Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers

Abstract

Background: Cancer/testis (CT) antigens are protein antigens normally expressed only in germ cells of testis, and yet are expressed in a proportion of a wide variety of human cancers. CT antigens can elicit spontaneous immune responses in cancer patients with CT-positive cancers, and CT antigen-based therapeutic cancer vaccine trials are ongoing for "CT-rich" tumors. Although some previous studies found breast cancer to be "CT-poor", our recent analysis identified increased CT mRNA transcripts in the ER-negative subset of breast cancer.

Methodology/principal findings: In this study, we performed a comprehensive immunohistochemical study to investigate the protein expression of eight CT genes in 454 invasive ductal carcinomas, including 225 ER/PR/HER2-negative (triple-negative) carcinomas. We found significantly more frequent expression of all eight CT antigens in ER-negative cancers, and five of them--MAGEA, CT7, NY-ESO-1, CT10 and CT45, were expressed in 12-24% of ER-negative cancers, versus 2-6% of ER-positive cancers (p<0.001 to 0.003). In comparison, GAGE, SAGE1 and NXF2 were only expressed in 3-5% of ER-negative and 0-2% of ER-positive cancers. ER-negative cancers were also more likely to simultaneously co-express multiple CT antigens, with 27% (34/125) of ER-negative, CT-positive tumors expressing three or more CT antigens. HER2 status had no consistent effect on CT expression, and triple-negative carcinomas showed similar frequencies of MAGEA and NY-ESO-1 expression as ER-negative/HER2-positive carcinomas. More frequent CT expression was also found in tumors with higher nuclear grade (p<0.001 to p = 0.01) and larger in size (>2 cm).

Conclusions/significance: CT antigens are preferentially expressed in hormone receptor-negative and high-grade breast cancer. Considering the limited treatment options for ER/PR/HER2 triple-negative breast cancer, the potential of CT-based immunotherapy should be explored.

Figure 1. Immunohistochemical analysis of CT antigen expression in breast cancer.

Eight CT antigens were analyzed– MAGEA (A), NY-ESO-1 (B), CT7 (C), CT10 (D), CT45 (E), GAGE (F), NXF2 (G) and SAGE1 (H). Of these, MAGEA, NY-ESO-1, CT7 and GAGE showed mixed nuclear and cytoplasmic staining, whereas CT10, CT45, NXF2 and SAGE1 were purely or predominantly nuclear proteins. (Magnifications: 400X).

Figure 2. Variations in cellular and subcellular distributions of CT antigens in breast cancer.

(A, B): NY-ESO-1 staining of two ER-negative carcinomas, showing mixed nuclear and cytoplasmic staining in (A) and pure cytoplasmic staining in (B). (C, D): MAGEA staining of two ER-negative carcinomas, showing predominantly nuclear (C) and cytoplasmic (D) staining, respectively. (E, F): CT7 staining in two cases, showing diffuse positivity in >90% of tumor cells in (E), as compared to (F) which showed only scattered positive cells and many tumor cells were negative. (Magnifications: 400X).

Figure 3. Distribution of immunohistochemical reactivity percentages among CT-positive breast cancer.

The immunoreactivity in each positive case was given an extent score (1, <10% cells positive; 2, 10-50% cells positive; 3, >50% cells positive) and an intensity score (1, +; 2, ++; 3, +++). A combined score of 2 is considered weak positive, 3 to 4 as moderate, and 5 to 6 as strong positive. Most CT-positive cases showed moderate to strong reactivity, but CT7 and CT10 had more weak positive cases, see text.

Figure 4. Distribution of CT antigen expression percentages in ER-positive and ER-negative breast cancer.

Comparisons were carried out for each CT antigen between ER+HER2- and ER-HER2- cases and between all ER+ and ER- cases. Significant differences were found for all comparisons (P<0.005).

Figure 5. Distribution of cases expressing one, two or multiple CT antigens among all CT–positive cases in percentage.

Significantly greater proportion of ER negative tumors expressed ≥3 antigens when compared to ER+ tumors (P = 0.026).

Figure 6. Distribution of CT antigen expression percentages in HER2 positive and HER2 negative breast cancer.

Comparisons were carried out for each CT antigen between the ER-HER2+ and the ER-HER2- (triple-negative) cases and between all HER2+ and HER2- cases. Significant differences were found for both comparisons for CT7 and CT45 only (P≤0.01), with CT7 more frequently expressed in HER2-positive tumors, whereas CT45 more frequently in HER2-negative tumors.