High microvessel and lymphatic vessel density predict poor prognosis in patients with esophageal squamous cell carcinoma

Patients and specimens

Surgical specimens and paraffin-embedded tissues were derived from ESCC patients, and included 100 ESCC and 100 cancer-adjacent normal (CAN) tissues. None of the preoperative tumor patients had undergone radiotherapy or chemotherapy. Between 2018 and 2021, we conducted research on ESCC patients at the Department of Pathology, First Affiliated Hospital of Shihezi University in Xinjiang, China. Based on the World Health Organization histological tumor classification criteria, two pathologists confirmed a diagnosis of ESCC. According to the National Comprehensive Cancer Network (NCCN) guidelines (Ajani et al., 2023), 67 patients were clinically staged I–II and 33 had stage III–IV disease. The survival status of all patients was ascertained via telephone until December 2022. The median follow-up for surviving patients was 30 months (range, 1–78 months). Overall survival (OS) was defined as the interval between surgery and death or between surgery and the last follow-up in surviving patients. Each participant provided written, informed consent before registering for the study. The study was approved by the Science and Technology Ethical Committee of the First Affiliated Hospital, Shihezi University School of Medicine (201803801) in accordance with the ethical guidelines of the Declaration of Helsinki.

Immunohistochemistry

Paraffin sections were cut (4-μm thick) and deparaffinized with xylene. The sections were dehydrated in xylene and rehydrated using a graded series of ethanol solutions. After dewaxing, the sections were incubated in 3% peroxide-methanol solution at room temperature, and then repaired at 100 °C. The sections were incubated at room temperature for 30 min, and then washed with phosphate-buffered saline (PBS). Then, the sections were incubated with antibodies against CD34 (mouse monoclonal, 1:400; Zsbio) and D2-40 (mouse monoclonal, 1:100; Zsbio) for 8 h. Subsequently, the sections were washed thoroughly with PBS, and primary antibody binding was visualized using an EnVision kit (DAKO, Glostrup, Denmark) following the manufacturer’s instructions. Finally, the sections were faintly counterstained with hematoxylin and mounted with glycerol gelatin.

Immunoreactivity evaluation

CD34 was used as a microvascular endothelial cell marker for the MVD and MV count; CD34 positive expression in the cell membrane and/or cytoplasm appeared as brownish yellow particles. MVs were identified as follows: CD34-stained endothelial cells were surrounded by blood vessels, with a diameter of less than eight red blood cells are counted as MVs. The immunostained sections were scanned under light microscopy (OLYMPUS BX51) at low magnification (40×) and the areas with the greatest number of distinctly highlighted MVs (“hot spots”) were selected. MVD was determined by counting all immunostained MVs at 200× magnification. Five hot spots were chosen in each section. The mean values of the MVs in the five fields were analyzed statistically.

D2-40 was used as a lymphatic endothelial cell marker for the LVD; D2-40 positive expression was observed in the cytoplasm as brownish yellow particles. The immunostained sections were scanned under light microscopy (OLYMPUS BX51) at low magnification (40×), and hot spots with the greatest number of distinctly highlighted LVs were selected. LVD was determined by counting all immunostained LVs at 200× magnification. Five hot spots were chosen in each section (Sharma et al., 2023). The mean values of the LVs in the five fields were analyzed statistically.

Statistical analysis

All statistical analyses were performed using SPSS version 20 (IBM, Endicott, NY, USA). The difference in CD34 and D2-40 expression between the ESCC and CAN groups was compared using a nonparametric test. The clinical parameters between the high MVD and LVD group and low MVD and LVD group were compared using the χ² test. For classification analysis, mild expressions were divided in to two groups according to CD34 and D2-40 expression. The correlation between MVD and LVD co-expression, and the ESCC clinical parameters, were evaluated using Spearman rank correlation. Survival curves were estimated using the Kaplan–Meier method. The expression of both CD34 and D2-40, the correlation between clinicopathological parameters, and the OS rate were evaluated in univariate analysis. Multivariate analysis was performed using a Cox proportional risk regression model. The p-value was calculated using EPi Info; p < 0.05 was considered significant.

ESCC tissue exhibits greater MVD and LVD than CAN tissues

CD34 and D2-40 were used as MV and LV endothelial cell markers, respectively (Figs. 1, 2). Immunohistochemistry staining was used to demonstrate the distribution of MVs and LV in ESCC, and the MVD and LVD were quantified and analyzed. The average MVD in ESCC tissues was 16/HPF (high-power fields) (range, 3–33/HPF), which was significantly higher than that in CAN tissues (range, 2–19/HPF) (p < 0.001). The average LVD in ESCC tissues was 6/HPF (range, 2–14/HPF), which was also significantly higher than that in CAN tissues (range, 0–7/HPF) (p < 0.001) (Fig. 3).

Correlation between the distribution of MV and LV and the clinicopathological characteristics of ESCC

To analyze the role of MV and LV in ESCC progressions, the clinicopathological parameters of the patients were collected and tissue sections were examined. Patients with lymphatic metastasis had significantly higher MVD compared with patients without lymphatic metastasis (p < 0.05). Patients with advanced ESCC had significantly higher MVD than patients with early-stage ESCC (p < 0.05). At the same time, patients with lymphatic metastasis had significantly higher LVD than patients without lymphatic metastasis (p < 0.05). Moreover, LVD in advanced ESCC was significantly higher than that in early-stage ESCC (p < 0.05). There was no significant correlation between the distribution of MV, LV and age, sex, differentiation, invasion depth (all, p > 0.05) (Table 1).

Correlation between MVD and LVD and their synergistic role in the evaluation of clinical progression of ESCCs

To analyze the association between MVs and LVs in patients with ESCC, we performed a correlation analysis of ESCC samples, and found a significant positive correlation between MVD and LVD (p < 0.001) (Fig. 4). The above findings indicate a strong correlation between increases in MVD and LVD with lymph node metastasis and clinical staging of ESCC. There exists a significant linear relationship between MVs and LVs. Is the combined effect of MVD and LVD more valuable for assessing ESCC invasion and clinical progression? Based on the optimal cut-off numbers (MVD: 16, LVD: 6), we divided the cases into four groups: high MVD, high LVD, low MVD and low LVD to evaluate the synergistic effect of MVD and LVD in the invasion and clinical progression of ESCC. Analysis showed that lymph node metastasis and clinical progression were significantly increased in patients with high MVD and low LVD compared with patients with low MVD and LVD (p < 0.05), but were independent of other clinicopathological parameters (sex, age, degree of differentiation, depth of invasion, p > 0.05) (Table 2). It is suggested that the combination of high MVD and high LVD is associated with tumor invasion and lymphatic metastasis, and can be used as an important parameter to evaluate the biological behavior and metastasis trend of ESCC patients.

Correlation between MVD, LVD and the prognosis of patients with ESCC

To further investigate the prognostic significance of MVD and LVD in patients with ESCC, Kaplan-Meier survival analysis was employed to assess the correlation between MVD, LVD, and overall survival. The findings revealed a significantly lower overall survival period in patients with high MVD compared to those with low MVD (p < 0.05). Similarly, patients with high LVD exhibited significantly lower overall survival period than those with low LVD (p < 0.05).

Moreover, patients with high-density MVD and LVD demonstrated markedly reduced overall survival period compared to those with low-density MVD and LVD (p < 0.001). Overall survival period in non-synergistic groups for MVD and LVD (High MVD + Low LVD or High LVD + Low MVD) fell between that of high-density and low-density groups. However, no statistically significant difference was observed among the three groups (p > 0.05) (Fig. 5). Cox univariate regression analysis indicated that depth of invasion, lymph node metastasis, clinical stage, high MVD, and high LVD were all significantly negatively correlated with the prognosis of ESCC (p < 0.05). Multivariate analysis revealed that only clinical stage emerged as an independent prognostic factor for ESCC patients and displayed a significant negative correlation with overall survival period (p < 0.05) (Table 3).