogate of tumor burden in newly diagnosed GBM. Tumor tissue samples from our cohort have been subjected to targeted next generation sequencing (NGS), and baseline plasma samples have been sent to Guardant Health for NGS. Plasma NGS results and concordance with matched tissue NGS will be included at time of presentation.

2181. Yield and Impact of Molecular Diagnostics for Pathogen Detection in Pediatric Patients: 16/18S rRNA PCR and Noninvasive Assays

BackgroundMolecular diagnostic tests can identify bacterial and fungal pathogens from clinical samples. Nucleic acid detection tests include 16S and 18S rRNA gene PCR (16/18S PCR) and plasma next-generation sequencing (NGS). Other assays (fungal galactomannan and 1,3-β-d-glucan) detect structural factors. Our objective was to assess the utilization, yield, and impact of molecular diagnostics in pediatric patients who had samples sent for 16/18S PCR.MethodsSterile site fluid or tissue specimens were collected as part of standard care at Lurie Children’s Hospital, cultured, and sent to Northwestern Memorial Hospital for 16/18S PCR as clinically indicated. Medical records were reviewed for diagnostics, antibiotics, and clinical course.ResultsFrom 1/2016–8/2018, 236 samples were sent for 16 and/or 18S PCR from 183 patients. 83% had a concurrent ID consult. 16S PCR was done on 215 samples, 42 (20%) were positive, and 36 yielded species identification (Table 1). Antibacterial agents were administered prior to specimen collection in 73% and did not affect likelihood of positive 16S PCR. 18S PCR was sent on 163 samples; 12 (7.4%) were positive (Table 2) of which 10 were from immunocompromised hosts. 40% of patients were on antifungals prior to sample acquisition. 16/18S PCR impacted antimicrobial decision-making in 70 cases (30%). A pathogenic fungus was detected by PCR but not culture in 2 cases. Time to positivity of fungal culture was 1–15 days. Fungal culture was positive in 5 cases with-negative 18S PCR. Seventeen patients had positive serum 1,3-ß-D-glucan and/or galactomannan: 3 of which had positive 18S PCR, 5 with fungal growth, 5 presumed infection based on imaging, 1 Nocardia, and 3 noninfectious etiology. Plasma NGS was sent on 45 cases, was positive in 34, and affected clinical management in 10.Conclusion16S PCR can identify bacterial pathogens in the setting of negative culture and impact clinical care. Abscess, bronchial/pleural fluid, and brain/organ tissue were high yield specimens. 18S PCR can provide expeditious fungal identification in cases of suspected invasive disease, but fungal culture and serum molecular testing increase diagnostic yield. No single fungal test is comprehensive. Plasma NGS had relatively high yield and clinical impact in selected patients. Disclosures All authors: No reported disclosures.

1409. Next-Generation Sequencing-based Detection of Angiostrongylus cantonensis (AC) Using Microbial Cell-free DNA Sequencing of Plasma in Atypical Cases of Rat Lungworm Meningitis Presenting with Ascending Paralysis

BackgroundAC or Rat Lungworm meningitis usually presents as a self-limited illness with headache and sensory changes, rarely progressing to coma, death, or permanent brain damage. It is usually diagnosed by eosinophils in the CSF. Once limited to Asia and the tropical Pacific AC transmission via slugs and snails documented on US mainland in 2018. We describe 2 unusual, severe examples of AC infection in infants presenting with ascending paralysis and initial CSF without eosinophilia suggesting Guillain–Barre syndrome (GBS).MethodsConventional lab testing of serum and CSF, brain and spine MRI, AC PCR by Hawaii Department of Health, and mcfDNA next-generation sequencing (NGS) of plasma (Karius).ResultsTwo infants, aged 8 and 11 months, presented with fever, lower extremity weakness, and ascending paralysis. An initial evaluation in both included normal brain/spine imaging and CSF with modest lymphocytic pleocytosis without eosinophils. Paralysis progressed despite IVIG. Case 1: 11-month male: Admitted on fever day 5. Paralysis progressed to respiratory failure requiring ventilation for 20 days. Illness day 16: MRI showed spinal cord swelling C3-C7, Brain normal. CSF#3: WBC 269 28% eos, ↑ protein. Visible 8 mm long young adult worms, PCR positive for AC. Rx high-dose corticosteroids, albendazole for >4 weeks. Day 29 illness MRI: Cerebral infarct L frontal lobe, worm tracks medulla, inflammation cauda equina. Slow improvement over 5 months. Case 2: 8-month-old female: Admitted fever day 8. Weakness progressed to arms and trunk. Day 10 illness: CSF #2: Visible worms present, WBC 84, 26% eos. PCR positive for AC. Rx: High-dose steroids and albendazole x4 weeks. MRI spine illness day 30: inflammation cauda equina. Weakness improved by illness day 37. McfDNA sequencing of plasma detected AC in acute stage peaks of 123 and 12 molecules/microliter in cases 1 and 2. Serial mcfDNA testing showed a decline in the AC DNA level in plasma which correlated with treatment and clinical response.ConclusionAC infection may mimic GBS or transverse myelitis. AC diagnosis may require repeat CSF testing. NGS detection of AC in plasma holds promise as rapid, noninvasive diagnosis and assessment of response to therapy. High-dose steroids with albendazole may be effective even in severe AC.Disclosures All authors: No reported disclosures.

2886. Retrospective Case–Control Study of the Performance of the Karius Test, a Plasma Microbial Cell-free DNA Next-generation Sequencing test, to Detect Invasive Mold Infections in Hematopoietic Cell Transplant Recipients with Pneumonia

BackgroundPulmonary invasive mold infections (IMI) cause significant morbidity and mortality after hematopoietic cell transplant (HCT). Noninvasive diagnostic options are limited, particularly for non-Aspergillus (non-Asp) molds. Given differences in activity and toxicities of antifungals, early diagnosis and targeting of specific IMI is important. We evaluated the performance of the Karius Test, a plasma microbial cell-free DNA next-generation sequencing (NGS) test, for detecting IMI in HCT recipients.MethodsWe conducted a retrospective case–control study of 24 HCT recipients with proven non-Asp pulmonary IMI, 51 probable/proven Aspergillus pulmonary IMI, and 20 controls with nonfungal pulmonary infections. All subjects had plasma obtained within 14 days of diagnosis. Workup included bronchoalveolar lavage (BAL) and/or biopsy, with fungal stains/culture and galactomannan testing of BAL and serum. Plasma cell-free DNA was extracted and NGS performed (Karius, Redwood City, CA). Human reads were removed and remaining sequences aligned to a curated database including over 300 fungi. Organisms present above a predefined significance threshold were reported. A higher sensitivity research-use only pipeline was also used. Analysis of sequencing data was blinded to all clinical data.ResultsWe identified pathogenic molds in 19/24 (79%) of subjects with proven non-Asp IMI, including Mucor, Rhizomucor, Scedosporium, Rhizopus, and Cunninghamella spp. In Aspergillus proven/probable IMI, A. fumigatus was identified in 13.7% (7/51) of subjects. In 3 other subjects with proven/probable aspergillosis, we also identified Rhizomucor miehei and R.pusillus, and Cunninghamella, consistent with clinical findings. The use of an optimal-sensitivity pipeline identified an additional 9 subjects with Aspergillus spp. and other pathogenic molds, increasing detection of molds to 37.3% (19/51). Specificity for molds in negative samples was 100% (20/20).ConclusionThe Karius plasma NGS test is a noninvasive means of detecting IMI with high sensitivity for non-Aspmolds in HCT recipients with pulmonary disease. Further assay optimization may increase sensitivity for Aspergillus. This may be a useful adjunctive test for diagnosing IMI, and larger studies should be conducted.Disclosures All Authors: No reported Disclosures.

Clinical Application of Next-Generation Sequencing as A Liquid Biopsy Technique in Advanced Colorectal Cancer: A Trick or A Treat?

Owing to its advantages over prior relevant technologies, massive parallel or next-generation sequencing (NGS) is rapidly evolving, with growing applications in a wide range of human diseases. The burst in actionable molecular alterations in many cancer types advocates for the practicality of using NGS in the clinical setting, as it permits the parallel characterization of multiple genes in a cost- and time-effective way, starting from low-input DNA. In advanced clinical practice, the oncological management of colorectal cancer requires prior knowledge of KRAS, NRAS, and BRAF status, for the design of appropriate therapeutic strategies, with more gene mutations still surfacing as potential biomarkers. Tumor heterogeneity, as well as the need for serial gene profiling due to tumor evolution and the emergence of novel genetic alterations, have promoted the use of liquid biopsies—especially in the form of circulating tumor DNA (ctDNA)—as a promising alternative to tissue molecular analysis. This review discusses recent studies that have used plasma NGS in advanced colorectal cancer and summarizes the clinical applications, as well as the technical challenges involved in adopting this technique in a clinically beneficial oncological practice.