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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]

Background: Biomarkers to predict the recurrence risk are required to optimize perioperative treatment. Adjuvant chemotherapy for patients with human epidermal growth factor 2-positive (HER2-positive) early breast cancer is decided by pathological responses of neoadjuvant chemotherapy (NAC). However, whether pathological responses are appropriate biomarkers is unclear. Currently, there are several studies using minimal residual disease (MRD) as a predictor of prognosis in solid tumors. However, there is no standard method for detecting MRD. Objectives: This study aimed at prospectively evaluating the relationship between MRD detection and recurrence in Asian patients with HER2-positive early breast cancer. Design: Prospective, observational, single-group, and exploratory. This study will include 60 patients from 2 institutions in Japan and the Philippines. The invasive disease-free survival (IDFS) rates of the MRD-positive and MRD-negative groups are compared in patients with HER2-positive early breast cancer who undergo surgery after receiving NAC. Methods and analysis: Circulating tumor DNA (ctDNA) levels of patients will be evaluated 6 times: before NAC, after NAC, after surgery, and annually after surgery for 3 years. We will analyze the genetic profile of blood and tissue samples using the Todai OncoPanel (TOP) and the methylation level of DNA. The primary endpoint is IDFS. Secondary endpoints include overall survival (OS) and disease-free survival (DFS). Patient enrollment began in June 2022, and new participants are still being recruited. Ethics: This study has been approved by the National Cancer Center Hospital Certified Review Board in March 2022 and has been approved by the Research Ethics Board of the participating center. Discussion: Our findings will contribute to determining whether MRD detection using TOP is useful for predicting the recurrence of HER2-positive early breast cancer. If this is proven, MRD detected by TOP could be used in the future as a biomarker to assist in the de-/escalation of treatment strategies in the next interventional trial, thereby avoiding overtreatment in patients at low risk, and in the addition of intensive treatment modalities for those in patients at high risk. [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]

The detection of circulating tumor DNA (ctDNA) in blood is significant for non‐invasive cancer diagnosis and treatment monitoring. Herein, MBene nanosheets is synthesized and compared with MXene via Density Functional Theory (DFT) calculations and fluorescence kinetic evaluations, for first time, revealing MBene's exceptional DNA adsorbability and discrimination to single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA). Then, a sensitive fluorescence biosensor for ctDNA detection is developed, demonstrating impressive performance. To facilitate point‐of‐care testing (POCT) ctDNA, a paper‐based microfluidic chip incorporated a delay area and a mixing channel is designed. The constricted‐expanded structure channel optimization is guided by numerical simulations and experiments. A WeChat mini program named “ctDNA Detection” is designed for readout assay. Furthermore, cell and mice serum samples are analyzed, with Magnetic Bead@Graphene Oxide (MB@GO) and clutch probes for magnetic pre‐enrichment. The results accuracy is confirmed by its consistency with standard qPCR results (AUC = 1). The successful detection of ctDNA in post‐surgery mouse models underscored the biosensor's potential for cancer treatment monitoring. Thus, this research not only advanced the understanding of the MBene‐DNA interaction in biosensing, but also can pave the way for novel applications in bioimaging and nanopore‐based nucleic acid sequencing, leveraging the digital transformation of DNA base‐MBene adsorption differences. [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: Lazertinib is a potent, irreversible, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with significant efficacy in patients with EGFR T790M-mutated non-small cell lung cancer (NSCLC). This is the final overall survival (OS) report from the phase 1/2 LASER201 study in patients with advanced NSCLC with disease progression on or after prior EGFR TKI therapy. Methods: Eligible patients were aged ≥ 20 years, with advanced EGFR-mutated NSCLC and previous therapy with EGFR TKI. Patients in this integrated analysis received oral lazertinib 240 mg/day. Endpoints included efficacy and safety; exploratory analyses included associations between circulating EGFR-mutant tumor DNA (ctDNA) and efficacy parameters. Results: This integrated analysis included 78 patients in Korea who received second- or later-line lazertinib. The median OS was 38.9 months; estimated survival rates at 12, 24, and 36 months were 89.5%, 73.9%, and 52.8%, respectively. The cumulative 12-month incidence of central nervous system progression was 9.4%. EGFR-mutant ctDNA was detected in 46 patients (62.2%) at baseline. The presence of ctDNA at baseline significantly predicted progression-free survival (PFS), disease control rate (DCR), and OS. PFS, response rate, and DCR were significantly associated with EGFR-mutant ctDNA clearance at cycle 3; PFS and OS were significantly associated with ctDNA clearance at cycle 5. The safety profile of lazertinib 240 mg/day was consistent with previous findings. Conclusions: Lazertinib is a promising treatment option for patients with EGFR T790M-positive NSCLC following disease progression on prior EGFR-directed TKIs. Patients in LASER201 experienced prolonged OS, regardless of their EGFR mutation, brain metastases, or prior brain radiation status. Clearance of plasma EGFR mutations after lazertinib was associated with patient outcomes. Trial registration: ClinicalTrials.gov identifier NCT03046992. [ABSTRACT FROM AUTHOR]

Background: Melanoma is the most aggressive skin cancer with ability to recur also after early‐stage tumor surgery. The aim was to identify early‐stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. Methods: Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre‐amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. Results: The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild‐type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). Conclusion: We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. Significance: There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early‐stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre‐ and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage. [ABSTRACT FROM AUTHOR]

Cancer continues to pose significant challenges to the medical community. Early detection, accurate molecular profiling, and adequate assessment of treatment response are critical factors in improving the quality of life and survival of cancer patients. Accumulating evidence shows that circulating tumor DNA (ctDNA) shed by tumors into the peripheral blood preserves the genetic and epigenetic information of primary tumors. Notably, DNA methylation, an essential and stable epigenetic modification, exhibits both cancer- and tissue-specific patterns. As a result, ctDNA methylation has emerged as a promising molecular marker for noninvasive testing in cancer clinics. In this review, we summarize the existing techniques for ctDNA methylation detection, describe the current research status of ctDNA methylation, and present the potential applications of ctDNA-based assays in the clinic. The insights presented in this article could serve as a roadmap for future research and clinical applications of ctDNA methylation. [ABSTRACT FROM AUTHOR]

Over the past decades, liquid biopsy, especially circulating tumor DNA (ctDNA), has received tremendous attention as a noninvasive detection approach for clinical applications, including early diagnosis of cancer and relapse, real-time therapeutic efficacy monitoring, potential target selection and investigation of drug resistance mechanisms. In recent years, the application of next-generation sequencing technology combined with AI technology has significantly improved the accuracy and sensitivity of liquid biopsy, enhancing its potential in solid tumors. However, the increasing integration of such promising tests to improve therapy decision making by oncologists still has complexities and challenges. Here, we propose a conceptual framework of ctDNA technologies and clinical utilities based on bibliometrics and highlight current challenges and future directions, especially in clinical applications such as early detection, minimal residual disease detection, targeted therapy, and immunotherapy. We also discuss the necessities of developing a dynamic field of translational cancer research and rigorous clinical studies that may support therapeutic strategy decision making in the near future. [ABSTRACT FROM AUTHOR]

The advent of liquid biopsies has brought significant changes to the diagnosis and monitoring of non-small cell lung cancer (NSCLC), presenting both promise and challenges. Molecularly targeted drugs, capable of enhancing survival rates, are now available to around a quarter of NSCLC patients. However, to ensure their effectiveness, precision diagnosis is essential. Circulating tumor DNA (ctDNA) analysis as the most advanced liquid biopsy modality to date offers a non-invasive method for tracking genomic changes in NSCLC. The potential of ctDNA is particularly rooted in its ability to furnish comprehensive (epi-)genetic insights into the tumor, thereby aiding personalized treatment strategies. One of the key advantages of ctDNA-based liquid biopsies in NSCLC is their ability to capture tumor heterogeneity. This capability ensures a more precise depiction of the tumor's (epi-)genomic landscape compared to conventional tissue biopsies. Consequently, it facilitates the identification of (epi-)genetic alterations, enabling informed treatment decisions, disease progression monitoring, and early detection of resistance-causing mutations for timely therapeutic interventions. Here we review the current state-of-the-art in ctDNA-based liquid biopsy technologies for NSCLC, exploring their potential to revolutionize clinical practice. Key advancements in ctDNA detection methods, including PCR-based assays, next-generation sequencing (NGS), and digital PCR (dPCR), are discussed, along with their respective strengths and limitations. Additionally, the clinical utility of ctDNA analysis in guiding treatment decisions, monitoring treatment response, detecting minimal residual disease, and identifying emerging resistance mechanisms is examined. Liquid biopsy analysis bears the potential of transforming NSCLC management by enabling non-invasive monitoring of Minimal Residual Disease and providing early indicators for response to targeted treatments including immunotherapy. Furthermore, considerations regarding sample collection, processing, and data interpretation are highlighted as crucial factors influencing the reliability and reproducibility of ctDNA-based assays. Addressing these challenges will be essential for the widespread adoption of ctDNA-based liquid biopsies in routine clinical practice, ultimately paving the way toward personalized medicine and improved outcomes for patients with NSCLC. [ABSTRACT FROM AUTHOR]

B‐cell acute lymphoblastic leukaemia (B‐ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Managing B‐ALL remains challenging due to its heterogeneity and relapse risk. This study aimed to delineate the molecular features of paediatric B‐ALL and explore the clinical utility of circulating tumour DNA (ctDNA). We analysed 146 patients with paediatric B‐ALL who received systemic chemotherapy. The mutational landscape was profiled in bone marrow (BM) and plasma samples using next‐generation sequencing. Minimal residual disease (MRD) testing on day 19 of induction therapy evaluated treatment efficacy. RNA sequencing identified gene fusions in 61% of patients, including 37 novel fusions. Specifically, the KMT2A‐TRIM29 novel fusion was validated in a boy who responded well to initial therapy but relapsed after 1 year. Elevated mutation counts and maximum variant allele frequency in baseline BM were associated with significantly poorer chemotherapy response (p = 0.0012 and 0.028, respectively). MRD‐negative patients exhibited upregulation of immune‐related pathways (p < 0.01) and increased CD8+ T cell infiltration (p = 0.047). Baseline plasma ctDNA exhibited high mutational concordance with the paired BM samples and was significantly associated with chemotherapy efficacy. These findings suggest that ctDNA and BM profiling offer promising prognostic insights for paediatric B‐ALL management. [ABSTRACT FROM AUTHOR]

Introduction Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC). Methods In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (n  = 1329) from 298 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023. Results Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, P  < .0001) as well as during the surveillance period (median DFS: 11.40 months for ctDNA-positive vs NR for ctDNA-negative; HR: 12.38, P  < .0001). Additionally, DFS was significantly better with KRAS wildtype status followed by KRAS G12R (HR: 0.99, P  = .97), KRAS G12D (HR: 1.42, P  = .194), and worse with KRAS G12V (HR: 2.19, P  = .002) status. In multivariate analysis, ctDNA detection at surveillance was found to be the most significant prognostic factor for recurrence (HR: 24.28, P  < .001). Conclusions Perioperative tumor-informed ctDNA detection in PDAC is feasible across all stages and is associated with patient survival outcomes. [ABSTRACT FROM AUTHOR]

Purpose: RAS (KRAS/NRAS) mutational status on a tumor biopsy is mandatory to guide the best treatment in metastatic colorectal cancer (mCRC). Determining the RAS mutational status by tumor-tissue biopsy is essential in guiding the optimal treatment decision for mCRC. RAS mutations are negative predictive factors for the use of EGFR monoclonal antibodies. Cell-free DNA (cfDNA) analysis enables minimally invasive monitoring of tumor evolution. Methods/patients: PERSEIDA was an observational, prospective study assessing cfDNA RAS, BRAF and EGFR mutations (using Idylla™) in first-line mCRC, RAS wild-type (baseline tumor-tissue biopsy) patients (cohort 2). Plasma samples were collected before first-line treatment, after 20 ± 2 weeks, and at disease progression. Results: 117 patients were included (103 received panitumumab + chemotherapy as first-line treatment). At baseline, 7 (6.8%) patients had RAS mutations, 4 (3.9%) BRAF mutations and no EGFR mutations were detected (cfDNA, panitumumab + chemotherapy subpopulation [panitumumab + Ch]). The baseline RAS mutational status concordance between tissue and liquid biopsies was 94.0% (93.2%, panitumumab + Ch). At 20 weeks, only one patient in the study (included in the panitumumab + Ch) had an emerging cfDNA RAS mutation. No emerging BRAF or EGFR mutations were reported. At disease progression, 6 patients had emergent mutations not present at baseline (RAS conversion rate: 13.3% [6/45]; 15.0% [6/40], panitumumab + Ch). Conclusions: The concordance rate between liquid and solid biopsies at baseline was very high, as previously reported, while our results suggest a considerable emergence of RAS mutations during disease progression. Thus, the dynamics of the genomic landscape in ctDNA may provide relevant information for the management of mCRC patients. [ABSTRACT FROM AUTHOR]

Purpose: We evaluated the prevalence of homologous recombination deficiencies (HRD) to determine the efficacy of different techniques and clinical characteristics of patients. Methods: This retrospective study included patients with metastatic prostate cancer who underwent molecular testing at our hospital between 2016 and 2022. We used tumor tissue, ctDNA, and lymphocytes for somatic or germline testing. We analyzed the clinical characteristics and survival outcomes. Results: 144 patients were tested (113 somatic, 21 germline, and 10 both). Technical issues prevented the analysis of 23 prostatic samples (18.7%). 12 (8.3%) patients had HRD. BRCA2 was the most frequent mutation (66.7%). Patients with HRD were younger (57.5 years). Patients with BRCA mutations had poorer survival (31.9 vs 56.3 months, p = 0.048). Conclusion: In our institution, 8.3% of the patients had HRD. Tumor tissue analysis failed in 18.7% of tests. ctDNA analysis is an alternative detection method. BRCA mutations are correlated with poor prognosis. [ABSTRACT FROM AUTHOR]

Background: Liquid biopsy is a method that could potentially improve the management of thyroid cancer (TC) by enabling the detection of circulating tumor DNA and RNA (ctDNA, ctRNA). The BRAFV600E mutation appears to be the most representative example of a biomarker in liquid biopsy, as it is the most specific mutation for TC and a target for molecular therapeutics. The aim of this review is to summarize the available data on the detection of the BRAFV600E mutation in liquid biopsy in patients with TC. Methods: A comprehensive analysis of the available literature on the detection of the BRAFV600E mutation in liquid biopsy in TC was performed. Thirty-three papers meeting the inclusion criteria were selected after full-text evaluation. Results: Eleven papers discussed correlations between BRAF mutation and clinicopathological characteristics. Nine studies tested the utility of BRAFV600E detection in the assessment of residual or recurrent disease. Seven studies investigated BRAF-mutated circulating tumor nucleic acids (ctNA) as a marker of response to targeted therapy. In seven studies the method did not detect the BRAFV600E mutation. Conclusions: This review shows the potential of BRAFV600E-mutated ctNA detection in monitoring disease progression, particularly in advanced TC. The diagnostic value of BRAFV600E-mutated ctNA detection appears to be limited to advanced TC. The choice of the molecular method (quantitative PCR [qPCR], droplet digital polymerase chain reaction [ddPCR], and next-generation sequencing [NGS]) should be made based on the turnaround time, sensitivity of the test, and the clinical indications. Despite the promising outcomes of some studies, there is a need to verify these results on larger cohorts and to unify the molecular methods. [ABSTRACT FROM AUTHOR]

Background: Comprehensive genomic profiling (CGP) has gained an important role in patients with advanced prostate cancer following the introduction of PARP inhibitors in daily clinical practice. Here, we report an overview of CGP results, specifically of BRCA1 and BRCA2 HRD-repair system genes, from patients with prostate cancer analyzed in our institution, and we compare our results with those available from more recent scientific literature. Methods: The study cohort consisted of 70 patients. Somatic DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue using a MagCore Genomic DNA FFPE One-Step Kit for MagCore System. The DNA was quantified by EasyPGX® Real-Time qPCR and EasyPGX® Analysis Software (version 4.0.13). Tissue somatic DNA libraries were prepared with Myriapod® NGS BRCA1-2 panel-NG035 and sequenced in a Mi-Seq® System. The sequence alignment in hg19 and the variant calling were performed using Myriapod® NGS Data Analysis Software version 5.0.8 NG900-SW 5.0.8 with a software detection limit (LoD) of 95%. Variants with a coverage of 500 and VAF% ≥ 5 were evaluated. Results: Tumor tissue NGS was unsuccessful in 46/70 patients (66%). Mutations of the BRCA2 gene were detected in 4 of the samples: (1) BRCA2 ex10 c.1244A>G p.His415Arg VAF = 51.03%; (2) BRCA2 ex11 c.5946delT p.Ser1982fs VAF = 72.1%; (3) BRCA2 ex11 c.3302A>G p.His1101Arg VAF = 52.9%; and (4) BRCA2 ex11 c.3195_3198delTAAT p.Asn1066fs VAF = 51.1%. Conclusions: The results from our internal overview seem to support the data and to confirm the performance of the technical issues reported in the literature. Considering the advanced age of our patients, with 84% of men over the age of 65, the application of alternative and less invasive procedures such as liquid biopsy, could be a more suitable solution for some cases. [ABSTRACT FROM AUTHOR]

Simple Summary: Peripheral blood analyses can offer a minimally invasive view into systemic immunity during cancer and can lead to the identification of biomarkers for cancer screening and therapeutic management. While a limited number of studies have reported blood transcriptome in breast cancer (BC) using RNA-seq analysis, our study is the first that aimed to identify potential BC biomarkers by analyzing transcriptome at an isoform level in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women. Our approach has led to the identification of an isoform of the RASGEF1A gene, the ENST00000374459 transcriptional variant, as a promising blood mRNA biomarker for distinguishing BC and healthy subjects. Additionally, our association analysis with clinicopathological characteristics revealed that lower ENST00000374459 expression in PBMCs of breast cancer patients was associated with higher proliferation and circulating tumor DNA (ctDNA) shedding, thereby linking expression of this isoform in blood immune cells to cancer progression and spreading. Background: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant (p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid biopsy's use in the field of head and neck cancer has garnered interest due to providing an efficient and insightful alternative or complement to the present standards for the diagnosis and monitoring of patients. Certain biomarkers have been associated with specific cancer diagnoses, responses to treatment, and risk of recurrence, including circulating tumor DNA. Analysis of circulating tumor DNA and viral DNA via liquid biopsy has linked certain mutations and methylation patterns to various patient outcomes across several types of head and neck cancer. The present paper has reviewed the recent literature examining liquid biopsy's clinical utility in head and neck oncology and has discussed the clinical implications of aspects of circulating tumor DNA, among other biomarkers. Recent data have shown a continued rise in the worldwide annual incidence and mortality rates of head and neck cancers. The present standard for diagnosis and monitoring for disease recurrence or progression involves clinical examination, imaging, and invasive biopsy techniques of lesions suspected of being malignant. In addition to limitations relating to cost, time, and patient discomfort, these methodologies have inherent inaccuracies for detecting recurrence. In view of these limitations, the analysis of patient bodily fluid samples via liquid biopsy proposes a cost-effective and convenient alternative, which provides insight on the biogenetic and biomolecular underpinnings of oncologic disease processes. The monitoring of biomarkers for head and neck cancer via liquid biopsy, including circulating tumor DNA, circulating tumor cells, and circulating cell-free RNA, has shown clinical utility in the screening, diagnosis, prognostication, and monitoring of patients with various forms of head and neck cancer. The present review will provide an update on the current literature examining the use of liquid biopsy in head and neck cancer care and the clinical applicability of potential biomarkers, with a focus on viral and non-viral circulating tumor DNA. Possible future avenues for research to address specific shortcomings of liquid biopsy will be discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: The diagnosis and treatment of ovarian cancer still pose many problems; so, it is important to search for effective biomarkers that will allow for the detection of changes in the early stages of the disease. There is more and more talk about the use of circulating tumor DNA in oncology as a promising biomarker and an aid in tailoring individual therapy to patients. We describe what changes we can observe in ctDNA in patients with ovarian cancer and how we can use this information in diagnosis and therapy. Ovarian cancer is the deadliest of all gynecological diseases because its diagnosis and treatment still pose many problems. Surgical excision, hormone therapy, radiation, chemotherapy, or targeted therapy for eradicating the main tumor and halting the spread of metastases are among the treatment options available to individuals with ovarian cancer, depending on the disease's stage. Tumor DNA that circulates in a patient's bodily fluids has been studied recently as a possible novel biomarker for a number of cancers, as well as a means of quantifying tumor size and evaluating the efficacy of cancer therapy. The most significant alterations that we could find in the ctDNA of ovarian cancer patients—such as chromosomal instability, somatic mutations, and methylation—are discussed in this review. Additionally, we talk about the utility of ctDNA in diagnosis, prognosis, and therapy response prediction for these patients. [ABSTRACT FROM AUTHOR]

Background: Circulating tumour DNA (ctDNA) is a subset of cell free DNA (cfDNA) released by tumour cells into the bloodstream. Circulating tumour DNA has shown great potential as a biomarker to inform treatment in cancer patients. Collecting ctDNA is minimally invasive and reflects the entire genetic makeup of a patient's cancer. ctDNA variants in NGS data can be difficult to distinguish from sequencing and PCR artefacts due to low abundance, particularly in the early stages of cancer. Unique Molecular Identifiers (UMIs) are short sequences ligated to the sequencing library before amplification. These sequences are useful for filtering out low frequency artefacts. The utility of ctDNA as a cancer biomarker depends on accurate detection of cancer variants. Results: In this study, we benchmarked six variant calling tools, including two UMI-aware callers for their ability to call ctDNA variants. The standard variant callers tested included Mutect2, bcftools, LoFreq and FreeBayes. The UMI-aware variant callers benchmarked were UMI-VarCal and UMIErrorCorrect. We used both datasets with known variants spiked in at low frequencies, and datasets containing ctDNA, and generated synthetic UMI sequences for these datasets. Variant callers displayed different preferences for sensitivity and specificity. Mutect2 showed high sensitivity, while returning more privately called variants than any other caller in data without synthetic UMIs – an indicator of false positive variant discovery. In data encoded with synthetic UMIs, UMI-VarCal detected fewer putative false positive variants than all other callers in synthetic datasets. Mutect2 showed a balance between high sensitivity and specificity in data encoded with synthetic UMIs. Conclusions: Our results indicate UMI-aware variant callers have potential to improve sensitivity and specificity in calling low frequency ctDNA variants over standard variant calling tools. There is a growing need for further development of UMI-aware variant calling tools if effective early detection methods for cancer using ctDNA samples are to be realised. [ABSTRACT FROM AUTHOR]

Despite recent advances in cancer diagnostics and treatment, the mortality associated with lung cancer is still the highest in the world. Late-stage diagnosis, often accompanied by metastasis, is a major contributor to the high mortality rates, emphasizing the urgent need for reliable and readily accessible diagnostic tools that can detect biomarkers unique to lung cancer. Circulating factors, such as circulating tumor DNA and extracellular vesicles, from liquid biopsy have been recognized as diagnostic or prognostic markers in lung cancer. Numerous clinical studies are currently underway to investigate the potential of circulating tumor DNA, circulating tumor RNA, exosomes, and exosomal microRNA within the context of lung cancer. Those clinical studies aim to address the poor diagnostics and limited treatment options for lung cancer, with the ultimate goal of developing clinical markers and personalized therapies. In this review, we discuss the roles of each circulating factor, its current research status, and ongoing clinical studies of circulating factors in non-small cell lung cancer. Additionally, we discuss the circulating factors specifically found in lung cancer stem cells and examine approved diagnostic assays designed to detect circulating biomarkers in lung cancer patients. [ABSTRACT FROM AUTHOR]

Natural drug functionalised silver (Ag) nanoparticles (NPs) have gained significant interest in pharmacology related applications due to their therapeutic efficiency. We have synthesised silver nanoparticle using hesperetin as a reducing and capping agent. This work aims to discuss the relevance of the hesperetin functionalised silver nanoparticles (H-AgNPs) in the field of nano-medicine. The article primarily investigates the anticancer activity of H-AgNPs and then their interactions with calf thymus DNA (ctDNA) through spectroscopic and thermodynamic techniques. The green synthesised H-AgNPs are stable, spherical in shape and size of 10 ± 3 nm average diameter. The complex formation of H-AgNPs with ctDNA was established by UV–Visible absorption, fluorescent dye displacement assay, isothermal calorimetry and viscosity measurements. The binding constants obtained from these experiments were consistently in the order of 104 Mol−1. The melting temperature analysis and FTIR measurements confirmed that the structural alterations of ctDNA by the presence of H-AgNPs are minimal. All the thermodynamic variables and the endothermic binding nature were acquired from ITC experiments. All these experimental outcomes reveal the formation of H-AgNPs-ctDNA complex, and the results consistently verify the minor groove binding mode of H-AgNPs. The binding constant and limit of detection of 1.8 μM found from the interaction studies imply the DNA detection efficiency of H-AgNPs. The cytotoxicity of H-AgNPs against A549 and L929 cell lines were determined by in vitro MTT cell viability assay and lactate dehydrogenase (LDH) assay. The cell viability and LDH enzyme release are confirmed that the H-AgNPs has high anticancer activity. Moreover, the calculated LD50 value for H-AgNPs against lung cancer cells is 118.49 µl/ml, which is a low value comparing with the value for fibroblast cells (269.35 µl/ml). In short, the results of in vitro cytotoxicity assays revealed that the synthesised nanoparticles can be considered in applications related to cancer treatments. Also, we have found that, H-AgNPs is a minor groove binder, and having high DNA detection efficiency. [ABSTRACT FROM AUTHOR]

Transplant oncology is an expanding area of cancer therapy that specifically emphasizes the use of liver transplantation (LT) as the preferred treatment for patients with manageable, but unresectable, tumors. The management and optimization of overall survival strategies, accompanied by an arguably decent quality of life, have been at the forefront of liver oncology treatment, as a plurality of all primary liver cancers are identified as either hepatocellular carcinoma (HCC) or cholangiocarcinoma (CCA), which are classified as highly aggressive malignancies and frequently remain asymptomatic until they progress to advanced stages, rendering curative procedures, such as resection, impractical. This has led to an increase in utilization of neoadjuvant interventions conducted prior to surgery, which has yielded favorable outcomes. Though this treatment modality has prompted further investigations into the efficacy of immune checkpoint inhibitors (ICPIs) as standalone treatments and in combination with locoregional treatments (LRTs) to bridge more patients into curative eligibility. This multidisciplinary methodology and treatment planning has seen multiple successful trials of immunotherapy regimes and combinate treatments, setting the groundwork for increasing eligibility through downstaging and "bridging" previously ineligible patients within stringent LT criteria. Surveillance after LT is a crucial component of transplant oncology. The emergence of circulating tumor DNA (ctDNA) has provided a novel approach to identifying the recurrence of cancer in its early stages. Recent research has focused on liquid biopsy, a technique that effectively identifies the dynamics of cancer. This is another innovation to demonstrate the rate at which transplant oncology is rapidly advancing, making the focus of care feel disorienting. Modalities of care are constantly evolving, but when a field is changing as rapidly as this one, it is imperative to reorient to the data and the needs of the patients. In this commentary, we reflect on the update's utilization of ICPIs in neoadjuvant settings as well as the updates on the utilization of liquid biopsy in post-LT follow-up surveillance. [ABSTRACT FROM AUTHOR]

Background: Liquid biopsy is a minimally invasive procedure investigating tumor mutations. Methods: In our retrospective study, we investigated whether molecular therapy monitoring of patients receiving neoadjuvant radio(chemo)therapy on a daily routine is possible in 17 patients with locally advanced rectal cancer. Six patients received short-course radiotherapy (5 × 5 Gy) with subsequent surgery, six patients were treated according RAPIDO protocol with short-course radiotherapy followed by chemotherapy (FOLFOX4) and subsequent surgery and five patients received conventional neoadjuvant radiochemotherapy with 5-FU followed by surgery. Response was assessed by Dworak. Liquid biopsies were taken before and immediately after neoadjuvant radiotherapy to isolate and ultradeeply sequence cell free DNA with a panel of 127 genes. Somatic mutations were determined bioinformatically by comparison with normal DNA from leukocytes to distinguish them from germline variants or aging mutations. Results: In 12 patients (71%) at least one somatic mutation was detected. In 8/12 patients a decrease and in 4/12 an increase or mixed response in ctDNA was seen. Statistical correlation between ctDNA analysis and clinical response could not be seen. Conclusion: ctDNA is responding to neoadjuvant therapy and liquid biopsy is easily integrated into a daily routine. As part of translational research this protocol leaves room for further investigations. [ABSTRACT FROM AUTHOR]

Introduction: Hotspots (HS) mutations in the PIK3CA gene may lead to poorer oncological outcomes and endocrine resistance in advanced breast cancer (BC), but their prognostic role in early‐stage disease remains controversial. The overall agreement within plasma and tissue methods has not been well explored. Our aim was to correlate tissue and plasma approaches and to analyze the prognostic impact of PIK3CA mutations (PIK3CAm) in HR+/HER2− BC. Methods: A retrospective and unicentric analysis of PIK3CA mutational status in tissue and plasma samples by Cobas®PIK3CA Mutation Kit in patients with HR+/HER2− BC. Results: We analyzed 225 samples from 161 patients with luminal BC. PIK3CA mutations were identified in 62 patients (38.5%), of which 39.6% were found in tissue and 11.8% in plasma. In advanced disease, plasma and tissue correlation rate was performed in 64 cases, with an overall agreement of 70.3%. Eighty patients were treated with CDK4/6 inhibitors + endocrine therapy. We observed a moderately worse progression‐free survival (PFS) in PIK3CAm versus wild‐type (WT) (24 m vs. 30 m; HR = 1.39, p = 0.26). A subanalysis was carried out based on exons 9 and 20, which showed a statistically poorer PFS in PIK3CAm exon 9 versus 20 population (9.7 m vs. 30.3 m; HR = 2.84; p = 0.024). Furthermore, detection of PIK3CAm in plasma was linked to a worse PFS vs PIK3CAm detection just in tissue (12.4 vs. 29.3; HR = 2.4; p = 0.08). Conclusions: Our findings suggest the PIK3CA evaluation in tissue as the diagnostic method of choice, however, additional investigations are required to improve the role of liquid biopsy in the PIK3CA assessment. PIK3CAm show worse outcomes in advanced luminal BC, especially in exon 9 mutation carriers, despite visceral involvement, prior exposure to endocrine therapy or detection of PIK3CAm in plasma, with an unclear prognosis in early‐stage disease. Nonetheless, this should be validated in a prospective cohort study. [ABSTRACT FROM AUTHOR]

We analysed whether temporal heterogeneity of ctDNA encodes evolutionary patterns in ovarian cancer. Targeted sequencing of 275 cancer-associated genes was performed in a primary tumor biopsy and in ctDNA of six longitudinal plasma samples from 15 patients, using the Illumina platform. While there was low overall concordance between the mutational spectrum of the primary tumor biopsies vs. ctDNA, TP53 variants were the most commonly shared somatic alterations. Up to three variant clusters were detected in each tumor biopsy, likely representing predominant clones of the primary tumor, most of them harbouring a TP53 variant. By tracing these clusters in ctDNA, we propose that liquid biopsy may allow to assess the contribution of ancestral clones of the tumor to relapsed abdominal masses, revealing two evolutionary patterns. In pattern#1, clusters detected in the primary tumor biopsy were likely relapse seeding clones, as they contributed a major share to ctDNA at relapse. In pattern#2, similar clusters were present in tumors and ctDNA; however, they were entirely cleared from liquid biopsy after chemotherapy and were undetectable at relapse. ctDNA private variants were present among both patterns, with some of them mirroring subclonal expansions after chemotherapy. We demonstrate that tracing the temporal heterogeneity of ctDNA, even below exome scale resolution, deciphers evolutionary trajectories in ovarian cancer. Furthermore, we describe two evolutionary patterns that may help to identify relapse seeding clones for targeted therapy. [ABSTRACT FROM AUTHOR]

Amivantamab-lazertinib significantly prolonged progression-free survival (PFS) versus osimertinib in patients with epidermal growth factor receptor (EGFR)-mutant advanced non-small-cell lung cancer [NSCLC; hazard ratio (HR) 0.70; P < 0.001], including those with a history of brain metastases (HR 0.69). Patients with TP53 co-mutations, detectable circulating tumor DNA (ctDNA), baseline liver metastases, and those without ctDNA clearance on treatment have poor prognoses. We evaluated outcomes in these high-risk subgroups. This analysis included patients with treatment-naive, EGFR -mutant advanced NSCLC randomized to amivantamab-lazertinib (n = 429) or osimertinib (n = 429) in MARIPOSA. Pathogenic alterations were identified by next-generation sequencing (NGS) of baseline blood ctDNA with Guardant360 CDx. Ex19del and L858R ctDNA in blood was analyzed at baseline and cycle 3 day 1 (C3D1) with Biodesix droplet digital polymerase chain reaction (ddPCR). Baseline ctDNA for NGS of pathogenic alterations was available for 636 patients (amivantamab-lazertinib, n = 320; osimertinib, n = 316). Amivantamab-lazertinib improved median PFS (mPFS) versus osimertinib for patients with TP53 co-mutations {18.2 versus 12.9 months; HR 0.65 [95% confidence interval (CI) 0.48-0.87]; P = 0.003} and for patients with wild-type TP53 [22.1 versus 19.9 months; HR 0.75 (95% CI 0.52-1.07)]. In patients with EGFR -mutant, ddPCR-detectable baseline ctDNA, amivantamab-lazertinib significantly prolonged mPFS versus osimertinib [20.3 versus 14.8 months; HR 0.68 (95% CI 0.53-0.86); P = 0.002]. Amivantamab-lazertinib significantly improved mPFS versus osimertinib in patients without ctDNA clearance at C3D1 [16.5 versus 9.1 months; HR 0.49 (95% CI 0.27-0.87); P = 0.015] and with clearance [24.0 versus 16.5 months; HR 0.64 (95% CI 0.48-0.87); P = 0.004]. Amivantamab-lazertinib significantly prolonged mPFS versus osimertinib among randomized patients with [18.2 versus 11.0 months; HR 0.58 (95% CI 0.37-0.91); P = 0.017] and without baseline liver metastases [24.0 versus 18.3 months; HR 0.74 (95% CI 0.60-0.91); P = 0.004]. Amivantamab-lazertinib effectively overcomes the effect of high-risk features and represents a promising new standard of care for patients with EGFR- mutant advanced NSCLC. [Display omitted] • Amivantamab-lazertinib significantly improved PFS in patients with detectable ctDNA at baseline versus osimertinib. • Amivantamab-lazertinib significantly improved PFS in patients without ctDNA clearance at C3D1 versus osimertinib. • Amivantamab-lazertinib significantly improved PFS in patients with TP53 co-mutations versus osimertinib. • Amivantamab-lazertinib significantly improved PFS in patients with liver metastases versus osimertinib. • Eighty-nine percent of patients with analyzable ctDNA by NGS from MARIPOSA had at least one high-risk baseline feature. [ABSTRACT FROM AUTHOR]

Understanding progression mechanisms and developing new targeted therapies is imperative in pancreatic ductal adenocarcinoma (PDAC). In this study, 80 metastatic PDAC patients were prospectively recruited and divided into discovery (n=37) and validation (n=43) cohorts. Tumor and plasma samples taken at diagnosis were pair analyzed using whole exome sequencing (WES) in patients belonging to the discovery cohort alone. The variant allele frequency (VAF) of KRAS mutations was measured by ddPCR in plasma at baseline and response assessment in all patients. Plasma WES identified at least one pathogenic variant across the cohort, uncovering oncogenic mechanisms, DNA repair, microsatellite instability, and alterations in the TGFb pathway. Interestingly, actionable mutations were mostly found in plasma rather than tissue. Patients with shorter survival showed enrichment in cellular organization regulatory pathways. Through WES we could identify a specific molecular profile of patients with liver metastasis, which exhibited exclusive mutations in genes related to the adaptive immune response pathway, highlighting the importance of the immune system in liver metastasis development. Moreover, KRAS mutations in plasma (both at diagnosis and persistent at follow-up) correlated with shorter progression free survival (PFS). Patients presenting a reduction of over 84.75 % in KRAS VAF at response assessment had similar PFS to KRAS-negative patients. Overall, plasma WES reveals molecular profiles indicative of rapid progression, potentially actionable targets, and associations between adaptive immune response pathway alterations and liver tropism. [ABSTRACT FROM AUTHOR]

Summary: Currently, the main clinical application of liquid biopsy (LB) in breast cancer (BC) is the circulating tumor DNA (ctDNA)-based detection of treatment targets in metastatic or advanced disease. In this short review we focus on clinically relevant applications in BC and give a brief overview of potential future uses. [ABSTRACT FROM AUTHOR]

Background/Objectives: Immunotherapy has changed the therapeutic approach for various solid tumors, especially lung tumors, malignant melanoma, renal and urogenital carcinomas, demonstrating significant antitumor activity, with tolerable safety profiles and durable responses. However, not all patients benefit from immunotherapy, underscoring the need for predictive biomarkers that can identify those most likely to respond to treatment. Methods: The integration of predictive biomarkers into clinical practice for immune checkpoint inhibitors (ICI) holds great promise for personalized cancer treatment. Programmed death ligand 1 (PD-L1) expression, tumor mutational burden (TMB), microsatellite instability (MSI), gene expression profiles and circulating tumor DNA (ctDNA) have shown potential in predicting ICI responses across various cancers. Results: Challenges such as standardization, validation, regulatory approval, and cost-effectiveness must be addressed to realize their full potential. Predictive biomarkers are crucial for optimizing the clinical use of ICIs in cancer therapy. Conclusions: While significant progress has been made, further research and collaboration among clinicians, researchers, and regulatory institutes are essential to overcome the challenges of clinical implementation. However, little is known about the relationship between local and systemic immune responses and the correlation with response to oncological therapies and patient survival. [ABSTRACT FROM AUTHOR]

Purpose: Small bowel adenocarcinoma (SBA) is a rare malignancy of the gastrointestinal tract, and its unique location within the small intestine presents difficulties in obtaining tissue samples from the lesions. This limitation hinders the research and development of effective clinical treatment methods. Circulating tumor DNA (ctDNA) analysis holds promise as an alternative approach for investigating SBA and guiding treatment decisions, thereby improving the prognosis of SBA. Methods: Between January 2017 and August 2021, a total of 336 tissue or plasma samples were obtained and the corresponding mutation status in tissue or blood was evaluated with NGS. Results and conclusions: The study found that in SBA tissues, the most commonly alternated genes were TP53, KRAS, and APC, and the most frequently affected pathways were RTK–RAS–MAPK, TP53, and WNT. Notably, the RTK–RAS–MAPK pathway was identified as a potential biomarker that could be targeted for treatment. Then, we validated the gene mutation profiling of ctDNA extracted from SBA patients exhibited the same characteristics as tissue samples for the first time. Subsequently, we applied ctDNA analysis on a terminal-stage patient who had shown no response to previous chemotherapy. After detecting alterations in the RTK–RAS–MAPK pathway in the ctDNA, the patient was treated with MEK + EGFR inhibitors and achieved a tumor shrinkage rate of 76.33%. Our study utilized the largest Chinese SBA cohort to uncover the molecular characteristics of this disease, which might facilitate clinical decision making for SBA patients. [ABSTRACT FROM AUTHOR]

Abstract 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.

Purpose: to analyze the frequency of GNAQ/GNA11 mutations in circulating tumor DNA and tumor tissue, and the frequency of genotypes of polymorphic marker C3435T of ABCB1 gene in patients with iris melanoma. Material and methods. The study included 139 patients with uveal melanoma (UM) followed in 2011–2023. The experimental group included 46 patients with iris melanoma (n = 20, group I) and ciliary body involvement (n = 26, group II), who underwent a molecular genetic study. The comparison group III consisted of 30 UM patients managed in 2012. Morphologically, uveal melanoma was verified in all cases. Results. No mutations in the GNAQ/GNA11 genes were identified in group I. In group II, one heterozygous mutation in the GNAQ/GNA11 genes was detected in 2 patients (7.7 %). No significant associations with clinical or pathomorphological features were found (p > 0.1). In the comparison group III, mutations in the GNAQ/ GNA11 genes were detected in 27 patients (90 %). When comparing the frequency of heterozygous mutations in the GNAQ/ GNA11 genes significant differences between the experimental groups and the comparison group were revealed (F = 0.0000001, χ2 = 56.45). The CC genotype of the C3435T polymorphic marker of the ABCB1 gene was found in 90 % (F = 0.026418, χ2 = 5.36, significantly more frequently than in group III), in group II — in 92.3 % (F = 0.006183, χ2 = 7.75, significantly more frequently than in group III), in group III it was found in 60 %. The TT genotype was not detected in any group. Conclusion. This study has shown that the frequency of mutations in GNAQ and GNA11 genes, the frequency of CT genotype of ABCB1 polymorphic marker C3435T gene in iris melanoma is significantly lower than that in choroid melanoma, indicating a relatively favorable tumor behaviour.

Abstract Background Lazertinib is a potent, irreversible, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with significant efficacy in patients with EGFR T790M-mutated non-small cell lung cancer (NSCLC). This is the final overall survival (OS) report from the phase 1/2 LASER201 study in patients with advanced NSCLC with disease progression on or after prior EGFR TKI therapy. Methods Eligible patients were aged ≥ 20 years, with advanced EGFR-mutated NSCLC and previous therapy with EGFR TKI. Patients in this integrated analysis received oral lazertinib 240 mg/day. Endpoints included efficacy and safety; exploratory analyses included associations between circulating EGFR-mutant tumor DNA (ctDNA) and efficacy parameters. Results This integrated analysis included 78 patients in Korea who received second- or later-line lazertinib. The median OS was 38.9 months; estimated survival rates at 12, 24, and 36 months were 89.5%, 73.9%, and 52.8%, respectively. The cumulative 12-month incidence of central nervous system progression was 9.4%. EGFR-mutant ctDNA was detected in 46 patients (62.2%) at baseline. The presence of ctDNA at baseline significantly predicted progression-free survival (PFS), disease control rate (DCR), and OS. PFS, response rate, and DCR were significantly associated with EGFR-mutant ctDNA clearance at cycle 3; PFS and OS were significantly associated with ctDNA clearance at cycle 5. The safety profile of lazertinib 240 mg/day was consistent with previous findings. Conclusions Lazertinib is a promising treatment option for patients with EGFR T790M-positive NSCLC following disease progression on prior EGFR-directed TKIs. Patients in LASER201 experienced prolonged OS, regardless of their EGFR mutation, brain metastases, or prior brain radiation status. Clearance of plasma EGFR mutations after lazertinib was associated with patient outcomes. Trial registration ClinicalTrials.gov identifier NCT03046992.

Bin Liu,&ast; Bingtian Zhao,&ast; Yan Yin, Yan Jiang, Xue Feng, Lei Wang, Liang Zhai, Guangxin Liu, Dongsheng Shi, Jianwen Qin Department of Respiratory and Critical Medicine, Tianjin Chest Hospital, Tianjin, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Dongsheng Shi; Jianwen Qin, Tianjin Chest Hospital, No. 261, Taierzhuang South Road, Jinnan District, Tianjin, 300051, People’s Republic of China, Email shi_ds@126.com; qinjianwen2005@aliyun.com; 5020200603@nankai.edu.cnPurpose: DNA methylation plays a regulatory role in the oncogenesis and tumor progression and is valuable in the diagnosis and prognosis of cancer. While circulating tumor DNA (ctDNA) is widely used in the detection of oncogenic mutations and the guidance of treatment in advanced non-small cell lung cancer (NSCLC), studies of ctDNA methylation remains insufficient. We aim to investigate the methylation profiles of ctDNA in patients with advanced NSCLC undergoing EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy and to discover novel biomarkers with predictive or prognostic value.Patients and Methods: We recruited 49 patients with EGFR-mutated advanced NSCLC undergoing EGFR-TKI as first-line treatment. Utilizing next-generation sequencing, we examined the somatic mutations and methylation signatures within the tumor-associated genomic regions of ctDNA from pre-treatment blood. Subsequently, we explored the association of these molecular features with the patients’ response to therapy and their progression-free survival (PFS).Results: Genomic mutation profiling revealed no significant association of PFS or best overall response (BOR) and ctDNA status. Evaluation of ctDNA methylation showed a negative correlation between the methylation of small nucleolar RNA (snoRNA) genes and PFS (R=− 0.31, P=0.043). Furthermore, high-level methylation of SNORD3F was associated with poorer PFS (mPFS 346d vs 243d, HR 0.49, 95% CI 0.24– 0.93, P=0.029).Conclusion: Our study explored the prognostic value of ctDNA methylation in patients with advanced NSCLC undergoing targeted therapies and first revealed the predictive role of SNORD3F.Keywords: ctDNA, EGFR, DNA methylation, lung cancer, snoRNA, SNORD3F

Abstract The assessment of ctDNA has emerged as a minimally invasive avenue for molecular diagnosis and real-time tracking of tumor progression in NSCLC. However, the evaluation of ctDNA by amplicon-based NGS has been not endorsed by all the healthcare systems and remains to be fully integrated into clinical routine practice. To compare tissue single-gene with plasma multiplexed testing, we retrospectively evaluated 120 plasma samples from 12 consecutive patients with advanced non-squamous NSCLC who were part of a prospective study enrolling treatment-naïve patients and in which tissue samples were evaluated using a single-gene testing approach. While the plasma ctDNA detection of EGFR and BRAF mutations had an acceptable level of concordance with the archival tissue (85%), discordance was seen in all the patients in whom ALK alterations were only detected in tissue samples. Among six responders and six non-responders, early ctDNA mutant allelic frequency (MAF) reduction seemed to predict radiologic responses and longer survival, whereas increasing MAF values with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early indicator of molecular and radiologic progression. This report using an amplicon-based NGS assay on ctDNA underscores the real-life need for plasma and tissue genotyping as complementary tools in the diagnostic and therapeutic decision-making process.

Abstract Background Circulating tumour DNA (ctDNA) is a subset of cell free DNA (cfDNA) released by tumour cells into the bloodstream. Circulating tumour DNA has shown great potential as a biomarker to inform treatment in cancer patients. Collecting ctDNA is minimally invasive and reflects the entire genetic makeup of a patient’s cancer. ctDNA variants in NGS data can be difficult to distinguish from sequencing and PCR artefacts due to low abundance, particularly in the early stages of cancer. Unique Molecular Identifiers (UMIs) are short sequences ligated to the sequencing library before amplification. These sequences are useful for filtering out low frequency artefacts. The utility of ctDNA as a cancer biomarker depends on accurate detection of cancer variants. Results In this study, we benchmarked six variant calling tools, including two UMI-aware callers for their ability to call ctDNA variants. The standard variant callers tested included Mutect2, bcftools, LoFreq and FreeBayes. The UMI-aware variant callers benchmarked were UMI-VarCal and UMIErrorCorrect. We used both datasets with known variants spiked in at low frequencies, and datasets containing ctDNA, and generated synthetic UMI sequences for these datasets. Variant callers displayed different preferences for sensitivity and specificity. Mutect2 showed high sensitivity, while returning more privately called variants than any other caller in data without synthetic UMIs – an indicator of false positive variant discovery. In data encoded with synthetic UMIs, UMI-VarCal detected fewer putative false positive variants than all other callers in synthetic datasets. Mutect2 showed a balance between high sensitivity and specificity in data encoded with synthetic UMIs. Conclusions Our results indicate UMI-aware variant callers have potential to improve sensitivity and specificity in calling low frequency ctDNA variants over standard variant calling tools. There is a growing need for further development of UMI-aware variant calling tools if effective early detection methods for cancer using ctDNA samples are to be realised.

BACKGROUND: The DYNAMIC trial investigated the use of circulating tumor DNA (ctDNA) to guide adjuvant treatment decisions in stage II colon cancer. Despite the DYNAMIC trials assertion that a ctDNA-guided approach could minimize the use of adjuvant treatment without compromising recurrence-free survival (RFS), we raised concerns regarding the trials methodology and the practical implications of its findings in a Debate article. Here, we expand upon these concerns in a response to a correspondence by the authors of the DYNAMIC trial. MAIN BODY: We dispute the choice of a large non-inferiority margin in the DYNAMIC trial, simply because an 8.5 percentage points decrease in recurrence-free survival could result in significant harm to patients. We challenge the authors comparisons of the DYNAMIC trial outcomes with observational studies. Such comparison is subject to selection bias and changes over time that limit their relevance. The prognostic role of ctDNA do not automatically imply that more treatment in patients with ctDNA positivity would improve outcomes, which we highlight. In real-world settings, we anticipate a potential rise in chemotherapy use due to clinicians utilizing ctDNA alongside existing clinicopathologic factors, rather than using ctDNA as an entire replacement. Lastly, a key concern in DYNAMIC was an 350% higher use of oxaliplatin in the ctDNA arm compared with standard management (9.5% versus 2.7%, respectively), which poses a risk for long-term neuropathy. CONCLUSION: We look forward improvements in patient selection in the adjuvant setting, but we maintain our reservations about the DYNAMIC trial and the real-life implementation of its results. As an alternative to exploring de-escalation strategies with large margins non-inferiority trials, we propose that superiority trials in stage II patients could be a more effective strategy.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor‐positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth‐regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA‐mutated ctDNA were detected 4–17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real‐time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth‐related genes may contribute to combined CDK4/6i and endocrine therapy resistance. [ABSTRACT FROM AUTHOR]

Transplant oncology is an expanding area of cancer therapy that specifically emphasizes the use of liver transplantation (LT) as the preferred treatment for patients with manageable, but unresectable, tumors. The management and optimization of overall survival strategies, accompanied by an arguably decent quality of life, have been at the forefront of liver oncology treatment, as a plurality of all primary liver cancers are identified as either hepatocellular carcinoma (HCC) or cholangiocarcinoma (CCA), which are classified as highly aggressive malignancies and frequently remain asymptomatic until they progress to advanced stages, rendering curative procedures, such as resection, impractical. This has led to an increase in utilization of neoadjuvant interventions conducted prior to surgery, which has yielded favorable outcomes. Though this treatment modality has prompted further investigations into the efficacy of immune checkpoint inhibitors (ICPIs) as standalone treatments and in combination with locoregional treatments (LRTs) to bridge more patients into curative eligibility. This multidisciplinary methodology and treatment planning has seen multiple successful trials of immunotherapy regimes and combinate treatments, setting the groundwork for increasing eligibility through downstaging and “bridging” previously ineligible patients within stringent LT criteria. Surveillance after LT is a crucial component of transplant oncology. The emergence of circulating tumor DNA (ctDNA) has provided a novel approach to identifying the recurrence of cancer in its early stages. Recent research has focused on liquid biopsy, a technique that effectively identifies the dynamics of cancer. This is another innovation to demonstrate the rate at which transplant oncology is rapidly advancing, making the focus of care feel disorienting. Modalities of care are constantly evolving, but when a field is changing as rapidly as this one, it is imperative to reorient to the data and the needs of the patients. In this commentary, we reflect on the update’s utilization of ICPIs in neoadjuvant settings as well as the updates on the utilization of liquid biopsy in post-LT follow-up surveillance.

Abstract Background Triple negative breast cancer (TNBC) is an aggressive subtype with poor prognosis. We aimed to determine whether circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) could predict response and long-term outcomes to neoadjuvant chemotherapy (NAC). Methods Patients with TNBC were enrolled between 2017–2021 at The University of Texas MD Anderson Cancer Center (Houston, TX). Serial plasma samples were collected at four timepoints: pre-NAC (baseline), 12-weeks after NAC (mid-NAC), after NAC/prior to surgery (post-NAC), and one-year after surgery. ctDNA was quantified using a tumor-informed ctDNA assay (SignateraTM, Natera, Inc.) and CTC enumeration using CellSearch. Wilcoxon and Fisher’s exact tests were used for comparisons between groups and Kaplan–Meier analysis used for survival outcomes. Results In total, 37 patients were enrolled. The mean age was 50 and majority of patients had invasive ductal carcinoma (34, 91.9%) with clinical T2, (25, 67.6%) node-negative disease (21, 56.8%). Baseline ctDNA was detected in 90% (27/30) of patients, of whom 70.4% (19/27) achieved ctDNA clearance by mid-NAC. ctDNA clearance at mid-NAC was significantly associated with pathologic complete response (p = 0.02), whereas CTC clearance was not (p = 0.52). There were no differences in overall survival (OS) and recurrence-free survival (RFS) with positive baseline ctDNA and CTC. However, positive ctDNA at mid-NAC was significantly associated with worse OS and RFS (p = 0.0002 and p = 0.0034, respectively). Conclusions Early clearance of ctDNA served as a predictive and prognostic marker in TNBC. Personalized ctDNA monitoring during NAC may help predict response and guide treatment.

Current advances in the understanding of the lung cancer landscape have drastically changed the approach to treating a patient with lung carcinoma. The field has progressed from analyzing single gene to using advanced techniques like next-generation sequencing and microarray technology. While a tumor tissue sample is considered the gold standard, it has several limitations. The limitations of invasive procedures, long processing periods, inaccessibility, and sample inadequacy are being addressed by sampling biofluids, termed ‘liquid biopsy,’ which offers a less invasive and more accessible way to obtain tumor-related information. Liquid biopsy has transformed the care of lung cancer patients by directly targeting somatic alterations from tumors. This article provides insights into the biology, technical aspects, limitations, and practical applications of ‘liquid biopsy,’ focusing on cell-free DNA and circulating tumor DNA in the context of lung cancer.

Abstract Background Colon cancer ranks as the second most lethal form of cancer globally. In recent years, there has been active investigation into using the methylation profile of circulating tumor DNA (ctDNA), derived from blood, as a promising indicator for diagnosing and monitoring colon cancer. Results We propose a liquid biopsy-based epigenetic method developed by utilizing 49 patients and 260 healthy controls methylation profile data to screen and monitor colon cancer. Our method initially identified 901 colon cancer-specific hypermethylated (CaSH) regions in the tissues of the 49 cancer patients. We then used these CaSH regions to accurately quantify the amount of circulating tumor DNA (ctDNA) in the blood samples of these same patients, utilizing cell-free DNA methylation profiles. Notably, the methylation profiles of ctDNA in the blood exhibited high sensitivity (82%) and specificity (93%) in distinguishing patients with colon cancer from the control group, with an area under the curve of 0.903. Furthermore, we confirm that our method for ctDNA quantification is effective for monitoring cancer patients and can serve as a valuable tool for postoperative prognosis. Conclusions This study demonstrated a successful application of the quantification of ctDNA among cfDNA using the original cancer tissue-derived CaSH region for screening and monitoring colon cancer.

Abstract Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis.

The application of adjuvant treatment has significantly enhanced the survival of patients with resectable non-small cell lung cancer (NSCLC) carrying driver gene mutations. However, adjuvant-targeted therapy remains controversial for some NSCLC patients carrying rare gene mutations such as RET, as there is currently a lack of confirmed randomized controlled trials demonstrating efficacy. In this report, we describe the case of a 58-year-old man with stage IIIA NSCLC who underwent complete lobectomy with selective lymph node dissection. Postoperative next-generation sequencing revealed that the patient harbored a rare KIF13A-RET fusion. The patient elected to receive adjuvant treatment with pralsetinib monotherapy and underwent serial circulating tumor DNA (ctDNA) monitoring after surgery. During follow-up, despite experiencing dose reduction and irregular medication adherence, the patient still achieved a satisfactory disease-free survival (DFS) of 27 months. Furthermore, ctDNA predicted tumor recurrence 4 months earlier than imaging techniques. The addition of bevacizumab to the original regimen upon recurrence continued to be beneficial. Pralsetinib demonstrated promising efficacy as adjuvant therapy, while ctDNA analysis offered a valuable tool for early detection of tumor recurrence. By leveraging targeted therapies and innovative monitoring techniques, we aim to improve outcomes and quality of life for NSCLC patients in the future.

Abstract Detecting aberrant cell-free DNA (cfDNA) methylation is a promising strategy for lung cancer diagnosis. In this study, our aim is to identify methylation markers to distinguish patients with lung cancer from healthy individuals. Additionally, we sought to develop a deep learning model incorporating cfDNA methylation and fragment size profiles. To achieve this, we utilized methylation data collected from The Cancer Genome Atlas and Gene Expression Omnibus databases. Then we generated methylated DNA immunoprecipitation sequencing and genome-wide Enzymatic Methyl-seq (EM-seq) form lung cancer tissue and plasma. Using these data, we selected 366 methylation markers. A targeted EM-seq panel was designed using the selected markers, and 142 lung cancer and 56 healthy samples were produced with the panel. Additionally, cfDNA samples from healthy individuals and lung cancer patients were diluted to evaluate sensitivity. Its lung cancer detection performance reached an accuracy of 81.5% and an area under the receiver operating characteristic curve of 0.87. In the serial dilution experiment, we achieved tumor fraction detection of 1% at 98% specificity and 0.1% at 80% specificity. In conclusion, we successfully developed and validated a combination of methylation panel and a deep learning model that can distinguish between patients with lung cancer and healthy individuals.