Immunohistochemical analysis of BMI-1 and EZH2 expression in HRS cells and germinal center follicular lymphocytes in the lymph node. A and B: Staining of Reed-Sternberg (HRS) cells for BMI-1 (A) and EZH2 (B). HRS cells express both BMI-1 and EZH2, whereas infiltrating lymphocytes stain for BMI-1 but not for EZH2. Note that dividing cells express BMI-1 in the cytoplasm, whereas EZH2 remains associated with condensed chromosomes (indicated by * in A and B). C, E, and G: Expression of BMI-1 in germinal centers of the lymph node. D, F, and H: Expression of EZH2 in germinal centers of the lymph node. C and D: Overview of lymph node germinal center. E and F: Detail of germinal center showing dark zone (DZ) and mantle zone (MZ). G and H: Detail of germinal center showing light zone (LZ) and MZ. Cb, centroblast; cc, centrocyte; LN, lymph node; MΦ, macrophage. Note that the mutually exclusive expression of BMI-1 and EZH2 is particularly notable in the MZ, DZ, and MΦ staining profiles (C−H), whereas HRS cells stain for both PcG proteins (A and B). Original magnifications, ×400 (A and B), ×200 (C and D), and ×630 (E−H).

A−I: Expression of BMI-1 and EZH2 in HRS cells (A−F) and LN (G−I). BMI-1 (A and D; red fluorescence) and EZH2 (B and E; green fluorescence) are detected in HRS cells, whereas infiltrating lymphocytes are BMI-1⁺ (A and B) and generally do not express EZH2 (B and E). Coexpression of BMI-1 and EZH2 produces a yellow nuclear signal (C and F). These nuclei belong to HRS cells since these express CD30 (blue signal in D−F). Occassional EZH2-expressing cells with a compact nucleus also express CD30 (E), but are BMI-1⁻; these are probably activated lymphocytes. Note that EZH2⁺ lymphocytes in the infiltrate may be remaining centroblasts from lymph node follicles. CD30⁻/BMI-1⁺ cells with a large nucleus are probably macrophages (MΦ in F). In follicular lymphocytes (G−I), BMI-1 is mainly detected in the LZ (G), whereas EZH2 is primarily detected in the DZ (H). Coexpression of BMI-1 and EZH2 is sometimes observed at the interface of LZ and DZ, but occurs at a very low frequency (I). Expression of EZH2 and Mib-1/Ki-67 in HRS cells (J−L) and LN (M−O): Expression of EZH2 occurs in both HRS cells (J; green fluorescence) and LN centroblasts situated in the DZ (M), and coincides with detection of Mib-1/Ki-67 (K and N). Combination of the red and green fluorescent signal produces yellow nuclei (L and O), supportive of Mib-1/Ki-67 and EZH2 coexpression in HRS cells and centroblasts. Expression of BMI-1 and Mib-1/Ki-67 in HRS cells and LN: BMI-1 expression occurs in HRS cells and the surrounding infiltrate (P). HRS cells also express Mib-1/Ki-67 (Q, green fluorescence), producing a yellow signal in the HRS cell when red and green fluorescence are combined (R). By contrast, BMI-1 (S, red fluorescence) and Mib-1/Ki-67 expression (T, green fluorescence) are separated in follicular lymphocytes (U) and infiltrating lymphocytes surrounding an HRS cell (R). For abbreviations, see Figure 1 ▶ legend. Original magnifications, ×400.