Expression of MAGE-A and NY-ESO-1 cancer/testis antigens in medullary breast cancer: a retrospective immunohistochemical study

Aim To immunohistochemically evaluate the expression of MAGE-A1, MAGE-A, and NY-ESO-1 cancer/testis (C/T) tumor antigens in medullary breast cancer (MBC) tumor samples and to analyze it in relation to the clinicopathological features. Methods This retrospective study included samples from 49 patients: 40 with typical MBC and 9 with atypical MBC. Tumor specimens were obtained from patients operated on in the University Hospital for Tumors and the Sisters of Mercy University Hospital, Zagreb, Croatia, from 1999 to 2005. Standard immunohistochemistry was used on archival paraffin-embedded MBC tissues. Results MAGE-A1, MAGE-A, and NY-ESO-1 antigens were expressed in 33% (16/49), 33% (16/49), and 22% (11/49) of patients, respectively. No difference between the groups with and without C/T tumor antigen expression in age at diagnosis, tumor size, axillary lymph node metastasis, adjuvant therapy, and HER-2 expression was identified. Significantly more patients died in the MAGE-A-positive group than in the MAGE-A-negative group (P = 0.010), whereas a borderline significance was found between MAGE-A1-positive and the MAGE-A1-negative group (P = 0.079) and between NY-ESO-1-positive and NY-ESO-1-negative group (P = 0.117). Overall survival, as evaluated by the Kaplan-Meier curves, was lower in MAGE-A1- (P = 0.031), MAGE-A- (P = 0.004), NY-ESO-1-positive groups (P = 0.077). Conclusion Expression of C/T antigens may represent a marker of potential prognostic relevance in MBC.

Cancer/testis (C/T) antigens are a subgroup of tumor-associated antigens expressed in normal testis germ line cells and trophoblast, and in various malignancies of different histological types. They were discovered in the last two decades by a combination of immunological and molecular biology techniques. Most genes that encode these antigens are localized on the X-chromosome, frequently as multigene families and are referred to as CT-X genes or CT-X antigens (18)(19)(20)(21)(22)(23). Biological functions of C/T genes and C/T antigens in both germ lines and tumors remain poorly understood. Due to their tumor-associated expression pattern and limited presence in normal tissues, C/T antigens appear to be valuable targets for immunotherapy of cancer. The best-studied C/T antigens are those of the MAGE-A family and the NY-ESO-1 antigen (18)(19)(20)(21)(22)(23). Our initial reports on C/T antigens expression detected by immunohistochemistry in breast invasive ductal carcinomas of no special type (24,25) has been confirmed by other studies (26,27). However, these studies have not been performed on special or relatively rare histological types of breast cancers, such as the MBC.
We have recently reported clinicopathological features of MBCs in 48 patients who were operated on in our two hospitals between 1999 and 2005 (28). The present study includes immunohistochemical analysis of the expression of C/T antigens MAGE-A, MAGE-A1, and NY-ESO 1 in these MBC samples.

PAtIeNtS ANd MethodS
This retrospective study included samples from 49 patients: 40 with typical and 9 with atypical MBC (28).  11 (22) *At that time, adjuvant trastuzumab was not a standard therapy for the heR-2 +++ positive patients, ie, a therapy covered by our national health insurance system. from pathological reports from the Departments of Pathology of the two hospitals. At the time of diagnosis, patients had nonmetastatic MBC (M0). Adjuvant therapy data were obtained from patients' medical reports from the hospitals' Oncology Departments ( Table 1). The patients' survival data were obtained at the end of 2008 from the Croatian National Cancer Registry and through personal contacts with patients and their physicians. The data on the diseasefree survival were not available. The study protocol was approved by the Ethics Committee of the hospitals (28).
For routine histological analysis, the resected breast tissue was fixed immediately after surgery in 10% buffered formalin and later embedded in paraffin. From paraffin-embedded tumor samples, 4-μm thick sections were cut and stained with hematoxylin and eosin, and reviewed by the experienced pathologist (VS, BK, IN) in order to establish the diagnosis. Immunohistochemical staining was performed by appropriate monoclonal antibodies (mab) in accordance with the manufacturer's instructions, as previously reported (19,24,25,28 There was no difference between groups with and without C/T tumor antigen expression in these clinicopathologic parameters. Compared with MAGE-A-negative group, significantly greater number of patients died in the MAGE-Apositive group (P = 0.010), whereas borderline significance   Table 2). In particular, MAGE-A1-positive group had a significantly lower overall survival (P = 0.031, log-rank test) than MAGE-A1-negative group (Figure 2A). Similarly, multi MAGE-A positivity was also associated with a significantly lower overall survival (P = 0.004) ( Figure 2B). A similar trend was also detectable for NY-ESO-1 (P = 0.077), although the difference did not reach significance ( Figure 2C).
Our study suggested that the studied C/T antigens may be used in MBC as tumor markers of potential prognostic relevance. Due to the relative rarity of this type of breast cancer, in order to obtain a final confirmation of this observation, the expression of these C/T antigens needs to be investigated on a greater number of tumor samples. Interestingly, however, a recent publications by Grigoriadis et al (27) and Curigliano et al (32) have pointed out that the expression of CT-X antigens is more frequent in the ER-negative subgroup of breast cancers, including triple-negative and basal-like breast cancers. However, expression of CT-X antigens, to our knowledge, has not been studied specifically in MBC. In studies on squamous non-small-cell lung carcinomas (33), transitional cell carcinomas of the urinary bladder (34), and gynecologic (35,36) and gastric neoplasms (37), expression of C/T antigens has been found to be correlated with patients' shorter tumor-specific survival.
It is still unclear whether C/T antigen expression contributes to tumorigenesis or represents an epiphenomenon in the process of cellular transformation related to the global genome hypomethylation (20)(21)(22)(38)(39)(40)(41) frequently occurring in highly aggressive cancers. However, our data reinforce the notion that C/T antigen specific immunization, possibly in the early stages of the disease, ie, after surgery, might be clinically relevant in selected groups of patients (19,20,23 declaration of authorship: BM conducted data acquisition, interpreted the results, drafted and critically revised the manuscript. AJ planned and designed the study, interpreted the results, drafted and critically revised the manuscript, and gave the final approval. GCS interpreted the results, drafted and critically revised the manuscript, and gave the final approval. VŠ conducted data acquisition and performed data analysis and interpretation. MG conducted data acquisition and performed data analysis and interpretation. RŠ interpreted the results and drafted the manuscript. NŠ interpreted the results and drafted the manuscript. MBK performed data analysis and interpretation of results, and drafted the manuscript. IN performed data analysis and interpretation of results, and drafted the manuscript. BK interpreted the results and drafted the manuscript.