http://orcid.org/0000-0002-2513-4721
Figures
Abstract
Objective
To study the prognostic value of baseline leukocytosis or neutrophiliain two retrospective cohorts of stage III Non-Small Cell Lung Cancer (NSCLC) patients.
Materials and methods
Clinical records of consecutive previously untreated NSCLC patients in our Institution between June 2001 and September 2016 for stage III NSCLC were collected. The prognostic value of pretreatment leucocyte disorders was examined, with focus on patterns of relapse and survival. Leukocytosis and neutrophilia were defined as a leukocyte count or a neutrophil count exceeding 10 and 7 G/L, respectively.
Results
We identified 238 patients, displaying baseline leukocytosis or neutrophilia in 39% and 40% respectively. Most were diagnosed with adenocarcinoma (48%), and stage IIIB NSCLC (58%). 3-year actuarial overall survival (OS) and progression-free survival (PFS) were 35% and 27% respectively. Local relapses were reported in 100 patients (42%), and distant metastases in 132 patients (55%). In multivariate analysis, leukocytosis, neutrophilia, and induction chemotherapy regimen based on carboplatin/paclitaxel were associated with worse OS and PFS (p<0.05). Neutrophilia independently decreased Locoregional Control (LRC) (HR = 2.5, p<0.001) and Distant Metastasis Control (DMC) (HR = 2.1, p<0.001). Neutrophilia was significantly associated with worse brain metastasis control (p = 0.004), mostly in adenocarcinoma patients (p<0.001).
Conclusion
In stage III NSCLC patients, treated with concurrent cisplatin-based chemoradiation, baseline leukocytosis and neutrophilia were associated with worse OS, PFS, LRC, and DMC. In addition with previously available markers, this independent cost-effective biomarker could help to stratify stage III NSCLC population with more accuracy.
Citation: Schernberg A, Mezquita L, Boros A, Botticella A, Caramella C, Besse B, et al. (2018) Neutrophilia as prognostic biomarker in locally advanced stage III lung cancer. PLoS ONE 13(10): e0204490. https://doi.org/10.1371/journal.pone.0204490
Editor: Jung Weon Lee, Seoul National University College of Pharmacy, REPUBLIC OF KOREA
Received: January 5, 2018; Accepted: September 10, 2018; Published: October 10, 2018
Copyright: © 2018 Schernberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Non-small cell lung cancer (NSCLC) represents 83% of patients diagnosed with lung cancer [1]. Despite advances in diagnosis and treatment management, patients prognosis remains poor, with 5-year overall survival (OS) of 27% for early or locally advanced and 4% for metastatic disease [1].
There is a lack of validated prognostic biomarkers in stage-III NSCLC. Tumor stage, performance status, smoking status, age, and gender (PS) are the historical ones [2]. Few are used in clinical practice to guide treatment and determine prognosis. In parallel with -omics recent fields of study, e.g. genomics or proteomics, affordable and easy to access markers have been investigated in NSCLC. Carcinoembryonic antigen (CEA), cytokeratin fragment 21–1 (Cyfra 21–1), and inflammation biomarkers have been found to be associated with patients outcome [3,4]. In metastatic NSCLC, molecular characterization has led to the definition of new subgroups, such as epidermal growth factor receptor (EGFR)-mutated NSCLC, and anaplastic lymphoma kinase (ALK)-rearranged NSCLC, among others that need specific treatments and strategies [5]. Certain mutations are predictive of clinical activity: the tyrosine kinase inhibitors targeting EGFR and ALK have shown striking efficacy in clinical trials, and are currently the standard of care in the clinic, regardless of others risk factors [5]. Still, search for prognostic factors is warranted, especially if they are inexpensive.
Studies revealed relations between systemic inflammation and immunology in the development and progression of various cancers [6]. Neutrophils are the most abundant white blood cells and play a key role in inflammation. They also dominates the immune cell composition in NSCLC [7]. Tumour reactive lymphocyte T cells are also frequently present [7].
The aims of the current study were to assess the clinical utility leukocytosis and neutrophilia in patients diagnosed with stage-III NSCLC, and to compare their accuracy with established prognostic markers.
Materials and methods
Patients and tumors
We examined clinical records of consecutive previously untreated and histologically confirmed stage III NSCLC registered in our institution, between June 2001 and September 2016. Only non-operated patients were included, whether with unresectable tumor, surgical contraindications or an impaired performance status. The participants provided consent for their medical records to be used in this research, and data was accessed anonymously. This study was approved approved by Gustave Roussy's Scientific Commission of Clinical Trials (CSET).
All patients had been referred to a multidisciplinary lung tumor board prior to treatment initiation. Explorations at diagnosis included physical examination, endoscopy with biopsy, computed tomography (CT) exploring cervical and thoracic regions, with or without brain magnetic-resonance imaging (MRI) and positron-emission tomography (PET-CT). Disease staging was defined according to the UICC’s lung cancer TNM staging classification, 7th edition.We excluded patients treated in a palliative intent with hypofractionated chemoradiation, patients who received neoadjuvant chemotherapy, patients treated for an immune disease or under steroids, and patients with acute infection defined as defined as the use of antibiotic therapy during the chemoradiotherapy treatment.
In our population, 145 (61%) patients prospectively registered through MSN study (NCT02105168) were included with 136 additional patients. Patients who had chemotherapy before blood count in our institution (n = 16) were excluded from the survival analysis. Patients who had an incomplete set of blood values (n = 27) were excluded as well, leading to 238 patients.
Complete blood count analysis
Pretreatment blood samples taken in the week preceding the first chemotherapy cycle were used for the current analysis. Leukocytosis and neutrophilia, defining biological inflammation, were defined as blood count ≥ 10 G/L and 7 G/L respectively, while anemia was defined as hemoglobin count < 12.0 g/dL. Thrombocytosis, lymphopenia and monocytosis were defined as platelets count ≥ 400 G/L, lymphocytes count < 1 G/L, and monocytes count ≥ 1 G/L respectively. These cut-off points were chosen because they have been recognized as standard pathological definitions.
Treatment characteristics and follow-up
All patients had a similar follow-up protocol, with a CT performed every 3 months for the first 2 years, then every 6 months. A PET scan was also performed if there was suspicion of recurrence.
Statistical analysis
Pearson χ2-square test and analysis of variance were used to determine any associations between the variables. Factors associated with tumor relapse were examined. Survival times were defined as the time between the diagnosis and the first event (time of death for OS, time of recurrence or death for PFS, time of loco-regional recurrence LRC, time of distant metastasis for DMC, and time of brain metastasis for brain metastasis control) estimated by the Kaplan Meier method. Patients were censored at the time of the most recent follow-up visit. Survival curves were compared using the log-rank test for the univariate analysis. Multivariate analyses were performed for variables with p value < 0.2 in univariate analysis, according to the Cox proportional hazards model. Neutrophilia and monocytosis were not tested in the same model with leukocytosis, they are subpopulation of leucocytes. Variables with a P value of greater than 0.05 were excluded. Statistical analyses were performed using R (version 3.3.2).
Results
Demographics and treatment characteristics
In the population, the median age of the patients at baseline was 60 years (range 25–85). Most were diagnosed with adenocarcinoma (48%), and stage IIIB NSCLC (58%). Most patients received induction chemotherapy (99%), 146 patients (61%) and 71 patients (30%) underwent further chemoradiation or chest radiotherapy alone respectively. Radiotherapy could not be completed in 2 patients due to tumor local progression. No patients underwent surgery following chemoradiotherapy.
At the time of analysis, 157 patients (66%) had died, with a median OS of 19.9 months (range 1.3–148.2). With a median PFS of 10.6 months (range 1.2–138.2), 196 patients (82%) relapsed. Local relapses were reported in 100 patients (42%), and distant metastases in 132 patients (55%). Eighty-one patients (34%) were still alive without evidence of tumor recurrence at time of analysis, with a median follow-up time of 28.4 months (range 1.7–148.2).
The clinicopathological and treatment features are displayed in Tables 1 and S1.
Relationship between baseline leukocytosis or neutrophilia and patient characteristics
On initial blood count, median leukocytes and neutrophils counts were 9.1 G/L (2.9–101.7) and 6.2 G/L (1.5–95.6) respectively. Leukocytosis and neutrophilia were found in 93 (39%) and 95 (40%) patients respectively. Neutrophilia was associated with lower PS (p<0.001). The 8 patients (100%) who did not undergo radiotherapy due to rapid local tumor progression had baseline leukocytosis and neutrophilia.
Analyzing total population with 180 assessable responses to induction chemotherapy, there was a correlation between leukocytosis or neutrophilia and increased proportion of patients displaying progression disease or stable disease, vs. partial or complete response (p<0.001, Fig 1).
RECIST response assessment after induction chemotherapy in stage-III patients, regarding leukocytosis (A) or neutrophilia (B).
Impact of leukocyte disorders as predictors of survival
In univariate analysis factors significantly associated with worse OS were leukocytosis (p<0.001), neutrophilia (p<0.001), PS (p = 0.001), TNM-N3 positive status (p = 0.004), stage IIIB vs. IIIA (p = 0.013), non-adenocarcinoma histology (squamous cell carcinoma, others; p = 0.036), concomitant chemotherapy (p = 0.014) and induction chemotherapy regimen others than cisplatin and vinorelbine (p<0.001). The radiotherapy technique (3D vs. IMRT) had no influence on patients’ OS (p = 0.412).
At 3-year follow-up, estimated OS was 44% (95%CI: 36–54%) for patients that had not initial leukocytosis vs. 22% (95%CI: 14–35%) if they had; 3-years PFS was 24% (95%CI: 17–32%) for patients that had not initial leukocytosis vs. 9% (95%CI: 4–19%) if they had. Similarly, estimated 3-years OS was 45% (95%CI: 37–55%) for patients who had not initial neutrophilia vs. 21% (95%CI: 13–34%) if they had and 3-years PFS was 24% (95%CI: 17–33%) for patients that had not initial neutrophilia vs. 8% (95%CI: 4–18%) if they had. Kaplan-Meier curves with univariate analysis regarding leukocytosis or neutrophilia for locoregional control, and distant metastasis control are displayed in S1 and S2 Figs. Moreover, analyzing distant relapses, neutrophilia was significantly associated with worse brain metastasis control (p = 0.004), mostly in adenocarcinoma patients (p<0.001) (S3, S4, S5 and S6 Figs).
Using multivariate analysis, neutrophilia was independently associated with worse OS with hazard ratio (HR) of 2.3 (95% confidence interval (CI): 1.6–3.3, p<0.001) and worse PFS with HR of 1.9 (95%CI: 1.4–2.6, p<0.001). Similar, leukocytosis was independently associated with worse OS with HR of 2.3 (95%CI: 16–3.3, p<0.001) and worse PFS with HR of 1.8 (95%CI: 1.3–2.5, p<0.001) (Fig 2 and Table 2).
Estimated overall survival in patients with or without leukocytosis (A), progression free survival in patients with or without leukocytosis (B), overall survival in patients with or without neutrophilia (C) or progression free survival in patients with or without neutrophilia (D).
In multivariate analysis, PS (p = 0.044) and induction chemotherapy other than cisplatin and vinorelbin (p<0.001) were the only features associated with worse survival. Stage IIIB vs. IIIA was not independently related with worse OS or PFS in this population, but was associated with poor DMC. Neutrophilia was also independently associated with worse LRC (HR = 2.5, p<0.001) and DMC (HR = 2.1, p<0.001) (Table 3). Non-adenocarcinoma histologies were independently associated with improved DMC (p<0.05).
Discussion
Several histopathological features and molecular biomarkers have been studied as potential prognostic factors. Still, in locally-advanced NSCLC patients, the only ones which are commonly used in clinical decision-making are patient’s performance status and tumour stage. In our study we hypothesized that a simple biological feature such as neutrophilia could be prognostic in locally advanced NSCLC. Both leukocytosis and neutrophilia were associated with poor OS and PFS, in a large set of patients. Moreover, there was a strong association between neutrophilia, LRC and poor response to induction chemotherapy, when evaluated.
Tumor-related leukocytosis and neutrophilia, established after excluding obvious causes (infection, bone marrow metastasis, and corticosteroid use), may result from hematopoietic colony-stimulating factors and inflammatory cytokines direct from solid tumors, comprising granulocyte colony-stimulating factor (G-CSF) among others [8,9]. Since the first report of G-CSF-producing malignant lung cancer in 1977, studies in cervical, head and neck, lung, gastric, bladder…, among others, described this particular biological characteristic, related with patient outcome [10–12].
Myeloid-derived suppressor cells (MDSCs) expands during cancer and inflammation, associated with a remarkable ability to suppress T-cell responses [13]. Debate persists as to which these suppressive properties are maintained by their tumour-infiltrating counterparts [14]. MDSCs both can exert antitumoral or protumoral activity. TGF-β within the tumor microenvironment induces a population of Tumor-Associated Neutrophils (TANs) with a protumor N2 phenotype [15]. TGF-β blockade slows tumor growth through activation of CD8+ T cells, macrophages, and TANs with an antitumor N1 phenotype [15]. Radiation therapy may also induces the N1 polarization [16]. A flow cytometry panel measuring 51 immune cell populations recently determined neutrophils as the most prevalent immune cell type in NSCLC microenvironment, that could partially explains why ~80% of NSCLC patients initially fail immune checkpoint inhibitor therapy [7]. Moreover, the neutrophil (and not the monocyte) population negatively correlated with CD8+ content, while increased levels CD8 + tumor-infiltrating lymphocytes (TILs) have been previously associated with better outcome in NSCLC [7,17]. In the present study, local relapses were reported in 100 patients (42%), and distant metastases in 132 patients (55%); both leukocytosis and neutrophilia decreased locoregional control (p<0.001).
There are evidences that neutrophils protect tumor cells through metastasis process [18]. Neutrophils facilitate intermediate steps of the invasion-metastasis cascade, suppressing natural killer cell activity and enhancing the extravasation of tumor cells, mainly through the secretion of various matrix metalloproteinases (MMPs) [19]. Such protection from attack by innate and adaptive immune system offers a clear advantage to tumor cells in transit [20]. Neutrophil NETosis, the formation of Neutrophil extracellular traps (NETs), is a unique form of cell survival mechanism characterized by the release of networks of extracellular fibers primarily composed of DNA from neutrophils which entangle pathogens [20,21]. Such entangled circulating tumor cells may be more apt to survive intraluminally, adhere to endothelial cells, and extravasate [20]. NETs were displayed as potential candidate pharmaceutical targets in cancer patients [21]. In accordance with these findings, in the present study neutrophilia independently decreased DMC (p<0.001). Moreover, prior study recently associated elevated neutrophil to lymphocyte ratio (NLR) with poor brain metastasis control, mostly in adenocarcinoma patients [22]. In accordance, the present study displayed similar findings, with neutrophilia decreasing brain metastasis control, mostly in adenocarcinoma patients.
To translate a given neutrophilia into a personalized prognosis or treatment plan is challenging [23]. Prospective longitudinal measurements of white blood count in individual patients is mandatory to maximize the clinical utility of systemic neutrophil scores [23]. Thus far, a limited number of studies have attempted this approach. Characterization of neutrophil polarization in different tumour types and stages is needed to maximize their prognosis significance and utility as potential therapeutic modalities [23]. Another question is whether parallel scoring of patient serum levels of neutrophil-activating and polarizing soluble mediators (IL-1β, IL-17, G-CSF, GM-CSF and/or TGFβ) increases the prognostic or predictive power of neutrophil measurement [23].
Neutrophil-targeting agents are being developed for the treatment of inflammatory and autoimmune diseases [23]. In patients with chronic obstructive pulmonary disease, CXCR2 antagonist decreases absolute neutrophil counts, reduces biological inflammation and disease symptoms [24]. Inhibition of CXCL8–CXCR1/2 signaling by CXCL8 antibodies, or small molecules targeting CXCR1 and/or CXCR2, also decreases tumor growth and progression in tumor mouse models [25]. CXCR2 inhibition in a preclinical metastatic breast cancer model enhanced response of both tumor and micrometastases to chemotherapy treatment [26]. Clinical trials evaluating reparixin, a CXCR1 and CXCR2 inhibitor, are ongoing in cancer patients [27]. Inhibitors of NE are also being tested and have shown some promise in mouse models of lung cancer [28] Another neutrophil-associated pathway under intense investigation is the IL-23 –IL-17 axis [23]. Inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis [29]. Targeting neutrophil Alox5 inhibition have been reported to limit metastatic progression in preclinical models [29].
A promising therapeutic approach is the combination of T cell checkpoint inhibitor immunotherapy with neutrophil manipulation [23]. Experimental studies have shown that anti-programmed cell death protein 1 (PD1) and anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) synergizes with anti-CXCR2 or anti-Ly6G, respectively, to delay tumour growth [30,31]. However, targeting neutrophils can be associated with side effects as neutrophils are critical for host defense against infection [25]. Alternative approach is the idea that neutrophil resolution of inflammation could be induced within the tumor microenvironment, possibly resolved by neutrophil reverse migration rather than whole neutrophil targeting all circulating neutrophils [25].
Finally, not all neutrophils have protumor effects. Tumor associated N2 neutrophils are characterized by high expression of CXCR4, VEGF, and gelatinase B/MMP9 and can be induced on exposure to high TGF-β levels [25]. By contrast, N1 neutrophils are induced on TGF-β blockade and express immunoactivating cytokines and chemokines and low levels of arginase and are able to kill cancer cells [25]. Understanding how neutrophils are polarized and if and how they can be reprogrammed will be crucial to developing successful cancer therapies [25]. TGF-β inhibitors in oncology have therefore moved towards the use of combinatorial therapies with considerable promise for the clinic [32]. Clinical trials are ongoing [33].
The present study is one of the largest locally-advanced cohort associating both leukocytosis and neutrophilia with OS in NSCLC to our knowledge [34,35]. The relatively small sample size and the retrospective nature of our study, despite a prospective patients registration through MSN study (NCT02105168), should be acknowledged as potential limitations. In addition, although there is a postulated pathological inflammatory mechanism in carcinogenesis, infection and concomitant corticosteroids intakes alters the prognostic utility of these parameters. However, the process of validation in a second independent case series, in addition with independent metastatic cohort, supports baseline leukocyte and neutrophil absolute counts as predictors of survival in patients with stage-III NSCLC.
Conclusion
This study recognized baseline leukocytosis and neutrophilia to be independent prognosis markers for OS, PFS and DMC in stage-III NSCLC. This accessible biomarker may guide patients' management.
Supporting information
S1 Fig. Estimated locoregional control in patients with or without leukocytosis.
https://doi.org/10.1371/journal.pone.0204490.s002
(TIF)
S2 Fig. Estimated locoregional control in patients with or without neutrophilia.
https://doi.org/10.1371/journal.pone.0204490.s003
(TIF)
S3 Fig. Estimated distant metastasis control in patients with or without leukocytosis.
https://doi.org/10.1371/journal.pone.0204490.s004
(TIF)
S4 Fig. Estimated distant metastasis control in patients with or without neutrophilia.
https://doi.org/10.1371/journal.pone.0204490.s005
(TIF)
S5 Fig. Estimated brain metastasis control in patients with or without neutrophilia.
https://doi.org/10.1371/journal.pone.0204490.s006
(TIF)
S6 Fig. Estimated brain metastasis control in patients with or without neutrophilia, in adenocarcinoma patients.
https://doi.org/10.1371/journal.pone.0204490.s007
(TIF)
S1 File. Data lung.csv.
Anonymized analyzed data.
https://doi.org/10.1371/journal.pone.0204490.s008
(CSV)
References
- 1. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, et al. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin 2016;66:271–89. pmid:27253694
- 2. Paesmans M, Sculier JP, Libert P, Bureau G, Dabouis G, Thiriaux J, et al. Prognostic factors for survival in advanced non-small-cell lung cancer: univariate and multivariate analyses including recursive partitioning and amalgamation algorithms in 1,052 patients. The European Lung Cancer Working Party. J Clin Oncol Off J Am Soc Clin Oncol 1995;13:1221–30. pmid:7738625
- 3. Carvalho S, Troost EGC, Bons J, Menheere P, Lambin P, Oberije C. Prognostic value of blood-biomarkers related to hypoxia, inflammation, immune response and tumour load in non-small cell lung cancer–A survival model with external validation. Radiother Oncol 2016;119:487–94. pmid:27139126
- 4. Ding N, Pang Z, Shen H, Ni Y, Du J, Liu Q. The Prognostic Value of PLR in Lung Cancer, a Meta-analysis Based on Results from a Large Consecutive Cohort. Sci Rep 2016;6:34823. pmid:27703265
- 5. Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, et al. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol 2013;8:823–59. pmid:23552377
- 6. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7. pmid:12490959
- 7. Kargl J, Busch SE, Yang GHY, Kim K-H, Hanke ML, Metz HE, et al. Neutrophils dominate the immune cell composition in non-small cell lung cancer. Nat Commun 2017;8:14381. pmid:28146145
- 8. Wilcox RA. Cancer-associated myeloproliferation: old association, new therapeutic target. Mayo Clin Proc 2010;85:656–63. pmid:20592171
- 9. Mabuchi S, Matsumoto Y, Isohashi F, Yoshioka Y, Ohashi H, Morii E, et al. Pretreatment leukocytosis is an indicator of poor prognosis in patients with cervical cancer. Gynecol Oncol 2011;122:25–32. pmid:21514632
- 10. Banerjee R, Roxin G, Eliasziw M, Joseph K, MacLean A, Buie WD, et al. The Prognostic Significance of Pretreatment Leukocytosis in Patients With Anal Cancer Treated With Radical Chemoradiotherapy or Radiotherapy: Dis Colon Rectum 2013;56:1036–42. pmid:23929012
- 11. Chen M-H, Chang PM-H, Chen P-M, Tzeng C-H, Chu P-Y, Chang S-Y, et al. Prognostic significance of a pretreatment hematologic profile in patients with head and neck cancer. J Cancer Res Clin Oncol 2009;135:1783–90. pmid:19551407
- 12. Garcia-Arias A, Cetina L, Candelaria M, Robles E, DueñAs-GonzáLez A. The prognostic significance of leukocytosis in cervical cancer. Int J Gynecol Cancer 2007;17:465–70. pmid:17309562
- 13. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 2009;9:162–74. pmid:19197294
- 14. Gros A, Turcotte S, Wunderlich JR, Ahmadzadeh M, Dudley ME, Rosenberg SA. Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma. Clin Cancer Res 2012;18:5212–23. pmid:22837179
- 15. Fridlender ZG, Sun J, Kim S, Kapoor V, Cheng G, Ling L, et al. Polarization of Tumor-Associated Neutrophil Phenotype by TGF-β: “N1” versus “N2” TAN. Cancer Cell 2009;16:183–94. pmid:19732719
- 16. Takeshima T, Pop LM, Laine A, Iyengar P, Vitetta ES, Hannan R. Key role for neutrophils in radiation-induced antitumor immune responses: Potentiation with G-CSF. Proc Natl Acad Sci 2016;113:11300–5. pmid:27651484
- 17. Schalper KA, Brown J, Carvajal-Hausdorf D, McLaughlin J, Velcheti V, Syrigos KN, et al. Objective Measurement and Clinical Significance of TILs in Non-Small Cell Lung Cancer. JNCI J Natl Cancer Inst 2015;107:dju435–dju435. pmid:25650315
- 18. Liang W, Ferrara N. The Complex Role of Neutrophils in Tumor Angiogenesis and Metastasis. Cancer Immunol Res 2016;4:83–91. pmid:26839309
- 19. Spiegel A, Brooks MW, Houshyar S, Reinhardt F, Ardolino M, Fessler E, et al. Neutrophils Suppress Intraluminal NK Cell-Mediated Tumor Cell Clearance and Enhance Extravasation of Disseminated Carcinoma Cells. Cancer Discov 2016;6:630–49. pmid:27072748
- 20. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging Biological Principles of Metastasis. Cell 2017;168:670–91. pmid:28187288
- 21. Erpenbeck L, Schön MP. Neutrophil extracellular traps: protagonists of cancer progression? Oncogene 2016. pmid:27941879
- 22. Koh YW, Choi J-H, Ahn MS, Choi YW, Lee HW. Baseline neutrophil–lymphocyte ratio is associated with baseline and subsequent presence of brain metastases in advanced non-small-cell lung cancer. Sci Rep 2016;6:38585. pmid:27924837
- 23. Coffelt SB, Wellenstein MD, de Visser KE. Neutrophils in cancer: neutral no more. Nat Rev Cancer 2016;16:431–46. pmid:27282249
- 24. Rennard SI, Dale DC, Donohue JF, Kanniess F, Magnussen H, Sutherland ER, et al. CXCR2 Antagonist MK-7123. A Phase 2 Proof-of-Concept Trial for Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2015;191:1001–11. pmid:25695403
- 25. Powell DR, Huttenlocher A. Neutrophils in the Tumor Microenvironment. Trends Immunol 2016;37:41–52. pmid:26700397
- 26. Acharyya S, Oskarsson T, Vanharanta S, Malladi S, Kim J, Morris PG, et al. A CXCL1 Paracrine Network Links Cancer Chemoresistance and Metastasis. Cell 2012;150:165–78. pmid:22770218
- 27. Coffelt SB, Wellenstein MD, de Visser KE. Neutrophils in cancer: neutral no more. Nat Rev Cancer 2016;16:431–46. pmid:27282249
- 28. Houghton AM, Rzymkiewicz DM, Ji H, Gregory AD, Egea EE, Metz HE, et al. Neutrophil elastase–mediated degradation of IRS-1 accelerates lung tumor growth. Nat Med 2010;16:219–23. pmid:20081861
- 29. Wculek SK, Malanchi I. Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature 2015;528:413–7. pmid:26649828
- 30. Kim K, Skora AD, Li Z, Liu Q, Tam AJ, Blosser RL, et al. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc Natl Acad Sci 2014;111:11774–9. pmid:25071169
- 31. Highfill SL, Cui Y, Giles AJ, Smith JP, Zhang H, Morse E, et al. Disruption of CXCR2-Mediated MDSC Tumor Trafficking Enhances Anti-PD1 Efficacy. Sci Transl Med 2014;6:237ra67–237ra67. pmid:24848257
- 32. Connolly EC, Freimuth J, Akhurst RJ. Complexities of TGF-β Targeted Cancer Therapy. Int J Biol Sci 2012;8:964–78. pmid:22811618
- 33. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, et al. Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther 2015;147:22–31. pmid:25444759
- 34. Kasuga I, Makino S, Kiyokawa H, Katoh H, Ebihara Y, Ohyashiki K. Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma. Cancer 2001;92:2399–405. pmid:11745296
- 35. Tomita M, Shimizu T, Hara M, Ayabe T, Onitsuka T. Preoperative leukocytosis, anemia and thrombocytosis are associated with poor survival in non-small cell lung cancer. Anticancer Res 2009;29:2687–90. pmid:19596947