Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

<div><p>Objectives</p><p>Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.</p><p>Methods</p><p>Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K₂EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).</p><p>Results</p><p>Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.</p><p>Conclusion</p><p>Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.</p></div

Experimental study cohorts.

<p>Experimental setup for cfDNA BCT vs K₂EDTA performance experiments. Cohort I: Time point experiments at room temperature including DNA quantification and mutation analysis using BEAMing and Safe-SeqS. Cohort II: BEAMing analysis of blood samples spiked with synthetic double-stranded mutant DNA fragments at different allele frequencies. Cohort III: BEAMing analysis of samples collected from colorectal cancer (CRC) patients. Cohort IV: Experiment evaluating effects of extreme storage temperatures on DNA quantity. Cohort V: Experimental evaluation of recommended temperature range.</p

<i>In Vivo</i> Activity and Pharmacokinetics of Nemorosone on Pancreatic Cancer Xenografts

<div><p>Pancreatic cancer is one of the leading cancer-related causes of death in the western world with an urgent need for new treatment strategies. Recently, hyperforin and nemorosone have been described as promising anti-cancer lead compounds. While hyperforin has been thoroughly investigated <i>in vitro</i> and <i>in vivo</i>, <i>in vivo</i> data for nemorosone are still missing. Thus, we investigated the growth-inhibitory potential of nemorosone on pancreatic cancer xenografts in NMRI nu/nu mice and determined basic pharmacokinetic parameters. Xenograft tumors were treated with nemorosone and gemcitabine, the current standard of care. Tumor sections were subjected to H&E as well as caspase 3 and Ki-67 staining. Nemorosone plasma kinetics were determined by HPLC and mass spectrometry. Induction of CYP3A4 and other metabolizing enzymes by nemorosone and hyperforin was tested on primary hepatocytes using qRT-PCR. At a dose of 50 mg/kg nemorosone per day, a significant growth-inhibitory effect was observed in pancreatic cancer xenografts. The compound was well tolerated and rapidly absorbed into the bloodstream with a half-life of approximately 30 min. Different metabolites were detected, possibly resembling CYP3A4-independent oxidation products. It is concluded that nemorosone is a potential anti-cancer lead compound with good bioavailability, little side-effects and promising growth-inhibitory effects, thus representing a valuable compound for a combination therapy approach.</p> </div

Genomic DNA release and obtained plasma volume after 3 days of storage within temperature range recommended by the manufacturer (study cohort V).

<p>Blood collected in cfDNA BCTs (n = 8) was stored for 3 days at the indicated temperature and subsequently analyzed using the genomic DNA release assay based on the 402:96 bp LINE-1 ratio (A). Shown are box plots with 1.5 x IQR applied to create whiskers. Statistically significant differences from the reference condition (20°C) were determined by one-way ANOVA and are marked with an * (p ≤ 0.05). (B) Obtained mean plasma volume with SD for indicated storage conditions.</p

Effect of extreme storage temperatures on plasma separation and DNA yield in study cohort IV.

<p>(A) Representative image of cfDNA BCTs centrifuged after 3 days of storage at RT, 4°C and 40°C. RT storage resulted in expected plasma separation with defined buffy coat layer and clear yellow plasma fraction. Extreme temperature conditions resulted in an expanded cellular interface layer or hemolytic plasma at 4°C or 40°C, respectively. (B) Effect of extreme temperatures on genomic DNA release (402 bp LINE-1 qPCR fragment). Statistically significant differences between K₂EDTA and cfDNA BCT storage conditions determined by one-way ANOVA are marked with ** (p ≤ 0.01). Shown are box plots with 1.5 x IQR applied to create whiskers. (C) Obtained mean plasma volume with SD for indicated storage conditions.</p

Analysis of cfDNA yield and genomic DNA release for study cohort I.

<p>(A) DNA yield was assessed for cfDNA from blood samples stored at room temperature (18°C– 22°C) in K₂EDTA tubes vs cfDNA BCTs (healthy donors, n = 60). Plasma was prepared after indicated storage conditions. Extracted DNA was analyzed for overall yield by qPCR amplifying a 96 bp LINE-1 fragment. (B) Illustration of the DNA yield ratio between long (402 bp) and short (96 bp) LINE-1 fragments (n = 60). Increased ratios compared to K₂EDTA reference would indicate genomic DNA release. Shown are box plots with 1.5 × interquartile range (IQR) applied to create whiskers and outliers. Statistical analysis using one-way ANOVA revealed no significant difference between conditions.</p

Hematoxylin-eosine and immunohistochemical staining of tumor sections.

<p>Sections of 5 µm of nemorosone-treated and control tumors were stained with hematoxylin-eosine (H&E; <b>A</b> and <b>B</b>) or immunohistochemically analyzed with antibodies directed against active caspase 3 (<b>C</b> and <b>D</b>) or Ki-67 (<b>E</b> and <b>F</b>). Treated tumors demonstrated reduced tumor mass due to apoptosis/necrosis (black arrows) and a lower number of proliferating cells as compared to the control (dark-brown cells).</p

Hyperforin and nemorosone.

<p>Closely related chemical structures of hyperforin (left) and nemorosone (right), two polycyclic polyprenylated acylphloroglucinols.</p

Tumor volume and body weight development in MIA-PaCa-2 xenograft mice.

<div><p>Mice were treated with daily i.p. injections of 50 mg/kg nemorosone, vehicle only or 120 mg/kg gemcitabine at the indicated time points (green dots). Tumor volume was measured 2-3 times per week using a digital caliper. Values represent the mean ± SD of 8 animals per group.</p> <p>* p < 0.05, ** p < 0.01 (compared to the vehicle control).</p></div

Induction of selected genes involved in metabolism.

<p>Primary human hepatocytes were treated with the indicated concentrations of rifampicin, hyperforin, nemorosone or vehicle only. RNA was extracted after 48 h and subjected to qRT-PCR analysis to detect expression changes of selected genes involved in drug metabolism. Expression values are relative to the vehicle control and represent means ± SD of triplicate measurements.</p