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Pre-treatment inflammatory indexes as predictors of survival and cetuximab efficacy in metastatic colorectal cancer patients with wild-type RAS

This study aims at evaluating the prognostic significance of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and systemic immune-inflammation indexes (SII) in metastatic colorectal cancer (mCRC) patients treated with cetuximab. Ninety-five patients receiving cetuximab for mCRC were categorized into the high or low NLR, PLR, LMR, and SII groups based on their median index values. Univariate and multivariate survival analysis were performed to identify the indexes’ correlation with progression-free survival (PFS) and overall survival (OS). In the univariate analysis, ECOG performance status, neutrphil counts, lymphocyte counts, monocyte counts, NLR, PLR, and LDH were associated with survival. Multivariate analysis showed that ECOG performance status of 0 (hazard ratio [HR] 3.608, p < 0.001; HR 5.030, p < 0.001, respectively), high absolute neutrophil counts (HR 2.837, p < 0.001; HR 1.922, p = 0.026, respectively), low lymphocyte counts (HR 0.352, p < 0.001; HR 0.440, p = 0.001, respectively), elevated NLR (HR 3.837, p < 0.001; HR 2.467, p = 0.006) were independent predictors of shorter PFS and OS. In conclusion, pre-treatment inflammatory indexes, especially NLR were potential biomarkers to predict the survival of mCRC patients with cetuximab therapy.


Discussion
Increasing evidence suggested that the inflammatory reaction plays an important role in tumor development [29][30][31][32] . Accordingly, serum blood cells such as neutrophils, lymphocytes, platelets and monocytes have been assessed in different malignancies and found to be able to predict prognosis and response to treatment [33][34][35] . Furthermore, several studies have reported that inflammatory indexes including NLR, PLR, LMR and SII were potential prognostic markers for various tumors [36][37][38][39][40][41][42] . Such parameters were also associated with survival in mCRC patients, including those receiving bevacizumab or palliative chemotherapy 25,43 . In our study, we observed that pre-treatment inflammatory indexes were potential prognostic factors for survival in mCRC patients receiving cetuximab. The results of this study suggested that the elevated pre-treatment neutrophil counts, lymphocyte counts and NLR were independent predictors for PFS and OS in mCRC patients receiving cetuximab. PLR, LMR and SII showed no significant association with survival. In addition to inflammatory indexes, we analyzed the associations between patients' clinical factors (gender, age, ECOG performance status, tumor localization, liver metastasis, lung metastasis, pathological differentiation, M stage, chemotherapy regimen, LDH, CA 19-9, CA-125) and survival. We demonstrated that ECOG performance status was an independent influence factor for both PFS and OS. We also found that patients with low pre-treatment LDH had better PFS but no significant difference in patients' OS was observed. However, other characteristics such as gender, age, tumor localization, liver metastasis, lung metastasis, pathological differentiation, M stage and chemotherapy regimen, CA 19-9, and CA-125 showed no significant associations with survival.
Neutrophils promote tumor development through facilitating the secretion of circulating growth factors such asinterlukin-1, interlukin-6 and VEGF while lymphocytes play a significant role in anti-tumor response by promoting cytotoxic cell death and inhibiting tumor cell proliferation and migration 14,[44][45][46][47] . Additionally, neutrophils suppress lymphocytes activities, and therefore suppress the anti-tumor immune response 39 . Tumor-associated macrophages which are derived from circulating monocytes, promote tumor growth, migration, invasion, and metastasis 14,48,49 . Thus, neutrophils and monocytes could promote tumor progression, whereas lymphocytes play an important role in the anti-tumor immunity of the host 14,47 . The role of inflammation in cancer progression is supported by studies which showing that many inflammatory diseases increase the risk of tumors, while aspirin and other non-steroidal anti-inflammatory drugs reduce the risk 14,[50][51][52][53] . Previous studies suggested that low NLR and high LMR correlated with favorable survival in various cancers, including colorectal cancer, esophagus cancer, gastric cancer and pancreatic cancer 16,[36][37][38][39]42,[54][55][56] . The results of this study confirmed that pre-treatment NLR was an independent predictor for PFS and OS. A prognostic factor with RR > 2 is considered useful practical value, which indicated that elevated NLR was a powerful predictive indicator of poor outcome 57 . This study indicated that LMR was not significantly associated with survival. However, univariable analysis showed a tendency of improved PFS and OS in patients with high LMR which was not an independent prognostic factor. As a result, further studies are expected to confirm the prognostic value of LMR. Several studies reported that platelets were related to the angiogenesis and tumor invasion through the increasing production of vascular epidermal growth factor in cancer microenvironment 58,59 . In turn, malignant tumor cells induce platelets aggregation and promote the development of cancer-associated thrombosis 60,61 . As a result, platelets recruited to the tumor microenvironment consequently allow tumor cells to evade immune surveillance and to be protected from physical clearance 61,62 . Thus, cancer progression is not only caused by the intrinsic properties of tumor cells but also stimulated by systemic and local inflammatory reactions. In fact, the role of PLR in the prognosis of CRC patients is still controversial. Several studies supported the positive role of pretreatment PLR as a good marker for CRC patients while several studies did not approve this conclusion 22 NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio; SII, systemic immune-inflammation index; LDH, lactate dehydrogenase, CA19-9, carbohydrate antigen 19-9; CA-125, carbohydrate antigen-125; PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval. a 94 were available; b 90 were available; c 52 were available. A p value < 0.05 was considered statistically significant. tumor, small cell lung cancer, hepatocellular carcinoma and gastric cancer [65][66][67] . In the present study, PLR showed significant correlation with PFS but not with OS in univariate analysis. However, no statistically significant correlation was observed about the elevated PLR and poor survival in terms of HR value in the multivariate analysis. Elevated SII indicates high neutrophil counts, high platelet counts and low lymphocyte counts. In this study, we did not confirm the associations of SII with survival. Thus, further studies should be performed to investigate the prognostic value of PLR and SII for the efficacy of cetuximab in mCRC patients. The limitation of this study lies in its retrospective nature. In addition, our single-center study with a limited number of patients (n = 95) might cause selection bias. Thus, a larger study population, multi-center studies and longer follow-up are needed to validate these results.
In conclusion, this study demonstrated that ECOG performance status, neutrophil counts, lymphocyte counts and NLR were independent predictors for PFS and OS in mCRC patients, while serum level of LDH was independent predictors for PFS but not for OS. Pre-treatment inflammatory indexes, especially NLR were potential biomarkers to predict the survival of mCRC patients with cetuximab therapy, which would hopefully establish a convenient and inexpensive approach to predict of the efficacy of cetuximab in the treatment of metastatic colorectal cancer.

Materials and Methods
Patients and inflammatory indexes. We reviewed 7207 colon cancer patients treated at West China hospital between January 2009 and December 2015. Patients who met the following criteria were included: (a) patients with pathological diagnosis of CRC, (b) patients with wild-type RAS mCRC, (c) patients receiving first-line treatment (chemotherapy plus cetuximab), and (d) patients with available and complete basic characteristics, laboratory data and follow-up information. Patients with clinical evidence of acute and chronic inflammation, autoimmune diseases, hematological disorders, or underwent radiotherapy, prior steroid therapy were excluded. The following variables were collected and evaluated in this study: gender, age, ECOG performance status, tumor localization, liver metastasis, lung metastasis, pathological differentiation, M stage and chemotherapy regimen. Laboratory tests results included levels of neutrophil counts, lymphocyte counts, platelet counts, LDH, CA19-9, and CA-125. All of the data were retrieved from electronic patient record system. Laboratory data were obtained within 10 days prior to the initial administration of cetuximab. Blood cell counting was performed with Sysmex hematology analyzers. Patients were staged according to the American Joint Committee on Cancer tumor-node-metastasis (TNM) classification system. NLR and PLR were defined as the absolute counts of neutrophils and platelets respectively, divided by the absolute lymphocyte count. LMR was calculated by dividing the absolute lymphocyte count by the absolute monocyte count. SII was calculated as platelet count × neutrophil count/lymphocyte count. All patients were followed every month in the first year, every 3 months in the second year and every 6 months thereafter. The start date of follow-up was the date of patients receiving the first dose of cetuximab, and the end of follow-up was December 2016 or death. This study was approved by the Ethics Administration Office of West China Hospital, Sichuan University. An exemption from informed consent in our study was also approved by this Ethics Administration Office. In addition, all methods in this study were performed in accordance with the relevant guidelines and regulations.
Statistical analysis. Group comparisons on disease-specific variables were performed using Chi-square test for categorical variables and Student's t test for continuous variables. All p-values were based on two-sided testing, and differences were considered statistically significant when p value is less than 0.05. PFS was defined as the duration from patients primarily receiving cetuximab to the date when radiological evidence of recurrence observed. Patients who died but without progression were not censored to the PFS evaluation. OS was defined as the time interval from patients primary received cetuximab to death from any cause or to the last date of follow-up.
Patients were divided into two groups based on the median index value of NLR (2.34), PLR (142.00), LMR (4.00) and SII (460.66), respectively. NLR ≥2.34, PLR ≥142.00, LMR ≥4.00, and SII ≥460.66 were considered as elevated levels. The cut-off value of neutrophil counts, lymphocyte counts, monocyte counts and platelet counts were their median value, respectively. In the univariate analysis, the log-rank test (at a significance level of 5%) was used to compare the PFS and OS between two groups. Survival curves were estimated using the Kaplan-Meier method. All variables with statistic significance in univariate analysis were further evaluated in the multivariate analysis. We investigated the association of multiple variables with survival using the Cox proportional hazard regression analysis. Estimated hazard ratios (HRs) and their two-sided 95% Confidence Intervals (95% CI) were calculated using the Cox proportional hazard model. All statistical analyses were carried out with SPSS version 22.0 (IBM Corporation, Armonk, NY, USA).
Ethical approval. This study was approved by the Ethics Administration Office of West China Hospital, Sichuan University.