Abstract. Background: Tumor-infiltrating lymphocytes (TILs) have been shown to be of prognostic significance in a variety of tumors. Not only T-cell, but also B-cell In recent years, studies concerning the microenvironment of tumors, especially tumor-infiltrating lymphocytes (TILs), have been abundant. This microenvironment has been shown to influence overall survival (OS), as well as relapse times, and probability of tumor recurrence (1, 2). However, most studies have focused on the relationship between T-cell infiltration and patients' outcome, while the role of tumor infiltrating B-lymphocytes (TIL-Bs), as well as plasma cells in this setting, is still poorly understood and less commonly investigated.Not only the presence, but also the distribution pattern has been described as being crucial to prognosis: In carcinomas, immune cells are predominantly found in the stroma (3) and the formation of dense, follicle-like lymphocyte infiltrates (so-called tertiary lymphatic structures (TLSs), which arise in non-lymphoid tissues, often in the context of chronic inflammation) has been reported to have the strongest impact on prognosis and survival (2, 4, 5).T-lymphocytes (especially cytotoxic T-cells), as well as Blymphocytes, increase and establish the activation of antitumoral immunity (as an adaptive mechanism), leading to increased cell death in tumors and resulting in improved prognosis in patients with high levels of TILs (2, 6). Tumorassociated plasma cells have been described to have a significant impact on prognosis through modulation of the humoral immune response (7).Up to date, most reports in the literature state a positive effect of TILs in respect to OS, relapse-free survival and association with favorable prognostic factors, as well as lower T-stage (8). High B-cell density is said to be associated with improved outcome in gastric, breast and colorectal cancer (9-11), as well as squamous cell carcinoma of the tongue (12), melanoma and hepatocellular carcinoma (5, 13). The same correlation has been shown for pancreatic adenocarcinomas, though here the impact was only measurable if cells were arranged in the pattern of TLSs (4,14). High proportions of TILs have also been shown to be predictive of response to neoadjuvant therapy in breast cancer patients (15). 5339

Inhibition of the immunoregulatory programmed cell death-protein-1 (PD-1)/PD-ligand (PD-L)-pathway by PD-1 blocking antibodies has resulted in high response rates in patients with relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). Nevertheless, the majority of r/r cHL patients relapse on anti-PD-1 treatment. To address the unsolved question of mechanisms of resistance to anti-PD-1 blockade in cHL we retrospectively examined sequential biopsies of nine r/r cHL-patients with progressive disease or relapse during anti-PD-1 treatment. We found a striking increase of PD-1-positive T-cells in four and a slight increase in the other cases with progressive disease during anti-PD-1 treatment, whereas a strong PD-L1 expression on Hodgkin-and Reed Sternberg cells (HRSC) prior to anti-PD-1 blockade and at relapse/progression was detected. The observation from our case series indicates that upregulation of PD-1 on T-cells in the microenvironment (ME) contributes the development of resistance. This hypothesis needs to be further explored by analysis of larger patient cohorts.Classical HL is characterized by rare HRSC, which are embedded in an anergic inflammatory cellular ME.1 Constitutional and high expression of PD-L1 by HRSC indicates a role of PD-1/PD-L1 interaction in cHL. 2-5Inhibition of PD-1/PD-L1 interaction by anti-PD1 blockade has shown impressive response rates in r/r cHL. Although durable responses to anti-PD-1 treatment have been documented, the majority of patients eventually relapse. [4][5][6] To address the important, but so far unsolved issue of mechanisms of resistance to PD-1 blockade, we retrospectively assessed sequential biopsies of r/r cHL patients developing progressive disease (PD) or relapse on anti-PD-1 treatment with regard to microenvironmental changes and PD-1 as well as PD-L1 expression.In nine cHL patients' formalin fixed paraffin embedded biopsy specimen prior to and a second biopsy at relapse/progression under anti-PD-1-treatment were available including three patients with three available biopsies (two patients with two biopsies before and one patient with two biopsies under anti-PD-1-treatment). All patients gave their informed consent for this study which had been approved by our local ethics committee.Biopsy specimens were stained for CD30, CD15, EBER or LMP1, CD20, and CD3 to characterize HRSC and to confirm the diagnosis using established protocols ( were performed on a Leica-Bond automated stainer to characterize the ME and to quantify PD-1-and PD-L1-expression. The total number of PD-1, PD-L1, CD8 and CD4 positive microenvironmental cells was counted by two expert pathologists in three high-power fields (HPF, 1000-fold magnification using oil immersion) and the average value was used for further analysis (table 1 supplements The time interval between the most recent application of the anti-PD-1-antibody and the biopsy at progressive disease or relapse ranged from 6 days to 6 months with seven of eight sequential biopsies being taken within 6 weeks after documentation of PD.B...

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