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 . In contrast, Madjd et al. investigated HLA class I expression in breast cancer using a HC-10 antibody , 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 . 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%)  and esophageal cancer (43%) , and osteosarcoma (55%) , but less than the downregulation in lung cancer (70%) . 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 . 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 . 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 . 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 . 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 . 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.  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.