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Circulating tumor cells (CTCs) are gaining momentum as a diagnostic tool and therapeutic target. CTC clusters are more metastatic, but harder to study and characterize, because they are rare and the methods of isolation are mostly focused on single CTCs. This review highlights the recent advances to our understanding of tumor cell clusters with the emphasis on their composition, origin, biology, methods of detection, and impact on metastasis and survival. New approaches to therapy, based on cluster characteristics are also described.

Background: Decisions regarding the staging, prognosis, and treatment of patients with head and neck squamous cell carcinomas (HNSCCs) are made after determining their p16 expression levels and human papillomavirus (HPV) infection status. Methods: We investigated the prognostic roles of p16-positive and p16-negative circulating tumor cells (CTCs) and their cell counts in HNSCC patients. We enrolled patients with locally advanced HNSCCs who received definitive concurrent chemoradiotherapy for final analysis. We performed CTC testing and p16 expression analysis before chemoradiotherapy. We analyzed the correlation between p16-positive and p16-negative CTCs and HPV genotyping, tissue p16 expression status, response to chemoradiotherapy, disease-free survival, and overall survival. Results: Forty-one patients who fulfilled the study criteria were prospectively enrolled for final analysis. The detection rates of p16-positive (&gt, 0 cells/mL blood) and p16-negative (≥3 cells/mL blood) CTCs were 51.2% (n = 21/41) and 70.7%, respectively. The best responses of chemoradiotherapy and the p16 positivity of CTCs are independent prognostic factors of disease progression, with hazard ratios of 1.738 (95% confidence interval (CI): 1.031–2.927), 5.497 (95% CI: 1.818–16.615), and 0.176 (95% CI: 0.056–0.554), respectively. The p16 positivity of CTCs was a prognostic factor for cancer death, with a hazard ratio of 0.294 (95% CI: 0.102–0.852). Conclusions: The p16-positive and p16-negative CTCs could predict outcomes in HNSCC patients receiving definitive chemoradiotherapy. This non-invasive CTC test could help stratify the risk and prognosis before chemoradiotherapy in clinical practice and enable us to perform de-intensifying therapies.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis. Methods: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests. Results: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02–10.14, p = 0.016, PROM1: HR 4.23, 95% CI 0.65–27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72–24.14, p = 0.025, PROM1: HR 4.77, 95% CI 0.29–78.94, p = 0.032). Conclusions: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.

Simple Summary Dickkopf-related protein 1 (DKK1), an antagonist of the canonical Wnt pathway has been the subject of research for many years. Especially in gastrointestinal cancers, research suggests a pivotal role of DKK1. In order to understand the role of DKK1 in esophageal cancer, we analyzed blood samples of esophageal cancer patients for their DKK1 levels and retrospectively analyzed the clinicopathological data. In our study cohort, we observed a negative prognostic role of high DKK1 serum levels with respect to overall survival in esophageal cancer patients. These data may suggest serum DKK1 as a novel biomarker for improved risk stratification and treatment monitoring in esophageal cancer patients. Abstract Dickkopf-related protein 1 (DKK1), an antagonist of the canonical Wnt pathway, has received tremendous attention over the past years as its dysregulation is said to be critically involved in a wide variety of gastrointestinal cancers. However, the potential clinical implications of DKK1 remain poorly understood. Although multimodal treatment options have been implemented over the past years, esophageal cancer (EC) patients still suffer from poor five-year overall survival rates ranging from 15% to 25%. Especially prognostic factors and biomarkers for risk stratification are lacking to choose the most beneficial treatment out of the emerging landscape of different treatment options. In this study, we analyzed the serum DKK1 (S-DKK1) levels of 91 EC patients prior to surgery in a single center study at the University Medical Center Hamburg-Eppendorf by enzyme-linked immunosorbent assay. High levels of S-DKK1 could be especially observed in patients suffering from esophageal adenocarcinoma which may promote the hypothesis of a crucial role of DKK1 in inflammation. S-DKK1 levels of ≥5800 pg/mL were shown to be associated with unfavorable five-year survival rates and the presence of CTCs. Interestingly, significantly lower S-DKK1 levels were detected in patients after neoadjuvant treatment, implying that S-DKK1 may serve as a useful biomarker for treatment monitoring. Multivariate analysis identified S-DKK1 as an independent prognostic marker with respect to overall survival in EC patients with a hazard ratio of 2.23. In conclusion, our data implicate a negative prognostic role of DKK1 with respect to the clinical outcome in EC patients. Further prospective studies should be conducted to implement S-DKK1 into the clinical routine for risk stratification and treatment monitoring.

Simple Summary Despite a substantial increase in publications in recent years, liquid biopsy from blood, cerebrospinal fluid (CSF), and other body fluids is usually not routinely used in cancer diagnostics and tumor monitoring. In this regard, brain tumors represent an additionally challenging group of tumors due to the blood–brain barrier as a potential suppressor of migrating tumor cells and their property of rarely metastasizing via the blood. Surprisingly, however, circulating tumor cells (CTCs) have been found in 20% of glioblastoma patients, which may allow for monitoring of tumor progression and response to therapies based on the genetic profiling of such tumors. Genetic biomarkers from CTC, circulating tumor DNA (ctDNA), extracellular vesicles, and microRNA (miRNA) are discussed. Here, we review the recent developments and future potential of liquid biopsy in brain tumors. Abstract Two decades of “promising results” in liquid biopsy have led to both continuing disappointment and hope that the new era of minimally invasive, personalized analysis can be applied for better diagnosis, prognosis, monitoring, and therapy of cancer. Here, we briefly highlight the promises, developments, and challenges related to liquid biopsy of brain tumors, including circulating tumor cells, cell-free nucleic acids, extracellular vesicles, and miRNA; we further discuss the urgent need to establish suitable biomarkers and the right standards to improve modern clinical management of brain tumor patients with the use of liquid biopsy.

Circulating tumor cells (CTCs) are known to be heterogeneous and clustered with tumor-associated cells, such as macrophages, neutrophils, fibroblasts, and platelets. However, their molecular profile and clinical significance remain largely unknown. Thus, we aimed to perform a comprehensive gene expression analysis of single CTCs and CTC clusters in patients with pancreatic cancer and to identify their potential clinical relevance to provide personalized medicine. Epitope-independent, rapid (&gt, 3 mL of whole blood/min) isolation of single CTCs and CTC clusters was achieved from a prospective cohort of 16 patients with unresectable pancreatic cancer using a centrifugal microfluidic device. Forty-eight mRNA expressions of individual CTCs and CTC clusters were analyzed to identify pancreatic CTC phenotype. CTC clusters had a larger proportion of mesenchymal expression than single CTCs (p = 0.0004). The presence of CTC clusters positively correlated with poor prognosis (progression-free survival, p = 0.0159, overall survival, p = 0.0186). Furthermore, we found that most CTCs in these patients (90.7%) were cloaked with platelets and found the presence of a positive correlation between the increase in CTC clusters and rapid disease progression during follow-ups. Efficient CTC cluster isolation and analysis techniques will enhance the understanding of complex tumor metastasis processes and can facilitate personalized disease management.

Neutrophils are the most abundant immune cell in the circulation of human and act as gatekeepers to discard foreign elements that have entered the body. They are essential in initiating immune responses for eliminating invaders, such as microorganisms and alien particles, as well as to act as immune surveyors of cancer cells, especially during the initial stages of carcinogenesis and for eliminating single metastatic cells in the circulation and in the premetastatic organs. Since neutrophils can secrete a whole range of factors stored in their many granules as well as produce reactive oxygen and nitrogen species upon stimulation, neutrophils may directly or indirectly affect carcinogenesis in both the positive and negative directions. An intricate crosstalk between tumor cells, neutrophils, other immune cells and stromal cells in the microenvironment modulates neutrophil function resulting in both anti- and pro-tumor activities. Both the anti-tumor and pro-tumor activities require chemoattraction towards the tumor cells, neutrophil activation and ROS production. Divergence is seen in other neutrophil properties, including differential secretory repertoire and membrane receptor display. Many of the direct effects of neutrophils on tumor growth and metastases are dependent on tight neutrophil–tumor cell interactions. Among them, the neutrophil Mac-1 interaction with tumor ICAM-1 and the neutrophil L-selectin interaction with tumor-cell sialomucins were found to be involved in the neutrophil-mediated capturing of circulating tumor cells resulting in increased metastatic seeding. On the other hand, the anti-tumor function of neutrophils was found to rely on the interaction between tumor-surface-expressed receptor for advanced glycation end products (RAGE) and Cathepsin G expressed on the neutrophil surface. Intriguingly, these two molecules are also involved in the promotion of tumor growth and metastases. RAGE is upregulated during early inflammation-induced carcinogenesis and was found to be important for sustaining tumor growth and homing at metastatic sites. Cathepsin G was found to be essential for neutrophil-supported lung colonization of cancer cells. These data level up the complexity of the dual role of neutrophils in cancer.

Breast cancer (BC) is a disease characterized by high degrees of heterogeneity at morphologic, genomic, and genetic levels, even within the same tumor mass or among patients. As a consequence, different subpopulations coexist and less represented clones may have a selective advantage, significantly influencing the outcome of BC patients. Circulating tumor cells (CTCs) represent a rare population of cells with a crucial role in metastatic cascade, and in recent years have represented a fascinating alternative to overcome the heterogeneity issue as a “liquid biopsy”. However, besides the raw enumeration of these cells in advanced epithelial tumors, there are no CTC-based assays applied in the clinical practice to improve personalized medicine. In this review, we report the latest findings in the field of CTCs for intra-tumoral heterogeneity unmasking in BC, supporting the need to deepen their analysis to investigate their role in metastatic process and include the molecular characterization in the clinical practice. In the future, CTCs will be helpful in monitoring patients during treatment, as well as to better address therapeutic strategies.

Simple Summary Prostate cancer research has been recently characterized by the discovery of several prognostic and predictive molecular factors, which ultimately improve patients’ management. In this review, we present the clinical impact of such factors and the methods to detect them, both on tissue and blood, in advanced prostate cancer patients. The aim of this review is ultimately to depict the role of these molecular factors in the era of precision oncology. Abstract Prostate cancer (PCa) therapy has been recently revolutionized by the approval of new therapeutic agents in the metastatic setting. However, the optimal therapeutic strategy in such patients should be individualized in the light of prognostic and predictive molecular factors, which have been recently studied: androgen receptor (AR) alterations, PTEN-PI3K-AKT pathway deregulation, homologous recombination deficiency (HRD), mismatch repair deficiency (MMRd), and tumor microenvironment (TME) modifications. In this review, we highlighted the clinical impact of prognostic and predictive molecular factors in PCa patients’ outcomes, identifying biologically distinct subtypes. We further analyzed the relevant methods to detect these factors, both on tissue, i.e., immunohistochemistry (IHC) and molecular tests, and blood, i.e., analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Moreover, we discussed the main pros and cons of such techniques, depicting their present and future roles in PCa management, throughout the precision medicine era.

Circulating tumor cells (CTCs) are single cells or clusters of cells within the circulatory system of a cancer patient. While most CTCs will perish, a small proportion will proceed to colonize the metastatic niche. The clinical importance of CTCs was reaffirmed by the 2008 FDA approval of CellSearch®, a platform that could extract EpCAM-positive, CD45-negative cells from whole blood samples. Many further studies have demonstrated the presence of CTCs to stratify patients based on overall and progression-free survival, among other clinical indices. Given their unique role in metastasis, CTCs could also offer a glimpse into the genetic drivers of metastasis. Investigation of CTCs has already led to groundbreaking discoveries such as receptor switching between primary tumors and metastatic nodules in breast cancer, which could greatly affect disease management, as well as CTC-immune cell interactions that enhance colonization. In this review, we will highlight the growing variety of isolation techniques for investigating CTCs. Next, we will provide clinically relevant context for CTCs, discussing key clinical trials involving CTCs. Finally, we will provide insight into the future of CTC studies and some questions that CTCs are primed to answer.

Simple Summary During the development and progression of lung tumors, processes such as necrosis and vascular invasion shed tumor cells or cellular components into various fluid compartments. Liquid biopsies consist of obtaining a bodily fluid, typically peripheral blood, in order to isolate and investigate these shed tumor constituents. Circulating tumor cells (CTCs) are one such constituent, which can be isolated from blood and can act as a diagnostic aid and provide valuable prognostic information. Liquid-based biopsies may also have a potential future role in lung cancer screening. Circulating tumor DNA (ctDNA) is found in small quantities in blood and, with the recent development of sensitive molecular and sequencing technologies, can be used to directly detect actionable genetic alterations or monitor for resistance mutations and guide clinical management. While potential benefits of liquid biopsies are promising, they are not without limitations. In this review, we summarize the current state and limitations of CTCs and ctDNA and possible future directions. Abstract Lung cancer is a leading cause of cancer-related deaths, contributing to 18.4% of cancer deaths globally. Treatment of non-small cell lung carcinoma has seen rapid progression with targeted therapies tailored to specific genetic drivers. However, identifying genetic alterations can be difficult due to lack of tissue, inaccessible tumors and the risk of complications for the patient with serial tissue sampling. The liquid biopsy provides a minimally invasive method which can obtain circulating biomarkers shed from the tumor and could be a safer alternative to tissue biopsy. While tissue biopsy remains the gold standard, liquid biopsies could be very beneficial where serial sampling is required, such as monitoring disease progression or development of resistance mutations to current targeted therapies. Liquid biopsies also have a potential role in identifying patients at risk of relapse post treatment and as a component of future lung cancer screening protocols. Rapid developments have led to multiple platforms for isolating circulating tumor cells (CTCs) and detecting circulating tumor DNA (ctDNA); however, standardization is lacking, especially in lung carcinoma. Additionally, clonal hematopoiesis of uncertain clinical significance must be taken into consideration in genetic sequencing, as it introduces the potential for false positives. Various biomarkers have been investigated in liquid biopsies; however, in this review, we will concentrate on the current use of ctDNA and CTCs, focusing on the clinical relevance, current and possible future applications and limitations of each.

Simple Summary Lung cancer is the leading cause of cancer death worldwide. Detecting lung malignancies promptly is essential for any anticancer treatment to reduce mortality and morbidity, especially in high-risk individuals. The use of liquid biopsy to detect circulating biomarkers such as RNA, microRNA, DNA, proteins, autoantibodies in the blood, as well as circulating tumor cells (CTCs), can substantially change the way we manage lung cancer patients by improving disease stratification using intrinsic molecular characteristics, identification of therapeutic targets and monitoring molecular residual disease. Here, we made an update on recent developments in liquid biopsy-based biomarkers for lung cancer early diagnosis, and we propose guidelines for an accurate study design, execution, and data interpretation for biomarker development. Abstract Lung cancer burden is increasing, with 2 million deaths/year worldwide. Current limitations in early detection impede lung cancer diagnosis when the disease is still localized and thus more curable by surgery or multimodality treatment. Liquid biopsy is emerging as an important tool for lung cancer early detection and for monitoring therapy response. Here, we reviewed recent advances in liquid biopsy for early diagnosis of lung cancer. We summarized DNA- or RNA-based biomarkers, proteins, autoantibodies circulating in the blood, as well as circulating tumor cells (CTCs), and compared the most promising studies in terms of biomarkers prediction performance. While we observed an overall good performance for the proposed biomarkers, we noticed some critical aspects which may complicate the successful translation of these biomarkers into the clinical setting. We, therefore, proposed a roadmap for successful development of lung cancer biomarkers during the discovery, prioritization, and clinical validation phase. The integration of innovative minimally invasive biomarkers in screening programs is highly demanded to augment lung cancer early detection.

Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.

Prostate cancer is the second most common male malignancy, with a highly variable clinical presentation and outcome. Therefore, diagnosis, prognostication, and management remain a challenge, as available clinical, imaging, and pathological parameters provide limited risk assessment. Thus, many biomarkers are under study to fill this critical gap, some of them based on epigenetic aberrations that might be detected in liquid biopsies. Herein, we provide a critical review of published data on the usefulness of DNA methylation and circulating tumor cells in diagnosis and treatment decisions in cases of prostate cancer, underlining key aspects and discussing the importance of these advances to the improvement of the management of prostate cancer patients. Using minimally invasive blood tests, the detection of highly specific biomarkers might be crucial for making therapeutic decisions, determining response to specific treatments, and allowing early diagnosis.

Simple Summary Breast cancer, the most common malignant tumor among women worldwide, remains an incurable disease once it has spread to the brain. Past research has shown that a primary breast cancer’s biology is an important determining factor predisposing its ability to form brain metastases. This review summarizes our current understanding of which genes, mutations, and molecules cause this increased ability to spread to and survive in the brain, specifically focusing on the different stages of this process. This knowledge may help us develop more effective, tumor-specific therapies and, as such, increase the chance of recovery for patients with breast cancer brain metastases. Abstract Breast cancer (BC) is the most frequent cause of cancer-associated death for women worldwide, with deaths commonly resulting from metastatic spread to distant organs. Approximately 30% of metastatic BC patients develop brain metastases (BM), a currently incurable diagnosis. The influence of BC molecular subtype and gene expression on breast cancer brain metastasis (BCBM) development and patient prognosis is undeniable and is, therefore, an important focus point in the attempt to combat the disease. The HER2-positive and triple-negative molecular subtypes are associated with an increased risk of developing BCBM. Several genetic and molecular mechanisms linked to HER2-positive and triple-negative BC breast cancers appear to influence BCBM formation on several levels, including increased development of circulating tumor cells (CTCs), enhanced epithelial-mesenchymal transition (EMT), and migration of primary BC cells to the brain and/or through superior local invasiveness aided by cancer stem-like cells (CSCs). These specific BC characteristics, together with the ensuing developments at a clinical level, are presented in this review article, drawing a connection between research findings and related therapeutic strategies aimed at preventing BCBM formation and/or progression. Furthermore, we briefly address the critical limitations in our current understanding of this complex topic, highlighting potential focal points for future research.

In cancer, many analytes can be investigated through liquid biopsy. They play fundamental roles in the biological mechanisms underpinning the metastatic cascade and provide clinical information that can be monitored in real time during the natural course of cancer. Some of these analytes (circulating tumor cells and extracellular vesicles) share a key feature: the presence of a phospholipid membrane that includes proteins, lipids and possibly nucleic acids. Most cell-to-cell and cell-to-matrix interactions are modulated by the cell membrane composition. To understand cancer progression, it is essential to describe how proteins, lipids and nucleic acids in the membrane influence these interactions in cancer cells. Therefore, assessing such interactions and the phospholipid membrane composition in different liquid biopsy analytes might be important for future diagnostic and therapeutic strategies. In this review, we briefly describe some of the most important surface components of circulating tumor cells and extracellular vesicles as well as their interactions, putting an emphasis on how they are involved in the different steps of the metastatic cascade and how they can be exploited by the different liquid biopsy technologies.

Simple Summary Malignant pleural effusion is a common complication arising as the natural progression of many tumors, such as lung cancer. When this occurs, the common protocol consists of analyzing the pleural fluid for the presence of malignant cells. However, on many occasions no malignant cells are found despite a clear suspicion of cancer. Thus, the current diagnostic methodology is imperfect and more precise methods for the identification of malignancy are needed. Nonetheless, these methods are often invasive, which may be counterproductive, especially for patients with poor health condition. These concerns have made clinicians consider alternative non-invasive strategies to diagnose cancer using the generally abundant pleural fluid (e.g., liquid biopsy). Thus, a liquid sample can be analyzed for the presence of cancer footprints, such as circulating malignant cells and tumor nucleic acids. Herein, we review the literature for studies considering pleural fluid as a successful source of liquid biopsy. Abstract Liquid biopsy is emerging as a promising non-invasive diagnostic tool for malignant pleural effusions (MPE) due to the low sensitivity of conventional pleural fluid (PF) cytological examination and the difficulty to obtain tissue biopsies, which are invasive and require procedural skills. Currently, liquid biopsy is increasingly being used for the detection of driver mutations in circulating tumor DNA (ctDNA) from plasma specimens to guide therapeutic interventions. Notably, malignant PF are richer than plasma in tumor-derived products with potential clinical usefulness, such as ctDNA, micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs), and circulating tumor cells (CTC). Tumor-educated cell types, such as platelets and macrophages, have also been added to this diagnostic armamentarium. Herein, we will present an overview of the role of the preceding biomarkers, collectively known as liquid biopsy, in PF samples, as well as the main technical approaches used for their detection and quantitation, including a proper sample processing. Technical limitations of current platforms and future perspectives in the field will also be addressed. Using PF as liquid biopsy shows promise for use in current practice to facilitate the diagnosis and management of metastatic MPE.

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations. While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.

Background: This study used NeuN transgenic (NTTg) mice with spontaneous breast tumor development to evaluate the dynamic changes of circulating tumor cells (CTCs) prior to and during tumor development. Methods: In this longitudinal, clinically uninterrupted study, we collected 75 μL of peripheral blood at the age of 8, 12, 16, and 20 weeks in the first group of five mice, and at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability in the second group of four mice. Diluted blood samples were run through a modified mouse-CMx chip to isolate the CTCs. Results: The CTC counts of the first group of mice were low (1 ± 1.6) initially. The average CTC counts were 16 ± 9.5, 29.0 ± 18.2, and 70.0 ± 30.3 cells per 75 μL blood at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability, respectively. There was a significant positive correlation between an increase in CTC levels and tumor vascular density (p-value &lt, 0.01). This correlation was stronger than that between CTC levels and tumor size (p-value = 0.076). The captured CTCs were implanted into a non-tumor-bearing NTTg mouse for xenografting, confirming their viability and tumorigenesis. Conclusion: Serial CTCs during an early stage of tumor progression were quantified and found to be positively correlated with the later tumor vascular density and size. Furthermore, the successful generation of CTC-derived xenografts indicates the tumorigenicity of this early onset CTC population.

Simple Summary Information describing circulating tumor cells (CTCs) holds promise for clinical applications. However, conventional CTCs enumeration could ignore the CTCs more relevant to cancer metastasis. Thus, negative selection CTC enumeration was proposed, by which information on the numbers of CTCs and CD45neg EpCAMneg cells can be obtained. By combining this approach with the conventional biomarker carcinoembryonic antigen (CEA), this study aimed to explore whether any combination of these biomarkers could improve the predictive performance for colorectal cancer (CRC) or its status. Results revealed that a combination of the two cell populations showed improved performance (AUROC: 0.893) for CRC prediction over the use of only one population. Compared with CEA alone, the combination of the three biomarkers increased the performance (AUROC) for advanced CRC prediction from 0.643 to 0.727. Compared with that of CEA alone for metastatic CRC prediction, the AUROC was increased from 0.780 to 0.837 when the CTC count was included. Abstract Conventional circulating tumor cell (CTC) enumeration could ignore the CTCs more relevant to cancer metastasis. Thus, negative selection CTC enumeration was proposed, by which information on two cellular biomarkers (numbers of CTCs and CD45neg EpCAMneg cells) can be obtained. By combining this approach with the conventional biomarker carcinoembryonic antigen (CEA), this study aimed to explore whether any combination of these biomarkers could improve the predictive performance for colorectal cancer (CRC) or its status. In this work, these two cell populations in healthy donors and CRC patients were quantified. Results revealed that enumeration of these two cell populations was able to discriminate healthy donors from CRC patients, even patients with non-advanced CRC. Moreover, the combination of the two cell populations showed improved performance (AUROC: 0.893) for CRC prediction over the use of only one population. Compared with CEA alone, the combination of the three biomarkers increased the performance (AUROC) for advanced CRC prediction from 0.643 to 0.727. Compared with that of CEA alone for metastatic CRC prediction, the AUROC was increased from 0.780 to 0.837 when the CTC count was included. Overall, this study demonstrated that the combination of these two cellular biomarkers with CEA improved the predictive performance for CRC and its status.

Simple Summary Androgen receptor splice variants (AR-Vs) play an important role in prostate cancer progression, especially as a putative resistance mechanism against AR-targeted therapies. Recent technological advances have enabled detection of AR-Vs in many types of human specimens including circulating tumor cells. Here, we discuss the biology of AR-Vs, the clinical utility of AR-Vs as prognostic and predictive biomarkers, and AR-Vs as potential therapeutic targets with a special focus on AR-V7. Abstract Over the past decade, advances in prostate cancer research have led to discovery and development of novel biomarkers and effective treatments. As treatment options diversify, it is critical to further develop and use optimal biomarkers for the purpose of maximizing treatment benefit and minimizing unwanted adverse effects. Because most treatments for prostate cancer target androgen receptor (AR) signaling, aberrations affecting this drug target are likely to emerge following the development of castration-resistant prostate cancer (CRPC), and it is conceivable that such aberrations may play a role in drug resistance. Among the many AR aberrations, we and others have been studying androgen receptor splice variants (AR-Vs), especially AR-V7, and have conducted preclinical and clinical studies to develop and validate the clinical utility of AR-V7 as a prognostic and potential predictive biomarker. In this review, we first describe mechanisms of AR-V generation, regulation and their functions from a molecular perspective. We then discuss AR-Vs from a clinical perspective, focusing on the significance of AR-Vs detected in different types of human specimens and AR-Vs as potential therapeutic targets.

Simple Summary Although immunotherapy has dramatically revolutionized non-small cell lung cancer (NSCLC) treatment, not all the patients will benefit from this innovative therapy. The identification of potential biomarkers able to predict efficacy and toxicity of immunotherapy represents an urgent need for tailored treatment regimens. Liquid biopsy is a minimally invasive and economical tool that could provide important information about patients’ selection and treatment monitoring. Currently, several blood biomarkers are under investigation (circulating immune and tumor cells, soluble immunological mediators, peripheral blood cells). Prospective clinical trials are needed to validate their use in clinical practice. Abstract Immune checkpoint inhibitors (ICIs) targeting the programmed cell death (PD)-1 protein and its ligand, PD-L1, and cytotoxic T-lymphocyte-associated antigen (CTLA)-4, have revolutionized the management of patients with advanced non-small cell lung cancer (NSCLC). Unfortunately, only a small portion of NSCLC patients respond to these agents. Furthermore, although immunotherapy is usually well tolerated, some patients experience severe immune-related adverse events (irAEs). Liquid biopsy is a non-invasive diagnostic procedure involving the isolation of circulating biomarkers, such as circulating tumor cells (CTC), cell-free DNA (cfDNA), and microRNAs (miRNAs). Thanks to recent advances in technologies, such as next-generation sequencing (NGS) and digital polymerase chain reaction (dPCR), liquid biopsy has become a useful tool to provide baseline information on the tumor, and to monitor response to treatments. This review highlights the potential role of liquid biomarkers in the selection of NSCLC patients who could respond to immunotherapy, and in the identification of patients who are most likely to experience irAEs, in order to guide improvements in care.

Simple Summary Metastasis is the main cause of cancer-related death. Circulating cancer stem cells have recently attracted attention because they have higher tumorigenicity than non-stem-like circulating tumor cells. Despite the strong scientific evidence for circulating cancer stem cells and secondary tumor formation, the exact mechanisms behind the generation and characteristics of circulating cancer stem cells are not yet fully understood because of their extreme scarcity. This review aims to introduce the recent advances in the detection and analysis of circulating tumor cells and circulating cancer stem cells. Abstract Detecting circulating tumor cells (CTCs) has been considered one of the best biomarkers in liquid biopsy for early diagnosis and prognosis monitoring in cancer. A major challenge of using CTCs is detecting extremely low-concentrated targets in the presence of high noise factors such as serum and hematopoietic cells. This review provides a selective overview of the recent progress in the design of microfluidic devices with optical sensing tools and their application in the detection and analysis of CTCs and their small malignant subset, circulating cancer stem cells (CCSCs). Moreover, discussion of novel strategies to analyze the differentiation of circulating cancer stem cells will contribute to an understanding of metastatic cancer, which can help clinicians to make a better assessment. We believe that the topic discussed in this review can provide brief guideline for the development of microfluidic-based optical biosensors in cancer prognosis monitoring and clinical applications.

The effectiveness of cancer treatment strongly depends on the early detection of the disease. Currently, the most common diagnostic method, tissue biopsy, takes time and can be damaging to the patient. Circulating cancer biomarkers such as circulating tumor DNA, micro-RNA (miRNA), tumor proteins, exosomes, and circulating tumor cells have repeatedly demonstrated their viability as targets for minimally invasive cancer detection through liquid biopsies. However, among other things, achieving a great sensitivity of detection is still challenging due to the very low concentration of biomarkers in fluid samples. This review will discuss how the recent advances in nanoparticle-based biosensors are overcoming these practical difficulties. This report will be focusing mainly on optical transduction mechanisms of metal nanoparticles (M-NPs), quantum dots (QDs), and upconversion nanoparticles (UCNPs).

Simple Summary Detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging issue, since they adapt to the biochemical and physical landscape of the bloodstream. We approached the issue of CTC identification on a biophysical level. For the first time, we recorded the mechanical deformation profiles of potential CTCs, which were isolated from the blood of breast cancer patients, at the force regime of the deforming blood flow. Mechanical fingerprints of CTCs were significantly different from healthy white blood cells. We used machine learning to further evaluate the differences and identify discrimination criteria. Our results suggest that mechanical characterization of CTCs at low forces is a promising path towards CTC detection. Abstract Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

Malignant melanoma (MM) is a highly heterogenic tumor whose histological diagnosis might be difficult. This study aimed to investigate the diagnostic and prognostic utility of the conventional pan-melanoma cocktail members (HMB-45, melan-A and tyrosinase), in conjunction with SOX10 and SOX11 immunohistochemical (IHC) expression. In 105 consecutive cases of MMs and 44 of naevi, the IHC examination was performed using the five-abovementioned markers, along with microphthalmia transcription factor (MITF), S100, and Ki67. Correlation with the clinicopathological factors and a long-term follow-up was also done. Survival analysis was performed with Kaplan–Meier curves and compared with TCGA public datasets. None of the 44 naevi expressed SOX11, but its positivity was seen in 52 MMs (49.52%), being directly correlated with lymphovascular invasion, the Ki67 index, and SOX10 expression. HMB-45, SOX10, and tyrosinase, but not melan-A, proved to differentiate the naevi from MMs successfully, with high specificity. Triple MITF/SOX10/SOX11 co-expression was seen in 9 out of 15 negative conventional pan-melanoma-cocktail cases. The independent prognostic value was proved for the conventional pan-melanoma cocktail (triple positivity for HMB-45, melan-A, and tyrosinase) and, independently for HMB-45 and tyrosinase, but not for melan-A, SOX10, or SOX11. As consequence, to differentiate MMs from benign naevi, melan-A should be substituted by SOX10 in the conventional cocktail. Although the conventional pan-melanoma cocktail, along with S100 can be used for the identification of melanocytic origin of tumor cells and predicting prognosis of MMs, the conventional-adapted cocktail (triple positivity for HMB-45, SOX10, and tyrosinase) has a slightly higher diagnostic specificity. SOX11 can be added to identify the aggressive MMs with risk for lymphatic dissemination and the presence of circulating tumor cells.