lymphocyte ratio predicts poor clinical outcome in soft tissue sarcoma patients

The American Journal of Surgery (2015) 210, 111-116

Clinical Science

The derived neutrophil/lymphocyte ratio predicts poor clinical outcome in soft tissue sarcoma patients Joanna Szkandera, M.D.a,b,*, Armin Gerger, M.D.a,b, Bernadette Liegl-Atzwanger, M.D.c, Michael Stotz, M.D.a,b, Hellmut Samonigg, M.D.a, Joerg Friesenbichler, M.D.d, Tatjana Stojakovic, M.D.e, Andreas Leithner, M.D.d, Martin Pichler, M.D.a a

Division of Clinical Oncology, Department of Medicine, bResearch Unit Genetic Epidemiology and Pharmacogenetics, Division of Clinical Oncology, Department of Medicine, cInstitute of Pathology, d Department of Orthopaedic Surgery and eClinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria KEYWORDS: Derived neutrophil/ lymphocyte ratio; Soft tissue sarcoma; Prognostic marker; Inflammation

Abstract BACKGROUND: Inflammation plays an important role in tumor proliferation and survival in cancer patients. The aim of this study was to investigate the prognostic impact of the pre-operative–derived neutrophil/lymphocyte ratio (dNLR) in a large cohort of soft tissue sarcoma (STS) patients after curative surgical resection. METHODS: The impact of preoperative dNLR on disease-free survival (DFS) and overall survival (OS) in retrospectively evaluated 340 STS patients was assessed using Kaplan–Meier curves and Cox proportional models. RESULTS: Applying receiver operating curve analysis, we determined a cut-off value of 2.39 for the dNLR to be optimal for discrimination of patients’ survival in the whole cohort. Kaplan–Meier curves revealed a dNLR greater than or equal to 2.39 as a marker for decreased DFS (P 5 .031) and OS (P 5 .007, log-rank test) in STS patients. In multivariate analysis, increased dNLR was significantly associated with poor OS (hazard ratio 1.60, 95% confidence interval 1.07 to 2.40, P 5 .022). CONCLUSIONS: This study demonstrates that preoperative dNLR might represent a well-correlated surrogate marker for the widely validated NLR. The dNLR is easily obtainable and can provide important information for individual risk assessment in clinical trials. Ó 2015 Elsevier Inc. All rights reserved.

The authors declare no conflicts of interest. * Corresponding author. Tel.: 143-316-385-82025; fax: 143-316-38513355. E-mail address: [email protected] Manuscript received April 17, 2014; revised manuscript September 22, 2014 0002-9610/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjsurg.2014.10.021

Soft tissue sarcomas (STSs) are a heterogeneous group of tumors that arise predominantly from the embryonic mesoderm.1 STS has more than 70 distinct histological subtypes, with leiomyosarcoma, liposarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, and myxofibrosarcoma being among the most common subtypes.2

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STS occurs rarely and accounts for approximately 1% of malignancies in adults and 2% of cancer mortality.3,4 The treatment of STS is largely dictated by tumor size, histologic grade, histologic subtype, tumor depth and site, and age at diagnosis.5 Despite improvements in local control rates with wide local resections and radiation therapy, metastasis and death remain a significant problem in 50% of patients who present with high-risk STSs.1 Patients typically demonstrate a median survival ranging from 11 to 18 months from diagnosis of advanced disease.6,7 Hence, readily available and economically feasible clinical tools to identify cancer patients at high risk of tumor recurrence and death are required for improving survival data. Inflammation is a critical component of tumor progression. It is now becoming clear that the tumor microenvironment, which is largely orchestrated by inflammatory cells, is an essential participant in the neoplastic process, promoting proliferation, survival, and migration of tumor cells.8,9 In recent years, there has been raising interest in the use of systemic inflammatory markers as prognostic factors in malignancies. Particularly, the neutrophil/lymphocyte ratio (NLR) represents a marker of systemic inflammatory response and has been widely reported as a prognostic factor in various types of cancer, including colon cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, and STS.10–14 However, in most clinical trials, only white cells and neutrophil counts without specification of the absolute lymphocyte count have been documented. To offer an inflammation-based biomarker for these clinical trials, Procter et al recently developed a so-called derived NLR (dNLR), which consists of neutrophil count divided by (white cell count minus neutrophil count). The authors of this study evaluated the prognostic value of the dNLR on clinical outcome in different cancer types, and demonstrated that the dNLR had similar prognostic value to the well-established NLR.15 To the best of our knowledge, no study has been previously published with a special focus on the prognostic value of the dNLR in STS patients. Therefore, the aim of this study was to evaluate the prognostic impact of the preoperative dNLR on disease-free survival (DFS) and overall survival (OS) in a large cohort of STS patients to decipher the usefulness of this potential prognosticator in clinical trials.

Clinical Oncology, Medical University of Graz, providing follow-up examinations in regular intervals (3-month intervals in years 1 to 3, 6-month intervals in years 4 to 5, and 12-month intervals in years 6 to 15 after diagnosis). The laboratory data, including preoperative blood neutrophil and leukocyte count, were obtained by preoperative determination 1 to 3 days before surgery was performed. Follow-up investigations included clinical check-up and radiological analyses (computed tomography alternating with X-ray of the chest, local magnetic resonance imaging, and abdominal ultrasound). Clinicopathological data including histopathological diagnosis and tumor grade were retrospectively obtained from the patient’s history. For this study, all histological specimens were centrally reviewed by an independent experienced soft tissue pathologist (B.L.A.). All sarcomas were diagnosed according to the current World Health Organization classification of soft tissue and bone tumors.2 Tumors were graded according to the French Federation of Cancer Centres Sarcoma Group grading system if possible or tumor grade was defined by tumor entity.16 Malignant fibrous histiocytomas have been reclassified according to the current diagnostic criteria.2,17 This study has been approved by the Institutional Review Board of the Medical University of Graz (25-050 ex 12/13).

Patients and Methods Subjects Three hundred forty patients with histologically confirmed STS and available laboratory parameters, who have been operated between March 1998 and August 2013 at the Department of Orthopaedic Surgery, Medical University of Graz, were included in this retrospective study. Follow-up was performed until September 2013. All patients were included in the follow-up program of the Department of Orthopaedic Surgery and the Division of

Statistical analyses The primary endpoint of this study was OS, which was calculated from the date of diagnosis to the date of death from any cause. The secondary endpoint was DFS (time between diagnosis and local recurrence or distant metastases). We seek an ideal cut-off value for the continuous dNLR by applying receiver operating curve analysis as previously reported.18 The relationship between dNLR and other clinicopathological parameters was studied by nonparametric tests (chi-square test and Mann–Whitney U test). Patients’ clinical endpoints were calculated using Kaplan–Meier curves and compared by the log-rank test. Backward stepwise multivariate Cox proportion analysis was performed to determine the influence of age, sex, tumor grade, tumor site and size, and dNLR on DFS and OS. Hazard ratios estimated from the Cox analysis were reported as relative risks with corresponding 95% confidence intervals. All statistical analyses were performed using the Statistical Package for Social Sciences version 20.0 (SPSS, Inc, Chicago, IL). A 2-sided P value of less than .05 was considered statistically significant.

Results Overall, there were 175 male and 165 female patients with STS. Two hundred nineteen patients had grade 3 sarcomas according to the French Federation of Cancer Centres Sarcoma Group grading system, 62 patients were histologically classified as having grade 1, and 59 patients had grade

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Table 1 The relation between clinicopathological parameters and pre-operative–derived neutrophil/lymphocyte ratio of patients with soft tissue sarcoma (n 5 340) Characteristics Age at operation (years) ,65 R65 Sex Female Male Tumor depth Superficial Deep Tumor grade G1 1 G2 G3 Tumor size (cm) ,5 R5 Tumor site Upper extremity Lower extremity Thoracic/trunk Retroabdominal/intraabdominal Head/neck

dNLR , 2.39

dNLR R 2.39

P value

138 86

58 58

.040

108 116

57 59

.872

85 139

28 88

.010

85 139

36 80

.207

63 161

24 92

.328

60 129 30 2

23 66 23 3

.268

3

1

dNLR 5 derived neutrophil/lymphocyte ratio.

2 tumors. The 340 patients were histologically classified as follows: 100 myxofibrosarcomas, 80 liposarcomas, 35 leiomyosarcomas, 29 synovial sarcomas, 11 malignant peripheral nerve sheath tumors, and 85 other histological subtypes. The primary tumor sites were localized at the upper extremities (n 5 83), lower extremities (n 5 195), thorax/trunk (n 5 53),

retroperitoneal/intra-abdominal (n 5 5), and head/neck (n 5 4). The tumor depth was defined as superficial in 113 patients and deep in 227 patients, respectively. First, we correlated the dNLR with the NLR and found a highly significant correlation for both these parameters (Spearman correlation .930, P , .001). Applying receiver operating curve analysis, we determined a cut-off value of 2.39 as optimal to discriminate between survival and death. Consequently, we separated our STS patients into 2 groups according to low dNLR (,2.39) or high dNLR (R2.39) and tested the association between preoperative dNLR and other clinicopathological factors. High dNLR was significantly associated with older age and deep tumor location (P , .05), whereas no association with sex and tumor grade, size, and site could be found (Table 1). Of the 340 STS patients, 69 (20.3%) developed disease recurrence within the follow-up period. Twenty patients (6%) presented with local recurrence. Overall, 98 (28.8%) patients died of any cause. Among the 340 STS patients, disease recurrence was diagnosed in 39 of the 224 (17.4%) patients with low and in 30 of the 116 (25.9%) patients with high dNLR (P 5 .031). Overall deaths occurred in 54 (24.1%) patients with low and in 44 (37.9%) patients with high dNLR, respectively (P 5 .007). Fig. 1 shows the Kaplan–Meier curve for DFS and reveals that dNLR greater than or equal to 2.39 is a consistent factor for decreased DFS in STS patients (P 5 .031, log-rank test). Additionally, we demonstrated that dNLR greater than or equal to 2.39 is significantly associated with a reduced OS in our study cohort (P 5 .007, log-rank test; Fig. 2). To determine the independent prognostic value of the dNLR for DFS and OS in relation to well-established prognostic factors, multivariate analyses using Cox proportional models were performed. In the multivariate analysis that included age, sex, tumor grade, depth, size, and site, and the dNLR, we identified tumor grade (P 5 .039) and tumor size (P 5 .025) as independent prognostic factors for DFS, whereas dNLR showed no statistically significant value (P 5 .082) (Table 2). For OS, tumor grade (P , .001) and dNLR (P 5 .022) were identified as independent prognostic factors for poor clinical outcome (Table 2).

Comments

Figure 1 Kaplan–Meier curve for disease-free survival regarding high (R2.39) versus low (,2.39) dNLR ratio. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

In this study, we demonstrate for the first time that determination of preoperative dNLR can be useful to predict OS in a large cohort of STS patients. To date, several studies have evaluated the influence of neutrophils and lymphocytes on the prognosis of various types of cancer, and reported a poor clinical outcome in patients with an elevated NLR.10–14 In STS patients, we recently showed that elevated preoperative NLR in the peripheral blood is associated with decreased time-torecurrence and OS in 260 STS patients following curative surgery.14 Accordingly, a smaller study suggested an association between the NLR and OS in STS, also reporting

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Figure 2 Kaplan–Meier curve for overall survival regarding high (R2.39) versus low (,2.39) dNLR ratio. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

worse outcome in patients with elevated pretreatment NLR.19 However, in many clinical trials, only leucocytes and neutrophil counts are documented. Therefore, Proctor et al investigated the prognostic value of the dNLR, which is mainly derived from the count of neutrophils and lymphocytes, in a population-based database of patients with different cancer types. The authors reported that the

Table 2

dNLR has a comparable effect on cancer prognosis as the highly correlated NLR, showing a poor clinical outcome in patients with elevated dNLR.15 However, before a prognostic marker can be implemented into a clinical setting for patients’ surveillance or recruitment strategies to treatment modalities, an independent external validation is a prerequisite. The significance of the external validation of prognostic factors has been extensively discussed by Altmann and Royston, and its importance before implementation into clinical practice has been underlined by Bleeker et al and others.20–22 In line with the findings by Proctor et al, Absenger et al reported that the dNLR may be an independent prognostic marker for time-to-recurrence and OS in patients with stage II and III colon cancer.15,18 Furthermore, Troppan et al23 validated the prognostic value of dNLR in a large cohort of 290 diffuse large B-cell lymphoma patients and found an independent significant association between high dNLR and decreased 5-year DFS and 5-year OS. In patients with upper tract urothelial carcinoma, a high pretreatment dNLR showed subsequently higher cancerspecific as well as overall mortality after surgery compared with those with a low pretreatment dNLR.24 To the best of our knowledge, in STS patients, such an external validation of the dNLR has not been reported. In this study, we determined a cut-off value of 2.39 for the dNLR to be optimal for our study cohort of 340 STS patients and demonstrated for the first time that elevated dNLR correlates significantly and independently with poor OS. Taken together, various reports underline the prognostic impact of dNLR in different types of cancer.15,18,23,24 In the

Multivariate Cox proportional analysis regarding disease-free survival and overall survival

Parameter Age at operation (years) ,65 R65 Sex Female Male Tumor site Extremities Trunk Tumor depth Superficial Deep Tumor grade G1 1 G2 G3 Tumor size (cm) ,5 R5 dNLR ,2.39 R2.39

Multivariate analysis for DFS

Multivariate analysis for OS

HR (95% CI)

P value

HR (95% CI)

P value

1 (referent) 1.35 (.83–2.19)

.231

1 (referent) 1.48 (.98–2.22)

.061

1 (referent) .96 (.59–1.56)

.874

1 (referent) .83 (.55–1.26)

.380

1 (referent) 1.05 (.80–1.40)

.713

1 (referent) 1.00 (.78–1.28)

.993

1 (referent) .98 (.57–1.68)

.939

1 (referent) 1.25 (.77–2.01)

.368

1 (referent) 1.77 (1.03–3.03)

.039

1 (referent) 3.79 (2.20–6.52)

1 (referent) 2.17 (1.10–4.26)

.025

1 (referent) 1.74 (.99–3.02)

.051

1 (referent) 1.54 (.95–2.51)

.082

1 (referent) 1.60 (1.07–2.40)

.022

,.001

CI 5 confidence interval; DFS 5 disease-free survival; dNLR 5 derived neutrophil/lymphocyte ratio; HR 5 hazard ratio; OS 5 overall survival.

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dNLR and cancer prognosis

last decade, extensive efforts have been made to identify molecular biomarkers, which predict clinical outcome in cancer patients, but excessive costs and technical factors preclude their clinical use.25,26 In contrast, dNLR represents an easily determinable, cost-effective, and highly reproducible marker that components are frequently measured in the routinely tested blood count panel. The easily accessible peripheral blood is more and more attractive for clinically useful biomarkers, as the measurement is relatively noninvasive.27 Blood-based biomarkers seem to be helpful for prognosis purposes, which might assist clinicians to adopt follow-up schedules and therapeutic strategies for high-risk patients. The results of this study indicate that STS patients with high preoperative dNLR might be considerable as candidates for additional, more aggressive treatment approaches or more stringent preventive strategies, but this has to be validated in prospective clinical trials. The mechanisms underlying this association remain speculative. The dNLR is postulated as an indicator of the inflammatory status.15 Neutrophils are the most common leucocyte subset in the bloodstream. According to previous published literature, the role of leucocytes, specifically the neutrophils, in cancer progression is widely discussed. Tumor cells produce various cytokines and chemokines, such as interleukin-6, interleukin-8, granulocyte colony-stimulating factor and macrophage migration inhibitory factor, which activate and recruit neutrophils from the peripheral blood into the tumor stroma, stimulating tumor progression.28–30 Neutrophils, in turn, promote tumor growth and angiogenesis by releasing several mediators, such as vascular endothelial growth factor and matrix metalloproteinase-9, that were demonstrated to induce a switch into an angiogenic state in tumor cells.31,32 Furthermore, neutrophils have also been shown to generate reactive oxygen species, nitric oxide, and arginase, which interfere with the T-cell function.33,34 Inflammatory cells, such as neutrophils, have essential effects on tumor development. In the neoplastic process, these cells are powerful tumor promoters, producing a stimulating tumor microenvironment that allows a more aggressive tumor behavior by facilitating genomic instability and favoring tumor growth and microspread, ultimately supporting tumor progression.8 On the other hand, it is widely accepted that lymphocytes are crucial for providing cancer immune surveillance.35,36 It has been reported that increased numbers of lymphocytes within the tumor stroma are associated with better survival in several malignancies, indicating that the immune system may initially induce a tumorrelated immune response in controlling the growth of the tumor.37–39 However, it remains unclear why the immune response against the tumor is not sustained. A possible explanation is that immune escape often develops during tumor progression and tumor-specific lymphocyte response might be inhibited.40 Taken together, these data suggest that the ratio of neutrophils

115 to lymphocytes reflects the status of homeostasis between cancer progression and antitumor activity. This study has certain limitations. It is limited by its retrospective nature and a mixture of various histologic types of STS. For the endpoint DFS, we did not observe a significant difference in the multivariate analysis. Comorbidities including cardiovascular, metabolic, or infectious diseases are also influenced by the leucocytes and neutrophil counts, which logically are reflected by the OS endpoint.41 On the other hand, in addition to the biological variables, from the statistical viewpoint, we observed a higher event rate in the OS endpoint than in the DFS endpoint. The P value in univariate as well as multivariate analyses for DFS is rather borderline with regard to the significance level of P equal to .05. A mathematical explanation for these findings is also the lack of power to detect significant differences for the rare number of events in disease recurrence. In conclusion, our study provides the first evidence that a high dNLR, calculated on the basis of leucocyte and neutrophil counts, is an independent prognostic factor for reduced OS in a large cohort of STS patients. Our results need further prospective evaluation in a larger population to validate the prognostic value of this inflammatory biomarker.

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