The CST1 gene, which encodes the S-type Cystatin SN peptide, belongs to the Cystatin (CST) superfamily, the members of which control the proteolytic activities of cysteine proteases . Studies have indicated that proteases are involved in both primary and metastatic tumor growth [26, 27]. The CST1 gene is known to play a crucial role in human gastrointestinal tract cancer, including colon cancer and gastric cancer [6, 27]. To the best of our knowledge, our study is the first to systematically evaluate the expression and clinicopathologic significance of Cystatin SN in ESCC. Our findings have indicated that Cystatin SN serves as an independent prognostic factor in ESCC patients.
In our study, the immunostaining of ESCC samples revealed that Cystatin SN is predominantly located in the cytoplasm. Previous studies showed that Cystatin SN was present primarily in the cytosolic region of gastric cancer cells,  but it was detected in the cytomembrane of colorectal cancer cells . Those observations indicate that the expression of Cystatin SN in different cancers may be tissue–specific . However, we failed to show any significant correlations between Cystatin SN expression and patients clinicopathological parameters. In contrast, some studies revealed that overexpression of Cystatin SN correlated with descending pathological TNM stage for gastric and colorectal cancer. We believe that can explained by the following factors. First, the biology character of Cystatin SN may have tissue specificity. Second, the enrolled ESCCs is only II, III stages and belongs to a single institution’s database, which may results in a selection bias and low statistical power to detect meaningful relationships. Therefore, this concludion merits additional research.
In the studies mentioned above, Cystatin SN, which was proved to contribute to cell proliferation, has been reported as an oncogene in colorectal and gastric caicinoma [6, 21]. Howere, in our study, we found that ESCC patients with positive expression of Cystatin SN had significantly longer DFS and OS than those with negative Cystatin SN expression. We believe that can be explained by two particular factors. Firstly, histology differences such as squamous cell carcinoma and adenocarcinoma might explain the observed phenomena. Secondly, the heterogeneity of biomarkers might also result in the discrepancies in the findings between the previous literatures and our study. These results are similar to other members of the CST superfamily in a number of previous reports. Cystatin C is a nonglycosylated 13 kDa basic protein, consisting of 120 amino acids. It belongs to the cystatin superfamily of cysteine proteinase inhibitors. Strojan et al  demonstrated significantly longer survival in squamous cell carcinoma of the head and neck patients with high Cystatin C than in those with low Cystatin C. However, in colorectal cance , the patients with high levels of Cystatin C exhibited a significantly higher risk of death than those with lower levels. Alterations in secretion may result in higher extracellular and lower intracellular levels of Cystatin C and, therefore, the reverse correlation of Cystatin C with patients’ survival is to be expected. On the other hand, one has to be aware that cysteine proteases and consequently their inhibitors are also involved in biological processes other than tissue remodeling during the progression of primary tumors, such as the regulation of inflammatory and immune responses  or apoptosis , so that different level of Cystatin C may lead to various clinical outcomes. Moreover, in the subgroup analysis, Cystatin SN expressions distinguish the DFS or OS, especially in the groups of pN0 and stage II patients. Our results suggested that Cystatin SN in ESCC maybe play an significant role in the early stage of carcinogenesis. The underlying mechanism need further study.
The TNM stage is the most powerful and widely accepted predictor of survival for ESCCs . However, many patients with the same stage of disease have different outcomes, indicating the TNM stage may be insufficient to distinguish ESCC patients’ survival . An increasing number of studies have focused on the use of biomarkers to predict patients’ survival and select patients who will benefit from adjuvant treatments. To date, the metastasis and invasiveness of several tumors have been shown to be associated with members of the CST superfamily, such as Cystatin C (encoded by CST3), CystatinD (encoded by CST5), Cystatin F (encoded by CST7), Cystatin M (encoded by CST6) and Cystatin S (encoded by CST4), which have been described and investigated [11, 13–18]. Our study focused on the relationship between one of CST superfamily members and the survival of cancer patients. In our study, Cystatin SN expression, combined with the N status and differentiation, serve as independent and significant predictors in surgically resected ESCCs. Consistent with the findings reported by the previous studies, we also suggested some factors, including age, gender, tumor location, surgery and pT status, were not the independently significant predictive factors for ESCC survival, in spite of some other different points. And some crowd confounding factors may influence the foundings, of course, it needs additional studies. On the other hand, the Cystatin SN expression was shown to distinguish the DFS or OS in a subgroup analysis, especially in the subgroups of pN0 and stage II patients. Therefore, our results indicate that Cystatin SN expression combined with clinicopathological parameters may serve as an extra factor for identifying ESCC patients with a higher risk of tumor recurrence and metastasis.
Unfortunately, one limitation of out study in the lack of confirmation on Cystatin SN expression status by quantitative methods like Reverse Transcription-Polymerase Chain Reaction (RT-PCR), which could, in conjunction with results of IHC, further refine the prognostic value of this biomarker. Also, our study is a retrospective study, relied exclusively on a single-institutional database. Additional mechanistic investigations into this area will be vital to facilitate our understanding of the biological significance of Cystatin SN.