Correlation of Neutrophil/Lymphocyte Ratio and Chemotherapy Outcome in Metastatic Colorectal Cancer

UNIVERSITY OF BELGRADE FACULTY OF MEDICINE Dr Vladimir Vukov Correlation of neutrophil/lymphocyte relations and outcome after chemotherapy in metastatic colorectal cancer -work in close specialisation- Mentor: prof. dr Svetislav Jeli

Neutrophil lymphocyte ratio - cancer biomarker of inflammation Inflammation represents a key component of a tumor microenvironment Develop and growth of a tumor are considered to be the result of interaction between tumor and stromal tissue together with blood vessels and immunological/inflammatory cells of a host Systemic changes detected in patients with malignant solid tumors are leukocytosis and neutrophilia These conditions are significantly correlated with a stadium of disses and with bad prognosis.

Neutrophil lymphocyte ratio - cancer biomarker of inflammation Cancer caused myelopoiesis affects the immune response of the organism, which has an integral effect on tumorigenesis and metastasis Qualitative changes of myelopoiesis, provided by a neoplastic process, lead to the accumulation of immature myeloid cells, which acts immunosuppressive Patients with metastatic colorectal cancer, have neutrophil/lymphocyte ratio, as an independent, survival factor, Chua and coworkers have been promoting the ratio of neutrophil/lymphocyte, as a useful potential biomarker for the systemic inflammatory response, in the prediction of clinical outcome of patients with metastatic KRK-treated first-line chemotherapy When chemotherapy normalizes high neutrophil/lymphocyte ratio, survival is prolonged.

GOALS OF STUDY To show the absolute number of leukocyte and relative number of neutrophils and lymphocytes before and after systematic chemotherapy of metastatic colorectal cancer (MKRK-a) To show the influence of sHT on (NLR) Show whether there is a difference in the absolute number of white blood cells, then in the relative number of neutrophils and lymphocytes, as well as the NLR before/after SHT MKRK among subjects who are after surgery treated with adjuvant chemotherapy (AHT) and those who did not To show if there is a relation between changes in NLR before/after SHT and final outcome of a disease.

MATERIALS AND METHODS Study conducted in Service of medical oncology in KBC Zemun, from 2011 to 2013 Included 46 volunteers with average age of life 63 11 (34-82) , of which (54.3%) were women and the rest included man (45.7%) The main inclusion criteria for the study was the existence of metastatic colon cancer (MKRK), and possibility of systemic chemotherapy (SHT) Subjects were divided into two groups, where the criterium was adjusting of adjuvant chemotherapy (AHT), after surgical treatment \ To the first group (n = 19) aHT was applied after the operation of the local and locally advanced KRK. During the time these patients have developed metastatic disease and they were treated SHT The second group (n = 27) included subjects who already suffered from metastatic disease and they were immediately treated with sHT All respondents voluntarily agreed to participate.

MATERIALS AND METHODS All the patients were diagnosed with metastatic disease (stage IV / Dukes D) either at the time of presentation or during controlling (including during application aHT) Combinations that were used for the chemotherapeutic agents according to the protocols for MKRK FU / FA, are that FOLFOX, as well as 5-fluorouracil / folic acid/irinotecan (FOLFIRI) Subjects were treated with 2 (2.2%) 3 (2.2%) 4 (13.0%) 5 (2.2%), ie. 6 (37%) Cycles of chemotherapy combination for metastatic disease was determined depending on whether it was the initial chemotherapy for metastatic disease or systemic chemotherapy after the first sHT

MATERIALS AND METHODS Evaluation of the effects of used sHT mKRK is provided by regular controlling of the damaged organs with methods such as;(CT examinations of the chest, abdomen and pelvis, X-ray of the heart and lungs, abdominal EHO) The outcome of the last cycle, SHT MKRK times according to RECIST criteria was defined as progression of the disease (Eng. Progression of disease - PD), a stable disease (Eng. Stable disease -SD), as well as partial remission (Eng. Partial remission - PR)

MATERIALS AND METHODS Patients were tested in an absolute number of white blood cells, absolute neutrophil count and lymphocyte count and the ratio of the number of neutrophils and lymphocytes (HT) before the initiation of SHT and during the evaluation of the therapeutic effect upon completion of the last cycle SHT Hematologic analysis of cytologic parameters are examined with the Coulter Hematology Analyzer HmX, produced by "Beckman Coulter," Service Laboratory KBC Zemun Then, the value of the ratio of the neutrophil / lymphocyte (NLR) is obtained by calculation

MATERIALS AND METHODS In statistical analysis were used descriptive and analytical statistics As methods of descriptive statistics were used relative numbers, measures of central tendency and variability measures. For measures of central tendency were used the mean and median, a measure of variability in the standard deviation, and coefficient of variation interval. The methods of analytical statistics tests were used for assessing the significance of connections and differences. Since tests for assessing the significance of correlation test was used single linear correlation (Pearson) for the parameter, and rank correlation test (Spearman) for nonparametric data. For the evaluation of the significance of difference was used 2 test in the case of categorical data, that is, in the case of the interval of data that do not follow a normal distribution rank sum test (Mann-Whitney) for independent samples, ie. test for matched pairs (Wilcoxon) for dependent samples. To standardize the level of statistical significance value was 0.05 and the high statistical significance 0.01. In the analysis, we used the statistical package SPSS for Windows 17.0

RESULTS Study included volunteers with average age of life 63 11 (34- 82) Statistically significant differences in age between the groups (t = 0.689, df = 44, p> 0.05) is not detected Graphic 1. illustrates distribution of subjects by gender MU KI ENSKI

RESULTS Graphic 2. Distribution KRK primary site in the studied population 18 16 cekum 14 ascedens 12 descedens 10 8 sigma 6 rektum 4 2 0 grupa 1 grupa 2

RESULTS Graph. 3. Distribution of metastatic disease in the study population 18 16 14 jetra 12 multipla lokalizacija plu a 10 8 6 4 2 0 grupa 1 grupa 2

RESULTS Graph. 4. Distribution of metastatic disease by gender 16 14 12 mu karci 10 ene 8 6 4 2 0 jetra multipla lokalizacija plu a

RESULTS Table 7. frequency distribution of respondents by Dukes classification in the group of patients treated with adjuvant HT Histological stage of the disease n % 5 26.3 Dukes B 14 73.7 Dukes C 19 100

RESULTS Table 8. adjuvant chemotherapy protocols Protocols Adj.HT n % 16 84.2 5FU/LV 2 10.5 FOLFOX 1 5.3 Xeloda 19 100

RESULTS Table 9. Protocols SHT in the study population Protocol systematic n % HT 12 26.1 5FU/LV 32 69.6 FOLFOX 2 4.3 FOLFIRI 46 100

RESULTS Graph 4. Illustrates respondents by the number of cycles of systemic chemotherapy II ciklusa FOLFIRI IV ciklusa FOLOX 5FU/LV VI ciklusa 0 5 10 15 20 25 30 35

RESULTS Evaluation of the outcome of systemic HT was performed after the last cycle of HT and was defined as the progression of the disease in 22 (47.8%), stable disease in 11 (23.9%) and partial regression in 13 (28.3%) subjects There were no statistically significant differences between the two groups of subjects reviewing the outcome of the ( 2 = 1.176; df2; p> 0.05)

RESULTS Table. 10. The absolute number of leukocytes, the number of neutrophils relaltivni and lymphocytes, as well as before and after the NLR SHT. Variables .HT After systematic HT P 7.6 1.8 7.0 2.1 Aps. Number of Le p>0.05 (4.5-13.0) (3.6-12.0) [X SD(min-max)(x109cell/ l)] 62 10 62 11 Rel. Number of Neut p>0.05 (40-85) (38-86) [X SD(min-max)(%)] 27 28 13 Rel. Number of Ly p>0.05 (6-50) (10-90) [Med(min-max)(%)] 2.25 2.31 NLR p>0.05 (0.8-12.5) (0.8-9.1) [Med(min-max)]

RESULTS Table. 11 Hematocitological data and NLR ratio by Group Variables Group 1 (n=19) Abs. Number of Le before sHT 7.07 1.74 (4.5-11.0) Group 2 (n=27) 7.97 1.76 (5.2-13.0) [X SD(min- max)(x109cell/l)] Rel. Number of Neut before sHT 63.1 9.7 (40-85) 62.0 10.5 (44-78) [X SD(min-max)(%)] Rel. Number of Ly before sHT 27 (6-50) 28(14-50) [Med(min-max)(%)] NLR before sHT 2.5 (0.8-12.5) 2.2 (0.8-5.5) [Med(min-max)] Abs. Number of Le after sHT [X SD(min- max)(x 109cell./l)] Rel. Number of Neut after sHT [X SD(min- max)(%)] Rel. Number of Ly after sHT 7.11 2.04 (4.2-11.0) 6.99 2.12(3.6-12.0) 64.9 11.0 (47-86) 60.8 10.5 (38-83) 25 (10-90) 30 (10-47) [Med(min-max)(%)] NLR after sHT 2.6 (1.3-9.1) 2.1 (0.8-8.3) [Med(min-max)]

RESULTS Table. 12. Illustrates frequency distribution of respondents by SHT protocols in group 1 n % protocol sHT 1 0.19 5FU/LV 16 84.21 FOLFOX 2 10.53 FOLFIRI 19 100

RESULTS Table 15 Illustrates distribution of frequency of respondents by SHT cycle in group 1 Number of cycle sHT n % 1 5.3 2 3 15.8 4 15 78.9 6 19 100 Table 16. frequency distribution of respondents by the outcome after SHT in group 1 Outcome after sHT n % 10 52.6 PD 3 15.8 SD 6 31.6 PR 19 100

RESULTS There was statistically significant effect changes NLR connection relationship before and after the SHT and outcome of the disease ( =-0.924; p<0.01) in group 1 None of the participants with PD had a reduction NLR after SHT, even as one respondent to the PR had an increase in NLR

RESULTS Table 17. Shows frequency distribution of respondents by protocols systemic HT in group 2 ProtoCol sHT n % 11 40.7 5FU/LV 16 59.3 FOLFOX 27 100 Table 17. Shows frequency distribution of respondents by protocols systemic HT in group 2 Number of cycles sHT n % 1 3.7 2 4 11.1 4 22 81.5 6 27 100

RESULTS Table. 19. Shows frequency distribution of respondents by the outcome after the SHT group 2 Outcome after sHT n % 12 44.5 PD 8 29.6 SD 7 25.9 PR 27 100

RESULTS There was statistically significant effect changes NLR connection relationship before and after the SHT and outcome of the disease ( =-0.893; p<0.01) in group 2 None of the participants with PD had a reduction NLR after SHT, even as one respondent to the PR had an increase in NLR

RESULTS Chart no.5. The distribution of patients into groups according to the outcome of treatment SHT 12 10 8 6 PD 4 SD 2 0 Grupa 2 PR Grupa 1 PR SD PD

RESULTS Table 20. NLR summary values change before / after SHT by groups of respondents to the outcome of treatment OUTCOME NLR before/after sHT GROUP 1 GROUP 2 NLR before sHT 2.861 2.626 PD NLR after sHT 4.318 3.868 NLR beores HT 3.383 2.920 SD NLR after sHT 2.350 2.425 NLR before sHT 4.371 3.701 PR NLR after sHT 2.098 1.624

RESULTS Table. 21. The statistical significance of changes in monitored variables before and after SHT in relation to disease outcome in all subjects Monitored variables according to PD (n=22) SD (n=11) PR (n=13) disease outcome Aps. Number of leucocytes t= - 2.431 t= 3.806 t= 3.008 (p<0.05) (p<0.01) (p<0.01) before/after sHT Rel. Number of neutrophils t= - 8.225 t= 1.749 t= 7.214 before/after sHT (p<0.01) (N.S) (p<0.01) Rel. Number of lymphocyte before / after SHT Z= - 3.398 Z= - 2.194 Z= - 3.183 (p<0.01) (p<0.05) (p<0.01) Z= - 2.268 Z= - 4.107 Z= - 3.184 NLR before/after sHT (p<0.05) (p<0.01) (p<0.01)

RESULTS Evaluation after carrying out SHT showed that there was 22 PD, 13 PR, 11 SD metastatic disease in total Compared to the monitored variables is indicated by a high and significance of changes NLR relations before/after SHT and outcomes in patients who have achieved progression and partial remissions of metastatic disease Patients who achieved disease stabilization changes NLR relationship with respect to the outcome of the disease exists but is not produced highly significant

DISCUSSION The migration of leukocytes is considered to be one of the key factors in the inflammatory response in the tumor. Tumor released specific chemokines that control this migration and the function of these cells after their arrival at the tumor microenvironment There is a growing body of evidence that carcinogenesis and tumor progression results in qualitative and quantitative changes of myelopoiesis Cancer caused myelopoiesis is characterized by the accumulation of myeloid cells at different stages of differentiation, resulting in suppression of host immunity and the promotion of tumor angiogenesis

DISCUSSION Myeloid cells are the most common hematopoietic cells with a nucleus inside of the human body and they represent are a group of cell populations with different functions Three groups of extremely differentiated myeloid cells (macrophages, dendritic cells, and granulocytes) are essential for the normal function and native and gained by the immunity Tumor microenvironment alters myeloid cells and is converted them into a potent immunosuppressive cells These cells are referred to as myeloid-derived suppressor cells ( "myeloid-derived suppressor cells" - MDSC), have a strong immunosuppressive effect and derive from the bone marrow Circulating neutrophils contain and secrete most of circulating growth factors which are thought to play a crucial role in tumor development.

DISCUSSION Neutrophil/lymphocyte ratio from blood samples is identified as a potentially useful marker for prognosis outcomes in the different disease, including cardiovascular disorders and gastrointestinal tumors Higher values of circulating neutrophils carry adverse effect on the bearing tumors, as indicated by a negative correlation between the number of circulating neutrophils and survival of patients NLR may be regarded as the balance between the pro- inflammatory status of the tumor and the anti-tumor immune status of the host

DISCUSSION Patients with high NLR have relative lymphocytes and neutrophils which means that if the balance is changed in favor of pro-inflammatory response of the tumor it is associated with a bad outcome NLR is examining as a predictor of prognosis in different types of cancer Neutrophil/lymphocyte ratio as a marker of systemic inflammation has not been studied in the context of the outcome after the treatment by systemic chemotherapy in metastatic CRK

CONCLUSIONS The prospective study has been performed on medical oncology Department KBC Zemun on the study population of 46 patients with MKRK. All of the patients were primarily had a surgical treatment. Observing was conducted over groups were mean age 63 11 years. The first group of patients (Dukes' B and C, n = 19), carried out is the treatment of AHT. During follow-up, due to disease progression in MKRK, treatment SHT-om. The second group of patients (Dukes' D, n = 27) was started immediately SHT. After conducting SHT there were 22 progressions, 13 partial remissions and 11 stabilization of metastatic disease There was no statistically significant difference between the general characteristics of the patient and the monitored parameters hematocitological (absolute number of leukocytes, the relative number of neutrophils and lymphocytes) Change of NLR relationship significantly varies with disease: subjects with PD had an increase NLR, while respondents with a PR had a reduction in NLR.