Clinicopathologic demographics of the ESCC patients

The present study included 146 patients with ESCC, of which 104 (71.23%) were men and 42 (28.77%) were women. The patient ages at the time of surgery ranged from 36 years to 81 years, with a median age of 62 years. The proportion of smokers among ESCC patients was 99 (67.81%), whereas 47 (32.19%) were non-smokers. The proportions of drinkers and non-drinkers were 82 (56.16%) and 64 (43.84%), respectively. Of the 146 ESCC cases, 19 (13.01%) were well differentiated, 83 (55.48%) were moderately differentiated, and 44 (30.14%) were poorly differentiated [16]. Lymph node metastasis was present in 64 (43.84%) of the patients. Furthermore, 94 (64.38%) of the patients were at the I or II clinical stage, whereas 52 (35.62%) were at stage III or IV. The proportions of the upper, middle, and lower tumor locations were 15 (10.27%), 80 (54.80%), and 51 (34.93%), respectively. The invasion depth shows that 6 (4.11%) were Tis and T1, whereas 140 (95.89%) were T2, T3, and T4.

C20orf54 protein expression decreased in ESCC and ESIN compared with the non-tumoral squamous epithelium tissue

A total of 108 non-tumoral control tissues, 94 LGESIN, 54 HGESIN, and 146 primary ESCC tissue samples were used in the present study. IHC staining demonstrates that C20orf54 was localized in the cytoplasm (Fig. 1). The distributions of the staining patterns of C20orf54 were significantly different among these four groups (Table 1). We observed that the rate of C20orf54 expression had a decreasing tendency as the cell type progressed from normal (non-tumoral) to increasing levels of metastasis (LGESIN followed by HGESIN and ESCC). Positive staining for C20orf54 was generally observed within the non-tumoral control tissues, but weak or no C20orf54 staining was mostly detected in LGESIN, HGESIN, and ESCC tissues. Approximately, 74.07% (80 of 108) of the control cells was highly stained (strong staining and medium staining) for C20orf54, whereas the value sharply decreased to 45.74% (43 of 94), 33.33% (18 of 54), and 11.64% (17 of 146) in LGESIN, HGESIN, and ESCC tissues, respectively (both P < 0.05 compared with the non-tumoral control tissues). The positive rate in ESCC was the least among the four groups, and the value was significant (P < 0.05) (Fig. 2).

Fig. 1
figure 1

Representative images of the IHC staining of C20orf54 protein expression, as well as hematoxylin and eosin staining, in different esophageal lesion tissues. C20orf54 primarily expressed in the cytoplasm (original magnification, AD: ×40, EL: ×200). A, E Strong staining of C20orf54 in non-tumoral esophageal tissue. B, F Moderate staining of C20orf54 in LGESIN. C, G Weak staining of C20orf54 in HGESIN. D, H C20orf54 staining is negative in ESCC. Hematoxylin and eosin staining is shown for non-tumoral esophageal tissue (I), LGESIN (J), HGESIN (K), and ESCC (L)

Table 1 Expression of C20orf54 in non-tumoral, ESCC, LGESIN and HGESIN tissues
Fig. 2
figure 2

Expression levels of C20orf54 in different esophageal lesion tissues. The box plot shows significantly higher expression of C20orf54 in non-tumoral esophageal tissues than that in LGESIN, HGESIN, and ESCC tissues. The C20orf54 expression was significantly lower in ESCC than in LGESIN and HGESIN (*P < 0.001)

Association of C20orf54 protein expression in ESCC tissues with clinicopathological characteristics

We also evaluated the relationship between the C20orf54 expression and clinicopathological characteristics of ESCC, including age, gender, histological grade, lymph node metastasis status, TNM stage, tumor location, invasion depth, smoking status, and drinking status. Notably, we can infer that the C20orf54 defective expression in tumor cells appears to be significantly associated with poor differentiation (P = 0.014), lymph node metastasis (P = 0.021), and invasion depth (P = 0.019). These data suggest that the downregulated expression of C20orf54 can be involved in tumor metastasis and aggressiveness and contribute to tumor development. However, the other parameters such as age, gender, TNM stage, tumor location, smoking status, and drinking status had no significant relationship with C20orf54 expression (Table 2).

Table 2 The correlation between C20orf54 protein expression and clinicopathologic factors in ESCC

C20orf54 can be a potential diagnostic biomarker of ESCC and ESIN

We then used ROC curves to evaluate the C20orf54 IHC scores determined for each of the ESCC, HGESIN, LGESIN, and control tissues. We found that the ESCC, HGESIN, and LGESIN tissues were easily distinguished from the controls with ROC AUC values of 0.879 (95% CI, 0.835–0.922), 0.770 (95% CI, 0.693–0.843), and 0.710 (95% CI, 0.638–0.780), respectively (Fig. 3). We next used cutoff levels to determine the sensitivity and specificity values for each cancerous tissue type that optimized the diagnostic accuracy rate, as well as minimized the false-negative and false-positive rates (Table 3). The sensitivity and specificity values for ESCC were 74.1% and 88.4% (cutoff score of 5), whereas HGESIN and LGESIN were 49.1% and 94.4% (cutoff score of 7.5), and 49.1% and 89.4% (cutoff score of 7.75), respectively. Thus, these results support the conclusion that C20orf54 can be a potential diagnostic biomarker for ESCC and ESIN.

Fig. 3
figure 3

ROC curve analysis of the C20orf54 IHC scores for detecting LGESIN (A), HGESIN (B), and ESCC (C) tissues from the controls

Table 3 High sensitivity, specificity, and AUC values of C20orf54 in ESCC, HGESIN, and LGESIN

C20orf54 defective expression predicts poor prognosis in ESCC

We examined the correlation between the C20orf54 expression and postoperative survival of patients who underwent curative surgery, and parameters including differentiation, lymph node metastasis, invasion depth, TNM stage, tumor location, smoking status, and drinking status. The mean follow-up period of postoperative survival was 20 months, ranging from 1 to 116 months after treatment. Furthermore, we found no difference in survival time using the Kaplan-Meier analysis. Thus, we found a tendency of patients who expressed higher C20orf54 to experience a longer overall survival time than patients with lower C20orf54 expression (Fig. 4A). Moreover, the postoperative mortality rate was higher in patients with low C20orf54 expressed than in those with high expression. Other parameters, such as lymph node metastasis, invasion depth, TNM stage, and tumor location, showed significant differences in survival time (Fig. 4, both P < 0.05).

Fig. 4
figure 4

Survival analyses for C20orf54 expression and other clinical parameters in ESCC patients. The Kaplan-Meier survival curves that show the association among ESCC patient survival times with the C20orf54 expression level (A, P = 0.068), differentiation (B, P = 0.213), L/N metastasis (C, P = 0.001), TNM stage (D, P = 0.007), invasion depth (E, P = 0.001), location (F, P = 0.031), smoking status (G, P = 0.906), and drinking status (H, P = 0.715)

Finally, a multivariate Cox regression analysis was performed for the C20orf54 expression, and all clinicopathological factors were included in the univariate analysis to explore whether additional prognostic information was involved in the IHC analysis. Statistically, the C20orf54 detective expression was an insignificant factor in the multivariate Cox regression model. These data generally indicate that C20orf54 expression was an insignificant independent prognostic factor for poor prognosis in ESCC, but it was unsuitable for TNM stage and invasion depth (P = 0.040, HR = 2.519; P = 0.009, HR = 5.817, Table 4).

Table 4 Univariate and multivariate survival analyses of clinicopathological characteristics

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