Cell-free Plasma Research Articles

P3.02b-101 EGFR T790M Resistance Mutation in NSCLC: Real-Life Data of Austrian Patients Treated with Osimertinib: Topic: EGFR RES

Somatic mutations in the epidermal growth factor receptor (EGFR) are detected in approximately 13% of the Austrian non-small cell lung cancer (NSCLC) patients. The EGFR T790M resistance mutation located on Exon 20 is the most common mechanism of drug resistance to EGFR tyrosine kinase inhibitors (TKI) in these patients. The mutation can be detected by re-biopsy as well liquid biopsy. Osimertinib (AZD9291), a 3rd generation EGFR-TKI, showed a highly clinical activity in these patients. We report about our experience with Osimertinib in EGFR-mutated NSCLC patients, who became resistant to first or second generation TKI's due to EGFR T790M mutation. From April 2015 to June 2016 we administered osimertinib 80 mg daily to 82 patients who had disease progression after previous treatment with an EFGR TKI. The T790M mutation status was assessed by re-biopsy and/or liquid biopsy. For liquid biopsies, blood samples were collected in EDTA-containing vacutainer tubes and processed within 2 hours after collection. Cell-free plasma DNA was extracted by using the QIAamp circulating nucleic acid kit (Qiagen) according to the manufacturer’s instructions. Mutation status was assessed with QX-100™ Droplet Digital™ PCR System (Bio-Rad). The T790M mutation status was assessed in 48 patients by liquid biopsy only and in 13 patients by re-biopsy of the tumor. In 21 patients the T790M mutation was detected by both methods. 70 (85%) patients showed a clear clinical and radiographic response. Out of these, 70 patients, 14 (17%) patients reached a complete remission, 56 (68%) patients showed partial response and in 5 (6%) patients, a stable disease after treatment with osimertinib was observed. Five patients had symptomatic brain metastasis initially without any further option of local treatment, and showed a clear a clear clinical benefit and a partial remission radiographically. Osimertinib was well tolerated. No clinically relevant significant side effects were reported. Osimertinib was highly active in our patients, while showing good safety profile. Therefore, re-biopsy or liquid biopsy should be performed in clinical routine to detect the T790M mutation. With the above described method, liquid biopsy could replace re-biopsy in clinical practice in the future.

P3.02b-032 Association between EGFR T790M Mutation Copy Numbers in Cell-Free Plasma DNA and Response to Osimertinib in Advanced NSCLC: Topic: EGFR Biomarkers

Patients with advanced EGFR-mutated non-small-cell lung cancer (NSCLC) who developed the T790M resistance mutation during treatment with EGFR tyrosine kinase inhibitors (TKIs) benefit from treatment with third-generation EGFR TKIs such as osimertinib. Treatment with osimertinib requires the confirmation of the presence of the T790M mutation by re-biopsy of the tumor or by analysis of cell-free plasma DNA from blood samples (liquid biopsy). The purpose of our study was to compare T790M mutation copy numbers in cell-free plasma DNA with response to osimertinib. From April 2015 to June 2016, we included 44 patients with advanced T790M-positive NSCLC who received osimertinib after previous disease progression with an EFGR TKI and in whom response to osimertinib was evaluable. T790M mutation status was assessed by droplet digital PCR in cell-free plasma DNA. The threshold for T790M positivity was >1 copy/mL. The T790M mutation status was assessed in all patients by liquid biopsy and in 18 patients also by re-biopsy of the tumor. All 44 patients were T790M-positive in the liquid biopsy. Two out of 18 (11%) patients had a T790M-negative re-biopsy. Thirty-seven patients (86%) showed a response to treatment with osimertinib: 13 (29.5%) complete responses (CR), 24 (54.5%) partial responses (PR), one (2%) stable disease (SD), and six (14%) progressive disease (PD) (Table 1). We observed no statistically significant association between response to osimertinib and T790M copy numbers (p=0.54; Table 1). The median T790M copy numbers across response categories were: CR 25 copies/mL (range 1.7-38092 copies/mL), PR 14 copies/mL (range 1.6-7282 copies/mL), SD+PD 6 copies/mL (range 1.8-475 copies/mL).Table 1ResponseCopies/mLCRPRSDPD<105 (39%)11 (46%)0 (0%)4 (67%)≥108 (62%)13 (54%)1 (100%)2 (33%) Open table in a new tab Patients benefited from osimertinib treatment independent of T790M copy numbers in the blood samples. Although limited by low numbers, we observed a trend towards better response to osimertinib in patients with ≥10 T790M copies/mL.

Does detection of DDX4 mRNA in cell-free seminal plasma represents a reliable noninvasive germ cell marker in patients with nonobstructive azoospermia?

This study aimed to investigate the potential application of DDX4 gene expression in cell-free seminal mRNA as a noninvasive biomarker for the identification of the presence of germ cells in men with nonobstructive azoospermia and to correlate this factor with testicular biopsy. Male reproductive organ-specific genes were chosen: DDX4, which is a germ cell-specific gene and transglutaminase 4, which is a prostate-specific gene that was used as a control gene. Thirty-nine azoospermic males and twenty-eight normospermic fertile males (serving as a control group) participated in the study. Histopathological examination of testicular biopsies categorised azoospermic males into 20.5% with maturation arrest, 17.9% with incomplete Sertoli cell-only syndrome and 61.5% with complete Sertoli cell-only syndrome. Using real-time polymerase chain reaction, positivity for DDX4 gene was detected in 17 of 39 males with NOA which was due to maturation arrest in 35.3% (n=6/17) of cases, due to incomplete Sertoli cell only in 23.5% (n=4/17) and due to complete Sertoli cell only in 41.2% (n=7/17). The study recommends joint utilisation of molecular transcripts as noninvasive biomarkers and histopathological examination of testicular biopsies in management of cases with azoospermia of the nonobstructive type.

Frequency and significance of parvovirus B19 infection in patients with rheumatoid arthritis.

The present study aims to clarify the possible involvement of parvovirus B19 (B19V) infection in rheumatoid arthritis (RA) pathogenesis by investigating the presence of B19V infection markers (genomic sequences and virus-specific antibodies) in association with the level of cytokines and RA clinical activity and aggressiveness. A total of 118 RA patients and 49 age- and sex-matched healthy volunteers were enrolled in the study. Nested PCR was used to detect B19V sequences in whole blood and cell-free plasma DNA, ELISA to detect virus-specific antibodies and cytokine levels in plasma and recomLine dot blot assay for antibodies to separate B19V antigens. The detection frequency of B19V DNA was higher in patients with RA (25.4 %) in comparison with healthy persons (18.4 %). B19V DNA in cell-free plasma (B19+p) was detected significantly often in RA patients in comparison with healthy controls (13.6 vs 2 %; P=0.0002). RA B19+p patients had higher disease activity and aggressiveness, decreased haemoglobin and increased erythrocyte sedimentation rates. IL-6 plasma levels were significantly higher in RA patients than in controls. Within the RA patients’ group the IL-6 level was significantly increased in B19+p patients with disease activity scores of DAS28>5.2, high C-reactive protein and low haemoglobin. Contrary to the healthy controls, the majority of RA B19+p patients did not have antibodies to VP-1S (VP1u) and VP-N (N-terminal half of structural proteins VP1 and VP2), which correspond to the epitopes of neutralizing antibodies. These results indicate that B19V infection at least in some patients is involved in RA pathogenesis.

ISY-24-3 - ESR1 gene alterations related to endocrine resistance in metastatic breast cancer

Estrogen receptor (ER) positive breast cancer can be treated by endocrine therapy; however a certain population of ER-positive patients becomes resistant to endocrine therapies. Expression of the ESR1 gene encoding ER was upregulated in response to estrogen deprivation (LTED cells), which was usually seen in the metastatic breast cancer (MBC) with resistance to aromatase inhibitor. We found that RNAs from ESR1 gene locus were induced both from coding and non-coding regions in LTED cells using both total RNA-sequencing and mRNA-seq analyses. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating non-coding RNAs). Depletion of one of Eleanors, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. The activation of the ESR1 gene locus by the Eleanors is critical for cancer cell adaptation to estrogen deprivation and can be a new therapeutic and diagnostic targets in endocrine therapy resistance. Recent evidences have shown that the key potential mechanisms of the resistance to aromatase inhibitors based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. We initially developed a droplet digital PCR-based method for the sensitive detection of ESR1 mutations, Y537S, Y537N, Y537C, and D538G, which showed ESR1 mutations occurred in 2.5% of primary breast cancer specimens (n = 270) and in 20% of MBC specimens (n = 55). Furthermore, the patients with ESR1 mutations from cell free (plasma) DNA tended to have a poorer response to post-treatment, compared with wild-type patients. Taken together, ESR1 gene alterations including non-coding RNA are profoundly related to the endocrine resistance in metastatic breast cancer and could be a predictor of the usage to combination with the signal transduction inhibitor, such as mTOR inhibitor, PI3K inhibitor.

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.

BackgroundCirculating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application.MethodsWe designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients.ResultsCompared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R2=0.916, P<0.0001).ConclusionsThis duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.

A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon

Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

A mobile phone-based approach to detection of hemolysis

Preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome are pregnancy-related complications with high rates of morbidity and mortality. HELLP syndrome, in particular, can be difficult to diagnose. Recent work suggests that elevated levels of free cell hemoglobin in blood plasma can, as early as the first trimester, potentially serve as a diagnostic biomarker for impending complications. We therefore developed a point-of-care mobile phone–based platform that can quickly characterize a patient's level of hemolysis by measuring the color of blood plasma. The custom hardware and software are designed to be easy to use. A sample of the whole blood (~10µL or less) is first collected into a clear capillary tube or microtube, which is then inserted into a low-cost 3D-printed sample holder attached to the phone. A 5–10min period of quiescence allows for gravitational sedimentation of the red blood cells, leaving a layer of yellowish plasma at the top of the tube. The phone camera then photographs the capillary tube and analyzes the color components of the cell-free plasma layer. The software converts these color values to a concentration of free hemoglobin, based on a built-in calibration curve, and reports the patient's hemolysis level: non-hemolyzed, slightly hemolyzed, mildly hemolyzed, frankly hemolyzed, or grossly hemolyzed.. The accuracy of the method is ~1mgdL−1. This phone-based point-of-care system provides the potentially life-saving advantage of a turnaround time of about 10min (versus 4+hours for conventional laboratory analytical methods) and a cost of approximately one dollar USD (assuming you have the phone and the software are already available).

Effect of HHV-6 and HHV-7 Infection on the Posttransplant Process and the Development of Complications in Patients after Autologous Stem Cell Transplantation / HHV-6 Un HHV-7 Infekcijas Ietekme Uz Pēctransplantācijas Klīnisko Gaitu Un Komplikāciju Attīstību Pacientiem Pēc Autologas Cilmes Šūnu Transplantācijas

Abstract The relationship between HHV-6 and HHV-7 reactivation and development of post-autologous peripheral stem cell transplantation complications was examined. The presence of viral genomic sequences in whole peripheral blood and cell free plasma was determined by nested PCR, HHV-6 and HHV-7 load by real-time PCR, virus specific antibodies and cytokines in serum by ELISA, and HHV-6 variants by restriction endonuclease analysis. Clinical features, reactivation of viruses and serum TNF- α, and IL-6 concentrations were determined in seventy-six patients with Roseolovirus infection before and after transplantation. Anti-HHV-6 antibodies were found in 62 of 76 (81.6%) patients before transplantation. A significantly higher rate of single HHV-7 infection was found in patients with viral infection in comparison with single HHV-6 infection (p = 0.0003) and concurrent (HHV-6 and HHV-7) infection (p = 0.0017). Complications after transplantation developed in 30.3% of patients and reactivation of viruses was detected in all of these patients. Significant increase of HHV-6 and HHV-7 reactivation with simultaneous increase of pro-inflammatory cytokines serum levels suggests that both viruses may be involved in the development of complications after autologous peripheral blood stem cell transplantation via their immunomodulatory ability. The kinetics of the Roseolovirus reactivation may reflect the potential role of HHV-7 as a co-factor for HHV-6 activation.

Increased Levels of Plasma Epstein Barr Virus DNA Identify a Poor-Risk Subset of Patients With Advanced Stage Cutaneous T-Cell Lymphoma

Outcomes in advanced stage (AS) cutaneous T-cell lymphomas (CTCL) are poor but with great variability. Epstein-Barr virus (EBV) is associated with a subset of non-Hodgkin lymphomas. Frequency of plasma EBV-DNA (pEBVd) detection, concordance with EBV RNA (EBER) in tumor tissue, codetection of plasma cytomegalovirus DNA (pCMVd), and prognostic effect in AS CTCL are unknown. Patients (n= 46; 2006-2013) with AS CTCL (≥IIB) were retrospectively studied. pEBVd and pCMVd were longitudinally measured using quantitative real-time polymerase chain reaction. EBER in situ hybridization (ISH) was performed on tumor samples. Survival from time of diagnosis (ToD) and time of progression to AS was assessed. Plasma EBV-DNA and pCMVd were detected in 37% (17 of 46) and 17% (8 of 46) of AS CTCL patients, respectively. pCMVd detection was significantly more frequent in pEBVd-positive (pEBVd(+)) than pEBVd(-) patients (35% vs. 7%; P= .038). Tumor tissue for EBER-ISH was available in 14 of 17 pEBVd(+) and 22 of 29 pEBVd(-) patients; 12 of 14 (85.7%) pEBVd(+) patients were EBER(+) versus 0 of 22 pEBVd(-) patients. Frequency of large cell transformation (LCT) tended to be greater in pEBVd(+) patients, but was not significant (10 of 14 pEBVd(+) vs. 10 of 23 pEBVd(-); P= .17). No notable differences in rates of increased levels of serum lactate dehydrogenase (LDH) were observed (17 of 17 pEBVd(+) vs. 27 of 29 pEBVd(-)). pEBVd detection was associated with significantly worse survival from ToD (P= .021) and time of progression to AS (P= .0098). Detection of cell-free plasma EBV-DNA was highly concordant with the presence of EBERs in tumor tissue, predicted survival independent of LDH and LCT, and should be further studied as a biomarker in AS CTCL.

Particulate suspension Jeffrey fluid flow in a stenosed artery with a particle-free plasma layer near the wall

The present article concerns the problem of blood flow through an artery with an axially asymmetric stenosis (constriction). The two-layered macroscopic model consisting of a cell-rich core of suspension of all the erythrocytes described as a particle-fluid suspension (Jeffrey fluid) and a peripheral zone of cell-free plasma (Newtonian fluid). The analytical expressions for flow characteristics such as fluid phase and particle phase velocities, flow rate, wall shear stress, and resistive force are obtained. It is of interest to mention that the magnitudes of wall shear stress and flow resistance increase with red cell concentration but the flow resistance decreases with increasing shape parameter. One of the important observations is that when blood behaves like a Jeffrey fluid, the flowing blood experiences lesser wall shear stress and flow resistance than in the case of blood being characterized as a Newtonian fluid in both the particle-fluid suspension and particle- free flow studies. The rheology of blood as Jeffrey fluid and the introduction of plasma layer thickness cause significant reduction in the magnitudes of the flow characteristics.

Abstract 3133A: Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma

Abstract BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and children and adolescents represent half of new cases diagnosed each year. During and after treatment there is no non-invasive test to assess disease response and early relapse, respectively. Monitoring of circulating tumor material as a “liquid biopsy” has potential to provide this critical information in OS, as it has proven useful in other solid tumors. We hypothesized that circulating tumor DNA (ctDNA) can be extracted from mouse plasma and identified using next generation sequencing. This could ultimately be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We overcome the challenge of identifying tumor DNA in a background of host DNA by first using primary tumor material to identify genomic aberrations in a targeted gene region known to be altered in &amp;gt;95% of patients with OS. We then use next generation sequencing to identify in circulation the rare tumor DNA based on known aberrations expected to be present when there is a tumor burden. MATERIALS AND METHODS: Human osteosarcoma cell lines 143b and M17 were grown in culture and introduced into SCID mice via tail vein, subcutaneous flank, and tibial plateau injections. Terminal bleeds were performed 30 minutes after tail vein injection and at max tumor growth respectively. DNA was extracted from primary tumor cell lines as well as from the plasma of injected mice. We then performed next generation sequencing (NGS) with the Illumina HiSeq 2000 using custom designed probes capturing genes commonly altered in osteosarcoma including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. DNA extracted from the plasma of a SCID mouse that did not receive tumor cell injection was used as a negative control. RESULTS: Gene coverage of approximately 80% was obtained for targeted genes to a depth ranging from 250x to 2000x coverage for tumor cell lines. Cell free circulating tumor DNA was identified in plasma of mice injected with 143b mice via tail vein and flank injections but not from tumor cell free plasma negative control. Over 1000 mutations were identified, most notably c.467G&amp;gt;C p.R156P, a well documented osteosarcoma mutation, which was present in the 143b cell line as well as the DNA extracted from the plasma of tail vein and flank injected mice. CONCLUSIONS: Circulating cell free tumor DNA can be successfully extracted from SCID mouse plasma and identified using next generation sequencing of target genes. Based on these mouse findings, we anticipate that this will serve as a non-invasive biomarker of disease burden and response to therapy, as well as a biomarker to assess recurrence. The pilot work provides rationale to expand these findings into clinical trials that can prospectively validate our methods and findings. Our approach has the potential to improve outcomes for a childhood cancer frequently associated with poor survival. Citation Format: Michael Fremed, Sajida Piperdi, Wendong Zhang, Shahina Maqbool, Brent Calder, Raquel Castellanos, Jonathan Gill, Michael Roth, Bang Hoang, David Geller, Richard Gorlick, Daniel Weiser. Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma. [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 3133A.

Abstract 3151: Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients

Abstract Introduction: Cancer-associated mutations are present in circulating cell free plasma tumor DNA (ptDNA). We have previously reported mutation profiles of DNA extracted from CTC (CTC-DNA) from two patients with MBC (#2 and 24 in table). Here, we report an expanded cohort with an updated gene panel. Methods: We studied seven patients (two previously reported, along with five additional patients) with MBC who were enrolled in Mi-CTC-ONCOSEQ, had ≥5 CTC/7.5 ml whole blood (WB), and had at least one CTC with high quality DNA determined by the Ampli1™ quality control kit. CTC were enriched from WB with CellSearch© and purified from white blood cells (WBC) (DEPArray™). DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1™ WGA) and genotyped by multiplexed PCR-based next generation sequencing with the Oncomine Comprehensive Panel (OCP) on the Ion Torrent Proton. Exome sequencing of research biopsies of metastatic tissue was performed using an Illumina HiSeq 2500 platform. Previously reported patients (#2 and 24) sequenced with a beta version of the OCP were re-run, and updated results are provided. Results: Six of seven patients were ER positive. Patients #2, 12, and 24 had CTC with mutations also found in the research biopsy (table). Novel alterations were found in comparison to research biopsy in five of the seven patients (table). In two patients (#19, 24), two potential actionable mutations (PTCH1 and NOTCH1) were found in CTC-DNA but not in tissue-DNA. No mutations were detected in any WBC. Conclusions: We demonstrate the ability to purify CTC, and to isolate and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Mutations found in tissue as well as novel mutations were found in CTC-DNA. Two potential actionable mutations were identified in CTC, but not in tissue, opening potentially new therapeutic opportunities. We conclude that mutational analysis of CTC-DNA and of tissue may be complementary. Prioritized mutations in CTCsPt #Gene (Mutation)# CTC Single (S) Pooled (P)# with mutation (variant fraction)# without mutation# not evaluable (insufficient coverage)# WBC (all pooled)# with mutationPresent in Biopsy?2CDH1 (p.Q641X)7 (S)5 (1.00)NA230YCDH1 (S70F)7 (S)5 (1.00)NA230YESR1 (p.Y537S)7 (S)4 (0.46)2130YESR1 (unreported mutation)7 (S)1 (0.56)5130N8NA3 (P)*NANA3 (P)40NA12PIK3CA (H1047R)1 (S)1 (0.85)NANA10YTP53 (p.R248Q)1 (S)1 (0.72)NANA10Y14HNF1A (p.W206C)3 (P)+ (0.17)NANA40N17BRCA2 (p.Q1931X)4 (P)+ (0.10)NANA40N19PTCH1 (p.E1242X)3 (P)+ (0.28)NANA30N24CDH1 (p.I584fs)5 (P)+ (0.79)NANA40Y4 (P)+ (0.68)4 (P)+ (0.77)CDH1 (p.E841X)5 (P)0NANA40N4 (P)+ (0.14)4 (P)0TP53 (p.152_156del)5 (P)+ (0.94)NANA40Y4 (P)+ (0.29)4 (P)+ (0.36)NOTCH1 (p.S2492X)5 (P)0 +NANA40N4 (P)(0.17)4 (P)0Legend: NA = not applicable; + = mutation present in pooled CTC; Y = Yes; N = No; *CTC-DNA from the pool of 3 CTC had low and high quality. Citation Format: Costanza Paoletti, Andi K. Cani, Kimberly Aung, Elizabeth P. Darga, Emily M. Cannell, Daniel H. Hovelson, Maryam Yazdani, Allen R. Blevins, Nahomi Tokudome, Paul J. Baratta, Jose’ M. Larios, Dafydd G. Thomas, Martha E. Brown, Christina Gersch, Anne F. Schott, Daniel Robinson, Arul M. Chinnaiyan, Farideh Bischoff, Daniel F. Hayes, James M. Rae, Scott A. Tomlins. Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [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 3151.

Abstract 3107: A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors

Abstract . Introduction Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size. The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring. Materials and methods The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation (“drop-off”). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment. Results Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected. CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%). Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy. Conclusion The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914). Citation Format: Pieter A. Boonstra, Marco Tibbesma, Lisette J. Bosman, Ron H.J. Mathijssen, Frits van Coevorden, Neeltje Steeghs, Albert J.H. Suurmeijer, Arja ter Elst, Jourik A. Gietema, Anna K.L. Reyners, Ed M.D. Schuuring, Dutch GIST Consortium. A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors. [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 3107.

Potential confounding factors in measurement of specific cell-free seminal mRNAs and microRNAs derived from human reproductive organs.

Cell-free seminal RNA (cfs-RNA) is mixed transcripts derived from male reproductive organs, and is potential biomarker for the research and diagnosis of male reproductive-related diseases. However, some clinical factors, including age, asymptomatic Ureaplasma urealyticum (UU) infection, scrotal heat stress, abstinence period, and the storage condition of semen samples, may interfere with sperm parameters and the measurement of seminal biomarkers. Accordingly, this study was designed to evaluate the effect of above clinical factors on the measurement of cfs-RNA, aiming to lay a foundation for its research use and potential clinical application. Semen samples were collected according to the selected clinical factors. Cell-free seminal plasma was obtained by centrifugation and total RNA was extracted with TRIzol LS. Selective male reproductive organ-specific cfs-mRNAs and cfs-miRNAs were quantified by quantitative real-time PCR. The concentration and total amount of cfs-mRNAs and cfs-miRNAs in one ejaculate were calculated and compared. ACTB, DDX4 (testis-specific), WFDC9 (epididymis-specific), and miR-514a-3p (testis-specific) significantly increased after scrotal heat stress. SEMG1 (seminal vesicle-specific) showed declining tendency with the prolonged abstinence period. Age, asymptomatic UU infection, and the storage condition showed no significant impact on the measurement of cfs-RNA. These results indicate that scrotal heat stress significantly interfere with the selected cfs-RNA derived from the testis and epididymis, and abstinence period may affect the yield of cfs-mRNA from seminal vesicle, while other clinical factors has no significant impact on the measurement. Thus, heat exposure and abstinence period should be considered for the cfs-RNA measurement in its research or clinical application.

Diagnosis of Sepsis with Cell-free DNA by Next-Generation Sequencing Technology in ICU Patients

Bacteremia is a common serious manifestation of disease in the intensive care unit (ICU), which requires quick and accurate determinations of pathogens to select the appropriate antibiotic treatment. To overcome the shortcomings of traditional bacterial culture (BC), we have adapted next-generation sequencing (NGS) technology to identify pathogens from cell-free plasma DNA. In this study, 78 plasma samples from ICU patients were analyzed by both NGS and BC methods and verified by PCR amplification/Sanger sequencing and ten plasma samples from healthy volunteers were analyzed by NGS as negative controls to define or calibrate the threshold of the NGS methodology. Overall, 1578 suspected patient samples were found to contain bacteria or fungi by NGS, whereas ten patients were diagnosed by BC. Seven samples were diagnosed with bacterial or fungal infection both by NGS and BC. Among them, two samples were diagnosed with two types of bacteria by NGS, whereas one sample was diagnosed with two types of bacteria by BC, which increased the detectability of bacteria or fungi from 11 with BC to 17 with NGS. Most interestingly, 14 specimens were also diagnosed with viral infection by NGS. The overall diagnostic sensitivity was significantly increased from 12.82% (10/78) by BC alone to 30.77% (24/78) by NGS alone for ICU patients, which provides more useful information for establishing patient treatment plans. NGS technology can be applied to detect bacteria in clinical blood samples as an emerging diagnostic tool rich in information to determine the appropriate treatment of septic patients.

Vascular Fragility and the Endothelial Glycocalyx in the Tissues Lining the Healthy Gingival Crevice.

The aim of this study is to investigate vascular fragility and endothelial glycocalyx (ECG) in images of the healthy gingival crevice (GC). The ECG is a surface layer lining the luminal walls of blood vessels composed of proteoglycans and glycoproteins with other components. Degradation of the layer has been shown to drastically affect microvascular flow, permeability, and immune function. Videomicroscopic images from tissues lining the healthy GC in 20 healthy volunteers were analyzed to determine the presence of microhemorrhage related to branched dilated microvessels. Imaging of capillary ECG in human labial mucosa was achieved by the use of a 1-mm-diameter 0.5 numerical aperture gradient index objective lens and one-sided oblique illumination using light at 450 nm. This allows resolution of red blood cells within the capillary and the endothelial cell wall of the lumen. The cell-free plasma gap between the red cell and endothelial wall represents the ECG. In the healthy GC there was no evidence of microhemorrhage in participants that had only simple capillary loops (seven of 20). Microhemorrhage was present in the majority of participants with branched dilated microvessels (11 of 13) (McNemar Test for equal distribution, P = 0.05). A cell-free plasma gap representing the ECG could be imaged in the superficial tissues lining the labial sulcus. No such cell-free plasma gap could be detected in the branched dilated microvessels in tissues lining the GC. There is an association between the presence of vascular microhemorrhage and branched dilated microvessels in the tissues lining the clinically healthy GC. Loss of ECG might be part of the inflammatory process in these tissues.

Suspension model for blood flow through a catheterized arterial stenosis with peripheral layer of plasma free from cells

The present article describes the blood flow in a catheterized artery with radially symmetric and axially asymmetric stenosis. To understand the effects of red cell concentration, plasma layer thickness and catheter size simultaneously, blood is considered by a two-layered model comprising a core region of suspension of all the erythrocytes (particles) supposed to be a particle-fluid mixture and a peripheral zone of cell-free plasma. The analytical expressions for flow features, such as fluid phase and particle phase velocities, flow rate, wall shear stress and resistive force are obtained. It is witnessed that the presence of the catheter causes a substantial increase in the frictional forces on the walls of arterial stenosis and catheter, shear stress and flow resistance, in addition to that, have occurred due to the presence of red cells concentration (volume fraction density of the particles) and the absence of peripheral plasma layer near the wall of the stenosed artery. The introduction of an axially asymmetric nature of stenosis and plasma layer thickness causes significant reduction in flow resistance. One can notice that the two-phase fluid (suspension model) is more profound to the thickness of peripheral plasma layer and catheter than the single-phase fluid.

HPV cell free DNA as a marker of response to chemoradiation for HPV-positive oropharngeal cancers.

6083Background: Circulating cell free plasma DNA(cfDNA) can be a sensitive biomarker of tumor burden that is accessible by simple phlebotomy. Virally induced cancers present unique advantages for c...