In this study, we investigated several molecules which are associated with tumor immunity using immunohistochemical staining techniques and explored their potential as biomarkers for ICI therapy. We found that high infiltration of CD8-positive T cells in tumor tissue, high expression of CD68-positive tumor-associated macrophages, and low expression of CD4-positive T cells was significantly associated with good response to ICI treatment and survival. These findings may represent a potential biomarker of ICI therapy for patients with RCC, respectively. Furthermore, the combined scoring of the IHC expression status has a powerful potential to be a surrogate marker for a more accurate prediction of ICI treatment efficacy.
CD8-positive T cells constitute a central component of anti-tumor immunity in various malignancies, including RCC.15 Previous reports have generally suggested that infiltration of CD8-positive T cells is associated with poor prognosis in RCC, such as high grade, high stage, and high recurrence rates.15,16 Regarding ICI treatment, high expression of CD8-positive T cells in tumors has been shown to positively associate with favorable clinical outcomes after ICI treatment.17,18 Exploratory data on CD8 infiltration from the randomized phase III trial JAVELIN RENAL 101 indicated that high CD8 infiltration was associated with poor PFS for patients treated with sunitinib but not for patients treated with the avelumab-axitinib combination.19 However, there are several opposite reports in RCC.20,21 This situation demonstrates the peculiarity of the tumor immune environment in RCC. Our results also do not assess in detail the subtypes of infiltrating CD8-positive T cells. Since there are cells termed exhausted T cells22,23, which do not have sufficient anti-tumor effects, and in fact, there are subtypes that act pro-tumor.24
Giraldo et al highlighted tertiary lymphoid structure(TLS) and dendritic cells in mRCC tissues with high CD8-positive T cells. They reported the importance of their role in effector T-cells activation25. We evaluated the correlation between the presence of TLS and ICI treatment by using CD20 as a marker, however, no significant positive association was observed.
CD68 is a protein that is highly expressed on monocytes, blood circulating macrophages, and tissue-infiltrating macrophages.26 Tumor-associated macrophages (TAMs) have been reported to be strongly involved in cancer growth in many malignant tumors, and reports from lung cancer27, gastric cancer28, pancreatic cancer29 and breast cancer30 have shown that high CD68 expression is positively associated with high tumor grade and T stage and is a poor prognostic factor31. Few studies report that a high density of CD68-positive cells is associated with higher tumor grade, higher T stage, and poor prognosis32. Based on the mechanism of activation, TAMs can be divided into two types: M1 type, which acts mainly as an anti-tumor growth, and M2 type, which acts mainly as a pro-tumor growth31,33. M1 type produces inflammatory cytokines such as IL-6 and IL-12, in contrast, M2 type expresses PD-1 ligand and induces the production of anti-inflammatory cytokines such as IL-10 and TGF-β as well as the proliferation of regulatory T cells34,35. Regarding RCC, TAMs are considered to activate cells involved in tumor immunity, such as Tregs, via CXCL20- and IL-1032,36,37. Furthermore, high TIM-3 expression in RCC tumors is strongly associated with increased TAMs infiltration, indicating that TIM-3 expression on tumor cells may be regulated by intercellular interactions with TAMs38. There are only a few reports on the therapeutic effect of ICI and tumor infiltration of TAM.
In the present study, our data showed that high expression of CD68, a marker of macrophages, was significantly associated with a longer prognosis after ICI treatment. These results deviated from earlier reports of high TAM expression and poor prognosis for RCC. Several recent studies have reported that high TAM expression is a favorable prognostic factor for ICI treatment. Voss et al. reported that M2 macrophage infiltration into tumor tissue was associated with continuous clinical response to anti-PD-1 antibody, by WES and RNAseq analysis39. Furthermore, it was very interesting that this trend was not observed in the TKI treatment group. Results of the post hoc analysis of the NIVOREN trial showed that infiltration of CD163-positive macrophages was associated with favorable PFS in patients treated with nivolumab40. Therefore, it is possible that tumor-associated macrophages, although a poor prognostic factor in RCC typically, may act beneficially under ICI treatment. It remains unclear how ICI provides an antitumor effect on TAMs, and further investigation is required.
High expression of CD4-positive T cells, especially regulatory T cells (Tregs), has been associated with poor prognosis in some malignancies.41,42 It has also been reported to be associated with inferior ICI treatment response.43,44 Our data demonstrated a trend of significantly increased fraction of non-responders in the high CD4-positive T cell expression group. Although there was no association observed between Foxp3 expression, a marker specific for Tregs, and response, it was suggested that the status of CD4 expression could be a biomarker of ICI treatment response in RCC.
There are several limitations in this study. First, it is a relatively small observational study. Compared to the subanalysis data of large clinical trials, the number of cases is limited, and further development into a collaborative study at other institutions with a larger number of cases is expected in the future. In addition, stained images were not analyzed by an objective image analysis software. When strict cutoff values are established with digital data, even immune molecules with previously ambiguous results may become candidates for predicting ICI response. Finally, the heterogeneity of kidney cancers must also be considered. Because the FFPE samples were derived from representative tumor tissue in each patient, the response to ICI treatment and infiltrating immune cells could vary by tumor site in RCC with multiple genotypes.
In summary, we evaluated the relationship between the expression status of various immune-related molecules in RCC and the ICI treatment from multiple perspectives, using the immunohistochemistry technic. We determined that high-expression CD8 and CD68, and low-expression CD4 were associated with more favorable ICI treatment response and prolonged prognosis. Each immune cell and tumor cell balances pro- and anti-tumor via complex crosstalk in the tumor microenvironment. In addition, immune cells regulate their differentiation and proliferation through cytokines and chemokines45,46. Therefore, the approach shown here enables a more accurate selection of patients who can benefit from ICI treatment.