Expression of fatty acid‐binding protein‐4 in gastrointestinal stromal tumors and its significance for prognosis

Abstract Background Fatty acid‐binding proteins (FABPs) have been found to be involved in tumorigenesis and development. However, the role of FABP4, a member of the FABPs, in GISTs (Gastrointestinal stromal tumors) remains unclear. This study aimed to investigate the expression of FABP4 and its prognostic value in GISTs. Methods FABP4 expression in 125 patients with GISTs was evaluated by immunohistochemical analysis of tissue microarrays. The relationship between FABP4 expression and clinicopathological features and prognosis of GISTs was analyzed. Results Multiple logistic regression analysis showed that expression of FABP4 correlated with tumor size and mitotic index. Furthermore, FABP4 level, tumor size, mitotic index, and high AFIP‐Miettinen risk were independent prognostic factors in GISTs. The Kaplan‐Meier survival curve showed that the 5‐year survival rate of patients with high‐FABP4 expression GISTs was lower. Conclusions These results suggested that high‐FABP4 expression might be a marker of malignant phenotype of GISTs and poor prognosis.


| INTRODUC TI ON
Gastrointestinal stromal tumor (GIST) is defined as a stromal tumor of spindle or epithelioid cells that is primary in the gastrointestinal tract, greater omentum, and mesentery with a KIT (CD117 stem cell factor receptor) positive stain. It is a distinct tumor from the typical smooth muscle and neurogenic tumor, and it is a separate disease but it can differentiate into either smooth muscle or nerve. GISTs are the most common mesenchyme-derived tumors of the gastrointestinal tract. They are different from interstitial cells of Cajal. 1,2 GISTs are characterized by the biological characteristics of benign or malignant tumors. Complete radical resection is the most effective treatment for localized GISTs and potentially resectable GISTs. 3 However, some patients have no chance of surgery due to the late stage of the tumor or special lesions such as the low rectal GISTs that require combined anal resection. 4 The emergence of specific 2 | MATERIAL S AND ME THOD

| Research sample
The study included 125 patients with GISTs who were treated at the

| Tissue microarray construction and IHC analysis
A tissue microarray (TMA) system (Quick-Ray, UT06; UNITMA) was used from the Department of Clinical Pathology, at the Affiliated Hospital of Nantong University. TMA was performed following a previously described protocol. 27 Based on the results of HE staining of pathological sections, the representative cancer nests were selected and labeled on the corresponding donor paraffin blocks.
Then, TMA was analyzed by IHC, and the primary antibody was replaced with phosphate-buffered saline (PBS) for negative control.
The sections were dewaxed and rehydrated, in xylene and gradient ethanol, respectively. They were then incubated with primary antibody against FABP4 (1:100 dilution, Proteintech, 12802-1-AP) overnight at 4°C. Further, they were washed with PBS three times and incubated with the secondary antibody (horseradish peroxidaseconjugated anti-mouse antibody) for 1 h at room temperature.
Immunoreactivity was evaluated using a Vectastain Elite ABC kit (Vector Laboratories). The films were viewed in a double-blind manner by two experienced pathologists, and the ratio and strength of FABP4-positive cells were evaluated. From weak to strong, the dyeing strength was divided into four grades: 0, 1, 2, and 3. The ratio of positive cells was scored as follows: 0%-25% for 0 points, 26%-50% for 1 point, 51%-75% for 2 points, and 76%-100% for 3 points. The FABP4 immunohistochemistry score (IHS) was based on the ratio of positive cells multiplied by the strength score, with the IHS of 3 or less being defined as low expression and greater than or equal to 4 as high expression. [27][28][29] SPSS 22.0 statistical software (SPSS) was used for data analysis.

| Statistical analysis
The clinical significance of FABP4 expression in GISTs and its various clinical parameters was analyzed by univariate and multivariate statistical analysis, using chi-square test. Kaplan-Meier was used to draw the survival curve, and log-rank test was used to compare the survival rate. Independent risk factors were identified using the Cox model for univariate and multivariate analyses. A p value <0.05 indicated a statistically significant difference.

| Clinical features
A total of 125 patients with GISTs were enrolled in this study

| Expression level and location of FABP4 in GIST
In this study, immunohistochemical analysis was used to analyze the

| Correlation between FABP4 expression and clinicopathological components
This study further investigated the relationship between FABP4 ex-

| Survival analysis
The Kaplan-Meier survival curve showed that the 5-year OS (p < 0.001) and DFS (p < 0.001) were significantly lower in patients with high-FABP4 expression GIST compared with patients with low and no FABP4 expression (Figure 2A,B). And the median survival of the 125 patients was 47 months. The univariate analysis revealed that the prognosis of patients with GIST was related to FABP4 expression (p < 0.001), tumor size (p = 0.026), mitotic index (p = 0.027), and AFIP-Miettinen risk levels (p < 0.001). The multivariate analysis suggested that poor prognosis in patients with GIST was significantly associated with high-FABP4 expression (p = 0.021) and high AFIP-Miettinen risk (p < 0.001) ( Table 2).

| DISCUSS ION
The main function of FABP4 is to control lipid metabolism; participate in inflammatory response, cell growth, and differentiation; and regulate apoptosis. cells revealed that the proliferation was significantly inhibited. 35 Therefore, whether FABP4 played a role in promoting or suppressing cancer growth is controversial.
Studies showed that lipid biosynthesis increased dramatically in highly invasive GISTs to meet the needs of rapidly proliferating tumor cells. 36

Fatty acid synthase (FASN) is expressed in highly invasive
GISTs, can be used as a predictor of survival alone, and may serve as a therapeutic target for GISTs. 37  The clinical studies focused on GIST tumor size, primary site, mitotic index, number of lesions, and, so forth, as indicators to determine the degree of GIST risk and prognosis. However, some patients with disease progression could not be judged very well using these indicators. For example, tumors measuring less than 2 cm had distant metastasis, and some patients with a high level of malignancy had long-term survival. Therefore, finding molecules that affect the progress of GISTs is very important to judge the degree of GIST risk and prognosis. Studies found that the expression of miR-148b-3p in GIST increased with increasing risk, while the low expression of miR-148b-3p in high-risk GIST suggested that recurrence and metastasis were more likely to occur. 38 Studies suggested that the level of DKK4 was significantly higher in high-risk patients with GIST than in low-risk patients and could be used as a prognostic indicator. 39  Note: *p < 0.05.