Multiplex Immunofluorescence reveals specific subsets of immune cell populations expressing CD137/TNFRSF9 as predictors of unfavorable outcomes in Hodgkin Lymphoma

The tumor microenvironment (TME) is a crucial determinant of tumor growth, progression, and resistance to chemotherapy in cancer, with classical Hodgkin's lymphoma (cHL) being one of the most representative examples. The major component of the tumor mass in cHL is a rich and vast immune microenvironment, comprising a diverse repertoire of cell populations, many of which remain unknown. The specific contribution of the different functional cell subsets in the TME, such as T lymphocytes, monocytic populations, or dendritic cells, is also undiscovered. Currently, around 30% of advanced-disease patients or some atypical cases of primary refractory tumors do not respond to treatment or relapse shortly afterwards. Response predicting features of TME have been recently identified, which suggest a rationale for alternative therapeutic strategies. All existing evidence indicates that HRS cells actively coordinate and remodel their complex TME and that its diverse components influence the therapeutic response in cHL patients. The sensitivity of current technology enables analysis of specific functional phenotypes in the TME as diagnostic and predictive biomarkers. One example is the quantitative dissection of the TME with in situ multispectral imaging using formalin-fixed, paraffin-embedded (FFPE) tissues, with which we could analyze simultaneously CD68, CD137, CD30, CD3, PDL1, and PD1 markers to identify specific cell subsets. Remarkably, some cell populations expressing CD137 were associated with unfavorable responses in cHL patients, such as the activated monocytic cells (CD68+ CD137+), the tumor-reactive T cells (CD3+ CD137+), and the tumor CD30+ CD137+ PDL1+ cell subset. By contrast, the CD3+ CD137- T-cell population was associated with better prognosis, suggesting that CD137 could be a new prognostic marker in cHL for poor outcomes. Moreover, some cell subsets that could not be studied until now due to the lack of appropriate technology were found to be more abundant in the first stages of the disease, whereas others were less abundant than in stage IV. For instance, we could study exhausted T cells (CD3+ PD-L1+ PD-1+) and other related cells that were largely unexplored in cHL. Finally, these findings were validated with independent series of patient samples, including immunohistochemical studies with specific antibodies and gene-expression analysis.