Establishment of using serum YKL-40 and SCCA in combination for the diagnosis of patients with esophageal squamous cell carcinoma

Abstract

Background: Elevated serum YKL-40 levels have been observed in various cancers. We evaluated the diagnostic performance of serum YKL-40 alone or in combination with the CEA, CYFRA21-1 and SCCA tumor markers for patients with esophageal squamous cell carcinoma (ESCC).

Methods: YKL-40 was detected in ESCC cell lines and tissues by real-time RT-PCR, Western blotting and ELISA. YKL-40 protein expression was determined in 20 ESCC tumor tissues using immunohistochemistry. Serum YKL-40 was measured by ELISA in 126 healthy donors, 59 patients with benign esophageal diseases and 150 patients with ESCC. Serum CEA, CYFRA21-1 and SCCA were determined by electrochemiluminescence.

Results: YKL-40 mRNA and protein were observed in ESCC cancer cell lines, tissues and cell culture media, respectively. YKL-40 expression was observed in 17 of 20 ESCC samples (85%). Serum YKL-40 concentration was significantly elevated in patients with ESCC (Range: 6.95-502.10 ng/ml) compared with patients with benign diseases (Range: 1.21-429.30 ng/ml; P = 0.038) and healthy controls (Range: 2.56-132.26 ng/ml; P < 0.001). ROC curves demonstrated that serum YKL-40 has a sensitivity of 72.70%, a specificity of 84.13% and an AUC of 0.874 for the diagnosis of ESCC, which was superior to CEA (Sen: 8.00%; Spe: 96.80%, AUC = 0.652), CYFRA21-1 (Sen: 40.00%; Spe: 92.06%, AUC = 0.746) and SCCA (Sen: 32.67%; Spe: 94.44%, AUC = 0.789). The YKL-40 and SCCA combination was better for diagnosing ESCC (Sen: 82.00%, Spe: 79.37%, PPV: 82.55 and NPV: 78.74; AUC = 0.917) than the YKL-40 and CEA combination (Sen: 74.00%, Spe: 83.20%, PPV: 84.09 and NPV: 72.73; AUC = 0.877), the YKL-40 and CYFRA21-1 combination (Sen: 82.00%, Spe: 77.78%, PPV: 81.46% and NPV: 78.40%; AUC = 0.897) or the CEA, CYFRA21-1 and SCCA combination (Sen: 56.67%, Spe: 84.80%, PPV: 81.73 and NPV: 61.99; AUC = 0.831). Associations between serum YKL-40 levels and the clinic characteristics of ESCC were not significant, with the exception of age (p = 0.001).

Conclusions: ESCC tumor cells and tissues express YKL-40. Serum YKL-40 may be a potential biomarker for ESCC. Serum YKL-40 in combination with SCCA significantly increases the sensitivity of detecting ESCC.

Figure 1

Expression of YKL-40 mRNA or protein in ESCC cell lines, tissues and location in tissue. Expression of mRNA and protein in immortalized esophageal epithelial cell line (NE-3) and esophageal carcinoma cell lines was analyzed by real-time PCR and Western Blotting, respectively (A, B) and in six pairs of matched ESCC and noncancerous tissues (D, E). Expression level was normalized by GAPDH and α-tublin, respectively. Error bars represent standard deviations (SD) calculated from three parallel experiments. Protein level in supernatant was measured by ELISA (C). Location of YKL-40 was determined by immunohistochemistry (F). The normal esophageal epithelial tissue showed no expression of YKL-40 (F a-b, 200 × and 400×). The ESCC tissues showed low (F c-d), medium (F e-f) and high (F g-h) expression of YKL-40 (200× and 400×).

Figure 2

YKL-40 concentration in serum in the test cohort. Left side, the serum YKL-40 in ESCC patients, benign disease patients, healthy controls and early-stage ESCC patients are plotted as a distribution. P value was calculated using Kruskal-Wallis test. Right side, YKL-40 serum levels in different groups.

Figure 3

Diagnostic outcomes for serum YKL-40, CEA, CYFRA21-1 or SCCA in the diagnosis of ESCC. A. ROC curves of the serum YKL-40 levels of 150 ESCC patients and 126 controls. The estimated area under the ROC curve was observed as AUC = 0.874. B. ROC curves for the diagnostic strength to identify ESCC using YKL-40, CEA, CYFRA21-1 or SCCA (CEA: AUC = 0.652; CYFRA21-1 = 0.746; SCCA = 0.789).

Figure 4

Diagnostic outcomes for serum YKL-40, CEA, CYFRA21-1 or SCCA combination in the diagnosis of ESCC. A. ROC curves for the diagnostic strength to identify ESCC using CEA and YKL-40+CEA (CEA: AUC=0.652; YKL-40+CEA: AUC=0.877). B. ROC curves for the diagnostic strength to identify ESCC using CYFRA21-1 and YKL-40+CYFRA21-1 (CYFRA21-1: AUC=0.746; YKL-40+CYFRA21-1: AUC=0.897). C. ROC curves for the diagnostic strength to identify ESCC using SCCA and YKL-40+SCCA (SCCA: AUC=0.789; YKL-40+SCCA: AUC=0.917). D. ROC curves for the diagnostic strength to identify ESCC using YKL-40+CEA, YKL-40+CYFRA21-1, YKL-40+SCCA and CEA+CYFRA21-1+SCCA (YKL-40+CEA: AUC=0.877; YKL-40+CYFRA21-1: AUC=0.897; YKL-40+SCCA: AUC=0.917; CEA+CYFRA21-1+SCCA: AUC=0.831).