Down-regulation of Human Leukocyte Antigen class I heavy chain in tumors is associated with a poor prognosis in advanced esophageal cancer patients

Abstract

The HLA class I antigen processing machinery (APM) plays a crucial role in the anticancer immune response. The aim of this study was to assess the clinical significance of APM components in esophageal cancer. A total of 11 esophageal cancer cell lines were evaluated by Western blot analysis for 13 HLA class I APM components. There was a different expression pattern among cancer cell lines for HLA class I heavy chain (HLA-HC), β2 microglobulin, Tapasin, TAP-1, TAP-2, LMP-7 and LMP-10. Immunohistochemical staining utilizing a tissue microarray method for HLA class I APM expression showing different expression patterns among cell lines was performed for 95 surgical specimens from patients with esophageal cancer. Prognostic factors were the down-regulation of HLA-HC, and the up-regulation of β2 microglobulin and TAP-1 in the cancer tissues. Multivariate analysis using a Cox regression model indicated that the down-regulation of HLA-HC, and up-regulation of TAP-1 in cancer tissues are independent, unfavorable prognostic factors (hazard ratio, 2.361 and 2.297; P=0.0141 and 0.0145, respectively). Although there was no significant difference in survival for selected p-stage I and II patients (n=54) in all APM components, only down-regulation of HLA-HC was an unfavorable prognostic factor by a Cox regression model for selected p-stage III and IV patients (n=41). In conclusion, the current results suggest that the down-regulation of HLA-HC in tumors is especially associated with a poor prognosis among advanced esophageal cancer patients.

Figure 1

Western blot analysis for HLA class I antigen processing machinery (APM): HLA class I heavy chain (HLA-HC), β2 microglobulin, Tapasin, Calnexin, Calreticulin, ERp57, TAP-1, TAP-2, LMP-7, LMP-10, MB-1, Delta, and Z. Lysates of the cultured human esophageal cancer cell lines (TE2, 4, 5, 6, 8, 9, 10, 13, 14, HEC46, and SGF7) and homogenized normal human esophageal mucosa tissue (NHET) were subjected to Western blot analysis. Lysates of NHET were used as a positive control for APM component. The difference of expression among cell lines was found in seven APM molecules (HLA-HC, β2 microglobulin, Tapasin, TAP-1, TAP-2, LMP-7, LMP-10).

Figure 2

Western blot analysis for the HLA class I APM that had a difference of expression among cell lines with lysates from CB17/severe combined immunodeficiency (SCID) mouse xenograft models. (A) lysates from implanted tumors derived from human esophageal cancer cell lines (TE4, 8, 14, HEC46, and SGF7) and NHET were subjected to Western blot analysis. Each esophageal cancer cell line was implanted subcutaneously into the right flanks of CB17/SCID mice. The expression with lysates from xenografts was almost identical to the expression with lysates from cell lines. (B) Lysates from implanted tumors derived from human esophageal carcinoma cell line (TE8), human lung cancer cell line (LCD), NHET and normal human lung tissue (NHLT) were subjected to Western blot analysis. The expression with lysates from TE8 xenograft was positive in all components, and the expression with lysates from LCD was negative. These xenografts were used to decide the condition of immunohistochemical staining. These experiments were done twice, and the same results were obtained.

Figure 3

Representative immunohistochemical staining pattern for APM components in xenografts and human esophagus. TE8 xenograft and LCD xenograft, which were positive and negative, respectively, by Western blotting, were stained with mAb (HLA class I heavy chain (HLA-HC), β2 microglobulin, Tapasin, TAP-1, TAP-2, LMP-7, LMP-10). TE8 xenograft was used as a positive control for APM components. LCD xenograft was used as a negative control. The intensity of staining was scored as 1, 2, or 3 indicating weak, clear, or strong expression respectively. Original magnification, ×200 (xenograft), ×100 (carcinoma lesion and normal esophageal mucosa).

Figure 4

Kaplan-Meier analysis of overall survival according to the expression of APM components. HLA class I heavy chain (HLA-HC) and LMP-7 expression was classified into two groups according to the score (score 1 vs. score 2 and score 3). Score 1 was described as (−). Score 2 and score 3 were described as (+). β2 microglobulin, Tapasin, TAP-1, TAP-2, and LMP-10 expression was classified (score 1 and score 2 vs. score 3). Score 1 and score 2 were described as (−). Score 3 was described as (+). Differences in overall survival were analyzed with the log rank test.