Pushing the Boundaries of Liquid Biopsies for Early Precision Intervention.

Liquid biopsies are emerging as powerful minimally invasive approaches that have the potential to solve several long-standing problems spanning the continuum of cancer care: early detection of cancer, minimal residual disease tracking, and refinement of the heterogeneity of clinical responses together with therapeutic response monitoring in the metastatic setting. Existing challenges driven by technical limitations and establishment of the clinical value of liquid biopsies represent fields of active research that call for convergence science approaches to bridge scientific discovery with clinical care.

MIR145 Core Promoter Methylation in Pretreatment Cell-Free DNA: A Liquid Biopsy Tool for Muscle-Invasive Bladder Cancer Treatment Outcome.

PURPOSE: The lack of personalized management of bladder cancer (BlCa) results in patients' lifelong post-treatment monitoring with invasive interventions, underlying the urgent need for tailored and minimally invasive health care services. On the basis of our previous findings on miR-143/145 cluster methylation in bladder tumors, we evaluated its clinical significance in pretreatment cell-free DNA (cfDNA) of patients with BlCa. MATERIALS AND METHODS: Methylation analysis was performed in our screening cohort (120 patients with BlCa; 20 age-matched healthy donors) by bisulfite-based pyrosequencing. Tumor recurrence/progression for patients with non-muscle-invasive bladder cancer, and progression and mortality for patients with muscle-invasive bladder cancer (MIBC) were used as clinical end point events in survival analysis. Bootstrap analysis was applied for internal validation of Cox regression models and decision curve analysis for assessment of clinical benefit on disease prognosis. RESULTS: Decreased methylation of MIR145 core promoter in pretreatment cfDNA was associated with short-term disease progression (multivariate Cox: hazard ratio [HR], 2.027 [95% CI, 1.157 to 3.551]; P = .010) and poor overall survival (multivariate Cox: HR, 2.098 [95% CI, 1.154 to 3.817]; P = .009) of patients with MIBC after radical cystectomy (RC). Multivariate models incorporating MIR145 promoter methylation in cfDNA with tumor stage clearly ameliorated patients' risk stratification, highlighting superior clinical benefit in MIBC prognostication. CONCLUSION: Reduced pretreatment cfDNA methylation of MIR145 core promoter was markedly correlated with increased risk for short-term progression and worse survival of patients with MIBC after RC and adjuvant therapy, supporting modern personalized and minimally invasive prognosis. Methylation profiling of MIR145 core promoter in pretreatment cfDNA could serve as a minimally invasive and independent predictor of MIBC treatment outcome and emerge as a promising marker for blood-based test in BlCa.

Cancer-derived exosomes as novel biomarkers in metastatic gastrointestinal cancer.

Gastrointestinal cancer (GIC) is the most prevalent and highly metastatic malignant tumor and has a significant impact on mortality rates. Nevertheless, the swift advancement of contemporary technology has not seamlessly aligned with the evolution of detection methodologies, resulting in a deficit of innovative and efficient clinical assays for GIC. Given that exosomes are preferentially released by a myriad of cellular entities, predominantly originating from neoplastic cells, this confers exosomes with a composition enriched in cancer-specific constituents. Furthermore, exosomes exhibit ubiquitous presence across diverse biological fluids, endowing them with the inherent advantages of non-invasiveness, real-time monitoring, and tumor specificity. The unparalleled advantages inherent in exosomes render them as an ideal liquid biopsy biomarker for early diagnosis, prognosticating the potential development of GIC metastasis.In this review, we summarized the latest research progress and possible potential targets on cancer-derived exosomes (CDEs) in GIC with an emphasis on the mechanisms of exosome promoting cancer metastasis, highlighting the potential roles of CDEs as the biomarker and treatment in metastatic GIC.

Liquid biopsy techniques and lung cancer: diagnosis, monitoring and evaluation.

Lung cancer stands as the most prevalent form of cancer globally, posing a significant threat to human well-being. Due to the lack of effective and accurate early diagnostic methods, many patients are diagnosed with advanced lung cancer. Although surgical resection is still a potential means of eradicating lung cancer, patients with advanced lung cancer usually miss the best chance for surgical treatment, and even after surgical resection patients may still experience tumor recurrence. Additionally, chemotherapy, the mainstay of treatment for patients with advanced lung cancer, has the potential to be chemo-resistant, resulting in poor clinical outcomes. The emergence of liquid biopsies has garnered considerable attention owing to their noninvasive nature and the ability for continuous sampling. Technological advancements have propelled circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), tumor metabolites, tumor-educated platelets (TEPs), and tumor-associated antigens (TAA) to the forefront as key liquid biopsy biomarkers, demonstrating intriguing and encouraging results for early diagnosis and prognostic evaluation of lung cancer. This review provides an overview of molecular biomarkers and assays utilized in liquid biopsies for lung cancer, encompassing CTCs, ctDNA, non-coding RNA (ncRNA), EVs, tumor metabolites, TAAs and TEPs. Furthermore, we expound on the practical applications of liquid biopsies, including early diagnosis, treatment response monitoring, prognostic evaluation, and recurrence monitoring in the context of lung cancer.

Emerging role of exosomes as a liquid biopsy tool for diagnosis, prognosis & monitoring treatment response of communicable & non-communicable diseases.

ABSTRACT: From an initial thought of being used as a cellular garbage bin to a promising target for liquid biopsies, the role of exosomes has drastically evolved in just a few years of their discovery in 1983. Exosomes are naturally secreted nano-sized vesicles, abundant in all types of body fluids and can be isolated intact even from the stored biological samples. Being stable carriers of genetic material (cellular DNA, mRNA and miRNA) and having specific cargo (signature content of originating cells), exosomes play a crucial role in pathogenesis and have been identified as a novel source of biomarkers in a variety of disease conditions. Recently exosomes have emerged as a promising 'liquid biopsy tool'and have shown great potential in the field of non-invasive disease diagnostics, prognostics and treatment response monitoring in both communicable as well as non-communicable diseases. However, there are certain limitations to overcome which restrict the use of exosome-based liquid biopsy as a gold standard testing procedure in routine clinical practices. The present review summarizes the current knowledge on the role of exosomes as the liquid biopsy tool in diagnosis, prognosis and treatment response monitoring in communicable and non-communicable diseases and highlights the major limitations, technical advancements and future prospects of the utilization of exosome-based liquid biopsy in clinical interventions.

A comprehensive guide to extract information from extracellular vesicles: a tutorial review towards novel analytical developments.

In the medical field, extracellular vesicles (EVs) are gaining importance as they act as cells mediators. These are phospholipid bilayer vesicles and contain crucial biochemical information about their mother cells being carrier of different biomolecules such as small molecules, proteins, lipids, and nucleic acids. After release into the extracellular matrix, they enter the systemic circulation and can be found in all human biofluids. Since EVs reflect the state of the cell of origin, there is exponential attention as potential source of new circulating biomarkers for liquid biopsy. The use of EVs in clinical practice faces several challenges that need to be addressed: these include the standardization of lysis protocols, the availability of low-cost reagents and the development of analytical tools capable of detecting biomarkers. The process of lysis is a crucial step that can impact all subsequent analyses, towards the development of novel analytical strategies. To aid researchers to support the evolution of measurement science technology, this tutorial review evaluates and discuss the most commonly protocols used to characterize the contents of EVs, including their advantages and disadvantages in terms of experimental procedures, time and equipment. The purpose of this tutorial review is to offer practical guide to researchers which are intended to develop novel analytical approaches. Some of the most significant applications are considered, highlighting their main characteristics divided per mechanism of action. Finally, comprehensive tables which provide an overview at a glance are provided to readers.

[Call for attention to the value and challenge of liquid biopsy in diagnosis and treatment for peritoneal metastasis of gastric cancer].

Peritoneal metastasis is the common route of metastasis in gastric cancer and is a major cause of death in advanced gastric cancer. Early intervention with comprehensive treatment can effectively improve the prognosis of some patients with peritoneal metastasis. However, early peritoneal metastasis in gastric cancer is predominantly micro-metastasis, which cannot be effectively evaluated by imaging studies. Moreover, the detection of disseminated cancer cells in peritoneal lavage suffers from a low detection rate and significant heterogeneity. In recent years, the development and application of new liquid biopsy technologies such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have provided new means to assess potential peritoneal metastasis at the cellular and molecular levels, gradually becoming research hotspots in this field. This review will summarize the relevant progress of liquid biopsy in peritoneal metastasis, which holds significant importance for improving the prognosis of gastric cancer patients in China.

Nanobiotechnology augmented cancer stem cell guided management of cancer: liquid-biopsy, imaging, and treatment.

Cancer stem cells (CSCs) represent both a key driving force and therapeutic target of tumoral carcinogenesis, tumor evolution, progression, and recurrence. CSC-guided tumor diagnosis, treatment, and surveillance are strategically significant in improving cancer patients' overall survival. Due to the heterogeneity and plasticity of CSCs, high sensitivity, specificity, and outstanding targeting are demanded for CSC detection and targeting. Nanobiotechnologies, including biosensors, nano-probes, contrast enhancers, and drug delivery systems, share identical features required. Implementing these techniques may facilitate the overall performance of CSC detection and targeting. In this review, we focus on some of the most recent advances in how nanobiotechnologies leverage the characteristics of CSC to optimize cancer diagnosis and treatment in liquid biopsy, clinical imaging, and CSC-guided nano-treatment. Specifically, how nanobiotechnologies leverage the attributes of CSC to maximize the detection of circulating tumor DNA, circulating tumor cells, and exosomes, to improve positron emission computed tomography and magnetic resonance imaging, and to enhance the therapeutic effects of cytotoxic therapy, photodynamic therapy, immunotherapy therapy, and radioimmunotherapy are reviewed.

Prognostic Value of Liquid-Biopsy-Based Biomarkers in Upper Tract Urothelial Carcinoma.

Currently, there are no reliable prognostic factors to determine which upper tract urothelial carcinoma (UTUC) patients will progress after radical nephroureterectomy (RNU). We aim to evaluate whether liquid-biopsy-based biomarkers (circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA)) were able to predict clinical outcomes in localized UTUC patients undergoing RNU. Twenty patients were prospectively enrolled between 2021 and 2023. Two blood samples were collected before RNU and three months later. CTCs and cfDNA were isolated and evaluated using the IsoFlux system and Quant-iT PicoGreen dsDNA kit, respectively. Droplet digital PCR was performed to determine ctDNA status. Cox regression analysis was performed on CTCs, cfDNA, and ctDNA at two different follow-up time points to examine their influence on tumor progression and cancer-specific survival (CSS). During a median follow-up of 18 months, seven (35%) patients progressed and three (15%) died. Multivariate analysis demonstrated that cfDNA levels three months after RNU are a significant predictor of tumor progression (HR = 1.085; p = 0.006) and CSS (HR = 1.168; p = 0.029). No associations were found between CTC enumeration and ctDNA status with any of the clinical outcomes evaluated. The evaluation of cfDNA levels in clinical practice could improve the disease management of UTUC patients.

Unlocking Precision Medicine: Liquid Biopsy Advancements in Renal Cancer Detection and Monitoring.

Renal cell carcinoma (RCC) remains a formidable diagnostic challenge, especially in the context of small renal masses. The quest for non-invasive screening tools and biomarkers has steered research towards liquid biopsy, focusing on microRNAs (miRNAs), exosomes, and circulating tumor cells (CTCs). MiRNAs, small non-coding RNAs, exhibit notable dysregulation in RCC, offering promising avenues for diagnosis and prognosis. Studies underscore their potential across various biofluids, including plasma, serum, and urine, for RCC detection and subtype characterization. Encouraging miRNA signatures show correlations with overall survival, indicative of their future relevance in RCC management. Exosomes, with their diverse molecular cargo, including miRNAs, emerge as enticing biomarkers, while CTCs, emanating from primary tumors into the bloodstream, provide valuable insights into cancer progression. Despite these advancements, clinical translation necessitates further validation and standardization, encompassing larger-scale studies and robust evidence generation. Currently lacking approved diagnostic assays for renal cancer, the potential future applications of liquid biopsy in follow-up care, treatment selection, and outcome prediction in RCC patients are profound. This review aims to discuss and highlight recent advancements in liquid biopsy for RCC, exploring their strengths and weaknesses in the comprehensive management of this disease.

Plasma extracellular vesicles in meningioma patients following radiotherapy as liquid biopsy- a prospective explorative biomarker study (ARO 2023-05/AG-NRO-07).

BACKGROUND: While surgical resection remains the primary treatment approach for symptomatic or growing meningiomas, radiotherapy represents an auspicious alternative in patients with meningiomas not safely amenable to surgery. Biopsies are often omitted in light of potential postoperative neurological deficits, resulting in a lack of histological grading and (molecular) risk stratification. In this prospective explorative biomarker study, extracellular vesicles in the bloodstream will be investigated in patients with macroscopic meningiomas to identify a biomarker for molecular risk stratification and disease monitoring. METHODS: In total, 60 patients with meningiomas and an indication of radiotherapy (RT) and macroscopic tumor on the planning MRI will be enrolled. Blood samples will be obtained before the start, during, and after radiotherapy, as well as during clinical follow-up every 6 months. Extracellular vesicles will be isolated from the blood samples, quantified and correlated with the clinical treatment response or progression. Further, nanopore sequencing-based DNA methylation profiles of plasma EV-DNA will be generated for methylation-based meningioma classification. DISCUSSION: This study will explore the dynamic of plasma EVs in meningioma patients under/after radiotherapy, with the objective of identifying potential biomarkers of (early) tumor progression. DNA methylation profiling of plasma EVs in meningioma patients may enable molecular risk stratification, facilitating a molecularly-guided target volume delineation and adjusted dose prescription during RT treatment planning.

tRNA-derived small RNAs in human cancers: roles, mechanisms, and clinical application.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are a new type of non-coding RNAs (ncRNAs) produced by the specific cleavage of precursor or mature tRNAs. tsRNAs are involved in various basic biological processes such as epigenetic, transcriptional, post-transcriptional, and translation regulation, thereby affecting the occurrence and development of various human diseases, including cancers. Recent studies have shown that tsRNAs play an important role in tumorigenesis by regulating biological behaviors such as malignant proliferation, invasion and metastasis, angiogenesis, immune response, tumor resistance, and tumor metabolism reprogramming. These may be new potential targets for tumor treatment. Furthermore, tsRNAs can exist abundantly and stably in various bodily fluids (e.g., blood, serum, and urine) in the form of free or encapsulated extracellular vesicles, thereby affecting intercellular communication in the tumor microenvironment (TME). Meanwhile, their abnormal expression is closely related to the clinicopathological features of tumor patients, such as tumor staging, lymph node metastasis, and poor prognosis of tumor patients; thus, tsRNAs can be served as a novel type of liquid biopsy biomarker. This review summarizes the discovery, production, and expression of tsRNAs and analyzes their molecular mechanisms in tumor development and potential applications in tumor therapy, which may provide new strategies for early diagnosis and targeted therapy of tumors.

A cfDNA methylation-based tissue-of-origin classifier for cancers of unknown primary.

Cancers of Unknown Primary (CUP) remains a diagnostic and therapeutic challenge due to biological heterogeneity and poor responses to standard chemotherapy. Predicting tissue-of-origin (TOO) molecularly could help refine this diagnosis, with tissue acquisition barriers mitigated via liquid biopsies. However, TOO liquid biopsies are unexplored in CUP cohorts. Here we describe CUPiD, a machine learning classifier for accurate TOO predictions across 29 tumour classes using circulating cell-free DNA (cfDNA) methylation patterns. We tested CUPiD on 143 cfDNA samples from patients with 13 cancer types alongside 27 non-cancer controls, with overall sensitivity of 84.6% and TOO accuracy of 96.8%. In an additional cohort of 41 patients with CUP CUPiD predictions were made in 32/41 (78.0%) cases, with 88.5% of the predictions clinically consistent with a subsequent or suspected primary tumour diagnosis, when available (23/26 patients). Combining CUPiD with cfDNA mutation data demonstrated potential diagnosis re-classification and/or treatment change in this hard-to-treat cancer group.

Liquid biopsy for gastric cancer: Techniques, applications, and future directions.

After the study of circulating tumor cells in blood through liquid biopsy (LB), this technique has evolved to encompass the analysis of multiple materials originating from the tumor, such as nucleic acids, extracellular vesicles, tumor-educated platelets, and other metabolites. Additionally, research has extended to include the examination of samples other than blood or plasma, such as saliva, gastric juice, urine, or stool. LB techniques are diverse, intricate, and variable. They must be highly sensitive, and pre-analytical, patient, and tumor-related factors significantly influence the detection threshold, diagnostic method selection, and potential results. Consequently, the implementation of LB in clinical practice still faces several challenges. The potential applications of LB range from early cancer detection to guiding targeted therapy or immunotherapy in both early and advanced cancer cases, monitoring treatment response, early identification of relapses, or assessing patient risk. On the other hand, gastric cancer (GC) is a disease often diagnosed at advanced stages. Despite recent advances in molecular understanding, the currently available treatment options have not substantially improved the prognosis for many of these patients. The application of LB in GC could be highly valuable as a non-invasive method for early diagnosis and for enhancing the management and outcomes of these patients. In this comprehensive review, from a pathologist's perspective, we provide an overview of the main options available in LB, delve into the fundamental principles of the most studied techniques, explore the potential utility of LB application in the context of GC, and address the obstacles that need to be overcome in the future to make this innovative technique a game-changer in cancer diagnosis and treatment within clinical practice.

[Progress of circulating tumor DNA in the clinical management of colorectal cancer].

Despite the great progress in the treatment of colorectal cancer (CRC), the current standard treatment protocols still have many limitations, and there is an urgent need for more effective biomarkers for personalized patient treatment. Circulating tumor DNA (ctDNA), as a dynamic, non-invasive liquid biopsy approach, overcomes the limitations of tissue biopsy in detecting tumor heterogeneity and molecular evolution. Current evidence from several studies suggests that ctDNA shows great promise in stratifying recurrence risk, guiding treatment decisions, and monitoring early recurrence. In addition, ctDNA can improve the efficiency of clinical research and drug development. However, the lack of standardisation of pre-ctDNA test variables and analysis procedures and the high technical costs limit its promotion and development. In this review, we summarize the available evidence on ctDNA in the clinical management of CRC and present its limitations and strategies for improvement.

Cancer diagnosis using label-free SERS-based exosome analysis.

Exosomes, carrying distinctive biomolecules reflective of their parent cell's status and origin, show promise as liquid biopsy biomarkers for cancer diagnosis. However, their clinical translation remains challenging due to their relatively low concentration in body fluids. Surface-Enhanced Raman spectroscopy (SERS) has recently gained significant attention as a label-free and sensitive technique for exosome analysis. This review explores label-free SERS for exosome detection, covering exosome isolation and characterization methods, advancements in SERS substrates, and fingerprint analysis techniques using machine learning. Furthermore, we emphasize the challenges and offer insights into the future prospects of SERS-based exosome analysis to enhance cancer diagnosis.

A Quest for Survival: A Review of the Early Biomarkers of Pancreatic Cancer and the Most Effective Approaches at Present.

Pancreatic cancer (PC) is the most lethal type of cancer; it has the lowest 5-year survival rate among all other types of cancers. More than half of PC cases are diagnosed at an advanced stage due to PC's insidious and non-specific symptoms. Surgery remains the most efficacious treatment option currently available, but only 10-20% of PC cases are resectable upon diagnosis. As of now, the sole biomarker approved by the United States Food and Drug Administration (US-FDA) for PC is carbohydrate antigen 19-9 (CA19-9); however, its use is limited for early diagnosis. An increasing number of studies have investigated a combination of biomarkers. Lately, there has been considerable interest in the application of a liquid biopsy, including the utilization of microRNAs (miRNAs), circulating tumor DNA (ctDNA), and circulating tumor cells (CTCs). Screening for PC is indicated for high-risk patients; studies on new diagnostic models combined with biomarkers for early detection have also shown promising results in terms of the ability of these models and biomarkers to aid clinicians in deciding on whether to start screening. This review seeks to provide a concise overview of the advancements in relation to existing biomarkers and explore novel strategies for the early detection of PC.

The Circulating Biomarkers League: Combining miRNAs with Cell-Free DNAs and Proteins.

The potential of liquid biopsy for the prognosis and diagnosis of diseases is unquestionable. Within the evolving landscape of disease diagnostics and personalized medicine, circulating microRNAs (c-miRNAs) stand out among the biomarkers found in blood circulation and other biological fluids due to their stability, specificity, and non-invasive detection in biofluids. However, the complexity of human diseases and the limitations inherent in single-marker diagnostics highlight the need for a more integrative approach. It has been recently suggested that a multi-analyte approach offers advantages over the single-analyte approach in the prognosis and diagnosis of diseases. In this review, we explore the potential of combining three well-studied classes of biomarkers found in blood circulation and other biofluids-miRNAs, DNAs, and proteins-to enhance the accuracy and efficacy of disease detection and monitoring. Initially, we provide an overview of each biomarker class and discuss their main advantages and disadvantages highlighting the superiority of c-miRNAs over the other classes of biomarkers. Additionally, we discuss the challenges and future directions in integrating these biomarkers into clinical practice, emphasizing the need for standardized protocols and further validation studies. This integrated approach has the potential to revolutionize precision medicine by offering insights into disease mechanisms, facilitating early detection, and guiding personalized therapeutic strategies. The collaborative power of c-miRNAs with other biomarkers represents a promising frontier in the comprehensive understanding and management of complex diseases. Nevertheless, several challenges must be addressed before this approach can be translated into clinical practice.