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 quanti-
tatively 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 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 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
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The distinction between benign follicular hyperplasia
(FH) and follicular lymphoma (FL) can sometimes be chal-
lenging for surgical pathologists. The bcl-2 oncoprotein, a
26-kd protein that prolongs cell survival by inhibiting apop-
tosis,1 has been a particularly useful target for distinguishing
FH from FL by immunohistochemical means.2,3 The overex-
pression 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 interfollic-
ular 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 labo-
ratory 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 find-
ings 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

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. Approxi-
mately 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 deter-
mined 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
Hematopathology / ORIGINAL ARTICLE
Molecular Analysis
Analysis of bcl-2 gene rearrangement was performed
according to the method of Liu et al9 with minor modifica-
tions. 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 break-
point region and minor cluster region were used as described
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by Liu et al.9 A 200-ng aliquot of genomic DNA was ampli-
fied 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 chlo-
ride, 200 µmol/L of dNTP ([deoxynucleoside triphosphate]
Amersham Pharmacia Biotech, Piscataway, NJ), and 0.5
µmol/L of each primer. Amplification products were elec-
trophoresed through a 1.75% agarose gel (Ultrapure, Gibco-
BRL, Gaithersburg, MD) and transferred by semidry elec-
troblotting onto nylon membranes. They then were probed
with 3′ tailed digoxigenin-labeled oligonucleotides, and ampli-
fied 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 repre-
sent mostly germinal center, mantle, and T cells, respec-
tively.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
103 T 103 T
M

bcl-2
bcl-2

102 102

GC

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101 101

100 100

100 101 102 103 104 100 101 102 103 104

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.

60
bcl-2 Expression (MFI)

10

0.5

T Cells B Cells B Cells T Cells B Cells

Mantle Germinal
Center

Follicular Hyperplasia 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

 Hematopathology / ORIGINAL ARTICLE

cells) in cases of FL demonstrated a significantly higher bcl- A Polytypic

2 expression (MFI = 7.1 ± 2.3) than the equivalent popula- B Cells

tion in cases of FH (MFI = 4.7 ± 1.6; P = .02).

In 5 cases, there was only partial involvement of the 1023
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 deter-

lambda
mined 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

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chain expression ❚Figure 3A❚ and ❚Figure 3B❚. The intensely L
staining CD20+ cells, corresponding to the lymphoma 0
0 1023
elements, showed higher bcl-2 expression than the less CD20
intense CD20+ normal B cells ❚Figure 3C❚. The bcl-2 MFI of B Polytypic
the neoplastic cells in these FL cases with partial lymphoma B Cells

involvement (23.66 ± 16.35) was not different from that of

the more advanced cases (21.53 ± 13.85). 1023
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

kappa
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 0 CD20 1023

By using a relatively simple flow cytometric technique C

that used simultaneous staining of cell surface CD20 and
104
intracellular bcl-2, we demonstrated a pattern of bcl-2
expression in FL that is distinct from that of FH. CD20 was Polytypic
103 B Cells
chosen because it is a universal marker of peripheral B cells
and is differentially expressed in mantle and germinal center
bcl-2

102 L
B cells.7 Other markers preferentially expressed in germinal
center cells could be used but are less discriminatory.8 In FL, 101
the CD20+ malignant cells showed strong bcl-2 expression.
This expression was higher than that of any cell subpopula- 100
tion of FH and strikingly different from the intensely CD20+ 100 101 102 103 104
germinal center cells, which typically do not express bcl-2. CD20
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 ❚Figure 3❚ Lymph node partially involved by follicular
of bcl-2 in the malignant cells was as high as that of lymphoma (FL). The node contains normal B cells as well as
neoplastic cells in nodes fully involved by FL. FL cells. In A and B, simultaneous surface CD20 and
The distinction between FH and FL sometimes may be immunoglobulin light chain staining identifies normal (poly-
extremely difficult. Molecular techniques and immunohis- typic) B cells (vertical boxes) consisting of a mixture of
tology can aid in establishing a firm diagnosis, but these kappa- and lambda-bearing cells. Lymphoma cells (L)
techniques are not sufficiently specific, may be subject to a express (faintly) kappa but no lambda immunoglobulin.
C, A high level of bcl-2 is shown in the more intensely
variety of methodologic artifacts, and fail to recognize some
CD20-expressing lymphoma cells.
cases of FL. Immunophenotyping by flow cytometry can
provide additional and often diagnostic information and is

© American Society of Clinical Pathologists 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 conve-
nience, 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 expres-
sion by flow cytometry adds an additional piece of confirma-
tory 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 approxi-
mately 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 mono-
typic 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 inter-
leukins 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 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 FL may express bcl-2, as
demonstrated by immunohistochemical methods in paraffin-
embedded 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
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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.
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