Value of ctDNA in surveillance of adjuvant chemosensitivity and regimen adjustment in stage III colon cancer: a protocol for phase II multicentre randomised controlled trial (REVISE trial)

Introduction
The standard of care for stage III colon cancer is 3 or 6 months of double-drug regimen chemotherapy following radical surgery. However, patients with positive circulating tumour DNA (ctDNA) exhibit a high risk of recurrence risk even if they receive standard adjuvant chemotherapy. The potential benefit of intensified adjuvant chemotherapy, oxaliplatin, irinotecan, leucovorin and fluoropyrimidine (FOLFOXIRI), for ctDNA-positive patients remains to be elucidated.

Methods and analysis
This multicentre phase II randomised controlled trial aims to investigate the utility of ctDNA in monitoring chemosensitivity and to preliminarily assess whether intensified chemotherapy with FOLFOXIRI can increase ctDNA clearance and improve survival outcomes. A total of 60 eligible patients with stage III colon cancer exhibiting postoperatively positive ctDNA before and after two cycles of oxaliplatin and capecitabine (XELOX) will be randomly assigned to continue five additional cycles of XELOX (control arm) or switch to eight cycles of FOLFOXIRI (experimental arm). This sequential approach is designed to escalate treatment for patients with persistent ctDNA positivity while avoiding overtreatment in those who may respond well to standard chemotherapy. The primary endpoint is the change in ctDNA concentration, defined as the difference between the ctDNA concentration measured after two cycles of XELOX and after the completion or termination of chemotherapy. Secondary endpoints include the ctDNA clearance rate, 2-year disease-free survival, distant metastasis-free survival, chemotherapy-related side effects and quality of life.

Ethics and dissemination
This trial has been approved by the Ethics Committee of the West China Hospital, Sichuan University (approval number: 20231998). The findings will be disseminated through peer-reviewed publications and presentations at scientific conferences.

Trial registration number
ClinicalTrials.gov (NCT06242418, registered on 27 January 2024).

Read More

Is the Multitarget Stool DNA Test Just a Better “FIT” for Colorectal Cancer Screening?

Both the fecal immunochemical test (FIT) and multitarget stool DNA test (mt-sDNA) are stool-based screening tests for colorectal cancer (CRC) that are reported qualitatively as positive or negative, even though both tests are quantitative, producing a precise value on a continuous scale. The FIT quantifies the globin portion of human hemoglobin in either per milliliter of buffer or per gram of stool, while the mt-sDNA test generates a score from a multivariable algorithm that measures human hemoglobin as well as specific abnormalities in human DNA. For both tests, the threshold (ie, the cut point) determines how the test performs, ie, its sensitivity and specificity. Lowering the threshold increases sensitivity for CRC, advanced precancerous lesions (APLs), or both and would likely decrease specificity, resulting in a higher false-positive rate and more (unnecessary) colonoscopies.

Read More

Activation of Imprinted Gene PW1 Promotes Cardiac Fibrosis After Ischemic Injury

Circulation, Ahead of Print. BACKGROUND:Cardiac fibrosis, characterized by excessive extracellular matrix (ECM) deposition in the myocardium, is an important target for heart disease treatments.Pw1(paternally expressed gene 3) is an imprinted gene expressed from the paternal allele, and de novo purine biosynthesis (DNPB) is a crucial pathway for nucleotide synthesis. However, the roles of PW1 and DNPB in ECM production by cardiac fibroblasts during myocardial ischemia are not yet understood.METHODS:To induce myocardial damage, we performed left anterior descending coronary artery ligation. We generatedPw1CreER-2A-eGFPandPw12A-CreERknock-in mouse lines to evaluate the expression of the 2Pw1alleles in normal and injured hearts. Bisulfite sequencing was used to analyze the DNA methylation of thePw1imprinting control region. We identified the phosphoribosylformylglycinamidine synthase (Pfas) gene, encoding the DNPB enzyme PFAS, as a direct target of PW1 using chromatin immunoprecipitation sequencing and real-time quantitative polymerase chain reaction. The role of DNPB in ECM production and cardiac fibrosis after injury was examined in vitro using cultured cardiac fibroblasts and in vivo withPfas-deficient mice.RESULTS:Our study demonstrates that myocardial infarction reduces DNA methylation at the imprinting control region of the maternally imprinted genePw1, triggering a switch from monoallelic imprinting to biallelic expression ofPw1in cardiac fibroblasts. In activated cardiac fibroblasts, increasedPw1expression promotes purine biosynthesis and induces ECM production by transcriptionally activating the DNPB factorPfas. We identified that DNPB is essential for ECM production in activated fibroblasts and that loss ofPfasin fibroblasts limits cardiac fibrosis and improves heart function after injury.CONCLUSIONS:This study demonstrates thatPw1imprinting is disrupted after injury and reveals a novel role for the downstream target PFAS in ECM production and cardiac fibrogenesis. Targeting the PW1/PFAS signaling pathway presents a promising therapeutic strategy for improving cardiac repair after injury.

Read More

METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

Circulation, Ahead of Print. BACKGROUND:Mitochondrial dysfunction is a key factor in the development of atherogenesis. METTL4 (methyltransferase-like protein 4) mediates N6- methyldeoxyadenosine (6mA) of mammalian mitochondrial DNA (mtDNA). However, the role of METTL4-mediated mitoepigenetic regulation in atherosclerosis is still unknown. This study aims to investigate the potential involvement of METTL4 in atherosclerosis, explore the underlying mechanism, and develop targeted strategies for treating atherosclerosis.METHODS:Expression levels of mtDNA 6mA and METTL4 were determined in atherosclerotic lesions. We explored the mechanism of METTL4 involvement in atherosclerosis usingMettl4Mac-KO-Apoe-/-andMettl4MUT-Apoe-/-mice and cell models, as well as bone marrow transplantation. Natural compound libraries were screened to identify potent METTL4 antagonists. In addition, bioinspired proteolysis targeting chimera technology targeting macrophages within plaques was used to increase the efficacy of the METTL4 antagonist.RESULTS:The expression levels of mtDNA 6mA and METTL4 were significantly increased in plaque macrophages.Mettl4Mac-KO-Apoe-/-mice displayed suppressed mtDNA 6mA levels and atherosclerotic progression, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Mechanistically, elevated METTL4 expression reduces MT-ATP6 expression by suppressing its transcription, thereby impairing the activity of mitochondrial respiration chain complex V. This disruption leads to the accumulation of excess protons in the mitochondrial intermembrane space, causing mitochondrial dysfunction. Consequently, mtDNA is released into the cytoplasm, ultimately triggering inflammasome activation. All results were reversed by the mutation in the METTL4 methyltransferase active site.Mettl4MUT-Apoe-/-mice showed suppressed mtDNA 6mA levels and atherosclerotic progression and repaired mitochondrial function of macrophage, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Pemetrexed was identified as the first METTL4 antagonist to effectively alleviate atherosclerotic progression. Furthermore, we generated a proteolysis targeting chimera drug based on pemetrexed that specifically targeted METTL4 in macrophages within plaques, showing a promising therapeutic effect on atherosclerosis.CONCLUSIONS:This study revealed a novel mechanism by which mtDNA 6mA orchestrated mitochondrial function–related gene expression in macrophages, thereby promoting atherosclerosis. Through various experimental techniques, such as gene manipulation, pharmacological inhibition, and proteolysis targeting chimera, this study demonstrated that mtDNA 6mA and its specific enzyme METTL4 hold potential as therapeutic targets for atherosclerosis.

Read More