In the present study, we developed an immunohistogram based on immunohistochemical analysis of resected primary cancer tissues as a potential clinically available biomarker for ICI therapy in patients with urological cancer. We confirmed that the immune-hot group, which suggests the existence of a functional cancer immunity cycle, was significantly associated with higher DCR, longer PFS, and CSS after ICI therapy. Some patients were classified into the immune-cold group and did not respond to ICI therapy even though their tumors showed infiltration of CD8-positive T cells, suggesting that the evaluation of the entire cancer immunity cycle, not a single biomarker, is important in the prediction of ICI efficacy. The immunohistogram visualizes the status of each step in the cancer-immunity cycle and therefore is useful not only for predicting the efficacy of ICI therapy but also for discussing treatment targets to improve ICI efficacy. To our knowledge, this is the first study to evaluate the utility of an immunohistogram as a predictive biomarker for ICI therapy.
The feasibility of immunohistochemical analyses in pathological diagnostic workflows has prompted extensive efforts to establish the utility of immunohistochemical analyses of PD-L1 as a potential biomarker for ICI therapy. In pivotal clinical trials of ICI therapy for urological cancer, the predictive ability of PD-L1 and related biomarkers have been studied using IHC. In RCC trials, which evaluated ICIs in combination with conventional molecular-targeted agents, high PD-L1 expression was associated with a treatment benefit of ICIs[3–5]. In UC trials, the survival benefit of avelumab maintenance therapy was shown in the PD-L1-high population in the JAVELIN Bladder 100 studies, while the 2nd line pembrolizumab study (Keynote-045) reported a similar response irrespective of PD-L1 combined positive score. As shown in the above-mentioned studies, PD-L1 classification combining the expression on tumor and immune cells might be useful for predicting ICI efficacy. However, due to differences in the antibodies used, staining methods, and scoring algorithms, there is a significant concern about the interchangeability and comparability of data for clinical use. Furthermore, PD-L1 classification might combine predictive and prognostic information, which may have led to inconsistency in the ability of PD-L1 expression to predict outcomes of ICI therapy in previous trials.
The importance of tumor-infiltrating lymphocytes (TILs) in cancer immunity has been demonstrated by numerous oncological studies. TILs include various functionally divergent components, such as active or exhausted CD8-positive T cells, regulatory T cells, and natural killer (NK) cells. In RCC, it has been reported that an increased number of CD8-positive T cells in the TME is associated with a better prognosis, while an increase in exhausted T cells, which also belong to CD8-positive T cells, was associated with shorter survival. Similarly, higher CD8-positive T cells in bladder cancer are associated with better outcomes. However, the predictive ability of TILs for the efficacy of ICI therapy has not yet been established. Based on the expression of PD-L1 and the presence or absence of TILs, Teng et al. proposed classifying TME into four groups. We applied this classification to surgically treated upper tract urothelial cancer (UTUC) patients and confirmed its prognostic ability. Although the predictive ability of this classification for the efficacy of ICI therapy has not been well studied, combining multiple factors may improve its predictive ability. In this regard, the concept of immunohistogram, which involves not only TILs and PD-L1 but also the status of HLA molecules, could be useful to develop better biomarkers are predictive of the efficacy of ICI therapy.
In addition to PD-L1 and TILs, the expression of HLA molecules in the TME is an important factor in predicting the functional cancer immunity cycle. HLA-DR, expressed on antigen-presenting cells such as macrophages, B cells, and dendritic cells, presents cancer antigens to helper T cells to stimulate the immune system. Therefore, we used HLA-DR as a marker for step 3 (priming and activation) in the cancer immunity cycle. In this study, all patients with positive HLA-DR also had infiltration of CD8-positive, TIA-1 positive T cells and expression of HLA-class I molecules in tumor cells and responded to ICI therapy. This suggests that the cancer immunity cycle probably functioned properly in these patients. In contrast, patients with negative HLA-DR tended to show a lower number of CD8-positive, TIA-1 positive T cells, or excluded or desert infiltration patterns, and did not respond to ICI therapy, suggesting an impaired cancer immunity cycle in these patients. The function of HLA-class I is to display peptide fragments of cancer antigens and trigger the attack by cytotoxic T cells. Therefore, the expression of HLA-class I in tumor cells is a prerequisite for cytotoxic T cell-based cancer therapy, and we assume HLA-class I to be a marker for step 6 (recognition of cancer cells by T cells) in the cancer immunity cycle. In this study, the patients with negative HLA-class 1 showed negative HLA-DR and a small number of CD8-positive, TIA-1 positive T cells, suggesting an impaired cancer immunity cycle.
A major concern in the development of an immunohistogram using primary tumor tissue is the possible discrepancy in the TME between the primary tumor and metastatic sites. As tumor cells with high antigenicity are under attack by anti-tumor host immunity, tumor cells with impaired HLA-class I expression can escape from the immune system and become predominant in recurrent or metastatic tumors. We have previously referred to this phenomenon as “adaptive immune escape” and reported this phenomenon in an RCC patient. Although we observed no patients with negative HLA-class I but positive HLA-DR in their primary tumor, evaluation of recurrent or metastatic tumors using IHC should be performed in future trials to confirm the adaptive immune escape phenomenon. On the other hand, we observed a phenomenon opposite to adaptive immune escape in the current study, three patients responded to ICI therapy despite their primary tumor having an immune-cold pattern. Since these three patients had negative HLA-DR and the excluded pattern of T-cell infiltration, this immunohistogram pattern might be associated with a discrepancy in the TME between primary tumor and recurrent or metastatic tumors, which warrants further confirmation.
Our study had several limitations. First, this was a retrospective study with small sample size; therefore, it was subjected to a probable selection bias. Second, this study is a pan-cancer analysis that includes both RCC and UC. A cancer type-specific analysis with an extended cohort is warranted in the future. As the cancer immunity cycle can be applied to all cancer types, pan-cancer analyses might be an optimal method for preliminary analyses, as is the case in previous immunogram studies. Third, we used archival tissue of resected primary cancer to develop cancer immunohistogram; therefore, patients who had received tumor biopsy only or those who did not undergo biopsy before ICI therapy were excluded from the study.
In conclusion, immunogram, which could be developed only with immunohistochemical analyses, was associated with DCR and survival of urological cancer patients treated with ICI therapy and could be a clinically accessible potential biomarker for predicting the efficacy of ICI therapy. A more precise and tumor-specific model of the immunohistogram should be investigated in future studies.