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PURPOSE: In addition to the existing biomarkers HER2 and PD-L1, FGFR2b has become an area of interest for the development of new targeted-based treatment. Given that clinical evaluation of FGFR2 targeted therapy is underway, we sought to elucidate the genomic landscape of FGFR2amp in gastroesophageal cancer (GEC) using a circulating tumor DNA (ctDNA) platform. MATERIALS AND METHODS: We retrospectively evaluated the Guardant Health database from 2017 to 2022 for patients with GECs with Guardant360 ctDNA next-generation sequencing (NGS) performed. We assessed co-occurring genetic alterations for patients who harbored FGFR2amp versus FGFR2null. We also explored real-world evidence database with Guardant Health, publicly available genomic databases (MSK cohort using cBioPortal), and pooled clinical data from large-volume cancer centers for FGFR2amp GECs. RESULTS: Less than 4% of patients with GEC in the Guardant Health database were identified to be FGFR2amp. The most commonly co-occurring gene mutations were TP53, CTNNB1, CDH1, and RHOA. Upon interrogation of the MSK cohort, these same genes were not significant on tissue NGS in the FGFR2amp cohort of GEC. In the pooled institutional cohort, we noted that FGFR2amp tumors were most commonly involving the gastroesophageal junction (GEJ). The overall survival of these patients was noted at 13.1 months. CONCLUSION: FGFR2 is a validated target in GECs, and the contexture of FGFR2amp will be important in defining patient subgroups with responses to FGFR2-directed therapy. Using ctDNA to provide a more detailed genomic landscape in patients with GECs will allow the advancement of targeted therapy in the near future for these aggressive cancers.

Accurate diagnosis of lung cancer is important for treatment decision-making. Tumor biopsy and histologic examination are the standard for determining histologic lung cancer subtypes. Liquid biopsy, particularly cell-free DNA (cfDNA), has recently shown promising results in cancer detection and classification. In this study, we investigate the potential of cfDNA methylome for the noninvasive classification of lung cancer histologic subtypes. We focused on the two most prevalent lung cancer subtypes, lung adenocarcinoma and lung squamous cell carcinoma. Using a fragment-based marker discovery approach, we identified robust subtype-specific methylation markers from tumor samples. These markers were successfully validated in independent cohorts and associated with subtype-specific transcriptional activity. Leveraging these markers, we constructed a subtype classification model using cfDNA methylation profiles, achieving an AUC of 0.808 in cross-validation and an AUC of 0.747 in the independent validation. Tumor copy-number alterations inferred from cfDNA methylome analysis revealed potential for treatment selection. In summary, our study demonstrates the potential of cfDNA methylome analysis for noninvasive lung cancer subtyping, offering insights for cancer monitoring and early detection.SignificanceThis study explores the use of cfDNA methylomes for the classification of lung cancer subtypes, vital for effective treatment. By identifying specific methylation markers in tumor tissues, we developed a robust classification model achieving high accuracy for noninvasive subtype detection. This cfDNA methylome approach offers promising avenues for early detection and monitoring.

Liquid biopsy is emerging as a pivotal tool in precision oncology, offering a noninvasive and comprehensive approach to cancer diagnostics and management. By harnessing biofluids such as blood, urine, saliva, cerebrospinal fluid, and pleural effusions, this technique profiles key biomarkers including circulating tumor DNA, circulating tumor cells, microRNAs, and extracellular vesicles. This review discusses the extended scope of liquid biopsy, highlighting its indispensable role in enhancing patient outcomes through early detection, continuous monitoring, and tailored therapy. While the advantages are notable, we also address the challenges, emphasizing the necessity for precision, cost-effectiveness, and standardized methodologies in its broader application. The future trajectory of liquid biopsy is set to expand its reach in personalized medicine, fueled by technological advancements and collaborative research.

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

Biomarkers for evaluating tumor response to therapy and estimating the risk of disease relapse represent tremendous areas of clinical need. To evaluate treatment efficacy, tumor response is routinely assessed using different imaging modalities like positron emission tomography/computed tomography or magnetic resonance imaging. More recently, the development of circulating tumor DNA detection assays has provided a minimally invasive approach to evaluate tumor response and prognosis through a blood test (liquid biopsy). Integrating imaging- and circulating tumor DNA-based biomarkers may lead to improvements in the prediction of patient outcomes. For this mini-review, we searched the scientific literature to find original articles that combined quantitative imaging and circulating tumor DNA biomarkers to build prediction models. Seven studies reported building prognostic models to predict distant recurrence-free, progression-free, or overall survival. Three discussed building models to predict treatment response using tumor volume, pathologic complete response, or objective response as endpoints. The limited number of articles and the modest cohort sizes reported in these studies attest to the infancy of this field of study. Nonetheless, these studies demonstrate the feasibility of developing multivariable response-predictive and prognostic models using regression and machine learning approaches. Larger studies are warranted to facilitate the building of highly accurate response-predictive and prognostic models that are generalizable to other datasets and clinical settings.

In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.

PURPOSE: VitreoDx is an experimental device enabling push-button collection of a neat vitreous liquid biopsy incidental to an intravitreal injection. We explored the ability of the device to collect a sample usable for proteomic biomarker discovery and testing. DESIGN: Pilot study using ex vivo human eyes. METHODS: Non-vitrectomized, human eyes from nine donors 75-91 years of age were refrigerated in BSS and used within 5 days of death. Four VitreoDx devices fitted with 25G needles, and four staked needle insulin syringes with 30G needles, were inserted at equal intervals through the pars plana of each eye and held in place by a fixture. The sampling mode of each VitreoDx device was triggered to attempt to acquire a liquid biopsy up to 70 μL. The plunger of each insulin syringe was retracted to attempt to obtain a liquid biopsy with a maximum volume of 50 μL. Samples acquired with the VitreoDx were extracted to polypropylene cryovials, refrigerated to -80 ºC, and sent for offsite proteomic analysis by proximity extension assay with a focus on panels containing approved and pipelined drug targets for neovascular disease and inflammatory factors. RESULTS: Of the attempted liquid biopsies with the novel 25G VitreoDx, 92% (66 of 72) resulted in successful acquisition (>25 μL) while 89% (64 of 72) attempted by a traditional 30G needle resulted in a successful acquisition. Sample volume sufficient for proteomics array analysis was acquired by the VitreoDx for every eye. Detectable protein was found for 151 of 166 unique proteins assayed in at least 25% of eyes sampled by VitreoDx. CONCLUSIONS: The high acquisition rate achieved by the prototype was similar to that achieved in previous clinical studies where a standard syringe was used with a 25G needle to biopsy vitreous fluid directly prior to standard intravitreal injection. Successful aspiration rates were likewise high for 30G needles. Together, these suggest that it is possible to routinely acquire liquid vitreous biopsies from patients who typically receive intravitreal injections with an injection device using a standard size needle without a vitreous cutter. Protein analysis shows that proteins of interest survive the sampling mechanism and may have potential to direct care in the future.

Background: The heterogeneous and complex genetic landscape of NSCLC impacts the clinical outcomes of patients who will eventually develop resistance to osimertinib. Liquid biopsy (LB) analysis as a minimally invasive approach is a key step to efficiently identify resistance mechanisms and adjust to proper subsequent treatments. Materials and methods: In the present study, we combined plasma-cfDNA and CTC analysis from 30 NSCLC patients in samples collected before treatment and at the progression of disease (PD). We detected molecular alterations at the DNA mutation (EGFR, PIK3CA, KRAS G12C, BRAF V600E), DNA methylation (RASSF1A, BRMS1, FOXA1, SLFN1, SHISA3, RARβ,, WIF-1, RASSF10 and APC), gene expression (CK-19, CK-18, CK-8, AXL, TWIST-1, PD-L1, PIM-1, Vimentin, ALDH-1 , and B2M) and chromosomal level (HER2 and MET amplification) as possible resistance mechanisms and druggable targets. We also studied the expression of PD-L1 in single CTCs using immunofluorescence. Results: In some cases, T790M resistance EGFR mutation was detected at baseline in CTCs but not in the corresponding plasma cfDNA. PIK3CA mutations were detected only in plasma-cfDNA but not in corresponding CTCs. KRAS G12C and BRAF V600E mutations were not detected in the samples analyzed. MET amplification was detected in the CTCs of two patients before treatment whereas HER2 amplification was detected in the CTCs of three patients at baseline and in one patient at PD. DNA methylation analysis revealed low concordance between CTCs and cfDNA, indicating the complementary information obtained through parallel LB analysis. Results from gene expression analysis indicated high rates of vimentin-positive CTCs detected at all time points during osimertinib. Moreover, there was an increased number of NSCLC patients at PD harboring CTCs positive in PD-L1. AXL and PIM-1 expression detected in CTCs during treatment suggesting new possible therapeutic strategies. Discussion: Our results reveal that comprehensive liquid biopsy analysis can efficiently represent the heterogeneous molecular landscape and provide prominent information on subsequent treatments for NSCLC patients at PD since druggable molecular alterations were detected in CTCs. [ABSTRACT FROM AUTHOR]

In recent years, advancements in medical treatment and imaging technologies have revolutionized the assessment of tumor response. However, the Response Evaluation Criteria in Solid Tumors (RECIST) has long been established as the gold standard for evaluating tumor treatment. As treatment modalities evolve, the need for continuous refinement and adaptation of RECIST becomes increasingly apparent. This review explores the historical evolution, current applications, limitations, and future directions of RECIST. It discusses the challenges of distinguishing true progression from pseudo-progression in ICIs (immune checkpoint inhibitors), the integration of advanced imaging tools, and the necessity for RECIST criteria tailored to specific therapies like neoadjuvant treatments. The review highlights the ongoing efforts to enhance RECIST's accuracy and reliability in clinical decision-making and the potential for developing new standards to better evaluate treatment efficacy in the rapidly evolving landscape of oncology. [ABSTRACT FROM AUTHOR]

Cancer is highly diverse and heterogeneous. Accurate and rapid analysis of the characteristics of individual cancer cells, using a complex array of big data that includes various clinicopathological features and molecular mechanisms, is crucial for advancing precision medicine. In recent years, experts in biomedical sciences and data sciences have explored the potential of artificial intelligence (AI) to analyze such extensive data sets. The next phase of AI-based medical research on cancer should focus on the practical applications of AI tools and how they can be effectively used in actual medical research settings. Recently, translational research that leverages AI and comprehensive genetic analysis data has emerged as a significant research focus. This field represents an opportunity for groundbreaking discoveries to be shared globally. To further precision medicine in clinical practice, it is vital to develop sophisticated AI tools for cancer research. These tools should not only identify potential therapeutic targets through comprehensive genetic analysis but also predict therapeutic outcomes in clinical settings. [ABSTRACT FROM AUTHOR]

Introduction: Intra‐amniotic inflammation is causally linked to spontaneous preterm labor. The gold standard for the diagnosis of intra‐amniotic inflammation is the determination of an amniotic fluid profile obtained from transabdominal amniocentesis, which is invasive. Cervicovaginal fluid fetal fibronectin (fFN) is a widely‐used predictive biomarker for spontaneous preterm labor. The aims of this study are to determine (1) whether a quantitative cervicovaginal fluid fFN test can be used to identify the presence of intra‐amniotic inflammation; and (2) an appropriate cut‐off value of a cervicovaginal fluid fFN concentration for the identification of intra‐amniotic inflammation. Material and Methods: This prospective cohort study included 78 patients with preterm labor and intact membranes who had a sample collected for quantitative cervicovaginal fluid fFN measurement and underwent transabdominal amniocentesis. Intra‐amniotic inflammation was defined as an amniotic fluid interleukin‐6 concentration ≥2.6 ng/mL. Clinicians were masked from the results of cervicovaginal fluid fFN and amniotic fluid interleukin‐6 concentrations. Logistic regression analysis was used to determine which factors were significant predictors of intra‐amniotic inflammation. The diagnostic indices of the cervicovaginal fluid fFN test for the identification of intra‐amniotic inflammation were calculated. Results: (1) Frequency of intra‐amniotic inflammation was 26.9% (21/78); (2) the higher the cervicovaginal fluid fFN concentration, the greater the risk of intra‐amniotic inflammation (p < 0.001); (3) cervicovaginal fluid fFN concentration ≥125 ng/mL had an area under the curve of 0.91 (95% confidence interval: 0.83–0.96) for the identification of intra‐amniotic inflammation with 100% sensitivity, 100% negative predictive value, 82.46% specificity and a positive likelihood ratio of 5.7; and (4) cervicovaginal fluid fFN cut‐off of 125 ng/mL had a significant higher predictive performance than the traditional cut‐off (50 ng/mL) for the identification of intra‐amniotic inflammation. Conclusions: Quantitative cervicovaginal fluid fFN with a cut‐off of 125 ng/mL had a high sensitivity and a negative predictive value as well as a positive likelihood ratio for the identification of intra‐amniotic inflammation. Its high sensitivity and negative predictive value can be used to decrease an index of suspicion of intra‐amniotic inflammation. This test may be useful as an initial assessment test to select appropriate patients for amniocentesis to determine intra‐amniotic inflammation. [ABSTRACT FROM AUTHOR]

The transformative role of artificial intelligence (AI) and multiomics could enhance the diagnostic and prognostic capabilities of liquid biopsy (LB) for lung cancer (LC). Despite advances, the transition from tissue biopsies to more sophisticated, non‐invasive methods like LB has been impeded by challenges such as the heterogeneity of biomarkers and the low concentration of tumour‐related analytes. The advent of multiomics – enabled by deep learning algorithms – offers a solution by allowing the simultaneous analysis of various analytes across multiple biological fluids, presenting a paradigm shift in cancer diagnostics. Through multi‐marker, multi‐analyte and multi‐source approaches, this review showcases how AI and multiomics are identifying clinically valuable biomarker combinations that correlate with patients' health statuses. However, the path towards clinical implementation is fraught with challenges, including study reproducibility and lack of methodological standardization, thus necessitating urgent solutions to solve these common issues. [ABSTRACT FROM AUTHOR]

Traditional tumor diagnosis methods rely on tissue biopsy, which can be invasive and unsuitable for long-term monitoring of tumor dynamics. The advent of liquid biopsy has notably improved the overall management of patients with cancer. Liquid biopsy techniques primarily involve detection of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). The present review focuses on ctDNA because of its significance in tumor diagnosis, monitoring and treatment. The use of ctDNA-based liquid biopsy offers several advantages, including non-invasive or minimally invasive collection methods, the ability to conduct repeated assessment and comprehensive insights into tumor biology. It serves crucial roles in disease management by facilitating screening of high-risk patients, dynamically monitoring therapeutic responses and diagnosis. Furthermore, ctDNA can be used to demonstrate pseudo-progression, monitor postoperative tumor status and guide adaptive treatment plans. The present study provides a comprehensive review of ctDNA, exploring its origins, metabolism, detection methods, clinical role and the current challenges associated with its application. [ABSTRACT FROM AUTHOR]

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug‐resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell‐derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO‐1 were identified on extracellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma‐derived extracellular vesicles but also Ewing sarcoma cells demonstrated proteomic expression of CD99 and ENO‐1 on their surface membranes. ENO‐1+CD63+ extracellular vesicle detection was reduced after tumor resection while both CD99+CD63+ and ENO‐1+CD63+ extracellular vesicles were detected in serum from Ewing sarcoma‐bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO‐1+CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P < 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO‐1+CD81+ extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignancy due to the difficulty in diagnosis and poor prognosis because of the high recurrence rate, necessitating reliable biomarkers to improve the diagnosis and prognosis. However, the existing markers have limitations. We previously identified extracellular vesicles (EVs) recognized by O‐glycan‐binding lectins (Amaranthus caudatus agglutinin [ACA]) as a novel diagnostic biomarker for PDAC using an EV‐counting system (ExoCounter). This retrospective study analyzed changes in ACA‐positive EVs in perioperative PDAC serum and its association with prognosis using ExoCounter. Absolute EV levels in the pre‐ and postoperative sera of 44 patients who underwent curative pancreatectomy for PDAC were quantified using ExoCounter. The carbohydrate antigen 19‐9 levels declined in most samples postoperatively, and presented no correlation with poor prognosis. In contrast, ACA‐positive EVs increased in serum at 7 days postoperatively in 27 of 44 patients (61.4%). We therefore divided participants with ACA‐positive EVs before and after surgery into elevation and decline groups. The overall survival (OS) and recurrence‐free survival (RFS) of patients with higher ACA‐positive EVs were significantly shorter than those with lower ACA‐positive EVs (26.1 months vs. not reached, P = 0.018; 11.9 vs. 38.6 months, P = 0.013). Multivariable analysis revealed that ACA‐positive EV elevation in postoperative serum was an independent prognostic factor for poor OS (hazard ratio [HR] = 3.891, P = 0.023) and RFS (HR = 2.650, P = 0.024). The detection of ACA‐positive EVs in perioperative serum may be used to predict the prognosis of PDAC in the early postoperative period. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) comprises two clinically relevant molecular subtypes that are currently determined using tissue biopsies, which are spatially biased and highly invasive. We used whole transcriptome sequencing of 10 plasma samples with tumor‐informed subtypes, complemented by proteomic analysis for minimally invasive identification of PDAC subtype markers. Data were validated in independent large cohorts and correlated with treatment response and patient outcome. Differential transcript abundance analyses revealed 32 subtype‐specific, protein‐coding cell‐free RNA (cfRNA) transcripts. The subtype specificity of these transcripts was validated in two independent tissue cohorts comprising 195 and 250 cases, respectively. Three disease‐relevant cfRNA‐defined subtype markers (DEGS1, KDELC1, and RPL23AP7) that consistently associated with basal‐like tumors across all cohorts were identified. In both tumor and liquid biopsies, the overexpression of these markers correlated with poor survival. Moreover, elevated levels of the identified markers were linked to a poor response to systemic therapy and early relapse in resected patients. Our data indicate clinical applicability of cfRNA markers in determining tumor subtypes and monitoring disease recurrence. [ABSTRACT FROM AUTHOR]

In the over 80 years since androgens were found to play a pivotal role in prostate cancer (PCa) progression, androgen deprivation therapy (ADT) has been a cornerstone in treating advanced PCa. Castration‐resistant PCa persists, however, with some of these tumors evolving to androgen receptor (AR)‐independent forms like neuroendocrine PCa. The development of novel diagnostic and therapeutic approaches to PCa is therefore crucial. This review provides an overview of recent basic research in PCa, focusing on two main areas: PCa cells and their tumor microenvironments. The first section describes current knowledge on the intricate mechanisms of AR signaling pathways, emphasizing the roles of coactivators and chromatin state alterations in gene regulation. Genomic analyses have revealed recurrent mutations and copy number alterations critical for precision medicine. Liquid biopsy has become a promising tool for real‐time tumor monitoring, identifying genetic alterations in circulating‐tumor DNA or extracellular vesicles. The second section describes the tumor microenvironment of PCa, highlighting its immunosuppressive landscape and the potential of combining ADT with immunotherapy. Advanced techniques, including single‐cell RNA sequencing and spatial transcriptomics offer insights into cellular heterogeneity and interactions within the tumor microenvironment, paving the way for novel therapeutic strategies. Integration of these diverse research areas will provide a comprehensive understanding of the current state and future directions of PCa research, underscoring the importance of personalized medicine and the dynamic nature of cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Uveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines. Methods: Four primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared. Results: In all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA. Conclusions: This data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool. [ABSTRACT FROM AUTHOR]

Glioblastoma remains the most prevalent and aggressive primary malignant brain tumor in adults, characterized by limited treatment options and a poor prognosis. Previous drug repurposing efforts have yielded only marginal survival benefits, particularly those involving inhibitors targeting receptor tyrosine kinase and cyclin-dependent kinase-retinoblastoma pathways. This limited efficacy is likely due to several critical challenges, including the tumor's molecular heterogeneity, the dynamic evolution of its genetic profile, and the restrictive nature of the blood-brain barrier that impedes effective drug delivery. Emerging diagnostic tools, such as circulating tumor DNA and extracellular vesicles, offer promising non-invasive methods for real-time tumor monitoring, potentially enabling the application of targeted therapies to more selected patient populations. Moreover, innovative drug delivery strategies, including focused ultrasound, implantable drug-delivery systems, and engineered nanoparticles, hold potential for enhancing the bioavailability and therapeutic efficacy of treatments. [ABSTRACT FROM AUTHOR]

Introduction: Approximately 50% of patients diagnosed with colorectal cancer develop colorectal cancer liver metastases (CRLM). Although curative intent liver resection provides 5-year survival of 40-50%, up to 70% of patients develop recurrence of CRLM. Detection of minimal residual disease (MRD) is essential for timely, optimized treatment. This study evaluated the feasibility and utility of using circulating tumor DNA (ctDNA) to identify MRD and predict disease recurrence. Methods: Patients with CRLM that underwent liver resection and had known KRAS or PIK3CA mutations were retrospectively identified. Serial blood samples were collected every 3 months following surgery for disease surveillance. ctDNA was isolated from the samples and analyzed with digital PCR (dPCR). Results: KRAS and PIK3CA mutations were identified by dPCR in 29 patients over 115 timepoints. In patients with detectable ctDNA at time of liver resection, 81% (13/16) developed disease recurrence, while 46% (6/13) of the patients with undetectable ctDNA recurred (p=0.064). Presence of ctDNA was detected in 27.6% (8/29) of the initial postoperative samples. Radiologic recurrence was later diagnosed in 100% (8/8) of these patients, while 52% (11/21) who had undetectable ctDNA postoperatively recurred (p=0.026). Detectable ctDNA postoperatively was associated with a shorter disease-free survival (DFS) of 9 months vs 13 months in patients who had undetectable ctDNA (HR 2.95, 95% CI 1.16-7.49; p=0.02). Conclusion: Liquid biopsy using dPCR can identify low levels of ctDNA, enabling early detection of disease recurrence. Additionally, the presence of ctDNA postoperatively was predictive of recurrence. This study corroborates current literature and provides rational for moving toward a clinical trial using ctDNA and dPCR to detect MRD after CRLM resection. [ABSTRACT FROM AUTHOR]

Background: We developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples. Methods: This study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance. Results: Eighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25). Conclusion: The ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer is a public health issue and was the third leading cause of cancer-related death worldwide in 2022. Early diagnosis can improve prognosis, making screening a central part of colorectal cancer management. Blood-based screening, diagnosis and follow-up of colorectal cancer patients are possible with the study of cell-free circulating tumor DNA. This study aimed to identify novel DNA methylation biomarkers of colorectal cancer that can be used for the follow-up of patients with colorectal cancer. Methods: A DNA methylation profile was established in the Gene Expression Omnibus (GEO) database (n = 507) using bioinformatics analysis and subsequently confirmed using The Cancer Genome Atlas (TCGA) database (n = 348). The in silico profile was then validated on local tissue and cell-free DNA samples using methylation-specific digital PCR in colorectal cancer patients (n = 35) and healthy donors (n = 35). Results: The DNA methylation of COL25A1 and METAP1D was predicted to be a colorectal cancer biomarker by bioinformatics analysis (ROC AUC = 1, 95% CI [0.999–1]). The two biomarkers were confirmed with tissue samples, and the combination of COL25A1 and METAP1D yielded 49% sensitivity and 100% specificity for cell-free DNA. Conclusion: Bioinformatics analysis of public databases revealed COL25A1 and METAP1D DNA methylation as clinically applicable liquid biopsies DNA methylation biomarkers. The specificity implies an excellent positive predictive value for follow-up, and the high sensitivity and relative noninvasiveness of a blood-based test make these biomarkers compatible with colorectal cancer screening. However, the clinical impact of these biomarkers in colorectal cancer screening and follow-up needs to be established in further prospective studies. [ABSTRACT FROM AUTHOR]

Background: For early detection and postoperative monitoring of upper tract urothelial carcinoma (UTUC), the traditional detection method was limited to its invasiveness and insufficient sensitivity. We aim to use urine tumour DNA (utDNA) for detecting minimal residual disease (MRD), early diagnosis and perioperative monitoring in UTUC. Method: We previously established a utDNA multidimensional bioinformatic valuation model, named utLIFE, using low‐coverage whole‐genome sequencing and targeted deep sequencing. This prospective cohort enrolled 93 patients diagnosed with UTUC without metastasis. We collected morning urine samples on the day of surgery and the discharge day after the operation for utLIFE testing. In addition, we also enrolled 80 healthy controls to further validate the specificity of the utLIFE model in the study. Results: The utLIFE of preoperative samples could discriminate UTUC with high specificity (96.25%, 77/80), and high sensitivity (96.77%, 90/93) regardless of stage and grade. The sensitivity of utLIFE was significantly higher than urine cytology (p < 0.001) and fluorescence in situ hybridisation (FISH) (p < 0.001) (N = 19), especially in early‐stage and low‐grade UTUC. Postoperative utLIFE scores were significantly decreased compared with those of preoperative samples (79 vs. 36, p < 0.001), indicating its association with tumour burden. For special pathology types, utLIFE performed less well in sensitivity and perioperative alteration. Conclusion: In conclusion, we established a bioinformatic utDNA valuation model, utLIFE, which was validated to be a rapid and noninvasive approach with high sensitivity for early detection and MRD monitoring for UTUC. [ABSTRACT FROM AUTHOR]

Background/Objectives: Currently, the prediction of disease recurrence after radical prostatectomy (RP) in localized prostate cancer (PCa) relies on clinicopathological parameters, which lack accuracy in predicting clinical outcomes. This study focused on evaluating the utility of cfDNA levels and fragmentation patterns as prognostic biomarkers in progressive prostate-specific antigen (PSA) patients, including those with persistent PSA and biochemical recurrence (BR), after primary treatment in localized PCa patients. Methods: Twenty-nine high-risk localized PCa patients were enrolled in the study between February 2022 and May 2023. Blood samples were obtained before robotic RP. cfDNA concentration and fragment size were quantified using the Quant-it PicoGreen dsDNA Assay kit and Agilent 2200 TapeStation System, respectively. Results: The mean PSA value at diagnosis was 9.4 ng/mL. Seven patients (24.1%) had stage pT2 and 22 (75.9%) pT3. Nine patients (31%) had detectable PSA at the first PSA control six weeks after surgery, and four patients (20%) had BR during a mean follow-up of 18.4 months. No associations were found between cfDNA levels or fragmentation patterns and clinicopathological data. Although not statistically significant, patients with detectable PSA levels post-surgery exhibited higher cfDNA levels and shorter fragments compared with those with undetectable PSA. Conclusions: Our study indicated a tendency toward more fragmented cfDNA levels in PCa patients with persistent PSA. Strikingly, biochemical recurrent PCa patients exhibited similar cfDNA levels and fragmentation patterns compared to non-recurrent patients. Further studies exploring liquid biopsy-derived biomarkers in localized PCa patients are needed to elucidate their clinical utility in predicting PSA persistence. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) has a rising incidence and poor prognosis due to late diagnosis and limited treatments. Currently, treatment is based solely on TNM staging, without considering molecular tumor characterization. In the present study, we validated a combined amplification refractory mutation system (ARMS) and high-resolution melting analysis (HRMA) technique for detecting mutations in codon 12 of KRAS in PDAC tumor and plasma samples and assessed its prognostic value. We included 88 newly diagnosed PDAC patients, treated with either surgery, chemotherapy, or best supportive treatment only. Both tumor and plasma samples were analyzed, and the most frequent mutations were G12D (36%) and G12V (25%). KRAS mutations G12D and/or G12C in tumors and plasma were associated with lower progression-free survival (PFS) and overall survival (OS) independently of disease stage or treatment performed. ARMS–HRMA offers a rapid, cost-effective method for detecting KRAS mutations and can aid in prognosis and treatment decisions. Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) incidence is rising, and prognosis remains poor due to late diagnosis and limited effective therapies. Currently, patients are treated based on TNM staging, without molecular tumor characterization. This study aimed to validate a technique that combines the amplification refractory mutation system (ARMS) with high-resolution melting analysis (HRMA) for detecting mutations in codon 12 of KRAS in tumor and plasma, and to assess its prognostic value. Methods: Prospective study including patients with newly diagnosed PDAC with tumor and plasma samples collected before treatment. Mutations in codon 12 of KRAS (G12D, G12V, G12C, and G12R) were detected using ARMS–HRMA and compared to Sanger sequencing (SS). Univariate and multivariate analyses were used to evaluate the prognostic significance of these mutations. Results: A total of 88 patients, 93% with ECOG-PS 0–1, 57% with resectable disease. ARMS–HRMA technique showed a higher sensitivity than SS, both in tumor and plasma (77% vs. 51%; 25 vs. 0%, respectively). The most frequent mutation was G12D (n = 32, 36%), followed by G12V (n = 22, 25%). On multivariate analysis, patients with G12D and/or G12C mutations, either in tumor or plasma, had lower PFS (HR 1.792, 95% CI 1.061–3.028, p = 0.029; HR 2.081, 95% CI 1.014–4.272, p = 0.046, respectively) and lower OS (HR 1.757, 95% CI 1.013–3.049, p = 0.045; HR 2.229, 95% CI 1.082–4.594, p = 0.030, respectively). Conclusions: ARMS–HRMA is a rapid and cost-effective method for detecting KRAS mutations in PDAC patients, offering the potential for stratifying prognosis and guiding treatment decisions. The presence of G12D and G12C mutations in both tumor and plasma is associated with a poorer prognosis. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) remains a malignancy with one of the highest mortality rates. One limitation in the diagnosis and treatment of PDAC is the lack of an early and universal biomarker. Extensive research performed recently to develop new assays which could fit this role is available. In this review, we will discuss the current landscape of liquid biopsy in patients with PDAC. Specifically, we will review the various methods of liquid biopsy, focusing on circulating tumor DNA (ctDNA) and exosomes and future opportunities for improvement using artificial intelligence or machine learning to analyze results from a multi-omic approach. We will also consider applications which have been evaluated, including the utility of liquid biopsy for screening and staging patients at diagnosis as well as before and after surgery. We will also examine the potential for liquid biopsy to monitor patient treatment response in the setting of clinical trial development. [ABSTRACT FROM AUTHOR]

Background: Tumor genomic profiling has a significant impact on the selection of targeted therapy. Circulating tumor DNA (ctDNA) has emerged as a noninvasive, and reproducible assay compared with tissue biopsy. We aimed to evaluate its utility in identifying mutations and guiding targeted therapy for lung cancer. Methods: A total of 173 lung cancer patients underwent next-generation sequencing (NGS) using a targeted enrichment panel covering 20 lung cancer-related genes. The performance of the ctDNA NGS assay in identifying genetic mutations or alterations was compared with tissue biopsy and droplet digital PCR (ddPCR). The treatment response to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapies based on the ctDNA assay results was also assessed. Results: The ctDNA was detected in 61.85% of patients. Tissue mutations were detected in paired ctDNA in 38.57% of cases, while ctDNA mutations were detected in paired tissues in 89.1% of cases. The ctDNA increased the number of advanced non-small cell lung cancer (NSCLC) patients who received NCCN-recommended genetic testing by 12%. The concordance between ddPCR and ctDNA was relatively high reaching 99.43%. EGFR T790M/C797S c.G2390C and EGFR T790M/C797S c.T2389A were detected in tissue and ctDNA, respectively, in patient 01015. Moreover, ctDNA assay identified the EGFR T790M mutation, which was missed by tissue biopsy in patient 01149, who developed drug resistance after 1 year of EGFR-TKI therapy. Of the 17 patients who received EGFR-TKI targeted therapies based on the ctDNA NGS results, 12 patients achieved a partial response and two patients had stable disease. Conclusions: The results demonstrated that the ctDNA assay could partially overcome tumor heterogeneity in detecting mutations and provide complementary information on tumor genomic profiles. Moreover, the presence of EGFR mutations in ctDNA could offer valuable guidance for selecting appropriate EGFR-TKI treatment for advanced lung cancer patients. However, it is important to note that the ctDNA NGS assay has certain limitations in fully identifying all genomic alterations present in the tumor. [ABSTRACT FROM AUTHOR]

Purpose: Although cystoscopy is a reliable tool for detecting bladder cancer, it poses a high burden on patients and entails high costs. This highlights the need for non-invasive and cost-effective alternatives. This study aimed to validate a previously developed urinary methylation marker panel containing GHSR and MAL. Methods: We enrolled 134 patients who underwent cystoscopy because of hematuria, including 63 individuals with primary bladder cancer and 71 with non-malignant findings. Urine samples were self-collected at home and sent via regular mail. Subsequently, DNA was extracted and the hypermethylation of GHSR and MAL was evaluated using quantitative methylation-specific polymerase chain reaction. The performance of methylation markers was assessed using area-under-the-curve (AUC) analysis and sensitivity and specificity based on pre-established cut-off values. Results: Validation of the marker panel GHSR/MAL resulted in an AUC of 0.87 at 79% sensitivity and 80% specificity. Sensitivity was comparable to the previous investigation (P > 0.9), though specificity was significantly lower (P = 0.026). Sensitivity was higher for high-grade tumors compared to low-grade tumors (94% vs. 60%, P = 0.002). Conclusion: Validation of the GHSR/MAL methylation marker panel on at home collected urine samples confirms its robust performance for bladder cancer detection in a hematuria population, and underscores the diagnostic potential for future clinical application. [ABSTRACT FROM AUTHOR]

Monitoring tumor evolution and predicting survival using non-invasive liquid biopsy is an unmet need for glioblastoma patients. The era of proteomics and metabolomics blood analyzes, may help in this context. A case–control study was conducted. Patients were included in the GLIOPLAK trial (ClinicalTrials.gov Identifier: NCT02617745), a prospective bicentric study conducted between November 2015 and December 2022. Patients underwent biopsy alone and received radiotherapy and temozolomide. Blood samples were collected at three different time points: before and after concomitant radiochemotherapy, and at the time of tumor progression. Plasma samples from patients and controls were analyzed using metabolomics and proteomics, generating 371 omics features. Descriptive, differential, and predictive analyses were performed to assess the relationship between plasma omics feature levels and patient outcome. Diagnostic performance and longitudinal variations were also analyzed. The study included 67 subjects (34 patients and 33 controls). A significant differential expression of metabolites and proteins between patients and controls was observed. Predictive models using omics features showed high accuracy in distinguishing patients from controls. Longitudinal analysis revealed temporal variations in a few omics features including CD22, CXCL13, EGF, IL6, GZMH, KLK4, and TNFRSP6B. Survival analysis identified 77 omics features significantly associated with OS, with ERBB2 and ITGAV consistently linked to OS at all timepoints. Pathway analysis revealed dynamic oncogenic pathways involved in glioblastoma progression. This study provides insights into the potential of plasma omics features as biomarkers for glioblastoma diagnosis, progression and overall survival. Clinical implication should now be explored in dedicated prospective trials. Key points: Circulating omic features distinguishes patients with glioblastoma from healthy subjects. Plasma proteomic features change over time in patients undergoing radiochemotherapy. Certain plasma proteins are correlated with survival. [ABSTRACT FROM AUTHOR]

A common feature of advanced solid tumors is their ability to metastasize and colonize distant organs, including the Central Nervous System (CNS), which encompasses brain and leptomeningeal metastases (LM). While cerebrospinal fluid cytopathological analysis remains a gold standard diagnostic tool, it only provides limited insights into the biology of tumor cells; thus, it is urgent to develop minimally invasive biomarkers that enable a comprehensive quantitative and molecular characterization of disseminated cells, therapy response assessment, and disease monitoring. Liquid biopsy methods have been swiftly developed for some readily accessible bodily fluids such as plasma and urine; circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from these sources have been rapidly implemented into clinical trial design, disease monitoring, and treatment assignment across different tumor types. However, the filter imposed by the brain blood barrier (BBB) hampers the release of tumorderived cells and molecules from CNS metastases. Crucially, cerebrospinal fluid (CSF) liquid biopsy methods offer a unique and unparallel source to develop liquid biopsy methodologies in patients with CNS-disseminated disease, including the characterization of CTCs and ctDNA arising specifically from brain and leptomeningeal metastasis. These technologies have enabled a deeper understanding of tumor cell and molecular dynamics, including the reconstruction of clonal evolution in the brain microenvironment through longitudinal sapling. Here, we discuss the current challenges and opportunities that CSF liquid biopsy methods face for the implementation of these approaches into clinical settings. [ABSTRACT FROM AUTHOR]

Cell-derived extracellular vesicles (EVs), which carry diverse biomolecules such as nucleic acids, proteins, metabolites, and lipids reflecting their cell of origin, are released under both physiological and pathological conditions. EVs have been demonstrated to mediate cell-to-cell communication and serve as biomarkers. EV-associated DNA (EV-DNA) comprises genomic and mitochondrial DNA (i.e., gDNA and mtDNA) fragments. Some studies have revealed that EV-DNA can represent the full nuclear genome and mitochondrial genome of parental cells. Furthermore, DNA fragments loaded into EVs are stable and can be transferred to recipient cells to regulate their biological functions. In this review, we summarized and discussed EV-DNA research advances with an emphasis on EV-DNA detection at the population-EV and single-EV levels, gene transfer-associated biological functions, and clinical applications as biomarkers for disease liquid biopsy. We hope that this review will provide potential directions or guidance for future EV-DNA investigations. [ABSTRACT FROM AUTHOR]

Background: Today, the prognostic tools available at the time of diagnosis in colorectal cancer (CRC) are limited. Better prognostic tools are a prerequisite for personalised treatment. This study aimed to investigate whether circulating tumour DNA (ctDNA) markers found in plasma before clinical diagnosis of CRC could contribute to the prediction of poor prognosis. Methods: This observational cohort study included patients diagnosed with CRC stage I-III within 24 months following participation in the Trøndelag Health Study (n = 85). Known methylated ctDNA biomarkers of CRC were analysed by PCR in plasma. Outcomes were overall survival (OS), recurrence-free survival (RFS) and poor prognosis (PP). Candidate clinical and methylated ctDNA predictors of the outcomes were identified by Cox regression analyses. Results: Methylated GRIA4 (HR 1.96 (1.06–3.63)), RARB (HR 9.48 (3.00–30.00)), SLC8A1 (HR 1.97 (1.03–3.77)), VIM (HR 2.95 (1.22–7.14)) and WNT5A (HR 5.83 (2.33–14.56)) were independent predictors of OS, methylated RARB (HR 9.67 (2.54–36.81)), SDC2 (HR 3.38 (1.07–10.66)), SLC8A1 (HR 2.93 (1.01–8.51)) and WNT5A (HR 6.95 (1.81–26.68)) were independent predictors of RFS and methylated RARB (HR 6.11 (1.69–22.18)), SDC2 (HR 2.79 (1.20–6.49)) and WNT5A (HR 5.57 (3.04–15.26)) were independent predictors of PP (p < 0.05). Conclusions: Prediagnostic ctDNA markers are promising contributors to predicting poor prognosis in CRC, potentially becoming one of the tools guiding more personalised treatment. [ABSTRACT FROM AUTHOR]

Background: Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. Methods: We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. Results: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. Conclusions: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays. [ABSTRACT FROM AUTHOR]

Liquid biopsy is a minimally invasive method that uses biofluid samples instead of tissue samples for cancer diagnosis. Exosomes are small extracellular vesicles secreted by donor cells and act as mediators of intercellular communication in human health and disease. Due to their important roles, exosomes have been considered as promising biomarkers for liquid biopsy. However, traditional methods for exosome isolation and cargo detection methods are time-consuming and inefficient, limiting their practical application. In the past decades, many new strategies, such as microfluidic chips, nanowire arrays and electrochemical biosensors, have been proposed to achieve rapid, accurate and high-throughput detection and analysis of exosomes. In this review, we discussed about the new advance in exosome-based liquid biopsy technology, including isolation, enrichment, cargo detection and analysis approaches. The comparison of currently available methods is also included. Finally, we summarized the advantages and limitations of the present strategies and further gave a perspective to their future translational use. [ABSTRACT FROM AUTHOR]

Background: A blood-based diagnostic test is a promising strategy for colorectal cancer (CRC). The MethyDT test (IColohunter), which detects methylation levels of NTMT1 and MAP3K14-AS1, exhibited potential in discriminating CRC, but its clinical performance needs to be validated in large-scale populations. Methods: A multicenter, double-blinded, cross-sectional study that enrolled 1194 participants was performed. Plasma samples were collected to detect methylation levels of NTMT1 and MAP3K14-AS1 using quantitative methylation-specific PCR with the MethyDT test, and the accuracy was further evaluated by Sanger sequencing. Results: The sensitivities of the MethyDT test for detecting CRC, early stages of CRC (I and II), advanced adenoma (AA), and high-grade intraepithelial neoplasia (HGIN) were 91.2% (95% confidence interval [CI], 88.4–94.0), 87.4% (95% CI, 82.5–92.2), 43.5% (95% CI, 35.7–51.4), and 72.7% (95% CI, 57.5–87.9), respectively. The specificities for participants with non-AA, interfering diseases (ID), and no evidence of disease (NED) were 85.0% (95% CI, 78.8–91.3), 93.7% (95% CI, 91.4–95.9) and 97.3% (95% CI, 90.5–99.7), respectively, and its overall specificity for all-controls was 92.4% (95% CI, 90.3–94.4). The consistency of the MethyDT test with pathology for CRC was high with a kappa value of 0.830 (95% CI, 0.795–0.865). Additionally, the MethyDT test was comparable to Sanger sequencing for detecting methylation with kappa values > 0.97. Conclusions: The MethyDT test demonstrates excellent sensitivity and specificity for CRC and high consistency with Sanger sequencing for methylation, suggesting it may serve as a potential noninvasive diagnostic tool for the detection of CRC. Trial registration: This clinical trial has been registered in ClinicalTrials.gov (NCT05508503). [ABSTRACT FROM AUTHOR]

While cell‐free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell‐based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC‐derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional in vitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult‐to‐treat cancers such as SGC. [ABSTRACT FROM AUTHOR]

Introduction: The presence of minimal residual disease (MRD) after curativeintent surgery for early-stage cancers is associated with disease recurrence. Circulating tumour deoxyribonucleic acid (ctDNA) has emerged as a promising biomarker for MRD assessment in patients with colorectal cancer (CRC) who have undergone surgery or completed adjuvant therapy. MRD tests are already available for use in clinics; however, treatment decisions following MRD results obtained in routine practice are infrequently described. Methods: In this observational study, we report on the real-world clinical use of Guardant Reveal, a validated tissue-free MRD assay, in the first 215 consecutive patients (279 samples) with CRC tested in Asia and the Middle East. Results: Overall, 22% of patients had ctDNA detected in their first MRD test, and the frequency of ctDNA positivity increased with increasing tumour stage. 132 samples were tested with an earlier version of Guardant Reveal, one that assessed both genomic and epigenomic features. An updated version of the assay assesses only ctDNA methylation data and was used for the remaining 147 samples. In patients with stage II CRC, 71% of tests were ordered within 12 weeks after tumour resection, while for patients with stage III disease, 69% of tests were ordered after completion of all curative-intent treatment. Discussion: Clinical cases utilizing tissue-free MRD assessment are described. [ABSTRACT FROM AUTHOR]

Background: Lung cancer (LC) is the most common form of cancer in the world. Of the proteins involved in cell differentiation and proliferation, the epidermal growth factor receptor (EGFR) is among the most significant. Amino acids play a crucial role in cell physiology as metabolic regulators. The benefits of liquid biopsies are their non-invasive nature, ease of collection, and ability to depict the entire tumor's status. The present study is designed to detect the relation between the EGFR exon 19 747–750 deletion mutation and lung cancer and investigate the patterns of alterations of plasma-free amino acids (PFAA) in lung cancer patients of different histopathological types and stages as biomarkers for early detection of lung cancer. Methods: The study sample comprised 60 lung cancer patients and 60 age- and sex-matched healthy individuals as the control group. Polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) were used to examine the EGFR exon 19 747–750 deletion mutation, and an AA analyzer was used to quantify the plasma free amino acid (PFAA) profile. Results: Compared with controls, LC patients had significantly higher levels of three AAs and significantly lower levels of fifteen AAs. Thirteen AAs varied significantly between stages I and II. In the lung cancer group, the percentage of cases of mutant EGFR exon-19 deletion increased to 30% from 13.3% in the control group. The histological forms of lung cancer did not significantly differ in this rise. Valine and citrulline plasma levels were substantially greater in the mutant than in the wild-type. Lysine, histidine, and methionine were the independent predictors of the LC group in multivariate analysis. Conclusion: Lung cancer development is influenced by the EGFR exon 19 747–750 deletion mutation, and the prognosis and early prediction of lung cancer are greatly affected by the amino acid profile concentrations. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related deaths in Canada, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Timely access to comprehensive molecular profiling is critical for selecting biomarker-matched targeted therapies, which lead to improved outcomes in advanced NSCLC. Tissue biopsy samples are the gold standard for molecular profiling; however, several challenges can prevent timely and complete molecular profiling from being performed, causing delays in treatment or suboptimal therapy selection. Liquid biopsy offers a minimally invasive method for molecular profiling by analyzing circulating tumour DNA (ctDNA) and RNA (cfRNA) in plasma, potentially overcoming these barriers. This paper discusses the outcomes of a multidisciplinary working group in Ontario, which proposed three eligibility criteria for liquid biopsy reimbursement: (1) insufficient tissue for complete testing or failed tissue biomarker testing; (2) suspected advanced NSCLC where tissue biopsy is not feasible; and (3) high-risk patients who may deteriorate before tissue results are available. The group also addressed considerations for assay selection, implementation, and economic impact. These discussions aim to inform reimbursement and implementation strategies for liquid biopsy in Ontario's public healthcare system, recognizing the need for ongoing evaluation as technology and evidence evolve. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell‐free plasma or serum. Because available one‐step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2‐step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV‐ and tumour‐biomarkers in pure FPLC‐derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases‐associated biomarkers in pure EVs, supporting the feasibility of using single‐vesicle analysis for liquid biopsy. [ABSTRACT FROM AUTHOR]

Using microRNAs (miRNAs) as potential circulating biomarkers in diagnosing and treating glioblastoma (GBM) has garnered a lot of scientific and clinical impetus in the past decade. As an aggressive primary brain tumor, GBM poses challenges in early detection and effective treatment with significant current diagnostic constraints and limited therapeutic strategies. MiRNA dysregulation is present in GBM. The intricate involvement of miRNAs in altering cell proliferation, invasion, and immune escape makes them prospective candidates for identifying and monitoring GBM diagnosis and response to treatment. These miRNAs could play a dual role, acting as both potential diagnostic markers and targets for therapy. By modulating the activity of various oncogenic and tumor-suppressive proteins, miRNAs create opportunities for precision medicine and targeted therapies in GBM. This review centers on the critical role and function of circulating miRNA biomarkers in GBM diagnosis and treatment. It highlights their significance in providing insights into disease progression, aiding in early diagnosis, and potential use as targets for novel therapeutic interventions. Ultimately, the study of miRNA would contribute to improving patient outcomes in the challenging landscape of GBM management. [ABSTRACT FROM AUTHOR]

Cancer is a prominent global cause of mortality, primarily due to delayed detection leading to limited treatment options. Current screening methods are mostly invasive and involve complex lengthy processes with high costs. Moreover, each screening typically focuses on a single type of cancer. This imposes a growing need for innovative, precise, and minimally invasive methods for early cancer detection. With the current advances in assay technologies and data science, multi-cancer early detection (MCED) tests are gaining increased interest in the research community as they offer potential for earlier diagnosis and improved patient outcomes. Different approaches are followed for MCED, and multiple machine learning methods are considered. In this paper, we systematically explore various MCED studies and their applied machine learning (ML) models for different types of biomarker data. We discuss the strengths and limitations of different study designs and compare their performance. Future directions are proposed, emphasizing the importance of integrating multi-omics data, enhancing model transparency, and fostering collaborative efforts to develop robust, cost effective and clinically applicable MCED tools. [ABSTRACT FROM AUTHOR]

Problem: Preeclampsia is a heterogeneous syndrome of diverse etiologies and molecular pathways leading to distinct clinical subtypes. Herein, we aimed to characterize the extracellular vesicle (EV)‐associated and soluble fractions of the maternal plasma proteome in patients with preeclampsia and to assess their value for disease prediction. Method of Study: This case–control study included 24 women with term preeclampsia, 23 women with preterm preeclampsia, and 94 healthy pregnant controls. Blood samples were collected from cases on average 7 weeks before the diagnosis of preeclampsia and were matched to control samples. Soluble and EV fractions were separated from maternal plasma; EVs were confirmed by cryo‐EM, NanoSight, and flow cytometry; and 82 proteins were analyzed with bead‐based, multiplexed immunoassays. Quantile regression analysis and random forest models were implemented to evaluate protein concentration differences and their predictive accuracy. Preeclampsia subgroups defined by molecular profiles were identified by hierarchical cluster analysis. Significance was set at p < 0.05 or false discovery rate‐adjusted q < 0.1. Results: In preterm preeclampsia, PlGF, PTX3, and VEGFR‐1 displayed differential abundance in both soluble and EV fractions, whereas angiogenin, CD40L, endoglin, galectin‐1, IL‐27, CCL19, and TIMP1 were changed only in the soluble fraction (q < 0.1). The direction of changes in the EV fraction was consistent with that in the soluble fraction for nine proteins. In term preeclampsia, CCL3 had increased abundance in both fractions (q < 0.1). The combined EV and soluble fraction proteomic profiles predicted preterm and term preeclampsia with an AUC of 78% (95% CI, 66%–90%) and 68% (95% CI, 56%–80%), respectively. Three clusters of preeclampsia featuring distinct clinical characteristics and placental pathology were identified based on combined protein data. Conclusions: Our findings reveal distinct alterations of the maternal EV‐associated and soluble plasma proteome in preterm and term preeclampsia and identify molecular subgroups of patients with distinct clinical and placental histopathologic features. [ABSTRACT FROM AUTHOR]

Background and Aims: Cancer has grabbed the attention of scientists and medical professionals all over the world much more than any other disease. In the past few decades, the medical field has improved quite a lot but progress in the path to find a solution for cancer is very less. As the popularity of invasive technologies is diminishing in cancer treatment, scientists have come up withminimally invasive or non‐invasive alternatives, among which liquid biopsy, by far is the most suitable. Methods: Liquid biopsy is used to analyse nucleic acids, subcellular components and circulating tumour cells in various biological fluids for diagnosis of cancer. It can also be used to know the efficacy of cancer drugs in a patient by analysing multiple samples. Outcomes: Liquid biopsy is becoming standard of care as it allows biopsy of those samples in which solid tumour biopsies are not possible. The diversity of sampling procedures, such as collection of urine for urothelial carcinoma or bladder or prostate cancer and phlebotomy for other types of cancer, make liquid biopsy one of the best methods for diagnosis of cancer. Conclusion: This review aims in discussing the several techniques used for the detection of cancer biomarkers and some clinical manifestations due to the changes in the biomarkers which are analysed by liquid biopsy. [ABSTRACT FROM AUTHOR]

Background: High-grade serous carcinoma (HGSC) is the most lethal of gynecological cancers in developed countries. It usually presents late with non-specific symptoms and most cases are diagnosed at an advanced stage, with 5-year overall survival being around 40%. Biomarkers for screening and early diagnosis of this aggressive disease are, thus, a research priority. Extracellular vesicles (EVs) that reflect the cell of origin and that can be isolated from local fluid and plasma by minimally invasive liquid biopsy are such promising biomarkers. Besides EV concentration and molecular profile, which have been the main focus of research for many years, recent studies have also called attention to EV size distribution. The aim of our study was to evaluate the potential of EV concentration and size distribution in local fluid and plasma as diagnostic biomarkers for HGSC. Methods: Paired pretreatment ascites and plasma samples from 37 patients with advanced HGSC and paired pretreatment free peritoneal fluid (FPF) and plasma samples from 40 controls with benign ovarian pathology (BOP) were analyzed using nanoparticle tracking analysis (NTA). Results: We observed a significant difference in EV concentration in local fluid, but not in plasma, between HGSC patients and the control group. We also found a significant difference in EV size distribution in both local fluid and plasma between HGSC patients and the control group. The receiver operating characteristics (ROC) curve analysis of EV characteristics showed excellent diagnostic performance for the mode, D10, and D50 in local fluid and acceptable diagnostic performance for EV concentration and mean EV size in local fluid, as well as for the mode and D10 value in plasma. Conclusions: The results of our study show that EV concentration in local fluid and more importantly EV size distribution in both local fluid and plasma are significantly changed in the presence of HGSC. Future research of size-dependent molecular profiling of EVs could help identify novel diagnostic biomarkers for HGSC. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroendocrine neoplasms, primarily found in the gastroenteropancreatic tract, are classified as neuroendocrine tumours and neuroendocrine carcinomas, depending on the grade. Liquid biopsy offers a less invasive alternative to tissue biopsy in detecting circulating tumour components in body fluids. This systematic review evaluates liquid biopsy applications in neuroendocrine neoplasms, approaching its various types in different settings, such as diagnosis and characterisation; prognostic and predictive value; monitoring treatment response and prediction of recurrence; and finally, current applications and future perspectives. Overall, liquid biopsy holds potential for managing neuroendocrine neoplasms, and its use should be standardised. Neuroendocrine neoplasms incidence has been increasing, arising the need for precise and early diagnostic tools. Liquid biopsy (LB) offers a less invasive alternative to tissue biopsy, providing real-time molecular information from circulating tumour components in body fluids. The aim of this review is to analyse the current evidence concerning LB in NENs and its role in clinical practice. We conducted a systematic review in July 2024 focusing on LB applications in NENs, including circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), micro RNA (miRNA), messenger RNA (mRNA) and extracellular vesicles. Sixty-five relevant articles were analysed. The LB showed potential in diagnosing and monitoring NENs. While CTCs face limitations due to low shedding, ctDNA provides valuable information on high-grade neoplasms. MiRNA and mRNA (e.g., the NETest) offer high sensitivity and specificity for diagnosis and prognosis, outperforming traditional markers like chromogranin A. The LB has significant potential for NEN diagnosis and monitoring but lacks widespread clinical integration due to limited prospective studies and guidelines, requiring further validation. Advances in sequencing technologies may enhance the clinical utility of LB in NENs. Future research should focus on refining LB methods, standardising protocols and exploring applications in high-grade NENs. [ABSTRACT FROM AUTHOR]

Simple Summary: A liquid biopsy (LB) is now considered to be a valuable diagnostic tool for advanced and metastatic nonsquamous nonsmall cell lung cancer (NS-NSCLC) patients. The analysis of circulating tumor DNA (ct-DNA) for NS-NSCLC has been shown to increase the chance of identifying the presence of targetable mutations and has been adopted by many clinicians around the world. However, a tissue biopsy (TB) is still the "golden standard" for lung cancer diagnosis and molecular profiling, which is essential for decision-making regarding treatment of NS-NSCLC. Both approaches should be considered as complementary and their sequential or simultaneous use for next-generation sequencing (NGS) can be questionable at tumor diagnosis and/or at tumor progression. The targeted therapies and immunotherapies in thoracic oncology, particularly for NS-NSCLC, are associated with an increase in the number of predictive biomarkers to be assessed in routine clinical practice. These treatments are administered thanks to marketing authorization for use in daily practice or are evaluated during clinical trials. Since the molecular targets to be identified are more and more complex and numerous, it is now mandatory to use NGS. NGS can be developed from both tissue and fluid (mainly blood). The blood tests in oncology, so-called "liquid biopsies" (LB), are performed with plasmatic circulating free DNA (cf-DNA) and are complementary to the molecular testing performed with a TB. LB use in lung cancer is associated with international guidelines, but additional algorithms could be set up. However, even if useful for better care of patients, notably with advanced and metastatic NS-NSCLC, until now LB are not often integrated into daily practice, at least in Europe and notably in France. The purpose of this review is to describe the different opportunities and algorithms leading to the identification of the molecular signature of NS-NSCLC, using both tissue and liquid biopsies, and to introduce the principle limitations but also some perspectives in this field. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterised by late diagnosis and poor prognosis, with current diagnostic and prognostic strategies proving insufficient. Liquid biopsy techniques, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes, and proteomics, present potential avenues for enhancing PDAC diagnosis, prognosis, and management. A review of literature from 2019 to 2024 identified 49 relevant studies focusing on these biomarkers. Investigations into ctDNA, particularly in detecting mutant KRAS, have shown some diagnostic and prognostic potential, though its efficacy in early-stage disease remains limited. CTCs, whilst highly specific, exhibit similarly restricted diagnostic utility in early disease, and studies have yielded inconsistent prognostic findings. Exosomal research identifies diverse biomarkers with promising diagnostic and prognostic value. Importantly, proteomics demonstrates superior accuracy in PDAC diagnosis, including early-stage detection, and significant prognostic capacity, especially when combined with CA19-9 analysis. This review highlights the significance of continued research into liquid biopsy techniques to improve PDAC management and outcomes. Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterised by late diagnosis and poor prognosis. Despite advancements, current diagnostic and prognostic strategies remain limited. Liquid biopsy techniques, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), circulating tumour exosomes, and proteomics, offer potential solutions to improve PDAC diagnosis, prognostication, and management. A systematic search of Ovid MEDLINE identified studies published between 2019 and 2024, focusing on liquid biopsy biomarkers for PDAC. A total of 49 articles were included. ctDNA research shows some promise in diagnosing and prognosticating PDAC, especially through detecting mutant KRAS in minimal residual disease assays. CTC analyses had low sensitivity for early-stage PDAC and inconsistent prognostic results across subpopulations. Exosomal studies revealed diverse biomarkers with some diagnostic and prognostic potential. Proteomics, although relatively novel, has demonstrated superior accuracy in PDAC diagnosis, including early detection, and notable prognostic capacity. Proteomics combined with CA19-9 analysis has shown the most promising results to date. An update on multi-cancer early detection testing, given its significance for population screening, is also briefly discussed. Liquid biopsy techniques offer promising avenues for improving PDAC diagnosis, prognostication, and management. In particular, proteomics shows considerable potential, yet further research is needed to validate existing findings and comprehensively explore the proteome using an unbiased approach. [ABSTRACT FROM AUTHOR]

Simple Summary: Salivary gland tumors (SGTs) are rare and complex, making them difficult to diagnose and treat. This work focuses on the role of exosomes, extracellular vesicles secreted by almost all cell types, in the development and progression of SGT. Exosomes are crucial in cell-to-cell communication and play significant roles in tumor biology, including modulating the tumor environment, aiding metastasis, and affecting immune responses. A better understanding of exosome biology can lead to their use as biomarkers for diagnosis and prognosis, as well as targets for treatment, potentially transforming SGT management and improving patient outcomes. Exosome-based liquid biopsies could offer non-invasive, real-time diagnostics and enhance patient care through precision medicine. This review explores the advancements in salivary exosome analysis, highlighting its potential for non-invasive cancer detection and the development of innovative diagnostic techniques. Backgroud: Salivary gland tumors (SGTs) are rare and diverse neoplasms, presenting significant challenges in diagnosis and management due to their rarity and complexity. Exosomes, lipid bilayer vesicles secreted by almost all cell types and present in all body fluids, have emerged as crucial intercellular communication agents. They play multifaceted roles in tumor biology, including modulating the tumor microenvironment, promoting metastasis, and influencing immune responses. Results: This review focuses on the role of exosomes in SGT, hypothesizing that novel diagnostic and therapeutic approaches can be developed by exploring the mechanisms through which exosomes influence tumor occurrence and progression. By understanding these mechanisms, we can leverage exosomes as diagnostic and prognostic biomarkers, and target them for therapeutic interventions. The exploration of exosome-mediated pathways contributing to tumor progression and metastasis could lead to more effective treatments, transforming the management of SGT and improving patient outcomes. Ongoing research aims to elucidate the specific cargo and signaling pathways involved in exosome-mediated tumorigenesis and to develop standardized techniques for exosome-based liquid biopsies in clinical settings. Conclusions: Exosome-based liquid biopsies have shown promise as non-invasive, real-time systemic profiling tools for tumor diagnostics and prognosis, offering significant potential for enhancing patient care through precision and personalized medicine. Methods like fluorescence, electrochemical, colorimetric, and surface plasmon resonance (SPR) biosensors, combined with artificial intelligence, improve exosome analysis, providing rapid, precise, and clinically valid cancer diagnostics for difficult-to-diagnose cancers. [ABSTRACT FROM AUTHOR]

Background: The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRASG12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC). Results: We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRASG12D from KRASWT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRASG12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients' pancreatic ultrasound-guided fine-needle aspiration fluid. Conclusion: CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation. [ABSTRACT FROM AUTHOR]