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Prognostic significance of bcl-xL gene expression and apoptotic cell counts in follicular
lymphoma
Wei-Li Zhao1, Marjan Ertault Daneshpouy1, Nicolas Mounier1, Josette Brière1,
Christophe Leboeuf1, Louis-François Plassa2, Elisabeth Turpin2, Jean-Michel Cayuela3,
Jean-Claude Ameisen4, Christian Gisselbrecht1, and Anne Janin1,5
1
ERM-0220, Institut Universitaire d'Hématologie, Université Paris VII, Hôpital Saint-
Louis, AP-HP, 75475 Paris, France.
2
Service de Biochimie B and CNRS UPR-9051, Hôpital Saint-Louis, AP-HP, 75475
Paris, France.
3
INSERM U462, Laboratoire d'Hématologie, Hôpital Saint-Louis, AP-HP, 75475 Paris,
France.
4
EMI-U 9922 INSERM/Université Paris VII, IFR 02, Hôpital Bichat-Claude-Bernard,
75877 Paris, France
5
Corresponding author: Anne Janin, ERM-0220, Institut Universitaire d'Hématologie,
Université Paris VII, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75475 Paris
Cedex
10,
France.
Tel:
0033-1-42499933.
Fax:
0033-1-42494922.
E-mail:
[email protected]
This work was supported in part by INSERM, Université Paris VII and ARC
subvention 9052.
Word count: Text 1137, Abstract 136
Key words: bcl-xL, follicular lymphoma, prognosis
1
Copyright (c) 2003 American Society of Hematology
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Abstract
Bcl-xL, a member of the Bcl-2 family, exerts an anti-apoptotic effect on lymphocytes. To
assess its clinical significance in follicular lymphoma patients, real-time quantitative RT-PCR
analysis of bcl-xL gene expression was investigated in whole lymph node sections and laser
microdissected lymphoma cells of 27 patients. Compared to 10 patients with reactive
follicular hyperplasia, bcl-xL gene was overexpressed in follicular lymphoma patients with a
higher level in microdissected lymphoma cells. Bcl-xL gene level correlated with the number
of apoptotic lymphoma cells labeled by TUNEL assays (r=-0.7736). Clinically, high bcl-xL
level was significantly associated with multiple extranodal involvement (P=0.0020), elevated
lactate dehydrogenase level (P=0.0478), and high-risk international prognostic index
(P=0.0235). Moreover, bcl-xL gene overexpression was linked to short overall survival
(P=0.0129). The value of bcl-xL gene expression as a prognostic marker in follicular
lymphoma should thus be considered.
Corresponding author’s email: [email protected]
Acknowledgments: The authors thank Christian Bastard for critical review of the manuscript,
Janet Jacobson for English editing, Luc Legrès and Allison Desveaux for engineering
assistance, Sylvie Corre for data management, and Laboratoire Photo d’Hématologie for
illustrations.
2
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Introduction
Follicular lymphoma is characterized primarily by defects in cell apoptosis rather than cell
proliferation.1 Bcl-2, involved in follicular lymphoma, prevents cell apoptosis by blocking
the mitochondrial pathway.2,3 The long isoform of bcl-x, bcl-xL, acts as another important
anti- apoptotic factor of bcl-2 family and is expressed in follicular lymphoma.4
Experimentally, functional differences exist between bcl-2 and bcl-xL. In bcl-2-deficient mice
there is massive death of mature lymphocytes, while immature lymphocytes undergo
apoptosis in bcl-x-deficent mice.5 In vitro, bcl-2 maintains survival of resting T cells when
bcl-xL prevents apoptosis of activated T cells.6 Importantly, when bcl-xL is downregulated,
even constitutive level of bcl-2 is maintained, follicular lymphoma cells undergo apoptosis. 7
Therefore, bcl-xL seems to play a key role in follicular lymphoma. However, the relation
between bcl-xL gene expression and clinical features in patients with follicular lymphoma has
not yet been reported.
In the present study, we assessed bcl-xL gene expression and apoptotic cell counts in 27
follicular lymphoma patients using real-time quantitative reverse transcriptase-polymerase
chain reaction (RT-PCR) and terminal deoxytransferase-catalyzed DNA nick end labeling
(TUNEL) assays. Bcl-xL gene overexpression in lymphoma cells was related to low number
of apoptotic cells. Both factors reflect poor disease outcome in follicular lymphoma patients.
Patients and Methods
Patients
Twenty-seven follicular lymphoma patients, 16 males and 11 females, aged from 21 to 82
years (median 51 years), with available frozen tumor specimen at diagnosis were included in
this study. Histological diagnoses were established according to the WHO classifications.8
Ten age- and sex-matched patients with reactive follicular hyperplasia were referred as
controls.
Written informed consent was obtained from all patients for this study, in accordance with
our Institution's regulation.
Tissue samples
Lymph nodes, surgically removed for diagnostic purposes, were immediately cut into two
parts: one was fixed in formaldehyde and further processed for paraffin embedding, the other
was snap frozen and stored in –80°C. A section was cut from each tissue block for
systemically light microscopic control.
3
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Laser microdissection
Seven-µm frozen sections were prepared, immediately fixed in 70% ethanol, and stained
with hematoxylin-eosin. The sections were dehydrated through a graded series of ethanol and
xylene and air-dried. For each patient, approximately 1500 lymphoma cells, corresponding to
an average surface of 450,000µm2, were laser microdissected (PALM, Bernried, Germany)
and catapulted into tubes for RNA extraction.
RNA extraction and cDNA synthesis
Total RNA was extracted from 10 frozen sections of 10 µm thickness using the acidguanidinium thiocyanate-phenol-chloroform method.9 First-strand cDNA was synthesized
from 1µg of total RNA using Superscript II reverse transcriptase (Invitrogen Corporation, CA,
USA) and random hexamers according to the manufacturer's instructions.
Total RNA from microdissected lymphoma cells was independently extracted and
immediately reverse transcribed following the same protocol.
Real-time quantitative RT-PCR
Quantitative PCR was performed on ABI PRISM 7700 system using the Pre-Developed
TaqMan Assay Reagent specific to human bcl-xL and human transcription factor IID/TATA
binding factor (TBP) gene expression quantification (PE Applied Biosystems, Warrington,
UK), according to the manufacturer's instructions. TBP gene was used as an endogenous
control.
Quantification results were expressed in terms of the threshold cycle value (CT) according
to the baseline adjusted to 0.05. Jurkat cell, which expresses bcl-xL gene,10 was considered as
calibrator. The comparative CT method (PE Applied Biosystems) was used to quantify
relative bcl-xL expression compared to Jurkat cell. Briefly, the CT values were averaged for
each triplicate. The differences between the mean CT values of bcl-xL and those of TBP was
calculated CTsample=CT bcl-xL - CT TBP, and the same as CT for Jurkat cell ( CTcalibrator
=CT bcl-xL – CT TBP). Final results, the sample/calibrator ratio, expressed as N-fold
differences of bcl-xL expression in the samples compared with Jurkat cell, were determined
as 2 –(
CTsample- CTcalibrator)
.
TUNEL assay
4
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Cell apoptosis was confirmed by in situ detection of fragmented DNA, using TUNEL
assay,
11
on deparaffinized 5-µm-thick sections, treated with proteinase K (20 µg/mL) for 15
minutes at room temperature.
The number of apoptotic lymphoma cells was assessed blindly by 2 pathologists, who did
not know the bcl-xL levels, on an Olympus Provis AX 70 microscope, with wide-field
eyepiece number 26.5. At ×400 magnification, this wide-field eyepiece provided a field size
of 0.344mm2. Results are expressed as the mean number of cells per field at ×400
magnification.
Statistical analyses
Patient characteristics were compared using Chi-square and Fisher’s exact tests for
categorical variables, and Wilcoxon’s test for continuous variables. Overall survival was
measured from the date of diagnosis to either death from any cause or the stopping date of
December 31, 2002. When the stopping date was not reached, the data were censored at the
date of the last follow-up evaluation. Survival functions were estimated using the KaplanMeier method and compared by Log-rank test. Multivariate survival analysis was performed
using a Cox regression model. Differences were considered significant when the two-sided P
value was < 0.05. All statistical analyses were performed using SAS 8.2 (SAS Institute Inc,
Cary, NC, USA) and Splus 2000 (MathSoft Inc, Berkeley, CA, USA) softwares.
Results and Discussion
Follicular lymphoma cells overexpressed bcl-xL
Compared to the patients with reactive follicular hyperplasia, bcl-xL gene was
overexpressed in whole lymph node sections [median 5.5 (range 1.2-28.1) vs 2.3 (1.6-3.2),
P=0.0115].
Since lymph node sections contain not only lymphoma cells, but also stromal cells,
lymphoma cells were further isolated by laser microdissection. The higher level of bcl-xL in
microdissected lymphoma cells compared with whole lymph node sections [median 11.0
(range 1.9-59.6) vs 5.5 (1.2-28.1), P=0.0379] demonstrated that bcl-xL was expressed by
lymphoma cells.
Bcl-xL gene overexpression corresponded to low numbers of apoptotic lymphoma cells
5
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The number of the apoptotic lymphoma cells on TUNEL assays (Figure 1) ranged from 5
to 108 (median 38) and inversely correlated with bcl-xL gene expression level both on lymph
node sections (r=-0.7552) and microdissected lymphoma cells (r=-0.7736).
This is in accordance with experimental data showing that bcl-xL gene prevents apoptosis.2
Bcl-xL-overexpressing mice showed enhanced survival of B cells,
12
while bcl-x-deficient
5
mice had extensive lymphocyte apoptosis. In vitro, B lymphocytes expressing high level of
bcl-xL were resistant to Fas-induced cell apoptosis.13 In contrast, downregulation of bcl-xL
enhanced TGF -induced apoptosis of cultured B lymphoma cells.14
Bcl-xL reflected poor disease outcome in follicular lymphoma
Bcl-xL overexpression and low numbers of apoptotic lymphoma cells were associated with
multiple extranodal sites, elevated LDH level, and high risk international prognostic index in
the 27 follicular lymphoma patients (Table 1). Moreover, both factors were significantly
related to short overall survival (Figure 1).
To our knowledge, this is the only study of bcl-xL gene expression on disease outcome in
follicular lymphoma patients. Experimental data on B lymphoma cells demonstrated that bclxL gene overexpression caused resistance to apoptosis induced by anti-IgM or by
chemotherapeutic agents.15,16 This mechanism might favor lymphoma development and result
in poor prognosis in follicular lymphoma patients. Further studies are needed to assess the
value of bcl-xL as a therapeutic target in high risk follicular lymphoma patients.
6
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References
1. Reed CJ. Apoptosis and cancer: strategies for integrating programmed cell death.
Semin Hematol. 2000;37:9-16.
2. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and
cell death. Blood. 1996;88:386-401.
3. Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in
human follicular lymphoma. Science. 1985;228:1440-1443.
4. Xerri L, Parc P, Brousset P, Schlaifer D, Hassoun J, Reed JC, et al. Predominant
expression of the long isoform of Bcl-x (Bcl-xL) in human lymphomas. Br J Haematol.
1996;92:900-906.
5. Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Negishi I, et al. Massive
cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science.
1995;267:1506-1510.
6. Ma A, Pena JC, Chang B, Margosian E, Davidson L, Alt FW, et al. Bclx regulates
the survival of double-positive thymocytes. Proc Natl Acad Sci U S A. 1995;92:4763-4767.
7. Ghia P, Boussiotis VA, Schultze JL, Cardoso AA, Dorfman DM, Gribben JG, et al.
Unbalanced expression of bcl-2 family proteins in follicular lymphoma: contribution of CD40
signaling in promoting survival. Blood. 1998;91:244-251.
8. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J.
Lymphoma classification--from controversy to consensus: the R.E.A.L. and WHO
Classification of lymphoid neoplasms. Ann Oncol. 2000;11:3-10.
9.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.
10. Gao G, Dou QP. G(1) phase-dependent expression of bcl-2 mRNA and protein
correlates with chemoresistance of human cancer cells. Mol Pharmacol. 2000;58:1001-1010.
11. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death
in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119:493-501.
12. Grillot DA, Merino R, Pena JC, Fanslow WC, Finkelman FD, Thompson CB, et
al. bcl-x exhibits regulated expression during B cell development and activation and
modulates lymphocyte survival in transgenic mice. J Exp Med. 1996;183:381-391.
13. Wurster AL, Rodgers VL, White MF, Rothstein TL, Grusby MJ. Interleukin-4mediated protection of primary B cells from apoptosis through Stat6-dependent up-regulation
of Bcl-xL. J Biol Chem. 2002;277:27169-27175.
7
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14.
Saltzman A, Munro R, Searfoss G, Franks C, Jaye M, Ivashchenko Y.
Transforming growth factor-beta-mediated apoptosis in the Ramos B-lymphoma cell line is
accompanied by caspase activation and Bcl-XL downregulation. Exp Cell Res. 1998;242:244254.
15. Lam EW, Choi MS, van der Sman J, Burbidge SA, Klaus GG. Modulation of E2F
activity via signaling through surface IgM and CD40 receptors in WEHI-231 B lymphoma
cells. J Biol Chem. 1998;273:10051-10057.
16. Simonian PL, Grillot DA, Nunez G. Bcl-2 and Bcl-XL can differentially block
chemotherapy-induced cell death. Blood. 1997;90:1208-1216.
8
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Figure 1 TUNEL assays on two follicular lymphoma cases.
9
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Figure legand
Figure 1 TUNEL assays on two follicular lymphoma cases.
Case 1 (A and B) Follicular lymphoma with high level of bcl-xL gene expression.
A: A malignant follicle (surrounded by dotted line) is negative. TUNEL assay x 200.
B: Higher magnification of the malignant follicle: Typical malignant centrocytic cells, with a
cleaved nucleus (arrows) are negative. TUNEL assay x 700.
Case 2 (C and D) Follicular lymphoma with low level of bcl-xL gene expression.
C: A malignant follicle (surrounded by dotted line) is formed of cells stained by TUNEL
assay, when normal lymphocytes around the follicle (arrows) are negative. TUNEL assay x
300.
D: Higher magnification of the malignant follicle: Typical malignant centrocytic cells, with a
cleaved nucleus (arrows) stained. TUNEL assay x 800.
E: Kaplan-Meier survival curve for follicular lymphoma patients according to bcl-xL gene
expression. The overall survival of the patients with bcl-xL gene level above the median (red
line) was significantly shorter than that of patients with bcl-xL level below and equal to the
median level (green line).
F: Kaplan-Meier survival curve for follicular lymphoma patients according to the number of
apoptotic cells. The overall survival of the patients with number of apoptotic lymphoma cells
less than median (red line) was significantly shorter than that of patients with number of
apoptotic cells more than or equal to the median (green line).
10
Table 1 Bcl-xL gene expression, apoptotic lymphoma cell counts and their relation to clinical characteristics in follicular lymphoma patients (n=27)
No. (%)
Characteristics
No. apoptotic lymphoma cells
in lymph node sectionsA
in microdissected cellsA
by TUNEL assay
Median
Range
P value
Median
Range
P value
Median
Range
P value
0.6384
9.1
1.9-59.6
0.9685
40
5-108
0.1596
12.5
5.4-45.9
29
5-58
8.1
2.9-15.2
45
38-96
12.5
1.9-59.6
28
5-108
8.1
1.9-44.7
46
5-108
28.9
5.4-59.6
21
5-58
8.1
1.9-45.9
45
5-108
18.9
6.7-59.6
24
5-58
8.1
1.9-44.7
46
5-108
28.3
5.4-59.6
22
5-58
0-1
17 (63%)
5.4
1.2-28.1
2-4
10 (37%)
6.8
3.2-22.4
I/II
5 (19%)
3.5
1.5-5.4
III/IV
22 (81%)
6.9
1.2-28.1
No. extranodal involvement 0-1
16 (59%)
4.5
1.2-26.5
>1
11 (41%)
13.9
3.2-28.1
<=normal
17 (63%)
5.1
1.2-26.8
>normal
10 (37%)
11.9
3.2-28.1
16 (59%)
4.7
1.2-26.8
11 (41%)
13.2
3.2-28.1
Ann Arbor Stage
Serum LDH
IPI Low risk-Intermediate low risk
Intermediate high risk-High risk
A
bcl-xL gene expression
0.0873
0.0028
0.0459
0.0360
0.1365
0.0020
0.0478
0.0235
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Performance status
bcl-xL gene expression
0.1832
0.0040
0.0280
0.0143
- Sample/Calibrator Ratio
TUNEL - terminal deoxytransferase-catalyzed DNA nick end labeling
LDH - lactate dehydrogenase
IPI - international prognostic index
11
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Prepublished online September 11, 2003;
doi:10.1182/blood-2003-06-1901
Prognostic significance of bcl-xL gene expression and apoptotic cell
counts in follicular lymphoma
Wei-Li Zhao, Marjan Ertault Daneshpouy, Nicolas Mounier, Josette Briere, Christophe Leboeuf,
Louis-Francois Plassa, Elisabeth Turpin, Jean-Michel Cayuela, Jean-Claude Ameisen, Christian
Gisselbrecht and Anne Janin
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