The Prognostic Value of A Preoperative Lymphocyte Count and C-Reactive Protein Ratio In Osteosarcoma


 Background: Systemic inflammatory response and nutritional status are closely related to tumor development, and both have been recognized as predictors of tumors. Our study investigated the effect on the prognosis of osteosarcoma by analyzing the ratio of lymphocytes to C-reactive protein (LCR) before surgery.Methods: Patients who were diagnosed with osteosarcoma and underwent surgery in the First Affiliated Hospital of Guangxi Medical University from 2012 to 2019 were included in this retrospective study. The albumin (g/L) +5 × total lymphocyte count (PNI), neutrophil/lymphocyte count (NLR), platelet/lymphocyte count (PLR) and platelet × neutrophil/lymphocyte count (SII) were calculated from preoperative peripheral white blood cells, C-reactive protein and serum albumin. The optimal cutoff values of LCR, PNI, NLR, PLR and SII were determined by receiver operating characteristic (ROC) analysis. According to the Optimal cutoff values, LCR, PNI, NLR, PLR and SII were divided into high and low groups. The Kaplan-Meier method was used to compare the overall survival (OS) between the high and low LCR groups. Univariate analysis was used to determine the influence of age, gender, tumor size, Enneking stage and neoadjuvant chemotherapy on the prognosis of osteosarcoma.The independent predictors of OS were determined by Cox multivariate analysis.Results: The optimal cutoff values for LCR, PNI, NLR, PLR and SII were 0.093, 48.4, 1.23, 157.03 and 314.27, respectively. A low preoperative LCR was significantly correlated with tumor metastasis, stage, NLR, PLR and SII. However, a low preoperative PNI was significantly associated with tumor metastasis, stage, and PLR.Kaplan-Meier survival analysis indicated that the postoperative OS was significantly correlated with preoperative LCR and PNI (P < 0.05). Univariate analysis showed that Enneking stage, metastasis and preoperative LCR, PNI, NLR, PLR and SII were important factors affecting OS (P < 0.05). For multivariate analysis, the results revealed that the preoperative LCR (HR, 0.401; 95% CI, 0.199-0.807; P = 0.01) and Enneking stage (HR, 2.717; 95%CI, 1.067-6.919; P = 0.036) is an independent prognostic factor affecting the postoperative OS of osteosarcoma.Conclusions: The high preoperative LCR is strongly associated with longer survival time in patients with osteosarcoma. Enneking stage and preoperative LCR may be important parameters for the prognosis of osteosarcoma.


Introduction
Osteosarcoma is a common malignant bone tumors, mainly occurring in children and adolescents, with an annual incidence of approximately 4/1000000, accounting for approximately 45% of all bone and joint tumors [1] . Although surgery combined with chemotherapy and other multidisciplinary therapies can signi cantly improve the prognosis of patients compared with that of the previous treatments alone, The 5-year survival rate for patients with osteosarcoma is still less than 70%.The latest study found that the prognostic factors of osteosarcoma include tumor grade, histological subtypes, metastasis, alkaline phosphatase and lncRNAs, tp73-as1, circRNAs, calcitonin 3 (CNN3) and other proteins [2,3] .Although these factors have some advantages in predicting the prognosis of osteosarcoma, they also have some disadvantages, such as a complicated process, expensive price or being di cult to monitor continuously. Therefore, nding effective prognostic indicators for patients with osteosarcoma has been the focus of researchers in recent years.
The systemic in ammatory response has been recognized as the seventh characteristic of cancer [4] .
In ammatory cells and in ammatory factors widely exist in the microenvironment of osteosarcoma, which can induce the expression of various cytokines and chemokines, promote the proliferation of tumor cells and induce angiogenesis, and change the body's response to hormones and chemotherapy drugs, thus promoting the development, metastasis and drug resistance of tumors [5] .Taking the relationship between in ammation and tumor into consideration, in recent years, more and more studies have found that in ammation-related markers are prognostic factors for colorectal cancer, gastric cancer, pancreatic cancer and other malignant tumors [6][7][8][9] . Recently, some scholars have found that in ammatory indicators such as NLR, PLR and SII are correlated with the prognosis of osteosarcoma. However, there are still few reports on the in uence of preoperative a LCR on the prognosis of patients with osteosarcoma. Therefore, in this study, preoperative LCR and PNI were analyzed in patients with osteosarcoma to investigate the effect of preoperative LCR on the prognosis of patients.

Patients
Patients with osteosarcoma diagnosed and operated on in the First A liated Hospital of Guangxi Medical University from 2012 to 2019 were included in this retrospective study. Inclusion criteria are as follows: (1) postoperative histopathological diagnosis of osteosarcoma, (2) no anticancer treatment, such as chemotherapy, radiotherapy or blood transfusion, was received before surgery, and (3) with detailed medical data and laboratory results. Exclusion criteria are as follows: (1) preoperative infection, high fever, autoimmune disease or blood system disease, (2) history of other malignant tumors, (3) receipt of nonsteroidal anti-in ammatory drugs or antibiotics, or (4) incomplete medical records. Finally, 101 patients met the above criteria to be included. Our study was conducted with the consent of Ethics Committee of Guangxi Medical University and the informed consent of each patient.

Data Collection And De nition
Through the hospital information system, we collected the gender, age, tumor size, stage, metastasis, neoadjuvant chemotherapy, pathological fracture, laboratory examination and other basic clinical data of all patients. The results of blood routine and liver function tests were obtained by drawing blood within 1 week before the operation. The de nitions of LCR, PNI, NLR, PLR and SII were shown as follows: LCR = lymphocyte counts/ C-reactive protein; PNI = albumin (g/L) + 5 × total lymphocyte count (10 9 /L); NLR = neutrophil/lymphocyte-count; PLR = platelet/lymphocyte count; SII = platelet × neutrophil/lymphocyte count. According to receiver operating characteristic curve (ROC), the optimal truncation values of the LCR, NLR, PLR, SII and PNI are obtained when the Youden index is the largest. LCR and PNI values greater than the corresponding cutoff values were de ned as high LCR, high PNI, high NLR, high PLR, and high SII (HLCR, HPNI, HNLR, HPLR, and HSII respectively).

Follow-up
After the operation, all patients were followed up regularly by re-examination and telephone calls. Overall survival (OS) was de ned from the time of de nite histopathological diagnosis until the date of last follow-up or death in this study. Follow-up was conducted every 3 months for the rst 4 years, followed by every 6 months to the most recent follow-up. Follow-up included a thorough history, physical examination, laboratory tests (routine blood, blood biochemistry, tumor markers, etc.), MRI, chest CT, etc.
Statistical analysis SPSS 24.0 Software was used for data analysis. ROC analysis was used to obtain the optimal cutoff values for continuous prognostic variables (LCR, PNI, NLR, PLR, and SII) by the maximal Youden index. The χ2 test was used to compare the differences between groups, the Kaplan-Meier was used to draw the survival curve, and the log-rank test was used to compare the survival differences between the two groups. In terms of prognostic analysis, both a univariate analysis and multivariate analysis were investigated by Cox hazard regression model. The factors were supposed to be remarkably signi cant. A two-tailed p < 0.05 was considered to be statistically signi cant.     Fig. 2b).

Clinical Parameters Of Patients With Osteosarcoma
Univariate Cox regression analysis showed that tumor stage (P < 0.001), metastasis (P < 0.001), LCR (P < 0.001), PNI (P = 0.001), NLR (P = 0.018), PLR (P = 0.002) and SII (P < 0.001) were signi cantly associated with OS. Therefore, these factors were incorporated into a Cox regression analysis for multifactor analysis. The results showed that the tumor stage (HR, 2.717; 95%CI, 1.067-6.919; P = 0.036) and preoperative LCR (HR, 0.401; 95% CI, 0.199-0.807; P = 0.01) is an independent prognostic factors for OS after surgery. (Table 3)  [7,8,10,11] . However, the effect of preoperative LCR and PNI on the prognosis of patients with osteosarcoma is not yet clear, and there are few relevant reports. In this study, preoperative peripheral blood in ammatory and nutritional indicators were used to evaluate the prognosis of patients with osteosarcoma in order to obtain a comprehensive and objective predictor and provide relevant theoretical basis for timely treatment. We detected and analyzed the preoperative peripheral blood count and albumin levels of all patients with osteosarcoma, and nally found that LCR and PNI were important factors affecting the postoperative survival rate of patients with osteosarcoma, among which LCR and tumor stage were independent predictors of prognosis.
There is increasing evidence that systemic immune in ammation and nutrition are related to clinical indicators and prognosis of tumor patients [12][13][14] . Our study found that LLCR and LPNI were related to late Enneking stage and more prone to tumor metastasis, which was the same as previous studies.
In ammatory responses can lead to increased neutrophils, thrombocytopenia, lymphocytopenia, elevated C-reactive protein, and decreased serum albumin. As a leukocyte subgroup, both neutrophils and platelets can promote tumorigenesis, proliferation and metastasis by secreting chemokines and vascular endothelial factors, such as vascular endothelial growth factor (VEGF) [4,15,16] . The rapid increase in CRP is mainly related to pro-in ammatory factors, such as interleukin (IL)-1, TNF-α, IL-6 and IL-8, and these pro-in ammatory factors regulate cell proliferation and angiogenesis by activating intracellular pathways, such as the pathway mediated by the nuclear factor NF-κB that activates B cells, thereby promoting the development and metastasis of malignant tumors [17][18][19] . Serum albumin, which is produced by the liver, is an important nutritional indicator of the body and plays an important role in improving the body's immunity, suppressing the in ammatory response and anti-tumor effects [20] . However, an increase of CRP is often accompanied by a decrease of serum albumin concentration, an increase of tissue consumption, and the continuous loss of body weight, which leads to malnutrition of the body and further aggravation of the disease [21,22] . In contrast, lymphocytes are crucial in the body's immunity. During the development of tumor cells, lymphocytes suppress tumor growth and invasion by secreting cytokines such as TNF-α and interferon. Therefore, lymphocytosis often indicates that the host's immune response to tumor is impaired [23][24][25] .In summary, patients with LLCR and LPNI who had an immunosuppressed state and malnutrition had shorter OS time. In addition, our results suggest that patients with LLCR may have HNLR, HPLR and HSII, and the degree of in ammation in vivo may be more severe. At present, it has been reported that systemic in ammatory markers such as preoperative LCR, PNI, NLR, PLR and SII can be independent predictors of prognosis of gastric cancer, colorectal cancer, lung cancer and osteosarcoma [26][27][28][29] . As the most prevalent malignant bone tumor, the prognostic factors of osteosarcoma, such as local recurrence, metastasis, tumor stage and pathological fracture, have been reported in a large number of clinical studies. [1,28,[30][31][32] . However, there are few studies on the correlation between preoperative LCR and PNI and the prognosis of patients with osteosarcoma. In this paper, the relationship between preoperative LCR, PNI, NLR, PLR and SII and the postoperative OS rate of 101 patients with osteosarcoma was analyzed to explore the important factors affecting the postoperative OS of patients with osteosarcoma. Univariate analysis by the Cox hazard regression model showed that tumor stage (P < 0.001), metastasis (P < 0.001), LCR (P < 0.001), PNI (P = 0.001), NLR (P = 0.018), PLR (P = 0.002) and SII (P < 0.001) were signi cantly associated with postoperative OS in patients with osteosarcoma. However, after multivariate analysis by the Cox hazard regression model, only tumor stage (HR, 2.717;95%CI, 1.067-6.919; P = 0.036) and preoperative LCR (HR, 0.401;95% CI,0.199-0.807; P = 0.01) were independent prognostic factors for OS after surgery. However, Huang et al. [28] considered that PNI and SII were also independent prognostic factors for patients with osteosarcoma, which was different from the results of this study. These differences may be due to the small sample size and heterogeneity of the study population. For example, of the 126 patients with osteosarcoma included in the study by Huang et al, 31.7% received neoadjuvant chemotherapy. In our study according to the results of the univariate analysis, NLR, PLR, SII, PNI and metastasis were the important factors in uencing the postoperative OS in patients with osteosarcoma, but by the multivariate Cox hazards regression model analysis to eliminate confounding factors, the effect of the above factors on the postoperative OS of patients with osteosarcoma decreased obviously.
There are differences in research results related to osteosarcoma in recent years [1,28,[30][31][32] . The reasons for this result may be as follows: 1. the sample size included in this study is small, 2. The included indicators are too few, 3. the mutual in uence of different indicators. There are some limitations to our study. First, the number of cases included in this study is small, with only 101 cases. Second, our study is a single-center retrospective study, which may have a large selection bias, which needs to be further con rmed by a multicenter, large sample size study or prospective study. In addition, there are many factors affecting the prognosis of osteosarcoma, and still need more studies to determine the correlation between different in uencing factors. It is still necessary to further explore the relationship between systemic in ammation and nutritional immunity and the prognosis, recurrence and metastasis of osteosarcoma.

Conclusion
The higher preoperative LCR is strongly associated with the longer survival time in patients with osteosarcoma. Tumor staging and preoperative LCR may be important parameters for the prognosis of osteosarcoma. Declarations