Anemia, Iron Deficiency, and Iron Regulators in Pancreatic Ductal Adenocarcinoma Patients: A Comprehensive Analysis

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Very insightful study depicting the real picture of anemia in patients with pancreatic ductal adenocarcinoma (PDAC). The resubmitted manuscript substantially improved and it's now ready for publication.

Very insightful study depicting the real picture of anemia in patients with pancreatic ductal adenocarcinoma (PDAC). The resubmitted manuscript substantially improved and it's now ready for publication.

Author Response

Dear Reviewer,

We greatly appreciate the time and effort you invested in reviewing our article. Your constructive feedback has not only strengthened the article itself but has also contributed to our growth as researchers and authors. Your thorough examination has prompted us to explore new angles and address potential concerns, resulting in a more robust and well-rounded work. Please find in the attachment the new version of the manuscript after comments from all Reviewers. 

Reviewer 2 Report (Previous Reviewer 2)

The authors have correctly addressed my comments and the new version of the paper is greatly improved.

The quality of English language is good.

Author Response

Dear Reviewer,

We greatly appreciate the time and effort you invested in reviewing our article. Your constructive feedback has not only strengthened the article itself but has also contributed to our growth as authors. Your thorough examination has prompted us to address potential concerns, resulting in a more robust and well-rounded work. Please find in the attachment the new version of the manuscript after comments from all Reviewers. 

Reviewer 3 Report (Previous Reviewer 3)

The authors addressed almost all the reviewer's requests. The manuscript has been improved, above all the section "Abstract" and "Discussion" and this reviewer appreciated that. However, this reviewer has still some suggestions and there are some text editing.

Major:

- For figure 5 and 6, please use a bigger font for the written part, since it is difficult to read properly;

- In the Results section 3.2, it would be better first to show the IHC representative image and then the graphs with %. Thus it would be more appreciable to invert Fig 3 with Fig 4.

Minor:

- Line 36: put bracket after "ZIP14"; remove the two dots(:) after "storage"

- Line 84: remove the two dots(:) after "storage"

- Line 173: remove the word "analysis" at the beginning of the line

- Line 309: add the dot (.) after the bracket

- Line 341: remove the two dots(:) after "proteins"

- Line 494: add the dots (:) after FTH

- Line 495: add the dots (:) after FTL

Author Response

Dear Reviewer, 

We greatly appreciate the time and effort you invested in reviewing our article. Your meticulous attention to detail has undoubtedly enriched the content and improved the coherence of our article. Your critical evaluation has inspired us to delve deeper into our research and present our findings in the most comprehensive manner possible.

Here are the answers to your last comments: 

- For figure 5 and 6, please use a bigger font for the written part, since it is difficult to read properly;

We increased the size of the figures, it should be more clear and easier to read now.

- In the Results section 3.2, it would be better first to show the IHC representative image and then the graphs with %. Thus it would be more appreciable to invert Fig 3 with Fig 4.

Addressed

- Line 36: put bracket after "ZIP14"; remove the two dots(:) after "storage"

- Line 84: remove the two dots(:) after "storage"

- Line 173: remove the word "analysis" at the beginning of the line

- Line 341: remove the two dots(:) after "proteins"

- Line 494: add the dots (:) after FTH

- Line 495: add the dots (:) after FTL

All the above comments were addressed.

- Line 309: add the dot (.) after the bracket
we couldn’t identify the error in the indicated place, we hope the Current Oncology Editors will provide some assistance with this comment.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In this cohort study, the authors investigated factors leading to development of anemia in 103 patients with pancreatic ductal adenocarcinoma. They performed both laboratory and immunoistochemical evaluation, by dosing markers of iron metabolism direcly within the bioptic culture. The idea is really interesting, but the small sample size, the lack of correction for potential confounders and some unusual findings (probably due to lack of sufficient statistical power) are major reasons fo rejecting this manuscript for publication. Here below you can find some points to be addressed (I suggest to avoid using stratification in the presence of small numbered cohorts, or to report the main clinical and laboratory (other than anemia) differences (in term of comorbidities) between the 2 groups. Furthermore, drugs other than chemotherapy may affect iron status and red blood cell production, but are not reported in descriptive analyses.

a) Major issues:

a1) Given the small sample size of the study, stratification  by type of chemotherapy (aadjuvant vs palliative) can lead to low statistical power which can bias evidence derived from study findings. Furthermore, even when statistical analysis led to significant differences between the 2 groups (e.g. folate concentration), lack of correction for potential confounders (which can't be performed given the low number of patients) does not allow to draw any conclusion from this study findings (e.g. we can't exclude that the found differences are simply causal or due to unmeasured confounders).

a2) Data on CKD are not reliable as only 16 patients had CKD (in this regard, the trend of decreased hemoglobin in this group, despite not significant, may be due to insufficient statistical power).

a3) Drugs and comorbidities (other than cancer and CKD) may significantly affect hemoglobin levels as well as iron metabolism but were not investigated.

b) Figures: are barely visible. Improve their resolution and figure space ( by decreasing bar height and increasing font size). 

Minor revisions for spells and typos are needed.

Author Response

Dear Reviewer, 

We would like to thank you for the work you have put into reviewing our article, it is evident that you put considerable effort into comprehensively evaluating our work, and we are genuinely thankful for the time and energy you invested in this endeavor. Your contributions have undoubtedly elevated the overall quality of our publication, and we are deeply grateful for your commitment to advancing scientific knowledge. Tha manuscript was heavily modified after your and other Reviewers' comments, and we hope you would consider this manuscript suitable for publication.

1a) Major issues:

a1) Given the small sample size of the study, stratification  by type of chemotherapy (aadjuvant vs palliative) can lead to low statistical power which can bias evidence derived from study findings. Furthermore, even when statistical analysis led to significant differences between the 2 groups (e.g. folate concentration), lack of correction for potential confounders (which can't be performed given the low number of patients) does not allow to draw any conclusion from this study findings (e.g. we can't exclude that the found differences are simply causal or due to unmeasured confounders).

We appreciate your comments, and indeed, we are aware of a critical limitation of our study, that is a low number of patients; nevertheless, in the study design, we wanted to show differences in iron and other micronutrient deficiencies between the patient’s with less advanced tumors (adjuvant group) and more advanced tumors (palliative group) that would give clinicians an important insight on how to tackle those different populations. Therefore, we decided to maintain the stratification. Nevertheless, we are aware that studies on a bigger number of patients are necessary. In any case, we improved the manuscript according to your and other reviewer’s comments, which led to the improvement of our work, which you might find interesting.

a2) Data on CKD are not reliable as only 16 patients had CKD (in this regard, the trend of decreased hemoglobin in this group, despite not significant, may be due to insufficient statistical power).

Addressed: we underlined this problem in the study’s limitation section.

a3) Drugs and comorbidities (other than cancer and CKD) may significantly affect hemoglobin levels as well as iron metabolism but were not investigated.

Addressed: we underlined this problem in the study’s limitation section. 

1. b) Figures: are barely visible. Improve their resolution and figure space ( by decreasing bar height and increasing font size).

We addressed this issue.

Thank you again for your work and insight, 

Authors

Reviewer 2 Report

The paper reports the results of a retrospective study of PDAC patients in the effort to evaluate possible correlations between selected iron markers and anemia/iron deficiency in relation to palliative vs adjuvant chemotherapy groups and CKD. Although the data suggest a possible correlation only for ZIP14, it may still be worth to investigate iron status in PDAC patients. The authors correctly outline and discuss the limitations of their study. The paper is written clearly but the two following points should be addressed by the authors:

1) The figures are illegible, please substantially increase font size of all figures.

2) Describe the GEPIA analysis in methods section

Minor point:

1) lines 256, 258 and 259, MIT 'content' may be more adequate than MIT 'expression'

Quality of English language is good, only minor checks are needed

Author Response

Dear Reviewer, we would like to express our sincere gratitude for your commitment to reviewing our scientific publication. Your work put an indelible impact on the quality and credibility of our research.

We addressed all your comments, but the manuscript itself was heavily modified after the remarks of other Reviewers. We hope you will find it easier to read with more concise messages for the clinicians 

1) The figures are illegible, please substantially increase font size of all figures.

Addressed

2) Describe the GEPIA analysis in methods section

Addressed, together with the CEPIA database results moved to the results section.

Minor point:

1) lines 256, 258 and 259, MIT 'content' may be more adequate than MIT 'expression'

Addressed

Reviewer 3 Report

Comments:

In the present manuscript, Osmola M et al., investigated the prevalence of iron deficiency and iron deficiency anemia in patients with pancreatic duodenal adenocarcinoma (PDAC) with the effort to shed light on the mechanisms leading to the iron deficiency condition.

The work sounds interesting, even if, as mentioned by the authors in the Discussion section, there are some critical aspects and limits, and it did not completely elucidate and demonstrate the mechanisms leading to the iron deficiency condition in PDAC.

The article is properly written; however, the reviewer has some suggestions.

Major suggestions

-       Abstract could be improved, explaining better the aims results and brief conclusion.

-       Paragraph 3.1.4: the reviewer was wondering if you have assessed the analysis of kidneys status and to define the severity of kidney damage, verifying not only the serum creatinine but also other markers of inflammations.

-       For figure 1,2,4 please use a bigger font for the written part because it is barely possible to properly read the words on the graphs. In general, the authors should introduce the figures or tables just below the description.

-       Table 3: It could be easier and more intuitive for the reader if the authors would remove from the table the IHC data on HEP, FPN, ZIP14, MIT and introduce instead a graph for each protein above the IHC representative images (Fig. 3).

-       Figure 3: It would be easier for the reader to add on the left of the images the protein analyzed by IHC (reported once on the left) and also on the upper part it would be appreciable to write “NEGATIVE; WEAK; STRONG; C+”. Images could be also enlarged. Report the bar for each image (some are missing) and add in the legend the description of the bar.

-       As mentioned by the authors, due to the limit of the sample available it was not possible to perform too many analyses, however this reviewer was wondering if you have some IHC data or if you have the possibility to analyze the ferritin and Transferrin Receptor (TfR1) (by IHC) and iron (by Perl’s Prussian Blue staining) in the PDAC tissues. It would add another piece to the puzzle to further complete the characterization.

-       Lines 244-259: it would be appreciable to introduce the correlation graphs (for example as scatter plot) between HEP-FPN and ZIP14-MIT with the statistic mentioned in the text. This would help the reader to better follow the description.

-       Figure 4: please add on the top of Kaplan-Meier curves the respective analysis (HEP, FPN; ZIP14; MIT).

-       Please add in “Material and Methods” section the description of the analysis showed using GEPIA database.

-       Figure 5: the results related to the analysis from GEPIA database should be moved in the “Results” section, in a dedicated paragraph, removing the detailed part from the discussion. It could be of interest, if IHC could not be performed on PDAC tissues, to analyze TfR1 and ferritins using GEPIA database.

-       The correlation between ZIP14 and Mitochondrial mass is highly of interest and the IHC staining for ZIP14 on PDAC tissue was performed for the first time in this study. Since this part is one of the strengths of the present work, the reviewer would suggest the author to improve this point in the discussion section, further describing and pointing out the importance of these data, the biological significance, with the effort to speculate the possible diagnostic, prognostic, and therapeutic translation.

Minor suggestions

-       Line 45: “I” for iron is missing.

-       Line 119: define the antibody dilution as for the other antibodies (es. 1:2000).

-       Please add the product codes for the antibodies.

-       Vitamin B12: please check and write it always in the same way in the whole text.

-       Define the extent name for PAAD (Fig. 5).

-       Please, when possible, try to fit figures or tables plus their respective legends in the same page.

-       Line 257: please move the dot after the bracket.

-       Line 330: please move the dot after the brackets.

English was clear and properly written. It was easy to read and follow the written part in the text. There are only few typing errors.

Author Response

 Dear Reviewer, we are writing to express our sincere gratitude for the meticulous review you conducted on our scientific publication. we would like to thank you for your insightful comments, thoughtful suggestions, constructive feedback, and the work, you put to review our work. We addressed almost each of your remarks, and we hope that now you will find our work more clear, and elaborated, and find the manuscript suitable for publication, as it adds an interesting point to the discussion of iron deficiency and pancreatic cancer.

Major issues:

Abstract could be improved, explaining better the aims results and brief conclusion.

addressed

-       Paragraph 3.1.4: the reviewer was wondering if you have assessed the analysis of kidneys status and to define the severity of kidney damage, verifying not only the serum creatinine but also other markers of inflammations.

The severity of kidney damage (creatinine concentration and eGFR) was assessed and is presented in Table 1. Out of 103 patients, only 16 had CKD, which makes the data difficult to analyze and draw conclusions from. As you mentioned, inflammation can affect iron status, and markers of inflammation were not assessed in our study (e.g., C-reactive protein, CRP); nevertheless, elevated ferritin level in the whole cohort already reflects inflammation in these patients. Which is described in the discussion (study’s limitation part).

- For figure 1,2,4 please use a bigger font for the written part because it is barely possible to properly read the words on the graphs. In general, the authors should introduce the figures or tables just below the description.

Addressed

- Table 3: It could be easier and more intuitive for the reader if the authors would remove from the table the IHC data on HEP, FPN, ZIP14, MIT and introduce instead a graph for each protein above the IHC representative images (Fig. 3).

We removed the table, and added a separate figure (Figure 3), where we present the results on the graph, as you suggested. In our opinion, adding them to the IHC images makes it is too blurred, so we decided to put it separately.

- Figure 4 (former Figure 3): It would be easier for the reader to add on the left of the images the protein analyzed by IHC (reported once on the left) and also on the upper part it would be appreciable to write “NEGATIVE; WEAK; STRONG; C+”. Images could be also enlarged. Report the bar for each image (some are missing) and add in the legend the description of the bar.

we added the description, enlarged the images, as you mentioned, so the figure is easier to read. The magnification (formerly shown on the bars), is described in the legend. 

- As mentioned by the authors, due to the limit of the sample available it was not possible to perform too many analyses, however this reviewer was wondering if you have some IHC data or if you have the possibility to analyze the ferritin and Transferrin Receptor (TfR1) (by IHC) and iron (by Perl’s Prussian Blue staining) in the PDAC tissues. It would add another piece to the puzzle to further complete the characterization.

We performed Perl’s Prussian Blue stating in the PDAC tissues to look for the iron deposits in the tissue samples which revealed no iron deposits within pancreatic cancer cells. The iron depositis were only present in the surrounding macrophages (added on the figure)

We added information from the GEPIA database for Transferrin receptor (TfR1) and ferritin chains, unfortunately, we can not perform IHC staining in the tissue. IHC staining will have to be performed in further studies in this field.

- Lines 244-259: it would be appreciable to introduce the correlation graphs (for example as scatter plot) between HEP-FPN and ZIP14-MIT with the statistic mentioned in the text. This would help the reader to better follow the description.

Because the results are semi-quantitative (1-2-3), the results cannot be presented as scatter plots, so we were unable to address this demand.

-  Figure 4: please add on the top of Kaplan-Meier curves the respective analysis (HEP, FPN; ZIP14; MIT).

Addressed

-       Please add in “Material and Methods” section the description of the analysis showed using GEPIA database.

Addressed

- Figure 5: the results related to the analysis from GEPIA database should be moved in the “Results” section, in a dedicated paragraph, removing the detailed part from the discussion. It could be of interest, if IHC could not be performed on PDAC tissues, to analyze TfR1 and ferritins using GEPIA database. 

Addressed

- The correlation between ZIP14 and Mitochondrial mass is highly of interest and the IHC staining for ZIP14 on PDAC tissue was performed for the first time in this study. Since this part is one of the strengths of the present work, the reviewer would suggest the author to improve this point in the discussion section, further describing and pointing out the importance of these data, the biological significance, with the effort to speculate the possible diagnostic, prognostic, and therapeutic translation.

Addressed, we improved the discussion section

Minor suggestions: 

- lines 256, 258 and 259, MIT 'content' may be more adequate than MIT 'expression' – addressed.

-       Line 45: “I” for iron is missing.

Addressed

-       Line 119: define the antibody dilution as for the other antibodies (es. 1:2000).

Dilutions are provided in the text for every antibody.

-       Please add the product codes for the antibodies.

Addressed

-       Vitamin B12: please check and write it always in the same way in the whole text.

Addressed

-       Define the extent name for PAAD (Fig. 6, former fig. 5).

Addressed

-       Please, when possible, try to fit figures or tables plus their respective legends in the same page.

Thank you for this remark, the manuscript is in the word file tamplate provided by the publisher, and in the final version, the tables will be well visible and as single image.

-       Line 257, Line 330: please move the dot after the bracket.

Addressed

thank you for you work. 

Authors