For the enrichment and molecular characterization of circulating tumor cells (CTCs) from whole human blood.
AdnaTest LungCancer allows molecular characterization of CTCs by combining AdnaTest CTC-Select with AdnaTest LungCancerDetect using the Combination of Combinations Principle (COCP). AdnaTest CTC-Select is a highly specific immunomagnetic cell-selection system for enriching circulating tumor cells from peripheral blood. AdnaTest LungCancerDetect allows sensitive analysis of epithelial-mesenchymal transition (EMT) and lung cancer-associated gene expression in immunomagnetically enriched tumor cells by reverse transcription and PCR.
AdnaTest LungCancer is a highly specific immunomagnetic cell selection system for enriching circulating tumor cells from peripheral blood which includes a highly sensitive RT-PCR for detecting lung cancer-associated mRNA markers. The specificity of the detection of AdnaTest LungCancer is 95%. Using tumor markers for lung cancer in spiking experiments, 30 tumor cells in 5 ml blood are detected at a recovery rate of 100% (Table below: AdnaTest LungCancer specificity and recovery).
In approximately 80% of all CTCs from lung cancer samples, EMT and tumor stem cell metabolism is observable. Analyzed CTCs were positive for at least one of the EMT markers: Twist1, Akt2 and PI3KCA. The AdnaTest LungCancer provides – in addition to anti-EpCam - antibodies against Her2 and EGFR, resulting in a strong increase of performance (see "Increased sensitivity in lung cancer"). To cover the aforementioned tumor markers, the AdnaTest LungCancer contains primer mixes for both the detection of EMT and lung cancer associated markers.
Findings with detection systems based solely on EpCam as capturing target reached only 15% positivity, whereas the AdnaTest CTC-Select uses anti-EpCam, anti-Her2 and anti-EGFR antibodies in combination. EpCam expression analysis enables differentiation of epithelial-like cells, EMT-like cells and cells with tumor stem cell characteristics.
Leukocyte reduction is critical to minimize leukocyte bias.
The detection of tumor stemness-related markers like ALDH1, or EMT-related markers like Twist1, Akt2 or PI3KCα requires a significantly higher purity to eliminate a bias from contaminating leukocytes. The AdnaWash Leukocyte Reducer is a special buffer developed to reduce the leukocyte content during CTC enrichment. Using a CD45 endpoint PCR assay clearly demonstrates that the number of leukocytes is dramatically reduced using AdnaWash in the cell capturing procedure instead of PBS (see “Leukocyte reduction using AdnaWash”).
The AdnaTest’s successful CTC detection is based on the Combination of Combinations Principle (COCP). Each AdnaTest has a unique combination of tumor associated markers and an optimized combination of antibodies for cell selection. The combination of highly specific immunomagnetic cell selection system (using the optimized antibody combination, see “A CTC captured by three antibodies coupled to magnetic beads” ) and highly sensitive RT-PCR technology (using a combination of mRNA tumor markers) provides the highest degrees of analytical specificity and sensitivity (see "Increased sensitivity in lung cancer").
The AdnaTest uses a two-step process (Select and Detect) to generate results within 5 hours (see “AdnaTest CTC-Select: Immunomagnetic cell selection with multiple tumor associated antibodies.” and “AdnaTest LungCancerDetect for analysis of various cancer-associated tumor markers.”). AdnaTest CTC-Select enables the immunomagnetic enrichment of tumor cells from whole blood via epithelial- and tumor-associated antigens. Antibodies against epithelial- and tumor-associated antigens are conjugated to magnetic beads for labeling of tumor cells. Labeled cells are extracted by a magnetic particle concentrator (AdnaMag-L and AdnaMag-S), and subsequently lysed. (see “AdnaTest Select: Immunomagnetic cell selection with multiple tumor-associated antibodies labeled to magnetic beads”). The cell lysate is then used in AdnaTest LungCancerDetect, step 2 of the procedure, to isolate mRNA.
AdnaTest LungCancerDetect contains Oligo (dT) 25-coated beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA that can subsequently be used as template for tumor cell detection and characterization by multiplex PCR (See "AdnaTest LungCancerDetect for analysis of various cancer-associated tumor markers.”).
Multiplex PCR provides tumor-associated gene expression profiles for a variety of relevant tumor markers to ensure that the selected cells are cancer cells (see “AdnaTest LungCancerDetect results of samples using an Agilent 2100 Bioanalyzer”). The kit contains one primer mix to amplify cDNA target fragments of Twist, PI3Kα, Akt2 and a housekeeping gene as control (actin) (see "AdnaTest LungCancer specificity and recovery."). The PrimerMix LungDetect amplifies ALDH1 and EpCam (GA733-2) (below). The primers generate fragments of the following sizes:
Importantly, the AdnaTest is an open platform that allows the profiling of additional custom selected mRNA targets for comprehensive biomarker discovery.
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