Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells
AR-V7 expression is predictive of resistance to anti-androgen therapy, but detecting it alongside full-length AR in the same circulating tumor cell has meant running separate assays; this AACR 2018 poster reports a prototype 6-parameter immunofluorescence assay that stains both receptors at once and agrees with the independent assays it was checked against.
Presented by RareCyte and the University of Washington.
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- One assay reads AR and AR-V7 in the same circulating tumor cell, which the authors report is the first they are aware of to do so. It adds the two receptor markers to the 4 core identification parameters of the RareCyte CTC-4 panel — nuclear dye, CD45, pan-cytokeratin and EpCAM — for a 6-parameter read, and an antibody denaturing step lets two rabbit monoclonals be amplified in sequence on one slide.
- The simultaneous assay agreed with the independent assays run beside it. Five assays were applied to equivalent volumes of one advanced prostate cancer patient sample and identified a mean of 36 CTCs each; only three of the five stained AR and only three stained AR-V7, and the combined AR / AR-V7 assay is one of each three. The two independent AR assays read 86% and 61% AR-positive CTCs, against 66% for the combined assay; the two independent AR-V7 assays read 26% and 39% AR-V7-positive, against 26%.
- Every AR-V7-positive CTC was also AR-positive, and the cell-line controls expressed what they were expected to express. In the combined assay 10 of 38 CTCs were AR-V7-positive (26%) and all of them were AR-positive, while 15 were AR-positive only (39%); spike-in models matched their reported phenotypes, with 22RV1 expressing both receptors, LNCaP expressing AR but not AR-V7, and PC3 expressing neither.
