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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 624-631
Reduced Expression of Adhesion Molecules and Cell Signaling
Receptors by Chronic Lymphocytic Leukemia Cells With 11q Deletion
By
Sabine Sembries,
Heike Pahl,
Stephan Stilgenbauer,
Hartmut Döhner, and
Folke Schriever
From the Medizinische Klinik, mit Schwerpunkt Hämatologie und
Onkologie, Charité, Campus Virchow-Klinikum, Humboldt
Universität, Berlin, Germany; Institut für Experimentelle
Anaesthesiologie, Klinikum der Albert-Ludwigs-Universität,
Freiburg, Germany; and Medizinische Klinik und Poliklinik V,
Ruprecht-Karls-Universität, Heidelberg, Germany.
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ABSTRACT |
Deletions in chromosome bands 11q22-q23 were recently shown to be
one of the most frequent chromosome aberrations in B-cell chronic
lymphocytic leukemia (B-CLL). Patients suffering from B-CLL with 11q
deletion are characterized by extensive lymphadenopathy, rapid disease
progression, and short survival times. Phenotypic and functional
characteristics of B-CLL cells with 11q deletion that may help to
explain the pathophysiology of this entity are yet unknown. In the
present study, B-CLL cells with (n = 19) and without (n = 19) 11q
deletion were analyzed for their expression of functionally relevant
cell surface molecules (n = 57). B-CLL cells with 11q
deletion carried significantly lower levels of the adhesion molecules
CD11a/CD18 (integrin  L/ 2), CD11c/CD18 (integrin
X/2), CD31 (PECAM-1), CD48, and CD58 (LFA-3).
Furthermore, B-CLL cells with 11q deletion expressed less the cell
signaling receptors CD45 (leukocyte common antigen [LCA]), CD6, CD35
(complement receptor 1), and CD39. Reduced CD45 levels and low-level
expression of CD49d correlated with decreased overall survival. B-CLL
cells with or without 11q deletion did not differ in their growth
fractions, expression levels of transcription factor NF- B, or their
response to mitogenic stimuli. Decreased levels of functionally
relevant adhesion molecules and of cell signaling receptors may
contribute to the pathogenesis of the subgroup of B-CLL characterized
by 11q22-q23 deletion.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DELETIONS IN chromosome bands 11q22-q23
were recently shown to be one of the most frequent chromosome
aberrations in B-cell chronic lymphocytic leukemia
(B-CLL).1,2 In an interphase cytogenetic study of 214 patients with B-CLL, such 11q deletions were found in 20% of the cases
and were the second most common chromosome aberration after 13q14
deletions.2 Patients with 11q deletion had a characteristic
clinical picture. These patients were younger than other B-CLL
patients, they had extensive lymphadenopathy, and they suffered from
B-symptoms more frequently. Importantly, the presence of 11q deletion
was an independent prognostic factor predicting rapid disease
progression and short survival times. The critical region of these
deletions has been delineated to a 2- to 3-Mb sized genomic segment in
bands 11q22.3-q23.1.3 This genomic region likely contains a
novel tumor-suppressor gene that is important for the development and
progression of this clinically relevant subgroup of B-CLL. Known
candidate genes within this region include radixin (RDX), which
has homology to the neurofibromatosis-type 2 (NF2) tumor-suppressor
gene,4 and the ataxia telangiectasia mutated (ATM)
gene.5 Evidence that the ATM gene functions as a
tumor-suppressor gene comes from murine knock-out models and from the
recent observation of biallelic mutations of the gene in T-cell
prolymphocytic leukemia.6-8
The characteristic extensive lymphadenopathy of B-CLL cases with 11q
deletions points towards functional aberrations of these leukemic
cells. Massive infiltration of secondary lymphoid organs likely
involves the action of multiple adhesion molecules regulating homophilic and heterophilic binding processes. It has recently been
shown that B-CLL cells posses several different adhesion pathways and
these appear to vary with the stage of the disease.9 We
therefore investigated whether B-CLL cells with or without 11q deletion
differ in their expression pattern of functionally relevant adhesion
molecules and of other cell signaling receptors.
The present study demonstrates that B-CLL cells with 11q deletion
express significantly lower levels of several adhesion molecules and
proteins regulating relevant cell functions.
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MATERIALS AND METHODS |
Antibodies.
Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies
(MoAbs) against CD5 (BL1a), CD11a (integrin L,
LFA-1; 25.3.1), CD21 (CR2, EBVR; BL13), CD23 (9P.25), CD29
(integrin 1; K20), CD49d (integrin 4, VLA4; HP2/1), CD54
(ICAM-1; 84H10), CD80 (B7.1; MAB104), FMC7 (FMC7), phycoerythrin
(PE)-conjugated anti-CD19 (J4.119), and PE-conjugated mouse IgG1
(isotype control; 679.1Mc7) were purchased from Immunotech (Hamburg,
Germany). Unconjugated MoAbs against the cell surface markers CD6 (SPVL
14), CD10 (CALLA; ALB1), CD11b (integrin M, MAC-1;
BEAR1), CD11c (integrin X, p150, 95; BU15), CD18
(integrin 2; 7E4), CD20 (B9E9), CD22 (SJ.10.1H11), CD24 (ALB9), CD26
(BA5), CD27 (LT27), CD30 (HRS-4), CD31 (PECAM-1; 5.6E), CD32 (2E1),
CD35 (CR1; J3.D3), CD37 (BL14), CD38 (T16), CD39 (AC2), CD40 (MAB89),
CD40L (TRAP1), CD44 (J-173), CD46 (J4-48), CD48 (J4-57), CD49b
(integrin 2, VLA2; Gi9), CD49c (integrin 3, VLA3; M-KID2),
CD49e (integrin 5, VLA5; SAM), CD49f (integrin 6, VLA6;
GoH3), CD50 (ICAM-3; HP2/19), CD51 (integrin V; AMF7), CD58 (LFA-3; AICD58), CD61 (integrin 3; SZ.21), CD62L (L-selectin; DREG65), CD69 (TP1/55.3.1), CD70 (HNE51), CD71 (YDJ.1.2.2.), CD72 (J3.109), CD77 (38-13), CD79b (CB3-1), CD81 (TAPA-1; JS64), CD95 (APO-1, Fas; CH11), CD102 (ICAM-2; B-T1), CD103 (HML-1; 2G5.1), surface
IgM (sIgM; AF6), and surface IgD (sIgD; JA11) were acquired from
Immunotech. Anti-CD86 [2331 (FUN-1)], anti- (G20-193), and anti- (JDC-12) were obtained from Pharmingen (San Diego, CA). The
isotype control mouse IgM (R4A3-22-12) was purchased from Coulter Clone
(Coulter Corp, Miami, FL). Anti-CD43 (DF-T1) and anti-CD45 [LCA;
T29/33.(1)] were obtained from Dako (Glostrup, Denmark).
FITC-conjugated rabbit-antimouse F(ab )2 fragments
(Dako) served as the secondary antibody for unconjugated MoAbs.
Contaminating T cells, monocytes, and natural killer (NK) cells were
quantified using MoAbs against CD3 (FITC-conjugated; UCHT-1), CD14
(RMO52), and CD56 (T199), respectively (all purchased from Immunotech). Expression of the nuclear proliferation antigen Ki-67 was determined using a FITC-conjugated MoAb (MIB-1) from Dako. IgG1-FITC (Pharmingen) served as isotype control.
Proper function of the MoAbs was verified using defined cell
preparations as positive controls for each antibody used
(Fig 1): Burkitt lymphoma cell line Daudi
(CD10, CD24, CD37, CD71, CD79b, CD102, surface IgM, and light
chain); Burkitt lymphoma cell line Raji (CD20, CD21, CD22, CD40, CD45,
CD54, CD80, CD81, CD86, and Ki-67); CLL cell line EHEB (obtained from
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany; CD23, CD30, CD39, CD43, CD44, CD46, CD48, CD50,
CD58, CD70, CD77, and FMC7); normal activated peripheral blood T
lymphocytes at day 14 of culture with 300 U/mL interleukin-2
(IL-210; CD5, CD6, CD11a, CD18, CD26, CD27, CD29, CD38,
CD40L, CD49b, CD49d, CD49e, CD69, CD95, and CD103); normal platelets
(CD51 and CD61), normal granulocytes (CD62L), and normal monocytes
(CD11b, CD11c, CD31, and CD32) were analyzed in peripheral blood by
setting the gates according to the characteristic pattern of these
cells in forward and light scatter analysis; normal peripheral blood B lymphocytes (CD35, CD72, and surface IgD) were identified using a
PE-conjugated anti-CD19 MoAb; leukemic cells expressing lambda light
chain were isolated from a patient with prolymphocytic leukemia ( light chain); a greater than 97% cytokeratin-positive primary colon
carcinoma cell line (CD49c and anti-CD49f) was generously donated by B. Trojaneck (Charité, Berlin, Germany).
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| Fig 1.
Expression pattern of cell surface antigens (n = 57) by
B-CLL cells with ( ) and without ( ) 11q deletion and by positive
control cell preparations ( ; see Materials and Methods for details).
Marked by the box diagram are the 10th, 25th, 50th (median), 75th, and
the 90th percentiles. Antigens were combined into groups with high
(++), low (+), and no ( ) expression.
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To compare the survival times of patients who differ in the CD45 and
CD49d intensities on the B-CLL cells, discerning levels for these
antigens were defined. Cut-off level of CD49d was the mean fluorescence
that discriminated negative from positive antigen expression (mean
fluorescence of 2.8; Fig 1). Cut-off level of CD45 was defined as the
mean fluorescence that discerned patients who were alive from those who
died during follow-up (mean fluorescence of 400;
Fig 2A). Thus, B-CLL cells could be defined
as CD49dlow-positive (>2.9 mean fluorescence) and
CD49dnegative ( 2.9 mean fluorescence),
CD45high-positive (>400 mean fluorescence), and
CD45low-positive (400 mean fluorescence).
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| Fig 2.
B-CLL cells of patients with differing survival differ in
their expression levels of CD45 and CD49d. (A) Mean fluorescence of
CD45 and CD49d on B-CLL cells of all patients (with or without 11q
deletion) and on B-CLL cells with 11q deletion separated into groups of
cells of () patients who are alive and () patients who died.
Marked by the box diagram are the 10th, 25th, 50th (median), 75th, and
the 90th percentiles. ( ) Values below or above the 10th and 90th
percentile, respectively. (B) Kaplan-Meier curves of the overall
survival of all patients with B-CLL (with or without 11q deletion)
separated into groups of CD45high-positive (>425 mean
fluorescence), CD45low-positive (425 mean fluorescence),
CD49dlow-positive (>2.9 mean fluorescence), and
CD49dnegative (2.9 mean fluorescence) expression levels
on the B-CLL cells. Tick marks represent censored data on patients who
were alive or lost to follow-up.
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Patients and cells.
Mononuclear cells from 38 patients with B-CLL were analyzed. At the
time of analysis for the present study, 5 patients had Rai stage 0, 4 stage 1, 20 stage 2, 3 stage 3, and 6 stage 4 disease. Diagnosis of
B-CLL was confirmed by flow cytometric assessment of coexpression of
CD5, CD19, CD23, and surface Ig (low) on the B-CLL cells. Interphase
cytogenetic analysis was performed as described
previously.2 Nineteen cases had 11q deletion, whereas the
remaining 19 cases had a normal karyotype or other chromosome aberrations. As of June 1998, of the 38 patients whose B-CLL cells were
examined, 25 patients are alive and 12 patients have died. No survival
data of 1 patient could be obtained due to loss to follow-up. Mean
follow-up was 74 months (range, 12 to 216 months).
Immunophenotyping.
Expression of cell surface antigens (n = 57) by B-CLL cells was
determined using a FACScan (Becton Dickinson, Heidelberg, Germany).
B-CLL cells were specifically detected and phenotyped by examining
coexpression of CD19 and the antigen under investigation with two-color
flow cytometry.
Detection of nuclear proliferation antigen Ki-67.
For detection of Ki-67, B-CLL cells were fixed at room temperature
using Reagent A (Fix & Perm Cell Permeabilisation Kit; An der Grub
GmbH, Kaumberg, Austria) and fixed at 4°C in precooled absolute
methanol. Cells were washed once in cold phosphate-buffered saline
(PBS), permeabilized with Reagent B (Fix & Perm Cell Permeabilisation Kit), and incubated with FITC-labeled MIB-1 MoAbs or the isotype control, respectively. Cells were washed in cold PBS and analyzed for
expression of Ki-67 by flow cytometry.
Analysis of DNA content.
DNA content of B-CLL cells was determined by fixing the cells for 2 hours at 20°C in cold 75% ethanol. Raji cells were used as
positive controls. Cells were washed in PBS (0.1% NaN3,
1% heat-inactivated fetal calf serum [FCS; GIBCO BRL, Eggenstein, Germany]) and treated with cold 0.25% Triton X-100 in PBS for 5 minutes on ice. Subsequently, cells were resuspended in PBS (10 µg/mL
propidium iodide [PI], 0.1% RNase A), incubated at 4°C for 20 minutes, and analyzed by flow cytometry.
Electrophoretic mobility shift assays.
Activity of transcription factor NF-B in B-CLL cells was determined
by a previously described assay using a 32P-labeled
oligonucleotide probe containing a high-affinity NF-B binding
site.11,12 The identity of the specific NF-B-DNA complex in this assay has been previously determined both by antibody supershift and by competition assay.11,12 Jurkat cells
expressing high levels of active NF-B were prepared as positive
controls by treatment for 15 minutes with 200 U/mL tumor necrosis
factor- (TNF-). Briefly, total cell extracts were prepared using
a high-salt detergent buffer (Totex; 20 mmol/L HEPES, pH 7.9, 350 mmol/L NaCl, 20% [wt/vol] glycerol, 1% [wt/vol] NP-40, 1 mmol/L
MgCl2, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 0.5 mmol/L
dithiothreitol [DTT], 0.1% phenylmethyl sulfonyl fluoride [PMSF],
and 1% aprotinin). Cells were harvested by centrifugation, washed once
in ice-cold PBS (Sigma, Deisendorf, Germany) and
resuspended in four cell volumes of Totex buffer. The cell lysate was
incubated on ice for 30 minutes and then centrifuged for 5 minutes at
13,000g at 4°C. The protein content of the supernatant was
determined and equal amounts of protein (10 to 20 µg) were added to a
reaction mixture containing 20 µg bovine serum albumin (BSA; Sigma),
2 µg poly(dI-dC) (Boehringer Mannheim, Mannheim,
Germany), 2 µL buffer D+ (20 mmol/L HEPES, pH 7.9, 20%
glycerin, 100 mmol/L KCl, 0.5 mmol/L EDTA, 0.25% NP-40, 2 mmol/L DTT,
0.1% PMSF), 4 µL buffer F (20% Ficoll 400, 100 mmol/L HEPES, 300 mmol/L KCl, 10 mmol/L DTT, 0.1% PMSF), and 100,000 cpm (Cerenkov) of a
32P-labeled oligonucleotide in a final volume of 20 µL.
Samples were incubated at room temperature for 25 minutes. NF-B
oligonucleotides (Promega, Madison, WI) were labeled using
 -[32P]-ATP (3,000 Ci/mmol; Amersham, Arlington
Heights, IL) and T4 polynucleotide kinase (New England
Biolabs, Beverly, MA). The film was scanned and the relative amounts of
the NF-B DNA complexes were quantified using the NIH-Image
software. Based on these analyses, a negative or a
positive score for NF-B-activity could be assigned to each B-CLL
sample.
Cell stimulation.
B-CLL cells (2 × 105/well), with (n = 9) and without
(n = 10) 11q deletion, were cultured in 200 µL RPMI 1640 medium (10%
FCS) in a 96-well plate. Cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA; Sigma), 200 U/mL recombinant IL-2
(rIL-2; PromoCell GmbH, Heidelberg, Germany), 10 mg/mL
antihuman IgM F(ab)2 fragments (Dako), or antihuman IgM
F(ab)2 fragments in combination with rIL-2. B-CLL cells
stimulated with PMA served as positive control and cells cultured in
medium witout mitogens were used as negative control. B-CLL cells were
incubated in triplicate for 48 hours at 37°C. Response to the
stimuli was quantified using a colorimetric proliferation assay (EZ4U;
Biomedica GmbH, Wien, Austria) based on MTT
(tetrazolium13). This assay was performed according to the instructions of the manufacturer. Absorbance was
measured at 450 nm using a MRX microplate reader (Dynatech, Denkendorf,
Germany).
Statistical methods.
Significance of the differences of the mean fluorescence between B-CLL
cells was determined using the Mann-Whitney-U test. Overall survival
was measured from the time of diagnosis until death from any cause.
Significance of the difference of the overall survival of patients was
determined using the log-rank test. P values less than .05 were
considered significant.
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RESULTS |
Differential expression pattern of cell surface antigens by B-CLL cells
with or without 11q deletion.
To define the expression pattern of functionally relevant cell surface
antigens on B-CLL cells, two-color flow cytometry was performed with a
large panel of MoAbs (n = 57) and an anti-CD19 MoAb. Differential
staining results obtained with each antibody were validated using
defined positive control cell preparations. The mean fluorescence of
each marker was determined as a measurement of the relative amount of
the antigen on the cell surface (Fig 1). All cells had an antigen
pattern characteristic for B-CLL cells, ie, they coexpressed CD19, CD5,
and CD23 and had low levels of cell surface Igs. Markers could be
divided into groups of high (++), low (+), and no () expression
on B-CLL cells (Fig 1). B-CLL cells were most intensively stained by
MoAbs against the B-cell antigens CD20 and CD24 and by antibodies
against CD37, CD43, CD44, CD45, and CD50. In contrast, a panel of
functionally important molecules, such as CD40L, B7.1 (CD80), B7.2
(CD86), and Fas/Apo-1 (CD95), was not found on B-CLL cells (Fig 1).
B-CLL cells of most patients expressed the integrins 3 (CD49c) and
5 (CD49e) but lacked the corresponding integrin 1 chain (CD29).
Among all 57 antigens examined, 12 markers showed a differential
expression pattern on B-CLL cells with or without 11q deletion (Fig 1).
CD6, the integrins L (CD11a), X (CD11c),
and 2 (CD18), PECAM-1 (CD31), the complement receptor 1 (CD35),
CD39, the leukocyte common antigen (CD45), CD48, and LFA-3 (CD58) were
expressed at significantly lower levels on 11q-deleted B-CLL cells than
on B-CLL cells that lacked a 11q deletion. As shown in
Table 1, the differences of the expression
levels of these 12 antigens were significant. CD62L and CD71 were also
differentially expressed but were found only on a few of the B-CLL
cases examined.
Flow cytometric analyses also showed morphological differences between
B-CLL cells with and without 11q deletion. As determined by the forward
scatter (FSC), B-CLL cells with 11q deletion were on the average 11%
smaller than B-CLL cells without 11q deletion (P < .05).
Correlation of levels of antigen expression with survival of the
patients.
To determine the possible relevance of the differential antigen
expression of B-CLL cells for the clinical course of the disease, the
overall survival times of the patients were determined. Patients with
11q deletion had a significantly reduced survival compared with
patients without 11q deletion (P = .0038; data not shown). Among all antigens examined, expression levels of CD45 and CD49d correlated significantly with overall survival of the patients (Fig 2A
and B). B-CLL cells of patients who died during follow-up had lower
levels of CD45 (P = .017) and higher levels of CD49d (P = .0051) than the leukemic cells of patients who were still alive (Fig
2A). When only cases with 11q deletion were analyzed, CD45 levels and
survival no longer correlated, whereas CD49d was still higher expressed
on cells of patients who died (P = .0055; Fig 2A).
To further evaluate the clinical relevance of the CD45 and CD49d
levels, all patients were divided into groups depending on the
expression intensity of these antigens on the B-CLL cells (see also
Materials and Methods). As shown in Fig 2B, patients with
CD45low-positive B-CLL cells had a significantly reduced
overall survival compared with patients with
CD45high-positive leukemic cells (P = .032). Furthermore, patients with CD49dlow-positive B-CLL
cells had shorter survival times than patients with
CD49dnegative B-CLL cells (P = .0012).
Growth fraction, cell cycle position, and NF-B
expression of B-CLL cells with or without 11q deletion.
B-CLL cells with or without 11q deletion did not differ in their growth
fraction or cell cycle position. A total of 0.7% of B-CLL
cells with (n = 9) and 1.3% of B-CLL cells without 11q deletion (n = 8) expressed the nuclear proliferation antigen Ki-67. Accordingly, 98.9% of B-CLL cells with and 98.5% without 11q deletion were found
to be in G0/G1-phase of the cell cycle. In contrast, of the Raji cells
serving as positive control, 97% were positive for Ki-67, 50% in
G0/G1-phase, 30% in S-phase, and 20% in G2/M-phase of the cell cycle.
We further investigated whether 11q deletion in B-CLL cells was
correlated with a differential expression of the transcription factor
NF-B. NF-B is a central mediator of the human immune response
regulating the expression of various immune-modulatory molecules, among
them adhesion receptors such as intercellular adhesion molecule (ICAM)
1, vascular adhesion molecule (VCAM) 1, and endothelial adhesion
molecule (ELAM).13 Using a 32P-labeled
oligonucleotide probe containing a high-affinity NF-B binding site,
B-CLL cells of different patients were shown to contain various
expression levels of NF-B (Fig 3).
However, no correlation was observed between the level of
NF-B-expression and the presence or absence of a 11q deletion.
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| Fig 3.
DNA-binding activity of the transcription factor NF-B
by B-CLL cells with or without 11q deletion detected using a
32P-labeled oligonucleotide probe containing a
high-affinity NF-B binding site. Shown are also the scores of
NF-B activity. The Jurkat cells stimulated with TNF- served as
positive control (+) and unstimulated Jurkat cells were used as
negative control () for NF-B-activity. The solid arrowhead
indicates the position of NF-B DNA complexes. () Nonspecific
activity binding to the probe. The open arrowhead shows unbound
oligonucleotide.
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Stimulatory response of B-CLL cells with or without 11q deletion.
We investigated the response of B-CLL cells with or without 11q
deletion to various mitogenic stimuli (Fig
4). All B-CLL cells were stimulated most strongly by PMA, whereas
anti-IgM alone or in combination with rIL-2 exerted an intermediate
stimulation of the B-CLL cells. rIL-2 alone resulted in a low-grade
stimulation. As shown in Fig 4, no significant difference was noted in
the cellular response of B-CLL cells with or without 11q deletion to
these mitogenic substances.
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| Fig 4.
In vitro response of B-CLL cells with () and without
() 11q deletion to mitogenic stimuli. The control cells received
medium without mitogens. Percentiles are marked by the box diagram as
described in the legend to Fig 2A.
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DISCUSSION |
Recent cytogenetic studies have suggested that deletions in chromosome
bands 11q22-q23 identify a new subset of B-CLL, characterized by
extensive lymph node involvement, rapid disease progression, and short
survival times.2,3 The present study demonstrates a
differential expression pattern of functionally relevant adhesion molecules and cell signaling receptors on B-CLL cells with or without
11q deletion.
Coexpression of CD19, CD5, CD23, and cell surface Igs confirmed that
all leukemic cells were truly B-CLL cells. The finding that the
integrins L/2 (CD11a/CD18) and X/2
(CD11c/CD18) are relatively weakly expressed by all B-CLL cells is
consistent with earlier reports.14-16 Furthermore,
expression of CD43,17 CD44,15,16 and
CD5018 and lack of CD51 and CD61 expression19
by B-CLL cells is in agreement with published findings. Therefore,
results obtained by the technique used here to analyze antigen
expression levels correlate well with findings of other investigators.
The underlying cause for the observation that B-CLL cells expressed
CD49c and CD49e (integrin 3 and 5, respectively) but lacked the
corresponding CD29 (integrin 1 chain) is unclear. Yet, this finding
is consistent with earlier reports. Overexpression of
integrin--chains contrasted to low or negative expression levels of
integrin-1-chains in CLL, whereas acute lymphocytic leukemia and
multiple myeloma cells had a balanced expression pattern of these
adhesion molecules.20 Furthermore, lymphoid cells within
the mantle zone of the lymphoid follicle that may represent normal
counterparts of B-CLL cells were found to have increased CD49d levels
and decreased expression of CD29.21 As suggested by
Möller et al,20 these findings may indicate that on
these lymphoid cells integrin 4 chains may associate with other
-integrins than 1-integrins.
Phenotypic analyses confirmed the working hypothesis of the present
study that B-CLL cells with or without 11q deletion differentially express functionally relevant cell surface molecules. All of these markers CD6, CD11a, CD11c, CD18, CD31, CD35, CD39, CD45, CD48, and CD58
were found at lower levels on cells with 11q deletion than on leukemic
cells that lacked this chromosomal aberration. These antigens exert a
broad variety of cellular functions. Yet, a property common to all of
these molecules except for the complement receptor
CD3522is that they are able to mediate cell adhesion.
CD6 belongs, together with CD5, to the scavenger-receptor-cystein-rich
(SRCR) super family23,24 and acts as a receptor for the
activated leukocyte cell adhesion molecule (ALCAM). Binding via CD6
appears to influence the fate of B-CLL cells, because ligation of CD6
protects these cells from anti-IgM-induced apoptosis by increasing the
Bcl-2/Bax ratio.25
The integrins L/2 (CD11a/CD18) and
X/2 (CD11c/CD18) are essential for mediating
leukocyte migration.26,27 The importance of these adhesion
molecules for the clinical course of the B-CLL disease has not been
clarified unambiguously. Lack of integrin 2 chains was shown to be
associated with more favorable clinical features.28,29
Consistently, expression of integrin 215 and integrin
X30 correlated with more advanced disease
stages and with a diffuse bone marrow infiltration, respectively. In contrast, other publications demonstrated that low levels of integrin 216,31 and integrin X32 on
B-CLL cells were associated with higher mortality. Data presented in
the current study are in agreement with these latter reports. We
detected lower expression levels of integrin L/2
within the group of B-CLL cells with 11q deletion that previously was
shown to correlate with extensive lymphadenopathy and early disease
progression.2
PECAM-1 (CD31) is a broadly expressed adhesion molecule mediating
homophilic and heterophilic intercellular binding.33,34 Similarly, CD39 is an activation-dependent molecule that mediates rapid
integrin L/2-dependent and -independent homotypic
adhesion.35,36 So far, the relevance of PECAM-1 and CD39
for the clinical presentation of a B-CLL has not been determined.
It was interesting to note that two ligands for the T-cell adhesion
molecule CD2, ie, CD48 and CD58, were expressed at lower levels on
B-CLL cells with 11q deletion. These receptors are essential for
inducing antigen-dependent and antigen-independent immune and
inflammatory T-cell responses.26,27
In addition, CD45, the transmembrane protein tyrosine phosphatase
expressed on nucleated hematopoietic cells, was reported to initiate
cell adhesion. Triggering via CD45 induces an
integrin-L2/ICAM-1- and -2-mediated cell
aggregation.37 This homotypic adhesion leads also to a
coclustering of CD45 and integrin L2.37
It is not clear whether the decreased expression levels of adhesion
molecules on B-CLL cells with 11q deletion can help to explain the
pathophysiology of this subentity. Possibly, the altered antigen
pattern of B-CLL cells with 11q deletion could influence the migratory
properties of these cells within lymphoid organs and in peripheral
blood. This might participate in the development of the increased
lymphadenopathy that is typical for B-CLL cases with 11q deletion.
In addition, the differential expression of antigens by 11q-deleted
B-CLL cells may impair the cellular immune defense, because some of
these molecules are relevant for the recognition of target cells by
cytolytic T cells.26 Downregulation of CD58 by T-cell leukemia38,39 and Burkitt lymphoma cells40 has
been correlated with unsusceptibility to killing by cytotoxic
lymphocytes. B-CLL cells with or without 11q deletion did not express
B7.1 (CD80), B7.2 (CD86), and Fas/Apo-1 (CD95). B7.1 and B7.2 are
costimulatory molecules essential for inducing a T-cell response and
Fas/Apo-1 is an important receptor for cytotoxic T cells leading to
programmed cell death (apoptosis) of the target cells.41,42
In addition, even in the presence of B7-1 or B7-2 on tumor cells, CD48
appears to be required for the generation of T-cell-specific antitumor immunity.43 Taken together, absence or decreased expression of molecules mediating T-cell cytolysis on B-CLL cells with 11q deletion might contribute to the impairment of the cellular immune defense against these leukemic cells.
Our observation that patients with 11q deletion had a reduced overall
survival has been previously reported.2 Data of the present
study support the notion that differential expression levels of CD45
and CD49d on B-CLL cells may also influence the clinical outcome of the
disease. Patients with CD45low-positive or
CD49dlow-positive B-CLL cells had a significantly reduced
overall survival compared with patients with
CD45high-positive and CD49dnegative leukemic
cells, respectively. When only B-CLL cases with 11q deletion were
analyzed, levels of CD49d but not those of CD45 were still relevant for
poor survival. Most likely, lower CD45 levels correlated with reduced
survival of all patients examined, because this antigen alteration was
the most significant marker of B-CLL cells with 11q deletion.
Therefore, reduction of CD45 levels does not appear to be a marker for
poor survival independently of 11q deletion. In contrast, differential
expression of CD49d further divided the B-CLL cases with 11q deletion
into groups with differing overall survival. Thus, low-level CD49d
expression may represent a factor of poor survival that is independent
of 11q deletion. Support for the latter interpretation comes from the
observation that expression of CD49d correlated with advanced disease
stages.9 In addition, expression of 1, 2, and 3 integrins on B-CLL cells was shown to be associated with poor prognosis
and with splenomegaly.28
We did not observe significant differences in the growth fraction, cell
cycle position, expression of the transcription factor NF-B, or
stimulatory response of B-CLL cells with or without 11q deletion. The
current study sets the basis for further investigations aiming to
define which in vitro detectable function of B-CLL cells with 11q
deletion is altered and is responsible for the poor clinical outcome of
this entity.
It is yet unclear if there is a unifying cause for the decreased
expression levels of the functionally diverse receptors on B-CLL cells.
Conceivably, loss of a putative tumor-suppressor gene by the 11q
deletion could represent a common defect impairing several cellular
pathways in B-CLL cells. Further identification of these cell signaling
aberrations in B-CLL cells with 11q deletion might help to explain the
pathogenesis of this clinically relevant B-CLL subentity.
| |
FOOTNOTES |
Submitted April 2, 1998;
accepted September 15, 1998.
Supported by grants from the Wilhelm Sander Stiftung (94.042.1), the
Tumorzentrum Heidelberg/Mannheim (I/I.1), the Deutsche Forschungsgemeinschaft (DFG Pa 611/1-2 and SFB 364 project C2), and the
Deutsche Krebshilfe (10-0917-DöI). F.S. is supported by a
research fellowship of the DFG (Schr 318/3-1).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Folke Schriever, MD, Virchow-University
Hospital, Department of Hematology and Oncology, Augustenburger Platz
1, 13353 Berlin, Germany; e-mail: folke.schriever{at}charlte.de.
| |
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Abstract
Introduction