ALBERT

Description: Abstract 1925 Poster Board I-948 Eukaryotic initiation factor 4E binding protein 1 (4E-BP1) regulates the function of eukaryotic initiation factor 4E (eIF-4E) which mediates nucleo-cytoplasmic transport and cap-dependent translation of specific transcripts. eIF-4E expression itself is elevated in several carcinomas and hematopoietic malignancies. Hypophosphorylated 4E-BP1 binds eIF-4E and inhibits cap-dependent translation. Phosphorylated 4E-BP1 dissociates from eIF-4E, thereby allowing translation of several cap-dependent transcripts responsible for cell cycling, survival and angiogenesis. Hyper-phosphorylation of 4E-BP1 may thus be associated with oncogenesis and the malignant phenotype; its influence on the response to chemotherapy and prognosis of diffuse large B cell lymphoma (DLBCL) is unknown. We examined diagnostic tissues from 85 patients with DLBCL for 4E-BP1 expression and phosphorylation, and correlated these with clinical outcomes. Samples were stained for total and phosphorylated 4E-BPI using immunohistochemistry (IHC). Slides were scored for percent cells positive for the two markers (0% to 100%) and the intensity of expression (0 to 3+) by hematopathologists (A.R. and H.M.) blinded to the clinical data. An Expression Score was calculated from their product ([% cells positive × intensity]/10), thus ranging from 0-30. In parallel, expression of bcl-2 was determined by IHC on the same samples. Patients had a median age of 75 years (range, 25-91). The majority received CHOP-based chemotherapy with/without rituximab. Total 4E-BP1 was expressed uniformly (by 97% of the cases). However, there was marked heterogeneity in the level of phosphorylation of 4E-BP1 (none [Expression Score = 0] in 35%, uniformly high [Expression Score = 30] in 34%, and variable [Expression Score = 1-29] in the remainder [31%]). We therefore examined if the level of phosphorylation of 4E-BP1 varied with clinical features at diagnosis, and whether it correlated with response to chemotherapy and survival. The level of expression or phosphorylation of 4E-BP1 did not correlate with the clinical stage, IPI score or bcl-2 expression at diagnosis, and did not influence the likelihood of achieving complete remission with CHOP-based chemotherapy. However, in patients with low IPI scores (IPI 0-2), the level of phosphorylation of 4E-BP1 correlated significantly with overall survival (OS). In these patients, the OS was significantly different (P = 0.031) in patients with low levels of phosphorylation of 4E-BP1 (Expression Score = 0-15) compared to those with higher levels of 4E-BP1 phosphorylation (Expression Score = 16-30). The median survival was 〉146.4 months vs 20.6 months, and the 5-year survival was 73% vs 31%, in patients with low and high levels of 4E-BP1 phosphorylation, respectively. The difference in survival between the two groups was not a consequence of differences in age or IPI score at diagnosis or inclusion of rituximab in the treatment regimen. The level of phosphorylation of 4E-BP1 did not have any further impact on the survival of patients with high IPI scores (IPI 4-5). We conclude that while the large majority of DLBCL express the translation regulatory protein 4E-BP1, the level of phosphorylation (indicative of biological activity) of this protein varies widely. Importantly, the level of phosphorylation of 4E-BP1 further stratifies DLBCL patients who would be expected to have a favorable prognosis based on low IPI scores, and identifies a subset of these patients who do poorly following standard CHOP-based chemotherapy. Since hyper-phosphorylation of 4E-BP1 increases the activity of eIF-4E, this study provides the rationale for trials incorporating novel targeted agents that inhibit eIF-4E into the therapeutic regimen, specifically in DLBCL patients with low IPI scores and high levels of 4E-BP1 phosphorylation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3919 Poster Board III-855 Identifying pathogenic mechanisms that contribute to the development of lymphomas and influence clinical behavior is critical for developing targeted therapies, and selecting patients who may benefit from such drugs. An important level of control of gene expression occurs during initiation of cap-mediated mRNA translation by the eukaryotic initiation factor-4F (eIF-4F) trimolecular complex (eIF-4E, eIF-4G and eIF-4A), in which eIF-4E is rate limiting and oncogenic. eIF-4F hyperactivity plays a key role in human cancers by mediating expression of proteins critical for cell growth, transformation and tumorigenesis. eIF-4F activity is controlled by repressor eIF-4 binding proteins (BPs). 4E-BP1 activity is regulated by phosphorylation. Hypo/non-phosphorylated 4E-BP1 is active, binds eIF-4E and impedes eIF-4F formation, blocking translation and inducing apoptosis. Phosphorylation of 4E-BP1 (p4E-BP1) releases bound eIF-4E, which initiates cap-dependent translation. Because only limited information is available on the expression and phosphorylation of 4E-BP1 in lymphomas, and since agents (e.g., antisense oligonucleotides and small molecules) that target eIF-4E have been developed, we examined the frequency and level of expression of 4E-BP1 and its phosphorylation in various subtypes of mature B cell non-Hodgkin's lymphomas (BCL). Forty-six BCLs (12 follicular [FL], 13 diffuse large B-cell [DLBCL], 7 mantle cell, 5 extranodal marginal zone, and 9 small lymphocytic [SLL] lymphomas), 4 FL with incipient/partial lymph node involvement, and 11 reactive lymphoid tissues were examined using immunohistochemistry for total and phosphorylated 4E-BP1. Staining intensity was graded as from 0 to 3+. Western immunoblotting (WB) was performed on lysates of 5 mature BCLs (2 FL, 3 DLBCL) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E and 4E-BP1 (total and phosphorylated) expression. In reactive lymphoid tissues, there was regional and cellular specificity of expression of 4E-BP1, with either lack of, or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T-cells, 2+ expression in follicular dendritic cells and paracortical zone Langerhan cells, and 3+ expression in mantle and marginal zones. p4E-BP1 expression was inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells and no expression in the mantle and marginal zone cells or T-cells, and 2+ or 3+ immunoreactivity in follicular dendritic cells and paracortical zone Langerhan cells. In BCLs, a consistently high level (2+ or 3+) of cytoplasmic 4E-BP1 expression was seen in neoplastic lymphocytes in 45/46 (98%) cases. In contrast, p4E-BP1 was moderately or strongly expressed in 19/46 (41%) cases of BCL, being negative in 17 (37%) cases, and only dimly expressed in the remaining 10 (22%) cases. Three of 4 cases with incipient/partial involvement by FL were easily distinguishable from reactive germinal centers by strong, diffuse staining with 4E-BP1 (and 1+ staining in the 4th case) in neoplastic follicles, distinct from negative/weak staining of adjacent reactive germinal centers. In SLL, slightly higher 4E-BP1 expression was noted in proliferation centers in comparison to surrounding small mature lymphocytes. WB confirmed that non-phosphorylated and p4E-BP1 were expressed in reactive nodes, FL and DLBCL. Other components of the eIF-4F complex including eIF-4G, total and p-eIF-4E and total 4E-BP1 were detectable in whole tissue lysates from BCL samples. We conclude that (a) while 4E-BP1 is almost uniformly expressed in various subtypes of BCL, its level of phosphorylation (indicative of activity) varies widely and has regional and cellular specificity, and (b) 4E-BP1 expression may identify minimal/early lymphomatous involvement in tissues. We speculate that 4E-BP1 phosphorylation may influence the biological behavior of BCLs, since in other investigations we found that the level of phosphorylation of 4E-BP1 correlates with survival after CHOP-based chemotherapy in DLBCL. Our findings support therapeutic trials targeting the eIF-4E pathway in many BCL subtypes, particularly in patients where immunostaining identifies high levels of 4E-BP1 phosphorylation. Disclosures: No relevant conflicts of interest to declare.