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

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

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

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

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

Detectability of low frequency mutations in study cohort II.

<p>Blood from healthy donors (n = 8) was spiked with synthetic double-stranded mutant DNA fragments of different allele frequencies in a background of 60,000 genome equivalents of fragmented human genomic wild-type DNA and subsequently stored at RT. DNA was extracted and analyzed by BEAMing after the indicated storage time. Mean and standard deviation of the detected mutant fraction is shown. (A) <i>PIK3CA</i> c.3140A>G spike (0.1%), (B) <i>EGFR</i> c.2369C>T spike (0.5%), (C) <i>KRAS</i> c.34G>A spike (1%).</p

Response to gemcitabine dependent on time and growth rate.

<p>BxPC-3 and JoPaca-1 cells we treated with increasing concentrations of gemcitabine for 72, 96, and 120 hours. Cell viability was normalized to the non-treated controls. Data points represent averaged results of six replicate experiments with standard deviations indicated by error bars. A. Increasing incubation time with gemcitabine results in increased cytotoxicity for JoPaca-1. B. Cell viability was compared between 3.79 and 4.28 cell divisions for BxPC-3 and JoPaca-1, respectively. JoPaca-1 shows a higher tolerance towards gemcitabine than BxPC-3 even after extended incubation of 0.49 cell divisions.</p

Immunofluorescence of cytokeratin 19 and the tumour marker mesothelin in cell culture.

<p>Phase contrast images are presented that show mesothelin and cytokeratin 19 as detected in fixed JoPaca-1, BxPC-3 and HPDE c7 cells. Both proteins were marked with primary and FITC-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Isotype controls were negative. A. Cytokeratin 19 is expressed at the growing front of JoPaca-1 cells. B. JoPaca-1 and the established cell line BxPC-3 both express mesothelin while the normal pancreatic ductal cell line HPDE c7 does not.</p

Micro- and macroenvironment of primary and xenograft tumours.

<p>Shown here are H&E stains of primary (A) and xenograft (B) tissue slices. A. Smooth muscle tissue of the duodenum was infiltrated by tumour cells of the primary tumour. B. Invasive front of xenograft tumours with areas of infiltrative tumour expansion into the adjacent normal pancreas tissue (arrow heads). C. One out of three NSG mice developed metastasis during initial experiments when 1 Mio cells were injected orthotopically (arrow heads).</p

Karyogram and common chromosomal aberrations.

<p>A representative karyogram of JoPaca-1 is shown here. JoPaca-1 is a tetra- to penta-ploidic cell line. Below the overview picture, the five most commonly observed aberrations in 26 karyograms are presented. Translocations could be identified by hybridization of differently coloured probes resulting in dual-coloured chromosomes. Inversion of chromosome 13 (i13) is the fusion of two q-arms at the kinetochore. The Y-chromosome is often lost in tumour cells as it is in this representation.</p