Stesura Seveso 97Archivio Italiano di Urologia e Andrologia 2019; 91, 2 ORIGINAL PAPER Increased neutrophil/lymphocyte ratio in testicular cancer Aytaç Şahin, Tuncay Toprak, Musab Ali Kutluhan, Yasin Vural, Ahmet Ürkmez, Ayhan Verit SBU Fatih Sultan Mehmet Training and Research Hospital, Istanbul. Objective: Testicular cancers, which are less common than other cancers, are important in terms of being seen in young people. Physical examination, imaging, laboratory and tumor markers are used for diagnosis. There are some studies of some blood parameters that can be involved in inflammation and tumorogenesis. We retrospective- ly compared hematological values measured in our patients who were diagnosed with testicular tumor in comparison with patients with similar age group who underwent varicocelectomy repair. Materials and methods: This cross-sectional retrospective study included 120 patients who underwent radical inguinal orchiec- tomy for testicular tumor between January 2010 and December 2018, and 171 patients who underwent varicocelectomy as a control group. Patients with an active infection and hematologi- cal disorders were excluded from the study. We evaulated hematological parameters including neutrophil (NEU), lympho- cyte (LYM), platelet (PLT) count, and mean platelet volume. The study was conducted on 291 patients. divided in two groups: tumor (n = 120) and varicocele (n = 171). Results: There was no statistically significant difference between the groups in terms of PLT / lymphocyte ratio and mean platelet volume (MPV) levels (p > 0.05). The neutrophil /lymphocyte ratio (NLR) of the tumor group was significantly higher than the varicocele group (p = 0.001; p < 0.05). There was a statistically significant difference between the tumor stages in terms of PLT / Lymphocyte ratios (p = 0.006; p < 0.05). Conclusions: There was only a statistically significant increase in NLR values in the testicular tumor group compared to the varicocele group. Larger, randomized controlled studies are needed at this field. KEY WORDS: Testis; Cancer; Mean platelet volume (MPV); Neutrophil/lymphocyte ratio (NLR). Submitted 13 January 2019; Accepted 2 April 2019 Summary No conflict of interest declared. diagnosis more effective treatment schedules can be applied contributing to better survival. At this point, simple, inexpensive and easily applicable markers can be useful in the clinical approach. There are some studies that some blood parameters can be associated to inflam- mation and tumorogenesis. Studies have shown that inflammatory response is closely related to tumorigene- sis and tumor invasion (2). Interactions occur between the tumor and inflammation according to complex and various mechanisms. At each stage of carcinogenesis; inflammation has an important role (3). Changes in sys- temic inflammatory response can be assessed by hema- tological parameters. For example, changes in C-reactive protein (CRP) and neutrophil to lymphocyte ratio (NLR) show signs of systemic inflammatory response in various malignancies (4). There are also reports that elevated NLR is associated with poor prognosis in some urothelial cancers (5). The vast majority of studies have reported that the increase in NLR is associated with poor progno- sis in many malignant tumors. For this reason, NLR can be used not only as a marker of systemic inflammatory response, but also in various tumor types and inflamma- tory conditions (6). In order to predict cancer prognosis and inflammatory conditions, there is a growing interest in simple blood methods such as NLR. NLR, lymphocyte- monocyte ratio (LMR), platelet-lymphocyte ratio (PLR) and mean platelet volume (MPV) can be used as factors to determine the prognosis of patients in various clinical situations (7). Platelets are seedless cells derived from megakaryocytes in the bone marrow. Platelets, an ele- ment of the immune system, also play a role in cancer formation, progression and metastatization. It is known that activated platelets have critical roles in tumor prolif- eration, neoangiogenesis and release of mitogenic medi- ators in the microenvironment of cells that exhibit tumoral behavior, although their production, maturation and clearance from circulation are still not fully elucidat- ed (8). Yun ZY et al. Reported that decreased MPV may be a marker of poor prognosis in renal cell cancer (9). Because it is known that MPV is an index of bioactive platelets activated for any reason and incorporated into the inflammation process, rather than platelet count (10). These markers, which are easily applicable in prac- tice, were retrospectively analyzed in our patients who were diagnosed with testicular tumor and compared with the values of patients with similar age group of patients who underwent varicocelectomy repair. DOI: 10.4081/aiua.2019.2.97 INTRODUCTION Testicular cancers, which are less common than other cancers, are important because they are often seen in young people. It is the most common solid organ cancer in men between the ages of 15-35 while it contitutes 1- 1.5% of all male cancers. In developed countries there is an increase incidence for testicular cancer (1). Both testes can be easily examined and results of early diag- nosis of testicle tumours are very favorable enhancing the importance of early diagnosis and treatment of tes- ticular tumors. Physical examination, imaging, laborato- ry and tumor markers are used for diagnosis. With early Archivio Italiano di Urologia e Andrologia 2019; 91, 2 A. Şahin, T. Toprak, M. Ali Kutluhan, Yasin Vural, A. Ürkmez, A. Verit 98 MATERIALS AND METHODS This cross-sectional retrospective study included 120 patients who underwent radi- cal inguinal orchiectomy for testicular tumor between January 2010 and December 2018, and 171 patients who underwent varicocelectomy as a control group. Patients with acute infections, chronic inflammatory disease, malignancies or hematological disorders, those using anti- coagulant treatment, and subjects with a history of hormonal treatment in the last 12 months or blood product administration in the last month were excluded. Hematological parameters were evaluated with peripheral blood samples taken preop- eratively. These hematological parameters include neu- trophil (NEU), lymphocyte (LYM), platelet count, and mean platelet volume (MPV). The staging of patients with testicular tumors was performed by examining the computed tomography and by measurement of beta human chorionic gonadotropin, alpha feto- protein and lactate dehydrogenase (LDH) as tumor markers. Statistical analysis To evaluate the findings obtained in this study, IBM SPSS Statistics 22 for statistical analysis (SPSS IBM, Turkey) program was used. Conformity of the parameters to the normal distribution was evaluated by the Shapiro Wilks test. Descriptive sta- tistical values were computed (mean, standard deviation, frequency) and the comparison of quantitative data were done by Kruskal Wallis test and the Mann Whitney U test was used for the determination of the group causing the difference. Mann-Whitney U test was used for the two-group comparisons of the parameters that did not show normal distribution, and Student's t test was used for the parameters with normal distribution. The cut-off point was chosen based on the ROC curve analysis. A p < 0.05 was considered significant. RESULTS The study was conducted on 291 patients with ages ranging from 1 to 85 years. The mean age was 34.25 ± 16.56 years. The cases were divided into two groups: tumor (n = 120) and varicocele (n = 171). There was no statistically significant difference between the groups in terms of PLT/lymphocyte ratio and MPV levels (p > 0.05). The neutrophil/lymphocyte ratio of the tumor group was significantly higher than the varicocele group (p = 0.001; p < 0.05) (Table 1, Figure 1). There was a statistically significant difference between the tumor stages in terms of PLT/Lymphocyte ratios (p = 0.006; p < 0.05). Paired comparisons demonstrated that PLT/lymphocyte ratio of pT3 group was significantly higher than pT1 and pT2 (p1 = 0.002; p2 = 0.003; p < 0.05). There was no significant difference between pT1 and pT2 stages (p > 0.05). There was no statistically significant difference in neu- trophil/lymphocyte ratio and MPV levels between tumor stages (p > 0.05) (Table 2). The ROC curve for neutrophil/lymphocyte ratio (NLR) was plotted in the diagnosis of testicular tumor. The area Table 1. Evaluation of groups in terms of PLT/Lymphocyte, Neutrophil/Lymphocyte ratio and MPV. Tumor Varicocele p Mean ± SD (median) Mean ± SD (median) PLT/lymphocyte 128.91 ± 95.19 (110.4) 125.45 ± 63.3 (110.8) 0.9071 Neutrophil/lymphocyte 4.22 ± 3.54 (3.5) 3.49 ± 2.79 (2.7) 0.001*, 1 MPV 8.05 ± 1.46 8.28 ± 1.56 0.2142 1 Mann Whitney U Test; 2 Student t test; * p < 0.05. Table 2. Evaluation of groups in terms of PLT/Lymphocyte, Neutrophil/Lymphocyte ratio and MPV. pT1 (n = 60) pT2 (n = 43) pT3 (n = 5) p Mean ± SD (median) Mean ± SD (median) Mean ± SD (median) PLT/Lymphocyte 115.57 ± 58.01 (108.1) 140 ± 135.35 (110.4) 231.27 ± 74.21 (212.5) 0006* Neutrophil/lymphocyte 3.83 ± 2.58 (3.4) 4.62 ± 4.94 (3.5) 5.78 ± 1.87 (6.3) 0.108 MPV 8.26 ± 1.62 (7.9) 7.92 ± 1.19 (7.8) 7.24 ± 0.82 (7.2) 0.107 Kruskal Wallis Test; * p < 0.05. NOTE: Since the number of patients with pT3 was 5, Kruskal Wallis test was used despite the normal distribution of MPV. Figure 1. Stone expulsion duration in the groups. under the curve is 0.612 and the standard deviation is 0.03. The area under the ROC curve was significantly higher than 0.5 (p = 0.001; p < 0.05). The cut-off point for NLR in the diagnosis of testicular tumor is > 3.16. The sensitivity of this value was 63.87% and the speci- ficity was 63.16% (Figure 2). DISCUSSION Inflammation plays an important role in tumor develop- ment and progression. The relationship between inflam- mation and cancer has long been known. In 1863, Virchow put forward the hypothesis that cancer occurs in the areas of chronic inflammation, and that some irritants increase cell proliferation along with inflammation leading to tissue injury (11). Although the effect of this proliferation is clear, cells alone do not cause cancer. Continuous cell proliferation, inflammatory cells, growth factors, activated stroma and DNA-damage enhancing agents increase or promote neoplastic risk. Neutrophils mediate inflammation through various bio- chemical mechanisms such as release of arachidonic acid metabolites and platelet aggravating factors (12). Neutrophilia could represent a consequence of ectopic production of myeloid growth factors as part of a para- neoplastic syndrome (13) or, more likely, a nonspecific response to cancer-related inflammation secondary to tissue destruction and cytokine releases. Lymphopenia is associated with cortisol induced stress response (12). High NLR occurring as a result of the added effect of increased NEU response to LYM suppression can sup- port the development of cancer by inhibiting the antitu- mor immune response (14). Experimental data have shown that active neutrophils can stimulate tumor growth directly and indirectly (15). NLR and platelet/lymphocyte ratio (PLR) have also been shown to be reliable markers of systemic inflammation by many studies (16). According to the type of malig- nancy, inflammatory and immune responses to systemic tumor cells and secreted peptides can vary. Today, sys- temic inflammatory response indicators such as cytokine, CRP, albumin, serum amyloid A and leuko- cytes have gained importance in the patients with malig- nancy and it has been thought that they can be inde- pendent prognostic factors (17). The immune system has a positive and negative effect on cancer development and progression. It can eliminate tumor cells or increase the metastatic ability and invasion capacities of active malig- nant cells, leading to tumor progression. The excess of circulating NEUs is thought to play an important role in tumor progression and angiogenesis. Therefore, increased number of NEUs should be associ- ated with poor prognosis (18). MPV represents the mean platelet size in the blood. It can be altered in various dis- eases such as cancer, thrombosis, sepsis, respiratory dis- tress syndrome, and acute appendicitis (19). PLTs are frequently observed in the cancer microenvironment and are thought to stimulate proliferation and transformation of cancer cells by platelet derived growth factor (PDGF) release (20). In the study of Russell et al., It was reported that increased PDGF alpha receptor expression was asso- ciated with bone metastasis in castration-resistant PCa (21). Even in the current literature, anti-platelet therapy has been reported to have a role in PCa adjuvant thera- py (22). MPV measurement is a useful method in deter- mining the presence of these activated PLTs (9). A high MPV means that your platelets are larger than average. This is sometimes a sign that you're producing too many platelets. Platelets are produced in the bone marrow and released into the bloodstream. Larger platelets are usual- ly young and more recently released from the bone mar- row. Smaller platelets are more likely to have been in cir- culation for a few days. When someone has a low platelet count and a high MPV level, it suggests that the bone marrow is rapidly pro- ducing platelets. This may be because older platelets are being destroyed, so the bone marrow is trying to com- pensate. Increased MPV is associated with platelet acti- vation, which can happen when platelets encounter tumor byproducts. Still, a high MPV doesn't mean you have cancer. The diagnostic role of mean platelet volume (MPV) is reported in various malignant tumors such as ovary (23), pancreas (24), and colon (25) cancers, the diagnostic and prognostic role of MPV cannot be pre- cisely demonstrated for testicular tumors. In a study conducted by Gokcen K et al., 36 patients with testicular tumors were investigated. WBC, NEU, PLR, and NLR values were significantly higher in testicular tumors however MPV was significantly lower than the control group p < 0.05). Also differences between hematological parameters of patients with testicular cancer according to the stages were examined, and differences were observed between mean corpuscular volume (MCV), mean corpuscu- lar hemoglobin (MCH) and mean platlet volume (MPV) (p < 0.05). MCV was significantly higher in Stage 1 compared 99Archivio Italiano di Urologia e Andrologia 2019; 91, 2 Increased neutrophil/lymphocyte ratio in testicular cancer Figure 2. ROC curve for NLR in the diagnosis of testicular tumor. Cut off point determination for NLR in the diagnosis of testicular tumors. Archivio Italiano di Urologia e Andrologia 2019; 91, 2 A. Şahin, T. Toprak, M. Ali Kutluhan, Yasin Vural, A. Ürkmez, A. Verit 100 to Stage 2 or 3 tumour (p = 0.035 and p = 0.025, respec- tively). MCH was significantly higher in Stage 1 com- pared to Stage 3 (p = 0.022). MPV was significantly lower in Stage 1 compared to Stage 3 (p = 0.016) (26). In our study, the neutrophil/lymphocyte ratio of the tumor group was significantly higher than the varicocele group (p = 0.001; p < 0.05), but there was no statistically sig- nificant difference between the groups in terms of PLT/lymphocyte ratio and MPV levels (p > 0.05). On the other hand we found that there was no statistically sig- nificant difference between the tumor stages in terms of Neutrophil/lymphocyte ratio and MPV levels (p > 0.05). In contrast, there was a statistically significant difference in terms of PLT/lymphocyte ratios (p: 0.006; p < 0.05). As a result of paired comparisons PLT/lymphocyte ratio of pT3 group was significantly higher than pT1 and pT2 (p1: 0.002; p2: 0.003; p < 0.05). There was no significant difference between pT1 and pT2 stages (p > 0.05). Limited numbers of reports are available on immune resistance in patients with testicular cancer. Considerable evidence supports the view that the bio- logical behavior of tumors and in particular, their capac- ity to metastasize are in part determined by immunolog- ical factors requiring participation of T lymphocytes, B lymphocytes, macrophages and natural killer cells. Immunological reactivity has been analyzed in a wide spectrum of solid tumors and a vast literature indicates a correlation between depressed cell-mediated immunity and the stage of the disease. On the contrary, there is little evidence about the role of immunological factors in the development and spread of testicular tumors. CONCLUSIONS In this study, there was only statistically significant increase in NLR values in the testicular tumor group compared to the varicocele group. There was no statistically significant result for MPV and PLR. In the evaluation of patients with testicular tumors according to their stages, the PLT/lym- phocyte ratio of the pT3 group was found to be signifi- cantly higher than the pT1 and pT2 stages. Although there are many studies on hematological parameters related to other cancers, there is limited data for testicular tumors in the literature. The limitations of our study were that it was a retrospec- tive one with limited study group and had not a prognos- tic predictive design. Larger, randomized controlled stud- ies are needed at this field. REFERENCES 1. Borghesi M, Brunocilla E, Schiavina R, et al. Role of testis spar- ing surgery in the conservative management of small testicular masses: oncological and functional perspectives. Actas Urol Esp. 2015; 39:57-62. 2. Gregory AD, Houghton AM. Tumor associated neutrophils: New targets for cancer therapy. Cancer Res. 2011; 71:24116. 3. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140:88399. 4. Duan H, Zhang X, Wang FX, et al. Prognostic role of neutrophil lymphocyte ratio in operable esophageal squamous cell carcinoma. World J Gastroenterol. 2015; 21:55917. 5. Viers BR, Boorjian SA, Frank I, et al. Pretreatment neutrophil to lymphocyte ratio is associated with advanced pathologic tumor stage and increased cancer specific mortality among patients with urothe- lial carcinoma of the bladder undergoing radical cystectomy. Eur Urol. 2014; 66:115764. 6. Templeton AJ, McNamara MG, Šeruga B, et al. Prognostic role of neutrophil to lymphocyte ratio in solid tumors: A systematic review and metaanalysis. J Natl Cancer Inst. 2014; 106:dju124. 7. Lee JS, Kim NY, Na SH, et al. Reference values of neutrophil-lym- phocyte ratio, lymphocyte-monocyte ratio, platelet-lymphocyte ratio, and mean platelet volume in healthy adults in South Korea. Medicine (Baltimore) 2018; 97:e11138. 8. Goubran HA, Stakiw J, Radosevic M, et al. Platelet-cancerinter- actions. Semin ThrombHemost. 2014; 40:296-305. 9. Yun ZY, Zhang X, Liu ZP, et al. Association of decreased mean platelet volume with renal cell carcinoma. Int J Clin Oncol. 2017; 22;1076-1080. 10. Gasparyan AY, Ayvazyan L, Mikhailidis DP, et al.Meanplateletvolume: a link between thrombosis and inflamma- tion. Curr Pharm Des. 2011; 17:47-58. 11. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001; 357:539-545. 12. Tamhane UU, Aneja S, Montgomery D, et al. Association between admission neutrophil to lymphocyte ratio and outcomes in patients with acute coronary syndrome. Am J Cardiol. 2008; 102:653-7. 13. Vassilatou E, Fisfis M, Morphopoulos G, et al. Papillary thyroid carcinoma producing granulocyte-macrophage colony-stimulating factor is associated with neutrophilia and eosinophilia. Hormones (Athens). 2006; 5:303-9. 14. Schaider H, Oka M, Bogenrieder T, et al. Differential response of primary and metastatic melanomas to neutrophils attracted by IL 8. Int J Cancer. 2003; 103:33543 15. Fridlender ZG, Sun J, Kim S et al. Polarization of tumor-asso- ciated neutrophil phenotype by TGF-beta: “N1”versus “N2” TAN. Cancer Cell. 2009; 16:183-94. 16. Guthrie GJ, Charles KA, Roxburgh CS, et al. The systemic inflammation-based neutrophil lymphocyte ratio: experience in patients with cancer. Crit Rev Oncol Hematol. 2013; 88:218-30. 17. Moore MM, Chua W, Charles KA, Clarke SJ. Inflammation and cancer: Causes and consequences. Clin Pharmacol Ther. 2010; 87:5048. 18. Kusumanto YH, Dam WA, Hospers GA, et al. Platelets and granulocytes, in particular the neutrophils, form important com- partments for circulating vascular endothelial growth factor. Angiogenesis. 2003; 6:2837. 19. Albayrak Y, Albayrak A, Albayrak F, et al. Mean platelet vol- ume: a new predictor in confirming acute appendicitis diagnosis. Clin Appl Thromb Hemost. 2011; 17:362-6. 20. Ustach CV, Taube ME, Hurst NJ, et al. A potential oncogenic activity of platelet-derived growth factor d in prostate cancer pro- gression. Cancer Res. 2004; 64:1722-9. 21. Russell MR, Liu Q, Fatatis A. Targetingthe {alpha} receptor for platelet-derived growth factor as a primary or combination therapy in a preclinical model of prostate cancer skeletal metastasis. Clin Cancer Res. 2010; 16:5002-10. 22. Mezouar S, Frere C, Darbousset R, et al. Role of platelets in can- cerandcancer-associated thrombosis: Experimental and clinical evi- dences. Thromb Res. 2016; 139: 65-76. 23. Kemal Y, Demirag G, Ekiz K, et al. Mean platelet volume could be a useful biomarker for monitoring epithelial ovarian cancer. J Obstet Gynaecol. 2014; 34:515-8. 24. Karaman K, Bostanci EB, Aksoy E, et al. The predictive value of mean platelet volume in differential diagnosis of non functional pan- creatic neuroendocrine tumors from pancreatic adenocarcinomas. Eur J Intern Med. 2011; 22:e95-8. 25. Kilincalp S, Çoban S, Akinci H, et al. Neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, and mean platelet volume as poten- tial biomarkers for early detection and monitoring of colorectal ade- nocarcinoma. Eur J Cancer Prev 2015; 24:328-3. 26. Gokcen K, Dundar G, Gulbahar H, et al. Can routine peripher- al blood counts like neutrophil to lymphocyte ratio be beneficial in prediagnosis of testicular cancer and its stages? J Res Med Sci. 2018; 23:64. 101Archivio Italiano di Urologia e Andrologia 2019; 91, 2 Increased neutrophil/lymphocyte ratio in testicular cancer Correspondence Aytaç Şahin MD (Corresponding Author) draytacsahin@gmail.com Tuncay Toprak, MD drtuncay55@hotmail.com Musab Ali Kutluhan, MD dr.musab151@hotmail.com Yasin Vural, MD yasin_vural@windowslive.com Ahmet Urkmez, MD ahmeturkmez@hotmail.com Ayhan Verit, Prof. veritayhan@yahoo.com Urology Clinic SBU Fatih Sultan Mehmet Training and Research Hospital Atasehir, İstanbul 34752 Turkey