Personalized, tumor-informed, circulating tumor DNA assay for detecting minimal residual disease in non-small cell lung cancer patients receiving curative treatments.

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). CONCLUSION: 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.

Early real-world experience monitoring circulating tumor DNA in resected early-stage non-small cell lung cancer.

OBJECTIVE: The study objective was to evaluate the impact of monitoring circulating tumor DNA on the detection and management of recurrence in patients with resected early-stage non-small cell lung cancer. METHODS: Between October 2021 and March 2023, postoperative circulating tumor DNA was monitored in patients with non-small cell lung cancer (N = 108). Longitudinal blood samples (n = 378 samples) were collected for prospective circulating tumor DNA analysis at 3-month intervals after curative-intent resection. A tumor-informed assay was used for the detection and quantification of circulating tumor DNA. The primary outcome measure was a circulating tumor DNA-positive result. The secondary outcome measure was changes in practice after a circulating tumor DNA-positive result. RESULTS: The mean age of the patients in this cohort was 68.1 years. Of the 108 patients, 12 (11.1%) were circulating tumor DNA positive at least at 1 timepoint postsurgery, of whom 8 (66.7%) had a clinically evident recurrence and the remaining 4 had limited clinical follow-up. Of the 10 patients with recurrent disease, 8 demonstrated circulating tumor DNA positivity and the remaining 2 patients had brain-only metastases. Postoperative clinical care was altered in 100% (12/12) of circulating tumor DNA-positive patients, with 58.3% (7/12) receiving an early computed tomography scan and 100% (12/12) receiving an early positron emission tomography computed tomography scan as part of their surveillance strategy. Among the patients who received an early positron emission tomography scan, 66.6% (8/12) were positive for malignant features. CONCLUSIONS: Routine monitoring of tumor-informed circulating tumor DNA after curative intent therapy improved patient risk stratification and prognostication.

ctDNA-based detection of molecular residual disease in stage I-III non-small cell lung cancer patients treated with definitive radiotherapy.

Background: Sensitive and reliable biomarkers for early detection of recurrence are needed to improve post-definitive radiation risk stratification, disease management, and outcomes for patients with unresectable early-stage or locally advanced non-small cell lung cancer (NSCLC) who are treated with definitive radiation therapy (RT). This prospective, multistate single-center, cohort study investigated the association of circulating tumor DNA (ctDNA) status with recurrence in patients with unresectable stage I-III NSCLC who underwent definitive RT. Methods: A total of 70 serial plasma samples from 17 NSCLC patients were collected before, during, and after treatment. A personalized, tumor-informed ctDNA assay was used to track a set of up to 16 somatic, single nucleotide variants in the associated patient's plasma samples. Results: Pre-treatment ctDNA detection rate was 82% (14/17) and varied based on histology and stage. ctDNA was detected in 35% (6/17) of patients at the first post-RT timepoint (median of 1.66 months following the completion of RT), all of whom subsequently developed clinical progression. At this first post-RT time point, patients with ctDNA-positivity had significantly worse progression-free survival (PFS) [hazard ratio (HR): 24.2, p=0.004], and ctDNA-positivity was the only significant prognostic factor associated with PFS (HR: 13.4, p=0.02) in a multivariate analysis. All patients who developed clinical recurrence had detectable ctDNA with an average lead time over radiographic progression of 5.4 months, and post-RT ctDNA positivity was significantly associated with poor PFS (p<0.0001). Conclusion: Personalized, longitudinal ctDNA monitoring can detect recurrence early in patients with unresectable NSCLC patients undergoing curative radiation and potentially risk-stratify patients who might benefit most from treatment intensification.

Circulating Tumor DNA Monitoring on Chemo-immunotherapy for Risk Stratification in Advanced Non-Small Cell Lung Cancer.

PURPOSE: Chemoimmunotherapy (chemoIO) is a prevalent first-line treatment for advanced driver-negative non-small cell lung cancer (NSCLC), with maintenance therapy given after induction. However, there is significant clinical variability in the duration, dosing, and timing of maintenance therapy after induction chemoIO. We used circulating tumor DNA (ctDNA) monitoring to inform outcomes in patients with advanced NSCLC receiving chemoIO. EXPERIMENTAL DESIGN: This retrospective study included 221 patients from a phase III trial of atezolizumab+carboplatin+nab-paclitaxel versus carboplatin+nab-paclitaxel in squamous NSCLC (IMpower131). ctDNA monitoring used the FoundationOne Tracker involving comprehensive genomic profiling of pretreatment tumor tissue, variant selection using an algorithm to exclude nontumor variants, and multiplex PCR of up to 16 variants to detect and quantify ctDNA. RESULTS: ctDNA was detected (ctDNA+) in 96% of pretreatment samples (median, 93 mean tumor molecules/mL), and similar ctDNA dynamics were noted across treatment arms during chemoIO. ctDNA decrease from baseline to C4D1 was associated with improved outcomes across multiple cutoffs for patients treated with chemoIO. When including patients with missing plasma or ctDNA- at baseline, patients with ctDNA- at C4D1 (clearance), had more favorable progression-free survival (median 8.8 vs. 3.5 months; HR, 0.32;0.20-0.52) and OS (median not reached vs. 8.9 months; HR, 0.22; 0.12-0.39) from C4D1 than ctDNA+ patients. CONCLUSIONS: ctDNA monitoring during induction chemoIO can inform treatment outcomes in patients with advanced NSCLC. Importantly, monitoring remains feasible and informative for patients missing baseline ctDNA. ctDNA testing during induction chemoIO identifies patients at higher risk for disease progression and may inform patient selection for novel personalized maintenance or second-line treatment strategies.

BESPOKE IO protocol: a multicentre, prospective observational study evaluating the utility of ctDNA in guiding immunotherapy in patients with advanced solid tumours.

INTRODUCTION: Immunotherapy (IO) has transformed the treatment paradigm for a wide variety of solid tumours. However, assessment of response can be challenging with conventional radiological imaging (eg, iRECIST), which do not precisely capture the unique response patterns of tumours treated with IO. Emerging data suggest that circulating tumour DNA (ctDNA) can aid in response assessment in patients with solid tumours receiving IO. The short half-life of ctDNA puts it in a unique position for early treatment response monitoring. The BESPOKE IO study is designed to investigate the clinical utility of serial ctDNA testing to assess treatment response using a tumour-informed, bespoke ctDNA assay (Signatera) and to determine its impact on clinical decision-making with respect to continuation/discontinuation, or escalation/de-escalation of immunotherapy in patients with advanced solid tumours. METHODS AND ANALYSIS: The BESPOKE IO is a multicentre, prospective, observational study with a goal to enroll over 1500 patients with solid tumours receiving IO in up to 100 US sites. Patients will be followed for up to 2 years with serial ctDNA analysis, timed with every other treatment cycle. The primary endpoint is to determine the percentage of patients who will have their treatment regimen changed as guided by post-treatment bespoke ctDNA results along with standard response assessment tools. The major secondary endpoints include progression-free survival, overall survival and overall response rate based on the ctDNA dynamics. ETHICS AND DISSEMINATION: The BESPOKE IO study was approved by the WCG Institutional Review Board (Natera-20-043-NCP BESPOKE Study of ctDNA Guided Immunotherapy (BESPOKE IO)) on 22 February 2021. Data protection and privacy regulations will be strictly observed in the capturing, forwarding, processing and storing patients' data. Natera will approve the publication of any study results in accordance with the site-specific contract. TRIAL REGISTRATION NUMBER: NCT04761783.

The Utility of Circulating Tumor DNA (ctDNA) Monitoring in Cancer Patients Who Are Pregnant or Planning to Become Pregnant.

The number of pregnant women with cancer is on the rise. These patients and their providers encounter complex medical management decisions. Standard-of-care systemic therapy and radiological imaging can impair fetal development and affect viability. Conversely, insufficient monitoring and treatment can lead to cancer progression, compromising the health of the patient. Personalized and tumor-informed circulating tumor DNA (ctDNA) testing (Signatera™, bespoke mPCR NGS assay) is a validated, noninvasive blood test that can accurately assess cancer progression and tumor response to treatment ahead of radiological imaging, across solid tumors. In this case series of four patients, we explore the clinical utility of longitudinal ctDNA testing in the medical management of pregnant patients with solid tumors, to aid in informed decision-making for patients and providers.

Circulating tumor DNA (ctDNA) serial analysis during progression on PD-1 blockade and later CTLA-4 rescue in patients with mismatch repair deficient metastatic colorectal cancer.

Immune checkpoint inhibitors have shown great promise in treating patients with mismatch repair deficient/microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC). Although single-agent pembrolizumab has been approved for first-line treatment of dMMR/MSI-H metastatic CRC, combination therapy with cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) inhibition (ipilimumab/nivolumab) has reported higher response rates. It is unclear whether patients who progress on PD-1 inhibition will respond to CTLA-4 blockade. Here, we report a case series of three patients with dMMR/MSI-H mCRC, where a durable and ongoing response to nivolumab with ipilimumab was achieved after initial progression with pembrolizumab monotherapy. Blood-based biomarkers such as carcinoembryonic antigen and CA 19-9 were employed to assess treatment response and monitor disease progression along with circulating tumor DNA (ctDNA). Our findings indicate ctDNA's potential to accurately monitor response to therapy and detect disease progression, as validated by standard imaging. This case series demonstrates that CTLA-4 rescue is worthy of additional investigation as a treatment strategy after progression on PD-1 blockade in patients with dMMR/MSI-high mCRC. Our data support the utilization and expansion of clinical studies with combination therapies and using ctDNA kinetics as early dynamic marker for therapy response assessment.

ctDNA Clearance and Radiographic Resolution of Disease in Response to Dual Checkpoint Inhibition in Metastatic Microsatellite Stable Colorectal Cancer with a High Tumor Mutation Burden.

Immunotherapy (IO) has increasingly been demonstrated to provide therapeutic benefit to patients with metastatic colorectal cancer (mCRC). However, only a subset of mCRC tumors respond to IO. Monitoring response with tumor biomarkers like carcinoembryonic antigen (CEA) has been challenging in patients with microsatellite stable (MSS) mCRC due to low expression of CEA (CEA/lo). Noninvasive blood-based biomarkers such as circulating tumor DNA (ctDNA) can inform early treatment response and augment radiographic monitoring. We describe a case study of a patient with chemotherapy-refractory CEA/lo MSS mCRC, with metastatic disease present in a cardiophrenic lymph node. The patient was given 2 cycles of combination IO (ipilimumab/nivolumab). Response was monitored by ctDNA using a multiplex PCR next-generation sequencing assay, CEA, and CT scan. After IO administration, ctDNA levels rapidly declined, becoming undetectable. This was concurrent with radiographic resolution of the lymph node metastasis. Serial monitoring of CEA during this same period was uninformative, with no significant changes observed. Significant decline in ctDNA identified metastatic response to IO in a patient with CEA/lo, MSS mCRC and was concurrently validated by CT scan. This case study provides evidence that ctDNA can be used as a prospective surrogate for radiographic tumor response.

Early Detection of Circulating Tumor DNA Postoperatively Enables Discovery of Resectable Metastatic Disease in a Patient with Colon Cancer.

Currently, serum carcinoembryonic agent (CEA) along with contrast-enhanced imaging and colonoscopy are used for evaluation of recurrence of colorectal cancer. However, CEA is an unreliable and nonspecific biomarker that may fail to rise and signal relapse. Analysis of circulating tumor DNA (ctDNA) in patients offers a minimally invasive method to assess risk of relapse several months ahead of conventional clinical means. Here, we report the case of a colon adenocarcinoma with postoperative liver metastasis diagnosed early by ctDNA measurement, using a personalized NGS-mPCR assay. While ctDNA levels continued to rise, CEA levels tested negative. Metastatic relapse to the liver was promptly confirmed by PET/CT scan. The patient underwent a successful metastasectomy with curative intent. Following surgery, the patient exhibited no evidence of disease and ctDNA levels remained negative. Our case report suggests that the early detection of postoperative molecular residual disease by means of ctDNA measurement can accurately predict mCRC relapse in cases where CEA levels fail to increase. Close monitoring of ctDNA levels during the postoperative period can allow for earlier intervention and more favorable outcomes in relapsing mCRC patients.

Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling.

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

Recent advancements in understanding endogenous heart regeneration-insights from adult zebrafish and neonatal mice.

Enhancing the endogenous regenerative capacity of the mammalian heart is a promising strategy that can lead to potential treatment of injured cardiac tissues. Studies on heart regeneration in zebrafish and neonatal mice have shown that cardiomyocyte proliferation is essential for replenishing myocardium. We will review recent advancements that have demonstrated the importance of Neuregulin 1/ErbB2 and innervation in regulating cardiomyocyte proliferation using both adult zebrafish and neonatal mouse heart regeneration models. Emerging findings suggest that different populations of macrophages and inflammation might contribute to regenerative versus fibrotic responses. Finally, we will discuss variation in the severity of the cardiac injury and size of the wound, which may explain the range of outcomes observed in different injury models.

Epicardial Epithelial-to-Mesenchymal Transition in Heart Development and Disease.

The epicardium is an epithelial monolayer that plays a central role in heart development and the myocardial response to injury. Recent developments in our understanding of epicardial cell biology have revealed this layer to be a dynamic participant in fundamental processes underlying the development of the embryonic ventricles, the coronary vasculature, and the cardiac valves. Likewise, recent data have identified the epicardium as an important contributor to reparative and regenerative processes in the injured myocardium. These essential functions of the epicardium rely on both non-cell autonomous and cell-autonomous mechanisms, with the latter featuring the process of epicardial Epithelial-to-Mesenchymal Transition (EMT). This review will focus on the induction and regulation of epicardial EMT, as it pertains to both cardiogenesis and the response of the myocardium to injury.