Purpose: To investigate whether circulating tumor DNA (ctDNA) assessment in patients with muscle-invasive bladder cancer predicts treatment response and provides early detection of metastatic disease. Experimental Design: We present full follow-up results (median follow-up: 68 months) from a previously described cohort of 68 neoadjuvant chemotherapy (NAC)-treated patients who underwent longitudinal ctDNA testing (712 plasma samples). In addition, we performed ctDNA evaluation of 153 plasma samples collected before and after radical cystectomy (RC) in a separate cohort of 102 NAC-naïve patients (median follow-up: 72 months). Total RNA sequencing of tumors was performed to investigate biological characteristics of ctDNA shedding tumors. Results: Assessment of ctDNA after RC identified metastatic relapse with a sensitivity of 94% and specificity of 98% using the expanded follow-up data for the NAC-treated patients. ctDNA dynamics during NAC was independently associated with patient outcomes when adjusted for pathologic downstaging (HR = 4.7; P = 0.029). For the NAC-naïve patients, ctDNA was a prognostic predictor before (HR = 3.4; P = 0.0005) and after RC (HR = 17.8; P = 0.0002). No statistically significant difference in recurrence-free survival for patients without detectable ctDNA at diagnosis was observed between the cohorts. Baseline ctDNA positivity was associated with the Basal/Squamous (Ba/Sq) subtype and enrichment of epithelial-to-mesenchymal transition and cell cycle–associated gene sets. Conclusions: ctDNA is prognostic in NAC-treated and NAC-naïve patients with more than 5 years follow-up and outperforms pathologic downstaging in predicting treatment efficacy. Patients without detectable ctDNA at diagnosis may benefit significantly less from NAC, but additional studies are needed.

Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (SDM) of arginine, a posttranslational modification involved in oncogenesis and embryonic development. However, the role and mechanisms by which PRMT5 modulates Th cell polarization and autoimmune disease have not yet been elucidated. Here, we found that PRMT5 promoted SREBP1 SDM and the induction of cholesterol biosynthetic pathway enzymes that produce retinoid-related orphan receptor (ROR) agonists that activate RORγt. Specific loss of PRMT5 in the CD4⁺ Th cell compartment suppressed Th17 differentiation and protected mice from developing experimental autoimmune encephalomyelitis (EAE). We also found that PRMT5 controlled thymic and peripheral homeostasis in the CD4⁺ Th cell life cycle and invariant NK (iNK) T cell development and CD8⁺ T cell maintenance. This work demonstrates that PRMT5 expression in recently activated T cells is necessary for the cholesterol biosynthesis metabolic gene expression program that generates RORγt agonistic activity and promotes Th17 differentiation and EAE. These results point to Th PRMT5 and its downstream cholesterol biosynthesis pathway as promising therapeutic targets in Th17-mediated diseases.

The prognostic role of circulating-tumor DNA (ctDNA)-based molecular residual disease (MRD) detection and its utility for postsurgical risk-stratification has been reported in colorectal cancer. In this study, we explored the use of ctDNA-based MRD detection in patients with colorectal liver metastases (CLM), for whom the survival benefit of adjuvant chemotherapy (ACT) after surgical resection remains unclear. Patients with CLM without extrahepatic disease from GALAXY study (UMIN000039205) were included. The disease-free survival (DFS) benefit of ACT was evaluated in MRD-positive and -negative groups after adjusting for age, gender, number and size of liver metastases, RAS status, and previous history of oxaliplatin for primary cancer. ctDNA was detected using a personalized, tumor-informed 16-plex mPCR-NGS assay. ctDNA-based MRD status was evaluated 2 to 10 weeks after curative surgery, before the start of ACT. Among 6,061 patients registered in GALAXY, 190 surgically resected CLM patients without any preoperative chemotherapy were included with a median follow-up of 24 (1-48) months. ctDNA positivity in the MRD window was 32.1% (61/190). ACT was administered to 25.1% (48/190) of patients. In the MRD-positive group, 24-month DFS was higher for patients treated with ACT (33.3% vs. not reached, adjusted HR: 0.07, P <0.0001); whereas no benefit of ACT was seen in the MRD-negative group (24-month DFS: 72.3% vs. 62.2%, adjusted HR: 0.68, P =0.371). Multivariate analysis showed that the size of liver metastases (HR: 3.94, P =0.031) was prognostic of DFS in the MRD-positive group. However, in the MRD-negative group, none of the clinicopathological factors were prognostic of DFS. Our data suggest that ACT may offer notable clinical benefits in MRD-positive patients with CLM. MRD status-based risk-stratification could be potentially incorporated in future clinical trials for CLM.

Surgery is the only curative therapeutic option for resectable extrahepatic cholangiocarcinoma, but recurrence is common, and prognosis is poor. There is an unmet clinical need for improved decision-making regarding adjuvant chemotherapy (ACT). Herein, we evaluated the usefulness of monitoring longitudinal circulating tumor DNA (ctDNA) for molecular residual disease (MRD) in patients from the STAMP trial, which compared the efficacy of adjuvant capecitabine (CAP) vs. gemcitabine plus cisplatin (GemCis). Between July 2017 and November 2020, 101 patients were randomized 1:1 to receive GemCis (n = 50) or CAP (n = 51). Efficacy outcomes were analyzed with an extended follow-up of 19 months from the previous report. From a biomarker cohort of 89 patients, longitudinal plasma samples (n = 254) were prospectively collected post-surgery before ACT, and on-ACT at 12 and 24 weeks from cycle 1 day 1 (C1D1). ctDNA was evaluated using a personalized, tumor-informed, 16-plex PCR next-generation sequencing assay and was correlated with clinical outcomes. In the extended follow-up analysis, median disease-free survival (DFS) and overall survival did not significantly differ between the CAP and GemCis groups. Significantly inferior DFS was associated with ctDNA positivity before ACT (hazard ratio [HR] 1.8; p = 0.029), on-ACT at 12 weeks from C1D1 (HR 7.72; p <0.001), on-ACT at 24 weeks from C1D1 (HR 5.24; p <0.001), and anytime post-surgery (HR 3.81; p <0.001). Analysis of pre-treatment to on-treatment ctDNA dynamics revealed that serially ctDNA-negative patients exhibited a significantly longer DFS compared to those with sustained ctDNA positivity (HR 6.7; p <0.001) or those who turned ctDNA positive (HR 5.8; p <0.001). In patients with resected extrahepatic cholangiocarcinoma, ctDNA status and dynamics predicted recurrence during adjuvant therapy, and may help optimize clinical decision-making. The findings from this study highlight the critical role of ctDNA as a prognostic biomarker and monitoring tool for patients with resected extrahepatic cholangiocarcinoma. By demonstrating the superiority of ctDNA to predict disease recurrence compared to conventional biomarkers such as cancer antigen 19-9 and carcinoembryonic antigen, this study underscores its potential in guiding decision-making during adjuvant chemotherapy. These results may be crucial to refine post-surgical treatment strategies and improve patient outcomes. The practical application of ctDNA monitoring could lead to more personalized treatment approaches, enabling timely interventions based on molecular residual disease status. NCT03079427.

Despite curative-intent radical cystectomy (RC), patients with muscle-invasive bladder cancer (MIBC) are at high risk of recurrence. Biomarkers are urgently needed to refine prognostication and selection of appropriate perioperative systemic therapies. Our aim was to evaluate the prognostic and predictive value of tumor-informed circulating tumor DNA (ctDNA) results in a multicenter cohort of patients with bladder cancer who underwent RC. We performed a retrospective analysis of real-world data for a commercial ctDNA test (Signatera; Natera, Austin, TX, USA) performed in 167 patients (852 plasma samples) before RC and during molecular residual disease (MRD; adjuvant decision) and surveillance windows. We assessed the correlation between recurrence and ctDNA status before and after RC using Cox regression analysis. During study-defined postoperative MRD and surveillance windows, detectable ctDNA was associated with shorter disease-free survival (DFS) when compared to undetectable ctDNA (MRD: hazard ratio 6.93; p < 0.001; surveillance: hazard ratio 23.02; p < 0.001). Of note, patients with undetectable ctDNA did not appear to benefit from adjuvant therapy (p = 0.34). Detectable ctDNA in the pre-RC (p = 0.045), MRD (p = 0.002), and surveillance (p < 0.001) windows was the only risk factor independently associated with shorter DFS. Limitations include the retrospective and nonrandomized nature of the study. ctDNA testing in patients with bladder cancer undergoing RC was prognostic and potentially predictive. Identification of patients at high risk of recurrence may aid in patient counseling and decision-making. We found that outcomes for patients with muscle-invasive bladder cancer are strongly linked to detection of tumor DNA in blood samples. The results show the value of tumor-informed testing for tumor DNA in blood for decisions on the best treatment for each individual patient.

Patients with locally advanced resectable (LAR) gastric/gastroesophageal junction (G/GEJ) adenocarcinomas have a high recurrence risk despite pre- and post-operative treatment. In the PLAGAST prospective study (NCT02674373), we investigated the ability of circulating tumor DNA (ctDNA) to predict treatment response and improve risk stratification. Plasma samples were prospectively collected before neoadjuvant therapy (NAT), during-NAT, post-NAT, and post-surgery. The primary endpoint was recurrence-free survival (RFS), and the secondary endpoints were overall survival (OS), tumor regression grade (TRG), and pathological tumor stage. ctDNA positivity decreased over these four therapeutic timelines (69.6%, 51.2%, 26.8%, and 20%, respectively). ctDNA-positivity was associated with significantly worse outcomes during-NAT (RFS: HR = 6.17, P = 0.002; OS: HR = 4.71, P = 0.022), post-NAT (RFS: HR = 5.26, P = 0.001; OS: HR = 7.35, P = 0.001) and after surgery (RFS: HR = 12.94, P < 0.0001; OS: HR = 14.54, P < 0.0001). Patients with early ctDNA clearance during NAT had better outcomes compared to those who cleared ctDNA post-NAT, while patients who remained ctDNA-positive pre-, during-, and post-NAT had worse outcomes (RFS: HR = 18.57, P = 0.01; OS: HR = 16.06, P = 0.007). Our data suggests that longitudinal ctDNA monitoring is prognostic of patient outcomes and may guide therapeutic decision-making in patients with LAR G/GEJ adenocarcinoma.