Preoperative Lymphocyte-to-monocyte Ratio and CA125 Level as Risk Factors for Advanced Ovarian Cancer

Background: Detailed descriptions of the relationship between lymphocyte-to-monocyte ratio alone and combined with CA125 (COLC) and advanced stage of ovarian cancer (OC) have been lacking to date. This study is to analyze the relationship between LMR, CA125 and COLC and advanced stage of OC. Methods: A retrospective clinicopathologic review was performed. The receiver-operating characteristic (ROC) curves of LMR, CA125, and COLC staging OC were constructed. Furthermore, a binary logistic regression model was used to assay the independent risk factors. Results: A total of 225 patients with OC were identied in this cohort. Eighty-ve patients with OC were diagnosed at an early stage, and 140 OC patients were diagnosed at an advanced stage. The median of LMR at the early stage was higher than the advanced stage (4.39 vs. 2.78), and the median of CA125 was lower than the advanced stage (80 U/mL vs. 251.25 U/mL). Multivariate logistic regression indicated that LMR (OR=0.314, 95% condence interval [CI]: 0.143–0.687, P=0.004) and CA125 (OR=4.045, 95%CI: 1.883–8.692, P<0.001) were associated with OC staging. Furthermore, the area under the curve of COLC was higher than that of LMR (0.779 vs. 0.732) or CA125 (0.779 vs. 0.708) in staging OC. The specicity of COLC was higher than that of LMR (87.11% vs. 70.61%) or CA125 (87.11% vs. 61.21%) in staging OC. Conclusions: LMR alone or in combination with CA125 might be associated with OC staging. Besides, as a predictive factor, COLC may have high specicity in staging OC.


Background
Ovarian cancer (OC) is the third most common gynecologic malignancy with high mortality [1]. Owing to the tissue and anatomical characteristics [2], a number of patients are diagnosed with advanced disease (FIGO III-IV, version 2014), by which time it is di cult to achieve optimal debulking surgery (ODS).
Preoperative staging of OC is bene cial in terms of the choice of treatment. However, the effective index that can accurately re ect the preoperative stage of OC patients is not clear.
The lymphocyte-to-monocyte ratio (LMR) represents the lymphocyte levels divided by the monocyte levels in OC. The LMR has recently been evaluated for its magnitude of survival in various solid cancers [3][4][5].
However, few studies have evaluated the magnitude of LMR on OC staging. Cancer antigen 125 (CA125) is a mucin-type glycoprotein, produced by the MUC16 gene, associated with the cellular membrane [6]. Previous studies have shown that CA125 was associated with the prognosis of OC [7]. However, the relationship between CA125 and OC staging is still not completely understood and shows inconsistent ndings.
Thus, we assessed the magnitude of LMR alone and in combination with CA125 (COLC) to attempt to identify preoperative factors to stage OC.

Patients
In this retrospective study, data of 225 patients with OC diagnosed in the a liated Nanchong Central Hospital of North Sichuan Medical College from January 2008 to May 2020 were collected. The inclusion criteria were as follows: (1)all patients underwent primitive surgery under the chief physician; (2) pathological diagnosis was used as the gold standard for assessing the stage of OC, and the pathology reports were obtained after evaluation and issuing by two senior pathologists; (3) the complete blood count and serum CA125 values were collected before initiation of anti-tumor therapy; (4) su cient data could be extracted for the fourfold Table. The exclusion criteria were as follows: patients who had received anti-tumor therapy such as radiotherapy and chemotherapy before the operation, and those that had complications due to other malignant tumors except OC.

Samples And Marker Assays
Preoperative blood samples collected in appropriate collection tubes were transported to the hospital clinical laboratory center at room temperature to determine whole blood cell count and CA125 level.
Samples were retrieved and serum CA125 concentrations were measured by two-step immunoassay for the quantitative determination with exible assay protocols [8]. All biomarkers were assayed in a central laboratory within 3 days before surgery, and the lab personnel were blinded to the clinical data.

Data analysis
Clinical variables such as age; BMI; FIGO stage; histological type and grade; malignant ascites; lymph node metastases; and clinical characteristics (white blood cell, lymphocyte, and monocyte count and serum CA125 levels) within 3 days before surgery were retrospectively analyzed. The LMR was calculated based on the ratio of peripheral blood lymphocyte count to monocyte count [4]. If the patient had several preoperative test results of blood parameters, the most recent results before surgery were selected for analysis.
Descriptive statistics were used to show the baseline characteristics of participants in this study. Chi square test and binary logistic regression were used to analyze the relationship between clinicopathological features and OC staging. Areas under the receiver operating characteristic curves (ROCs; MedCalc Software bvba [ver. 15.2.1], Ostend, Belgium) were used to assess LMR, CA125, and COLC. The optimal cut-off values were determined according to ROC. BMI was categorized as > 24 or ≤ 24 [9]. Age was classi ed as ≥ 50 or < 50 (years) [7]; white blood cell as > 6.4 or ≤ 6.4 [4]. The de nition of COLC was based on the Glasgow prognostic score (GPS) ( Table 5). Statistical analyses were performed using SPSS software (ver. 20.0; IBM Corp, Armonk, NY, USA). A two-sided P value of < 0.05 was considered to indicate statistical signi cance.

Roc Curves Of The Lmr And Ca125
The ROC curve of LMR staging OC is shown in Fig. 1a. The optimal cut-off value of LMR was 3.67, and the sensitivity and speci city were 70.17% and 70.61%, respectively. Patients with OC were divided into two groups based on this cut-off value (LMR > 3.67, n = 101, 44.89%; LMR ≤ 3.67, n = 124, 55.11%).

Clinical Characteristics Of Oc Between Early And Advanced Stage
Clinicopathological features between early and advanced stage of OC were compared by chi-square test. The median of LMR in early stage cancer was higher than that in advanced stage cancer (4.39 vs. 2.78), and the median of CA125 was lower in the early than advanced stage of cancer (80 U/mL vs. 251.25 U/mL). Results showed that BMI ≥ 24, histologic serous and G1, malignant ascites (+), lymph node metastases (+), LMR ≤ 3.67, CA125 > 95.7 U/mL were all associated with advanced stage of OC (P < 0.05) ( Table 2).   The usefulness of COLC in staging OC As shown above, both LMR and CA125 were biomarkers to stage OC. However, whether COLC had the same e cacy needed further investigation. The de nition of COLC was as per the GPS ( Table 5). The ROC curve of COLC in staging OC was constructed (Fig. 1c). The capacities of LMR, CA125, and COLC in staging OC patients were compared by ROC curves. The result showed that the sensitivity and speci city of COLC were 64.41% and 87.11%, respectively, and the AUC of COLC was 0.779 (95%CI: 0.719−0.831, P < 0.001; Fig. 1c), which was higher than that of LMR (87.11% vs. 70.61%; Fig. 1a, 1c) or CA125 (87.11% vs. 61.21%, Fig. 1b, 1c).

Discussion
In this study, we attempted to determine preoperative indicators to stage OC to help attain ODS, reduce surgical complications and economic burden, and better evaluate the prognosis of OC patients. Multivariate logistic regression analysis showed that LMR was signi cantly associated with stage of OC (Table 3): the lower the LMR, the higher the stage, and the worse the prognosis, which was consistent with the conclusion of previous research [10]. Our study also showed that serum CA125 was associated with stage of OC (Table 3); however, the speci city was lower than LMR (Figs. 1a, 1b). To better analyze the factors associated with OC staging, we recommended COLC to improve the accuracy, which achieved a higher AUC and speci city than LMR or CA125, indicating that COLC might have a higher speci city associated with OC staging.
Our study also showed that ascites was associated with stage of OC (Table 3), one probable mechanisms is that constitutive expression of STAT3 in malignant ascites plays a role in ovarian tumor progression and metastasis [11]. Besides, Matt et al (2019) showed that ascites might be associated with CA125 by regulating MUC16 expression at a posttranscriptional level through an Akt-dependent pathway, accordingly, we matched samples with presence or absence of malignant ascites, and the results showed that both LMR and CA125 were signi cantly associated with OC staging for presence or absence of malignant ascites (Table 4).
LMR and CA125 are calculated directly or computed from blood and can be easily measured, in addition to being practical and inexpensive metrics. At present, several studies have been conducted with respect to the application of LMR and CA125 in OC. LMR and CA125 could likely serve as clinically useful indicators of metastasis and survival in OC patients [4,7,12]. CA125 also was reported as a risk factor in the diagnosis of OC [6]. However, few studies have been conducted with respect to the magnitude of LMR an CA125 on staging OC. Our study preliminarily showed that LMR, CA125, and COLC might be risk factors for OC staging.
The mechanisms underlying the capacity of LMR in OC have not yet been elaborated. We tried to explain the possible mechanisms. First, the association between lymphocytes and malignancies has been well established. Tumor in ltrating lymphocytes in OC can prevent cancer cells from spreading and metastasis by establishing a defense barrier [13]. The decrease of peripheral blood lymphocyte count may lead to weak and insu cient tumor immune response, thereby promoting tumor progression and metastasis [14] and resulting in rapid disease progression and late surgical pathological stage in OC patients. Second, in ammation can make monocytes migrate from the bone marrow into peripheral blood [15]. After being recruited into tumor tissue, monocytes can differentiate into tumor-associated macrophages (TAMs) [16]. TAMs can not only play an immunosuppressive role in a variety of tumor microenvironments, including OC, but can also promote tumor cell in ltration, growth and neovascularization, and metastasis [17,18]. Therefore, to an extent, peripheral blood mononuclear cell counts can re ect the formation or existence of TAMs. Besides, the LMR often represents the relative decrease of lymphocyte count and (or) the relative increase of monocyte count, which can re ect the balance of anti-tumor immunity and pro-tumor in ammatory response [12]: low-level LMR often represents the dominant role of pro-tumor in ammatory response, indicating a high level of malignancy and rapid progress in OC. Further, in this case, the possibility of advanced stage OC that is di cult to treat with relatively poor prognosis is higher; on the contrary, high-level LMR indicates that the anti-tumor immune system of patients with OC is more active, indicating that the disease progression of OC is slow, with the possibility of early stage OC and relatively better prognosis.
Rising and falling levels of serum CA125 correlate with the progression and regression of high-grade serous ovarian carcinomas [19], making CA125 a possible factor for OC staging. However, some researchers have also suggested that because of its low speci city and the observed increased levels in different physiological situations, CA125 is not considered as a very good differentiating biomarker for ovarian tumors [6]. Therefore, we recommended the combination of CA125 and LMR (COLC) to stage OC.
Our results showed that the AUC and speci city of COLC were higher than those of LMR or CA125 to stage OC (Fig. 1), implying that COLC might improve the accuracy in staging OC and provide guidelines for selection of treatment. As a new biomarker, COLC might play a role in immune surveillance and provide novel approaches and strategies for treatment of OC.
To our knowledge, this is the rst study to evaluate the relationship among LMR, CA125, COLC, and stage of OC. However, our study also has some limitations. Firstly, the retrospective design meant that we cannot conclusively state that LMR, CA125, and COLC are risk factors for advanced stage of OC.
However, our study provides a potential clinical strategy for staging advanced OC. Secondly, the potential bias in testing serum CA125 and peripheral blood level cannot be completely eliminated. Nonetheless, we believe that the in uence of LMR and CA125 level on the risk of advanced stage of OC will be of interest to clinicians to improve the accuracy in preoperative staging and make clinical strategies. Moreover, LMR is an in ammatory marker that could be affected by autoimmune status and other diseases; hence, we allowed a 30-min interval for blood collection to exclude any possible treatment or drug interference in the results.

Conclusions
LMR alone and in combination with preoperative CA125 might be used as independent risk factors for staging OC and may provide a new direction for making clinical decisions for OC.

Declarations
Ethics approval and consent to participate Our study was approved by the Ethics Committee of the a liated Nanchong Central Hospital of North Sichuan Medical College. Informed consent could not be obtained from every patient given the retrospective study design. Therefore, we posted a notice about our study design and contact information at a public location in Nanchong Central Hospital of North Sichuan Medical College.

Consent for publication
Not applicable, as all results presented in this manuscript were aggregated.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Con ict of Interest
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.