BACKGROUND: Optimizing functional outcomes and securing long-term remissions are key goals in managing patients with locally advanced rectal cancer. In this proof-of-concept study, we set out to further optimize neoadjuvant therapy by integrating the radiosensitizer trifluridine/tipiracil and explore the potential of cell free tumor DNA (ctDNA) to monitor residual disease.
METHODS: About 10 patients were enrolled in the phase I dose finding part which followed a 3 + 3 dose escalation design. Tipiracil/trifluridine was administered concomitantly to radiotherapy. ctDNA monitoring was performed before and after chemoradiation with patient-individualized digital droplet PCRs.
RESULTS: No dose-limiting toxicities were observed at the maximum tolerated dose level of 2 × 35 mg/m² trifluridine/tipiracil. There were 9 grade 3 adverse events, of which 8 were hematologic with anemia and leukopenia. Chemoradiation yielded a pathological complete response in 1 out of 8 assessable patients, downstaging in nearly all patients, and 1 clinical complete response referred for watchful waiting. Three of 4 assessable patients with residual tumor cells at pathological assessment remained liquid biopsy positive after chemoradiation, but 1 turned negative.
CONCLUSIONS: In this exploratory phase I trial, the novel combination of neoadjuvant trifluridine/tipiracil and radiotherapy proved to be feasible, tolerable, and effective. However, the application of liquid biopsy as a potential marker for therapeutic de-escalation in the neoadjuvant setting requires additional research and prospective validation. The trial was registered at ClinicalTrials.gov: NCT04177602.

Marine ecosystems are under escalating threats from myriad environmental stressors, necessitating a deeper understanding of their impact on biodiversity and the health of sentinel organisms. In this study, we carried out a spatiotemporal multi-omic analysis of liquid biopsies collected from mussels (Mytilus spp.) in marine ecosystems of a national park. We delved into the epigenomic, transcriptomic, glycomic, proteomic, and microbiomic profiles to unravel the intricate interplay between ecosystem biodiversity and mussels\' biological response to their environments. Our analysis revealed temporal fluctuations in the alpha diversity of the circulating microbiome associated with human activities. Analysis of the hemolymphatic circulating cell-free DNA (ccfDNA) provided information on the biodiversity and the presence of potential pathogens. Epigenomic analysis revealed widespread hypomethylation sites within the mitochondrial (mtDNA). Comparative transcriptomic and glycomic analyses highlighted differences in metabolic pathways and genes associated with immune and wound healing functions. This study demonstrates the potential of multi-omic analysis of liquid biopsy in sentinel to provide a holistic view of human activities\' environmental impacts on marine coastal ecosystems. Overall, this approach has the potential to enhance the effectiveness and efficiency of various conservation efforts, leading to more informed decision-making and better outcomes for biodiversity and ecosystem conservation.

UNASSIGNED: Oncology clinical trial enrollment is strongly recommended for patients with cancer who are not eligible for established and approved therapies. Many trials are specific to biomarker-targeted therapies, which are typically managed as specialty pharmacy services. Comprehensive genomic profiling (CGP) of advanced cancers has been shown to detect biomarkers, guide targeted treatment, improve outcomes, and result in the clinical trial enrollment of patients, which is modeled to offset pharmacy costs experienced by US payers, yet payer policy coverage remains inconsistent. A common concern limiting coverage of CGP by payers is the potential of identifying biomarkers beyond guideline-recommended treatments, which creates a perception that insurance companies are being positioned to \"pay for research.\" However, these biomarkers can increase clinical trial eligibility, and specialty pharmacy management may have an interest in maximizing the clinical trial enrollment of members.
UNASSIGNED: To investigate if clinical trial enrollment following liquid biopsy CGP for non-small cell lung cancer (NSCLC) is clinically and/or economically impactful from a payer claims perspective.
UNASSIGNED: Clinical and economic outcomes were studied using a real-world clinical genomic database (including payer claims data) from patients with NSCLC who enrolled in clinical trials immediately following liquid biopsy CGP (using Guardant360) and matched NSCLC patient controls also tested with liquid biopsy CGP.
UNASSIGNED: Real-world overall survival was significantly (log-rank P < 0.0001) better for patients enrolled in clinical trials with similar costs of care, albeit with more outpatient encounters among those enrolled compared with matched controls.
UNASSIGNED: The results, together with previous analyses, suggest that, in addition to the clinical benefits associated with targeted therapies directed by CGP and other testing approaches, payers and specialty pharmacy managers may consider clinical trial direction and enrollment as a clinical and economic benefit of liquid biopsy CGP and adopt this into coverage decision frameworks and formularies.

Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the PDGFRA D842V mutation and SEPT9 hypermethylated gene. We utilized ddPCR to analyze the predominant PDGFRA mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists\' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating SEPT9 gene hypermethylation. We successfully detected the PDGFRA D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated SEPT9 gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.

Lung cancer is a major global health concern with a low survival rate, often due to late-stage diagnosis. Liquid biopsy offers a non-invasive approach to cancer detection and monitoring, utilizing various features of circulating cell-free DNA (cfDNA). In this study, we established two models based on cfDNA coverage patterns at the transcription start sites (TSSs) from 6X whole-genome sequencing: an Early Cancer Screening Model and an EGFR mutation status prediction model. The Early Cancer Screening Model showed encouraging prediction ability, especially for early-stage lung cancer. The EGFR mutation status prediction model exhibited high accuracy in distinguishing between EGFR-positive and wild-type cases. Additionally, cfDNA coverage patterns at TSSs also reflect gene expression patterns at the pathway level in lung cancer patients. These findings demonstrate the potential applications of cfDNA coverage patterns at TSSs in early cancer screening and in cancer subtyping.

BACKGROUND: Esophageal cancer (EC) is a highly lethal disease lacking early detection approaches. We previously identified that OTOP2 and KCNA3 were specifically hypermethylated in circulating cell-free DNA from patients with EC. We then developed a blood-based methylation assay targeting OTOP2 and KCNA3 (named \"IEsohunter\") for esophageal cancer noninvasive detection. This double-blinded, multicenter, prospective study aimed to comprehensively evaluate its clinical diagnostic performance.
METHODS: Participants with EC, high-grade intraepithelial neoplasia (HGIN), other malignancies, benign gastrointestinal lesions, or no abnormalities were prospectively enrolled from 5 tertiary referral centers across China. Peripheral blood samples were collected, followed by plasma cell-free DNA methylation analysis using the IEsohunter test based on multiplex quantitative polymerase chain reaction adopting an algorithm-free interpretation strategy. The primary outcome was the diagnostic accuracy of IEsohunter test for EC.
RESULTS: We prospectively enrolled 1116 participants, including 334 patients with EC, 71 with HGIN, and 711 controls. The areas under the receiver operating characteristic curves of the IEsohunter test for detecting EC and HGIN were 0.903 (95% CI 0.880-0.927) and 0.727 (95% CI 0.653-0.801), respectively. IEsohunter test showed sensitivities of 78.5% (95% CI 69.1-85.6), 87.3% (95% CI 79.4-92.4), 92.5% (95% CI 85.9-96.2), and 96.9% (95% CI 84.3-99.8) for stage I-IV EC, respectively, with an overall sensitivity of 87.4% (95% CI 83.4-90.6) and specificity of 93.3% (95% CI 91.2-94.9) for EC detection. The IEsohunter test status turned negative (100.0%, 47/47) after surgical resection of EC.
CONCLUSIONS: The IEsohunter test showed high diagnostic accuracy for EC detection, indicating that it could potentially serve as a tool for noninvasive early detection and surveillance of EC.

BACKGROUND: Pancreatic cancer, predominantly characterized by ductal adenocarcinoma (PDAC) accounts for 90% of cases and is the fourth leading cause of cancer-related deaths globally. Its incidence is notably increasing. This poor prognosis is primarily due to late-stage diagnosis (approximately 70% to 80% of patients are diagnosed at an advanced stage), aggressive tumor biology, and low sensitivity to chemotherapy. Consequently, it is crucial to identify and develop a simple, feasible and reproducible blood-based signature (i.e., combination of biomarkers) for early detection of PDAC.
METHODS: The PANLIPSY study is a multi-center, non-interventional prospective clinical trial designed to achieve early detection of PDAC with high specificity and sensitivity, using a combinatorial approach in blood samples. These samples are collected from patients with resectable, borderline or locally advanced, and metastatic stage PDAC within the framework of the French Biological and Clinical Database for PDAC cohort (BACAP 2). All partners of the BACAP consortium are eligible to participate. The study will include 215 PDAC patients, plus 25 patients with benign pancreatic conditions from the PAncreatic Disease Cohort of TOuLouse (PACTOL) cohort, and 115 healthy controls, totaling 355 individuals. Circulating biomarkers will be collected in a total volume of 50 mL of blood, divided into one CellSave tube (10 mL), two CELL-FREE DNA BCT® preservative tubes (18 mL), and five EDTA tubes (22 mL in total). Samples preparation will adhere to the guidelines of the European Liquid Biopsy Society (ELBS). A unique feature of the study is the AI-based comparison of these complementary liquid biopsy biomarkers. Main end-points: i) to define a liquid biopsy signature that includes the most relevant circulating biomarkers, ii) to validate the multi-marker panel in an independent cohort of healthy controls and patients, with resectable PDAC, and iii) to establish a unique liquid biopsy biobank for PDAC study.
CONCLUSIONS: The PANLIPSY study is a unique prospective non-interventional clinical trial that brings together liquid biopsy experts. The aim is to develop a biological signature for the early detection of PDAC based on AI-assisted detection of circulating biomarkers in blood samples (CTCs, ctDNA, EVs, circulating immune system, circulating cell-free nucleosomes, proteins, and microbiota).
BACKGROUND: ClinicalTrials.gov Identifier: NCT06128343 / NCT05824403. Registration dates: June 8,2023 and April 21, 2023.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

Blue mussels are often abundant and widely distributed in polar marine coastal ecosystems. Because of their wide distribution, ecological importance, and relatively stationary lifestyle, bivalves have long been considered suitable indicators of ecosystem health and changes. Monitoring the population dynamics of blue mussels can provide information on the overall biodiversity, species interactions, and ecosystem functioning. In the present work, we combined the concept of liquid biopsy (LB), an emerging concept in medicine based on the sequencing of free circulating DNA, with the Oxford Nanopore Technologies (ONT) platform using a portable laboratory in a remote area. Our results demonstrate that this platform is ideally suited for sequencing hemolymphatic circulating cell-free DNA (ccfDNA) fragments found in blue mussels. The percentage of non-self ccfDNA accounted for >50 % of ccfDNA at certain sampling Sites, allowing the quick, on-site acquisition of a global view of the biodiversity of a coastal marine ecosystem. These ccfDNA fragments originated from viruses, bacteria, plants, arthropods, algae, and multiple Chordata. Aside from non-self ccfDNA, we found DNA fragments from all 14 blue mussel chromosomes, as well as those originating from the mitochondrial genomes. However, the distribution of nuclear and mitochondrial DNA was significantly different between Sites. Similarly, analyses between various sampling Sites showed that the biodiversity varied significantly within microhabitats. Our work shows that the ONT platform is well-suited for LB in sentinel blue mussels in remote and challenging conditions, enabling faster fieldwork for conservation strategies and resource management in diverse settings.

BACKGROUND: Early diagnosis of hepatocellular carcinoma (HCC) can significantly improve patient survival. We aimed to develop a blood-based assay to aid in the diagnosis, detection and prognostic evaluation of HCC.
METHODS: A three-phase multicentre study was conducted to screen, optimise and validate HCC-specific differentially methylated regions (DMRs) using next-generation sequencing and quantitative methylation-specific PCR (qMSP).
RESULTS: Genome-wide methylation profiling was conducted to identify DMRs distinguishing HCC tumours from peritumoural tissues and healthy plasmas. The twenty most effective DMRs were verified and incorporated into a multilocus qMSP assay (HepaAiQ). The HepaAiQ model was trained to separate 293 HCC patients (Barcelona Clinic Liver Cancer (BCLC) stage 0/A, 224) from 266 controls including chronic hepatitis B (CHB) or liver cirrhosis (LC) (CHB/LC, 96), benign hepatic lesions (BHL, 23), and healthy controls (HC, 147). The model achieved an area under the curve (AUC) of 0.944 with a sensitivity of 86.0% in HCC and a specificity of 92.1% in controls. Blind validation of the HepaAiQ model in a cohort of 523 participants resulted in an AUC of 0.940 with a sensitivity of 84.4% in 205 HCC cases (BCLC stage 0/A, 167) and a specificity of 90.3% in 318 controls (CHB/LC, 100; BHL, 102; HC, 116). When evaluated in an independent test set, the HepaAiQ model exhibited a sensitivity of 70.8% in 65 HCC patients at BCLC stage 0/A and a specificity of 89.5% in 124 patients with CHB/LC. Moreover, HepaAiQ model was assessed in paired pre- and postoperative plasma samples from 103 HCC patients and correlated with 2-year patient outcomes. Patients with high postoperative HepaAiQ score showed a higher recurrence risk (Hazard ratio, 3.33, p < .001).
CONCLUSIONS: HepaAiQ, a noninvasive qMSP assay, was developed to accurately measure HCC-specific DMRs and shows great potential for the diagnosis, detection and prognosis of HCC, benefiting at-risk populations.