The NF-κB pathway is activated and frequently targeted by genetic lesions in SMZLs. (A) Immunohistochemical staining of SMZL biopsy samples with anti-NFKB1 (p50) antibody. Nuclear localization of NF-κB denotes active signaling as opposed to inactive cytoplasmic localization. Original magnification ×400. (B) Prevalence of constitutive NF-κB activation scored by immunohistochemistry in SMZLs overall and according to mutation status of NF-κB genes. (C) Western blot analysis showing NFKB2 processing and MAP3K14 expression in purified primary tumor cells from 8 SMZL cases carrying wild-type (Wt) or aberrant NF-κB genes. Case 11230 was run in a different gel using the same conditions, as indicated by the black dividing line. The RPMI-8226 and LP-1 (both multiple myeloma) and JJN3 (plasma cell leukemia) cell lines were used as positive controls for NFKB2 processing. The KMS-12PE cell line (multiple myeloma) was used as negative control for NFKB2 processing. The RPMI-8226 and JJN3 cell lines were used as positive controls for MAP3K14 expression. The LP-1 and KMS-12PE cell lines were used as negative controls for MAP3K14 expression. Actin was used as loading control. (D) Venn diagram illustrating the absence of overlap between NF-κB gene somatic mutations. (E) Genetic lesions (including mutations and CNAs) of NF-κB genes are largely mutually exclusive. In the heat map, rows correspond to identical genes, and columns represent individual patients color-coded on the basis of gene status (green, wild-type; red, mutations of IKBKB, mutations or gain of MAP3K14, mutations and/or deletion of TRAF3, TNFAIP3, and BIRC3).