Characteristics of Peripheral Blood Lymphocyte Populations in Patients with Locally Advanced Unresectable Non-Small Cell Lung Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title

 

-The study examines different subsets of lymphocytes in blood of  patients with unrespectable non-small cell lung cancer. Therefore, it is suggested that the title be used with the term lymphocytes instead of immune cells.

 

Abstract:

-It is not clear what markers or methods were used to examine the cells studied.

-It is well known that advanced cancer patients have deficits in lymphocyte function and number. Therefore, this conclusion is not noble and restricted to this study.

 

 

Introduction:

- the introduction is  not thorough (citing 12 references) and should include a hypothesis statement for clarity.

-The role and importance of the lymphocytes studied (specially circulating lymphocytes)  in lung cancer should be added.

 

 

Material and methods:

-The authors should explain why they only used patients with advanced stages (stages IIIB-IIIC).

-Histologic tumor sub-type in lung cancer patients is unclear.
-Authors should explain what markers they used to differentiate each lymphocyte subset.

NK cells are identified by positive expression of CD16/CD56 and negative expression of CD3 markers on their surface. The authors should explain why they did not use the CD16 marker to differentiate NK cells.

 

Results:

-In Figures 1 and 2, it should be specified whether the mean ± SD or mean ± SEM is used.

 

-In figure3, the authors should explain gating strategy. Please use a figure text similar to Erfani et al PMID: 22608141 DOI: 10.1016/j.lungcan.2012.04.011

Discussion:

 

--The authors should explicitly highlight how their work provides novelty or advances beyond existing literature.

-The discussion is adequate but overly reliant on animal studies or other cancers.

-Focus solely on peripheral blood, without examination of the tumor microenvironment (TME). While the authors reference the TME (as stated in introduction), no relevant data are provided.
-Acknowledge the limitations of your work (e.g.  Small sample size) and recommendations for Improvement (e.g. functional assays) in Discussion.

Author Response

Dear Sir, we thank You for carefully studying our research and for your important comments and suggestions, which allowed us to significantly improve the text.

 

Comments and Suggestions for Authors

Title

• The study examines different subsets of lymphocytes in blood of patients with unrespectable non-small cell lung cancer. Therefore, it is suggested that the title be used with the term lymphocytes instead of immune cells

Answer: We completely agree with you and propose a new option: “Characteristics of peripheral blood lymphocyte populations in patients with unresectable non-small cell lung cancer”.

 

Abstract:

• It is not clear what markers or methods were used to examine the cells studied.

Answer: We have made additions to the Abstract and in the METHODS section we indicate: “We analyzed the frequency of circulating  lymphocytes (CD45+ cells) population including Т-cell (CD3, CD4, CD8), В-cells (Cd19, CD20) , NK-cells (CD3-CD16+CD56+ cell), and NKT-cells (Cd3+CD56+CD16+ cells)  in 80 patients with lung cancer and 40 healthy controls using 8-color flow cytometry.

• It is well known that advanced cancer patients have deficits in lymphocyte function and number. Therefore, this conclusion is not noble and restricted to this study.

Answer: We absolutely agree that it is well known that patients with advanced cancer have decreased lymphocyte function and count. There may be several reasons for this: the influence of the tumor, the influence of previous therapy, and age. Of course, in this small study we do not claim outstanding conclusions, but only state and confirm the fact of changes among immunocompetent blood cells in primary patients diagnosed with non-small cell lung cancer III b, IIIc stage. We have not found any similar examples in the open literature and we also ask the distinguished reviewer to provide specific examples that would confirm the presence of immune system dysfunction at the level of populations of immunocompetent cells (actually lymphocytes) in patients with non-small cell lung cancer at the diagnostic stage, so that we could compare the data from other studies with our own.

Introduction:

• The introduction is not thorough (citing 12 references) and should include a hypothesis statement for clarity.

Answer: We absolutely agree with the distinguished reviewer that any study should contain a formulation of a hypothesis, which determines the purpose of the study and formulated the hypothesis: « This study characterizes blood lymphocyte populations in treatment-naïve patients with unresectable NSCLC with stage IIIB, IIIC NSCLC in order to identify possible signs of secondary immunodeficiency. A deeper understanding of this baseline immune status is crucial for optimizing immunology-based clinical strategies, such as immune checkpoint inhibitors (ICI) and adoptive cell therapy »

• The role and importance of the lymphocytes studied (specially circulating lymphocytes) in lung cancer should be added.

Answer: We have added this information to the introduction.

Material and methods:

• The authors should explain why they only used patients with advanced stages (stages IIIB-IIIC).

Answer: This is a pilot study to explore the possibilities of improving the immediate treatment outcomes of patients diagnosed with unresectable NSCLC, which implies the inclusion of patients with only stages IIIB-IIIC of the disease (actually unresectable NSCLC)

 

• Histologic tumor sub-type in lung cancer patients is unclear.

Answer: As described in the materials and methods, the study included not only patients with squamous cell carcinoma, but also with adenocarcinoma: «Histologically, squamous cell carcinoma was the most common type of cancer in the study group, affecting 65% of patients (52 out of 80), followed by adenocarcinoma in 33.8% (27 out of 80) of patients, and a rare type of pleomorphic tumor was found in one patient».

• Authors should explain what markers they used to differentiate each lymphocyte subset.

Answer: The main antigens are presented in materials and methods. We have added a more detailed description of the immunofluorescence technique for the identification of lymphocyte populations («The composition of lymphocyte populations in the peripheral blood from the healthy individuals, and the patients with lung cancer before any treatment, was determined using flow cytometry. Lymphocyte populations were evaluated in a direct immunofluorescence reaction using monoclonal antibodies to the main antigens of human lymphocytes conjugated with fluorochromes (CD3-FITC; CD8-PE; CD4-PerCP; CD19-PE, CD20-FITC CD56-PE-Cy7;CD16-FITC CD45-APC-H7). The results were evaluated by flow cytometry using a 6-color BD FACS Canto II system (BD Biosciences, USA). Briefly, 1.5 mL of peripheral blood collected in EDTA was diluted in 0.4 mL flow cytometry buffer (PBS containing 10% v/v heat inactivated fetal calf serum, FCS). For each analysis, of 100 µL cell suspension were incubated with the fluorescent-labeled monoclonal antibodies for 20 min in the dark. After washing with flow cytometry buffer, erythrocytes were lysed by incubating with 2 mL of BD FACS™ Lysing solution (1:9 dilution in double distilled (dd) H2O, 349202-BD Biosciences) for 10 min in the dark at room temperature. After another washing step, the probes were analyzed in a FACSCanto II™ flow cytometer. Flow cytometry data were processed with Kaluza software.»).

• NK cells are identified by positive expression of CD16/CD56 and negative expression of CD3 markers on their surface. The authors should explain why they did not use the CD16 marker to differentiate NK cells.

Answer: It is absolutely true that two main NK cell markers usually used to identify NK cell populations, and we used both. We thank you for this comment and will correct the inaccuracy in the text.

 

Results: 

• In Figures 1 and 2, it should be specified whether the mean ± SD or mean ± SEM is used.

Answer: Figures 1 and 2 show the average mean with the standard error of the mean

• In figure3, the authors should explain gating strategy. Please use a figure text similar to Erfani et al PMID: 22608141 DOI: 10.1016/j.lungcan.2012.04.011

Answer: changed the text according to your recommendations

Discussion:

 

-Acknowledge the limitations of your work (e.g. Small sample size) and recommendations for Improvement (e.g. functional assays) in Discussion.

• The authors should explicitly highlight how their work provides novelty or advances beyond existing literature.

Answer: In our study, changes in blood lymphocyte populations are shown on our own extensive clinical material, including 80 primary, untreated patients diagnosed with unresectable non-small cell lung cancer, which, in our opinion, may be significant in terms of predicting response to therapy. The study is not organized by any single type of limophocytes, but is complex and includes all the main types of circulating immune cells – T cells and their subpopulations, NK cells and NKT cells, as well as B cells and their special population of B1a lymphocytes, which is practically unexplored and constitutes the scientific novelty of the study.

• The discussion is adequate but overly reliant on animal studies or other cancers.

Answer: There are practically no clinical studies devoted to the study of the subpopulation composition of lymphocytes in a special cohort of patients with unresectable lung cancer (and these are patients who receive chemoradiotherapy) in the literature, isolated studies in advanced lung cancer have been conducted on smaller samples. We were unable to find data on the B1a population in lung cancer in the public domain.

• Focus solely on peripheral blood, without examination of the tumor microenvironment (TME). While the authors reference the TME (as stated in introduction), no relevant data are provided.

Answer: According to your recommendation, we have excluded references to the tumor microenvironment from the text.

 

• Acknowledge the limitations of your work (e.g. Small sample size) and recommendations for Improvement (e.g. functional assays) in Discussion.

Answer: We also added the limitations of our work, which relate to functional tests, studies of T-regulatory cells and their subpopulations, and the assessment of the humoral  immunity (cytokines and the compliment system) in this cohort of patients.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Grivtsova et al. have performed an important study characterizing the immune cell composition in the blood of patients with unresectable non-small cell lung cancer. However, the manuscript presents very valuable findings a couple of major and minor clarifications should be addressed before this manuscript can be published

 

 

Major comments

• Please clarify the gating strategy and present it as a table/scheme/gating diagram with gate definitions. It is important to clarify how each cell population was identified. CD4 and CD8 cells have to be CD3+; however, this is somehow not clear across the manuscript
• The age differences between the patients and the  control group are 20 years. Authors should address in the discussion the possible higher presence of CD3+CD8+ double-positive T cells due to the age gap and how it can affect the results and the conclusions of this study.
• Please clarify how the secondary immunodeficiency was diagnosed and what diagnostic criteria were used
• Please discuss the observed increase in the peripheral blood NKT cells in NSCLC,  provide the references of this pattern and justify why it is different from previously observed published literature.
• Please clarify and discuss in more detail the double-positive T cells and their role in the prognosis and outcomes. The route of double-positive T cells is complex and should be discussed across different cancer types in the manuscript
• Please provide a section describing the clinical outcomes of the patients who were included of the study. What were the treatment protocols (describe it in the method section) and what were the clinical outcomes? Prognosis/overall survival or progression-free survival. What were the treatment responses? Provide the history of the patients as a table and complement it if possible, with BMI, tumor size,  smoking history, comorbidities, time from diagnosis to blood sampling, and medication. This information is crucial for this study in order to be able to understand the possible effect on the immune cell composition for the patients with unresectable non-small cell lung cancer

 

Minor comments

• Please standardize the nomenclature across the manuscript: T lymphocytes vs T cells vs T-Cells

• ⁃ Please provide the results of CD4toCD8 ration as a figure or table

Author Response

Dear Sir, we thank You for carefully studying our research and for your important comments and suggestions, which allowed us to significantly improve the text.

 

Comments and Suggestions for Authors

Major comments:

• - Please clarify the gating strategy and present it as a table/scheme/gating diagram with gate definitions. It is important to clarify how each cell population was identified. CD4 and CD8 cells have to be CD3+; however, this is somehow not clear across the manuscript

Answer: We have added a gating strategy to the materials and methods and in the caption to Figure 4.

 

• - The age differences between the patients and the control group are 20 years. Authors should address in the discussion the possible higher presence of CD3+CD8+ double-positive T cells due to the age gap and how it can affect the results and the conclusions of this study.

Answer: In the DISCUSSION, we pointed out the problem of increasing double positive T cells in older age groups in healthy individuals. “Despite the marked diversity, double-positive CD4+CD8+ T cells usually account for less than 1% of the total number of T cells found in the blood of healthy people. It has been shown that the number of double-positive T cells in the blood of older people (for people over 65 years of age) is higher than in younger people ….In our study, an increase in the number of double positive T cells in 80% of cases was detected in people under 60 years of age, which suggests that their increase is not related to age, but rather is a consequence of the development of tumor-associated inflammation”

 

• - Please clarify how the secondary immunodeficiency was diagnosed and what diagnostic criteria were used

Answer: We also understand how complex and ambiguous the role of the population of double positive T cells is in pathology and in the norm. These cells, like NKT cells, require very thorough studies of their role in carcinogenesis.  This study did not aim to study the fundamental aspects of carcinogenesis, its purpose was purely practical – to identify the characteristics of populations of circulating peripheral blood lymphocytes in a very clearly selected cohort with locally advanced (non-metastatic!) unresectable stage IIIB-IIIC NSCLC, that is, patients who are indicated for chemoradiotherapy. Such disorders are one of the signs of secondary immunodeficiency and their correction in the future may affect the effectiveness of specific treatment of this cohort of patients. It is possible that it is somewhat incorrect to use the term "secondary immunodeficiency", since most patients had only weakness from clinical signs, and no more than 30% of patients complained of frequent viral infections. Nevertheless, we believe that even isolated disorders in the immune system without clinical manifestations may indicate the presence of secondary immunodeficiency in cancer patients.

 

• Please discuss the observed increase in the peripheral blood NKT cells in NSCLC, provide the references of this pattern and justify why it is different from previously observed published literature.

Answer: Thank you, we have added relevant information.: «NKT cells show the characteristics of innate as well as adaptive immune cells. NKT cell activation leads to rapid production of inflammatory cytokines and modulates the function of several effectors and regulatory immune cells both in mice and humans [Nair S, Dhodapkar MV. Natural killer T cells in Cancer immunotherapy. Front Immunol. 2017;8:1178. doi: 10.3389/fimmu.2017.01178.]. According to the nature of the activating ligand, NKT cells are classified into two groups; type-I and type-II NKT cells. Individual studies have shown that the number of NKT cells in the peripheral blood of patients with multiple myeloma and breast cancer is reduced compared to healthy donors. There is also a reduced number of circulating NKT cells in patients with squamous cell carcinoma of the head and neck, which is associated with poorer patient survival [Crough T, Purdie DM, Okai M, Maksoud A, Nieda M, Nicol AJ. Modulation of human Valpha24(+)Vbeta11(+) NKT cells by age, malignancy and conventional anticancer therapies. Br J Cancer. 2004;91(11):1880–1886. doi: 10.1038/sj.bjc.6602218. Molling JW, Langius JA, Langendijk JA, Leemans CR, Bontkes HJ, van der Vliet HJ, et al. Low levels of circulating invariant natural killer T cells predict poor clinical outcome in patients with head and neck squamous cell carcinoma. J Clin Oncol. 2007;25(7):862–868. doi: 10.1200/JCO.2006.08.5787.]

In locally advanced small cell lung cancer, on the contrary, it was observed an increase in the number of circulating NKT cells [31]. In this study, patients exhibited a relative increase in the total number of NKT cells with the CD3+CD56+/CD16+ immunophenotype, as well as an increase in both relative and absolute numbers of NK cells (CD3−CD56+CD16+) compared with healthy volunteers. These NKT cells findings are consistent with the data obtained for ovarian cancer and breast cancer from a study by Tabakov D.V. et al [30], as well as for epithelial cancers from earlier studies [31]. With regard to NK cells, similar da-ta have been obtained by Shubina I. et al. in ovarian cancer [32]. However, this study included not only patients with primary disease, but also those undergoing chemotherapy.

It is possible that the role of these cells should be considered only in the context of individual types of cancer and the stage of the disease.»

 

• - Please clarify and discuss in more detail the double-positive T cells and their role in the prognosis and outcomes. The route of double-positive T cells is complex and should be discussed across different cancer types in the manuscript

Answer: The article provides brief information: «These cells can stimulate inflammation due to their increased secretion of IL-10, interferon gamma (IFN-γ) and TGF-β. In the case of certain types of malignant neoplasms, such as Hodgkin's lymphoma, melanoma, breast cancer and hepatocellular carcinoma, higher numbers of double-positive CD4+CD8+ T cells have been found. The increased prevalence of these cells in advanced stages of cancer, along with their cytotoxic properties and cytokine profile, suggest that they may be important in the progression and spread of cancer. However, further research is needed to fully understand their role and potential as a target for therapy».

 

• Please provide a section describing the clinical outcomes of the patients who were included of the study. What were the treatment protocols (describe it in the method section) and what were the clinical outcomes? Prognosis/overall survival or progression-free survival. What were the treatment responses? Provide the history of the patients as a table and complement it if possible, with BMI, tumor size, smoking history, comorbidities, time from diagnosis to blood sampling, and medication. This information is crucial for this study in order to be able to understand the possible effect on the immune cell composition for the patients with unresectable non-small cell lung cancer

Answer: Thank you very much for your recommendation. We also would like to draw your attention to the fact that our study did not aim to correlate the population composition of blood lymphocytes with clinical outcomes. Therefore, it was not necessary for us to overload the study with clinical data for each individual patient (80 people). However, taking into account your comment, we will now add a table with general patient characteristics to the Materials and Methods section. We are currently working on this. Undoubtedly, the relationship between immunological blood parameters and oncological outcomes is important and we will evaluate it in future studies.

 

Minor comments

• Please standardize the nomenclature across the manuscript: T lymphocytes vs T cells vs T-Cells

Answer: Thank you very much, we standardized and corrected everything for T cells.

 

• ⁃ Please provide the results of CD4toCD8 ration as a figure or table

Answer: We have added this information in the manuscript: «The ratio of CD4+ to CD8+ T cells in patients was 1.55, while in donors it was 1.79.».

 

Parameter	Patients, mean (n=80)	Donors, mean (n=40)
СD4+ (%%)	42,555	48,750
СD8+ (%%)	27,528	27,200
ration	1,55	1,79
СD4+ (per ml)	812	854,538
СD8+ (per ml)	530	478,575
ration	1,53	1,79

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations to you on your brilliant study about the immune cell composition in the blood of unresectable NSCLC. The manuscript was well written in acceptable language and layout. The following are my comments.

(1) Why did you select squamous cell carcinoma as the dominant studied candidiate ? As it was not the normal incidence in our clinical practice ?

(2) How did you select the donors ?

(3) There was great discrepancy of age between the patients and the donors. Will it affect the study results ?

(4) In the text, what was meant by' unrecoverable' NSCLC ?

Author Response

Dear Sir, thank you for your careful reading of our research and your favorable review.

 

Comments and Suggestions for Authors

Congratulations to you on your brilliant study about the immune cell composition in the blood of unresectable NSCLC. The manuscript was well written in acceptable language and layout. The following are my comments.

 

(1) Why did you select squamous cell carcinoma as the dominant studied candidiate ? As it was not the normal incidence in our clinical practice ?

Answer: Our study included patients not only with squamous cell carcinoma, but also with adenocarcinoma, as indicated in the materials and methods.: “Histologically, squamous cell carcinoma was the most common type of cancer in the study group, affecting 65% of patients (52 out of 80), followed by adenocarcinoma in 33.8% (27 out of 80) of patients, and a rare type of pleomorphic tumor was found in one patient”.

 

(2) How did you select the donors ?

Answer: Blood samples for comparison were taken at the appropriate time interval at our donation center from people who regularly donate blood to cancer hospital patients who have confirmed the absence of any infectious processes and chronic diseases by laboratory methods.

 

(3) There was great discrepancy of age between the patients and the donors. Will it affect the study results ?

Answer: In fact, the age difference was sufficient, and this could have influenced the significance of differences in individual populations (for example, the number of DP T cells, however, an increase in this indicator in our patients was noted in most cases in people under 60 years of age. We have described this fact in the latest edition of the manuscript.

(4) In the text, what was meant by' unrecoverable' NSCLC ?

Answer: This may be a typo; we will carefully proofread the text and correct it.  This study examines a cohort of patients diagnosed with locally advanced, unresectable non-small cell lung cancer.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Gritsova et al have carefully and thoughtfully addressed my major and minor concerns. I thank the authors for their hard work and for implementing my suggestions. I recommend accepting the manuscript in current form. Great job.