A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).

Longitudinal Molecular Profiling of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma.

PURPOSE: Liquid biopsies in metastatic renal cell carcinoma (mRCC) provide a unique approach to understand the molecular basis of treatment response and resistance. This is particularly important in the context of immunotherapies, which target key immune-tumor interactions. Unlike metastatic tissue biopsies, serial real-time profiling of mRCC is feasible with our noninvasive circulating tumor cell (CTC) approach. METHODS: We collected 457 longitudinal liquid biopsies from 104 patients with mRCC enrolled in one of two studies, either a prospective cohort or a phase II multicenter adaptive immunotherapy trial. Using a novel CTC capture and automated microscopy platform, we profiled CTC enumeration and expression of HLA I and programmed cell death-ligand 1 (PD-L1). Given their diametric immunological roles, we focused on the HLA I to PD-L1 ratio (HP ratio). RESULTS: Patients with radiographic responses showed significantly lower CTC abundances throughout treatment. Furthermore, patients whose CTC enumeration trajectory was in the highest quartile (> 0.12 CTCs/mL annually) had shorter overall survival (median 17.0 months v 21.1 months, P < .001). Throughout treatment, the HP ratio decreased in patients receiving immunotherapy but not in patients receiving tyrosine kinase inhibitors. Patients with an HP ratio trajectory in the highest quartile (≥ 0.0012 annually) displayed significantly shorter overall survival (median 18.4 months v 21.2 months, P = .003). CONCLUSION: In the first large longitudinal CTC study in mRCC to date to our knowledge, we identified the prognostic importance of CTC enumeration and the change over time of both CTC enumeration and the HP ratio. These insights into changes in both tumor burden and the molecular profile of tumor cells in response to different treatments provide potential biomarkers to predict and monitor response to immunotherapy in mRCC.

BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity.

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.

Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer.

PURPOSE: Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy. MATERIALS AND METHODS: A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board-approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings. RESULTS: Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]). CONCLUSION: We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.

Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma.

Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC-specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD-L1 and HLA-I expression and correlated with patient response to therapy. CTC enumeration and expression of PD-L1 and HLA-I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker.

Centrosome amplification is a frequent event in circulating tumor cells from subjects with metastatic breast cancer.

Centrosome amplification (CA) is a common phenomenon in cancer, promotes genomic stability and cancer evolution, and has been reported to promote metastasis. CA promotes a stochastic gain/loss of chromosomes during cell division, known as chromosomal instability (CIN). However, it is unclear whether CA is present in circulating tumor cells (CTCs), the seeds for metastasis. Here, we surveyed CA in CTCs from human subjects with metastatic breast cancer. CTCs were captured by CD45 exclusion and selection of EpCAM-positive cells using an exclusion-based sample preparation technology platform known as VERSA (versatile exclusion-based rare sample analysis). Centriole amplification (centrin foci> 4) is the definitive assay for CA. However, determination of centrin foci is technically challenging and incompatible with automated analysis. To test if the more technically accessible centrosome marker pericentrin could serve as a surrogate for centriole amplification in CTCs, cells were stained with pericentrin and centrin antibodies to evaluate CA. This assay was first validated using breast cancer cell lines and a nontransformed epithelial cell line model of inducible CA, then translated to CTCs. Pericentrin area and pericentrin area x intensity correlate well with centrin foci, validating pericentrin as a surrogate marker of CA. CA is found in CTCs from 75% of subjects, with variability in the percentage and extent of CA in individual circulating cells in a given subject, similar to the variability previously seen in primary tumors and cell lines. In summary, we created, validated, and implemented a novel method to assess CA in CTCs from subjects with metastatic breast cancer. Such an assay will be useful for longitudinal monitoring of CA in cancer patients and in prospective clinical trials for assessing the impact of CA on response to therapy.