Introduction. Acta Medica Mediterranea, 2018, 34: 1383 FERYAL KARACA 1, CIGDEM USUL AFSAR 2 1

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1 Acta Medica Mediterranea, 2018, 34: 1383 THE EFFECT OF PRETREATMENT NEUTROPHIL/LYMPHOCYTE RATIO AND PLATELET/LYM- PHOCYTE RATIO ON PATHOLOGICAL RESPONSE AND SURVIVAL IN PATIENTS WITH LOCALLY ADVANCED RECTAL CANCER FERYAL KARACA 1, CIGDEM USUL AFSAR 2 1 Health Sciences University, Adana Numune Education and Research Hospital, Department of Radiation Oncology, Adana, Turkey - 2 Acibadem Mehmet Ali Aydinlar University, Department of Internal Medicine and Medical Oncology, Istanbul, Turkey ABSTRACT Introduction: Rectal carcinoma is one of the leading causes of cancer death today. In our study, we retrospectively reviewed 357 patients with locally advanced rectal cancer who applied to our hospital between 2009 and Materials and methods: Chemoradiotherapy (CRT) was applied to the patients before surgery. The ratio of neutrophil/lymphocyte (NLR) and platelet/lymphocyte (PLR) were examined at the diagnosis in relation to pathological response and surveillance. The Kaplan-Meier method was used for estimating the survival function from lifetime data. In addition, Cox regression model was also used to explore the relationship between the survival of a patient and several explanatory variables. Results: As the tumor diameter increases, the NLR increases, p value is <0.01 and statistically significant. NLRs were not different between the tumors of T1 and T2, but for T3 and T4 tumors it was higher and more different than others (T3 = , T4 = ). NLR was higher in patients with surgical border continuity, p = 0.01 and statistically significant. A decrease of 1 unit of NLR would increase the likelihood of a pathological good response (1/0.962) by 1.04 fold. PLR, however, is not statistically significant; it has been observed that a decrease in the 1-unit PLR tends to increase the likelihood of a good response (1/0.996) in the pathological stage by fold. While the patients who received partial response to the treatment were living at 59 months, the patients who received good response were living at 71 months. Although p value was 0.383, a 12-month median difference in life expectancy was significant. Conclusion: It has been shown that high NLR in colorectal cancer and many other cancers is a negative prognostic factor. However, there are few articles evaluating the NLR and PLR only for rectum cancer. As in other cancers, a high PLR is associated with a pathological response in rectal cancer and poor prognosis. Keywords: rectal cancer, locally advanced, survival, NLR, PLR. DOI: / _2018_5_211 Received January 30, 2017; Accepted March 20, 2018 Introduction Colorectal cancers (CRC) are common among gastrointestinal cancers and have a very high mortality. It is the third most common cancer, and the fourth most common cancer-related death (1). Four to five % of cancer development is related to the patient s age, habits, environmental factors, and the patient's chronic diseases (1). Surgery is the primary treatment for rectal cancer, and the survival rate for rectal cancer without surgery is quite poor (2). Many risk factors related to the surveillance of the rectum cancer are known as pathology of the tumor, subtype, clinical stage and pathological stage. After the operation of locally advanced rectal carcinoma, the number of positive lymph nodes, tumor diameter, lymphovascular invasion, spread to adjacent tissues, perineural invasion, grade and surgical margin in the pathology reports of patients can be counted as other factors affecting the prognosis (3).

2 1384 Feryal Karaca, Cigdem Usul Afsar When the inflammatory cells transform to malignant cells, inflammation increases and transformation and angiogenesis increase in malignant cells. In this way, the response to inflammation and inflammatory cells increases the recurrence and metastasis of tumor cells (4). Nowadays it is known that inflammatory cells can provide microenvironment through cytokine chemokines and enzymes through various mediators (5). Inflammation in CRC, causes ten times more malignant cell development than other cancers. For example, chronic inflammatory conditions such as Crohn s disease and ulcerative colitis increase the incidence of cancer (6,7). Neutrophil/lymphocyte ratio (NLR) and platelet/thrombocyte ratio (PLR) have been described as poor prognostic factors in many cancer types. At every one unit increase in NLR, recurrence risk increases by 15% in localized non-clear cell renal carcinoma (8). It has also been shown in gastric cancer patients (9,10). The high NLR in colon cancer is a poor prognostic factor in the course of the disease (10,11). In many studies, patients with a high NLR have been reported to have poor prognosis (12). PLR was examined and found to be important for predicting prognosis of colorectal carcinoma in response to systemic inflammatory response (13). Materials and methods Patients with locally advanced rectal cancer who referred to our hospital between 2009 and 2016 were included in the study. Our study was retrospective and patients with metastatic rectal cancer were excluded from the study. A total of 357 patients, 219 male and 138 female, were included in the study. Whole blood cell count (WBC), carcinoembryonic antigen (CEA), liver and kidney function tests were performed at the time of diagnosis to all patients. Clinical staging was performed with magnetic resonance imaging (MRI), computed tomography (CT) and positron emission tomography (PET-CT) before treatment. Clinical correlations of all patients according to neutrophil, lymphocyte and thrombocyte levels were performed. After neoadjuvant chemoradiotherapy (CRT), the effect of adjunctive NLR and PLR to pathological response was investigated. Since having an operation can change neutrophil, leukocyte, and platelet counts, the blood values of the patients were taken at first admission before CRT. The characteristics of the patients are shown in Table 1. Gender Woman 138 Male 219 Total 357 Mean age at diagnosis (female) 54.8 Mean age at diagnosis (male) 55.2 Tumor localization Upper rectum 121 Middle rectum 180 Lower rectum 56 Radiological Stage T2 3 T3 179 T4 175 N0 3 N1 179 N2 175 M0 375 Clinical stage 2C 1 3A 2 3B 174 3C 180 R0 resection 340 R1 resection 17 Radiotherapy dose 45 Gy Gy Gy 17 Table 1: Characteristics of the patients. The clinical stages of the patients were based on the 2010 American Joint Committee on Cancer (AJCC) 7th edition. The blood values at the time of diagnosis are shown in Table 2. N Minimum Maximum Mean Standart deviation WBC 357 2,26 14,07 8,4675 2,73940 NE % ,60 98,00 68, ,10368 LY % 357 1,00 47,70 17, ,33514 LY 357,37 4,09 1,2916,90539 NE 357,60 11,30 5,1737 2,44227 Thrombocyte ,20 480,00 256, ,91727 % BA 357,00,90,3586,25119 Table 2: Blood values of patients at diagnosis. (WBC: white blood cell; NE: neutrophil; LY: lymphocyte) Chemotherapy During the treatment course, capecitabine 2x625 mg/m 2 /day or 5-fluorouracile (5-FU) infusion 225 mg/m 2 weekly was administered as radiosensitizers.

3 The effect of pretreatment neutrophil/lymphocyte ratio and platelet/lymphocyte ratio on pathological Radiotherapy In the clinical phase, 175 patients with T4 disease were treated with 5 Gy of primary boost and 50 Gy of radiotherapy. The remaining 165 patients received 45 Gy of radiotherapy. A total of 60 Gy was completed in 17 patients who had sustained postoperative surgery. Radiotherapy was given to the primary tumor and all pelvic lymph nodes in patients with locally advanced rectum cancer, which were diagnosed as middle and upper rectum. Radiotherapy was given to the inguinal lymph nodes in addition to the above areas in patients with lower rectum cancer. Follow-Up WBC, CEA, liver and kidney function tests were performed in all patients in the first 2 years and at 3 months intervals after surgery. Imaging was performed with whole abdominal MRI and thorax CT. In the next 3 years these examinations were made with 6 months intervals. Patients who have completed 5 years have been invited to check once in a year and have been evaluated with all these tests. Annual colonoscopies were performed and biopsies of the patients who were thought to have local recurrence were obtained. Patients were divided into 3 groups as disease-free survivors, dead patients, and patients still living without local recurrence and metastasis. According to postoperative pathology reports, 37 patients with stage 0 and 155 patients with stage 1 were evaluated as complete response to CRT and the remaining 165 patients were evaluated as partial response to CRT. Post-operative pathologies of the patients are shown in Table 3. Statistical analysis Descriptive statistics for the continuous variables (characteristics) were presented as mean, standard deviation, minimum and maximum values while count and percent for the categorical variables. Pearson correlation coefficients were calculated for determination between the variables. Survival analysis with Kaplan-Meier method was performed to determine the median survival time for the groups. Cox regression model was also used to explore the relationships between the survival of a patient and several explanatory variables. In addition, multiple logistic regression analysis was also carried out to determine the relationships between response level (Yanit düzeyi) and explanatory variables. Statistical significance level was considered as 5% and Statistical Package for the Social Sciences (SPSS) version 20 (IBM) statistical program was used for all statistical computations. Results Histopathology Adeno Cancer 270 Mucinous Cancer 87 Grade Pathologic T stage T0 31 T1 142 T2 68 T3 90 T4 36 Pathologic N stage N0 260 N1 91 N2 6 M0 357 Pathological stage Stage 0 37 Stage Stage 2A 52 Stage 2B 4 Stage 2C 26 Stage 3A 58 Stage 3B 25 Number of positive lymph nodes Table 3: Patient characteristics according to pathology. NLR and PLRs at diagnosis were assessed by postoperative pathological response. According to the pathology results, 192 patients with stage 0 and stage 1 who were treated with CRT was the best response group and 165 patients with stage 2A, stage 2B, stage 2C, stage 3A, stage 3B was with partial response.

4 1386 Feryal Karaca, Cigdem Usul Afsar Accordingly, as the NLR increases, the tumor diameter increases by 62.7%. As the tumor diameter increases, it increases the NLR, p value is < 0.01 and statistically significant. Correlation of tumor diameter with CEA of the patients was or 18.1%, indicating that the tumor diameter was also increased in patients with high CEA. Accordingly, p value is <0.01 and statistically significant. Tumor diameter, NLR, WBC, % neutrophil count and % lymphocyte count were all correlated. WBC and % lymphocyte ratio were negatively correlated with tumor size, and NLR and % of neutrophils were negatively correlated with the resected lymph nodes (Table 4). First CEA Second CEA CEA at diagnosis Tumor diameter Tumor diameter,181** ** Resected lymph nodes Resected lymph nodes ,185** 1 NLR,112* ,627** WBC ,187** 0.045,121* 1 NLR WBC NE % LY % NE %,121* ,263** ,563** LY % -,108* ,543** ,681** -,112* -,594** 1 T2 but T3 and T4 were higher and more different than others (T3 = , T4 = ) (Table 5). The mean PLR in N0 and N1 cases was found to be and ; therefore no difference was observed between them. However, the mean PLR of N2 patients was , which was lower than in the N0 and N1 cases. Statistically, p = was found to be significant. The NLR was in patients with N0, in N1, and in N2. According to these values, p = was found and no statistically significant difference was found (Table 6). Correlation was also observed between patients with positive surgical margins and patients with negative surgical margins according to the postoperative pathology report. The correlation between PLR values of patients with positive and negative surgical margins was statistically significant at p = At the time of diagnosis, the surgical margin of patients with high PLR persisted. When the ratio of NLR was examined, it was found that this ratio was higher in patients with surgical border continuity, p = 0.01 and statistically significant (Table 7). Table 4: Correlation coefficient among the variables. *p < 0.05; **p < 0.01; (CEA: carcinoembryonic antigen; NLR: neutrophil/lymphocyte ratio; WBC: white blood cell; NE: neutrophil; LY: when the NLR is increased by 1 unit, the prob- According to this detailed statistical analysis, lymphocyte) ability of pathological good response will When compared to the pathologic T stage, no increase by times. In other words, a decrease difference was observed. When the PLRs were correlated between the T1 and T2 patients, PLRs were pathological good response (1/0.962) by 1.04 fold. of 1 unit of NLR would increase the likelihood of a higher when compared to the other T stages, and a PLR, however, is not statistically significant; it has significant difference was observed in the T4 been observed that a decrease in the 1-unit PLR patients. NLR was not different between the T1 and tends to increase the likelihood of a good response PLR NLR N Mean Std. Deviation Std. Error Minimum Maximum p ,6169 ab * ,4901 d ,3699 cd ,9253 c ,6569 a Total ,8284 b ,1635 d ,7325 d ,0186 c ,4391 a Total Table 5: Correlation of PLR and NLR according to pathological T stage. *Different T lower cases represent statistically significant differences; (1/0.996) in the pathological stage by fold. Among patients with clinical stage T4 and patients with T3; in the T3 category, the likelihood of a good pathologic response is 2.43-fold greater than in the T4 category. In other words, those in the T3 category will have a 2.43-fold more likely response than those in the T4 category. In those with clinical stage 3B, the likelihood of a good pathologic response is times greater than in those of the 3C category. In other words, those in category 3B will get a good response with a probability times greater than those in category 3C. When the age of diagnosis increases by 1 year, the probability of receiving a good pathologic response will increase by fold. In other words, the age at diagnosis 1 year earlier will increase the likeli-

5 The effect of pretreatment neutrophil/lymphocyte ratio and platelet/lymphocyte ratio on pathological hood of a good pathological response (1/0.949) by 1.05 fold. PLR NLR N Mean Std. Deviation Std. Error Minimum Maximum p ,4569 a ,5816 a ,7480 b Total Total Table 6: Correlation of the post operative lymph nodes. PLR NLR Surgical margin negative Surgical margin positive N Mean Std. Deviation Std. Error Minimum Maximum ,6439 b ,9587 a Total Surgical margin negative Surgical margin positive 340 5,4846 b ,2961 a Total Table 7: Correlation according to surgical margins. B S.E. Wald df p OR 95% C.I.for OR Lower The mean PLR of 165 patients receiving partial response according to pathologic stage was , and the mean PLR of 192 patients receiving good response was According to this, p = 0.05 and statistically significant. When we look to NLR it was in the partial response group, and it was in the good response group. This was statistically significant (p = 0.026) (Table 9). Upper NLR PLR T size (T3) Clinical stage 3B Stage Age CT Constant Table 8: Results of logistics regression analysis for good response. (NLR: neutrophil/lymphocyte ratio; PLR: platelet/lymphocyte ratio; T: tumor; CT: chemotherapy) Survival Analysis According to the pathologic stage, the median life span was found to be 8 months, while the median life span was found to be 10 months when there was no recurrence. The difference between them is statistically significant as p = There was a difference between the survival curves of the patients who received good response and the curve of the patients who received the partial response. Patients who received good response, with life continuing after 80 months without recurrence, whereas recurrences are observed in approximately 18 months of partial response patients. As seen from the survival graphs, the statistical difference between the survival curves of the partial responders and those who were well responded was statistically significant (Figure 1). While the patients who received the partial response to the treatment were living 59 months, the patients who received good response were living 71 months. Although p = 0.383, a 12-month median difference in life expectancy was significant (Figure 2). Discussion Many theories have been put forward regarding the formation of tumor pathogenesis and metastases as well as the continuing survival of patients without metastasis, and recurrence free survival. It is known that changes in cancer cells may occur depending on the change in the inflammatory response. Inflammation is most easily evaluated by measuring the counts of lymphocytes, neutrophils, platelets and acute phase reactants. Several studies have shown that NLR at the time of diagnosis is poor prognostic factor (14). In our study, the blood counts of patients with locally advanced rectal cancer were taken before CRT. A total of 367 neoadjuvant CRT patients were observed for 7 years. According to the stages of the patients after surgery, 192 patients with stage 0 and 1 were the best responded group and the stage 2 and 3 patients were with partial response. At the time of diagnosis, the ratio of NLR and CEA values were evaluated. Postoperative pathology reports showed a significant relationship between tumor size and high CEA at diagnosis.

6 1388 Feryal Karaca, Cigdem Usul Afsar Patients with high CEA were also found to have higher pathologic tumor size (p < 0.01). PLR NLR N Mean Std. Deviation Std. Error Minimum Maximum Partial Response Good response Total Partial Response Good response Total Table 9: Correlation of NLR and PLR in well-responding and partially responding patients to treatment. Disease-free survival Pathological stage Estimate Median Std. Error 95% Interval Confidence Log Rank Lower Bound Upper Bound p Partial Response Good response Overall Table 10: Disease-free survival Fig. 1: Recurrence-free survival of well-responding and partially responding patients. Fig. 2: Chart showing overall survival of patients It was observed that patients with high NLR at diagnosis also affected the tumor size in the postoperative pathology and increased the tumor diameter in parallel with this ratio (p < 0.01) (Table 4). Regarding T-stage, NLR was higher in patients with T3 and T4 tumors (p < 0.01), and PLR was higher when compared with other T (p = 0.01). At the time of diagnosis, the surgical margin of patients with high PLR persisted. When NLR was taken into consideration, it was found that this ratio was higher in patients with surgical continuity and p = 0.01, which was statistically significant (Table 7). When the patients were analyzed according to the pathologic stage, partial response and good response, PLR was statistically significant (p = 0.05). When the NLR is examined, the correlation between partial response and good response was statistically significant (p = 0.026) (Table 9). In our study, a correlation was found between the age at diagnosis and NLR and it was observed that increase in age for one year increases pathological response by 1.05 times. Neutrophils have previously been shown to effect tumor proliferation, angiogenesis, by the way of proangiogenesis (15). In our study, it was observed that patients with high NLR had a negative effect on postoperative pathology. In patients with surgical border continuity according to pathology, T4 patients had a high NLR. In other words, when the patients were divided into two groups with good response and partial response, the percentage of NLR with partially response patients were higher than that of well response patients. We have observed in our study that the PLR is also prognostic. Again, we observed that this ratio was lower in patients who had good response than those who had partial response. Up to now, no identifiable studies about PLR have been found. We have observed this by long-running follow-up. We know that lymphocytes play an important role in tumor suppression. Reduced lymphocyte ratio plays a role in tumor proliferation and angiogenesis. Accordingly, high NLR can cause adverse factors as mentioned above in cancer cells (15-17). It is known that platelets can form a pregenerative environment within the tumor microenvironment. This suggests that platelets may play a role in proinflammatory procedure, thus leading to adverse effects on tumor proliferation as we have

7 The effect of pretreatment neutrophil/lymphocyte ratio and platelet/lymphocyte ratio on pathological already mentioned. However, there has been no study showing that the PLR is so important in tumor proliferation. In our retrospective study, it was observed that the PLR was higher in patients with partial response. Accordingly, it is seen that this value is important in tumor proliferation. In the same way, it is observed that patients with good response to treatment have a low NLR and they are still living without recurrence (Figure 1). Well-response patients who had lower NLRs had a longer recurrence-free life according to figure 3, but those with higher NLRs were found to have earlier recurrences. A 12-month difference was found to be very important for the best and partial responders (Figure 2). In this study, we found that the NLR and PLRs were significant in pathological response and for survival. Fig. 3: Disease-free survival chart (Treatment shows that recurrence in patients with good response is 80 months and recurrence in patients with partial response is 40 months). Conclusion It has been observed that lymphocytes enhance tumor proliferation and angiogenesis during the studies done until today, and this was seen as a poor prognostic factor. In our study, higher NLR was found to be an important factor both for pathologic response and prognosis. There have been no studies showing that PLR affects tumorigenesis. Our study is the first study to show that the pathologic response is partial in patients with high PLR and that affected the survival adversely. References 1) Mármol I, Sánchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi MJ. Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer. Int J Mol Sci 2017 Jan 19; 18(1). pii: E197. doi: /ijms ) Roxburgh CS, McMillan DC. Cancer and systemic inflammation: treat the tumour and treat the host. Br J Cancer 2014; 110(6): ) Büttner S, Lalmahomed ZS, Coebergh van den Braak RR, Hansen BE, Coene PP, Dekker JW, et al. Completeness of pathology reports in stage II colorectal cancer. Acta Chir Belg 2017 Jan 24: 1-7. doi: / ) Lu H, Ouyang W, Huang C. Inflammation: a key event in cancer development. Mol Cancer Res 2006; 4(4): ) Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420: OpenUrl 6) Seril DN, Lia J, Yang GY, Yang CS. Oxidative stress and ulcerative colitis-associated carcinogenesis: studies in humans and animal models. Carcinogenesis 2003; 24: ) Moody GA, Jayanthi V, Probert CS, Mac Kay H, Mayberry JF. Long term therapy with sulphasalazine protects against colorectal cancer in ulcerative colitis; a retrospective study of colorectal cancer risk and compliance with treatment in Leicestershire. Eur J Gastroenterol Hepatol 1996; 8: ) de Martino M, Pantuck AJ, Hofbauer S, Waldert M, Shariat SF, Belldegrun AS, Klatte T. Prognostic impact of preoperative neutrophil-to-lymphocyte ratio in localized nonclear cell renal cell carcinoma. J Urol 2013; 190(6): ) Graziosi L, Marino E, De Angelis V, Rebonato A, Cavazzoni E, Donini A. Prognostic value of preoperative neutrophils to lymphocytes ratio in patients resected for gastric cancer. Am J Surg 2015; 209(2): ) Shibutani M, Maeda K, Nagahara H, Noda E, Ohtani H, NishiguchiY, et al. A high preoperative neutrophilto-lymphocyteratio is associated with poor survival in patients with colorectal cancer. Anticancer Res 2013; 33(8): ) empleton AJ, McNamara MG, Šeruga B, Vera-Badillo FE, Aneja P, Ocana A, et al. Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis. J Natl Cancer Inst 2014; 106(6): ) Azab B, Mohammad F, Shah N, Vonfrolio S, Lu W, Kedia S, et al. The value of the pretreatment neutrophil lymphocyte ratio vs. platelet lymphocyte ratio in predicting the long-term survival in colorectal cancer. Cancer Biomarkers 2014; 14: ) Guthrie GJ, Charles KA, Roxburgh CS, Horgan PG, McMillan DC, Clarke SJ. The systemic inflammationbased neutrophil-lymphocyte ratio: experience in patients with cancer. Crit Rev Oncol Hematol 2013; 88(1): ) Tazzyman S, Lewis CE, Murdoch C. Neutrophils: key mediators of tumour angiogenesis. Int J Exp Pathol 2009; 90(3): ) Proctor MJ, Morrison DS, Talwar D, Balmer SM, Fletcher CD, O'Reilly DS, et al. A comparison of inflammation-based prognostic scores in patients with cancer. A glasgow inflammation outcome study, Eur J Cancer 2011; 47(17): ) Alexandrakis MG, Passam FH, Moschandrea IA, Christophoridou AV, Pappa CA, Coulocheri SA, Kyriakou DS. Levels of serum cytokines and acute

8 1390 Feryal Karaca, Cigdem Usul Afsar phase proteins in patients with essential and cancerrelated thrombocytosis. Am J Clin Oncol 2003; 26(2): ) Fridlender ZG, Albenda SA. Tumor-associated neutrophils: friend or foe? Carcinogenesis 2012; 33(5): Corresponding author CIGDEM USUL AFSAR, MD, Assoc. Prof Acıbadem Mehmet Ali Aydınlar University, Department of Internal Medicine and Medical Oncology, Acıbadem Bakirkoy Hospital, Zeytinlik Mah. Halit Ziya Usaklıgil Cad. No: 1, 34140, Bakirkoy Istanbul cigdemusul@yahoo.com (Turkey)