Endoscopic liquid biopsies of gastric fluid in a large patient cohort reveal DNA content as a candidate tumor biomarker in gastric cancer
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eLife Assessment
This paper reports a valuable finding that gastric fluid DNA content can be used as a potential biomarker for human gastric cancer. The evidence supporting the claims of the authors is solid, although an inclusion of explanations for the methodological limitations, moderate diagnostic performance, and the unexpected survival correlation would have strengthened the study. The work will be of interest to medical biologists working in the field of breast cancer.
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Abstract
Abstract
Background
Human gastric cancer remains a diagnostic and therapeutic challenge worldwide. Improved prognostic biomarkers may support treatment planning, including surgery, neoadjuvant and adjuvant therapies. We offer a new liquid biopsy approach, integrated with esophagogastroduodenoscopy (EGD) to evaluate gastric fluid recovered from a large patient cohort (n=1,056 patients), demonstrating the value of a simple DNA concentration analysis to provide diagnostic support and prognosis in gastric cancer.
Methods
We performed an exploratory study to evaluate gastric fluid DNA (gfDNA) concentration in patients with normal gastric mucosa or diagnosed with peptic diseases, preneoplastic conditions, or cancer. Key variables including sex, gastric fluid pH, use of proton-pump inhibitors, tumor subtype, clinical stage, and disease outcomes were considered.
Results
gfDNA concentrations were significantly increased in gastric cancer versus all groups (mean 26.86 ng/µL; 95% CI: 20.05 to 33.79; p=3.61e-12) and as compared to non-malignant controls (normal mucosa and peptic diseases; mean 10.77 ng/µL; 95% CI: 9.23 to 12.33; p=9.55e-13), and preneoplastic conditions (mean 10.10 ng/µL; 95% CI: 7.59 to 12.60; p=1.10e-5). gfDNA concentrations were higher in advanced tumors (T3; mean 25.66 ng/µL; 95% CI: 19.46 to 31.85) compared to early-stage disease (T2 and below; mean 15.12 ng/µL; 95% CI: 9.73 to 20.50; p=5.97e-4). Our findings also suggest gfDNA prognostic value for gastric cancer diagnosed subjects. In this subset, patients with gfDNA concentrations >1.28 ng/µL showed longer progression-free survival (p=0.009), which correlated with increased tumor-infiltrating immune cells (p=0.001), remaining significant after adjusting for tumor stage (p=0.014).
Conclusions
While high gfDNA indicates gastric cancer in a general population and may contribute to disease management, its diagnostic clues should be integrated with further work up such as tissue biopsies, to ensure comprehensive and accurate clinical assessment. However, in patients with established gastric cancer, gfDNA is a potential prognostic marker, and subjects with high gfDNA content might paradoxically have better outcomes. The elevated presence of immune cell infiltrates—compared to those observed in peptic diseases and pre-neoplastic lesions (including their associated gastric cavity DNA shedding)—may serve as a critical missing link to resolve this empirical paradox.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
The study analyzes the gastric fluid DNA content identified as a potential biomarker for human gastric cancer. However, the study lacks overall logicality, and several key issues require improvement and clarification. In the opinion of this reviewer, some major revisions are needed:
(1) This manuscript lacks a comparison of gastric cancer patients' stages with PN and N+PD patients, especially T0-T2 patients.
We are grateful for this astute remark. A comparison of gfDNA concentration among the diagnostic groups indicates a trend of increasing values as the diagnosis progresses toward malignancy. The observed values for the diagnostic groups are as follows:
Author response table 1.
The chart below presents the statistical analyses of the same diagnostic/tumor-stage groups …
Author response:
Public Reviews:
Reviewer #1 (Public review):
The study analyzes the gastric fluid DNA content identified as a potential biomarker for human gastric cancer. However, the study lacks overall logicality, and several key issues require improvement and clarification. In the opinion of this reviewer, some major revisions are needed:
(1) This manuscript lacks a comparison of gastric cancer patients' stages with PN and N+PD patients, especially T0-T2 patients.
We are grateful for this astute remark. A comparison of gfDNA concentration among the diagnostic groups indicates a trend of increasing values as the diagnosis progresses toward malignancy. The observed values for the diagnostic groups are as follows:
Author response table 1.
The chart below presents the statistical analyses of the same diagnostic/tumor-stage groups (One-Way ANOVA followed by Tukey’s multiple comparison tests). It shows that gastric fluid gfDNA concentrations gradually increase with malignant progression. We observed that the initial tumor stages (T0 to T2) exhibit intermediate gfDNA levels, which in this group is significantly lower than in advanced disease (p = 0.0036), but not statistically different from non-neoplastic disease (p = 0.74).
Author response image 1.
(2) The comparison between gastric cancer stages seems only to reveal the difference between T3 patients and early-stage gastric cancer patients, which raises doubts about the authenticity of the previous differences between gastric cancer patients and normal patients, whether it is only due to the higher number of T3 patients.
We appreciate the attention to detail regarding the numbers analyzed in the manuscript. Importantly, the results are meaningful because the number of subjects in each group is comparable (T0-T2, N = 65; T3, N = 91; T4, N = 63). The mean gastric fluid gfDNA values (ng/µL) increase with disease stage (T0-T2: 15.12; T3-T4: 30.75), and both are higher than the mean gfDNA values observed in non-neoplastic disease (10.81 ng/µL for N+PD and 10.10 ng/µL for PN). These subject numbers in each diagnostic group accurately reflect real-world data from a tertiary cancer center.
(3) The prognosis evaluation is too simplistic, only considering staging factors, without taking into account other factors such as tumor pathology and the time from onset to tumor detection.
Histopathological analyses were performed throughout the study not only for the initial diagnosis of tissue biopsies, but also for the classification of Lauren’s subtypes, tumor staging, and the assessment of the presence and extent of immune cell infiltrates. Regarding the time of disease onset, this variable is inherently unknown--by definition--at the time of a diagnostic EGD. While the prognosis definition is indeed straightforward, we believe that a simple, cost-effective, and practical approach is advantageous for patients across diverse clinical settings and is more likely to be effectively integrated into routine EGD practice.
(4) The comparison between gfDNA and conventional pathological examination methods should be mentioned, reflecting advantages such as accuracy and patient comfort.
We wish to reinforce that EGD, along with conventional histopathology, remains the gold standard for gastric cancer evaluation. EGD under sedation is routinely performed for diagnosis, and the collection of gastric fluids for gfDNA evaluation does not affect patient comfort. Thus, while gfDNA analysis was evidently not intended as a diagnostic EGD and biopsy replacement, it may provide added prognostic value to this exam.
(5) There are many questions in the figures and tables. Please match the Title, Figure legends, Footnote, Alphabetic order, etc.
We are grateful for these comments and apologize for the clerical oversight. All figures, tables, titles and figure legends have now been double-checked.
(6) The overall logicality of the manuscript is not rigorous enough, with few discussion factors, and cannot represent the conclusions drawn.
We assume that the unusual wording remark regarding “overall logicality” pertains to the rationale and/or reasoning of this investigational study. Our working hypothesis was that during neoplastic disease progression, tumor cells continuously proliferate and, depending on various factors, attract immune cell infiltrates. Consequently, both tumor cells and immune cells (as well as tumor-derived DNA) are released into the fluids surrounding the tumor at its various locations, including blood, urine, saliva, gastric fluids, and others. Thus, increases in DNA levels within some of these fluids have been documented and are clinically meaningful. The concurrent observation of elevated gastric fluid gfDNA levels and immune cell infiltration supports the hypothesis that increased gfDNA—which may originate not only from tumor cells but also from immune cells—could be associated with better prognosis, as suggested by this study of a large real-world patient cohort.
In summary, we thank Reviewer #1 for his time and effort in a constructive critique of our work.
Reviewer #2 (Public review):
Summary:
The authors investigated whether the total DNA concentration in gastric fluid (gfDNA), collected via routine esophagogastroduodenoscopy (EGD), could serve as a diagnostic and prognostic biomarker for gastric cancer. In a large patient cohort (initial n=1,056; analyzed n=941), they found that gfDNA levels were significantly higher in gastric cancer patients compared to non-cancer, gastritis, and precancerous lesion groups. Unexpectedly, higher gfDNA concentrations were also significantly associated with better survival prognosis and positively correlated with immune cell infiltration. The authors proposed that gfDNA may reflect both tumor burden and immune activity, potentially serving as a cost-effective and convenient liquid biopsy tool to assist in gastric cancer diagnosis, staging, and follow-up.
Strengths:
This study is supported by a robust sample size (n=941) with clear patient classification, enabling reliable statistical analysis. It employs a simple, low-threshold method for measuring total gfDNA, making it suitable for large-scale clinical use. Clinical confounders, including age, sex, BMI, gastric fluid pH, and PPI use, were systematically controlled. The findings demonstrate both diagnostic and prognostic value of gfDNA, as its concentration can help distinguish gastric cancer patients and correlates with tumor progression and survival. Additionally, preliminary mechanistic data reveal a significant association between elevated gfDNA levels and increased immune cell infiltration in tumors (p=0.001).
Reviewer #2 has conceptually grasped the overall rationale of the study quite well, and we are grateful for their assessment and comprehensive summary of our findings.
Weaknesses:
(1) The study has several notable weaknesses. The association between high gfDNA levels and better survival contradicts conventional expectations and raises concerns about the biological interpretation of the findings.
We agree that this would be the case if the gfDNA was derived solely from tumor cells. However, the findings presented here suggest that a fraction of this DNA would be indeed derived from infiltrating immune cells. The precise determination of the origin of this increased gfDNA remains to be achieved in future follow-up studies, and these are planned to be evaluated soon, by applying DNA- and RNA-sequencing methodologies and deconvolution analyses.
(2) The diagnostic performance of gfDNA alone was only moderate, and the study did not explore potential improvements through combination with established biomarkers. Methodological limitations include a lack of control for pre-analytical variables, the absence of longitudinal data, and imbalanced group sizes, which may affect the robustness and generalizability of the results.
Reviewer #2 is correct that this investigational study was not designed to assess the diagnostic potential of gfDNA. Instead, its primary contribution is to provide useful prognostic information. In this regard, we have not yet explored combining gfDNA with other clinically well-established diagnostic biomarkers. We do acknowledge this current limitation as a logical follow-up that must be investigated in the near future.
Moreover, we collected a substantial number of pre-analytical variables within the limitations of a study involving over 1,000 subjects. Longitudinal samples and data were not analyzed here, as our aim was to evaluate prognostic value at diagnosis. Although the groups are imbalanced, this accurately reflects the real-world population of a large endoscopy center within a dedicated cancer facility. Subjects were invited to participate and enter the study before sedation for the diagnostic EGD procedure; thus, samples were collected prospectively from all consenting individuals.
Finally, to maintain a large, unbiased cohort, we did not attempt to balance the groups, allowing analysis of samples and data from all patients with compatible diagnoses (please see Results: Patient groups and diagnoses).
(3) Additionally, key methodological details were insufficiently reported, and the ROC analysis lacked comprehensive performance metrics, limiting the study's clinical applicability.
We are grateful for this useful suggestion. In the current version, each ROC curve (Supplementary Figures 1A and 1B) now includes the top 10 gfDNA thresholds, along with their corresponding sensitivity and specificity values (please see Suppl. Table 1). The thresholds are ordered from-best-to-worst based on the classic Youden’s J statistic, as follows:
Youden Index = specificity + sensitivity – 1 [Youden WJ. Index for rating diagnostic tests. Cancer 3:32-35, 1950. PMID: 15405679]. We have made an effort to provide all the key methodological details requested, but we would be glad to add further information upon specific request.
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eLife Assessment
This paper reports a valuable finding that gastric fluid DNA content can be used as a potential biomarker for human gastric cancer. The evidence supporting the claims of the authors is solid, although an inclusion of explanations for the methodological limitations, moderate diagnostic performance, and the unexpected survival correlation would have strengthened the study. The work will be of interest to medical biologists working in the field of breast cancer.
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Reviewer #1 (Public review):
The study analyzes the gastric fluid DNA content identified as a potential biomarker for human gastric cancer. However, the study lacks overall logicality, and several key issues require improvement and clarification. In the opinion of this reviewer, some major revisions are needed:
(1) This manuscript lacks a comparison of gastric cancer patients' stages with PN and N+PD patients, especially T0-T2 patients.
(2) The comparison between gastric cancer stages seems only to reveal the difference between T3 patients and early-stage gastric cancer patients, which raises doubts about the authenticity of the previous differences between gastric cancer patients and normal patients, whether it is only due to the higher number of T3 patients.
(3) The prognosis evaluation is too simplistic, only considering staging …
Reviewer #1 (Public review):
The study analyzes the gastric fluid DNA content identified as a potential biomarker for human gastric cancer. However, the study lacks overall logicality, and several key issues require improvement and clarification. In the opinion of this reviewer, some major revisions are needed:
(1) This manuscript lacks a comparison of gastric cancer patients' stages with PN and N+PD patients, especially T0-T2 patients.
(2) The comparison between gastric cancer stages seems only to reveal the difference between T3 patients and early-stage gastric cancer patients, which raises doubts about the authenticity of the previous differences between gastric cancer patients and normal patients, whether it is only due to the higher number of T3 patients.
(3) The prognosis evaluation is too simplistic, only considering staging factors, without taking into account other factors such as tumor pathology and the time from onset to tumor detection.
(4) The comparison between gfDNA and conventional pathological examination methods should be mentioned, reflecting advantages such as accuracy and patient comfort.
(5) There are many questions in the figures and tables. Please match the Title, Figure legends, Footnote, Alphabetic order, etc.
(6) The overall logicality of the manuscript is not rigorous enough, with few discussion factors, and cannot represent the conclusions drawn
-
Reviewer #2 (Public review):
Summary:
The authors investigated whether the total DNA concentration in gastric fluid (gfDNA), collected via routine esophagogastroduodenoscopy (EGD), could serve as a diagnostic and prognostic biomarker for gastric cancer. In a large patient cohort (initial n=1,056; analyzed n=941), they found that gfDNA levels were significantly higher in gastric cancer patients compared to non-cancer, gastritis, and precancerous lesion groups. Unexpectedly, higher gfDNA concentrations were also significantly associated with better survival prognosis and positively correlated with immune cell infiltration. The authors proposed that gfDNA may reflect both tumor burden and immune activity, potentially serving as a cost-effective and convenient liquid biopsy tool to assist in gastric cancer diagnosis, staging, and follow-up.
Reviewer #2 (Public review):
Summary:
The authors investigated whether the total DNA concentration in gastric fluid (gfDNA), collected via routine esophagogastroduodenoscopy (EGD), could serve as a diagnostic and prognostic biomarker for gastric cancer. In a large patient cohort (initial n=1,056; analyzed n=941), they found that gfDNA levels were significantly higher in gastric cancer patients compared to non-cancer, gastritis, and precancerous lesion groups. Unexpectedly, higher gfDNA concentrations were also significantly associated with better survival prognosis and positively correlated with immune cell infiltration. The authors proposed that gfDNA may reflect both tumor burden and immune activity, potentially serving as a cost-effective and convenient liquid biopsy tool to assist in gastric cancer diagnosis, staging, and follow-up.
Strengths:
This study is supported by a robust sample size (n=941) with clear patient classification, enabling reliable statistical analysis. It employs a simple, low-threshold method for measuring total gfDNA, making it suitable for large-scale clinical use. Clinical confounders, including age, sex, BMI, gastric fluid pH, and PPI use, were systematically controlled. The findings demonstrate both diagnostic and prognostic value of gfDNA, as its concentration can help distinguish gastric cancer patients and correlates with tumor progression and survival. Additionally, preliminary mechanistic data reveal a significant association between elevated gfDNA levels and increased immune cell infiltration in tumors (p=0.001).
Weaknesses:
The study has several notable weaknesses. The association between high gfDNA levels and better survival contradicts conventional expectations and raises concerns about the biological interpretation of the findings. The diagnostic performance of gfDNA alone was only moderate, and the study did not explore potential improvements through combination with established biomarkers. Methodological limitations include a lack of control for pre-analytical variables, the absence of longitudinal data, and imbalanced group sizes, which may affect the robustness and generalizability of the results. Additionally, key methodological details were insufficiently reported, and the ROC analysis lacked comprehensive performance metrics, limiting the study's clinical applicability.
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