The follow-up of living patients (with or without events) was censored at their last follow-up date. the final cohort showed characteristics much like those of the original 95 patients. All main diagnostic tumor biopsies were examined and reclassified according to the World Health Business classification of tumors of the hematopoietic and lymphoid tissues . The current study was performed in rigid accordance with local ethical guidelines and recommendations of the Declaration of Helsinki (Seoul revision, 2008). In this retrospective study involving archived materials, no individual patient identification, or BVT 2733 study-driven clinical interventions were performed. Clinical and follow-up data BVT 2733 were obtained from clinical records including histopathological subtype, age, gender, presence or absence of B symptoms, clinical stage according to the Cotswolds modification of the Ann Arbor staging system , lactate dehydrogenase level, EBV status, and main treatment. Most patients were treated with four to eight cycles of ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) chemotherapy. Additional radiotherapy was administered in cases of pre-therapeutic heavy or localized residual masses. Relapsed or refractory patients received either salvage chemotherapy or high-dose chemotherapy with autologous stem-cell transplantation. Immunohistochemistry and in situ hybridization Tissue blocks of untreated patients were obtained from the Department of Pathology at two hospitals. Paraffin-embedded lymph node specimens of 86 patients were available. Immunohistochemical staining for CSF-1R, PD-1 and FOXP3 was performed as explained below. Briefly, 5-m-thick sections were transferred onto poly-L-lysine-coated adhesive slides and dried at 65C for 90 min. After standard heat-induced epitope retrieval for 2 to 3 3 min in citrate buffer (pH 7.4), the samples were incubated with antibodies against PD-1 (dilution 1:50; R&D Systems, Germany), FOXP3 (dilution 1:100; Abcam, UK), and CSF-1R (dilution 1:100; Abcam, UK) overnight at 4C. The sections were incubated with biotinylated anti-goat and anti-mouse/rabbit immunoglobulins, and DAB was used as a substrate. The positive index was estimated by counting the number of positive cells in five randomly selected high power fields (HPF) at 4010 magnification. For FOXP3+ quantification, the results were considered high when more than 25% of the cells were positive among the total cells. Among the percentage of non-HRS BVT 2733 cells expressing CSF-1R, we selected more than 30% of CSF-1R-positive cells as the cut-off value for BVT 2733 defining high-and low-CSF-1R groups. Cases stained with anti-PD-1 were scored according to the intensity of cytoplasmic and/or membranous positivity. It was considered positive when more than 20% of all cell populace was stained. hybridization analysis for Epstein-Barr computer virus encoded RNA (EBER) was performed. EBER was considered as positive in case of dark-blue nuclear staining. Statistical analysis Overall survival (OS) was defined as the interval between the date of diagnosis and death from any cause. The follow-up of living patients (with or without events) was censored at their last follow-up date. Progression-free survival (PFS) was defined as the interval between the date of treatment and the date of disease progression, relapse, or death from any cause. Cumulative OS and PFS were analyzed by Kaplan-Meier method, and comparisons were made using the log-rank test. Multivariate prognostic analyses were performed for OS and PFS using the Cox proportional hazards regression model. All values were two-sided, and a value 0.05 was considered as significant. Statistical analyses were performed ENOX1 using the SPSS 17.0. Results Patient characteristics The main clinical and histopathological characteristics are summarized in Table 1. The median age of HL patients was 31.5 years (ranging from 7 to 82 years) and 53 patients.