LAUSR

Hodgkin lymphoma (HL) is an immunologically active lymphoid neoplasm composed of a few (usually 1-10%) neoplastic Hodgkin and Reed-Sternberg (HRS) cells or lymphocyte-predominant (LP) cells and >90% non-neoplastic cells, mainly Tand B-lymphocytes, plasma cells, macrophages, eosinophils and fibroblasts. The substantial amount of reactive cells in HL is supposed to be the net effect of a complex signaling network of cytokines and chemokines secreted by either the HRS cells or nonneoplastic cells. One component of this network is transforming growth factor beta (TGF-β), which is produced by HRS cells and cancer-associated fibroblasts. TGF-β unfolds its immunosuppressive impact by stimulating tumor-infiltrating T-lymphocytes (TIL) to differentiate into anergic, tumor-promoting, regulatory T cells (Treg). Additionally, TGF-β inhibits natural killer cells one of the key components of the innate anticancer immunity. Interestingly and still poorly understood, the HRS cells themselves seem to remain unaffected by the tumor-suppressive properties of TGF-β. Recent studies on diffuse large B-cell lymphoma (DLBCL) revealed a previously unknown tumor-suppressive signaling axis involving SMAD1 as a downstream messenger of TGF-β. SMAD1 functions as an intracellular signal transducer between extracellular TGF-β and the nucleus, where it modulates the transcription of target genes. This signaling cascade was shown to be recurrently inactivated in DLBCL, mainly by hypermethylation of five promoter regions surrounding the SMAD1 transcription start site, which finally generates a significant growth advantage for lymphoma cells. In the course of these investigations, we noted that SMAD1 was not expressed in HRS cells of screened HL cases. This led us to hypothesize that the absence of SMAD1 expression in HRS cells may mechanistically be linked to their resistance to the tumor-suppressive effects of TGF-β. In order to further elucidate this finding, we analyzed 132 well-characterized archival tissue-microarrayed cases, and 11 conventional routine lymphadenectomy specimens from patients suffering from all subtypes of classic HL (77 nodular sclerosis [NS]; 48 mixed cellularity [MC]; 7 lymphocyte-rich [LR]; 5 lymphocyte-depleted; and 6 unclassifiable classic HL) and 14 routine samples from patients suffering from nodular lymphocyte-predominant HL (NLPHL). We analyzed all these instances for immunohistochemical expression of SMAD1. Importantly, to guarantee retained antigenicity, only cases containing (physiologically) SMAD1-positive endothelia were considered. We found that all NLPHL (14/14 cases; 100%) and the great majority of classic HL (138/143 cases; 97%) displayed SMAD1-negative LP and HRS cells, respectively (Figure 1A and B). Single HRS cells stained faintly for SMAD1 in five cases only (2 NS; 2 MC; and 1 LR classic HL). With respect to non-neoplastic cells, 65/143 classic HL (45%) showed moderate (15-49% of TIL) up to abundant (≥50% of TIL) amounts of SMAD1 positive surrounding TIL, thus being potentially susceptible to the suppressive influence of TGF-β (Figure 1A and B); in NLPHL, 11/14 (79%) cases displayed abundant SMAD1-expressing TIL, including TIL involved in rosetting around LP cells (Online Supplementary Figure S1). The presence of abundant SMAD1-expressing TIL did not correlate with disease stage, patients’ age, gender, presence of B symptoms, association with Epstein-Barr virus (EBV) or outcome, while showing significant correlations with the NS subtype (45/77 NS cases, i.e. 58%, compared to 20/66 non-NS cases, i.e. 30%, P=0.025 χ test) and with the amount of FOXP3-positive Treg (Rho=0.351, P=0.000053 Spearman correlation), which both, in turn, may be directly linked to the effects of TGF-β, promoting sclerosis and a shift towards Treg differentiation. In contrast, surrounding plasma cells seemed to lack SMAD1 expression, potentially rendering them insensitive to the pro-apoptotic and anti-proliferative signals of TGF-β. With regard to plasma cells, this largely fits with the newly described negative prognostic impact of their increased numbers in classic HL. To strengthen our hypothesis, we investigated the promoter methylation status of the SMAD1 gene in six different HL cell lines, including one NLPHL cell line (DEV) exactly as described elsewhere. Methylation analysis by bisulfite sequencing was successful for three regions of