A Novel Highly Specific and Sensitive Digital Droplet PCR Assay for the Detection of Androgen Receptor and Its Variants

The aberrant functioning of androgen receptor (AR) signaling is a central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant prostate cancer (mCRPC). Androgen deprivation therapy (ADT) is usually employed, along with the use of AR signaling inhibitors such as abiraterone and enzalutamide. Patients often develop resistance to these therapies, leading to the next line of treatment, taxane chemotherapy. However, many patients also develop resistance to taxanes, ultimately resulting in death.

Prostate cancer drug resistance is due to the expression of AR splice variants (AR-Vs) that lack the ligand-binding domain and are constitutively active in the nucleus. Expression of AR-V7, in circulating tumor cells (CTCs) isolated from the blood of patients has been associated with resistance to AR inhibitors. Hence, assessment of the presence and/or amounts of different AR-Vs has clinical utility. Clinical trial results indicate that response to taxane treatment is significantly associated with lower AR expression in CTCs, and that this was predictive of the biochemical response to taxane treatment for the individual patient.

The inventors have devised sensitive assay methods for capturing CTCs, extracting mRNAs and detecting and quantifying full-length AR (AR-FL), AR-V7 and AR-V567 variants via parallel digital droplet PCR assays.

• This assay is sensitive when using mRNA from as little as half of a cell (0.5 cell)
• This assay is also highly specific for each AR-V transcript and avoids co-detection of other similar variants, such as AR-V9 that is structurally similar to AR-V7
• This assay is superior to currently available methods since:
• 1) It can adequately and specifically detect AR-V567, and a variety of AR-Vs, and AR-FL
• 2) Does not require pre-amplification of transcripts
• 3) Does not require reference genes or standard curves
• 4) Separate assay mixtures can be used instead of standard multiplex assay procedures, thus improving assay sensitivity.

Potential Applications

These methods can detect AR and AR variants from prostate cancer patients who may be in need of treatment.