5-7,18,19) Nevertheless, only limited information is available on the expression of the eIF-4E regulatory protein 4E-BP1 in non-Hodgkin lymphoma.
In the present study, we evaluate the expression and regional localization of 4E-BP1 and phosphorylated 4EBP1 in reactive lymphoid tissues and various subtypes of B-cell lymphoma.
Using standard procedures, formalin-fixed, paraffin-embedded sections were immunostained with rabbit monoclonal anti-4E-BP1 (clone 53H11, Cell Signaling Technology, Danvers, Massachusetts) at 1:3000 dilution and with citrate-based antigen retrieval and antiphosphorylated 4E-BP1 ([Thr37/46], clone 236B4, Cell Signaling Technology) at 1:100 dilution and with EDTA-based antigen retrieval on a Bondmax automated immunostainer (Leica Biosystems, Bannockburn, Illinois).
Western immunoblot analysis was performed on lysates of 5 mature B-cell lymphomas (2 FLs, 3 DLBCLs) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E, and 4E-BP1 (total and phosphorylated) expression, as previously described by us.
In reactive lymphoid tissues, there was regional and cellular specificity of 4E-BP1 expression, with either lack of or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T cells, 1+ to 2+ expression in follicular and interdigitating dendritic cells, and 3+ expression in mantle and marginal zones (Table 1; Figure 2, A through C).
In marked contrast to reactive lymphoid tissues, a consistently high level (2+ to 3+) of cytoplasmic 4E-BP1 expression was seen within the neoplastic lymphocytes in 49 of 50 (98%) B-cell lymphomas (Table 1; Figure 2, D through M).
Western immunoblot confirmed that nonphosphorylated and phosphorylated 4E-BP1 were expressed in reactive nodes, FL, and DLBCL (Figure 2, N).
Interestingly, in reactive lymphoid tissues, the levels of phosphorylated 4E-BP1 expression were inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells, no expression in the mantle and marginal zone cells or the paracortical T-cells, and 1+ or 2+ immunoreactivity in the follicular and interdigitating dendritic cells (Table 1; Figure 3, A through C).
The level of expression of phosphorylated 4E-BP1 in Bcell lymphomas was extremely variable, being moderate or strong in 23 of 50 (46%) B-cell lymphomas, negative in 18 (36%), and dim in 9 (18%) cases (Table 1; Figure 3, D through H).
An interesting finding was that 4E-BP1 was expressed in the neoplastic cells in all 12 cases of lymphoma (2 FLs, 6 DLBCLs, and 4 Burkitt lymphomas) that lacked BCL2 expression (Table 2; Figure 5).
We demonstrate that 4E-BP1 expression demonstrates regional specificity in reactive tissues that may be useful in distinguishing germinal centers from neoplastic lymphocytic infiltrates.
We also found that all 4 cases with in situ or partial involvement by FL were readily distinguishable from reactive germinal centers by the presence of strong and diffuse staining with 4E-BP1 in the neoplastic follicles in 3 cases and 1+ staining in the fourth case versus negative or dim staining in the germinal centers.