βcatenin is a marker of poor clinical characteristics and suppressed immune infiltration in testicular germ cell tumors

Testicular-germ cell tumors (GCTs) are chemotherapy sensitive malignancies [1, 2]. Little was known regarding immune mechanisms in this disease until recently. The significance of PD-1/PD-L1 pathway as well as the role of systemic-immune inflammation was previously described in our works [3, 4]. Additionally, changes in cytokine signalling was observed by our team in association with poor prognosis of GCTs [5, 6]. The role of immune check-point inhibition with anti-PD-1/anti-PD-L1/anti-CTLA4 agents has been established in numerous malignancies [7‐10]. Data suggest that a T-cell inflamed microenvironment seems to be a predisposing factor for the efficacy of check-point inhibition covering only a subset of patients who benefit greatly from modern immune therapy [11, 12]. An activation of WNT/βcatenin (WNTβ) signalling pathway has been recently suggested as an intrinsic inhibitory mechanism for tumor T-cell infiltration in malignant melanoma pre-clinical model [13]. WNTβ pathway is known to be activated in early stages of embryonic development and it is involved in the regulation of differentiation of pluripotent cells [14, 15]. It has also been associated with carcinogenesis [16], cell proliferation and migration and the process of epithelial-mesenchymal transition [17]. Mutations in WNTβ have been identified in malignancies, such as hepatocellular, breast, colorectal and other cancer [18], however, the constitutive receptor stimulation can account for hyperactive WNTβ signalling in the absence of activating mutations as well [19]. Potapov et al. described the abundance of βcatenin in embryonal carcinoma identified by immunohistochemistry in 39 cases of testicular embryonal carcinomas [20]. Two studies reported differential expression in seminomas versus non-seminomas, but no correlation to resistance to chemotherapy was found [21, 22]. However, the role of βcatenin and WNTB signalling and its clinical implications in GCTs have not been comprehensively explored. In this retrospective study, we aimed to evaluate the role of βcatenin in GCTs and find correlations with systemic immune-inflammation and PD-L1 expression in tumor and tumor infiltrating lymphocytes (TILs).

Patients’ characteristics are shown in Table 1. Majority of patients had a non-seminoma histology. A testicular tumor was the most common primary and more than two thirds of patients were within good risk according to the IGCCCG (International Germ Cell Cancer Collaborative Group). Tumor specimens from 247 patients before administration of systemic therapy included 50 pure seminomas, 128 non-seminomas (86 embryonal carcinomas, 19 yolk sac tumors, 1 choriocarcinoma, 22 teratomas) and 69 mixed GCTs. (Additional file 1: Table S1). Eight cases of seminomas were clinically considered as non-seminomas based on the positivity of alpha-fetoprotein.

In this translational study, we assessed the significance of the βcatenin expression in patients with GCT prior to administration of chemotherapy. We performed a survival analysis and correlated its’ expression to clinical and pathological characteristics. We identified a significant difference in the expression of βcatenin between seminomas and non-seminomas. A study by Fritsch et al. reported activated WNTβ and differential βcatenin expression in female paediatric GCT patients [29]. All non-seminoma subtypes in our study have shown strong staining for βcatenin with the highest expressions seen in embryonal carcinoma and yolk sac tumor. This finding can be attributed to the pluripotency of embryonal cancer cells that are known to be malignant counterparts of embryonic stem cells and act as stem cells in the development of GCTs. However, the implication for yolk sac tumor is unclear [30, 31]. Raggioli et al. demonstrated that βcatenin was vital for the maintenance of pluripotency associated genes in mouse embryonic stem cell lines. In addition, removal of βcatenin led to a strong increase of cell death [32]. Our study suggests the loss of βcatenin during the development from the progenitor cell, as only a weak or no expression was seen in seminomas. We hypothesize it can be responsible for different biology and treatment sensitivity compared to non-seminoma GCTs. The loss of other markers of pluripotency, such as SOX2 and CD30 was well described in the past [33]. Our further analysis uncovered differences in βcatenin expression in association with poor-risk clinical features. Primary testicular and retroperitoneal tumors had lower expression of βcatenin in comparison with PMNSGCTs which represent the worst prognostic group of GCT patients [34]. However, this sub-analysis is underpowered due to small number of PMNSGCTs in the cohort. The underlying connection to βcatenin and WTNβ signalling suggest that patients with tumors that have higher abilities for pluripotency belong to poorer risk category and produce more tumor markers. Although βcatenin assessed in our cohort of chemotherapy naïve patients did not show a prognostic significance in terms of PFS and OS, further studies assessing the βcatenin in relapsed or platinum refractory patients are needed to explore its’ clinical significance.

Our subsequent analysis evaluated βcatenin and its possible associations with the tumor immune microenvironment. Our previous works have shown prognostic significance of PD-L1 expressed on tumor cells and TILs and systemic inflammation in GCTs [3, 4, 23]. Herein we identified the positive correlation of βcatenin to PD-L1 expression on tumor cells, but not on TILs. High PD-L1 expression on TILs was linked to a better outcome, whereas high PD-L1 expression on tumor cells was predictive of poor outcome in GCTs. The role of βcatenin and its’ interaction with PD-1/PD-L1 pathway is not clear. Its’ association with PD-L1 on tumor cells but not on TILs may suggest the independence of PD-L1 positive TILs from WNTβ signalling. Moreover, the association with immune inflamed microenvironment was confirmed in inverse correlation with SII suggesting that patients with low inflammatory activity had activated WNTβ signalling. Spranger et al. observed a lack of T-cell infiltrate in autochtonous mouse models, which led to resistance to anti-PD-L1/anti-CTLA4 monoclonal antibody therapy. Subsequently, authors were able to restore T-cell infiltration in mice by introduction of injected dendritic cells harvested from transgenic mice [13]. We did not observe a higher expression of βcatenin in patients without TILs in tumor specimens, perhaps due to insufficient size of our cohort. Our subsequent ingenuity pathway analysis uncovered important roles of CTNNB1 and CD274 genes in the process of differentiation, proliferation and function of CD8+ and CD4+ T cells. The increase in number of CD8+ positive cells resulting from aberrant mutated CTNNB1 is seemingly conflicting with other described effects of WNT signalling, however, Cohen et al. described that these cells failed to be activated due to a lack of cross-presentation resulting from increased splenic CD8α dendritic cell activity [35]. Furthermore, animal model experiments performed by Augustin et al. in mouse embryonic stem cells with overexpressed WNT secretion factor Evi/WIs, showed an increased tumor growth and impaired immune cell recruitment in the presence of enhanced Evi expression. Authors observed reduced T-cell and B-cell infiltration especially in Evi overexpressing teratomas, thus, showing the negative effect of WNT signalling on immune surveillance [36].

Our study has some strengths and limitations. The large patient population creates a relatively balanced population for the biomarker assessment. Additionally, the availability of the PD-L1 expression and the SII data from the same set of patients creates a unique cohort with the possibility to uncover associations among these. Limitations include the retrospective nature of the analysis and underrepresentation of extragonadal germ cell tumors. The membranous expression of βcatenin does not provide clear evidence of WNT pathway signalling, thus the expression of a target gene would provide more information. Our selection of βcatenin to explore as a biomarker in this hypothesis generating study may be biased, therefore we plan a more comprehensive proteomic analysis in the future.

In conclusion, this is the first translational study to assess the biological, prognostic and clinical significance of βcatenin in GCTs of adult men. We uncovered the differences between seminomas and non-seminomas. We have shown associations with several clinical characteristics and systemic immune-inflammation and PD-L1 on tumor cells, thus providing the insights into immunobiology of GCTs. More studies are needed to validate this exploratory trial and to explore the possible therapeutic implications.