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Diagnostic Significance of CD20 and FMC7

Expression in B-Cell Disorders

Article in American Journal of Clinical Pathology · November 2003

DOI: 10.1309/FNGC-YEMJ-E3MA-E5L2 · Source: PubMed

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Hematopathology / CD20 AND FMC7 EXPRESSION IN B-CELL DISORDERS
Diagnostic Significance of CD20 and FMC7 Expression
in B-Cell Disorders
Julio Delgado, MD, Estella Matutes, MD, PhD, Alison M. Morilla, Ricardo M. Morilla, MSc,
Kwasi A. Owusu-Ankomah, Furheen Rafiq-Mohammed, MSc, Ilaria del Giudice, MD,
and Daniel Catovsky, DMSc, FRCPath, FRCP, FMedSci
Key Words: Chronic lymphocytic leukemia; CLL; Lymphomas; Scoring; CD20; FMC7; Diagnosis
DOI: 10.1309/FNGCYEMJE3MAE5L2
Abstract
We analyzed by flow cytometry the expression of
CD20 and FMC7 in cell suspensions from 932 patients,
including 630 cases of chronic lymphocytic leukemia
(CLL), 23 cases of other B-cell leukemias, and 279
cases of B-cell non-Hodgkin lymphoma (B-cell NHL).
CD20 was positive in 94.5% of cases; FMC7 was
positive in 35.7%. There was a correlation between
CD20 and FMC7 expression in patients with B-cell
NHL (P < .001) but not CLL (P = .1). We also tested a
scoring system in which FMC7 was replaced by CD20
and compared it with our current scoring system for
CLL. With this modification, the accuracy of the scoring
system for differentiating CLL from other non-CLL
disorders fell from 94.4% to 81.5%. In CD20+ CLL, the
intensity of CD20 expression correlated with FMC7
and low scores (P < .001 for both comparisons). We
suggest that the particular conformation of CD20
recognized by FMC7 is manifested only in cells with
strong CD20 expression, which is not the case for CLL.
FMC7 is of greater diagnostic value than CD20 for
distinguishing CLL from other B-cell disorders; we
recommend its continued use for this purpose.
754 Am J Clin Pathol 2003;120:754-759
754 DOI: 10.1309/FNGCYEMJE3MAE5L2
The monoclonal antibody FMC7 identifies a 105-kd
glycoprotein expressed in most normal mature B lympho-
cytes.1,2 Immunoblotting studies have demonstrated that
FMC7 reacts with a carbohydrate determinant or a determi-
nant of the protein that does not depend on its tertiary struc-
ture, since it withstands denaturation.1 In a diagnostic setting,
FMC7 is a reliable marker for distinguishing chronic
lymphocytic leukemia (CLL) from other B-cell lymphopro-
liferative disorders, 3,4 and, for that reason, it has been
included in the immunological scoring system used to distin-
guish CLL from other B-cell conditions.3,5,6
The monoclonal antibody CD20 recognizes a 35- to 37-
kd nonglycosylated phosphoprotein that is expressed in
mature B cells.7 Its role in the differential diagnosis of B-cell
disorders is lower than FMC7 because CD20 is positive in
virtually all cases.8
A recent report indicated that FMC7 binds to a particular
conformation of the multimeric CD20 complex. This state-
ment is based on the results of blocking studies that showed
mutual inhibition of FMC7 and CD20 and on the expression
of both FMC7 and CD20 in myeloid cells transfected with
plasmids containing CD20-encoded complementary DNA.9
Two studies have shown that FMC7 expression parallels that
of CD20 in cells from patients with a variety of malignant and
nonmalignant conditions.10,11 The authors suggested that the
use of FMC7 for diagnosis may be unnecessary, provided that
CD20 intensity also is considered. However, these conclusions
were based on a small number of cases and the use of many
samples from tissues or bone marrow specimens.10
The aim of the present study was to analyze the
expression of CD20 and FMC7 in cells from a large series
of B-cell disorders to reexamine the possibility that the
© American Society for Clinical Pathology
 Hematopathology / ORIGINAL ARTICLE

diagnostic value of CD20 is greater than or equal to that of antibodies were located in the left lower quadrant. A marker
FMC7. We also compared the standard scoring systems3,5,6 was considered positive when expressed in more than 30% of
with a modified scoring system in which FMC7 was cells above the control result. When estimating the reactivity
replaced by CD20, providing evidence that the latter is less of CD5 on the surface of B cells, CD5/CD19 coexpression and
accurate for distinguishing between CLL and other B-cell the percentage of CD2+ T cells were taken into account.
disorders using flow cytometry. The intensity of CD20, CD22, CD79b, and surface
immunoglobulins (sIg) by κ and λ expression was estimated in
a dot plot display selecting the logarithmic scale in the fluores-
cence axis and compared with the isotype control. The criteria
Materials and Methods
adopted were as follows: weak, positive peak within the first
Samples from 932 patients with B-cell disorders were logarithmic percentile; moderate, peak within the second
analyzed retrospectively. These included 721 peripheral blood percentile; and strong or very strong, peak within or more than
samples, 185 bone marrow specimens, 17 spleen or lymph node 3 percentiles.3
cell suspensions, and 9 samples from other fluids, comprising All cases were given a score according to their reactivity
630 cases of CLL, 23 cases of other B-cell leukemias, and 279 with CD5, CD23, and FMC7 and the intensity of sIg, CD79b,
cases of B-cell non-Hodgkin lymphomas (B-cell NHL) with and/or CD22 expression. A value of 1 or 0 was given
lymphocytosis ❚Table 1❚. All cases had a clonal B-cell popula- according to whether each marker was typical (CD5+,
tion, with a median of 80% on gated cells (range, 11%-99%). CD23+, FMC7–, weak expression of sIg, and weak or absent
The diagnosis was based on clinical features, cell expression of membrane CD22 and/or CD79b) or atypical for
morphologic features according to the French-American- CLL3,5,6 ❚Table 2❚. A modified scoring system also was tested
British proposals,12 immunologic markers,3 and histologic by replacing FMC7 with CD20 (1 point when negative, 0
features according to the World Health Organization classifi- points when positive).
cation.13 In certain diseases, additional tests were performed, All statistical analyses were performed using SPSS
ie, conventional cytogenetics, fluorescence in situ hybridiza- version 10.0 (SPSS, Chicago, IL). Contingency tables were
tion analysis for the t(11;14) and trisomy 12,14 and cyclin D1 analyzed by χ2 or Fisher exact tests as appropriate. Logistic
immunostaining,15 to confirm the diagnosis, particularly in regression was used to assess statistically significant differ-
cases of B-cell NHL and atypical CLL. ences between both scoring systems. In all statistical calcula-
Whole peripheral blood and bone marrow samples were tions, a P value of less than .05 was considered significant.
incubated at room temperature for 10 minutes with the appro-
priate amount of each antibody, exposed to an ammonium
chloride–based erythrocyte lysing solution, washed in phos- Results
phate-buffered saline, and resuspended in FACSFlow balanced
Correlation Between CD20 and FMC7 Reactivity
electrolyte solution (Becton Dickinson, Mountain View, CA).
Single and/or double immunostainings with fluorescein Considering all cases, CD20 was expressed in 94.5%,
isothiocyanate/phycoerythrin directly conjugated monoclonal while FMC7 was expressed in 35.7% ❚Table 3❚. There were
antibodies were used. These monoclonal antibodies were
CD5/CD19, CD23, CD22, CD20/CD79b, FMC7/CD2 and
κ/λ light chains. When a diagnosis of hairy cell leukemia was ❚Table 1❚
suspected, CD11c, CD25, and CD103 also were used.16 All Diagnosis of B-Cell Disorders
monoclonal antibodies were supplied by Caltag (Burlingame, Disease No. (%)
CA) except CD79b and CD103, which were provided by
Immunotech (Marseille, France) and Immunoquality Prod- Chronic lymphocytic leukemia 630 (67.6)
Typical 560 (60.1)
ucts (Groningen, the Netherlands), respectively. Fluorescein Atypical 70 (7.5)
isothiocyanate– and phycoerythrin-conjugated mouse Other mature B-cell leukemias 23 (2.5)
B-cell prolymphocytic leukemia 1 (0.1)
immunoglobulins of the IgG1 subclass (Caltag) were used as Hairy cell leukemia 21 (2.3)
negative controls in every experiment. Hairy cell leukemia variant 1 (0.1)
B-cell non-Hodgkin lymphomas 279 (29.9)
Immunophenotypic analysis was performed on a Splenic with villous lymphocytes 60 (6.4)
FACScalibur flow cytometer (Becton Dickinson) using a Mantle cell 31 (3.3)
lymphocyte gate by forward and side scatter characteristics of Follicular lymphoma 22 (2.4)
Lymphoplasmacytic 11 (1.2)
the cells. Two-parameter dot plots and quadrant statistics were Large cell 11 (1.2)
generated by CellQuest software (Becton Dickinson). Quad- Other 144 (15.5)
Total 932
rants were set in such a way that cells labeled with the control

© American Society for Clinical Pathology Am J Clin Pathol 2003;120:754-759 755

755 DOI: 10.1309/FNGCYEMJE3MAE5L2 755
Delgado et al / CD20 AND FMC7 EXPRESSION IN B-CELL DISORDERS

❚Table 2❚ Analysis of CD20 as Part of the Scoring System

Scoring System for the Diagnosis of Chronic Lymphocytic According to the scoring system from Matutes et al,3
Leukemia6*
modified by Moreau et al5 and updated by Matutes and
Points Polliack,6 91.7% of CLL cases scored 4 or 5, and only a
1 0
minority (2.5%) had a low score (1-2). In contrast, most B-
cell NHL and B-cell leukemia cases other than CLL had
CD5 + – scores ranging from 0 to 2 ❚Table 4❚.
CD23 + –
FMC7 – + When CD20 was used instead of FMC7 in the scoring
sIg Weak Moderate to strong system, only 72.7% of CLL cases had scores of 4 or 5, 21.6%
CD79b and CD22 Weak Moderate to strong
scored 3, and 5.7% had low scores (0-2) ❚Table 5❚. Thus, the
+, positive; –, negative. proportion of CLL cases with a score of 3 rose significantly,
* Scores in chronic lymphocytic leukemia range from 3 to 5; in other B-cell

disorders, they range from 0 to 2.
no differences in the percentage of CD20+ cells in cases of
CLL (95.4%) compared with other B-cell leukemias (100%
[23/23]) and B-cell NHLs (92.1%).
In contrast, cells from 85.7% of CLL cases were nega-
tive for FMC7, whereas most B-cell NHL and other B-cell
leukemia cases were positive for FMC7 (79.2% and 96%
[22/23], respectively) ❚Image 1❚. These differences were
statistically significant (P < .001; Fisher exact test).
CLL cases with typical morphologic features more
frequently were negative for FMC7 than those with atypical
morphologic features (88.2% vs 66% [46/69]). This differ-
ence was statistically significant (P < .001).
A correlation was found between CD20 and FMC7 expres-
sion in cases of B-cell NHL when a 30% cutoff level for posi-
tivity was used (P < .001; Table 3). However, in CLL cases, there
was no correlation between CD20 and FMC7 positivity (P = .1;
Table 3), even when atypical CLL was analyzed separately.
from 5.7% to 21.6% when using CD20 for scoring (P = .001;
Tables 4 and 5).
By using the 5 standard markers, the accuracy of the
scoring system for differentiating between CLL and non-CLL
was 94.4% (95% confidence interval, 92.9%-95.9%) when a
cutoff value of 4 points or higher was used. When replacing
FMC7 with CD20, the accuracy was 81.5% (95% confi-
dence interval, 79.0%-83.9%) (P < .001).
CD20 Intensity
Analysis of CD20 intensity in CD20+ cases showed that
cells from 83.5% of the CLL cases had weak or moderate
CD20 intensity compared with 13.6% of B-cell NHL cases and
17% of other B-cell leukemia cases (4/23) (P < .001) ❚Table 6❚.
Within the CD20+ CLL cases, CD20 intensity corre-
lated with FMC7 expression; FMC7+ cases had a stronger
CD20 intensity than FMC7– CLL cases (P < .001; χ2 test)
❚Table 7❚. The intensity of CD20 also correlated with the
scores. Cases with high scores had moderate or weak CD20
intensity, while those with low scores had strong CD20
intensities (P < .001; χ2 test).

❚Table 3❚
Comparison of CD20 and FMC7 Reactivity for Each B-Cell Disorder*

FMC7 CD20

Disease Negative Positive Negative Positive P

Chronic lymphocytic leukemia (n = 630) 540 (85.7) 90 (14.3) 29 (4.6) 601 (95.4) .104
Typical (n = 560) 494 (88.2) 66 (11.8) 28 (5.0) 532 (95.0) .233
Atypical (n = 70) 46 (66) 24 (34) 1 (1) 69 (99) 1.0
Other mature B-cell leukemias (n = 23) 1 (4) 22 (96) 0 (0) 23 (100) —
B-cell prolymphocytic leukemia (n = 1) 0 (0) 1 (100) 0 (0) 1 (100) —
Hairy cell leukemia (n = 21) 1 (5) 20 (95) 0 (0) 21 (100) —
Hairy cell leukemia variant (n = 1) 0 (0) 1 (100) 0 (0) 1 (100) —
B-cell non-Hodgkin lymphomas (n = 279) 58 (20.8) 221 (79.2) 22 (7.9) 257 (92.1) <.001
Splenic with villous lymphocytes (n = 60) 11 (18) 49 (82) 4 (7) 56 (93) .001
Mantle cell (n = 31) 3 (10) 28 (90) 2 (6) 29 (94) .006
Follicular lymphoma (n = 22) 4 (18) 18 (82) 1 (5) 21 (95) .182
Lymphoplasmacytic (n = 11) 5 (45) 6 (55) 2 (18) 9 (82) .182
Large cell (n = 11) 2 (18) 9 (82) 1 (9) 10 (91) .182
Other (n = 144) 33 (22.9) 111 (77.1) 12 (8.3) 132 (91.7) <.001
Total (n = 932) 599 (64.3) 333 (35.7) 51 (5.5) 881 (94.5) <.001
* Data are given as number (percentage). Fisher exact test P values apply to each comparison.

756 Am J Clin Pathol 2003;120:754-759 © American Society for Clinical Pathology

756 DOI: 10.1309/FNGCYEMJE3MAE5L2
 Hematopathology / ORIGINAL ARTICLE

Discussion A B

Immunophenotyping is essential for the diagnosis and 104 104

subclassification of B-cell disorders. Although there is some
103 103
overlap on membrane marker expression in the various B-

CD79b PE
CD2 PE
cell disorders, some of them have a distinct immunopheno- 102 102
typic profile (eg, CLL, hairy cell leukemia). In this context,
101 101
the CLL scoring system was devised to distinguish CLL
from other B-cell disorders. 3,5 Although early reports 100 100
suggested that most CLL cases were CD79b–,5,17 the use of a 100 101 102 103 104 100 101 102 103 104
Anti-FMC7 FITC CD20 FITC
phycoerythrin-conjugated CD79b monoclonal antibody has
shown that CD79b is expressed weakly in most CLL C D
cases6,18 and, thus, should be considered as typical of CLL (1
point) when negative or weakly positive. 104 104
FMC7 was included in the score because of its discrimi-
103 103
native power between CLL (negative) and other B-cell

CD79b PE
CD2 PE
conditions. Indeed, FMC7 and sIg expression proved to be 102 102
the most reliable markers for discriminating CLL from other
101 101
B-cell neoplasms.3 It has been reported that FMC7 binds to a
multimeric CD20 complex.9 Because of this, a few reports 100 100
suggested that CD20 might well replace FMC7 for discrimi- 100 101 102 103 104 100 101 102 103 104
Anti-FMC7 FITC CD20 FITC
nating CLL from non-CLL diagnoses.10,11
In the present study, we analyzed the diagnostic value ❚Image 1❚ A and B, Dot plots of a case of chronic lymphocytic
of the expression of CD20 as a discriminative marker leukemia showing weak expression of CD20 and CD79b and
(CLL vs non-CLL) and compared it with FMC7 in a large negative FMC7 expression. C and D, Dot plots of a case of B-
group of cases involving a variety of B-cell disorders. Our cell lymphoma showing strong expression of CD20, CD79b,
data show that in B-cell NHL, CD20 expression correlates and FMC7. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
well with that of FMC7, as with other B-cell markers (eg,
CD22 and CD79b). In contrast, such a correlation was not ❚Table 4❚
found in CLL when considering the percentage of positive Scores in Chronic Lymphocytic Leukemia and Other B-Cell
Disorders
cells; only within the CD20+ CLL cases did the intensity
of CD20 correlate with the expression of FMC7. Never- No. (%) of Cases
theless, assessment of antigen density instead of propor- Chronic Lymphocytic Other B-Cell B-Cell Non-Hodgkin
tion of positive cells may be more subjective and unreli- Score* Leukemia (n = 630) Leukemias (n = 23) Lymphoma (n = 279)†
able owing to reagent and operator variability. When 5 390 (61.9) 0 (0) 0 (0.0)
FMC7 was replaced by CD20 in the scoring system, 4 188 (29.8) 0 (0) 0 (0.0)
results were less discriminative as 27.3% of CLL cases 3 36 (5.7) 0 (0) 8 (2.9)
2 10 (1.6) 1 (4) 45 (16.1)
had scores of 3 or lower. It seems that CD20 expression 1 6 (1.0) 12 (52) 139 (48.7)
(assessed as negative or positive) does not improve the 0 0 (0.0) 10 (43) 87 (31.2)
accuracy of the scoring system. * According to Matutes and Polliack6 using CD79b and CD22 intensity of expression.
† Cases of B-cell non-Hodgkin lymphoma with scores of 3 comprised essentially
mantle cell lymphoma known to be CD5+.

❚Table 5❚
The Scoring System (FMC7 Replaced by CD20)

No. (%) of Cases

Score Plus CD20 Chronic Lymphocytic Other B-Cell B-Cell Non-Hodgkin

Minus FMC7 Leukemia (n = 630) Leukemias (n = 23) Lymphoma (n = 279)

5 19 (3.0) 0 (0) 0 (0.0)

4 439 (69.7) 0 (0) 0 (0.0)
3 136 (21.6) 0 (0) 5 (1.8)
2 30 (4.8) 0 (0) 41 (14.7)
1 6 (1.0) 13 (57) 122 (43.7)
0 0 (0.0) 10 (43) 111 (39.8)

© American Society for Clinical Pathology Am J Clin Pathol 2003;120:754-759 757

757 DOI: 10.1309/FNGCYEMJE3MAE5L2 757
Delgado et al / CD20 AND FMC7 EXPRESSION IN B-CELL DISORDERS

❚Table 6❚
Analysis of CD20 Intensity in CD20+ Cases According to Different B-Cell Disorders*

CD20 Intensity

Disease Weak Moderate Strong or Very Strong Total

Chronic lymphocytic leukemia 117 (19.5) 385 (64.1) 99 (16.5) 601

Typical 104 (19.5) 357 (67.1) 71 (13.3) 532
Atypical 13 (19) 28 (41) 28 (41) 69
Other mature B-cell leukemias 0 (0) 4 (17) 19 (83) 23
B-cell prolymphocytic leukemia 0 (0) 0 (0) 1 (100) 1
Hairy cell leukemia 0 (0) 4 (19) 17 (81) 21
Hairy cell leukemia variant 0 (0) 0 (0) 1 (100) 1
B-cell non-Hodgkin lymphomas 1 (0.4) 34 (13.2) 222 (86.4) 257
Splenic with villous lymphocytes 0 (0) 6 (11) 50 (89) 56
Mantle cell 0 (0) 0 (0) 29 (100) 29
Follicular lymphoma 0 (0) 1 (5) 20 (95) 21
Lymphoplasmacytic 0 (0) 2 (22) 7 (78) 9
Large cell 0 (0) 5 (50) 5 (50) 10
Other 1 (0.8) 20 (15.1) 111 (84.1) 132
Total 118 (13.4) 423 (48.0) 340 (38.6) 881
* Data are given as number (percentage). P < .001; χ2 test; P value applies to comparison of CD20 intensity with chronic lymphocytic leukemia, other B-cell leukemias, and
B-cell non-Hodgkin lymphomas.

❚Table 7❚ expressed in CLL,8,19 and that many other proteins, like L-

Correlation of CD20 Intensity With FMC7 Expression and selectin and leukocyte common antigen, are glycosylated
Scores6 Within CD20+ Chronic Lymphocytic Leukemia Cases*
aberrantly in CLL,21,22 it is possible that the abnormal glyco-
CD20 Intensity sylation pattern of CD20 observed in CLL may account for
Strong or the absence of FMC7 expression in this disease. Although
Weak Moderate Very Strong Total FMC7 is a better discriminating marker for CLL and B-cell
FMC7 Reactivity
NHL by flow cytometry in cell suspensions, FMC7 cannot
Negative 115 (22.5) 335 (65.4) 62 (12.1) 512 be used in tissue sections studied by immunohistochemical
Positive 2 (2) 50 (56) 37 (42) 89 analysis. Therefore, analysis of CD20 is useful to determine
Score
0-2 0 (0) 3 (19) 13 (81) 16 the possible use of rituximab in patients with B-cell disor-
3 3 (8) 19 (53) 14 (39) 36 ders and is recommended for use in immunohistochemical
4-5 114 (20.8) 363 (66.1) 72 (13.1) 549
analysis and flow cytometry for clinical purposes.
* Data are given as number (percentage). P < .001; χ2 test; P value refers to
comparison of CD20 intensity with FMC7 expression and with chronic
lymphocytic leukemia scores. From the Academic Department of Haematology and
Cytogenetics, the Royal Marsden NHS Trust and Institute of
Cancer Research, London, England.
Second, we assessed the intensity of CD20 expression in Address reprint requests to Dr Matutes: Academic Dept of
all CD20+ malignant neoplasms. As previously reported, Haematology and Cytogenetics, the Royal Marsden NHS Trust,
CLL cells showed weaker CD20 expression compared with Fulham Rd, London SW3 6JJ, England.
those in B-cell NHLs and other B-cell leukemias.8,19,20 The
weak expression of CD20 in CLL parallels, to some extent,
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