B2M validation by immunohistochemistry and correlation with subtype and EBV status. (A) Hematoxylin and eosin (H&E) staining and immunohistochemical staining for B2M and for MHC-I are shown for 2 representative cases of cHL. Case 1 has wild-type B2M sequences, whereas case 7 is mutated for B2M, indicating that this genomic alteration can be determined by lack of B2M expression in HRS cells. Correspondingly, case 1 shows clear Golgi and membrane localization of MHC-I, whereas staining is diffuse in the cytoplasm in case 7, indicating mislocalization. Original magnifications ×20 (H&E) and ×60 (B2M and MHC-I). (B) There was a significant correlation between the lack of B2M expression and the NS subtype of cHL, and between the presence of B2M expression and the MC subtype of cHL. Cases classified as “Others” include 1 case of lymphocyte-rich cHL and cases with features of both NS and MC, making the distinction challenging. (C) A cohort of patients with HIV infection and cHL was evaluated for B2M expression; however, the relationship of histologic subtype and B2M expression did not reach statistical significance in this cohort. (D) The presence of EBV in the HRS cells was assessed by in situ hybridization for Epstein-Barr encoding region. EBV-negative cases were more frequently also negative for B2M; however, among the EBV-positive cases, both B2M-positive and B2M-negative cases were identified. IC, immunocompetent; neg, negative; pos, positive.