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Jul 1, 2013 (Vol. 33, No. 13)

Blood-Based Tumor Sequence

Direct Sequence Analysis of Tumor Cells Recovered from Whole Blood

  • Click Image To Enlarge +
    Figure 1. The LiquidBiopsy CTC Flow cell microfluidic device: This device allows magnetophoretic extraction of ferrofluid labelled target cells from a sample arrayed in sheath flow displayed in a cartoon (A). Magnets applied to the device will extract tumor cells as they pass through the device. The captured cells can be examined under the microscope and easily recovered for molecular analysis (B).

    Advances in precision medicine have emerged from a new and growing understanding of the genetic changes that occur within cancer cells. New therapies that can specifically target cancer-driving mutations have improved treatment outcomes in breast, lung, and prostate cancer. The result has been a much more targeted attack of the tumor containing the mutated protein or pathway, and a reduction of side effects on a patient’s normal cells and tissues.

    Personalized cancer treatment requires access to tumor samples throughout the treatment cycle so that as cancer changes, so does the approach to therapy. Patients can now benefit from the longitudinal monitoring of mutations that are changing in response to treatment. Until recently, detecting mutations that could benefit from targeted drugs has required sampling cells recovered from diseased tissue.

    In practice, this means that treatment decisions are made on a tissue biopsy that is representative of the cancer at one point in time and at one location in the body. Clearly, the advent of targeted therapies is driving a need for access to the genetic material of the tumor. To effectively monitor patients on a longitudinal scale, these samples are needed more often and with greater ease of collection than a traditional solid tumor biopsy can provide.

    There is a population of tumor cells circulating in the bloodstream that can address this need for closer monitoring—the circulating tumor cell (CTC). Metastasis, the development of secondary malignant growths at distant sites, arises when an accumulation of genetic errors sufficiently deregulate growth control causing the cancer cells to become mobile. These cells, carrying genetic material that reflects the tumor, migrate from their primary site through the bloodstream or the lymphatic system to other, distant sites in the body.

  • It is the metastatic events that lead to the majority of cancer-associated mortality. By extracting CTCs from a patient’s blood, the much-needed longitudinal analysis to track possible disease progression and the likelihood of metastases is made possible—even after there has been a surgical removal of the primary tumor. The challenge to date has been to reliably recover this rare CTC population in a manner that allows sensitive molecular analysis.

    For molecular analysis, CTCs need to be effectively and reliably separated from the tens of millions of normal lymphocytes and the hundreds of millions of red blood cells found in whole blood—a significant technical hurdle.

  • LiquidBiopsy

    Click Image To Enlarge +
    Figure 2. Yield and detection of mutations in breast cancer cells recovered on the Liquid Biopsy platform: MCF7 breast cancer cells were added to normal blood and purified on the Liquid Biopsy platform (A). Efficiency of recovery was evaluated by enumerating target cells in the CTC flow cell (B). The presence of the E545K mutation in the PIK3ca gene of recovered breast cancer cells was determined by demonstrating a signal lower than a negative control threshold (red dashed line). (C) This data shows the positive signal in each spike-in experiment (spike samples = open circles). MCF7 gDNA positive control (blue square), WT human DNA negative control samples (green circle) were included. Threshold for detection was determined by testing 12 negative control chip runs (red circles) and establishing a threshold two standard deviations below the mean for the negative controls (dashed line). (D) As an alternate readout, MCF7-spiked samples were recovered on the LiquidBiopsy platform and processed for evaluation using a next-generation sequencing panel. The table indicates the mutant frequency for two spiked samples that had the PIK3ca mutation detected using a next-generation Ion Torrent sequencing platform.

    Cynvenio Biosystems’ LiquidBiopsy platform leverages a technology that was developed to sort molecular libraries with upwards of 1014 individual components. By that measure, isolating between 10 and 1,000 tumor cells from a blood draw is a significantly more straightforward task.

    The LiquidBiopsy platform works by attaching nanometer-sized paramagnetic beads to target cells in blood using monoclonal antibodies. The platform then applies ultra-high magnetic gradients within a microfluidic CTC sheath flow cell to extract from the blood only those cells that were annotated with the paramagnetic beads (Figure 1). The population of target tumor cells is thus recovered free of all but about 0.001% of the normal blood cells.

  • By eliminating the normal cells so efficiently, CTCs extracted with the LiquidBiopsy platform can be molecularly analyzed with ease with a number of downstream technologies including NGS, expression analysis, PCR, as well as traditional microscopy and pathology, etc. The utility of this is demonstrated by ease of examining a blood sample for the presence of cells containing a mutation for which a drug is available.

    By examining blood samples containing a small number of spiked tumor cells known to contain a druggable mutation in the gene for PIK3ca, the platform demonstrates robust detection of the mutation using either PCR (Figure 2C) or next-generation sequencing (Figure 2D) based readouts. Therefore, for the first time, the LiquidBiopsy platform provides a practical means to analyze the genetic sequence of tumor cell populations directly purified from whole blood.

  • Examination of a sample from a patient with breast cancer shows that mutations can be detected in the CTC population and that some, but not all, of these mutations are detectable in a biopsy sample (Table 1). Therefore the CTC population reflects an aspect of the disease that is recoverable only from a blood sample.

  • Conclusion

    Click Image To Enlarge +
    Table 1. Examination of mutations found in the CTC population and a matched biopsy sample from a patient with breast cancer. Some but not all mutations are discoverable in both sequences. In addition, there are examples of mutations found in biopsy (shown here) or CTC LiquidBiopsy only (not detected in this sample).

    The LiquidBiopsy isolation and analysis technology has proven itself a powerful tool to effectively identify mutations in patient CTC samples. The platform has immediate applications in the clinical trial setting where, for the first time, CTC technology can address the need for better access to, and tracking of, biomarkers in near real-time. LiquidBiopsy can be used to identify candidates for specific targeted therapy trials, thus accelerating trial recruitment. The molecular readout from a CTC sample can also serve to mine patient samples for biomarkers that are predictive of a favorable outcome.

    Finally, when a specific mutation is being addressed, the prevalence of the mutation in a LiquidBiopsy sample can be serially monitored with ease. Thus the design, identification, and longitudinal analysis of patient cohorts can be accelerated with impacts on both efficacy of treatment as well as discovery and longitudinal validation processes.



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