Combination of high anti-SKI and low anti-TMED5 antibody levels is preferable prognostic factor in esophageal carcinoma

Abstract

Given that esophageal cancer is highly malignant, the discovery of novel prognostic markers is eagerly awaited. We performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified SKI proto-oncogene protein and transmembrane p24 trafficking protein 5 (TMED5) as antigens recognized by serum IgG antibodies in patients with esophageal carcinoma. SKI and TMED5 proteins were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens. The serum anti-SKI antibody (s-SKI-Ab) and anti-TMED5 antibody (s-TMED5-Ab) levels were significantly higher in 192 patients with esophageal carcinoma than in 96 healthy donors. The presence of s-SKI-Abs and s-TMED5-Abs in the patients' sera was confirmed by western blotting. Immunohistochemical staining showed that the TMED5 protein was highly expressed in the cytoplasm and nuclear compartments of the esophageal squamous cell carcinoma tissues, whereas the SKI protein was localized predominantly in the nuclei. Regarding the overall survival in 91 patients who underwent radical surgery, the s-SKI-Ab-positive and s-TMED5-Ab-negative statuses were significantly associated with a favorable prognosis. Additionally, the combination of s-SKI-Ab-positive and s-TMED5-Ab-negative cases showed an even clearer difference in overall survival as compared with that of s-SKI-Ab-negative and s-TMED5-Ab-positive cases. The s-SKI-Ab and s-TMED5-Ab biomarkers are useful for diagnosing esophageal carcinoma and distinguishing between favorable and poor prognoses.

Keywords: SKI; TMED5; antibody biomarker; esophageal carcinoma; overall survival.

FIGURE 1

Comparison of the serum anti‐SKI antibody (s‐SKI‐Ab) and serum anti‐TMED5 antibody (s‐TMED5‐Ab) levels and receiver operating characteristic (ROC) curve analysis between healthy donors (HDs) and surgically treated 91 esophageal carcinomas. The s‐SKI‐Ab (A) and s‐TMED5‐Ab (B) levels in patients with esophageal carcinoma and HDs were examined by AlphaLISA and shown in box–whisker plots. Box plots represent 25, 50, and 75 percentiles. The upper and lower horizontal lines represent the 90 percentile. ***p < 0.001; evaluated using the Mann–Whitney U‐test. The ROC curve analysis for SKI‐Ab and TMED5‐Ab are shown in panels (C) and (D), respectively. The numbers in (C) and (D) represent the area under the curve (AUC), 95% confidence interval (CI), cutoff level, specificity and sensitivity, and p‐values.

FIGURE 2

The result of western blotting and immunohistochemistry with esophageal carcinoma patients. (A) Representative results by western blotting are shown. GST, GST‐SKI, and GST‐TMED5 proteins were electrophoresed through SDS‐PAGE gels followed by staining with Coomassie Brilliant Blue (CBB) (I), or western blotting using anti‐GST (αGST) (II), sera of patients [EC#61 (III), EC#62 (VI), EC#64 (V)], or a healthy donor serum [HD#13 (VI)]. The arrows indicate the GST‐SKI, GST‐TMED5 and GST positions. The left vertical axis indicates the molecular weight. In the figure, the single asterisk ‘*’ represents the degradation products, whereas the double asterisks ‘**’ represent non‐specific reaction for EC#61. The immunohistochemical staining for SKI (B) and TMED5 (C) of esophageal carcinoma is shown.

FIGURE 3

Comparison of the overall survival between the positive and negative s‐SKI‐Ab and s‐TMED5‐Ab cases. The overall survival of surgically treated esophageal carcinoma is presented in the Kaplan–Meyer plots for s‐SKI‐Ab (A) and s‐TMED5‐Ab (B). The s‐SKI‐Ab and s‐TMED5‐Ab levels are divided into high and low groups. The cutoff level was assessed using ROC curve analysis between the survival and mortality cases of esophageal carcinoma using X‐tile software (SKI cutoff: 102888; TMED5 cutoff: 179720). Statistical analyses were performed by using the log‐rank test in each group. (C) The prognosis when using a combination of the s‐SKI‐Ab and s‐TMED5‐Ab levels. All s‐TMED5‐Ab‐positive cases were included in the s‐SKI‐Ab positive group, and the combinations were categorized into three (C). A flow chart of s‐SKI‐Ab and s‐TMED5‐Ab level was created to allow easy understanding of the prognosis (D). First, the s‐TMED5‐Ab level of the esophageal carcinoma patients was measured before treatment. Only the s‐TMED5‐Ab‐negative cases will undergo s‐SKI‐Ab measurement in the next step. Only the s‐SKI‐Ab‐positive cases were expected to have a favorable prognosis.