Diagnostic and prognostic values of upregulated SPC25 in patients with hepatocellular carcinoma

SPC25 expression data and clinicopathological characteristics

The gene expression profiles of 374 human HCC tissues and 50 non-neoplastic tissues, as well as the clinical data of patients, such as age at diagnosis, sex, histologic grade, pathological stage, vascular invasion, family history of cancer, survival status, and survival time were downloaded from TCGA Liver Hepatocellular Carcinoma (TCGA-LIHC; https://portal.gdc.cancer.gov/) database. The mRNA expression levels of SPC25 in TCGA cohort were extracted. The expression levels of SPC25 in HCC and non-tumorous tissues were comparatively analyzed in R (version 3.6.3) using the limma package (Ritchie et al., 2015). Next, the expression data and clinical data were merged using the patients’ unique identification numbers. In total, 370 patients were included in the final analysis. The relationship between SPC25 expression and clinical parameters was analyzed in these patients. The patients were classified into high expression and low expression groups based on the median SPC25 expression level in patients with HCC. The differences in the overall survival rates between the two groups were analyzed using R (version 3.6.3) ‘survival’ package (https://CRAN.R-project.org/package=survival). Furthermore, ESTIMATE (Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data) algorithm was used to estimate the tumor purity, and the presence of infiltrating stromal/immune cells in tumor microenvironment for each sample. We analyzed the relationships between SPC25 expression and immune cell infiltration (Yoshihara et al., 2013).

Statistical analysis

The correlation between SPC25 expression and clinicopathological characteristics was evaluated using the Chi-square test and logistic regression and was represented with box plots. The area under the curve (AUC) value obtained from the receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic effectiveness of SPC25 in discriminating HCC tissues from normal tissues in both cohorts using the pROC package (Robin et al., 2011). An AUC value greater than 0.85 is considered to have outstanding predictive value (Metz, 1978). Kaplan–Meier curves with P-value calculated by the log-rank test were used to compare the differences in overall survival rates. The univariate Cox regression analysis was performed to identify the possible prognostic variables. The multivariate Cox analysis was performed to verify the effect of SPC25 expression level on prognosis along with other clinical factors. The variables with P < 0.05 in the univariate analysis were adjusted in the multivariate analysis. A nomogram was established based on the independent clinicopathological factors in TCGA cohort using the ‘rms’ package (https://CRAN.R-project.org/package=rms). The nomogram was validated by analyzing discrimination and calibration curves in both cohorts. The concordance index (C-index) was used to evaluate the discrimination of the nomogram. The decision curve analysis (DCA, https://CRAN.R-project.org/package=rmda) was performed to test the clinical utility of the model between the SPC25-based nomogram, the AJCC staging system, and alpha-fetoprotein levels. The differences were considered statistically significant when the P-value was less than 0.05. All statistical analyses were performed using the R software (V.3.6.3, http://www.r-project.org).

Validation of the diagnostic and prognostic values of SPC25 using an external database

To obtain reliable results, the mRNA expression profiles of 240 HCC cases and 260 adjacent nontumor tissues with clinical data downloaded from the ICGC database (https://dcc.icgc.org/projects/LIRI-JP) were analyzed following the same methodology used to analyze TCGA cohort. Among the ICGC cohort, 202 adjacent nontumor tissues from patients with cancer and 240 primary tumor samples from patients with HCC were included in this study to validate the results of TCGA cohort analysis.

SPC25 expression in patients with varied clinicopathological parameters

The correlation between SPC25 expression and clinicopathological characteristics in patients with HCC was represented using the box plots. The clinical data of patients in the internal validation group and the external validation group were shown in Table 1. As shown in Fig. 1, high SPC25 expression was associated with patient age (P = 0.025, Fig. 1A), pathological stage (P = 0.004, Fig. 1C), histologic grade (P < 0.0001, Fig. 1D), survival status (P = 0.00029, Fig. 1E), and family history of cancer (P = 0.01, Fig. 1G). However, SPC25 expression was not significantly correlated with sex and vascular invasion (all P > 0.05, Figs. 1B and 1F). The differences in the SPC25 expression level in HCC tissues and non-tumorous tissues were represented using a boxplot. The expression level of SPC25 in HCC tissues was significantly upregulated when compared with that in normal tissues (Fig. 2A, P < 0.0001). Next, the expression level of SPC25 in 50 paired HCC tissues and non-neoplastic tissues from TCGA cohort was analyzed. The expression level of SPC25 in HCC tissues was upregulated when compared with that in non-neoplastic tissues (Fig. 2B, P < 0.0001).

Logistic regression analysis revealed that upregulated SPC25 expression in HCC was closely associated with age (odds risk [OR] = 0.568 for ≥65 vs <65, 95% CI [0.372–0.863], P = 0.0083), pathological stage (OR = 1.957 for stage III + stage IV vs stage I + stage II, 95% CI [1.202–3.223], P = 0.0075), histologic grade (OR= 4.936 for grade 3 vs grade 1, 95% CI [2.506–10.104], P < 0.001; OR= 12.187 for grade 4 vs grade 1, 95% CI [2.827–85.344], P = 0.0026), and patients survival status (OR = 1.958 for dead vs. alive, 95% CI [1.272–3.036], P = 0.0024) (Table 2).

Diagnostic and prognostic values of upregulated SPC25 expression in patients with HCC

The prognostic value of SPC25 in patients with HCC was evaluated. The Kaplan–Meier curve demonstrated that high SPC25 expression was significantly associated with unfavorable overall survival (Fig. 2C, P < 0.001). To assess the diagnostic value of SPC25, we generated an ROC curve using the SPC25 expression data from 370 HCC tissues and 50 non-neoplastic tissues. As shown in Fig. 2D, SPC25 had an excellent diagnostic value in discriminating HCC tissues from normal tissues with an AUC of 0.969 (95% CI [0.94.8–0.984]), sensitivity of 92.78% (95% CI [89.7–95.2]%), and specificity of 94.00 (95% CI [83.5–98.7]%).

We further explored the expression level of SPC25 in other gastrointestinal tract malignances. As shown in Fig. 3, the expression levels of SPC25 in cholangiocarcinoma, colon adenocarcinoma, pancreatic adenocarcinoma, rectum adenocarcinoma, LIHC, and stomach adenocarcinoma were significantly higher than those in non-neoplastic tissues. To confirm the prognostic value of SPC25 expression, univariate and multivariate analyses were performed. The univariate analysis revealed that age, sex, pathological stage, and SPC25 expression were significantly correlated with overall survival (all P < 0.05). After adjusting for other prognostic variables, the multivariate analysis revealed that SPC25 expression was independently associated with overall survival (hazard ratio [HR] = 1.76, 95% CI [1.100–2.815], P = 0.0183). All other independent risk variables, including age and pathological stage, and the corresponding HR and 95% CI are listed in Table 3.

Nomogram construction and validation

Based on the results from multivariate Cox analysis, age, pathological stage, and SPC25 expression were identified as independent prognostic factors for overall survival. Nomograms for predicting 1-, 3-, and 5- year overall survival rates were established based on these independent risk factors in TCGA cohort (Fig. 4A). The internal validation in TCGA cohort demonstrated that the C-index of nomogram for overall survival was 0.647 (95% CI [0.582–0.711]), which was higher than that of the AJCC staging (C-index = 0.558; 95% CI [0.494–0.622]) and alpha-fetoprotein levels (C-index = 0.425, 95% CI [0.350–0.501]). In the ICGC cohort, the C-index for nomogram to predict overall survival was 0.805 (95% CI [0.745–0.864]), which was higher than that of AJCC staging (C-index = 0.705, 95% CI [0.634–0.780]). The calibration plots in TCGA cohort (Fig. 4B) and ICGC cohort (Fig. 4C) demonstrated an excellent agreement between the nomogram prediction and observed estimates for 1-, 3-, and 5-year overall survival rates. In the DCA analysis, the nomogram presented preferable net benefit with a wider range of threshold probabilities when compared with the AJCC stage system and alpha-fetoprotein levels in both cohorts (Figs. 4D and 4E). These results suggest that the SPC25-based nomogram is superior to the AJCC stage system in predicting HCC prognosis.

Validation of the diagnostic and prognostic values of SPC25 in the ICGC cohort

External validation further confirmed that the expression of SPC25 in HCC tissues was higher than that in adjacent tissues (Fig. 5A, P < 0.0001). The Kaplan-Meir analysis revealed that the high SPC25 expression group had an unfavorable prognosis when compared with the low SPC25 expression group (Fig. 5B, P < 0.001). The diagnostic value of SPC25 also demonstrated excellent performance in the ICGC cohort with an AUC of 0.945 (95% CI [0.920–0.965], Fig. 5C). The univariate analysis indicated that SPC25 expression, sex, and pathological stage were correlated with overall survival (all P < 0.005, Fig. 5D). After adjusting for other risk factors, the multivariate analysis results confirmed that SPC25 expression was independently correlated with survival in patients with HCC (HR = 6.495, 95% CI [2.646–15.946], P < 0.001, Fig. 5E). These results indicate that SPC25 is a novel diagnostic and prognostic biomarker for HCC.

Correlation of SPC25 with the proportion of tumor-infiltrating immune cells

Furthermore, the correlation of SPC25 expression with the immune microenvironment was explored among 21 kinds of immune cell profiles in LIHC samples. The results from the correlation analyses demonstrated that a total of 3 kinds of tumor-infiltrating immune cells were correlated with the expression of SPC25 (Fig. 6). Among them, activated CD4 T memory cells were positively correlated with SPC25 expression (Fig. 6A); two kinds of tumor-infiltrating immune cells were negatively correlated with SPC25 expression, including regulatory T cells (Tregs), and resting memory CD4 + T cells (Figs. 6B–6C). These results further indicated that the levels of SPC25 affected the immune activity of immune microenvironment to a certain extent.