Lymphocyte-Activation Gene-3 Expression and Prognostic Value in Neoadjuvant-Treated Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive cancer phenotype characterized by the lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). It accounts for 15% to 20% of all breast cancer subtypes [1]. Chemotherapy is the only therapeutic method used for TNBC because of the lack of effective endocrine and targeted therapies.

Systemic neoadjuvant therapy is the treatment of choice for patients with locally advanced breast cancer. Pathologically complete response (pCR) is an indicator of good prognosis after neoadjuvant therapy. Previous clinical trials evaluating the efficacy of neoadjuvant chemotherapy (NACT) revealed that patients with TNBC are more sensitive to chemotherapy and show higher rates of pCR than patients with other breast cancer subtypes [2]. Therefore, identifying novel biological markers for the prediction of pCR rates or the survival of non-pCR patients is required for patients with TNBC.

Previous studies have shown that the immune microenvironment plays a key role in cancer progression and response to therapies. Tumor-infiltrating lymphocytes (TILs) have prognostic and predictive values in both adjuvant chemotherapy and NACT in breast cancer, especially in triple-negative and HER2-overexpressing breast cancers [3, 4]. CD8⁺TILs, a subtype of TILs, are a good prognostic factor for TNBC and are related to pCR after NACT [5].

Immune checkpoints play vital roles in tumor immune escape. Cancer immunotherapy, regarded as one of the most important scientific breakthroughs, can reverse tumor immune escape by suppressing immune checkpoints. Programmed death-1 (PD-1) is a well-known immunological checkpoint and the PD-1/programmed death ligand-1 (PD-L1) axis can mediate a key immune modulatory pathway. It has recently been shown that immune checkpoint inhibitors of PD-1/PD-L1 are associated with chemotherapy response in melanoma and lung cancer and may stimulate TILs [6, 7]. However, less than 25% of patients benefit from anti-PD-1/PD-L1 therapy and this treatment has not been evaluated in breast cancer. Therefore, studies exploring the role and relationship of immune checkpoints and TILs in TNBC are required to highlight the urgent need for novel immunotherapy strategies and validated immune biomarkers for guiding patient selection.

Lymphocyte-activation gene-3 (LAG-3), designated as CD223, is another important checkpoint of the immunoglobulin superfamily [8]. LAG-3 plays a negative regulatory role on T cell proliferation and activation and may exert synergistic effects with PD-1/PD-L1 to mediate immune exhaustion. Co-expression of LAG-3 and PD-1 on tolerized TILs indicates their possible contribution to tumor-mediated immune suppression [9]. Recent studies have shown the co-expression of LAG-3 and PD-1 in 15% of patients with TNBC [10]; however, the relationship between these immune checkpoints and their predictive effects remains unclear. Therefore, studies investigating the correlation of LAG-3, PD1/PD-L1, and CD8⁺TIL expression in TNBC with prognosis are needed to reveal the immunosuppressive effects of LAG-3 and evaluate its efficacy in patients with TNBC.

In this study, we retrospectively evaluated the expression of LAG-3 in TNBC patients treated with NACT and investigated its correlation with clinical characteristics, patient prognosis, and response to chemotherapy. We also analyzed the relationship between LAG-3 and PD-1, PD-L1, and CD8⁺TILs to find TNBC patients who may benefit from the addition of immunotherapy to standard chemotherapeutic regimens.

LAG-3 controls T cell proliferation and inhibits cell cycle progression, leading to a dysfunctional state [15]. It plays a role in anticancer immunity and has been studied in many cancer types. As early as 2003, Cappello et al. [16] observed that anti-LAG-3 treatment may control breast cancer in mouse models. LAG-3 expression is significantly upregulated in CD8⁺TILs of patients with hepatocellular carcinoma [17]. Recent evidence suggested that co-expression of LAG-3 and PD-1 may increase tumor-mediated immunosuppression [9]. The inhibition of both LAG-3 and PD-1 pathways resulted in longer survival than PD-1 inhibition alone [18]. In addition, the expression of these two immune checkpoints is correlated with the presence of TILs [10]. These results with immune checkpoint inhibitors have renewed interest in immune checkpoints in the tumor microenvironment and their relationship with TILs and chemotherapy responses.

Many studies have focused on LAG-3; however, little is known about the role of LAG-3 in TNBC, especially in NACT. In this work, we demonstrated LAG-3 expression pre- and post-NACT in TNBC and also described the correlations between LAG-3 and PD-1, PD-L1, and CD8⁺TILs.

In our study, we showed that the levels of these four markers increased at different degrees following NACT. These results are similar to those of previously published studies [10, 19]. Furthermore, one-to-one correspondence of each marker between biopsy and resected specimens indicated that the immune marker expression on TILs increased significantly after NACT. Mesnage et al. [20] showed that chemotherapy may lead to the activation of immunosuppressive mechanisms. Preclinical data showed upregulation of PD-L1 in ovarian cancer cell lines under chemotherapy [21]. In addition, increased infiltration by CD8⁺ and CD4⁺ T cells and relatively stable numbers of co-inhibitory molecules were detected after NACT [22]. The effect of chemotherapy on tumor control will be weakened under an immunosuppressive microenvironment. Therefore, the addition of immunotherapy to chemotherapy may be a good choice, as NACT induces immune antigen exposure, especially for non-pCR patients. Our research provides strong evidence to support this hypothesis. At present, some case reports and preclinical studies have demonstrated the efficacy of combining chemoradiotherapy with immunotherapy [23, 24, 25].

A previous study has shown that in luminal B/HER2-negative breast cancer, high quantities of intratumoral CD8⁺TILs serve as a predictor of pCR after NACT and represent a good prognostic factor for overall survival [26]. In the present work, we also observed that expression of LAG-3 in pre-NACT specimens showed significant effects on pCR. At the same time, univariate and multivariate logistic analyses showed the possible factors that may affect pCR. In univariate analysis, multiple factors (nodal status, NACT cycles, quantity of CD8⁺TILs, and expression of LAG-3 and PD-L1) were predictors of pCR after NACT. On the other hand, only the nodal status and quantity of CD8⁺TILs were significant in predicting pCR according to multivariate analysis. Nevertheless, high expression of LAG-3 was associated with a high pCR rate. After inhibiting PD-1, increased CD8⁺ T cell infiltration was observed in tumors and effector and memory T cells in the peripheral blood [27]. LAG-3 inhibition may show the same result. Therefore, the addition of anti-LAG-3 therapy to NACT may increase the number of CD8⁺TILs, thereby increasing the pCR rate.

Recent studies have revealed that LAG-3, in combination with PD-1, plays a negative regulatory role in T cells by mediating a state of exhaustion and antitumor immune response in many tumor samples [8]. In mice, LAG-3 and PD-1 exhibit synergistic effects that can alleviate large tumor burdens, whereas only delayed tumor growth was observed in single-knockout mice [28]. In an immunotherapeutic model, LAG-3 expression is upregulated with anti-PD-L1 therapy, possibly by a compensatory measure [18]. This may explain the combinatorial blockade. We also observed a correlation between high expression levels of LAG-3, PD-1, and PD-L1 in pre-NACT specimens, whereas high expression of LAG-3 on TILs showed significant differences with nodal status and PD-1 in post-NACT specimens. However, the interactions of related regulatory pathways remain poorly understood [29].

Although pCR status suggests good prognosis, studies have shown that residual lesions have become a useful tool for NACT in breast cancer patients [30]. In the present study, survival analysis of patients following NACT demonstrated that high expression of LAG-3 in residual tissues suggests poor prognosis; however, such expression cannot be used as a prognostic indicator (p>0.05). Meanwhile, the combination of LAG-3 and CD8⁺TILs or PD-L1 expression showed poor prognosis for DFS. In the Kaplan-Meier analyses, LAG-3-high/PD-1-high, LAG-3-high/CD8⁺TILs-low, and LAG-3-high/PD-L1-high indicated the poorest prognosis. Although LAG-3-high/PD-1-high was associated with the poorest prognosis, it showed no statistical significant difference. In contrast, Bottai et al. [10] demonstrated that co-expression of PD-1 and LAG-3 on TILs in the tumor microenvironment may be associated with good prognosis in TNBC. This finding may be inconsistent with the selected population in the present study. We selected patients who underwent neoadjuvant therapy, whereas Bottai et al. [10] studied postoperative patients. This difference in findings may also be caused by tumor heterogeneity. Therefore, a more comprehensive analysis should be performed using whole-tissue sections to reduce sampling bias caused by tumor heterogeneity.

We further confirmed prognostic factors in TNBC by univariate and multivariate Cox proportional hazard regression analyses. Nodal status, CD8⁺TILs, and LAG-3-high/PD-L1-high showed prognostic values in multivariate analysis. This result is similar to findings in a study on non-small-cell lung cancer [19]. Thus, LAG-3, especially in combination with other immune checkpoints, is comprehensive and accurate in predicting patient survival in TNBC following NACT. The finding provides the basis for screening candidates for immune combination therapy. The combination of immunotherapy with chemotherapy may be more effective for those with poor prognosis.

Our results show that LAG-3 may be a viable candidate for targeted monotherapy and has significant potential, in combination with other immune factors, for the prediction of prognosis. However, our study presents some limitations. For example, our sample size is small. We must further expand the sample size to verify our results. The interaction of LAG-3 with other immune molecules must also be further validated with cytology. There are no clinical research reports investigating whether changes in LAG-3 occur in patients receiving poor anti-PD-1/PD-L1 treatment or identifying the possible signaling pathways connecting these molecules. These problems should be resolved in future studies.

In summary, we demonstrated that CD8⁺TILs are sensitive predictive markers in response to NACT. Moreover, tumor immunity was activated by increased immune cells after NACT. In residual tissues, high expression of LAG-3, especially in combination with low quantities of CD8⁺TILs or high expression of PD-L1, was associated with poor prognosis. These results will have an important bearing on future studies on immune maintenance therapy, immunotherapy combined with NACT, and evaluation of the efficacy of and prognosis of tumors for immunotherapy following NACT. Lastly, future studies should analyze the molecular mechanisms of LAG-3 and its synergistic effects with other immune checkpoints in TNBC.