Pathological and Clinical Implications of Tumor Microenvironment Evaluated With Multiplex Immunohistochemistry in Testicular Embryonal Carcinoma

Introduction: Testicular germ cell tumors (TGCTs) are the most common cancer in young men. Their development is closely linked to embryogenesis, yet much about their pathogenesis remains unknown. TGCTs exhibit exceptional sensitivity to cisplatin-based chemotherapy, achieving high cure rates even in metastatic disease. Further research is needed to deepen our understanding of TGCTs and refine risk stratification. In this pilot study, we focused on embryonal carcinoma (EC), a subtype with increased risk of relapse and considered a key step in TGCT reprogramming. Patients and methods: We analyzed the tumor microenvironment (TME) in 49 EC samples using bright-field multiplex immunohistochemistry (BF-mIHC), which enables the simultaneous evaluation of multiple biomarkers, making it particularly useful for TME characterization. We evaluated B-cells (CD20), T-cells (CD3), and tumor-associated macrophages (TAMs, CD68) and established cutoffs that correlated with reprogramming phase, clinical stage, and relapse. Results: High TAM levels (CD68 + > 83/mm²) were strongly associated with phase I of reprogramming (pure EC or EC mixed only with seminoma), while low TAM characterized phase II (P<.001), suggesting a role in stemness maintenance through epigenetic regulation. High CD68 (> 46/mm²) and CD3 (> 125.5/mm²) correlated with metastatic disease (P<.001 and P=.026, respectively), whereas high CD20 (> 38.5/mm²) and CD3 (> 83/mm²) were linked to lower relapse risk (P=.014 and P=.017, respectively). Important limits are small sample size and few relapse events. Conclusions: These findings highlight the relevance of TME in TGCT biology and prognosis. The observed associations suggest TME may influence tumor plasticity, metastatization and relapse risk, warranting validation in larger studies.