Review Reports - Plasma 5-Fluorouracil Exposure, Clinical Outcomes, and Therapeutic Drug Monitoring in Advanced Colorectal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a prospective observational study evaluating the relationship between plasma exposure of 5-fluorouracil (5-FU), clinical outcomes, and the feasibility of therapeutic drug monitoring (TDM) in patients with advanced or recurrent colorectal cancer. The study further explores the applicability of a rapid immunochromatographic assay as a practical alternative to conventional pharmacokinetic measurement methods. The topic is clinically relevant and timely, as inter-individual variability in 5-FU pharmacokinetics remains a critical limitation of body surface area (BSA)-based dosing. The study attempts to bridge an important gap by linking pharmacokinetic exposure (AUC) with toxicity, tumor response, and survival outcomes, while also proposing a feasible bedside monitoring approach. The manuscript is generally well-structured and provides meaningful preliminary insights. However, given the pilot nature, small sample size (n = 15), and exploratory statistical analysis, several methodological and interpretational issues need to be addressed to strengthen the scientific rigor and clarity of the study.

The manuscript lacks a formal justification of the sample size. A discussion on statistical power and limitations due to small cohort size should be included.
Approach of using median concentrations across eight cycles to derive a “representative AUC” requires stronger justification and methodological validation.
Important clinical variables (e.g., treatment regimens, metastatic burden, prior therapies) are not controlled for, limiting causal interpretation.
Multivariate analysis or at least adjusted exploratory models, should be considered to support conclusions better.
The assay comparison is based on only nine samples, which is insufficient for robust validation.
In discussion of PFS and OS needs to be more cautious and clearly framed as exploratory.
Manuscript does not clearly define how measured AUC values would translate into actionable dose modifications.
The study includes only 15 patients, which significantly limits statistical power and generalizability. The authors should explicitly acknowledge this limitation and, if possible, provide a rationale for the chosen sample size or include a post hoc power estimation.
Use of median plasma concentrations across eight cycles to calculate a representative AUC is unconventional. The authors should provide a clear methodological justification and discuss potential biases introduced by this approach.

The cohort includes patients with varying disease stages, metastatic burden, and treatment histories. This heterogeneity may confound the relationship between AUC and outcomes. A stratified or adjusted analysis is recommended.
manuscript reports trends in progression-free survival (PFS) without statistical significance (p = 0.36). These findings should be clearly described as hypothesis-generating rather than suggestive of clinical benefit.
While the association between AUC and toxicity is statistically significant, the sample size is small. The authors should discuss whether specific toxicities correlate with higher or lower AUC values in a more mechanistic context.
Comparison between the immunochromatographic method and the My-5FU assay is based on only nine samples, which is insufficient for validation. Additional data or acknowledgment of this limitation is required.
The manuscript highlights the feasibility of TDM but does not provide a clear framework for dose adjustment based on AUC values. Including a proposed dosing algorithm or referencing established protocols would improve clinical relevance.
Although DPYD genotyping is discussed, it was not incorporated into the study. The authors should clarify whether any pharmacogenetic screening was performed and discuss its potential impact on results.

The manuscript addresses an important topic in oncology and provides promising preliminary data on the role of 5-FU therapeutic drug monitoring. However, methodological clarifications, stronger statistical support, and more cautious interpretation are required before the study can be considered for publication.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the English language requires improvement for clarity and readability. Several sentences are overly long and complex, and minor grammatical errors are present throughout.

Author Response

Thank you very much for your detailed and constructive comments. We have addressed all points raised by Reviewer 1, including the pilot sample-size rationale, representative AUC methodology, treatment-regimen heterogeneity, multivariable analysis limitations, specific adverse-event analysis, Bland–Altman method comparison, dose-adjustment framework, DPYD limitation, and cautious interpretation of survival outcomes. Please see the attached point-by-point response to Reviewer 1.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors explored whether achieving a predefined target area under the concentration–time curve for plasma 5-fluorouracil was associated with favorable clinical outcomes and evaluated the feasibility of a simple plasma 5-fluorouracil measurement kit. Fifteen patients with unresectable advanced or recurrent colorectal cancer who received continuous-infusion of 5-fluorouracil-based chemotherapy were prospectively evaluated. The research design is appropriate, and the introduction has sufficient background. I want to suggest the following:

In Figure 1, the ligand should contain the directions for readers, such as the sampling times in dotted lines, and the study initiation time, termination time, etc.
Figure 2a showed pigment levels 1~8; however, only 1~6 concentration levels were presented. Better to present the concentration levels beside the pigment levels, for example, Lv8 0ng/mL;
In Figure 2b, the plot should have the X and Y axes. A better way to express the linear equation, if possible, slope, and intercept with the R2 value.
My-5FU® Kits look highly reproducible, Wherther authors measured the kits with known concentration and plotted in the regression equation to confirm the accuracy and %RSD.
Table 2: Please mention the target AUC range Unit (mg·h/L);
Table 3&4 with the percentile would be valuable information.
Similar in Table 4: the target AUC range Unit (mg·h/L);
Figure 4b should contain the X and Y axes.

Author Response

Thank you very much for your helpful suggestions. We have revised the figures and tables accordingly, including Figure 1, Figure 2, Tables 2–4, and Figure 4. We also clarified the limitations of assay reproducibility testing and provided editable figure source data. Please see the attached point-by-point response to Reviewer 2.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This pilot study offers a pragmatic and clever solution to the long-standing "Goldilocks" problem of chemotherapy dosing; finding that narrow window where the drug is effective without being overly toxic. By introducing a rapid, bedside assay, you’ve addressed the turnaround time gap that usually makes real-time dose adjustments a logistical nightmare for oncologists. While the cohort is small, the decision to track exposure across multiple cycles provides a much more stable and authentic representative profile than the typical single-point snapshot.

The "Outside target range" group is currently too heterogeneous. By grouping patients with insufficient exposure together with those showing excessive exposure, the distinct clinical drivers of toxicity and poor tumor control become blurred. Separating these would clarify if toxicities are tied to supratherapeutic levels and lack of efficacy to subtherapeutic levels.
While the correlation between the novel assay and the laboratory standard is promising, the degree of variance suggests a significant margin of uncertainty for clinical dosing. Including a Bland-Altman plot is essential to evaluate the actual bias and define the limits of agreement between the two methods.
Survival outcomes must be strictly framed as exploratory. The observed numerical trends are interesting for a pilot study, but they do not reach statistical significance and should be presented as hypothesis-generating rather than definitive evidence of a survival benefit.
It is important to clarify whether patients were screened for genetic enzyme deficiencies, such as DPD. In a small cohort, even a single outlier with impaired metabolism can disproportionately skew the safety data and the average exposure results.
Please provide more technical detail on the assay’s origin and its specific limits of detection. Defining whether this is an in-house prototype or a commercial version under development will help clinicians understand its practical boundaries and reliability in a busy ward.

Major Revision. The manuscript addresses a critical clinical need with an innovative tool, but it requires a more nuanced breakdown of the exposure data and a more rigorous statistical validation of the new assay before it is ready for publication.

Author Response

Thank you very much for your constructive assessment and suggestions. We have separated the outside-target-range group into underexposure and overexposure subgroups, added Bland–Altman analysis, reframed survival findings as exploratory, clarified that DPYD screening was not performed, and added technical details and limitations of the prototype IC assay. Please see the attached point-by-point response to Reviewer 3.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript evaluates the association between plasma 5-fluorouracil (5-FU) exposure and clinical outcomes in advanced or recurrent colorectal cancer, while also assessing the feasibility of an immunochromatographic assay for therapeutic drug monitoring (TDM). The topic is clinically relevant, particularly given the increasing interest in pharmacokinetically guided chemotherapy.

The study provides preliminary evidence supporting the clinical utility of achieving target AUC levels and proposes a practical tool for real-time monitoring. However, several methodological limitations—primarily the small sample size and exploratory design—significantly restrict the strength of the conclusions. While the manuscript is well-structured and clearly written, important issues should be addressed before consideration for publication.

Major Comments

-The study includes only 15 patients, which severely limits statistical power and generalizability. Although the authors acknowledge this limitation, the conclusions are still somewhat overstated.

The authors should clearly emphasize the exploratory nature of the findings throughout the manuscript (not only in the discussion).
Consider reframing conclusions to avoid causal implications (e.g., replace “may be associated” with “suggests a potential association”).

-The cohort is heterogeneous (e.g., stage III vs IV, prior surgery, metastatic burden).

Please clarify whether treatment regimens (e.g., FOLFOX, FOLFIRI, bevacizumab use) were uniform or variable.
If heterogeneous, discuss how this may confound the relationship between AUC and outcomes.
A table summarizing treatment regimens would strengthen the manuscript.

-The use of median concentrations across eight cycles to calculate a “representative AUC” is interesting but unconventional.

Provide stronger justification and references supporting this approach.
Discuss potential bias: this method may mask intra-patient variability and dilute clinically relevant fluctuations.
Consider including cycle-specific analyses, if available.

Several key outcomes (e.g., tumor control, PFS) are not statistically significant, yet are discussed as meaningful trends.
The p-value for PFS (p = 0.36) and OS (p = 0.76) indicates no statistical difference.

Clearly distinguish statistically significant vs. descriptive findings.
Avoid overinterpretation of non-significant results.
Consider reporting confidence intervals to provide more context.

-The IC assay is a key novelty of the study but is insufficiently validated.

Only nine samples were used for comparison with My-5FU.
An R² of 0.76 indicates moderate correlation but not strong agreement.

-Although DPYD genotyping is discussed, it was not performed in this cohort.

This is a significant limitation given current clinical practice.
The authors should explicitly discuss how absence of pharmacogenetic data may influence interpretation.

Author Response

Thank you very much for your careful evaluation. We have revised the manuscript to emphasize the pilot and exploratory nature of the study, added treatment-regimen information, clarified representative AUC methodology and its limitations, avoided overinterpretation of non-significant findings, added Bland–Altman analysis, and discussed the absence of DPYD genotyping. Please see the attached point-by-point response to Reviewer 4.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all the comments and revised the manuscript accordingly, so the manuscript has been accepted in its present form.

Reviewer 3 Report

Comments and Suggestions for Authors

I commend the authors for their thorough and constructive point-by-point response to the previous critique. The revisions significantly strengthen the manuscript by adding transparency, improving statistical rigor, and providing a more accurate clinical interpretation of the data. By directly addressing the limitations of the prototype assay and the exploratory nature of the clinical findings, the authors have elevated the scientific integrity of this pilot study.

The addition of a supplementary analysis separating the non-target patients into distinct under- and over-exposure subgroups effectively clarifies the separate drivers of toxicity and treatment failure.
Integrating a Bland–Altman analysis satisfies the requirement for a rigorous comparison between the novel assay and the laboratory standard, and the candid discussion regarding the wide limits of agreement is highly appropriate.
Tempering the language surrounding survival outcomes ensures that the findings are correctly framed as exploratory and hypothesis-generating, rather than definitive conclusions.
Explicitly acknowledging the absence of genetic screening provides essential context for the safety data and protects against overinterpreting the pharmacokinetic variations.
The added technical details clarifying that the assay is an in-house prototype under development manage clinical expectations well and appropriately highlight the need for future validation.

Verdict: Accept. The authors have successfully resolved all major concerns with commendable rigor and transparency. The revised manuscript is now fully optimized for publication and will serve as a highly valuable, honest framework for future research into rapid therapeutic drug monitoring for oncology.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have adequately addressed the major concerns and implemented the requested revisions satisfactorily. The manuscript has significantly improved in terms of clarity, organization, and scientific presentation. In particular, the critical discussion, structural organization of the therapeutic strategies, and overall readability have been strengthened.

The revised version now provides a more coherent and comprehensive overview of synergistic UPR-targeting strategies in cancer therapy and represents a valuable contribution to the field.

Therefore, in its current form, the manuscript is acceptable for publication.