AdnaTest ProstateCancerPanel AR-V7

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.

The AdnaTest uses a two-step process (Select and Panel) to generate results within 6 hours (see “AdnaTest ProstateCancerSelect: Immunomagnetic cell selection with multiple tumor associated antibodies” and “AdnaTest ProstateCancerPanel AR-V7: qRT-PCR of AR-V7 and various prostate cancer associated tumor markers.”).

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 (“AdnaTest ProstateCancerSelect: Immunomagnetic cell selection with multiple tumor associated antibodies.”). 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. See "AdnaTest ProstateCancerPanel AR-V7: qRT-PCR of AR-V7 and various prostate cancer associated tumor markers."

Recovery

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 (See “AR-V7 Panel gene dilutions”). The positive control has been used as template for standard sample generation.
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The resulting Ct values for this dilution series were clearly linear, correlated to the copy numbers for each gene at each dilution step (See “AR-V7 Panel gene dilutions”) 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 (see the table below).

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. See "Gene profile in clinical samples."