Assessing Reporting Quality and Pre-Analytical Standards in Extrachromosomal Circular DNA Studies in Cancer: A...

Background/Objectives: eccDNA is a promising cancer biomarker in liquid biopsy. However, the reliability and reproducibility of eccDNA studies rely on the standardization of pre-analytical handling and processing of eccDNA, as well as transparent methodological reporting. Although evidence-based guidelines for cell-free DNA handling provide clear recommendations for plasma/serum processing, the extent to which eccDNA studies report and adhere to these key procedures remains uncertain. Methods: We systematically reviewed 14 studies (2017-2025) assessing eccDNA in plasma or serum from cancer patients. Each study was evaluated against 22 checklist items summarized from the NCI Biospecimen Collection and Processing Guideline, categorized into biospecimen collection, blood processing, and eccDNA processing. Items were classified as "reported," "not reported," or "deviated from NCI guideline," and missing/deviation rates were calculated. Results: Publication was largely post-guideline (13/14 after 2020). Reporting gaps were widespread. Items with 100% missing were venipuncture site (14/14), freeze-thaw cycles (14/14), and fitness for downstream analysis/pre-assay QC (14/14). Other frequently missing elements included blood volume (10/14; 71%), collection tube type (7/14; 50%), date/time from draw to processing (11/14; 79%), second centrifugation parameters (8/14; 57%), storage temperature before freezing (6/14; 43%), frozen storage duration (13/14; 93%), and post-thaw handling (13/14; 93%). By contrast, the extraction method was consistently reported (0% missing), and most reported quantification (21% missing). Deviations from the NCI guideline were uncommon when items were reported, such as a blood processing delay (2/14; 14%) and the use of circulating nucleic acid kits for eccDNA extraction (1/14; 7%). Conclusions: Inconsistent reporting of pre-analytical procedures limits the reproducibility, transparency, and clinical translation of eccDNA research. We propose a concise reporting checklist informed by the NCI guideline. By focusing on the most frequently underreported aspects of key pre-analytical eccDNA procedures, the checklist provides researchers with an efficient and succinct methodological reporting framework that will enhance transparency and promote standardization in future eccDNA studies.