Resistance to AR Signaling Inhibitors

There are multiple mechanisms driving resistance to FDA approved Androgen Receptor Signaling inhibitors (ARSi), such as Zytiga® (Abiraterone Acetate) and Xtandi® (Enzalutamide). Numerous novel therapeutics are being developed for metastatic castration resistance prostate cancer (mCRPC) to target these specific mechanisms of resistance which may be exploited for the development of therapy selection devices or utilized to demonstrate therapeutic pharmacodynamics and efficacy during the drug development process.

At Epic Sciences, we work with leading academic and government investigators to develop strategies for identifying resistance mechanisms and creating non-invasive tests that aid in the selection and monitoring of patients during clinical trials. These include tests to measure the following:

Constitutively Active AR Signaling

Valuable in the clinical development of 3rd generation AR inhibitor/degraders

AR C Terminal Loss: Measure of AR N terminal and AR C terminal on single CTCs from mCRPC patients

Additional AR splice variants beyond AR-V7 may also contribute to resistance to AR inhibitors. To identify other mechanisms of ligand binding domain alterations (such as AR-V567es), we developed a single CTC analysis for simultaneous protein expression of the AR N and AR C terminus to pinpoint patients who harbor a C terminus loss (AR ligand binding domain alteration) and would therefore demonstrate resistance to AR inhibition.

Figure 1. Expression of AR N and AR C-Terminus via the 5 channel biomarker assay on the Epic Sciences platform.

AR/PI3K Reciprocal Feedback

Valuable in the development of PI3K/AKT inhibitors

AR/PTEN CTC Analysis

AR resistance can occur through reciprocal feedback between PI3K and AR signaling. To measure cooperative AR/PI3K signaling we have developed single CTC phenotype and genotypic tools to assess AR protein expression, AR amplification and PTEN deletions within single CTCs.

Figure 2. Copy Number Variation plot of a single CTC from an mCRPC patient expressing homozygous PTEN loss in conjunction with AR gain. 

Figure 3. NextGen sequencing results of mCRPC patients indicate variety of single CTCs that exhibit coinciding AR amplification and PTEN deletion.

Tumor Heterogeneity

Valuable in the clinical development of targeted therapy and combination therapy

CTC Phenotypic Heterogeneity

A subset of mCRPC patients harbor high tumor heterogeneity at any line of therapy, in which multiple drivers of disease are present. This could potentially thwart a narrow, targeted therapeutic approach.

To assess this phenomenon, high resolution digital pathology was used to identify patients with a high degree of phenotypic heterogeneity in their circulating tumor cells. In a 221 sample cohort presented at ASCO GU 2016, patients with high phenotypic CTC phenotypic heterogeneity demonstrated worse overall survival rates on AR signaling inhibitors than patients with low phenotypic heterogeneity (HR = 5.5, p < 0.00001). Multivariate analyses revealed a 68% reduction of risk of death on taxane therapy for patients with high phenotypic heterogeneity.

Figure 4. KM curve demonstrates how patients with high heterogeneity show poorer overall survival on AR signaling inhibitors and achieve better outcomes when treated with taxane chemotherapy.

AR Independence

Valuable in design of clinical trials and measures of resistance 

Neuroendocrine Prostate Cancer (NEPC) CTC Classifier

CTCs from patients with NEPC determined through metastatic biopsy that confirmes Small Cell Prostate Cancer Pathology or NE expressing tumor cells have a unique morphologic phenotype. While analysis of this cell type is specific to NEPC patients, ~10% of mCRPC patients have sub-clonal populations of NEPC CTCs. These patients demonstrate a worse response to AR inhibitors and are more likely to harbor visceral metastases.

Figure 5. Cell image of a “Small CTC” exhibiting small morphology and positive for Cytokeratin expression. Prevalence of these cells are indicative of neuroendocrine disease.

Cell Type-K Classifier

The presence of a single specific CTC phenotype (Cell Type K, shown left) was found to be predictive of resistance to both AR Tx and taxane chemotherapy. The KM curves below show the overall survival rate of patients with a prevalence of this CTC phenotype after they are given either standard of care drugs, AR Tx (HR=6.4) or taxane chemotherapy (HR=5.2).

Figure 6. Cell image of CTC subtype Cell Type K and KM curves of Cell Type K positive patients given either AR Tx or Taxane chemotherapy.