Expression and Phosphorylation of Eukaryotic Translation Initiation Factor 4E Binding Protein 1 in B-Cell Lymphomas and Reactive Lymphoid Tissues

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1 Expression and Phosphorylation of Eukaryotic Translation Initiation Factor 4E Binding Protein 1 in B-Cell Lymphomas and Reactive Lymphoid Tissues Dhatri Kodali, MD; Ajay Rawal, MD; Mary J. Ninan, MD; Manish R. Patel, MD; Hector Mesa, MD; Dennis Knapp, BA; Bertram Schnitzer, MD; Robert A. Kratzke, MD; Pankaj Gupta, MD N Context. Cap-mediated messenger RNA translation controlled by the eukaryotic initiation factor 4F (eif-4f) complex plays a key role in human cancer. eif-4f activity is controlled by a repressor binding protein (4E-BP1), which promotes translation when phosphorylated. Objective. To examine the level of expression and phosphorylation of 4E-BP1 in various subtypes of B-cell lymphoma and reactive lymphoid tissues. Design. Archival formalin-fixed, paraffin-embedded B-cell lymphoma samples and reactive lymphoid tissues were immunostained and examined for expression of 4E- BP1 and phosphorylated 4E-BP1. Expression of components of the eif-4f complex and unphosphorylated and phosphorylated 4E-BP1 was confirmed using Western immunoblotting on lysates of frozen lymphoma samples and reactive tissues. Identifying pathogenic mechanisms that contribute to the development and clinical behavior of lymphomas is critical for developing targeted therapies and for selecting patients who might benefit from such agents. An important level of control of eukaryotic gene expression occurs during initiation of cap-mediated messenger RNA translation by the eukaryotic initiation factor 4F (eif-4f) trimolecular complex (composed of eif-4e, eif-4g, and eif-4a), in which eif-4e is rate limiting and oncogenic. 1 3 eif-4f activity is controlled by repressor eif-4 binding proteins (BPs). The activity of 4E-BP1, one of the major 4E- BPs, is regulated by phosphorylation. Hypophosphorylated or nonphosphorylated 4E-BP1 is active, binds eif-4e, Accepted for publication April 9, 1. From the Hematology-Oncology Section, Departments of Medicine (Drs Kodali, Ninan, Patel, and Gupta) and Pathology (Drs Rawal and Mesa and Mr Knapp), Veterans Administration Medical Center, Minneapolis, Minnesota; the Division of Hematology, Oncology and Transplantation, Departments of Medicine (Drs Kodali, Ninan, Patel, Kratzke, and Gupta) and Pathology (Drs Rawal and Mesa), University of Minnesota, Minneapolis; and the Department of Pathology, University of Michigan, Ann Arbor (Dr Schnitzer). The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the annual meeting of the American Society of Hematology, New Orleans, Louisiana, December 7, 9. Reprints: Pankaj Gupta, MD, Hematology/Oncology Section 111E, VA Medical Center, One Veterans Dr, Minneapolis, MN ( gupta13@umn.edu). Results. Immunohistochemical analysis demonstrated weak to undetectable 4E-BP1 staining within benign, reactive germinal centers (N = 1). In contrast, 4E-BP1 was consistently expressed (moderate to strong staining) in 98% of various subtypes of mature B-cell lymphoma (N = 5). 4E-BP1 expression was also demonstrable in all 4 lymph nodes with in situ or partial involvement by follicular lymphoma and in all 1 cases of BCL-negative lymphoma. The level of phosphorylation of 4E-BP1 in lymphomas, evaluated by immunohistochemistry, was heterogeneous. Conclusions. The immunohistochemical expression pattern of 4E-BP1 exhibits regional and cellular specificity in reactive lymphoid tissues and may offer a diagnostic tool for distinguishing reactive follicles from neoplastic B-cell proliferations. (Arch Pathol Lab Med. 11;135: ) and impedes eif-4f formation, thus blocking translation and inducing apoptosis (Figure 1). Phosphorylation of 4E- BP1 releases bound eif-4e and allows interaction with the eif-4g scaffolding protein, resulting in initiation of capdependent translation. Overactivity of eif-4f (caused by overexpression of its components and/or phosphorylation of 4E-BP1) plays a key role in human cancers by mediating expression of proteins critical for cell growth, survival, transformation, and metastases and for angiogenesis (including cyclin D1, c-myc, BCL, survivin, defender against cell death 1, Bax inhibitor 1, bcl-x L, myeloid cell leukemia differentiation protein 1, ornithine decarboxylase, matrix metalloproteinase 9, osteopontin, collagenase IV, vascular endothelial growth factor, and fibroblast growth factor ). 1 8 In several solid tumors, the levels of expression and phosphorylation of 4E-BP1 correlate with histologic grade, aggressiveness, metastases, and survival.,3,9 Inhibition of the eif-4f complex may be of therapeutic value in cancer. 8 eif-4e antisense oligonucleotides 1 and small molecule pharmacomimetics of 4E-BP1 11 suppress the oncogenic properties of cancer cell lines and xenografts. Inhibition of expression of eif-4e and its relocation induces clinical response in acute myeloid leukemia. 1 Rapamycin analogs that inhibit the mammalian target of rapamycin dependent 4E-BP1 phosphorylation demonstrate antitumor activity in mantle cell lymphoma (MCL) and other hematologic malignancies. 13 Inhibition of Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al 365

2 Figure 1. Phosphorylation status of 4E-BP1 regulates cap-dependent translation initiation. Several signaling pathways converge on the mtor/4e-bp1/eif-4e axis. mtor activation induces phosphorylation of 4E-BP1 and the release of active eif-4e, which participates in the formation of the eif-4f translation initiation complex that initiates capdependent translation of diverse proteins involved in cell proliferation, survival and differentiation, and angiogenesis. This simplified depiction does not show the several additional proteins and signaling intermediates that further influence the activity of this axis. Abbreviations: 4E, eukaryotic initiation factor 4E (eif-4e); 4E-BP1, eif-4e binding protein 1; mrna, messenger RNA; mtor, mammalian target of rapamycin; VEGF, vascular endothelial growth factor. translation initiation may also enhance sensitivity to chemotherapy in lymphoma. 14 We have shown that transfection with wild-type 4E-BP1 or the constitutively active mutant 4E-BP1 A37/A46 decreases tumorigenicity and enhances chemotherapy-induced apoptosis in lung cancer cell lines 15 and mesothelioma. 16 Phosphorylation of 4E-BP1 ( funnel factor ) is thus a convergence point for diverse signaling cascades that induce oncogenesis and tumor progression and may represent a valuable therapeutic target in diverse malignancies. eif-4e plays an important role in the pathogenesis of murine lymphoma 17 and clinical behavior of lymphomas in humans. In non-hodgkin lymphomas, the level of expression of eif-4e correlates with aggressiveness, vascular endothelial growth factor and cyclin D1 expression, and resistance to chemotherapy. 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. 4E-BP1 is expressed in MCL, and this pathway is associated with cyclin D1 translation in MCL cell lines.,1 The frequency, level of expression, and phosphorylation of 4E-BP1 in the other subtypes of de novo non-hodgkin lymphoma has not been reported. In the present study, we evaluate the expression and regional localization of 4E-BP1 and phosphorylated 4E- BP1 in reactive lymphoid tissues and various subtypes of B-cell lymphoma. MATERIALS AND METHODS Tissue Samples Fifty mature B-cell lymphomas (1 follicular lymphomas [FLs], 9 small lymphocytic lymphomas, 7 MCLs, 5 extranodal marginal zone lymphomas, 13 diffuse large B-cell lymphomas [DLBCLs], and 4 Burkitt lymphomas), 1 reactive lymphoid tissue samples (8 lymph nodes and tonsillar tissues with reactive follicular hyperplasia), and 4 additional cases of FL with in situ or partial lymph node involvement were obtained from the institutional pathology archives. All cases were independently reviewed and classified according to current World Health Organization diagnostic criteria, and immunohistochemistry (IHC) was analyzed by hematopathologists (A.R. and H.M.). This study was approved by the institutional Human Subjects Committee. Immunohistochemistry Using standard procedures, formalin-fixed, paraffin-embedded sections were immunostained with rabbit monoclonal anti- 4E-BP1 (clone 53H11, Cell Signaling Technology, Danvers, Massachusetts) at 1:3 dilution and with citrate-based antigen retrieval and antiphosphorylated 4E-BP1 ([Thr37/46], clone 36B4, Cell Signaling Technology) at 1:1 dilution and with EDTA-based antigen retrieval on a Bondmax automated immunostainer (Leica Biosystems, Bannockburn, Illinois). Staining intensity was graded as, 1+, +, or3+, denoting none, weak, moderate, and strong staining, respectively, as described previously. 9 Selected samples were also evaluated by IHC for expression of BCL. Sections were immunostained with a mouse monoclonal anti-bcl antibody (clone E17, Cell Marque, Rocklin, California) at 1:1 dilution with EDTA-based antigen retrieval on a Bondmax automated immunostainer. Staining was interpreted as positive or negative. Western Immunoblotting Western immunoblot analysis was performed on lysates of 5 mature B-cell lymphomas ( FLs, 3 DLBCLs) and reactive lymph nodal tissues for eif-4g (total), eif-4e, and 4E-BP1 (total and phosphorylated) expression, as previously described by us. 15,16 Briefly, 5-mg protein from tissue lysates were separated on 8% to 15% gradient or 15% sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and subjected to Western immunoblot using specific antibodies to eif-4g (kindly provided by Nahum Sonenberg, PhD, McGill University, Montreal, Canada), eif-4e and 4E-BP1 (Cell Signaling Technology), and b-actin (Sigma, St Louis, Missouri). Membranes were incubated with primary antibodies overnight at 4uC, exposed to anti-rabbit (1:5) and anti-mouse (1:3) horseradish peroxidase conjugated antibodies as appropriate for 1 hour at room temperature. Proteins were detected and quantified using enhanced chemiluminescence (Peirce, Rockford, Illinois). RESULTS In reactive lymphoid tissues, there was regional and cellular specificity of 4E-BP1 expression, with either lack of or minimal ( to 1+) cytoplasmic expression in follicular center cells and paracortical T cells, 1+ to + expression in follicular and interdigitating dendritic cells, and 3+ expression in mantle and marginal zones (Table 1; Figure, A through C). In marked contrast to reactive lymphoid tissues, a consistently high level (+ to 3+) of cytoplasmic 4E-BP1 expression was seen within the neoplastic lymphocytes in 49 of 5 (98%) B-cell lymphomas (Table 1; Figure, D through M). Low intensity of 4E-BP1 staining (1+) was present in only one FL. Additional nuclear expression was seen in 4 cases of B-cell lymphoma (3 FLs and 1 DLBCL). Among cases of small lymphocytic lymphoma, a slightly brighter intensity of 4E-BP1 expression was noted within the proliferation centers in comparison with the surrounding small mature lymphocytes. Three of 4 additional cases with in situ or partial involvement by FL were easily distinguishable from reactive germinal centers by presence of strong and diffuse staining for 4E-BP1 in the neoplastic follicles, distinct from negative to weak staining pattern of adjacent reactive germinal centers, with the 366 Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al

3 Table 1. Eukaryotic Initiation Factor 4E Binding Protein 1 (4E-BP1) and Phosphorylated 4E-BP1 Expression by Immunohistochemistry in Reactive Lymphoid Tissues and B-Cell Lymphomas Diagnosis Total Cases, No. Intensity of 4E-BP1 Expression, No. of Cases Intensity of Phospho-4E-BP1 Expression, No. of Cases Germinal centers in reactive lymphoid tissue Mantle/marginal zones in reactive lymphoid tissue Follicular lymphoma Small lymphocytic lymphoma Mantle cell lymphoma Extranodal marginal zone lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma 4 fourth case demonstrating 1+ immunoreactivity in neoplastic follicles. Western immunoblot confirmed that nonphosphorylated and phosphorylated 4E-BP1 were expressed in reactive nodes, FL, and DLBCL (Figure, N). Western immunoblot also detected at least separate bands of 4E- BP1 that were phosphorylated to different degrees (the hyperphosphorylated and phosphorylated bands labeled a and b, respectively, in Figure, N); this finding is consistent with the known sequential phosphorylation of 4E-BP1 on several amino acid residues. Because Western immunoblot was done on whole sample lysates, it detected expression of both nonphosphorylated and phosphorylated 4E-BP1 in reactive lymphoid tissues; IHC showed that these were expressed in different regions of the reactive lymphoid tissues. Other components of the eif-4f complex including eif-4g, total and phospho-eif- 4E, and 4E-BP1 were detectable in whole tissue lysates from B-cell lymphoma samples, and their levels of expression were not different from reactive tissues (not shown). 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 + immunoreactivity in the follicular and interdigitating dendritic cells (Table 1; Figure 3, A through C). The level of expression of phosphorylated 4E-BP1 in B- cell lymphomas was extremely variable, being moderate or strong in 3 of 5 (46%) B-cell lymphomas, negative in 18 (36%), and dim in 9 (18%) cases (Table 1; Figure 3, D through H). Evaluation of the intensity of staining for 4E- BP1 and phosphorylated 4E-BP1 in each of the 5 individual B-cell lymphoma samples (Figure 4) showed that, whereas 4E-BP1 was consistently expressed, its level of phosphorylation (indicative of biologic activity) varied markedly in each of the subtypes of lymphoma examined. An interesting finding was that 4E-BP1 was expressed in the neoplastic cells in all 1 cases of lymphoma ( FLs, 6 DLBCLs, and 4 Burkitt lymphomas) that lacked BCL expression (Table ; Figure 5). Among these, all except one case of FL showed moderate to strong (+ to 3+) staining for 4E-BP1. COMMENT We demonstrate that 4E-BP1 expression demonstrates regional specificity in reactive tissues that may be useful in distinguishing germinal centers from neoplastic lymphocytic infiltrates. The diagnostic utility of the expression pattern of 4E-BP1 stems from our observation that the reactive germinal centers were essentially negative for 4E- BP1, whereas 98% of lymphomas demonstrated moderate to strong 4E-BP1 expression. This may be especially useful in interpreting lymphoid infiltrates in small tissue specimens. 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. In addition, 4E-BP1 appears to be of value in the identification of neoplastic lymphoid proliferations even in BCL-negative lymphomas. In contrast to the consistent expression of 4E-BP1 in lymphomas, the level of phosphorylation of 4E-BP1 was variable; therefore, we do not suggest a role of phosphorylated 4E-BP1 staining for diagnostic purposes. For evaluation of phosphorylated 4E-BP1, we used an antibody that detects phosphorylation at Thr37/46 because (1) phosphorylation on these residues by mammalian target of rapamycin or other upstream pathways is a prerequisite for subsequent hyperphosphorylation at Thr7 and Ser65 as well as for blocking binding to eif- 4E and () we showed that mutation at Thr37/46 creates a 4E-BP1 form that is constitutively functionally active and inhibits tumorigenesis. 15,16 Wang et al 18 showed that constitutively increased expression of eif-4e and eif-a may play an important role in development of lymphomas and correlates with biologic aggressiveness. The differential staining pattern of eif-4e and -a in physiologically proliferating follicular center cells of reactive follicles (high in germinal centers and low in mantle and paracortical zones) was similar to that seen for phosphorylated 4E-BP1 in the present study. Wang et al 18 did not report on 4E-BP1 or phosphorylated 4E-BP1 expression. Our findings, together with that report, 18 are consistent with the known effect of phosphorylated 4E-BP1 on cap-mediated translation (eif-4f activation). One previous study 3 reported absent () or weak (1+) 4E-BP1 expression in 53% and 47% of 3 lymphoma samples, respectively. In contrast, we observed moderate to strong (+ or 3+) IHC staining for 4E-BP1 in most (98%) of our specimens and confirmed this with Western immunoblot. Several factors may be responsible for the observed difference in 4E-BP1 staining between the previous study report and the present study. First, the study by Xu et al 3 examined 4E-BP1 expression on tissue Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al 367

4 Figure. Expression of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in reactive lymph nodes and mature B-cell lymphomas. A, Florid reactive follicular hyperplasia in a patient with human immunodeficiency virus/aids with negative follicular center cells in the germinal centers (asterisk) and paracortical T-cell zone (arrow) and strong (3+) expression in the mantle zone (arrowhead) (original magnification 35). B, High magnification view of A, showing 1+ to + staining in follicular dendritic cells (arrowheads) within the germinal center (original magnification 34). C, High magnification view of A, showing 1+ to + staining in interdigitating dendritic cells (arrow) in the paracortical zone (original magnification 34). D, Small lymphocytic lymphoma with strong expression in the diffuse lymphocytic infiltrate (original magnification 3). E, Extranodal marginal zone lymphoma with negative reactive germinal center (asterisk), 3+ expression in the mantle zone (arrowhead), and 1+ to + expression in the interfollicular neoplastic monocytoid B-cells (arrow) (original magnification 3). F, Mantle cell lymphoma with 3+ expression in a mantle zone pattern (between arrowheads) (original magnification 31). G, Follicular lymphoma with diffuse + expression in the malignant 368 Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al

5 Figure 3. Expression of phosphorylated eukaryotic initiation factor 4E binding protein 1 in reactive lymph nodes and mature B-cell lymphomas. A, Florid reactive follicular hyperplasia in a patient with human immunodeficiency virus/aids with 3+ expression in follicular center cells in the germinal centers (asterisk); negative paracortical (arrow) and mantle zones (arrowheads) (original magnification 35). B, High magnification view of A, showing 3+ staining in the follicular center cells and negative mantle zone (original magnification 34). C, High-magnification view of A, showing 1+ to + staining in interdigitating dendritic cells (arrow) in the paracortical zone (original magnification 34). D, Follicular lymphoma with 3+ expression in neoplastic follicles (asterisk) (original magnification 31). E, Follicular lymphoma lacking expression in the neoplastic follicles (asterisks) (original magnification 31). F, Diffuse large B-cell lymphoma with diffuse 3+ expression (original magnification 31). G, Diffuse large B-cell lymphoma lacking expression in the neoplastic lymphocytes (original magnification 31). H, Burkitt lymphoma in a bone marrow with + cytoplasmic expression (original magnification 34). microarrays, which likely provided relatively small areas of lymphoma for evaluation; second, the distribution of subtypes of lymphoma in the microarray set (not provided) may have been different than in the present study; and finally, the primary anti 4E-BP1 antibody used in that study (P-1; sc-9977, Santa Cruz Biotechnology Inc, Santa Cruz, California) resulted in the complete lack of 3+ staining (and + staining in only 7% of cases) of all the various solid tumor types in that report, suggesting that the P-1 antibody may not be optimal for the detection of 4E-BP1 by IHC. Xu et al3 did not report on the regional or cellular distribution of 4E-BP1 in different subytpes of non-hodgkin lymphoma or the expression of phosphorylated 4E-BP1. In our study, 7 of 7 MCLs showed 4E-BP1 expression by IHC, consistent with a previous report in this lymphoma subtype. Our studies also support and extend the recent report of detection of phosphorylated 4E-BP1 (Ser65) in AIDS-related lymphomas.4 A high level of expression of unphosphorylated 4E-BP1 in neoplastic lymphocytes that we observed in some cases r follicles (asterisk) (original magnification 3). H, Lymph node with in situ follicular lymphoma (arrow) (hematoxylin-eosin, original magnification 34). I, Diffuse BCL staining in the neoplastic follicle (arrow) from H, distinct from the adjacent benign follicles with negative reactive germinal centers (arrowheads) (original magnification 35). J, Diffuse 4E-BP1 expression in the neoplastic follicle (arrow) from H, in contrast to the adjacent benign follicles with negative germinal centers (arrowheads) (original magnification 34). K, Diffuse large B-cell lymphoma with 3+ cytoplasmic expression (4E-BP1, original magnification 34). L, Follicular lymphoma with 3+ nuclear expression (4E-BP1, original magnification 34). M, Burkitt lymphoma in a bone marrow with + cytoplasmic expression (4E-BP1, original magnification 34). N, Western immunoblotting demonstrating expression of hyperphosphorylated (a), phosphorylated (b), and nonphosphorylated (g) 4E-BP1. Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al 369

6 of B-cell lymphoma may appear counterintuitive because lack of phosphorylation would downregulate cap-mediated translation of proteins that mediate malignant transformation, angiogenesis, metastases and cell proliferation, and survival.1 8 The level of expression and phosphorylation of 4E-BP1 thereby affects not only apoptosis but a variety of biologic phenomena in malignant cells; conversely, apoptosis of malignant cells is the consequence of the net effect of multiple aberrant signaling pathways. To enhance cap-mediated translation of oncogenic proteins, the malignant cell first needs to upregulate the transcription and translation of 4E-BP1 then to stimulate sequential phosphorylation on several amino acid residues, which is mediated by several independent proteins and enzymes. The extent to which this occurs (expression and phosphorylation of 4E-BP1) has been reported to vary markedly in several tumor types.,3,9 Consistent with these reports, we found that the extent to which 4E-BP1 is phosphorylated varies widely in individual cases of different subtypes of B-cell lymphoma. As would be expected, the level of phosphorylation of 4EBP1 appears to correlate with the aggressiveness of B-cell lymphomas because in other investigations we found that phosphorylation of 4E-BP1 correlates with survival after standard chemotherapy in DLBCL.5 Patients in whom 4E- Figure 4. Correlation between expression of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and phosphorylated 4E-BP1 in B-cell lymphomas. The relative immunohistochemical expression of 4E-BP1 and phosphorylated 4E-BP1 in each of 5 cases of B-cell lymphoma is shown. Expression was graded as to 3+, as described in Methods. Because there were no cases where 4E-BP1 was not expressed, the x-axis (4E-BP1) does not include a negative () category. The various subtypes of lymphoma are indicated as follows: follicular lymphoma, open circles; small lymphocytic lymphoma, filled triangles; mantle cell lymphoma, open triangles; extranodal marginal zone lymphoma, filled squares; diffuse large B-cell lymphoma, open squares; Burkitt lymphoma, filled circles. Table. Eukaryotic Initiation Factor 4E Binding Protein 1 (4E-BP1) Expression in BCL-Negative B-Cell Lymphomas Intensity of 4E-BP1 Expression, No. of Cases Diagnosis Follicular lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma Total Total Cases, No Figure 5. Expression of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in BCL-negative B-cell lymphomas. A, Follicular lymphoma lacking BCL expression in the neoplastic follicles (asterisks) (original magnification 31). B, Diffuse large B-cell lymphoma lacking BCL expression in the large neoplastic cells (original magnification 3). C, Burkitt lymphoma lacking BCL expression (original magnification 3). D, Follicular lymphoma (the same case as in A) with + 4E-BP1 expression in the neoplastic follicles (asterisks) (original magnification 31). E, Diffuse large B-cell lymphoma (the same case as in B) with + 4E-BP1 expression (original magnification 3). F, Burkitt lymphoma (the same case as in C) with + 4E-BP1 expression (original magnification 3). 37 Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al

7 BP1 was largely unphosphorylated or had low levels of phosphorylation in the DLBCL lymphocytes experienced significantly better survival than those in whom 4E-BP1 was highly phosphorylated. In summary, our findings indicate that (1) although 4E- BP1 is almost uniformly expressed in various subtypes of B-cell lymphoma, its level of phosphorylation (indicative of activity) varies; () 4E-BP1 expression has regional and cellular specificity in reactive lymphoid tissues with potentially diagnostic utility in distinguishing reactive follicles (nil to minimal expression) versus neoplastic lymphoid proliferations (moderate to strong expression) including BCL-negative lymphomas; and (3) 4E-BP1 expression may identify minimal or early lymphomatous involvement in tissues. We also speculate that 4E-BP1 phosphorylation may influence the biologic behavior of at least some subtypes of B-cell lymphoma. 5 Studies on larger numbers of patients will help further determine heterogeneity of 4E-BP1 and phosphorylated 4E-BP1 expression and their prognostic impact in other subtypes of lymphoma. We thank Gloria A. Niehans, MD, for providing the frozen tissue samples for Western immunoblotting. This study was supported by the Veterans Health Administration. References 1. Ma XM, Blenis J. Molecular mechanisms of mtor-mediated translational control. Nat Rev Mol Cell Biol. 9;1(5): Armengol G, Rojo F, Castellvi J, et al. 4E-binding protein 1: a key molecular funnel factor in human cancer with clinical implications. Cancer Res. 7; 67(16): Thumma SC, Kratzke RA. Translational control: a target for cancer therapy. Cancer Lett. 7;58(1): Graff JR, Zimmer SG. Translational control and metastatic progression: enhanced activity of the mrna cap-binding protein eif-4e selectively enhances translation of metastasis-related mrnas. Clin Exp Metastasis. 3;(3): Yang SX, Hewitt SM, Steinberg SM, Liewehr DJ, Swain SM. Expression levels of eif4e, VEGF, and cyclin D1, and correlation of eif4e with VEGF and cyclin D1 in multi-tumor tissue microarray. Oncol Rep. 7;17(): Wendel HG, Silva RL, Malina A, et al. Dissecting eif4e action in tumorigenesis. Genes Dev. 7;1(4): Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eif4e in oncogenesis and cancer therapy. Nature. 4;48(698): Graff JR, Konicek BW, Carter JH, Marcusson EG. Targeting the eukaryotic translation initiation factor 4E for cancer therapy. Cancer Res. 8;68(3): Graff JR, Konicek BW, Lynch RL, et al. eif4e activation is commonly elevated in advanced human prostate cancers and significantly related to reduced patient survival. Cancer Res. 9;69(9): Graff JR, Konicek BW, Vincent TM, et al. Therapeutic suppression of translation initiation factor eif4e expression reduces tumor growth without toxicity. J Clin Invest. 7;117(9): Moerke NJ, Aktas H, Chen H, et al. Small-molecule inhibition of the interaction between the translation initiation factors eif4e and eif4g. Cell. 7; 18(): Assouline S, Culjkovic B, Cocolakis E, et al. Molecular targeting of the oncogene eif4e in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin. Blood. 9;114(): Rizzieri DA, Feldman E, Dipersio JF, et al. A phase clinical trial of deforolimus (AP3573, MK-8669), a novel mammalian target of rapamycin inhibitor, in patients with relapsed or refractory hematologic malignancies. Clin Cancer Res. 8;14(9): Bordeleau ME, Robert F, Gerard B, et al. Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model. J Clin Invest. 8;118(7): Jacobson BA, Alter MD, Kratzke MG, et al. Repression of cap-dependent translation attenuates the transformed phenotype in non-small cell lung cancer both in vitro and in vivo. Cancer Res. 6;66(8): Jacobson BA, De A, Kratzke MG, et al. Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eif4f complex in mesothelioma. Br J Cancer. 9;11(3): Ruggero D, Montanaro L, Ma L, et al. The translation factor eif-4e promotes tumor formation and cooperates with c-myc in lymphomagenesis. Nat Med. 4;1(5): Wang S, Rosenwald IB, Hutzler MJ, et al. Expression of the eukaryotic translation initiation factors 4E and alpha in non-hodgkin s lymphomas. Am J Pathol. 1999;155(1): Zhao Y, Liu W, Zhou S, Zhou J, Sun H. Relationship between eukaryotic translation initiation factor 4E and malignant angiogenesis in non-hodgkin lymphoma. J Huazhong Univ Sci Technol Med Sci. 5;5(6): Peponi E, Drakos E, Reyes G, Leventaki V, Rassidakis GZ, Medeiros LJ. Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma. Am J Pathol. 6;169(6): Kawamata N, Chen J, Koeffler HP. Suberoylanilide hydroxamic acid (SAHA; vorinostat) suppresses translation of cyclin D1 in mantle cell lymphoma cells. Blood. 7;11(7): Gingras AC, Raught B, Gygi SP, et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 1;15(1): Xu G, Zhang W, Bertram P, Zheng XF, McLeod H. Pharmacogenomic profiling of the PI3K/PTEN-AKT-mTOR pathway in common human tumors. Int J Oncol. 4;4(4): El-Salem M, Raghunath PN, Marzec M, et al. Activation of mtorc1 signaling pathway in AIDS-related lymphomas. Am J Pathol. 9;175(): Ninan M, Rawal A, Mesa H, Knapp D, Gupta P. Translation regulatory protein 4E-binding protein (BP)-1 phosphorylation identifies diffuse large B cell lymphoma patients with low IPI scores who experience short survival after chemotherapy [abstract 195]. Blood. 9;114():764. Submissions Now Accepted for CAP 11 Abstract Program Abstracts and case studies are now being accepted for the College of American Pathologists (CAP) 11 meeting, which will be held September 11th through the 14th in Grapevine, Texas. Submissions for the CAP 11 Abstract Program will be accepted through Friday, April 1, 11. Accepted submissions will appear in the September 11 issue of the Archives of Pathology & Laboratory Medicine. Visit the CAP 11 Web site at for specific abstract program information. Arch Pathol Lab Med Vol 135, March 11 4E-BP1 Expression in B-Cell Lymphomas Kodali et al 371