Proportions of Bene cial Factors in MLR, NLR, PLR and D-dimer in Preoperative Peripheral Blood of Patients With Early Stage Lung Cancer as Predictors of Patient Survival After Surgery

jun wang The Second A liated Hospital of Harbin Medical University huawei li The Second A liated Hospital of Harbin Medical University ran xu The Second A liated Hospital of Harbin Medical University tong lu The Second A liated Hospital of Harbin Medical University jiaying zhao The Second A liated Hospital of Harbin Medical University Pengfei zhang The Second A liated Hospital of Harbin Medical University lidong qu The Second A liated Hospital of Harbin Medical University Shengqiang zhang The Second A liated Hospital of Harbin Medical University Jida guo The Second A liated Hospital of Harbin Medical University Linyou zhang (  lyzhang@hrbmu.edu.cn ) The Second A liated Hospital of Harbin Medical University


Abstract Objective
The purpose of this paper is to predict the following items. preoperative baseline monocyte-to-lymphocyte ratio (MLR) neutrophil-to-lymphocyte ratio (NLR) Platura-to-lymphocyte ratio (PLR) and dimeric brin fragment D (D-dimer) associated with clinical outcome in patients with Early Lung Cancer (LC).

Methods
We performed a retrospective analysis of 376 patients with LC. Progression-free survival (PFS) and overall survival (OS) were assessed by Kaplan-Meier, and univariate and multivariate Cox regression analyses were performed to identify prognostic factors. Finally, multivariate Cox regression analysis was used to evaluate the in uence of favorable factors on patients' OS and PFS combined with the basic clinical characteristics of the patient

Results
Among the variables screened by univariate Cox regression, MLR < 0.22, NLR < 1.99, PLR < 130.55 and D-Dimer < 70.5 (ng/ml) were signi cantly associated with both better OS and PFS. In multivariate Cox regression analysis, it was determined that MLR and D-Dimer had a better independent correlation with OS (p = 0.009, p = 0.05, respectively), while MLR was only better independently associated with PFS (P = 0.005). Furthermore, according to the number of favorable factors, patients with none of these factors had a signi cantly worse prognosis than patients with at least one of these factors.

Conclusion
Baseline characteristics of low MLR, low NLR, low PLR and low D-dimer were associated with better outcomes.

Background
With the increasing morbidity and mortality in China, malignant tumors have become the main cause of death, among which lung cancer is the most common cancer and the main cause of death 1,2 . With the development of science and technology, the treatment of lung cancer also presents a variety of methods including surgery, chemotherapy and immunotherapy.However, due to the new coronavirus epidemic, some operations cannot be performed in time. Nevertheless, studies have shown that the bene t of delayed surgical treatment for patients with early-stage non-small cell lung cancer is still better than immediate radiotherapy 3 . Nevertheless, there is still a wide variation in OS and PFS in post-operative patients. Currently, the prediction of survival in postoperative lung cancer patients relies mainly on tumor node metastases (TNM) staging 4 . Although genetic and some molecular tests have shown great promise in predicting patient prognosis 5 , their huge nancial burden makes it di cult to be widely available in most otherwise a uent cancer families. Therefore, we are working to identify novel circulating biomarkers that can successfully predict patient outcomes during routine preoperative testing.
In recent years, many studies have demonstrated the important role of peripheral blood markers in the prognosis of patients with various tumors. Patients with bladder cancer with a low NLR 6 had signi cantly better clinical survival outcomes than those with high NLR 6 .In patients with HER2+ breast cancer, lower NLR and lower MLR show longer survival status 6,7 . It was reported that Plasma D-dimer was regarded as a prognostic marker for various types of malignancies, which included non-small-cell lung carcinoma (NSCLC) 8 . In addition, peripheral blood biomarkers have shown good prognostic ability in a variety of solid tumors including melanoma 9 , colorectal cancer 10 , esophageal cancer 11 and pancreatic cancer 12 . The above studies strongly suggest that single peripheral blood biomarkers have good prognostic ability in patients with malignancies, but whether there is a superimposed effect or whether these peripheral blood parameter indicators interact with each other needs further elucidation.

Patients
This study retrospectively reviewed the medical records of all LC patients with stage I and stage II that who were treated with standard lobectomy at the Department of Thoracic Surgery, Second A liated Hospital of Harbin Medical University, Heilongjiang Province, China, from January 2015 to July 2017.
Inclusion criteria: (1) preoperative imaging suggestive of a mass con ned to a single lung lobe; (2) no distant metastases; (3) no preoperative adjuvant medication; (4) no hematological malignancies; (5) complete clinical and follow-up information; and (6) survival for at least 30 days postoperatively. Patients were followed up every three months after surgery via outpatient clinics or over the phone, with the last follow-up visit for all patients on June 30, 2020. This paper has been approved by the Ethics Committee of the Second A liated Hospital of Harbin Medical University. Because the study was retrospective, informed consent from patients was not required. Patient data con dentiality rules are consistent with the Declaration of Helsinki.

Data collection
Peripheral blood biomarkers including neutrophil count (10^9/L), monocyte count (10^9/L), lymphocyte count (10^9/L), platelet count (10^9/L) and D-dimer (ng/ml) were collected from the electronic medical record within 3 days prior to the procedure, as well as the patient's age, gender, BMI, underlying disease history, pathology pro le and ECOG at that time scores and other basic clinical information. NLR and MLR were calculated as follows: NLR equals neutrophil count/lymphocyte count; Second, MLR equals monocyte count/lymphocyte count and PLR equals platelet count/lymphocyte count. Survival rates were analyzed by PFS and OS.

Statistical analysis
According to receiver operating Characteristic (ROC) curve analysis, the critical values of MLR, NLR, PLR and D-dimer can be determined and the highest Yorden index (de ned as sensitivity + speci city -1) can be used to predict OS. This study used SPSS version 25.0 (IBM Corp., Armonk, NY, USA) for baseline statistics of patient clinical data. Moreover, OS and PFS were analyzed according to the Kaplan-Meier estimate by the log-rank test. Univariate and multivariate Cox regression were used to determine 95% con dence interval (CI) and risk ratio (HR). Clinical characterizations, including smoking status (former or never), age, multivariate cox regression was used to analyze the performance of patients in the Eastern tumor cooperative group. A P value less than 0.05 was considered statistically signi cant. P <0.05 on both sides was considered statistically signi cant.

Patient demographics and clinical characteristics
As shown in Table 1, 376 patients with early LC who underwent surgery were nally screened out. The results showed that there were 218 cases (58%) in males and 158 cases (42%) in females. The median age was 60(53,65) years. In our population, the majority of patients had an ECOG physical status of 0-1 (320/376, 85.1%) and the greatest number of patients had lung adenocarcinoma (n=233/376, 62%).

Multivariate Model for Survival of Patients
Then, according to the quantitative analysis results of favorable factors, including MLR<0.22, NLR<1.99, PLR<130.55 and D-DIMER <70.5(ng/ mL).As shown in Figure 2, Sixty-eight patients (18%) showed no signi cant reduction in PFS and OS compared to those who had 1 and 2(group II, at 71 and 93, respectively) or three and four (group III, at 102 and 42, respectively) (Kaplan Meier analysis and survival rates compared to P < 0.0001, respectively). Multivariate COX analysis including clinically important Covariates (age, gender, BMI, ECOG ps, etc.) con rmed that the number of favorable factors was closely associated to PFS and OS (Table 5).

Discussion
To date, biomarkers remain a major focus of research in the eld of Oncology, whether in the diagnosis of disease, the assessment of e cacy of treatment or the prognosis of patients. The connection between in ammation and cancer was rst explored in 1863 by Rudolf Virchow et al. 15 . Since then, more and more studies have further con rmed the value of in ammatory markers in the diagnosis and prognosis evaluation of various malignant tumors. The in ammatory response is an organism's antagonistic response to noxious stimuli, both exogenous and endogenous 16,17 .In ammation associated with cancer has a dramatic impact on the tumor microenvironment, which consists of tumor cells and in ammatory cells that release various cytokines and chemotactic factors 18 . Among them, NLR, PLR, MLR and D-dimer are closely related to in ammation and immune status of cancer patients, and have been applied to predict the prognosis of patients with various solid tumors 9-12 . NLR, PLR and MLR, as indicators of in ammation, are obtained from peripheral blood neutrophilic granulocyte, platelets and monocytes compared to lymphocytes, respectively. Therefore, the ratios between them can similarly indicate the role of in ammatory mediators in tumors.
The function of in ammatory mediators in the tumor microenvironment is not fully understood, but there are several potential mechanisms that could provide a simple explanation for their role. (1) Lymphocytes are an important component of an organism's immune system, playing a major role in the body's immune surveillance and serving as a protective prognostic factor for patients with malignancies 19 . CD8 cytotoxic T lymphocytes (CTL) are the primary immune cells that target tumors. During cancer progression, CTL become dysfunctional and suppressed due to immune-related tolerance and immunosuppression within the tumor microenvironment (TME) 20 . (2) The role of neutrophilic granulocyte in the body is to regulate immunity by producing tumor necrosis factor (TNF)-α, a cytokine that impairs CD8T cell activity and increases vascular permeability, thus suppressing the immune system by inhibiting the immune activity of lymphocytes and ultimately leading to progression and metastasis 21,22 . (3) Monocytes play an important role as a protective immune factor in suppressing tumor growth by inducing the recruitment and function of lymphocytes in TME and interacting with adaptive immunity, especially in peripheral blood where monocytes are involved in paracrine signaling and produce many in ammatory cytokines and chemokines, including tumor necrosis factor α 23 . (4) Platelets in plasma play a vital part in tumor hematogenous metastasis and are a prerequisite for it. The mechanism of action may be that platelets protect tumor cells from shear and NK cell attack 24 . (5) Although the mechanism of plasma D-Dimer in tumor development is still unclear, some studies have reported that elevated plasma D-Dimer levels in breast cancer patients are associated with progesterone receptor expression, TNM staging and metastasis in breast cancer 25 .
Although a number of studies have demonstrated that NLR, PLR, MLR and D-Dimer can be used as potential prognostic biomarkers in patients with a variety of solid tumors, these studies have more or less analyzed only one or two of these in ammatory indicators as markers. MLR and D-dimer were also proposed in our study as independent predictive markers of prognosis in patients with surgically treated lung cancer. However, we are not aware of any studies that have examined whether there is superposition between these markers or whether there is an interaction between them. For this reason, we divided these markers into three groups according to the number of bene cial factors, and the results of a multifactorial COX regression show that patients in the group without a single bene cial factor had the worst prognosis compared to the other two groups, while patients in the group with the most bene cial factors had the best prognosis. It is reasonable to believe that the number of bene cial peripheral blood biomarkers described above could be a valid predictor of the prognostic status of patients. There are some shortcomings in our study, as a retrospective study, selection bias may occur in the investigation and therefore the results need to be further clari ed in a multicenter prospective study. In addition, only patients with operable early-stage lung cancer were selected, while it is unknown whether patients with inoperable advanced lung cancer have a superimposed effect in these tumor markers, and further studies are needed. 2. Exemption of informed consent will not adversely affect the rights and health of subjects.Note: The patient only needs to accept the normal diagnosis and treatment process of the disease, and any medical treatment and rights will not be affected.

Conclusion
3. The subjects' privacy and personally identi able information are protected.Note: The personal information of patients in the study is con dential. Information that can identify patients will not be disclosed to members other than the research team. All study members and study sponsors are required to keep patient identities con dential. The patient le will only be available to researchers. In order to ensure that the research is carried out in accordance with regulations, when necessary, the government management department or the members of the ethics committee can review the patient data in the research unit according to the regulations. When the results of this research are published, no personal information about the patients will be disclosed.
4. If informed consent is required, the research will not be possible (patients have the right to know that their medical records/specimen may be used for research, and their refusal or disagreement to participate in the research is not the reason why the research cannot be implemented or the informed consent is exempted).Note: Patients have the right to know that their tissue samples may be used for research and have the right to refuse to participate in the research.
5. This study does not use medical records and specimens that patients/subjects have speci cally refused to use in the past.
. Research using human body materials or data with identi able information has failed to nd the subject, and the research project does not involve personal privacy or commercial interests.
We believe that our research meets the above conditions. This study has been reviewed by the Ethics Committee of the Second A liated Hospital of Harbin Medical University, with approval number:KY2021-254.

Consent for publication
This study is a retrospective study, and the ethics committee's exemption consent application has been passed, and the relevant data is only used in this study Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due [Our ethics committee stipulates that the information that can identify patients will not be disclosed to members other than the research team] but are available from the corresponding author on reasonable request.