Utility of Blood Biomarkers as a Predictor of Pathological Lymph Node Metastasis in Clinical Stage T1N0 Esophageal Squamous Cell Carcinoma

Background Accurate preoperative evaluation of lymph node (LN) metastasis is important for determining the treatment strategy for supercial esophageal cancer. There have been reports on the clinical signicance of blood biomarkers, such as the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and lymphocyte-monocyte ratio (LMR), as predictors of LN metastasis for different cancers. Methods neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; SD, deviation

clinical N0 esophageal cancer develop metastasis to the regional LNs after esophagectomy [12]. Therefore, accurate preoperative assessment of regional LN metastasis and assessing the depth of tumor invasion are important to determine the appropriate treatment strategy for super cial esophageal cancer. Although the accuracy of the assessment of LN metastasis has been improved by advances in diagnostic imaging, it remains inadequate and needs to be improved by use of other diagnostic tools.
Lymphocytes act as tumor suppressors and are widely used as indicators of immunocompetence [13,14]. Neutrophils, platelets, and monocytes are indicators of in ammation, which is associated with increased tumor growth, invasion, and angiogenesis, and can be used to evaluate tumor progression [15][16][17]. The neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and lymphocyte-monocyte ratio (LMR) are indicators of nutritional status and the in ammation associated with increased tumor growth, invasion, and angiogenesis and can be used to assess tumor progression and immune status.
Changes in NLR, PLR, and LMR values may re ect broader changes in the tumor microenvironment and are reportedly associated with poor survival in patients with many types of solid tumors [18][19][20][21][22].
Furthermore, there have been reports on the usefulness of these biomarkers as predictors of LN metastasis for different cancers in recent years [23][24][25][26] but not for esophageal cancer.
Therefore, we hypothesized that these biomarkers may also be useful tools for detecting microscopic metastasis of cancer cells to the LNs for esophageal cancer with an early tumor stage, which is di cult to predict by imaging. The aim of this study was to investigate the usefulness of NLR, PLR, and LMR as predictors of pathological LN metastasis and prognosis in patients with clinical stage T1N0M0 esophageal squamous cell carcinoma (ESCC).

Patients
Patients with pretreatment clinical stage T1N0M0 ESCC who underwent esophagectomy with R0 resection at Hiroshima University Hospital between April 2003 and August 2021 were enrolled (n = 185). Table 1 shows the patient demographic and clinicopathological characteristics. The study was approved by the Institutional Review Board of Hiroshima University (E-2225).  Endoscopic treatment LN metastasis was extremely rare in mucosal lesions involving the epithelium and lamina propria, and endoscopic mucosal or submucosal dissection was performed as the initial treatment unless endoscopic resection was technically di cult. Patients whose pathological results after endoscopic treatment indicated that the lesion was deeper than the muscularis mucosa or had a positive vertical margin were eligible for additional surgical resection and were included in this study.

Surgical treatment
All patients underwent open transthoracic (n = 112) or thoracoscopic esophagectomy (n = 73) and LN dissection in at least two elds (thoracic and abdominal). Esophageal cancer in the upper or middle third of the thoracic esophagus was treated by cervical lymphadenectomy. A gastric tube was subsequently raised for cervical anastomosis with the esophagus. The reconstruction path was in the retrosternal (n = 89) or posterior mediastinal (n = 88) regions or in the region before the chest wall (n = 8). Three experts in esophageal surgery performed these procedures.

Adjuvant chemotherapy
Patients with stage II or higher disease based on postoperative pathology results received adjuvant chemotherapy, which consisted of cisplatin plus 5-uorouracil. Cisplatin 80 mg/m 2 was administered via intravenous infusion on day 1 and 5-uorouracil 800 mg/m 2 was administered from days 1 to 5 by intravenous injection. Two courses were administered at 3-week intervals.

NLR, PLR, and LMR data
Blood samples were collected preoperatively. In patients who underwent additional surgical resection after endoscopic treatment, blood samples were collected before the endoscopy procedure to avoid the effects of in ammation associated with invasive endoscopic treatment. The NLR was de ned as the absolute neutrophil count divided by the absolute lymphocyte count, PLR as the platelet count divided by the absolute lymphocyte count, and LMR as the absolute lymphocyte count divided by the absolute monocyte count.

Statistical analysis
The results are presented as the number (percentage) or as the mean, unless otherwise stated. Survival was analyzed using Kaplan-Meier curves, which were compared using the log-rank test. Relapse-free survival (RFS) was de ned as the interval between the date of surgery and either the rst event (recurrence or death from any cause) or the most recent follow-up. Overall survival (OS) was de ned as the interval between the date of surgery and either death due to any cause or the last follow-up visit.
Optimal cutoff values for NLR, PLR, and LMR were determined from receiver-operating characteristic curves. Multivariate Cox regression analysis was performed to identify independent predictors of OS and RFS. A backward stepwise method was used to select variables for multivariate analysis. All statistical analyses were performed using the JMP Pro 15 software (SAS Institute Inc., Cary, NC, USA). A P-value <0.05 was considered statistically signi cant.

Results
Blood sampling data Table 2 presents the blood sampling data. The mean NLR, PLR, and LMR (± standard deviation) values were 2.1 ± 1.1, 128.4 ± 48.7, and 5.7 ± 2.6, respectively. Values are shown as mean ± standard deviation. LMR, lymphocyte-to-monocyte ratio; NLR, neutrophilto-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; SD, standard deviation Clinical characteristics of patients according to pathological LN metastasis Table 3 shows the clinical characteristics of patients according to the detection of pathological LN metastasis. The features that showed signi cant between-group differences according to pathological LN metastasis were the length of the primary tumor, depth of tumor invasion on EUS, NLR, PLR, and LMR.   Multivariate analysis showed that the length of the primary tumor, depth of tumor invasion on EUS, and NLR were statistically signi cant predictors of LN metastasis (Table 6).  Fig. 1a), as was the ve-year OS (90.9% vs. 72.4%, P < 0.0001; Fig. 1b).

Discussion
LN metastasis is an independent and important prognostic factor in patients with esophageal cancer.
Despite their importance, the preoperative clinical LN diagnosis and the nal pathologic evaluation may not be in agreement. The presence of false-negative LNs obscures the actual status of the cancer and the choice of treatment. EUS, CT, and FDG-PET are often used to diagnose LN; however, past studies have reported their diagnostic performance to be unsatisfactory. EUS, when combined with ne needle aspiration, has been shown to improve the sensitivity for diagnosis of LN metastasis from 84.7-96.7% [4][5][6]. However, this method can only evaluate LNs proximal to the esophagus and gastric wall. FDG-PET has the disadvantage of low spatial resolution, which makes it di cult to distinguish between LNs adjacent to the tumor and the tumor itself. Furthermore, FDG-PET is not suitable for diagnosis of small LN metastases. FDG-PET has been reported to detect tumors with a diameter of more than 6 mm or an area of more than 33 mm 2 [7,8]. Therefore, diagnosis of small LN metastases is di cult. CT is a noninvasive modality that is widely used to evaluate invasion of esophageal cancer to the LNs. Detection of metastatic LNs on CT depends primarily on size, and the sensitivity of CT for detection of metastasis is inadequate unless LNs are larger than 10 mm [7,9,10]. The short-axis diameter alone is insu cient to detect normal-sized metastatic LNs and to distinguish between reactive hyperplasia and metastatic hypertrophy [11]. Previous studies have reported false-negative rates of 11-56% for LN metastases [12].
In our study, LN metastasis was found in 24.3% of cases (21.1% for pN1 and 3.2% for pN2) and included many false-negative results.
Lymphocytes act as tumor suppressors and are widely used as indicators of immunity [13,14]. Neutrophils, platelets, and monocytes are indicators of in ammation, and systemic in ammation plays an important role in all stages of tumorigenesis. In ammation is thought to contribute to tumor initiation through genetic mutations, genomic instability, and epigenetic changes. It also activates tissue responses that induce proliferation of precancerous cells and enhance their survival. In ammation also stimulates angiogenesis, causes immunosuppression, and encourages a microenvironment in which malignant cells can survive and ultimately promote metastatic spread [15][16][17]. NLR, PLR, and LMR from these blood components have been used as indicators of the nutritional status of patients and are also attracting attention as biomarkers for assessment of immune status and tumor progression. Changes in NLR, PLR, and LMR may re ect widespread changes in the tumor microenvironment and have been recently identi ed as prognostic markers for cancer [18][19][20][21][22]. Compared with other biomarkers, these have the advantages of being inexpensive and rapid to detect with no additional costs. Apart from their prognostic value, these biomarkers are reportedly useful for predicting recurrence, the e cacy of preoperative The group with a high NLR also had a signi cantly poorer RFS and OS, further con rming that NLR is a useful prognostic factor. Interestingly, in subgroup analysis, even in the pN0 group, which is considered to have a good prognosis, the group with a high NLR had a signi cantly poorer RFS and OS. The NLR may also be an important predictor of recurrence in patients with pN0 disease. Therefore, it may be a prognostic factor even in very early-stage esophageal cancer. It is possible that the immune system in patients with a high NLR value, i.e., those with impaired immunity because of cancer, may not be able to destroy microscopic cancer cells that are not clinically recognized as LN metastasis, leading to postoperative recurrence. However, further research is needed to elucidate this problem.
This study had several limitations. First, it had a retrospective design. Second, the number of patients was somewhat small, and the study was conducted at a single institution. Therefore, larger prospective studies are required in the future to con rm our ndings regarding the usefulness of the NLR as a predictor of pathological LN metastasis and the prognosis in patients with clinical stage T1N0 ESCC.

Conclusions
Among the various blood biomarkers investigated in this study, the NLR was the most useful predictor of pathological LN metastasis. Furthermore, the NLR was strongly correlated with prognosis and may be useful in predicting recurrence. NLR may be a useful tool in the evaluation of LN metastasis, which is In the study, all methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Institutional Review Board of Hiroshima University (approval no. E-2225). Informed consent was obtained from all subjects or their legal guardians for participating in the current study.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and analysed during the current study available from the corresponding author on reasonable request.  ve-year RFS and (b) the ve-year OS were better in the group with a low NLR than in the group with a high NLR (P < 0.0001). For patients without pathological LN metastasis, (c) the ve-year RFS and (d) the ve-year OS were better in the group with a low NLR than in the group with a high NLR (P < 0.0001). LN, lymph node; NLR, neutrophil-to-lymphocyte ratio; OS, overall survival; RFS, relapse-free survival.