Plasma Next-generation Sequencing Research Articles

Efficacy of vemurafenib in a heavy smoker with BRAF-mutated lung adenocarcinoma: A case report and literature review.

At present, research on BRAF gene mutations appears to be mainly focused on melanoma rather than non-small-cell lung cancer (NSCLC). We herein describe the case of a patient with BRAF V600E-mutated advanced NSCLC, whose symptoms were relieved and computed tomography imaging revealed partial response to vemurafenib following failure of chemotherapy. This case demonstrates the promising prospects of BRAF inhibitor treatment in patients with BRAF-mutated NSCLC. Targeted therapies have significantly modified the treatment of NSCLC. However, tumor tissue is frequently hard to obtain, whereas the coincidence rate of gene mutations between the plasma and tumor tissue is 60-80%. Therefore, in cases where tumor tissue is difficult to obtain, plasma next-generation sequencing may be used to detect gene mutations, which can overcome the limitations of gene detection. Furthermore, due to the tumor heterogeneity, different patients exhibit different gene mutation abundance. Research has demonstrated that mutation abundance is associated with the therapeutic efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors. However, the association between BRAF mutation abundance and the therapeutic effect of BRAF inhibitors requires further verification.

Real world results of liquid biopsy in stage 3/4 solid tumors and potential ''clinical actionability."

2555Background: Next-generation sequencing (NGS) of plasma circulating cell-free DNA (cfDNA) and cfRNA, also known as a “liquid biopsy”, is a minimally invasive technique available in the clinic. H...

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer

BackgroundGenomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methodsPlasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. ResultsA total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2=0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. ConclusionsThis blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Discrimination of Germline EGFR T790M Mutations in Plasma Cell-Free DNA Allows Study of Prevalence Across 31,414 Cancer Patients.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non-small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA.Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested.Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001).Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351-9. ©2017 AACR.

Highlights of This Issue

HDAC inhibitors have been shown to modulate immune response in preclinical models. To explore the immunomodulatory activity of HDAC in the clinical setting, Pili and colleagues designed a phase I/II clinical study with the selective class I inhibitor entinostat and high-dose IL2 in patients with metastatic clear cell renal cell carcinoma. The combination resulted in greater objective response rate and increased median progression-free survival compared to historical data. This proof of principle provides the rationale for further clinical testing of this epigenetic modulator and immunotherapies as rational combination strategies.PARP inhibitors (PARPi) have emerged as an intriguing treatment strategy for patients with Ewing sarcoma (ES), but have yet to demonstrate responses in clinical trials. Heske and colleagues performed a high-throughput matrix drug screen to identify novel, synergistic drug combinations in ES and found that combining PARPi with NAMPT inhibitors, which block the rate-limiting step in NAD+ production, was a rational approach to enhancing PARP inhibition. This combination resulted in robust synergy in vitro through decreased PAR activity and increased DNA damage, and retained efficacy in vivo, suggesting that this combination may be a promising strategy for ES.Hu and colleagues identify a novel application of plasma cell-free DNA (cfDNA) genotyping, widely used as a cancer diagnostic, to study the germline genetics of cancer patients. Germline and somatic EGFR T790M mutations are used as a clinical model to develop methods for differentiating germline and somatic variants within plasma cfDNA. Using a database of 31,414 subjects undergoing plasma next-generation sequencing (NGS), they demonstrate that germline EGFR T790M mutations are enriched in lung cancer, but not other cancer types. Awareness is needed of the potential for clinically important incidental germline findings from plasma NGS.Hyperactivation of the Wnt/β-catenin and PI3K/Akt signaling pathways has limited clinical benefit mostly due to unknown resistance mechanisms and the lack of predictive biomarkers of the drug response. Kang and colleagues investigated molecular crosstalk between the Wnt/β-catenin and PI3K/Akt pathways via ICAT, a negative regulator of Wnt/β-catenin signaling. The authors provide evidence for a rational stratification of patients hyperactivated by the Wnt/β-catenin and PI3K/Akt signaling pathways using PLD1 and ICAT as predictive targets of the drug response. These results may assist in the clinical development of a PLD1 inhibitor for treatment of CRC patients carrying APC and PI3KCA mutations.

The SEP-SEQ Trial: Clinical Validation of the Karius Plasma Next-Generation Sequencing Test for Pathogen Detection in Sepsis

BackgroundSepsis is a leading cause of death and can be caused by a wide range of potential pathogens. In up to 40% of cases, a causative pathogen is never identified. There is a need for improved diagnostic tests that can accurately identify the breadth of potential pathogens to inform effective antimicrobial therapy.MethodsWe enrolled a prospective cohort of patients presenting to the hospital with signs and symptoms of sepsis. Plasma samples were collected for NGS testing at time of initial blood culture. Extracted plasma cell-free DNA was sequenced, human sequences removed and remaining reads aligned against a pathogen database consisting of viruses, bacteria, and eukaryotic pathogens. Relative abundance was estimated; pathogens present at high statistical significance were identified. NGS results were compared with a composite reference standard of all microbiology testing performed within 7 days of admission and clinical diagnosis.ResultsOf 286 patients enrolled, plasma NGS identified potential pathogens in 60.1% (172 of 286) of septic subjects including DNA viruses, bacteria (including fastidious/unculturable bacteria like Mycobacterium tuberculosis), and fungi. In contrast, 15.7% (45 of 286) subjects had a positive initial blood culture and 38.1% (109 of 286) had a potential infectious etiology identified using a composite microbiology laboratory standard. The NGS plasma assay had a positive agreement of 86.7% (39 of 45) and 79.5% (78 of 98) compared with initial blood culture (after excluding contaminants) and the composite laboratory reference standard, respectively. After clinical adjudication, 81.4% (140 of 172) of the positive plasma NGS results were deemed to be consistent with the septic event. Of the remaining 32 subjects, 15 had NGS results that were plausible causes of sepsis but clinical were insufficient to confirm this.ConclusionsWith a single blood draw, the Karius plasma NGS assay identified a broad range of pathogens in septic patients three times more often than blood culture and more often than all microbiology tests combined. This plasma NGS test can identify a viruses, bacteria, and eukaryotic pathogens which can provide valuable information to help clinicians better target antimicrobial therapy for patients with sepsis.Disclosures H. Seng, Karius, Inc.: Employee, Salary. D. Hollemon, Karius, Inc.: Employee, Salary. D. Hong, Karius, Inc.: Employee, Salary. T. Blauwkamp, Karius, Inc.: Employee, Salary. M. Kertesz, Karius, Inc.: Employee, Salary. S. Yang, Karius, Inc.: Research Contractor, Research grant.

Detection of fetal duplication 16p11.2q12.1 by next-generation sequencing of maternal plasma and invasive diagnosis

Objective: The objective of study is to report the feasibility of non-invasive prenatal screening (NIPS) combined with invasive detection by chromosomal analysis in identifying fetal duplication, providing clinical performance of NIPS on copy number variations (CNVs) detection. Material and methods: NIPS was offered to a 35-year-old pregnant woman. Amniocentesis was performed to confirm the positive screening result. Fetal sample was detected by karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA). Parental karyotyping was also conducted. Results: NIPS result was positive for chromosome 16, indicating an extra copy of chromosome 16. FISH and chromosomal karyotyping revealed that the fetus had a marker chromosome derived from chromosome 16. CMA further demonstrated an approximately 19-Mb duplication in chromosome 16. The final fetal karyotype was 47,XY,+mar. ish der (16)(D16Z3+).arr 16p11.2q12.1 (30 624 186–49 696 337 × 3). Ultrasound scan and MRI showed some structure malformations. Conclusions: A protocol for CNVs detection by combining a series of genetic methods was presented in this study and a novel marker duplication 16p11.2q12.1 was reported. With the ability to identify subchromosomal deletions and duplications in fetus, NIPS could reduce the possibility of invasive diagnosis. The followed confirmation test for positive sample is necessary and ensures the accuracy of the diagnosis.

Abstract LB-249: Examination of analytical factors impacting concordance of plasma-tumor testing by next-generation sequencing (NGS)

Abstract The increased usage of circulating tumor DNA (ctDNA) sequencing for oncology clinical research demonstrates a critical need for sensitive and specific testing. While we have observed a high degree of concordance between single tumor mutations in tumor and plasma, several recent studies have highlighted a lack of concordance between plasma and tumor panel NGS gene panel testing due to biological and technical factors. To explore further these factors and benchmark ctDNA NGS testing services, a set of matched plasma, tumor, and normal samples from 24 subjects were acquired from three biobanking companies. Replicate 2 ml-plasma samples were tested by four ctDNA sequencing companies, and matching tumor/normal samples were tested by two tumor sequencing companies. Concordance was measured by comparing plasma mutations to tumor mutations as well as comparing mutations among the same plasma tested by the ctDNA companies. While our experience with NGS of matched samples from clinical trials typically identifies ~30% of patients with no detectable mutation and therefore likely not shedding tumor DNA, with the retrospectively collected commercial samples ~60% lacked detectable high confidence mutations, likely due to quality control issues with sample collection. We also found variation in the concordance of ctDNA mutation detection rates among the four vendors, due to significant differences in DNA yield and assay sensitivity. While factors such as tumor heterogeneity and timing of plasma-tumor collection can lower concordance rates, the majority of discordance in our study was due to technical rather than biological variation. Assay analytical variance and the impact of reporting false positive variants are key factors that need to be addressed as plasma-based NGS testing is more widely incorporated into translational and clinical research. Examples illustrating the complexity of the analyses and giving support for confidence in ctDNA testing results will be given. Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Tristan Lubinski, Aleksandra Markovets, Kenneth Thress, Robert McEwen, Gaia Schiavon, David Whitston, Barrett Nuttall, J. Carl Barrett. Examination of analytical factors impacting concordance of plasma-tumor testing by next-generation sequencing (NGS) [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 LB-249. doi:10.1158/1538-7445.AM2017-LB-249

Abstract 5692: Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR

Abstract Background: Non-invasive genotyping of cell-free DNA (cfDNA) provides physicians with the ability to identify genomic alterations, evaluate response to treatment, and uncover potential mechanisms of resistance. Here, we analyze the concordance of next-generation sequencing (NGS) and droplet digital PCR (ddPCR) in detecting and quantifying actionable mutations from cfDNA of lung, colorectal, and breast cancer patients. Methods: We studied a cohort of 221 advanced cancer cfDNA samples with completed Guardant360 digital sequencing results. Variant allele frequencies (% AFs) ranged from less than 0.2% to greater than 90% for common driver and resistance mutations. Blinded to plasma NGS results, we performed ddPCR using our validated assays for EGFR L858R, exon19 del, T790M (113 presumed lung cases); BRAF V600E, KRAS G12X (77 colorectal cases); ESR1 D538G, Y537C and PIK3CA H1047R (31 breast cases). Following unblinding, qualitative and quantitative accuracy were compared. Results: Qualitative results (Table 1) show a high concordance between NGS and ddPCR, with 98.3% (576/586) of all assays run being concordant. 9/22 of samples that had an AF below 0.4% were discordant, while only one sample above 0.4% AF was discordant. AFs calculated by NGS and ddPCR were strongly correlated by linear regression (R between 0.951 and 0.999). Notably, the slope of the ddPCR AF versus NGS AF graph for EGFR ex19del was 0.725, which improved to 0.95 with the adoption of an alternate calling method. Conclusions: This interlaboratory comparison of actionable mutations in cfDNA from lung, colorectal, and breast cancers by orthogonal genotyping platforms demonstrates a substantial concordance in detection and quantification between NGS and ddPCR. Our cross-platform study establishes the feasibility of standardization of qualitative and quantitative measurements of cfDNA-detected alterations. Table 1LungEGFR L858REGFR Ex19 delEGFR T790M(n=113)G360+G360-G360+G360-G360+G360-ddPCR+580510530ddPCR-055458456BreastESR1 Y537CESR1 D538GPIK3CA H1047R(n=31)G360+G360-G360+G360-G360+G360-ddPCR+1022020ddPCR-03009029ColorectalBRAF V600EKRAS G12X(n=78)G360+G360-G360+G360-ddPCR+220571ddPCR-055118 Citation Format: Bryan C. Ulrich, Rebecca J. Nagy, Justin I. Odegaard, Richard B. Lanman, Geoffrey R. Oxnard, Cloud P. Paweletz. Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR [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 5692. doi:10.1158/1538-7445.AM2017-5692

Early detection of competing resistance mutations using plasma next-generation sequencing (NGS) in patients (pts) with EGFR-mutant NSCLC treated with osimertinib.

11529 Background: In pts with EGFR+ NSCLC, genotyping of plasma cell-free DNA (cfDNA) has become a routine option for non-invasive detection of EGFRT790M. We hypothesized that serial NGS of cfDNA would allow early detection of co-existent resistance mutations during osimertinib treatment. Methods: Serial plasma samples were collected from pts with advanced EGFR+ NSCLC and T790M+ acquired resistance treated with osimertinib. Up to 4 specimens were analyzed, blinded to tumor genotype: baseline, initial 2 follow-ups, and progression. Plasma NGS was performed using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes. Diagnostic accuracy was compared to tumor genotype (including NGS when available) and droplet digital PCR (ddPCR) of cfDNA. Results: 94 specimens from 26 pts underwent plasma NGS. Studying 26 baseline specimens, plasma NGS was more sensitive than ddPCR for known EGFR driver mutations (100% vs 88.5%). In 8 pts with pretreatment tumor NGS, 5 of 6 TP53 mutations were detected; one plasma positive/tissue negative result was seen across 36 genes, a PIK3CA mutation (0.6% AF) which was confirmed using ddPCR (99.6% specificity). Quantitative concordance of AF compared to ddPCR was high (R = 0.94). 21 pts had detectable EGFR driver mutations at resistance. Among 6 pts with maintained T790M, 4 acquired C797S and 2 of these additionally acquired low level KRAS mutations (G13D, Q61K). Among 15 pts with loss of T790M, 7 had competing non- EGFR alterations identified: MET amp, PIK3CA E545K, BRAF V600E (n = 2), HER2 amp, KRAS G12S, and FGFR1 amp. Resistance mutations detected at AF &gt; 0.3% were confirmed with ddPCR. In 3 pts, a competing resistance mutation (1 KRAS, 2 BRAF) could be detected in plasma NGS pretreatment and reemerged as putative drivers at time of resistance. Conclusions: In this retrospective blinded validation, tagged amplicon-based plasma NGS was more sensitive than ddPCR with high specificity and quantitative concordance. In a subset of cases, serial plasma NGS can detect emergence of competing resistance mutations, creating an opportunity for the study of osimertinib-based targeted therapy combinations.

Liquid biopsy in the clinic: A prospective study of plasma circulating tumor DNA (ctDNA) next generation sequencing (NGS) in patients with advanced non-small cell lung cancers to match targeted therapy.

11536 Background: Liquid biopsy for plasma ctDNA NGS is a rapidly evolving science. Plasma ctDNA assays are commercially available and are increasingly adopted in the community with a paucity of evidence-based guidance. We set out to study the optimal timing and utility of plasma ctDNA NGS in clinic. Methods: Pts with advanced NSCLC who were driver unknown, defined as not having prior tissue NGS or clinical concern for tumor heterogeneity that may affect treatment decisions, were eligible. Peripheral blood was collected in a Streck tube (10mL), DNA extracted, and subjected to a bias-corrected hybrid-capture 21 gene targeted NGS assay in a CLIA lab with unique reads at 3000x and sensitive detection at variant allele frequency above 0.1% (ResolutionBio Bellevue, WA). Pts also had concurrent tissue NGS via MSK IMPACT. Clinical endpoints included detection of oncogenic drivers in plasma, ability to match pts to targeted therapy, concordance and turnaround time of plasma and tissue NGS. Results: Forty-one pts were prospectively accrued. Plasma ctDNA detected an oncogenic driver in 39% (16/41) of pts, of whom 17% (7/41) were matched to targeted therapy; including pts matched to clinical trials for HER2 exon 20 insertionYVMA, BRAF L597Q and MET exon14. Mean turnaround time for plasma was 7 days (4-12) and 28 days (20-43) for tissue. Plasma ctDNA was detected in 56% (23/41) of pts; detection was 40% (8/20) if blood was drawn on active therapy and 71% (15/21) if drawn off therapy, either at diagnosis or progression (Odds ratio 0.28, 95% CI 0.06 - 1.16; p = 0.06). All pts had concurrent tissue NGS; of the 10 samples resulted, there was 100% driver concordance between tissue and plasma in pts drawn off therapy. Conclusions: In pts who were driver unknown or who had clinical concern for tumor heterogeneity, plasma ctDNA NGS identified a variety of oncogenic drivers with a short turnaround time and matched them to targeted therapy. Plasma ctDNA detection was more frequent at diagnosis of metastatic disease or at progression. A positive finding of an oncogenic driver in plasma is highly specific, but a negative finding may still require tissue biopsy.

Abstract P1-07-15: Detection of molecular alterations in breast cancer through next generation sequencing of both tumor tissue and circulating tumor DNA: The UC San Diego Moores Cancer Center experience

Abstract Background: Next generation sequencing (NGS) analysis of actionable molecular alterations has the potential to guide cancer treatment, especially for patients with advanced cancer who have progressed on standard treatment. In this study, we analyzed tumor biopsies and peripheral blood from 62 patients with advanced breast cancer by two different NGS clinical-grade assays for molecular alterations in tumor tissue or in circulating tumor DNA (ctDNA). We used these results to determine if these specimens have potentially “actionable” alterations that could guide cancer therapy. Methods: From 2014 to 2016, 62 patients with advanced breast cancer had plasma sent for ctDNA analysis (Guardant360 assay; 54 to 70 genes) Thirty-eight of these patients (61%) also had tumor biopsies evaluated by NGS (FoundationOne®; 182 to 315 genes). Alterations were defined as mutations, insertions, deletions, truncations, or rearrangements or amplifications/copy number variations. Patients that harbored multiple alterations in the same gene were not counted as having separate alterations; however, if a gene amplification and an alteration were found in the same gene these were counted as separate events. Variants of unknown significance (VUS) and synonymous mutations were excluded from both assays. Data were collected and analyzed according to a UCSD Institutional Review Board approved protocol. Results: The median age of our patients at the time of ctDNA analysis was 55 years (range, 44 to 84 years); the median age at the time of tissue biopsy for NGS was 52 years (range, 39 to 82 years). One patient was male. The most common receptor status was estrogen receptor (ER) and progesterone receptor (PR) positive, human epidermal growth factor receptor 2 (HER2) non-amplified or negative (neg)(N=44; 71%), followed by triple negative breast cancer (ERnegPRnegHer2neg) (N=10, 16%), triple positive (N=6, 1%) and finally ERnegPRnegHER2positive (N=2, 0.03%). One patient of 38 (2%) had no tumor alteration detected and 19 of 62 had no ctDNA alterations (31%). In 38 breast cancer patients with tumor NGS results, alterations were detected in 79 unique genes, with the most frequent being TP53 (37% of patients), PIK3CA (24%) and GATA3 (24%) genes. In the 62 patients with ctDNA analysis, 31 unique genes had at least one alteration, with the most frequent being TP53 (36% of patients) and PIK3CA (23%) and EGFR amplification (11%) (GATA3 was not analyzed in the ctDNA assay). Both assays had a high rate of detection for potentially actionable mutations: 41 out of 62 patients (66%) by ctDNA and 34 out of 38 (89%) by tumor NGS. No two patients harbored identical genomic profiles by either tumor NGS or plasma ctDNA analysis except for 1 patient who had no alterations detected by either assay. Conclusions: Plasma and tissue NGS analysis appear to be complementary assays that yield a high percentage of potentially actionable alterations in patients with advanced breast cancer. Studies of the clinical impact of NGS-guided therapy in breast cancer are warranted. Citation Format: Shatsky RA, Parker BA, Schwab R, Helsten T, Boles SG, Subramanian R, Piccioni D, Kurzrock R. Detection of molecular alterations in breast cancer through next generation sequencing of both tumor tissue and circulating tumor DNA: The UC San Diego Moores Cancer Center experience [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-15.

Direct Detection and Quantification of Bacterial Cell-free DNA in Patients with Bloodstream Infection (BSI) Using the Karius Plasma Next Generation Sequencing (NGS) Test

BackgroundBlood cultures can have low sensitivity if a patient is pretreated with antibiotics. A molecular diagnostic for bloodstream infection (BSI) that can also quantitate pathogen DNA could be a useful tool in detecting and monitoring culture-negative infections.MethodsWe prospectively enrolled 75 patients (50 with culture confirmed BSI due to Staphylococcus aureus [n = 21] or Gram-negative bacilli [n = 29] at baseline, and 25 with negative blood cultures) to evaluate the Karius plasma next generation sequencing (NGS) test to detect BSI. Blood samples from patients with confirmed BSI were collected for the study within one day of positive blood culture and then every 2–3 days. Cell-free DNA (cfDNA) was extracted from plasma and underwent NGS in the Karius CLIA/CAP laboratory (Redwood City, CA). After removal of human sequences, remaining reads were aligned against a curated pathogen database. Organisms present at a significance-level above a predefined threshold were reported. Quantity of cfDNA for each reported pathogen was expressed as molecules per microliter (MPM).ResultsWhen compared with baseline blood culture, the plasma NGS test had a positive agreement of 80% (40/50) and negative agreement of 84% (21/25). Overall, serially collected samples were positive by plasma NGS testing significantly longer than blood culture (mean 6.0 days vs. 2.4 days, respectively; P < 0.0001. Figure). Patients with BSI were positive longer by NGS testing than blood culture for both S. aureus (mean 6.9 days vs. 4.0 days, respectively; P < 0.005) and gram-negative bacilli (mean 5.4 days vs. 1.3 days, respectively; P < 0.001). Pathogen cfDNA in BSI patients, quantified as MPM, declined over time during treatment. S. aureus MPM declined more slowly than gram-negative MPM and was significantly higher than gram-negative MPM at day 6 (P < 0.001).ConclusionThe Karius plasma NGS test can directly detect pathogens in patients with BSI. Pathogen cfDNA signal in plasma remains positive longer than blood culture and combined with quantification of pathogen cfDNA, could be a useful biomarker to aid in diagnosis and monitoring of infections, particularly in those with sterile blood cultures.Disclosures L. Wanda, Karius, Inc: Collaborator and Employee, Salary; F. Ruffin, Karius, Inc.: Collaborator and Research Contractor, Salary; J. Hill-Rorie, Karius, Inc: Collaborator, Research support and Salary; D. Hollemon, Karius, Inc: Employee, Salary; H. Seng, Karius, Inc: Employee, Salary; D. Hong, Karius, Inc.: Employee, Salary; T. Blauwkamp, Karius, Inc: Employee, Salary; M. Kertesz, Karius, Inc: Employee, Salary; V. Fowler Jr., Karius, Inc: Grant Investigator, Grant recipient Pfizer, Novartis, Galderma, Novadigm, Durata, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., Cerexa, Tetraphase, Trius, MedImmune, Bayer, Theravance, Cubist, Basilea, Affinergy, Janssen, xBiotech, Contrafect: Consultant, Consulting fee NIH, MedImmune, Cerexa/Forest/Actavis/Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Cubist/Merck; Medical Biosurfaces; Locus; Affinergy; Basilea; Contrafect; Karius: Grant Investigator, Grant recipient Green Cross, Cubist, Cerexa, Durata, Theravance; Debiopharm: Consultant, Consulting fee UpToDate: Royalties, Royalties

OA06.01 Clinical Utility of Circulating Tumor DNA (ctDNA) Analysis by Digital next Generation Sequencing of over 5,000 Advanced NSCLC Patients

Detection of actionable genomic alterations is now required for NCCN guideline-compliant work-up of NSCLC adenocarcinoma. Next-generation sequencing (NGS) of ctDNA, if sufficiently sensitive and specific, could provide a non-invasive, comprehensive genotyping platform relevant to clinical decision-making when tissue is insufficient or at time of progression on targeted therapies. A highly accurate, deep-coverage (15,000x) ctDNA plasma NGS test targeting 54-70 genes (Guardant360) was used to genotype 5,206 advanced-stage NSCLC patients accrued between 6/2014 – 4/2016. The frequency and distribution of somatic alterations in key genes were compared to those described in TCGA (Pearson and Spearman correlations). The clinical impact of ctDNA testing was evaluated by identification of resistance mechanisms emergent at progression on targeted therapies, and through analysis of additional driver mutations detected by ctDNA at baseline in 362 consecutive NSCLC patients with tissue mutation data available. The positive predictive value (PPV) of ctDNA sequencing was assessed in 229 patients with known tumor driver alterations. ctDNA alterations were detected in 86% of cases; EGFR mutations in 25%, KRAS mutations in 17%, MET amplification in 4%, BRAF mutations in 3% and other rare but potentially actionable alterations in 9%. Mutation patterns among driver oncogenes were highly consistent with those from TCGA (Pearson r=0.92, 0.99, 0.99 for EGFR, KRAS, and fusion breakpoint location). PPV of ctDNA-detected variants was 100% for EGFRL858R, 98% for EGFRE19del, 96% for ALK, RET, or ROS1 fusions, and 100% for KRASG12/G13/Q61 mutations. In 362 cases with tissue information available, 63% (229/362) were tissue quantity-insufficient or undergenotyped (QNS/UG). ctDNA analysis identified driver mutations in 51 of the 229 QNS/UG cases, a 38% increase in detection rate over tissue alone. Among 1,111 EGFR-mutant cases, resistance mutations were identified at progression at frequencies consistent with published literature: EGFRT790M 47%, MET amp 5%, ERBB2 amp 5%, FGFR3 fusions 0.4%, ALK/other fusions 1%, BRAF mutations 1.8%, PTEN inactivation 2.5%, NF1 inactivation 3%, RB1 inactivation 3%, KRAS mutations 1.9%. In 143 consecutive NSCLC patients with detailed follow-up and serial analysis seen at the UC Davis Cancer Center, informative driver mutations were observed in 48 (34%). This series represents the largest NSCLC ctDNA study to date. Genotypic patterns of truncal mutations were highly consistent with TCGA in terms of frequency and distribution. At baseline, ctDNA augmented tissue analysis by identifying additional, actionable mutations when tissue was QNS/UG. ctDNA NGS conducted at progression identified emergent resistance mutations that could inform subsequent courses of therapy.

The DISCOVER Trial: Application of the Karius Plasma Next-Generation Sequencing Test for Pathogen Detection in Stem-Cell Transplant Patients

BackgroundThe diagnosis of infections in stem-cell transplant (SCT) patients is challenging. There is a need for improved diagnostics that can quickly and accurately detect a broad range of viruses, bacteria, and fungi that can infect these patients to guide targeted therapy.MethodsWe enrolled 20 patients in a prospective study evaluating the Karius plasma next-generation sequencing (plasma NGS) test to detect infections in SCT patients. Patients had baseline plasma samples drawn prior to transplant followed by weekly collections during engraftment and at onset of febrile episodes. Samples were transferred to the Karius CLIA/CAP laboratory (Redwood City, CA) where cell-free DNA was extracted from plasma and NGS performed. Human sequences were removed and remaining reads were aligned against a curated pathogen database. Organisms present at a significance level above a predefined threshold were reported.ResultsCytomegalovirus (CMV) was identified in 12/20 patients. Using a nearest-neighbor method, 61 pairs of observations were identified for a comparison of plasma NGS and CMV qPCR collected within 1 day of each other. Positive agreement (PPA) between plasma NGS and CMV qPCR was 84.1% (37/44). PPA was 100% (20/20) when values below the lower limit of quantitation (<137 IU/ml) for CMV qPCR were excluded. In addition to CMV, plasma NGS detected EBV, HHV6B, BK virus and Torque teno viruses. Plasma NGS also detected pathogens prior to conventional tests being ordered in two patients with subsequently confirmed acute infections; Staphylococcus aureus was detected one day prior to blood culture in one patient and Chlamydia trachomatis 30 days prior to targeted testing in a second patient. These two cases highlight the potential of plasma NGS to detect pathogens in SCT patients early when used as a monitoring tool.ConclusionKarius has developed a novel NGS plasma test that can simultaneously identify pathogens in SCT patients. The test had 100% concordance with CMV qPCR above the lower level of quantitation. Further work is ongoing to determine the lower limits of detection for the plasma NGS test. Using NGS to monitor SCT patients for infection could permit earlier detection of pathogens, enabling earlier targeted therapy for this vulnerable population.Disclosures H. Seng, Karius, Inc: Employee, Salary; D. Hollemon, Karius, Inc: Employee, Salary; D. Hong, Karius, Inc.: Collaborator, Research support and Salary; T. Blauwkamp, Karius, Inc: Employee, Salary; M. Kertesz, Karius, Inc: Employee, Salary; J. Eng, Karius, Inc.: Collaborator, Salary; K. Wong, Karius, Inc.: Collaborator, Salary; P. Chin-Hong, Karius, Inc.: Collaborator, Research support

Abstract 4342: Ultra-deep next generation sequencing (NGS) of plasma cell-free DNA (cfDNA) from patients with advanced lung cancers: results from the Actionable Genome Consortium

Abstract Introduction: Noninvasive genotyping using plasma cfDNA from cancer patients has the potential to obviate the need for some biopsies while also characterizing disease heterogeneity. This study was undertaken to develop an ultra-deep plasma NGS panel for patients with non-small cell lung cancers (NSCLC). Methods / Results: Plasma was prospectively collected from 51 patients with advanced, progressive NSCLC and a known oncogenic driver from prior tumor genotyping. We performed ultra-deep NGS on extracted cfDNA using a customized Illumina library preparation, hybrid capture panel covering 37 lung cancer related genes (complete exons and partial introns), and ultra-deep sequencing (HiSeq4000). Mean sequencing depth was ∼50,000X (150 million, 150bp reads per sample). After specialized consensus-based error correction for low allele frequency (AF) genomic alterations, the median unique DNA molecules per position were ∼3,500. The mean sequence error rate was reduced by 20-fold to 0.002%, enabling the confident call of a driver mutation as low as 0.03%. In a subset of cases, paired plasma droplet digital PCR (ddPCR) was performed for common EGFR and KRAS mutations using a validated assay. Blinded to tumor genotype, plasma NGS detected SNVs (EGFR, KRAS, BRAF), indels (EGFR, ERBB2), and fusions (ALK, ROS1) as well as significant copy number gains (CNG) (ERBB2, MET). Sensitivity of cfDNA for the detection of known oncogenic drivers was 88% (45/51). A single false positive driver mutation was identified in a case with a known EGFR mutation in tumor; plasma NGS found both EGFR exon 19 del (0.88% AF) and KRAS G12D (2.65% AF), and plasma ddPCR confirmed the presence of both mutations (2.2% and 2.0% AF). Evaluation for an occult second primary is ongoing. In 22 EGFR, ALK, or ROS1 cases with acquired resistance to targeted therapy, plasma genotyping detected a range of potential resistance mechanisms: EGFR T790M and C797S, ALK F1174C, ERBB2 CNG, MET CNG. In 16 cases with paired resistance biopsies, concordance for EGFR T790M status was 94% (15/16). 18 cases with known EGFR or KRAS mutations underwent paired ddPCR. In 14 cases the driver mutation was detected using both assays with high concordance of the%AF (r = 0.91). The remaining 4 cases were negative with ddPCR but 3 were positive with NGS at low AF (0.04%, 0.08%, and 0.29%), and the specificity for each driver was 100%. Conclusions: Ultra-deep plasma NGS can detect a wide range of oncogenic drivers in NSCLC and may be more sensitive than established ddPCR assays. In the setting of acquired resistance to targeted therapy, plasma NGS reliably captured EGFR T790M and additional somatic alterations as potential resistance mechanisms. Citation Format: Bob T. Li, Filip Janku, Pasi A. Janne, Gordon B. Mills, Kiran Madwani, Ryan S. Alden, Cloud P. Paweletz, Marc Ladanyi, Alex Aravanis, Byoungsok Jung, Sante Gnerre, Amy J. Sehnert, David B. Solit, Gregory J. Riely, Geoffrey R. Oxnard. Ultra-deep next generation sequencing (NGS) of plasma cell-free DNA (cfDNA) from patients with advanced lung cancers: results from the Actionable Genome Consortium. [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 4342.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.

Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.

THU-264 - Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.

Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection.

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.

Abstract B104: Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA)

Abstract Introduction: Genomic analysis of cfDNA is emerging as a powerful tool for noninvasive characterization of advanced cancer. Some cfDNA assays are quantitative, allowing analysis of genomic subpopulations. Here, we sought to use quantitative cfDNA analysis to distinguish somatic and germline variants, gaining insight into cancer biology as well as inherited risk without needing paired germline DNA. To do this, we studied EGFR mutations in cfDNA aiming to distinguish those with somatic T790M, generally acquired after resistance to targeted therapy, from those with germline T790M, a rare allele associated with inherited lung cancer risk. Methods &amp; Results: We first explored an institutional cohort of lung cancer patients undergoing cfDNA genotyping using droplet digital PCR (ddPCR). We identified 64 patients positive for T790M and a concurrent EGFR driver mutation by ddPCR. For the vast majority of cases, the mutant allelic fraction (MAF) of T790M was lower than the MAF of the driver mutation, resulting in a T790M/driver ratio of 0.001-1. For two cases, T790M was the predominant mutation, with T790M/driver ratios of 4.2 and 54.6; MAF of T790M was 49% and 53% for these cases. Under an IRB-approved protocol (NCT01754025), we performed sequencing of DNA extracted from PBMCs which confirmed germline T790M in both. We then submitted cfDNA from 3 patients with lung cancer and germline T790M for blinded NGS using the 68-gene Guardant360 assay. In each, NGS identified over 90 coding and non-coding variants. T790M was identified at a high MAF (56%, 50%, 49%) in the range of other recognized SNPs, while an EGFR driver mutation (L858R or L861Q) was identified at a lower level (23%, 4%, 1%) in the range of coding TP53 mutations. Each case was treated with a third-generation EGFR kinase inhibitor targeting T790M. NGS of post-treatment cfDNA demonstrated the expected response in the EGFR driver mutation (0.2%, 0%, 0%) but minimal change in the high MAF T790M (50%, 49%, 49%), consistent with persistent shed of germline DNA on therapy. To broadly screen for germline T790M carriers, we queried a cohort of 1082 lung cancer patients who had undergone NGS of cfDNA using Guardant360. 74 were positive for EGFR T790M (median MAF 3%, range 0.2%-51%). 58 also harbored a second EGFR driver mutation (median MAF 5%), with 23 also having EGFR amplification (median MAF 16%). Four cases were identified with high MAF T790M in the expected range of SNPs (51%, 49%, 49%, 48%), but no driver EGFR mutations were identified at this high MAF. These 4 cases have been referred for germline testing on a prospective study of germline T790M (NCT01754025). Conclusions: We have identified that quantitative cfDNA analysis with NGS can identify germline mutations through differentiation of high MAF germline variants from lower MAF somatic variants. In this cohort, 4 of 74 cases (5%) positive for T790M using NGS of cfDNA were consistent with a germline mutation. This ability to differentiate germline from somatic variants differentiates NGS of cfDNA from NGS of tumor specimens, where these distinctions are challenging without paired germline DNA. Diagnostic labs performing plasma NGS will need to be vigilant to identify these potential germline alterations, and will need strategies for reporting these to providers and patients as appropriate. Citation Format: Geoffrey R. Oxnard, Adrian G. Sacher, Ryan S. Alden, Nora B. Feeney, Jennifer C. Heng, Rebecca J. Nagy, Richard B. Lanman, Cloud P. Paweletz, Pasi A. Janne. Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA). [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 B104.