Early Diagnosis and Prognostic Prediction of Colorectal Cancer through Plasma Methylation Regions

Reviewer #1 (Public review):

Summary:

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. Colonoscopy and fecal immunohistochemical testing are among the early diagnostic tools that have significantly enhanced patient survival rates in CRC. Methylation dysregulation has been identified in the earliest stages of CRC, offering a promising avenue for screening, prediction, and diagnosis. The manuscript entitled "Early Diagnosis and Prognostic Prediction of Colorectal Cancer through Plasma Methylation Regions" by Zhu et al. presents that a panel of genes with methylation pattern derived from cfDNA (27 DMRs), serving as a noninvasive detection method for CRC early diagnosis and prognosis.

Strengths:

The authors provided evidence that the 27 DMRs pattern worked well in predicting CRC distant metastasis, and the methylation score remarkably increased in stages III-IV. Additionally, compared with the traditional tumor marker CEA, 27 DMRs prediction showed a superior sensitivity, highlighting the potential clinical application.

Weaknesses:

The major concerns are the design of DMRs screening, the relatively low sensitivity of this DMRs' pattern in detecting early-stage of CRC, the limited size of the cohorts, and the lack of comparison with the traditional diagnosis test.

Comments on revisions:

All my concerns have been cleared, and I have no further questions.

Reviewer #2 (Public review):

In this study, the authors aimed to develop cfDNA markers for comprehensive diagnosis, metastatic assessment, and prognostic prediction of colorectal cancer (CRC). Through integrative analysis of public 450K DNA methylation datasets and in-house targeted bisulfite sequencing (BS-seq) data from CRC and paired normal tissues, as well as plasma samples, they identified a signature comprising 27 differentially methylated regions (DMRs). This signature was subsequently validated for three clinical applications: cancer detection, metastasis prediction, and prognosis assessment.

Strengths:

The 27-DMR signature demonstrates value for both diagnosis and prognosis of CRC. Additionally, the datasets generated in this study serve as a valuable resource for the research community.

Weaknesses:

The validation cohorts for cancer detection and metastasis prediction were relatively small, which may limit the generalizability of the findings. The cancer detection model's performance does not surpass some published methods or commercial products.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. Colonoscopy and fecal immunohistochemical testing are among the early diagnostic tools that have significantly enhanced patient survival rates in CRC. Methylation dysregulation has been identified in the earliest stages of CRC, offering a promising avenue for screening, prediction, and diagnosis. The manuscript entitled "Early Diagnosis and Prognostic Prediction of Colorectal Cancer through Plasma Methylation Regions" by Zhu et al. presents that a panel of genes with methylation pattern derived from cfDNA (27 DMRs), serving as a noninvasive detection method for CRC early diagnosis and prognosis.

Strengths:

The authors provided evidence that the 27 DMRs pattern worked well in predicting CRC distant metastasis, and the methylation score remarkably increased in stage III-IV.

Weaknesses:

The major concerns are the design of DMR screening, the relatively low sensitivity of this DMR pattern in detecting early-stage CRC, the limited size of the cohorts, and the lack of comparison with the traditional diagnosis test.

We sincerely thank the reviewer for their thorough evaluation and constructive feedback on our manuscript. We are encouraged that the reviewer found our 27-DMR panel promising for predicting distant metastasis and for its performance in late-stage CRC. We have carefully considered the weaknesses pointed out and have made revisions to address these concerns, which we believe have significantly strengthened our paper.

We agree with the reviewer that achieving high sensitivity for early-stage disease is the ultimate goal for any noninvasive screening test. Detecting the minute quantities of cfDNA shed from early-stage tumors is a well-recognized challenge in the field. Although the sensitivity of our current panel for early-stage CRC is modest, its core strengths, lie in its capability to also detect advanced adenomas and its excellent performance in assessing CRC metastasis and prognosis. Furthermore, we have now added a direct comparative analysis of our 27-DMR panel against the most widely used clinical serum biomarker for CRC, carcinoembryonic antigen (CEA), using samples from the same patient cohorts. Our results demonstrate that 27-DMR methylation score significantly outperforms CEA in diagnostic accuracy for early-stage CRC (64% vs. 18%) (Table s7). And in the Discussion section, we have also acknowledged our limitations and suggest that future studies are warranted to combine the cfDNA methylation model with commonly used clinical markers, such as CEA and CA19-9, with the aim of improving the sensitivity for early diagnosis.

We acknowledge the reviewer's concern regarding the cohort size and validation in larger, prospective, multi-center cohorts is essential before this panel can be considered for clinical application. We have explicitly stated this as a limitation of our study in the Discussion section and have highlighted the need for future large-scale validation studies (Page 18, Lines 367-373). We once again thank the reviewer for their insightful comments, which have allowed us to substantially improve our manuscript. We hope that the revised version is now suitable for publication.

Reviewer #2 (Public review):

This work presents a 27-region DMR model for early diagnosis and prognostic prediction of colorectal cancer using plasma methylation markers. While this non-invasive diagnostic and prognostic tool could interest a broad readership, several critical issues require attention.

Major Concerns:

(1) Inconsistencies and clarity issues in data presentation

(a) Sample size discrepancies

The abstract mentions screening 119 CRC tissue samples, while Figure 1 shows 136 tissues. Please clarify if this represents 119 CRC and 17 normal samples.

We sincerely thank the reviewer for this careful observation and for pointing out the inconsistency. We apologize for the error and the confusion it caused. Regarding Figure 1: The reviewer is correct. The number 136 in the original Figure 1 was an error. This was due to an inadvertent double-counting of the tumor samples that were used in the differential analysis against adjacent normal tissues. The actual number of tissue samples used in this analysis is 89. We have now corrected this value in the revised Figure 1.

Regarding the Abstract: The 119 CRC tissue samples mentioned in the abstract represents the total number of unique tumor samples analyzed across all stages of our study. This number is composed of two cohorts: the initial 15 pairs of tissues used for preliminary screening, and the subsequent 89 tissue samples used for validation, totaling 119 samples. We have ensured all sample numbers are now consistent throughout the revised manuscript.

The plasma sample numbers vary across sections: the abstract cites 161 samples, Figure 1 shows 116 samples, and the Supplementary Methods mentions 77 samples (13 Normal, 15 NAA, 12 AA, 37 CRC).

We sincerely thank the reviewer for their meticulous review and for identifying these inconsistencies in the plasma sample numbers. We apologize for this oversight and the lack of clarity.

Figure 1 & Supplementary Methods (77 samples): The number 116 in the original Figure 1 was a clerical error. The correct number is 77, which is the cohort used for our differential methylation analysis. This number is now consistent with the Supplementary Methods. This cohort is composed of 13 Normal, 15 NAA, 12 AA, and 37 CRC samples. The figure has been revised accordingly.

Abstract (161 samples): The total of 161 plasma samples mentioned in the abstract is the sum of two distinct sample sets used for different stages of our analysis: The 77 samples (13 Normal, 15 NAA, 12 AA, 37 CRC) used for the differential analysis. An additional 84 samples (33 Normal, 51 CRC) which served as the training set for the LASSO regression model. We have now clarified these distinctions in the text and ensured consistency across the abstract, figures, and methods sections.

(b) Methodological inconsistencies

The Supplementary Material reports 477 hypermethylated sites from TCGA data analysis (Δβ>0.20, FDR<0.05), but Figure 1 indicates 499 sites.

The manuscript states that analyzing TCGA data across six cancer types identified 499 CRC-specific methylation sites, yet Figure 1 shows 477. Please also explain the rationale for selecting these specific cancer types from TCGA.

We sincerely thank the reviewer for their sharp observation and for highlighting these inconsistencies. We apologize for this clerical error, which occurred when labeling the figure. The numbers 477 and 499 in Figure 1 were inadvertently swapped and the text in Supplementary Material is correct. We have now corrected this error throughout the manuscript to ensure clarity and consistency. We deeply regret the confusion this has caused.

Regarding the rationale for selecting the cancer types:

The selection of colorectal, esophageal, gastric, lung, liver, and breast cancers was based on the following strategic criteria to ensure the stringent identification of CRC-specific markers. Firstly, esophageal, gastric, liver, and colorectal cancers all originate from the gastrointestinal tract and share developmental and functional similarities. Comparing CRC against these closely related cancers allowed us to filter out general GI-tract-related methylation patterns and isolate those that are truly unique to colorectal tissue. Secondly, we included lung and breast cancer as they are two of the most common non-GI malignancies worldwide with distinct tissue origins. This helps ensure our identified markers are not just pan-cancer methylation events but are specific to CRC, even when compared against highly prevalent cancers from different lineages. Finally, these six cancer types have some of the largest and most complete datasets available in the TCGA database, including high-quality methylation data. This provided a robust statistical foundation for a reliable cross-cancer comparison. We hope this explanation clarifies our methodology. Thank you again for your valuable feedback.

"404 CRC-specific DMRs" mentioned in the main text while "404 MCBs" in Figure 1, the authors need to clarify if these terms are interchangeable or how MCBs are defined.

We sincerely thank the reviewer for pointing out this important inconsistency in terminology. We apologize for the confusion this has caused and for the error in Figure 1. The two terms are closely related in our study. The final 404 markers are technically DMRs that were identified through an analysis of MCBs. To avoid confusion, we have decided to unify the terminology. The manuscript has now been revised to consistently use "DMRs", which is the most accurate final descriptor. The label in Figure 1 has been corrected accordingly.

(2) Methodological documentation

The Results section requires a more detailed description of marker identification procedures and justification of methodological choices.

Figure 3 panels need reordering for sequential citation.

We thank the reviewer for this valuable suggestion. We agree that the original Results section lacked sufficient detail regarding the marker identification procedures and the justification for our methodological choices. To address this, we have substantially rewritten the "Methylation markers selection" subsection. This revised section provides a clear, step-by-step narrative of our marker discovery. The revised text now integrates the specific methodological details and statistical criteria. For instance, we now explicitly describe the three-pronged approach for the initial TCGA data mining and the specific criteria (Δβ, FDR, log2FC) for each, and the analysis methodology such as Wilcoxon test and LASSO regression analysis. We believe this detailed narrative now provides the necessary description and justification for our methodological choices directly within the results, significantly improving the clarity and logical flow of our manuscript. This revision can be found on (Page 9-11, Lines 180-195, 202-213). We hope these changes fully address the reviewer's concerns.

We thank the reviewer for pointing out the citation order of the panels in Figure 3. This was a helpful suggestion for improving the clarity of our manuscript. We have now reordered the panels in Figure 3 to ensure they are cited sequentially within the text. These adjustments have been made in the "Development and validation of the CRC diagnosis model" subsection of the Results (Page 11, lines 224-230). We appreciate the reviewer's attention to detail.

(3) Quality control and data transparency

No quality control metrics are presented for the in-house sequencing data (e.g., sequencing quality, alignment rate, BS conversion rate, coverage, PCA plots for each cohort).

The analysis code should be publicly available through GitHub or Zenodo.

At a minimum, processed data should be made publicly accessible to ensure reproducibility.

We sincerely thank the reviewer for their valuable and constructive feedback regarding quality control and data transparency. We fully agree that these elements are crucial for ensuring the robustness and reproducibility of our research. As the reviewer suggested, we have made all processed data and the key quality control metrics for each sample including sequencing quality scores, bisulfite (BS) conversion rates, and sequencing coverage publicly available to ensure the reproducibility of our findings. The analysis was performed using standard algorithms as detailed in the Methods section. While we are unable to host the code in a public repository at this time, all analysis scripts are available from the corresponding author upon reasonable request. The data has been deposited in the National Genomics Data Center (NGDC) and is accessible under the accession number OMIX009128. This information is now clearly stated in the "Data and Code Availability" section of the manuscript. We thank the reviewer again for pushing us to improve our manuscript in this critical aspect.

Reviewer #3 (Public review):

Summary:

This article provides a model for early diagnosis and prognostic prediction of Colorectal Cancer and demonstrates its accuracy and usability. However, there are still some minor issues that need to be revised and paid attention to.

Strengths:

A large amount of external datasets were used for verification, thus demonstrating robustness and accuracy. Meanwhile, various influencing factors of multiple samples were taken into account, providing usability.

Weaknesses:

There are notable language issues that hinder readability, as well as a lack of some key conclusions provided.

We are very grateful to the reviewer for their positive assessment of our study and for the constructive feedback provided. We are particularly encouraged that the reviewer recognized the strengths of our work, especially the robustness demonstrated through extensive external validation and the practical usability of our model. Regarding the weaknesses, we have taken the comments very seriously and have thoroughly revised the manuscript. We sincerely apologize for the language issues that hindered readability in our initial submission. To address this, the entire manuscript has undergone a comprehensive round of professional language polishing and editing. We have carefully reviewed and revised the text to improve clarity, flow, and grammatical accuracy. Besides, we agree that the conclusions could be stated more explicitly. To rectify this, we have substantially revised the final paragraph of the Discussion and the Conclusion section (Page 14-18, lines 279-305, 319-334, 346-348, 358-360, 367-379). We now more clearly summarize the main findings of our study, emphasize the clinical significance and potential applications of our model, and provide clear take-home messages. We thank you again for your time and insightful comments, which have been invaluable in improving the quality of our paper. We hope the revised manuscript now meets the standards for publication.

Reviewer #1 (Recommendations for the authors):

Detail comments are outlined below:

(1) In this study, the authors have highlighted methylated cfDNA as a noninvasive approach for CRC early diagnosis. However, the small size of cohorts for plasma screening, particularly the sample number of NAA and AA , may cause bias in the selection of DMRs. This bias may lead to inappropriate DMRs for early diagnosis. Furthermore, the similar issues for the training set with a high percentage of late-stage CRC, no AA or NAA samples were included. This absence may be the key factor in screening changed methylated cfDNA that can predict the early stages of CRC.

We are very grateful to the reviewer for this insightful methodological critique. We agree that cohort composition and sample size are critical factors in the development of robust biomarkers, and we appreciate the opportunity to clarify our study design and the interpretation of our results.

We agree with the reviewer that the number of precancerous lesion samples (NAA and AA) in our initial plasma screening cohort was limited. This is a valid point. However, it is important to contextualize the role of this step within our overall multi-stage marker selection funnel. The markers evaluated in this plasma cohort were not discovered from this small sample set alone. They were the result of a rigorous pre-selection process based on large-scale public TCGA data and our own tissue-level sequencing. This robust, tissue-based validation ensured that only the most promising CRC-specific markers were advanced for plasma testing. Therefore, while the plasma cohort was modest in size, its purpose was to confirm the circulatory detectability of markers already known to have a strong tissue-of-origin signal, thereby mitigating the potential bias from a smaller discovery set.

Our primary aim was to first build a model that could robustly and accurately identify a definitive cancer-specific methylation signal. By training the model on clear-cut invasive cancer cases versus healthy controls, we could isolate the most powerful and specific markers for established malignancy. Our working hypothesis was that these strong cancer-specific methylation patterns are initiated during the precursor stages and would therefore be detectable, albeit at lower levels, in precancerous lesions. Unfortunately, the panel could only identify a limited proportion of precancerous lesions (48.4% in the NAA group and 52.2% in the AA group). We fully agree with the reviewer's sentiment that including a larger and more balanced set of precancerous lesions in future training cohorts could potentially optimize a model specifically for adenoma detection. We have now explicitly added this point to our Discussion section, highlighting it as an important direction for future research (Page 18, lines 367-373).

(2) The sensitivity of 27 DMRs in the external validation set (for NAA, AA and CRC 0-Ⅱare 48.4%. 52.2% and 66.7%, respectively) were much lower compared with previously published studies, like ColonES assay (DOI: 10.1016/j.eclinm.2022.101717) and ColonSecure test (DOI: 10.1186/s12943-023-01866-z). The 27 DMRs from the layered screening process did not show superior performance in a small population of an external validation cohort. Therefore, it is unlikely that this DMR pattern will be applicable to the general population in the future.

We sincerely thank the reviewer for their insightful comments and for providing a thorough comparison with the highly relevant ColonES and ColonSecure assays. This has given us an important opportunity to clarify the unique contributions and specific clinical applications of our 27-DMR panel.

We acknowledge the reviewer's point that the sensitivities of our panel for precancerous lesions (NAA: 48.4%, AA: 52.2%), while substantial, are numerically lower than those reported by the excellent ColonES assay (AA: 79.0%). However, it is important to clarify that while the ColonES and ColonSecure tests are outstanding benchmarks designed primarily for early detection and screening, the primary objective and contribution of our study were slightly different. Our model demonstrated an exceptional ability to predict distant metastasis with an AUC of 0.955 and a strong capacity for predicting overall prognosis with an AUC of 0.867. Our goal was to develop a multi-functional, biologically-rooted biomarker panel that not only contributes to early detection but, more importantly, provides crucial information for post-diagnosis patient management, including staging, risk stratification, and prognostication, from a single preoperative sample. We believe this ability to preoperatively identify high-risk patients who may require more aggressive treatment or intensive surveillance is the key contribution of our work. It provides a distinct clinical utility that complements, rather than directly competes with, pure screening assays.

We agree with the reviewer that our external validation was performed on a limited cohort, and we have acknowledged this as a limitation in our Discussion section. However, the purpose of this validation was to provide a proof-of-concept for the panel's performance across its multiple functions. The promising and exceptionally high-performing results in the prognostic domain strongly warrant further validation in larger, prospective, multi-center cohorts.

(3) The 27 DMRs pattern worked well in predicting CRC distant metastasis, and the methylation score remarkably increased in stage III-IV. In contrast, the increase of AA and 0-II groups was very mild in the validation cohort. This observation raises concerns regarding the study design, particularly in the context of the layered screening process and sample assigning.

We sincerely thank the reviewer for this insightful and critical comment. We agree with the reviewer's observation that the methylation score increased more remarkably in late-stage (III-IV) CRC compared to the milder increase in adenoma (AA) and early-stage (0-II) CRC in the validation cohort. However, the observed pattern is biologically plausible and consistent with the nature of colorectal cancer progression. Carcinogenesis is a multi-step process involving the gradual accumulation of genetic and epigenetic alterations. The methylation changes we identified are likely associated with tumor progression and metastasis. Therefore, it is expected that advanced, metastatic cancers (Stage III-IV), which have undergone significant biological changes, would exhibit a much stronger and more robust methylation signal compared to pre-cancerous lesions (adenomas) or early-stage, non-metastatic cancers (Stage 0-II). The "mild" increase in early stages reflects the initial, more subtle epigenetic alterations, while the "remarkable" increase in late stages reflects the extensive changes required for invasion and metastasis. We believe this graduated increase actually strengthens the validity of our methylation signature, as it mirrors the underlying biological progression of the disease. We hope this response and the corresponding revisions address the reviewer's comments.

(4) The authors did not provide the 27 DMRs prediction efficacy comparison with other noninvasive CRC assays, like a CEA and a FIT test.

Thank you for this valuable suggestion. We agree that comparing our model with established non-invasive assays is crucial for demonstrating its clinical potential. Following your advice, we have now included a direct comparison of the diagnostic performance between our model and the traditional tumor marker, carcinoembryonic antigen (CEA), using the external validation cohort. The results show that our model has a significantly higher sensitivity for detecting early-stage colorectal cancer and adenomas compared to CEA. This detailed comparison has been added as Table s7 in the supplementary materials, and the corresponding description has been incorporated into the Results section of our manuscript (Page 12, lines 234-236). Regarding the Fecal Immunochemical Test (FIT), we unfortunately could not perform a direct statistical comparison because very few individuals in our cohort had undergone FIT. A comparison based on such a small sample size would lack statistical power and might not yield meaningful conclusions. We have acknowledged this as a limitation of our study in the Discussion section.We believe these additions and clarifications have substantially strengthened our manuscript. Thank you again for your constructive feedback.

(5) The authors did not explicitly describe how they assigned the plasma samples to the distinct sets, nor did they specify the criteria for the plasma screen set, training set, and validation set. The detailed information for the patient grouping should be listed.

Responce: Thank you for this essential feedback. We agree that a transparent and detailed description of the sample allocation process is crucial for the manuscript. We apologize for the previous lack of clarity and have now revised the Methods section to address this. Our patient cohorts were assigned to the screening, training, and validation sets based on a chronological splitting strategy. Specifically, samples were allocated based on the date of collection in a consecutive manner. This approach was chosen to minimize selection bias and to provide a more realistic, forward-looking assessment of the model's performance, simulating a prospective validation scenario. The screening set comprised 89 tissue samples and 77 plasma samples collected between June to December 2020. The primary purpose of this set was for the initial discovery and screening of potential methylation markers. The training set and validation set included 165 plasma samples collected from December 2020 to July 2022. The external validation cohort comprised 166 plasma samples collected from from July 2022 to December 2022. The subsection titled "Study design and samples" within the Methods section of the revised manuscript, which now contains all of this detailed information (Page 6, lines 116-133). We believe this detailed explanation now makes our study design clear and transparent. Thank you again for helping us improve our manuscript.

Reviewer #2 (Recommendations for the authors):

The manuscript requires significant language editing to improve clarity and readability. We recommend that the authors seek professional editing services for revision.

Thank you for your constructive comments on the language of our manuscript. We apologize for any lack of clarity in the previous version. To address this, we have performed a thorough revision of the manuscript. The text has been carefully reviewed and edited by a native English-speaking colleague who is an expert in our research field. We have focused on correcting all grammatical errors, improving sentence structure, and refining the phrasing throughout the document to enhance readability. We are confident that these extensive revisions have significantly improved the clarity of the manuscript. We hope you will find the current version much easier to read and understand.

Reviewer #3 (Recommendations for the authors):

(1) However, I think the abstract part of the article is too detailed and should be more concise and shortened. It is not necessary to show detailed values but to summarize the results.

Thank you for this valuable suggestion. We agree that the previous version of the abstract was overly detailed and that a more concise summary would be more effective for the reader. Following your advice, we have substantially revised the abstract. We have removed the specific numerical values (such as detailed statistics) and have instead focused on summarizing the key findings and their broader implications (Page 3, lines 54-60, 64-66, 70-72). The revised abstract is now shorter and provides a clearer, high-level overview of our study's background, methods, main results, and conclusions. We believe these changes have significantly improved its readability and impact. We hope you will find the current version more appropriate.

(2) Figure 4, the color in the legend and plot are not the same, and should be revised.

Thank you for your careful attention to detail and for pointing out the color inconsistency in Figure 4. We apologize for this oversight. We have now corrected the figure as you suggested, ensuring that the colors in the legend perfectly match those in the plot. The revised Figure 4 has been updated in the manuscript. We appreciate your help in improving the quality of our figures.

(3) Please pay attention to the article format, such as the consistency of fonts and punctuation marks. (For example, Lines 75 and Line 230).

Thank you for your meticulous review and for pointing out the inconsistencies in our manuscript's formatting. We sincerely apologize for these oversights and any inconvenience they may have caused. Following your feedback, we have carefully corrected the specific issues you highlighted. Furthermore, we have conducted a thorough proofread of the entire manuscript to ensure consistency in all fonts, punctuation marks, and overall adherence to the journal's formatting guidelines. We appreciate your help in improving the presentation and professionalism of our paper.