Immune Cell-Specific and Isoform-Selective Regulation of CD44 in Pancreatic Ductal Adenocarcinoma Links Lymph Node Variant Loss and Exosomal CD44 to Clinical Outcome in Pancreatic Ductal Adenocarcinoma
, Frederik Johannes Hansen 1,†, Melanie Litau 1, Isabelle Kuchenreuther 1, Johanne Mazurie 1, Finn Niklas Clausen 1, Sebastian Klöckner 1, Franziska Czubayko 1, Nadine Weisel 1, Bettina Klösch 1, Talida Andert-Veres 2, Stefanie Kröber 2, Susanne Merkel 1, Andreas R. R. Weiss 1, Maximilian Brunner 1, Christian Krautz 1, Robert Grützmann 1, Georg F. Weber 1,* and Paul David 1,*add
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Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsSee attached file
Comments for author File: 
Comments.pdf
Author Response
We sincerely thank the reviewer for the careful and detailed evaluation of our manuscript. The constructive comments have helped us clarify methodological aspects, strengthen our analyses, and improve the overall clarity and transparency of the study. We have revised the manuscript accordingly and provided additional stratified and multivariate analyses where appropriate. We believe that these revisions have substantially enhanced the scientific rigor and presentation of our work. We are grateful for the reviewer’s thoughtful feedback and consideration.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript analyzes immune-cell–specific regulation of CD44 in PDAC and links lymph-node CD44 variant loss and exosome-associated CD44 to clinical outcome. The authors combine patient-derived samples with exosome profiling and TCGA-based analyses. They conclude that CD44 shows isoform-specific and compartment-specific behavior associated with metastasis, survival, immune-checkpoint profiles, and predicted drug resistance.
The study addresses a relevant clinical question and integrates experimental and bioinformatic data in a comprehensive way.
However, several issues need to be addressed.
1) The title should specify immune-cell–specific regulation, as “compartment-specific” is too vague.
2) The Introduction should better justify the immune-cell focus.
3) Exact sample numbers (n) must be clearly stated in every figure panel and experiment.
4) Normality testing must be reported to justify the use of parametric tests.
5) Multivariate survival analyses are required to determine whether CD44 variants and exo-CD44 are independent prognostic factors.
6) Figure 5 is visually too small and dense.
Recommendation: Major revision.
Author Response
We thank the reviewer for the thorough and constructive evaluation of our manuscript. The comments have helped us improve the clarity, methodological transparency, and overall quality of the study. We have carefully revised the manuscript and addressed all concerns in detail.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper deals with CD44 expression and pancreatic ductal adenocarcinoma, PDAC CD44 was analyzed for its standard form and variants in different matrices revealing a compartment-specific regulation in PDAC which could be correlated to clinical outcome
- In line 97 the FACS sample should be defined for its composition. Thus, before line 138
- Line 154 and 161: you should delete two - in patients and proportion respectively
- Line 163 H2SO4 should de be written with subscript instead of apex
Author Response
We sincerely appreciate the reviewer’s thoughtful and detailed feedback. The suggestions provided have strengthened the manuscript, and we have revised the text accordingly to enhance clarity and rigor. We are grateful for the reviewer’s valuable input.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors
One of the main issues with your article was the statistic methodology you used. That is considering all your population (universe) as a single coherent cohort. As said previously, there are at least two different cohorts you cannot intermingle. That is those who were chemotherapy naive and those who received neoadjuvant chemo. These two populations should have different CD44 expression.
According to the text you added in the paper you partially solved the bias, although not entirely.
I am not going to obstruct the publication of your paper based on this issue.
There are other two issues with which I do not agree but again I will not make a major problem out of them.
1) Ethical issue: you performed lymph node resection in 12 cases of patients who underwent cholecistectomy or hernia, when they did not need this kind of added procedure. I do not think your patients consent provided permission for these added procedures. Furthermore, If you included the issue in the consent, I do not think you explained the patients that the lymph node extraction only served the purpose of publishing a paper.
2) Your level of failed surgeries (23 over 65) is two fold higher than what international and European standards admit. This standard is around 15% while your standard is around 34%.
As proof of concept:
Mayo Clinic series (2017–2021): In a retrospective review of 1,004 patients with radiographically localized PDAC undergoing staging laparoscopy, the overall positive laparoscopy rate (indicating unresectable disease due to gross metastases in 140 cases and/or positive peritoneal cytology in 96 cases) was 18%. For strictly upfront resectable cases, the yield was lower (~11% in subgroup analyses). Staging laparoscopy remains valuable in the modern era to avoid non-therapeutic operations. Primary source: "Yield of Staging Laparoscopy for Pancreatic Cancer in the Modern Era: Analysis of More than 1,000 Consecutive Patients" (Annals of Surgery, 2023). Dutch Pancreatic Cancer Audit (DPCA) data (2013–2017, with extensions and validations into recent years): Among patients with potentially resectable or borderline resectable PDAC, occult metastases were detected in approximately 9–10% during exploration or staging laparoscopy (liver ~61%, peritoneum ~31%). A 2024 validation/modeling study based on this audit confirmed similar rates and developed predictors for occult disease. Broader recent reviews and meta-context (2024–2025): Rates of occult metastases or unresectability discovered intraoperatively in apparently resectable cases range from 7–12% (aborted resections) to 10–20% overall. Modern multidetector CT/MRI has lowered rates from older eras (>30% in some historical data), but small peritoneal/liver deposits remain challenging.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have adequately addressed my comments and improved the manuscript accordingly. I therefore recommend acceptance in its current form.
