The use of cell free DNA (cfDNA) for mutational screening of multiple myeloma.

Multiple myeloma (MM) is an incurable haematological malignancy which relies heavily on bone marrow biopsies for disease monitoring and prediction of treatment response. In recent years, liquid biopsy derived cell-free DNA (cfDNA) has emerged as alternative for invasive biopsies. This pilot study aimed to evaluate the feasibility of using cfDNA for the detection of oncogenic mutations in the mitogen-activated protein kinase (MAPK) pathway genes NRAS, KRAS, and BRAF in MM patients. Matched peripheral blood and bone marrow aspirates were collected from thirteen MM patients at various disease stages. cfDNA was isolated using the Qiagen Circulating Nucleic Acid Kit while bone marrow DNA was extracted using the Maxwell Promega platform. The presence of NRAS, KRAS, and BRAF mutations was analysed by ddPCR and compared between the cfDNA and gDNA samples. Although our data come from a small patient cohort, mutations were detected, which supports cfDNA utility for mutational screening and prognostication in MM.

From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

Circulating Tumour Cell Associated MicroRNA Profiles Change during Chemoradiation and Are Predictive of Response in Locally Advanced Rectal Cancer.

Locally advanced rectal cancer (LARC) has traditionally been treated with trimodality therapy consisting of neoadjuvant radiation +/- chemotherapy, surgery, and adjuvant chemotherapy. There is currently a clinical need for biomarkers to predict treatment response and outcomes, especially during neoadjuvant therapy. Liquid biopsies in the form of circulating tumour cells (CTCs) and circulating nucleic acids in particular microRNAs (miRNA) are novel, the latter also being highly stable and clinically relevant regulators of disease. We studied a prospective cohort of 52 patients with LARC, and obtained samples at baseline, during treatment, and post-treatment. We enumerated CTCs during chemoradiation at these three time-points, using the IsofluxTM (Fluxion Biosciences Inc., Alameda, CA, USA) CTC Isolation and detection platform. We then subjected the isolated CTCs to miRNA expression analyses, using a panel of 106 miRNA candidates. We identified CTCs in 73% of patients at baseline; numbers fell and miRNA expression profiles also changed during treatment. Between baseline and during treatment (week 3) time-points, three microRNAs (hsa-miR-95, hsa-miR-10a, and hsa-miR-16-1*) were highly differentially expressed. Importantly, hsa-miR-19b-3p and hsa-miR-483-5p were found to correlate with good response to treatment. The latter (hsa-miR-483-5p) was also found to be differentially expressed between good responders and poor responders. These miRNAs represent potential predictive biomarkers, and thus a potential miRNA-based treatment strategy. In this study, we demonstrate that CTCs are present and can be isolated in the non-metastatic early-stage cancer setting, and their associated miRNA profiles can potentially be utilized to predict treatment response.

Melatonin Increases Proliferation and Decreases Apoptosis of GC-1 spg Cells by Upregulating the Expression of circTec.

Melatonin has been shown to be beneficial for the motility of human sperm, although its mechanism remains to be uncovered. Circular RNAs (circRNAs) have been shown to regulate cellular function in many diseases. However, there has been no relevant research on the effect of melatonin on sperm circRNAs. In this study, we aimed to explore the changes in circRNAs after melatonin treatment of GC-1 spg cells and identify key functional circRNAs. The results showed that melatonin enhanced the proliferation and reduced the apoptosis of GC-1 spg cells. A total of 1423 circRNAs were found to be significantly differentially expressed between groups with and without melatonin treatment. Of these circRNAs, 702 were upregulated and 721 were downregulated. circTec was one of the upregulated circRNAs. Suppressing the expression of circTec significantly reduced cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway activation but promoted melatonin-treated GC-1 spg cell apoptosis. In conclusion, melatonin increased the expression of circTec to exert its physiological effects on GC-1 spg cells, possibly by activating the mTOR signaling pathway. These results enhance our understanding of the biological function of circTec and its regulation by melatonin in spermatogenesis and infertility.

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells.

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers.

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called "liquid biopsies". Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.

Molecular Biomarkers in Glioblastoma: A Systematic Review and Meta-Analysis.

Background: Glioblastoma (GBM) is a highly aggressive cancer with poor prognosis that needs better treatment modalities. Moreover, there is a lack of reliable biomarkers to predict the response and outcome of current or newly designed therapies. While several molecular markers have been proposed as potential biomarkers for GBM, their uptake into clinical settings is slow and impeded by marker heterogeneity. Detailed assessment of prognostic and predictive value for biomarkers in well-defined clinical trial settings, if available, is scattered throughout the literature. Here we conducted a systematic review and meta-analysis to evaluate the prognostic and predictive significance of clinically relevant molecular biomarkers in GBM patients. Material and methods: A comprehensive literature search was conducted to retrieve publications from 3 databases (Pubmed, Cochrane and Embase) from January 2010 to December 2021, using specific terms. The combined hazard ratios (HR) and confidence intervals (95% CI) were used to evaluate the association of biomarkers with overall survival (OS) in GBM patients. Results: Twenty-six out of 1831 screened articles were included in this review. Nineteen articles were included in the meta-analyses, and 7 articles were quantitatively summarised. Fourteen studies with 1231 GBM patients showed a significant association of MGMT methylation with better OS with the pooled HR of 1.66 (95% CI 1.32−2.09, p < 0.0001, random effect). Five studies including 541 GBM patients analysed for the prognostic significance of IDH1 mutation showed significantly better OS in patients with IDH1 mutation with a pooled HR of 2.37 (95% CI 1.81−3.12; p < 0.00001]. Meta-analysis performed on 5 studies including 575 GBM patients presenting with either amplification or high expression of EGFR gene did not reveal any prognostic significance with a pooled HR of 1.31 (95% CI 0.96−1.79; p = 0.08). Conclusions: MGMT promoter methylation and IDH1 mutation are significantly associated with better OS in GBM patients. No significant associations were found between EGFR amplification or overexpression with OS.

Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m6A deposition to activate the PI3K/AKT signaling.

Melatonin is a key neuroendocrine hormone that promotes spermatogenesis and sperm motility, but the underlying mechanisms remains poorly understood. In this study, we aimed to investigate the possible roles of m6A (N6--methyl-adenosine) in mediating melatonin-regulated spermatogonia activity alterations. In this study, mouse-derived GC-1 spermatogonia (spg) cell line was used as the in vitro cellular model. The viability, proliferation rates and apoptosis of spermatogonia were detected via CCK-8, Edu staining and flow cytometry respectively. Total m6A level was quantitated by dot blot, while mRNA and proteins contents in spermatogonia were measured by qRT-PCR and western blot respectively. Differentially expressed mRNAs were characterized by deep RNA sequencing method. Results showed that melatonin significantly promoted viability and proliferation rate while inhibited apoptosis in the GC-1 spg cells. The total m6A levels in GC-1 spg cells were also greatly increased by melatonin treatment, accompanied by remarkable expressional elevation of the m6A writer KIAA1429. Moreover, the regulation of GC-1 spg cell viability, proliferation and apoptosis by melatonin were greatly abrogated by KIAA1429 silencing but effectively strengthened by KIAA1429 overexpression. In addition, KIAA1429 overexpression regulates multiple biological process and signaling pathways in spermatogonia such as the PI3K/AKT signaling. The PI3K inhibitor LY294002 effectively mitigated the regulation of spermatogonia activity by KIAA1429 overexpression under melatonin treatment. Taken together, melatonin promotes spermatogonia activity via enhancing KIAA1429 expression and m6A RNA methylation to activate the downstream PI3K/AKT signaling pathway.

Prognostic and Predictive Value of Liquid Biopsy-Derived Androgen Receptor Variant 7 (AR-V7) in Prostate Cancer: A Systematic Review and Meta-Analysis.

In advanced prostate cancer, access to recent diagnostic tissue samples is restricted and this affects the analysis of the association of evolving biomarkers such as AR-V7 with metastatic castrate resistance. Liquid biopsies are emerging as alternative analytes. To clarify clinical value of AR-V7 detection from liquid biopsies, here we performed a meta-analysis on the prognostic and predictive value of androgen receptor variant 7 (AR-V7) detected from liquid biopsy for patients with prostate cancer (PC), three databases, the Embase, Medline, and Scopus were searched up to September 2021. A total of 37 studies were included. The effects of liquid biopsy AR-V7 status on overall survival (OS), radiographic progression-free survival (PFS), and prostate-specific antigen (PSA)-PFS were calculated with RevMan 5.3 software. AR-V7 positivity detected in liquid biopsy significantly associates with worse OS, PFS, and PSA-PFS (P <0.00001). A subgroup analysis of patients treated with androgen receptor signaling inhibitors (ARSi such as abiraterone and enzalutamide) showed a significant association of AR-V7 positivity with poorer OS, PFS, and PSA-PFS. A statistically significant association with OS was also found in taxane-treated patients (P = 0.04), but not for PFS (P = 0.21) or PSA-PFS (P = 0.93). For AR-V7 positive patients, taxane treatment has better OS outcomes than ARSi (P = 0.01). Study quality, publication bias and sensitivity analysis were integrated in the assessment. Our data show that liquid biopsy AR-V7 is a clinically useful biomarker that is associated with poor outcomes of ARSi-treated castrate resistant PC (CRPC) patients and thus has the potential to guide patient management and also to stratify patients for clinical trials. More studies on chemotherapy-treated patients are warranted. Systematic Review Registration: PROSPERO, CRD42021239353.

Harnessing Liquid Biopsies to Guide Immune Checkpoint Inhibitor Therapy.

Immunotherapy (IO), involving the use of immune checkpoint inhibition, achieves improved response-rates and significant disease-free survival for some cancer patients. Despite these beneficial effects, there is poor predictability of response and substantial rates of innate or acquired resistance, resulting in heterogeneous responses among patients. In addition, patients can develop life-threatening adverse events, and while these generally occur in patients that also show a tumor response, these outcomes are not always congruent. Therefore, predicting a response to IO is of paramount importance. Traditionally, tumor tissue analysis has been used for this purpose. However, minimally invasive liquid biopsies that monitor changes in blood or other bodily fluid markers are emerging as a promising cost-effective alternative. Traditional biomarkers have limitations mainly due to difficulty in repeatedly obtaining tumor tissue confounded also by the spatial and temporal heterogeneity of tumours. Liquid biopsy has the potential to circumvent tumor heterogeneity and to help identifying patients who may respond to IO, to monitor the treatment dynamically, as well as to unravel the mechanisms of relapse. We present here a review of the current status of molecular markers for the prediction and monitoring of IO response, focusing on the detection of these markers in liquid biopsies. With the emerging improvements in the field of liquid biopsy, this approach has the capacity to identify IO-eligible patients and provide clinically relevant information to assist with their ongoing disease management.

Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR.

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

The impact of diagnosis related group payment on the performance of public hospitals.

OBJECTIVE: To investigate the practical value of diagnosis related groups (DRGs) according to payment for assessing the performance of public hospitals. METHODS: According to a random number table, 2400 patients were chosen from 3928 inpatients admitted for treatment in our hospital. Based on nodes implemented in the DRGs, these patients were assigned to the control group and the experimental group (1200 patients in each group). In the control group, patients didn't receive assistance with DRG payment (a clinical performance management approach was carried out based on the type of disease and cost), while patients in the experimental group received DRG. Bed turnover rate, hospitalization time, average cost, mortality, and subjective satisfaction were obtained and compared between the two groups. RESULTS: Compared with the control group, bed turnover rate, hospitalization time, average cost, and mortality in the experimental group were all significantly decreased (P<0.05), while subjective satisfaction was increased (P<0.05). CONCLUSION: DRG payment is beneficial for reduced clinical hospitalization time, cost, and mortality, and improved bed utilization rate and subjective satisfaction, which is worthy of clinical promotion.

PD-L1 Detection on Circulating Melanoma Cells.

The advent of personalized medicines targeting cell signaling pathways has radically improved melanoma patient outcomes. More recently, immune-modulating therapies disrupting the PD-1/PD-L1 axis have become a powerful tool in the treatment of a range of melanoma, showing a profound improvement in the overall survival outcomes. However, immune checkpoint inhibitors (ICIs) are associated with considerable toxicities and appear to only be efficacious in a subset of melanoma patients. Therefore, there is an urgent need to identify biomarkers that can determine if patients will or will not respond to ICI therapy. Here, we describe an optimized method for analyzing PD-L1 expression on circulating melanoma cells following immunomagnetic enrichment from patient blood samples.

Single-Cell Analysis of BRAFV600E and NRASQ61R Mutation Status in Melanoma Cell Lines as Method Generation for Circulating Melanoma Cells.

Molecular testing of tumor biopsies allows for the identification of the key mutations driving a patient's cancer. However, this is limited to singular nodes and may not accurately reflect cancer heterogeneity. Circulating tumor cell (CTC) analyses offer a noninvasive method of interrogating the genomic profile of patient-derived tumor material. To date, molecular analysis of CTCs has relied on the characterization of bulk or pooled CTC lysates, limiting the detection of minor tumorigenic CTC subclones. Here, we show a workflow enabling BRAFV600E/NRASQ61R mutation detection from single cultured melanoma cells by combining micromanipulation and genomic material amplification methods. This workflow can be directly integrated into circulating tumor cell analysis applications.

Rosacea is associated with conjoined interactions between physical barrier of the skin and microorganisms: A pilot study.

BACKGROUND: Rosacea is a common condition characterized by transient or persistent central facial erythema, and often papules and pustules. Currently, the role of bacterium in the development and progression of rosacea remains controversial. This study aimed to investigate the difference in the physiological conditions and microorganisms between the lesional and non-lesional areas of papulopustular rosacea. METHODS: Twenty-five French patients with papulopustular rosacea were enrolled in this pilot study. Each patient was subjected to clinical assessment, and the skin barrier function was tested in lesional and non-lesional areas. In addition, samples from the lesional and non-lesional areas were collected for bacterial culturing. RESULTS: Of all subjects included in the study, a lower skin conductivity was measured in lesional areas than in non-lesional areas (43.5 ± 12.4 vs. 57.2 ± 11.6 U, P < .05), and a higher transepidermal water loss (TEWL) value was found in lesional areas than in non-lesional areas (17.2 ± 5.9 vs. 14.2 ± 4.1 g/(m2  h), P < .05). We found a lower TEWL in lesions in rosacea patients with bacterial dysbiosis than in those with bacterial balance (P < .05). In addition, there were significant differences in the skin conductivity and TEWL between lesional and non-lesional areas in patients with bacterial dysbiosis (P < .001), and no significant differences were seen in patients with bacterial balance (P < .05). CONCLUSION: The results of the present study demonstrate that the physiological features of rosacea are closely associated with the interactions between the host and the microorganisms.

The Prospect of Identifying Resistance Mechanisms for Castrate-Resistant Prostate Cancer Using Circulating Tumor Cells: Is Epithelial-to-Mesenchymal Transition a Key Player?

Prostate cancer (PCa) is initially driven by excessive androgen receptor (AR) signaling with androgen deprivation therapy (ADT) being a major therapeutic approach to its treatment. However, the development of drug resistance is a significant limitation on the effectiveness of both first-line and more recently developed second-line ADTs. There is a need then to study AR signaling within the context of other oncogenic signaling pathways that likely mediate this resistance. This review focuses on interactions between AR signaling, the well-known phosphatidylinositol-3-kinase/AKT pathway, and an emerging mediator of these pathways, the Hippo/YAP1 axis in metastatic castrate-resistant PCa, and their involvement in the regulation of epithelial-mesenchymal transition (EMT), a feature of disease progression and ADT resistance. Analysis of these pathways in circulating tumor cells (CTCs) may provide an opportunity to evaluate their utility as biomarkers and address their importance in the development of resistance to current ADT with potential to guide future therapies.

Distinguishing rosacea from sensitive skin by reflectance confocal microscopy.

BACKGROUND: The updated standard classification and pathophysiology of rosacea have provided clear and meaningful evaluation parameters; however, differentiating rosacea from sensitive skin (SS) remained an obstacle for dermatologists around the world, especially in China. Herein, we aimed to find a better characteristic to distinguish rosacea from SS by using reflectance confocal microscopy (RCM). METHOD: Forty rosacea patients and 143 healthy subjects were recruited in this study. Firstly, a SS questionnaire and a lactic acid sting test were conducted among healthy subjects. Next, two major groups were divided out, including a SS group (40 subjects) and a normal skin control group (NS, 60 subjects). The cutaneous structures of face and fossa cubitalia were imaged by RCM. RESULTS: We found that more parakeratosis, honeycomb pattern, spongiform edema, and dermal papillae (P < .05) in rosacea patients than that of the NS group, whereas there were no significant differences, were found in rosacea patients and the SS group. Strikingly, we found that rosacea patients have a larger depth of honeycomb pattern than that of SS subjects (P < .05). But, the epidermal thickness of rosacea did not differ from that of SS groups. There was also no significant difference of epidermal thickness and honeycomb structure depth between rosacea patients and NS group. CONCLUSION: From the RCM images of parakeratosis, honeycomb pattern, spongiform edema, and dermal papillae, we found that RCM might be a faithful tool to distinguish rosacea from NS group. The depth of honeycomb structure of SS was more superficial than rosacea patients, whereas no significant difference between rosacea patients and NS group. RCM may provide a new method for evaluating the development of rosacea although it failed to distinguish rosacea and SS effectively.

Plasma next generation sequencing and droplet digital PCR-based detection of epidermal growth factor receptor (EGFR) mutations in patients with advanced lung cancer treated with subsequent-line osimertinib.

BACKGROUND: Gene mutation analysis from plasma circulating tumor DNA (ctDNA) can provide timely information regarding the mechanism of resistance that could translate to personalised treatment. We compared concordance rate of next generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) in the detection of the EGFR activating and T790M mutation from plasma ctDNA with diagnostic tissue biopsy-based assays. The second objective was to test whether putative osimertinib resistance associated mutations were detectable from plasma using NGS. METHODS: From January 2016 to December 2017, we prospectively collected plasma samples from patients prior to commencement of second- or third-line osimertinib therapy and upon disease progression, in a single tertiary hospital in South Western Sydney, Australia. Amplicon-based NGS and ddPCR assays were used to detect activating epidermal growth factor receptor (EGFR) and T790M mutations in 18 plasma samples from nine patients; all patients were required to have tissue biopsies with known EGFR status. RESULTS: High concordance of allelic fractions were seen in matched plasma NGS and ddPCR for activating EGFR mutations and T790M mutations (R2 = 0.92, P < 0.0001). Using tissue biopsies as reference standard, sensitivity was 100% for NGS and 94% for ddPCR. Several possible osimertinib resistance associated mutations, including PIK3CA, BRAF and TP53 mutations, were detected by NGS in samples upon progression on osimertinib therapy. CONCLUSION: ddPCR assays for EGFR mutations appear to be as sensitive and highly concordant as amplicon-based NGS. NGS has the ability to detect novel resistance mutations.

Detection of AR-V7 in Liquid Biopsies of Castrate Resistant Prostate Cancer Patients: A Comparison of AR-V7 Analysis in Circulating Tumor Cells, Circulating Tumor RNA and Exosomes.

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker.