Clinical implication of HLA class I expression in breast cancer

The antitumor activity via cytotoxic T lymphocytes (CTL) or tumor antigen has been clarified in the oncological field. Activation of anti-tumor CTL requires the recognition of immunogenic epitopes presented on various types of human leukocyte antigen (HLA) class I molecules on the tumor [1‐4]. The concept of immune surveillance is maintaining the relationship between tumor-associated antigens (TAA) complexing with the HLA class I and tumor-specific cytotoxic T cells. These activated tumor-specific cytotoxic T cells can eliminate cancer cells specifically. The loss of HLA class I on the tumor is believed to lead to malfunction of recognition by the CD8+ T cells. It is already known that malignancies exhibit altered or lost expression of histocompatible antigens on the tumor cells [5‐7]. Loss of HLA class I antigens appears to be a significant mechanism by which tumor cells escape specific immune attack and causes problems in the design of antitumor immunotherapy [7]. The loss of HLA class I antigens on tumor cells has been reported in several human tumors [5‐7], and the loss of HLA class I molecules has been discussed in the context of tumor aggressiveness, such as differentiation of histology [8‐10], invasiveness, and metastatic potential [5, 11].

The non-covalent association with β 2-microglobulin (β 2 m) is essential for the structural stability and optimal function of HLA class I molecules [12]. Thus, several authors have used immunostaining of β 2 m for the analysis of overall surface expression of HLA class I molecules [9, 11, 13]. However, there are often difficulties in evaluating immunostaining using anti-MHC class I monoclonal antibodies (mAbs), such as W6/32, HC-10, or HC-A2, as these type of antibodies are not ideal for the immunostaining of formalin-fixed, paraffin-embedded tissue. Moreover, these antibodies are not fully recognized whole HLA class I properly. Recently, EMR8-5, a monoclonal antibody against HLA class I heavy chains (HLA-A, B, C), has been validated in HLA class I immunohistochemistry [14‐16], and used to investigate HLA class I expression in osteosarcoma [14], non-small cell lung cancer [15], and renal cell carcinoma [16]. The clinical implication of the HLA class I expression has been discussed and reviewed in breast cancer and it is not clarified [17‐21]. Moreover, there have been no studies on HLA class I expression of breast cancer by EMR8-5. The present study assessed HLA class I expression in invasive breast cancer by immunohistochemistry using the EMR8-5 antibody, analyzed associations with clinicopathological factors, and discussed the clinical implication of HLA class I-positive breast cancer.

The monoclonal antibody EMR8-5 can recognize all of HLA A, B, and C heavy chain even in formalin-fixed tissue. In this context, EMR8-5 can recognize whole HLA molecules, and its validity was supported by the immunostaining performed in our current studies. Cordon et al. reported that HLA class I positivity examined using the conventional HLA class I antibody W6/32, which also recognizes all HLA class I antigens, was 30% of HLA class I positivity in breast cancer, similar to that shown in our present study [24]. In contrast, Madjd et al. investigated HLA class I expression in breast cancer using a HC-10 antibody [24], and demonstrated that HLA class I negativity correlated with a better postoperative outcome. These results conflicted with the data in our studies. This discrepancy may be explained by the fact that whereas the HC10 mAb scarcely reacts with HLA-A alleles, the anti- HLA class I heavy chain mAb EMR8-5 can detect all recombinant proteins of HLA-A, B, and C alleles by immunoblot analysis [25]. In addition, EMR8-5 can be applied to paraffin-fixed specimens, so in this context it is an ideal antibody for evaluating cancerous HLA class I antigen expression.

In our current study, the downregulation of HLA class I expression in breast cancer was 66%, which was more than that in gastric (32%) [22] and esophageal cancer (43%) [26], and osteosarcoma (55%) [14], but less than the downregulation in lung cancer (70%) [15]. The degree of HLA class I loss may be affected by organ specificity. For example, Ishigami et al. speculated that highly preserved HLA class I expression in gastric cancer is partly due to exogenous stimulation from gastritis or bacterial infection of Helicobacter pylori [22]. The differences in HLA class I expression in breast cancer may also be explained by the possible inflammation and proteolysis that can occur at the sites of breast cancer origin. These are important steps linked both to HLA loss and cancer aggressiveness. In this study, there was little HLA class I positivity in normal mammary gland tissue. In contrast, HLA class I antigens were preserved in early breast cancer, and cancerous HLA class I antigens were newly expressed or reduced according to the tumor extension. Although T1 tumors had 41% HLA class I positivity, T3-4 tumors only showed 15% positivity. According to tumor extension, preservation of HLA class I of the tumor was reduced. This clinical trait was also reported in other types of malignancies, such as gastrointestinal cancer [22, 26] and sarcoma [14]. It is possible that, in the process of tumor extension, tumors that lost HLA class I survived and escaped from antigen-specific CTL-mediated lysis leading to tumor dissemination and metastasis. However, these results do not fully explain the relationship with metastasis, therefore we need to perform more analyses comparing the results among in situ, lobular and ductal breast cancers on key parameters, such as VEGF, MMP etc.

In the current study, the downregulation of HLA class I expression was significantly associated with lymphatic and nodal invasion. Mizukami et al. showed that when HLA class I-positive esophageal cancer metastasized to the lymph node, tumor cells completely lost HLA class I expression in this system [22]. Zia et al. investigated the immunological characteristics of isolated cancer cells (ITC) in bone marrow and found that ITCs with the HLA class I downregulation phenotype were often derived from poorly differentiated primary breast carcinomas which was associated with a short survival period in breast cancer [27]. Therefore, cancerous HLA class I downregulation seems to be conducive to metastasis to other organs.

Patients with positive HLA class I expression showed a better DFS in comparison with those with downregulation of HLA class I expression. This result differs from that for esophageal cancer [26]. In breast cancer, the average OS is generally better than those in other malignancies; DFS is often used to evaluate aggressiveness of biological markers in breast cancer. In this context, significant differentiation in DFS seems to be meaningful.

It has been clarified that HLA molecule inactivity depends not only on the expression of HLA class I molecules themselves, but also on the post-transcriptional course that mainly affects β2-microglobulin gene expression. Aptsiauri et al. [28] showed that if apparent tumor cells expressed HLA class I, various types of HLA class I alterations were found in malignancies and in the molecular mechanisms that underlie these defects. In this context, the HLA class I molecules preserved in these breast cancers may exhibit altered expression and dysfunction as antigen presentation molecules. It seems to be difficult to precisely evaluate HLA class I expression, however, in the current study, we evaluate HLA expression including in β2 microglobulin expression using the EMR8-5 antibody.

The downregulation of HLA class I expression frequently occurred in breast cancer, in a similar manner to what has been seen in several other cancers, and it may also be associated with tumor progression and relapse. Therefore HLA class I status may be useful with other well-known prognostic factors like nodal involvement and hormone status to evaluate postoperative outcomes in breast cancer.