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April 01, 2017 (Vol. 37, No. 7)

CTCs Can Make Better Samples Than Biopsies

Think of Cancer as Criminal Activity, and You’ll Understand the Need to Properly Secure Evidence from Liquid Biopsies

  • Although circulating tumor cells (CTCs) and cell-free DNA (cfDNA) may be “bagged” in liquid biopsy samples, they may not stay intact through all the phases of an analytical workflow. If these biomarkers suffer degradation—during storage, transportation, nucleic acid extraction, or other sample preparation steps—they may skew assays toward erroneous or equivocal results. Plainly, this is no way to investigate a patient’s cancer, make a case for an individualized therapy, or monitor attempts at correction—to say nothing of guarding against treatment resistance or cancer recurrence.

    The care and handling of liquid biopsy samples is something of a chain-of-custody problem. Fortunately, various links in this chain received special attention at the International Molecular Med Tri-Con event, which was recently held in San Francisco. This Cambridge Health Institute event was not solely devoted to liquid biopsies, but it did include a channel (“Circulating Tumor Cells and Liquid Biopsy”) and a symposium (“Circulating Cell-Free DNA”) of direct relevance to our subject.

    Several of the event’s presentations are summarized below. All were selected to highlight key points in the analytical workflow, from the preanalytical “securing evidence” stage to the cancer management “post-judgment modification” stage. Preanalytical processing is vital for obtaining reliable downstream test results. CTC and cfDNA assays require that liquid biopsy samples “withstand stressors of preanalytical variables introduced in clinical settings,” asserted Landon Olp, Ph.D., R&D scientist, Streck.

    “Isolated CTCs can be a better sample than a tumor biopsy,” insisted Steve Crouse, chief commercial officer, Vortex Biosciences. CTCs sloughed off from multiple locations during cancer progression represent multiple cell populations from both primary and metastatic tumors, capturing cancer heterogeneity.

    Tumor cells release cfDNA into the bloodstream during cell death. “But cfDNA does not exist as freely dissociated DNA,” warned Hamid Khoja, Ph.D., principal scientist, Covaris. For example, cfDNA may be bound around nucleosomes and chaperone proteins, and it must be carefully extracted for downstream analysis.

    Analysis technologies such as next-generation sequencing are not without error. “The need for sensitivity and accuracy is critical,” advised Dale Yuzuki, market development director, SeraCare Life Sciences. “Without standards, people won’t know if a test and claimed sensitivity [are] real or not.”

    Phillip G. Febbo, M.D., CMO, Genomic Health, added that “you have to understand the performance of your assay with respect to clinical samples and the likely allele frequencies you might see in those clinical samples.”

  • Stabilizing cfDNAs in Sample Tubes

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    Streck’s Cell-Free DNA BCT simplifies collection, storage, and shipping of whole blood samples for circulating tumor biomarker assays. The stabilizing reagent included in the Cell-Free DNA BCT enables nucleated blood cells to withstand the stressors of preanalytical sample handling up to 14 days postphlebotomy—protecting the original proportions of cell-free DNA and circulating tumor cells in the sample.

    “There are many preanalytical variables, but sample storage and transportation probably have the greatest impact on cfDNA assays,” remarked Streck’s Dr. Olp.

    Most doctors’ offices can’t perform cfDNA tests in house. Blood samples are collected, shipped to a central testing lab, and often batched for cost savings. “It could take a week or longer from the time a blood sample is drawn until tested using a cfDNA assay,” lamented Dr. Olp.

    Shipping and storage places a high level of stress on the sample. Blood cells lyse in a standard EDTA blood collection tube; releasing cellular DNA into the plasma, the cfDNA space.

    “The already rare mutant cfDNA, your biomarker, becomes even more diluted,” informed Dr. Olp. “In essence, your needle in a haystack becomes a needle in a field of haystacks, and the assay results can be unreliable due to the instability of the sample.”

    To resolve this problem, Streck provides a specialized blood collection tube (BCT), the Cell-Free DNA BCT®. It contains a reagent that can stabilize nucleated cells right in the tube, enabling the sample to withstand the stressors of the preanalytical variables for up to 14 days at room temperature. The release of cellular DNA from white blood cells breaking up is prevented, and fragile CTCs don’t degrade.

    “Stabilization allows Cell-Free DNA BCT’s to be applicable for both cfDNA and CTC biomarkers,” emphasized Dr. Olp. “The original proportions of cfDNA and CTCs present in the sample will remain consistent for up to two weeks, giving you time before you begin processing that sample in a liquid biopsy assay.”

  • Trapping CTCs in Microvortices

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    With Vortex Biosciences’ VTX-1 Liquid Biopsy System, a blood sample flows down microchannels in a microfluidic chip, which contains chambers that sustain microvortices. These microvortices trap and retain the larger and more deformable circulating tumor cells (CTCs) while allowing blood cells to flow past. Once isolated, the CTCs can be released for downstream analysis.

    “The automated VTX-1 Liquid Biopsy System from Vortex Biosciences evaluates both the physical properties of cells and the characteristics of molecular markers,” noted Crouse. Many CTC isolation technologies bind EpCAM, an epithelial marker, to capture CTCs. Yet, many cancers don’t express EpCAM or lose expression during metastasis. Other technologies use a filter, which only targets the larger CTCs and can get clogged.

    The VTX-1 utilizes microfluidics to efficiently isolate CTCs directly from whole blood. “The VTX-1 flows blood down a microchannel which widens along a portion of its length, creating a reservoir. Between the narrowings at the inlet and outlet, microvortices form, similar to the vortices that may form in a river and trap leaves in looping paths,” explained Crouse. The VTX-1 vortices trap the larger and more deformable cells such as CTCs, stably holding them in a swirling current while the rest of the blood cells flow past.

    “Collecting based on physical properties captures a diverse population of cancer cells and, we believe, a more clinically relevant sample than affinity-based approaches,” remarked Crouse. Unlike affinity-based or filter-based collections, cells are not stuck onto anything like channels, membranes, or magnetic beads.

    Since the VTX-1-captured cells stay in suspension and experience pressures similar to those they would encounter in circulation, they remain intact and similar to how they were in the bloodstream. CTCs are released from the Vortex microfluidic chip and are immediately available for genomic, RNA, protein, or live-cell assays, allowing for a holistic picture of the cancer.

    The VTX-1, which accommodates 0.2–16 mL samples, has several exciting capabilities. For example, it can capture CTCs from the tiny liquid biopsy samples of patient-derived xenograft (PDX) mouse models, “where the VTX-1 offers an opportunity to get access to cancer biology in a way that wasn’t available before,” exclaimed Crouse.

    The VTX-1 enables synergistic CTC and cfDNA approaches. “A Streck Cell-Free DNA BCT can be used to ensure cfDNA remains stable. After pulling off the plasma (containing cfDNA), you can process the remaining blood through the VTX-1 and collect the CTCs,” explained Crouse. This enables mutation profiling of both the CTCs and cfDNA from a blood tube, potentially providing a more informed understanding of the patient’s cancer.

  • Soundly Standardizing Extraction

    Clinical decisions are made based on quantitative cfDNA results. “Although analytical technologies become more sensitive every year, the sample preparation workflows feeding these instruments are often low tech, antiquated, and inefficient,” cautioned Covaris’ Dr. Khoja.

    The Covaris Adaptive Focused Acoustics™ (AFA) platform normalizes diverse sample specimens and standardizes preanalytical sample preparation. AFA uses focused acoustic energy to actively control cavitation. Cavitation is caused by the conversion of acoustical to highly tuned mechanical, sheer force energy. “That’s how you get a variety of beneficial effects, such as DNA shearing and extraction, cell lysis, and solvation of a boundary layer,” explained Dr. Khoja.

    AFA minimizes chemical variability introduced during cfDNA extraction and purification, while maximizing cfDNA yields from precious clinical samples. These features enable the power of sensitive analytical instruments to be realized so that these devices may facilitate the development of more sensitive and relevant next-generation clinical applications.

    AFA is highly controllable. The non-contact isothermal AFA technology is very tunable, such that DNA can be randomly sheered to many different lengths (100 bp–5 kb, depending on the library preparation needs). “The uniformity of coverage obtained with an AFA-prepped sample enables lower sequencing depth, speeding sequencing and reducting its cost,” remarked Dr. Khoja.

    The cfDNA concentration is highly variable between patients, tumor types, stages, and treatment conditions. Standardization of all preanalytical protocols, including blood collection, is critical for optimal analytical results. “We recommend Streck Cell-Free DNA BCT because it brings a level of control over the collection, stabilization, and shipping of precious blood samples,” stated Dr. Khoja.

    Covaris develops kits for standardizing cfDNA extraction. The truXTRAC cfDNA™ kits are designed for actively dissociating and extracting cfDNA from histone-cfDNA and other protein-cfDNA complexes occurring in cfDNA BCT-stabilized plasma, and they enable scalable and automatic high-throughput sample processing.

    “DNA sequencing confirmed that truXTRAC cfDNA-processed samples had higher library complexity, more thoroughly mapped reads, greater coverage uniformity, and better variant sensitivity than cfDNA samples obtained from passive extraction methods,” concluded Dr. Khoja.