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Immunohistochemical Expression of the Transcription Factor DP-1 and Its Heterodimeric Partner E2F-1 in Non-Hodgkin Lymphoma

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Immunohistochemical Expression of the Transcription Factor DP-1 and Its Heterodimeric Partner E2F-1 in Non-Hodgkin Lymphoma Applied Immunohistochemistry & Molecular Morphology 10(4): 322–326, 2002 © 2002 Lippincott Williams & Wilkins, Inc., Philadelphia Immunohistochemical Expression of the Transcription Factor DP-1 and Its Heterodimeric Partner E2F-1 in Non-Hodgkin Lymphoma *Joel A. Chan, M.D., *Maria Olvera, M.D., †Raymond Lai, M.D. Ph.D., *Win Naing, M.D., *Sherif A. Rezk, M.D., and *Russell K. Brynes, M.D. DP-1 is a G1 cell cycle-related protein that forms heterodimers portant for cell cycle progression from G1 to S phase. Of with E2F, a family of transcriptional factors regulating the the six members identified thus far, E2F-1 is the most expression of genes important for G1 to S progression. Al- though the exact role of DP-1 is not well understood, it has extensively studied (1–7). When E2F-1 is released from been shown to stabilize DNA binding of E2F proteins. By the retinoblastoma protein, it forms a heterodimer with a immunohistochemistry, the authors examined the expression of member of the DP family (8). To date, two members of DP-1 in lymphoid tissues, including 8 cases of reactive follic- the DP family have been identified and cloned in mam- ular hyperplasia and 69 cases of B-cell non-Hodgkin lym- malian cells: DP-1 and DP-2 (9,10). DP-1 is the more phoma. The expression of the cell cycle-related proteins E2F-1 and Ki-67 was also assessed. Scoring was based on the pro- prevalent form (11). The exact biologic role of the DP portion of labeled nuclei (1–10%, 11–25%, 26–50%, and > family is unknown, although it has been shown that DP-1 50%). In reactive follicular hyperplasia, staining for DP-1, stabilizes DNA binding of E2F-1 and thereby facilitates E2F-1, and Ki-67 was largely confined to the germinal centers. E2F-1–dependent cell cycle progression (12). The E2F- All 25 cases of follicular lymphoma, regardless of grade, had a high proportion (> 50%) of DP-1–positive cells but a lower 1/DP-1 heterodimer has been shown to regulate genes proportion of cells marking for E2F-1 and Ki-67 (P < 0.001). involved in DNA synthesis (i.e., dihydrofolate reductase, had high DP-1 thymidine kinase, and DNA polymerase ␣) and those (24 ס The diffuse large B-cell lymphomas (n and Ki-67 scores but low E2F-1 scores (P < 0.001). Small involved in cell growth (i.e., N-myc, c-myc, IGF-1, and ס ס lymphocytic (n 10), marginal zone (n 3), and mantle cell cyclin A) (13–17). contained relatively low proportions of (5 ס lymphomas (n cells labeled for all three markers. Precursor B-cell lympho- Given the importance of the E2F-1/DP-1 heterodimer displayed high proportions of cells in cell cycle control, it is conceivable that abnormalities (2 ס blastic lymphoma (n positive for DP-1, Ki-67, and E2F-1 (> 50% in both cases). in either protein might contribute to the deregulation of Except in follicular center cell lesions, DP-1 expression gen- cellular processes involved in tumor growth. Many stud- erally correlated with that of Ki-67. However, the expression of DP-1 was discordant with that of E2F-1 in benign and malig- ies have been performed to examine the oncogenic po- nant follicular center cells, suggesting that DP-1 may have tential of E2F-1. For example, ectopic expression of functions other than facilitating E2F-1–dependent gene regula- E2F-1 in cell lines can override retinoblastoma protein- tion and cell cycle progression in these neoplasms. imposed cell arrest and propel cells from quiescence into Key Words: B-cell non-Hodgkin lymphoma—DP-1—E2F- S phase (18,19). In an interleukin-3–dependent myeloid 1—Ki-67. cell line, E2F-1 can replace or overcome the requirement Applied Immunohistochemistry & Molecular Morphology for growth factors and promote cell cycle progression 10(4): 322–326, 2002. (20). In previous studies, we demonstrated that E2F-1 expression is increased in some types of non-Hodgkin lymphoma (NHL), such as mantle cell lymphomas (21), Cell cycle progression from G1 to S phase is regulated and in approximately 30% of multiple myelomas (22). It by complex protein–protein interactions. One of these is known that DP-1 is broadly but not uniformly ex- involves the retinoblastoma protein, which acts as a sup- pressed during murine embryogenesis (23). However, pressor of cell proliferation by binding and inhibiting the deregulation and oncogenic properties of DP-1 have E2F proteins. The E2F family of transcriptional factors not been examined. modulates the expression of a broad array of genes im- In this study, we demonstrate that DP-1 can be de- tected using immunohistochemical techniques applied to Manuscript received February 14, 2002; accepted March 12, 2002. routinely fixed and processed, paraffin-embedded tissue From the *Department of Pathology, Los Angeles County and Uni- versity of Southern California Medical Center, Los Angeles, California, sections. We used this method to survey the expression and †Department of Pathology, University of Texas M.D. Anderson patterns of DP-1 and its heterodimeric partner E2F-1 in Cancer Center, Houston, Texas. follicular hyperplasia and in B-cell NHL. To evaluate Address correspondence and reprint requests to Russell K. Brynes, M.D., USC Department of Pathology, 2011 Zonal Ave., HMR 209, Los whether DP-1 is linked to E2F-1 expression and to cell Angeles, CA 90033. proliferation, we also examined the expression of Ki-67. 322 TRANSCRIPTION FACTOR DP-1 IN NON-HODGKIN LYMPHOMA 323 MATERIALS AND METHODS All cases were estimated independently by two au- thors (J.A.C. and R.K.B.). On the basis of these results, Tissues four grades of positivity were defined: 1 to 10%, 11 to Sixty-nine morphologically and immunophenotypi- 25%, 26 to 50%, and greater than 50% cells. These cat- cally defined cases of NHL were collected from the he- egories are similar to those used in our previous studies matopathology and consultation files of the Department (21,22). Discrepancies in the assignment of grade and of Pathology at the Los Angeles County and University staining intensity were reconciled by joint review using a of Southern California Medical Center, Los Angeles, double-headed microscope. The differences in scores CA. Each case was diagnosed according to the World between DP-1 and Ki-67 or E2F-1 were assessed us- Health Organization classification (24). The distribution ing Fisher exact test. Results with P <0.05 were consid- of chosen cases paralleled that of the major NHL sub- ered significant. types seen in the United States (25), with follicular lym- phoma and diffuse large B-cell lymphoma making up most of the cases studied. We also assessed eight cases of RESULTS reactive follicular hyperplasia in four tonsils and four Follicular Hyperplasia lymph nodes. All cases were fixed in 10% buffered for- Nearly all (> 90%) of the follicular center cells in malin, routinely processed, and embedded in paraffin. cases of follicular hyperplasia were strongly positive for DP-1 (Fig. 1), but only rare lymphoid cells in the mantle Immunohistochemistry zone were strongly positive. Similarly, only a relatively DP-1 was detected with the monoclonal antibody small proportion of the small and large lymphoid cells in 1DP06 (Ab6, Neomarkers, Fremont, CA) at a dilution of the interfollicular areas was strongly positive for DP-1. 1:25. The monoclonal antibody reactive with E2F-1 pro- In tonsillar tissues, nuclei in the basal layer of the squa- tein (clone KH 95; Santa Cruz Biotechnology, Santa mous epithelium were strongly positive. In tonsillar and Cruz, CA), was used at 1:30 dilution. For detection of the nodal tissues, the endothelial cells were often strongly Ki-67 antigen, a nuclear-associated proliferation antigen positive for DP-1. expressed in G1 through S phase, the monoclonal anti- The results for E2F-1 and Ki-67 staining patterns were body MIB-1 (Immunotech, Westbrook, ME), was used at similar to those of our previous study (21). Staining for a dilution of 1:50. Control sections were immunostained both E2F-1 and Ki-67 was predominantly found in the under identical conditions, substituting buffer solution germinal centers and was strongest in the polarized dark for the primary antibody. zones. Expression of Ki-67 was observed in greater than Tissue sections were mounted onto charged slides 50% of cells of all eight cases. In comparison, only three (Surgipath, Richmond, IL), baked at 56°C for 60 min- of eight cases stained for E2F-1 showed greater than utes, deparaffinized with xylene, and rehydrated with 50% of cells positive, and this positivity was restricted to graded ethanols to distilled water. Sections were placed a small subset of the centroblasts. Table 1 shows the in 0.01 mol/L citrate buffer at pH 6.0 and heated to distribution of scores for the three markers studied. boiling twice in a microwave oven for 5 minutes per cycle. Non-Hodgkin Lymphoma -mar ,(10 ס Reactivity was demonstrated by an avidin-biotin im- Cases of small lymphocytic lymphoma (n Ј Ј -and mantle cell lym ,(3 ס munoperoxidase detection system employing 3 ,3 diami- ginal zone lymphoma (n demonstrated relatively low scores for all (5 ס nobenzidine-tetrahydrochloride dihydrate (Vector Labo- phoma (n ratories, Burlingame, CA) as the chromogen. three markers. In small lymphocytic lymphoma, staining for DP-1 was predominantly restricted to the prolympho- Evaluation of Positive Cells cytes in the proliferation centers (Fig. 2). Mantle cell The tissue sections were initially scanned at low lymphoma tended to have intermediate scores for all power to identify evenly labeled areas.
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