Abstract. Materials and Methods

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1 Hematopathology / ANAYSIS OF BC-2 IN YMPHOMA AND HYPERPASIA Follicular ymphoma Can Be Distinguished From Benign Follicular Hyperplasia by Flow Cytometry Using Simultaneous Staining of Cytoplasmic bcl-2 and Cell Surface Dennis B. Cornfield, MD, Debra M. Mitchell, MD, Nidal M. Almasri, MD, John B. Anderson, MT, Kim P. Ahrens, BS, Elaine O. Dooley, MT, and Raul C. Braylan, MD Key Words: Flow cytometry; bcl-2; Follicular lymphoma; Follicular hyperplasia Abstract The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (F) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of F, using surface expression as a discriminator of the various lymphoid compartments. ymph node cell suspensions from 12 cases of FH and 17 cases of F were analyzed by flow cytometry using a combined surface and intracellular bcl-2 staining. T cells in FH demonstrated the same bcl-2 expression as the + mantle cells, but the bright + germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of F showed greater bcl- 2 expression than the T cells of the same tumors and all cell populations of FH. This difference was particularly significant between the neoplastic B cells of F and the germinal center cells of FH. The combined analysis of and bcl-2 should be useful for the differential diagnosis between FH and F and particularly applicable to limited samples or when B-cell clonality is in question. Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined. The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (F) can sometimes be challenging for surgical pathologists. The bcl-2 oncoprotein, a 26-kd protein that prolongs cell survival by inhibiting apoptosis, 1 has been a particularly useful target for distinguishing FH from F by immunohistochemical means. 2,3 The overexpression of bcl-2 in most cases of F results in its intense staining in the nodules of F, in contrast to the opposite pattern in FH, in which bcl-2 is localized most prominently in the small, nondividing lymphocytes of the mantle zone and, to a lesser extent, the B and T lymphocytes of the interfollicular areas. 4-6 It has been shown that monoclonal antibody to is a useful marker for separating mantle B-cells from germinal center B cells. 7 We found that, by using a relatively simple flow cytometric technique that uses dual staining with monoclonal antibodies to bcl-2 and, F displays a pattern of bcl-2 expression that is distinct from that of FH. Materials and Methods Selection of Samples All samples were submitted to the flow cytometry laboratory of Shands Hospital, University of Florida, Gainesville, for diagnostic purposes. FH samples consisted of lymph nodes displaying benign hyperplastic changes. F samples comprised lymph nodes (14), a small intestinal mass (1), a pharyngeal mass (1), and a parotid mass (1) and were selected based on histologic and flow cytometric findings that included the following: (1) typical morphologic features of F, including a nodular architecture on formalin-fixed H&E-stained tissue; (2) flow cytometric 258 Am J Clin Pathol 2;114: American Society of Clinical Pathologists

2 Hematopathology / ORIGINA ARTICE evidence of clonality as demonstrated by a monotypic population of B lymphocytes expressing kappa or lambda immunoglobulin light chain (15 cases). In 2 cases of F, no immunoglobulin light chain expression was demonstrated on B cells. Of the 17 cases of F, 16 expressed CD1, in keeping with previous findings. 8 Cell Suspension Preparation and Staining Single-cell suspensions were prepared by gentle mechanical tissue mincing. Flow cytometric analysis was performed using a panel of monoclonal antibodies routinely applied in our laboratory for the diagnosis and characterization of lymphoproliferative disorders. Approximately 1 6 cells were exposed to an appropriate dilution of fluorescein isothiocyanate or phycoerythrin-conjugated monoclonal antibody in 96-well microtiter plates (Pro-Bind U-Bottom, Becton Dickinson abware, Franklin akes, NJ). The plates were placed on ice in the dark for 15 minutes, and the cells in the wells were washed twice with phosphate-buffered saline (PBS) and resuspended in 1 µ of a 1:1 dilution of propidium iodide in 1.12% sodium citrate buffer before analysis. In addition, a combined surface and intracellular bcl-2 staining was performed. Approximately 1 6 cells were first exposed to 1 µ of phycoerythrin-conjugated anti- (eu16, Becton Dickinson Immunocytometry Systems, San Jose, CA) on ice for 15 minutes in the dark. Cells were washed twice with PBS and then exposed to the reagents in the Fix and Perm Cell Permeabilization Kit (Caltag, Burlingame, CA), according to the manufacturer s instructions. Cells subsequently were incubated with 1 µ of fluorescein isothiocyanate conjugated anti bcl-2 or the appropriate immunoglobulin control (Pharmingen, San Diego, CA) at room temperature for 15 minutes. After washing with PBS, the cells were resuspended in 5 µ of PBS. A FACScan or a FACSCalibur (Becton Dickinson Immunocytometry Systems) was used for cell analysis, and data analysis was performed by using ysis or CellQuest software (Becton Dickinson Immunocytometry Systems). The fluorescence intensity of the and bcl-2 expressions was determined visually on bivariate plots; bcl-2 expression was quantitated using the mean fluorescence intensity (MFI), calculated as the ratio of mean channel number of bcl-2 expression to that of the isotype-matched control for the -defined population of interest. MFI is expressed as mean ± SD. Immunohistochemical Staining Immunohistochemical staining was performed on formalin-fixed tissue using an anti bcl-2 antibody and an enzyme detection kit (DAKO, Carpinteria, CA) according to the manufacturer s instructions. Molecular Analysis Analysis of bcl-2 gene rearrangement was performed according to the method of iu et al 9 with minor modifications. In short, DNA was extracted from tissue or cell suspensions fixed in a 5% ethanol-rpmi mixture using a nonorganic method. 1 DNA from paraffin-embedded tissues was extracted with xylene through graded alcohols. Primers for the bcl-2 major breakpoint region, minor cluster region, and immunoglobulin heavy chain joining region (JH); consensus segments; and internal probes for the major breakpoint region and minor cluster region were used as described by iu et al. 9 A 2-ng aliquot of genomic DNA was amplified in a Perkin Elmer Cetus 96 thermocycler with 1.25 U of Taq polymerase (Perkin Elmer, Norwalk, CT) in a 5-µ reaction volume containing tris(hydroxymethyl)- aminomethane-hydrochloride buffer, ph 8.3 (Perkin Elmer) and final concentrations of 1. mmol/ of magnesium chloride, 2 µmol/ of dntp ([deoxynucleoside triphosphate] Amersham Pharmacia Biotech, Piscataway, NJ), and.5 µmol/ of each primer. Amplification products were electrophoresed through a 1.75% agarose gel (Ultrapure, Gibco- BR, Gaithersburg, MD) and transferred by semidry electroblotting onto nylon membranes. They then were probed with 3 tailed digoxigenin-labeled oligonucleotides, and amplified bands were detected by chemiluminescence (protocol of Boehringer-Mannheim, Indianapolis, IN). Results All cases in this series demonstrated bcl-2 expression in the lymphoma cells by immunohistochemical staining. By flow cytometry, the analysis of in most FH samples revealed distinct populations of bright +, dim +, and cells Figure 1A. These subpopulations represent mostly germinal center, mantle, and T cells, respectively. 7 In contrast, the presence of neoplastic cells uniformly expressing in F produced an obliteration of the 2 distinct B-cell populations noted in FH, resulting in a single + population Figure 1B. The bcl-2 expression in the B lymphocytes of FH (Figure 1A) was also different from that seen in the cases of F (Figure 1B). As shown in Figure 2, in FH, the intensity of bcl-2 expression in T cells (MFI = 4.7 ± 1.6) was similar to that of mantle cells (MFI = 5.4 ± 2.6), but germinal center cells, depicted by their intense expression, showed a near absence of bcl-2 (MFI = 1.5 ±.6). In F, the neoplastic B cells showed bcl-2 content (MFI = 22.2 ± 14.1) that was significantly higher than that of the T-cell population in the same tumor (MFI = 7.1 ± 2.3; P <.2), as well as all other cell populations of FH, particularly the FH germinal center cells (P <.1). The population of cells (mostly T American Society of Clinical Pathologists Am J Clin Pathol 2;114:

3 Cornfield et al / ANAYSIS OF BC-2 IN YMPHOMA AND HYPERPASIA A B T M 1 3 T bcl bcl GC Figure 1 Correlated analysis of surface and intracellular bcl-2 in a representative case of follicular hyperplasia (A) and follicular lymphoma (B). A, identifies 3 distinct populations with increasing levels of expression, corresponding mostly to T cells, mantle B cells, and germinal center B cells, respectively. Germinal center (GC) cells show virtually no expression of bcl-2, equivalent to the isotype-matched control (not shown). Higher bcl-2 expression is seen in T cells (T) and in mantle B cells (M). The follicular lymphoma case (B) displays 2 major populations: T lymphocytes (T) and + lymphoma cells () (recognized as such by their expression of a single immunoglobulin light chain, not shown). The malignant B cells express a high level of bcl-2. 6 bcl-2 Expression (MFI) T Cells Mantle Germinal Center T Cells Follicular Hyperplasia Follicular ymphoma Figure 2 Mean fluorescence intensity (MFI) for bcl-2 in the different lymphoid populations of follicular hyperplasias and follicular lymphomas. Significant differences in MFI were observed between the follicular lymphoma B cells and the T cells within the same tumors (P <.2) and the germinal center cells in follicular hyperplasias (P <.1). Also, the T cells of follicular lymphomas demonstrated a significantly higher bcl-2 expression than the T cells in follicular hyperplasias (P =.2). In cases of partial lymphoma involvement, the MFI of the bcl-2 expression corresponds only to the neoplastic component. Error bars represent SD. 26 Am J Clin Pathol 2;114: American Society of Clinical Pathologists

4 Hematopathology / ORIGINA ARTICE cells) in cases of F demonstrated a significantly higher bcl- 2 expression (MFI = 7.1 ± 2.3) than the equivalent population in cases of FH (MFI = 4.7 ± 1.6; P =.2). In 5 cases, there was only partial involvement of the node by F, and neoplastic B cells were admixed with numerous normal B cells. These 2 B-cell populations were different in expression, and their nature was determined by their corresponding surface immunoglobulin light chain distribution. Thus, normal B cells showed a polytypic distribution (a mixture of kappa- and lambda-bearing cells), whereas neoplastic B cells exhibited a single or no light chain expression Figure 3A and Figure 3B. The intensely staining + cells, corresponding to the lymphoma elements, showed higher bcl-2 expression than the less intense + normal B cells Figure 3C. The bcl-2 MFI of the neoplastic cells in these F cases with partial lymphoma involvement (23.66 ± 16.35) was not different from that of the more advanced cases (21.53 ± 13.85). Molecular analysis revealed the presence of a bcl-2 gene translocation in 1 (59%) of 17 cases of F in this series. The intensity of bcl-2 expression in lymphoma cells of cases with a molecular translocation (25.5 ± 16.32) did not differ significantly from that of cases not demonstrating such a translocation (17.37 ± 9.35; P =.21). A B lambda kappa Polytypic Polytypic Discussion By using a relatively simple flow cytometric technique that used simultaneous staining of cell surface and intracellular bcl-2, we demonstrated a pattern of bcl-2 expression in F that is distinct from that of FH. was chosen because it is a universal marker of peripheral B cells and is differentially expressed in mantle and germinal center B cells. 7 Other markers preferentially expressed in germinal center cells could be used but are less discriminatory. 8 In F, the + malignant cells showed strong bcl-2 expression. This expression was higher than that of any cell subpopulation of FH and strikingly different from the intensely + germinal center cells, which typically do not express bcl-2. The differences in bcl-2 expression between normal and F cells was clearly illustrated also in the few cases of partial involvement by lymphoma, in which the level of expression of bcl-2 in the malignant cells was as high as that of neoplastic cells in nodes fully involved by F. The distinction between FH and F sometimes may be extremely difficult. Molecular techniques and immunohistology can aid in establishing a firm diagnosis, but these techniques are not sufficiently specific, may be subject to a variety of methodologic artifacts, and fail to recognize some cases of F. Immunophenotyping by flow cytometry can provide additional and often diagnostic information and is C bcl Polytypic Figure 3 ymph node partially involved by follicular lymphoma (F). The node contains normal B cells as well as F cells. In A and B, simultaneous surface and immunoglobulin light chain staining identifies normal (polytypic) B cells (vertical boxes) consisting of a mixture of kappa- and lambda-bearing cells. ymphoma cells () express (faintly) kappa but no lambda immunoglobulin. C, A high level of bcl-2 is shown in the more intensely -expressing lymphoma cells. American Society of Clinical Pathologists Am J Clin Pathol 2;114:

5 Cornfield et al / ANAYSIS OF BC-2 IN YMPHOMA AND HYPERPASIA particularly useful when limited biopsy material precludes adequate morphologic assessment. It is also of value when fine-needle aspiration of lymphoid material is thought to be preferable to surgical biopsy for reasons of safety and convenience, as in elderly patients with retroperitoneal lymphadenopathy. The flow cytometry distinction of F from FH is based largely on the identification of a B-cell population with immunoglobulin light chain restriction and expression of CD1. 8 Our results show that the analysis of bcl-2 expression by flow cytometry adds an additional piece of confirmatory data that, in difficult or inconclusive cases, can help establish the diagnosis of F. This is useful when tumor cells fail to express immunoglobulin light chain, as observed in 2 of our cases, which showed molecular, morphologic, and immunohistochemical evidence of F but failed to express monoclonal light chain immunoglobulin by flow cytometry. Both cases demonstrated bcl-2 expression in the intense + cell population. It should be noted that approximately 1% to 15% of Fs do not overexpress bcl-2. 2,5 In these cases, evaluation of bcl-2 by flow cytometry would not be expected to provide useful information, although other potential results generated by flow cytometry, such as monotypic light chain expression or strong CD1 expression by a B-cell population, might suggest the diagnosis. The finding that T lymphocytes express higher levels of bcl-2 in F than in FH was unexpected. The factors that govern bcl-2 expression in reactive T lymphocytes are largely unknown. More than 8% of normal T cells express bcl-2 protein, and this expression can be maintained for at least several weeks under continued mitogenic stimulation. 11 It has been shown that viability factors such as interleukin-2 can induce endogenous bcl-2 messenger RNA expression in interleukin-2 dependent cells like the cytotoxic T-cell line CT2. 12 It might be postulated that the presence of interleukins or other factors in the local environment of F or even direct contact between the neoplastic cells of F and tumor-infiltrating T lymphocytes results in stimulation of T- cell bcl-2 messenger RNA and protein expression. At present, the mechanisms responsible for the enhanced bcl-2 protein expression that we observed in T lymphocytes remain entirely conjectural. B-cell lymphomas other than F may express bcl-2, as demonstrated by immunohistochemical methods in paraffinembedded tissue. 3,5 Thus, the simple detection of this protein would not help in identifying specific lymphoma types. Flow cytometry has the advantage of being highly quantitative and is an excellent means of measuring products at the cellular level, but the cytometric quantitation of bcl-2 protein in lymphomas has received virtually no attention in the medical literature. In this regard, it would be of interest to determine whether different types of bcl-2-expressing lymphomas exhibit measurable differences in bcl-2 content that may aid in their classification. It has been shown that bcl-2 expression is an adverse prognostic factor with regard to overall and disease-free survival in diffuse large cell lymphomas. 13,14 Its prognostic value in F is much less clear, with some observers claiming a poor overall prognosis 15 or freedom from progression after chemotherapy 16 in bcl-2 positive cases, and others 17 claiming no such relationship. Whether quantitative measurement of bcl-2 expression, rather than assessing only positivity or negativity, would add any useful prognostic information remains to be determined. From the Department of Pathology and aboratory Medicine, and Shands Hospital, University of Florida College of Medicine, Gainesville, F. Address reprint requests to Dr Braylan: Hematopathology Section, Dept of Pathology and aboratory Medicine, Box 1275, University of Florida College of Medicine, Gainesville, F References 1. Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 199;348: Utz G, Swerdlow SH. Distinction of follicular hyperplasia from follicular lymphoma in B5-fixed tissues: comparison of MT2 and bcl-2 antibodies. Hum Pathol. 1993;24: Wang T, asota J, Hanau CA, et al. 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