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.
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.
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.
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.