The therapeutic landscape of castration-resistant prostate cancer (CRPC) has rapidly expanded. There is a need to develop noninvasive biomarkers to guide treatment. We established a highly sensitive method for analyzing androgen receptor gene (AR) copy numbers (CN) and mutations in plasma circulating cell-free DNA (cfDNA) and evaluated the AR statuses of patients with CRPC. AR amplification was detectable in VCaP cell line (AR amplified) genomic DNA (gDNA) diluted to 1.0% by digital PCR (dPCR). AR mutation were detectable in LNCaP cell line (AR T878A mutated) gDNA diluted to 0.1% and 1.0% by dPCR and target sequencing, respectively. Next, we analyzed AR status in cfDNA from 102 patients. AR amplification and mutations were detected in 47 and 25 patients, respectively. As a biomarker, AR aberrations in pretreatment cfDNA were associated with poor response to abiraterone, but not enzalutamide. In serial cfDNA analysis from 41 patients, most AR aberrations at baseline diminished with effective treatments, whereas in some patients with disease progression, AR amplification or mutations emerged. The analysis of AR in cfDNA is feasible and informative procedure for treating patients with CRPC. cfDNA may become a useful biomarker for precision medicine in CRPC.

Second-generation androgen receptor axis-targeted (ARAT) agents, namely abiraterone and enzalutamide, enable stronger blockade of the androgen receptor (AR) axis and longer survival of men with castration-resistant prostate cancer (CRPC). However, the extent of the improved survival remains insufficient and the majority of patients eventually develop resistance to these novel agents. Some patients develop resistance against ARAT treatment through mechanisms termed “complete AR independence” or “AR indifference”, and no longer require activation of the AR axis. However, a considerable proportion of CRPC patients remain persistently dependent on AR or its downstream signaling pathways. Ligand-independent activation of the AR, an AR axis-dependent mechanism, is mediated by truncated forms of ARs that lack the ligand-binding domain (LBD), arising as products of AR splicing variants or nonsense mutations of AR. Post-translational modifications of ARs can also contribute to ligand-independent transactivation of the AR. Other mechanisms for AR axis activation are mediated by pathways that bypass the AR. Recent studies revealed that the glucocorticoid receptor can upregulate a similar transcription program to that of the AR, thus bypassing the AR. ARAT agents are essentially ineffective for CRPC driven by these AR-independent mechanisms. This review article describes recent efforts to overcome these refractory machineries for the development of next-generation AR axis blockade in CRPC.

Purpose: Although cell-free DNA (cfDNA) testing is expected to drive cancer precision medicine, little is known about the significance of detecting low-frequency variants in circulating cell-free tumor DNA (ctDNA) in castration-resistant prostate cancer (CRPC). We aimed to identify genomic profile including low-frequency variants in ctDNA from patients with CRPC and investigate the clinical utility of detecting variants with variant allele frequency (VAF) below 1%. Experimental Design: This prospective, multicenter cohort study enrolled patients with CRPC eligible for treatment with abiraterone or enzalutamide. We performed targeted sequencing of pretreatment cfDNA and paired leukocyte DNA with molecular barcodes, and ctDNA variants with a VAF ≥0.1% were detected using an in-house pipeline. We investigated progression-free survival (PFS) and overall survival (OS) after different ctDNA fraction cutoffs were applied. Results: One hundred patients were analyzed (median follow-up 10.7 months). We detected deleterious ATM, BRCA2, and TP53 variants even in samples with ctDNA fraction below 2%. When the ctDNA fraction cutoff value of 0.4% was applied, significant differences in PFS and OS were found between patients with and without defects in ATM or BRCA2 [HR, 2.52; 95% confidence interval (CI), 1.24–5.11; P = 0.0091] and TP53 (HR, 3.74; 95% CI, 1.60–8.71; P = 0.0014). However, these differences were no longer observed when the ctDNA fraction cutoff value of 2% was applied, and approximately 50% of the samples were classified as ctDNA unquantifiable. Conclusions: Detecting low-frequency ctDNA variants with a VAF <1% is important to identify clinically informative genomic alterations in CRPC.

Although immune checkpoint inhibitors have shown benefit for advanced urothelial carcinoma (aUC) patients, prognostication of treatment efficacy and response duration remains a clinical challenge. We evaluated the expression of immune markers in the tumor microenvironment and assessed their associations with response to and survival after pembrolizumab treatment in 26 aUC patients. High levels of CD8⁺ tumor-infiltrating lymphocytes (TILs) were associated with favorable objective responses (23.0% vs. 15.3%, p = 0.0425), progression-free survival (median, 8.8 vs 2.1 months; hazard ratio (HR), 0.24; 95% confidence interval (CI), 0.07–0.66, p = 0.0060), and overall survival (median, >24.0 vs. 5.3 months; HR, 0.17; 95% CI, 0.04–0.56, p = 0.0034) compared with low levels. High interferon-gamma (IFNγ) expression levels were associated with longer post-progression survival (median, 4.9 vs. 1.0 months; HR, 0.18; 95% CI, 0.04–0.59, p = 0.0027) compared with low expression. Multivariate analysis incorporating clinical prognosticators demonstrated that the coincidence of low CD8⁺ T cells/IFNγ was an independent factor for unfavorable overall survival after pembrolizumab treatment (HR, 4.07; 95% CI, 1.36–12.73; p = 0.0125). The combination of low CD8⁺ TILs and IFNγ expression was an independent prognostic factor with predictive ability equivalent to previously reported clinical prognosticators.

The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and is thought to originate from renal tubular epithelial cells. Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles that are secreted into extracellular spaces by nearly all cell types, including cancer cells and non-cancerous cells. EVs are involved in multiple steps of RCC progression, such as local invasion, host immune modulation, drug resistance, and metastasis. Therefore, EVs secreted from RCC are attracting rapidly increasing attention from researchers. In this review, we highlight the mechanism by which RCC-derived EVs lead to disease progression as well as the potential and challenges related to the clinical implications of EV-based diagnostics and therapeutics.