Ultra-short cell-free DNA fragments enhance cancer early detection in a multi-analyte blood test combining mutation, protein and fragmentomics

• Published in: Clinical chemistry and laboratory medicine Vol. 62; no. 1; pp. 168 - 177
• Main Authors: Wang, Fenfen, Li, Xinxing, Li, Mengxing, Liu, Wendi, Lu, Lingjia, Li, Yang, Chen, Xiaojing, Yang, Siqi, Liu, Tao, Cheng, Wen, Weng, Li, Wang, Hongyan, Lu, Dongsheng, Yao, Qianqian, Wang, Yingyu, Wu, Johnny, Wittkop, Tobias, Faham, Malek, Zhou, Huabang, Hu, Heping, Jin, Hai, Hu, Zhiqian, Ma, Ding, Cheng, Xiaodong
• Format: Journal Article
• Language: English
• Published: Germany De Gruyter 26.01.2024; Walter De Gruyter & Company
• Subjects: Biomarkers; Biomarkers, Tumor - genetics; Blood; Blood tests; Cancer; cell-free DNA; Cell-Free Nucleic Acids; circulating tumor DNA; Circulating Tumor DNA - genetics; Deoxyribonucleic acid; DNA; DNA sequencing; Early Detection of Cancer; Epigenetics; fragmentomics; Fragments; Gene expression; Humans; Morbidity; multi-cancer early detection; Mutation; Neoplasms - diagnosis; Neoplasms - genetics; Ovarian cancer; Prediction models; Proteins; Sensitivity; single-strand DNA sequencing; Technology; ultra-short fragments; cell-free DNA; multi-cancer early detection; single-strand DNA sequencing; fragmentomics; ultra-short fragments; circulating tumor DNA
• ISSN: 1434-6621; 1437-4331; 1437-4331
• DOI: 10.1515/cclm-2023-0541

Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early cancer detection, however, it is still at its infancy and requires technical improvement. We sought to apply a single-strand DNA sequencing technology, for measuring genetic and fragmentomic features of cfDNA and evaluate the performance in detecting multiple cancers. Blood samples of 364 patients from six cancer types (colorectal, esophageal, gastric, liver, lung, and ovarian cancers) and 675 healthy individuals were included in this study. Circulating tumor DNA mutations, cfDNA fragmentomic features and a set of protein biomarkers were assayed. Sensitivity and specificity were reported by cancer types and stages. Circular Ligation Amplification and sequencing (CLAmp-seq), a single-strand DNA sequencing technology, yielded a population of ultra-short fragments (<100 bp) than double-strand DNA preparation protocols and reveals a more significant size difference between cancer and healthy cfDNA fragments (25.84 bp vs. 16.05 bp). Analysis of the subnucleosomal peaks in ultra-short cfDNA fragments indicates that these peaks are regulatory element "footprints" and correlates with gene expression and cancer stages. At 98 % specificity, a prediction model using ctDNA mutations alone showed an overall sensitivity of 46 %; sensitivity reaches 60 % when protein is added, sensitivity further increases to 66 % when fragmentomics is also integrated. More improvements observed for samples representing earlier cancer stages than later ones. These results suggest synergistic properties of protein, genetic and fragmentomics features in the identification of early-stage cancers.