Blood collection tubes – the impact on ctDNA analysis reproducibility in breast cancer

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Cell-free circulating tumor DNA (ctDNA) is fragmented DNA from tumors (160–180 base pairs) that is found in the cell-free part of whole blood (i.e., plasma and serum) (1, 2). ctDNA can be distinguished from normal cell-free DNA (cfDNA) by the presence of somatic mutations (2). Analysis of ctDNA has the potential to be a ‘liquid biopsy’, i.e., a minimally invasive collection of blood or blood-based fluids that can be used to characterize cancer mutations (1). Given the fact that tumor tissue biopsy collection is invasive, takes a substantial amount of time to perform and can be expensive, the use of cell-free circulating tumor cells represents an alternative or complimentary method (1).

Breast cancer is the most prevalent cancer in women worldwide (3). For individuals with breast cancer, several mutations can be detected in their ctDNA. These include somatic single nucleotide variants (SNVs), copy number alterations (CNA) and structural variants (SVs), which can potentially be used to monitor tumor burden, determine therapeutic response and resistance, detect minimal residual disease, as well as help to provide insight into tumor heterogeneity and clonal evolution (1, 4).

To analyze ctDNA mutations in individuals with breast cancer, highly sensitive digital PCR technologies (dPCR) have been developed and are being investigated (1). Hrebien, S. et al. (2016), investigated the reproducibility of dPCR assays in ctDNA analysis of advanced breast cancer was studied (5). The research focused on the difference between immediate and delayed blood collection tube processing and the effects on reproducibility. One potential challenge with using ctDNA as a diagnostic tool is the need to process EDTA tubes within a short time after blood collection (5). Because analyzing blood directly is not always possible, this study focused on whether the use of preservative tubes would provide the same ctDNA data as EDTA tubes. Paired blood samples from 71 metastatic breast cancer participants were collected and placed in either EDTA K2 blood collection tubes, which were processed within 2 hours, or Streck Cell-Free DNA blood collection tubes, processed between 48–72 hours. cfDNA was then isolated from the plasma using QIAGEN’s QIAamp Circulating Nucleic Acid Kit. Subsequently, the ctDNA was analyzed with dPCR assays for mutations in PIK3CA, ESR1 and ERBB2, and AKT1 E17K. Results showed that with the Streck tubes ctDNA analysis was highly reproducible for mutation detection, but there was some limitation in the detection of low abundance mutations. They also found that the feasibility of shipping blood samples at room temperature in Streck preservative tubes could be advantageous to refrigerated transport of EDTA tubes, making clinical use easier.

One issue observed with the Streck tubes was that while these tubes preserved the total amount of circulating tumor DNA, in some samples, there were elevated amounts of total DNA compared to EDTA tubes samples. While the increase in high molecular weight germline DNA did not affect the sensitivity of ctDNA analysis by dPCR, the investigators stated that it could potentially affect ctDNA sequencing by reducing sensitivity (5).

QIAGEN has recently developed PAXgene Blood ccfDNA Tubes for collection and stabilization of blood for downstream ccfDNA processing. These tubes are shatter proof plastic (instead of glass), which prevents hazardous breakage, and meet OSHA safety standards. PAXgene Blood ccfDNA Tubes stabilize both ccfDNA and blood cell integrity so that no genomic DNA is released to compete with ccfDNA isolation (6). In addition, PAXgene Blood ccfDNA Tubes are better for investigators conducting epigenomic methylation studies and assays using bisulfate treatments. Furthermore, PAXgene tubes don’t have the risk of cross-linking modifications of DNA that makes Streck tubes less desirable for methylation based assays.

When PAXgene Blood ccfDNA Tubes are used in conjunction with QIAGEN’s new QIAamp MinElute ccfDNA Kit for ccfDNA purification, which has faster spin-column technology, investigators can achieve the best results for their downstream applications. In addition, using PAXgene tubes with the QIAamp kit requires no protocol modifications. However, if Streck tubes are used, an additional elongated proteinase K lysis step may need to be implemented. Lastly, the QIAamp MinElute ccfDNA Kit can also be partially automated on a QIAcube instrument for efficient, labor-saving results.

 

References

  1. 1. De Mattos-Arruda, L., Caldas, C. (2016) Cell-free circulating tumour DNA as a liquid biopsy in breast cancer. Mol. Oncol. 10, 464–474. Link

2. Diaz, L.A. Jr., Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. J. Clin. Oncol. 32, 579–586. Link

3. World Cancer Research Fund International webpage. Link

4. De Mattos-Arruda, L. et al. (2013) Circulating tumour cells and cell-free DNA as tools for managing breast cancer. Nat. Rev. Clin. Oncol. 10, 377–389. Link

5. Hrebien, S. et al. (2016) Reproducibility of digital PCR assays for circulating tumor DNA analysis in advanced breast cancer. PLoS One 11, doi: 10.1371/journal.pone.0165023. Link

6. Bowen, R.A., Hortin, G.L., Csako, G., Otañez, O.H., Remaley, A.T. (2010) Impact of blood collection devices on clinical chemistry assays. Clin. Biochem. 43, 4–25. Link

 

Nesrin Soetkamp

Nesrin Soetkamp, PhD is a Content Marketing Manager at QIAGEN. She received her PhD in Physiology and Biophysics from Georgetown University, where she studied the molecular mechanisms of taxane resistance in breast cancer. Prior to entering the biotech industry, Dr. Soetkamp worked as a postdoctoral fellow at the National Institutes of Health. At the NIH, she developed new methods to distinguish malignant adrenocortical tumors from benign tumors and normal tissue in the adrenal glands/cortex using epigenetic methylation patterns.

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