Peripheral Blood Lymphocyte Percentage May Predict Chemotolerance and Survival in Patients with Advanced Pancreatic Cancer. Association between Adaptive Immunity and Nutritional State

Round 1

Reviewer 1 Report

The manuscript is an original research about the role of the percentage of lymphocytes in WBC as a potential predictive factor for chemotolerance and survival in patients with advanced pancreatic cancer and the possible association between percentage of lymphocytes and body weight.

It is well structured, but a careful reading is recommended.

The title could be improved and something is missing between line 185 and line 186:

 (c) Relationships between BW and lymphocytes, neutrophils, N/L ratio, chemotolerance and clinical

BW, but not BMI, correlated with L% (Figure 3a, Pearson r = + 0.34, p = 0.033) but not with LAB (Figure 3b,

The sample is too small to draw generalizable conclusions, but the topic is interesting and the findings pave the way for further studies in this field.

The topic is in line with the journal.

I would recommend the publication of the manuscript after revision.

Author Response

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Reviewer 2 Report

In the submitted manuscript by Boschi et al, the authors demonstrated that blood lymphocytes expressed as a percentage of Total White Blood Cells (L% TWBC) could be used to determine the chemotolerance, patient survival time. Their finding revealed that patients with L%<29.7% possessed 13-fold higher risk for poor clinical outcomes despite given chemotherapy. However, we marked that the majority patients in the cohort were between 66≤x≤79 (n=30). It is a well-established fact that Lymphopenia is common in elderly patients. Our concern is whether the low L% and its correlation with chemotolerance/survival is more age-dependent than disease status itself. We would like the authors to defend this concern. In addition, we have few other questions that need to be addressed as well.   

1. Please check the following sentence and clarify what was meant by ‘linfo-mediated’:

‘A possible impact of nutritional status on 44 chemotherapy and survival seems to be linfo-mediated‘

2. The abstract starts with the result. We suggest to briefly mention the rationale/background of the study before summarizing the findings.
3. Please cite the appropriate reference for the following statement:

‘Surgery is the most important therapeutic option for localized PC. 56 Despite the progress made in recent decades, the recurrence rate five years after resection is 80%, and the 57 survival rate is 22%.‘

4. The ‘Objective‘ section in Matierial and Method fits better under ‚introduction‘ section.
5. We would suggest the author to classify the values (of Neutrophils (absolute and percentage), Lymphocytes (absolute and percentage), Neutrophils Lymphocytes ratio and number of Chemotherapy cycles) shown in Table 3 according to patients‘ response to first line chemotherapy (Complete / Partial response, Stable disease or Progressive disease. Presenting the data in such a way could correlate the inaflammtion status with the disease status. It would also expose the heterogeneity of Lymphocyte Percentage at different phases of disease progression.
6. Please provide a title to each Table or Figure.
7. For Figure 3-6, please also correlate the parameters to the Body Mass Index, BMI to understand how the deviating BMI afftects inflammation status.

Author Response

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Reviewer 3 Report

This manuscript was well written with an interesting concept and fascinating outcomes. The only question I would have is why the lower limit of normal was chosen for investigating lymphopenia (<1500) instead of the recognized definition of lymphopenia which is <1000?

Author Response

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Round 2

Reviewer 2 Report

Reviewer #2

In the submitted manuscript by Boschi et al, the authors demonstrated that blood lymphocytes expressed as a percentage of Total White Blood Cells (L% TWBC) could be used to determine the chemotolerance, patient survival time. Their finding revealed that patients with L%<29.7% possessed 13-fold higher risk for poor clinical outcomes despite given chemotherapy. However, we marked that the majority patients in the cohort were between 66≤x≤79 (n=30). It is a well-established fact that Lymphopenia is common in elderly patients. Our concern is whether the low L% and its correlation with chemotolerance/survival is more age-dependent than disease status itself. We would like the authors to defend this concern. In addition, we have few other questions that need to be addressed as well.   

We added “Strengths and limitations” to the text (line 377)

The knowledge of lymphocyte subsets may better identify subjects whose lymphopenia is prevalently age- or disease related. Indeed, from immunologic point of view, aging is characterized by quantitative and qualitative alterations of both adaptive and innate immune cells (43) (Pinti Marcello. et al. Eur J Immunol 2016; 46; 2286 - 2301). The current study cannot distinguish whether lymphopenia was related prevalently to age rather than PC. We postulate that in the study patients Lymphopenia could prevalently due to the disease as we did not observe different severity of lymphopenia between patients with < 65years and those with > 65 years. Anyway, the relationship between cancer and immunity should always take into consideration the age- related alterations of immunity.

R: We appreciate how the authors logically defended our concern.

1. Please check the following sentence and clarify what was meant by ‘linfo-mediated’:

‘A possible impact of nutritional status on chemotherapy and survival seems to be linfo-mediated‘

We corrected the word “linfo-mediated” with “lymphocyte-mediated…” (line 49)

R: Thanks for the correction.

1. The abstract starts with the result. We suggest to briefly mention the rationale/background of the study before summarizing the findings.

We corrected the initial part of the abstract as follows (line 35):

Pancreatic Carcinoma (PC) cells have the ability to induce patient immuno-suppression and to escape immunosurveillance. Low circulating lymphocytes are associated with advanced stages of PC and reduced survival.

R: We accept the correction.

• Please cite the appropriate reference for the following statement:

‘Surgery is the most important therapeutic option for localized PC. Despite the progress made in recent decades, the recurrence rate five years after resection is 80%, and the survival rate is 22%.‘

Two appropriate references have been added to the Introduction section (line 64) (references 8-9) and in References section.

R: We noted that in the Reference section, the reference orders are wrong and ref. number 11 is missing. We found it after ref. no. 24. Please kindly check them. Also make sure to use Endnote or similar reference tracking tool for correct placing of reference in the manuscript.

4    The ‘Objective‘ section in Matierial and Method fits better under ‚introduction‘ section

The “d       The Objectives” section has been moved from Materials to Introduction (line 101)

R: Thanks for the rearrangement.

• We would suggest the author to classify the values (of Neutrophils (absolute and percentage), Lymphocytes (absolute and percentage), Neutrophils Lymphocytes ratio and number of Chemotherapy cycles) shown in Table 3 according to patients‘ response to first line chemotherapy (Complete / Partial response, Stable disease or Progressive disease. Presenting the data in such a way could correlate the inaflammtion status with the disease status. It would also expose the heterogeneity of Lymphocyte Percentage at different phases of disease progression.

We added panel d) to table 3 and added sentences to the Results section (line 198)

Table 3d. reports absolute neutrophils, neutrophils %, absolute lymphocytes, lymphocytes %, neutrophils / lymphocytes ratio, and chemotherapy cycles in patients after stratification for their response to first line chemotherapy (PR partial response, SD stable disease, PD progressive disease). The table indicates the association between clinical outcomes and circulating lymphocytes.

R: In the result section, please also mention the statistical significance/difference among PR, SD and PD groups for lymphocytes total and lymphocyte percentage.

• Please provide a title to each Table or Figure.

We have added the titles

R: Thanks for including the title. However, a title should represent the main finding of the figure. Please rephrase them accordingly. For example, instead of writing, ‘Figure 4. Neutrophils/lymphocytes ratio as a function of weight‘, we suggest to state, ‘Figure 4. Body weight being negatively associated with the Neutrophils/lymphocytes ratio‘.

• For Figure 3-6, please also correlate the parameters to the Body Mass Index, BMI to understand how the deviating BMI afftects inflammation status.

As suggested, we correlated BMI with the parameters considered for BW and added the following to the Results section, paragraph c) (line 219):

BMI was negatively correlated with N/L ratio (figure 7) (r= - 0.34, p= 0.035) but not with absolute neutrophils (r= -0.12, p=0.45), L% (r=+0.26, p=0.11) and L_AB (r= +0.27, p= 0.09), number of chemotherapy cycles (r= - 0.09, p=0.56).

Therefore, in the Discussion paragraph LYMPHOPENIA, CHEMOTOLERANCE, CLINICAL OUTCOMES AND BW we added the following (line 382):

The fact that both BW and BMI negatively correlate with N/L ratio but not with absolute neutrophils and BW alone with L% may suggest that a good nutrition may positively influence immune response by improving adaptive immune cells and favouring the shift of innate to adaptive immunity.

R: We thank the authors for the additional analysis. However, please also provide the negative data as graphs in a supplementary section.

Author Response

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