With respect to research of ICB therapy YSTs was few, and was often included in the study of testicular germ cell tumors. This study is the first comprehensive and detailed assessment of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes for evaluating the potency of ICB therapy in YSTs. Remarkably, the presence and frequency of TLSs in YSTs were also first reported in the study. The observed results demonstrated that only a small proportion of patients, which had tumor-infiltrating lymphocytes and the expression of immune checkpoint molecules present, may be efficacious for ICB agents.
That the expression levels of PD-1/PD-L1 and CTLA4 are significant predictive biomarkers in cancer immunotherapy, has already been confirmed by extensive studies[23–25]. The expression of CTLA4 has been almost exclusively studied in the TILs, but increasing evidence has shown its expression on tumors with clinical response to ICB therapy. Notably, a high frequency of both CTLA4T+ and CTLA4L+ was found in our study, suggesting that anti-CTLA4 targeted therapy may be improve the prognosis of YSTs patients. However, this idea should be applied with caution, as until now, CTLA4 expression in YSTs has rarely been reported in the published literature. Also, the existence of patients with negative expression and positive responses has been previously reported and confirmed that the expression of CTLA4 is not an adequate biomarker to select candidate patients for ICB. The results by Hamid et al revealed significant associations between positive response and high baseline expression of Foxp3, and between positive response and increase in TILs between baseline and 3 weeks after start of treatment. In addition, Mastracci et al also demonstrated that high TIL score and density of CD3+, CD8+ T cells, and CTLA4L+ were significantly associated with a better response to anti-CTLA4 targeted therapy in patients with melanoma. These results suggested that the expression of CTLA4+ (CTLA4T+ or CTLA4L+) in combination with TILs may be optimal marker for selecting patients who could be candidates for anti-CTLA4 therapy. In our study, although a high frequency of both CTLA4T+ and CTLA4L+ was found; only a small subset of patients exhibited the immune-inflamed phenotype or with TLSs, suggesting that anti-CTLA4 therapy is not optimal choice for most YSTs patients.
In our study, five patients (21.74%) had positive PD-1 expression, indicating that YSTs exhibited a low frequency of PD-1 expression. As to PD-L1, that PD-L1T and PD-L1L had different prognostic values highlighted the importance of separately evaluating their expression. Nevertheless, the study of PD-L1 not only was few in YSTs, but also has been assessed without distinguishing between TCs and lymphocytes in the most studies. In our study, the PD-L1T and PD-L1L were observed, and the results showed that nine cases (39.1%) had positive PD-L1T, and four cases (17.4%) had positive PD-L1L. Fankhauser et al found that 19 (40%) patients with YSTs exhibited positive expression of PD-L1 without distinguishing between TCs and lymphocytes, the results were consistent with the expression with PD-L1T in our study. Consistent with our study, one study showed that five of 26 patients exhibited positive PD-L1L, and another study found that no positive expression was detected in four YST patients[30, 31]. These results demonstrated that the frequency of PD-L1L expression was low in YSTs.
The up-regulation of immune checkpoint molecules has been associated with an effective clinical response to ICB treatment; however, recent studies indicate that tumor-infiltrating lymphocytes is also crucial to positive responses[29, 32, 33]. In the study, we found that the immune-desert phenotype prevailed among YSTs, while only a minority exhibited the immune-inflamed phenotype. The immune-excluded phenotype featured massive infiltration of immune cells; however, the immune cells do not penetrate the parenchyma and instead are present tumor septa regions. The septa impose restrictions on the killing activity of T cells in response to TCs. Hence, with anti-PD-1/PD-L1 agent treatment, tumor-infiltrating T cells in tumors of the immune-excluded phenotype can demonstrate evidence of activation and proliferation but not infiltration, and thus, responses are likely to be uncommon. Due to the paucity of T cells in both the nest and septa regions of the tumor, the immune-desert phenotype represents a non-inflamed TME. Unsurprisingly, these tumors rarely respond to anti-PD-L1/PD-1 immunotherapy.
In Boldrini's research, among 28 YST cases, more than half of the cases featured the immune-desert phenotype, and 39% of the cases featured the immune-inflamed phenotype, consistent with our results. Compared with that study, we further assessed TLSs, that are involved in the TILs and contribute to ICB therapy response[13, 35]. To date, no assessment of TLSs in YSTs has been reported. In our study, five patients had TLSs, and the composition of TLSs was also assessed. Strikingly, multiple immune cells were present in the TLSs, including CD4+, CD8+ and Foxp3+ T cells, PD-1+ cells, PD-L1+ cells, and CTLA4+ cells. We further analyzed the relationship of TLSs with both TILs and the expression of PD-1/PD-L1 and CTLA4. The density of TILs and the expression of PD-1/PD-L1 were higher in patients with TLSs than in those without TLSs. The above results illustrated that TLSs have a key role in the immune microenvironment and may improve responses to ICB therapy in YSTs.
Due to both the density of TILs and the immune checkpoint molecules expression were strongly associated with the response to ICB therapy, their relations were also explored in our study. Consistent with another study, the expression of PD-1 was proportional to the density of TILs in the study. However, the differences in tumor-infiltrating CD8+ T cells failed to reach statistical significance (P = 0.1024 for CD8N and P = 0.0519 for CD8S), likely due to the small sample sizes. Similar to the results for PD-1, a higher density of TILs was also found in patients with PD-L1 positivity. In addition, we also found a higher density of TILs in patients with positive CTLA4L; nonetheless, no association was demonstrated between CTLA4T and the density of TILs.
Interestingly, we found that compared with tumors located in extragonadal sites or the ovaries, YSTs located in the testes usually have an exceptionally higher TLSs and expression of PD-1/PD-L1 and CTLA4, as well as more tumor-infiltrating T cells. The results suggested that YSTs located in the testes could profit from immunotherapeutic strategies using ICB, but a larger sample size is needed to confirm this finding.
We identified the immune phenotype of tumor-infiltrating T cells, a low frequency of PD-1/PD-L1 expression, a high frequency of CTLA4 expression, the presence of TLSs and relationships between these factors in YSTs. These features indicated that only a few YSTs patients may be benefit form ICB therapy; alternatively, converting the tumor from “cold” to “hot” could be a useful strategy prior to immunotherapy for most patients. A major limitation of our study might be the lack of neoadjuvant ICB trial data. Nevertheless, our comprehensive and detailed description of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes in YSTs forms a solid basis for ICB therapy.
In conclusion, ICB therapy could be a promising new treatment approach in YSTs, however, only a few YSTs patients may be benefit from these, and assessing TILs and the expression of immune checkpoint molecules for stratification is an indispensable part. Excitingly, patients with tumors located in testes exhibited a higher density of TILs and higher expression of immune checkpoint molecules, and might be efficacious for ICB therapy.