Expression of Immunoglobulin Transcription Factors in Primary Intraocular Lymphoma and Primary Central Nervous System Lymphoma

Sarah E. Coupland,1 Christoph Loddenkemper,1 Justine R. Smith,2 Rita M. Braziel,2 Frederic Charlotte,3,4 Ioannis Anagnostopoulos,1 and Harald Stein1

PURPOSE. Several B-cell-associated transcription factors and commitment to B-cell lineage, the lymphocyte migrates from their coactivators, including BCL-6, BSAP/PAX5, BOB.1/OBF.1, the bone marrow into the peripheral lymphoid tissue, passing Oct.2, MUM1/IRF4, and PU.1, have been detected in peripheral through various stages of differentiation that can be defined by B-cell lymphomas. There are limited data on their expression in genotypic and phenotype changes. These include the rear- centrally located lymphoid neoplasms, such as primary intraoc- rangement of immunoglobulin heavy (IgH) and light (IgL) ular lymphoma (PIOL) or primary central nervous system lym- chains, the expression of various antigens on the cell surface, phoma (PCNSL). PIOL is a rare non-, con- as well as affinity maturation in the germinal centers.1 It has sidered a subtype of PCNSL. Both are usually diffuse, large been demonstrated that different transcription factors are ex- B-cell lymphoma (DLBCL), rarely manifest outside the CNS, and pressed at various time points during B-cell maturation, and carry a poor prognosis. indeed are essential for the regulation of expression 2–5 METHODS. Tissue biopsy specimens were examined from eight during both early and late B-cell development. The physio- cases of PIOL and 42 cases of HIV-negative PCNSL, as well as 50 logical and clinical relevance of these transcription factors cases of peripheral DLBCL, for the above-mentioned transcrip- becomes apparent on disruption of or alterations of the tion factors and for immunoglobulin heavy and light chains, that them. using immunohistochemistry. To date, several B-cell-associated transcription factors and their coactivators have been detected in paraffin-embedded RESULTS. Immunoglobulin expression was demonstrated in 46 sections of human tissues. These include BCL-6,6 BOB.1/OBF.1 (92%) of 50 cases of PIOL/PCNSL but in only 27 (54%) of 50 (also known as OCA-B),7 Oct.2,7 MUM1/IRF4,8 BSAP/PAX5,9,10 cases of peripheral DLBCL. Positivity for BOB.1/OBF.1 and and PU.1 (also known as Spi-1).11,12 The relationship of these Oct.2 was observed in all immunoglobulin-expressing PIOL transcription factors to B-cell differentiation and the germinal and PCNSL. BSAP/PAX5 expression occurred in 98% of PIOL/ center is demonstrated in Figure 1. Most studies investigating PCNSL, and MUM1/IRF4 immunoreactivity in 45 (90%) of 50 of these molecules have been performed on specimens taken these cases. PU.1 expression was observed in only 10% of the from patients with peripheral B-cell lymphomas7,12–18 and, PIOL/PCNSL group in contrast to 23 (46%) of 50 peripheral recently, in some lymphoid tumors of non-B lineage.19 The DLBCLs. Aberrant coexpression of MUM1/IRF4, PAX5, MUM1/ literature, however, contains little/limited data on the expres- IRF4, and BCL-6 was observed in most PIOLs/PCNSLs. sion of these transcription factors in extranodal “central” lym- CONCLUSIONS. These data provide further support to the notion phoid neoplasms, defined herein as primary intraocular lym- that peripheral and centrally located DLBCLs differ in clinical, phoma (PIOL)20 or primary central nervous system lymphoma immunophenotypic, and genotypic features, despite their sim- (PCNSL).21 ilar morphologic characteristics. PIOL and PCNSL tumor cells PIOL is a rare, high-grade, malignant non-Hodgkin B-cell are most likely to be derived from mature B-cells that have lymphoma, involving the retina, the vitreous, and occasionally undergone the germinal center reaction. (Invest Ophthalmol the optic nerve.22–26 It is considered a subtype of PCNSL, Vis Sci. 2005;46:3957–3964) DOI:10.1167/iovs.05-0318 which is defined as a lymphoma limited to the craniospinal axis without evidence of systemic disease.27 Most PIOL and PCNSL imilar to other developmental processes, the differentiation are diffuse, large B-cell lymphomas (DLBCLs), according to the Sof hematopoietic stem cells into B cells involves the sequen- updated World Health Organization (WHO) lymphoma classi- tial expression of structural and regulatory genes. After its fication,28 and are associated with a poor prognosis.29,30 Al- though there has been progress in the comprehension of the molecular pathology of PIOL,23,31,32 the understanding of the From the 1Department of Pathology, Charite´ University Medicine, histogenesis of PIOL remains limited, in part because of the Campus Benjamin Franklin, Berlin, Germany; the 2Casey Eye Institute paucity of tumor specimens available for examination. Because and 3Department of Pathology, Oregon Health and Science University, the expression of the B-lymphocyte-associated transcription Portland, Oregon; and the 4Department of Pathology, Pitie-Salpetriere, factors could help define the cellular origin of PIOL, we used Paris, France. standard immunohistochemical techniques to investigate the Supported by a Research to Prevent Blindness Career Develop- expression of Ig and the transcription factors BSAP/PAX5, ment Award and National Eye Institute Grant R01 EY014909 (JRS). BOB.1/OBF.1, Oct.2, MUM1/IRF4, and PU.1 in PIOL and PC- Submitted for publication March 14, 2005; revised May 11, 2005; NSL and compared the results with those obtained in periph- accepted August 26, 2005. Disclosure: S.E. Coupland, None; C. Loddenkemper, None; J.R. eral DLBCL. Smith, None; R.M. Braziel, None; F. Charlotte, None; I. Anagnos- topoulos, None; H. Stein, None The publication costs of this article were defrayed in part by page METHODS charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Tissue Samples Corresponding author: Sarah E. Coupland, Department of Pathol- ogy, Charite´-Universita¨tsmedizin Berlin, Campus Benjamin Franklin, A series of eight cases of PIOL were collected, and slides were retro- Hindenburgdamm 30, D-12200 Berlin, Germany; spectively reviewed from the consultation files of the Reference Center [email protected]. for Hematopathology, Pathology Department of Charite´ University

Investigative Ophthalmology & Visual Science, November 2005, Vol. 46, No. 11 Copyright © Association for Research in Vision and Ophthalmology 3957

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TABLE 1. Primary Antibodies

Antibody Clone Company/Source Cell Population(s)

CD20 L26 DakoCytomation, Denmark B lymphocytes CD3 F7.2.38 DakoCytomation T lymphocytes CD10 56C6 Novocastra, Newcastle-upon- B lymphocytes (centrocytes, centroblasts); Tyne, UK granulocytes BSAP/PAX5 2h BD-Pharmingen, San Diego, CA Nonplasmacellular differentiated B cells BOB.1 Sc-955 Santa Cruz, Santa Cruz, CA B lymphocytes Oct.2 Sc-233 Santa Cruz B lymphocytes IRF4 MUM1p DakoCytomation Plasmacellular differentiated B cells; T cells; melanocytes BCL-6 PG-B6p DakoCytomation B lymphocytes (centrocytes, centroblasts); extrafollicular blasts PU.1 G148-74 BD-Pharmingen B-lymphocytes; macrophages IgL Polyclonal DakoCytomation Plasma cells, plasmacellular differentiated B cells IgH Polyclonal DakoCytomation Plasma cells, plasmacellular differentiated B cells

Medicine, Campus Benjamin Franklin (Charite´ CBF) and of the Depart- PU.1 detection. CD10 was examined in these experiments to help ment of Pathology, Pitie-Salpetriere, Paris. The specimens consisted of enable interpretation of the results with respect to B-cell differentiation enucleated eyes (n ϭ 2) and chorioretinal biopsies (n ϭ 6). In addition, and the germinal center. To demonstrate Ig expression, the streptavi- stereotactic biopsies of 34 (Charite´ CBF) and 8 (Casey Eye Institute and din-biotin-peroxidase method was used.36 Alkaline phosphatase was Department of Pathology) cases of PCNSL were reviewed. The results revealed by fast red as the chromogen, whereas peroxidase was devel- were compared with those obtained from 50 cases of peripheral oped in diaminobenzidine, giving a brown reaction product. Frozen (nonmediastinal) DLBCL (Charite CBF). The experiments were per- sections for the validation of Ig expression in reactive lymphoid tissue formed concurrently under the same experimental conditions; the were air dried overnight and fixed in acetone for 10 minutes, and the results of 36 of 50 cases of peripheral DLBCL has been published.14 All binding of the polyclonal antibodies (DakoCytomation) to IgH and IgL patients were HIV negative. All lymphomas were diagnosed according chains ␥, ␮, ␦, ␬, and ␭ was evaluated. Appropriate negative and to the WHO Classification of Tumors.28 positive tissue control experiments were performed with each inves- The tissue biopsy specimens had been fixed in 4% buffered forma- tigation. The research was approved by the respective institutional lin and embedded in paraffin. In addition, hyperplastic tonsil, reactive review boards. All human tissue was managed in accordance with the lymphoplasmacellular infiltrates in the lacrimal gland, eye enucleated guidelines set forth in the Declaration of Helsinki. for malignant choroidal melanoma, and cerebral tissue obtained from autopsies (average postmortem period, 15 hours) were evaluated. RESULTS Immunohistology Reactive and Normal Tissues For immunostaining of paraffin-embedded tissues, tissue sections (4 ␮m) were cut from the paraffin blocks, dewaxed, rehydrated, and In agreement with results in previous studies,7,14,18,19 the ex- subjected to heat-induced epitope retrieval methods before incubation pression of transcription factors in reactive lymphoid tissue with the appropriate antibodies. Sections were immersed either in reflected the maturation stage of the B-cells in the different sodium citrate buffer solution at pH 6.0 or in EDTA solution (pH 8.0), germinal center areas (Fig. 1). and were subsequently heated in a pressure cooker, according to Immunoglobulin Expression. The palatine tonsils and standard practices.33 After they were rinsed in running water and the interstitial tissue of the lacrimal glands contained an infil- Tris-buffered saline, the sections were incubated with the primary trate of small, bland, mixed T and B lymphocytes with reactive antibodies listed in Table 1. For detection of PAX5, BOB.1/OBF.1, lymphoid follicles. The small B cells of the mantle of the Oct.2, MUM1/IRF4, and CD10, the alkaline-phosphatase anti-alkaline secondary follicles were strongly immunoreactive for IgD and phosphatase complex (APAAP) was used as a stain,34 whereas the En IgM (Figs. 2A, 2B). The marginal zone B cells, in contrast, Vision (DakoCytomation, Glostrup, Denmark) method35 was used for showed strong positivity for IgM, but only weak expression of

FIGURE 1. The Ig transcription fac- tors and their relationship to B-cell differentiation and the germinal cen- ter.

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pigment epithelium of the ciliary body; occasional B-cells within the uvea; and the ganglion cell, inner plexiform, and outer plexiform layers of the retina (Fig. 3A). In normal cere- bral tissue, no BOB.1/OBF.1 immunoreactivity was observed. Oct.2. The nuclei of all lymphoid cells in the dark zone of the germinal centers in the tonsils and reactive lymphoid infil- trates were strongly positive for Oct.2 (Fig. 2E). In the light zone, most cells stained with a similar intensity. Follicle mantle lymphocytes displayed weak to moderate Oct.2 positivity. In the interfollicular zone, nuclear Oct.2 expression was present in scattered blasts and occasional small lymphocytes, as well as in plasma cells, which displayed a polytypical expression of IgL ␬ and ␭ (Fig. 1). In the eyes enucleated for malignant choroidal

FIGURE 2. Expression distribution of (A) IgM, (B) IgD, (C) PAX5 (BSAP), (D) BOB.1/OBF.1, (E) Oct.2, (F) MUM1/IRF4, (G) BCL-6, and (H) PU.1 in reactive lymphoid tissues. (A, B, H) Stained with PAP (brown); (C–G) stained with APAAP (red). Original magnification, ϫ200.

IgD, consistent with previous studies.14 Most germinal center B cells of the dark zone of the follicles were negative or only weakly positive for Ig , with the exception of B cells showing plasmacellular differentiation. BSAP/PAX5. In the lymphoid tissues of the tonsils and the lacrimal glands, strong BSAP/PAX5 expression was observed in the lymphocytes within the germinal centers and the mantle zone, as well as in intraepithelial lymphocytes (Fig. 2C). Only occasional cells were immunoreactive in the interfollicular zone. In the eyes enucleated for choroidal melanoma, occa- sional lymphocytes in the choroid and iris were positive for BSAP/PAX5. No BSAP/PAX5 immunoreactivity was observed in normal cerebral tissue. BOB.1/OBF.1. Strong immunoreactivity for BOB.1/OBF.1 was observed easily in all B-cell populations encountered in reactive lymphoid tissues. The strongest expression occurred FIGURE 3. (A) Expression of the Ig BOB.1/OBF.1 in the nuclei of the germinal center cells (Fig. 2D). in the normal retina. (B) Occasional reactive lymphocytes in the In the eyes enucleated for malignant choroidal melanoma, choroid expressed Oct.2 (✱). (C) Positivity of malignant uveal mela- BOB.1/OBF.1 nuclear staining was present in the basal epithe- noma cells for MUM1/IRF4. APAAP staining (red); original magnifica- lial layer of the cornea; the equatorial cells of the lens; the tion: (A, B) ϫ400; (C) ϫ200.

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TABLE 2. Summary of the Expression of Immunoglobulin Transcription Factors and their Coactivators in PIOL/PCNSL versus Peripheral DLBCL

Ig BSAP/PAX5 BOB.1/OBF.1 Oct.2 MUM1/IRF4 BCL-6 CD10 PU.1

PIOL/PCNSL 46 (92)* 49 (98) 50 (100) 50 (100) 49 (98) 43 (86) 6 (12) 5 (10)† Perpiheral DLBCL‡ 27 (54) 47 (94) 50 (100) 50 (100) 45 (90) 35 (70) 12 (24) 23 (46)

Data are expressed as the number of specimens (percentage) expressing each factor, N ϭ 50. * All PIOL expressed immunoglobulin. † All PIOL were negative for PU.1. ‡ Some of these results have been published.14

melanoma, Oct.2 expression was limited to occasional lympho- Admixed macrophages demonstrated a strong staining for cytes in the choroid and iris (Fig. 3B). In the nonneoplastic PU.1, in contrast to the reactive plasma cells, which were brain, Oct.2 expression was absent. negative for this transcription factor. In normal cerebral and MUM1/IRF4. Some marginal-zone B cells showed weak ocular tissues, no PU.1 immunoreactivity was observed. nuclear immunoreactivity for MUM1/IRF4 (Fig. 2F). Most ger- minal center cells were negative for MUM1/IRF4, although PIOL and PCNSL several germinal center cells in the light zone displayed strong nuclear and moderate cytoplasmic positivity for MUM1/IRF4. The findings of the expression patterns of transcription factors Admixed MUM1/IRF4-positive plasma cells were present in the in PIOL and PCNSL are summarized in Table 2. First, there subepithelial areas in the tonsils, in the interstitial tissue of the were no differences in the staining patterns or staining inten- lacrimal glands, and occasionally in the reactive germinal cen- sity of the transcription factors in PIOL tumor cells in the ter. In the eyes enucleated for malignant choroidal melanoma, vitreous when compared with those in the chorioretinal biopsy MUM1/IRF4 was observed in occasional melanocytes in the specimens or in the enucleated eye. Secondly, there were no choroid, and demonstrated variable immunoreactivity in the fundamental differences in the staining patterns between PIOL malignant uveal melanoma cells (Fig. 3C). In normal cerebral and PCNSL; consequently, they are grouped together in Table tissue, no MUM1/IRF4 immunoreactivity was observed. 2, and the results compared with peripheral, diffuse, large BCL-6. Strong BCL6 expression was observed in the germi- B-cell lymphomas. For the sake of completeness, however, the nal center cells (Fig. 2G). Occasional extrafollicular blasts were expression patterns of the Ig transcription factors in PIOL and immunoreactive for BCL6. In ocular tissues, some basal epithe- PCNSL are demonstrated separately in Figures 4 and 5, respec- lial cells of the conjunctival and corneal epithelium, demon- tively. strated moderate nuclear expression of BCL-6. Normal cerebral In nearly all (92%) of PIOL and PCNSL cases, monoclonal tissue was negative for BCL-6. expression of an IgL and/or IgH was demonstrated (Figs. 4A, PU.1. PU.1 expression was observed in the nuclei of cen- 4B, 5A, 5B). All PIOLs were Ig positive. Moderate to strong troblasts and centrocytes with a moderate intensity (Fig. 2H). positivity for BOB.1/OBF.1 and Oct.2 was observed in all Ig-

FIGURE 4. (A) Expression of IgM by PIOL. (B) Negativity of the same tu- mor cells for IgD; inset: intrinsic pos- itive control in the form of a reactive expressing IgD. Strong expression in PIOL cells of (C) PAX5/ BSAP, (D) BOB.1/OBF.1, (E) Oct.2, and (F) MUM1/IRF4. (G) Variable im- munoreactivity of PIOL for BCL6 was observed. Negativity of the PIOL tu- mor cells for (H) PU.1 and for (I) CD10 (✱, neutrophilic granulocyte). PAP staining, brown; APAAP staining, red; original magnification, ϫ200.

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FIGURE 5. (A) Expression of IgM by PCNSL. (B) Negativity of the same tumor cells for IgD; inset: intrinsic positive control in the form of a re- active plasma cell expressing IgD. Strong immunoreactivity of the PCNSL tumor cells for (C) PAX5/BSAP, (D) BOB.1/OBF.1, (E) Oct.2, and (F) MUM1/IRF4. (G) BCL-6 was variably and partially expressed in PCNSL. The PCNSL tumor cells were nega- tive for (H) PU.1 and (I) CD10. PAP staining, brown; APAAP staining, red; original magnification, ϫ200.

positive and Ig-negative PIOLs and PCNSLs (Figs. 4D, 4E, 5D, tors were chosen because of their roles in the regulation of 5E). Both BSAP/PAX5 and MUM1/IRF4 immunoreactivity oc- during B-cell development. This is the first curred in 49 (98%) of 50 PIOL/PCNSL specimens, whereas study in which the Ig transcription factor expression in PIOL BCL-6 expression was observed in 43 (86%) of 50 of the cases and PCNSL has been investigated in detail. In agreement with (Figs. 4C, 4F, 5C, 5F). Weak PU.1 immunoreactivity was previous findings,14,37 we observed simultaneous expression present in only 5 (10%) of the PIOL/PCNSL group; it is note- of BOB.1/OBF.1 and Oct.2 in all reactive and normal B-cells worthy that all PIOLs were PU.1 negative (Figs. 4H, 5H). CD10 that expressed Ig. Recently published data demonstrate that expression was observed in only 6 (12%) of 50 PIOL/PCNSL Oct.2 and BOB.1/OBF.1 transcription factors are coexpressed (Table 2; Figs. 4I, 5I). in almost all categories of peripheral B-cell neoplasms, with the exception of the classic Hodgkin lymphoma.7,11,14,15,38 In the Peripheral Diffuse, Large B-Cell Lymphoma present study, we demonstrated a consistent expression of BOB.1/OBF.1 and Oct.2 in all Ig-expressing PIOLs, PCNSLs, and A moderate to strong intensity of BOB.1/OBF.1 and a strong peripheral DLBCLs. This observation adds support to the no- intensity of Oct.2 was observed in all DLBCLs (Figs. 6D, 6E; tion that Oct.2 and BOB.1/OBF.1 are imperative for Ig expres- Table 2). Moderate to strong immunoreactivity for MUM1/IRF4 sion, not only in normal, but also in neoplastic B cells.39 was seen in 45 (90%) of 50 of peripheral DLBCLs, BCL-6 positivity in 35 (70%) of 50 cases (Fig. 6G), and BSAP/PAX5 One of the most interesting observations in this study was expression in 42 (84%) of 50 of cases (Fig. 6C). In contrast, the finding that Ig expression was absent in neoplastic B cells, weak immunoreactivity for PU.1 was observed in 23 (46%) of despite the presence of Oct.2 and BOB.1/OBF.1. This pattern 50 peripheral DLBCLs (Fig. 6I). Immunoglobulin expression at of expression was noted in 46% of peripheral DLBCLs com- the protein level was observed in only 27 (54%) of 50 cases of pared with only 8% of PIOL/PCNSLs. The difference between peripheral DLBCL (Figs. 6A, 6B), consistent with previous these two groups of essentially morphologically similar lym- findings.14 Finally, CD10 expression was observed in 12 (24%) phomas is striking and suggests that, in contrast to PIOL/ of 50 peripheral DLBCLs (Fig. 6H). PCNSL tumor cells, a significant proportion of peripheral DL- BCLs arise from germinal-center B cells that have either lost the ability to express Ig or have downregulated Ig expression. The DISCUSSION consistent and strong expression of BOB.1/OBF.1 and Oct.2 in the Ig-negative PIOL/PCNSL and peripheral DLBCL indicates, To gain new insights into the cellular origin and the maturation however, that other mechanisms, possibly “crippling muta- stage of PIOL and PCNSL, we investigated the expression of the tions” within the transcription control regions and/or coding Ig transcription factors PAX5, BOB.1/OBF.1, Oct.2, PU.1, sequences,40 are involved to account for the lack of Ig tran- BCL-6, and MUM1/IRF4, were investigated and compared with scription in these tumors. An alternative could be epigenetic those patterns in peripheral DLBCLs. These transcription fac- silencing of the Ig heavy chain gene, as recently described in

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FIGURE 6. (A) Expression of IgM in a peripheral DLBCL. (B) A second peripheral DLBCL showing no immu- noreactivity for IgM. Similar results were obtained for IgD, ␬, and ␭ stain- ing. Strong expression of peripheral DLBCL for (C) PAX5/BSAP, (D) BOB.1/OBF.1, (E) Oct.2, (F) MUM1/ IRF4, (G) BCL-6, (H) CD10, and (I) PU.1. PAP staining, brown; APAAP staining, red; original magnification, ϫ200.

classic Hodgkin lymphoma.41 To clarify this, further investiga- that does not correspond to the mutually exclusive pattern tions are needed. found in normal germinal centers.8 The dysregulation of these Another finding of interest of the present study was the transcription factors in PIOL/PCNSL is further undermined by absence of immunostaining of PU.1 in the majority (90%) of the coexpression of BSAP/PAX5 and MUM1/IRF4 by the neo- Ig-positive PIOL/PCNSL. This contrasted with a weak immuno- plastic B-cells, again an abnormal phenotype not found in the reactivity of this transcription factor in 23 (46%) of 50 periph- normal counterpart.47 These findings, together with the detec- eral DLBCL cases and a corresponding lack of expression in tion of numerous IgH gene somatic mutations in PIOL32,48 and 54% of cases. PU.1 is a hematopoietic transcription factor PCNSL,49–52 provide further evidence for the hypothesis that belonging to the Ets family. It is identical with the Spi-1 onco- PIOL and PCNSL are derived from mature B cells that have gene, which is implicated in spleen focus-forming, virus-in- undergone a prolonged interaction in the microenvironment of duced murine erythroleukemias. PU.1 regulates the transcrip- the germinal center or are at the late germinal center stage of tion of genes relevant to the development of B-cells, differentiation. This may explain the extensive loss of CD10 macrophages, and myeloid cells.4,42,43 Moreover, it binds to (only 12% PIOL and PCNSL cases positive versus 24% of pe- several promoters, such as MUM1/IRF4, and regulates the ex- ripheral DLBCL cases) and the variable expression of both pression of genes required for terminal B-cell differentiation. It BCL-6 and MUM1/IRF4 in PIOL.20 is reported to be expressed in immature and mature B lympho- Germinal centers arise in peripheral lymphatic tissue (e.g., cytes, being lost only in plasma cells and their precursors.44 tonsil, mucosa-associated lymphatic tissue, and lymph nodes) The physiological absence or downregulation of PU.1 expres- as a result of antigen stimulation, and they are responsible for sion has been reported in some plasma-cell–derived lympho- the production of B cells with high-affinity antigen receptors. mas,45 whereas most other B-cell neoplasms are PU.1 positive. The memory B cells and long-lived plasma cells, which emerge Most of the PIOLs and PCNSLs investigated in the present study from the germinal centers, are attracted by homing receptors, demonstrated some degree of plasmacellular differentiation, including chemokine ligands, in peripheral lymphatic possibly explaining their PU.1 negativity. Of note, there was no tissue (e.g., the gastrointestinal tract) and the bone marrow, correlation between PU.1 negativity and Ig expression in either causing their migration to the respective tissues. Because the the PIOL/PCNSL or peripheral DLBCL specimens. Therefore, it eye and the CNS in humans is devoid of a germinal center is unlikely that PU.1 is responsible for the lack of Ig expression structure, our and others’ findings imply that PIOL/PCNSL are in Ig negative PIOLs/PCNSLs or DLBCLs. tumors that arise initially in an extraneural germinal center Finally, the majority of the cases examined expressed environment. Subsequent localization to the retina and/or to MUM1/IRF4. Positivity was observed in 49 (98%) of 50 PIOL/ the CNS may involve the development of a “neurotropic” PCNSL and in 47 (94%) of 50 DLBCL. In keeping with our cellular phenotype, the influence of chemokine receptors and preliminary findings in PIOL,20 with data examining PCNSL21 their ligands,53,54 and/or the incomplete destruction of the and with previous data on peripheral DLBCL,8,46 MUM1/IRF4 neoplastic cells by the host immune system. was often coexpressed with BCL-6 in both central and periph- In summary, our data indicate that (1) PIOLs and PCNSLs eral DLBCL by the same neoplastic cells, an aberrant feature demonstrated Ig expression more frequently than did periph-

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eral DLBCL (92% vs. 54%); (2) similar to peripheral DLBCLs, all 15. Marafioti T, Mancini C, Ascani S, et al. Leukocyte-specific phos- PIOLs and PCNSLs expressed BOB.1/OBF1 and Oct.2; (3) an phoprotein-1 and PU.1: two useful markers for distinguishing T- aberrant coexpression of BCL-6 and MUM1/IRF4 and of MUM1 cell-rich B-cell lymphoma from lymphocyte-predominant and PAX5 was present in most PIOLs, PCNSLs, and DLBCLs; (4) Hodgkin’s disease. Haematologica. 2004;89:957–964. nearly all PIOLs and PCNSLs were immunonegative for PU.1, 16. Nagy M, Chapuis B, Matthes T. Expression of transcription differing from peripheral DLBCLs, 46% of which were positive factors Pu.1, Spi-B, Blimp-1, BSAP and oct-2 in normal human for this transcription factor. The latter finding may be ex- plasma cells and in multiple myeloma cells. Br J Haematol. 2002;116:429–435. plained by the greater degree of plasmacellular differentiation, 17. Pileri SA, Gaidano G, Zinzani PL, et al. 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