For the enrichment and molecular characterization of circulating tumor cells (CTCs) from whole human blood; For 12 enrichments of tumor cells from whole blood and subsequent detection of prostate cancer-associated gene expression including AR-V7 expression.

Features

Efficient isolation and detection of CTCs from whole blood

Molecular characterization of CTCs

High specificity and sensitivity

Product Details

AdnaTest ProstateCancerPanel AR-V7 allows molecular characterization of CTCs using the Combination of Combinations Principle (COCP). AdnaTest ProstateCancerPanel AR-V7 is a highly specific immunomagnetic cell-selection system for enriching circulating tumor cells from peripheral blood. This allows sensitive analysis of prostate cancer-associated gene expression including AR-V7 detection in immunomagnetically enriched tumor cells by reverse transcription and PCR.

Performance

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 and highly sensitive RT-PCR technology provides the highest degrees of analytical specificity and sensitivity.

Principle

The AdnaTest uses a two-step process (Select and Panel) to generate results within 6 hours .

AdnaTest ProstateCancerSelect enables the immunomagnetic enrichment of tumor cells via epithelial and tumor associated antigens. Antibodies against these antigens are conjugated to magnetic beads for labeling of tumor cells in whole blood. Labeled cells are extracted by a magnetic particle concentrator (AdnaMag-L and AdnaMag-S) and are subsequently lysed. The cell lysate is used for further analysis with AdnaTest ProstateCancerPanel AR-V7.

AdnaTest ProstateCancerPanel AR-V7 contains Oligo (dT)25 Beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA, which is subsequently used as template for preamplification and tumor cell detection and characterization by qRT-PCR. The AdnaPanel PrimerMixes allow amplification of four tumor associated antigens and two control genes. Furthermore, the kit contains PrimerMixes for amplification of an internal control and an inhibition control. 

Procedure

The AdnaTest ProstateCancerSelect is used for the enrichment of CTCs from whole blood in prostate cancer research. Subsequently, the AdnaTest ProstateCancerPanel AR-V7 is used for molecular characterization by analyzing gene expression of AR-V7 and further prostate cancer associated genes. In spiking experiments, 2 tumor cells in 5 ml of whole blood are detected at a recovery rate of 95%.

General cell recovery using AdnaTest ProstateCancerPanel AR-V7

                                                        Recovery [%] n

 2 tumor cells spiked into 5 ml blood 38/40 (95%) 40

Two cancer cells (Prostate cancer cell line LnCap) were spiked into blood samples (5 ml) from healthy donors (n=40) to determine the recovery rates achieved with the AdnaTest ProstateCancerPanel AR-V7 as summarized in above.

Specificity

Fourteen male healthy donors were analyzed with the AdnaTest ProstateCancerPanel AR-V7 to determine the rate of false positives/specificity at the given cut offs.

Specificity determination

                                  PSA     PSMA   ARwt AR-V7

 Cut-off                        35.00    35.00   35.00 35.00

 Positives after cut-off   0       0       1   0

 Specificity                 100%    100%      93% 100%

The AdnaTest ProstateCancerPanel AR-V7 could demonstrate in this performance evaluation a specificity of 100% for all tumor associated genes (PSA, PSMA, AR-V7) except ARwt where it reached 93% (details in table above). Also, GAPDH reached 100% specificity and CD45 79%, respectively, whereas these 2 genes are not used for CTC characterization as such, hence a specificity below 90% for those two target genes is acceptable.

qRT-PCR linearity and efficiency

Perfect linearity and efficiency of the qRT-PCR reactions for each of the genes contained in the AdnaTest ProstateCancerPanel AR-V7 was proven in a serial dilution series of standards. The positive control has been used as template for standard sample generation.

The resulting Ct values for this dilution series were clearly linear, correlated to the copy numbers for each gene at each dilution step getting down to ~500 copies. The qRT-PCR efficiency of all the tumor cell related genes was higher than 90% and the corresponding amplification factors ranged above 1.9 which all in all can be taken as a prove for very effective and sensitive qRT-PCR design .

qRT-PCR efficiency

                                                          PSA     PSMA  AR          AR-V7

Slope over the range                        -3.55    -3.58 -3.56 -3.48

Efficiency: -1+10(-1/slope)                 91%     90% 91%          94%

Amplification factor: 10^(1/-slope) 1.91     1.90 1.91  1.94

Field test results

The AdnaTest ProstateCancerPanel AR-V7 workflow was tested in 12 castration-resistant prostate cancer (CRPC) samples. 

Applications

Isolation of CTCs from whole human blood

Molecular characterization of CTCs

Biomarker discovery

mRNA profiling

Liquid biopsy

Cancer research