Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR

Abstract

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

Keywords: amplification; androgen receptor; cell free DNA; liquid biopsy; mutation; prostate cancer.

Figure 1

Two-dimensional plot of multiplexed ddPCR AR-Amp-1/T877A assay to detect both the point mutation T877A as well as AR CN. A combination of LNCaP and MFM-233 gDNA was used to represent the various resulting droplet populations. The droplet populations indicated by various colors demonstrate good separation of different variants. Note that the appearance of combination populations is dependent on the amount of DNA input and chance of co-localization of templates within the same droplets.

Figure 2

Two-dimensional plot of multiplexed AR-Amp-2 ddPCR assay. The individual droplet clusters demonstrated good separation of different amplicons as shown in the representative 2D plot. Combination populations are also present: 1: AR-X2 + MYM; 2: AR-X1 + MYM; 3: TBP + AR-X2; 4: AR-X1 + X2 + MYM; 5: AR-X1 + TBP; 6: AR-X2 + TBP + MYM; 7: AR-X1 + X2 + TBP; 8: AR-X1 + TBP + MYM; 9: AR-X1 + X2 + TBP + MYM. Note that the appearance of combination populations is dependent on DNA input and chance of co-localization of templates within the same droplets.

Figure 3

AR CN of 6–8 healthy controls and 47 PC patients. CN was calculated as described in the Materials and Methods section. Circles: AR-Amp-1/T877A; squares: AR-Amp-2; open circles/squares: healthy blood donors; solid circles/squares: patients; error bars in red: mean ± SEM.

Figure 4

Side-by-side scatter dot plot demonstrating cross-validation of AR CN between AR-Amp-1/T877A (solid circles) and AR-Amp-2 (solid squares) separated into HSPC vs. CRPC patient groups. Patient sample data are sorted by CN value measured by the AR-Amp-1/T877A method. Arrows indicate patient samples with identified T877A mutation, double arrows indicate patients with large scale Chromosome X CN increase.

Figure 5

Chr X CN of 6 healthy controls and 47 PC patients as determined with AR-Amp-2 method. The threshold was set at 2. Chr X: X chromosome.