Efficient Detection of BRAF Mutation in Plasma of Patients after Long-term Storage of Blood in Cell-Free DNA Blood Collection Tubes

To the Editor: The improvement of sensitive techniques has allowed tumor-specific aberrations in circulating tumor DNA (ctDNA)1 to be detected, including mutations in oncogenes and tumor suppressor genes, microsatellite instability, and DNA methylation (1). Therefore, ctDNA is a potential alternative source of tumor DNA that could be used for noninvasive identification of biomarkers at various time points during the course of the disease. In this context, digital PCR has been used to detect BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations in ctDNA from melanoma patients treated with a BRAF inhibitor (2), showing that BRAF mutation detection in ctDNA is useful as a follow-up to treatment response in patients with advanced melanoma (2). Sensitive techniques such as allele-specific amplification, digital PCR, and next-generation sequencing are used in most studies, but preanalytical procedures are also important. It is now clear that plasma should be preferred (3). Blood samples should be processed in the hours immediately after collection to prevent release of genomic DNA from white blood cells, which interferes with the detection of somatic mutations in tumor DNA (4) and limits the use of ctDNA. Cell-free DNA BCT® (Streck) is a direct-draw whole blood collection tube intended for the collection, stabilization, and transportation of ctDNA. These tubes have been shown to prevent genomic DNA contamination that may occur owing to nucleated cell disruption during sample storage (5) and are frequently used for fetal ctDNA analysis. However, there has been no study evaluating these tubes for the detection of gene mutations in ctDNA of cancer patients. We drew blood samples from melanoma patients with a BRAF mutation (V600E or V600K) in their tumor into K3EDTA-containing tubes (Greiner Bio-One) and BCT tubes. None of the tubes used contained a gel separator. Aliquots were removed at specified time points, and plasma was collected after centrifugation (2000g, 10 min, room temperature). We extracted circulating DNA using the PureLink Virus Kit and an iPrep Purification Instrument (Life Technologies). To detect BRAF alterations, we used the approved Therascreen BRAF RGQ kit (Qiagen) according to the manufacturer's recommendations; samples were considered positive for a BRAF mutation when the change in quantification cycle (ΔCq) value (Cq of the mutation specific PCR minus Cq of the control PCR) was <7. Blood samples were drawn and aliquots prepared at 2 h (reference) and 4, 7, and 10 days. The results demonstrated a large increase in ΔCq for EDTA tubes (Fig. 1) due to a lower Cq value for the control PCR, indicating a higher concentration of DNA released from normal blood cells (from 1 ng/mL at baseline to 6, 44, and 110 ng/mL after 4, 7, and 10 days, respectively), whereas the mutation-specific PCR remained unchanged. These results indicated that EDTA-containing tubes could not be used for such long times. When using BCT tubes, only 2 of 10 samples recorded an increase in ΔCq over time. Both samples were collected from the same patient, and the reason for this increase is being investigated. On the other hand, stable ΔCq values were obtained with BCT tubes for 8 of 10 samples tested (Fig. 1). Blood samples can thus be used for gene mutation detection after up to 10 days of storage at room temperature in BCT tubes. We then determined whether shipping would influence these results. Blood was drawn from 6 patients into BCT tubes. An aliquot was removed and processed immediately, and the remaining blood in the BCT tubes was sent to our laboratory by ordinary mail (room temperature). Samples were then tested 4–6 days after collection. Identical ΔCq values were obtained for all the samples tested, indicating that shipping did not decrease ctDNA stability. The BCT tubes contain a proprietary stabilization cocktail. To determine whether these tubes could also be used for biochemical analysis, we compared the results obtained from several tests. Identical results were obtained for sodium, albumin, prealbumin, and lactate dehydrogenase; however, these tubes could not be used to measure potassium, chloride, calcium, or the S100 protein. In conclusion, we demonstrate that BCT tubes can be used for efficient detection of gene mutation in ctDNA, even after long-term storage at room temperature. These collection devices can be shipped at room temperature, providing flexibility for offsite analysis without preliminary centrifugation or cryopreservation. circulating tumor DNA change in quantification cycle.