Identification and validation of LINC02381 as a biomarker associated with lymph node metastasis in esophageal squamous cell carcinoma

Introduction

Esophageal cancer is a common malignant tumor with a high incidence rate and high mortality, ranking 11th and 7th among cancers, respectively (1). Esophageal cancer mainly includes pathological types of esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (2). More than 50% of esophageal cancer cases occur in China, with ESCC being the predominant pathological type, accounting for over 90% of these cases. Among malignancies in China, esophageal carcinoma ranks 7th and 5th in incidence and mortality rate, respectively (3). Lymph node metastasis (LNM) is the most common form of metastasis in ESCC, and research indicates that even in early stages of the primary tumor, LNM is very common (4). A study has demonstrated that LNM is a key contributor to the poor prognosis of patients with ESCC (5). In addition, the treatment strategy for ESCC differs considerably depending on whether LNM is present, and it has been reported that if the primary tumor is at the T1a stage, endoscopic mucosal resection is preferable (6). This endoscopic approach for T1a lesions can avoid esophagectomy, which can be performed safely but is associated with some postoperative risks and lifestyle modifications (7). The treatment of ESCC varies by stage. Patients with ESCC and positive lymph nodes experience a survival benefit with preoperative neoadjuvant chemoradiation (8,9) or neoadjuvant immunotherapy (10). Therefore, accurate preoperative clinical staging is crucial for the treatment of these patients. The related research suggests that despite the use of effective diagnostic methods before surgery, rates of occult nodal metastases can range from 16% to 39%, even for clinical early-stage disease (11). The main preoperative diagnostic methods for LNM in ESCC are contrast-enhanced computed tomography (CT) or positron emission tomography-CT (PET-CT), but the rate of missed diagnoses remains high. In one study, the accuracy of preoperative contrast-enhanced CT in diagnosing LNM was reported to range from 46% to 58% (12). If LNM occurs after esophageal cancer surgery, it can seriously worsen the prognosis and quality of life of patients. For patients with regional and distant metastasis, the 5-year survival rate is 23% and 5%, respectively (13).

Biomarkers in ESCC are potentially very useful for diagnosing the development of cancer and presence of metastases (14). Studies on predicting LNM in ESCC have been extensively studied, and more recent research has identified biomarkers with high accuracy that are critical to treatment (15). The predictive biomarkers of ESCC examined thus far chiefly include microRNAs, with miR-20b-5p being a potential biomarker for predicting LNM in patients with T1 ESCC (16). Another study suggested that blood circulating tumor DNA (ctDNA) can serve as an independent prognostic biomarker for the LNM or distant metastasis of ESCC (17). Blood biomarkers, including neutrophil:lymphocyte, platelet:lymphocyte, and lymphocyte:monocyte ratios, have also been reported to be closely related to LNM in ESCC. Constructing a predictive model based on combined clinical features to predict early LNM may be a valuable strategy with promising application prospects (18).

Long noncoding RNAs (lncRNAs) are a group of non-protein-coding transcripts with a length greater than 200 nucleotides (19). The investigation of lncRNAs in cancer metastasis may lead to the identification of novel biomarkers and therapeutic targets (20). Research suggests that the HERES lncRNA holds substantial potential as a therapeutic target for ESCC, which may be related to defects in Wnt signaling pathways (21). In a study on the lncRNAs related to LNM in ESCC, VESTAR lncRNA was found to regulate lymphangiogenesis and the LNM of ESCC by enhancing VEGFC messenger RNA (mRNA) stability (22).

Our preliminary research suggests that lncRNAs may be associated with LNM in ESCC (23). LINC02381 has been previously reported to be located in the 12q13.13 region (24) and may be associated with cell proliferation, migration, and invasion in breast cancer, exerting its carcinogenic effects via the miR-1271-5p-FN1 axis to activate the PI3K/AKT pathway (25). Research on colorectal cancer and LINC02381 suggests that LINC02381 might have suppressive effects on human colorectal cancer tumorigenesis via regulation of the PI3K signaling pathway (26). In cervical cancer, LINC02381 is upregulated and acts as an oncogene, promoting cell viability and migration by targeting miR-133b (27). Meanwhile, studies related to glioma suggest that LINC02381 can promote the transcription of CBX5 through its interaction with CEBPβ (28).

However, no research exists concerning the association between LINC02381 and ESCC, especially as it relates to LNM. In our previous study (23), we used RNA sequencing (RNA-seq) and found that lncRNAs were significantly elevated in ESCC tissues with LNM. In the present study, we employed quantitative real-time polymerase chain reaction (qRT-PCR) and further found that LINC02381 was significantly elevated in ESCC tissues and closely related to the overall prognosis of patients. A subsequent series of validation experiments further supported LINC02381 as a biomarker for LNM in patients with ESCC. We present this article in accordance with the TRIPOD and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2402/rc).

Methods

Patient selection and clinical samples

Patients who had clinically localized ESCC (cT1-3N0M0) and who underwent esophagectomy for ESCC at Gaozhou People’s Hospital between January 2019 and June 2023 were reviewed. These patients underwent upper endoscopy before surgery, and the pathology of biopsies confirmed ESCC. They additionally underwent standardized lymph node dissection surgery, and LNM was confirmed by postoperative pathology. Patients were excluded if they were administered any type of neoadjuvant therapy (including radiation, chemotherapy, immunotherapy, hormone therapy, or other systemic therapy, primary tumor staging carcinoma in situ, or unknown primary tumor stage). Our study included 186 patients with ESCC tissues and 65 paired normal esophageal epithelial tissues. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The procedures involving human participants were approved by the Medical Ethics Committee of the Gaozhou People’s Hospital (No. GYLLPJ-2023145). Written informed consent was provided by all patients.

RNA extraction and qRT-PCR

Cell/Tissue Total RNA Kit (cat. no. 19221ES50; Yeasen, Shanghai, China) was used to extract total RNA from tissue or cultured cell lines, and then reverse transcription was applied with Hifiar III 1st Strand cDNA Synthesis SuperMix for qPCR (genomic DNA digesting plus) and complementary DNA (cDNA) synthesis kit (cat. no. 11141ES60; Yeasen). We conducted qRT-PCR by using Hieff UNICON Universal Blue qPCR SYBR Master Mix (cat. no. 11184ES08; Yeasen). We standardized our expression data to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which can control the variability of expression levels. We repeated our experiments for three times independently, and all the primers are listed in Table S1.

Cell lines

Our study included the following cell lines: human esophageal epithelial cells HET-1A and human ESCC cell lines TE-1, KYSE180, KYSE410, and KYSE510. Human esophageal epithelial cells HET-1A were also maintained in Roswell Park Memorial Institute (RPMI) 1640 (cat. no. 72400047; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium containing 10% fetal bovine serum (FBS; cat. no. A3161002C; Gibco). TE-1, KYSE180, and KYSE410 cells were maintained in RPMI 1640 medium containing 10% FBS. KYSE510 cells were maintained in RPMI 1640 and Ham’s F-12 Nutrient Mixture (1:1; cat. no. PM150810; Wuhan Pricella Biotechnology Co., Ltd., Wuhan, China) containing 10% FBS. The abovementioned cell lines were cultured at 37 ℃ in a 5% CO₂ atmosphere.

Small interfering RNA (siRNA) transfection

Three targets of the LINC02381 siRNAs and the negative control (NC) siRNA were purchased from RiboBio (Guangzhou, China) (the sequences are shown in Table S2). Transient transfections were performed in an antibiotic-free medium using Lipofectamine RNAiMAX Reagent (cat. no. 13778150; Invitrogen, Thermo Fisher Scientific) following the manufacturer’s protocol. As the expression of LINC02381 was significantly increased, LINC02381 silencing was performed by transfecting the siRNAs in the KYSE180 and KYSE510 cell lines for 48 hours. The knockdown efficiency of LINC02381 was determined via qRT-PCR.

Cell Counting Kit-8 (CCK-8) cell viability assay

KYSE180 and KYSE510 cells were collected 48 hours after transfection, and a concentration of 1,000 cells/200 µL of mixture medium was seeded into a 96-well plate. We seeded 1,000 cells into a 96-well plate, and then culture for 24, 48, and 72 hours. We added CCK-8 (cat. no. K1018-5; APExBIO, Houston, TX, USA) stock arrangement to each well, and then incubated at 37 ℃ with 5% CO₂ for 4 hours. Optical density (OD) values were then detected at a wavelength of 450 nm. Cell growth curves were plotted using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) based on the data obtained from the CCK-8 assays.

Colony formation assay

We used ESCC cell lines KYSE180/KYSE510 to perform a colony formation assay. We firstly seeded 1,000 cells/2 mL into a six-well plate the culture medium being changed weekly. Then we washed these cells with phosphate-buffered saline (PBS) after 2 weeks, fixed with paraformaldehyde for 30 minutes, and stained with 2% crystal violet for 30 minutes, finally we calculated the colony number. We repeated these experiments independently three times.

Wound healing migration assay

We used the tip of a P20 pipette to wound fused monolayer cells for the measurement of wound healing migration. After washing cells three times with PBS (time 0 hours), we obtained microscope images. The cells were then cultured in a standard medium, and the wound healing rates after 24 hours were recorded. We selected one representative image after a total of three separate fields were captured. We repeated these experiments independently three times.

Transwell assays

We seeded 50,000 KYSE180/KYSE510 cells into the upper chamber in the migration assay. After 48-hour culture, the chambers of transient transfection groups were stained with crystal violet. The mean number obtained from assays was used in the final analysis. We repeated these experiments independently three times.

Statistical analyses

We used GraphPad, R v. 4.4.1 (The R Foundation for Statistical Computing), and SPSS 26 software (IBM Corp., Armonk, NY, USA) to perform statistical analyses. For comparison of nonparametric variables, a χ² test was performed. For the comparison of parametric variables with normal distribution, a two-tailed unpaired or paired Student t-test was used. Quantitative data are presented as the mean ± standard deviation (SD). For the assessment of the overall survival (OS) and disease-free survival (DFS) of patients with ESCC, we used Kaplan-Meier (KM) analysis and log-rank test method. To screen for significant LNM predict variables, we firstly performed univariate logistic regression analyses, and included the factors with P<0.05 in the univariate analysis into multivariate logistic regression analysis. We then used multivariate analysis to identify independent risk factors for predicting LNM. We selected predictors according to both statistical significance (P<0.05) and clinical relevance. Primary tumor staging, tumor differentiation, and LINC02381 expression were finally included in our risk model. We used receiver operating characteristic (ROC) curves to evaluate predictive value, and the area under the curve (AUC) was applied to determine the discriminative values of variables for LNM. In addition to these numeric measures, we also used the calibration curve to display the calibration aspects of our final model. A nomogram based on the independent predictors for LNM identified by multivariate logistic regression analysis was constructed using the R package for regression modeling strategies.

Results

LINC02381 was significantly upregulated in ESCC with LNM

In our previous study (23), we performed RNA-seq on 10 pairs of ESCC tissues and adjacent normal tissues, and identified 26 significantly altered RNAs by using a Venn diagram of differentially expressed genes (DEGs) between T and N and between T3N+ and T3N−. The intersection included LINC02381 and lncRNA GAS6-AS1 (Figure 1A). To further validate our RNA-seq results, the LINC02381 expression in 65 ESCC tissues and paired normal esophageal tissues was detected via qRT-PCR (Figure 1B), which indicated that LINC02381 expression level was higher in ESCC tissues. LINC02381 expression in patients with LNM (LN+) was also significantly higher than that in patients without LNM (LN−) (Figure 1C). To eliminate interference from the number of lymph nodes resected, we calculated the number of lymph nodes dissected between the two groups and saw no difference (Figure 1D). These results indicated an elevation of LINC02381 expression in ESCC tissue, and an even higher increase in patients with LNM.

Figure 1 LINC02381 was significantly upregulated in lymph node-positive ESCC. (A) Venn diagram of our previous published RNA-seq data showing the DEGs between T and N and between T3N+ and T3N−, with the intersection including LINC02381 (23). (B) LINC02381 mRNA expression in the ESCC tissues and paired normal esophageal tissues as detected by qRT-PCR. (C) qRT-PCR analysis of LINC02381 expression in LN− and LN+. (D) Number of lymph node dissections in LN− and LN+. ns, no significance; ***, P<0.001. T, tumor tissues; N, normal tissues; T3N−, primary tumor stage T3 patients without lymph node metastasis; T3N+, primary tumor stage T3 patients with lymph node metastasis; LN−, patients without lymph metastasis; LN+, patients with lymph node metastasis; ESCC, esophageal squamous cell carcinoma; RNA-seq, RNA sequencing; DEG, differentially expressed gene; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction.

High expression of LINC02381 was associated with poor prognosis

A total of 186 patients with ESCC who underwent esophagectomy between January 2019 and June 2023 were reviewed. KM survival analysis was used to analyze OS and DFS data, and qRT-PCR analysis was used to examine LINC02381 expression in these patients. According to a median expression cutoff value, a high expression level of LINC02381 was negatively correlated with OS (Figure 2A) and DFS (Figure 2B). Meanwhile, patients with LNM had a worse prognosis, which was negatively correlated with OS (Figure 2C) and DFS (Figure 2D). We further found that patients with high expression levels of LINC02381 and LNM (LN+ and high LINC02381) had the worst prognosis. Meanwhile, patients without LNM and a low expression of LINC02381 (LN− and low LINC02381) had the longest OS (Figure 2E) and DFS (Figure 2F). Collectively, these data indicated that the expression level of LINC02381, especially when combined with LNM, was closely correlated with the poor prognosis of these patients.

Figure 2 The survival curve showing that high LINC02381 expression and lymph node metastasis was associated with significantly poorer survival. (A,B) Kaplan-Meier curve of OS and DFS in patients with high or low LINC02381 expression. (C,D) Kaplan-Meier curve of OS and DFS in ESCC patients with lymph node metastasis and those without lymph node metastasis. (E,F) Kaplan-Meier curve of OS and DFS in ESCC patients with lymph node metastasis and high LINC02381 expression (LN+ and high LINC02381), no lymph node metastasis and high LINC02381 expression (LN− and high LINC02381), lymph node metastasis and low LINC02381 expression (LN+ and low LINC02381), and no lymph node metastasis and low LINC02381 expression (LN− and low LINC02381). LN−, patients without lymph metastasis; LN+, patients with lymph node metastasis; OS, overall survival; DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma.

Clinical characteristics and development of the predictive model

The 186 ESCC samples were divided into two groups according to the median value determined by qRT-PCR analysis and were compared with the χ² test. Increased expression of LINC02381 in ESCC was significantly associated with higher differentiation grade (P=0.01) and TNM staging (P<0.001). Meanwhile, patients with a high expression of LINC02381 had higher rates of LNM. However, this was not related to the age, gender, or primary tumor invasion depth (Table 1). The 186 ESCC samples were also divided into two groups according to LNM status, which showed that patients with LNM had higher rates of more advanced tumor staging (P<0.001) and higher differentiation grade (P<0.001). However, it was not related to age or gender (Table 2). Univariable and multivariable logistic regression were conducted to determine whether LINC02381 expression was related to LNM. The univariate analysis showed that LINC02381 expression, tumor differentiation grade, and tumor stage were significantly associated with LNM. Finally, the variables independently associated with LNM and included in the final model were tumor differentiation grade, tumor stage, and LINC02381 expression [odds ratio (OR) =11.212; 95% confidence interval (CI): 4.870–25.812; P<0.001; Table 3].

ROC curve and nomogram for predicting LNM of ESCC

To verify the ability of LINC02381 expression to predict LNM, LINC02381 expression levels were used in ROC curve construction and yielded an AUC value of 0.821 (P<0.001) (Figure 3A). We then incorporated LINC02381 expression level, tumor grade, and clinical tumor stage into ROC curve analysis, which increased the AUC value to 0.913 (P<0.001) (Figure 3B). Importantly, we constructed a nomogram for predicting the probability of LNM, and the predictive variables (including LINC02381 expression level, tumor grade, and clinical tumor stage) and corresponding point scales are presented in Figure 3C. Finally, we drew a calibration curve for the predictive accuracy measures in this model, which indicated comparable performance to the original (Figure 3D). Overall, these data indicated that our model is an effective method for predicting LNM in ESCC.

Figure 3 ROC curves and nomogram for predicting pathologic positive lymph node status. (A) ROC analysis of LINC02381 expression for predicting lymph node metastasis (AUC =0.821). (B) ROC analysis of our model incorporating LINC02381 expression, tumor grade, and clinical T stage (AUC =0.913). (C) Nomogram for predicting the likelihood of pathologic positive lymph node status in patients with ESCC. (D) Calibration curve of the nomogram. T stage, tumor stage; ROC, receiver operating characteristic; AUC, area under the curve; ESCC, esophageal squamous cell carcinoma.

Knockdown of LINC02381 inhibited the growth and metastasis of ESCC

To clarify the role of LINC02381 in ESCC proliferation and metastasis, we first used qRT-PCR to detect the expression of LINC02381 in a normal esophageal epithelial cell line and ESCC cell lines. The results showed that the expression of LINC02381 in three ESCC cell lines (KYSE180, KYSE410, and KYSE510) was significantly increased compared with that in the normal esophageal epithelium cell line Het-1A (Figure 4A). Knockdown of LINC02381 expression in KYSE180 and KYSE510 cells was conducted with independent siRNAs, si#1 and si#2, due to their superior knockdown effect (Figure 4B,4C). The CCK-8 assay was used to detect the growth of ESCC cells, which showed that knockdown of LINC02381 significantly inhibited the growth of KYSE180 (Figure 4D) and KYSE510 (Figure 4E) cells. Colony formation assays showed that knocking down the expression of LINC02381 significantly reduced the colony number and size as compared to the control condition in KYSE180 (Figure 4F) and KYSE510 (Figure 4G) cells. We also performed Transwell assay to characterize the biological function of LINC02381 in ESCC metastasis, which indicated that knockdown of LINC02381 significantly restrained the migration abilities of KYSE180 (Figure 4H) and KYSE510 (Figure 4I) cells. Wound healing assays further demonstrated that LINC02381 silencing inhibited cellular motility in KYSE180 (Figure 4J) and KYSE510 (Figure 4K) cells.

Figure 4 Knockdown of LINC02381 inhibited the growth and metastasis of ESCC. (A) qRT-PCR analysis of LINC02381 expression level in normal esophageal epithelium cell line Het-1A and ESCC cell lines (TE-1, KYSE180, KYSE410, and KYSE510). (B,C) Knockdown of LINC02381 expression in KYSE180 and KYSE510 cell using siRNA, si#1, si#2 and si#3. (D,E) The cell growth of ESCC cells (KYSE180 and KYSE510) as determined by CCK-8 assay. (F,G) Crystal violet staining of the colony formation assays performed in LINC02381-knockdown and control cells (KYSE180 and KYSE510) (magnification: ×1). (H,I) Crystal violet staining of the migration assays. Silencing of LINC02381 could restrain the migratory ability in KYSE180 and KYSE510 cells as compared with siRNA-negative control cells (magnification: ×100). (J,K) Wound healing assay of the LINC02381 knockdown in KYSE180 and KYSE510 cells (magnification: ×100). ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; si#1, si-LINC02381-1; si#2, si-LINC02381-2; si#3, si-LINC02381-3; OD, optical density; ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; CCK-8, Cell Counting Kit-8.

Discussion

LNM is a critical factor in the progression and prognosis of ESCC, with the overall 5-year survival rate of patients with ESCC being less than 30%, mainly due to LNM occurring even when primary tumors are discovered at an early stage (29). The anatomy of the esophagus accounts for this propensity for LNM, as the submucosa contains a rich lymphatic plexus. An effective early prediction method of LNM in ESCC would be a powerful addition to the management of patients and lower the chances of understaging a patient clinically (22). However, the research on lncRNA as a biomarker for predicting LNM in esophageal cancer is limited. In our preliminary research, certain lncRNAs, including LINC02381, were found to be significantly upregulated in sample ESCC tissues from patients with LNM (23). A similar study has suggested that lncRNA can serve as an important biomarker for LNM in ESCC (30). To identify the key lncRNAs associated with LNM in ESCC, RNA-seq data analysis of 10 pairs of ESCC tissues and paired normal epithelium were intersected, yielding several key lncRNAs significantly altered in patients with LNM (23). A similar comparative method was reported for the identification of methylation signatures for LNM detection in ESCC (15).

The results of qRT-PCR in our study showed that LINC02381 expression in 65 ESCC tissues was significantly elevated as compared to the paired normal esophageal tissues, which is consistent with the expression of most tumors, including LINC02381 in osteosarcoma (31), breast cancer (25), and glioma (32). However, this was inconsistent with other tumor research, which reported LINC02381 to be downregulated in colorectal cancer and in different cancerous cell lines (26). Although the expression levels of LINC02381 have been reported in other tumors, we found LINC02381 to be significantly upregulated in patients with ESCC and LNM. Further mechanistic studies are needed to determine the precise role of LINC02381 in cell development. The differences in expression in different solid organ tumors, however, may provide direction for future experiments.

lncRNAs are closely related to the prognosis of patients. For instance, one study reported that the high expression of VESTAR was associated with poor OS in patients with ESCC (22). Our KM survival curve analysis of OS and DFS data also indicated that a high expression of LINC02381 was significantly correlated with poor prognosis. Moreover, patients with LNM had a shorter OS and DFS, and importantly, those with a high expression of LINC02381 and LNM had the worst prognosis, which corresponds with other research on osteosarcoma (31) and glioma (32). The upregulated expression of LINC02381 was closely related to differentiation grade and LNM, and lymph node-positive status was also significantly associated with higher primary tumor stage and differentiation grade, which is consistent with the other study (33). Univariate logistic regression analysis was performed to identify variables associated with LNM, which suggested tumor differentiation grade and tumor stage to be associated with LNM, which is in line with other findings (34). As LINC02381 expression was found to be closely correlated with LNM, multivariable logistic regression was conducted to develop our model, which indicated LINC02381 expression to be an independent risk factor for LNM of ESCC.

After LINC02381 expression, tumor differentiation grade, and tumor stage were incorporated into the risk model, the model demonstrated greater sensitivity and accuracy as indicated by the ROC curve analysis. This in accordance with other studies that have combined multiple variables to achieve higher diagnostic efficacy (15,16). Nomograms have been extensively used in oncology to estimate the prognosis of patients according to the relevant clinical parameters of a given cancer type (35). In our study, we developed and internally validated a nomogram to predict the likelihood of LNM in patients with ESCC, which indicated that our model had good predictive accuracy. This tool was developed using relevant, clinically available tumor-related factors including tumor differentiation grade, tumor stage, and expression level of LINC02381.

LINC02381 is associated with tumor growth and metastasis in various types of tumors (26,31). In our in vitro experiments, the knockdown of LINC02381 significantly inhibited the growth and metastasis of ESCC in vitro. These findings confirm the functional role of LINC02381 in ESCC and are in line with those reported for other tumor types (25,31).

The mechanism of action of LINC02381 in tumors remains largely unknown, especially in ESCC, which may differ from other tumors. One study reported that LINC02381 could downregulate CDCA4 via sponging miR-503e5p (31). In another study, the miR-1271-5p-FN1 axis was demonstrated to activate the PI3K/AKT pathway, with LINC02381 being shown to potentially aggravate breast cancer (25). The ability of LINC02381 to function as a competing endogenous RNA (ceRNA) has been examined extensively in various tumors; for instance, it has been found that LINC02381 functions as a ceRNA to exert its oncogenic effect in glioma through the IGF1R signaling pathway (32). However, another study reported that LINC02381 may have suppressive effects on colorectal cancer tumorigenesis partly by regulating the PI3K signaling pathway (26).

Our study included several limitations that should be addressed. First, the specific signaling pathway of LINC02381 affecting LNM of ESCC was not identified and should be investigated further. Second, we employed a retrospective dataset including 186 patients with ESCC undergoing esophagectomy, and prospective research may be needed to address this. Third, our model was not externally validated, but a bootstrap approach with a calibration curve was used to address this, which has been shown to provide stable estimates with low bias (36). Therefore, the role of LINC02381 should be explored further as a candidate biomarker for the LNM of ESCC, as this may represent an effective method for the early diagnosis of LNM.

Conclusions

To our knowledge, this is the first study to identify LINC02381 as a potential predictive biomarker of LNM in ESCC and to demonstrate the in vitro effects of LINC02381 on the growth and metastasis of ESCC. We developed an effective predictive model with good accuracy and specificity and used a nomogram to visualize the prediction of pathological LNM. This may serve as a powerful tool for developing improved staging and informing decision-making in clinical practice, especially for those who require neoadjuvant therapy.

Acknowledgments

We thank Dr. Luigi Bonavina (University of Milan Medical School, IRCCS Policlinico San Donato, Milano, Italy) for the critical comments and valuable advice on this study.

Footnote

Reporting Checklist: The authors have completed the TRIPOD and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2402/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2402/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2402/prf

Funding: This work was supported by the 2024 Medical Scientific Research Foundation of Guangdong Province, China (No. B2024342).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2402/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with Declaration of Helsinki (as revised in 2013), and the procedures involving human participants were approved by the Medical Ethics Committee of the Gaozhou People’s Hospital (No. GYLLPJ-2023145). Written informed consent was provided by all patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)