Liquid biopsy encompasses a variety of molecular approaches to detect circulating tumor DNA (ctDNA) and has become a powerful tool in the diagnosis and treatment of solid tumors. Current applications include comprehensive genomic profiling for identifying targetable mutations and therapeutic resistance mechanisms, with emerging applications in minimal residual disease detection and treatment response monitoring. Increasingly, the potential for liquid biopsy in guiding treatment decisions is under active investigation through prospective clinical trials using ctDNA-adaptive interventions in patients with early-stage and metastatic cancers. Limitations arise on the basis of the sensitivity and feasibility of individual liquid biopsy assays; nonetheless, emerging technologies set the stage for improving these shortcomings. As the global oncology community continues to ascertain the clinical value of liquid biopsy across the continuum of patient care, this minimally invasive approach heralds a significant advancement in the promise of precision oncology.

Liquid biopsies offer the possibility to evaluate cancer patients using noninvasive approaches. Circulating cell-free DNA (ccfDNA) is 1 of the most used and promising sources. Detecting tumor DNA among ccfDNA (ctDNA) can be used for early cancer detection, treatment response assessment, prognosis, and predictive evaluations. Providing analyses that can increase the quality of patient treatment is very much a joint effort between laboratory scientists and clinicians. With its use approaching clinical practice, it is important for clinicians to be familiar with the basic concepts and analyses behind ctDNA results in a similar way as laboratory scientists should have knowledge of the clinical needs to provide relevant analyses. In this Perspective, we describe the whole process of ctDNA analyses, from the preanalytical standards to reporting/analyzing results, and highlight some important factors that need to be addressed in the process of implementing them to clinical practice.

Molecular testing plays a critical role in guiding optimal treatment decisions for lung cancer patients across a variety of clinical settings. While guidelines for biomarker testing exist in other jurisdictions, to date no best practice guidelines have been developed for the Australian setting. To address this need, the Royal College of Pathologists of Australasia collaborated with the Thoracic Oncology Group of Australasia to identify state-based pathologists, oncologists and consumer representatives to develop consensus best practice recommendations. Sixteen recommendations were established encompassing appropriate biomarkers, lung cancer subtype, tumour stage, specimen types, assay selection and quality assurance protocols that can inform and standardise best practice in molecular testing of lung cancer. These multidisciplinary evidence-based recommendations are designed to standardise and enhance molecular testing practices for lung cancers and should help ensure laboratories provide high-quality molecular testing of lung cancer for all Australians, including those from regional or remote communities.

The ability to detect specific DNA, including single nucleotide variants (SNVs), with high sensitivity is essential for advancing genetic research, diagnostics, and personalized medicine. This study presents a novel method for ultrasensitive DNA detection, combining ligation-rolling circle amplification (L-RCA) with CRISPR-Cas12a. While L-RCA systems have been widely used for nucleic acid detection, the sensitivity of conventional L-RCA generally reaches approximately 100 pM. Here, we demonstrate that the sensitivity of RCA-Cas12a systems can be markedly enhanced by incorporating multiple CRISPR target regions into the padlock probe. This method achieves remarkable sensitivity, detecting DNA at concentrations as low as 1 aM (6 copies per reaction), and is capable of identifying single nucleotide variants (SNVs) with allele fractions as low as 1 %. Unlike many current complex RCA-Cas12a strategies, this approach is simple and does not require advanced labeling or instrumentation, making it a promising tool for ultrasensitive DNA detection in various applications.

Breast cancer (BC) might change its receptor status during malignant progression, resulting in challenging clinical care. The project, developed during two residential meetings by expert Italian oncologists and pathologists, was aimed at optimizing metastatic BC management. A 17-point survey was administered to healthcare workers in centers of three regions, essentially to assess the perception of metastasis biopsy on treatment choice in different BC molecular subtypes, the appropriate timing for performing tissue and liquid biopsy in metastatic BC, the selection of target genes and the use of multidisciplinary approaches in tumour management. Nineteen centers participated in the survey. The first important finding was that most centers manage more than 150 patients yearly, and 95% have a pathology department. Some questions received a substantial agreement: tissue biopsy of metastatic BC was largely considered as mandatory, generally performed at onset of metastatic disease. Liquid biopsy is not used by all centers, confirming its strict dependency on the available technologies. The answers on the therapeutic line change in case of HER2 + disease becoming triple negative confirmed an attitude consistent with clinical practice. The survey also revealed that half of the interviewed oncologists discuss with multidisciplinary team only for selected cases. The survey revealed that while a shared consensus exists on the importance of metastasis biopsy, some areas still deserve attention to achieve standardized approaches: interdisciplinary collaboration and more effective communication between specialists and patients are necessary to optimize the diagnostic and therapeutic journey of women with metastatic BC.

Tebentafusp, a bispecific fusion protein consisting of affinity-enhanced T cell receptor fused to anti-CD3 effector, has shown overall survival (OS) benefits across all RECIST response categories, including progressive disease (PD). In a phase 2 trial (NCT02570308) for advanced uveal melanoma (mUM), 35 % of PD patients experienced ≥ 0.5 log ctDNA reduction, resulting in a median overall survival (OS) of ∼17 months, compared to ∼8 months in the non-ctDNA reduction group.

A total of 34 of 127 2L+ mUM patients with PD were split into two groups based on absence or presence of ctDNA reduction (≥0.5 log reduction). Lesions from CT and MRI scans were analyzed using radiomics features at baseline and week eight, yielding two machine-learning-derived patient signatures (16 features). Performance of per-patient analysis (n = 32) and per-lesion analysis (n = 148) was assessed using ROC AUC (95 % confidence interval [CI]).

In the per-patient analysis, a volumetric signature classified patients into groups with ROC AUC of 0.71 [0.53-0.90] with 63 % specificity and 81 % sensitivity at the optimal threshold (0.57). In the per-lesion analysis, a radiomic signature reached an ROC AUC of 0.70 [0.58-0.81] with 66 % specificity and 74 % sensitivity at the optimal threshold (0.53). Group B had lower baseline tumor lesion volume (ROC AUC=0.65), distinct baseline (ROC AUC=0.66), and change by week eight (ROC AUC=0.66/0.69 on CT/MRI) in tumor heterogeneity.

Radiomic analysis accurately predicted ctDNA reduction in PD patients at both the patient and lesion level. The most influential predictor was decreased tumor heterogeneity observed on CT/MRI.

For patients with resectable colorectal liver metastases (CRLM), the efficacy of adjuvant chemotherapy remains a subject of debate. Several studies have concluded that postoperative circulating tumor DNA (ctDNA) is a marker of minimal residual disease (MRD) and is a useful prognostic factor in patients with nonmetastatic colorectal cancer. However, few studies have explored its application in cases involving metastases. This was an observational study that included CRLM patients who underwent primary and liver tumor resection. By examining targeted sequencing of 50 genes commonly mutated in CRC, we identified at least one somatic mutation in each patient's metastatic liver tumor. Blood samples were obtained before and 1-month after surgery. Fifty-three patients were included, and recurrence was diagnosed in 39 patients. Of those, 13 patients experienced early relapse. ctDNA was detected in 45 patients before surgery and 11 after. All MRD-positive patients experienced recurrence. Among them, nine had early recurrence. MRD-positive patients had poorer recurrence free survival (RFS, p < 0.0001) and overall survival (OS, p < 0.0005). Nine of 13 patients with early recurrence had MRD; however, two of 40 patients without early recurrence also had MRD (p < 0.0001). Among 42 MRD-negative patients, adjuvant chemotherapy had no impact of RFS (p = 0.84) or OS (p = 0.54). MRD proved valuable in predicting the risk of postoperative recurrence in patients with CRLM, particularly because MRD positivity emerged as a significant risk factor for early recurrence. Furthermore, it appears that adjuvant chemotherapy may not effectively improve the prognosis for MRD-negative patients.

Advances in targeted therapies for patients with non-small-cell lung cancer have substantially improved the outcomes of those with actionable alterations in certain oncogenic driver genes. However, acquired resistance to these targeted therapies remains a major challenge. Understanding the mechanisms underlying acquired resistance will be crucial for the development of strategies that might either overcome this effect or delay the onset. Circulating tumour DNA, owing to the need for only minimally invasive sampling and a potential role as both a prognostic and predictive biomarker, is increasingly being used in both research and clinical practice. Several studies have explored the landscape of acquired resistance to targeted therapies using this approach. However, the methodologies of the published studies vary widely, and several major challenges remain in addressing the practical difficulties associated with these methods. These challenges currently limit the depth of research insight provided by the available data. In this Perspective, we review clinical reports describing the use of circulating tumour DNA to detect mechanisms of acquired resistance to targeted therapies, predominantly in patients with advanced-stage non-small-cell lung cancer, and highlight key unresolved questions with the aim of moving towards more-informative research studies.

Predictive biomarker identification in cancer treatment has traditionally relied on pre-defined analyses, limiting discoveries to expected biomarkers and potentially overlooking novel ones predictive of therapy response. In this work, we develop a novel machine-learning approach capable of exploring full landscape of mutations and combinations and identify potentially new predictive biomarkers for chemoimmunotherapy. Utilizing the liquid biopsy dataset from 313 non-small cell lung cancer (NSCLC) patients in the Phase 3 Impower150 trial (NCT02366143), we developed the HRdiffRF algorithm with a novel hazard ratio-splitting criterion. Predictive mutations and combinations were identified for overall survival (OS) improvement with atezolizumab plus bevacizumab plus carboplatin and paclitaxel (ABCP) compared to bevacizumab plus carboplatin and paclitaxel (BCP). Our analysis confirms the predictive role of KRAS mutations and reveals the predictive value of PTPRD and SMARCA4 mutations in chemoimmunotherapy efficacy. Unlike other KRAS wild-type NSCLC patients, NSCLC patients with KRAS wild-type status and mutations in FAT1, ERBB2, or PTPRD may benefit from chemoimmunotherapy, while NTRK3 and GNAS mutations could negatively impact survival. Patients harboring concurrent KRAS and KEAP1 mutations may not benefit from chemoimmunotherapy. These findings highlight the complex genetic factors influencing treatment response for chemoimmunotherapy in NSCLC. In summary, the proposed machine-learning tool identified potential predictive biomarkers for first-line chemoimmunotherapy in NSCLC and can be readily applied to other tumor types and studies. It can also be extended to explore predictive biomarkers beyond mutations.

Cell-free DNA (cfDNA) is changing the face of liquid biopsy as a minimally invasive tool for disease detection and monitoring, with its main applications in oncology and prenatal testing, and rising roles in transplant patient monitoring. However, the processes of cfDNA biogenesis, fragmentation, and clearance are complex and require further investigation. Evidence suggests that cfDNA production relates to mechanisms of cell death and DNA repair, both of which further influence fragment size and its applicability as a biomarker. An emerging domain, cfDNA fragmentomics is being explored for advancing the field of diagnostics using non-mutational signatures such as fragment size ratios and methylation patterns. Thus, this review examines structural diversity in cfDNA with various fragment sizes. In examining these cfDNA subsets, we discuss their distinct biological origins and potential clinical utility. Development of sequencing methodologies has broadened the application of cfDNA in diagnosing cancers and organ-specific pathologies, as well as directing personalized therapies. This has been achieved by identifying and uncovering different subsets of cfDNA in biofluids using different methodologies and biofluids. Different cfDNA subsets provide important insights regarding genomic and epigenetic features, enhancing the understanding of gene regulation, tissue-specific functions, and disease progression. Advancement of these key areas further asserts increasing clinical relevance for the use of cfDNA as a biomarker. Continued exploration of cfDNA subsets is expected to drive further innovation in liquid biopsy and its integration into routine clinical practice.

Liquid biopsy has already proven effective in aiding diagnosis, risk stratification and treatment personalization in several malignancies, and it could represent a practice-changing tool also in biliary tract cancer, even though clinical applications are currently still limited. It is promising for early diagnosis, especially in high-risk populations, and several studies on circulating free DNA (cfDNA), circulating tumour cells and differential microRNA (miRNA) profiles in this setting are ongoing. Circulating tumour DNA (ctDNA) also appears as a feasible noninvasive biomarker in the curative setting, in detecting minimal residual disease after resection and in monitoring disease recurrence. As of today, it can be particularly valuable in biliary tract cancer for genomic profiling, with a good concordance with tissue samples for most molecular alterations. CtDNA analysis may especially be considered in clinical practice when the tumour tissue is not sufficient for next-generation sequencing, or when urgent therapeutic decisions are needed. Moreover, it offers the possibility of providing a real-time picture to monitor treatment response and dynamically identify resistance mutations, potentially representing a way to optimize treatment strategies.

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising method for detection of minimal or molecular residual disease (MRD) among patients with breast cancer.

In this narrative review, we provide a summary of currently available studies assessing use of ctDNA in detection of MRD in patients after completion of curative therapy. Additionally, we discuss limitations of present studies, future considerations, and an overview of ongoing trials evaluating the clinical utility of MRD-directed therapy interventions.

While the clinical utility of MRD-directed therapy guidance remains under investigation, collective data from studies overwhelmingly confirm the prognostic value of ctDNA status across various stages and subtypes of breast cancer. Results from ongoing clinical trials in the coming years will provide more clarity on the overall clinical benefit of MRD-directed interventions for breast cancer patients.

Circulating tumor DNA (ctDNA) is a promising biomarker for predicting minimal residual disease (MRD) and guiding treatment decisions in patients with colorectal cancer (CRC). This study aimed to examine the study designs and settings of ongoing clinical trials that use ctDNA to guide treatment decisions and to determine the best timing for detecting MRD in non-metastatic CRC. We searched PubMed, Embase, Web of Science, Cochrane Library, and clinicaltrials.gov for English language records. The ctDNA settings from the clinical trials were categorized by randomization to ctDNA testing, treatment options based on ctDNA results, and the timing of ctDNA testing relative to adjuvant therapy. For prospective studies, a network meta-analysis using a frequentist approach was conducted to examine the pairwise associations between different ctDNA timing strategies and MRD, defined as recurrence, relapse, and progression. The main approaches in ctDNA-based interventional trial designs were categorized as ctDNA-guided treatment, ctDNA-by-treatment, ctDNA-guided surveillance, and ctDNA-enriched adjuvant therapy for guiding treatment decisions, including both escalation and de-escalation strategies, and surveillance. Overall, both preoperative and postoperative ctDNA detection were linked to higher risks of progression, with pooled hazard ratios (95% confidence intervals) of 5.23 (2.10-13.00) and 7.95 (5.30-11.91), respectively. Among the timing strategies, ctDNA testing after adjuvant therapy was the most effective for identifying high-risk patients, strongly suggesting the presence of residual disease. This study comprehensively reviewed the clinical settings of ctDNA testing in ongoing trials and provided evidence supporting the selection of post-adjuvant therapy as the optimal timing for ctDNA testing.

Breast cancer is one of the most prevalent cancers and has emerged as a major global challenge. Circulating tumor DNA (ctDNA), a liquid biopsy method, overcomes the accessibility limitations of tissue-based testing and is widely used for monitoring minimal residual disease and molecular relapse, predicting prognosis, evaluating the response of neoadjuvant therapy, and optimizing treatment decisions in non-metastatic breast cancer. However, the application of ctDNA still faces many challenges. Here, we survey the clinical applications of ctDNA in non-metastatic breast cancer and discuss the significant biological and technical challenges of utilizing ctDNA. Importantly, we investigate potential avenues for addressing the challenges. In addition, emerging technologies, including fragmentomics detection, methylation sequencing, and long-read sequencing, have clinical potential and could be a future direction. Proper utilization of machine learning facilitates the identification of meaningful patterns from complex fragment and methylation profiles of ctDNA. There is still a lack of clinical trials focused on the subsets of ctDNA (e.g., circulating mitochondrial DNA), ctDNA-inferred drug-resistant clonal evolution, tumor heterogeneity, and ctDNA-guided clinical decision-making in non-metastatic breast cancer. Due to regional differences in the number of registered clinical trials, it is essential to enhance communication and foster global collaboration to advance the field.

Peritoneal carcinomatosis (PC) represents a challenge in the management of metastatic colorectal cancer (mCRC) because of the difficulties in diagnosis, tumor burden assessment, and in selecting the optimal treatments. A critical limitation is the lack of robust prognostic and predictive biomarkers, largely relying on serum markers (e.g., carcinoembryonic antigen) or the peritoneal carcinomatosis index (PCI) for disease extent. Circulating tumor DNA (ctDNA)-genomic fragments shed by tumor cells into the bloodstream-is now recommended by international guidelines for mCRC management. Its potential extends to PC, where it may enhance diagnostic, therapeutic, and follow-up strategies. However, PC from CRC (PC-CRC) is associated with lower ctDNA levels and detection rates compared to other metastatic sites, posing a challenge for its clinical utility. To address these limitations, peritoneal fluid analysis has emerged as a promising alternative, with peritoneal tumor DNA (ptDNA) detected at higher concentrations in this anatomical space. Integrating ctDNA and ptDNA may offer a deeper understanding of PC-CRC biology and provide more precise tools for managing this complex disease. This approach has the potential to revolutionize the treatment paradigm for PC-CRC, bringing precision medicine even to this subgroup of patients traditionally associated with poor outcomes. This review aims to evaluate the diagnostic, prognostic, and therapeutic implications of ctDNA and ptDNA in PC-CRC, highlighting current limitations and future directions.

Liquid biopsies, indicating the sampling of body fluids rather than solid-tissue biopsies, have the potential to revolutionize cancer care through personalized, noninvasive disease detection and monitoring. Circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) are promising blood-based biomarkers in bladder cancer. Results from several studies have shown the clinical potential of ctDNA and CTCs in bladder cancer for prognostication, treatment-response monitoring, and early detection of minimal residual disease and disease recurrence. Following successful clinical trial evaluation, assessment of ctDNA and CTCs holds the potential to transform the therapeutic pathway for patients with bladder cancer - potentially in combination with the analysis of urinary tumour DNA - through tailored treatment guidance and optimized disease surveillance.

Fragmentomics analysis of plasma autosomal DNA has shown promise in cancer diagnostics. Here we evaluated the clinical utility of plasma Epstein-Barr virus (EBV) DNA fragmentomics analysis for nasopharyngeal carcinoma (NPC) screening. Among our prospective cohort of approximately 20,000 subjects that underwent two rounds of screening, we analyzed the first-round blood samples of subjects who tested positive for EBV DNA via polymerase chain reaction (PCR) (n = 558). We found that those who subsequently developed NPC in the second round exhibited a distinctive mononucleosomal size pattern, an NPC-associated end motif (specifically, a depletion of CC-motif) and aberrations in methylation identified through fragmentomics-based methylation analysis (FRAGMA). Subjects with these aberrant fragmentomics features and higher quantity of EBV DNA had a relative risk of 87.1 times greater for developing NPC in the second round compared to subjects tested negative for EBV DNA on PCR. These results demonstrate plasma DNA fragmentomics could predict future cancer risk.

The advent of personalised and precision medicine has radically modified the management and the clinical outcome of cancer patients. However, the expanding number of predictive, prognostic, and diagnostic biomarkers has raised the need for simple, noninvasive, quicker, but equally efficient tests for molecular profiling. In this complex scenario, the adoption of liquid biopsy, particularly circulating tumour DNA (ctDNA), has been a real godsend for many cancer patients who would otherwise have been denied the benefits of targeted treatments. Undeniably, ctDNA analysis has several advantages over conventional tissue-based analysis. One advantage is that it can guide treatment decision making, especially when tissue samples are scarce or totally unavailable. Indeed, a simple blood test can inform clinicians on patients' response or resistance to targeted therapies, help them monitor minimal residual disease (MRD) after surgical resections, and facilitate them with early cancer detection and interception. Finally, an equally important advantage is that ctDNA analysis can help decipher temporal and spatial tumour heterogeneity, a mechanism highly responsible for therapeutic resistance. In this review, we gathered and analysed current evidence on the clinical usefulness of ctDNA analysis in solid tumours.

Metastatic breast cancer (BC) is the main cause of cancer-related mortality in women worldwide. HR + /HER2- BC patients are treated with endocrine therapy (ET), but therapeutic resistance is common. The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with ET was approved for metastatic BC patients and extended the median progression-free survival to 24 months. This therapy is not always effective, and in every patient, resistance ultimately occurs, but the underlying resistance mechanisms remain unclear. To address this gap, we explored circulating tumour cells (CTCs) as biomarkers to assess treatment response and resistance in metastatic HR + /HER2- BC patients receiving CDK4/6i plus ET.

In total, 53 HR + /HER2- metastatic BC patients who received a CDK4/6i plus ET as first-line treatment were analysed, including samples from internal and external validation cohorts. CTCs were isolated using the negative enrichment approach RosetteSep (STEMCELL Technologies) or positive immunomagnetic selection targeting EpCAM, EGFR, and HER2 (AdnaTest EMT-2/StemCell Select™, QIAGEN). RNA was extracted from CTCs and PBMCs for nCounter analysis (Pancancer pathways panel) in a discovery phase. Subsequent validation was performed by RT-qPCR.

CTC gene expression analysis revealed that non responder patients (those who experienced disease progression before 180 days) exhibited elevated PRKCB (p-value: 0.011), MAPK3 (p-value: 0.006) and STAT3 (p-value: 0.008) expression, while responders showed increased CDK6 (p-value: 0.011) and CCND1 (p-value: 0.035) expression at baseline. CTC transcriptional characterization revealed a gene expression signature (STAT3highPRKCBhighCDK6low) that accurately classified HR + /HER2- metastatic BC patients who responded to CDK4/6i plus ET, regardless of the CTC isolation method (AUC > 0.8). CTC characterization at progression also identified biomarkers linked to therapy resistance, including the epigenetic regulators EZH2 and HDAC6 and the cell cycle regulator CDC7, which could guide the selection of subsequent therapy lines. The expression of the CDK4 and STAT3 genes in CTCs was associated with progression-free survival and overall survival, respectively. Likewise, the presence of ≥ one CTC after one cycle of therapy predicts a worse prognosis.

CTC gene expression provides information about treatment outcomes in HR + /HER2- metastatic BC patients receiving CDK4/6i plus ET and could guide personalized strategies and improve prognosis.

In patients with solid tumors undergoing neoadjuvant immune checkpoint inhibitor (ICI) therapy, identifying biomarkers to predict pathologic complete response (pCR) preoperatively could enhance treatment modulation. Circulating tumor DNA (ctDNA) clearance is a potential predictor of pCR, though its analytical and clinical validity has yet to be established. This systematic review and meta-analysis aims to assess the role of ctDNA clearance as a predictor of pCR in patients with solid tumors treated with neoadjuvant ICIs.

A systematic search of PubMed, EMBASE and conference proceedings up to 5 August 2024 was carried out to identify phase Ib, II or III clinical trials investigating ctDNA clearance and pCR in patients with solid tumors and detectable ctDNA, undergoing neoadjuvant therapy with ICIs. Using a bivariate model, we estimated the pooled sensitivity and specificity of ctDNA clearance in predicting pCR, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio, with 95% confidence intervals (CIs).

Thirteen trials involving 380 patients with detectable ctDNA at baseline were included. ctDNA was assessed with a tumor-informed approach in 11 (85%) trials. Overall, 38% of patients achieved pCR and 73% had ctDNA clearance before/at the surgery. Pooled sensitivity was 0.98 (95% CI 0.86-1.00), specificity was 0.53 (95% CI 0.37-0.69), positive likelihood ratio was 2.09 (95% CI 1.48-2.93), negative likelihood ratio was 0.04 (95% CI 0.01-0.26), diagnostic odds ratio was 57.36 (95% CI 8.12-405.12). Significant heterogeneity was observed across studies (I2 ∼70% for all metrics), indicating considerable variability in the diagnostic performance.

The lack of ctDNA clearance may identify patients unlikely to have a pCR. Instead, the confirmatory power of ctDNA clearance is limited by low specificity and high heterogeneity due to the variability of the assays, and warrants further study. Therefore, clinicians should not rely on the use of ctDNA clearance to inform treatment decisions in the neoadjuvant setting.

Liquid biopsy is a laboratory test used to detect and analyze circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and other tumor-derived components, in a blood sample. In the context of breast cancer (BC), liquid biopsies hold significant promise for guiding the use of immune checkpoint inhibitors and immune-based combinations, offering real-time insights into tumor dynamics, treatment response, and resistance mechanisms. This review explores the role of liquid biopsy in BC immunotherapy, focusing on its applications, benefits, issues, and current and future research directions.

The implementation of circulating tumor DNA (ctDNA) in the diagnostic routine may enable non-invasive predictive biomarker testing and treatment optimization in patients who lack a suitable tumor specimen, have failed previous molecular analysis or are clinically ineligible for (re-)biopsy procedures. As the interpretation and reporting are more complex for ctDNA than conventional tissue-based NGS, there is a need for specific guidelines. These will offer support for the reporting of ctDNA test results and will facilitate optimal communication of liquid biopsy findings between diagnostic laboratories and the medical oncology team. Aiming to generate guidelines based on real-world experiences and broad perspectives, we organized a European Liquid Biopsy Society (ELBS) ctDNA workshop, in which forty-four experts and key stakeholders from different molecular diagnostics laboratories, oncology and pathology departments, as well as an IVDR specialist, convened to address significant challenges associated with the reporting of liquid biopsy test results. This report delineates the resulting consensus recommendations for ctDNA test reporting with underlying rationale and background information.

The utility of capturing heterogeneity by circulating tumor DNA (ctDNA) genotyping combined with tissue analysis or applying it in a sequential manner remains uncertain.

We assessed the clinical value of ctDNA genotyping using data from 2,187 patients with advanced solid tumors enrolled in SCRUM-Japan MONSTAR-SCREEN-1, a nationwide cancer genome screening project, which examined ctDNA from longitudinally collected blood samples and tumor tissue samples (UMIN 000036749).

Among 667 patients with both baseline ctDNA and tissue genotyping results, 51 (7.6%) had actionable biomarkers identified exclusively through ctDNA genotyping. The most frequent targets of genotype-matched therapy guided by solely ctDNA were immune checkpoint, estrogen receptor, and poly(ADP-ribose) polymerase (PARP). Comparison of objective response rates (ORRs) and progression-free survival (PFS) between patients treated based on tissue versus ctDNA alone showed no significant difference, with ORRs of 34.0% versus 23.1% (P = .54) and a median PFS of 11.5 versus 13.8 months (hazard ratio [HR], 1.4 [95% CI, 0.72 to 2.80]), respectively. Among 924 patients undergoing sequential ctDNA genotyping, the detection of actionable biomarkers increased from 63.2% to 72.5% following subsequent ctDNA. Targets for genotype-matched therapy guided by subsequent ctDNA alone commonly included PARP, immune checkpoint, and BRAF. The ORR was 23.2% and 26.7% (P = .75), and the median PFS was 5.2 and. 3.7 months (HR, 1.5 [95% CI, 0.79 to 2.80]) for genotype-matched therapy based on initial versus subsequent ctDNA alone, respectively.

Combining ctDNA with tissue analysis, followed by sequential ctDNA assessments, effectively enhances the identification of actionable biomarkers. This strategy facilitates clinically beneficial, genetically informed therapies, underscoring its significant value in precision oncology.

Genomic profiling is a major component for first-line treatment decisions in patients with NSCLC and the timeliness of biomarker testing is essential to improve time to treatment initiation (TTI) or avoid inappropriate treatment.

The phase III LIquid Biopsy for the Early detection of LUng cancer Lesion trial (NCT03721120) included patients with radiological suspicion of advanced lung cancer. They were randomized (1:1), the control arm receiving diagnostic procedures according to each center's practice, and the liquid biopsy arm with additional testing performed at the first visit using the InVisionFirst-Lung assay. Treatment initiation and type were defined according to the European Society for Medical Oncology guidelines. Primary endpoint was the time from randomization to initiation of appropriate treatment on the basis of informative genomic and pathological results in the intention-to-treat population.

A total of 319 patients were enrolled (liquid biopsy [LB]: 161; control: 158). The median age was 68 years, 28.8% were non-smokers, 18.1% had a performance status of 2 or higher, and 56.7% had adenocarcinoma. In the LB arm, 81% of patients had circulating tumor DNA findings. The mean TTI was not significantly reduced (LB: 29.0 d; control 34 d (p = 0.26)). Sensitivity analyses found a shorter TTI in patients from the LB arm who received systemic treatment (LB: 29.1 d; control: 38.9 d, p = 0.01), in patients with advanced non-squamous NSCLC (LB: 29.5 d; control: 40.3 d, p = 0.01), and in patients with first-line targetable alterations (LB: 21d; control 37.4 d) (p = 0.004). Time to contributory genomic results was significantly reduced (LB: 17.9 d; control: 25.6 d, p < 0.001).

Early liquid biopsy testing did not significantly shorten the TTI in unselected patients referred for suspected advanced lung cancer. Nevertheless, it could reduce the TTI in patients eligible for systemic treatment, particularly for those with actionable alterations.

At present, no reliable blood-based biomarkers have been established for patients with endometrial cancer. Liquid biopsies, which can detect circulating tumor DNA (ctDNA), provide a non-invasive way to assess prognosis, monitor tumor evolution and treatment response. We aimed to examine the feasibility and performance of ctDNA as a prognostic tool in a multi-center cohort of EC patients with matched tumor samples.

Blood plasma samples were collected preoperatively from 83 patients at three European cancer centers. Circulating cell-free DNA (cfDNA) was isolated and analyzed using the Oncomine™ Pan-Cancer cell-free assay. Tumor tissue from 56 of the 83 patients was subjected to whole-exome sequencing, and clinical data were collected for oncological outcome assessment.

The mean input of cfDNA was 8.17 ng (range 1.47-29.12 ng). Sixteen (19.3 %) patients were considered ctDNA positive with mutations in one or more genes. Most alterations detected in plasma were concordant with mutations found in the matched tumor for the paired cases. The preoperative presence of ctDNA was associated with a significantly higher rate of recurrence (37.5 % vs 11.9 %, P = 0.024). Although eight of the 14 (57 %) patients with recurrence were negative for ctDNA at diagnosis, positive ctDNA status remained an independent predictor of recurrence also when controlling for other known histopathologic risk factors (HR 5.49, 95 % CI 1.5-20, P = 0.010).

Our results demonstrated the feasibility of using an off-the-shelf gene panel to detect ctDNA in patients with endometrial cancer. ctDNA positivity was significantly associated with worse oncological outcomes.

Although the 5-year relative survival rates for resectable solid tumors have improved over the past few years, the risk of postoperative recurrence necessitates effective monitoring strategies. Recent advancements in molecular residual disease (MRD) testing based on circulating tumor DNA (ctDNA) analysis have shown considerable promise in the context of predicting recurrence; however, significant barriers to widespread clinical implementation remain-mainly, low awareness among healthcare professionals, high costs, and lack of standardized assays and comprehensive evidence. This position paper, led by the Japan Society of Clinical Oncology, aims to establish a common framework for the appropriate clinical use of MRD testing in a tumor type-agnostic manner. It synthesizes currently available evidence, reviews region-specific clinical trends, addresses critical clinical questions related to MRD testing, and offers recommendations to guide healthcare professionals, biotechnology and pharmaceutical companies, and regulatory authorities. These recommendations were developed based on a voting process involving 15 expert members, ensuring a consensus-driven approach. These findings underscore the importance of collaborative efforts among various stakeholders in enhancing the clinical utility of MRD testing. This project aimed to foster consensus and provide clear guidelines to support the advancement of precision medicine in oncology and improve patient outcomes in the context of perioperative care.

Liquid biopsy gained significant interest in the area of cancer management. This study aims to evaluate the effectiveness of molecular testing using ctDNA (circulating tumor DNA) to; detect genetic alterations, screen for abnormalities, identify mutations associated with treatment sensitivity or resistance and guide therapy decision for several types of cancer in patients with metastasis. A total of 85 samples were collected from 74 patients recruited at our center, as part of their routine clinical follow-up. 17 different cancer types were analyzed. Genetic testing was conducted in patients with metastasis after failure of standard treatments. Sequencing was conducted in plasma-ctDNA samples; and when it was possible on the tumor tissue as well. Our analysis revealed that 88% (65 patients) of patients were eligible for treatment guidance using liquid biopsy. Among them, 64% (47 patients) received an FDA-approved drug, and treatment decisions were based on molecular testing using ctDNA. Somatic gene mutations were detected in 89% (66 patients) of the patients tested; 81% (60 patients) of patients had at least two mutations, 8% (6 patients) had only one mutation and 11% (8 patients) had no detected mutations. Interestingly, among the genes tested, BRCA2, EGFR, MSH6, and NF1 were the most frequently mutated in our patients. Our study highlights the potential benefits of personalized medicine through a non-invasive genetic testing across patients with metastasis regardless of the cancer types. Moreover, our study identified the frequent occurrence of specific gene mutations across various types of cancer, which paves the way for considering targeted therapies that could be applicable to multiple cancer types, rather than being restricted to just a few.

Circulating tumor DNA (ctDNA) is a promising non-invasive tool that has been demonstrated to be a clinically useful biomarker in several tumor types for risk stratification, prognosis, and early detection of recurrence. However, there are limited data on the clinical utility of ctDNA in endometrial cancer (EC) compared with other solid tumors. The evolution of EC management through the integration of molecular characterization into the treatment algorithm has intensified the need to develop more effective predictive biomarkers to optimize treatment and reduce clinical toxicities. Given its non-invasive nature and its ability to represent and complement tumor multiclonal spatial and temporal heterogeneity, ctDNA could act as a valid surrogate for tissue sampling. In addition to plasma ctDNA detection being associated with clinicopathologic features of tumor aggressiveness at pre-operative assessment, an association with reduced disease-free survival and overall survival has been observed in patients with detectable ctDNA. Moreover, the half-life of ctDNA is significantly shorter than CA125, and plasma levels are reported to be completely cleared from the blood within 1 week from surgical debulking. Therefore, ctDNA may serve as a dynamic biomarker for occult microscopic residual disease when assessed within the first 4 to 8 weeks after eradicative surgery. Few studies have reported high sensitivity of ctDNA in detecting disease recurrence at longitudinal follow-up, although there are limited data comparing ctDNA and traditional serum biomarkers (CA125 and HE4) in identifying recurrence. In the perspective of personalized oncology, ctDNA may potentially help improve adjuvant therapeutic management by escalating/de-escalating treatment based on ctDNA detection after surgery, during maintenance, or in the recurrent/metastatic setting, in addition to acting as a sensitive biomarker for early detection of recurrence. Several challenges hinder the use of ctDNA in EC, including the lack of standardized protocols, the low mutational burden, tumor heterogeneity, and background normal DNA, which limit assay sensitivity and specificity. In addition, the high cost of ctDNA analysis, particularly, next-generation sequencing, restricts its accessibility. Future trials should focus on cost-effective approaches to ensure sustainability and efficient resource allocation.

Background: Circulating tumor DNA (ctDNA) may be released from neoplastic cells into biological fluids through apoptosis, necrosis, or active release. In patients with non-small-cell lung cancer (NSCLC), ctDNA analysis is being introduced in clinical practice only for advanced disease management. Nevertheless, an interesting and promising field of application is the analysis of ctDNA in the management of early stage non-small-cell lung cancer, both for evaluation before treatment, such as diagnosis and screening, and for prediction of histology or pathological features. Methods: A thorough review of the literature published between 2000 and 2024 was performed on PubMed, utilizing the advanced search feature to narrow down titles and abstracts containing the following keywords: ctDNA, early stage, and NSCLC. A total of 20 studies that met all inclusion criteria were chosen for this review. Results: In this review, we summarize the increasing evidence suggesting that ctDNA has potential clinical applications in the management of patients with early stage NSCLC. ctDNA levels in early stage cancers are very low, posing many technical challenges in improving the detection rate and sensitivity, especially in clinical practice, if it is to be implemented for early detection. Presently, the main limitation of ctDNA experimental and clinical studies, especially in early stage settings, is the lack of definitive standardization and consensus regarding methodology, the absence of systematically validated analyses, and the lack of adoption of sensitive approaches. Conclusions: Possible applications of this analyte open up new fields of diagnosis, treatment, and follow up, which are less invasive and more precise than other approaches currently in use, especially in early stage NSCLC patients.

Colorectal cancer (CRC), a global health concern, requires effective treatments. Anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies are used for RAS wild-type and BRAFV600 mutation-negative metastatic CRC (mCRC) but are not indicated for RAS mutant CRC. Evidence suggests CRC patients with sub-clonal RAS/BRAF mutations in tumor tissue may benefit from anti-EGFRs. We assessed the outcomes of patients with sub-clonal RAS/BRAF mutated advanced/mCRC receiving anti-EGFRs using liquid-based GuardantINFORM real-world clinical-genomic analysis.

GuardantINFORM analyzed US patients with advanced CRC with BRAFV600/KRAS/NRAS mutations who received anti-EGFR therapies within 90 days after a Guardant360 test. Primary endpoints were time-to-next treatment (TTNT) and overall survival (OS) (compared across RAS/BRAF mutation clonality cut-offs of 0.3-0.8 using the Cox proportional hazards model).

In GuardantINFORM, 446 patients initiated anti-EGFR therapy within 90 days after the Guardant360 test, and 11%, 9%, and 1% had BRAFV600E, KRAS, or NRAS mutations, respectively; median distribution of RAS/BRAF clonality was 0.84 (IQR, 0.57-1.00). The data show that patients harboring sub-clonal RAS/BRAF mutations benefited from anti-EGFR therapy to a degree similar to patients without RAS/BRAF mutations. For cut-offs of 0.3 to 0.8, sub-clonal RAS/BRAF had similar TTNT to patients without RAS/BRAF mutations, while clonal RAS/BRAF had a significantly shorter TTNT. For cut-offs of 0.3 to 0.7, sub-clonal RAS/BRAF had similar OS to RAS/BRAF mutations not detected, while clonal RAS/BRAF had a significantly shorter OS.

Consistent with tumor tissue biopsy data, patients with CRC harboring sub-clonal RAS/BRAF mutations as assessed by liquid biopsy may derive benefit from anti-EGFR therapy, warranting further investigation.

Targeted therapies have radically altered the prognosis of patients with non-small cell lung cancer (NSCLC). Although the MET pathway was characterized in 1984, the treatment paradigm for patients with MET alterations has only recently changed. Genomic alterations in MET are found in 3%-5% of patients with NSCLC, and can include MET exon 14 (METex14) skipping, MET-activating mutations, and MET amplification. These alterations lead to the prolonged activation of the cellular MET receptor and downstream proliferation pathways that drive cell survival and migration. This review explores the history and pathophysiology of the MET pathway by focusing on METex14 skipping, and highlights insights gained since its discovery. Both unsuccessful and successful treatments that have emerged alongside the evolution of next-generation sequencing are examined, as well as current approved therapies and future options that target potential resistance mechanisms.

Liquid biopsy is a promising, non-invasive diagnostic tool for cancer, offering rapid and cost-effective genomic analysis. It provides a less invasive alternative to traditional tissue biopsies, with potential benefits in monitoring disease progression and detecting minimal residual disease (MRD). However, its clinical integration faces challenges, including utility assessment and workflow adaptation. This study evaluates the value of liquid biopsy in Taiwan from a clinical physician's perspective.

A survey was conducted with 16 physicians specializing in thoracic medicine and hematologic oncology. Participants responded to a 5-point Likert scale to evaluate the timing of liquid biopsy adoption, willingness to incorporate it into clinical practice, and agreement on its role in managing specific clinical conditions.

Forty percent of physicians preferred liquid biopsy when tissue samples were unavailable. The inclusion of liquid biopsy under National Health Insurance (NHI) was a key factor in its adoption. Hematologic oncologists showed a stronger preference for liquid biopsy, particularly for MRD testing, compared to their counterparts in thoracic medicine (hematologic oncology vs. thoracic medicine: 4.2 ± 0.83 vs. 3.1 ± 0.60; p value = 0.01). Younger physicians valued turnaround time, while senior physicians prioritized test brand, with a focus on report speed.

Physicians are generally less inclined to replace tissue biopsies with liquid biopsy, but hematologic oncologists show more flexibility. Test brand plays a role in physician decision-making, and the inclusion of liquid biopsy under NHI coverage is vital for its broader adoption in Taiwan.