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. 2022 Oct 4;12:16566. doi: 10.1038/s41598-022-20975-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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(A) Classifier trained on 5hmC levels in enhancer regions shows equivalent performance on the training (dotted line) and validation sets (solid line) and high performance across all CRC stages versus controls, with AUCs ranging from 88.6 to 93.6%. Cut-of-values are reported in Supp. Table 1. (B) The IchorCNA tumor fraction was positively correlated with tumor stage in validation samples. The correlation with the CRC classifier score was lower in early-stage samples (stages 1 and 2), with higher p-values than in later-stage samples. (C) CRC classifier score on validation samples with ichorCNA values ≤ 3% tumor fraction, demonstrating that the 5hmC-based classifier maintains robust performance on samples with a low tumor fraction. The corresponding table presents the percentage of samples on either side of the classification threshold (0.5), demonstrating that the classifier performs similarly across CRC stages. (D) The top significant biological pathways identified that relate to the pathway relationship of genes regulated by enhancer features in the 5hmC classifier indicate a global immune response to tumorigenesis.