The clinical value of peripheral immune cell counts in pancreatic cancer.

BACKGROUND: Elevated neutrophil-lymphocyte ratio (NLR) is linked to poor overall survival (OS) in pancreatic cancer. We aim to investigate the association of the various hematologic markers, in particular NLR among others, with distant metastases, a common feature in pancreatic cancer.
METHODS: Clinical data from 355 pancreatic cancer patients managed at King Hussein Cancer Center (Amman-Jordan) have been reviewed. We examined the relationship between absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute eosinophilic count (AEC), absolute monocytic count (AMC), NLR, monocyte to lymphocyte ratio (MLR) and platelet to lymphocyte ratio (PLR) with the presence of baseline distant metastases and OS. Receiver Operating Characteristic (ROC) curve analysis was plotted to identify the NLR optimum cutoff value indicative of its association with distant metastases.
RESULTS: On univariate and multivariate analyses patients whom on presentation had high NLR (≥3.3) showed more baseline distant metastases compared to patients with low NLR (<3.3), (p-value: <0.0001 and <0.0001, respectively). Patients with high baseline ANC (≥5500/μL), AMC (≥600/μL), MLR (≥0.3) had more baseline distant metastases in comparison to patients with lower values (p-value: 0.02, 0.001, and <0.0001, respectively). High ANC, NLR, MLR, and PLR and low ALC were associated with poorer OS, (p-value: <0.0001, <0.0001, <0.0001, 0.04, and 0.01, respectively).
CONCLUSION: This study presents additional evidence of the association of some of the hematologic markers; in particular ANC, NLR, AMC, and MLR, with baseline distant metastases and poor outcome in pancreatic cancer. Whether these immune phenomena can help in identifying patients at higher risk for the subsequent development of distant metastases is unknown.

Introduction

Pancreatic cancer is the most fatal malignancy worldwide with approximately 500,000 deaths each year [1]. More than half of patients had distant metastases at the time of presentation. Despite recent progress in the management of advanced-stage pancreatic cancer, the ultimate prognosis is still dismal [2].

Various studies have associated Inflammation to carcinogenesis, metastases and survival outcomes in cancer. [1, 2] Nonetheless, how inflammation is linked to metastasis remains elusive. Hematologic parameters, in particular neutrophil-lymphocyte ratio (NLR), were repeatedly shown to negatively impact survival outcome in various tumors including pancreatic cancer [3-8]. Additionally, studies have related high NLR to baseline presence and subsequent development of distant metastases in various tumors including non-small cell lung cancer, gastric cancer, and colorectal cancer among others [9-11]. Theories were proposed in an attempt to explain the relationship between inflammation and metastasis. A high circulating neutrophil count, which would result in a high NLR, might promote tumor cells’ metastasis via releasing various proteases and specific growth factors like vascular endothelial growth factor (VEGF) [13]. Moreover, circulating lymphocytes induce cytotoxic cell death, and therefore are actively involved in the elimination of tumor cells, in addition to delaying tumor cell migration, thus explaining the relationship between low ALC and poor outcomes [14, 15].

In this retrospective study, we aim to investigate the relationship between the various peripheral hematologic cells, in particular the different types of inflammatory cells with distant metastases in pancreatic cancer. Our main objectives are to answer the following questions; Do pancreatic cancer patients with high NLR have a high incidence of distant metastases? Would they benefit from more frequent surveillance investigation including imaging? And finally, would they potentially benefit from prophylactic strategies?

Methods

Initial step in data collection from the electronic medical records

This is a retrospective chart review study approved by the Institutional Review Board (IRB) at King Hussein Cancer Center (KHCC). Once the IRB approval was granted, a request was made to the Center’s Cancer Registry to obtain the full details of patients diagnosed and managed i.e. full abstract, with pancreatic carcinoma from July 2006- December 2018 inclusive. The Electronic Medical Records (EMRs) at KHCC were then searched for particular hematologic parameters. The IRB policy that governs the ethical approval for studies on pre-existing i.e. data is already documented in the EMR and/ or Center’s Cancer Registry at the time of data collection does not mandate patient’s informed consent in the absence of direct interaction/ intervention. Positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) scans were reviewed to detect the presence of baseline distant metastases. Clinical data including age, gender, location of the primary tumor, and sites of baseline distant metastases are summarized in Table 1. We excluded all patients who received only part of treatment at our center and patients who were on steroids before obtaining a complete blood count (CBC). Using steroids was expected to confound the results of the study as it leads to leukocytosis and specifically neutrophilia [16, 17].

Table 1

Characteristics of pancreatic cancer patients.

Patients features	No. of patients (%)
Age > 60	167 (47%)
Age< = 60	188 (53%)
Gender
Male	210 (59%)
Female	145 (41%)
Location
Head of pancreas	184 (52%)
Others (body, tail. . .etc.)	110 (31%)
Unknown	61 (17%)
Baseline Distant Metastases
Present	206 (58%)
Absent	149 (42%)
Clinical TNM Stage
I & II	43 (12%)
III	106 (30%)
IV	206 (58%)
Hematologic Parameters:	Median, (Mean)
ANC	5500 (6550)
ALC	1680 (1900)
AEC	140 (190)
AMC	600 (670)
NLR	3.3 (4.3)
MLR	0.3 (0.4)
PLR	0.15 (0.2)

Complete blood count (CBC) with the differential white cell count was collected before the initiation of any cancer-specific treatment (systemic treatment or radiation). The pre-treatment baseline NLR, MLR and PLR were calculated using these formulas; NLR = ANC/ALC, MLR = AMC/ALC and PLR = Platelet Count/ALC.

Determination of the NLR cut-off value and relation to other variables

The Receiver Operating Characteristic (ROC) curve was operated to determine the best NLR cut-off value, for assessing its association with the presence of baseline distant metastases, matching the most extreme joint sensitivity and specificity. The association between NLR, age, gender and the anatomic location of the primary tumor within the pancreas with the presence of baseline distant metastases was tested. Univariate and multivariate logistic regression analyses were used to examine the association between the various variables and the presence of baseline distant metastases. A p-value of ≤ 0.05 was determined as the cut off value for significance association.

The association between baseline hematological markers and distant metastasis

Our analysis proceeded stepwise. In the first phase, we examined the association between baseline NLR with the presence of distant metastases. In the second phase, we examined the association between other hematologic parameters including ANC, ALC, AEC, AMC, MLR and PLR with the baseline presence of distant metastases.

The association between baseline hematological markers and clinical variables

In the third phase, we examined the association between baseline presence of distant metastases with the clinical variables like age, gender, and location of the primary tumor.

In the fourth phase, we performed a multivariate analysis that included the collected variables (age, gender, and location of the primary tumor) with NLR as a dichotomous variable, to identify correlation with baseline distant metastases.

The association between baseline hematological markers and overall survival

In the fifth phase, we examined the association between the hematologic parameters including ANC, ALC, AEC, AMC, NLR, MLR & PLR with the OS. In the last phase, we examined the association between NLR as a categorical variable with both the presence of baseline distant metastases and the OS.

Descriptive analysis of the studied patients’ information was performed. Categorical data, such as age group, gender and other factors were presented as counts and percentages. The mean, standard deviation and range were calculated for the continuous data including age, ANC and other factors. In general, differences in proportions were tested with χ2 test or Fisher’s exact test, and differences in continuous variables were tested with Student’s t-test or a non-parametric test (Wilcoxon Rank Test) depending on the assumptions required for each test. Multivariate analysis was done for gender, age, site (pancreas head vs others), NLR cutoff (NLR>3.286 vs NLR< = 3.286) with probability modeled on (stage = 'IV'), using the logistic regression model. Kaplan-Meier method was used to estimate OS curves, and log-rank test was used to compare patients' survival times between factors groups. The OS time was calculated from the diagnosis date to death from any cause. Survival was expressed as median with a 95% confidence interval (CI). Multivariate analysis was done by using Cox proportional hazards regression model. Receiver operating characteristic curve (ROC curve) was performed for NLR ANC, AMC, and MLR with stage and survival. A significance criterion of p< = 0.05 was used in the analysis. All analyses were performed using SAS version 9.4 (SAS Institute Inc, Cary, NC).

Results

The clinical characteristics of 355 pancreatic cancer patients are summarized in Table 1. More than half of the patients were males; (59% versus 41.0%, with a male to female ratio of 1.4:1). The median age at diagnosis was 60 years. The median OS for all patients was 6.6 months. More than half of the patients had distant metastases at time of presentation (n = 206, 58%). The mean of baseline NLR was 4.3, and the median was 3.3. The cutoff value of NLR for its association with baseline distant metastases was determined to be 3.3 using the ROC curve, where the area under the curve (AUC) had a value of 0.609 (S1 Fig).

The relationship between the peripheral count of various immune cells, their ratios and the baseline presence of distant metastases are shown in Table 2. Patients with elevated baseline NLR (≥3.3) were more likely to have distant metastases at time of presentation in comparison to patients with low baseline NLR (<3.3), (p-value <0.0001, Odds Ratio (OR): 1.7, CI: 2.6–4.0). High baseline ANC (≥5500/μL), high AMC (≥600 /μL), and high MLR (≥0.3) were associated with baseline distant metastases (p-value: 0.02, 0.001, and <0.0001 respectively). ALC, AEC, and PLR were not associated with the presence of baseline distant metastases in pancreatic cancer (p-value: 0.65, 0.38 and 0.21, respectively).

Table 2

The association between hematologic indices with the presence of baseline distant metastases.

	Baseline distant metastases
	Present	Absent	p- value	OR	95% CI
Baseline ANC≥5500	113 (64%)	63 (36%)	0.024	1.65	(1.0–2.5)
Baseline ANC<5500	92 (52%)	85 (48%)
Baseline ALC≥1680	99 (57%)	75 (43%)	0.65	0.9	(0.6–1.4)
Baseline ALC<1680	106 (59%)	73 (41%)
Baseline AMC≥ 600	117 (66%)	59 (34%)	0.001	2.0	(1.3–3.0)
Baseline AMC< 600	88 (50%)	89 (50%)
Baseline AEC≥ 143	83 (62%)	51 (38%)	0.38	1.2	(0.7–2.0)
Baseline AEC< 143	76(57%)	58 (43%)
Baseline NLR≥3.3	126 (69%)	56 (31%)	< .0001	2.6	(1.7–4.0)
Baseline NLR<3.3	79 (46%)	92 (54%)
Baseline MLR≥0.3	130 (68%)	62 (32%)	< .0001	2.4	(1.5–3.7)
Baseline MLR<0.3	75 (46%)	86 (54%)
Baseline PLR≥0.15	108 (61%)	68 (39%)	0.2	1.3	(0.85–2.0)
Baseline PLR<0.15	97 (55%)	80 (45%)

Table 3 shows the univariate and multivariate analyses assessing the association of NLR and clinical variables with baseline distant metastases. The location of the primary tumor was significantly associated with the baseline presence of distant metastases; patients with the primary tumor located in the head of the pancreas had less baseline distant metastases compared to patients with the primary tumor located in other sites (body, tail, and overlapping), (p-value: <0.0001). Age and gender were not associated with baseline presence of distant metastases in the univariate analysis (p-value: 0.29 and 0.53, respectively). In spite of that, we involved both factors in the multivariate analysis because of their clinical relevance. In the multivariate analysis, age, location of the primary tumor, and NLR, but not gender were significantly associated with the baseline presence of distant metastases (p-value: 0.02, <0.0001, 0.0003, and 0.9, respectively). Furthermore, the association with baseline metastasis remained significant even when NLR was evaluated as a categorical variable. (Table 4)

Table 3

Univariate and multivariate analyses for the association of different variables with the presence of baseline distant metastases.

	Baseline Distant Metastases		Univariate		Multivariate
Variables	Present	Absent	P value	OR (95% CI)	P value	OR (95% CI)
Age			0.29		0.02
Age≤60	114 (61%)	74 (39%)		1.2 (0.8–1.9)		1.8 (1.0–2.9)
Age>60	92 (55%)	75 (45%)
Gender			0.53		0.9
Male	119 (57%)	91 (43%)		0.8 (0.5–1.3)		0.9 (0.6–1.6)
Female	87 (60%)	58 (40%)
Location			< .0001		< .0001
Head	85 (46%)	99 (54%)		0.3 (0.19–0.53)		0.3 (0.18–0.54)
Others	80 (73%)	30 (27%)
Unknown	41 (67%)	20 (33%)
Baseline NLR			< .0001		0.0003
NLR>3.3	126 (69%)	56 (31%)		2.6 (1.7–4.0)		2.5 (1.5–4.1)
NLR≤3.3	79 (46%)	92 (54%)

Table 4

The association between NLR (categorical variable) and the presence of baseline distant metastases.

NLR	Distant Mets Present	Distant Mets Absent	Odds Ratio	95% Confidence Intervals
NLR (<2.0)	39 (50%)	39 (50%)	1.0	N/A
NLR (2.0–3.2)	45 (45%)	54 (55%)	0.833	(0.460–1.510)
NLR (3.2–5.0)	60 (70%)	26 (30%)	2.307	(1.217–4.373)
NLR (>5.0)	61 (68%)	29 (22%)	2.103	(1.124–3.935)

The results of the survival analysis are summarized in Table 5. High baseline ANC, NLR, MLR, PLR, and low ALC were associated with poor OS, (p-value: <0.0001, <0.0001, <0.0001, 0.04, and 0.01, respectively) (Figs 1–5). The ROC curve was utilized to determine if NLR, ANC, ALC, MLR, or PLR is a better predictor of overall survival. The curve showed that NLR has the highest AUC followed by ANC, PLR, MLR and then ALC which indicates that NLR is a better predictor of overall survival in comparison to these tested variables (S2 Fig). High NLR was also associated with poor OS as a categorical variable (Table 6 & Fig 6). Baseline AEC and AMC were not associated with the OS, (p-value: 0.54 and 0.23, respectively).

Table 5

The results of the survival analysis.

	Median of OS	Number of patients	p-value	HR	95% CI
ANCv5500	5 months	176 (50%)	< .0001	1.5	(1.2–1.9)
ANC < 5500	10 months	177 (50%)
ALC≥ 1680	9 months	174 (49%)	0.01	0.7	(0.6–0.9)
ALC < 1680	5 months	179 (51%)
AEC≥143	8 months	134 (50%)	0.54	0.9	(0.7–1.2)
AEC < 143	8 months	134 (50%)
AMC≥ 600	6 months	176 (50%)	0.2	1.3	(0.9–1.4)
AMC < 600	8 months	177 (50%)
NLR≥ 3.3	4 months	176 (50%)	< .0001	1.9	(1.5–2.5)
NLR < 3.3	11 months	177 (50%)
MLR≥ 0.3	5 months	192 (54%)	< .0001	1.5	(1.2–1.9)
MLR < 0.3	9 months	161 (46%)
PLR≥ 0.15	5 months	176 (50%)	0.04	1.2	(1.0–1.5)
PLR < 0.15	9 months	177 (50%)

Fig 1

Kaplan Meier curve for overall survival for patients with ANC≥ 5500.

Fig 5

Kaplan Meier curve for overall survival for patients with PLR≥ 0.15.

Table 6

The relationship between NLR (categorical variable) and OS.

NLR	Overall Survival (mons)	p-value	Hazard Ratio	95% Confidence Intervals
NLR (<2.0)	9.7	0.0001	1.0	N/A
NLR (2.0–3.2)	11.3		0.979	(0.701–1.368)
NLR (3.2–5.0)	5.2		1.770	(1.262–2.483)
NLR (>5.0)	3.0		2.164	(1.548–3.027)

Fig 6

Kaplan Meier curve for overall survival for patients with NLR as categorical variable.

Discussion

In this study we assessed the association of NLR, ANC, ALC, AEC, AMC, MLR and PLR with the presence of baseline distant metastases and OS in pancreatic cancer patients, as well as the potential predictive value of NLR for detecting distant metastases. An elevated baseline NLR (≥ 3.3) was an independent factor associated with the baseline presence of distant metastases in pancreatic cancer, as depicted in both the univariate and multivariate analyses, after adjusting for important covariates (p = < .0001, and p = 0.0003, respectively) (Table 2). Patients with elevated baseline ANC (≥5500/μL), AMC (≥600/μL), MLR (≥0.3) had more distant metastases in comparison to patients with low baseline ANC (<5500/μL), AMC (<600/μL), MLR (<0.3) (p-value: 0.02, 0.001 and <0.0001, respectively).

Several studies have investigated the prognostic value of these inflammatory markers in pancreatic cancer, in which NLR, PLR, MLR were reported to be associated with poor OS. The cutoff value of NLR reported in these studies varies widely; ranging from 2.3 to 5[12]. In our cohort, the cutoff value of NLR using the ROC curve is 3.3. To our knowledge, this appears to be the first study exploring the association between these different inflammatory markers with the presence of baseline distant metastatic lesions in pancreatic cancer.

Using a simple test such as a complete blood count (CBC), to foresee the likelihood of the development distant metastases might have a direct impact on the management of pancreatic cancer patients through early detection of distant metastatic lesions. The presence of high NLR in pancreatic cancer patients’ CBC tests could stimulate surveillance for distant metastases through more frequent imaging, and consideration for prophylactic measures.

In patients with different types of malignant tumors, NLR correlates with the extent of therapeutic success in response to chemotherapy and immunotherapy [13-17]. Moreover, other blood-based markers i.e. circulating tumor cells and tumor-DNA were reported to be associated with survival in pancreatic cancer [8]. This might represent a prospect to link these markers together and study their potential associations.

Treatment plans to limit distant metastases in pancreatic cancer include chemotherapy, radiation, and surgery. Future research can include investigating the association of NLR to the response of distant metastases to the various treatment modalities. A major advancement in the management of advanced-stage pancreatic cancer will be achieved if NLR is confirmed to be a predictive marker of distant metastases’ response to available and future novel treatments.

We acknowledge that the retrospective nature of the data, which was collected from a single institution, represents a limitation to this study. Moreover, there was no assessment of the exact treatments administered and of any other factors that might alter NLR, such as infections. Nonetheless, we believe that our study adds more evidence to current literature on the potential usefulness of hematological markers in predicting distant metastases in pancreatic cancer, and which might potentially be useful in assessing the response to various treatment modalities including novel therapies.

Conclusion

In pancreatic cancer, elevated baseline NLR, ANC, AMC, and MLR appear to be independent predictive factors for the presence of baseline distant metastases. Prospective validation and how these inflammatory markers can be related to other markers to predict the likelihood of subsequent development of distant metastases warrants further investigation.

(XLS)

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Receiver-operating-characteristic (ROC) curve and the area under the curve (AUC) for NLR.

(TIF)

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ROC curve of NLR, ANC, ALC, MLR, and PLR for predicting overall survival.

(TIF)

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10 Feb 2020

PONE-D-20-00008

The Clinical Value of Peripheral Immune Cell Counts in Pancreatic Cancer

PLOS ONE

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Reviewer #1: This manuscript aims at evaluating the potential prognostic role in pancreatic cancer of the absolute count of some inflammatory cell types (neutrophils, lymphocytes, eosinophils and monocytes), as well as of two “absolute” values, such as monocyte-lymphocyte ratio and platelet-lymphocyte ratio.

This paper is well written and has a very good sample size (355 patients); the ROC curves /statistics have been properly used. In my opinion, there are some points to be improved before considering this paper for publication, as follows:

- In the abstract, mini-abstract and in text, the authors should tone down the considerations regarding immunotherapy. For example, they have written: “whether or not these immune phenomena can help in identifying patients who might respond to immunotherapy needs further investigation”. This consideration should be deleted, since does not have much sense. Indeed, immunotherapy in pancreatic cancer does not represent a possible therapeutic strategy in the vast majority of cases (>98%), due to the very rare presence of microsatellite instability and of high-tumor mutational burden (and also the lack of significant utility of PD-L1 as a predictive marker). The importance of this study, in the opinion of this reviewer, is giving further biological insights on the blood parameters of patients with pancreatic cancer and demonstrating that other blood related variables (other than the classical known) may be introduced into the clinical practice due to their potential diagnostic / prognostic value. All the implications and considerations about immunotherapy, thus, should be deleted or toned down.

- For investigating the “prognostic” role of the studied parameters, the authors used the baseline presence of metastasis. It may be of great help if they have also values (timing) on metastatic progression for calculating this “prognostic role” also as the effective metastatic risk.

- In the discussion, the authors should better explain the importance and future perspectives of blood analysis for patients with pancreatic cancer. Indeed, not only inflammatory-cells related values can be investigated, but also other parameters, such as tumor circulating cells and circulating tumor-DNA, may be of great importance. Please discuss more in depth this topic (I suggest this comprehensive reference: PMID: 31405192)

- In the discussion, please comment more in depth the prognostic value of neutrophil-to-lymphocyte ratio for invasive pancreatic cancers associated to IPMN. You may use for this the main reference on this topic, which from the group of Dr. Christopher Wolfgang from the Johns Hopkins University (Gemenetzis G et al.; Ann Surg. 2017; PMID: 27631774). This reference should be used also in the introduction when the authors presented similar moderators in different cancer types.

- Figure 1 and figure 7 should be put as “supplementary figures”. There are 6 figures (Kaplan-Meier curves) that are very important for the reader, but the two figures presenting the “ROC curves” are not so important for the reader. Indeed, they represent a “concept” very important for the methods, but not for the overall comprehension of the paper. Thus, please put them as supplementary (as usually happens).

Reviewer #2: The manuscript by Wang and co-authors titled “The clinical value of peripheral immune cell counts in pancreatic cancer” demonstrated the potential role of the absolute numbers enumeration of myeloid and lymphocytes as a potential biomarker to predict distant metastases. The major concern of this work is the novelty. Several reports have already reported these parameters (NLR, MLR etc) to identify PDAC patients with a poor prognosis induced by metastatic disease: Formica V. et al. Neutrophil/lymphocyte Ratio Helps Select Metastatic Pancreatic Cancer Patients Benefitting From Oxaliplatin, Cancer Biomark; 2016; Ventriglia J. et al. Neutrophil to Lymphocyte Ratio as a Predictor of Poor Prognosis in Metastatic Pancreatic Cancer Patients Treated with Nab-Paclitaxel plus Gemcitabine: A Propensity Score Analysis, Gastroenterology Research and Practice; 2018; Piciucchi M. et al. The Neutrophil/Lymphocyte Ratio at Diagnosis Is Significantly Associated with Survival in Metastatic Pancreatic Cancer Patients, Int J Mol Sci., 2017.

The authors did not comment their results in light to the published data, for example: the identified cutoff of NLR (3.3) is quite different compared to data from literature. Please comment this discrepancy.

More concerns are about patient data. As reported the patient cohort is based on 355 patients. Considering the percentage of absent metastases at baseline from Table 2, the reader can conclude that this experimental group is approximately composed by 178 patients (i.e 89 patients represent 50% of the group). However from Table 1, patients with baseline metastases (Liver+ lung + peritoneal) are listed as 260. Therefore, the analyzed patient cohort result to be composed by 438 patients. Please comment and clarify this aspect.

Minor concern. Some conclusions are overstated:

Abstract. “…these immune phenomena can help in identifying patients who might respond to immunotherapy”. In my knowledge there are not clinical conventional immune-based approaches for PDAC treatment.

Finally, the graphic quality is very poor.

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23 Mar 2020

Editorial Formatting comments:

All the suggestions regarding editorial formatting have been incorporated. All paragraphs were re-written to minimize any potential overlap with our previously published work.

Response to Reviewer 1:

Thank you for your review of our paper. We have answered each of your points below.

1. In the abstract, mini-abstract and in text, the authors should tone down the considerations regarding immunotherapy. For example, they have written: “whether or not these immune phenomena can help in identifying patients who might respond to immunotherapy needs further investigation”. This consideration should be deleted, since does not have much sense. Indeed, immunotherapy in pancreatic cancer does not represent a possible therapeutic strategy in the vast majority of cases (>98%), due to the very rare presence of microsatellite instability and of high-tumor mutational burden (and also the lack of significant utility of PD-L1 as a predictive marker). The importance of this study, in the opinion of this reviewer, is giving further biological insights on the blood parameters of patients with pancreatic cancer and demonstrating that other blood related variables (other than the classical known) may be introduced into the clinical practice due to their potential diagnostic / prognostic value. All the implications and considerations about immunotherapy, thus, should be deleted or toned down.

Response: Done. We have removed all the sentences that addressed the use of immunotherapy in pancreatic cancer.

2. For investigating the “prognostic” role of the studied parameters, the authors used the baseline presence of metastasis. It may be of great help if they have also values (timing) on metastatic progression for calculating this “prognostic role” also as the effective metastatic risk.

Response: We agree that investigating the predictive value of the baseline immune cell counts for the subsequent development of distant metastases would be of a great help, but since a small percentage of our cohort had no baseline distant metastases and no available data regarding the subsequent development of distant metastases in this subset of patients, this was not feasible.

3. In the discussion, the authors should better explain the importance and future perspectives of blood analysis for patients with pancreatic cancer. Indeed, not only inflammatory-cells related values can be investigated, but also other parameters, such as tumor circulating cells and circulating tumor-DNA, may be of great importance. Please discuss more in depth this topic (I suggest this comprehensive reference: PMID: 31405192)

Response: Done. We touched base with other potential markers of distant metastases in the discussion and cited the suggested reference as well.

4. In the discussion, please comment more in depth on the prognostic value of neutrophil-to-lymphocyte ratio for invasive pancreatic cancers associated to IPMN. You may use for this the main reference on this topic, which is from the group of Dr. Christopher Wolfgang from the Johns Hopkins University (Gemenetzis G et al.; Ann Surg. 2017; PMID: 27631774). This reference should be used also in the introduction when the authors presented similar moderators in different cancer types.

Response: Done. We have cited this reference in the introduction. Our aim in this study was to explore the predictive value of these inflammatory-based markers for the presence of distant metastases in pancreatic cancer. We can’t discuss other predictive values of these markers in this paper in depth as that would be irrelevant topics.

5. Figure 1 and figure 7 should be put as “supplementary figures”. There are 6 figures (Kaplan-Meier curves) that are very important for the reader, but the two figures presenting the “ROC curves” are not so important for the reader. Indeed, they represent a “concept” very important for the methods, but not for the overall comprehension of the paper. Thus, please put them as supplementary (as usually happens).

Response: Done. We made the ROC curves supplementary figures.

Response to Reviewer 2:

Thank you for your review of our paper. We have answered each of your points below.

1. The manuscript by Wang and co-authors titled “The clinical value of peripheral immune cell counts in pancreatic cancer” demonstrated the potential role of the absolute numbers enumeration of myeloid and lymphocytes as a potential biomarker to predict distant metastases. The major concern of this work is the novelty. Several reports have already reported these parameters (NLR, MLR etc) to identify PDAC patients with a poor prognosis induced by metastatic disease: Formica V. et al. Neutrophil/lymphocyte Ratio Helps Select Metastatic Pancreatic Cancer Patients Benefitting From Oxaliplatin, Cancer Biomark; 2016; Ventriglia J. et al. Neutrophil to Lymphocyte Ratio as a Predictor of Poor Prognosis in Metastatic Pancreatic Cancer Patients Treated with Nab-Paclitaxel plus Gemcitabine: A Propensity Score Analysis, Gastroenterology Research and Practice; 2018; Piciucchi M. et al. The Neutrophil/Lymphocyte Ratio at Diagnosis Is Significantly Associated with Survival in Metastatic Pancreatic Cancer Patients, Int J Mol Sci., 2017.

Response: We agree that there are articles in the literature addressing the prognostic utility of NLR. PLR...etc in pancreatic cancer. However, to our knowledge, this is the first study that delineate the predictive value of these markers for the presence of distant metastases in pancreatic cancer. Other reports focused mainly on the overall survival, progression free survival, response to chemotherapy, the surgical outcomes, or the clinical stage. Also, to our knowledge, this is the first study reporting the prognostic impact of NLR as categorical variable instead of dichotomous variable. Furthermore, to our knowledge, this is the first study that assess the predictive value of NLR for the presence of distant metastases in pancreatic cancer using multiple logistic regression model.

2. The authors did not comment their results in light to the published data, for example: the identified cutoff of NLR (3.3) is quite different compared to data from literature. Please comment this discrepancy.

Response: Done. We have discussed the differences between NLR cutoff values among the articles, including ours, in the discussion.

3. More concerns are about patient data. As reported the patient cohort is based on 355 patients. Considering the percentage of absent metastases at baseline from Table 2, the reader can conclude that this experimental group is approximately composed by 178 patients (i.e 89 patients represent 50% of the group). However, from Table 1, patients with baseline metastases (Liver+ lung + peritoneal) are listed as 260. Therefore, the analyzed patient cohort result to be composed by 438 patients. Please comment and clarify this aspect.

Response: Done. We added a sentence to the results and a row to the table #2 showing that 58% of the patients had distant metastases at time of presentation. In our cohort, 206 patients had distant metastases at time of presentation. We edited the results paragraph and the table #2 to avoid any confusion. Thanks for your comment.

4. Minor concern. Some conclusions are overstated:

Abstract. “…these immune phenomena can help in identifying patients who might respond to immunotherapy”. In my knowledge there are not clinical conventional immune-based approaches for PDAC treatment.

Response: We have edited the manuscript and removed all the sentences that talked about the use of immunotherapy in pancreatic cancer.

5. The graphic quality is very poor.

Response: We used the original graphs in the revised manuscript hoping they are clear now.

Submitted filename: Round 2 comments.docx

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7 Apr 2020

The Clinical Value of Peripheral Immune Cell Counts in Pancreatic Cancer

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27 May 2020

PONE-D-20-00008R1

The Clinical Value of Peripheral Immune Cell Counts in Pancreatic Cancer

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