In the past it was believed that cancer cells could autonomously proliferate and survive because of a variety of genetic abnormalities, but recently it has become clear that the peripheral environment (tumor microenvironment) greatly affects cancer cells and contributes to the formation of characteristics particular to cancer . Improvement of the immune microenvironment in tumor is needed because the immune microenvironment in tumor tissues affects not only the efficacy of immunotherapy, but also the efficacy and prognosis of chemotherapy and other modes of anticancer therapy [22, 23]. Tumor antigens are present on tumor cells, and they induce an antitumor immune response in the host. Moreover, tumors activate mechanisms to suppress antitumor immunity, particularly in the microenvironment of tumor tissue, and they utilize those mechanisms for self-proliferation . Furthermore, there are diverse activation and suppression signals for antitumor immunity, and they are integrated to adjust the T-cell activation process . In the group of immunoadjuvant molecules that regulate T-cell activation, inhibitory molecules such as CTLA-4, PD-1, PD-L1/2, LAG-3, and TIM-3 function as immunologic checkpoints [14, 18]. Furthermore, immunotherapy utilizing a blocking antibody to inhibit the signals of the immunological checkpoints has shown promising therapeutic efficacy in a clinical setting [5, 7, 8]. Meanwhile, activators include OX-40, and it has been reported that administration of an anti-OX-40 agonistic antibody may enhance the antitumor immune response . In cancers such as malignant melanoma, renal cell carcinoma, and breast cancer, a correlation has been suggested between PD-1 and PD-L1 expression levels and both the malignancy of cancer and extent of a poor prognosis [9, 11, 22]. However, there have been few reports that have examined the level of expression of immunoadjuvant molecules such as LAG-3, TIM-3, and OX-40 clinically, and therefore monitoring of the tumor immune microenvironment has been conducted in NAC breast cancer patients using TILs. In this study, the LAG low-expression group had a significantly higher pCR rate than the high LAG expression group in NAC breast cancer patients, and low-LAG expression contributed to an extension of the disease-free survival interval.
LAG-3 is a molecule with a structure similar to CD4, and it appears on the cell surface when T-cells are activated [13, 15, 42]. The signal transduction pathway of LAG-3 is still unclear, but it is believed to function as a molecule that not only suppresses the proliferation and activation of T-cells, but that also plays a key role as an immune checkpoint similar to PD-1 and CTLA-4 . Basic study has demonstrated that the antitumor immune response in mice is enhanced by inhibiting the LAG-3 signal using an anti-LAG-3 antibody while concurrently administering an anti-PD-1 antibody . Furthermore, an anti-LAG-3 antibody was adapted for human use, and it was discovered that the use of this antibody in combination with paclitaxel in phase I and II clinical studies of breast cancer raised the response rate from 25–50% compared with groups treated with anticancer monotherapy . Paclitaxel is considered to improve immune escape in the host by suppressing Tregs, but expression of LAG-3 has also been found in Tregs. In other words, an anti-LAG-3 antibody in combination with paclitaxel may effectively act to relieve immunosuppression by suppressing Tregs. In the NAC regimen used in our study that treats paclitaxel as a key drug, it appears that these mechanisms enable immune response monitoring via LAG-3 expression.
On the other hand, TIM-3 has galectin-9 as a ligand and suppresses the activation of effector T-cells mediated by ligand-receptor interaction [16–18]. Clinical studies have reported that the level of expression of TIM-3 in renal cell carcinoma and head and neck cancer exhibits a negative correlation to prognosis [45, 46]. In our study, we found no correlation between TIM-3 expression and the therapeutic efficacy of NAC. TIM-3 is a molecule that contributes to the suppression of T-cell function synergistically with PD-1, and simply activating antitumor immunity by lowering TIM-3 expression alone may not improve the tumor immune microenvironment. Moreover, OX-40 is a member of the TNF receptor superfamily, and is expressed by activated T-cells, NK cells, and Tregs [19–21]. It has been reported in studies using a mouse model that administration of an anti-OX-40 agonistic antibody enabled rejection of fully established tumors . In our study, however, we found no correlation between OX-40 expression and therapeutic efficacy of NAC. Under the assumption that no potent antitumor effect will be obtained with anti-OX-40 agonistic antibody monotherapy, a search is now underway in basic study for a combination therapy with another drug . In other words, in the case of OX-40 as well, this finding indicates that a clear improvement of the tumor immune microenvironment cannot be obtained clinically through modulation of OX-40 expression alone. In addition, no correlations between expression of LAG-3, TIM-3, and OX-40 were found in this study.
In previous study, breast cancer was not considered as a cancer that develops as the result of an immune disorder . Recently, however, breast cancer has come to be viewed as an immunogenic tumor, and the highly malignant subtypes TNBC and HER2BC have a high level of immune activity [49, 50]. We believe that in these highly malignant breast cancers the tumor immune microenvironment can be reliably monitored by TILs because of the high immune activity. In our study, when breast cancer was stratified by intrinsic subtype and TILs were evaluated, the groups with low-LAG expression in highly malignant breast cancers had a significantly higher pCR rate, but no significant difference was found for HRBC.