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AdnaTest ProstateCancerPanel AR-V7

For the enrichment and molecular characterization of circulating tumor cells (CTCs) from whole human blood
  • Efficient isolation and detection of CTCs from whole blood
  • Molecular characterization of CTCs
  • High specificity and sensitivity
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.

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Cat No./ID: 396132
AdnaTest ProstateCancerPanel AR-V7
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For 12 enrichments of tumor cells from whole blood and subsequent detection of prostate cancer-associated gene expression including AR-V7 expression.
Cat No./ID: 399911
AdnaMag-S
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For 8 tubes, 1.5 ml
Cat No./ID: 399921
AdnaMag-L
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For 8 tubes, 15 ml
Cat No./ID: 399932
AdnaTube
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12 sample tubes containing EDTA. Use only with anticoagulated blood collected in ACDA blood collection tubes from BD (Becton Dickinson, 8.5 ml)
The AdnaTest ProstateCancerPanel AR-V7 is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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Figure 2: AdnaTest ProstateCancerSelect: Immunomagnetic cell selection with multiple tumor associated antibodies.
In the first step, the CTCs in the blood are enriched (AdnaTest Select). This is achieved using antibody-coated magnetic particles (beads). Several antibodies are used, which bind with high specificity and affinity to the corresponding cancer cells. The enriched cells are lysed and subsequently purified several times to extract mRNA.
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Figure 5: Gene profile in clinical samples.
Overall positivity rate was 75% (at least 1 one gene must be detected). PSMA expression was determined in 75% of the cases (9/12). All other genes seem to be co-expressed with PSMA but were found in lower frequencies. AR was present in 50% (6/12), AR-V7 in 33% (4/12) and PSA in 25% (3/12). With AR-V7 being identified through the presence of PSA and PSMA, false positives are reduced. Therefore, the findings indicate that AR-V7 detection is feasible and accurate as the CTCs identified through PSA and PSMA indicate AR-V7 in the sample.
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Figure 4: AR-V7 panel gene dilutions.
Standard dilution series starting with ~0.00014 ng/µl cDNA of each tumor related gene in the panel leads to a linear CT correlation versus cDNA copy numbers during qRT-PCR. A 0.14 ng/µl sample of the standard was diluted 1:10,000. This sample was further diluted ten-fold and 1:100 and resulting copy numbers where calculated. The linear correlation of CT values and the corresponding copy numbers was used to further calculate qRT-PCR efficiency as well as the amplification factor.
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Figure 3: AdnaTest ProstateCancerPanel AR-V7: qRT-PCR of AR-V7 and various prostate cancer associated tumor markers.
In a second step, the enriched cells are examined by qRT-PCR for AR-V7 expression and other tumor associated expression patterns. First mRNA strands are reverse transcribed into cDNA and subsequently, several tumor associated markers are analyzed using qRT-PCR.
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Figure 1: A CTC captured by three antibodies coupled to magnetic beads.
 
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 (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.
Principle
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."
Procedure
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."

 
Applications
  • Isolation of CTCs from whole human blood
  • Molecular characterization of CTCs
  • Biomarker discovery
  • mRNA profiling
  • Liquid biopsy
  • Cancer research

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