Hodgkin- and Reed-Sternberg cells (HRSC) proliferate in a presumably highly immunogenic tumor microenvironment (TME) containing abundant CD4+ and CD8+ T-cells. Even though anti-PD-1 directed treatment is clinically well established, the mechanism of action in classical Hodgkin lymphoma (cHL) is still incompletely understood. To elucidate features of a T-cell mediated immune response in cHL, we studied HRSC immunogenicity defined by human leukocyte antigen (HLA) expression, associated T-cell expansion, and TME composition. We analyzed T-cell Receptor (TCR) repertoires, bulk gene expression levels and whole-slide images of treatment-naïve cHL patients before (n=90) and on anti-PD-1 therapy (n=4) in the NIVAHL phase II trial (Bröckelmann et al. JAMA Oncol 2020). In independent cohorts of breast cancer (n=6), benign lymph nodes (n=8) and cHL treated with chemotherapy (n=18), a biopsy both at primary diagnosis and relapse was available. To describe the proliferative activity of T-cells in the TME, we used three measures: debiased Simpson’s clonality (Unbiased Clonality; UC), Percentage of Singletons (PoS), and clonal expansion. We identified significantly lower UC and higher PoS in pre-treatment cHL compared to breast cancer specimens. Moreover, cHL showed less expansion of TCR clones in follow-up biopsies than breast cancer. A pair-wise comparison of pre- and on-anti-PD-1 treatment cHL specimens did not show a significant difference in UC or PoS. However, an analysis of primary versus relapse (after chemotherapy) biopsies of cHL showed a trend to increased UC in relapse specimens. Next, we analyzed bulk gene expression and TCR repertoires with respect to HLA I and II status on HRSC. HLA status was not associated with UC or PoS. However, we observed a significant enrichment of CD8+ T-cells and up-regulation of cytotoxicity genes in the bulk TME of HLA I+ HRSC. This trend seems to persist for both CD8+ T-cells close and more distant to HRSC (see Müller-Meinhard et al., ISHL12 submission). In summary, we did not observe features of a T-cell activation (UC, PoS, clonal expansion) in cHL pre- and on-anti-PD-1 treatment biopsies. Our findings suggest a cHL TME that contains a polyclonal mass of T-cells not activated early during anti-PD-1 treatment. However, our analyses cannot exclude an increased clonality in cHL at later timepoints or in relapse biopsies after conventional chemotherapy.
Nicole Seifert, Sarah Reinke, Johanna Grund, Berit Müller-Meinhard, Andreas Engert, Paul J. Bröckelmann, Wolfram Klapper, Michael Altenbuchinger