Cell-Free DNA Blood Collection Tubes Research Articles

Two Commercially Available Blood-Stabilization Reagents Serve as Potent Inactivators of Coronaviruses.

The continued circulation of SARS-CoV-2 and the increasing frequency of coronavirus (CoV) outbreaks over the decades demonstrates the enduring threat that the CoV family poses. There remains a significant need to develop tools to monitor and prevent the spread of these viruses. We tested blood-stabilization reagents from two commercially available blood collection tubes (BCTs) for their ability to inactivate three different coronaviruses (MHV, OC-43, and SARS-CoV-2) and stabilize their RNA. Both Cell-Free DNA BCT® (cfDNA) and Cyto-Chex® BCT (CytoChex) reagents reduced infectious virus in the buffer to below the limit of detection within 18 h of treatment, with some conditions showing this effect in as little as 3 h. CytoChex had more potent activity than cfDNA as in all cases it more rapidly reduced the actively replicating virus to the limit of detection. Despite the rapid inactivation of the virus, both reagents effectively preserved viral RNA for 7 days. Finally, both reagents accelerated viral inactivation in blood compared to the control samples. These results indicate that cfDNA and CytoChex could be used to inactivate and preserve CoV RNA for detection and further testing.

Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.

Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study.

Background: Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Methods: Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. Results: MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Conclusions: Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells.

Abstract 3110: In depth comparison of cell free DNA blood collection tubes

Abstract Liquid biopsy continues to gain traction as a minimally invasive method to monitor biomarkers associated with malignancy and metastasis such as cell-free DNA (cfDNA). However, cell-free DNAs are present in low quantities and are prone to rapid degradation and contamination, which presents challenges for the detection of rare cfDNA targets. Higher molecular weight genomic DNA from poorly stabilized leukocytes can also serve to contaminate plasma, compromising liquid biopsy assay results by increasing the total amount of extracted DNA while diluting rare cfDNA targets; leading to sample rejection or false negative results. Efficient and robust preservation of cfDNA and prevention of leukocyte lysis is thus essential for the accuracy of downstream tests, and several blood collection tubes (BCTs) designed to stabilize cfDNA are now commercially available. The work presented here evaluates LBgard® Blood Tubes (Biomatrica), the Streck Cell-Free DNA BCTs® (Streck), and PAXgene Blood ccfDNA Tubes (PreAnalytix) for analysis of cfDNA. Healthy donor blood was collected into collection tubes, and the samples were either stored for several days at a range of temperatures or subjected to various shipping conditions. Several metrics were evaluated, including hemolysis, plasma recovery, cfDNA preservation, contamination with genomic DNA (gDNA), and quantitation of cfDNA targets. Differences were observed between the various tubes evaluated, especially when tested under real-world stress shipping conditions. Data reports performance metrics and evaluations across the multiple formulations of BCTs and highlights the criticality of collection tube selection in biomarker investigation and study. Citation Format: Joel Desharnais, Jacob M. Vasquez, Katya J. Reshatoff, Quyen Bui, Han-Mei Chen, Billyana Tsvetanova, Jerry Lu, Amber N. Murray, Gina L. Costa. In depth comparison of cell free DNA blood collection tubes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3110.

Hemolysis and Fetal Fraction in Cell-Free DNA Blood Collection Tubes for Noninvasive Prenatal Testing.

BackgroundLysis of maternal white blood cells in prenatal cell-free DNA (cfDNA) test samples increases the level of maternal DNA and consequently decreases fetal fraction.ObjectiveThe objective of this study was to determine whether hemolysis, traditionally used as a marker for cell lysis, is correlated with a decrease in fetal fraction in maternal blood samples collected in specialized cfDNA tubes for noninvasive prenatal testing.MethodsIn the first part of the study, blood from pregnant women was collected into three Roche Cell-Free DNA Collection Tubes. These replicate specimens from the same subject were evaluated for a visual difference in hemoglobin level as a measure of hemolysis. The specimens were then processed with the Harmony® prenatal test to measure fetal fraction using polymorphic digital analysis of selected regions (DANSR) assays. In a second part of the study, clinical laboratory samples with hemoglobin levels of ≥ 500 mg/dL were tracked through the laboratory and their fetal fraction compared with that of concurrently processed samples with lower hemoglobin levels.ResultsThere was no significant difference in fetal fraction in 339 paired samples, with a difference in hemoglobin levels ranging from 0 to 1000 mg/dL. There was strong correlation in fetal fraction between tubes, regardless of the differences in hemoglobin concentration. The fetal fraction distribution in 203 tracked clinical samples with hemoglobin levels ≥ 500 mg/dL was statistically equivalent to the distribution in a concurrent series of 12,705 samples.ConclusionHemolysis in maternal blood samples collected in specialized cfDNA tubes does not correlate with a decrease in fetal fraction; therefore, it should not be a cause for rejection of samples submitted for prenatal cfDNA testing.Electronic supplementary materialThe online version of this article (10.1007/s40291-020-00446-x) contains supplementary material, which is available to authorized users.

P3690Plasma levels of cardiac-specific micro-RNA mir-1 and mir-133a differ in patients with acute myocardial infarction and takotsubo cardiomyopathy

Abstract Background Takotsubo cardiomyopathy (TTC) is an acute, life-threatening condition which is typically induced by stress and manifested by chest pain and ECG changes. The TTC prevalence among patients (pts) with acute coronary syndrome is 1.7–2.2%. The mortality rate from TTC is up to 8%. TTC is clinically indistinguishable from acute myocardial infarction (AIM). Differential diagnosis of TTC and AIM remains an unresolved problem. Recent studies have shown the differences in profiles of plasma microRNAs inTTC and AIM. Purpose To evaluate the possibility of differentiating TTC and AIM using a PCR-based semi-quantitative analysis of mRNAs, the plasma levels of which had been shown to be increased in patients with AIM (miR-1, miR-208a, miR-133a, miR-499a) and TTC (miR-16, miR-26a). Methods Plasma from 38 pts was used: 13 pts with confirmed TTC (12 women, 1 man), 25 pts with AIM (9w, 16m). For 10 pts with AIM, blood was collected twice: at 6 and 24 hours after the heart attack. The control arm comprised 40 healthy people from the same age group (12w, 28m). Plasma was obtained using the Cell-Free DNA blood collection tubes. Nucleic acids were separated using a modified Boom method. A semi-quantitative assessment of the mRNA levels was performed using dCq method with stem-loop qRT-PCR. Normalization for spiked synthetic cel-miR-39 and endogenous miR-23a was performed. Control of hemolysis was performed by measuring the ratio of miR-451 (specific for RBCs) and miR-23a (absent from RBCs). The statistical significance of differences between mRNA levels was assessed using Mann–Whitney test. Results No significant differences in the levels of miR-16, miR-26a and miR-208a in TTC group and control group have been found. Pts with AIM significantly differed from pts with TTC (p=0.0038 and 0.0002, respectively) and control pts (p=3.1x10–9 and 2,66x10–10) with the increased levels of cardiac-specific miR-1 and miR-133a. As compared to plasma levels at 6 hours after the heart attack, at 24-hour point the levels of these mRNAs were markedly reduced in 9 of 10 pts (mean reduction was 22.3-fold for miR-133a and 7.5-fold for miR-1). The correlation between changes in the levels of these mRNAs was high (Spearman correlation coefficient=0.89). Significant differences in plasma levels of miR-133a between pts with AIM and TTC were maintained even if blood was collected after 24 hours (p=0.007). For miR-16 and miR-26a, no significant differences between pts with AIM and TTC were found. The results of analysis of these mRNAs are affected to a substantial degree by the residual hemolysis due to their high content in blood cells. Conclusions It was shown that measuring the plasma levels of mRNAs miR-1 and miR-133a allows to distinguish TTC from AIM by excluding the diagnosis of TTC. The differential diagnosis is possible only within several hours after acute clinical symptoms and requires proper normalization and full compliance with the technical specifications.

Validation of Cell-Free DNA Collection Tubes for Determination of EGFR Mutation Status in Liquid Biopsy from NSCLC Patients

IntroductionPrecision medicine has revolutionized the understanding and treatment of cancer by identifying subsets of patients who are amenable to specific treatments according to their molecular characteristics, as exemplified by epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Although tissue biopsy is the gold standard for determining molecular alterations in tumors, its limitations have prompted the development of new techniques for studying tumor biomarkers in liquid biopsies, such as mutation analysis in cell-free DNA (cfDNA). cfDNA analysis can accurately determine tumor progression and prognosis and more effectively identify appropriate targeted therapies. However, cfDNA is vulnerable, particularly during plasma sample shipping.ObjectiveWe compared the cell- and DNA-stabilizing properties of cell-free DNA blood collection tubes (BCTs) with those of the traditional shipping method (frozen plasma) for EGFR mutation testing using the cobas® EGFR Mutation Test v2 in a prospective cohort of 49 patients from three different Spanish hospitals.MethodsIn total, 98 NSCLC samples, two from each patient, were studied; five of the 49 cases were considered invalid by cobas® with one of the two shipping methods analyzed. After excluding these samples, we analyzed 88 samples from 44 patients. Considering the current methodology (frozen plasma) for sending samples as the gold standard, we evaluated the sensitivity and specificity of cfDNA BCT shipment.ResultsThe global agreement between the two methods was 95.4%, with 100% sensitivity and 94.6% specificity for the cfDNA BCTs. cfDNA BCTs had a positive predictive value of 81.8% and negative predictive value of 100%.ConclusioncfDNA BCTs have the same sensitivity for EGFR mutation analysis in liquid biopsy as the current methodology and very high specificity. They also have some additional advantages in terms of collection and further shipment. Therefore, cfDNA BCTs can be perfectly incorporated into the routine practice for EGFR mutation determination.FundingRoche Farma S.A., Spain.Electronic Supplementary MaterialThe online version of this article (10.1007/s40487-019-00099-9) contains supplementary material, which is available to authorized users.

Abstract 2279: Serial changes in tumor-derived whole-genome cfDNA fraction to identify early disease progression prior to imaging

Abstract Background: Response to cancer treatment is usually determined by clinical exam and imaging assessment. Here, we analyzed changes in tumor-derived whole-genome cell-free DNA (cfDNA) at baseline and after treatment initiation to determine response to treatment prior to routine imaging. Methods: We prospectively enrolled and serially collected blood from 54 patients with metastatic malignancies (21 lung, 20 breast, 13 other tumor types). Baseline blood samples were drawn prior to initiation of a new treatment and at one or two additional time points, after the first cycle (median 21 days) and the second cycle (median 42 days). 4 mL of plasma was separated from peripheral blood collected in Streck Cell-Free DNA Blood Collection Tubes, and cfDNA was isolated from plasma aliquots using Qiagen QIAmp extraction kits. To prepare sequencing libraries, a method optimized for whole genome sequencing (WGS) was used based on the Kapa HyperPrep chemistry. WGS was performed at approximately 25X depth on the Illumina HiSeq X. Based on a patient-specific profile of whole genome features, changes in the fraction of tumor-derived cfDNA were quantified over the initial course of treatment. Imaging was performed per standard practice with treatment response determined by RECIST. Results: Median number of prior treatment lines was 1 [range 0-6]. Patients were treated with the following therapies: chemotherapy (27), immunotherapy (14), hormone therapy (7), or targeted therapy (6). For the entire cohort, patients with predicted progression by cfDNA (n=11), indicated by an increase in tumor fraction at either post-treatment blood collection, had worse event-free survival compared to patients that did not show an increase (n=43) (hazard ratio 8.0, [95% CI 3.4-19.2], log-rank p=4.5 x 10-8). For the patients who were predicted to progress, the cfDNA assay preceded clinical evaluation by a median of 39 days. Median progression-free survival was 62 days for patients with predicted progression versus 232 days for others. All patients with predicted progression were later confirmed to progress at the first follow-up evaluation (11/11, 100% positive predictive value). For the remaining patients, 32 of 43 did not progress (74% negative predictive value). Therefore, sensitivity for the assay was 50% and specificity was 100%. Conclusions: Analyzing tumor-derived cfDNA early in the course of a new therapy holds promise to identify patients with early disease progression across a variety of tumor histologies and types of treatment. Early identification of patients who are not benefitting from treatment will enable initiation of other potentially effective therapies, and reduce unnecessary side effects and cost associated with these treatments. Further studies are warranted to validate these findings in larger cohorts and to confirm the histology and treatment-independent nature of the approach. Citation Format: Andrew A. Davis, Wade T. Iams, David Chan, Michael S. Oh, Robert W. Lentz, Neil Peterman, Alex Robertson, Abhik Shah, Rohith Srivas, Nicole Lambert, Tim Wilson, Peter George, Becky Wong, Ayse Tezcan, Ram Yalamanchili, Ken Nesmith, John C. Spinosa, Haluk Tezcan, Young Kwang Chae. Serial changes in tumor-derived whole-genome cfDNA fraction to identify early disease progression prior to imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2279.

Evaluation of pre-analytical factors affecting plasma DNA analysis

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.

Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer.

BackgroundOptimized blood collection tubes (BCT) have been developed to expand the utility of plasma cell‐free DNA (cfDNA) and are in clinical use. The appropriateness of plasma collected and stored in these tubes for proteomic analysis is unknown.MethodsPaired blood samples were collected in BCT and traditional K3EDTA (EDTA) tubes from healthy controls and from colorectal cancer (CRC) patients before and after surgery, and stored for between 45 min and 48 h at room temperature. Plasma proteins were analyzed following high‐abundant plasma protein depletion in quantitative discovery and targeted proteomics by liquid chromatography tandem‐mass spectrometry (LC‐MS/MS).ResultsBCT reduced cellular protein contamination in healthy controls over time, and increased the number of high confident low‐abundant protein identifications in CRC blood samples compared to matched samples collected in EDTA tubes. The known CRC plasma protein biomarker, carcinoembryonic antigen (CEA), showed elevated levels across patients pre‐operatively when collected and stored in BCT compared to EDTA tubes. Emerging CRC biomarkers, Dickkopf‐3 (DKK3) and Gelsolin (GSN), showed elevated levels pre‐operatively when collected in BCT.ConclusionsOptimized BCT are appropriate for low‐abundant plasma protein analysis and can be used with confidence for clinical proteomics.

Abstract 2731: Identifying circulating tumor DNA in patients with osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant bone tumor and primarily affects children and adolescents. During and after treatment there is no non-invasive test to assess disease response and early relapse. We hypothesize that circulating tumor DNA (ctDNA) can be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We effectively tested this hypothesis on a mouse model and have utilized our methods on a first cohort of human patients. Materials and Methods: Twenty blood samples with matched primary tumor material were obtained from 10 patients with OS who were at various stages of treatment or post treatment. Cell free DNA was isolated from blood collected in Cell-Free DNA blood collection tubes using the QIamp Circulating Nucleic Acid kit. We performed massively parallel sequencing (MPS) with the Illumina HiSeq 2500 in the Epigenomics Shared Facility (ESF) to a depth of at least 800x using custom designed probes (Roche NimbleGen SeqCap EZ) capturing genes commonly altered in OS including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. We aligned the fastq files with BWA, called the variants using Varscan and GATK HaplotypeCaller, and annotated the variants with SnpEff. Summary of Data: Using an initial threshold of 2% of variants, ctDNA was identified in two of the patients in our study. The first patient’s sample, collected at the completion of therapy, contained a missense mutation in the coding exon of DLG2 at position chr11 84027990 (2.07% of reads). This patient has no radiographic evidence of recurrent disease 12 months after completion of treatment. In a second patient with 4 samples drawn, a mutation was found in the intron region of RB1 at position chr13 48986157. The variant was 0.17% of reads upon completion of therapy, undectable at 3 months, 2.16% of reads at 6 months, and 1.5% of reads at 9 months. The patient is at high-risk for recurrence but is currently disease-free. Sequencing and analysis of serially collected patient samples, including from 3 patients with recurrent disease, and primary tumor is ongoing. Conclusions: We have identified pathogenic variants in the cell free DNA of patients with no radiologic evidence of OS but who are at high risk for relapse. This is the first step to utilizing a non-invasive test to assess tumor burden, response to treatment, and likelihood of recurrence. As part of future work, we will lower the threshold for calling variants and quantify ctDNA abundance in serially collected samples from treatment and during surveillance, and correlate the findings with clinical outcome. Citation Format: David M. Barris, Michael Fremed, Robert Durbin, Xusheng Zhang, Sajida Piperdi, Wendong Zhang, Shahina Maqbool, Jonathan Gill, Michael Roth, Bang Hoang, David Geller, Richard Gorlick, Daniel Weiser. Identifying circulating tumor DNA in patients with osteosarcoma [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 2731. doi:10.1158/1538-7445.AM2017-2731

Abstract 2758: Comparison of cell-free DNA blood collection tubes

Abstract Critical biomarkers such as cell-free DNA (cfDNA) derived from tumors and circulating tumor cells (CTCs) can be detected and analyzed from a simple blood draw. These analytes are fragile, prone to degradation, and present in extremely low quantities. Therefore, proper preservation of these analytes is necessary to ensure accuracy of test results. Several blood collection tubes are commercially available for cfDNA applications, and selecting the ideal blood collection tube for cfDNA impacts test results, sample collection logistics and costs. However, no comprehensive and systematic evaluation of performance among these tubes is available. This study evaluates five commercial blood collection tubes: LBgard™ Blood Tube (Biomatrica), Streck cfDNA BCT® (Streck), PAXgene Blood ccfDNA Tube (PreAnalytiX), Cell-Free DNA Collection Tube (Roche), and EDTA (BD). Healthy donor blood samples were collected in each tube type and incubated over several days at different temperatures. Total plasma DNA was subsequently isolated, and the yield, fold increase versus time 0 and quality of purified DNA were compared. cfDNA controls were also spiked into blood samples collected in each tube type and measured by droplet digital PCR to determine the mutant allele frequencies over time. Finally, the yield and quality of cfDNA isolated from stage IV colorectal cancer blood collected in LBgard Blood Tube, Streck cfDNA BCT and EDTA were compared. Additionally, the inhibition of hemolysis and CTC stabilization were also assessed. Our results show that LBgard Blood Tube, Streck cfDNA BCT, PAXgene ccfDNA Tube and Cell-Free DNA Collection Tube are superior to EDTA tubes in maintaining cfDNA yield and quality over 7 days at ambient temperature. LBgard Blood Tube out-performs all tested blood tubes in inhibiting genomic DNA release for the longest duration (14 days) and across the widest temperature range (4°C, 25°C and 37°C). LBgard Blood Tube shows equivalent inhibition of genomic DNA release to Streck cfDNA BCT for clinical samples, and both tubes out-perform EDTA blood tubes. LBgard Blood Tube also consistently shows better CTC stabilization and inhibition of hemolysis over Streck cfDNA BCT. This comprehensive and systematic study of blood collection tube performance provides a quantitative and exhaustive assessment of blood sample stability, allowing researchers to make informed decisions based on their sample stabilization needs. Citation Format: Cecille D. Browne, Margrith E. Mattmann, Marc J. Wycoco, Sheila N. Chen, Rajeswari Ravichandran, Joel Desharnais, Laura J. Varela, Jonathan D. Browne, Vasco Liberal, Florence Lee. Comparison of cell-free DNA blood collection tubes [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 2758. doi:10.1158/1538-7445.AM2017-2758

Abstract 3145: Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology

Abstract Liquid profiling based on circulating tumor DNA (ctDNA) extracted from blood has become a powerful diagnostic approach in oncology. Making ctDNA analysis broadly available requires shipping whole blood to a clinical laboratory while ensuring cell-free DNA (cfDNA) and blood cell integrity to prevent dilution of ctDNA with cellular genomic DNA. Since standard EDTA blood collection tubes cannot retain this integrity for a prolonged time, several laboratories have proposed Streck Cell-Free DNA blood collection tubes (Streck cfDNA BCTs) as an alternative for sample collection. Qualification data for the use of Streck cfDNA BCTs in oncology is limited and mainly based on blood collected from healthy individuals as well as data extrapolated from the prenatal field. However, the data generated from these samples may not represent the unique dynamics of clinical oncology specimens and therefore a study of true clinical oncology samples is required to support the use of Streck cfDNA BCT tubes in routine practice. In this study, we performed a clinical evaluation of cfDNA sample integrity from matched Streck cfDNA BCTs vs standard EDTA tubes from colorectal, pancreatic and non-small cell lung cancer patients (N > 50). Blood drawn into Streck cfDNA BCTs was either processed immediately or 3 days after phlebotomy. DNA quantification was followed by BEAMing digital PCR (OncoBEAM™) on KRAS, NRAS and EGFR mutations and compared to matching specimens collected in EDTA tubes. Our results suggest that cfDNA yield as well as the genomic DNA background is not affected by prolonged storage of clinical samples in Streck cfDNA BCTs for up to 3 days. In all sample sets containing mutated ctDNA, the detected mutational load was comparable between Streck cfDNA BCTs and EDTA tubes. In conclusion, this study represents a comprehensive clinical evaluation of Streck cfDNA BCTs vs EDTA tubes for ctDNA profiling. In conjunction with the findings of our previously presented Streck cfDNA BCT shipping condition studies, our data supports the compatibility of clinical oncology specimens collected in Streck cfDNA BCTs with the BEAMing technology. Citation Format: Inga Medina Diaz, Stefan Holdenrieder, Annette Nocon, Makbule Kobilay, Dirk Skowasch, Stefanie Held, Johanna Weiland, Claudia Stamm, Frank Diehl, Frank Holtrup. Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology. [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 3145.

Abstract 3160: Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer

Abstract Background: Advanced transitional cell carcinoma (TCC) of the bladder is a lethal malignancy with few available biomarkers to guide therapy. Recent evidence suggests that circulating tumor cells (CTCs) are detectable in TCC and may have a prognostic role; however, most studies to date have focused on CTC enumeration. We leveraged novel enrichment and capture technologies to isolate and recover ultra-pure CTC (upCTC) that can be used for single cell molecular analysis. Methods: Patients with metastatic bladder cancer were enrolled under an institutional review board approved pilot study. Blood samples (7.5 ml each) were drawn into EDTA or Cell-Free DNA blood collection tubes (Streck), and preliminary CTC enrichment was performed using one of 3 microfluidic platforms: i) EpCAM-based LiquidBiopsy system (Cynvenio); ii) size/deformability-based ClearCell FX system (Clearbridge); or iii) size/deformability-based Parsortix system (Angle). Enriched CTCs were labeled with Cytokeratin (CK), EpCAM, and CD45 immunofluorescent antibodies and Hoechst nuclear stain. Identification and recovery of upCTCs was performed on a DEPArray v2 system (Silicon BioSystems). Additional experiments using cell lines were performed to gauge the recovery of mRNA from rare cancer cells collected in EDTA vs. preservative tubes. Results: To date, samples were collected from 8 patients of whom 5 (∼63%) had detectable CTCs. All 3 enrichment platforms successfully yielded CTC fractions from which Hoechst+/CK+/CD45- upCTCs were subsequently identified and recovered on the DEPArray v2 system. Additional cells that were Hoechst+/CK-/CD45- were identified and collected, and cells also were gated by integral Hoechst intensity and collected for correlation to aneuploidy. Comparison of mRNA recovery from 10 cancer cells collected into EDTA vs. fixative tubes (CellSave, Cynvenio, Streck Cell-free RNA) demonstrated that gene expression readings by qPCR from rare fixed cells was feasible but associated with variable results, likely due to variable proteinase K reversal of mRNA-protein cross-linkages. Conclusion: We leveraged new techniques to develop and optimize a workflow for identification and collection of upCTCs from bladder TCC patients, and we established that gene expression profiling is also feasible, albeit currently is still best achieved using EDTA collection and not any of the available preservative tubes. Single upCTC molecular profiles (DNA alterations, gene expression) from patient samples generated using these protocols may help to elucidate mechanisms of disease progression and ultimately may advance the management of advanced bladder cancer. Citation Format: Gareth Morrison, Cory Hugen, Tong Xu, Yucheng Xu, Dorff Tanya, David Quinn, Sarmad Sadeghi, Amir Goldkorn. Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer. [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 3160.