Follicular Lymphoma Can Be Distinguished From Benign Follicular ...

Hematopathology / ANALYSIS OF BCL-2 IN LYMPHOMA AND HYPERPLASIA

Follicular Lymphoma Can Be Distinguished From Benign Follicular Hyperplasia by Flow Cytometry Using Simultaneous Staining of Cytoplasmic bcl-2 and Cell Surface CD20 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 (FL) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments. Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining. CD20– T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of FL showed greater bcl2 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 FL and the germinal center cells of FH. The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL 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.

258

Am J Clin Pathol 2000;114:258-263

The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) 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 FL by immunohistochemical means.2,3 The overexpression of bcl-2 in most cases of FL results in its intense staining in the nodules of FL, 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 CD20 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 CD20, FL 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. FL 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 FL, including a nodular architecture on formalin-fixed H&E-stained tissue; (2) flow cytometric © American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE

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 FL, no immunoglobulin light chain expression was demonstrated on B cells. Of the 17 cases of FL, 16 expressed CD10, 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 106 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 Labware, Franklin Lakes, 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 100 µL of a 1:100 dilution of propidium iodide in 1.12% sodium citrate buffer before analysis. In addition, a combined surface CD20 and intracellular bcl-2 staining was performed. Approximately 106 cells were first exposed to 10 µL of phycoerythrin-conjugated anti-CD20 (Leu16, 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 10 µL 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 500 µL of PBS. A FACScan or a FACSCalibur (Becton Dickinson Immunocytometry Systems) was used for cell analysis, and data analysis was performed by using Lysis or CellQuest software (Becton Dickinson Immunocytometry Systems). The fluorescence intensity of the CD20 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 CD20-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. © American Society of Clinical Pathologists

Molecular Analysis Analysis of bcl-2 gene rearrangement was performed according to the method of Liu et al9 with minor modifications. In short, DNA was extracted from tissue or cell suspensions fixed in a 50% ethanol-RPMI mixture using a nonorganic method.10 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 Liu et al.9 A 200-ng aliquot of genomic DNA was amplified in a Perkin Elmer Cetus 9600 thermocycler with 1.25 U of Taq polymerase (Perkin Elmer, Norwalk, CT) in a 50-µL reaction volume containing tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.3 (Perkin Elmer) and final concentrations of 1.0 mmol/L of magnesium chloride, 200 µmol/L of dNTP ([deoxynucleoside triphosphate] Amersham Pharmacia Biotech, Piscataway, NJ), and 0.5 µmol/L of each primer. Amplification products were electrophoresed through a 1.75% agarose gel (Ultrapure, GibcoBRL, 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 CD20 in most FH samples revealed distinct populations of bright CD20+, dim CD20+, and CD20– cells ❚Figure 1A❚. These subpopulations represent mostly germinal center, mantle, and T cells, respectively.7 In contrast, the presence of neoplastic cells uniformly expressing CD20 in FL produced an obliteration of the 2 distinct B-cell populations noted in FH, resulting in a single CD20+ 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 FL (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 CD20 expression, showed a near absence of bcl-2 (MFI = 1.5 ± 0.6). In FL, 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 < .0002), as well as all other cell populations of FH, particularly the FH germinal center cells (P < .0001). The population of CD20– cells (mostly T Am J Clin Pathol 2000;114:258-263

259

Cornfield et al / ANALYSIS OF BCL-2 IN LYMPHOMA AND HYPERPLASIA

A

B

104

104

L T

T

103

M bcl-2

bcl-2

103

102

102

GC 101

101

100

100 100

101

102

103

100

104

101

102

103

104

CD20

CD20

❚Figure 1❚ Correlated analysis of surface CD20 and intracellular bcl-2 in a representative case of follicular hyperplasia (A) and follicular lymphoma (B). A, CD20 identifies 3 distinct populations with increasing levels of CD20 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: CD20– T lymphocytes (T) and CD20+ lymphoma cells (L) (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.

bcl-2 Expression (MFI)

60

10

1

0.5 T Cells

B Cells Mantle

B Cells Germinal Center

Follicular Hyperplasia

T Cells

B Cells

Follicular Lymphoma

❚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 < .0002) and the germinal center cells in follicular hyperplasias (P < .0001). Also, the T cells of follicular lymphomas demonstrated a significantly higher bcl-2 expression than the T cells in follicular hyperplasias (P = .02). In cases of partial lymphoma involvement, the MFI of the bcl-2 expression corresponds only to the neoplastic component. Error bars represent SD.

260

Am J Clin Pathol 2000;114:258-263

© American Society of Clinical Pathologists


Polytypic B Cells

A

lambda

1023

L 0 0

1023

CD20 Polytypic B Cells

B

1023

kappa

cells) in cases of FL demonstrated a significantly higher bcl2 expression (MFI = 7.1 ± 2.3) than the equivalent population in cases of FH (MFI = 4.7 ± 1.6; P = .02). In 5 cases, there was only partial involvement of the node by FL, and neoplastic B cells were admixed with numerous normal B cells. These 2 B-cell populations were different in CD20 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 CD20+ cells, corresponding to the lymphoma elements, showed higher bcl-2 expression than the less intense CD20+ normal B cells ❚Figure 3C❚. The bcl-2 MFI of the neoplastic cells in these FL 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 10 (59%) of 17 cases of FL in this series. The intensity of bcl-2 expression in lymphoma cells of cases with a molecular translocation (25.50 ± 16.32) did not differ significantly from that of cases not demonstrating such a translocation (17.37 ± 9.35; P = .21).

L

0

Discussion


1023

CD20

C 104 Polytypic B Cells

103 bcl-2

By using a relatively simple flow cytometric technique that used simultaneous staining of cell surface CD20 and intracellular bcl-2, we demonstrated a pattern of bcl-2 expression in FL that is distinct from that of FH. CD20 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 FL, the CD20+ 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 CD20+ germinal center cells, which typically do not express bcl-2. The differences in bcl-2 expression between normal and FL 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 FL. The distinction between FH and FL 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 FL. Immunophenotyping by flow cytometry can provide additional and often diagnostic information and is

0

102

L

101 100 100

101

102

103

104

CD20

❚Figure 3❚ Lymph node partially involved by follicular lymphoma (FL). The node contains normal B cells as well as FL cells. In A and B, simultaneous surface CD20 and immunoglobulin light chain staining identifies normal (polytypic) B cells (vertical boxes) consisting of a mixture of kappa- and lambda-bearing cells. Lymphoma cells (L) express (faintly) kappa but no lambda immunoglobulin. C, A high level of bcl-2 is shown in the more intensely CD20-expressing lymphoma cells.

Am J Clin Pathol 2000;114:258-263

261

Cornfield et al / ANALYSIS OF BCL-2 IN LYMPHOMA AND HYPERPLASIA

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 FL from FH is based largely on the identification of a B-cell population with immunoglobulin light chain restriction and expression of CD10.8 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 FL. 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 FL but failed to express monoclonal light chain immunoglobulin by flow cytometry. Both cases demonstrated bcl-2 expression in the intense CD20+ cell population. It should be noted that approximately 10% to 15% of FLs 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 CD10 expression by a B-cell population, might suggest the diagnosis. The finding that T lymphocytes express higher levels of bcl-2 in FL than in FH was unexpected. The factors that govern bcl-2 expression in reactive T lymphocytes are largely unknown. More than 80% 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 CTLL2.12 It might be postulated that the presence of interleukins or other factors in the local environment of FL or even direct contact between the neoplastic cells of FL and tumor-infiltrating T lymphocytes results in stimulation of Tcell 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 FL 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 262

Am J Clin Pathol 2000;114:258-263

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 FL is much less clear, with some observers claiming a poor overall prognosis15 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 Laboratory Medicine, and Shands Hospital, University of Florida College of Medicine, Gainesville, FL. Address reprint requests to Dr Braylan: Hematopathology Section, Dept of Pathology and Laboratory Medicine, Box 100275, University of Florida College of Medicine, Gainesville, FL 32610.

References 1. Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990;348:334-336. 2. Utz GL, Swerdlow SH. Distinction of follicular hyperplasia from follicular lymphoma in B5-fixed tissues: comparison of MT2 and bcl-2 antibodies. Hum Pathol. 1993;24:1155-1158. 3. Wang T, Lasota J, Hanau CA, et al. Bcl-2 oncoprotein is widespread in lymphoid tissue and lymphomas but its differential expression in benign versus malignant follicles and monocytoid B-cell proliferations is of diagnostic value. APMIS. 1995;103:655-662. 4. Gaulard P, d’Agay MF, Peuchmaur M, et al. Expression of the bcl-2 gene product in follicular lymphoma. Am J Pathol. 1992;140:1089-1095. 5. Zutter M, Hockenbery D, Silverman GA, et al. Immunolocalization of the Bcl-2 protein within hematopoietic neoplasms. Blood. 1991;78:1062-1068. 6. Ngan BY, Chen-Levy Z, Weiss LM, et al. Expression in nonHodgkin’s lymphoma of the bcl-2 protein associated with the t(14;18) chromosomal translocation. N Engl J Med. 1988;318:1638-1644. 7. Gadol N, Peacock MA, Ault KA. Antigenic phenotype and functional characterization of human tonsil B cells. Blood. 1988;71:1048-1055. 8. Almasri NM, Iturraspe JA, Braylan RC. CD10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles. Arch Pathol Lab Med. 1998;122:539-544. 9. Liu J, Johnson RM, Traweek ST. Rearrangement of the BCL-2 gene in follicular lymphoma: detection by PCR in both fresh and fixed tissue samples. Diagn Mol Pathol. 1993;2:241-247. 10. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.



11. Aiello A, Delia D, Borrello MG, et al. Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells. Cytometry. 1992;13: 502-509. 12. Deng G, Podack ER. Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2–mediated gene transcription and deregulated expression of the protooncogene bcl-2. Proc Natl Acad Sci U S A. 1993;90:2189-2193. 13. Hermine O, Haioun C, Lepage E, et al. Prognostic significance of bcl-2 protein expression in aggressive nonHodgkin’s lymphoma. Groupe d’Etude des Lymphomes de l’Adulte (GELA). Blood. 1996;87:265-272. 14. Gascoyne RD, Adomat SA, Krajewski S, et al. Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin’s lymphoma. Blood. 1997;90:244-251.


15. Martinka M, Comeau T, Foyle A, et al. Prognostic significance of t(14;18) and bcl-2 gene expression in follicular small cleaved cell lymphoma and diffuse large cell lymphoma. Clin Invest Med. 1997;20:364-370. 16. Wendum D, Sebban C, Gaulard P, et al. Follicular large-cell lymphoma treated with intensive chemotherapy: an analysis of 89 cases included in the LNH87 trial and comparison with the outcome of diffuse large B-cell lymphoma. Groupe d’Etude des Lymphomes de l’Adulte. J Clin Oncol. 1997;15:1654-1663. 17. Pezzella F, Jones M, Ralfkiaer E, et al. Evaluation of bcl-2 protein expression and 14;18 translocation as prognostic markers in follicular lymphoma. Br J Cancer. 1992;65:87-89.

Am J Clin Pathol 2000;114:258-263

263