Refers to article:
Molecular Characterization of Enzalutamide-treated Bone Metastatic Castration-resistant Prostate Cancer
Accepted 8 May 2014
February 2015 (Vol. 67, Issue 2, pages 53 - 60)
There is a significant need for predicative markers that help clinicians better select between CYP17 inhibitors such as abiraterone (ABI) and androgen receptor (AR) antagonists such as enzalutamide (ENZ) as first-line therapy in castration-resistant prostate cancer (CRPC). Since only a minority of patients who progress on ABI or ENZ respond to an alternative AR pathway inhibitor, predictive signatures would also help identify patients who may benefit from sequencing of AR pathway inhibitors in the second-line space. To help define markers associated with clinical course in patients with metastatic CRPC, in this issue ofEuropean Urology, Efstathiou and al  reported on their collection of blood and bone marrow samples to characterize the effects of ENZ on the AR-signaling pathway and correlated them with clinical observations in a group of 60 patients with CRPC treated with ENZ. The authors are to be congratulated for the tremendous effort required to obtain and analyze serial bone biopsies in patients with metastatic CRPC, which builds on their prior work investigating mechanisms of sensitivity and resistance to ABI and . The results are informative and represent important contributions to the field, helping guide the design of future studies to better define predictive markers of ENZ resistance.
It is important to note that the study identifies experimentally defined mechanisms of ENZ resistance in human metastatic CRPC, associating ARwtsignaling with clinical benefit and AR splice variants with primary resistance, as well as adaptive feedback between AR and androgen biosynthesis. As in transition from castration-sensitive disease to CRPC, many mechanisms and pathways converge to support AR reactivation in ENZ resistance, including ligand-dependent activation by way of intratumoral steroidogenesis  , AR amplification or mutations  , ligand-independent AR splice variants, , and , and broad support by a host of adaptive survival signaling pathways and . For example, reciprocal feedback regulation between AR and phosphatidylinositide 3-kinase (PI3K) pathways leads to activation of Akt signaling following AR inhibition by reducing levels of the Akt phosphatase PHLPP. Conversely, PI3K inhibition activates AR signaling by relieving feedback inhibition of HER kinases, which leads to phosphorylation of AR. Inhibition of one activates the other, thereby enhancing survival  .
AR pathway inhibition also induces stress responses involving chaperones such as clusterin  and Hsp27  , which mediate cross-talk activation of the Akt and MAPK pathways and acquired treatment resistance. Castration- or ENZ-resistant cell lines may increase steroid enzyme expression that could support ligand-driven activation of AR  . Mutations in the ligand-binding domain (LBD) of the AR converts ENZ from an antagonist to an agonist similar to those reported years ago for other nonsteroidal antiandrogens such as flutamide  .
Another genomic mechanism of AR pathway inhibitor resistance, in addition to AR amplification and LBD mutations, includes expression of AR splice variants that are constitutively activated, , and . Unlike copy number and mutational analyses, which use comparative genomic hybridization (CGH) and/or DNA sequencing, AR variant detection requires RNA-based assays (using reverse transcription polymerase chain reaction [RT-PCR] or RNA sequencing) or immunostaining assays (using a specific antibody). The most significant AR variants, ARV7 and ARv567es, lose their C-terminal AR LBD but retain their nuclear localization signal encoded partly within the DNA-binding domain, resulting in ligand-independent nuclear localization and constitutive AR activation. ARV7 appears clinically relevant and associated with shorter survival in CRPC metastases. New drugs targeting the DNA and N-terminal regions of the AR to suppress this facet of AR activity are under development.
Studies by Efstathiou and colleagues, , and lay a foundation for defining predictor signatures to help guide sequencing of ABI and ENZ, combining AR, ARV7, glucocorticoid receptor (GR), and CYP17 immunostaining with liquid chromatography–mass spectrometry measured steroid levels  . For example, CYP17 tumor expression of >10% combined with intense homogeneous nuclear AR expression was associated with prolonged benefit to ENZ, suggesting that these biomarkers could help predict patients more likely to respond to ENZ. Conversely, ARV7 expression predicted for a higher chance of primary resistance. ARV7 was absent in patients with long responses to ENZ, while GR was expressed in 38% of patients with primary resistance but in only 17% of patients with a prolonged ENZ benefit. While the numbers were low, no correlation was seen with AR copy number.
While this analysis illustrates that biologically relevant molecular markers can be used to predict response, it requires bone biopsies and semiquantitative immunostaining, and it is feasible at relatively few boutique academic institutions. Other limitations reflect the descriptive analyses in a relatively small nonrandomized cohort from a single institution. Given limitations imposed by bone biopsy tissue analyses, complete molecular studies on acquired tissue could be assessed in only 23 of the patients (38%), which introduces potential selection and lead-time biases that could influence duplication in subsequent validation cohorts. Patients who derive benefit from therapy are more likely not to have as much tumor remaining on repeat biopsy. Significant heterogeneity in AR expression was observed among samples and patients, which although not surprising, does confound analyses of small samples.
Given biopsy tissue limitations in this study, it was not feasible to sequence for the presence of AR LBD mutations known to confer resistance to ENZ. Bone-directed aspirates or biopsies are invasive and undersample tumor heterogeneity. Other approaches, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offer the promise of a “liquid biopsy” that can be performed serially in a convenient fashion and be more scalable for widespread clinical use. For example, Azad et al.  recently reported the use of plasma ctDNA sampling to correlate gene copy number changes (using array CGH) and AR mutation status detected in ctDNA with changes found in biopsies of metastatic CRPC. This method proved more acceptable for patients, as only 6 of 51 consented patients were amenable for biopsy, and only 13 patients had sufficient CTCs for gene copy number analysis. Sufficient ctDNA was available for array CGH in 94% of the patients, and 62% had detectable copy number changes, including 8p loss, 8q gain, and AR gain in 29%, 40% and 54% of the patients, respectively. Clinical correlations showed that AR amplification, which was common in patients progressing on ENZ (76%), increased after initiation of ENZ and correlated with primary ENZ resistance. AR exon 8 sequencing also identified F876L and other novel AR LBD mutations that correlated with clinical outcomes. Indeed, ENZ-naive patients were identified with F876L AR LBD mutations, which may be useful in predicting primary ENA resistance.
The potential clinical utility of detecting ARV7 expression in CTC was also recently reported by Antonarakis et al.  . Using the AdnaGen platform and RT-PCR, detection of ARV7 in CRPC patients was associated with a lower response rate and a shorter time to progression with ABI or ENZ compared with patients who were found by the assay to be ARV7 negative.
While cross-resistance between ABI and ENZ is common, a small but significant number of patients may benefit from sequential treatment. In this regard, Efstathiou and colleagues, using bone biopsies, provide important contributions that will guide future studies of predictive markers of response to AR pathway inhibitors and precision medicine. To contain costs associated with expensive drugs with low response rates, we need predicative signatures to enrich the likelihood of response and to more precisely tailor the use of second-line therapies. Liquid biopsy approaches, perhaps combining CTC (for ARV7 expression) with ctDNA (for AR copy number and LBD mutations), are more convenient and scalable, especially when performed serially, and promise a quantifiable approach to circumvent the sampling and heterogeneity limitations of targeted biopsies.
Conflicts of interest
The authors have nothing to disclose.
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The Vancouver Prostate Centre and Department of Urological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
© 2014 European Association of Urology, Published by Elsevier B.V.