Cancer-testis antigens PRAME and NY-ESO-1 correlate with tumour grade and poor prognosis in myxoid liposarcoma

Abstract

Myxoid liposarcoma is the second most common liposarcoma. Although myxoid liposarcoma is relatively chemosensitive and thus a good candidate for chemotherapy, cases with relapsed or metastatic disease still have poor outcome. Here, we performed a gene microarray analysis to compare the gene expression profiles in six clinical myxoid liposarcoma samples and three normal adipose tissue samples, and to identify molecular biomarkers that would be useful as diagnostic markers or treatment targets in myxoid liposarcoma. This showed that the cancer-testis antigen PRAME was up-regulated in myxoid liposarcoma. We then performed immunohistochemical, western blotting and real-time polymerase chain reaction analyses to quantify the expression of PRAME and another cancer-testis antigen, NY-ESO-1, in clinical samples of myxoid liposarcoma (n = 93), dedifferentiated (n = 46), well-differentiated (n = 32) and pleomorphic liposarcomas (n = 14). Immunohistochemically, positivity for PRAME and NY-ESO-1 was observed in 84/93 (90%) and 83/93 (89%) of the myxoid liposarcomas, and in 20/46 (43%) and 3/46 (7%) of the dedifferentiated, 3/32 (9%) and 1/32 (3%) of the well-differentiated and 7/14 (50%) and 3/21 (21%) of the pleomorphic liposarcomas, respectively. High immunohistochemical expression of PRAME and/or NY-ESO-1 was significantly correlated with tumour diameter, the existence of tumour necrosis, a round-cell component of >5%, higher histological grade and advanced clinical stage. High PRAME and NY-ESO-1 expression correlated significantly with poor prognosis in a univariate analysis. The myxoid liposarcomas showed significantly higher protein and mRNA expression levels of PRAME and NY-ESO-1 (CTAG1B) than the other liposarcomas. In conclusion, PRAME and NY-ESO-1 (CTAG1B) were expressed in the vast majority of myxoid liposarcomas, and their high-level expression correlated with tumour grade and poor prognosis. Our results support the potential use of PRAME and NY-ESO-1 as ancillary parameters for differential diagnosis and as prognostic biomarkers, and indicate that the development of immunotherapy against these cancer-testis antigens in myxoid liposarcoma would be warranted.

Keywords: NY‐ESO‐1; PRAME; cDNA microarray; cancer‐testis antigen; liposarcoma; myxoid liposarcoma.

Figure 1

(a,b) Representative H&E stains of MLS. (a) Pure myxoid and (b) round cell components. (c–f) The immunohistochemical results of MLS are shown, indicating (c) PRAME intensity 1, (d) PRAME intensity 2, (e) PRAME intensity 3, (f) NY‐ESO‐1 intensity 1, (g) NY‐ESO‐1 intensity 2 and (h) NY‐ESO‐1 intensity 3. PRAME demonstrated predominantly nuclear staining. NY‐ESO‐1 showed nuclear and cytoplasmic staining. The magnification was ×400 in all panels.

Figure 2

The immunohistochemical results for other liposarcomas are shown, indicating (a) nuclear and cytoplasmic PRAME staining in a PLS, (b) heterogenous nuclear and cytoplasmic NY‐ESO‐1 staining in a PLS, (c) nuclear PRAME staining in a DDLS, (d) homogenous nuclear and cytoplasmic NY‐ESO‐1 staining in a DDLS, (e) nuclear PRAME staining in a WLS and (f) nuclear and cytoplasmic NY‐ESO‐1 in a WLS sample. NY‐ESO‐1 expression was increased in dedifferentiated compared to well‐differentiated components. The magnification was ×400 in all panels.

Figure 3

Immunohistochemical results for the liposarcoma subtypes and the coexpression of PRAME and NY‐ESO‐1 in MLSs. (a,b) PRAME (a) and NY‐ESO‐1 (b) expression by immunohistochemistry among the liposarcoma subtypes. The MLSs showed significantly higher PRAME and NY‐ESO‐1 expression compared to the other liposarcoma subtypes. (c,d) Different expression levels of PRAME (c) and NY‐ESO‐1 (d) between the dedifferentiated component and the well‐differentiated component in DDLS. PRAME expression was significantly higher in the dedifferentiated component (p = 0.0035). (e) In the three NY‐ESO‐1‐positive DDLSs, PRAME expression was increased in the dedifferentiated component compared to the well‐differentiated component. (f) Coexpression of PRAME and NY‐ESO‐1. Immunohistochemical expression of PRAME and NY‐ESO‐1 correlated significantly (p < 0.0001 by chi‐square test). IHC, immunohistochemistry; DDLS, dedifferentiated liposarcoma; MLS, myxoid liposarcoma; PLS, pleomorphic liposarcoma; WLS, well‐differentiated liposarcoma; Low, low expression by immunohistochemistry; High, high expression by immunohistochemistry.

Figure 4

Kaplan–Meier survival curves according to the results of the immunohistochemical study. (a,b) Relationships between PRAME and overall survival (a) and disease‐free survival (b). (c,d) Relationships between NY‐ESO‐1 and overall survival (c) and disease‐free survival (d). High PRAME expression correlated with poor prognosis, and high NY‐ESO‐1 expression correlated with shorter disease‐free survival (p < 0.05).

Figure 5

(a–c) Western blotting of PRAME and NY‐ESO‐1 (a) in MLS tumour samples and corresponding normal tissue, (b) in MLS tumour samples and (c) in MLS tumour samples and MLS cell lines. PRAME was detected in 11 of 19 tumour samples and NY‐ESO‐1 was detected in 18 of 19 tumour samples, but not in any normal tissues. The MLS cell lines showed varying degrees of PRAME and NY‐ESO‐1 expression. PRAME/Actin and NY‐ESO‐1/Actin were normalised to the expression of testis tissue in each membrane. (d,e) Western blot analysis was performed to compare the normal tissue with tumour samples. (d) PRAME and (e) NY‐ESO‐1 showed significantly higher expression in tumour samples than normal tissue (p < 0.05 by Mann–Whitney U‐test). (f) The samples that showed high PRAME expression in the immunohistochemical study showed significantly higher quantitative values of PRAME/Actin compared to the low expression samples (p = 0.0466). (g) The samples that showed high NY‐ESO‐1 expression in the immunohistochemical study showed higher NY‐ESO‐1/Actin compared to the low expression samples, but not significantly so (p = 0.0801). N, normal tissue; T, tumour; IHC immunohistochemistry; High, high expression by immunohistochemistry; Low, low expression by immunohistochemistry.

Figure 6

Relative fold expression of (a) PRAME and (b) CTAG1B measured by quantitative real‐time PCR and the corresponding immunohistochemical results. PRAME and CTAG1B expression were normalised by GAPDH, and further normalised to the expression of a testis sample. (a) PRAME expression was detected in all MLS samples, and 16/20 samples showed higher expression than the testis sample. PRAME was detected in 12/16 DDLS, 13/20 WLS and 2/3 PLS samples; however, all of them showed a low expression level. (b) All MLS samples showed significantly higher CTAG1B expression than the testis sample. CTAG1B was detected in 6/16 DDLS, 14/20 WLS and 2/3 PLS samples. One DDLS and one PLS sample showed higher CTAG1B expression than the testis sample, and the others showed low expression. (c, d) The expression levels of (c) PRAME mRNA and (d) CTAG1B mRNA among the liposarcoma subtypes. The mRNA levels of PRAME and CTAG1B in the MLSs were significantly higher than in the other liposarcoma subtypes (p < 0.05 by Steel‐Dwass test). (e, f) The association between the expression level of (e) PRAME mRNA and the immunohistochemical result, and (f) between CTAG1B mRNA and the NY‐ESO‐1 immunohistochemical result. The samples that showed high PRAME and NY‐ESO‐1 expression by immunohistochemistry showed higher mRNA expression than the samples that showed low expression by immunohistochemistry, but the differences were not significant. IHC, immunohistochemistry; H, high expression by immunohistochemistry; L, low expression by immunohistochemistry; −, negative expression by immunohistochemistry; ND, not detected, Low, low expression by immunohistochemistry; High, high expression by immunohistochemistry.