In patients with resected non-metastatic melanoma, the liquid biopsy for the assessment of molecular residual disease (MRD) by circulating tumour DNA (ctDNA) represents a promising tool to stratify the risk and to monitor tumour evolution. However, its validation requires the demonstration of analytical validity, clinical validity and utility. Indeed, the development of sensitive and specific assays can optimize prognostication and eventually help clinicians to modulate adjuvant treatments, in order to improve clinical outcomes. Data about ctDNA-guided prognosis stratification is emerging, but clinical trials assessing ctDNA-guided therapeutic decisions are still ongoing. This review aims to depict the role of ctDNA-based MRD assessment in patients with non-metastatic melanoma and to provide a roadmap to face challenges for its introduction into clinical practice.

Systemic treatment of resectable non-small cell lung cancer (NSCLC) is evolving with emerging neoadjuvant, perioperative, and adjuvant immunotherapy approaches. Circulating tumor DNA (ctDNA) detection at clinical diagnosis, during neoadjuvant therapy, or after resection may discern high-risk patients who might benefit from therapy escalation or switch. This Review summarizes translational implications of data supporting ctDNA-based risk determination in NSCLC and outstanding questions regarding ctDNA validity/utility as a prognostic biomarker. We discuss emerging ctDNA capabilities to refine clinical tumor-node-metastasis (TNM) staging in lung adenocarcinoma, ctDNA dynamics during neoadjuvant therapy for identifying patients deriving suboptimal benefit, and postoperative molecular residual disease (MRD) detection to escalate systemic therapy. Considering differential relapse characteristics in landmark MRD-negative/MRD-positive patients, we propose how ctDNA might integrate with pathological response data for optimal postoperative risk stratification.

Gene fusions and MET alterations are rare and difficult to detect in plasma samples. The clinical detection efficacy of molecular residual disease (MRD) based on circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC) with these mutations remains unknown. This prospective, non-intervention study recruited 49 patients with operable NSCLC with actionable gene fusions (ALK, ROS1, RET, and FGFR1), MET exon 14 skipping or de novo MET amplification. We analyzed 43 tumor tissues and 111 serial perioperative plasma samples using 1021- and 338-gene panels, respectively. Detectable MRD correlated with a significantly higher recurrence rate (P < 0.001), yielding positive predictive values of 100% and 90.9%, and negative predictive values of 82.4% and 86.4% at landmark and longitudinal time points, respectively. Patients with detectable MRD showed reduced disease-free survival (DFS) compared to those with undetectable MRD (P < 0.001). Patients who harbored tissue-derived fusion/MET alterations in their MRD had reduced DFS compared to those who did not (P = 0.05). To our knowledge, this is the first comprehensive study on ctDNA-MRD clinical detection efficacy in operable NSCLC patients with gene fusions and MET alterations. Patients with detectable tissue-derived fusion/MET alterations in postoperative MRD had worse clinical outcomes.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a prognostic and predictive biomarker for detection of minimal residual disease (MRD), monitoring treatment response, and early detection of recurrence in cancer patients. In this study, we explored the utility of ctDNA-based MRD detection to predict recurrence in a real-world cohort of primarily early-stage non-small cell lung cancer (NSCLC) patients treated with curative intent.
METHODS: Longitudinal plasma samples were collected post curative-intent treatment from 36 patients with stage I-IV NSCLC. A personalized, tumor-informed assay was used to detect and quantify ctDNA in plasma samples.
RESULTS: Of the 24 patients with plasma samples available during the MRD window (within 6 months of curative surgery and before adjuvant therapy), ctDNA was detectable in two patients. Patients with ctDNA-positivity during the MRD window were 15 times more likely to recur compared to ctDNA-negative patients (HR: 15.0, 95% CI: 1.0-253.0, p = 0.010). At any time post-curative intent treatment, ctDNA-positivity was associated with significantly poorer recurrence-free survival compared to persistently ctDNA-negative patients (p < 0.0001).
CONCLUSIONS: Our real-world data indicate that longitudinal, personalized, tumor-informed ctDNA monitoring is a valuable tool in patients with NSCLC receiving curative treatment to identify patients at high risk for recurrence.

Circulating tumor DNA (ctDNA) is the fraction of cell-free DNA in patient blood that originates from a tumor. Advances in DNA sequencing technologies and our understanding of the molecular biology of tumors have increased interest in exploiting ctDNA to facilitate detection of molecular residual disease (MRD). Analysis of ctDNA as a promising MRD biomarker of solid malignancies has a central role in precision medicine initiatives exemplified by our CIRCULATE-Japan project involving patients with resectable colorectal cancer. Notably, the project underscores the prognostic significance of the ctDNA status at 4 weeks post-surgery and its correlation to adjuvant therapy efficacy at interim analysis. This substantiates the hypothesis that MRD is a critical prognostic indicator of relapse in patients with colorectal cancer. Despite remarkable advancements, challenges endure, primarily attributable to the exceedingly low ctDNA concentration in peripheral blood, particularly in scenarios involving low tumor shedding and the intrinsic error rates of current sequencing technologies. These complications necessitate more sensitive and sophisticated assays to verify the clinical utility of MRD across all solid tumors. Whole genome sequencing (WGS)-based tumor-informed MRD assays have recently demonstrated the ability to detect ctDNA in the parts-per-million range. This review delineates the current landscape of MRD assays, highlighting WGS-based approaches as the forefront technique in ctDNA analysis. Additionally, it introduces our upcoming endeavor, WGS-based pan-cancer MRD detection via ctDNA, in our forthcoming project, SCRUM-Japan MONSTAR-SCREEN-3.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient\'s tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

UNASSIGNED: Circulating tumor DNA (ctDNA) has emerged as a biomarker that can define the risk of recurrence after curative-intent surgery for patients with colorectal cancer (CRC). However, beyond the predictive power of postoperative ctDNA detection, the efficacy and potential limitations of ctDNA detection urgently need to be fully elucidated in a large cohort of CRC.
UNASSIGNED: To define potentially cured CRC patients through ctDNA monitoring following surgery.
UNASSIGNED: A prospective, multicenter, observational study.
UNASSIGNED: We enrolled 309 patients with stages I-IV CRC who underwent definitive surgery. Tumor tissues were sequenced by a custom-designed next-generation sequencing panel to identify somatic mutations. Plasma was analyzed using a ctDNA-based molecular residual disease (MRD) assay which integrated tumor-genotype-informed and tumor-genotype-naïve ctDNA analysis. The turnaround time of the assay was 10-14 days.
UNASSIGNED: Postoperative ctDNA was detected in 5.4%, 13.8%, 15%, and 30% of patients with stage I, II, III, and IV disease, respectively, and in 17.5% of all longitudinal samples. Patients with positive postsurgery MRD had a higher recurrence rate than those with negative postsurgery MRD [hazard ratio (HR), 13.17; p < 0.0001], producing a sensitivity of 64.6%, a specificity of 94.8%, a positive predictive value (PPV) of 75.6%, and a negative predictive value (NPV) of 91.5%. Furthermore, patients with positive longitudinal MRD also had a significantly higher recurrence rate (HR, 14.44; p < 0.0001), with increased sensitivity (75.0%), specificity (94.9%), PPV (79.6%), and NPV (93.4%). Subgroup analyses revealed that adjuvant therapy did not confer superior survival for patients with undetectable or detectable MRD. In addition, MRD detection was less effective in identifying lung-only and peritoneal metastases.
UNASSIGNED: Postoperative ctDNA status is a strong predictor of recurrence independent of stage and microsatellite instability status. Longitudinal undetectable MRD could be used to define the potentially cured population in CRC patients undergoing curative-intent surgery.

OBJECTIVE: Molecular residual disease (MRD) is the main cause of postoperative recurrence of breast cancer. However, the baseline tumor genomic characteristics and therapeutic implications of breast cancer patients with detectable MRD after surgery are still unknown.
METHODS: In this study, we enrolled 80 patients with breast cancer who underwent next-generation sequencing-based genetic testing of 1,021 cancer-related genes performed on baseline tumor and postoperative plasma, among which 18 patients had detectable MRD after surgery.
RESULTS: Baseline clinical characteristics found that patients with higher clinical stages were more likely to have detectable MRD. Analysis of single nucleotide variations and small insertions/deletions in baseline tumors showed that somatic mutations in MAP3K1, ATM, FLT1, GNAS, POLD1, SPEN, and WWP2 were significantly enriched in patients with detectable MRD. Oncogenic signaling pathway analysis revealed that alteration of the Cell cycle pathway was more likely to occur in patients with detectable MRD (p=0.012). Mutational signature analysis showed that defective DNA mismatch repair and activation-induced cytidine deaminase (AID) mediated somatic hypermutation (SHM) were associated with detectable MRD. According to the OncoKB database, 77.8% (14/18) of patients with detectable MRD had U.S. Food and Drug Administration-approved mutational biomarkers and targeted therapy.
CONCLUSIONS: Our study reports genomic characteristics of breast cancer patients with detectable MRD. The cell cycle pathway, defective DNA mismatch repair, and AID-mediated SHM were found to be the possible causes of detectable MRD. We also found the vast majority of patients with detectable MRD have the opportunity to access targeted therapy.

The value of circulating tumor DNA (ctDNA) during chemoradiotherapy (CRT) remains unclear but is critical for detecting molecular residual disease (MRD). In this prospective study, we sequenced 761 blood samples from 139 patients with locally advanced non-small cell lung cancer treated with definitive radiation therapy (RT). ctDNA concentrations showed a significantly declining trend as CRT progressed at on-RT and after-RT time points versus baseline. Thirty-eight (27.3%) patients with early undetectable ctDNA at both on-RT (RT reached 40 Gy) and after-RT time points, indicating early response to CRT, had better survival outcomes for both with or without consolidation immune checkpoint inhibitors. Longitudinal undetectable MRD was found in 20.1% patients. The 2-year cancer-specific progression-free survival of these patients was 88.4%, corresponding to a potentially cured population. Further analysis revealed that pretreatment ctDNA variants serve as an essential MRD informed source. These data provide clinical insights for ctDNA-MRD detection.

[This corrects the article DOI: 10.3389/fonc.2023.1222716.].