Programmed cell death protein-1 (PD-1)-expression in the microenvironment of classical Hodgkin lymphoma at relapse during anti-PD-1-treatment

Classical Hodgkin lymphoma (cHL) is characterized by rare Hodgkin and Reed Sternberg cells (HRSC), which are embedded in an anergic inflammatory cellular microenvironment (ME). Constitutional and high expression of programmed cell death-protein-1 (PD-1)/PD-ligand (PDL) pathway by HRSC indicates a role of PD-1/PD-L1 interaction in cHL. Inhibition of PD-1/PD-L1 interaction by anti-PD-1 blockade has shown impressive response rates in relapsed/refractory (r/r) cHL. Although durable responses to anti-PD-1 treatment have been documented, the majority of patients eventually relapse. 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 antiPD-1 treatment with regard to microenvironmental changes and PD-1 as well as PD-L1 expression. In 9 cHL patients, formalin fixed paraffin embedded biopsy specimens taken prior to anti-PD-1-treatment, and a second biopsy at relapse/progression, were available. These included 3 patients with three available biopsies (2 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 (Online Supplementary Table S1). We determined HRSC phenotypes by conventional single stainings assessed by experienced hematopathologists. Stainings for CD4 (clone 4B12, Leica), CD8 (clone C8/144B), DAKO PD-1 (clone MRQ22 Cell Marque), and PD-L1 (clone E1L3N Cell Signaling) were performed on a Leica-Bond automated stainer to characterize the ME and to quantify PD-1and PD-L1-expression. The total number of PD-1, PD-L1, CD8 and CD4 positive microenvironmental cells was counted by 2 expert pathologists in three high-power fields (HPF, 1000-fold magnification using oil immersion) and the average value was used for further analysis (Online Supplementary Table S1). HPF were chosen with at least one HRSC in the center in order to analyze cell counts in the immediate vicinity of the neoplastic cells. RS cells and macrophages were differentiated by means of CD30and PAX5-staining and by standardized characterization of the distinct morphology of the RS-cell and macrophage nuclei. t-tests were applied for comparison of cell counts in the HRSC vicinity in the most current biopsy before and the biopsy at relapse under anti-PD-1 treatment. P≤0.05 was considered significant. Histopathological diagnosis of cHL was confirmed in all biopsies (Table 1A). Prior to immunmodulatory checkpoint treatment (ICT), cHL subtypes were nodular sclerosis in 2, mixed cellularity in 4, lymphocyte depleted in 1, and not classifiable in 2 patients. Patients’ characteristics, including prior lines of treatment, stage at start and duration of anti-PD-1-treatment until documentation of progressive disease (PD) are summarized in detail in Tables 1A and B. The time interval between the most recent application of the anti-PD-1-antibody and the biopsy at progressive disease or relapse ranged from six days to six months, with seven of eight sequential biopsies being taken within six weeks after documentation of PD.