This study demonstrated that the number of immune cells infiltrating PAs and MGMT expression were correlated with tumor aggressiveness based on imaging and molecular features. In addition, the positive rate of PD-L1 was positively associated with the Ki-67 index and p53 expression. Therefore, the above results reveal that there will be a novel and promising therapeutic approach for the management of aggressive PA.
Our findings confirmed that infiltrating TAMs and M2 polarization in PA were correlated with Knosp grades and MMP-9 expression. However, studies investigating M2 polarized macrophages in PA are rare. Macrophages become polarized based on changes in the environment, creating the different macrophage subtypes, M1 and M2. As is well-known, M1 macrophages generally have antitumor properties and enhanced antigen-presenting ability. In contrast, M2 macrophages show reduced antimicrobial and tumoricidal activity[28]. A study by Marques et al[29]. showed that M2 macrophages could promote neo-angiogenesis in pituitary neuroendocrine tumors (PitNET).The number of CD68 + macrophages infiltrating PAs was positively correlated with tumor size and Knosp grade, which indicates tumor aggressiveness[24]. TAMs could enwrap tumor-associated blood vessels and create an environment conducive to tumor progression by promoting tumor angiogenesis and secreting growth factors[30]. MMP-9, as a member of the zinc-dependent endopeptidase family, could promote angiogenesis and cancer invasion by degrading type IV collagen of basal membrane near the tumor cell and extracellular matrix (ECM)[31]. There are few studies on this topic, and further work is required to validate the specific mechanism.
It has been reported that the number of CD68 + macrophages in sparsely granulated GH-secreting adenomas is greater than in ACTH adenomas, and nonfunctional adenomas show more infiltrating CD68 + macrophages than ACTH adenomas[24]. Interestingly, only GH-secreting adenomas had the highest number of CD163 + macrophage infiltration, which may support the evidence that GH adenomas tend to have an aggressive behavior[32].
Controversies exist regarding the associations between CD8 + TILs and PA invasiveness or aggressiveness. It has been reported that T-lymphocytes (CD8, CD4, FOXP3) recruited by pituitary neuroendocrine tumors-derived chemokines determine the aggressive behavior[33]. A tendency for higher invasiveness or aggressiveness in PAs with more infiltration of CD8 + TILs has been observed[9, 34]. However, it was noted that the number of CD8 + lymphocytes was positively correlated with Knosp grades in our study.
In addition, the number of CD8 + TILs was positively correlated with the expression of MMP-9. MMP-9 plays a critical role in CD8 + T-cell infiltration into tissues and exerts a regulatory role on CD8 + T-cell activation[35, 36]. Our results revealed that PAs with aggressive behavior and/or MMP-9 expression could present a microenvironment highly infiltrated by CD8 + TILs, which may exert a specific effect on the invasion of PAs. Whether CD8 + TILs could encourage an immunosuppressive phenotype or not warrants further study.
There was a trend suggesting a higher positive rate of PD-L1 in aggressive PA and non-functional PA[9, 34]. However, the present results only confirmed that a higher expression of PD-L1 occurred in null cell PAs than the other PAs. In addition, it was reported that PD-L1 expression is positively associated with increased numbers of CD8 + TILs[34]. However, this result was not seen in this study. Interestingly, CD68 + macrophages were more infiltrated in the positive PD-L1 group. This result suggests that CD68 + macrophages may promote the expression of PD-L1, further encouraging the formation of an immunosuppressive microenvironment. In addition, it seems that patients with positive p53 expression and a high Ki-67 index would benefit most from anti-PD-L1 therapy. Although some studies on glioblastomas showed a correlation between PD-L1 and MGMT expression[37, 38], a similar result was not found in this study.
A previous study demonstrated that MMP-9 has potential as a marker for invasion but not for recurrence[39]. Survival analysis showed that patients with positive MMP-9 expression had worse PFS and a higher risk of tumor recurrence. In addition, incomplete resection was a significant independent predictor of recurrence of tumor in multivariate analyses, and patients with incomplete removal of the tumor had a significantly shorter PFS in this study.
The lower the MGMT expression, the better the response to the TMZ treatment in PA[40]. Our results suggested that there may be some interaction between the aggressiveness of the tumor and MGMT expression. A previous study showed that low-to-moderate MGMT immunoexpression occurred significantly more often in aggressive PAs[41].However, although it was not significantly different between aggressive and non-aggressive PAs in the imaging data, the level of MGMT immunopositivity was positively associated with MMP-9 staining extent. This result may further exemplify that aggressive PAs could be a suitable candidate for TMZ therapy, but it is necessary to provide more evidence to document this. In addition, the expression of MGMT did show linear relationships with infiltrating immune cells, which may provide evidence that it is possible to use immunotherapy in combination with TMZ to treat aggressive PA.
Together, our results demonstrate that the tumor immune microenvironment serves a vital role in the invasion of PA. However, the effect of immunotherapy on pituitary adenomas has only been reported in some cases, and the effect is uncertain. One patient with ACTH-secreting PA progressed rapidly after four cycles of anti-PD-L1 (pembrolizumab)[42]. Clinical trials showed that the therapeutic effect of a single immune checkpoint inhibitor is not satisfactory for GBM, and attention has shifted focus to immunotherapies combined with other treatments. This novel approach might be more beneficial for patients with GBM[43]. There are currently only two clinical trials of combined immunotherapy for pituitary tumors in clinicaltrials.gov. Combined immunotherapy (anti-PD-L1 and CTLA-4) for aggressive pituitary adenomas is still in phase 2 (Memorial Sloan Kettering cancer center, United States, NCT04042753). Another clinical trial of combined immunotherapy for rare pituitary tumors is still recruiting patients (National Cancer Institute, United States, NCT02834013). At present, there are no animal experiments or clinical studies on macrophage-targeting treatment for PA. In addition, the number of cases with low-expression of MGMT is relatively small. Unfortunately, a 2017 review reports that only approximately 42% of pituitary tumors show a radiological response to TMZ treatment[44].
In summary, we confirmed that there are significant differences in the infiltration of immune cells (CD8 + TILs, CD68 + and CD163 + macrophages) and the expression of PD-L1 and MGMT between aggressive and non-aggressive PA. Based on our results and combined with the experience of glioma immunotherapy, targeting macrophages / CD8 + TILs with anti-PD-L1 or other types of immune checkpoint inhibitors may provide a breakthrough in immunotherapy for aggressive PA. Whether immunotherapy based on TMZ treatment may benefit patients is expected to be confirmed by further preclinical and clinical trials.
Limitations
There were limitations to our study that should be acknowledged. First, the main limitation of this study is the low reproducibility of the immunohistochemical methods. However, immunohistochemistry is still the most important method for the diagnosis of PAs, based on the 2017 WHO classification of tumors of the pituitary gland[22]. Second, the conclusion that these data speak in favor of anti-lymphocytic or anti-macrophagic approaches in treatment is based on circumstantial evidence. Despite certain limitations of this retrospective study, the observed interesting clinical features have some enlightening significance and require further investigation.