Cell-free Plasma Research Articles

Different next-generation sequencing pipelines based detection of tumor DNA in cerebrospinal fluid of lung adenocarcinoma cancer patients with leptomeningeal metastases

BackgroundThe nucleic acid mutation status in intracranial metastasis is markedly significant clinically. The goal of the current study was to explore whether the tumor-associated mutations can be detected by different next-generation sequencing (NGS) pipelines in paired cerebrospinal fluid (CSF) and plasma samples from lung adenocarcinoma (LAC) patients with leptomeningeal metastases (LM).MethodsPaired CSF cell free DNA (cfDNA), CSF cells, plasma and formalin-fixed and paraffin-embedded (FFPE) samples of primary tumors were collected from 29 LAC patients with LM to detect the mutations by different NGS pipelines.ResultsDNA libraries were generated successfully for 79 various samples in total for NGS sequencing, of which mutations were detected in 7 plasma samples (24.14%), 12 CSF cfDNA samples (66.67%), and 10 CSF cells (76.9%) samples. For the 26 patients with detected mutations, 8/26(30.77%) had mutations in plasma, which was significantly lower than that those from CSF cfDNA (12/15, 80.00%), CSF cells (10/11, 90.91%) and FFPE samples (13/17, 76.47%). When the input DNA of CSF cells was less than 20 ng, the cHOPE pipeline of NGS identified the most mutations for epidermal growth factor receptor (EGFR).ConclusionsNGS-based detection of mutations in cfDNA or cells from CSF provided more information than from plasma samples from LAC patients with LM. In addition, the cHOPE pipeline performed better than the other three NGS pipelines when input DNA from CSF cells was low.

Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients

We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of colorectal cancer (CRC) patients. c-MYC GCN status was determined using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort 1 (192 CRC patients) and prospective cohort 2 (64 CRC patients). In cohort 1, c-MYC GCN gain was observed in 34 (17.5%) patients by SISH, and in 7 (3.6%) patients by ddPCR. c-MYC GCN by SISH significantly correlated with ddPCR results (ρ = 0.532, P < 0.001). Although 40 cases (20.7%) showed intratumoral genetic heterogeneity, it did not cause discordance in results obtained by the two methods. c-MYC GCN gain, by both SISH and ddPCR was independently correlated with worst prognosis (P = 0.002). In cohort 2, c-MYC GCN estimation in tissue by ddPCR was also significantly associated with results obtained by SISH (ρ = 0.349, P = 0.005), but correlated with plasma ddPCR with borderline significance (ρ = 0.246, P = 0.050). Additionally, detecting c-MYC GCN gain in plasma with ddPCR might have relatively low sensitivity but high specificity. Our study suggests that ddPCR can be a useful tool for detecting c-MYC GCN gain as a potential prognostic biomarker in CRC tissue samples; however, this will need further verification in plasma samples.

Community-acquired pneumonia in children: cell-free plasma sequencing for diagnosis and management

Community-acquired pneumonia (CAP) is a common cause of pediatric hospital admission. Empiric antibiotic therapy for hospitalized children with serious CAP now targets the most likely pathogen(s), including those that may demonstrate significant antibiotic resistance. Cell-free plasma next-generation sequencing (CFPNGS) was first made available for Pediatric Infectious Diseases physicians in June 1, 2017, to supplement standard-of-care diagnostic techniques. A retrospective chart review was performed for children hospitalized with CAP between June 1, 2017, and January 22, 2018, to evaluate the impact of CFPNGS. We identified 15 hospitalized children with CAP without other underlying medical conditions for whom CFPNGS was performed. CFPNGS identified a pathogen in 13 of 15 (86%) children compared with 47% for those using standard culture and PCR-based methods alone. Changes in antibiotic management were made in 7 of 15 (47%) of children as a result of CFPNGS.

Retrospective Case-Control Study of the Performance of Cell-Free Plasma Sequencing to Detect Invasive Mold Infections in Hematopoietic Cell Transplant Recipients with Pneumonia

Introduction Pulmonary invasive mold infections (IMI) remain a significant cause of morbidity and mortality after hematopoietic cell transplant (HCT). Non-invasive diagnostic options are limited, particularly for non-Aspergillus molds. Given differences in spectrum of activity and toxicities associated with mold-active therapy, early diagnosis and targeting of specific IMI is important. Objectives We evaluated the performance of the Karius cell-free plasma next-generation sequencing (NGS) test for detecting IMI in HCT recipients with pneumonia. Methods We conducted a retrospective case-control study of 57 HCT recipients with proven/probable pulmonary IMI (6 were non-Aspergillus) based on the 2008 EORTC criteria and 19 controls with non-fungal pulmonary infections. All subjects had frozen plasma samples obtained within 14 days of diagnosis (76% within 7 days). Diagnostic workup included computed tomography chest imaging and bronchoalveolar lavage (BAL) and/or biopsy. Fungal stains and culture were performed on BAL fluid and tissue samples, and galactomannan testing was performed on BAL and serum. Cell-free DNA was extracted from plasma 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. Analysis of sequencing data was blinded to all clinical data. Results Among 57 proven and probable cases of IMI, we identified pathogenic molds in 15 subjects (26%). Among the subset of 44 subjects with a plasma sample +/- 7 days from diagnosis, the rate of detection increased to 32% (14/44). Mold was not identified in any controls. In non-Aspergillus proven IMI, the Karius test identified 83% (5/6) of molds. In contrast, in Aspergillus proven/probable disease, Aspergillus fumigatus was identified in 13.7% (7/51) of subjects. All Aspergillus positives had a plasma sample available +/- 7 days from diagnosis. In three other subjects with Aspergillus proven/probable disease, we identified Rhizomucor miehei, Rhizomucor pusillus, and Cunninghamella, which could be consistent with the clinical findings. By optimizing the analysis pipeline for sensitivity, an additional 6 subjects with Aspergillus spp and other pathogenic molds were identified, increasing the detection rate of pathogenic molds to 31.4% (16/51). Conclusion The Karius cell-free plasma NGS test is a non-invasive means of detecting IMI, particularly non-Aspergillus molds in HCT recipients with pulmonary disease. This may be a useful adjunctive test for diagnosing IMI in specific clinical contexts, and larger studies should be conducted.

Plasma mitochondrial DNA and metabolomic alterations in severe critical illness

BackgroundCell-free plasma mitochondrial DNA (mtDNA) levels are associated with endothelial dysfunction and differential outcomes in critical illness. A substantial alteration in metabolic homeostasis is commonly observed in severe critical illness. We hypothesized that metabolic profiles significantly differ between critically ill patients relative to their level of plasma mtDNA.MethodsWe performed a metabolomic study with biorepository plasma samples collected from 73 adults with systemic inflammatory response syndrome or sepsis at a single academic medical center. Patients were treated in a 20-bed medical ICU between 2008 and 2010. To identify key metabolites and metabolic pathways related to plasma NADH dehydrogenase 1 (ND1) mtDNA levels in critical illness, we first generated metabolomic data using gas and liquid chromatography-mass spectroscopy. We performed fold change analysis and volcano plot visualization based on false discovery rate-adjusted p values to evaluate the distribution of individual metabolite concentrations relative to ND1 mtDNA levels. We followed this by performing orthogonal partial least squares discriminant analysis to identify individual metabolites that discriminated ND1 mtDNA groups. We then interrogated the entire metabolomic profile using pathway overrepresentation analysis to identify groups of metabolite pathways that were different relative to ND1 mtDNA levels.ResultsMetabolomic profiles significantly differed in critically ill patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma relative to those with an ND1 mtDNA level < 3200 copies/μl plasma. Several analytical strategies showed that patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma had significant decreases in glycerophosphocholines and increases in short-chain acylcarnitines.ConclusionsDifferential metabolic profiles during critical illness are associated with cell-free plasma ND1 mtDNA levels that are indicative of cell damage. Elevated plasma ND1 mtDNA levels are associated with decreases in glycerophosphocholines and increases in short-chain acylcarnitines that reflect phospholipid metabolism dysregulation and decreased mitochondrial function, respectively.

2050. Plasma Next-Generation Sequencing for Pathogen Detection in Pediatric Patients at Risk for Invasive Fungal Infection

BackgroundInvasive fungal infection (IFI) is a major cause of mortality and morbidity among immunocompromised patients. Microbiologic culture of biopsy samples remains the diagnostic gold standard. Noninvasive biomarker testing can provide clinically useful information, but does not give species-level identification. Next-generation sequencing (NGS) of cell-free plasma is a noninvasive approach for species-level identification of pathogens, and may guide specific treatment. We sought to describe the diagnostic utility of plasma NGS in high-risk immunocompromised pediatric patients, correlating results with standard microbiology studies.MethodsPlasma from at-risk immunocompromised patients with suspected IFI was tested using cell-free plasma NGS (Karius, Redwood City, CA). Human reads were removed, and remaining sequences aligned to a curated database including >1,000 pathogens. Organisms present above a predefined significance threshold were reported.ResultsForty evaluable patients were enrolled, the majority of whom had underlying oncologic diagnoses. Risk for IFI included prolonged febrile neutropenia (FN) in 22 patients, recrudescent FN in 7, concern for IFI on imaging in 8, and concern for IFI based solely on other clinical findings in 3. Six patients met established criteria for proven IFI, 1 for probable IFI, and 13 for possible IFI. NGS plasma testing identified a pathogen which was cultured from infected tissue or blood in 4 of 6 proven cases; one patient with localized cutaneous Rhizopus had negative NGS results. A patient with probable IFI (positive β-D-glucan) had P. jirovecii identified by NGS. Among 33 patients without proven or probable IFI, NGS testing identified a fungus in one (C. glabrata), no organism in 11, and potential alternative sources of fever in 16.ConclusionPlasma NGS testing can detect IFI from blood. The test identified fungi from proven IFI, and detected other pathogens in both probable and possible IFI cases. Many patients at risk received prolonged courses of antifungals despite negative testing, suggesting a possible future role for NGS testing in ruling out IFI. Future studies should more definitively evaluate the positive and negative predictive value for NGS testing in patients at risk of IFI.Disclosures R. Aquino, Karius, Inc.: Employee, Salary. D. Hollemon, Karius: Employee, Salary. D. Hong, Karius, Inc.: Employee, Salary.

156. Direct Detection and Quantification of Bacterial Cell-free DNA in Patients with Infective Endocarditis (IE) Using the Karius Plasma Next Generation Sequencing (NGS) Test

BackgroundThe variable clinical presentation of IE requires a diagnostic tool that accurately detects a wide range of organisms, including in culture-negative (CN) scenarios. A sensitive molecular diagnostic assay that quantitates pathogen DNA could be a useful tool to diagnose IE and evaluate response to antimicrobial therapy.MethodsWe prospectively enrolled 30 hospitalized adult patients evaluated for acute IE classified using the Duke Criteria. Residual plasma samples within 24 hours and/or fresh whole blood within 48–72 hours of enrollment blood culture were collected. Additional samples were collected every 2–3 days for up to 7 time points until discharge. Samples were shipped to the Karius laboratory (Redwood City, California) for testing. Cell-free DNA was extracted and NGS was performed. Human sequences were removed and remaining sequences were aligned to a curated pathogen database of over 1,000 organisms. Organisms present above a predefined statistical threshold were reported. Quantity of DNA for each reported pathogen was expressed as molecules per microliter.ResultsOf 29 patients eligible for analysis, 18 had prosthetic valves and 7 had implanted cardiac devices. Twenty-four patients had Definite IE. Twenty patients had positive blood cultures (including S. aureus, S. epidermidis, E. faecalis, S. agalactiae, Pantoea ananatis, S. sanguinis, C. albicans); NGS identified the same organism isolated in all 20 patients as well as E. cloacae complex, and E. faecalis in 2 of 4 CN Definite IE patients. For 1 CN patient with Possible IE, NGS identified E. coli. NGS and BC were negative for 4 patients with Rejected IE. NGS identified the IE etiology in patients pretreated with antibiotics up to 20 days prior to sample collection. Pathogen DNA signal was often observed in both initial and repeat plasma samples, while repeat blood cultures remained negative.ConclusionThis novel, cell-free pathogen quantitative NGS plasma assay accurately identified causative organisms in patients with IE, even when blood cultures were negative due to pretreatment with antibiotics. Pathogen DNA, detected in plasma longer than blood culture, is a promising biomarker to aid in the diagnosis and monitoring of IE, particularly culture-negative IE.Disclosures P. Shah, Karius Inc.: Collaborator and Research Contractor, Salary. F. Ruffin, Karius Inc.: Collaborator and Research Contractor, Salary. H. Seng, Karius Inc.: Employee, Salary. D. Hollemon, Karius Inc.: Employee, Salary. L. Winn, Karius Inc.: Collaborator and Research Contractor, Salary. C. Drennan, Karius Inc.: Collaborator and Research Contractor, Salary. K. L. Chan, Karius Inc.: Employee, Salary. H. Quach, Karius Inc.: Employee, Salary. T. Blauwkamp, Karius Inc.: Employee, Salary. G. Meshulam-Simon, Karius Inc.: Employee, Salary. D. Hong, Karius Inc.: Employee, Salary. V. G. Fowler Jr., Merck, Cerexa/Actavis, Pfizer, Advanced Liquid Logis, NIH, MedImmune, Basilea, Karius, Contrafect, Regneron, Genentech, Affinergy, Locus, Medical Surface, Inc., Achaogen, Astellas, Arsanis, Bayer, Cubist, Debiopharm, Durata, Grifols, Medicines Co, Novartis: Collaborator, Consultant and Scientific Advisor, Consulting fee, Research grant and Research support.

Circulating free plasma tumor DNA in patients with advanced gastric cancer receiving systemic chemotherapy

BackgroundAdvanced gastric cancers are usually associated with incurable conditions for which systemic treatments are indicated. Recent studies suggest that circulating cell-free plasma DNA of tumour origin (tDNA) is a promising non-invasive biomarker that can be used to predict the prognosis and monitor the efficacy of systemic treatments in patients with certain types of cancer. We conducted a pilot study to analyse the potential role of tDNA as a biomarker in patients with advanced gastric cancer.MethodsWe included 30 patients with locally advanced unresectable or metastatic gastric cancer. We obtained samples (10 mL of total blood) from each patient every 3 months and performed concomitant CT until disease progression or death. Total cell-free circulating DNA (cfDNA) samples were measured using GeneQuant RNA/DNA Calculator-Amersham Pharmacia Biotech (Biochrom) Ltd. The cfDNA was used to evaluate the ALU DNA sequences 247 and 115. The level of tDNA was calculated from the ratio of the expression of ALU DNA sequences and the concentration of total cell-free DNA. We utilized the RECIST criteria 1.1 to evaluate the tumour response.ResultsPatients with advanced gastric cancer had significantly higher concentrations of cfDNA compared with normal controls (p = 0.00015), which allowed us to conclude that the cfDNA in the patients originated from the tumour. We did not find any significant correlation between the level of tDNA and OS or tumour response. However, after the first cycles of chemotherapy (at 3 months), we observed that patients with lower tDNA levels had significantly longer DFS compared with those with higher levels (Cox Regression p = 0.0228).ConclusionsAt 3 months after the beginning of chemotherapy, the tDNA levels are correlated with DFS in patients with advanced gastric cancer who receive systemic chemotherapy. tDNA may be a specific, non-invasive and cost effective new biomarker for these patients.

Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

BackgroundMitochondrial diseases are a clinically heterogeneous group of diseases caused by mutations in either nuclear or mitochondrial DNA (mtDNA). The diagnosis is challenging and has frequently required a tissue biopsy to obtain a sufficient quantity of mtDNA. Less-invasive sources mtDNA, such as peripheral blood leukocytes, urine sediment, or buccal swab, contain a lower quantity of mtDNA compared to tissue sources which may reduce sensitivity. Cellular apoptosis of tissues and hematopoetic cells releases fragments of DNA and mtDNA into the circulation and these molecules can be extracted from plasma as cell-free DNA (cfDNA). However, entire mtDNA has not been successfully identified from the cell free fraction previously. We hypothesized that the circular nature of mtDNA would prevent its degradation and a higher sensitivity method, such as next generation sequencing, could identify intact cf-mtDNA from human plasma. MethodsPlasma was obtained from patients with mitochondrial disease diagnosed from skeletal muscle biopsy (n = 7) and healthy controls (n = 7) using a specially cfDNA collection tube (Streck Inc.; La Vista, NE). To demonstrate the presence of mtDNA within these samples, we amplified the isolated DNA using custom PCR primers specific to overlapping fragments of mtDNA. cfDNA samples were then sequenced using the Illumina MiSeq sequencing platform. ResultsWe confirmed the presence of mtDNA, demonstrating that the full mitochondrial genome is in fact present within the cell-free plasma fraction of human blood. Sequencing identified the mitochondrial haplogroup matching with the tissue specimen for all patients. ConclusionWe report the existence of full length mtDNA in cell-free human plasma that was successfully used to perform haplogroup matching. Clinical applications for this work include patient monitoring for heteroplasmy status after mitochondrially-targeted therapies or haplogroup monitoring as a measure of stem cell transplantation.

High cut-off membrane for in-vivo dialysis of free plasma hemoglobin in a patient with massive hemolysis

BackgroundThe possibility of clearing Cell-free Plasma Hemoglobin (CPH) from human plasma may appear attractive, especially when considering the noxious effects that CPH has on the immune function and the renal damage caused by its filtration. The existence of the so-called High Cut-Off (HCO) filters, possessing pores as big as 60 kDa, could potentially allow the clearance of the αβ dimers (31.3 kDa), the form in which the α2β2 hemoglobin tetramers (62.6 kDa) physiologically dissociate in plasma. We present herein the first reported case in which such an attempt was made.Case presentationThe patient was a 51-year-old man with hemolytic crisis due to glucose-6-phosphate dehydrogenase deficiency, further complicated by pigment-induced nephropathy. He underwent a 48-h CVVHD session, in which a HCO filter was used. The Sieving Coefficient (SC) for CPH was initially 0.08 and decreased to 0.02 after 24 h. This unexpected low SC was due to the initial high concentration of CPH (4.24 g/L). At such concentrations, the α2β2 tetramer poorly dissociates into the αβ dimer; but increases exponentially at concentrations lower than 1 g/L.ConclusionsClearance of CPH through a HCO filter is technically feasible but its performance markedly relies on the initial concentration of CPH. Critically ill patients with smoldering hemolysis, as it happens during septic shock or ECMO treatment, may benefit the most from the use of this membrane in order to clear CPH.

PS02.195: CIRCULATING TUMOUR DNA HAS UTILITY IN THE CLINICAL MANAGEMENT OF ESOPHAGEAL CANCER

Abstract Background Cell-free DNA, a consequence of normal cell death and apoptosis, is found in the blood of all individuals. In cancer patients, a proportion of circulating cell free DNA will be derived from tumour cells, and is termed circulating tumour DNA (ctDNA).We have developed a blood-based ‘liquid biopsy’ to detect ctDNA in plasma of esophageal cancer patients. Methods Tumour, buffy-coat (germline) and plasma samples were obtained from 28 esophageal cancer patients at the time of diagnosis. Serial blood samples were collected from 8 patients. Somatic DNA mutations in 9 genes (TP53, ARID1A, SMAD4, CDKN2A, SMARCA4, NRG1, APC, PIK3CA and KRAS) were evaluated in tumour biopsies and plasma using targeted sequencing. Tumour specific mutations were confirmed by droplet digital PCR, which was used to track patient-specific ctDNA in plasma from serially collected blood samples. Results Somatic mutations in at least one of the targeted genes were detected in 19/28 (68%) tumour biopsies. The same mutations were detected in ctDNA from plasma in 9/19 (48%) patients. Additional mutations that were not detected in the tumour biopsies were detected in plasma DNA from 2 patients, highlighting the issues of intra-tumoural heterogeneity and sampling bias of tumour biopsies. ctDNA was more frequently detected in patients with advanced disease where it represented a greater proportion of the total cell-free plasma DNA than in patients with localised disease. In patients with serial samples, levels of ctDNA correlated with response to treatment, and rises in ctDNA could be detected prior to clinical evidence of relapse. Conclusion Detection of ctDNA using targeted sequencing and digital PCR is feasible in the majority of esophageal cancer patients, particularly those with advanced disease. Dynamic changes in ctDNA levels reflect response to treatment and disease relapse as determined by conventional clinical imaging. We conclude that ctDNA levels can provide supplemental evidence regarding disease staging, as well as prognostic and predictive information that can inform therapeutic pathway decisions in esophageal cancer patients. Disclosure All authors have declared no conflicts of interest.

Phase II trial of gefitinib plus pemetrexed after relapse using first-line gefitinib in patients with non-small cell lung cancer harboring EGFR gene mutations

ObjectivesEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (i.e., EGFR-TKIs) improve the survival of lung cancer patients harboring EGFR mutations. Despite the initial efficacy of EGFR-TKIs, the disease progression caused by acquired resistance to these inhibitors is inevitable. T790M mutations represent a major resistance mechanism to EGFR-TKIs but can be overcome using osimertinib. The IMPRESS trial revealed that the continuation of EGFR-TKI beyond progressive disease (PD) concurrent with platinum-doublet chemotherapy was not beneficial. However, various clinical trials have suggested that EGFR-TKI beyond PD plus single-agent chemotherapy may be a possible treatment strategy. Materials and MethodsThis study was a single-arm phase II trial. Patients with EGFR-activating mutations (del19 and L858R) that progressed using first-line gefitinib treatment were enrolled and treated with gefitinib beyond PD plus pemetrexed 500 mg/m2 q3w. The primary endpoint was progression-free survival (PFS). Mutation-biased polymerase chain reaction quenching probe, which is the original method for detecting T790M mutations in cell-free plasma DNA, was used prior to treatment. ResultsThirty-six patients were enrolled between May 1, 2013, and March 31, 2016. One patient was excluded before starting the treatment. Among the 35 patients, 15 patients had del19 mutations, and 20 patients had L858R mutations; 33 patients were evaluable for response by using radiographic findings. The median PFS was 6.7 months (95% confidence interval: 4.4–7.7 months). Nineteen patients were T790M positive. No significant difference in PFS was found in a subgroup analysis of EGFR mutation status and T790M positivity. All toxicities were tolerable. ConclusionGefitinib plus pemetrexed treatment following relapse using gefitinib in patients with Non-small cell lung cancer harboring EGFR mutations demonstrated preferable PFS with mild toxicity. This combination therapy may be considered for platinum-unfit patients without T790M with disease progression using first-line gefitinib. (This clinical trial was registered in UMIN-CTGR as UMIN000010709).

Abstract 5592: HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method

Abstract In around 10% of gastric cancer, amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) is observed and trastuzumab is adopted as a target therapy agent for HER2-positive gastric cancer. Because of efficiency and accuracy, droplet digital PCR (ddPCR) is one of the best methods for measuring HER2 amplification, especially in cell-free plasma samples. In this study, we aimed to investigate the correlation between tissue and plasma HER2 status by using ddPCR method in gastric cancer patients. We collected formalin-fixed and paraffin-embedded tissue (FFPE) and cell-free plasma samples of 81 gastric cancer patients, including 30 HER2 immunohistochemistry (IHC)-negative (0 or 1+) cases and 51 HER2 IHC-positive cases (IHC 2+, n=20; IHC 3+, n=31), with informed consents. We performed ddPCR of a target gene HER2 and a reference gene EIF2C1 in both tissue and cell-free plasma samples, and gene copy number (GCN) of HER2 was calculated. The median HER2 GCN of tissue DNA ddPCR was 2.61 (1.47-49.00) and the median GCN of plasma ddPCR was 1.83 (0.97-7.67). HER2 IHC results showed moderate correlation with HER2 GCN of tissue ddPCR (r=0.455, p&amp;lt;0.001) and weak correlation with plasma ddPCR (r=0.321, p=0.003). A ratio &amp;gt;2.405, which showed the best discriminating level from a receiver operating characteristic curve, was defined as a cut-off for tissue HER2 amplification using ddPCR method. Comparing the HER2 amplification results of tissue DNA ddPCR to HER2 IHC results, the concordance rate was 86.4% with Cohen's kappa of 0.715. Sensitivity and specificity of tissue ddPCR were 86.3% and 86.7%, respectively. HER2 amplification by tissue ddPCR was significantly associated with advanced gastric cancer and lymphatic invasion (p&amp;lt;0.05). HER2 GCN of tissue ddPCR was weakly correlated with plasma ddPCR (r=0.250, p=0.025). Sensitivity and specificity of plasma ddPCR for HER2 IHC positivity were 35.3% and 93.3%, respectively. However, sensitivity and specificity of plasma ddPCR were 66.7% and 60.0% with another cut-off of 1.715. In 51 HER2 IHC-positive cases, intratumoral heterogeneity was observed in 100% (5/5) of both tissue and plasma ddPCR-negative cases, 100% (2/2) of tissue-negative and plasma positive cases, 64.3% (18/28) of tissue-positive and plasma-negative cases, and 50.0% (8/16) of both positive cases (p=0.026). In conclusion, HER2 GCN of plasma ddPCR showed a weak correlation with tissue ddPCR, and the sensitivity of plasma ddPCR was not high. Intratumoral heterogeneity is suggested to be one of the possible causes, and these findings may contribute to developing plasma HER2 testing useful in daily practice. Citation Format: Boram Kim, Soo Kyung Nam, Soo Hyun Seo, Sang-Hoon Ahn, Do Joong Park, Hyung-Ho Kim, Kyoung Un Park, Hye Seoung Lee. HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5592.

Abstract 1584: Comparison of circulating tumor cell (CTC) capture/identification methods and NanoString evaluation of gene expression in CTCs and cell-free circulating tumor mRNA (cctmRNA) in patients with metastatic lung cancer

Abstract Circulating tumor cells (CTC) and cell free circulating tumor mRNA (cctmRNA) can serve as biomarkers for prognosis and treatment response in lung and other cancers. A barrier to CTC use in some cancers is the low numbers of CTC captured, and the low percent of patients with detectable CTCs. Past comparisons of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients have shown larger cohorts of patients with detectable CTCs, and higher CTC numbers, for SCLCs. We compared methods for CTC capture/identification, and profiled mRNA from both the CTCs and cell free plasma RNA (cctmRNA) for to identify tumor-associated biomarkers. Peripheral blood was collected from 10 SCLC and 10 NSCLC patients prior to therapy, and from 4 normal controls. Capture using the standard EpCAM CellSearch reagent (unicapture) was run in parallel with EpCAM plus Her2, EGFR and MUC-1 combined ferrofluid (quadcapture) plus the use of anti-cytokeratin 7 (CK7) to assist in CTC identification. Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified 20% CTCs in NSCLC and 80% in SCLC. Overall, the quadcapture did not improve CTC capture efficiency. Exploratory NanoString analysis was performed on cell-free plasma from these patients and the ferrofluid isolated CTCs. Cancer-specific biomarkers were detected in mRNA from plasma and CTCs by NanoString included CCL5, CLU and SRGN. In summary, quadcapture ferrofluid reagent did not improve the capture efficiency, although the addition of CK7 may have the potential to identify additional CTCs. Nanostring analysis of cctmRNA and CTCs mRNA suggested potential new lung cancer biomarkers that require validation in larger studies. Citation Format: Yanis Boumber, Charu Aggarwal, Catherine Thrash-Bingham, Patricia Fittipaldi, Polina Guseva, Ramillya Vlasenkova, Chandra Rao, Jianming Pei, Brian L. Egleston, Hossein Borghaei, Massimo Cristofanilli, Ranee Mehra, Ilya Serebriiskii, R. Katherine Alpaugh. Comparison of circulating tumor cell (CTC) capture/identification methods and NanoString evaluation of gene expression in CTCs and cell-free circulating tumor mRNA (cctmRNA) in patients with metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1584.

Abstract 642: Comparison between in situ hybridization and digital polymerase chain reaction methods for detecting c-MYC gene copy number gain in tissue and cell-free plasma samples of colorectal cancer patients

Abstract Introduction: We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of CRC patients. Methods: c-MYC GCN was determined by using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort of 193 CRC patients (cohort 1) and prospective cohort of 64 CRC patients (cohort 2). Additionally, c-MYC GCN was analyzed by ddPCR method in cell-free plasma samples of cohort 2. Results: In cohort 1, c-MYC GCN gain, defined as mean c-MYC copies/nucleus ≥ 4.0 in SISH analysis, was observed in 34 (17.5%), and c-MYC GCN gain by ddPCR method in 7 (3.6%). c-MYC GCN by SISH was significantly correlated with ddPCR results (ρ= 0.532, P &amp;lt; 0.001). Forty cases (20.7%) showed intratumoral genetic heterogeneity, but it did not affect the concordance between SISH and ddPCR results (P &amp;gt; 0.05). c-MYC GCN gain by both SISH and ddPCR methods had the worst prognosis (P = 0.001). In cohort 2, c-MYC GCN by SISH was significantly associated with tissue ddPCR (ρ=0.349, P = 0.005), but not with plasma ddPCR (P = 0.620). Intratumoral genetic and regional heterogeneity of c-MYC status have been found in 29 (45.3%) and 8 (12.7%). c-MYC status in plasma was associated with tissue ddPCR, but borderline statistical significance (ρ= 0.246, P = 0.050). Conclusions: Our findings suggested that results by SISH and ddPCR methods were discordant. Therefore, detecting method should be carefully selected to determine c-MYC GCN status. Citation Format: Soo Hyun Seo, Kyu Sang Lee, Soo Kyung Nam, Kyoung Un Park, Heung-Kwon Oh, Duck-Woo Kim, Sung-Bum Kang, Hye Seung Lee. Comparison between in situ hybridization and digital polymerase chain reaction methods for detecting c-MYC gene copy number gain in tissue and cell-free plasma samples of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 642.

Hemodynamic Flow in a Vertical Cylinder with Heat Transfer : Two-phase Caputo Fabrizio Fractional Model

In blood, the concentration of red blood cells varies with the arterial diameter. In the case of narrow arteries, red blood cells concentrate around the centre of the artery and there exists a cell-free plasma layer near the arterial wall due to Fahraeus-Lindqvist effect. Due to non-uniformity of the fluid in the narrow arteries, it is preferable to consider the two-phase model of the blood flow. The present article analyzes the heat transfer effects on the two-phase model of the unsteady blood flow when it flows through the stenosed artery under an external pressure gradient. The direction of the artery is assumed to be vertical and the magnetic field is applied along the radial direction of the artery. Blood is considered as a non-Newtonian Casson fluid with uniformly distributed magnetic particles. Both the blood and magnetic particles are moving with distinct velocities. This two-phase problem is modelled using the Caputo-Fabrizio derivative approach and then solved for an exact solution using joint Laplace & Hankel transforms. Effects of pertinent parameters such as Grashoff number, Prandtl number, Casson fluid parameter and fractional parameters, and magnetic field on blood velocity and particle velocity have been shown graphically for both large and small values of time. Both velocity profiles increase with the increase of Grashoff number and Casson fluid parameter and reduce with the increase of magnetic field and Prandtl number. The behaviour of temperature is studied for different values of the fractional parameter.

Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease

Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The noninvasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.

Cell-Free Plasma DNA for Disease Stratification and Prognosis in Head and Neck Cancer.

Clinicians face many challenges in disease stratification and outcome prediction in head and neck squamous cancer cell (HNSCC) patients. Given the limitations of currently used clinical scoring, repetitive biopsies, and imaging techniques, liquid biopsy approaches may provide valuable additional diagnostic and prognostic information. A noninterventional, single-center observational study was performed with clinical data and plasma samples from HNSCC patients. Cell-free tumor DNA-derived copy number aberrations (CNAs) were determined in 116 patients by low-coverage next-generation sequencing (NGS). Significant CNAs were combined in a genome-wide copy number instability score (CNI), which was evaluated with respect to conventional clinical staging and patient outcome. Receiver-operating characteristic (ROC) curve analysis comparing the presurgery CNI in patients (n = 103) with that in tumor-free controls (n = 142) yielded an area under the ROC curve of 87.2% (95% CI, 79.4%-93.3%). At a specificity of 95%, the sensitivity to detect tumors varied between 46% (pT1) and 94% (pT4). A CNI above the median (i.e., >72) had a positive predictive value of 90% (95% CI, 79%-96%) for lymph node involvement (LNI), while the negative predictive value was 57% (95% CI, 43%-70%). For a CNI >72, overall survival (OS) was worse (hazard ratio, 4.89; 95% CI, 1.39-17.17; P = 0.01) with 62% and 90% survivors 3 years after surgery for a CNI >72 and ≤72, respectively. In multivariable models, the CNI was a superior predictor of OS compared to established disease features, including LNI. The CNI may assist in predicting LNI and prognosis in HNSCC with direct therapeutic implications concerning the need for neck dissection or more aggressive treatment.

Association between male Infertility and seminal plasma levels of growth hormone and insulin-like growth factor-1.

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) have been proposed to play a pivotal role in male infertility due to their anabolic effects. The aim of this study was to investigate possible associations between seminal plasma levels of GH and IGF-1 and sperm parameters. Fifty men participated in this study. Semen analysis was performed, while cell-free seminal plasma was collected following sperm centrifugation. Seminal plasma concentrations of IGF-1 and GH were determined by enzyme-linked immunosorbent assay (ELISA). Due to the presence of asthenozoospermia in all participants who presented with abnormal sperm parameters, the participants were further subdivided into normal (group A), asthenozoospermic (group B) and asthenozoospermic plus at least one additional abnormal parameter (group C). A marginally nonsignificant statistical difference (p=0.063) was revealed between the GH levels corresponding to the asthenozoospermic and the normal group with the latter presenting with higher GH levels. A statistically significant positive correlation (p<0.05) was noted between levels of GH and IGF-1 in group C. The above relationship has also been observed in men with low sperm concentration, vitality, volume and abnormal morphology. These novel findings require further investigation in order for the biological significance of those associations to be clarified.

Cancer patient-reported knowledge and preferences for liquid biopsies and blood biomarkers at a comprehensive cancer centre.

6587Background: Novel blood-based biomarkers, including cell-free DNA and plasma signatures, are becoming a reality in precision oncology. Yet, little is known about cancer patients’ perspectives o...