LOH in the HLA class I region at 6p21 is associated with shorter survival in newly diagnosed adult glioblastoma

Abstract

Purpose: Glioblastoma (GBM) shows downregulated expression of human leukocyte antigen (HLA) class I, thereby escaping from cytotoxic T cells and limiting the efficacy of immunotherapy. Loss of heterozygosity (LOH) of HLA class I (6p21) and/or β-2 microglobulin (B2m) (15q21) regions represents irreversible downregulation. In this study, we examined the prevalence of these LOH events and their relations with overall survival in GBM.

Experimental design: In a cross-sectional analysis on 60 adult patients with GBM, DNA from formalin-fixed, paraffin-embedded specimens were evaluated for 10 microsatellite regions of HLA class I, B2m, HLA class II, HLA class III, and 6q by PCR as well as immunohistochemical evaluation of HLA class I expression and CD8(+) T-cell infiltration.

Results: LOH in HLA class I, B2m, HLA class II, HLA class III, and 6q regions was present in 41.4%, 18.2%, 9.4%, 77.8%, and 36.0% of informative cases, respectively. LOH of HLA class I was associated with shorter overall survival (HR = 4.89, P = 0.0078). HLA class I was downregulated in 22% to 43% of cases based on immunohistochemistry. Cases that displayed negative staining were significantly younger. HLA class I expression correlated with intratumoral CD8(+) T-cell infiltration.

Conclusion: LOH in the HLA class I region is frequent in adult GBMs. The association of shorter survival with LOH in this region suggests a crucial role for these genes in immunosurveillance.

Figure 1

Immunohistochemical staining for HLA Class I expression in adult GBM. Representative images of immunohistochemistry using HCA2 shown at 10X magnification (A-D) and 20X (F). Each scale bar represents 100μm (A-D). Negative control using medulloblastoma, while endothelial cells serve as internal positive control (A). HCA2 staining showing negative (B), heterogeneous (C), and homogeneous (D) expression. Heterogeneous and homogeneous immunoreactivity are considered to be positive HLA I expression. A chart summary of HLA Class I staining (E). While some myeloid cells (CD11b⁺, dark blue) exhibit HLA Class I (brown) immunoreactivity (white arrow), most infiltrating myeloid cells (CD11b⁺, dark blue) in GBM are almost negative for HLA Class I (black arrow) (F).

Figure 1

Immunohistochemical staining for HLA Class I expression in adult GBM. Representative images of immunohistochemistry using HCA2 shown at 10X magnification (A-D) and 20X (F). Each scale bar represents 100μm (A-D). Negative control using medulloblastoma, while endothelial cells serve as internal positive control (A). HCA2 staining showing negative (B), heterogeneous (C), and homogeneous (D) expression. Heterogeneous and homogeneous immunoreactivity are considered to be positive HLA I expression. A chart summary of HLA Class I staining (E). While some myeloid cells (CD11b⁺, dark blue) exhibit HLA Class I (brown) immunoreactivity (white arrow), most infiltrating myeloid cells (CD11b⁺, dark blue) in GBM are almost negative for HLA Class I (black arrow) (F).

Figure 2

CD8⁺ T cells in adult GBM. Representative images are shown at 20X magnification (A-H). Each scale bar represents 125 μm. Intratumoral (A,C,E,G) and perivascular (B,D,F,H) infiltration. CD8 staining showing negative (A,B), weak (C,D), moderate (E,F), and strong (G,H) infiltration. Results of CD8⁺ T cell intratumoral and perivascular infiltration in adult GBM (I).

Figure 3

(A) Heat map of LOH in chromosome 6 and 15. (B-F) Kaplan-Meier Survival Curves according to LOH analyses. LOH of HLA Class I region (B) and survival (median 13.1 vs 8.6 months, p = 0.078). LOH of HLA Class II region (C) (p = 0.34) and Class III (D) (p= 0.37). LOH of B2m region (E) and survival (p = 0.083). LOH of 6q (F) and overall survival (p = 0.48). LOH = Loss of Heterozygosity. ROH = Retention of Heterozygosity.

Figure 3

(A) Heat map of LOH in chromosome 6 and 15. (B-F) Kaplan-Meier Survival Curves according to LOH analyses. LOH of HLA Class I region (B) and survival (median 13.1 vs 8.6 months, p = 0.078). LOH of HLA Class II region (C) (p = 0.34) and Class III (D) (p= 0.37). LOH of B2m region (E) and survival (p = 0.083). LOH of 6q (F) and overall survival (p = 0.48). LOH = Loss of Heterozygosity. ROH = Retention of Heterozygosity.